THE	
  EQUINE	
  PULMONARY	
  MICROVASCULATURE	
  AND	
  ITS	
  POTENTIAL	
  
	
  ROLE	
  IN	
  EXERCISE-­‐INDUCED	
  PULMONARY	
  HEMORRHAGE	
  
	
  
By	
  
	
  
Alice	
  Stack	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
A	
  DISSERTATION	
  
	
  
Submitted	
  to	
  	
  
Michigan	
  State	
  University	
  
in	
  partial	
  fulfillment	
  of	
  the	
  requirements	
  
for	
  the	
  degree	
  of	
  	
  
	
  
Comparative	
  Medicine	
  and	
  Integrative	
  Biology	
  –	
  Doctor	
  of	
  Philosophy	
  
	
  
2014	
  	
  

ABSTRACT	
  
	
  
STUDY	
  OF	
  THE	
  EQUINE	
  PULMONARY	
  MICROVASCULATURE	
  AND	
  ITS	
  	
  
ROLE	
  IN	
  EXERCISE-­‐INDUCED	
  PULMONARY	
  HEMORRHAGE	
  
	
  
By	
  
	
  
Alice	
  Stack	
  
	
  
Exercise-­‐induced	
  pulmonary	
  hemorrhage	
  (EIPH)	
  is	
  diagnosed	
  by	
  the	
  presence	
  of	
  frank	
  
blood	
  in	
  the	
  airways	
  following	
  a	
  bout	
  of	
  intense	
  exercise.	
  EIPH	
  affects	
  all	
  racehorses,	
  and	
  
has	
  been	
  diagnosed	
  in	
  other	
  athletic	
  species,	
  including	
  humans.	
  EIPH	
  is	
  associated	
  with	
  
impaired	
  racing	
  performance	
  and	
  significant	
  pulmonary	
  pathology	
  in	
  the	
  caudodorsal	
  lung,	
  
while	
  the	
  cranioventral	
  lung	
  is	
  spared.	
  Lesions	
  include	
  hemosiderin	
  accumulation,	
  
interstitial	
  and	
  septal	
  fibrosis,	
  angiogenesis,	
  capillary	
  wall	
  disruption	
  and	
  remodeling	
  of	
  
small-­‐caliber	
  (100	
  –	
  200	
  μm	
  diameter)	
  intralobular	
  pulmonary	
  veins.	
  High	
  pressures	
  in	
  the	
  
pulmonary	
  circulation	
  of	
  the	
  exercising	
  horse	
  cause	
  capillary	
  stress	
  failure,	
  resulting	
  in	
  the	
  
main	
  symptom:	
  hemorrhage.	
  Stress	
  failure	
  alone	
  does	
  not	
  account	
  for	
  all	
  EIPH	
  lesions,	
  and	
  
in	
  particular,	
  venous	
  remodeling.	
  Nor	
  does	
  it	
  explain	
  regional	
  predilection	
  of	
  EIPH	
  
pathology.	
  EIPH	
  pathogenesis	
  awaits	
  complete	
  explanation	
  at	
  this	
  time.	
  Capillary	
  pressure	
  
is	
  determined	
  in	
  part	
  by	
  resistance	
  to	
  flow	
  in	
  the	
  arteries	
  and	
  veins	
  that	
  supply	
  and	
  drain	
  a	
  
capillary	
  bed.	
  Decreased	
  arterial,	
  and	
  increased	
  venous	
  resistance	
  are	
  conditions	
  under	
  
which	
  capillary	
  pressures	
  will	
  increase,	
  and	
  may	
  approach	
  arterial	
  pressure	
  values.	
  	
  
I	
  hypothesize	
  that	
  a	
  combination	
  of	
  increased	
  blood	
  flow	
  during	
  exercise	
  to	
  
caudodorsal	
  lung,	
  coupled	
  with	
  exercise-­‐associated	
  alterations	
  in	
  vessel	
  tone	
  provides	
  
transient	
  but	
  sufficient	
  hemodynamic	
  stimuli	
  to	
  initiate	
  venous	
  remodeling	
  in	
  this	
  region	
  
only.	
  Therefore	
  the	
  impact	
  of	
  ongoing	
  venous	
  remodeling	
  would	
  be	
  to	
  reduce	
  venous	
  wall	
  
compliance,	
  thereby	
  increasing	
  venous	
  resistance	
  to	
  flow	
  and	
  increasing	
  capillary	
  
	
  

pressures	
  in	
  the	
  caudodorsal	
  lung.	
  Capillary	
  wall	
  stress	
  failure,	
  hemorrhage,	
  and	
  EIPH	
  
result.	
  	
  
The	
  characteristics	
  of	
  small	
  vessels	
  that	
  determine	
  capillary	
  pressure	
  include	
  
mechanical	
  and	
  reactivity	
  profiles	
  of	
  small	
  arteries	
  and	
  veins	
  that	
  work	
  to	
  effect	
  passive	
  
and	
  active	
  changes	
  in	
  vessel	
  diameter,	
  along	
  with	
  any	
  impact	
  of	
  remodeling	
  on	
  venous	
  wall	
  
compliance.	
  These	
  were	
  evaluated	
  in	
  three	
  studies.	
  The	
  important	
  findings	
  are	
  as	
  follows:	
  
First,	
  regional	
  differences	
  in	
  mechanical	
  properties	
  of	
  arteries	
  and	
  veins	
  exist	
  in	
  
control,	
  unraced	
  horses,	
  which	
  may	
  reflect	
  inhomogeneous	
  blood	
  flow	
  distribution	
  in	
  the	
  
equine	
  lung.	
  Racing	
  is	
  associated	
  with	
  increased	
  stiffness	
  of	
  caudodorsal	
  pulmonary	
  veins	
  
only,	
  despite	
  the	
  absence	
  of	
  severe	
  EIPH	
  pathology.	
  	
  
Second,	
  autonomic	
  control	
  of	
  small,	
  equine	
  pulmonary	
  arteries	
  and	
  veins	
  is	
  not	
  
consistent	
  across	
  lung	
  regions,	
  and	
  the	
  reported	
  differences,	
  when	
  extrapolated	
  to	
  in	
  vivo	
  
exercising	
  conditions	
  of	
  increased	
  sympathetic	
  input,	
  can	
  account	
  for	
  increased	
  capillary	
  
pressure	
  in	
  caudodorsal	
  lung.	
  	
  
Finally,	
  although	
  exercise	
  causes	
  significant	
  alterations	
  in	
  mRNA	
  expression	
  in	
  vein	
  
walls,	
  the	
  changes	
  do	
  not	
  support	
  initiation	
  of	
  a	
  remodeling	
  response	
  after	
  only	
  2	
  weeks	
  of	
  
intense	
  exercise.	
  	
  
These	
  data	
  contribute	
  to	
  the	
  understanding	
  of	
  EIPH	
  pathogenesis,	
  and	
  highlight	
  the	
  
pivotal	
  role	
  of	
  the	
  pulmonary	
  microvasculature,	
  in	
  particular	
  the	
  pulmonary	
  veins,	
  in	
  this	
  
significant,	
  ubiquitous	
  disease.	
  Furthermore,	
  regional	
  differences	
  in	
  mechanical	
  properties	
  
and	
  reactivity	
  profiles	
  of	
  pulmonary	
  vessels	
  of	
  this	
  caliber	
  are	
  not	
  reported	
  in	
  any	
  other	
  
species,	
  and	
  as	
  such,	
  these	
  findings	
  may	
  have	
  farther-­‐reaching	
  applications	
  in	
  the	
  field	
  of	
  
pulmonary	
  vascular	
  biology.	
  	
  

	
  

	
  
	
  
Copyright	
  by	
  
ALICE	
  STACK	
  
2014

	
  

	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
This	
  dissertation	
  is	
  dedicated	
  to	
  Geoff,	
  
in	
  recognition	
  of	
  the	
  many	
  thousands	
  of	
  miles	
  he	
  traveled.	
  
	
  
	
  
	
  
	
  

	
  

	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
v	
  	
  

ACKNKOWLEDGEMENTS	
  
	
  
	
  
	
  
I	
  would	
  like	
  to	
  express	
  my	
  sincere	
  gratitude	
  to	
  my	
  advisor	
  Dr.	
  Frederik	
  J.	
  Derksen	
  for	
  his	
  
unfaltering	
  enthusiasm,	
  mentorship	
  and	
  support.	
  
I	
  also	
  wish	
  to	
  acknowledge	
  and	
  thank	
  the	
  following	
  members	
  of	
  my	
  doctoral	
  committee	
  –	
  
Dr.	
  William	
  F.	
  Jackson,	
  Dr.	
  Kurt	
  J.	
  Williams	
  and	
  Dr.	
  N.	
  Edward	
  Robinson	
  for	
  their	
  guidance	
  
and	
  valuable	
  advice.	
  	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
vi	
  

TABLE	
  OF	
  CONTENTS	
  
	
  
	
  
	
  
	
  
LIST	
  OF	
  TABLES	
  	
  .........................................................................................................................................	
  x	
  
	
  
LIST	
  OF	
  FIGURES	
  	
  .....................................................................................................................................	
  xi	
  
	
  
KEY	
  TO	
  ABBREVIATIONS	
  	
  ...................................................................................................................	
  xiv	
  
	
  
	
  
CHAPTER	
  1	
  ...................................................................................................................................................	
  1	
  
Literature	
  Review	
  	
  .....................................................................................................................................	
  1	
  
	
  
Section	
  1:	
  Exercise-­‐induced	
  pulmonary	
  hemorrhage	
  .................................................	
  1	
  
	
  
History	
  	
  ..............................................................................................................................	
  1	
  
	
  
Epidemiology	
  ..................................................................................................................	
  2	
  
	
  
Risk	
  factors	
  for	
  EIPH	
  	
  ..................................................................................................	
  3	
  
	
  
Clinical	
  features	
  	
  ............................................................................................................	
  4	
  
	
  
Pathology	
  	
  ........................................................................................................................	
  4	
  
	
  
Pathogenesis	
  ...................................................................................................................	
  6	
  
	
  
Treatment	
  	
  .....................................................................................................................	
  10	
  
	
  
Summary	
  	
  ......................................................................................................................	
  	
  12	
  
	
  
Section	
  2:	
  The	
  pulmonary	
  microvasculature	
  	
  ...............................................................	
  13	
  
	
  
Vessel	
  anatomy	
  ............................................................................................................	
  15	
  
Vessel	
  wall	
  mechanical	
  properties	
  ......................................................................	
  17	
  
	
  
Regulation	
  of	
  pulmonary	
  vascular	
  tone	
  ............................................................	
  22	
  
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  
Autonomic	
  control	
  of	
  pulmonary	
  vascular	
  tone	
  ...........................................	
  23	
  
	
  
Regional	
  control	
  of	
  pulmonary	
  vascular	
  tone	
  ................................................	
  25	
  
	
  
U	
  46619	
  ...........................................................................................................................	
  26	
  
	
  
Phenylephrine	
  ..............................................................................................................	
  27	
  
	
  
Isoproterenol	
  ................................................................................................................	
  28	
  
	
  
Methacholine	
  ................................................................................................................	
  28	
  
	
  
Furosemide	
  ...................................................................................................................	
  29	
  
	
  
Summary	
  ........................................................................................................................	
  31	
  
	
  
Section	
  3:	
  Pulmonary	
  venous	
  remodeling	
  ......................................................................	
  32	
  
	
  
Venous	
  remodeling	
  in	
  EIPH	
  ...................................................................................	
  32	
  
	
  
Vascular	
  remodeling	
  .................................................................................................	
  33	
  
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  
Pulmonary	
  venous	
  remodeling	
  –	
  etiopathogenesis	
  ....................................	
  34	
  
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  
Role	
  of	
  venous	
  remodeling	
  in	
  EIPH	
  ....................................................................	
  36	
  
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  
Mechanisms	
  of	
  remodeling	
  ....................................................................................	
  38	
  
	
  
Section	
  4:	
  Experimental	
  technique	
  development	
  .......................................................	
  44	
  
	
  
Wire	
  myography	
  .........................................................................................................	
  44	
  
	
  
APPENDIX	
  .....................................................................................................................................	
  47	
  
	
  
	
  
	
  
	
  
vii	
  

CHAPTER	
  2	
  .................................................................................................................................................	
  51	
  
Lung	
  region	
  and	
  racing	
  affect	
  mechanical	
  properties	
  of	
  equine	
  pulmonary	
  	
  
microvasculature	
  ....................................................................................................................................	
  	
  51	
  
	
  
Abstract	
  	
  ........................................................................................................................................	
  51	
  
	
  
Introduction	
  	
  ...............................................................................................................................	
  52	
  
	
  
Materials	
  and	
  methods	
  ...........................................................................................................	
  54	
  
	
  
Animals	
  ...........................................................................................................................	
  54	
  
	
  
Tissue	
  acquisition	
  .......................................................................................................	
  55	
  
	
  
Vessel	
  dissection	
  .........................................................................................................	
  55	
  
	
  
Wire	
  myography	
  .........................................................................................................	
  56	
  
	
  
Vessel	
  histology	
  ...........................................................................................................	
  57	
  
	
  
Lung	
  histopathology	
  ..................................................................................................	
  58	
  
	
  
Data	
  analysis	
  .................................................................................................................	
  58	
  
	
  
Results	
  ............................................................................................................................................	
  59	
  
	
  
	
  
Vessel	
  diameters	
  .........................................................................................................	
  60	
  
	
  
	
  
Vessel	
  identification	
  ..................................................................................................	
  60	
  
	
  
	
  
Length-­‐tension	
  data	
  ...................................................................................................	
  60	
  
	
  
	
  
Length-­‐pressure	
  data	
  ................................................................................................	
  61	
  
	
  
	
  
Lung	
  histopathology	
  ..................................................................................................	
  61	
  
	
  
Discussion	
  .....................................................................................................................................	
  62	
  
	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  APPENDIX	
  ....................................................................................................................................	
  68	
  
	
  
CHAPTER	
  3	
  .................................................................................................................................................	
  75	
  
Regional	
  heterogeneity	
  in	
  reactivity	
  of	
  small	
  pulmonary	
  blood	
  vessels	
  in	
  the	
  horse	
  	
  
may	
  predict	
  exercise-­‐induced	
  pulmonary	
  hemorrhage	
  lesion	
  distribution	
  ..................	
  75	
  
	
  
Introduction	
  .................................................................................................................................	
  76	
  
	
  
Materials	
  and	
  methods	
  ...........................................................................................................	
  78	
  	
  
	
  
Animals	
  ...........................................................................................................................	
  78	
  
	
  
Tissue	
  acquisition	
  .......................................................................................................	
  79	
  
	
  
Vessel	
  dissection	
  .........................................................................................................	
  79	
  
	
  
Wire	
  myography	
  .........................................................................................................	
  80	
  
	
  
Vessel	
  normalization	
  .................................................................................................	
  80	
  
	
  
Vessel	
  wake-­‐up	
  ............................................................................................................	
  81	
  
	
  
Agonist	
  concentration-­‐response	
  curves	
  ...........................................................	
  81	
  
	
  
Vessel	
  fixation	
  ..............................................................................................................	
  82	
  
	
  
Immunohistochemistry	
  ...........................................................................................	
  85	
  
	
  
Endothelial	
  imaging	
  ...................................................................................................	
  83	
  
	
  
Vessel	
  histology	
  ...........................................................................................................	
  83	
  
	
  
Materials	
  .........................................................................................................................	
  84	
  
	
  
Statistical	
  analyses	
  .....................................................................................................	
  84	
  
	
  
Results	
  ............................................................................................................................................	
  85	
  
	
  
Vessels	
  .............................................................................................................................	
  85	
  
	
  
Vessel	
  dimensions	
  ......................................................................................................	
  86	
  
	
  
U	
  46619	
  ...........................................................................................................................	
  86	
  
	
  
Phenylephrine	
  ..............................................................................................................	
  86	
  
	
  
Isoproterenol	
  ................................................................................................................	
  87	
  
	
  
viii	
  

	
  

	
  
Furosemide	
  ...................................................................................................................	
  87	
  
	
  
Methacholine	
  ................................................................................................................	
  87	
  
	
  
Mechanisms	
  of	
  methacholine	
  reactivity	
  ...........................................................	
  88	
  
	
  
Endothelial	
  imaging	
  ...................................................................................................	
  89	
  
	
  
Histology	
  ........................................................................................................................	
  89	
  
	
  
Discussion	
  .....................................................................................................................................	
  89	
  
	
  
APPENDIX	
  .....................................................................................................................................	
  98	
  
	
  
CHAPTER	
  4	
  ..............................................................................................................................................	
  108	
  
Effects	
  of	
  exercise	
  on	
  markers	
  of	
  venous	
  remodeling	
  in	
  lungs	
  of	
  horses	
  .....................	
  108	
  
	
  
Abstract	
  ......................................................................................................................................	
  108	
  
	
  
Introduction	
  ..............................................................................................................................	
  109	
  
	
  
Materials	
  and	
  methods	
  ........................................................................................................	
  111	
  
Animals	
  ........................................................................................................................	
  111	
  
Experimental	
  protocol	
  ..........................................................................................	
  111	
  
Exercise	
  protocol	
  .....................................................................................................	
  112	
  
Pulmonary	
  wedge	
  resection	
  ...............................................................................	
  113	
  
Harvesting	
  of	
  pulmonary	
  veins	
  .........................................................................	
  115	
  
mRNA	
  extraction	
  ......................................................................................................	
  115	
  
Quantitative	
  real-­‐time	
  PCR	
  assays	
  ...................................................................	
  116	
  
Immunohistochemistry	
  ........................................................................................	
  118	
  
Collagen	
  content	
  analysis	
  .....................................................................................	
  119	
  
	
  
Statistical	
  analyses	
  ..................................................................................................	
  119	
  
	
  
Results	
  .........................................................................................................................................	
  120	
  
Discussion	
  ..................................................................................................................................	
  122	
  
	
  
APPENDIX	
  ..................................................................................................................................	
  128	
  
	
  
CHAPTER	
  5	
  ..............................................................................................................................................	
  132	
  
Conclusions	
  and	
  future	
  directions	
  .................................................................................................	
  132	
  
	
  
BIBLIOGRAPHY	
  ......................................................................................................................................	
  138	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
ix	
  

	
  

	
  

LIST	
  OF	
  TABLES	
  
	
  
	
  
	
  
Table	
  1:	
  Mean	
  vessel	
  diameters	
  at	
  start	
  point.	
  	
  Values	
  are	
  means	
  ±	
  SE,	
  n	
  =	
  number	
  of	
  
vessels.	
  Start	
  point	
  is	
  defined	
  as	
  the	
  smallest	
  diameter	
  at	
  which	
  a	
  vessel	
  
maintains	
  a	
  small	
  (<0.05	
  mN)	
  but	
  sustained	
  wall	
  tension.	
  CD,	
  caudodorsal;	
  
CV	
  cranioventral	
  ....................................................................................................................	
  74	
  
	
  
Table	
  2:	
  Primers	
  and	
  probes	
  used	
  for	
  detection	
  of	
  various	
  genes	
  in	
  pulmonary	
  vein	
  
samples	
  of	
  horses	
  via	
  qRT-­‐PCR	
  assay.	
  B2M	
  =	
  Beta-­‐2-­‐microglobulin.	
  EF-­‐1	
  =	
  
Elongation	
  factor-­‐1	
  alpha	
  ................................................................................................	
  131	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

x	
  	
  

LIST	
  OF	
  FIGURES	
  
	
  
	
  
	
  
Figure	
  1:	
  Equine	
  pulmonary	
  artery	
  on	
  a	
  pressure	
  myograph.	
  The	
  artery	
  is	
  secured	
  to	
  
2	
  glass	
  canulae	
  by	
  suture	
  material	
  .............................................................................	
  48	
  
	
  
Figure	
  2:	
  Schematic	
  of	
  a	
  vessel	
  mounted	
  on	
  wire	
  myograph.	
  Vessel	
  is	
  mounted	
  as	
  a	
  
cylinder	
  on	
  two	
  stainless	
  steel	
  wires.	
  Each	
  wire	
  is	
  attached	
  to	
  a	
  metal	
  jaw,	
  
one	
  of	
  which	
  can	
  be	
  moved	
  by	
  a	
  micrometer	
  screw	
  (right	
  of	
  image)	
  and	
  
the	
  other	
  is	
  connected	
  to	
  a	
  sensitive	
  force	
  transducer	
  ......................................	
  	
  49	
  
	
  
Figure	
  3:	
  Schematic	
  diagram	
  of	
  a	
  vessel	
  in	
  cross	
  section	
  mounted	
  on	
  two	
  myograph	
  
wires.	
  d	
  is	
  wire	
  diameter,	
  and	
  f	
  is	
  the	
  distance	
  between	
  the	
  wires.	
  These	
  
measurements	
  are	
  used	
  to	
  calculate	
  vessel	
  internal	
  circumference	
  (L)	
  
from	
  the	
  formula	
  L	
  =	
  (π	
  +	
  2)d	
  +	
  2f	
  ...............................................................................	
  50	
  
	
  
Figure	
  4:	
  Verhoeff-­‐Van	
  Geison	
  stained	
  pulmonary	
  artery	
  and	
  vein	
  for	
  histologic	
  
confirmation	
  of	
  identity	
  post-­‐myography.	
  Pulmonary	
  arteries	
  (A)	
  are	
  
thicker	
  walled	
  than	
  veins	
  (B)	
  and	
  possess	
  both	
  an	
  internal	
  (arrowhead)	
  
and	
  external	
  (arrow)	
  elastic	
  lamina	
  compared	
  to	
  veins	
  which	
  are	
  thin-­‐
walled	
  and	
  only	
  have	
  an	
  external	
  elastic	
  lamina	
  (arrow).	
  Bar	
  =	
  100µm	
  .....	
  69	
  
	
  
Figure	
  5:	
  Length-­‐tension	
  plots	
  for	
  arteries	
  and	
  veins	
  from	
  control,	
  unraced	
  (A)	
  and	
  
raced	
  (B)	
  horses.	
  Values	
  are	
  mean	
  ±	
  SE.	
  In	
  both	
  control	
  and	
  raced	
  horses,	
  
veins	
  are	
  stiffer	
  than	
  arteries.	
  P	
  <	
  0.05	
  is	
  considered	
  significant	
  ...................	
  70	
  
	
  
Figure	
  6:	
  Length-­‐tension	
  plots	
  for	
  caudodorsal	
  (CD)	
  and	
  cranioventral	
  (CV)	
  veins,	
  
and	
  for	
  CD	
  and	
  CV	
  arteries	
  from	
  unraced	
  horses	
  (A	
  and	
  B	
  respectively)	
  and	
  
from	
  raced	
  horses	
  	
  (C	
  and	
  D	
  respectively).	
  Values	
  are	
  mean	
  ±	
  SE.	
  In	
  both	
  
control	
  and	
  raced	
  horses,	
  veins	
  from	
  cranioventral	
  lung	
  are	
  stiffer	
  than	
  
veins	
  from	
  caudodorsal	
  lung,	
  and	
  arteries	
  from	
  caudodorsal	
  lung	
  are	
  
stiffer	
  than	
  arteries	
  from	
  cranioventral	
  lung.	
  P	
  <	
  0.05	
  is	
  considered	
  
significant	
  ................................................................................................................................	
  71	
  
	
  
Figure	
  7:	
  Length-­‐tension	
  plots	
  of	
  vessels	
  from	
  control	
  and	
  raced	
  horses.	
  Data	
  are	
  
from	
  caudodorsal	
  (CD)	
  and	
  cranioventral	
  (CV)	
  veins	
  (A	
  and	
  B	
  
respectively)	
  and	
  arteries	
  (C	
  and	
  D	
  respectively).	
  Values	
  are	
  means	
  ±	
  SE	
  
Veins	
  from	
  the	
  caudodorsal	
  region	
  of	
  lungs	
  of	
  raced	
  horses	
  are	
  stiffer	
  than	
  
those	
  from	
  unraced,	
  control	
  horses.	
  Arteries	
  from	
  the	
  cranioventral	
  region	
  
of	
  lungs	
  of	
  raced	
  horses	
  are	
  stiffer	
  than	
  those	
  from	
  unraced,	
  control	
  
horses.	
  p	
  <	
  0.05	
  is	
  considered	
  significant	
  .................................................................	
  72	
  
	
  
Figure	
  8:	
  Length-­‐pressure	
  plots	
  for	
  vessels	
  from	
  control	
  and	
  raced	
  horses.	
  Data	
  are	
  
from	
  caudodorsal	
  (CD)	
  and	
  cranioventral	
  (CV)	
  veins	
  (A	
  and	
  B	
  
respectively)	
  and	
  arteries(C	
  and	
  D	
  respectively).	
  Data	
  are	
  expressed	
  as	
  
	
  
xi	
  

mean	
  values.	
  Veins	
  from	
  the	
  caudodorsal	
  region	
  of	
  lungs	
  of	
  raced	
  horses	
  
are	
  stiffer	
  than	
  those	
  from	
  unraced,	
  control	
  horses.	
  Arteries	
  from	
  the	
  
cranioventral	
  region	
  of	
  lungs	
  of	
  raced	
  horses	
  are	
  stiffer	
  than	
  those	
  from	
  
unraced,	
  control	
  horses.	
  Hatched	
  regions	
  demarcate	
  the	
  range	
  of	
  in	
  vivo	
  
intravascular	
  pressures	
  from	
  rest	
  to	
  intense	
  exercise	
  (10	
  –	
  70	
  mmHg	
  and	
  
30	
  –	
  110	
  mmHg	
  for	
  veins	
  and	
  arteries	
  respectively).	
  P	
  <	
  0.05	
  is	
  considered	
  
significant	
  ................................................................................................................................	
  73	
  
Figure	
  9:	
  Cumulative	
  concentration	
  response	
  curves	
  for	
  U46619	
  for	
  all	
  arteries	
  and	
  
veins	
  (A),	
  caudodorsal	
  (CD)	
  and	
  cranioventral	
  (CV)	
  arteries	
  (B),	
  and	
  CD	
  
and	
  CV	
  veins	
  (C).	
  Values	
  are	
  means	
  ±	
  SE.	
  Veins	
  are	
  more	
  sensitive	
  to	
  U	
  
46619	
  than	
  arteries	
  (A);	
  regional	
  differences	
  in	
  responses	
  of	
  CD	
  and	
  CV	
  
arteries	
  do	
  not	
  exist	
  (p	
  =	
  0.25)(B)	
  whereas	
  CV	
  veins	
  are	
  more	
  sensitive	
  to	
  
U	
  46619	
  than	
  CD	
  veins	
  	
  (p	
  <0.0001)(C)	
  ....................................................................	
  	
  99	
  
	
  
Figure	
  10:	
  Cumulative	
  concentration	
  response	
  curves	
  for	
  isoproterenol	
  for	
  
caudodorsal	
  (CD)	
  and	
  cranioventral	
  (CV)	
  arteries	
  (A),	
  and	
  CD	
  and	
  CV	
  veins	
  
(B).	
  Values	
  are	
  means	
  ±	
  SE.	
  Concentration-­‐dependent	
  relaxation	
  is	
  greater	
  
in	
  CD	
  compared	
  to	
  CV	
  arteries	
  (p	
  <	
  0.0001)(A),	
  whereas	
  pre-­‐contracted	
  
veins	
  do	
  not	
  relax	
  in	
  response	
  to	
  isoproterenol,	
  in	
  both	
  CD	
  and	
  CV	
  regions	
  
(B)	
  ..........................................................................................................................................	
  101	
  
	
  
	
  
Figure	
  11:	
  Cumulative	
  concentration	
  response	
  curves	
  for	
  furosemide	
  for	
  all	
  arteries	
  
and	
  veins	
  (A),	
  caudodorsal	
  (CD)	
  and	
  cranioventral	
  (CV)	
  arteries	
  (B),	
  and	
  
CD	
  and	
  CV	
  veins	
  (C).	
  Values	
  are	
  means	
  ±	
  SE.	
  Mild	
  concentration-­‐dependent	
  
relaxation	
  to	
  furosemide	
  occurs	
  in	
  arteries,	
  and	
  to	
  a	
  lesser	
  degree	
  in	
  veins	
  
(A);	
  regional	
  differences	
  in	
  the	
  response	
  of	
  arteries	
  and	
  veins	
  to	
  
furosemide	
  do	
  not	
  exist	
  (p	
  =	
  0.07	
  and	
  p	
  =	
  0.19	
  for	
  arteries	
  and	
  veins	
  
respectively)(B	
  and	
  C	
  respectively).	
  DMSO	
  vehicle	
  (dark	
  triangle)	
  does	
  not	
  
affect	
  arteries	
  and	
  veins	
  (B	
  and	
  C	
  respectively)	
  ..................................................	
  102	
  
	
  
Figure	
  12:	
  Cumulative	
  concentration	
  response	
  curves	
  for	
  methacholine	
  for	
  
caudodorsal	
  (CD)	
  and	
  cranioventral	
  (CV)	
  arteries	
  (A),	
  and	
  CD	
  and	
  CV	
  
veins	
  (B).	
  Values	
  are	
  means	
  ±	
  SE.	
  Concentration-­‐dependent	
  relaxation	
  
occurs	
  in	
  CV	
  arteries,	
  and	
  concentration-­‐dependent	
  constriction	
  occurred	
  
in	
  CD	
  arteries	
  (A);	
  pre-­‐contracted	
  pulmonary	
  veins	
  relax	
  in	
  a	
  
concentration-­‐dependent	
  manner,	
  regardless	
  of	
  region	
  .............................	
  104	
  
	
  
Figure	
  13:	
  Cumulative	
  concentration	
  response	
  curves	
  for	
  CD	
  (A,	
  B,	
  C)	
  and	
  CV	
  (D,	
  E,	
  F)	
  
arteries	
  comparing	
  responses	
  to	
  methacholine	
  (MCh)	
  only,	
  with	
  responses	
  
to	
  MCh	
  applied	
  after	
  pre-­‐incubation	
  with	
  L-­‐NAME	
  (A	
  and	
  D),	
  indomethacin	
  
(B	
  and	
  E),	
  and	
  L-­‐NAME	
  and	
  indomethacin	
  (C	
  and	
  F).	
  Values	
  are	
  means	
  ±	
  
SE.	
  Pre-­‐incubation	
  with	
  L-­‐NAME	
  does	
  not	
  affect	
  CD	
  artery	
  constriction	
  in	
  
response	
  to	
  MCh	
  (A)	
  whereas	
  CV	
  artery	
  relaxation	
  is	
  partially	
  inhibited	
  by	
  
L-­‐NAME	
  (D).	
  Indomethacin	
  pre-­‐incubation	
  augments	
  CD	
  artery	
  
	
  
xii	
  

constriction,	
  and	
  partially	
  inhibits	
  CV	
  artery	
  relaxation	
  (B	
  and	
  E	
  
respectively).	
  Pre-­‐incubation	
  with	
  both	
  L-­‐NAME	
  and	
  indomethacin	
  caused	
  
enhanced	
  MCh-­‐induced	
  constriction	
  in	
  CD	
  arteries	
  (C)	
  and	
  a	
  mild	
  
contraction	
  followed	
  by	
  mild	
  relaxation	
  in	
  CV	
  arteries	
  (D)	
  .........................	
  105	
  

	
  
Figure	
  14:	
  Fluorescent	
  staining	
  of	
  CD-­‐31	
  on	
  endothelial	
  surface	
  of	
  equine	
  pulmonary	
  
artery.	
  	
  Regions	
  of	
  intact	
  endothelium	
  can	
  be	
  discerned	
  from	
  
endothelium-­‐denuded,	
  non-­‐stained	
  regions	
  (indicated	
  by	
  arrow-­‐heads).	
  
Scale	
  bar	
  =	
  100	
  μm	
  .........................................................................................................	
  107	
  
	
  
Figure	
  15:	
  Mean	
  ±	
  SEM	
  fold	
  changes	
  in	
  mRNA	
  expression	
  of	
  10	
  genes	
  in	
  pulmonary	
  
vein	
  samples	
  of	
  4	
  horses	
  after	
  a	
  2-­‐week	
  period	
  of	
  intense	
  exercise	
  versus	
  
expression	
  before	
  exercise.	
  *Expression	
  is	
  significantly	
  (P	
  <	
  0.05)	
  
different	
  between	
  pulmonary	
  vein	
  samples	
  collected	
  before	
  and	
  after	
  
exercise	
  ..............................................................................................................................	
  129	
  
	
  
Figure	
  16:	
  Least	
  square	
  mean	
  ±	
  SEM	
  percentage	
  of	
  collagen	
  in	
  samples	
  of	
  
cranioventral	
  (CV)	
  and	
  caudodorsal	
  (CD)	
  regions	
  of	
  lung	
  of	
  6	
  horses	
  
before	
  (black	
  bars)	
  and	
  after	
  (grey	
  bars)	
  a	
  2-­‐week	
  period	
  of	
  intense	
  
exercise.	
  Bars	
  indicate	
  no	
  significant	
  (P	
  <	
  0.05)	
  differences	
  between	
  lung	
  
samples	
  obtained	
  before	
  and	
  after	
  exercise	
  within	
  a	
  region.	
  *Mean	
  value	
  
for	
  pre-­‐	
  and	
  postexercise	
  tissue	
  samples	
  obtained	
  from	
  caudodorsal	
  
regions	
  of	
  lungs	
  are	
  significantly	
  (P	
  <	
  0.05)	
  higher	
  than	
  those	
  obtained	
  
from	
  cranioventral	
  regions	
  of	
  lungs	
  .......................................................................	
  130	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
xiii	
  

BAL	
  

	
  

KEY	
  TO	
  ABBREVIATIONS	
  
	
  
	
  
	
  
Broncho-­‐alveolar	
  lavage	
  

CD	
  

	
  

Caudodorsal	
  

CV	
  	
  

	
  

Cranioventral	
  

EIPH	
   	
  

Exercise-­‐induced	
  pulmonary	
  hemorrhage	
  

ET	
  

Endothelin	
  	
  

	
  

HRmax	
  	
   	
  

Maximum	
  heart	
  rate	
  	
  

MMP	
  	
   	
  

Matrix	
  metalloproteinase	
  	
  

O.D.	
  

	
  

Outer	
  diameter	
  

Pcap	
  

	
  

Pulmonary	
  capillary	
  pressure	
  

Pla	
  

	
  

Left	
  atrial	
  pressure	
  

Ppa	
  

	
  

Pulmonary	
  arterial	
  pressure	
  

Ptm	
  

	
  

Pulmonary	
  transmural	
  pressure	
  

Pwp	
  

	
  

Pulmonary	
  wedge	
  pressure	
  

PDGF	
  	
   	
  

Platelet-­‐derived	
  growth	
  factor	
  

PVR	
   	
  

Pulmonary	
  vascular	
  resistance	
  

qRT-­‐PCR	
  	
  	
  

Quantitative	
  real-­‐time	
  PCR	
  

TGF-­‐β	
   	
  

Transforming	
  growth	
  factor-­‐beta	
  	
  

TIMP	
   	
  

Tissue	
  inhibitor	
  of	
  metalloproteinase	
  

VEGF	
   	
  

Vascular	
  endothelial	
  growth	
  factor	
  	
  

	
  

	
  
xiv	
  

CHAPTER	
  1	
  
	
  Literature	
  Review	
  
	
  
Section	
  1:	
  Exercise-­‐induced	
  pulmonary	
  hemorrhage	
  
This	
  section	
  is	
  designed	
  to	
  provide	
  comprehensive	
  background	
  information	
  on	
  exercise-­‐
induced	
  pulmonary	
  hemorrhage	
  (EIPH).	
  Specifically,	
  deficits	
  in	
  our	
  understanding	
  of	
  
pathogenic	
  mechanisms	
  will	
  be	
  addressed,	
  thereby	
  providing	
  justification	
  for	
  further	
  study	
  of	
  
EIPH	
  pathogenesis.	
  
	
  
History	
  
Exercise-­‐induced	
  pulmonary	
  hemorrhage	
  (EIPH)	
  is	
  defined	
  as	
  the	
  presence	
  of	
  blood	
  in	
  the	
  
airways	
  after	
  an	
  intense	
  bout	
  of	
  exercise,	
  and	
  is	
  a	
  ubiquitous,	
  performance-­‐limiting	
  
condition	
  of	
  modern-­‐day	
  racehorses.	
  Post-­‐exercise	
  epistaxis	
  has	
  been	
  recognized	
  for	
  
hundreds	
  of	
  years	
  however.	
  
Markham’s	
  Masterpiece	
  containing	
  “all	
  knowledge	
  touching	
  the	
  curing	
  of	
  all	
  
diseases	
  of	
  horses”	
  that	
  was	
  first	
  published	
  in	
  1610,	
  has	
  a	
  chapter	
  dedicated	
  to	
  the	
  topic.	
  	
  
“Many	
  horses	
  (especially	
  young	
  horses)	
  are	
  often	
  subject	
  to	
  this	
  bleeding	
  at	
  the	
  nose,	
  which	
  I	
  
imagine	
  proceedeth	
  either	
  from	
  the	
  abundance	
  of	
  Blood,	
  or	
  that	
  the	
  Vein	
  which	
  endeth	
  in	
  that	
  
Place	
  is	
  either	
  broken,	
  fretted,	
  or	
  opened...	
  
...it	
  (the	
  Vein)	
  may	
  be	
  broken	
  by	
  some	
  violent	
  strain”	
  (130)	
  
The	
  Thoroughbred	
  stallion	
  Bartlet’s	
  Childers	
  was	
  born	
  in	
  1716	
  and	
  was	
  also	
  known	
  as	
  
“Bleeding	
  Childers”	
  in	
  his	
  younger	
  years.	
  The	
  horse	
  was	
  never	
  raced,	
  which	
  according	
  to	
  
racing	
  lore,	
  was	
  due	
  to	
  severe	
  EIPH.	
  	
  He	
  was	
  full	
  brother	
  to	
  Flying	
  Childers,	
  who	
  is	
  

1	
  	
  

described	
  in	
  the	
  General	
  Stud	
  Book	
  as	
  “the	
  fleetest	
  horse	
  that	
  was	
  ever	
  trained”(1).	
  Despite	
  
being	
  unraced,	
  Bartlet’s	
  Childers	
  was	
  bred	
  extensively,	
  and	
  “got	
  so	
  many	
  good	
  horses,	
  that	
  
he	
  is	
  ranked	
  with	
  the	
  first-­‐rate	
  stallions”	
  (1).	
  Indeed,	
  he	
  is	
  the	
  great-­‐grandsire	
  of	
  Eclipse	
  –	
  a	
  
hugely	
  influential	
  stallion	
  to	
  whom	
  more	
  than	
  95%	
  of	
  English	
  Thoroughbreds	
  are	
  
genetically	
  linked	
  (221).	
  
In	
  his	
  publication	
  “Epistaxis	
  in	
  the	
  Racehorse”	
  published	
  in	
  1974,	
  Cook	
  suspects	
  that	
  
the	
  epistaxis	
  observed	
  after	
  exercise	
  is	
  from	
  a	
  pulmonary	
  source,	
  as	
  he	
  used	
  a	
  rigid	
  
endoscope	
  to	
  rule	
  out	
  upper	
  airway	
  (rostral	
  to	
  the	
  larynx)	
  sources	
  of	
  hemorrhage(32).	
  The	
  
term	
  “exercise-­‐induced	
  pulmonary	
  hemorrhage”	
  was	
  first	
  used	
  in	
  1981	
  when,	
  through	
  use	
  
of	
  a	
  flexible	
  endoscope,	
  Pascoe	
  et	
  al	
  confirmed	
  that	
  post-­‐exercise	
  epistaxis	
  in	
  horses	
  was	
  in	
  
fact	
  of	
  pulmonary	
  origin	
  (162).	
  	
  
	
  
Epidemiology	
  
It	
  is	
  worth	
  noting	
  that	
  EIPH	
  is	
  not	
  limited	
  to	
  racehorses.	
  EIPH	
  has	
  been	
  described	
  in	
  horses	
  
that	
  participate	
  in	
  disciplines	
  other	
  than	
  racing,	
  such	
  as	
  polo	
  (220),	
  rodeo	
  (8),	
  and	
  also	
  in	
  
racing	
  greyhounds	
  (42),	
  and	
  camels	
  (4).	
  There	
  are	
  also	
  reports	
  of	
  EIPH	
  in	
  small	
  numbers	
  of	
  
(mostly	
  elite)	
  human	
  athletes,	
  specifically	
  after	
  running	
  (52),	
  cycling	
  (79)	
  and	
  swimming	
  
(227).	
  Due	
  to	
  an	
  absence	
  of	
  routine	
  post-­‐exertion	
  diagnostics,	
  the	
  true	
  prevalence	
  of	
  EIPH	
  
in	
  non-­‐racing	
  horses,	
  and	
  in	
  other	
  species	
  is	
  not	
  known.	
  However	
  in	
  all	
  reports,	
  the	
  level	
  of	
  
exercise	
  resulting	
  in	
  EIPH	
  can	
  be	
  considered	
  “intense.”	
  	
  
If	
  epistaxis	
  is	
  used	
  as	
  the	
  diagnostic	
  criterion,	
  reported	
  prevalence	
  of	
  EIPH	
  in	
  
Thoroughbreds	
  is	
  actually	
  low.	
  For	
  example,	
  in	
  Thoroughbreds	
  racing	
  in	
  South	
  Africa,	
  and	
  
Japan,	
  the	
  epistaxis	
  rate	
  is	
  less	
  than	
  0.2	
  %	
  (205,	
  226).	
  When	
  endoscopy	
  of	
  the	
  upper	
  

2	
  	
  

airways	
  including	
  the	
  trachea,	
  is	
  performed	
  within	
  2	
  hours	
  of	
  racing,	
  reported	
  EIPH	
  
prevalence	
  is	
  much	
  higher.	
  Based	
  on	
  a	
  single	
  post	
  race	
  endoscopic	
  examination,	
  between	
  
62	
  and	
  75	
  %	
  of	
  racing	
  Thoroughbreds,	
  Standardbreds	
  and	
  Quarterhorses	
  experience	
  EIPH	
  
(18,	
  68,	
  107,	
  172).	
  When	
  horses	
  are	
  evaluated	
  after	
  multiple	
  races,	
  it	
  is	
  reported	
  that	
  87%	
  
to	
  100%	
  of	
  Thoroughbreds	
  and	
  Standardbreds	
  have	
  evidence	
  of	
  EIPH	
  after	
  one	
  of	
  three	
  
races	
  (18,	
  107).	
  	
  
Post-­‐exercise	
  broncho-­‐alveolar	
  lavage	
  (BAL),	
  which	
  can	
  be	
  performed	
  up	
  to	
  a	
  
number	
  of	
  weeks	
  after	
  an	
  exercise	
  bout,	
  is	
  also	
  used	
  to	
  diagnose	
  EIPH	
  based	
  on	
  the	
  
presence	
  of	
  free	
  red	
  blood	
  cells	
  and	
  hemosiderin-­‐laden	
  macrophages	
  in	
  lavage	
  fluid.	
  	
  Using	
  
BAL	
  as	
  the	
  diagnostic	
  technique,	
  EIPH	
  prevalence	
  in	
  Thoroughbreds	
  approaches	
  100%	
  
(131).	
  
	
  
Risk	
  factors	
  for	
  EIPH	
  
Regardless	
  of	
  species,	
  or	
  the	
  type	
  of	
  racing,	
  EIPH	
  is	
  typically	
  associated	
  with	
  intense	
  
exercise.	
  Although	
  underlying	
  pathology,	
  for	
  example	
  atrial	
  fibrillation,	
  may	
  increase	
  an	
  
individual	
  animal’s	
  risk	
  of	
  experiencing	
  EIPH	
  (36),	
  this	
  is	
  not	
  a	
  common,	
  concurrent	
  clinical	
  
finding	
  in	
  horses	
  with	
  EIPH.	
  	
  
Under	
  normal	
  circumstances,	
  numerous	
  risk	
  factors	
  have	
  been	
  purported	
  to	
  
contribute	
  to	
  EIPH	
  risk.	
  Horses	
  are	
  at	
  increased	
  risk	
  of	
  EIPH	
  that	
  manifests	
  as	
  epistaxis	
  
when	
  they	
  race	
  over	
  fences	
  (versus	
  flat	
  racing),	
  and	
  when	
  they	
  are	
  older	
  (145,	
  205).	
  When	
  
risk	
  factors	
  associated	
  with	
  endoscopically-­‐diagnosed	
  EIPH	
  are	
  evaluated,	
  it	
  transpires	
  that	
  
increased	
  EIPH	
  risk	
  associated	
  with	
  increasing	
  age	
  is	
  probably	
  due	
  to	
  a	
  higher	
  number	
  of	
  

3	
  	
  

race-­‐starts	
  (and	
  presumably	
  days	
  in	
  training)	
  in	
  older	
  animals	
  compared	
  to	
  younger	
  ones	
  
(75).	
  
	
  
Clinical	
  features	
  
Other	
  than	
  epistaxis	
  (a	
  relatively	
  rare	
  occurrence),	
  and	
  anecdotal	
  reports	
  of	
  increased	
  
swallowing	
  post-­‐exercise,	
  EIPH	
  is	
  not	
  associated	
  with	
  clinically	
  detectable	
  symptoms.	
  	
  
However	
  it	
  has	
  long	
  been	
  believed	
  that	
  EIPH	
  is	
  associated	
  with	
  compromised	
  racing	
  
performance,	
  and	
  this	
  was	
  demonstrated	
  conclusively	
  in	
  2005	
  based	
  on	
  evaluation	
  of	
  a	
  
population	
  of	
  racehorses	
  in	
  Australia.	
  	
  This	
  report	
  employs	
  an	
  endoscopic	
  scoring	
  system	
  
which	
  has	
  been	
  widely	
  adopted	
  as	
  standard,	
  and	
  demonstrated	
  to	
  have	
  excellent	
  inter-­‐
observer	
  reliability	
  (69).	
  Using	
  this	
  system,	
  horses	
  with	
  no	
  blood	
  in	
  the	
  trachea	
  are	
  
assigned	
  grade	
  0,	
  and	
  horses	
  with	
  blood	
  covering	
  >90%	
  of	
  the	
  tracheal	
  surface	
  are	
  assigned	
  
grade	
  4.	
  Horses	
  with	
  mild	
  (grade	
  1)	
  or	
  no	
  EIPH	
  are	
  4	
  times	
  as	
  likely	
  to	
  win	
  and	
  almost	
  
twice	
  as	
  likely	
  to	
  place	
  in	
  a	
  race	
  compared	
  to	
  horses	
  with	
  EIPH	
  grade	
  2,	
  3,	
  or	
  4	
  (70).	
  	
  
	
  
Pathology	
  
A	
  series	
  of	
  publications	
  by	
  O’Callaghan	
  et	
  al,	
  contain	
  some	
  of	
  the	
  first	
  descriptions	
  of	
  EIPH-­‐
associated	
  gross	
  and	
  microscopic	
  pathology.	
  26	
  horses	
  that	
  were	
  retired	
  from	
  racing	
  due	
  to	
  
severe	
  EIPH	
  were	
  studied.	
  Gross	
  pathologic	
  changes	
  included	
  dark	
  blue-­‐black	
  discoloration	
  
of	
  caudodorsal	
  lung,	
  with	
  up	
  to	
  45%	
  of	
  lung	
  affected,	
  and	
  these	
  areas	
  were	
  firmer	
  than	
  
normal	
  lung	
  tissue	
  (150,	
  151).	
  Hemosiderin	
  accumulation,	
  angiogenesis	
  (due	
  to	
  bronchial	
  
circulation	
  proliferation),	
  bronchiolitis	
  and	
  extensive	
  fibrosis	
  are	
  also	
  described	
  (151,	
  152).	
  
These	
  findings	
  are	
  consistent	
  with	
  a	
  subsequent	
  publication	
  on	
  the	
  topic	
  (157).	
  However,	
  

4	
  	
  

in	
  two	
  publications	
  from	
  investigators	
  at	
  Michigan	
  State	
  University	
  that	
  evaluate	
  
pulmonary	
  pathology	
  of	
  racehorses	
  from	
  both	
  Singapore	
  and	
  North	
  America,	
  bronchiolitis	
  
is	
  not	
  reported	
  to	
  be	
  a	
  consistently	
  occurring	
  lesion	
  (231,	
  233).	
  A	
  likely	
  reason	
  for	
  this	
  
discrepancy	
  is	
  due	
  to	
  the	
  fact	
  that	
  inflammatory	
  airway	
  disease	
  is	
  common	
  in	
  young	
  horses	
  
in	
  training	
  (237),	
  and	
  reports	
  of	
  small	
  airway	
  pathology	
  in	
  EIPH-­‐affected	
  lung	
  actually	
  
reflect	
  coincident	
  disease	
  processes	
  whose	
  pathogeneses	
  are	
  unrelated.	
  	
  
A	
  novel	
  lesion	
  -­‐	
  that	
  of	
  remodeling	
  of	
  small	
  intra-­‐lobular	
  pulmonary	
  veins	
  is	
  
described	
  in	
  the	
  caudodorsal	
  lung	
  tissue	
  of	
  both	
  the	
  Singapore	
  and	
  North	
  American	
  cohorts	
  
however	
  (231,	
  233).	
  Remodeling	
  affects	
  pulmonary	
  veins	
  up	
  to	
  approximately	
  200	
  μm	
  
outer	
  diameter	
  (O.D.)	
  and	
  is	
  characterized	
  by	
  expansion	
  of	
  the	
  adventitia	
  by	
  mature	
  
collagen	
  in	
  affected	
  vessel	
  walls.	
  Collagen	
  is	
  also	
  found	
  between	
  the	
  external	
  elastic	
  lamina	
  
and	
  the	
  vein	
  lumen.	
  In	
  some	
  veins,	
  tunica	
  media	
  and	
  tunica	
  intima	
  hypertrophy	
  is	
  also	
  
observed,	
  and	
  in	
  severely-­‐affected	
  vessels,	
  luminal	
  area	
  appears	
  markedly	
  reduced	
  (231).	
  
Morphometric	
  analyses	
  support	
  these	
  observations.	
  In	
  lung	
  tissue	
  from	
  EIPH-­‐affected	
  
horses,	
  veins	
  are	
  significantly	
  thicker-­‐walled	
  in	
  dorsal	
  lung	
  and	
  walls	
  are	
  thickest	
  in	
  the	
  
most	
  caudodorsal	
  region	
  (38).	
  Veins	
  also	
  have	
  reduced	
  luminal	
  area	
  in	
  the	
  most	
  severely	
  
affected	
  lung	
  regions,	
  compared	
  to	
  vessels	
  from	
  less	
  affected,	
  or	
  normal	
  lung	
  tissue	
  (38).	
  	
  
A	
  recent	
  detailed,	
  systematic	
  evaluation	
  of	
  EIPH-­‐affected	
  lungs	
  supplies	
  more	
  
information	
  about	
  venous	
  remodeling	
  in	
  the	
  context	
  of	
  other	
  EIPH	
  pathologic	
  features.	
  
Venous	
  remodeling,	
  hemosiderin	
  accumulation	
  and	
  fibrosis	
  are	
  more	
  common,	
  and	
  most	
  
severe	
  in	
  caudodorsal	
  lung,	
  compared	
  to	
  cranial	
  and	
  ventral	
  lung	
  (233).	
  	
  	
  In	
  93%	
  of	
  1,400	
  
randomly	
  selected	
  samples,	
  venous	
  remodeling	
  was	
  present	
  with	
  hemosiderin,	
  and	
  in	
  the	
  
remaining	
  samples,	
  venous	
  remodeling	
  was	
  mild.	
  Also,	
  interstitial	
  fibrosis	
  was	
  rarely	
  

5	
  	
  

present	
  (0.3%	
  of	
  samples	
  only)	
  without	
  venous	
  remodeling.	
  These	
  data	
  strongly	
  implicate	
  
venous	
  remodeling	
  as	
  a	
  lesion	
  of	
  interest	
  in	
  EIPH,	
  and	
  likely	
  one	
  that	
  occurs	
  early	
  in	
  the	
  
disease	
  process,	
  and	
  is	
  central	
  to	
  EIPH	
  pathogenesis.	
  	
  
	
  
Pathogenesis	
  
A	
  number	
  of	
  theories	
  of	
  EIPH	
  pathogenesis	
  have	
  been	
  proposed	
  and	
  include	
  some	
  that	
  
implicate	
  airway	
  inflammation	
  as	
  a	
  cause	
  (153,	
  178),	
  and	
  another	
  that	
  attributes	
  
hemorrhage	
  to	
  lung	
  trauma	
  resulting	
  from	
  transmission	
  of	
  a	
  shock	
  wave	
  from	
  the	
  ground	
  
to	
  the	
  lung	
  during	
  galloping	
  (184).	
  However	
  neither	
  of	
  these	
  theories	
  can	
  adequately	
  
explain	
  the	
  constellation	
  of	
  lesions	
  that	
  are	
  associated	
  with	
  EIPH,	
  nor	
  the	
  distinct	
  
caudodorsal	
  distribution	
  of	
  EIPH	
  pathology.	
  	
  
In	
  the	
  early	
  1990s	
  it	
  was	
  reported	
  that	
  mean	
  pulmonary	
  artery	
  pressure	
  in	
  the	
  
galloping	
  horse	
  was	
  in	
  the	
  order	
  of	
  90	
  –	
  110	
  mmHg,	
  and	
  that	
  pulmonary	
  wedge	
  pressures	
  
and	
  left	
  atrial	
  pressures	
  were	
  56	
  and	
  70	
  mmHg	
  respectively	
  (88,	
  124).	
  These	
  values	
  deliver	
  
estimated	
  intravascular	
  capillary	
  pressures	
  of	
  72	
  –	
  83	
  mmHg	
  (124,	
  127).	
  At	
  the	
  same	
  time,	
  
a	
  breakthrough	
  publication	
  by	
  West	
  et	
  al	
  described	
  pulmonary	
  capillary	
  wall	
  disruption	
  in	
  
lung	
  tissue	
  from	
  horses	
  that	
  had	
  recently	
  exercised	
  on	
  a	
  treadmill.	
  Extravascular	
  red	
  cells	
  
were	
  found	
  both	
  in	
  the	
  pulmonary	
  interstitium	
  and	
  in	
  surrounding	
  alveolar	
  airspaces	
  
(230).	
  These	
  data	
  suggest	
  that	
  the	
  pulmonary	
  circulation	
  is	
  the	
  source	
  of	
  airway	
  
hemorrhage,	
  and	
  the	
  authors	
  went	
  on	
  to	
  propose	
  that	
  capillary	
  rupture	
  was	
  secondary	
  to	
  
the	
  high	
  pulmonary	
  capillary	
  pressures	
  during	
  exercise	
  (229).	
  Further	
  support	
  for	
  the	
  
capillary	
  stress-­‐failure	
  theory	
  was	
  provided	
  by	
  Birks	
  et	
  al	
  who	
  demonstrated	
  that	
  the	
  
estimated	
  threshold	
  for	
  breaking	
  strength	
  of	
  equine	
  pulmonary	
  capillaries	
  was	
  exceeded	
  at	
  

6	
  	
  

a	
  value	
  of	
  75	
  mmHg	
  transmural	
  pressure	
  (Ptm)	
  (17).	
  This	
  value	
  in	
  the	
  horse	
  is	
  higher	
  than	
  
that	
  of	
  rabbit	
  or	
  dog	
  pulmonary	
  capillaries	
  (16),	
  and	
  based	
  on	
  multiple	
  studies,	
  falls	
  within	
  
the	
  range	
  of	
  estimated	
  pulmonary	
  capillary	
  pressures	
  during	
  exercise	
  (124,	
  127)	
  
particularly	
  when	
  negative	
  alveolar	
  pressure	
  (a	
  component	
  of	
  Ptm)	
  is	
  taken	
  into	
  account	
  
(106).	
  	
  
As	
  a	
  point	
  of	
  interest,	
  the	
  reason	
  that	
  the	
  extremely	
  thin-­‐walled,	
  and	
  apparently	
  
fragile	
  capillary	
  can	
  withstand	
  very	
  high	
  intravascular	
  pressures	
  (up	
  to	
  a	
  certain	
  point)	
  is	
  
not	
  due	
  to	
  the	
  inherent	
  strength	
  of	
  its	
  wall,	
  but	
  rather	
  its	
  very	
  small	
  diameter	
  (5	
  –	
  8	
  μm)	
  
(210),	
  as	
  predicted	
  by	
  the	
  Laplace	
  relationship	
  between	
  wall	
  tension	
  (T),	
  intraluminal	
  
pressure	
  (P)	
  and	
  vessel	
  radius	
  (r)	
  
T	
  =	
  P	
  x	
  r	
  	
  
At	
  normal,	
  resting,	
  pulmonary	
  capillary	
  pressures,	
  the	
  tension	
  that	
  must	
  be	
  maintained	
  by	
  
the	
  capillary	
  wall	
  to	
  resist	
  distension	
  and	
  rupture	
  is	
  only	
  16	
  dynes/cm.	
  This	
  is	
  
approximately	
  3000	
  times	
  less	
  than	
  the	
  breaking	
  strength	
  of	
  a	
  piece	
  of	
  wet	
  tissue	
  paper	
  
(whose	
  breaking	
  strength	
  is	
  50,000	
  dynes/cm)	
  (20)!	
  	
  
Pulmonary	
  capillary	
  pressure	
  is	
  determined	
  in	
  part	
  by	
  the	
  resistance	
  to	
  flow	
  in	
  the	
  
arteries	
  and	
  veins	
  that	
  supply	
  and	
  drain	
  the	
  capillaries.	
  In	
  turn,	
  resistance	
  to	
  flow	
  is	
  
strongly	
  influenced	
  by	
  vessel	
  diameter.	
  Vessel	
  diameter	
  is	
  determined	
  by	
  the	
  vessel	
  wall	
  
structure	
  (a	
  so-­‐called	
  passive	
  factor)	
  that	
  acts	
  to	
  resist	
  distension,	
  and	
  vascular	
  reactivity	
  
(an	
  active	
  factor),	
  which	
  is	
  controlled	
  by	
  smooth	
  muscle	
  contraction/relaxation	
  (11).	
  
Increased	
  resistance	
  in	
  the	
  venous	
  compartment,	
  and/or	
  decreased	
  resistance	
  in	
  the	
  
arterial	
  compartment	
  are	
  both	
  conditions	
  under	
  which	
  pulmonary	
  capillary	
  pressure	
  will	
  
increase,	
  and	
  potentially	
  approach	
  arterial	
  values.	
  Regional	
  differences	
  in	
  pulmonary	
  vessel	
  

7	
  	
  

reactivity	
  are	
  reported	
  in	
  large-­‐caliber	
  pulmonary	
  arteries	
  in	
  the	
  pig,	
  and	
  the	
  horse	
  (164,	
  
176).	
  Should	
  regional	
  differences	
  in	
  the	
  determinants	
  of	
  small	
  arterial	
  and	
  venous	
  diameter	
  
exist	
  in	
  a	
  pattern	
  that	
  causes	
  highest	
  pulmonary	
  capillary	
  pressures	
  in	
  caudodorsal	
  lung,	
  
this	
  could	
  provide	
  a	
  reason	
  for	
  the	
  predilection	
  of	
  pathology	
  for	
  this	
  lung	
  region.	
  	
  
A	
  study	
  using	
  microspheres	
  demonstrated	
  that	
  pulmonary	
  blood	
  flow	
  distribution	
  
in	
  the	
  horse	
  is	
  not	
  gravitationally	
  dependent.	
  Blood	
  flow	
  is	
  highest	
  in	
  dorsal	
  regions,	
  
compared	
  to	
  ventral	
  ones,	
  and	
  in	
  caudal	
  lung	
  compared	
  to	
  cranial	
  (76).	
  Furthermore,	
  blood	
  
flow	
  within	
  isogravitational	
  planes	
  displays	
  significant	
  heterogeneity	
  (average	
  coefficient	
  
of	
  variation	
  30.7%).	
  A	
  recent	
  publication	
  from	
  our	
  laboratory	
  demonstrated	
  that	
  EIPH	
  
pathology	
  (venous	
  remodeling,	
  hemosiderin	
  and	
  interstitial	
  fibrosis)	
  distribution	
  matches	
  
that	
  of	
  pulmonary	
  blood	
  flow	
  (233).	
  It	
  is	
  also	
  worth	
  noting	
  that	
  exercise	
  does	
  not	
  change	
  
the	
  overall	
  pattern	
  of	
  blood	
  flow,	
  but	
  does	
  result	
  in	
  yet	
  further	
  increased	
  flow	
  to	
  dorsal	
  
lung	
  (15),	
  while	
  sparing	
  ventral	
  lung.	
  	
  A	
  possible	
  reason	
  for	
  redistribution	
  of	
  blood	
  to	
  this	
  
region	
  during	
  exercise	
  include	
  regional	
  differences	
  in	
  vascular	
  reactivity,	
  which	
  has	
  been	
  
demonstrated	
  in	
  large	
  (6	
  mm	
  O.D.)	
  equine	
  pulmonary	
  arteries	
  (164).	
  	
  	
  
That	
  EIPH	
  lesion	
  distribution	
  matches	
  blood	
  flow	
  distribution	
  (233)	
  is	
  particularly	
  
interesting	
  when	
  venous	
  remodeling	
  pathogenesis	
  is	
  considered.	
  Vascular	
  remodeling,	
  and	
  
specifically	
  pulmonary	
  venous	
  remodeling	
  is	
  a	
  classic	
  adaptive	
  response	
  to	
  increased	
  
intravascular	
  flow	
  and/or	
  pressure	
  (64,	
  86).	
  The	
  region	
  of	
  lungs	
  in	
  which	
  veins	
  remodel	
  in	
  
EPIH	
  is	
  the	
  region	
  that	
  also	
  receives	
  the	
  highest	
  flow	
  during	
  exercise	
  (15).	
  Remodeled	
  veins	
  
have	
  increased	
  wall	
  collagen	
  content	
  and	
  reduced	
  lumen	
  diameter	
  (38).	
  It	
  is	
  likely	
  that	
  
these	
  changes	
  decrease	
  venous	
  wall	
  compliance,	
  and	
  may	
  effectively	
  impede	
  capillary	
  
drainage	
  and	
  increase	
  pulmonary	
  capillary	
  pressure	
  as	
  a	
  result.	
  	
  

8	
  	
  

Whether	
  EIPH	
  pathology	
  can	
  be	
  reproduced	
  by	
  the	
  presence	
  of	
  blood	
  in	
  the	
  airway	
  
(i.e.	
  without	
  exercise)	
  has	
  been	
  investigated.	
  Neither	
  a	
  single	
  nor	
  repeated	
  instillations	
  of	
  
autologous	
  blood	
  into	
  the	
  small	
  airways	
  resulted	
  in	
  reproduction	
  of	
  EIPH-­‐associated	
  
interstitial	
  fibrosis	
  (39,	
  232).	
  A	
  reasonable	
  interpretation	
  of	
  these	
  data	
  is	
  that	
  capillary	
  
stress	
  failure	
  and	
  presumably,	
  interstitial	
  hemorrhage	
  are	
  required	
  for	
  development	
  of	
  the	
  
fibrotic	
  component	
  of	
  EIPH	
  pathology.	
  Simple	
  capillary	
  stress	
  failure	
  alone	
  does	
  not	
  take	
  
into	
  account	
  the	
  distinctive	
  regional	
  pattern	
  of	
  the	
  EIPH	
  lesion	
  however.	
  	
  
Taking	
  all	
  of	
  this	
  information	
  into	
  account	
  led	
  to	
  the	
  development	
  of	
  a	
  more	
  detailed	
  
theory	
  of	
  pathogenesis,	
  which	
  follows:	
  
During	
  intense	
  exercise	
  horses	
  experience	
  elevated	
  pulmonary	
  artery,	
  left	
  atrial	
  and	
  
pulmonary	
  capillary	
  pressures.	
  In	
  caudodorsal	
  regions	
  of	
  lung	
  that	
  already	
  experience	
  
highest	
  flow,	
  regional	
  differences	
  in	
  determinants	
  of	
  arterial	
  and	
  venous	
  vessel	
  diameter	
  
promote	
  even	
  higher	
  pulmonary	
  capillary	
  pressures.	
  Pulmonary	
  capillary	
  breaking	
  
strength	
  is	
  exceeded	
  resulting	
  in	
  stress	
  failure	
  of	
  some	
  capillaries,	
  and	
  extravasation	
  of	
  red	
  
cells	
  and	
  airway	
  hemorrhage.	
  During	
  both	
  training	
  and	
  racing,	
  repeated	
  episodes	
  of	
  high	
  
pulmonary	
  blood	
  flow	
  and	
  pressures,	
  particularly	
  in	
  the	
  highest	
  flow	
  regions	
  within	
  
caudodorsal	
  lung,	
  result	
  in	
  pulmonary	
  venous	
  remodeling.	
  Remodeled	
  pulmonary	
  veins	
  are	
  
less	
  compliant	
  than	
  normal	
  veins	
  and	
  failure	
  of	
  these	
  vessels	
  to	
  distend	
  normally	
  further	
  
increases	
  pulmonary	
  capillary	
  pressures,	
  which	
  in	
  turn	
  augments	
  stress-­‐failure	
  and	
  
hemorrhage.	
  The	
  increased	
  risk	
  of	
  EIPH	
  for	
  horses	
  with	
  more	
  race	
  starts	
  is	
  a	
  reflection	
  of	
  
ongoing	
  venous	
  pathology	
  that	
  results	
  from	
  transient	
  but	
  recurrent	
  exercise-­‐associated	
  
pulmonary	
  hypertension.	
  	
  

9	
  	
  

It’s	
  clear	
  that	
  there	
  remain	
  gaps	
  in	
  our	
  understanding	
  of	
  EIPH	
  pathogenesis	
  
however,	
  and	
  my	
  outlined	
  theory	
  of	
  pathogenesis	
  requires	
  more	
  detailed	
  investigations	
  to	
  
provide	
  corroboration	
  for	
  all	
  of	
  its	
  components,	
  in	
  particular	
  regional	
  control	
  of	
  pulmonary	
  
capillary	
  pressure,	
  and	
  the	
  mechanisms	
  and	
  physiologic	
  ramifications	
  of	
  venous	
  
remodeling.	
  The	
  overarching	
  aim	
  of	
  this	
  dissertation	
  therefore	
  is	
  to	
  further	
  investigate	
  the	
  
equine	
  pulmonary	
  microvasculature	
  in	
  order	
  to	
  better	
  elucidate	
  EIPH	
  pathogenic	
  
mechanisms.	
  	
  
To	
  reach	
  this	
  goal	
  I	
  investigated	
  the	
  effect	
  of	
  exercise	
  on	
  pulmonary	
  veins	
  
specifically,	
  as	
  these	
  vessels	
  are	
  remodeled	
  in	
  EIPH-­‐affected	
  lung	
  and	
  I	
  hypothesize	
  that	
  
this	
  remodeling	
  affects	
  venous	
  compliance.	
  Vein	
  wall	
  gene	
  and	
  protein	
  expression,	
  and	
  
pulmonary	
  venous	
  mechanical	
  characteristics	
  after	
  exercise	
  are	
  evaluated	
  in	
  studies	
  1	
  and	
  
3.	
  	
  
I	
  also	
  evaluated	
  the	
  mechanisms	
  that	
  control	
  small	
  pulmonary	
  artery	
  and	
  vein	
  tone	
  
in	
  the	
  horse	
  lung,	
  and	
  specifically	
  whether	
  or	
  not	
  these	
  mechanisms	
  exhibit	
  a	
  regionally	
  
heterogeneous	
  pattern.	
  The	
  rationale	
  for	
  this	
  component	
  is	
  that	
  these	
  vessels	
  directly	
  
impact	
  pulmonary	
  capillary	
  pressure.	
  Furthermore,	
  EIPH	
  pathology	
  has	
  a	
  distinct	
  regional	
  
distribution.	
  	
  In	
  order	
  to	
  understand	
  EIPH	
  pathogenesis,	
  our	
  understanding	
  of	
  how	
  the	
  tone	
  
of	
  these	
  vessels	
  is	
  regulated	
  during	
  exercise,	
  and	
  whether	
  regional	
  differences	
  in	
  these	
  
control	
  mechanisms	
  needs	
  to	
  be	
  investigated.	
  
	
  
Treatment	
  

	
  
10	
  

Many	
  pharmacologic	
  agents	
  have	
  been	
  used	
  to	
  treat	
  EIPH,	
  for	
  the	
  most	
  part	
  in	
  the	
  absence	
  
of	
  proof	
  of	
  efficacy.	
  For	
  the	
  purposes	
  of	
  this	
  summary,	
  discussion	
  will	
  be	
  limited	
  to	
  the	
  
most	
  widely	
  used	
  therapy,	
  and	
  the	
  only	
  one	
  with	
  evidence	
  to	
  support	
  its	
  use,	
  furosemide.	
  	
  
Furosemide	
  (4-­‐chloro-­‐N-­‐2[(furylmethyl)amino]-­‐5-­‐sulfamoylbenzoic	
  acid)	
  is	
  a	
  high-­‐
ceiling	
  or	
  loop	
  diuretic	
  that	
  is	
  licensed	
  in	
  the	
  United	
  States	
  for	
  use	
  in	
  horses	
  for	
  the	
  
treatment	
  of	
  edema	
  (e.g.	
  pulmonary	
  congestion,	
  ascites)	
  associated	
  with	
  cardiac	
  
insufficiency,	
  and	
  acute	
  non-­‐inflammatory	
  tissue	
  edema	
  
(http://www.accessdata.fda.gov/scripts/animaldrugsatfda/details.cfm?dn=034-­‐478).	
  	
  
Furosemide’s	
  use	
  as	
  a	
  pre-­‐race	
  treatment	
  in	
  horses	
  became	
  commonplace	
  in	
  the	
  1970s,	
  and	
  
the	
  drug	
  is	
  currently	
  used	
  in	
  excess	
  of	
  90%	
  of	
  race	
  starts	
  in	
  North	
  America	
  (74).	
  High	
  
quality	
  evidence	
  of	
  its	
  efficacy	
  in	
  EIPH	
  treatment	
  was	
  finally	
  provided	
  in	
  2009	
  when	
  results	
  
of	
  a	
  blinded,	
  placebo-­‐controlled,	
  crossover	
  study	
  of	
  167	
  racehorses	
  were	
  published.	
  Horses	
  
that	
  were	
  treated	
  with	
  a	
  saline	
  placebo	
  before	
  racing	
  were	
  approximately	
  4	
  times	
  as	
  likely	
  
to	
  develop	
  EIPH	
  than	
  those	
  horses	
  that	
  received	
  furosemide	
  (74).	
  Furosemide	
  did	
  not	
  
prevent	
  EIPH	
  in	
  this	
  population,	
  but	
  almost	
  70%	
  of	
  horses	
  that	
  received	
  furosemide	
  and	
  
experienced	
  EIPH	
  had	
  a	
  reduced	
  severity	
  score	
  of	
  one	
  or	
  more	
  grades	
  (69)	
  compared	
  to	
  the	
  
placebo	
  treatment	
  arm	
  (74).	
  	
  
Furosemide	
  is	
  associated	
  with	
  enhanced	
  racing	
  performance	
  in	
  both	
  Thoroughbred	
  and	
  
Standardbred	
  horses	
  (60,	
  192),	
  and	
  while	
  it	
  is	
  proposed	
  by	
  many	
  that	
  this	
  is	
  due	
  to	
  its	
  
amelioration	
  of	
  performance-­‐limiting	
  EIPH,	
  data	
  that	
  can	
  prove	
  this	
  association	
  have	
  not	
  
been	
  published	
  to	
  date.	
  	
  
The	
  mechanism	
  of	
  action	
  of	
  furosemide	
  in	
  EIPH	
  is	
  attributed	
  to	
  its	
  diuretic	
  effect,	
  and	
  the	
  
resulting	
  8%	
  reduction	
  in	
  plasma	
  volume	
  (72).	
  This	
  is	
  turn	
  is	
  associated	
  with	
  a	
  rapid	
  and	
  

	
  
11	
  

sustained	
  decrease	
  in	
  right	
  atrial	
  pulmonary	
  artery	
  pressure	
  (71,	
  158).	
  Pulmonary	
  wedge	
  
and	
  capillary	
  pressures	
  are	
  also	
  reduced	
  by	
  approximately	
  20	
  %	
  during	
  exercise	
  after	
  
treatment	
  with	
  furosemide	
  (54,	
  123).	
  As	
  these	
  effects	
  are	
  abolished	
  when	
  post-­‐furosemide	
  
fluid	
  losses	
  are	
  replaced	
  with	
  intravenous	
  fluids	
  before	
  exercise	
  (71),	
  the	
  effect	
  of	
  
furosemide	
  on	
  EIPH	
  is	
  probably	
  due	
  in	
  large	
  part	
  to	
  attenuation	
  of	
  intravascular	
  pulmonary	
  
pressures	
  during	
  exercise.	
  However,	
  it	
  is	
  reported	
  by	
  some	
  that	
  cardiac	
  output	
  during	
  
exercise	
  is	
  unaffected	
  by	
  furosemide	
  administration,	
  and	
  furthermore,	
  that	
  pulmonary	
  
blood	
  flow	
  distribution	
  is	
  altered	
  in	
  a	
  manner	
  that	
  spares	
  caudodorsal	
  lung	
  (43).	
  In	
  the	
  face	
  
of	
  similar	
  cardiac	
  output,	
  altered	
  blood	
  flow	
  distribution	
  has	
  to	
  be	
  attributed	
  to	
  changes	
  in	
  
vascular	
  reactivity.	
  Furosemide	
  is	
  reported	
  to	
  be	
  a	
  systemic	
  and	
  pulmonary	
  venodilator	
  in	
  
humans	
  and	
  dogs	
  respectively	
  (59,	
  170).	
  I	
  propose	
  therefore	
  the	
  effect	
  of	
  furosemide	
  on	
  
EIPH	
  is	
  attributable	
  at	
  least	
  in	
  part	
  to	
  its	
  action	
  on	
  pulmonary	
  veins,	
  and	
  not	
  solely	
  to	
  
plasma	
  volume	
  reduction.	
  This	
  is	
  investigated	
  in	
  the	
  second	
  study	
  described	
  in	
  this	
  
dissertation.	
  	
  	
  
	
  
Summary	
  
EIPH	
  is	
  a	
  highly	
  prevalent	
  condition	
  of	
  a	
  large	
  population	
  of	
  athletic	
  horses,	
  which	
  limits	
  
their	
  performance,	
  and	
  results	
  in	
  significant	
  pulmonary	
  pathology.	
  In	
  the	
  absence	
  of	
  
thorough	
  understanding	
  of	
  underlying	
  disease	
  mechanisms,	
  effective	
  therapies	
  and	
  
management	
  strategies	
  for	
  EIPH	
  will	
  remain	
  elusive.	
  	
  
	
  

	
  

	
  
12	
  

Section	
  2:	
  The	
  pulmonary	
  microvasculature	
  	
  
This	
  section	
  is	
  designed	
  to	
  highlight	
  why	
  detailed	
  study	
  of	
  the	
  pulmonary	
  microvasculature	
  is	
  
warranted	
  in	
  the	
  investigation	
  of	
  EIPH	
  pathogenic	
  mechanisms.	
  To	
  achieve	
  this,	
  vascular	
  wall	
  
mechanical	
  properties,	
  and	
  small	
  vessel	
  reactivity	
  will	
  be	
  discussed.	
  This	
  will	
  provide	
  context	
  
and	
  rationale	
  for	
  the	
  following	
  studies:	
  “Lung	
  region	
  and	
  racing	
  affect	
  mechanical	
  properties	
  
of	
  equine	
  pulmonary	
  microvasculature”	
  and	
  “Regional	
  differences	
  exist	
  in	
  autonomic	
  control	
  
of	
  equine	
  pulmonary	
  vascular	
  reactivity.”	
  
	
  
The	
  pulmonary	
  circulation	
  
The	
  earliest	
  descriptions	
  of	
  the	
  pulmonary	
  circulation	
  are	
  attributed	
  to	
  Michael	
  Servetus	
  
(b.	
  1511),	
  a	
  French	
  physician	
  and	
  theologian	
  who	
  was	
  burned	
  at	
  the	
  stake	
  for	
  heresy	
  in	
  
1553	
  (197).	
  He	
  referred	
  to	
  the	
  pulmonary	
  circulation	
  as	
  the	
  “vital	
  spirit...a	
  mixture	
  of	
  
inhaled	
  air	
  and	
  subtle	
  blood,”	
  and	
  refuted	
  the	
  commonly	
  held	
  belief	
  at	
  the	
  time	
  that	
  blood	
  
moved	
  across	
  the	
  septum	
  of	
  the	
  heart,	
  instead	
  proposing	
  its	
  actual	
  route	
  through	
  the	
  lungs.	
  	
  
Servetus’	
  reasoning	
  is	
  flawless,	
  but	
  proved	
  very	
  unpopular	
  at	
  the	
  time.	
  
“Many	
  facts	
  prove	
  the	
  reality	
  of	
  this	
  communication	
  of	
  the	
  blood	
  through	
  the	
  lungs...a	
  
confirmation	
  is	
  provided	
  by	
  the	
  huge	
  width	
  of	
  the	
  arterial	
  vein	
  [the	
  pulmonary	
  artery].	
  The	
  
arterial	
  vein	
  itself	
  would	
  never	
  have	
  been	
  constructed	
  this	
  way,	
  nor	
  would	
  it	
  be	
  so	
  wide,	
  and	
  it	
  
would	
  not	
  be	
  forwarding	
  such	
  a	
  powerful	
  jet	
  of	
  the	
  purest	
  of	
  blood	
  from	
  the	
  heart	
  to	
  the	
  lungs	
  
simply	
  in	
  order	
  to	
  provide	
  nourishment	
  for	
  the	
  lungs;	
  the	
  heart	
  would	
  never	
  have	
  placed	
  itself	
  
in	
  the	
  service	
  of	
  the	
  lungs	
  in	
  this	
  manner.”	
  
	
  

	
  
13	
  

Needless	
  to	
  say,	
  since	
  Servetus’	
  time,	
  our	
  understanding	
  of	
  the	
  pulmonary	
  
circulation	
  anatomy	
  and	
  function	
  has	
  advanced	
  considerably.	
  Evidence	
  that	
  the	
  pulmonary	
  
circulation	
  is	
  the	
  source	
  of	
  hemorrhage	
  in	
  EIPH	
  was	
  published	
  in	
  1993	
  by	
  John	
  West	
  and	
  
colleagues	
  (230),	
  and	
  this	
  group	
  of	
  investigators	
  was	
  the	
  first	
  to	
  propose	
  that	
  stress-­‐failure	
  
of	
  pulmonary	
  capillaries	
  occurs	
  secondary	
  to	
  the	
  high	
  intravascular,	
  and	
  specifically	
  high	
  
capillary	
  pressures	
  that	
  normally	
  occur	
  in	
  the	
  horse	
  during	
  exercise	
  (229).	
  	
  
Elucidation	
  of	
  the	
  factors	
  that	
  determine	
  this	
  capillary	
  pressure	
  therefore,	
  should	
  be	
  
considered	
  crucial	
  for	
  complete	
  understanding	
  of	
  EIPH	
  pathogenesis.	
  	
  
Both	
  pulmonary	
  arterial	
  and	
  left	
  atrial	
  pressures	
  determine	
  pulmonary	
  capillary	
  
pressure.	
  Pappenheimer	
  and	
  Soto-­‐Riviera	
  determined	
  capillary	
  pressure	
  in	
  an	
  isolated	
  
dog/cat	
  limb	
  by	
  use	
  of	
  the	
  isogravimetric	
  technique,	
  and	
  demonstrated	
  that	
  it	
  required	
  a	
  
significantly	
  smaller	
  increase	
  in	
  venous	
  pressure,	
  compared	
  with	
  arterial	
  pressure,	
  to	
  cause	
  
a	
  given	
  increase	
  in	
  filtration	
  (i.e.	
  pressure)	
  across	
  the	
  capillary	
  bed	
  (160).	
  Gaar	
  and	
  
colleagues	
  employed	
  the	
  same	
  technique	
  to	
  estimate	
  pulmonary	
  capillary	
  pressure,	
  and	
  
arterial	
  and	
  venous	
  resistance	
  to	
  flow	
  in	
  the	
  isolated	
  dog	
  lung	
  in	
  1967	
  (49).	
  They	
  
determined	
  that	
  under	
  normal	
  conditions	
  of	
  flow	
  (that	
  do	
  not	
  cause	
  capillary	
  filtration),	
  
pulmonary	
  arteries	
  and	
  veins	
  contribute	
  56	
  and	
  44%	
  respectively	
  to	
  pulmonary	
  vascular	
  
resistance,	
  and	
  that	
  capillary	
  pressure	
  Pcap	
  can	
  be	
  calculated	
  as	
  follows:	
  
Pcap	
  =	
  Pla	
  +	
  0.4	
  x	
  (Ppa	
  –	
  Pla)	
  
where	
  Pla	
  and	
  Ppa	
  are	
  left	
  atrial	
  and	
  pulmonary	
  artery	
  pressures	
  respectively	
  (141).	
  	
  
This	
  equation	
  does	
  not	
  hold	
  true	
  in	
  conditions	
  of	
  high	
  flow	
  rates	
  however,	
  such	
  as	
  are	
  
experienced	
  by	
  the	
  horse	
  whose	
  cardiac	
  output	
  increases	
  at	
  least	
  6	
  fold	
  during	
  exercise	
  
(44).	
  As	
  flow	
  is	
  increased,	
  resistance	
  in	
  the	
  arterial	
  compartment	
  decreases,	
  whereas	
  that	
  

	
  
14	
  

of	
  the	
  venous	
  compartment	
  increases.	
  At	
  10	
  times	
  normal	
  flow	
  in	
  an	
  isolated	
  dog	
  lung	
  lobe,	
  
the	
  (venous)	
  compartment	
  downstream	
  of	
  the	
  capillary	
  bed	
  contributes	
  86%	
  of	
  pulmonary	
  
resistance	
  to	
  flow	
  (238).	
  	
  	
  
	
  	
  

More	
  recent	
  descriptions	
  of	
  EIPH	
  pathology	
  clearly	
  indicate	
  that	
  pathogenic	
  

mechanisms	
  are	
  more	
  complex	
  than	
  simple	
  stress-­‐failure	
  (231),	
  and	
  that	
  pulmonary	
  
venous	
  remodeling,	
  whose	
  distribution	
  can	
  be	
  predicted	
  by	
  that	
  of	
  pulmonary	
  blood	
  flow,	
  
is	
  also	
  a	
  fundamental	
  process	
  underpinning	
  EIPH	
  pathogenesis	
  (233).	
  As	
  it	
  is	
  highly	
  likely	
  
that	
  venous	
  wall	
  remodeling	
  increases	
  resistance	
  to	
  flow	
  above	
  normal	
  in	
  a	
  vascular	
  
compartment	
  that	
  already	
  contributes	
  the	
  majority	
  of	
  pulmonary	
  vascular	
  resistance	
  in	
  
high	
  flow	
  states,	
  pulmonary	
  veins	
  in	
  particular	
  are	
  considered	
  worthy	
  of	
  further	
  
investigation	
  and	
  scrutiny	
  in	
  the	
  context	
  of	
  EIPH.	
  	
  
The	
  pulmonary	
  circulation,	
  and	
  specifically	
  the	
  arteries	
  and	
  veins	
  that	
  supply	
  and	
  
drain	
  the	
  pulmonary	
  capillaries,	
  therefore	
  form	
  the	
  central	
  focus	
  of	
  all	
  experiments	
  
described	
  in	
  this	
  dissertation.	
  
	
  
Vessel	
  anatomy	
  
EIPH-­‐associated	
  venous	
  remodeling	
  is	
  reported	
  in	
  equine	
  pulmonary	
  vessels	
  that	
  are	
  
between	
  100	
  and	
  200	
  μm	
  in	
  outer	
  diameter	
  (O.D.)	
  (231).	
  In	
  humans,	
  pulmonary	
  arteries	
  
measuring	
  less	
  than	
  100	
  μm	
  O.D.	
  are	
  (arbitrarily)	
  classified	
  as	
  arterioles	
  by	
  some	
  authors	
  
(the	
  term	
  is	
  avoided	
  by	
  others	
  (34)),	
  and	
  have	
  varying	
  amounts	
  of	
  smooth	
  muscle	
  (95).	
  In	
  
my	
  studies	
  on	
  equine	
  pulmonary	
  vasculature	
  I	
  selected	
  muscular	
  pulmonary	
  arteries	
  and	
  
veins	
  that	
  range	
  between	
  100	
  and	
  400	
  μm	
  O.D.	
  in	
  order	
  to	
  encompasses	
  vessels	
  within	
  the	
  

	
  
15	
  

diameter	
  range	
  that	
  are	
  immediately	
  upstream	
  from	
  pulmonary	
  arterioles,	
  and	
  veins	
  from	
  
within	
  the	
  diameter	
  range	
  that	
  is	
  reported	
  to	
  remodel.	
  
Based	
  on	
  the	
  Strahler	
  ordering	
  system,	
  wherein	
  order	
  1	
  is	
  the	
  smallest,	
  noncapillary	
  
branch	
  of	
  the	
  pulmonary	
  arterial	
  (or	
  venous)	
  network,	
  the	
  average	
  diameter	
  ratio	
  across	
  
multiple	
  species	
  for	
  increasing	
  order	
  is	
  1.65	
  ±	
  0.11	
  (210).	
  This	
  system	
  is	
  suitable	
  for	
  
application	
  to	
  the	
  pulmonary	
  circulation	
  due	
  to	
  the	
  irregular	
  branching	
  pattern,	
  and	
  many	
  
“generations”	
  of	
  vessels	
  can	
  be	
  attributed	
  to	
  the	
  same	
  order	
  if	
  their	
  diameters	
  are	
  similar.	
  
Human	
  lungs	
  have	
  17	
  orders	
  of	
  pulmonary	
  artery	
  branches	
  (80).	
  Based	
  on	
  a	
  starting	
  
diameter	
  of	
  approximately	
  15	
  μm	
  in	
  first	
  order	
  arterioles,	
  with	
  an	
  increase	
  in	
  diameter	
  of	
  
65	
  %	
  with	
  each	
  new	
  branch	
  order,	
  the	
  equine	
  pulmonary	
  vessels	
  (both	
  arteries	
  and	
  veins)	
  
used	
  in	
  these	
  studies	
  range	
  between	
  orders	
  5	
  and	
  7.	
  	
  
In	
  order	
  to	
  perform	
  experiments	
  on	
  small	
  caliber	
  pulmonary	
  arteries	
  and	
  veins,	
  
accurate	
  and	
  reliable	
  identification	
  of	
  these	
  vessels	
  in	
  the	
  pulmonary	
  parenchyma	
  during	
  
vessel	
  dissection	
  is	
  a	
  key	
  technique.	
  McLaughlin	
  and	
  colleagues	
  classified	
  the	
  equine	
  lung	
  
as	
  a	
  type	
  III	
  lung,	
  based	
  in	
  part	
  on	
  their	
  observation	
  that	
  the	
  pulmonary	
  veins	
  did	
  not	
  
always	
  travel	
  with	
  bronchi	
  and	
  pulmonary	
  arteries,	
  tending	
  instead	
  to	
  take	
  a	
  “more	
  direct,	
  
independent	
  course	
  to	
  the	
  hilum”	
  (132).	
  Pulmonary	
  arteries	
  on	
  the	
  other	
  hand,	
  are	
  
generally	
  accompanied	
  by	
  a	
  paired	
  airway,	
  and	
  share	
  a	
  common	
  connective	
  tissue	
  sheath	
  
(210).	
  These	
  spatial	
  descriptors	
  provide	
  the	
  basic	
  criteria	
  for	
  discerning	
  small	
  pulmonary	
  
arteries	
  from	
  intralobular	
  pulmonary	
  veins	
  in	
  equine	
  lung	
  tissue	
  during	
  dissection.	
  	
  
Histological	
  confirmation	
  of	
  vessel	
  identity	
  was	
  also	
  used	
  as	
  a	
  secondary	
  
identification	
  technique	
  in	
  these	
  experiments,	
  as	
  relying	
  exclusively	
  on	
  the	
  anatomic	
  
distribution	
  described	
  above	
  may	
  be	
  prone	
  to	
  error	
  for	
  the	
  following	
  reasons.	
  In	
  peripheral	
  

	
  
16	
  

equine	
  pulmonary	
  vessels	
  there	
  is	
  a	
  tendency	
  for	
  pulmonary	
  veins	
  to	
  be	
  more	
  closely	
  
associated	
  with	
  the	
  bronchovascular	
  bundle	
  in	
  Type	
  III	
  lungs	
  (132),	
  although	
  in	
  this	
  
author’s	
  experience,	
  veins	
  are	
  generally	
  discernable	
  from	
  arteries	
  in	
  such	
  a	
  triad,	
  in	
  that	
  
they	
  are	
  thinner-­‐walled,	
  and	
  not	
  as	
  adherent	
  to	
  the	
  airway	
  as	
  pulmonary	
  arteries	
  typically	
  
are.	
  Also,	
  supernumerary	
  arteries	
  have	
  been	
  described	
  in	
  the	
  lungs	
  of	
  humans,	
  sheep,	
  pigs,	
  
cows	
  and	
  rats	
  (41,	
  185).	
  These	
  are	
  arteries	
  that	
  branch	
  from	
  the	
  parent	
  pulmonary	
  artery	
  
at	
  90	
  degrees	
  and	
  travel	
  unaccompanied	
  by	
  an	
  airway	
  through	
  pulmonary	
  parenchyma	
  
(210).	
  Should	
  these	
  arteries	
  exist	
  in	
  horses,	
  they	
  could	
  be	
  mistaken	
  as	
  intraparenchymal	
  
pulmonary	
  veins	
  based	
  on	
  the	
  absence	
  of	
  an	
  adjacent	
  airway.	
  	
  
The	
  relatively	
  thick	
  tunica	
  media	
  of	
  muscular	
  pulmonary	
  arteries	
  such	
  as	
  those	
  
studied	
  is	
  contained	
  by	
  both	
  an	
  internal	
  and	
  an	
  external	
  elastic	
  lamina,	
  whereas	
  pulmonary	
  
veins	
  possess	
  only	
  an	
  external	
  elastic	
  lamina	
  that	
  separates	
  the	
  tunica	
  adventitia	
  from	
  a	
  
thin	
  tunica	
  media	
  (which	
  contributes	
  between	
  33	
  and	
  60%	
  less	
  to	
  wall	
  thickness	
  in	
  veins	
  
than	
  it	
  does	
  in	
  pulmonary	
  arteries)	
  (134,	
  175,	
  210).	
  Staining	
  of	
  vessels	
  segments	
  in	
  cross	
  
section	
  with	
  hematoxylin	
  and	
  eosin,	
  and	
  Verhoef-­‐Van	
  Gieson	
  (to	
  stain	
  elastin	
  specifically)	
  
was	
  utilized	
  to	
  discern	
  between	
  arteries	
  and	
  veins,	
  and	
  confirm	
  or	
  refute	
  their	
  dissection	
  
identity	
  based	
  on	
  tunica	
  media	
  thickness	
  and	
  number	
  of	
  laminae.	
  	
  
	
  
Vessel	
  wall	
  mechanical	
  properties	
  
As	
  early	
  as	
  1880,	
  Charles	
  Roy	
  published	
  results	
  of	
  experiments	
  demonstrating	
  that	
  arterial	
  
walls,	
  amongst	
  other	
  tissues,	
  did	
  not	
  conform	
  to	
  Hooke’s	
  Law	
  for	
  elastic	
  materials	
  (179).	
  In	
  
general	
  terms,	
  Hooke’s	
  law	
  states	
  that	
  strain	
  (deformation)	
  of	
  an	
  elastic	
  material	
  is	
  
proportional	
  to	
  the	
  stress	
  (force)	
  applied	
  to	
  it.	
  In	
  other	
  words,	
  with	
  the	
  application	
  of	
  

	
  
17	
  

increasing	
  loads,	
  true	
  elastic	
  materials	
  become	
  more	
  extensible.	
  In	
  contrast,	
  Roy	
  observed	
  
that	
  arteries	
  demonstrate	
  increasing	
  resistance	
  to	
  stretch	
  as	
  applied	
  stress	
  is	
  increased	
  
(179).	
  	
  
The	
  ability	
  of	
  a	
  vessel	
  to	
  resist	
  stretch	
  is	
  an	
  important	
  quality,	
  as	
  it	
  is	
  this	
  passive	
  
tension	
  (which	
  can	
  be	
  calculated	
  using	
  the	
  Laplace	
  relationship)	
  that	
  counteracts	
  the	
  
tendency	
  of	
  the	
  blood	
  pressure	
  to	
  distend	
  a	
  vessel	
  indefinitely	
  (or	
  to	
  rupture),	
  and	
  this	
  
maintenance	
  passive	
  tension	
  can	
  be	
  achieved	
  without	
  expenditure	
  of	
  energy	
  (20).	
  The	
  
main	
  structural	
  components	
  of	
  vessel	
  (other	
  than	
  capillary)	
  walls	
  are	
  endothelial	
  cells,	
  
vascular	
  smooth	
  muscle,	
  elastin	
  and	
  collagen.	
  The	
  contributions	
  of	
  endothelial	
  cells	
  and	
  
vascular	
  smooth	
  muscle	
  to	
  the	
  ability	
  of	
  a	
  vessel	
  to	
  passively	
  resist	
  stretch	
  are	
  considered	
  
minimal	
  (20),	
  however	
  the	
  role	
  of	
  elastin	
  and	
  collagen	
  in	
  conferring	
  this	
  property	
  on	
  a	
  
vessel	
  wall	
  have	
  been	
  investigated	
  in	
  some	
  detail.	
  In	
  1957	
  a	
  key	
  manuscript	
  published	
  by	
  
Roach	
  and	
  Burton	
  entitled	
  “the	
  reason	
  for	
  the	
  shape	
  of	
  the	
  distensibility	
  curves	
  of	
  arteries”	
  
explained	
  Roy’s	
  observations	
  using	
  an	
  eloquently-­‐designed	
  study	
  (177).	
  Using	
  human	
  iliac	
  
arteries,	
  these	
  investigators	
  generated	
  pressure-­‐volume	
  curves,	
  which	
  were	
  subsequently	
  
converted	
  into	
  tension-­‐length	
  curves	
  using	
  the	
  Laplace	
  relationship,	
  from	
  3	
  sets	
  of	
  vessels:	
  
these	
  comprised	
  of	
  control	
  vessels,	
  arteries	
  that	
  had	
  undergone	
  formic	
  acid	
  digestion	
  to	
  
selectively	
  remove	
  collagen,	
  and	
  arteries	
  that	
  undergone	
  a	
  trypsin	
  digestion	
  to	
  remove	
  all	
  
elastin	
  fibers.	
  By	
  subtraction,	
  and	
  comparison	
  with	
  control	
  vessels,	
  the	
  individual	
  
contributions	
  of	
  elastin	
  and	
  collagen	
  to	
  the	
  mechanical	
  behavior	
  of	
  an	
  arterial	
  wall	
  under	
  
stress	
  were	
  elucidated.	
  In	
  summary,	
  it	
  can	
  be	
  deduced	
  from	
  their	
  data	
  that	
  elastin	
  is	
  
responsible	
  for	
  wall	
  tension	
  at	
  low	
  pressures	
  (i.e.	
  the	
  lower,	
  flatter	
  portion	
  of	
  a	
  typical	
  
arterial	
  length-­‐tension	
  curve)	
  whereas	
  collagen	
  is	
  responsible	
  at	
  higher	
  pressures	
  (i.e.	
  in	
  

	
  
18	
  

the	
  steeper	
  portion	
  of	
  the	
  curve)	
  (177).	
  At	
  low	
  pressures,	
  collagen	
  remains	
  unstretched	
  and	
  
effectively	
  coiled,	
  however	
  with	
  increasing	
  pressures	
  collagen	
  is	
  stretched	
  and	
  contributes	
  
more	
  to	
  vessel	
  wall	
  tension.	
  Roach	
  and	
  Burton’s	
  data	
  speak	
  to	
  the	
  fact	
  that	
  the	
  Young’s	
  
(elastic)	
  modulus	
  of	
  collagen	
  is	
  many	
  100-­‐fold	
  higher	
  than	
  that	
  of	
  elastin,	
  indicating	
  that	
  
the	
  former	
  can	
  withstand	
  much	
  more	
  stress	
  without	
  significant	
  deformation	
  than	
  elastin	
  
can	
  (20).	
  
That	
  pulmonary	
  arteries	
  are	
  more	
  distensible	
  that	
  pulmonary	
  veins	
  has	
  been	
  
described	
  in	
  the	
  rabbit	
  (22),	
  the	
  dog	
  (121)	
  and	
  in	
  large	
  pulmonary	
  arteries	
  and	
  veins	
  in	
  
people	
  (9,	
  119).	
  This	
  difference	
  is	
  attributed	
  to	
  the	
  greater	
  proportion	
  of	
  collagen	
  in	
  venous	
  
compared	
  to	
  arterial	
  walls	
  (121).	
  
Distensibility	
  of	
  the	
  pulmonary	
  circulation	
  is	
  a	
  mechanical	
  property	
  that	
  reflects	
  the	
  
%	
  change	
  in	
  diameter	
  of	
  a	
  vessel	
  /	
  mmHg	
  pressure,	
  and	
  is	
  determined	
  in	
  large	
  part	
  by	
  
vessel	
  wall	
  structure.	
  Distensibility	
  is	
  denoted	
  by	
  the	
  distensibility	
  coefficient	
  α.	
  Overall	
  
distensibility	
  of	
  the	
  human	
  pulmonary	
  circulation	
  (arteries	
  and	
  veins)	
  is	
  estimated	
  at	
  0.02	
  
(i.e.	
  a	
  2%	
  change	
  in	
  vessel	
  diameter	
  for	
  each	
  mmHg	
  increase	
  in	
  pressure),	
  whereas	
  that	
  of	
  
the	
  horse	
  is	
  lower,	
  and	
  calculated	
  at	
  0.01	
  (174),	
  although	
  these	
  particular	
  figures	
  are	
  
calculated	
  from	
  in	
  vivo	
  pulmonary	
  arterial	
  and	
  pulmonary	
  wedge	
  pressures,	
  therefore	
  the	
  
impact	
  of	
  changes	
  in	
  vessel	
  diameter	
  resulting	
  from	
  active	
  (smooth	
  muscle-­‐mediated)	
  
alterations	
  in	
  vessel	
  tone	
  must	
  be	
  considered	
  superimposed	
  on	
  distensibility	
  determined	
  
by	
  mechanical	
  properties	
  of	
  the	
  vessel	
  walls.	
  That	
  being	
  said,	
  distensibility	
  of	
  isolated	
  
pulmonary	
  vessels	
  of	
  varying	
  size,	
  in	
  multiple	
  species	
  is	
  also	
  approximately	
  2%	
  (102),	
  and	
  
for	
  the	
  most	
  part,	
  this	
  figure	
  is	
  a	
  reflection	
  of	
  these	
  isolated	
  vessels’	
  mechanical	
  properties	
  
only.	
  This	
  is	
  an	
  interesting	
  observation	
  that	
  suggests	
  that	
  active	
  changes	
  in	
  vessel	
  tone	
  have	
  

	
  
19	
  

a	
  relatively	
  small	
  impact	
  on	
  overall	
  distensibility	
  of	
  the	
  pulmonary	
  circulation,	
  and	
  that	
  
mechanical	
  properties	
  of	
  vessels	
  are	
  worthy	
  of	
  consideration	
  in	
  studies	
  of	
  pulmonary	
  
circulation	
  hemodynamics.	
  
It	
  seems	
  paradoxical	
  that	
  the	
  distensibility	
  of	
  arteries	
  and	
  veins	
  of	
  the	
  pulmonary	
  
circulation	
  has	
  been	
  demonstrated	
  to	
  be	
  relatively	
  independent	
  of	
  vessel	
  diameter	
  in	
  
multiple	
  species	
  (5,	
  33,	
  94),	
  although	
  distensibility	
  between	
  vessels	
  in	
  same	
  size	
  range	
  does	
  
differ.	
  It	
  is	
  proposed	
  by	
  some	
  authors	
  that	
  the	
  constant	
  nature	
  of	
  this	
  parameter	
  effectively	
  
preserves	
  the	
  overall	
  distribution	
  pattern	
  of	
  cardiac	
  output	
  to	
  the	
  lungs	
  when	
  flow	
  is	
  
increased	
  (102).	
  The	
  reason	
  suggested	
  for	
  why	
  this	
  is	
  a	
  desirable	
  characteristic	
  is	
  that	
  if	
  
distensibility	
  of	
  an	
  individual	
  vessel	
  was	
  diameter	
  dependent,	
  for	
  example	
  higher	
  
distensibility	
  seen	
  only	
  in	
  larger	
  vessels,	
  when	
  cardiac	
  output	
  increased,	
  flow	
  would	
  
preferentially	
  be	
  directed	
  to	
  larger	
  vessels	
  over	
  smaller	
  ones	
  at	
  a	
  bifurcation	
  of	
  two	
  vessels	
  
of	
  different	
  diameters,	
  and	
  flow	
  (mal)redistribution	
  would	
  result	
  (94).	
  If	
  this	
  diameter-­‐
independent	
  characteristic	
  of	
  vascular	
  distensibility	
  is	
  also	
  true	
  of	
  the	
  pulmonary	
  arteries	
  
and	
  veins	
  of	
  the	
  horse,	
  it	
  could	
  explain	
  why	
  the	
  distribution	
  of	
  pulmonary	
  blood	
  flow	
  does	
  
not	
  change	
  a	
  great	
  deal	
  from	
  rest	
  to	
  exercise	
  in	
  horses	
  (15).	
  	
  
To	
  the	
  best	
  of	
  my	
  knowledge,	
  regional	
  differences	
  in	
  pulmonary	
  vessel	
  mechanical	
  
properties	
  have	
  not	
  been	
  reported	
  in	
  any	
  species	
  to	
  date,	
  and	
  without	
  such	
  a	
  precedent,	
  it	
  
is	
  difficult	
  to	
  predict	
  that	
  they	
  occur	
  in	
  the	
  horse.	
  However,	
  should	
  regional	
  heterogeneity	
  
in	
  equine	
  small	
  pulmonary	
  vessel	
  distensibility	
  exist,	
  resulting	
  differences	
  in	
  resistances	
  to	
  
flow	
  in	
  vessels	
  supplying	
  and/or	
  draining	
  pulmonary	
  capillaries	
  would	
  impact	
  regional	
  
pulmonary	
  capillary	
  pressures,	
  and	
  as	
  a	
  result,	
  perhaps	
  predict	
  regional	
  propensity	
  for	
  
EIPH	
  and	
  associated	
  pathology.	
  	
  

	
  
20	
  

Increases	
  in	
  collagen	
  content	
  of	
  a	
  vessel	
  are	
  associated	
  with	
  altered	
  mechanical	
  
properties	
  –	
  specifically	
  increased	
  stiffness	
  (12,	
  30).	
  	
  Increased	
  collagen	
  content	
  of	
  
remodeled	
  equine	
  pulmonary	
  veins	
  has	
  already	
  been	
  reported	
  (38),	
  and	
  this	
  change	
  occurs	
  
in	
  caudodorsal	
  but	
  not	
  in	
  cranioventral	
  lung	
  regions	
  (231,	
  233).	
  These	
  reports	
  describe	
  
remodeling	
  changes	
  in	
  horses	
  with	
  advanced/severe	
  EIPH,	
  and	
  reports	
  of	
  EIPH-­‐associated	
  
pathology	
  in	
  racehorses	
  that	
  lack	
  a	
  clinical	
  history	
  of	
  severe	
  EIPH	
  do	
  not	
  exist	
  at	
  this	
  time.	
  
Based	
  on	
  the	
  observations	
  in	
  the	
  2013	
  pathology	
  mapping	
  paper	
  that	
  demonstrate	
  
that	
  the	
  full	
  constellation	
  of	
  EIPH	
  lesions	
  (hemosiderin	
  and	
  fibrosis)	
  cannot	
  occur	
  without	
  
colocalized	
  venous	
  remodeling,	
  but	
  that	
  remodeling	
  can	
  occur	
  on	
  its	
  own	
  (233),	
  it	
  is	
  
proposed	
  that	
  remodeling	
  is	
  an	
  early	
  change	
  in	
  EIPH	
  pathogenesis.	
  It	
  is	
  reasonable	
  to	
  
suggest	
  therefore	
  that	
  alterations	
  in	
  venous	
  wall	
  stiffness	
  due	
  to	
  remodeling	
  processes	
  will	
  
be	
  detectable	
  in	
  horses	
  that	
  are	
  training	
  and	
  racing,	
  but	
  do	
  not	
  necessarily	
  have	
  severe	
  
EIPH	
  pathology.	
  In	
  the	
  event	
  that	
  vein	
  walls	
  in	
  caudodorsal	
  lung	
  are	
  stiffer	
  in	
  horses	
  that	
  
train	
  and	
  race	
  compared	
  to	
  cranioventral	
  veins,	
  and	
  compared	
  to	
  veins	
  from	
  horses	
  that	
  do	
  
not	
  race,	
  this	
  information	
  will	
  help	
  further	
  corroborate	
  the	
  pulmonary	
  vein	
  as	
  a	
  key	
  
component	
  of	
  early	
  EIPH	
  pathogenesis.	
  	
  Increased	
  stiffness	
  resulting	
  in	
  diminished	
  venous	
  
compliance	
  in	
  caudodorsal	
  lung	
  only	
  will	
  inhibit	
  pressure-­‐mediated	
  venous	
  dilation,	
  and	
  
cause	
  increased	
  resistance	
  to	
  blood	
  flow	
  in	
  those	
  veins.	
  This	
  in	
  turn	
  will	
  increase	
  
pulmonary	
  capillary	
  pressure	
  in	
  caudodorsal	
  lung,	
  and	
  increase	
  the	
  propensity	
  for	
  capillary	
  
rupture	
  and	
  EIPH	
  in	
  this	
  lung	
  region.	
  
	
  
In	
  summary,	
  investigation	
  of	
  the	
  mechanical	
  properties	
  of	
  small	
  pulmonary	
  vessels	
  
of	
  the	
  horse	
  have	
  not	
  heretofore	
  been	
  reported,	
  and,	
  based	
  on	
  the	
  outlined	
  rationale	
  –	
  

	
  
21	
  

alterations	
  in	
  these	
  properties	
  of	
  pulmonary	
  veins	
  could	
  be	
  implicated	
  in	
  both	
  
development	
  and	
  progression	
  of	
  EIPH.	
  
The	
  study	
  outlined	
  in	
  Chapter	
  3	
  of	
  this	
  dissertation,	
  was	
  designed	
  to	
  test	
  the	
  
following	
  hypotheses:	
  	
  
Mechanical	
  properties	
  of	
  small	
  pulmonary	
  arteries	
  and	
  veins	
  do	
  not	
  differ	
  by	
  region	
  in	
  
control,	
  unraced	
  horses.	
  Also,	
  pulmonary	
  veins,	
  but	
  not	
  pulmonary	
  arteries	
  from	
  horses	
  with	
  a	
  
recent	
  racing	
  history,	
  have	
  increased	
  wall	
  stiffness	
  compared	
  with	
  veins	
  from	
  horses	
  that	
  have	
  
never	
  raced,	
  and	
  this	
  change	
  is	
  limited	
  to	
  veins	
  from	
  caudodorsal	
  lung	
  only.	
  	
  
	
  
Regulation	
  of	
  pulmonary	
  vascular	
  tone	
  
In	
  general,	
  the	
  pulmonary	
  circulation	
  can	
  be	
  described	
  as	
  a	
  high-­‐flow,	
  low	
  pressure	
  system,	
  
that	
  functions	
  to	
  match	
  perfusion	
  to	
  ventilation.	
  This	
  is	
  achieved	
  by	
  both	
  passive	
  and	
  active	
  
factors	
  that	
  affect	
  pulmonary	
  vasculature.	
  Vascular	
  wall	
  mechanical	
  characteristics	
  that	
  are	
  
investigated	
  in	
  the	
  first	
  study	
  of	
  this	
  dissertation	
  are	
  among	
  the	
  passive	
  factors	
  that	
  exert	
  
control	
  over	
  pulmonary	
  vascular	
  resistance	
  (11).	
  However,	
  active	
  control	
  of	
  vascular	
  tone	
  
must	
  also	
  be	
  considered	
  an	
  important	
  variable	
  in	
  determining	
  pulmonary	
  vascular	
  
resistance	
  (PVR).	
  Tone	
  is	
  determined	
  by	
  the	
  state	
  of	
  contraction	
  of	
  vascular	
  smooth	
  muscle,	
  
which	
  affects	
  vessel	
  diameter,	
  and	
  resistance	
  to	
  flow	
  in	
  that	
  vessel	
  segment	
  as	
  a	
  result.	
  
Small	
  changes	
  in	
  vessel	
  diameter	
  exert	
  a	
  large	
  effect	
  on	
  resistance,	
  as	
  defined	
  by	
  the	
  Hagen-­‐
Poiseuille	
  relationship.	
  Of	
  particular	
  significance	
  to	
  EIPH	
  pathogenesis,	
  regulation	
  of	
  
resistance	
  to	
  flow	
  in	
  small	
  pulmonary	
  arteries	
  and	
  veins	
  directly	
  impacts	
  pulmonary	
  
capillary	
  pressure.	
  Active	
  regulation	
  of	
  the	
  pulmonary	
  circulation	
  is	
  achieved	
  by	
  means	
  of	
  a	
  
complex	
  combination	
  of	
  neural	
  and	
  humoral	
  influences	
  (11).	
  

	
  
22	
  

	
  
Autonomic	
  control	
  of	
  pulmonary	
  vascular	
  tone	
  
Neural	
  control	
  of	
  the	
  pulmonary	
  circulation	
  is	
  provided	
  by	
  the	
  autonomic	
  nervous	
  system	
  
which	
  directly	
  innervates	
  the	
  pulmonary	
  circulation,	
  and	
  also	
  exerts	
  its	
  effects	
  through	
  
release	
  of	
  circulating	
  (humoral)	
  vasoactive	
  factors	
  (34).	
  	
  
Sympathetic	
  nervous	
  system	
  activity	
  is	
  mediated	
  by	
  α	
  and	
  β-­‐adrenergic	
  receptors	
  
(11).	
  In	
  general,	
  activation	
  of	
  α-­‐adrenoreceptors	
  causes	
  vasoconstriction	
  of	
  pulmonary	
  
arteries	
  (83),	
  although	
  activation	
  of	
  α	
  2	
  receptors	
  on	
  pulmonary	
  artery	
  endothelium	
  cause	
  
relaxation	
  of	
  porcine	
  pulmonary	
  arteries	
  (165).	
  Both	
  β1	
  and	
  β2	
  receptors	
  have	
  been	
  
identified	
  on	
  pulmonary	
  vessels	
  (154),	
  although	
  β2	
  receptors	
  likely	
  predominate	
  (83),	
  and	
  
their	
  activation	
  results	
  in	
  vasodilation	
  (133).	
  	
  
The	
  extent	
  of	
  sympathetic	
  innervation	
  of	
  the	
  pulmonary	
  vasculature	
  varies	
  
significantly	
  between	
  species,	
  and	
  this	
  was	
  reviewed	
  in	
  detail	
  by	
  Barnes	
  and	
  Liu	
  in	
  1995	
  
(11),	
  however	
  to	
  the	
  best	
  of	
  my	
  knowledge,	
  whether	
  equine	
  pulmonary	
  vessels	
  (arteries	
  
and	
  veins)	
  of	
  the	
  caliber	
  evaluated	
  in	
  these	
  studies	
  are	
  innervated	
  with	
  postganglionic	
  
sympathetic	
  fibers	
  has	
  not	
  been	
  reported.	
  
	
  Muscarinic	
  receptors,	
  when	
  bound	
  by	
  acetylcholine,	
  mediate	
  parasympathetic	
  
control	
  of	
  vascular	
  tone,	
  and	
  4	
  of	
  the	
  5	
  subtypes	
  (M1	
  –	
  M4)	
  have	
  been	
  identified	
  in	
  
pulmonary	
  vessels	
  (11).	
  Acetylcholine	
  (Ach)	
  is	
  reported	
  to	
  cause	
  both	
  vasoconstriction	
  and	
  
vasodilation	
  of	
  pulmonary	
  arteries	
  (133).	
  Norel	
  et	
  al	
  reported	
  that	
  both	
  the	
  subtype	
  of	
  
muscarinic	
  receptor,	
  and	
  whether	
  the	
  receptor	
  was	
  located	
  on	
  the	
  vascular	
  smooth	
  muscle	
  
(for	
  contraction)	
  or	
  on	
  the	
  endothelium	
  (for	
  dilation)	
  were	
  factors	
  in	
  determining	
  a	
  vessel’s	
  
response	
  to	
  Ach	
  (148).	
  	
  

	
  
23	
  

As	
  is	
  the	
  case	
  with	
  sympathetic	
  innervation,	
  there	
  are	
  significant	
  differences	
  
between	
  species	
  in	
  the	
  extent	
  of	
  parasympathetic	
  innervation	
  of	
  the	
  pulmonary	
  
vasculature	
  (11),	
  and	
  whether	
  small	
  caliber	
  equine	
  pulmonary	
  arteries	
  and	
  veins	
  possess	
  
parasympathetic	
  nerves	
  is	
  not	
  reported	
  to	
  date.	
  	
  
Numerous	
  investigators	
  have	
  evaluated	
  the	
  effect	
  of	
  the	
  sympathetic	
  and	
  the	
  
parasympathetic	
  branches	
  of	
  the	
  autonomic	
  nervous	
  system	
  on	
  the	
  pulmonary	
  vasculature	
  
as	
  a	
  whole	
  circuit	
  (rather	
  than	
  individual	
  vessels),	
  and	
  in	
  essence,	
  unopposed	
  sympathetic	
  
stimulation	
  is	
  reported	
  to	
  increase	
  pulmonary	
  vascular	
  resistance	
  (91),	
  whereas	
  vagal	
  
stimulation	
  (in	
  the	
  face	
  of	
  adrenoreceptor	
  blockade)	
  results	
  in	
  dilation	
  of	
  the	
  pulmonary	
  
vascular	
  bed	
  (143).	
  However,	
  both	
  branches	
  of	
  the	
  autonomic	
  nervous	
  system	
  work	
  in	
  
concert	
  in	
  vivo.	
  Based	
  on	
  experiments	
  performed	
  in	
  conscious	
  dogs	
  in	
  which	
  complete	
  
autonomic	
  ganglion	
  blockade,	
  and	
  specific	
  cholinergic	
  and	
  adrenergic	
  receptor	
  blockers	
  
were	
  used,	
  the	
  net	
  effect	
  of	
  autonomic	
  nervous	
  system	
  activity	
  on	
  the	
  pulmonary	
  
circulation	
  at	
  rest	
  is	
  mild	
  vasodilation,	
  which	
  is	
  predominantly	
  mediated	
  by	
  sympathetic	
  β-­‐
adrenergic	
  activity	
  (140).	
  	
  
In	
  the	
  exercising	
  horse,	
  as	
  is	
  the	
  case	
  in	
  other	
  mammals,	
  sympathetic	
  activity	
  is	
  increased,	
  
while	
  parasympathetic	
  outflow	
  correspondingly	
  decreases	
  (133).	
  During	
  exercise	
  the	
  
horse’s	
  cardiac	
  output	
  increases	
  (44),	
  circulating	
  catecholamine	
  levels	
  increase	
  
approximately	
  10-­‐fold	
  (191)	
  and	
  pulmonary	
  vascular	
  resistance	
  (PVR)	
  decreases	
  (128).	
  
Although	
  recruitment	
  and	
  passive	
  distension	
  of	
  vessels	
  certainly	
  contributes	
  to	
  
reduced	
  PVR,	
  whether	
  the	
  sympathetic	
  nervous	
  system	
  could	
  play	
  a	
  role	
  in	
  the	
  PVR	
  
decrease	
  has	
  been	
  investigated.	
  In	
  sheep,	
  during	
  exercise,	
  both	
  α-­‐	
  and	
  β-­‐receptor	
  activation	
  
occurs,	
  however	
  the	
  net	
  effect	
  on	
  pulmonary	
  vascular	
  resistance	
  is	
  neutral	
  i.e.	
  PVR	
  during	
  

	
  
24	
  

exercise	
  in	
  the	
  face	
  of	
  α-­‐	
  and	
  β-­‐receptor	
  blockade	
  was	
  not	
  different	
  to	
  PVR	
  during	
  control	
  
runs	
  (93).	
  	
  Experiments	
  performed	
  in	
  exercising	
  swine	
  indicated	
  that	
  β-­‐adrenoreceptor-­‐
mediated	
  vasodilation	
  was	
  appreciable,	
  that	
  there	
  was	
  minimal	
  impact	
  of	
  α-­‐receptor	
  
activation,	
  and	
  some	
  vasodilation	
  occurred	
  secondary	
  to	
  muscarinic	
  receptor	
  activation	
  
(203).	
  
	
  
Regional	
  control	
  of	
  pulmonary	
  vascular	
  tone	
  
That	
  the	
  autonomic	
  nervous	
  system	
  plays	
  a	
  role	
  in	
  exerting	
  changes	
  in	
  pulmonary	
  
circulation	
  resistance	
  during	
  exercise	
  is	
  clear,	
  however	
  my	
  interest	
  in	
  the	
  autonomic	
  
control	
  of	
  vessel	
  tone	
  is	
  less	
  focused	
  on	
  overall	
  PVR	
  in	
  the	
  pulmonary	
  circulation,	
  but	
  
rather	
  on	
  regional	
  control	
  of	
  pulmonary	
  capillary	
  pressure.	
  As	
  already	
  discussed,	
  
resistance	
  to	
  flow	
  in	
  the	
  pulmonary	
  arteries	
  and	
  veins	
  supplying	
  and	
  draining	
  the	
  
pulmonary	
  capillaries	
  determine	
  capillary	
  pressures,	
  and	
  regional	
  differences	
  in	
  the	
  active	
  
control	
  of	
  small	
  pulmonary	
  vessel	
  tone	
  could	
  explain	
  why	
  EIPH	
  lesions	
  have	
  a	
  distinct	
  
predilection	
  for	
  caudodorsal	
  lung	
  (233).	
  
An	
  existing	
  precedent	
  for	
  regional	
  heterogeneity	
  of	
  small	
  pulmonary	
  vessel	
  
reactivity	
  in	
  any	
  species	
  does	
  not	
  exist	
  in	
  the	
  literature	
  at	
  present,	
  however	
  regional	
  
differences	
  in	
  reactivity	
  of	
  large	
  pulmonary	
  arteries	
  have	
  already	
  been	
  reported	
  in	
  both	
  the	
  
pig	
  (176),	
  and	
  in	
  the	
  horse	
  (164).	
  In	
  the	
  pig,	
  greater	
  relaxation	
  to	
  acetylcholine-­‐induced	
  
nitric	
  oxide	
  release	
  was	
  observed	
  in	
  large	
  (3.6	
  mm	
  O.D.)	
  dorsal	
  arteries,	
  compared	
  to	
  
ventral	
  (176).	
  Interestingly,	
  and	
  also	
  in	
  pigs	
  –	
  experimental	
  endotoxemia	
  is	
  reported	
  to	
  
increase	
  perfusion	
  to	
  dorsal	
  lung,	
  and	
  reduce	
  that	
  of	
  ventral	
  lung,	
  which	
  is	
  also	
  suggestive	
  
of	
  regional	
  differences	
  in	
  vascular	
  responses	
  of	
  large	
  pulmonary	
  vessels	
  to	
  a	
  vasoactive	
  

	
  
25	
  

stimulus	
  (51).	
  In	
  the	
  case	
  of	
  large	
  (6	
  mm	
  O.D.)	
  equine	
  pulmonary	
  arteries,	
  vessels	
  from	
  
caudodorsal	
  lung	
  relaxed	
  in	
  response	
  to	
  methacholine	
  (a	
  muscarinic	
  receptor	
  agonist)	
  in	
  
an	
  endothelium-­‐dependent	
  manner,	
  whereas	
  those	
  vessels	
  in	
  cranioventral	
  lung	
  
contracted	
  after	
  a	
  transient	
  relaxation,	
  again,	
  in	
  an	
  endothelium	
  dependent	
  manner	
  (164).	
  
When	
  applied	
  to	
  the	
  whole	
  lung,	
  these	
  data	
  provide	
  a	
  possible	
  explanation	
  (secondary	
  to	
  
anatomy)	
  for	
  the	
  heterogeneous	
  distribution	
  (caudodorsal	
  predilection)	
  of	
  pulmonary	
  
blood	
  flow	
  in	
  the	
  horse	
  lung	
  (15,	
  76).	
  	
  	
  
The	
  second	
  study	
  of	
  this	
  dissertation	
  was	
  designed	
  to	
  test	
  the	
  following	
  hypothesis:	
  	
  
Regional	
  differences	
  in	
  patterns	
  of	
  vascular	
  reactivity	
  to	
  adrenergic	
  and	
  cholinergic	
  
agonists	
  in	
  small	
  pulmonary	
  arteries	
  and	
  veins	
  exist,	
  and	
  do	
  so	
  in	
  a	
  manner	
  that	
  will	
  predict	
  
the	
  predilection	
  of	
  EIPH	
  pathology	
  for	
  the	
  caudodorsal	
  lung	
  region.	
  
Phenylephrine	
  and	
  isoproterenol	
  were	
  selected	
  as	
  the	
  alpha-­‐	
  and	
  beta-­‐adrenergic	
  
agonists	
  respectively,	
  and	
  the	
  muscarinic	
  agonist	
  methacholine	
  was	
  chosen	
  to	
  evaluate	
  
muscarinic	
  responses.	
  	
  
As	
  discussed	
  above,	
  whether	
  the	
  vessels	
  that	
  are	
  studied	
  possess	
  sympathetic	
  and	
  
parasympathetic	
  nerve	
  fibers	
  remains	
  ambiguous	
  at	
  this	
  time.	
  In	
  general	
  terms	
  however,	
  
any	
  response	
  to	
  an	
  agonist	
  will	
  be	
  interpreted	
  as	
  indication	
  that	
  the	
  receptor	
  in	
  question	
  is	
  
present	
  in	
  the	
  vessel	
  wall,	
  and	
  therefore	
  susceptible	
  to	
  binding	
  by	
  agonists	
  in	
  vivo,	
  whether	
  
the	
  source	
  of	
  that	
  agonist	
  is	
  from	
  an	
  adjacent	
  nerve	
  terminal,	
  or	
  the	
  circulating	
  blood.	
  	
  
	
  
U	
  46619	
  
In	
  order	
  to	
  evaluate	
  isoproterenol	
  and	
  methacholine,	
  both	
  of	
  which	
  are	
  expected	
  to	
  relax	
  
pulmonary	
  vessels,	
  pre-­‐contraction	
  of	
  vessels	
  to	
  provide	
  tone	
  is	
  necessary	
  to	
  evaluate	
  

	
  
26	
  

drug-­‐induced	
  relaxation.	
  The	
  thromboxane	
  A2	
  analog	
  U	
  46619	
  is	
  a	
  reliable	
  vasoconstrictor	
  
in	
  many	
  vessel	
  types,	
  and	
  was	
  selected	
  for	
  this	
  purpose.	
  	
  Thromboxane	
  A2,	
  an	
  arachidonic	
  
acid	
  metabolite	
  derived	
  from	
  endothelial	
  cells	
  and	
  platelets	
  (147,	
  168)	
  is	
  a	
  pulmonary	
  
vasoconstrictor	
  that	
  is	
  implicated	
  in	
  endotoxemia-­‐associated	
  pulmonary	
  hypertension	
  
(103)	
  and	
  other	
  pulmonary	
  diseases.	
  U	
  46619	
  mediates	
  its	
  effects	
  by	
  binding	
  to	
  G	
  12,13	
  
coupled	
  TP	
  (thromboxane)	
  receptors	
  on	
  vascular	
  smooth	
  muscle	
  cells,	
  which	
  results	
  in	
  
enhanced	
  calcium	
  sensitivity	
  of	
  myofilaments,	
  and	
  some	
  increases	
  in	
  intracellular	
  calcium	
  
concentration	
  and	
  (40,	
  194).	
  Enhanced	
  sensitivity	
  to	
  U	
  46619	
  in	
  pulmonary	
  veins	
  
compared	
  to	
  pulmonary	
  arteries	
  has	
  been	
  reported	
  in	
  multiple	
  species,	
  including	
  sheep,	
  
dogs	
  and	
  guinea	
  pigs	
  (10,	
  99,	
  187).	
  Ovine	
  pulmonary	
  veins	
  that	
  are	
  less	
  than	
  1	
  mm	
  in	
  
diameter	
  produce	
  significantly	
  more	
  thromboxane	
  A2	
  than	
  both	
  larger	
  veins,	
  and	
  
pulmonary	
  arteries	
  (84).	
  A	
  stable	
  analogue	
  of	
  thromboxane	
  A2	
  has	
  been	
  demonstrated	
  to	
  
increase	
  pulmonary	
  capillary	
  pressure	
  by	
  selective	
  venoconstriction	
  (189).	
  Based	
  on	
  
information	
  from	
  other	
  species,	
  any	
  significant	
  in	
  vivo	
  activity	
  of	
  thromboxane	
  A2	
  in	
  the	
  
horse	
  most	
  likely	
  involves	
  pulmonary	
  veins.	
  The	
  role	
  of	
  thromboxane	
  in	
  regulation	
  of	
  
vascular	
  tone	
  during	
  exercise	
  however,	
  is	
  thought	
  to	
  be	
  minimal	
  (144),	
  and	
  therefore	
  
thromboxane	
  A2	
  is	
  not	
  likely	
  to	
  be	
  a	
  contributing	
  factor	
  to	
  the	
  propensity	
  of	
  caudodorsal	
  
lung	
  to	
  develop	
  EIPH	
  lesions.	
  	
  
	
  
	
  Phenylephrine	
  
Binding	
  of	
  phenylephrine	
  to	
  the	
  Gq	
  protein	
  coupled	
  α1	
  adrenoreceptor	
  on	
  vascular	
  smooth	
  
muscle	
  cells	
  activates	
  phospholipase	
  C,	
  which	
  in	
  turn	
  mediates	
  a	
  signaling	
  cascade	
  that	
  

	
  
27	
  

both	
  causes	
  release	
  of	
  calcium	
  from	
  sarcoplasmic	
  reticulum	
  and	
  inhibition	
  of	
  myosin	
  light	
  
chain	
  phosphatase,	
  and	
  ultimately	
  –	
  smooth	
  muscle	
  contraction	
  (194).	
  	
  
Phenylephrine	
  is	
  a	
  reliable	
  pulmonary	
  arterial	
  constricting	
  agent	
  (83,	
  165),	
  and	
  has	
  already	
  
been	
  demonstrated	
  to	
  exert	
  vasoconstrictor	
  effects	
  on	
  large	
  (1.5	
  –	
  4	
  mm	
  in	
  diameter)	
  
equine	
  pulmonary	
  arteries	
  (117),	
  and	
  veins	
  (63).	
  
	
  
Isoproterenol	
  
When	
  bound	
  to	
  either	
  β	
  1	
  or	
  β	
  2	
  adrenoreceptors	
  on	
  vascular	
  smooth	
  muscle	
  cells,	
  
isoproterenol,	
  by	
  means	
  of	
  Gs	
  protein	
  coupling	
  increases	
  adenylate	
  cyclase	
  activity	
  and	
  
production	
  of	
  the	
  second	
  messenger	
  cAMP.	
  cAMP	
  in	
  turn	
  decreases	
  myosin	
  light	
  chain	
  
kinase	
  activity	
  (via	
  protein	
  kinase	
  A)	
  and	
  smooth	
  muscle	
  relaxation	
  results	
  (182).	
  More	
  
recently,	
  the	
  role	
  of	
  endothelial	
  β	
  adrenoreceptors	
  in	
  vasodilation	
  is	
  being	
  investigated,	
  and	
  
increased	
  nitric	
  oxide	
  synthesis/release	
  as	
  the	
  proposed	
  mechanism	
  (216,	
  228).	
  	
  
Isoproterenol	
  is	
  reported	
  to	
  cause	
  relaxation	
  in	
  both	
  pulmonary	
  arteries	
  (154)	
  and	
  
in	
  pulmonary	
  veins,	
  although,	
  the	
  response	
  of	
  rat	
  pulmonary	
  veins	
  to	
  isoproterenol	
  was	
  
affected	
  both	
  by	
  the	
  caliber	
  of	
  vessel	
  and	
  the	
  tension	
  (corresponding	
  to	
  pressure)	
  at	
  which	
  
vessels	
  were	
  normalized	
  before	
  the	
  experiment	
  (19).	
  	
  
	
  
Methacholine	
  
Methacholine	
  is	
  a	
  non-­‐selective	
  muscarinic	
  receptor	
  agonist	
  that	
  is	
  commonly	
  used	
  to	
  
mimic	
  acetylcholine	
  in	
  an	
  experimental	
  setting.	
  Muscarinic-­‐receptor	
  mediated	
  vasodilation	
  
is	
  typically	
  mediated	
  by	
  release	
  of	
  endothelium-­‐derived	
  relaxing	
  factors	
  (EDRFs)	
  such	
  as	
  
the	
  prototypical	
  relaxant	
  nitric	
  oxide	
  (NO)	
  and/or	
  prostanoids,	
  commonly	
  prostacyclin	
  

	
  
28	
  

(PGI2);	
  on	
  the	
  other	
  hand,	
  vasoconstriction	
  can	
  result	
  from	
  production	
  of	
  vasoconstrictor	
  
prostanoids	
  also	
  known	
  as	
  endothelial	
  derived	
  constricting	
  factors	
  (EDCFs)(236),	
  and/or	
  
direct	
  binding	
  of	
  vascular	
  smooth	
  muscle	
  muscarinic	
  receptors	
  (133).	
  Inhibition	
  of	
  nitric	
  
oxide	
  synthase	
  and/or	
  cyclooxygenase	
  are	
  commonly	
  used	
  techniques	
  employed	
  to	
  
investigate	
  the	
  relative	
  roles	
  of	
  nitric	
  oxide	
  and	
  arachidonic	
  acid	
  metabolites	
  respectively	
  
in	
  muscarinic-­‐receptor	
  mediated	
  vasomotion	
  (40,	
  164).	
  	
  
Muscarinic-­‐receptor	
  binding	
  can	
  cause	
  vasodilation	
  in	
  precontracted	
  pulmonary	
  
arteries	
  and	
  veins	
  (149),	
  and	
  again	
  –	
  heterogeneity	
  of	
  the	
  magnitude	
  of	
  responses	
  in	
  
vessels	
  of	
  different	
  diameters	
  is	
  reported	
  in	
  rats,	
  pigs	
  and	
  sheep	
  (99,	
  108,	
  239).	
  On	
  the	
  
other	
  hand,	
  acetylcholine	
  is	
  also	
  reported	
  to	
  cause	
  contraction	
  in	
  both	
  pulmonary	
  arteries	
  
(horse)	
  (164)	
  and	
  veins	
  (208).	
  As	
  already	
  mentioned,	
  experiments	
  by	
  Norel	
  et	
  al	
  support	
  
the	
  concept	
  that	
  whether	
  muscarinic	
  activation	
  results	
  in	
  constriction	
  or	
  relaxation	
  of	
  a	
  
blood	
  vessel	
  is	
  determined	
  by	
  the	
  relative	
  distribution	
  of	
  muscarinic	
  receptors	
  (or	
  various	
  
subtypes)	
  on	
  vascular	
  smooth	
  muscle	
  and	
  endothelium	
  (148).	
  	
  
	
  
Furosemide	
  
Furosemide	
  is	
  the	
  only	
  pharmacotherapeutic	
  used	
  in	
  the	
  treatment	
  of	
  EIPH	
  that	
  has	
  
demonstrable	
  (albeit	
  partial)	
  efficacy	
  (74).	
  Although	
  its	
  effects	
  are	
  most	
  often	
  attributed	
  to	
  
sustained	
  attenuation	
  of	
  pulmonary	
  capillary	
  pressures	
  (54,	
  123)	
  associated	
  with	
  
decreased	
  plasma	
  volume	
  (72),	
  the	
  drug’s	
  pulmonary	
  venodilator	
  properties	
  (59)	
  are	
  also	
  
of	
  interest	
  but	
  as	
  of	
  yet,	
  unstudied	
  in	
  the	
  horse.	
  
That	
  the	
  distribution	
  of	
  pulmonary	
  blood	
  flow	
  in	
  the	
  horse	
  both	
  at	
  rest	
  and	
  at	
  
exercise	
  is	
  altered	
  by	
  furosemide,	
  while	
  cardiac	
  output	
  is	
  not,	
  strongly	
  suggests	
  that	
  

	
  
29	
  

furosemide	
  has	
  a	
  direct	
  vasoactive	
  effect	
  on	
  the	
  equine	
  pulmonary	
  vasculature	
  (43).	
  
Indeed,	
  besides	
  pulmonary	
  vasoactive	
  properties	
  of	
  the	
  drug,	
  few	
  other,	
  plausible	
  
explanations	
  for	
  the	
  observation	
  that	
  furosemide	
  can	
  alter	
  pulmonary	
  blood	
  flow	
  
distribution	
  exist.	
  
If	
  vasoactivity	
  contributes	
  to	
  the	
  efficacy	
  of	
  the	
  drug	
  in	
  EIPH,	
  then	
  to	
  have	
  a	
  
protective	
  effect	
  on	
  pulmonary	
  capillary	
  pressures,	
  furosemide	
  should	
  dilate	
  pulmonary	
  
veins,	
  and	
  not	
  arteries.	
  This	
  selectivity	
  for	
  pulmonary	
  veins	
  and	
  not	
  arteries	
  has	
  been	
  
demonstrated	
  in	
  the	
  dog	
  (59).	
  	
  
For	
  these	
  reasons	
  the	
  following	
  hypothesis	
  was	
  also	
  investigated	
  in	
  the	
  second	
  
study	
  of	
  this	
  dissertation:	
  In	
  the	
  small	
  pulmonary	
  veins	
  of	
  the	
  horse,	
  furosemide	
  dilates	
  
pulmonary	
  veins	
  but	
  not	
  pulmonary	
  arteries	
  independent	
  of	
  lung	
  region.	
  	
  
There	
  does	
  not	
  appear	
  to	
  be	
  consensus	
  in	
  the	
  literature	
  regarding	
  the	
  mechanism	
  of	
  
action	
  of	
  furosemide	
  as	
  a	
  venodilator.	
  Pickkers	
  et	
  al	
  report	
  that	
  furosemide	
  effectively	
  
relaxed	
  pre-­‐contracted	
  dorsal	
  hand	
  veins,	
  but	
  that	
  the	
  effect	
  was	
  completely	
  abolished	
  by	
  
pretreatment	
  with	
  indomethacin,	
  which	
  implicates	
  local	
  prostanoid	
  synthesis	
  as	
  a	
  key	
  
mechanism	
  (170).	
  Prostanoid	
  involvement	
  of	
  furosemide’s	
  activity	
  as	
  a	
  vasodilator	
  is	
  also	
  
supported	
  by	
  data	
  from	
  isolated	
  dog	
  lung	
  lobe	
  preparations	
  (116).	
  Others	
  propose,	
  and	
  
have	
  data	
  to	
  support	
  a	
  direct	
  effect	
  of	
  furosemide	
  on	
  vascular	
  smooth	
  muscle,	
  by	
  inhibition	
  
of	
  chloride-­‐dependent	
  Na+/K+	
  co-­‐transport	
  resulting	
  in	
  reduced	
  intracellular	
  Na+,	
  the	
  
replacement	
  of	
  which	
  drives	
  Ca2+	
  out	
  of	
  the	
  cell	
  (59).	
  	
  
It	
  is	
  worth	
  noting	
  however	
  that	
  the	
  study	
  of	
  furosemide	
  activity	
  on	
  canine	
  
pulmonary	
  veins	
  encompassed	
  drug	
  concentrations	
  between	
  33	
  and	
  1000	
  times	
  as	
  high	
  as	
  
the	
  plasma	
  level	
  of	
  furosemide	
  in	
  horses	
  1	
  hour	
  after	
  administration	
  of	
  1	
  mg	
  /	
  kg,	
  which	
  is	
  

	
  
30	
  

an	
  effective	
  dose	
  in	
  EIPH	
  treatment	
  (24,	
  59).	
  Therefore	
  vasoactivity	
  would	
  probably	
  only	
  
occur	
  in	
  the	
  event	
  of	
  accumulation	
  of	
  active	
  drug	
  in	
  lung	
  tissue.	
  Significant	
  accumulation	
  of	
  
furosemide	
  in	
  lung	
  tissue	
  is	
  not	
  considered	
  likely,	
  as	
  furosemide	
  is	
  highly	
  protein	
  bound	
  in	
  
plasma,	
  and	
  excreted	
  before	
  significant	
  metabolism.	
  Indeed,	
  in	
  dogs,	
  at	
  1	
  minute	
  after	
  
maximal	
  diuresis	
  post-­‐furosemide	
  administration,	
  the	
  ratio	
  of	
  unaltered	
  furosemide	
  in	
  lung	
  
tissue	
  to	
  that	
  in	
  plasma	
  is	
  0.26,	
  indicating	
  that	
  little	
  to	
  no	
  accumulation	
  of	
  drug	
  occurs	
  in	
  
the	
  dog	
  lung	
  (31).	
  While	
  the	
  pharmacokinetics	
  of	
  furosemide	
  in	
  the	
  horse	
  are	
  documented	
  
(24),	
  to	
  the	
  best	
  of	
  my	
  knowledge,	
  equine	
  tissue	
  accumulation	
  studies	
  have	
  not	
  been	
  
reported.	
  	
  
	
  
	
  
Summary	
  
Little	
  is	
  known	
  about	
  the	
  mechanisms	
  of	
  control	
  of	
  vascular	
  tone	
  in	
  the	
  small	
  pulmonary	
  
arteries	
  and	
  veins	
  of	
  the	
  horse.	
  As	
  these	
  vessels	
  affect	
  pulmonary	
  capillary	
  pressures,	
  
knowledge	
  of	
  regional	
  differences	
  in	
  vessel	
  reactivity,	
  in	
  particular	
  under	
  the	
  high	
  
sympathetic/low	
  parasympathetic	
  outflow	
  conditions	
  of	
  exercise	
  could	
  contribute	
  further	
  
understanding	
  to	
  the	
  pathogenesis	
  of	
  EIPH.	
  

	
  
31	
  

Section	
  3:	
  Pulmonary	
  Venous	
  Remodeling	
  
This	
  section	
  is	
  designed	
  to	
  provide	
  further	
  information	
  on	
  vascular	
  remodeling,	
  focusing	
  
specifically	
  on	
  the	
  role	
  of	
  venous	
  remodeling	
  in	
  EIPH.	
  This	
  will	
  provide	
  both	
  context	
  and	
  a	
  
rationale	
  for	
  the	
  study	
  entitled	
  “Effects	
  of	
  exercise	
  on	
  markers	
  of	
  venous	
  remodeling	
  in	
  lungs	
  
of	
  horses.”	
  
	
  
Venous	
  remodeling	
  in	
  EIPH	
  
Remodeling	
  of	
  small	
  pulmonary	
  veins	
  as	
  a	
  component	
  of	
  EIPH	
  pathology	
  was	
  first	
  reported	
  
in	
  2008	
  (231).	
  Horses	
  that	
  were	
  retired	
  from	
  racing	
  due	
  to	
  severe	
  EIPH	
  were	
  studied,	
  and	
  
in	
  caudodorsal	
  lung,	
  remodeling	
  of	
  intralobular,	
  septal	
  and	
  sub-­‐pleural	
  veins	
  is	
  detailed	
  
along	
  with	
  other	
  classic	
  features	
  of	
  EIPH	
  pathology	
  such	
  as	
  hemosiderin	
  accumulation,	
  
angiogenesis	
  and	
  interstitial	
  fibrosis	
  (152,	
  157).	
  The	
  predominant	
  feature	
  of	
  remodeled	
  
vessels	
  is	
  expansion	
  of	
  the	
  adventitial	
  compartment	
  with	
  what	
  is	
  described	
  as	
  a	
  “prominent	
  
collar	
  of	
  mature	
  collagen”	
  (231).	
  Collagen	
  deposition	
  extends	
  to	
  the	
  media/intima	
  (i.e.	
  
between	
  the	
  external	
  elastic	
  lamina	
  and	
  vessel	
  lumen)	
  in	
  some	
  vessels,	
  causing	
  luminal	
  
obstruction.	
  This	
  reduction	
  in	
  lumen	
  area	
  was	
  further	
  defined	
  in	
  a	
  subsequent	
  study	
  that	
  
used	
  morphometric	
  techniques	
  to	
  confirm	
  that	
  the	
  luminal	
  perimeter	
  of	
  veins	
  in	
  
caudodorsal	
  lung	
  regions	
  with	
  the	
  most	
  severe	
  histopathology	
  was	
  significantly	
  less	
  than	
  in	
  
other,	
  less	
  severely	
  affected	
  regions	
  (38).	
  	
  
The	
  results	
  of	
  an	
  extensive	
  study	
  of	
  the	
  pathology	
  of	
  horses	
  that	
  raced	
  in	
  the	
  US	
  and	
  were	
  
retired	
  due	
  to	
  severe	
  EIPH	
  demonstrated	
  conclusively	
  that	
  EIPH	
  pathology,	
  including	
  
venous	
  remodeling,	
  was	
  most	
  common	
  and	
  most	
  severe	
  in	
  caudodorsal	
  lung	
  (233).	
  
Hemosiderin	
  and	
  remodeling	
  were	
  either	
  found	
  together,	
  or	
  mild	
  remodeling	
  was	
  found	
  on	
  

	
  
32	
  

its	
  own	
  without	
  hemosiderin.	
  Furthermore,	
  interstitial	
  fibrosis	
  almost	
  always	
  (in	
  99.7	
  %	
  of	
  
samples)	
  occurred	
  with	
  colocalized	
  venous	
  remodeling.	
  These	
  data	
  strongly	
  suggest	
  that	
  
remodeling	
  is	
  both	
  pivotal	
  in	
  the	
  disease	
  process,	
  and	
  is	
  an	
  earlier	
  pathologic	
  event	
  than	
  
fibrosis.	
  	
  
	
  
Vascular	
  remodeling	
  	
  
In	
  contrast	
  to	
  acute	
  changes	
  in	
  vessel	
  tone	
  such	
  as	
  those	
  induced	
  by	
  autonomic	
  agonists,	
  
vascular	
  remodeling	
  instead	
  describes	
  enduring	
  changes	
  in	
  vessel	
  wall	
  structure	
  that	
  
generally	
  occur	
  in	
  response	
  to	
  hemodynamic	
  stimuli	
  (67).	
  
Remodeling	
  responses	
  in	
  veins	
  from	
  the	
  systemic	
  circulation	
  have	
  been	
  studied	
  
extensively	
  as	
  a	
  result	
  of	
  the	
  common	
  practice	
  of	
  vein	
  grafting	
  in	
  coronary	
  artery	
  bypass	
  
procedures,	
  and	
  subsequent	
  issues	
  with	
  maintenance	
  of	
  long-­‐term	
  vein	
  graft	
  patency	
  due	
  
to	
  remodeling	
  (240)	
  .	
  When	
  systemic	
  veins	
  are	
  exposed	
  to	
  arterial	
  conditions	
  of	
  flow	
  
(resulting	
  in	
  shear	
  stress)	
  and	
  increased	
  transmural	
  pressure	
  (resulting	
  in	
  circumferential	
  
stress)	
  in	
  an	
  experimental	
  setting,	
  increased	
  wall	
  thickness	
  is	
  observed	
  as	
  early	
  as	
  one	
  to	
  
two	
  weeks	
  (28,	
  64).	
  	
  Venous	
  remodeling	
  and	
  resultant	
  stenosis	
  in	
  the	
  context	
  of	
  grafting	
  is	
  
generally	
  characterized	
  by	
  significant	
  neointima	
  formation,	
  a	
  process	
  that	
  is	
  modulated	
  by	
  
both	
  shear	
  stress	
  and	
  pressure	
  (14,	
  61).	
  	
  
In	
  contrast	
  to	
  systemic	
  veins,	
  remodeling	
  in	
  pulmonary	
  veins	
  is	
  dramatically	
  
understudied.	
  Extensive	
  investigations	
  into	
  remodeling	
  of	
  pulmonary	
  arteries	
  exist	
  
however,	
  and	
  are	
  performed	
  mainly	
  in	
  the	
  context	
  of	
  pulmonary	
  hypertension	
  in	
  humans.	
  
While	
  there	
  must	
  be	
  some	
  common	
  ground	
  between	
  pulmonary	
  arterial	
  and	
  venous	
  
remodeling	
  in	
  terms	
  of	
  surrounding	
  environment	
  and	
  remodeling	
  stimuli,	
  I	
  propose	
  that	
  

	
  
33	
  

pulmonary	
  artery	
  remodeling	
  is	
  not	
  necessarily	
  an	
  ideal	
  template	
  for	
  the	
  same	
  process	
  in	
  
pulmonary	
  veins	
  for	
  the	
  following	
  reasons.	
  Temporally,	
  embryonic	
  pulmonary	
  vein	
  
development	
  long	
  precedes	
  that	
  of	
  pulmonary	
  arteries	
  (37)	
  and	
  recent	
  molecular	
  
investigations	
  have	
  further	
  defined	
  arterial-­‐venous	
  heterogeneity	
  (3).	
  Data	
  from	
  Chapters	
  2	
  
and	
  3	
  of	
  this	
  dissertation	
  further	
  support	
  the	
  contention	
  that	
  pulmonary	
  veins	
  and	
  arteries	
  
are	
  clearly	
  distinct	
  from	
  one	
  another	
  in	
  both	
  their	
  anatomy	
  and	
  physiology	
  (196).	
  
	
  
Pulmonary	
  venous	
  remodeling	
  -­‐	
  etiopathogenesis	
  
To	
  the	
  best	
  of	
  my	
  knowledge,	
  and	
  with	
  the	
  exception	
  Chapter	
  1	
  of	
  this	
  dissertation,	
  there	
  
are	
  no	
  reports	
  of	
  pulmonary	
  venous	
  remodeling	
  that	
  is	
  associated	
  with	
  exercise	
  in	
  any	
  
other	
  species	
  (196).	
  There	
  are	
  however	
  accounts	
  of	
  such	
  remodeling	
  responses	
  to	
  
hemodynamic	
  stimuli	
  similar	
  to	
  those	
  experienced	
  by	
  the	
  exercising	
  horse	
  –	
  namely	
  
increased	
  pulmonary	
  blood	
  flow	
  (43)	
  and	
  intravascular	
  pulmonary	
  venous	
  pressures	
  (88,	
  
124).	
  
In	
  general,	
  two	
  types	
  of	
  hemodynamic	
  stresses	
  are	
  applied	
  to	
  vessel	
  walls	
  by	
  the	
  
blood	
  within	
  the	
  lumen;	
  namely	
  shear	
  stress	
  caused	
  by	
  blood	
  flow	
  and	
  circumferential	
  or	
  
hoop	
  stress	
  caused	
  by	
  pulse	
  pressure	
  (159).	
  Circumferential	
  stress	
  is	
  applied	
  to	
  all	
  
components	
  of	
  the	
  vessel	
  wall,	
  whereas	
  shear	
  stresses	
  mostly	
  affect	
  the	
  endothelium.	
  While	
  
alterations	
  in	
  shear	
  stress	
  are	
  widely	
  recognized	
  as	
  a	
  cause	
  of	
  vascular	
  remodeling	
  (217)	
  it	
  
is	
  difficult	
  to	
  estimate	
  whether	
  this	
  is	
  a	
  significant	
  stimulus	
  in	
  EIPH-­‐associated	
  venous	
  
remodeling,	
  in	
  large	
  part	
  because	
  very	
  little	
  is	
  known	
  about	
  in	
  vivo	
  flow	
  patterns	
  (and	
  
resulting	
  shear	
  stresses)	
  in	
  the	
  small	
  pulmonary	
  veins	
  of	
  any	
  species.	
  Remodeling	
  
associated	
  with	
  increased	
  shear	
  stress	
  typically	
  results	
  in	
  “outward	
  remodeling”	
  which	
  

	
  
34	
  

thickens	
  vessel	
  walls,	
  specifically	
  the	
  smooth	
  muscle	
  of	
  the	
  tunica	
  media	
  (21,	
  215),	
  but	
  
ultimately	
  results	
  in	
  vessel	
  diameter	
  expansion	
  to	
  promote	
  return	
  of	
  shear	
  stress	
  to	
  normal	
  
levels	
  (159).	
  In	
  contrast,	
  a	
  reduction	
  in	
  venous	
  diameter	
  reduction	
  is	
  reported	
  in	
  EIPH-­‐
associated	
  venous	
  remodeling	
  (38).	
  Additionally,	
  all	
  EIPH-­‐remodeled	
  vessels,	
  even	
  those	
  
that	
  are	
  mildly	
  affected,	
  demonstrate	
  adventitial	
  compartment	
  expansion	
  by	
  collagen,	
  in	
  
most	
  cases	
  without	
  expansion	
  of	
  the	
  smooth	
  muscle	
  of	
  the	
  tunica	
  media	
  (231,	
  233).	
  I	
  
propose	
  therefore	
  that	
  increased	
  shear	
  stress	
  in	
  response	
  to	
  increased	
  pulmonary	
  blood	
  
flow	
  during	
  exercise	
  is	
  not	
  the	
  predominant	
  stimulus	
  driving	
  development	
  of	
  pulmonary	
  
venous	
  remodeling.	
  The	
  rest	
  of	
  this	
  discussion	
  will	
  be	
  focus	
  mainly	
  on	
  increased	
  
intravascular	
  pressure	
  (and	
  resulting	
  circumferential	
  wall	
  stress)	
  as	
  a	
  stimulus	
  for	
  
remodeling.	
  	
  	
  
Experimental	
  data	
  exist	
  that	
  demonstrate	
  a	
  remodeling	
  response	
  of	
  pulmonary	
  
veins	
  to	
  increased	
  venous	
  pressure.	
  When	
  sheep	
  were	
  exposed	
  to	
  a	
  continuous	
  pulmonary	
  
arterial	
  air	
  embolus,	
  a	
  technique	
  that	
  increased	
  total	
  pulmonary	
  resistance	
  and	
  pulmonary	
  
vascular	
  pressures,	
  venous	
  wall	
  thickening	
  was	
  observed	
  within	
  4	
  days	
  of	
  exposure	
  (86).	
  	
  
Thickening	
  of	
  the	
  adventitia	
  was	
  noted	
  in	
  some	
  vessels	
  by	
  day	
  12.	
  	
  
In	
  left	
  heart	
  failure	
  pathologic	
  vascular	
  remodeling	
  is	
  usually	
  seen	
  in	
  the	
  pulmonary	
  
veins	
  before	
  arterial	
  changes	
  occur,	
  due	
  to	
  increased	
  “retrograde”	
  pressure	
  in	
  the	
  
pulmonary	
  circulation	
  (82,	
  213).	
  Mitral	
  stenosis	
  for	
  example,	
  results	
  in	
  significant	
  
thickening	
  of	
  small	
  pulmonary	
  vein	
  walls	
  due	
  to	
  fibrosis	
  of	
  the	
  intima	
  and	
  adventitia	
  (25)	
  in	
  
response	
  to	
  persistent	
  venous	
  	
  hypertension.	
  	
  
Pulmonary	
  venous	
  occlusive	
  disease	
  (PVOD)	
  is	
  a	
  rare	
  form	
  of	
  pulmonary	
  
hypertension	
  that	
  is	
  associated	
  with	
  high	
  mortality	
  (81).	
  PVOD	
  is	
  characterized	
  by	
  fibrotic	
  

	
  
35	
  

intimal	
  expansion	
  of	
  small	
  pulmonary	
  veins	
  and	
  venules,	
  and	
  in	
  some	
  patients	
  alveolar	
  
hemorrhage	
  and	
  hemosiderosis	
  (78).	
  PVOD	
  is	
  frequently	
  idiopathic,	
  and	
  the	
  initiating	
  
trigger	
  for	
  disease	
  development	
  remains	
  undetermined	
  for	
  many	
  patients	
  (81).	
  However,	
  
sustained	
  venous	
  hypertension	
  is	
  thought	
  to	
  be	
  a	
  significant	
  stimulus	
  for	
  ongoing	
  venous	
  
remodeling	
  in	
  PVOD,	
  as	
  the	
  vascular	
  changes	
  observed	
  resemble	
  those	
  described	
  in	
  mitral	
  
stenosis	
  and	
  other	
  causes	
  of	
  pulmonary	
  venous	
  hypertension.	
  	
  
	
  
Role	
  of	
  venous	
  remodeling	
  in	
  EIPH	
  
It	
  is	
  particularly	
  interesting	
  that	
  pulmonary	
  arteries	
  in	
  EIPH-­‐affected	
  lungs	
  are	
  not	
  
routinely	
  remodeled,	
  while	
  the	
  small	
  veins	
  are	
  significantly	
  affected	
  (231).	
  Cardiac	
  output	
  
and	
  pulmonary	
  artery	
  pressures	
  are	
  dramatically	
  increased	
  in	
  the	
  exercising	
  horse	
  (44,	
  
124),	
  yet	
  only	
  venous	
  pathology	
  is	
  observed.	
  Conditions	
  in	
  humans	
  in	
  which	
  venous	
  
pathology	
  either	
  occurs	
  exclusively,	
  or	
  to	
  a	
  greater	
  degree	
  than	
  arterial	
  remodeling	
  include	
  
left	
  heart	
  failure,	
  and	
  PVOD,	
  and	
  remodeling	
  in	
  both	
  is	
  attributed	
  in	
  some	
  degree	
  to	
  venous	
  
hypertension	
  specifically	
  (25).	
  	
  
A	
  galloping	
  horse	
  has	
  a	
  maximum	
  heart	
  rate	
  of	
  approximately	
  220	
  b.p.m.	
  and	
  a	
  
stroke	
  volume	
  of	
  approximately	
  1.5	
  L	
  (44).	
  	
  Left	
  heart	
  diastole	
  is	
  estimated	
  therefore	
  at	
  
0.14	
  seconds,	
  and	
  minute	
  volume	
  is	
  330	
  L/minute.	
  In	
  the	
  Operation	
  Everest	
  II	
  studies	
  
performed	
  on	
  elite	
  human	
  athletes	
  in	
  the	
  early	
  1990s,	
  investigators	
  report	
  a	
  “remarkably”	
  
close	
  relationship	
  of	
  right	
  atrial	
  and	
  pulmonary	
  artery	
  wedge	
  pressures	
  (Pwp)	
  during	
  
exercise,	
  although	
  it	
  is	
  noted	
  that	
  a	
  1	
  mmHg	
  rise	
  in	
  right	
  atrial	
  pressure	
  resulted	
  in	
  a	
  higher	
  
(1.4	
  mmHg)	
  rise	
  in	
  Pwp	
  (173)	
  which	
  further	
  loads	
  the	
  venous	
  compartment.	
  Supplying	
  the	
  
left	
  ventricle	
  with	
  1.5L	
  of	
  blood	
  within	
  0.14	
  s	
  requires	
  extremely	
  high	
  filling	
  pressures,	
  

	
  
36	
  

which	
  is	
  provided	
  by	
  the	
  right	
  heart	
  and	
  pulmonary	
  arterial	
  driving	
  pressure,	
  however	
  
maintenance	
  of	
  this	
  filling	
  pressure	
  during	
  exercise	
  also	
  results	
  in	
  necessary,	
  but	
  sustained	
  
“retrograde”	
  pressure	
  increases	
  in	
  the	
  pulmonary	
  venous	
  system	
  specifically.	
  I	
  also	
  
consider	
  it	
  plausible,	
  that	
  the	
  equine	
  pulmonary	
  arterial	
  system	
  positioned	
  as	
  it	
  is,	
  
immediately	
  downstream	
  from	
  the	
  right	
  ventricle,	
  is	
  inherently	
  better	
  adapted	
  to	
  manage	
  a	
  
hypertensive	
  stimulus	
  without	
  pathologic	
  consequences	
  than	
  the	
  venous	
  circulation,	
  and	
  if	
  
this	
  were	
  the	
  case,	
  it	
  could	
  go	
  someway	
  toward	
  explaining	
  the	
  “selective”	
  remodeling	
  
observed	
  in	
  pulmonary	
  veins	
  in	
  EIPH.	
  	
  
Whether	
  arteriovenous	
  shunting	
  occurs	
  during	
  exercise	
  such	
  as	
  is	
  reported	
  in	
  other	
  
species,	
  including	
  dogs	
  (202)	
  has	
  been	
  preliminarily	
  investigated	
  in	
  the	
  horse,	
  and	
  was	
  not	
  
demonstrable	
  through	
  the	
  use	
  of	
  microsphere	
  injection	
  (125).	
  Use	
  of	
  another	
  sensitive	
  
technique,	
  contrast	
  echocardiography	
  has	
  demonstrated	
  shunting	
  during	
  exercise	
  in	
  
human	
  subjects	
  (114),	
  and	
  if	
  this	
  phenomenon	
  occurs	
  in	
  exercising	
  horses,	
  it	
  may	
  result	
  in	
  
further	
  volume	
  loading	
  of	
  the	
  pulmonary	
  venous	
  circulation,	
  and	
  further	
  compound	
  
pulmonary	
  venous	
  hypertension.	
  	
  
Although	
  it	
  is	
  possible	
  that	
  venous	
  remodeling	
  is	
  a	
  result	
  of	
  EIPH,	
  to	
  the	
  best	
  of	
  my	
  
knowledge	
  no	
  other	
  such	
  conditions	
  (i.e.	
  venous	
  remodeling	
  in	
  response	
  to	
  local	
  
hemorrhage)	
  are	
  reported	
  in	
  the	
  literature.	
  Therefore	
  I	
  propose	
  that	
  venous	
  remodeling	
  
assumes	
  a	
  key	
  role	
  early	
  in	
  EIPH	
  pathogenesis,	
  as	
  follows:	
  	
  
Exercise-­‐associated	
  elevations	
  in	
  cardiac	
  output	
  result	
  in	
  dramatically	
  elevated	
  
pulmonary	
  artery	
  pressures	
  in	
  the	
  galloping	
  horse.	
  These	
  high	
  pressures	
  are	
  transmitted	
  to	
  
the	
  venous	
  circulation	
  in	
  order	
  to	
  provide	
  elevated	
  left	
  atrial	
  filling	
  pressures.	
  
Furthermore,	
  maintenance	
  of	
  high	
  cardiac	
  output	
  from	
  the	
  left	
  ventricle	
  during	
  exercise	
  

	
  
37	
  

contributes	
  to	
  sustained	
  retrograde	
  elevations	
  in	
  left	
  atrial,	
  and	
  therefore	
  pulmonary	
  
venous	
  pressure	
  during	
  exercise.	
  It	
  is	
  widely	
  accepted	
  that	
  left	
  atrial	
  pressures	
  are	
  
transmitted	
  upstream	
  without	
  dampening,	
  in	
  a	
  fully-­‐recruited	
  pulmonary	
  circulation	
  (141)	
  
With	
  repeated	
  training,	
  these	
  periods	
  of	
  venous	
  hypertension	
  result	
  in	
  venous	
  wall	
  
remodeling,	
  specifically	
  in	
  the	
  caudodorsal	
  lung,	
  which	
  is	
  the	
  region	
  in	
  which	
  blood	
  flow	
  
and	
  presumably,	
  therefore	
  intravascular	
  pressures	
  are	
  highest	
  (15).	
  Pulmonary	
  capillary	
  
breaking	
  strength	
  is	
  exceeded	
  in	
  some	
  capillaries	
  during	
  exercise,	
  and	
  hemorrhage	
  results.	
  
Those	
  capillaries	
  that	
  are	
  drained	
  by	
  remodeled	
  veins	
  are	
  even	
  more	
  susceptible	
  to	
  rupture	
  
during	
  exercise	
  as	
  venous	
  wall	
  compliance	
  is	
  reduced	
  and	
  in	
  some	
  cases,	
  venous	
  luminal	
  
area	
  is	
  diminished.	
  Those	
  capillaries	
  are	
  exposed	
  to	
  pressures	
  approaching	
  arterial	
  values	
  
and	
  rupture.	
  With	
  each	
  exercise	
  bout	
  the	
  injurious	
  cycle	
  is	
  repeated	
  and	
  compounded,	
  
ultimately	
  resulting	
  in	
  clinically	
  detectable	
  hemorrhage,	
  significant	
  pulmonary	
  pathology,	
  
and	
  potentially	
  impaired	
  performance.	
  	
  
To	
  validate	
  this	
  pathogenesis	
  hypothesis,	
  it	
  is	
  necessary	
  to	
  implicate	
  venous	
  
remodeling	
  as	
  an	
  early,	
  pivotal	
  event	
  in	
  EIPH	
  (rather	
  than	
  merely	
  a	
  result	
  of	
  local	
  
hemorrhage).	
  Therefore	
  I	
  designed	
  a	
  study	
  investigate	
  whether	
  intense	
  exercise	
  could	
  be	
  
associated	
  with	
  venous	
  remodeling	
  before	
  the	
  development	
  of	
  severe	
  EIPH	
  pathology.	
  	
  
The	
  following	
  hypothesis	
  was	
  investigated	
  in	
  Chapter	
  4	
  of	
  this	
  dissertation:	
  	
  
Two	
  weeks	
  of	
  intense	
  exercise	
  will	
  alter	
  mRNA	
  and	
  protein	
  expression	
  of	
  those	
  factors	
  known	
  
to	
  mediate	
  vascular	
  remodeling	
  in	
  the	
  pulmonary	
  veins	
  from	
  caudodorsal	
  but	
  not	
  
cranioventral	
  regions	
  of	
  lungs	
  of	
  horses	
  in	
  a	
  manner	
  that	
  favors	
  venous	
  remodeling.	
  	
  
	
  
Mechanisms	
  of	
  venous	
  remodeling	
  

	
  
38	
  

mRNA	
  and	
  protein	
  production	
  precede	
  histopathologic	
  change.	
  Therefore,	
  in	
  order	
  to	
  
evaluate	
  remodeling	
  as	
  early	
  in	
  its	
  development	
  as	
  possible,	
  mRNA	
  and	
  protein	
  levels	
  of	
  
factors	
  known	
  to	
  mediate	
  vascular	
  remodeling	
  were	
  studied	
  after	
  just	
  two	
  weeks	
  of	
  
intermittent	
  intense	
  exercise.	
  
Achieving	
  a	
  balance	
  between	
  detecting	
  the	
  earliest	
  effects	
  of	
  exercise	
  in	
  pulmonary	
  
vein	
  walls,	
  while	
  providing	
  a	
  stimulus	
  that	
  was	
  adequate	
  to	
  invoke	
  such	
  change	
  was	
  to	
  
prove	
  challenging.	
  Reports	
  from	
  other	
  species	
  (sheep	
  and	
  rabbits)	
  describe	
  structural	
  
alterations	
  in	
  remodeling	
  veins	
  as	
  early	
  as	
  4	
  (86)	
  to	
  7	
  (64)	
  days	
  after	
  initiation	
  of	
  a	
  
continuous	
  hypertensive	
  stimulus.	
  With	
  a	
  goal	
  of	
  detecting	
  pre-­‐structural	
  changes,	
  six	
  
episodes	
  of	
  an	
  intermittent	
  hypertensive	
  stimulus	
  over	
  a	
  14-­‐day	
  period	
  were	
  deemed	
  
sufficient.	
  	
  
As	
  venous	
  remodeling	
  in	
  EIPH	
  follows	
  a	
  distinctive	
  regional	
  pattern,	
  and	
  veins	
  from	
  
cranioventral	
  lung	
  are	
  unaffected	
  (233),	
  expression	
  of	
  mRNA	
  and	
  protein	
  of	
  the	
  following	
  
list	
  of	
  factors	
  were	
  studied	
  in	
  veins	
  from	
  2	
  distinct	
  regions	
  of	
  lung	
  –	
  caudodorsal,	
  and	
  
cranioventral:	
  Collagen	
  type	
  I,	
  tenascin-­‐C,	
  matrix	
  metalloproteinases	
  (MMP)	
  MMP-­‐1	
  and	
  
MMP-­‐9,	
  tissue	
  inhibitors	
  of	
  metalloproteinases	
  (TIMP)	
  TIMP-­‐1	
  and	
  TIMP-­‐2,	
  endothelin-­‐1	
  
(ET-­‐1),	
  platelet-­‐derived	
  growth	
  factor	
  (PDGF),	
  transforming	
  growth	
  factor	
  beta	
  (TGF-­‐	
  β)	
  
and	
  vascular	
  endothelial	
  growth	
  factor	
  (VEGF).	
  	
  
Until	
  recently	
  the	
  role	
  of	
  tunica	
  adventitia	
  in	
  vascular	
  remodeling	
  has	
  been	
  
somewhat	
  overlooked	
  in	
  favor	
  of	
  the	
  tunica	
  intima	
  and	
  tunica	
  media	
  and	
  their	
  predominant	
  
cell	
  types,	
  the	
  endothelial	
  and	
  smooth	
  muscle	
  cell	
  respectively.	
  In	
  the	
  field	
  of	
  pulmonary	
  
hypertension	
  research	
  there	
  is	
  mounting	
  evidence	
  that	
  the	
  adventitia	
  and	
  the	
  cells	
  
contained	
  therein	
  act	
  as	
  a	
  key	
  injury-­‐sensing	
  and	
  regulatory	
  center	
  for	
  pulmonary	
  arteries	
  

	
  
39	
  

(199,	
  200).	
  In	
  remodeled	
  veins	
  in	
  EIPH-­‐affected	
  lung,	
  the	
  tunica	
  adventitia	
  of	
  these	
  vessels	
  
is	
  the	
  most	
  commonly,	
  and	
  most	
  severely	
  affected	
  compartment	
  (231,	
  233).	
  For	
  this	
  
reason,	
  I	
  consider	
  it	
  likely	
  that	
  the	
  adventitia	
  of	
  pulmonary	
  veins	
  also	
  plays	
  a	
  significant	
  
role	
  in	
  mediating	
  the	
  remodeling	
  response	
  in	
  EIPH.	
  	
  
The	
  most	
  common	
  cell	
  type	
  in	
  the	
  tunica	
  adventitia	
  is	
  the	
  adventitial	
  fibroblast.	
  
When	
  activated,	
  the	
  adventitial	
  fibroblast	
  rapidly	
  differentiates	
  into	
  a	
  myofibroblast,	
  and	
  
fulfills	
  multiple	
  functions,	
  some	
  of	
  which	
  include	
  production	
  of	
  extracellular	
  matrix	
  
proteins	
  including	
  collagen	
  and	
  tenascin-­‐C,	
  and	
  a	
  variety	
  of	
  growth	
  factors	
  (201).	
  	
  
The	
  first	
  description	
  of	
  remodeled	
  veins	
  in	
  EIPH	
  details	
  marked	
  collagen	
  deposition	
  
in	
  the	
  adventitia	
  of	
  remodeled	
  vessels	
  (231).	
  This	
  observation	
  was	
  supported	
  statistically	
  
when	
  morphometric	
  analysis	
  demonstrated	
  increased	
  collagen	
  content	
  in	
  vessels	
  from	
  
affected	
  lung	
  regions	
  (38).	
  Collagen	
  is	
  commonly	
  increased	
  in	
  the	
  extracellular	
  matrix	
  of	
  
remodeled	
  veins	
  from	
  other	
  species	
  (humans	
  and	
  rabbits)	
  (25,	
  235).	
  For	
  these	
  reasons	
  
collagen	
  mRNA	
  and	
  protein	
  expression	
  were	
  evaluated	
  in	
  this	
  study.	
  	
  
Tenascin-­‐C	
  is	
  another	
  important	
  extracellular	
  matrix	
  protein	
  that	
  is	
  expressed	
  by	
  
remodeling	
  tissues	
  during	
  fetal	
  development	
  and	
  in	
  disease	
  states.	
  For	
  example,	
  tenascin-­‐C	
  
expression	
  is	
  upregulated	
  in	
  remodeling	
  veins	
  in	
  both	
  humans	
  (222)	
  and	
  rodents	
  (2).	
  The	
  
adventitial	
  fibroblast	
  is	
  a	
  major	
  source	
  of	
  tenascin-­‐C	
  in	
  injured	
  vessels	
  (223),	
  therefore	
  the	
  
expression	
  of	
  tenascin-­‐C	
  mRNA	
  and	
  protein	
  were	
  selected	
  for	
  evaluation	
  in	
  this	
  study.	
  	
  
The	
  proteolytic	
  matrix	
  metalloproteinases	
  MMP-­‐2	
  and	
  MMP-­‐9	
  are	
  sub-­‐classified	
  as	
  
gelatinases,	
  and	
  their	
  substrates	
  include	
  extracellular	
  matrix	
  (ECM)	
  components	
  such	
  as	
  
collagen	
  and	
  elastin	
  (218).	
  TIMPS	
  are	
  endogenous	
  inhibitors	
  of	
  MMP	
  activity,	
  and	
  bind	
  
MMPs	
  in	
  a	
  1:1	
  stoichiometry	
  (171).	
  Although	
  4	
  TIMPS	
  have	
  been	
  characterized	
  in	
  

	
  
40	
  

vertebrates,	
  TIMP-­‐1	
  binds	
  preferentially	
  with	
  MMP-­‐9,	
  and	
  TIMP-­‐2	
  with	
  MMP-­‐2	
  (26),	
  
therefore	
  TIMP-­‐1	
  and	
  TIMP-­‐2	
  were	
  chosen	
  as	
  candidates	
  for	
  study.	
  MMPs	
  and	
  TIMPs	
  act	
  in	
  
concert	
  to	
  modulate	
  the	
  turnover	
  of	
  extracellular	
  matrix	
  of	
  vessel	
  walls	
  in	
  both	
  health	
  and	
  
disease.	
  For	
  example,	
  pressure-­‐associated	
  vascular	
  remodeling	
  is	
  generally	
  associated	
  with	
  
increases	
  in	
  MMP-­‐2	
  and	
  MMP-­‐9	
  expression	
  (27,	
  29)	
  and	
  while	
  TIMP	
  expression	
  can	
  be	
  
either	
  unaltered	
  or	
  decreased	
  (235)	
  in	
  remodeled	
  vessels,	
  the	
  overall	
  MMP:TIMP	
  ratio	
  is	
  
frequently	
  increased	
  (23,	
  90).	
  Most	
  vascular	
  wall	
  cell	
  types,	
  including	
  adventitial	
  
fibroblasts,	
  produce	
  MMPs	
  (26).	
  It	
  has	
  been	
  demonstrated	
  that	
  increased	
  MMP	
  and/or	
  
decreased	
  TIMP	
  expression	
  is	
  necessary	
  for	
  the	
  migration	
  of	
  activated	
  fibroblasts	
  from	
  the	
  
adventitia	
  to	
  the	
  media	
  and	
  intima	
  (188)	
  thereby	
  allowing	
  extension	
  of	
  the	
  remodeling	
  
process	
  to	
  the	
  rest	
  of	
  vessel	
  wall.	
  Inward	
  remodeling	
  such	
  as	
  this	
  is	
  observed	
  in	
  the	
  more	
  
severely	
  remodeled	
  veins	
  of	
  EIPH-­‐affected	
  equine	
  lung	
  (233).	
  
Endothelin-­‐1	
  (ET-­‐1)	
  is	
  reported	
  to	
  be	
  a	
  potent	
  vasoconstrictor	
  of	
  both	
  pulmonary	
  
and	
  systemic	
  vessels	
  in	
  the	
  horse,	
  and	
  this	
  effect	
  is	
  predominantly	
  mediated	
  through	
  the	
  
ETA	
  receptor	
  (13).	
  In	
  other	
  species	
  however,	
  endothelin	
  has	
  been	
  implicated	
  in	
  vascular	
  
remodeling	
  processes	
  as	
  a	
  mitogenic	
  factor	
  for	
  smooth	
  muscle	
  (35)	
  and	
  as	
  a	
  comitogen	
  that	
  
augments	
  TGF-­‐β-­‐induced	
  collagen	
  I	
  production	
  (105).	
  Increased	
  ET-­‐1	
  expression	
  is	
  
reported	
  in	
  a	
  hypoxic	
  model	
  of	
  pulmonary	
  venous	
  remodeling	
  (204),	
  and	
  administration	
  of	
  
an	
  ETA	
  receptor	
  antagonist	
  is	
  reported	
  to	
  inhibit	
  pressure-­‐mediated	
  remodeling	
  in	
  porcine	
  
veins	
  (224).	
  Although	
  vascular	
  smooth	
  muscle	
  is	
  an	
  important	
  target	
  tissue	
  in	
  both	
  acute	
  
and	
  chronic	
  responses	
  to	
  endothelin,	
  endothelial	
  cells	
  are	
  the	
  predominant	
  source	
  of	
  ET-­‐1,	
  
the	
  majority	
  of	
  which	
  is	
  secreted	
  at	
  the	
  basolateral	
  cell	
  surface	
  (96).	
  	
  

	
  
41	
  

Platelet-­‐derived	
  growth	
  factor	
  (PDGF)	
  is	
  a	
  mitogenic	
  factor	
  that	
  is	
  produced	
  by	
  and	
  
acts	
  upon	
  many	
  cell	
  types,	
  including	
  vascular	
  smooth	
  muscle	
  cells,	
  and	
  fibroblasts	
  (65).	
  
PDGF	
  is	
  over-­‐expressed	
  in	
  small	
  remodeled	
  pulmonary	
  arteries	
  in	
  patients	
  with	
  idiopathic	
  
pulmonary	
  arterial	
  hypertension	
  compared	
  to	
  arteries	
  from	
  normal	
  subjects	
  (166).	
  
Upregulation	
  of	
  PDGF	
  mRNA	
  in	
  porcine	
  veins	
  that	
  are	
  exposed	
  to	
  arterial	
  conditions	
  of	
  
pressure	
  and	
  flow	
  is	
  also	
  reported	
  (48).	
  The	
  role	
  of	
  PDGF	
  in	
  vascular	
  remodeling	
  processes	
  
is	
  further	
  substantiated	
  by	
  experimental	
  and	
  clinical	
  reports	
  of	
  effective	
  reversal	
  of	
  
pulmonary	
  arterial	
  remodeling	
  by	
  treatment	
  with	
  the	
  tyrosine	
  kinase	
  inhibitor	
  imatinib,	
  a	
  
PDGF	
  receptor	
  antagonist	
  (53,	
  181).	
  
Transforming	
  growth	
  factor	
  beta	
  (TGF-­‐β)	
  is	
  a	
  pleiotropic	
  cytokine,	
  and	
  the	
  
prototypical	
  member	
  of	
  a	
  large	
  group	
  of	
  cytokines	
  whose	
  functions	
  span	
  development,	
  
health	
  and	
  disease	
  in	
  many	
  tissues	
  (58).	
  The	
  role	
  of	
  TGF-­‐β	
  in	
  pulmonary	
  artery	
  remodeling	
  
has	
  been	
  studied	
  extensively,	
  as	
  mutations	
  of	
  the	
  TGF-­‐β	
  receptor	
  BMPR2	
  are	
  strongly	
  
associated	
  with	
  familial	
  pulmonary	
  arterial	
  hypertension	
  (118).	
  The	
  role	
  of	
  TGF-­‐β	
  in	
  
pulmonary	
  venous	
  remodeling	
  remains	
  undefined,	
  however	
  it	
  is	
  implicated	
  in	
  remodeling	
  
of	
  systemic	
  veins.	
  Vein	
  grafts	
  that	
  have	
  undergone	
  multiple	
  stenoses	
  express	
  more	
  TGF-­‐β	
  
than	
  veins	
  that	
  have	
  never	
  remodeled	
  or	
  have	
  only	
  stenosed	
  once	
  (146),	
  and	
  treatment	
  
with	
  TGF-­‐β	
  antisense	
  mRNA	
  prevents	
  collagen	
  deposition	
  in	
  femoral	
  vein	
  grafts	
  in	
  rats	
  
(234).	
  Furthermore,	
  TGF-­‐β	
  is	
  recognized	
  to	
  play	
  a	
  key	
  role	
  in	
  the	
  differentiation	
  of	
  
fibroblasts	
  to	
  activated	
  myofibroblasts	
  (120).	
  
The	
  final	
  growth	
  factor	
  that	
  was	
  selected	
  for	
  evaluation	
  in	
  this	
  study	
  is	
  vascular	
  
endothelial	
  growth	
  factor	
  (VEGF).	
  VEGF	
  is	
  highly	
  expressed	
  in	
  the	
  lung	
  (219),	
  and	
  its	
  
production	
  by	
  endothelial	
  cells	
  can	
  be	
  induced	
  by	
  many	
  growth	
  factors	
  including	
  TGF-­‐β	
  

	
  
42	
  

(167).	
  	
  In	
  pulmonary	
  arterial	
  hypertension	
  VEGF	
  protein	
  has	
  been	
  demonstrated	
  in	
  
plexiform,	
  lumen-­‐obliterating	
  endothelial	
  lesions	
  (214).	
  Its	
  role	
  in	
  vascular	
  remodeling	
  is	
  
supported	
  by	
  the	
  observation	
  that	
  VEGF	
  blockade	
  prevents	
  neointima	
  formation	
  in	
  arteries	
  
(241).	
  
	
  
Summary	
  	
  
In	
  summary,	
  to	
  implicate	
  pulmonary	
  venous	
  remodeling	
  as	
  a	
  pivotal	
  and	
  early	
  change	
  in	
  
EIPH	
  pathogenesis,	
  a	
  study	
  was	
  designed	
  to	
  evaluate	
  the	
  early	
  expression	
  patterns	
  of	
  
extracellular	
  matrix	
  proteins,	
  their	
  modulating	
  enzymes,	
  and	
  various	
  growth	
  factors	
  in	
  
pulmonary	
  veins	
  from	
  2	
  lung	
  regions	
  of	
  horses	
  after	
  two	
  weeks	
  of	
  a	
  transient	
  hypertensive	
  
stimulus	
  (exercise).	
  	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  

	
  
43	
  

Section	
  4:	
  Experimental	
  technique	
  development	
  
This	
  section	
  provides	
  supplemental	
  information	
  on	
  why	
  wire	
  myography	
  was	
  chosen	
  as	
  a	
  key	
  
experimental	
  technique,	
  and	
  the	
  rationale	
  and	
  theory	
  behind	
  vessel	
  normalization	
  strategy.	
  
	
  	
  	
  
Wire	
  myography	
  
Before	
  wire	
  myography	
  was	
  determined	
  to	
  be	
  the	
  most	
  suitable	
  technique	
  to	
  evaluate	
  both	
  
the	
  mechanical	
  and	
  pharmacological	
  properties	
  of	
  small	
  caliber	
  equine	
  pulmonary	
  vessels,	
  
attempts	
  were	
  made	
  to	
  utilize	
  pressure	
  myography.	
  In	
  brief,	
  pressure	
  myography	
  involves	
  
mounting	
  each	
  end	
  of	
  and	
  securing	
  the	
  vessel	
  of	
  interest	
  on	
  two	
  patent	
  glass	
  canulae	
  that	
  
are	
  themselves	
  attached	
  to	
  a	
  sealed,	
  and	
  pressurized	
  system.	
  	
  The	
  vessel	
  walls	
  and	
  lumen	
  
can	
  be	
  monitored	
  continually,	
  and	
  responses	
  of	
  vascular	
  smooth	
  muscle,	
  and	
  the	
  resulting	
  
alterations	
  in	
  vessel	
  diameter	
  recorded	
  in	
  real	
  time.	
  Some	
  advantages	
  of	
  pressure	
  
myography	
  over	
  wire	
  myography	
  include	
  equal	
  and	
  constant	
  distribution	
  of	
  intraluminal	
  
pressure	
  conditions	
  to	
  the	
  vascular	
  wall,	
  which	
  better-­‐emulates	
  in	
  vivo	
  conditions,	
  and	
  also,	
  
intravascular	
  pressure	
  can	
  be	
  easily	
  and	
  rapidly	
  adjusted	
  by	
  means	
  of	
  a	
  fluid	
  column	
  that	
  
communicates	
  with	
  the	
  canulae-­‐vessel	
  system.	
  	
  
When	
  equine	
  pulmonary	
  arteries	
  between	
  100	
  and	
  400	
  μm	
  O.D.	
  were	
  mounted	
  on	
  
the	
  pressure	
  myograph	
  however	
  (Figure	
  1),	
  it	
  was	
  difficult	
  to	
  maintain	
  pressure	
  in	
  any	
  
vessel	
  segment	
  despite	
  careful	
  dissection	
  technique.	
  The	
  reason	
  for	
  this	
  is	
  attributed	
  to	
  the	
  
frequent,	
  and	
  asymmetrical	
  branching	
  pattern	
  of	
  the	
  pulmonary	
  vasculature.	
  For	
  example,	
  
supernumerary	
  arteries	
  branching	
  from	
  a	
  parent	
  artery,	
  usually	
  at	
  90	
  degrees,	
  can	
  be	
  
significantly	
  smaller	
  in	
  diameter	
  that	
  the	
  vessel	
  from	
  which	
  they	
  originate	
  (210).	
  Due	
  to	
  an	
  

	
  
44	
  

unacceptable	
  failure	
  rate	
  using	
  this	
  technique,	
  the	
  decision	
  was	
  made	
  to	
  switch	
  to	
  
conventional	
  wire	
  myography.	
  	
  
Mulvaney	
  and	
  Halpern	
  developed	
  wire	
  myography	
  in	
  the	
  1970s,	
  and	
  they	
  first	
  
reported	
  its	
  use	
  as	
  a	
  tool	
  to	
  investigate	
  lengths	
  of	
  small	
  arterial	
  resistance	
  vessels	
  obtained	
  
from	
  rat	
  mesentery	
  (138,	
  139).	
  Wire	
  myography	
  is	
  now	
  a	
  commonly	
  used	
  technique	
  for	
  the	
  
study	
  of	
  small	
  blood	
  vessel	
  characteristics.	
  	
  
The	
  wire	
  myograph	
  chamber	
  is	
  filled	
  with	
  physiologic	
  buffer	
  solution,	
  which	
  can	
  be	
  
warmed,	
  and	
  perfused	
  with	
  gas(es),	
  and	
  to	
  which	
  pharmacologic	
  agents	
  may	
  be	
  added.	
  
Small	
  vessels	
  (<	
  400	
  μm	
  O.D.)	
  are	
  mounted	
  as	
  a	
  cylinder	
  on	
  two	
  parallel	
  40	
  μm	
  O.D.	
  
stainless	
  steel	
  wires	
  in	
  the	
  chamber.	
  One	
  wire	
  is	
  attached	
  at	
  two	
  points	
  to	
  a	
  metal	
  jaw	
  that	
  
is	
  controlled	
  by	
  a	
  micrometer	
  screw,	
  and	
  the	
  second	
  wire	
  attached	
  to	
  an	
  identical	
  jaw	
  that	
  
is	
  attached	
  in	
  turn	
  to	
  a	
  sensitive	
  force	
  transducer.	
  (Figure	
  2).	
  In	
  this	
  arrangement,	
  the	
  
parallel	
  wires	
  can	
  be	
  distracted	
  from	
  one	
  another	
  by	
  use	
  of	
  the	
  micrometer,	
  thus	
  
controlling	
  vessel	
  internal	
  circumference	
  (L)	
  and	
  enabling	
  evaluation	
  of	
  the	
  tension	
  in	
  the	
  
vessel	
  wall	
  (T)	
  evaluated	
  as	
  various	
  stimuli	
  are	
  applied.	
  	
  
The	
  formula	
  used	
  to	
  calculate	
  internal	
  circumference	
  (L)	
  is	
  	
  	
  
L	
  =	
  (π	
  +	
  2)d	
  +	
  2f	
  
where	
  d	
  is	
  wire	
  diameter,	
  and	
  f	
  is	
  the	
  distance	
  between	
  the	
  wires.	
  The	
  wire	
  circumference	
  
(π	
  d)	
  accounts	
  for	
  the	
  2	
  outer	
  halves	
  of	
  the	
  wire	
  that	
  are	
  in	
  contact	
  with	
  the	
  vessel,	
  but	
  it	
  
does	
  not	
  account	
  for	
  half	
  of	
  wire	
  diameter	
  (d/2)	
  that	
  is	
  closest	
  to	
  the	
  center	
  of	
  the	
  vessel,	
  
and	
  that	
  contributes	
  to	
  the	
  total	
  diameter,	
  even	
  when	
  the	
  wires	
  are	
  touching	
  (or	
  f	
  =	
  0).	
  For	
  
one	
  “side”	
  side	
  of	
  the	
  vessel,	
  2	
  x	
  d/2	
  =	
  d.	
  For	
  both	
  “sides”	
  of	
  the	
  vessel	
  d	
  is	
  doubled.	
  (Figure	
  
3).	
  	
  

	
  
45	
  

	
  	
  

Assuming	
  a	
  thin-­‐walled	
  tube	
  condition	
  (which	
  is	
  the	
  case	
  in	
  small	
  caliber	
  vessels	
  

such	
  as	
  those	
  studied)	
  the	
  Laplace	
  relationship	
  can	
  be	
  applied,	
  and	
  the	
  equivalent	
  
transmural	
  pressure	
  (P)	
  calculated	
  from	
  wall	
  tension	
  (T)(which	
  is	
  read	
  by	
  the	
  pressure	
  
transducer)	
  and	
  vessel	
  circumference	
  (L)(as	
  determined	
  by	
  the	
  micrometer	
  screw).	
  
P	
  =	
  	
  T/(L/2π)	
  
A	
  degree	
  of	
  preexisting	
  tension	
  is	
  necessary	
  in	
  order	
  to	
  align	
  smooth	
  muscle	
  fibers	
  
for	
  optimal	
  development	
  of	
  force	
  when	
  stimulated	
  (7).	
  Before	
  experiments,	
  vessels	
  are	
  
normalized	
  to	
  similar	
  conditions	
  of	
  wall	
  tension	
  that	
  permit	
  comparisons	
  to	
  be	
  made	
  
between	
  vessels	
  of	
  different	
  diameter.	
  	
  
Many	
  investigators	
  elect	
  to	
  set	
  a	
  value	
  for	
  L	
  such	
  that	
  the	
  passive	
  resting	
  tension	
  on	
  
vessel	
  walls	
  is	
  equivalent	
  to	
  a	
  physiologically	
  relevant	
  transmural	
  pressure,	
  for	
  example	
  
100	
  mmHg	
  in	
  systemic	
  arteries.	
  	
  
In	
  the	
  pharmacology	
  experiments	
  outlined	
  in	
  Chapter	
  3	
  of	
  this	
  dissertation,	
  a	
  
different	
  approach	
  was	
  taken	
  for	
  the	
  following	
  reason	
  -­‐	
  maintenance	
  of	
  the	
  very	
  minimal	
  
tension	
  required	
  to	
  emulate	
  physiologic	
  pulmonary	
  venous	
  transmural	
  pressure	
  
(approximately	
  10	
  –	
  15	
  mmHg)	
  throughout	
  an	
  entire	
  experiment	
  proved	
  challenging,	
  
which	
  led	
  me	
  to	
  become	
  concerned	
  that	
  not	
  all	
  vessels	
  were	
  being	
  exposed	
  to	
  similar	
  
conditions	
  at	
  all	
  times.	
  	
  Normalization	
  was	
  therefore	
  performed	
  with	
  the	
  goal	
  of	
  
determining	
  each	
  vessel’s	
  optimal	
  T	
  for	
  subsequent	
  force	
  generation.	
  	
  Vessels	
  were	
  tested	
  
with	
  60	
  mM	
  K+	
  solution	
  over	
  a	
  range	
  of	
  passive	
  wall	
  tensions.	
  The	
  tension	
  at	
  which	
  the	
  
maximal	
  response	
  to	
  KCl	
  was	
  generated	
  was	
  determined	
  to	
  be	
  that	
  vessel’s	
  optimal	
  passive	
  
tension,	
  and	
  the	
  tension	
  at	
  which	
  all	
  subsequent	
  experiments	
  were	
  performed.	
  	
  	
  
	
  

	
  

	
  
46	
  

	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
APPENDIX	
  

	
  
47	
  

	
  

	
  
	
  
	
  
	
  
	
  

	
  
Figure	
  1	
  Equine	
  pulmonary	
  artery	
  on	
  a	
  pressure	
  myograph.	
  The	
  artery	
  is	
  secured	
  to	
  2	
  
glass	
  canulae	
  by	
  suture	
  material.	
  	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  
48	
  

	
  
Figure	
  2	
  Schematic	
  of	
  a	
  vessel	
  mounted	
  on	
  wire	
  myograph.	
  Vessel	
  is	
  mounted	
  as	
  a	
  cylinder	
  
on	
   two	
   stainless	
   steel	
   wires.	
   Each	
   wire	
   is	
   attached	
   to	
   a	
   metal	
   jaw,	
   one	
   of	
   which	
   can	
   be	
  
moved	
   by	
   a	
   micrometer	
   screw	
   (right	
   of	
   image)	
   and	
   the	
   other	
   is	
   connected	
   to	
   a	
   sensitive	
  
force	
  transducer.	
  	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  
49	
  

	
  
Figure	
  3	
  Schematic	
  diagram	
  of	
  a	
  vessel	
  in	
  cross	
  section	
  mounted	
  on	
  two	
  myograph	
  wires.	
  d	
  
is	
  wire	
  diameter,	
  and	
  f	
  is	
  the	
  distance	
  between	
  the	
  wires.	
  These	
  measurements	
  are	
  used	
  to	
  
calculate	
  vessel	
  internal	
  circumference	
  (L)	
  from	
  the	
  formula	
  L	
  =	
  (π	
  +	
  2)d	
  +	
  2f	
  
	
  
	
  
	
  

	
  

	
  
50	
  

	
  
CHPATER	
  2	
  
Lung	
  region	
  and	
  racing	
  affect	
  mechanical	
  properties	
  of	
  equine	
  pulmonary	
  
microvasculature	
  	
  
	
  
Alice	
  Stack,	
  Frederik	
  J.	
  Derksen,	
  Kurt	
  J.	
  Williams,	
  N.	
  Edward	
  Robinson,	
  William	
  F.	
  Jackson	
  
	
  
J	
  Appl	
  Physiol	
  (1985).	
  2014	
  Jun	
  12.	
  doi:	
  jap.00314.2014.	
  [Epub	
  ahead	
  of	
  print]	
  
	
  
Abstract	
  	
  
Exercise-­‐induced	
  pulmonary	
  hemorrhage	
  (EIPH)	
  is	
  a	
  performance-­‐limiting	
  condition	
  of	
  
racehorses	
  associated	
  with	
  severe	
  pathology,	
  including	
  small	
  pulmonary	
  vein	
  remodeling.	
  
Pathology	
  is	
  limited	
  to	
  caudodorsal	
  (CD)	
  lung.	
  Mechanical	
  properties	
  of	
  equine	
  pulmonary	
  
microvasculature	
  have	
  not	
  been	
  studied.	
  We	
  hypothesized	
  that	
  regional	
  differences	
  in	
  
pulmonary	
  artery	
  and	
  vein	
  mechanical	
  characteristics	
  do	
  not	
  exist	
  in	
  control	
  animals;	
  and	
  
that	
  racing	
  and	
  venous	
  remodeling	
  impact	
  pulmonary	
  vein	
  mechanical	
  properties	
  in	
  CD	
  
lung.	
  Pulmonary	
  arteries	
  and	
  veins	
  (range	
  of	
  internal	
  diameters	
  207	
  –	
  386	
  ±	
  67	
  (mean	
  ±	
  
s.d.)	
  µm)	
  were	
  harvested	
  from	
  8	
  control	
  and	
  7	
  raced	
  horses.	
  Using	
  a	
  wire	
  myograph	
  CD	
  and	
  
cranioventral	
  (CV)	
  vessels	
  were	
  stretched	
  in	
  10	
  µm	
  increments.	
  Peak	
  wall	
  tension	
  was	
  
plotted	
  against	
  changes	
  in	
  diameter	
  (length).	
  Length-­‐tension	
  data	
  were	
  compared	
  between	
  
vessel	
  type,	
  lung	
  region,	
  and	
  horse	
  status	
  (control	
  and	
  raced).	
  Pulmonary	
  veins	
  are	
  stiffer-­‐
walled	
  than	
  arteries.	
  CD	
  pulmonary	
  arteries	
  are	
  stiffer	
  than	
  CV	
  arteries,	
  while	
  CV	
  veins	
  are	
  
stiffer	
  than	
  CD	
  veins.	
  Racing	
  is	
  associated	
  with	
  increased	
  stiffness	
  of	
  CD	
  pulmonary	
  veins,	
  
and,	
  to	
  a	
  lesser	
  extent,	
  CV	
  arteries.	
  For	
  example,	
  at	
  305	
  µm,	
  tension	
  in	
  raced	
  and	
  control	
  CD	
  

	
  
51	
  

veins	
  is	
  27.74	
  ±2.91	
  and	
  19.67	
  ±	
  2.63	
  mN/mm	
  respectively	
  (mean	
  ±	
  s.e.m;	
  p	
  <	
  0.05,	
  
Bonferroni’s	
  multiple	
  comparisons	
  test	
  after	
  2-­‐way	
  ANOVA);	
  and	
  16.12	
  ±	
  2.04	
  and	
  15.07	
  ±	
  
2.47	
  mN/mm	
  in	
  raced	
  and	
  control	
  CV	
  arteries.	
  This	
  is	
  the	
  first	
  report	
  of	
  an	
  effect	
  of	
  region	
  
and/or	
  exercise	
  on	
  mechanical	
  characteristics	
  of	
  small	
  pulmonary	
  vessels.	
  These	
  findings	
  
may	
  implicate	
  pulmonary	
  vein	
  remodeling	
  in	
  EIPH	
  pathogenesis.	
  	
  
	
  
Introduction	
  
Exercise-­‐induced	
  pulmonary	
  hemorrhage	
  (EIPH)	
  is	
  defined	
  as	
  the	
  presence	
  of	
  frank	
  blood	
  
(of	
  pulmonary	
  origin)	
  in	
  the	
  airways	
  after	
  a	
  bout	
  of	
  intense	
  exercise	
  (150,	
  152,	
  162).	
  	
  The	
  
condition	
  has	
  been	
  reported	
  in	
  several	
  athletic	
  species,	
  including	
  humans	
  (52),	
  racing	
  dogs	
  
(42)	
  and	
  camels	
  (4),	
  however	
  it	
  is	
  most	
  commonly	
  described	
  in	
  horses	
  (18,	
  162).	
  The	
  
incidence	
  rate	
  of	
  EIPH	
  in	
  racehorses	
  exceeds	
  75%	
  (18,	
  172)	
  when	
  diagnosed	
  by	
  tracheo-­‐
bronchoendoscopic	
  examination	
  within	
  30	
  –	
  90	
  minutes	
  of	
  exercise	
  (69).	
  This	
  highly	
  
prevalent	
  condition	
  is	
  associated	
  with	
  impaired	
  racing	
  performance	
  in	
  Thoroughbred	
  
horses	
  (70).	
  	
  
EIPH	
  pathology	
  is	
  most	
  severe	
  and	
  most	
  common	
  in	
  caudodorsal	
  lung	
  regions,	
  while	
  
the	
  cranioventral	
  lung	
  is	
  spared	
  (150-­‐152,	
  157,	
  231,	
  233).	
  The	
  caudodorsal	
  lung	
  is	
  also	
  the	
  
region	
  to	
  which	
  blood	
  flow	
  is	
  preferentially	
  distributed	
  in	
  the	
  horse,	
  both	
  at	
  rest	
  (76),	
  and	
  
to	
  an	
  even	
  greater	
  degree,	
  during	
  exercise	
  (15).	
  The	
  distribution	
  and	
  severity	
  of	
  the	
  EIPH	
  
lesion	
  matches	
  the	
  distribution	
  of	
  pulmonary	
  blood	
  flow	
  (233)	
  .	
  	
  
Remodeling	
  of	
  small	
  (100	
  –	
  200	
  µm	
  O.D.)	
  intralobular	
  pulmonary	
  veins	
  is	
  a	
  
consistent	
  histologic	
  feature	
  of	
  the	
  EIPH	
  lesion	
  (231,	
  233)	
  and	
  is	
  characterized	
  by	
  
accumulation	
  of	
  adventitial	
  collagen,	
  and	
  in	
  more	
  severely	
  affected	
  veins,	
  hypertrophy	
  of	
  

	
  
52	
  

the	
  tunica	
  media	
  (231).	
  Pulmonary	
  arteries,	
  and	
  larger	
  pulmonary	
  veins	
  are	
  not	
  affected	
  in	
  
a	
  similar	
  manner.	
  Venous	
  remodeling,	
  along	
  with	
  other	
  EIPH-­‐associated	
  histopathologic	
  
changes	
  such	
  as	
  interstitial	
  fibrosis	
  and	
  hemosiderin	
  formation,	
  is	
  limited	
  to	
  the	
  
caudodorsal	
  lung	
  (231,	
  233).	
  In	
  randomly	
  sampled	
  sections	
  of	
  EIPH-­‐affected	
  lung,	
  the	
  
entire	
  spectrum	
  of	
  lesions	
  never	
  occurs	
  without	
  co-­‐localized	
  venous	
  remodeling,	
  although	
  
venous	
  remodeling	
  occurs	
  on	
  its	
  own	
  (233).	
  This	
  suggests	
  that	
  venous	
  remodeling	
  is	
  an	
  
early	
  and	
  key	
  feature	
  of	
  the	
  EIPH	
  lesion,	
  and	
  may	
  be	
  central	
  to	
  the	
  pathogenesis	
  of	
  the	
  
disease.	
  	
  
Little	
  is	
  known	
  about	
  regional	
  differences	
  in	
  mechanical	
  characteristics	
  of	
  
pulmonary	
  microvasculature	
  in	
  horses,	
  or	
  indeed	
  in	
  any	
  other	
  species.	
  To	
  further	
  
understand	
  the	
  pathogenesis	
  of	
  EIPH,	
  a	
  region-­‐specific	
  condition,	
  investigations	
  into	
  
regional	
  differences	
  in	
  vascular	
  biology	
  are	
  warranted.	
  	
  
It	
  has	
  been	
  reported	
  that	
  pulmonary	
  vascular	
  remodeling,	
  including	
  collagen	
  
deposition,	
  alters	
  the	
  mechanical	
  properties	
  of	
  vessels	
  (101,	
  207,	
  212).	
  If	
  pulmonary	
  vein	
  
remodeling,	
  such	
  as	
  that	
  observed	
  in	
  EIPH-­‐affected	
  lungs	
  (231,	
  233)	
  affects	
  wall	
  mechanics,	
  
this	
  will	
  have	
  functional	
  ramifications	
  on	
  upstream	
  capillaries.	
  Pulmonary	
  capillary	
  stress	
  
failure	
  has	
  been	
  described	
  in	
  the	
  lungs	
  of	
  horses	
  with	
  EIPH	
  (230)	
  and	
  is	
  widely	
  considered	
  
to	
  be	
  the	
  source	
  of	
  airway	
  hemorrhage,	
  occurring	
  secondary	
  to	
  dramatic	
  increases	
  in	
  
pulmonary	
  vascular	
  pressures	
  in	
  the	
  exercising	
  horse.	
  Pulmonary	
  capillary	
  pressures	
  
(which	
  are	
  determined	
  by	
  arterial	
  and	
  venous	
  pressures)	
  are	
  estimated	
  to	
  range	
  between	
  
72	
  (106)	
  and	
  83	
  (127)	
  mmHg	
  during	
  galloping,	
  and	
  transmural	
  pressures	
  in	
  excess	
  of	
  75	
  
mmHg	
  exceed	
  the	
  breaking	
  strength	
  of	
  equine	
  pulmonary	
  capillaries	
  (17).	
  Remodeled	
  
veins	
  are	
  thick-­‐walled	
  compared	
  to	
  normal,	
  unaffected	
  veins,	
  and	
  in	
  some	
  cases	
  have	
  

	
  
53	
  

reduced	
  luminal	
  area	
  (38).	
  Should	
  these	
  changes	
  reduce	
  venous	
  compliance	
  (i.e.	
  increase	
  
venous	
  wall	
  stiffness)	
  it	
  follows	
  that,	
  during	
  strenuous	
  exercise,	
  pulmonary	
  capillary	
  
pressure	
  will	
  increase	
  yet	
  further	
  and	
  potentially	
  augment	
  EIPH.	
  
The	
  purpose	
  of	
  the	
  study	
  reported	
  here	
  was	
  to	
  test	
  two	
  hypotheses.	
  First,	
  
mechanical	
  properties	
  of	
  small	
  pulmonary	
  arteries	
  and	
  veins	
  do	
  not	
  differ	
  by	
  region	
  
(cranioventral	
  compared	
  to	
  caudodorsal)	
  in	
  control,	
  unraced	
  horses.	
  	
  	
  Second,	
  pulmonary	
  
veins,	
  but	
  not	
  arteries	
  from	
  horses	
  that	
  have	
  a	
  recent	
  racing	
  history	
  have	
  increased	
  wall	
  
stiffness	
  compared	
  to	
  veins	
  from	
  horses	
  that	
  have	
  never	
  raced,	
  and	
  this	
  change	
  is	
  limited	
  to	
  
veins	
  in	
  the	
  caudodorsal	
  lung,	
  the	
  site	
  of	
  venous	
  remodeling.	
  Wire	
  myography	
  (62)	
  was	
  
utilized	
  to	
  evaluate	
  vessel	
  mechanics	
  in	
  these	
  experiments	
  as	
  the	
  equipment	
  is	
  custom-­‐
designed	
  for	
  small	
  diameter	
  vessels	
  such	
  as	
  those	
  of	
  interest	
  in	
  this	
  study.	
  	
  
Our	
  data	
  demonstrate	
  regional	
  differences	
  in	
  vessel	
  wall	
  stiffness	
  in	
  both	
  pulmonary	
  
arteries	
  and	
  veins	
  from	
  control,	
  unraced	
  horses.	
  Furthermore,	
  caudodorsal	
  veins	
  from	
  
raced	
  horses	
  are	
  stiffer	
  than	
  those	
  from	
  control,	
  unraced	
  horses.	
  This	
  finding	
  establishes	
  
the	
  first	
  link	
  between	
  descriptions	
  of	
  pulmonary	
  vein	
  remodeling	
  (231,	
  233)	
  in	
  the	
  horse	
  
lung,	
  and	
  the	
  physiologic	
  effects	
  this	
  change	
  is	
  proposed	
  to	
  exert	
  on	
  the	
  pulmonary	
  
vasculature	
  during	
  exercise.	
  	
  
	
  
Materials	
  and	
  Methods	
  
Animals	
  
For	
  this	
  study,	
  8	
  control	
  horses,	
  and	
  7	
  raced	
  horses	
  were	
  acquired	
  by	
  donation.	
  Control	
  
horses	
  (3	
  geldings,	
  5	
  sexually	
  intact	
  females,	
  6.6	
  ±	
  0.6	
  (age	
  ±	
  s.e.m.)	
  years)	
  were	
  of	
  various	
  
breeds	
  (2	
  Arabians,	
  and	
  1	
  each	
  of	
  Thoroughbred,	
  Standardbred,	
  paint,	
  Quarterhorse,	
  

	
  
54	
  

Hafflinger	
  and	
  crossbred)	
  and	
  did	
  not	
  have	
  a	
  race	
  history.	
  Race-­‐trained	
  horses	
  (3	
  geldings,	
  
1	
  sexually-­‐intact	
  male	
  and	
  3	
  sexually	
  intact	
  females,	
  6.3	
  ±	
  0.6	
  (age	
  ±	
  s.e.m.)	
  years)	
  were	
  all	
  
Thoroughbreds	
  with	
  a	
  race	
  record.	
  The	
  time	
  period	
  between	
  the	
  last	
  race	
  and	
  euthanasia	
  
was	
  305	
  ±	
  77	
  (mean	
  ±	
  s.d.)	
  days.	
  Raced	
  horses	
  had	
  on	
  average,	
  22	
  ±	
  18	
  race	
  starts	
  (mean	
  ±	
  
s.d.,	
  range:	
  1	
  –	
  54	
  race	
  starts)	
  and	
  were	
  donated	
  for	
  reasons	
  other	
  than	
  severe	
  EIPH	
  
(predominantly	
  career-­‐limiting	
  lameness).	
  The	
  Michigan	
  State	
  University	
  Institutional	
  
Animal	
  Care	
  and	
  Use	
  Committee	
  approved	
  all	
  experimental	
  procedures.	
  
	
  
Tissue	
  acquisition	
  
Horses	
  were	
  administered	
  intravenous	
  heparin	
  sodium	
  (50,000	
  IU/horse)	
  approximately	
  
15	
  minutes	
  prior	
  to	
  euthanasia,	
  which	
  was	
  carried	
  out	
  with	
  pentobarbital	
  sodium	
  (90	
  
mg/kg	
  IV).	
  Lung	
  tissue	
  sections	
  (approximately	
  4	
  cm3)	
  were	
  immediately	
  harvested	
  from	
  
both	
  the	
  caudodorsal	
  (CD)	
  and	
  cranioventral	
  (CV)	
  regions	
  of	
  the	
  caudal	
  (diaphragmatic)	
  
lobe	
  of	
  both	
  left	
  and	
  right	
  lungs.	
  Lung	
  tissue	
  was	
  placed	
  in	
  chilled	
  normal	
  saline	
  (0.9%	
  
sodium	
  chloride)	
  solution	
  for	
  transportation	
  to	
  the	
  laboratory.	
  Additional	
  tissue	
  from	
  CD	
  
regions	
  of	
  lungs	
  of	
  raced	
  horses	
  was	
  placed	
  in	
  10%	
  neutral	
  buffered	
  formalin	
  for	
  fixation	
  
and	
  histologic	
  assessment.	
  	
  
	
  
Vessel	
  dissection	
  
Fresh,	
  chilled	
  lung	
  tissue	
  was	
  sectioned	
  (approximately	
  0.5	
  cm	
  thick	
  slices)	
  and	
  pinned	
  to	
  a	
  
Sylgard	
  (Dow	
  Corning,	
  Midland,	
  MI)	
  pad	
  in	
  the	
  bottom	
  of	
  a	
  water-­‐jacketed	
  dissection	
  
chamber	
  (Radnoti,	
  Monrovia,	
  CA)	
  and	
  fully	
  immersed	
  in	
  chilled	
  Ca2+	
  -­‐free	
  physiologic	
  saline	
  
solution	
  (PSS)	
  containing	
  (in	
  mM)	
  140	
  NaCl,	
  5	
  KCl,	
  1	
  MgCl2,	
  10	
  HEPES,	
  10	
  glucose	
  (pH	
  7.4,	
  

	
  
55	
  

295	
  mOsm).	
  A	
  low	
  calcium	
  environment	
  was	
  chosen	
  to	
  minimize	
  vasospasm	
  that	
  can	
  occur	
  
during	
  vessel	
  manipulation.	
  
Sections	
  of	
  pulmonary	
  veins	
  and	
  arteries	
  ranging	
  in	
  length	
  from	
  0.34	
  to	
  1.39	
  mm	
  
and	
  between	
  100	
  and	
  400-­‐μm	
  diameter	
  were	
  carefully	
  dissected	
  from	
  tissue	
  based	
  on	
  the	
  
following	
  anatomic	
  criteria:	
  intralobular	
  pulmonary	
  veins	
  that	
  course	
  completely	
  alone	
  in	
  
the	
  parenchyma(231);	
  	
  pulmonary	
  arteries	
  were	
  collected	
  from	
  broncho-­‐vascular	
  triads.	
  
Within	
  a	
  triad	
  of	
  pulmonary	
  artery,	
  vein	
  and	
  conducting	
  airway,	
  pulmonary	
  arteries	
  were	
  
easily	
  distinguishable	
  from	
  the	
  vein	
  in	
  the	
  same	
  bundle.	
  They	
  were	
  stiff-­‐walled	
  (compared	
  
to	
  veins),	
  and	
  always	
  immediately	
  adjacent	
  to	
  a	
  conducting	
  airway,	
  while	
  veins	
  in	
  the	
  
bundle	
  were	
  more	
  distant	
  from	
  the	
  airway	
  (175).	
  Individual	
  vessels	
  were	
  kept	
  in	
  Ca2+-­‐free	
  
PSS	
  at	
  4°C	
  for	
  up	
  to	
  24	
  hours	
  until	
  mounted	
  on	
  the	
  myograph.	
  
	
  
Wire	
  myography	
  
Vessels	
  were	
  mounted	
  as	
  a	
  cylinder	
  on	
  2	
  stainless	
  steel	
  40-­‐μm	
  diameter	
  wires	
  and	
  the	
  
wires	
  secured	
  (one	
  to	
  a	
  micrometer	
  screw	
  and	
  the	
  other	
  to	
  a	
  force	
  transducer)	
  in	
  a	
  4-­‐
chamber	
  myograph	
  (DMT,	
  Aarhus,	
  Denmark).	
  Each	
  chamber	
  contained	
  5	
  ml	
  of	
  Ca2+-­‐free	
  
PSS,	
  and	
  all	
  vessels	
  were	
  submerged	
  throughout	
  the	
  experiment.	
  The	
  bath	
  fluid	
  was	
  heated	
  
slowly	
  to	
  37°C,	
  and	
  air	
  was	
  bubbled	
  gently	
  through	
  the	
  fluid	
  continuously.	
  	
  Vessel	
  length	
  
was	
  recorded	
  using	
  a	
  previously	
  calibrated	
  stereomicroscope,	
  and	
  the	
  micrometer	
  reading	
  
at	
  which	
  the	
  wires	
  were	
  barely	
  touching	
  and	
  parallel	
  to	
  one	
  another	
  was	
  recorded.	
  The	
  
myograph	
  force	
  transducer	
  was	
  used	
  in	
  conjunction	
  with	
  a	
  PowerLab	
  (ADInstruments,	
  
Colorado	
  Springs,	
  CO)	
  data	
  acquisition	
  unit	
  and	
  LabChart	
  (ADInstruments,	
  Colorado	
  
Springs,	
  CO)	
  software	
  platform.	
  

	
  
56	
  

	
  	
  

Wires	
  were	
  then	
  separated	
  by	
  use	
  of	
  the	
  micrometer	
  until	
  the	
  transducer	
  registered	
  

a	
  small	
  (<0.05	
  mN)	
  but	
  sustained	
  force.	
  The	
  micrometer	
  reading	
  was	
  again	
  recorded,	
  and	
  
this	
  was	
  designated	
  an	
  individual	
  vessel’s	
  start	
  point.	
  Vessel	
  diameter	
  was	
  calculated	
  and	
  
recorded	
  at	
  this	
  point.	
  From	
  that	
  point,	
  wires	
  were	
  separated	
  from	
  one	
  another	
  in	
  10-­‐μm	
  
increments.	
  The	
  peak	
  force	
  achieved	
  at	
  each	
  micrometer	
  adjustment	
  was	
  recorded,	
  and	
  
once	
  a	
  plateau	
  in	
  force	
  was	
  attained	
  the	
  wires	
  were	
  separated	
  again	
  for	
  the	
  next	
  force	
  
measurement.	
  Vessels	
  were	
  stretched	
  in	
  this	
  stepwise	
  manner	
  until	
  stretching	
  resulted	
  in	
  
no	
  further	
  increase,	
  or	
  a	
  decrease	
  in	
  force	
  was	
  recorded,	
  which	
  was	
  interpreted	
  as	
  vessel	
  
failure.	
  	
  
	
  
Vessel	
  histology	
  
Once	
  length-­‐tension	
  data	
  acquisition	
  was	
  complete,	
  a	
  subset	
  of	
  vessels	
  was	
  fixed	
  in	
  situ	
  on	
  
the	
  myograph	
  wires	
  in	
  10%	
  neutral	
  buffered	
  formalin.	
  	
  	
  Following	
  fixation,	
  the	
  vessels	
  were	
  
removed	
  from	
  the	
  wires	
  and	
  mounted	
  orthogonal	
  to	
  the	
  long	
  axis	
  in	
  Histogel	
  (American	
  
MasterTech,	
  Lodi,	
  CA)	
  specimen	
  processing	
  gel.	
  The	
  gel-­‐embedded	
  vessel	
  was	
  then	
  
embedded	
  in	
  paraffin	
  for	
  sectioning.	
  	
  6-­‐μm	
  sections	
  were	
  placed	
  on	
  glass	
  slides	
  and	
  stained	
  
with	
  hematoxylin	
  and	
  eosin	
  (H	
  and	
  E)	
  and	
  Verhoeff-­‐Van	
  Gieson	
  (VVG).	
  These	
  stained	
  
sections	
  were	
  used	
  to	
  confirm	
  vessel	
  identity	
  as	
  a	
  pulmonary	
  artery,	
  or	
  a	
  pulmonary	
  vein.	
  	
  
Slides	
  were	
  reviewed	
  by	
  a	
  board-­‐certified	
  veterinary	
  pathologist	
  (KJW)	
  who	
  was	
  blinded	
  to	
  
the	
  identity	
  of	
  the	
  vessel	
  based	
  on	
  anatomic/dissection	
  criteria.	
  Pulmonary	
  arteries	
  were	
  
identified	
  based	
  on	
  a	
  relatively	
  substantial	
  tunica	
  media	
  that	
  was	
  bounded	
  by	
  both	
  an	
  
internal	
  and	
  external	
  elastic	
  lamina	
  (134).	
  In	
  contrast,	
  pulmonary	
  veins	
  had	
  less	
  smooth	
  
muscle	
  in	
  the	
  tunica	
  media,	
  a	
  single	
  distinct	
  external	
  elastic	
  lamina	
  between	
  the	
  tunica	
  

	
  
57	
  

media,	
  and	
  tunica	
  adventitia	
  (134)	
  	
  and	
  an	
  absent	
  internal	
  elastic	
  lamina	
  between	
  the	
  
tunica	
  intima	
  	
  and	
  tunica	
  media	
  (175,	
  210)(Figure	
  4).	
  	
  	
  
	
  
Lung	
  histopathology	
  
Fixed	
  lung	
  tissue	
  samples	
  from	
  the	
  CD	
  regions	
  of	
  6	
  of	
  the	
  7	
  raced	
  horses	
  underwent	
  routine	
  
processing	
  and	
  embedding	
  in	
  paraffin	
  for	
  histological	
  examination.	
  	
  Following	
  sectioning,	
  
tissues	
  were	
  stained	
  with	
  H	
  and	
  E	
  and	
  VVG,	
  and	
  evaluated	
  by	
  KJW	
  for	
  the	
  presence	
  and	
  
severity	
  of	
  EIPH	
  vascular	
  pathology	
  using	
  previously	
  described	
  criteria	
  (231).	
  	
  
	
  
Data	
  Analysis	
  
Variability	
  between	
  vessels	
  was	
  similar	
  to	
  variability	
  between	
  animals,	
  therefore,	
  in	
  this	
  
study,	
  the	
  sampling	
  units	
  are	
  individual	
  vessels,	
  and	
  not	
  horses.	
  	
  
Vessel	
  internal	
  diameters	
  at	
  the	
  start	
  point	
  were	
  calculated	
  as	
  follows:	
  vessel	
  
internal	
  circumference	
  at	
  that	
  point	
  (C)	
  divided	
  by	
  π.	
  Vessel	
  internal	
  circumference	
  (C)	
  was	
  
calculated	
  as	
  twice	
  the	
  distance	
  between	
  the	
  wires,	
  plus	
  the	
  wire	
  circumference	
  plus	
  twice	
  
the	
  wire	
  diameter	
  (242).	
  The	
  effect	
  of	
  vessel	
  type	
  (artery	
  or	
  vein)	
  and	
  region	
  (caudodorsal	
  
or	
  cranioventral)	
  on	
  diameter	
  at	
  the	
  start	
  point	
  was	
  analyzed	
  within	
  racing	
  status	
  (control	
  
or	
  raced)	
  using	
  a	
  two-­‐way	
  ANOVA	
  with	
  Bonferroni’s	
  multiple	
  comparisons	
  test	
  (GraphPad	
  
Prism	
  6,	
  GraphPad	
  Software	
  Inc.,	
  La	
  Jolla,	
  CA).	
  	
  
Change	
  in	
  vessel	
  internal	
  diameter	
  from	
  that	
  vessel’s	
  diameter	
  at	
  start	
  point	
  (as	
  
previously	
  defined)	
  was	
  referred	
  to	
  as	
  length	
  (L)	
  and	
  expressed	
  in	
  μm.	
  L	
  for	
  each	
  vessel	
  
was	
  plotted	
  against	
  recorded	
  tension	
  (T).	
  T	
  was	
  defined	
  as	
  the	
  peak	
  force	
  registered	
  per	
  
unit	
  of	
  vessel	
  segment	
  length,	
  and	
  expressed	
  in	
  mN/mm.	
  	
  Vessels	
  were	
  analyzed	
  over	
  a	
  

	
  
58	
  

range	
  of	
  L	
  that	
  did	
  not	
  cause	
  failure	
  in	
  any	
  vessel	
  (0	
  -­‐	
  337.41	
  μm).	
  	
  Length-­‐tension	
  data	
  
were	
  compared	
  (a)	
  between	
  regions	
  (caudodorsal	
  and	
  cranioventral),	
  (b)	
  between	
  vessel	
  
type	
  (artery	
  and	
  vein),	
  and	
  (c)	
  between	
  racing	
  status	
  (control	
  and	
  raced)	
  within	
  region	
  and	
  
vessel	
  type	
  using	
  a	
  2-­‐way	
  ANOVA	
  with	
  Bonferroni’s	
  multiple	
  comparisons	
  test	
  (GraphPad	
  
Prism	
  6,	
  GraphPad	
  Software	
  Inc.,	
  La	
  Jolla,	
  CA).	
  	
  
	
  	
  

In	
  order	
  to	
  evaluate	
  changes	
  in	
  mechanical	
  properties	
  within	
  a	
  physiologically	
  

relevant	
  range,	
  tension	
  (T)	
  values	
  were	
  converted	
  to	
  equivalent	
  transmural	
  pressure	
  
values	
  (P)	
  by	
  application	
  of	
  the	
  Laplace	
  relationship	
  as	
  follows:	
  	
  
Pi	
  =	
  Ti	
  /	
  (Ci/2	
  *	
  π)	
  
where	
  Pi,	
  Ti	
  and	
  Ci	
  are	
  pressure,	
  tension	
  and	
  internal	
  circumference	
  respectively	
  at	
  a	
  given	
  
length	
  value,	
  i.	
  Over	
  the	
  physiologic	
  range	
  of	
  pressures	
  encountered	
  in	
  arteries	
  and	
  veins,	
  
pressure	
  (P)	
  values	
  in	
  mN/mm2	
  were	
  converted	
  to	
  mmHg,	
  and	
  plotted	
  against	
  the	
  change	
  
in	
  vessel	
  diameter	
  from	
  start	
  point,	
  or	
  length	
  (L).	
  Length-­‐pressure	
  data	
  from	
  arteries	
  and	
  
veins	
  were	
  compared	
  between	
  racing	
  status	
  (control	
  and	
  raced)	
  within	
  each	
  region	
  (CD	
  
and	
  DV)	
  using	
  a	
  2-­‐way	
  ANOVA	
  with	
  Bonferroni’s	
  multiple	
  comparisons	
  test	
  (GraphPad	
  
Prism	
  6,	
  GraphPad	
  Software	
  Inc.,	
  La	
  Jolla,	
  CA).	
  	
  
	
  
Results	
  
From	
  control	
  horses,	
  9	
  veins	
  and	
  10	
  arteries	
  were	
  harvested	
  from	
  caudodorsal	
  lung	
  
regions,	
  and	
  7	
  veins	
  and	
  9	
  arteries	
  from	
  cranioventral	
  lung.	
  From	
  raced	
  horses,	
  it	
  was	
  15	
  
veins	
  and	
  10	
  arteries	
  from	
  caudodorsal	
  lung	
  regions,	
  and	
  8	
  veins	
  and	
  10	
  arteries	
  from	
  
cranioventral	
  lung.	
  Seventy-­‐eight	
  vessels	
  were	
  included	
  in	
  all	
  subsequent	
  statistical	
  
analyses.	
  

	
  
59	
  

	
  
Vessel	
  diameters	
  
Vessel	
  diameters	
  are	
  shown	
  in	
  Table	
  1.	
  Diameters	
  did	
  not	
  differ	
  between	
  arteries	
  and	
  
veins	
  from	
  caudodorsal	
  and	
  cranioventral	
  lung	
  in	
  both	
  control	
  and	
  raced	
  horses	
  with	
  the	
  
following	
  exception:	
  arteries	
  from	
  the	
  caudodorsal	
  lung	
  region	
  were	
  larger	
  in	
  diameter	
  
than	
  veins	
  from	
  both	
  caudodorsal	
  and	
  cranioventral	
  lung	
  region	
  of	
  raced	
  horses.	
  	
  	
  
	
  
Vessel	
  identification	
  
Histology	
  was	
  performed	
  on	
  29	
  of	
  the	
  78	
  vessels	
  that	
  were	
  studied.	
  All	
  vessels	
  that	
  were	
  
identified	
  during	
  dissection	
  as	
  either	
  an	
  artery	
  or	
  a	
  vein	
  had	
  that	
  identity	
  confirmed	
  by	
  use	
  
of	
  histology	
  (Figure	
  4).	
  
	
  
Length-­‐tension	
  data	
  
When	
  data	
  from	
  the	
  CD	
  and	
  CV	
  vessels	
  were	
  combined,	
  pulmonary	
  veins	
  (n	
  =	
  16	
  and	
  n	
  =	
  23	
  
for	
  control	
  and	
  raced	
  respectively)	
  were	
  stiffer	
  (as	
  demonstrated	
  by	
  a	
  steeper	
  length-­‐
tension	
  curve)	
  than	
  pulmonary	
  arteries	
  (n	
  =	
  19	
  and	
  n	
  =	
  20	
  for	
  control	
  and	
  raced	
  
respectively).	
  This	
  observation	
  was	
  consistent	
  in	
  both	
  control	
  and	
  raced	
  horses	
  (Figure	
  5,	
  
A	
  and	
  B	
  respectively)	
  (p	
  <	
  0.0001).	
  
In	
  control	
  horses,	
  pulmonary	
  veins	
  from	
  the	
  cranioventral	
  lung	
  were	
  stiffer	
  than	
  
those	
  from	
  the	
  caudodorsal	
  lung	
  (p	
  <	
  0.0001),	
  whereas	
  pulmonary	
  arteries	
  from	
  
caudodorsal	
  lung	
  were	
  stiffer	
  than	
  arteries	
  from	
  cranioventral	
  lung	
  (p	
  <	
  0.0001)	
  (Figure	
  6,	
  
A	
  and	
  B	
  respectively).	
  This	
  regional	
  pattern	
  of	
  differences	
  in	
  vessel	
  stiffness	
  was	
  
maintained	
  in	
  vessels	
  from	
  raced	
  horses	
  (Figure	
  6,	
  C	
  and	
  D).	
  

	
  
60	
  

Vessels	
  from	
  raced	
  horses	
  were	
  compared	
  to	
  vessels	
  from	
  control	
  horses	
  within	
  
lung	
  region.	
  Cranioventral	
  veins	
  and	
  caudodorsal	
  arteries	
  were	
  not	
  affected	
  by	
  race	
  
training	
  (p	
  =	
  0.8078	
  and	
  p	
  =	
  0.4317	
  respectively)	
  (Figure	
  7,	
  B	
  and	
  C	
  respectively).	
  
However	
  caudodorsal	
  veins	
  from	
  raced	
  animals	
  were	
  significantly	
  stiffer	
  than	
  those	
  from	
  
control	
  animals	
  (p	
  <	
  0.0001)	
  (Figure	
  7,	
  A).	
  Cranioventral	
  arteries	
  from	
  raced	
  horses	
  were	
  
also	
  significantly	
  stiffer	
  than	
  those	
  from	
  control	
  horses	
  (p	
  =	
  0.0014)	
  (Figure	
  7,	
  D).	
  	
  
	
  
Length-­‐pressure	
  data	
  
Tension	
  values	
  were	
  converted	
  to	
  equivalent	
  transmural	
  pressure	
  values	
  encompassing	
  in	
  
vivo	
  physiologic	
  pressure	
  ranges	
  experienced	
  by	
  horses	
  at	
  rest	
  and	
  during	
  exercise:	
  0-­‐120	
  
mmHg	
  for	
  arteries,	
  and	
  0	
  –	
  80	
  mmHg	
  for	
  veins.	
  Length-­‐pressure	
  data	
  in	
  these	
  ranges	
  were	
  
compared	
  between	
  control	
  and	
  raced	
  horses	
  within	
  lung	
  region.	
  Cranioventral	
  veins	
  and	
  
caudodorsal	
  arteries	
  were	
  not	
  affected	
  by	
  race	
  training	
  (p	
  =	
  0.9416	
  and	
  p	
  =	
  0.0552	
  
respectively)	
  (Figure	
  8,	
  B	
  and	
  C	
  respectively).	
  However	
  caudodorsal	
  veins	
  and	
  
cranioventral	
  arteries	
  from	
  raced	
  animals	
  underwent	
  a	
  significantly	
  smaller	
  change	
  in	
  
internal	
  diameter	
  over	
  a	
  physiologic	
  pressure	
  range	
  than	
  vessels	
  from	
  control	
  animals	
  (p	
  <	
  
0.0001)	
  (Figure	
  8,	
  A	
  and	
  D	
  respectively).	
  	
  
	
  
Lung	
  histopathology	
  
Venous	
  remodeling	
  and	
  lung	
  pathology	
  consistent	
  with	
  exercise-­‐induced	
  pulmonary	
  
hemorrhage	
  changes	
  were	
  detected	
  in	
  all	
  caudodorsal	
  lung	
  sections	
  examined	
  from	
  raced	
  
horses.	
  The	
  venous	
  remodeling	
  was	
  consistent	
  with	
  that	
  previously	
  described	
  in	
  
association	
  with	
  EIPH	
  (231,	
  233).	
  	
  Briefly,	
  these	
  changes	
  consisted	
  of	
  mild	
  to	
  moderate	
  

	
  
61	
  

increases	
  in	
  adventitial	
  collagen	
  surrounding	
  small	
  veins	
  along	
  with	
  small	
  numbers	
  of	
  
hemosiderophages,	
  indicating	
  prior	
  hemorrhage	
  and	
  erythrophagocytosis	
  by	
  the	
  alveolar	
  
macrophages.	
  	
  
	
  
Discussion	
  
To	
  the	
  authors’	
  knowledge,	
  this	
  is	
  the	
  first	
  published	
  account	
  of	
  the	
  effect	
  of	
  lung	
  region,	
  
and/or	
  exercise	
  on	
  pulmonary	
  microvascular	
  mechanical	
  properties	
  in	
  any	
  species	
  to	
  date.	
  
Study	
  of	
  these	
  factors	
  in	
  equids	
  is	
  particularly	
  important	
  in	
  order	
  to	
  better	
  understand	
  
exercise-­‐induced	
  pulmonary	
  hemorrhage	
  (EIPH)	
  pathogenesis.	
  	
  	
  
The	
  predominant	
  tissue	
  types	
  that	
  determine	
  a	
  vessel’s	
  mechanical	
  characteristics	
  
are	
  collagen	
  and	
  elastin	
  (20,	
  177).	
  Elastin	
  contributes	
  most	
  resistance	
  to	
  stretching	
  at	
  
lower	
  tension	
  values,	
  whereas	
  collagen	
  provides	
  most	
  resistance	
  at	
  higher	
  tension	
  values	
  
(20,	
  177).	
  Collagen	
  is	
  minimally	
  distensible,	
  and	
  has	
  an	
  elastic	
  modulus	
  that	
  is	
  
approximately	
  400	
  times	
  that	
  of	
  elastin	
  (20).	
  
In	
  both	
  control	
  and	
  raced	
  horses,	
  pulmonary	
  veins	
  are	
  stiffer-­‐walled	
  than	
  
pulmonary	
  arteries,	
  and	
  we	
  propose	
  that	
  this	
  difference	
  is	
  due	
  to	
  the	
  greater	
  proportion	
  of	
  
collagen	
  in	
  vein	
  walls,	
  compared	
  to	
  arteries.	
  This	
  particular	
  finding	
  has	
  been	
  documented	
  
in	
  other	
  species.	
  In	
  the	
  dog,	
  intraparenchymal	
  pulmonary	
  veins	
  are	
  less	
  distensible	
  in	
  
response	
  to	
  increases	
  in	
  intravascular	
  pressure	
  than	
  equivalently-­‐sized	
  pulmonary	
  arteries	
  
(121).	
  Larger	
  pulmonary	
  veins	
  are	
  also	
  less	
  distensible	
  than	
  pulmonary	
  arteries	
  in	
  both	
  
rabbits	
  (22),	
  and	
  in	
  humans	
  (9,	
  119).	
  	
  
Mechanical	
  properties	
  of	
  small-­‐caliber	
  pulmonary	
  vessels	
  such	
  as	
  those	
  evaluated	
  in	
  
this	
  study	
  are	
  not	
  well	
  described	
  in	
  the	
  literature	
  to	
  date,	
  and	
  to	
  the	
  best	
  of	
  the	
  authors’	
  

	
  
62	
  

knowledge,	
  published	
  data	
  regarding	
  regional	
  differences	
  in	
  mechanical	
  properties	
  of	
  
pulmonary	
  arteries	
  and	
  veins	
  of	
  this	
  size	
  range	
  in	
  any	
  species	
  do	
  not	
  exist.	
  In	
  both	
  control	
  
and	
  raced	
  horses,	
  caudodorsal	
  arteries	
  are	
  stiffer-­‐walled	
  than	
  cranioventral	
  arteries.	
  Due	
  to	
  
the	
  absence	
  of	
  similar	
  observations	
  in	
  other	
  species,	
  this	
  finding	
  is	
  unexpected,	
  and	
  any	
  
proposed	
  rationale	
  for	
  this	
  observation	
  at	
  this	
  time	
  remains	
  speculative.	
  However,	
  in	
  light	
  
of	
  what	
  is	
  known	
  about	
  pulmonary	
  blood	
  flow	
  distribution	
  in	
  quadruped	
  species,	
  it	
  is	
  
considered	
  likely	
  that	
  these	
  differences	
  are	
  related	
  to	
  the	
  inhomogeneous	
  blood	
  flow	
  
distribution	
  patterns	
  that	
  are	
  observed	
  in	
  the	
  horse	
  (76)	
  and	
  in	
  other	
  mammals	
  (56,	
  57).	
  
Stiffer-­‐walled	
  pulmonary	
  arteries	
  in	
  caudodorsal	
  lung	
  may	
  represent	
  an	
  adaptive	
  response	
  
to	
  normal	
  preferential	
  distribution	
  patterns	
  of	
  blood	
  flow	
  to	
  this	
  region,	
  which	
  in	
  turn	
  is	
  
largely	
  due	
  to	
  the	
  anatomy	
  of	
  the	
  pulmonary	
  vascular	
  system	
  (77).	
  This	
  change	
  may	
  also	
  
serve	
  to	
  protect	
  pulmonary	
  capillaries	
  in	
  this	
  region	
  from	
  a	
  higher-­‐flow	
  state	
  during	
  
intense	
  exercise.	
  	
  
In	
  both	
  control	
  and	
  raced	
  horses,	
  cranioventral	
  veins	
  are	
  stiffer-­‐walled	
  than	
  
caudodorsal	
  veins.	
  Considering	
  an	
  example	
  from	
  the	
  equine	
  systemic	
  circulation,	
  veins	
  
within	
  the	
  foot	
  (i.e.	
  laminar	
  veins)	
  are	
  exposed	
  to	
  the	
  highest	
  intravascular	
  pressures	
  in	
  the	
  
limb	
  due	
  to	
  gravitational	
  forces,	
  and	
  are	
  thick-­‐walled	
  (6)	
  structures	
  that	
  are	
  difficult	
  to	
  
discern	
  from	
  laminar	
  arteries	
  (97).	
  It	
  is	
  possible	
  that	
  pulmonary	
  veins	
  from	
  the	
  
cranioventral	
  lung	
  are	
  stiffer-­‐walled	
  than	
  their	
  caudodorsal	
  counterparts	
  due	
  to	
  a	
  similar	
  
adaptive	
  response	
  to	
  the	
  hydrostatic	
  pressure	
  difference	
  between	
  the	
  dorsal	
  and	
  ventral	
  
lung.	
  	
  	
  
Although	
  vessel	
  mechanics	
  play	
  a	
  role	
  in	
  influencing	
  blood	
  flow	
  distribution,	
  vessel	
  
reactivity	
  must	
  also	
  be	
  considered	
  a	
  key	
  determinant	
  of	
  pulmonary	
  blood	
  flow.	
  

	
  
63	
  

Interestingly,	
  a	
  small	
  number	
  of	
  studies	
  report	
  regional	
  differences	
  in	
  pulmonary	
  vessel	
  
reactivity,	
  both	
  in	
  the	
  horse	
  (164)	
  and	
  in	
  the	
  pig	
  (51,	
  176).	
  Larger	
  dorsal	
  arteries	
  in	
  both	
  
pigs	
  (176),	
  and	
  horses	
  (164)	
  demonstrate	
  enhanced	
  endothelial-­‐mediated	
  vasorelaxation	
  
compared	
  to	
  vessels	
  from	
  ventral	
  regions.	
  Regional	
  patterns	
  of	
  vessel	
  mechanical	
  
properties,	
  along	
  with	
  vascular	
  reactivity	
  merit	
  further	
  and	
  more	
  detailed	
  investigation.	
  	
  
An	
  important	
  finding	
  of	
  this	
  study	
  was	
  that	
  caudodorsal	
  veins	
  from	
  raced	
  animals	
  
were	
  significantly	
  stiffer	
  than	
  those	
  from	
  control	
  animals.	
  This	
  increase	
  in	
  stiffness	
  is	
  most	
  
likely	
  a	
  consequence	
  of	
  venous	
  remodeling	
  in	
  raced	
  horses.	
  We	
  previously	
  reported	
  that	
  
small	
  pulmonary	
  veins	
  in	
  caudodorsal,	
  but	
  not	
  in	
  cranioventral	
  lung	
  remodel	
  in	
  EIPH-­‐
affected	
  horses,	
  while	
  equivalently-­‐sized	
  pulmonary	
  arteries	
  are	
  largely	
  unaffected	
  in	
  both	
  
regions	
  (231).	
  	
  EIPH-­‐associated	
  venous	
  remodeling	
  is	
  typified	
  by	
  extensive	
  adventitial	
  
collagen	
  expansion	
  (231)	
  resulting	
  in	
  reduced	
  lumen	
  area	
  (38).	
  	
  	
  In	
  studies	
  performed	
  on	
  
the	
  pulmonary	
  vasculature	
  of	
  other	
  species,	
  remodeling	
  is	
  also	
  associated	
  with	
  altered	
  
mechanical	
  properties	
  (101,	
  207,	
  212).	
  	
  
An	
  increase	
  in	
  stiffness	
  of	
  pulmonary	
  veins	
  has	
  important	
  physiologic	
  consequences.	
  
Increased	
  vein	
  stiffness	
  will	
  decrease	
  pressure-­‐induced	
  distension	
  of	
  the	
  veins	
  and	
  
increase	
  resistance	
  to	
  blood	
  flow,	
  particularly	
  during	
  exercise.	
  This,	
  in	
  turn,	
  should	
  increase	
  
pressure	
  in	
  upstream	
  pulmonary	
  capillaries.	
  If	
  pressure	
  increases	
  exceed	
  the	
  reported	
  
breaking	
  strength	
  of	
  these	
  vessels	
  (Ptm	
  75	
  mmHg	
  (17)),	
  stress-­‐failure	
  (230)	
  of	
  capillary	
  
walls	
  can	
  occur,	
  resulting	
  in	
  EIPH.	
  	
  	
  
Despite	
  the	
  difference	
  between	
  length-­‐tension	
  curves	
  of	
  pulmonary	
  arteries	
  
harvested	
  from	
  cranioventral	
  lung	
  in	
  raced	
  and	
  unraced	
  horses	
  being	
  small,	
  this	
  difference	
  
was	
  statistically	
  significant.	
  The	
  observation	
  that	
  cranioventral	
  pulmonary	
  arteries	
  were	
  

	
  
64	
  

stiffer	
  in	
  raced	
  horses	
  was	
  unexpected,	
  as	
  vascular	
  pathology	
  in	
  cranioventral	
  lung	
  is	
  not	
  
reported	
  in	
  existing	
  literature	
  on	
  the	
  topic	
  (152,	
  157,	
  231).	
  As	
  extensive	
  EIPH	
  pathology	
  in	
  
this	
  region	
  is	
  not	
  observed,	
  it	
  is	
  possible	
  that	
  mild	
  arterial	
  remodeling	
  changes	
  have	
  been	
  
overlooked	
  in	
  past	
  studies	
  and	
  further	
  more	
  detailed	
  studies	
  of	
  the	
  vasculature	
  of	
  this	
  
region	
  are	
  warranted	
  based	
  on	
  this	
  observation.	
  It	
  is	
  plausible	
  that	
  stiffening	
  of	
  
cranioventral	
  pulmonary	
  arteries	
  in	
  response	
  to	
  race	
  training	
  is	
  somewhat	
  protective	
  of	
  
capillaries	
  in	
  that	
  region,	
  and	
  that	
  through	
  this	
  mechanism,	
  this	
  observation	
  may	
  also	
  
explain	
  (at	
  least	
  in	
  part)	
  the	
  regional	
  nature	
  of	
  the	
  EIPH	
  lesion.	
  
Alterations	
  in	
  vessel	
  stiffness,	
  as	
  determined	
  by	
  analysis	
  of	
  length-­‐tension	
  curves,	
  
are	
  a	
  direct	
  result	
  of	
  changes	
  in	
  vessel	
  wall	
  structural	
  components	
  (119).	
  In	
  this	
  study,	
  we	
  
evaluated	
  tension	
  over	
  a	
  wide	
  range,	
  from	
  zero-­‐stress	
  to	
  tension	
  values	
  approaching	
  vessel	
  
breaking	
  point.	
  In	
  light	
  of	
  previous	
  studies	
  in	
  EIPH-­‐affected	
  horses	
  that	
  report	
  collagen	
  
deposition	
  in	
  remodeled	
  vein	
  walls	
  (38,	
  231,	
  233)	
  it	
  is	
  most	
  likely	
  that	
  the	
  observed	
  
increase	
  in	
  vessel	
  stiffness	
  is	
  due	
  to	
  an	
  increase	
  in	
  wall	
  collagen	
  content.	
  In	
  this	
  study	
  all	
  
vessels	
  were	
  stretched	
  to	
  near-­‐breaking	
  point,	
  which	
  distorted	
  vessel	
  wall	
  anatomy	
  greatly.	
  
For	
  this	
  reason,	
  morphometric	
  analyses	
  of	
  the	
  vessels	
  were	
  not	
  performed,	
  and	
  therefore	
  
we	
  do	
  not	
  provide	
  data	
  quantifying	
  collagen	
  deposition	
  in	
  affected	
  vessels.	
  Studies	
  using	
  a	
  
reduced	
  range	
  of	
  tension	
  application	
  to	
  correlate	
  collagen	
  content	
  and	
  mechanical	
  
properties	
  are	
  warranted.	
  	
  
Vessel	
  mechanics	
  were	
  analyzed	
  over	
  a	
  large	
  range	
  of	
  wall	
  tensions,	
  which	
  
undoubtedly	
  exceeds	
  any	
  wall	
  tension	
  changes	
  experienced	
  during	
  exercise	
  in	
  vivo.	
  
Extrapolation	
  of	
  the	
  effect	
  of	
  mechanical	
  changes	
  over	
  such	
  a	
  large	
  wall	
  tension	
  range	
  on	
  
vessels	
  in	
  the	
  living	
  horse	
  at	
  rest	
  or	
  during	
  exercise	
  is	
  difficult,	
  and	
  predicted	
  effects,	
  if	
  any,	
  

	
  
65	
  

should	
  not	
  be	
  overstated.	
  	
  In	
  an	
  effort	
  to	
  address	
  this,	
  we	
  converted	
  tension	
  values	
  to	
  
equivalent	
  transmural	
  pressure	
  values	
  using	
  the	
  Law	
  of	
  Laplace,	
  and	
  evaluated	
  length-­‐
pressure	
  relationships	
  over	
  a	
  range	
  encompassing	
  in	
  vivo	
  physiologic	
  pressure	
  ranges	
  
experienced	
  by	
  horses	
  at	
  rest	
  and	
  during	
  exercise:	
  0-­‐120	
  mmHg	
  for	
  arteries,	
  and	
  0	
  –	
  80	
  
mmHg	
  for	
  veins	
  (127).	
  This	
  analysis	
  demonstrated	
  an	
  identical	
  pattern	
  of	
  significant	
  effects	
  
of	
  racing	
  (stiffening	
  of	
  caudodorsal	
  veins	
  and	
  cranioventral	
  arteries)	
  to	
  that	
  found	
  in	
  the	
  
larger	
  (supra-­‐physiologic)	
  range	
  of	
  vessel	
  wall	
  tensions.	
  It	
  is	
  reasonable	
  to	
  extrapolate	
  the	
  
effect	
  of	
  these	
  data	
  to	
  vessels	
  in	
  vivo,	
  and	
  these	
  findings	
  lend	
  further	
  support	
  to	
  the	
  
hypothesis	
  that	
  pulmonary	
  venous	
  remodeling	
  in	
  the	
  caudodorsal	
  lung,	
  which	
  in	
  this	
  study	
  
manifests	
  as	
  an	
  increase	
  in	
  vessel	
  wall	
  stiffness,	
  may	
  be	
  a	
  component	
  of	
  EIPH	
  pathogenesis.	
  	
  
Previous	
  reports	
  of	
  EIPH	
  pathology	
  have	
  utilized	
  horses	
  with	
  career-­‐limiting	
  EIPH	
  
(157,	
  231,	
  233)	
  and	
  correspondingly	
  severe	
  pulmonary	
  pathologic	
  changes.	
  In	
  contrast,	
  
raced	
  horses	
  used	
  in	
  this	
  study	
  were	
  retired	
  from	
  racing	
  for	
  reasons	
  other	
  than	
  EIPH.	
  
Interestingly,	
  and	
  despite	
  an	
  absence	
  of	
  severe	
  EIPH	
  clinical	
  history,	
  these	
  horses	
  had	
  mild	
  
to	
  moderate	
  EIPH	
  pathology,	
  suggestive	
  of	
  underlying	
  prior	
  pulmonary	
  hemorrhage.	
  
Consistent	
  with	
  previous	
  reports	
  (231,	
  233),	
  caudodorsal	
  veins	
  of	
  raced	
  horses	
  in	
  this	
  
study	
  were	
  remodeled.	
  The	
  data	
  presented	
  in	
  this	
  paper	
  expand	
  this	
  observation	
  by	
  
demonstrating	
  that	
  this	
  remodeling	
  is	
  associated	
  with	
  increased	
  vein	
  wall	
  stiffness.	
  These	
  
alterations	
  in	
  vessel	
  mechanics	
  further	
  substantiate	
  the	
  contention	
  that	
  remodeling	
  of	
  the	
  
pulmonary	
  venous	
  system	
  of	
  the	
  equine	
  lung	
  is	
  an	
  early	
  response	
  to	
  strenuous	
  exercise,	
  
The	
  authors	
  propose	
  that	
  this	
  remodeling	
  may	
  have	
  a	
  role	
  in	
  the	
  pathogenesis	
  of	
  EIPH	
  
(233)	
  although	
  it	
  is	
  acknowledged	
  that	
  the	
  experiments	
  described	
  in	
  this	
  study	
  were	
  not	
  
designed	
  to	
  ascertain	
  whether	
  alterations	
  in	
  pulmonary	
  vein	
  stiffness	
  contribute	
  to	
  EIPH,	
  	
  

	
  
66	
  

or,	
  whether	
  the	
  changes	
  come	
  about	
  because	
  of	
  EIPH	
  earlier	
  in	
  these	
  horses’	
  racing	
  careers.	
  
Further	
  support	
  for	
  a	
  relationship	
  between	
  histologic	
  remodeling	
  changes	
  and	
  increased	
  
vein	
  stiffness	
  is	
  provided	
  by	
  the	
  observation	
  that	
  the	
  mechanical	
  properties	
  of	
  
cranioventral	
  veins	
  and	
  caudodorsal	
  arteries,	
  vessels	
  that	
  are	
  not	
  reported	
  to	
  remodel	
  in	
  
EIPH-­‐affected	
  horses	
  (231,	
  233),	
  were	
  unaffected	
  by	
  racing.	
  
In	
  conclusion,	
  the	
  findings	
  of	
  this	
  study	
  indicate	
  for	
  the	
  first	
  time,	
  that	
  regional	
  
differences	
  in	
  vessel	
  mechanics	
  exist	
  in	
  the	
  unraced	
  horse,	
  and	
  that	
  changes	
  in	
  pulmonary	
  
vein	
  wall	
  structure	
  occur	
  in	
  horses	
  that	
  have	
  undergone	
  race	
  training.	
  Furthermore,	
  these	
  
changes	
  occur	
  before	
  the	
  development	
  of	
  severe,	
  career-­‐limiting	
  EIPH.	
  	
  Altered	
  vessel	
  
mechanics	
  are	
  detectable	
  within	
  a	
  physiologically	
  applicable	
  range	
  of	
  vessel	
  wall	
  tensions.	
  
Therefore	
  this	
  finding	
  may	
  have	
  important	
  consequences	
  in	
  the	
  exercising	
  horse.	
  Increased	
  
vein	
  wall	
  stiffness	
  increases	
  pulmonary	
  capillary	
  pressures,	
  particularly	
  during	
  exercise,	
  
thereby	
  augmenting	
  EIPH.	
  These	
  data	
  highlight	
  pulmonary	
  vein	
  remodeling	
  as	
  a	
  lesion	
  of	
  
interest	
  in	
  EIPH	
  pathogenesis,	
  and	
  suggest	
  that	
  pulmonary	
  veins	
  could	
  be	
  an	
  interesting	
  
therapeutic	
  target	
  in	
  future	
  approaches	
  to	
  EIPH	
  management.	
  	
  
	
  

	
  

	
  
67	
  

	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

APPENDIX	
  
	
  

	
  
68	
  

	
  

	
  
	
  
Figure	
  4	
  Verhoeff-­‐Van	
  Geison	
  stained	
  pulmonary	
  artery	
  and	
  vein	
  for	
  histologic	
  
confirmation	
  of	
  identity	
  post-­‐myography.	
  	
  Pulmonary	
  arteries	
  (A)	
  are	
  thicker	
  walled	
  than	
  
veins	
  (B)	
  and	
  possess	
  both	
  an	
  internal	
  (arrowhead)	
  and	
  external	
  (arrow)	
  elastic	
  lamina	
  
compared	
  to	
  veins	
  which	
  are	
  thin-­‐walled	
  and	
  only	
  have	
  an	
  external	
  elastic	
  lamina	
  (arrow).	
  
Bar	
  =	
  100	
  µm.	
  	
  
	
  
	
  
	
  
	
  

	
  
69	
  

	
  
	
  

	
  
	
  
	
  
	
  
Figure	
  5	
  Length-­‐tension	
  plots	
  for	
  arteries	
  and	
  veins	
  from	
  control,	
  unraced	
  (A)	
  and	
  raced	
  
(B)	
  horses.	
  Values	
  are	
  mean	
  ±	
  SE.	
  In	
  both	
  control	
  and	
  raced	
  horses,	
  veins	
  are	
  stiffer	
  than	
  
arteries.	
  P	
  <	
  0.05	
  is	
  considered	
  significant.	
  	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  

	
  
70	
  

	
  
Figure	
  6	
  Length-­‐tension	
  plots	
  for	
  caudodorsal	
  (CD)	
  and	
  cranioventral	
  (CV)	
  veins,	
  and	
  for	
  
CD	
  and	
  CV	
  arteries	
  from	
  unraced	
  horses	
  (A	
  and	
  B	
  respectively)	
  and	
  from	
  raced	
  horses	
  	
  (C	
  
and	
  D	
  respectively).	
  Values	
  are	
  mean	
  ±	
  SE.	
  In	
  both	
  control	
  and	
  raced	
  horses,	
  veins	
  from	
  
cranioventral	
  lung	
  are	
  stiffer	
  than	
  veins	
  from	
  caudodorsal	
  lung,	
  and	
  arteries	
  from	
  
caudodorsal	
  lung	
  are	
  stiffer	
  than	
  arteries	
  from	
  cranioventral	
  lung.	
  P	
  <	
  0.05	
  is	
  considered	
  
significant.	
  	
  
	
  
	
  

	
  
71	
  

	
  
	
  
Figure	
  7	
  Length-­‐tension	
  plots	
  of	
  vessels	
  from	
  control	
  and	
  raced	
  horses.	
  Data	
  are	
  from	
  
caudodorsal	
  (CD)	
  and	
  cranioventral	
  (CV)	
  veins	
  (A	
  and	
  B	
  respectively)	
  and	
  arteries	
  (C	
  and	
  D	
  
respectively).	
  Values	
  are	
  means	
  ±	
  SE	
  Veins	
  from	
  the	
  caudodorsal	
  region	
  of	
  lungs	
  of	
  raced	
  
horses	
  are	
  stiffer	
  than	
  those	
  from	
  unraced,	
  control	
  horses.	
  Arteries	
  from	
  the	
  cranioventral	
  
region	
  of	
  lungs	
  of	
  raced	
  horses	
  are	
  stiffer	
  than	
  those	
  from	
  unraced,	
  control	
  horses.	
  p	
  <	
  0.05	
  
is	
  considered	
  significant.	
  	
  
	
  
	
  

	
  
72	
  

	
  
Figure	
  8	
  Length-­‐pressure	
  plots	
  for	
  vessels	
  from	
  control	
  and	
  raced	
  horses.	
  Data	
  are	
  from	
  
caudodorsal	
  (CD)	
  and	
  cranioventral	
  (CV)	
  veins	
  (A	
  and	
  B	
  respectively)	
  and	
  arteries(C	
  and	
  D	
  
respectively).	
  Data	
  are	
  expressed	
  as	
  mean	
  values.	
  Veins	
  from	
  the	
  caudodorsal	
  region	
  of	
  
lungs	
  of	
  raced	
  horses	
  are	
  stiffer	
  than	
  those	
  from	
  unraced,	
  control	
  horses.	
  Arteries	
  from	
  the	
  
cranioventral	
  region	
  of	
  lungs	
  of	
  raced	
  horses	
  are	
  stiffer	
  than	
  those	
  from	
  unraced,	
  control	
  
horses.	
  Hatched	
  regions	
  demarcate	
  the	
  range	
  of	
  in	
  vivo	
  intravascular	
  pressures	
  from	
  rest	
  to	
  
intense	
  exercise	
  (10	
  –	
  70	
  mmHg	
  and	
  30	
  –	
  110	
  mmHg	
  for	
  veins	
  and	
  arteries	
  respectively).	
  P	
  
<	
  0.05	
  is	
  considered	
  significant.	
  
	
  

	
  
73	
  

Vessel	
  
Type	
  

Region	
  

Horse	
  
Status	
  

n	
  

Vein	
  
Artery	
  

CD	
  
CD	
  

Control	
  
Control	
  

9	
  
10	
  

Vein	
  

CV	
  

Control	
  

7	
  

Artery	
  

CV	
  

Control	
  

9	
  

Vein	
  

CD	
  

Raced	
  

15	
  

Artery	
  

CD	
  

Raced	
  

10	
  

Vein	
  
Artery	
  

CV	
  
CV	
  

Raced	
  
Raced	
  

8	
  
10	
  

Diameter	
  at	
  
start	
  (μm)	
  
mean	
  ±	
  
s.e.m	
  
217.5	
  ±	
  
25.21	
  
280.0	
  ±38.81	
  
207.3	
  ±	
  
30.83	
  
314.5	
  ±	
  
37.42	
  
223.8	
  ±	
  
15.35	
  
386.3	
  ±	
  
29.28	
  
211.9	
  ±	
  
34.68	
  
330.3	
  ±	
  27.2	
  

	
  
	
  
Table	
  1	
  Mean	
  vessel	
  diameters	
  at	
  start	
  point.	
  	
  Values	
  are	
  means	
  ±	
  SE,	
  n	
  =	
  number	
  of	
  
vessels.	
  Start	
  point	
  is	
  defined	
  as	
  the	
  smallest	
  diameter	
  at	
  which	
  a	
  vessel	
  maintains	
  a	
  small	
  
(<0.05	
  mN)	
  but	
  sustained	
  wall	
  tension.	
  CD,	
  caudodorsal;	
  CV	
  cranioventral	
  
	
  

	
  

	
  
74	
  

CHAPTER	
  3	
  
Regional	
  heterogeneity	
  in	
  reactivity	
  of	
  small	
  pulmonary	
  blood	
  vessels	
  in	
  the	
  horse	
  
may	
  predict	
  exercise-­‐induced	
  pulmonary	
  hemorrhage	
  lesion	
  distribution	
  
	
  
Alice	
  Stack,	
  Frederik	
  J.	
  Derksen,	
  Kurt	
  J.	
  Williams,	
  N.	
  Edward	
  Robinson,	
  William	
  F.	
  Jackson	
  
	
  
Abstract	
  
Exercise-­‐induced	
  pulmonary	
  hemorrhage	
  in	
  horses	
  results	
  in	
  significant	
  caudodorsal	
  (CD)	
  
lung	
  region	
  pathology.	
  Capillary	
  stress	
  failure	
  and	
  hemorrhage	
  occur	
  secondary	
  to	
  high	
  
pulmonary	
  circulation	
  pressures	
  during	
  exercise,	
  but	
  reasons	
  for	
  CD	
  EIPH	
  lesion	
  
distribution	
  have	
  not	
  been	
  established.	
  Alterations	
  in	
  vascular	
  tone	
  of	
  small	
  pulmonary	
  
arteries	
  and	
  veins	
  impact	
  pulmonary	
  capillary	
  pressures.	
  	
  We	
  investigated	
  the	
  hypothesis	
  
that	
  regional	
  heterogeneity	
  in	
  active	
  control	
  mechanisms	
  of	
  small	
  pulmonary	
  vessels	
  exist,	
  
and	
  do	
  so	
  in	
  a	
  manner	
  that	
  predicts	
  EIPH	
  lesion	
  distribution.	
  Autonomic	
  control	
  of	
  vascular	
  
tone	
  changes	
  during	
  exercise,	
  therefore	
  the	
  vasoactive	
  autonomic	
  agonists	
  phenylephrine,	
  
isoproterenol	
  and	
  methacholine	
  were	
  investigated	
  using	
  wire	
  myography	
  on	
  vessels	
  
dissected	
  from	
  CD	
  and	
  cranioventral	
  (CV)	
  regions	
  of	
  12,	
  unraced	
  horses.	
  U	
  46619,	
  
furosemide,	
  and	
  mechanisms	
  of	
  methacholine	
  activity	
  using	
  L-­‐NAME	
  and	
  indomethacin	
  
pre-­‐incubation	
  steps,	
  were	
  also	
  investigated.	
  	
  Phenylephrine	
  did	
  not	
  cause	
  contraction	
  in	
  
any	
  vessels,	
  whereas	
  isoproterenol	
  relaxed	
  pre-­‐contracted	
  arteries	
  (CD	
  to	
  a	
  greater	
  degree	
  
than	
  CV)	
  but	
  not	
  veins.	
  Methacholine	
  caused	
  contraction	
  of	
  CD	
  arteries,	
  and	
  relaxation	
  of	
  
CV	
  arteries	
  and	
  all	
  veins	
  in	
  a	
  non-­‐region	
  dependent	
  manner.	
  L-­‐NAME	
  and	
  indomethacin	
  
inhibited	
  methacholine-­‐induced	
  relaxation	
  of	
  CV	
  arteries,	
  whereas	
  indomethacin	
  only	
  

	
  
75	
  

augmented	
  CD	
  artery	
  contraction.	
  Furosemide	
  caused	
  mild	
  relaxation	
  of	
  pulmonary	
  
arteries	
  and	
  veins	
  at	
  high	
  concentrations.	
  Extrapolation	
  of	
  these	
  data	
  to	
  the	
  in	
  vivo	
  effect	
  of	
  
increased	
  sympathetic	
  and	
  decreased	
  parasympathetic	
  tone	
  during	
  exercise	
  predicts	
  that	
  
highest	
  capillary	
  pressures	
  occur	
  in	
  CD	
  lung,	
  explaining	
  in	
  part	
  EIPH	
  lesion	
  distribution	
  and	
  
disease	
  pathogenesis.	
  	
  Regional	
  heterogeneity	
  in	
  small	
  pulmonary	
  vessel	
  reactivity	
  is	
  
unreported	
  in	
  other	
  species.	
  	
  
	
  
	
  
Introduction	
  
Exercise-­‐induced	
  pulmonary	
  hemorrhage	
  (EIPH),	
  which	
  is	
  diagnosed	
  when	
  blood	
  is	
  
identified	
  in	
  the	
  airways	
  after	
  an	
  intense	
  bout	
  of	
  exercise(162),	
  has	
  been	
  reported	
  in	
  
multiple	
  athletic	
  species	
  (1,	
  10),	
  including	
  humans(4,	
  42,	
  52),	
  but	
  it	
  is	
  most	
  prevalent	
  in	
  
racehorses	
  (18)	
  where	
  it	
  is	
  associated	
  with	
  impaired	
  racing	
  performance(70).	
  
An	
  early	
  theory	
  of	
  EIPH	
  pathogenesis	
  is	
  based	
  on	
  descriptions	
  of	
  capillary	
  wall	
  
disruption	
  in	
  lung	
  tissue	
  from	
  exercised	
  horses(230),	
  that	
  is	
  likely	
  a	
  result	
  of	
  the	
  high	
  
intravascular	
  pressures	
  that	
  occur	
  normally	
  in	
  the	
  pulmonary	
  vascular	
  system	
  of	
  the	
  
exercising	
  horse	
  (i.e.	
  capillary	
  stress	
  failure).	
  High	
  pulmonary	
  artery	
  and	
  venous	
  pressures	
  
result	
  in	
  capillary	
  pressures	
  in	
  the	
  order	
  of	
  80	
  mmHg	
  (106,	
  127),	
  exceeding	
  the	
  reported	
  
threshold	
  for	
  breaking	
  strength	
  (75	
  mmHg)	
  of	
  equine	
  pulmonary	
  capillaries	
  (17).	
  
Stress-­‐failure	
  alone	
  does	
  not	
  account	
  for	
  the	
  pathology	
  in	
  lungs	
  of	
  EIPH-­‐affected	
  
horses	
  however	
  (150,	
  233).	
  EIPH	
  lesions	
  include	
  remodeling	
  of	
  small	
  (100	
  –	
  200	
  μm	
  O.D.)	
  
pulmonary	
  veins	
  (231),	
  and	
  are	
  most	
  common	
  and	
  most	
  severe	
  in	
  the	
  caudodorsal	
  lung	
  
region	
  while	
  cranioventral	
  lung	
  remains	
  normal(150,	
  157,	
  231,	
  233).	
  Pulmonary	
  venous	
  

	
  
76	
  

remodeling	
  is	
  reported	
  as	
  a	
  consequence	
  of	
  elevated	
  intravascular	
  pressures	
  in	
  other	
  
species	
  (25,	
  86),	
  and	
  is	
  predicted	
  to	
  occur	
  in	
  response	
  to	
  elevated	
  venous	
  pressures	
  in	
  the	
  
exercising	
  horse,	
  particularly	
  in	
  the	
  caudodorsal	
  lung.	
  	
  
Caudodorsal	
  lung	
  is	
  the	
  region	
  to	
  which	
  blood	
  is	
  preferentially	
  distributed	
  in	
  both	
  
the	
  standing	
  (76),	
  and	
  galloping	
  horse	
  (15)	
  and	
  we	
  recently	
  confirmed	
  that	
  EIPH	
  pathology	
  
has	
  a	
  similar	
  distribution	
  to	
  that	
  of	
  pulmonary	
  blood	
  flow	
  (233).	
  Blood	
  flow	
  distribution	
  in	
  
the	
  lung	
  is	
  determined	
  by	
  vascular	
  anatomy,	
  which	
  is	
  fixed,	
  and	
  variable	
  factors	
  including	
  
vascular	
  reactivity	
  (55).	
  Regional	
  differences	
  in	
  endothelial-­‐dependent	
  reactivity	
  of	
  large	
  
pulmonary	
  arteries	
  of	
  the	
  horse	
  that	
  can	
  account	
  for	
  this	
  flow	
  distribution	
  pattern	
  have	
  
already	
  been	
  reported	
  (164),	
  however	
  reactivity	
  patterns	
  of	
  small	
  pulmonary	
  vessels	
  have	
  
not.	
  These	
  vessels	
  are	
  worthy	
  of	
  investigation	
  as	
  arterial	
  and	
  venous	
  resistances	
  in	
  the	
  
small	
  vessels	
  supplying	
  and	
  draining	
  the	
  capillaries	
  directly	
  affect	
  pulmonary	
  capillary	
  
pressure,	
  the	
  ultimate	
  cause	
  of	
  stress-­‐failure	
  and	
  hemorrhage.	
  For	
  example,	
  reduced	
  
arterial	
  resistance	
  and/or	
  increased	
  venous	
  resistance	
  will	
  further	
  increase	
  pulmonary	
  
capillary	
  pressure,	
  thereby	
  augmenting	
  the	
  risk	
  of	
  EIPH.	
  	
  
In	
  general,	
  sympathetic	
  nervous	
  system	
  activation	
  associated	
  with	
  exercise	
  results	
  
in	
  both	
  α-­‐	
  and	
  β-­‐	
  adrenoreceptor	
  activation	
  in	
  the	
  pulmonary	
  circulation	
  (93,	
  203),	
  while	
  
parasympathetic	
  activity	
  during	
  exercise	
  is	
  diminished	
  (133).	
  Accounts	
  of	
  regional	
  
heterogeneity	
  in	
  reactivity	
  profiles	
  of	
  large	
  pulmonary	
  vessels,	
  and	
  mechanical	
  properties	
  
of	
  small	
  pulmonary	
  vessels	
  in	
  the	
  horse	
  exist	
  (164,	
  196).	
  Regional	
  differences	
  in	
  vascular	
  
responses	
  to	
  increases	
  and/or	
  decreases	
  in	
  vasoactive	
  autonomic	
  agonists	
  would	
  exert	
  
varying	
  effects	
  on	
  pulmonary	
  capillary	
  pressures	
  in	
  the	
  lung.	
  	
  

	
  
77	
  

For	
  these	
  reasons,	
  we	
  investigated	
  the	
  hypothesis	
  that	
  regional	
  differences	
  in	
  
patterns	
  of	
  vascular	
  reactivity	
  to	
  adrenergic	
  and	
  cholinergic	
  agonists	
  in	
  small	
  pulmonary	
  
arteries	
  and	
  veins	
  exist,	
  and	
  do	
  so	
  in	
  a	
  manner	
  that	
  will	
  predict	
  the	
  predilection	
  of	
  EIPH	
  
pathology	
  for	
  the	
  caudodorsal	
  lung	
  region.	
  
EIPH	
  severity	
  and	
  incidence	
  is	
  ameliorated	
  in	
  part	
  by	
  pre-­‐race	
  administration	
  of	
  
furosemide	
  (73),	
  which	
  is	
  a	
  pulmonary	
  venodilator	
  in	
  dogs(59).	
  We	
  also	
  tested	
  the	
  
hypothesis	
  that,	
  in	
  horses,	
  furosemide	
  dilates	
  pulmonary	
  veins	
  but	
  not	
  pulmonary	
  arteries	
  
independent	
  of	
  lung	
  region.	
  	
  
To	
  test	
  these	
  hypotheses,	
  pharmacology	
  studies	
  using	
  wire	
  myography	
  were	
  
performed	
  on	
  small	
  pulmonary	
  arteries	
  and	
  veins	
  from	
  unraced	
  horses.	
  Both	
  adrenergic	
  
(phenylephrine	
  and	
  isoproterenol)	
  and	
  muscarinic	
  (methacholine)	
  agonists	
  were	
  tested,	
  
along	
  with	
  the	
  thromboxane	
  analog	
  U	
  46619,	
  and	
  furosemide.	
  Regional	
  differences	
  in	
  
responses	
  of	
  pulmonary	
  arteries	
  to	
  isoproterenol	
  and	
  methacholine,	
  and	
  responses	
  of	
  veins	
  
to	
  U	
  46619	
  are	
  reported.	
  Further	
  investigations	
  into	
  mechanisms	
  of	
  methacholine	
  activity	
  
in	
  pulmonary	
  arteries	
  were	
  performed	
  based	
  on	
  observed	
  differences.	
  Regional	
  differences	
  
in	
  reactivity	
  profiles	
  of	
  small	
  pulmonary	
  vessels	
  such	
  as	
  those	
  described	
  in	
  these	
  studies	
  
have	
  not	
  been	
  reported	
  in	
  any	
  species	
  to	
  date.	
  	
  
	
  
Materials	
  and	
  Methods	
  
Animals	
  
Nine	
  horses,	
  (7	
  geldings	
  and	
  2	
  mares),	
  8.33	
  ±	
  0.9	
  (average	
  age	
  ±	
  s.e.m.)	
  years,	
  were	
  used	
  to	
  
determine	
  regional	
  patterns	
  of	
  reactivity	
  to	
  5	
  drugs.	
  These	
  horses	
  were	
  of	
  various	
  breeds	
  
including	
  two	
  Quarterhorses,	
  two	
  Tennessee	
  Walkers,	
  three	
  Arabian-­‐crosses,	
  one	
  

	
  
78	
  

Thoroughbred	
  and	
  one	
  crossbred.	
  Three	
  additional	
  horses	
  (2	
  mares	
  and	
  one	
  gelding),	
  9.67	
  
±	
  2.03	
  (average	
  age	
  ±	
  s.e.m.)	
  years,	
  were	
  used	
  to	
  investigate	
  mechanisms	
  of	
  methacholine	
  
activity.	
  These	
  horses	
  were	
  of	
  various	
  breeds	
  including	
  two	
  paint-­‐crosses	
  and	
  one	
  
Thoroughbred.	
  Thoroughbred	
  horses	
  had	
  never	
  raced.	
  The	
  Michigan	
  State	
  University	
  
Institutional	
  Animal	
  Care	
  and	
  Use	
  Committee	
  approved	
  all	
  experimental	
  procedures.	
  
	
  
Tissue	
  acquisition	
  
Approximately	
  15	
  minutes	
  before	
  euthanasia,	
  intravenous	
  heparin	
  sodium	
  (50,000	
  
IU/horse)	
  was	
  administered	
  to	
  all	
  horses.	
  Euthanasia	
  was	
  performed	
  using	
  pentobarbital	
  
sodium	
  (90	
  mg/kg	
  IV).	
  Lung	
  tissue	
  sections	
  (approximately	
  4	
  cm3)	
  were	
  acquired	
  from	
  the	
  
caudodorsal	
  (CD)	
  and	
  cranioventral	
  (CV)	
  regions	
  of	
  the	
  caudal	
  (diaphragmatic)	
  lobe	
  of	
  both	
  
left	
  and	
  right	
  lungs.	
  Lung	
  tissue	
  was	
  kept	
  in	
  chilled	
  normal	
  saline	
  (0.9%	
  sodium	
  chloride)	
  
solution	
  until	
  vessel	
  dissection.	
  	
  
	
  
Vessel	
  dissection	
  
Approximately	
  0.5	
  cm	
  thick	
  slices	
  of	
  lung	
  tissue	
  were	
  pinned	
  to	
  a	
  Sylgard	
  (Dow	
  Corning,	
  
Midland,	
  MI)	
  pad	
  in	
  a	
  water-­‐jacketed	
  dissection	
  chamber	
  (Radnoti,	
  Monrovia,	
  CA)	
  and	
  fully	
  
submerged	
  in	
  chilled	
  Ca2+	
  -­‐free	
  physiologic	
  saline	
  solution	
  (PSS)	
  containing	
  (in	
  mM)	
  140	
  
NaCl,	
  5	
  KCl,	
  1	
  MgCl2,	
  10	
  HEPES,	
  10	
  glucose	
  (pH	
  7.4,	
  295	
  mOsm).	
  Ca2+	
  -­‐free	
  PSS	
  was	
  used	
  to	
  
minimize	
  vasospasm	
  that	
  can	
  occur	
  during	
  vessel	
  manipulation.	
  
Sections	
  of	
  pulmonary	
  veins	
  and	
  arteries	
  (identified	
  by	
  use	
  of	
  anatomic	
  criteria)	
  that	
  
ranged	
  in	
  length	
  from	
  0.38	
  mm	
  to	
  1.46	
  mm	
  were	
  gently	
  dissected	
  from	
  pulmonary	
  tissue.	
  
Pulmonary	
  arteries	
  were	
  harvested	
  from	
  broncho-­‐vascular	
  triads.	
  Pulmonary	
  arteries	
  were	
  

	
  
79	
  

identified	
  as	
  stiff-­‐walled	
  vessels	
  (compared	
  to	
  veins	
  in	
  the	
  same	
  triad)	
  that	
  were	
  
immediately	
  adjacent/adherent	
  to	
  the	
  conducting	
  airway	
  (175).	
  For	
  this	
  experiment,	
  veins	
  
were	
  not	
  harvested	
  from	
  the	
  bronchovascular	
  triad	
  and	
  intralobular	
  pulmonary	
  veins	
  only	
  
were	
  harvested.	
  They	
  are	
  found	
  alone	
  in	
  parenchyma,	
  and	
  are	
  not	
  associated	
  with	
  airways	
  
(231).	
  Once	
  dissected,	
  vessels	
  were	
  kept	
  in	
  Ca2+-­‐free	
  PSS	
  at	
  4°C	
  for	
  up	
  to	
  24	
  hours.	
  
	
  
Wire	
  myography	
  
Vessels	
  were	
  submerged	
  in	
  chilled	
  Ca2+-­‐free	
  PSS	
  and	
  mounted	
  as	
  a	
  cylinder	
  on	
  2	
  intra-­‐
luminal	
  stainless	
  steel	
  40-­‐μm	
  diameter	
  wires	
  in	
  a	
  4-­‐chamber	
  myograph	
  (DMT,	
  Aarhus,	
  
Denmark)	
  as	
  previously	
  reported	
  (196).	
  Bath	
  fluid	
  was	
  then	
  exchanged	
  for	
  room	
  
temperature	
  PSS	
  containing	
  	
  (in	
  mM)	
  140	
  NaCl,	
  5	
  KCl,	
  1.8	
  CaCl2,	
  1	
  MgCl2,	
  10	
  HEPES,	
  10	
  
glucose	
  (pH	
  7.4,	
  295	
  mOsm).	
  Vessel	
  length	
  was	
  recorded	
  using	
  a	
  previously	
  calibrated	
  
stereomicroscope.	
  The	
  bath	
  fluid	
  was	
  then	
  heated	
  slowly	
  to	
  37°C,	
  and	
  air	
  was	
  bubbled	
  
gently	
  through	
  the	
  fluid	
  continuously.	
  	
  The	
  myograph	
  force	
  transducer	
  was	
  used	
  in	
  
conjunction	
  with	
  a	
  PowerLab	
  (ADInstruments,	
  Colorado	
  Springs,	
  CO)	
  data	
  acquisition	
  unit	
  
and	
  LabChart	
  (ADInstruments,	
  Colorado	
  Springs,	
  CO)	
  software	
  platform.	
  
	
  
Vessel	
  normalization	
  
Optimal	
  passive	
  tension	
  values	
  for	
  equine	
  pulmonary	
  arteries	
  and	
  veins	
  have	
  not	
  been	
  
published.	
  Therefore	
  the	
  first	
  11	
  arteries	
  harvested	
  were	
  tested	
  in	
  order	
  to	
  determine	
  this	
  
characteristic.	
  Briefly,	
  resting	
  wall	
  tension	
  (T)	
  was	
  set	
  at	
  0.1	
  mN/mm,	
  and	
  PSS	
  was	
  
exchanged	
  for	
  60	
  mM	
  K+	
  PSS	
  (which	
  contained	
  (in	
  mM)	
  85	
  NaCl,	
  60	
  KCl,	
  1.8	
  CaCl2,	
  1	
  MgCl2,	
  
10	
  HEPES,	
  10	
  glucose	
  (pH	
  7.4,	
  295	
  mOsm)).	
  Vessels	
  were	
  allowed	
  to	
  develop	
  a	
  contraction	
  

	
  
80	
  

for	
  2	
  minutes,	
  the	
  maximum	
  T	
  value	
  reached	
  was	
  recorded,	
  and	
  the	
  vessel	
  was	
  washed	
  
with	
  PSS	
  until	
  T	
  returned	
  to	
  baseline.	
  After	
  5	
  minutes,	
  resting	
  wall	
  T	
  was	
  increased	
  by	
  0.2	
  
mN/mm	
  (to	
  0.3	
  mN/mm)	
  and	
  PSS	
  was	
  exchanged	
  for	
  60	
  mM	
  K+	
  PSS	
  as	
  before.	
  This	
  process	
  
was	
  repeated	
  until	
  an	
  increase	
  in	
  passive	
  wall	
  T	
  no	
  longer	
  resulted	
  in	
  an	
  increase	
  in	
  active	
  
T	
  upon	
  the	
  addition	
  of	
  60	
  mM	
  K+	
  PSS.	
  Pulmonary	
  veins	
  underwent	
  a	
  similar	
  procedure	
  
other	
  than	
  passive	
  wall	
  T	
  for	
  veins	
  was	
  increased	
  in	
  0.1	
  mN/mm	
  increments.	
  
Once	
  it	
  became	
  apparent	
  that	
  pulmonary	
  arteries	
  had	
  similar,	
  repeatable	
  optimal	
  T	
  
values	
  (determined	
  in	
  11	
  arteries	
  from	
  4	
  horses),	
  mean	
  optimal	
  T	
  was	
  calculated,	
  and	
  this	
  
value,	
  1.1	
  mN/mm,	
  was	
  used	
  to	
  normalize	
  all	
  subsequent	
  arteries.	
  Veins	
  had	
  a	
  greater	
  
range	
  of	
  optimal	
  T	
  values	
  (from	
  0.2	
  –	
  0.7	
  mN/mm),	
  and	
  therefore	
  this	
  value	
  was	
  
determined	
  with	
  60	
  mM	
  K+	
  PSS	
  for	
  each	
  vein	
  studied	
  in	
  subsequent	
  experiments.	
  Vessel	
  
diameter	
  was	
  measured	
  under	
  minimal	
  tension	
  (0.1	
  mN/mm	
  for	
  arteries	
  and	
  veins)	
  and	
  at	
  
optimal	
  tension	
  (1.1	
  mN/mm	
  for	
  arteries,	
  individual	
  wall	
  tension	
  for	
  veins)	
  conditions.	
  	
  
	
  
Vessel	
  wake-­‐up	
  
Once	
  normalized,	
  vessels	
  were	
  allowed	
  a	
  40-­‐minute	
  equilibration	
  period	
  at	
  their	
  optimal	
  T.	
  
Then	
  vessels	
  underwent	
  a	
  wake-­‐up	
  procedure	
  consisting	
  of	
  2	
  challenges	
  with	
  60	
  mM	
  K+	
  
PSS	
  with	
  a	
  5-­‐minute	
  interval.	
  	
  
	
  
Agonist	
  concentration-­‐response	
  curves	
  
Cumulative	
  concentration-­‐response	
  curves	
  (CCRC)	
  were	
  generated	
  for	
  each	
  drug,	
  with	
  the	
  
exception	
  of	
  phenylephrine	
  for	
  which	
  a	
  single	
  concentration	
  challenge	
  (10∧-­‐5	
  M)	
  was	
  
performed.	
  Concentration	
  ranges	
  were	
  initially	
  established	
  based	
  on	
  literature	
  from	
  other	
  

	
  
81	
  

species,	
  and	
  then	
  confirmed	
  in	
  equine	
  vessels	
  during	
  preliminary	
  experiments.	
  U	
  46619,	
  
methacholine	
  and	
  isoproterenol	
  concentrations	
  ranged	
  from	
  1	
  x	
  10∧-­‐9	
  to	
  3	
  x	
  10∧-­‐6	
  M.	
  
Furosemide	
  was	
  tested	
  over	
  10∧-­‐5	
  to	
  3	
  x	
  10∧-­‐4	
  M.	
  DMSO	
  (without	
  furosemide)	
  was	
  tested	
  
to	
  rule	
  out	
  vehicle	
  effects.	
  
In	
  order	
  to	
  evaluate	
  responses	
  to	
  vasodilator	
  agents	
  (methacholine,	
  isoproterenol,	
  
furosemide),	
  all	
  vessels	
  were	
  first	
  exposed	
  to	
  10∧-­‐6	
  M	
  U	
  46619,	
  a	
  concentration	
  that	
  was	
  
confirmed	
  as	
  reliably	
  producing	
  maximal	
  vasoconstriction	
  in	
  both	
  arteries	
  and	
  veins	
  during	
  
preliminary	
  experiments.	
  	
  
Multiple	
  drugs	
  (up	
  to	
  4)	
  were	
  tested	
  in	
  the	
  same	
  vessel	
  with	
  thorough	
  washes	
  
between	
  each	
  challenge.	
  The	
  order	
  in	
  which	
  drugs	
  were	
  tested	
  on	
  a	
  set	
  of	
  4	
  vessels	
  was	
  
randomized	
  using	
  a	
  random	
  list	
  generator.	
  	
  
For	
  the	
  second	
  component	
  of	
  this	
  study	
  in	
  which	
  mechanisms	
  of	
  methacholine-­‐
reactivity	
  were	
  investigated,	
  artery	
  wall	
  T	
  was	
  set	
  at	
  1.1	
  mN/mm.	
  30	
  minutes	
  after	
  vessel	
  
wake-­‐up,	
  a	
  methacholine	
  CCRC	
  was	
  performed	
  on	
  all	
  vessels.	
  After	
  washing,	
  vessels	
  were	
  
incubated	
  with	
  either	
  L-­‐NAME	
  (10∧-­‐4	
  M)	
  or	
  indomethacin	
  (10∧-­‐5	
  M)	
  for	
  30	
  minutes,	
  and	
  
the	
  methacholine	
  CCRC	
  was	
  repeated.	
  After	
  washing,	
  all	
  vessels	
  were	
  incubated	
  with	
  L-­‐
NAME	
  and	
  indomethacin	
  for	
  30	
  minutes	
  and	
  a	
  third	
  methacholine	
  CCRC	
  was	
  performed	
  in	
  
the	
  presence	
  of	
  both	
  inhibitors.	
  	
  
	
  
Vessel	
  fixation	
  
After	
  pharmacology	
  experiments	
  were	
  concluded,	
  vessels	
  were	
  cut	
  along	
  their	
  long	
  axis	
  
between	
  the	
  myograph	
  wires.	
  Vessels	
  were	
  then	
  pinned	
  out	
  with	
  the	
  endothelial	
  surface	
  

	
  
82	
  

exposed	
  onto	
  a	
  Sylgard	
  (Dow	
  Corning,	
  Midland,	
  MI)	
  pad	
  in	
  a	
  small	
  petri	
  dish	
  filled	
  with	
  
PBS.	
  Vessels	
  were	
  fixed	
  in	
  situ	
  using	
  10%	
  methanol-­‐free	
  formaldehyde	
  for	
  20	
  minutes.	
  
	
  
Immunohistochemistry	
  
After	
  washing	
  with	
  PBS,	
  immunoflourescent	
  staining	
  for	
  endothelium	
  (CD-­‐31)	
  was	
  carried	
  
out	
  on	
  the	
  wholemount	
  vessels	
  from	
  the	
  second	
  component	
  of	
  the	
  study.	
  Monoclonal	
  
mouse	
  anti-­‐human	
  CD-­‐31	
  primary	
  antibody	
  (Dako,	
  Carpinteria,	
  CA)	
  (1:40)	
  was	
  applied	
  to	
  
the	
  vessels	
  and	
  incubated	
  overnight	
  at	
  4°C.	
  After	
  blocking	
  (with	
  5%	
  normal	
  goat	
  serum	
  in	
  a	
  
1%	
  saponin	
  in	
  PBS	
  solution)	
  for	
  1	
  hour,	
  Alexa	
  Fluor	
  488-­‐conjugated	
  AffiniPure	
  goat	
  anti-­‐
mouse	
  IgG	
  secondary	
  antibody	
  (Jackson	
  ImmunoResearch	
  Laboratories,	
  West	
  Grove,	
  PA)	
  
(1:100)	
  was	
  applied	
  for	
  1	
  hour.	
  Vessels	
  were	
  then	
  mounted	
  on	
  slides	
  under	
  PBS.	
  	
  
	
  
Endothelial	
  Imaging	
  
Endothelium	
  was	
  examined	
  using	
  epiflourescent	
  microscopy.	
  Each	
  vessel	
  was	
  
photographed	
  at	
  20X	
  magnification,	
  and	
  depending	
  on	
  vessel	
  surface	
  area,	
  between	
  2	
  and	
  7	
  
images	
  per	
  vessel	
  acquired	
  and	
  saved.	
  	
  Endothelium-­‐covered	
  regions	
  were	
  outlined	
  using	
  
imaging	
  software	
  (Image	
  J,	
  http://imagej.nih.gov/ij/)	
  and	
  total	
  endothelium-­‐covered	
  area	
  
expressed	
  as	
  a	
  percentage	
  of	
  the	
  total	
  tissue	
  area	
  in	
  an	
  image.	
  	
  
	
  
Vessel	
  histology	
  
Following	
  fixation	
  or	
  imaging,	
  vessels	
  were	
  removed	
  from	
  the	
  petri	
  dish	
  or	
  slide	
  and	
  placed	
  
in	
  Histogel	
  (American	
  MasterTech,	
  Lodi,	
  CA)	
  specimen	
  processing	
  gel.	
  Gel-­‐embedded	
  
vessels	
  were	
  embedded	
  in	
  paraffin	
  and	
  sectioned.	
  6-­‐μm	
  sections	
  were	
  stained	
  with	
  

	
  
83	
  

hematoxylin	
  and	
  eosin	
  (H	
  and	
  E)	
  and	
  Verhoeff-­‐Van	
  Gieson	
  (VVG),	
  and	
  stained	
  tissues	
  were	
  
used	
  to	
  confirm	
  vessel	
  identity	
  as	
  either	
  a	
  pulmonary	
  artery	
  or	
  vein.	
  Characteristics	
  of	
  
pulmonary	
  arteries	
  include	
  a	
  substantial	
  tunica	
  media	
  bounded	
  by	
  an	
  internal	
  and	
  external	
  
elastic	
  lamina	
  (134).	
  In	
  contrast,	
  pulmonary	
  veins	
  have	
  less	
  smooth	
  muscle	
  and	
  only	
  an	
  
external	
  elastic	
  lamina	
  between	
  the	
  tunica	
  media	
  and	
  tunica	
  adventitia	
  (134).	
  The	
  internal	
  
elastic	
  lamina	
  between	
  the	
  tunica	
  intima	
  and	
  tunica	
  media	
  (175,	
  210)	
  is	
  absent.	
  	
  Vessel	
  
identification	
  was	
  carried	
  out	
  by	
  a	
  board-­‐certified	
  veterinary	
  pathologist	
  (KJW)	
  who	
  was	
  
blinded	
  to	
  the	
  identity	
  of	
  the	
  vessel	
  based	
  on	
  anatomic/dissection	
  criteria.	
  
	
  
Materials	
  	
  
U	
  46619	
  was	
  acquired	
  from	
  Tocris	
  Bioscience	
  (Minneapolis,	
  MN).	
  Acetyl-­‐β-­‐methylcholine	
  
chloride,	
  phenylephrine	
  hydrochloride,	
  isoproterenol	
  hydrochloride,	
  furosemide,	
  Nω-­‐Nitro-­‐
L-­‐arginine	
  methyl	
  ester	
  hydrochloride	
  (L-­‐NAME)	
  and	
  indomethacin	
  were	
  acquired	
  from	
  
Sigma-­‐Aldrich	
  (St.	
  Louis,	
  MO).	
  U	
  46619	
  and	
  furosemide	
  were	
  dissolved	
  in	
  DMSO,	
  
indomethacin	
  was	
  dissolved	
  in	
  20X	
  bicarbonate	
  buffer,	
  and	
  all	
  other	
  drugs	
  were	
  dissolved	
  
in	
  double-­‐distilled	
  water	
  to	
  make	
  stock	
  solutions.	
  Serial	
  dilutions	
  of	
  stock	
  solutions	
  were	
  
made	
  in	
  PSS.	
  	
  
	
  
Statistical	
  analyses	
  
Responses	
  of	
  a	
  vessel	
  to	
  vasoconstrictor	
  agents	
  (U	
  46619	
  and	
  phenylephrine)	
  were	
  
expressed	
  as	
  a	
  percentage	
  of	
  the	
  mean	
  of	
  that	
  vessel’s	
  maximal	
  contractions	
  in	
  response	
  to	
  
60	
  mM	
  K+	
  -­‐PSS	
  during	
  vessel	
  wake-­‐up.	
  	
  

	
  
84	
  

Once	
  a	
  stable	
  contraction	
  to	
  10∧-­‐6	
  M	
  U	
  46619	
  was	
  established,	
  all	
  responses	
  to	
  
vasodilator	
  agents	
  (methacholine,	
  isoproterenol	
  and	
  furosemide)	
  were	
  expressed	
  as	
  a	
  
percentage	
  of	
  that	
  contraction.	
  
Cumulative	
  concentration	
  response	
  curves	
  were	
  fit	
  (when	
  possible)	
  to	
  a	
  
log(agonist)	
  vs.	
  response	
  sigmoidal	
  curve	
  (Y=Bottom	
  +	
  (Top-­‐Bottom)/(1+10^((LogEC50-­‐
X)*HillSlope)))	
  and	
  curve	
  fits	
  were	
  then	
  compared	
  between	
  arteries	
  and	
  veins,	
  and	
  
between	
  lung	
  regions	
  (GraphPad	
  Prism	
  6,	
  GraphPad	
  Software	
  Inc.,	
  La	
  Jolla,	
  CA).	
  EC50	
  is	
  
that	
  concentration	
  of	
  agonist	
  that	
  gives	
  a	
  response	
  halfway	
  between	
  the	
  bottom	
  and	
  the	
  top	
  
(plateau	
  regions)	
  of	
  the	
  sigmoidal	
  curve.	
  All	
  data	
  are	
  expressed	
  as	
  mean	
  ±	
  s.e.m.	
  and	
  p	
  <	
  
0.05	
  is	
  considered	
  significant.	
  	
  
For	
  image	
  analysis,	
  percent	
  endothelium-­‐cover	
  in	
  an	
  image	
  was	
  averaged	
  for	
  each	
  
vessel.	
  	
  Mean	
  percent	
  endothelium-­‐cover	
  for	
  CD	
  and	
  CV	
  vessels	
  was	
  compared	
  using	
  an	
  
unpaired	
  t-­‐test	
  (GraphPad	
  Prism	
  6,	
  GraphPad	
  Software	
  Inc.,	
  La	
  Jolla,	
  CA)	
  and	
  statistical	
  
significance	
  declared	
  at	
  p	
  <	
  0.05.	
  	
  
	
  
	
  
Results	
  
Vessels	
  
Twenty-­‐nine	
  pulmonary	
  arteries	
  (13	
  from	
  CD	
  and	
  16	
  from	
  CV	
  lung	
  regions)	
  and	
  23	
  
pulmonary	
  veins	
  (11	
  from	
  CD	
  and	
  12	
  from	
  CV	
  lung	
  regions)	
  from	
  9	
  horses	
  were	
  used	
  to	
  
study	
  regional	
  patterns	
  of	
  reactivity	
  to	
  drugs,	
  and	
  30	
  pulmonary	
  arteries	
  (15	
  from	
  CD	
  and	
  
15	
  from	
  CV	
  lung	
  regions)	
  from	
  3	
  different	
  horses	
  were	
  used	
  to	
  investigate	
  mechanisms	
  of	
  
methacholine	
  activity.	
  	
  

	
  
85	
  

	
  
Vessel	
  dimensions	
  
Diameter	
  of	
  arteries	
  and	
  veins	
  under	
  minimal	
  tension	
  of	
  0.1	
  mN/mm	
  was	
  169.9	
  ±	
  10.91	
  
(mean	
  ±	
  s.e.m.)	
  and	
  128.3	
  ±	
  9.0	
  μm,	
  respectively.	
  Under	
  optimal	
  tension	
  arterial	
  diameter	
  
was	
  367	
  ±	
  16.23	
  (mean	
  ±	
  s.e.m.)	
  μm,	
  while	
  venous	
  diameter	
  was	
  205.8	
  ±	
  20.86	
  (mean	
  ±	
  
s.e.m.)	
  μm.	
  Using	
  the	
  Law	
  of	
  Laplace	
  to	
  convert	
  wall	
  tension	
  (in	
  mN/mm)	
  to	
  transmural	
  
pressure	
  values	
  (in	
  mmHg),	
  optimal	
  wall	
  tension	
  values	
  (1.1	
  mN/mm	
  for	
  arteries,	
  and	
  0.32	
  
mN/mm	
  for	
  veins)	
  were	
  equivalent	
  to	
  pressures	
  of	
  24.21	
  ±	
  1.1	
  and	
  14.05	
  ±	
  1.4	
  (mean	
  ±	
  
s.e.m.)	
  mmHg	
  in	
  arteries	
  and	
  veins	
  respectively.	
  	
  
	
  
U	
  46619	
  
U	
  46619	
  caused	
  concentration-­‐dependent	
  contraction	
  in	
  both	
  arteries	
  (n	
  =	
  16)	
  and	
  veins	
  (n	
  
=	
  15)	
  (Figure	
  9,	
  A),	
  with	
  the	
  greatest	
  response	
  observed	
  in	
  veins.	
  The	
  maximum	
  response	
  
of	
  veins	
  was	
  234.2	
  ±	
  15.06	
  %	
  of	
  maximum	
  response	
  to	
  KCl,	
  whereas	
  that	
  of	
  arteries	
  was	
  
104.5	
  ±	
  4.89	
  %.	
  Pulmonary	
  veins	
  were	
  more	
  sensitive	
  to	
  U	
  46619	
  than	
  pulmonary	
  arteries	
  
with	
  EC50	
  values	
  of	
  9.74	
  x	
  10∧-­‐8	
  M	
  and	
  2.73	
  x	
  10∧-­‐7	
  M	
  in	
  veins	
  and	
  arteries,	
  respectively.	
  
When	
  evaluated	
  by	
  region,	
  caudodorsal	
  (n	
  =	
  7)	
  and	
  cranioventral	
  (n	
  =	
  9)	
  arteries	
  did	
  not	
  
differ	
  in	
  their	
  responses	
  to	
  U	
  46619	
  (Figure	
  9,	
  B)(p	
  =	
  0.25),	
  while	
  a	
  regional	
  effect	
  was	
  
observed	
  in	
  pulmonary	
  veins	
  with	
  cranioventral	
  (n	
  =	
  8)	
  veins	
  demonstrating	
  enhanced	
  
sensitivity	
  compared	
  to	
  caudodorsal	
  veins	
  (n	
  =	
  7)(	
  Figure	
  9,	
  C)(p	
  <	
  0.0001).	
  	
  
	
  	
  
Phenylephrine	
  

	
  
86	
  

Pulmonary	
  arteries	
  (n	
  =	
  10)	
  and	
  pulmonary	
  veins	
  (n	
  =	
  9)	
  did	
  not	
  constrict	
  in	
  response	
  to	
  
10∧-­‐5	
  M	
  phenylephrine	
  (data	
  not	
  shown).	
  	
  
	
  
Isoproterenol	
  
Isoproterenol	
  caused	
  a	
  concentration-­‐dependent	
  relaxation	
  in	
  pulmonary	
  arteries	
  (n	
  =	
  11),	
  
while	
  pulmonary	
  veins	
  (n	
  =	
  14)	
  failed	
  to	
  respond.	
  A	
  significant	
  difference	
  between	
  the	
  
responses	
  of	
  cranioventral	
  (n	
  =	
  5)	
  and	
  caudodorsal	
  (n	
  =	
  6)	
  arteries	
  to	
  isoproterenol	
  was	
  
detected	
  (p	
  <	
  0.0001)(Figure	
  10,	
  A)	
  with	
  caudodorsal	
  arteries	
  demonstrating	
  enhanced	
  
relaxation	
  (relaxation	
  28.18	
  ±	
  3.74	
  %	
  and	
  48.67	
  ±	
  3.09	
  %	
  of	
  maximum	
  in	
  CD	
  and	
  CV	
  
arteries	
  respectively).	
  There	
  was	
  no	
  regional	
  difference	
  in	
  the	
  response	
  of	
  veins	
  to	
  
isoproterenol	
  (Figure	
  10,	
  B).	
  
	
  
Furosemide	
  	
  
Furosemide	
  caused	
  a	
  mild,	
  concentration-­‐dependent	
  relaxation	
  in	
  pulmonary	
  arteries	
  (n	
  =	
  
12)(relaxation	
  51.91	
  ±	
  24.31	
  %	
  of	
  maximum),	
  and	
  to	
  a	
  lesser	
  degree,	
  in	
  pulmonary	
  veins	
  (n	
  
=	
  12)(Figure	
  11,	
  A).	
  DMSO	
  vehicle	
  did	
  not	
  cause	
  relaxation	
  in	
  either	
  pulmonary	
  arteries	
  (n	
  
=	
  5)	
  or	
  veins	
  (n	
  =	
  10)(Figure	
  11,	
  B	
  and	
  C).	
  	
  Lung	
  region	
  did	
  not	
  affect	
  responses	
  of	
  arteries	
  
or	
  veins	
  to	
  furosemide	
  (p	
  =	
  0.07	
  and	
  p	
  =	
  0.19	
  for	
  arteries	
  and	
  veins	
  respectively)(Figure	
  
11,	
  B	
  and	
  C).	
  	
  
	
  
Methacholine	
  
Methacholine	
  caused	
  a	
  concentration-­‐dependent	
  relaxation	
  in	
  pulmonary	
  veins	
  (n	
  =	
  13)	
  
but	
  the	
  response	
  of	
  arteries	
  varied	
  by	
  region	
  (n	
  =	
  16).	
  When	
  arteries	
  were	
  evaluated	
  by	
  

	
  
87	
  

region,	
  all	
  cranioventral	
  arteries	
  (n	
  =	
  8)	
  demonstrated	
  a	
  concentration-­‐dependent	
  
relaxation	
  in	
  response	
  to	
  methacholine,	
  while	
  all	
  caudodorsal	
  arteries	
  (n	
  =	
  8)	
  constricted,	
  
also	
  in	
  a	
  concentration-­‐dependent	
  manner	
  (Figure12,	
  A).	
  The	
  response	
  of	
  pulmonary	
  veins	
  
(n	
  =	
  6	
  and	
  n	
  =	
  7	
  for	
  CD	
  and	
  CV	
  veins	
  respectively)	
  to	
  methacholine	
  was	
  not	
  affected	
  by	
  
region	
  (p	
  =	
  0.59)(Figure	
  12,	
  B).	
  	
  
	
  
Mechanisms	
  of	
  methacholine	
  reactivity	
  
Pre-­‐incubation	
  of	
  caudodorsal	
  pulmonary	
  arteries	
  (n	
  =	
  7)	
  with	
  L-­‐NAME	
  did	
  not	
  affect	
  their	
  
response	
  (contraction)	
  to	
  methacholine	
  (p	
  =	
  0.7)(Figure	
  13,	
  A).	
  Pre-­‐incubation	
  with	
  
indomethacin	
  however,	
  augmented	
  the	
  contraction	
  of	
  CD	
  arteries	
  (n	
  =	
  8)	
  when	
  compared	
  
to	
  methacholine	
  alone	
  (p	
  =<	
  0.0001)(Figure	
  5,	
  B).	
  Pre-­‐incubation	
  of	
  CD	
  arteries	
  (n	
  =	
  13)	
  
with	
  both	
  L-­‐NAME	
  and	
  indomethacin	
  also	
  resulted	
  in	
  an	
  augmented	
  contraction	
  (Figure	
  
13,	
  C)	
  but	
  the	
  CCRC	
  did	
  not	
  differ	
  from	
  vessels	
  incubated	
  with	
  indomethacin	
  alone	
  	
  (p	
  =	
  
0.79).	
  
Pre-­‐incubation	
  of	
  cranioventral	
  pulmonary	
  arteries	
  (n	
  =	
  8)	
  with	
  L-­‐NAME	
  diminished	
  their	
  
response	
  (relaxation)	
  to	
  methacholine	
  (p	
  <	
  0.0001)(Figure	
  13,	
  D).	
  Pre-­‐incubation	
  with	
  
indomethacin	
  also	
  resulted	
  in	
  reduced	
  relaxation	
  of	
  CV	
  arteries	
  (n	
  =	
  7)(Figure	
  13,	
  E).	
  	
  Pre-­‐
incubation	
  of	
  CV	
  arteries	
  (n	
  =	
  15)	
  with	
  both	
  L-­‐NAME	
  and	
  indomethacin	
  induced	
  a	
  mild	
  
contraction	
  and	
  abolished	
  relaxation	
  until	
  the	
  highest	
  concentrations	
  of	
  methacholine	
  were	
  
added	
  to	
  the	
  bath	
  (1	
  x	
  10-­‐6	
  and	
  3	
  x	
  10-­‐6	
  M)	
  (Figure	
  13,	
  F).	
  Bicarbonate	
  buffer	
  (used	
  to	
  make	
  
indomethacin	
  stock	
  solution)	
  at	
  the	
  concentration	
  used	
  in	
  the	
  experiments	
  did	
  not	
  affect	
  
PSS	
  pH,	
  making	
  a	
  vehicle	
  effect	
  improbable.	
  	
  
	
  

	
  
88	
  

Endothelial	
  imaging	
  
Endothelium-­‐covered	
  regions	
  were	
  clearly	
  distinguishable	
  from	
  denuded	
  areas	
  (Figure	
  
14).	
  Data	
  from	
  8	
  CD	
  and	
  11	
  CV	
  vessels	
  from	
  the	
  component	
  of	
  the	
  study	
  that	
  investigated	
  
methacholine	
  mechanisms	
  are	
  reported	
  (remaining	
  vessels	
  from	
  this	
  study	
  component	
  
could	
  not	
  be	
  imaged).	
  Values	
  for	
  percent	
  endothelium-­‐cover	
  in	
  a	
  vessel	
  were	
  43.84	
  ±	
  4.418	
  
and	
  41.4	
  ±	
  3.617	
  (mean	
  ±	
  s.e.m.)	
  for	
  CD	
  and	
  CV	
  vessels	
  respectively,	
  and	
  these	
  values	
  did	
  
not	
  differ	
  between	
  regions	
  (p	
  =	
  0.8426).	
  
	
  
Histology	
  
51	
  arteries	
  (from	
  both	
  study	
  components)	
  and	
  21	
  veins	
  were	
  submitted	
  for	
  histologic	
  
evaluation.	
  	
  All	
  vessels	
  (with	
  3	
  exceptions)	
  had	
  their	
  identity	
  (as	
  determined	
  when	
  they	
  
were	
  dissected)	
  confirmed	
  by	
  use	
  of	
  histology.	
  2	
  vessels	
  that	
  were	
  dissected	
  as	
  veins	
  were	
  
identified	
  as	
  arteries	
  using	
  histology,	
  and	
  data	
  were	
  included	
  in	
  the	
  artery	
  dataset.	
  1	
  artery	
  
could	
  not	
  be	
  confirmed	
  as	
  such	
  due	
  to	
  its	
  orientation	
  in	
  the	
  specimen	
  processing	
  gel,	
  
however	
  based	
  on	
  a	
  typical	
  arterial	
  reactivity	
  profile,	
  data	
  from	
  this	
  vessel	
  were	
  also	
  
included	
  in	
  the	
  study.	
  	
  
	
  
	
  
	
  
Discussion	
  
The	
  present	
  study	
  was	
  designed	
  to	
  investigate	
  whether	
  regional	
  differences	
  in	
  small	
  
pulmonary	
  vessel	
  reactivity	
  exist	
  in	
  the	
  horse,	
  and	
  whether	
  detected	
  differences	
  could	
  
predict	
  the	
  distinct	
  predilection	
  of	
  exercise-­‐induced	
  pulmonary	
  hemorrhage	
  (EIPH)	
  lesions	
  
for	
  caudodorsal	
  lung	
  (233).	
  To	
  the	
  best	
  of	
  the	
  authors’	
  knowledge,	
  this	
  study	
  reports	
  for	
  

	
  
89	
  

the	
  first	
  time	
  in	
  any	
  species,	
  that	
  regional	
  differences	
  in	
  reactivity	
  exist	
  in	
  small	
  caliber	
  
pulmonary	
  arteries	
  and	
  veins.	
  	
  
The	
  vessels	
  of	
  interest	
  in	
  this	
  investigation	
  are	
  small	
  (100	
  –	
  400	
  μm	
  O.D.)	
  pulmonary	
  
arteries	
  and	
  veins.	
  Evidence	
  of	
  regional	
  heterogeneity	
  in	
  mechanical	
  characteristics	
  of	
  
equine	
  pulmonary	
  vessels	
  of	
  this	
  caliber	
  has	
  recently	
  been	
  reported,	
  and	
  it	
  is	
  suggested	
  
that	
  these	
  differences	
  are	
  a	
  reflection	
  of	
  the	
  preferential	
  distribution	
  of	
  blood	
  flow	
  in	
  the	
  
equine	
  lung	
  to	
  caudodorsal	
  regions	
  (196).	
  	
  Pulmonary	
  veins	
  in	
  this	
  size	
  range	
  from	
  
caudodorsal	
  lung	
  are	
  remodeled	
  in	
  lungs	
  of	
  EIPH-­‐affected	
  horses,	
  and	
  are	
  stiffer-­‐walled	
  in	
  
horses	
  with	
  a	
  history	
  of	
  racing	
  (196,	
  231,	
  233).	
  Further	
  study	
  of	
  these	
  vessels	
  was	
  
undertaken	
  based	
  on	
  the	
  following	
  reasoning.	
  The	
  source	
  of	
  hemorrhage	
  in	
  EIPH	
  is	
  
pulmonary	
  capillary	
  stress	
  failure	
  (230),	
  resulting	
  from	
  elevated	
  pulmonary	
  circulation	
  
intravascular	
  pressures	
  in	
  horses	
  during	
  exercise	
  (124).	
  Resistance	
  to	
  flow	
  in	
  the	
  arteries	
  
and	
  veins	
  that	
  supply	
  and	
  drain	
  pulmonary	
  capillaries	
  determines	
  capillary	
  pressure.	
  
Therefore,	
  regional	
  arterial	
  dilation	
  resulting	
  in	
  transmission	
  of	
  high	
  arterial	
  pressures	
  to	
  
the	
  capillary	
  bed,	
  and/or	
  venous	
  constriction	
  during	
  exercise	
  would	
  increase	
  capillary	
  
pressure	
  and	
  render	
  capillaries	
  in	
  that	
  region	
  more	
  susceptible	
  to	
  stress	
  failure,	
  and	
  EIPH.	
  	
  
Before	
  evaluation	
  of	
  vessel	
  reactivity,	
  a	
  normalization	
  procedure	
  was	
  performed	
  in	
  
order	
  to	
  determine	
  the	
  degree	
  of	
  smooth	
  muscle	
  stretch	
  that	
  would	
  result	
  in	
  maximal	
  force	
  
development	
  for	
  subsequent	
  experiments	
  (7).	
  Optimal	
  passive	
  wall	
  tension	
  of	
  arteries	
  was	
  
significantly	
  larger	
  than	
  that	
  of	
  veins.	
  Mean	
  in	
  vivo	
  resting	
  pulmonary	
  arterial	
  pressures	
  are	
  
approximately	
  30	
  mmHg	
  (106,	
  124,	
  190),	
  while	
  in	
  vivo	
  pulmonary	
  artery	
  wedge	
  pressures	
  
(proxy	
  for	
  venous	
  pressures)	
  range	
  from	
  13.4	
  to	
  18	
  mmHg(124,	
  128,	
  190).	
  Conversion	
  of	
  
optimal	
  wall	
  tension	
  to	
  equivalent	
  pressure	
  values	
  demonstrated	
  that	
  these	
  studies	
  were	
  

	
  
90	
  

conducted	
  at	
  physiologically	
  relevant	
  tensions	
  (24.2	
  and	
  14.1	
  mmHg	
  in	
  arteries	
  and	
  veins	
  
respectively).	
  	
  
The	
  thromboxane	
  A2	
  analogue	
  U	
  46619	
  was	
  investigated	
  with	
  a	
  view	
  to	
  its	
  use	
  in	
  
subsequent	
  experiments	
  in	
  which	
  pre-­‐existing	
  tone	
  was	
  necessary	
  to	
  study	
  another	
  
vasoactive	
  agent	
  of	
  interest.	
  U	
  46619	
  caused	
  contractions	
  in	
  both	
  pulmonary	
  arteries	
  and	
  
veins,	
  but	
  pulmonary	
  veins	
  were	
  more	
  sensitive	
  to	
  U	
  46619	
  than	
  arteries.	
  While	
  no	
  
regional	
  differences	
  in	
  reactivity	
  were	
  observed	
  in	
  arteries,	
  cranioventral	
  veins	
  were	
  more	
  
sensitive	
  than	
  their	
  caudodorsal	
  counterparts.	
  Thromboxane	
  A2	
  is	
  an	
  arachidonic	
  acid	
  
metabolite	
  derived	
  from	
  endothelial	
  cells	
  and	
  platelets	
  (147,	
  168)	
  and	
  enhanced	
  sensitivity	
  
to	
  U	
  46619	
  in	
  pulmonary	
  veins	
  compared	
  to	
  pulmonary	
  arteries	
  has	
  also	
  been	
  reported	
  in	
  
other	
  species,	
  including	
  sheep,	
  dogs	
  and	
  guinea	
  pigs	
  (10,	
  99,	
  187).	
  The	
  role	
  of	
  thromboxane	
  
in	
  regulation	
  of	
  vascular	
  tone	
  during	
  exercise	
  however,	
  is	
  thought	
  to	
  be	
  minimal	
  (144),	
  and	
  
regional	
  patterns	
  of	
  equine	
  pulmonary	
  vein	
  sensitivity	
  to	
  this	
  agent	
  are	
  unlikely	
  to	
  be	
  
significant	
  in	
  the	
  context	
  of	
  EIPH.	
  	
  
Pulmonary	
  arterial	
  and	
  venous	
  tone	
  is	
  mediated,	
  at	
  least	
  in	
  part,	
  by	
  the	
  autonomic	
  
nervous	
  system,	
  and	
  during	
  exercise,	
  both	
  sympathetic	
  outflow	
  and	
  circulating	
  
concentrations	
  of	
  vasoactive	
  catecholamines	
  increase,	
  while	
  parasympathetic	
  activity	
  is	
  
diminished	
  (133,	
  203).	
  That	
  this	
  increase	
  in	
  sympathetic	
  activity	
  exerts	
  an	
  effect	
  on	
  the	
  
pulmonary	
  circulation	
  specifically	
  during	
  exercise	
  is	
  supported	
  by	
  data	
  from	
  both	
  sheep	
  
and	
  pigs	
  (93,	
  203).	
  For	
  these	
  reasons,	
  investigations	
  into	
  how	
  small	
  pulmonary	
  arteries	
  and	
  
veins	
  of	
  the	
  horse	
  are	
  affected	
  by	
  the	
  autonomic	
  nervous	
  system	
  during	
  exercise,	
  and	
  
whether	
  regional	
  heterogeneity	
  in	
  vessel	
  reactivity	
  exist	
  were	
  undertaken.	
  Specifically,	
  the	
  
selective	
  α1-­‐	
  and	
  non-­‐selective	
  β-­‐	
  adrenergic	
  receptor	
  agonists	
  phenylephrine	
  and	
  

	
  
91	
  

isoproterenol	
  respectively,	
  and	
  the	
  muscarinic	
  agonist	
  methacholine	
  were	
  studied.	
  In	
  the	
  
intensely	
  exercising	
  horse,	
  circulating	
  venous	
  concentrations	
  of	
  epinephrine	
  and	
  
norepinephrine	
  increase	
  from	
  0.9	
  and	
  0.7	
  to	
  153	
  and	
  148	
  nmol/L,	
  respectively	
  (191).	
  
These	
  concentrations	
  (approximately	
  1.5	
  x	
  10^-­‐7	
  M)	
  fall	
  within	
  the	
  concentration	
  range	
  
over	
  which	
  isoproterenol,	
  an	
  adrenergic	
  agonist,	
  was	
  tested.	
  	
  
Even	
  though	
  large	
  equine	
  pulmonary	
  arteries	
  (1.5	
  –	
  4	
  mm	
  in	
  diameter)(117),	
  and	
  
the	
  largest	
  pulmonary	
  veins	
  (63),	
  contract	
  in	
  response	
  to	
  phenylephrine,	
  indicating	
  the	
  
presence	
  of	
  α1-­‐receptors	
  on	
  the	
  smooth	
  muscle	
  cells	
  of	
  these	
  vessels,	
  phenylephrine	
  did	
  
not	
  cause	
  contraction	
  in	
  either	
  arteries	
  or	
  veins	
  in	
  the	
  present	
  study,	
  suggesting	
  an	
  absence	
  
of	
  the	
  target	
  receptor	
  in	
  smaller	
  caliber	
  vessels.	
  This	
  observation	
  is	
  not	
  unique	
  to	
  the	
  horse.	
  
For	
  example,	
  small	
  pulmonary	
  arteries	
  (100	
  –	
  300	
  μm	
  in	
  diameter)	
  of	
  the	
  rat	
  do	
  not	
  
respond	
  reliably	
  to	
  norepinephrine	
  (108),	
  and	
  the	
  response	
  of	
  ovine	
  pulmonary	
  arteries	
  to	
  
norepinephrine	
  is	
  attenuated	
  with	
  decreasing	
  vessel	
  diameter	
  (99).	
  
With	
  regard	
  to	
  the	
  distribution	
  of	
  beta-­‐adrenergic	
  receptors,	
  we	
  found	
  that	
  pre-­‐
contracted	
  equine	
  pulmonary	
  arteries	
  relaxed	
  in	
  response	
  to	
  the	
  non-­‐specific	
  β-­‐adrenergic	
  
receptor	
  agonist	
  isoproterenol,	
  while	
  pulmonary	
  veins	
  failed	
  to	
  respond.	
  Furthermore,	
  
isoproterenol-­‐induced	
  relaxation	
  was	
  of	
  greater	
  magnitude	
  in	
  the	
  caudodorsal	
  than	
  
cranioventral	
  arteries	
  (Figure	
  10).	
  This	
  is	
  noteworthy	
  because	
  at	
  this	
  time,	
  and	
  to	
  the	
  best	
  
of	
  the	
  authors’	
  knowledge,	
  there	
  are	
  no	
  other	
  reports	
  of	
  regional	
  differences	
  in	
  distribution	
  
of	
  beta-­‐adrenoreceptors	
  in	
  the	
  pulmonary	
  vasculature	
  of	
  any	
  species.	
  	
  
In	
  the	
  small	
  equine	
  pulmonary	
  vessels,	
  β-­‐adrenoreceptor-­‐mediated	
  vasodilation	
  of	
  
small	
  arteries	
  will	
  predominate	
  during	
  exercise	
  (there	
  was	
  no	
  evidence	
  of	
  α1-­‐	
  
adrenoreceptor-­‐mediated	
  constriction).	
  A	
  combination	
  of	
  a	
  generalized	
  failure	
  of	
  small	
  

	
  
92	
  

pulmonary	
  veins	
  to	
  dilate,	
  and	
  enhanced	
  dilation	
  of	
  caudodorsal	
  pulmonary	
  arteries	
  in	
  
particular	
  would	
  expose	
  capillaries	
  in	
  the	
  caudodorsal	
  lung	
  to	
  the	
  greatest	
  intravascular	
  
pressures	
  during	
  exercise,	
  perhaps	
  explaining,	
  at	
  least	
  in	
  part,	
  the	
  regional	
  distribution	
  of	
  
EIPH	
  in	
  the	
  horse	
  lung.	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  	
  
In	
  this	
  study,	
  small,	
  pre-­‐contracted	
  pulmonary	
  veins	
  of	
  the	
  horse	
  relaxed	
  in	
  
response	
  to	
  methacholine,	
  and	
  regional	
  differences	
  in	
  the	
  magnitude	
  of	
  this	
  response	
  were	
  
not	
  detected.	
  Pulmonary	
  arteries	
  on	
  the	
  other	
  hand,	
  demonstrated	
  opposite	
  effects	
  
depending	
  on	
  lung	
  region.	
  Arteries	
  from	
  caudodorsal	
  lung	
  contracted	
  while	
  those	
  from	
  
cranioventral	
  lung	
  dilated	
  in	
  response	
  to	
  methacholine.	
  	
  
In	
  general,	
  binding	
  of	
  acetylcholine	
  to	
  muscarinic	
  receptors	
  on	
  the	
  endothelium,	
  or	
  
on	
  the	
  smooth	
  muscle	
  of	
  blood	
  vessels,	
  affects	
  vessel	
  tone	
  by	
  causing	
  vasodilation	
  and	
  
vasoconstriction	
  respectively	
  (133,	
  203).	
  Whether	
  equine	
  blood	
  vessels	
  of	
  this	
  caliber	
  are	
  
directly	
  innervated	
  by	
  the	
  parasympathetic	
  nervous	
  system	
  has	
  not	
  been	
  reported	
  in	
  detail,	
  
however,	
  based	
  on	
  their	
  responses	
  to	
  the	
  acetylcholine	
  analog	
  methacholine,	
  it	
  is	
  
reasonable	
  to	
  infer	
  that	
  blood	
  vessels	
  studied	
  in	
  these	
  experiments	
  possess	
  muscarinic	
  
receptors	
  on	
  the	
  endothelium	
  and/or	
  smooth	
  muscle.	
  It	
  is	
  also	
  considered	
  unlikely	
  that	
  the	
  
regional	
  differences	
  in	
  responses	
  are	
  explained	
  by	
  regional	
  differences	
  in	
  endothelial	
  
coverage	
  of	
  vessels	
  consequent	
  to	
  vessel	
  injury	
  during	
  experimental	
  manipulation.	
  Percent	
  
endothelial	
  cover	
  in	
  a	
  subset	
  of	
  vessels	
  in	
  this	
  study	
  was	
  determined	
  based	
  on	
  the	
  presence	
  
of	
  the	
  endothelial-­‐specific	
  CD31	
  antigen,	
  and	
  did	
  not	
  differ	
  significantly	
  between	
  arteries	
  
from	
  either	
  lung	
  region.	
  	
  
Regional	
  differences	
  in	
  muscarinic-­‐receptor	
  mediated	
  vessel	
  reactivity	
  have	
  been	
  
reported	
  before	
  in	
  the	
  horse	
  (164),	
  and	
  in	
  the	
  pig	
  (176).	
  However	
  both	
  studies	
  report	
  on	
  

	
  
93	
  

vessels	
  that	
  are	
  much	
  larger	
  (4	
  –	
  6	
  mm	
  O.D.)	
  than	
  those	
  evaluated	
  in	
  this	
  study.	
  In	
  porcine	
  
pulmonary	
  arteries,	
  more	
  pronounced	
  relaxation	
  in	
  response	
  to	
  acetylcholine	
  was	
  
observed	
  in	
  dorsal	
  vessels,	
  compared	
  to	
  those	
  from	
  ventral	
  lung	
  (176),	
  and	
  large	
  equine	
  
pulmonary	
  arteries	
  from	
  caudodorsal	
  lung	
  also	
  relax,	
  while	
  those	
  from	
  cranioventral	
  lung	
  
contract	
  in	
  response	
  to	
  methacholine	
  (164).	
  The	
  contrasting	
  pattern	
  observed	
  in	
  small	
  
equine	
  pulmonary	
  arteries	
  is	
  of	
  particular	
  interest	
  when	
  considered	
  in	
  the	
  context	
  of	
  EIPH	
  
lesion	
  distribution.	
  If	
  parasympathetic	
  outflow	
  is	
  responsible	
  for	
  basal	
  maintenance	
  of	
  tone	
  
in	
  small	
  pulmonary	
  arteries	
  in	
  a	
  region-­‐dependent	
  manner,	
  perhaps	
  fulfilling	
  the	
  role	
  of	
  
protecting	
  capillaries	
  from	
  high	
  flow	
  rates	
  in	
  this	
  region,	
  then	
  diminished	
  parasympathetic	
  
activity	
  during	
  exercise	
  (133)	
  could	
  result	
  in	
  reduced	
  arterial	
  tone,	
  in	
  caudodorsal	
  lung	
  
specifically.	
  This,	
  along	
  with	
  a	
  possible	
  attenuation	
  of	
  muscarinic-­‐receptor	
  mediated	
  
pulmonary	
  venous	
  dilation	
  in	
  the	
  same	
  region	
  could	
  result	
  in	
  transmission	
  of	
  higher	
  
pressures	
  to	
  caudodorsal	
  pulmonary	
  capillaries	
  compared	
  to	
  capillaries	
  in	
  other	
  lung	
  
regions.	
  However,	
  when	
  extrapolating	
  these	
  data	
  to	
  in	
  vivo	
  conditions,	
  it	
  is	
  noted	
  that	
  all	
  
vessels	
  in	
  these	
  studies	
  were	
  pre-­‐contracted	
  with	
  U	
  46619,	
  and	
  responses	
  of	
  pulmonary	
  
vessels	
  to	
  acetylcholine	
  can	
  vary	
  depending	
  on	
  whether	
  vascular	
  tone	
  is	
  present	
  (10).	
  	
  
	
  	
  

Vasodilation	
  associated	
  with	
  muscarinic-­‐receptor	
  activation	
  is	
  typically	
  mediated	
  by	
  

endothelium-­‐derived	
  nitric	
  oxide	
  (NO)	
  and/or	
  prostanoids,	
  commonly	
  PGI2;	
  whereas	
  
vasoconstriction	
  can	
  result	
  from	
  generation	
  of	
  vasoconstrictor	
  prostanoids	
  termed	
  
endothelial	
  derived	
  constricting	
  factors	
  (EDCFs)	
  (236)	
  and/or	
  direct	
  binding	
  of	
  vascular	
  
smooth	
  muscle	
  muscarinic	
  receptors	
  (133).	
  In	
  order	
  to	
  further	
  investigate	
  the	
  role	
  of	
  nitric	
  
oxide	
  and	
  prostanoids	
  in	
  muscarinic-­‐receptor	
  mediated	
  equine	
  pulmonary	
  artery	
  
vasomotion,	
  a	
  subset	
  of	
  small	
  arteries	
  were	
  incubated	
  with	
  L-­‐NAME,	
  a	
  nitric	
  oxide	
  synthase	
  

	
  
94	
  

inhibitor	
  and/or	
  indomethacin,	
  a	
  cyclooxygenase	
  inhibitor,	
  before	
  treatment	
  with	
  
methacholine.	
  	
  
L-­‐NAME	
  did	
  not	
  affect	
  the	
  response	
  of	
  caudodorsal	
  vessels	
  to	
  methacholine,	
  while	
  
indomethacin	
  pre-­‐incubation	
  resulted	
  in	
  augmented	
  contraction.	
  These	
  data	
  indicate	
  that	
  
some	
  prostanoid-­‐mediated	
  dilation	
  was	
  occurring	
  in	
  caudodorsal	
  arteries,	
  but	
  was	
  masked	
  
by	
  the	
  magnitude	
  of	
  the	
  contraction.	
  L-­‐NAME	
  caused	
  attenuation	
  of	
  vasodilation	
  in	
  
cranioventral	
  arteries,	
  as	
  did	
  indomethacin.	
  Co-­‐incubation	
  with	
  both	
  inhibitors	
  prevented	
  
any	
  relaxation	
  of	
  cranioventral	
  vessels	
  until	
  the	
  highest	
  methacholine	
  concentrations	
  were	
  
applied.	
  These	
  data	
  implicate	
  roles	
  for	
  both	
  nitric	
  oxide	
  and	
  prostanoids	
  in	
  cranioventral	
  
artery	
  relaxation.	
  	
  
That	
  nitric	
  oxide	
  contributes	
  to	
  maintenance	
  of	
  basal	
  pulmonary	
  vasomotor	
  tone	
  
has	
  been	
  demonstrated	
  in	
  the	
  horse.	
  Supplemental	
  nitric	
  oxide	
  administration	
  causes	
  a	
  
significant	
  decrease	
  in	
  mean	
  peak	
  pulmonary	
  artery	
  pressure	
  in	
  exercising	
  horses,	
  
suggesting	
  that	
  the	
  pulmonary	
  vasculature	
  is	
  not	
  fully	
  dilated	
  during	
  exercise	
  (100,	
  135).	
  
Furthermore,	
  administration	
  of	
  L-­‐NAME	
  to	
  horses	
  at	
  rest	
  results	
  in	
  significant	
  increases	
  in	
  
pulmonary	
  arterial,	
  capillary	
  and	
  venous	
  pressures	
  (126).	
  Our	
  data	
  demonstrate	
  that	
  nitric	
  
oxide	
  could	
  play	
  a	
  role	
  in	
  small	
  pulmonary	
  artery	
  vasomotion	
  in	
  vivo,	
  at	
  least	
  in	
  the	
  
cranioventral	
  lung,	
  but	
  the	
  effect	
  of	
  these	
  specific	
  arteries	
  on	
  whole	
  lung	
  vascular	
  pressure	
  
data	
  is	
  not	
  known.	
  	
  
Cyclooxygenase	
  inhibitors	
  are	
  commonly	
  used	
  to	
  treat	
  musculoskeletal	
  
abnormalities	
  in	
  performance	
  horses	
  (137).	
  The	
  effect	
  of	
  cyclooxygenase	
  inhibition	
  on	
  
pulmonary	
  vasculature	
  as	
  an	
  off-­‐target	
  effect	
  of	
  non-­‐steroidal	
  anti-­‐inflammatory	
  
medications	
  may	
  merit	
  future	
  consideration.	
  	
  

	
  
95	
  

The	
  loop	
  diuretic	
  furosemide	
  is	
  commonly	
  administered	
  to	
  horses	
  before	
  racing	
  to	
  
control	
  EIPH	
  (193),	
  and	
  has	
  been	
  demonstrated	
  to	
  reduce	
  both	
  the	
  severity	
  and	
  the	
  
incidence	
  of	
  EIPH	
  in	
  Thoroughbreds	
  (74).	
  This	
  protective	
  effect	
  of	
  furosemide	
  is	
  commonly	
  
attributed	
  to	
  a	
  decrease	
  in	
  mean	
  pulmonary	
  artery	
  pressure	
  during	
  strenuous	
  exercise	
  (71,	
  
129)	
  as	
  a	
  result	
  of	
  a	
  diuresis-­‐associated	
  reduction	
  in	
  plasma	
  volume	
  (71).	
  However,	
  
furosemide	
  administration	
  to	
  horses	
  results	
  in	
  redistribution	
  of	
  pulmonary	
  blood	
  flow	
  
without	
  a	
  concomitant	
  drop	
  in	
  cardiac	
  output	
  (43).	
  This	
  information	
  suggests	
  that	
  
furosemide	
  also	
  affects	
  equine	
  pulmonary	
  vascular	
  reactivity.	
  	
  
	
  	
  

Furosemide	
  is	
  a	
  pulmonary	
  venodilator	
  in	
  the	
  dog,	
  but	
  does	
  not	
  dilate	
  pre-­‐

contracted	
  pulmonary	
  arteries	
  (59).	
  Therefore	
  we	
  investigated	
  the	
  hypothesis	
  that	
  
furosemide	
  would	
  dilate	
  pulmonary	
  veins	
  but	
  not	
  pulmonary	
  arteries	
  independent	
  of	
  lung	
  
region.	
  Contrary	
  to	
  our	
  hypothesis,	
  pre-­‐contracted	
  pulmonary	
  arteries	
  relaxed	
  in	
  response	
  
to	
  high	
  concentrations	
  of	
  furosemide	
  in	
  a	
  non-­‐region	
  dependent	
  manner,	
  and	
  mild	
  
relaxation	
  was	
  also	
  observed	
  in	
  veins	
  from	
  both	
  lung	
  regions.	
  The	
  clinical	
  relevance	
  of	
  this	
  
finding	
  must	
  be	
  considered	
  negligible	
  as	
  the	
  concentration	
  at	
  which	
  maximal	
  effects	
  were	
  
observed	
  in	
  the	
  present	
  study	
  is	
  1000	
  times	
  higher	
  than	
  plasma	
  levels	
  of	
  furosemide	
  in	
  
horses	
  one	
  hour	
  after	
  intravenous	
  administration	
  of	
  1	
  mg/kg	
  (24).	
  	
  
It	
  is	
  worth	
  noting	
  however,	
  that	
  the	
  pulmonary	
  veins	
  from	
  dogs	
  in	
  which	
  a	
  dilator	
  
effect	
  was	
  seen	
  were	
  larger	
  (1	
  –	
  1.2	
  mm	
  O.D.)	
  than	
  vessels	
  in	
  the	
  present	
  investigation(59).	
  
Significant	
  differences	
  in	
  reactivity	
  to	
  various	
  pharmacologic	
  agents	
  between	
  vessels	
  of	
  
different	
  size	
  have	
  been	
  reported	
  in	
  many	
  species	
  including	
  rats	
  (108),	
  pigs	
  (239)	
  and	
  
sheep	
  (99).	
  For	
  this	
  reason,	
  the	
  effect	
  of	
  furosemide	
  on	
  equine	
  pulmonary	
  veins	
  of	
  a	
  larger	
  
caliber	
  is	
  considered	
  worthy	
  of	
  future	
  investigation.	
  	
  

	
  
96	
  

Data	
  in	
  this	
  study	
  were	
  acquired	
  from	
  horses	
  that	
  had	
  not	
  raced.	
  Horses	
  that	
  have	
  
trained	
  and	
  raced	
  have	
  remodeled	
  pulmonary	
  veins	
  (233),	
  and	
  racing	
  is	
  associated	
  with	
  
increased	
  wall	
  stiffness	
  of	
  caudodorsal	
  veins	
  (196).	
  It	
  is	
  reported	
  in	
  pigs	
  that	
  remodeling	
  of	
  
pulmonary	
  vessels	
  and	
  associated	
  structural	
  changes	
  are	
  associated	
  with	
  alterations	
  in	
  
vessel	
  reactivity	
  (98).	
  Therefore,	
  remodeled	
  pulmonary	
  veins	
  such	
  as	
  those	
  seen	
  in	
  EIPH-­‐
affected	
  lung	
  (231)	
  may	
  react	
  differently	
  than	
  vessels	
  used	
  in	
  this	
  study.	
  Furthermore,	
  
exercise	
  training	
  has	
  been	
  demonstrated	
  to	
  improve	
  pulmonary	
  artery	
  dilation	
  in	
  response	
  
to	
  acetylcholine	
  (87)	
  in	
  pigs.	
  Future	
  investigations	
  into	
  whether	
  there	
  is	
  an	
  effect	
  of	
  
exercise	
  and	
  associated	
  remodeling	
  on	
  equine	
  pulmonary	
  vascular	
  reactivity	
  may	
  shed	
  
further	
  light	
  on	
  progression	
  of	
  EIPH	
  over	
  the	
  course	
  of	
  a	
  horse’s	
  athletic	
  career.	
  	
  
	
  
	
  
	
  
	
  

	
  
97	
  

	
  

	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
APPENDIX	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
98	
  

Figure	
  9	
  Cumulative	
  concentration	
  response	
  curves	
  for	
  U46619	
  for	
  all	
  arteries	
  and	
  veins	
  
(A),	
  caudodorsal	
  (CD)	
  and	
  cranioventral	
  (CV)	
  arteries	
  (B),	
  and	
  CD	
  and	
  CV	
  veins	
  (C).	
  Values	
  

	
  
99	
  

Figure	
  9	
  (cont’d)	
  are	
  means	
  ±	
  SE.	
  Veins	
  are	
  more	
  sensitive	
  to	
  U	
  46619	
  than	
  arteries	
  (A);	
  
regional	
  differences	
  in	
  responses	
  of	
  CD	
  and	
  CV	
  arteries	
  do	
  not	
  exist	
  (p	
  =	
  0.25)(B)	
  whereas	
  
CV	
  veins	
  are	
  more	
  sensitive	
  to	
  U	
  46619	
  than	
  CD	
  veins	
  	
  (p	
  <	
  0.0001)(C).	
  	
  	
  	
  
	
  

	
  

	
  
100	
  

	
  
	
  

Figure	
  10	
  Cumulative	
  concentration	
  response	
  curves	
  for	
  isoproterenol	
  for	
  caudodorsal	
  
(CD)	
  and	
  cranioventral	
  (CV)	
  arteries	
  (A),	
  and	
  CD	
  and	
  CV	
  veins	
  (B).	
  Values	
  are	
  means	
  ±	
  SE.	
  
Concentration-­‐dependent	
  relaxation	
  is	
  greater	
  in	
  CD	
  compared	
  to	
  CV	
  arteries	
  (p	
  <	
  
0.0001)(A),	
  whereas	
  pre-­‐contracted	
  veins	
  do	
  not	
  relax	
  in	
  response	
  to	
  isoproterenol,	
  in	
  both	
  
CD	
  and	
  CV	
  regions	
  (B).	
  	
  	
  	
  
	
  
	
  
	
  
	
  
	
  

	
  
101	
  

	
  
Figure	
  11	
  Cumulative	
  concentration	
  response	
  curves	
  for	
  furosemide	
  for	
  all	
  arteries	
  and	
  
veins	
  (A),	
  caudodorsal	
  (CD)	
  and	
  cranioventral	
  (CV)	
  arteries	
  (B),	
  and	
  CD	
  and	
  CV	
  veins	
  (C).	
  
Values	
  are	
  means	
  ±	
  SE.	
  Mild	
  concentration-­‐dependent	
  relaxation	
  to	
  furosemide	
  occurs	
  in	
  

	
  
102	
  

Figure	
  11	
  (cont’d)	
  arteries,	
  and	
  to	
  a	
  lesser	
  degree	
  in	
  veins	
  (A);	
  regional	
  differences	
  in	
  the	
  
response	
  of	
  arteries	
  and	
  veins	
  to	
  furosemide	
  do	
  not	
  exist	
  (p	
  =	
  0.07	
  and	
  p	
  =	
  0.19	
  for	
  arteries	
  
and	
  veins	
  respectively)(B	
  and	
  C	
  respectively).	
  DMSO	
  vehicle	
  (dark	
  triangle)	
  does	
  not	
  affect	
  
arteries	
  and	
  veins	
  (B	
  and	
  C	
  respectively).	
  
	
  

	
  

	
  
103	
  

	
  

	
  
	
  
Figure	
  12:	
  Cumulative	
  concentration	
  response	
  curves	
  for	
  methacholine	
  for	
  caudodorsal	
  
(CD)	
  and	
  cranioventral	
  (CV)	
  arteries	
  (A),	
  and	
  CD	
  and	
  CV	
  veins	
  (B).	
  Values	
  are	
  means	
  ±	
  SE.	
  
Concentration-­‐dependent	
  relaxation	
  occurs	
  in	
  CV	
  arteries,	
  and	
  concentration-­‐dependent	
  
constriction	
  occurred	
  in	
  CD	
  arteries	
  (A);	
  pre-­‐contracted	
  pulmonary	
  veins	
  relax	
  in	
  a	
  
concentration-­‐dependent	
  manner,	
  regardless	
  of	
  region	
  (B).	
  	
  	
  	
  
	
  
	
  

	
  
104	
  

	
  
Figure	
  13:	
  Cumulative	
  concentration	
  response	
  curves	
  for	
  CD	
  (A,	
  B,	
  C)	
  and	
  CV	
  (D,	
  E,	
  F)	
  
arteries	
  comparing	
  responses	
  to	
  methacholine	
  (MCh)	
  only,	
  with	
  responses	
  to	
  MCh	
  applied	
  
	
  
105	
  

Figure	
  13	
  (cont’d)	
  after	
  pre-­‐incubation	
  with	
  L-­‐NAME	
  (A	
  and	
  D),	
  indomethacin	
  (B	
  and	
  E),	
  
and	
  L-­‐NAME	
  and	
  indomethacin	
  (C	
  and	
  F).	
  Values	
  are	
  means	
  ±	
  SE.	
  Pre-­‐incubation	
  with	
  L-­‐
NAME	
  does	
  not	
  affect	
  CD	
  artery	
  constriction	
  in	
  response	
  to	
  MCh	
  (A)	
  whereas	
  CV	
  artery	
  
relaxation	
  is	
  partially	
  inhibited	
  by	
  L-­‐NAME	
  (D).	
  Indomethacin	
  pre-­‐incubation	
  augments	
  CD	
  
artery	
  constriction,	
  and	
  partially	
  inhibits	
  CV	
  artery	
  relaxation	
  (B	
  and	
  E	
  respectively).	
  Pre-­‐
incubation	
  with	
  both	
  L-­‐NAME	
  and	
  indomethacin	
  caused	
  enhanced	
  MCh-­‐induced	
  
constriction	
  in	
  CD	
  arteries	
  (C)	
  and	
  a	
  mild	
  contraction	
  followed	
  by	
  mild	
  relaxation	
  in	
  CV	
  
arteries	
  (D).	
  	
  
	
  

	
  

	
  
106	
  

	
  
	
  
Figure	
  14	
  Fluorescent	
  staining	
  of	
  CD-­‐31	
  on	
  endothelial	
  surface	
  of	
  equine	
  pulmonary	
  
artery.	
  	
  Regions	
  of	
  intact	
  endothelium	
  can	
  be	
  discerned	
  from	
  endothelium-­‐denuded,	
  non-­‐
stained	
  regions	
  (indicated	
  by	
  arrow-­‐heads).	
  Scale	
  bar	
  =	
  100	
  μm.	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
107	
  

CHAPTER	
  4	
  
	
  
Effects	
  of	
  exercise	
  on	
  markers	
  of	
  venous	
  remodeling	
  in	
  lungs	
  of	
  horses	
  
	
  
Alice	
  Stack,	
  Frederik	
  J.	
  Derksen,	
  Lorraine	
  M.	
  Sordillo,	
  Kurt	
  J.	
  Williams,	
  John	
  A.	
  Stick,	
  
Christina	
  Brandenberger,	
  Juan	
  P.	
  Steibel,	
  and	
  N.	
  Edward	
  Robinson.	
  
	
  
Am	
  J	
  Vet	
  Res.	
  2013	
  Sep;74(9):1231-­‐8.	
  doi:	
  10.2460/ajvr.74.9.1231.	
  
	
  
Abstract	
  
Objective:	
  To	
  determine	
  the	
  effects	
  of	
  2	
  weeks	
  of	
  intense	
  exercise	
  on	
  expression	
  of	
  markers	
  
of	
  pulmonary	
  venous	
  remodeling	
  in	
  caudodorsal	
  and	
  cranioventral	
  regions	
  of	
  lungs	
  of	
  
horses.	
  	
  
Animals:	
  6	
  horses.	
  
Procedures:	
  Tissue	
  samples	
  of	
  caudodorsal	
  and	
  cranioventral	
  regions	
  of	
  lungs	
  were	
  
obtained	
  before	
  and	
  after	
  conditioning	
  and	
  2	
  weeks	
  of	
  intense	
  exercise.	
  Pulmonary	
  veins	
  
were	
  isolated	
  and	
  assayed	
  via	
  quantitative	
  real-­‐time	
  PCR	
  to	
  determine	
  mRNA	
  expression	
  of	
  
matrix	
  metalloproteinase-­‐2	
  and	
  -­‐9,	
  tissue	
  inhibitor	
  of	
  metalloproteinase-­‐1	
  and	
  -­‐2,	
  collagen	
  
type	
  I,	
  tenascin-­‐C,	
  endothelin-­‐1,	
  platelet	
  derived	
  growth	
  factor,	
  transforming	
  growth	
  factor-­‐
β	
  (TGF-­‐β),	
  and	
  vascular	
  endothelial	
  growth	
  factor	
  (VEGF).	
  Protein	
  expression	
  of	
  collagen	
  
(via	
  morphometric	
  analysis)	
  and	
  tenascin-­‐C,	
  TGF-­‐β,	
  and	
  VEGF	
  (via	
  immunohistochemistry)	
  
was	
  determined.	
  	
  
Results:	
  Exercise-­‐induced	
  pulmonary	
  hemorrhage	
  was	
  detected	
  in	
  33.3%	
  of	
  horses	
  after	
  
exercise.	
  The	
  mRNA	
  expression	
  of	
  matrix	
  metalloproteinase-­‐2	
  and	
  -­‐9,	
  tissue	
  inhibitor	
  of	
  
metalloproteinase-­‐2,	
  TGF-­‐β,	
  and	
  VEGF	
  was	
  significantly	
  lower	
  in	
  pulmonary	
  veins	
  obtained	
  

	
  
108	
  

after	
  exercise	
  versus	
  those	
  obtained	
  before	
  exercise	
  for	
  both	
  caudodorsal	
  and	
  
cranioventral	
  regions	
  of	
  lungs.	
  Collagen	
  content	
  was	
  significantly	
  higher	
  in	
  tissue	
  samples	
  
obtained	
  from	
  caudodorsal	
  regions	
  of	
  lungs	
  versus	
  those	
  obtained	
  from	
  cranioventral	
  
regions	
  of	
  lungs	
  both	
  before	
  and	
  after	
  exercise.	
  Exercise	
  did	
  not	
  alter	
  protein	
  expression	
  of	
  
tenascin-­‐C,	
  TGF-­‐β,	
  or	
  VEGF.	
  	
  
Conclusions	
  and	
  Clinical	
  Relevance:	
  Results	
  of	
  this	
  study	
  indicated	
  2	
  weeks	
  of	
  intense	
  
exercise	
  did	
  not	
  alter	
  expression	
  of	
  marker	
  genes	
  in	
  a	
  manner	
  expected	
  to	
  favor	
  venous	
  
remodeling.	
  Pulmonary	
  venous	
  remodeling	
  is	
  complex	
  and	
  more	
  than	
  2	
  weeks	
  of	
  intense	
  
exercise	
  may	
  be	
  required	
  to	
  induce	
  such	
  remodeling.	
  	
  
	
  
	
  	
  
Introduction	
  
Exercise-­‐induced	
  pulmonary	
  hemorrhage	
  is	
  common	
  in	
  racehorses	
  after	
  intense	
  exercise;	
  
EIPH	
  is	
  detected	
  in	
  up	
  to	
  75%	
  of	
  such	
  horses	
  via	
  endoscopic	
  evaluation	
  of	
  respiratory	
  
tracts	
  (162,	
  172).	
  Horses	
  with	
  no	
  or	
  very	
  mild	
  EIPH	
  are	
  four	
  times	
  as	
  likely	
  to	
  win	
  a	
  race	
  as	
  
horses	
  with	
  moderate	
  or	
  severe	
  EIPH	
  (70),	
  suggesting	
  this	
  condition	
  has	
  negative	
  effects	
  on	
  
racehorse	
  performance.	
  
The	
  predominant	
  location	
  of	
  EIPH	
  lesions	
  in	
  horses	
  is	
  the	
  caudodorsal	
  regions	
  of	
  
lungs	
  (150,	
  157,	
  231).	
  A	
  distinctive	
  histopathologic	
  lesion	
  of	
  EIPH	
  is	
  remodeling	
  of	
  small-­‐
diameter	
  pulmonary	
  veins	
  (venous	
  remodeling)	
  (231).	
  Venous	
  remodeling	
  is	
  characterized	
  
by	
  collagen	
  deposition	
  in	
  walls	
  and	
  smooth	
  muscle	
  hypertrophy	
  of	
  veins	
  resulting	
  in	
  
thickening	
  of	
  walls	
  and	
  narrowing	
  of	
  lumens	
  (38).	
  Other	
  lesions	
  of	
  EIPH	
  include	
  pulmonary	
  

	
  
109	
  

interstitial	
  and	
  septal	
  fibrosis,	
  hemosiderin	
  accumulation	
  in	
  lung	
  tissue,	
  and	
  bronchial	
  
circulation	
  neovascularization(38,	
  152).	
  
Pulmonary	
  venous	
  remodeling	
  has	
  potentially	
  important	
  physiologic	
  effects	
  on	
  
vascular	
  pressures	
  in	
  lungs.	
  During	
  exercise,	
  horses	
  have	
  a	
  substantial	
  increase	
  in	
  
pulmonary	
  intravascular	
  pressures	
  (106,	
  127).	
  Estimated	
  pulmonary	
  capillary	
  pressures	
  in	
  
horses	
  are	
  between	
  17.8	
  mmHg	
  (190)	
  to	
  25	
  mmHg	
  (127)	
  at	
  rest	
  and	
  72.5	
  mmHg	
  (106)	
  to	
  
83.3	
  mmHg	
  (127)	
  during	
  exercise.	
  Such	
  transmural	
  pulmonary	
  capillary	
  pressures	
  can	
  
cause	
  blood	
  vessel	
  rupture	
  and	
  EIPH	
  (17).	
  A	
  decrease	
  in	
  the	
  lumen	
  size	
  of	
  pulmonary	
  veins	
  
would	
  further	
  increase	
  pulmonary	
  capillary	
  pressures.	
  Complete	
  pulmonary	
  venous	
  
occlusion	
  would	
  cause	
  capillary	
  pressures	
  equal	
  to	
  pulmonary	
  arterial	
  pressures,	
  which	
  
can	
  be	
  ˃	
  96.5	
  mmHg	
  (106,	
  127).	
  Remodeling	
  of	
  systemic	
  (in	
  rabbits,	
  rodents,	
  and	
  pigs)(2,	
  
28,	
  29,	
  64,	
  112,	
  235)	
  and	
  pulmonary	
  (in	
  humans	
  and	
  sheep)(25,	
  86)veins	
  can	
  develop	
  
when	
  such	
  blood	
  vessels	
  are	
  exposed	
  to	
  high	
  intravascular	
  pressures.	
  
	
  	
  

Results	
  of	
  studies	
  of	
  vasculature	
  in	
  humans	
  (146,	
  163,	
  222),	
  pigs,(29,	
  224)	
  and	
  

rodents(225,	
  234)	
  indicate	
  venous	
  remodeling	
  is	
  preceded	
  by	
  alterations	
  in	
  mRNA	
  
expression	
  of	
  proteins	
  that	
  are	
  important	
  in	
  the	
  remodeling	
  process.	
  These	
  proteins	
  
include	
  MMPs,	
  TIMPs	
  (29,	
  225),	
  collagen	
  (234),	
  tenascin-­‐C	
  (222),	
  and	
  various	
  growth	
  
factors	
  that	
  are	
  produced	
  by	
  fibroblasts	
  in	
  vein	
  walls	
  and	
  monocytes	
  and	
  macrophages	
  
(146,	
  163,	
  199,	
  224,	
  234).	
  The	
  objective	
  of	
  the	
  study	
  reported	
  here	
  was	
  to	
  determine	
  mRNA	
  
expression	
  of	
  MMP-­‐2,	
  MMP-­‐9,	
  TIMP-­‐1,	
  TIMP-­‐2,	
  collagen	
  type	
  I,	
  tenascin-­‐C,	
  ET-­‐1,	
  PDGF,	
  
TGF-­‐β,	
  and	
  VEGF	
  and	
  protein	
  expression	
  of	
  tenascin-­‐C,	
  TGF-­‐β,	
  and	
  VEGF	
  in	
  pulmonary	
  
veins	
  obtained	
  from	
  caudodorsal	
  and	
  cranioventral	
  regions	
  of	
  lungs	
  of	
  horses	
  before	
  and	
  
after	
  2	
  weeks	
  of	
  intense	
  exercise.	
  Because	
  the	
  amount	
  of	
  collagen	
  in	
  lung	
  parenchyma	
  of	
  

	
  
110	
  

horses	
  with	
  EIPH	
  is	
  greater	
  than	
  that	
  for	
  horses	
  without	
  EIPH	
  (primarily	
  in	
  the	
  
caudodorsal	
  regions	
  of	
  lungs(38)),	
  we	
  also	
  compared	
  collagen	
  content	
  in	
  parenchyma	
  of	
  
caudodorsal	
  and	
  cranioventral	
  regions	
  of	
  lungs	
  of	
  horses	
  before	
  and	
  after	
  2	
  weeks	
  of	
  
intense	
  exercise.	
  The	
  hypothesis	
  was	
  that	
  2	
  weeks	
  of	
  intense	
  exercise	
  would	
  alter	
  mRNA	
  
and	
  protein	
  expression	
  of	
  the	
  evaluated	
  factors	
  in	
  pulmonary	
  veins	
  of	
  caudodorsal	
  but	
  not	
  
cranioventral	
  regions	
  of	
  lungs	
  of	
  horses	
  in	
  a	
  manner	
  expected	
  to	
  favor	
  vascular	
  remodeling.	
  
In	
  addition,	
  we	
  hypothesized	
  that	
  exercise	
  of	
  horses	
  would	
  cause	
  an	
  increase	
  in	
  the	
  
collagen	
  content	
  of	
  caudodorsal	
  but	
  not	
  cranioventral	
  regions	
  of	
  lungs.9	
  
	
  
Materials	
  and	
  Methods	
  	
  	
  
Animals	
  
Seven	
  horses	
  (six	
  geldings	
  and	
  one	
  sexually	
  intact	
  female;	
  age	
  range,	
  2	
  to	
  4	
  years;	
  body	
  
weight	
  range,	
  350	
  to	
  473	
  kg)	
  of	
  non-­‐racing	
  breeds	
  were	
  purchased	
  for	
  use	
  in	
  this	
  study.	
  
These	
  horses	
  had	
  not	
  been	
  previously	
  trained	
  for	
  any	
  purpose	
  and	
  were	
  selected	
  for	
  
inclusion	
  in	
  the	
  study	
  because	
  it	
  was	
  unlikely	
  that	
  they	
  had	
  prior	
  EIPH	
  episodes.	
  Horses	
  
were	
  not	
  vigorously	
  exercised	
  for	
  at	
  least	
  two	
  months	
  before	
  the	
  study.	
  The	
  horses	
  were	
  
determined	
  to	
  be	
  healthy	
  on	
  the	
  basis	
  of	
  results	
  of	
  physical	
  examinations	
  and	
  
tracheobronchoendoscopy.	
  One	
  horse	
  was	
  excluded	
  from	
  the	
  study	
  because	
  of	
  lameness.	
  
Therefore,	
  the	
  study	
  was	
  completed	
  and	
  data	
  were	
  analyzed	
  for	
  6	
  horses.	
  The	
  Michigan	
  
State	
  University	
  Institutional	
  Animal	
  Care	
  and	
  Use	
  Committee	
  approved	
  this	
  study.	
  
	
  
Experimental	
  protocol	
  

	
  
111	
  

Pulmonary	
  wedge	
  resections	
  were	
  performed	
  via	
  a	
  thoracoscopic	
  technique	
  for	
  standing	
  
horses.	
  Before	
  undergoing	
  an	
  intense	
  exercise	
  protocol,	
  lung	
  samples	
  were	
  obtained	
  from	
  
cranioventral	
  and	
  caudodorsal	
  regions	
  of	
  left	
  or	
  right	
  lungs	
  (determined	
  via	
  a	
  
randomization	
  procedure)	
  of	
  each	
  horse.	
  Horses	
  were	
  then	
  returned	
  to	
  pasture	
  for	
  at	
  least	
  
6	
  months.	
  Subsequently,	
  horses	
  underwent	
  conditioning	
  and	
  intense	
  exercise	
  during	
  a	
  4-­‐
week	
  period.	
  After	
  completion	
  of	
  the	
  intense	
  exercise	
  protocol	
  (first	
  exercise	
  period),	
  
pulmonary	
  wedge	
  resections	
  were	
  performed	
  to	
  obtain	
  lung	
  samples	
  from	
  cranioventral	
  
and	
  caudodorsal	
  regions	
  of	
  right	
  or	
  left	
  of	
  horses	
  (lung	
  contralateral	
  to	
  the	
  lung	
  from	
  which	
  
samples	
  were	
  obtained	
  before	
  exercise);	
  the	
  mRNA	
  prepared	
  from	
  these	
  lung	
  samples	
  was	
  
of	
  poor	
  quality	
  and	
  low	
  quantity.	
  Therefore,	
  horses	
  were	
  rested	
  for	
  a	
  further	
  6	
  months	
  and	
  
the	
  exercise	
  protocol	
  was	
  repeated	
  (second	
  exercise	
  period).	
  Subsequently,	
  tissue	
  samples	
  
from	
  the	
  cranioventral	
  and	
  caudodorsal	
  regions	
  of	
  the	
  same	
  lung	
  (contralateral	
  to	
  the	
  lung	
  
from	
  which	
  samples	
  were	
  obtained	
  before	
  the	
  first	
  exercise	
  protocol)	
  were	
  collected	
  
during	
  general	
  anesthesia	
  of	
  horses.	
  Lung	
  samples	
  were	
  obtained	
  from	
  sites	
  that	
  had	
  not	
  
previously	
  undergone	
  surgery.	
  A	
  long	
  time	
  was	
  allowed	
  between	
  pulmonary	
  wedge	
  
resection	
  procedures	
  to	
  minimize	
  the	
  effects	
  of	
  previous	
  surgeries	
  on	
  gene	
  expression.	
  	
  
	
  
Exercise	
  protocol	
  
Horses	
  underwent	
  a	
  2-­‐week	
  period	
  of	
  conditioning	
  followed	
  by	
  a	
  2-­‐week	
  period	
  of	
  intense	
  
exercise	
  intended	
  to	
  simulate	
  race	
  training.	
  Horses	
  were	
  conditioned	
  5	
  days/week	
  for	
  2	
  
weeks	
  on	
  a	
  high-­‐speed	
  treadmill	
  with	
  a	
  0%	
  incline.	
  After	
  two	
  weeks	
  of	
  conditioning,	
  the	
  
HRmax	
  of	
  each	
  horse	
  was	
  determined	
  via	
  a	
  rapid	
  incremental	
  exercise	
  test	
  (206).	
  Briefly,	
  
heart	
  rates	
  were	
  determined	
  by	
  use	
  of	
  a	
  telemetric	
  system;	
  the	
  HRmax	
  was	
  determined	
  to	
  be	
  

	
  
112	
  

the	
  heart	
  rate	
  at	
  which	
  an	
  increase	
  in	
  treadmill	
  speed	
  did	
  not	
  result	
  in	
  an	
  increase	
  in	
  HR.	
  
The	
  treadmill	
  speed	
  corresponding	
  to	
  120%	
  of	
  HRmax	
  was	
  determined	
  via	
  extrapolation.	
  
	
  
After	
  the	
  2-­‐week	
  conditioning	
  period	
  horses	
  were	
  intensely	
  exercised	
  on	
  6	
  days	
  (intense	
  
exercise	
  days	
  1,	
  3,	
  5,	
  8,	
  10	
  and	
  12).	
  Each	
  exercise	
  session	
  included	
  a	
  4-­‐minute	
  warm-­‐up	
  
period	
  followed	
  by	
  exercise	
  at	
  a	
  treadmill	
  speed	
  corresponding	
  to	
  120%	
  of	
  HRmax	
  for	
  2	
  
minutes	
  or	
  until	
  the	
  horse	
  could	
  no	
  longer	
  maintain	
  its	
  position	
  on	
  the	
  treadmill.	
  Within	
  45	
  
to	
  90	
  minutes	
  after	
  the	
  end	
  of	
  the	
  final	
  exercise	
  session	
  of	
  the	
  first	
  exercise	
  period,	
  horses	
  
underwent	
  endoscopic	
  examination	
  of	
  the	
  trachea.	
  Endoscopic	
  examination	
  of	
  horses	
  was	
  
not	
  repeated	
  after	
  the	
  second	
  exercise	
  period	
  because	
  the	
  intensity	
  of	
  exercise	
  during	
  the	
  
first	
  period	
  was	
  determined	
  to	
  have	
  been	
  adequate	
  to	
  induce	
  EIPH.	
  An	
  established	
  grading	
  
system	
  (grade	
  0	
  =	
  no	
  blood	
  visible	
  in	
  trachea;	
  grade	
  4	
  =	
  >90%	
  of	
  tracheal	
  surface	
  covered	
  
in	
  blood)(69)	
  was	
  used	
  to	
  determine	
  EIPH	
  severity	
  in	
  study	
  horses.	
  
	
  
Pulmonary	
  wedge	
  resection	
  
During	
  each	
  pulmonary	
  wedge	
  resection	
  procedure,	
  2	
  lung	
  samples	
  were	
  obtained	
  from	
  
each	
  horse	
  (one	
  each	
  from	
  the	
  cranioventral	
  and	
  caudodorsal	
  regions	
  of	
  the	
  left	
  or	
  right	
  
lung)	
  (115).	
  Briefly,	
  each	
  horse	
  was	
  restrained	
  in	
  stocks	
  and	
  sedated	
  with	
  a	
  continuous	
  IV	
  
infusion	
  of	
  detomidine	
  hydrochloride	
  (initial	
  dose	
  of	
  6	
  µg/kg	
  followed	
  by	
  0.8	
  µg/kg/min).	
  
Mepivacaine	
  (20	
  to	
  30	
  ml	
  of	
  a	
  2%	
  solution)	
  was	
  injected	
  SC	
  and	
  in	
  intercostal	
  muscles	
  at	
  
each	
  surgery	
  site.	
  Intercostal	
  nerves	
  at	
  surgery	
  sites	
  were	
  blocked	
  at	
  the	
  level	
  of	
  vertebral	
  
transverse	
  processes	
  with	
  0.75%	
  bupivacaine	
  (5	
  mL/site).	
  Antimicrobial	
  drugs	
  (penicillin	
  
G	
  potassium	
  [22,000	
  IU/kg,	
  IV,	
  q	
  6	
  h]	
  and	
  gentamicin	
  sulfate	
  [6.6	
  mg/kg,	
  IV,	
  q	
  24h])	
  and	
  an	
  

	
  
113	
  

NSAID	
  (flunixin	
  meglumine	
  [1.1	
  mg/kg,	
  IV,	
  q	
  12	
  h])	
  were	
  administered	
  during	
  surgery	
  after	
  
lung	
  samples	
  had	
  been	
  obtained	
  (to	
  avoid	
  potential	
  effects	
  of	
  drugs	
  on	
  gene	
  expression).	
  
	
  	
  

For	
  thoracoscopy,	
  a	
  30-­‐degree	
  rigid	
  endoscope	
  (10	
  mm	
  x	
  58	
  cm)(	
  Hopkins	
  

telescope,	
  Karl	
  Storz	
  Veterinary	
  Endoscopy,	
  Goleta,	
  CA.),	
  video	
  camera	
  (Vetcam,	
  Karl	
  Storz	
  
Veterinary	
  Endoscopy,	
  Goleta,	
  CA),	
  light	
  cable,	
  and	
  250-­‐watt	
  xenon	
  light	
  source	
  (Stryker	
  
Quantum	
  3000,	
  Stryker	
  Endoscopy,	
  Kalamazoo,	
  MI)	
  were	
  used.	
  Pneumothorax	
  was	
  induced	
  
and	
  lungs	
  were	
  deflated	
  via	
  insertion	
  of	
  a	
  teat	
  cannula	
  into	
  the	
  pleural	
  space.	
  
	
  	
  	
  

Six	
  instrument	
  portals	
  were	
  made	
  in	
  the	
  thoracic	
  wall	
  (3	
  for	
  each	
  lung	
  sample	
  

collection	
  site	
  [one	
  each	
  for	
  an	
  endoscope,	
  forceps,	
  and	
  stapler]).	
  The	
  caudodorsal	
  lung	
  
sample	
  collection	
  site	
  was	
  accessed	
  via	
  intercostal	
  spaces	
  12,	
  13	
  and	
  15;	
  the	
  cranioventral	
  
site	
  was	
  accessed	
  via	
  intercostal	
  spaces	
  7	
  and	
  8.	
  Endoscopic	
  atraumatic	
  forceps	
  (10	
  mm	
  
atraumatic	
  Babcock	
  forceps,	
  Ethicon	
  Endo-­‐Surgery	
  Inc,	
  Cincinnati,	
  OH)	
  were	
  used	
  to	
  
manipulate	
  lungs.	
  	
  
An	
  endoscopic	
  stapler	
  (ETS45	
  Endoscopic	
  linear	
  cutter,	
  Ethicon	
  Endo-­‐Surgery	
  Inc,	
  
Cincinnati,	
  OH)	
  was	
  used	
  to	
  perform	
  pulmonary	
  wedge	
  resections.	
  Lung	
  samples	
  (approx	
  4	
  
cm	
  long)	
  were	
  obtained	
  from	
  each	
  site.	
  Lungs	
  were	
  reinflated	
  by	
  withdrawing	
  air	
  from	
  the	
  
thorax	
  and	
  skin	
  at	
  portal	
  sites	
  was	
  closed	
  with	
  sutures	
  in	
  a	
  simple	
  interrupted	
  pattern.	
  
Antimicrobial	
  and	
  NSAID	
  administration	
  was	
  continued	
  for	
  7	
  days	
  after	
  surgery.	
  	
  
	
  	
  

Pulmonary	
  wedge	
  resections	
  were	
  performed	
  within	
  24	
  hours	
  after	
  completion	
  of	
  

the	
  first	
  exercise	
  period	
  to	
  collect	
  lung	
  samples	
  from	
  the	
  lung	
  contralateral	
  to	
  the	
  lung	
  from	
  
which	
  tissue	
  samples	
  had	
  been	
  obtained	
  before	
  exercise.	
  Within	
  24	
  hours	
  after	
  completion	
  
of	
  the	
  second	
  exercise	
  period,	
  each	
  horse	
  was	
  anesthetized	
  (xylazine	
  hydrochloride	
  [1.1	
  
mg/kg,	
  IV]	
  followed	
  by	
  ketamine	
  hydrochloride	
  [2.2mg/kg,	
  IV])	
  and	
  placed	
  in	
  left	
  or	
  right	
  

	
  
114	
  

lateral	
  recumbency.	
  Lung	
  samples	
  were	
  obtained	
  via	
  thoracotomy	
  and	
  previous	
  surgery	
  
sites	
  were	
  avoided.	
  Immediately	
  after	
  lung	
  samples	
  were	
  obtained,	
  anesthetized	
  horses	
  
were	
  euthanized	
  with	
  pentobarbital	
  sodium	
  (90mg/kg,	
  IV).	
  
	
  
Harvesting	
  of	
  pulmonary	
  veins	
  
Immediately	
  after	
  collection,	
  lung	
  samples	
  were	
  divided	
  into	
  2	
  approximately	
  equal	
  pieces;	
  
one	
  was	
  placed	
  in	
  a	
  storage	
  solution	
  (RNAlater,	
  Ambion,	
  Life	
  Technologies,	
  Carlsbad	
  CA)	
  
and	
  kept	
  at	
  4°C	
  for	
  24	
  hours,	
  and	
  then	
  stored	
  until	
  use	
  at	
  -­‐	
  20°C.	
  The	
  other	
  piece	
  of	
  each	
  
lung	
  sample	
  was	
  fixed	
  in	
  10%	
  neutral	
  buffered	
  formalin	
  and	
  embedded	
  in	
  paraffin	
  for	
  
histologic	
  examination	
  and	
  morphometric	
  and	
  immunohistochemical	
  analyses;	
  6	
  µm-­‐thick	
  
sections	
  of	
  lung	
  tissue	
  were	
  placed	
  on	
  glass	
  slides	
  and	
  stained	
  with	
  H&E,	
  picrosirius	
  red,	
  
and	
  Verhoeff-­‐Van	
  Gieson	
  stains.	
  
For	
  lung	
  samples	
  in	
  storage	
  solution	
  (RNAlater),	
  intralobular	
  pulmonary	
  veins	
  
(length,	
  0.5	
  to	
  3	
  mm)	
  were	
  collected	
  by	
  use	
  of	
  a	
  dissecting	
  microscope	
  (Olympus	
  SZX16,	
  
Olympus	
  America	
  Inc,	
  Center	
  Valley,	
  PA).	
  During	
  preliminary	
  studies,	
  accurate	
  
identification	
  and	
  dissection	
  of	
  pulmonary	
  veins	
  from	
  peripheral	
  lung	
  tissue	
  had	
  been	
  
validated	
  via	
  histologic	
  techniques.	
  For	
  each	
  horse,	
  all	
  veins	
  harvested	
  for	
  each	
  lung	
  
collection	
  site	
  and	
  time	
  were	
  pooled	
  for	
  mRNA	
  extraction.	
  	
  	
  
	
  
mRNA	
  extraction	
  
Pulmonary	
  vein	
  samples	
  were	
  removed	
  from	
  storage	
  solutionf	
  and	
  placed	
  in	
  400	
  µL	
  of	
  lysis	
  
buffer	
  (Buffer	
  RLT,	
  Qiagen	
  Inc,	
  Valencia,	
  CA)(containing	
  β-­‐mercaptoethanol).	
  Pulmonary	
  
vein	
  samples	
  were	
  processed	
  with	
  a	
  tissue	
  grinder	
  (Kontes	
  Glass	
  Co	
  Duall	
  21,	
  Fischer	
  

	
  
115	
  

Scientific,	
  Pittsburgh,	
  PA).	
  Total	
  RNA	
  was	
  extracted	
  with	
  a	
  kit	
  (RNeasy	
  Micro	
  Kit,	
  Qiagen	
  
Inc,	
  Valencia,	
  CA)	
  and	
  a	
  homogenizer	
  (QIAshredder,	
  Qiagen	
  Inc,	
  Valencia,	
  CA);	
  DNase	
  
digestion	
  (RNase-­‐Free	
  DNase	
  Set,	
  Qiagen	
  Inc,	
  Valencia,	
  CA)	
  was	
  used	
  in	
  conjunction	
  with	
  
RNA	
  extraction	
  in	
  an	
  attempt	
  to	
  remove	
  genomic	
  DNA.	
  	
  
The	
  purity	
  and	
  concentration	
  of	
  RNA	
  in	
  each	
  sample	
  were	
  determined	
  with	
  a	
  
spectrophotometer	
  (NanoDrop	
  1000	
  Spectrophotometer,	
  NanoDrop	
  Products,	
  
Wilimington,	
  DE).	
  In	
  addition,	
  RNA	
  integrity	
  number	
  (183)	
  was	
  determined	
  by	
  use	
  of	
  a	
  
bioanalzyer	
  system	
  (2100	
  Bioanalyzer	
  with	
  RNA	
  Pico	
  6000	
  kit,	
  Aligent	
  Technologies,	
  Santa	
  
Clara,	
  CA).	
  To	
  ensure	
  adequate	
  purity	
  and	
  concentration	
  of	
  mRNA,	
  only	
  samples	
  with	
  260	
  
nm-­‐to-­‐280	
  nm	
  absorbance	
  ratios	
  between	
  1.9	
  and	
  2.2	
  were	
  used.	
  In	
  addition,	
  only	
  mRNA	
  
samples	
  with	
  an	
  RNA	
  Integrity	
  Number	
  ˃	
  5	
  were	
  used	
  (47).	
  	
  
As	
  a	
  result	
  of	
  these	
  criteria,	
  all	
  samples	
  obtained	
  after	
  the	
  first	
  exercise	
  period	
  and	
  
samples	
  obtained	
  from	
  2	
  horses	
  after	
  the	
  second	
  period	
  were	
  not	
  assayed.	
  Therefore,	
  
mRNA	
  samples	
  for	
  4	
  horses	
  prepared	
  from	
  lung	
  samples	
  obtained	
  after	
  the	
  second	
  exercise	
  
period	
  were	
  assayed.	
  Both	
  horses	
  with	
  EIPH	
  (endoscopic	
  diagnosis)	
  were	
  included	
  in	
  the	
  
final	
  analysis.	
  	
  
Then,	
  cDNA	
  was	
  synthesized	
  (High	
  Capacity	
  cDNA	
  Reverse	
  Transcription	
  Kit	
  with	
  
RANse	
  inhibitor,	
  Applied	
  Biosystems,	
  Life	
  Technologies,	
  Carlsbad,	
  CA)	
  	
  and	
  amplified	
  
(TaqMan	
  PreAmp	
  Master	
  Mix,	
  Applied	
  Biosystems,	
  Life	
  Technologies,	
  Carlsbad,	
  CA)	
  
because	
  of	
  low	
  cDNA	
  concentrations.	
  	
  	
  
	
  
Quantitative	
  real-­‐time	
  PCR	
  assays	
  

	
  
116	
  

The	
  qRT-­‐PCR	
  assays	
  were	
  performed	
  with	
  a	
  PCR	
  system	
  (7500	
  Fast	
  Real-­‐Time	
  PCR	
  system,	
  
Biosystems,	
  Life	
  Technologies,	
  Carlsbad,	
  CA)	
  operating	
  in	
  standard	
  mode	
  with	
  custom-­‐
designed	
  probes	
  (Table	
  2)(Informatics	
  pipeline	
  software,	
  Applied	
  Biosystems,	
  Life	
  
Technologies,	
  Carlsbad,	
  CA).	
  	
  
The	
  primer	
  design	
  variables	
  for	
  each	
  gene	
  were	
  tested	
  extensively,	
  resulting	
  in	
  
100%	
  PCR	
  efficiency	
  of	
  a	
  6-­‐log	
  dilution	
  range	
  for	
  mRNA	
  samples	
  free	
  of	
  PCR	
  inhibitors.	
  The	
  
qRT-­‐PCR	
  reactions	
  were	
  performed	
  in	
  triplicate	
  with	
  a	
  20	
  µL	
  reaction	
  mixture	
  for	
  each	
  
reaction	
  well;	
  reaction	
  mixtures	
  contained	
  10	
  µL	
  of	
  a	
  DNA	
  polymerase	
  and	
  dNTP	
  mixture	
  
(TaqMan	
  Gene	
  Expression	
  Master	
  Mix,	
  Applied	
  Biosystems,	
  Life	
  Technologies,	
  Carlsbad,	
  
CA),	
  1	
  µL	
  of	
  a	
  mixture	
  of	
  forward	
  and	
  reverse	
  primers	
  and	
  custom-­‐designed	
  probes	
  
(Custom	
  TaqMan	
  Gene	
  Expression	
  Assay	
  Mix,	
  Applied	
  Biosystems,	
  Life	
  Technologies,	
  
Carlsbad,	
  CA),	
  5	
  µL	
  of	
  amplified	
  cDNA,	
  and	
  4	
  µL	
  of	
  nuclease-­‐free	
  water.	
  	
  Expression	
  of	
  
MMP-­‐2,	
  MMP-­‐9,	
  TIMP-­‐1,	
  TIMP-­‐2,	
  collagen	
  type	
  I,	
  tenascin-­‐C,	
  ET-­‐1,	
  PDGF,	
  TGF-­‐β	
  and	
  VEGF	
  
were	
  determined	
  via	
  qRT-­‐PCR	
  assays.	
  	
  
The	
  qRT-­‐PCR	
  assays	
  were	
  performed	
  at	
  50o	
  C	
  for	
  2	
  minutes,	
  95o	
  C	
  for	
  10	
  minutes,	
  
and	
  40	
  cycles	
  of	
  95o	
  C	
  for	
  15	
  seconds	
  and	
  60o	
  C	
  for	
  1	
  minute.	
  	
  The	
  endogenous	
  control	
  
values	
  for	
  the	
  RT-­‐PCR	
  assay	
  were	
  mean	
  values	
  of	
  beta-­‐actin,	
  beta-­‐2-­‐microglobulin,	
  and	
  
elongation	
  factor-­‐1alpha	
  expression.	
  	
  
Fold	
  changes	
  in	
  gene	
  expression	
  were	
  calculated	
  via	
  the	
  2-­‐ΔΔCT	
  method	
  (113).	
  
Statistical	
  analyses	
  were	
  performed	
  with	
  ΔCT	
  values;	
  for	
  each	
  mRNA	
  sample	
  and	
  gene	
  of	
  
interest,	
  the	
  ΔCT	
  values	
  were	
  defined	
  as	
  the	
  mean	
  CT	
  (cycle	
  threshold)	
  value	
  of	
  the	
  gene	
  in	
  
a	
  sample	
  minus	
  the	
  mean	
  CT	
  value	
  of	
  the	
  control	
  genes	
  in	
  that	
  same	
  sample.	
  
	
  

	
  
117	
  

Immunohistochemistry	
  
The	
  6	
  µm-­‐thick	
  lung	
  tissue	
  sections	
  were	
  deparaffinized	
  in	
  xylene	
  and	
  rehydrated	
  in	
  a	
  
graded	
  series	
  of	
  concentrations	
  of	
  ethanol.	
  Lung	
  sections	
  were	
  incubated	
  overnight	
  at	
  4°C	
  
with	
  antibodies	
  against	
  tenascin-­‐C	
  (1:100)	
  (Tenascin-­‐C	
  (BC-­‐24):	
  sc-­‐59884,	
  SantaCruz	
  
Biotechnology,	
  Santa	
  Cruz,	
  CA),	
  TGF-­‐β	
  (1:100)	
  (TGF-­‐β	
  (V):	
  sc-­‐146,	
  and	
  blocking	
  peptide,),	
  
or	
  VEGF	
  (1:100)(VEGF	
  (147):	
  sc-­‐507,	
  SantaCruz	
  Biotechnology,	
  Santa	
  Cruz	
  CA).	
  	
  
To	
  ensure	
  antibody-­‐binding	
  specificity,	
  a	
  peptide	
  blocking	
  (sc-­‐146	
  and	
  sc-­‐507	
  
blocking	
  peptides,	
  SantaCruz	
  Biotechnology,	
  Santa	
  Cruz,	
  CA)	
  step	
  was	
  used	
  for	
  antibodies	
  
against	
  TGF-­‐β	
  and	
  VEGF,	
  and	
  nonspecific	
  rabbit	
  IgG	
  was	
  used	
  for	
  antibodies	
  against	
  
tenascin-­‐C.	
  For	
  each	
  antibody,	
  an	
  appropriate	
  positive	
  control	
  tissue	
  was	
  analyzed.	
  
Following	
  incubation	
  with	
  primary	
  antibodies,	
  lung	
  sections	
  were	
  incubated	
  with	
  
rabbit	
  (TGF-­‐β	
  and	
  VEGF)	
  or	
  mouse	
  (tenascin-­‐C)	
  biotinylated	
  secondary	
  antibody.	
  Then,	
  
slides	
  were	
  incubated	
  with	
  avidin-­‐biotin	
  conjugated	
  horseradish	
  peroxidase	
  (Vectastain	
  
Elite	
  ABC	
  System,	
  Vector	
  Laboratories	
  Inc,	
  Burlingame,	
  CA)	
  and	
  antibodies	
  were	
  detected	
  
with	
  a	
  peroxidase	
  substrate	
  (NovaRED	
  Peroxidase	
  Substrate	
  Kit,	
  Vector	
  Laboratories	
  Inc,	
  
Burlingame,	
  CA).	
  	
  
A	
  board-­‐certified	
  veterinary	
  pathologist	
  (KJW)	
  who	
  was	
  unaware	
  of	
  the	
  exercise	
  
status	
  of	
  horses	
  and	
  sample	
  locations	
  of	
  lung	
  tissue	
  sections	
  evaluated	
  all	
  slides	
  via	
  bright	
  
field	
  microscopy.	
  Pulmonary	
  vein	
  protein	
  expression	
  was	
  scored	
  as	
  0	
  (no	
  evidence	
  of	
  
protein	
  expression),	
  1	
  (mild	
  protein	
  expression	
  in	
  a	
  small	
  number	
  of	
  veins),	
  or	
  2	
  (strong	
  
protein	
  expression	
  in	
  most	
  [˃	
  50%]	
  veins).	
  	
  	
  
	
  
	
  
	
  
118	
  

Collagen	
  content	
  analysis	
  
Picrosirius	
  red	
  staining	
  and	
  polarized	
  microscopy	
  of	
  tissue	
  samples	
  is	
  commonly	
  used	
  for	
  
detection	
  and	
  quantification	
  of	
  collagen	
  (38,	
  169).	
  For	
  the	
  quantification	
  of	
  collagen	
  in	
  lung	
  
tissue	
  samples	
  in	
  the	
  present	
  study,	
  picrosirius	
  red–stained	
  slides	
  were	
  scanned	
  and	
  
digitalized	
  at	
  a	
  magnification	
  of	
  20X	
  with	
  a	
  virtual	
  slide	
  system	
  (VS120-­‐SL,	
  Olympus	
  
America	
  Inc,	
  Center	
  Valley,	
  PA).	
  Polarization	
  filters	
  were	
  used	
  to	
  enhance	
  the	
  appearance	
  of	
  
the	
  picrosirius	
  red	
  stain	
  in	
  images	
  of	
  lung	
  tissue	
  samples.	
  	
  
Automated	
  random	
  subsampling	
  was	
  performed	
  on	
  each	
  of	
  the	
  digitalized	
  slides	
  
with	
  stereology	
  software	
  (NewCAST	
  whole	
  slide	
  stereology	
  software,	
  Visiopharm,	
  
Hoersholm,	
  Denmark)(magnification,	
  20X),	
  and	
  50	
  images	
  per	
  slide	
  were	
  analyzed.	
  Some	
  
lung	
  sample	
  slides	
  had	
  pleural	
  tissue;	
  such	
  regions	
  were	
  excluded	
  from	
  analysis.	
  	
  
Morphological	
  determination	
  of	
  the	
  percentage	
  of	
  collagen	
  in	
  lung	
  tissue	
  samples	
  
was	
  performed	
  with	
  software	
  (http://www.stepanizer.com)	
  (211).	
  Briefly,	
  a	
  point	
  grid	
  
with	
  a	
  density	
  of	
  7	
  X	
  7	
  points/98,157	
  μm2	
  was	
  superimposed	
  over	
  images	
  and	
  all	
  points	
  
that	
  contacted	
  noncollagenous	
  lung	
  tissue	
  and	
  those	
  that	
  contacted	
  collagenous	
  tissue	
  were	
  
counted.	
  The	
  percentage	
  of	
  collagen	
  in	
  lung	
  tissue	
  samples	
  was	
  estimated	
  by	
  dividing	
  the	
  
number	
  of	
  points	
  that	
  contacted	
  collagen	
  by	
  the	
  total	
  number	
  of	
  points	
  counted.	
  
	
  
Statistical	
  analyses	
  
The	
  ΔCT	
  values	
  were	
  evaluated	
  for	
  normality	
  and	
  transformed	
  as	
  needed	
  for	
  statistical	
  
analysis.	
  	
  
The	
  resulting	
  data	
  were	
  analyzed	
  with	
  the	
  following	
  model	
  (PROC	
  MIXED,	
  SAS	
  
Institute	
  Inc,	
  Cary,	
  NC)	
  to	
  determine	
  effect	
  of	
  exercise	
  on	
  the	
  expression	
  of	
  each	
  gene:	
  	
  

	
  
119	
  

Yijkl	
  	
  =	
  µ	
  +	
  Sitek	
  +	
  Statusl	
  +	
  Site×Status	
  +	
  Horsei	
  +	
  eijkl	
  
	
  where	
  Yijkl	
  is	
  the	
  normalized	
  gene	
  expression	
  of	
  a	
  gene	
  of	
  interest	
  for	
  horse	
  i	
  in	
  sample	
  j	
  
that	
  corresponds	
  to	
  lung	
  site	
  k	
  (caudodorsal	
  or	
  cranioventral)	
  and	
  status	
  l	
  (before	
  or	
  after	
  
exercise);	
  µ	
  is	
  the	
  mean	
  value	
  for	
  the	
  population;	
  and	
  eij	
  is	
  the	
  residual.	
  Horse	
  effects	
  were	
  
assumed	
  to	
  be	
  random	
  to	
  account	
  for	
  within	
  horse	
  measurement	
  correlations;	
  residuals	
  
within	
  each	
  horse	
  were	
  heteroskedastic	
  for	
  lung	
  samples	
  obtained	
  before	
  and	
  after	
  
exercise,	
  indicating	
  there	
  were	
  different	
  variances	
  for	
  those	
  groups.	
  This	
  model	
  is	
  
practically	
  equivalent	
  to	
  using	
  a	
  joint	
  mixed	
  model	
  analysis	
  of	
  test	
  and	
  control	
  genes	
  (198).	
  
For	
  immunohistochemistry	
  data,	
  the	
  Wilcoxon	
  Signed-­‐Rank	
  Test	
  (NCSS	
  Statistical	
  
Software,	
  Kaysvill,	
  UT)	
  for	
  nonparametric	
  data	
  was	
  used	
  for	
  analyses.	
  The	
  pre-­‐	
  and	
  
postexercise	
  scores	
  were	
  compared	
  for	
  each	
  lung	
  sample	
  collection	
  site.	
  	
  
For	
  collagen	
  content	
  data,	
  a	
  3-­‐factor	
  (2-­‐factor	
  repeated	
  measures)	
  analysis	
  of	
  
variance	
  (PROC	
  MIXED,	
  SAS	
  Institute	
  Inc,	
  Cary,	
  NC)	
  was	
  used	
  for	
  analyses	
  with	
  site	
  
(caudodorsal	
  or	
  cranioventral)	
  and	
  time	
  (before	
  or	
  after	
  exercise)	
  as	
  fixed	
  factors	
  and	
  
horse	
  as	
  the	
  random	
  factor.	
  Bonferroni’s	
  correction	
  for	
  multiple	
  comparisons	
  was	
  used.	
  A	
  
normal	
  distribution	
  of	
  errors	
  was	
  determined	
  via	
  the	
  Shapiro-­‐Wilks’	
  test.	
  Collagen	
  content	
  
data	
  were	
  reported	
  as	
  least	
  square	
  means	
  ±	
  SEM.	
  	
  
Values	
  of	
  P	
  <	
  0.05	
  were	
  considered	
  significant.	
  	
  
	
  
Results	
  
Two	
  of	
  6	
  horses	
  that	
  finished	
  the	
  study	
  had	
  tracheobronchoscopic	
  evidence	
  of	
  pulmonary	
  
hemorrhage	
  within	
  90	
  minutes	
  after	
  the	
  end	
  of	
  the	
  final	
  high-­‐intensity	
  exercise	
  session	
  

	
  
120	
  

during	
  the	
  first	
  exercise	
  period;	
  therefore,	
  33.3%	
  of	
  horses	
  had	
  EIPH	
  at	
  that	
  time.	
  The	
  EIPH	
  
severity	
  grade	
  for	
  both	
  of	
  those	
  horses	
  was	
  1	
  of	
  4	
  (69).	
  
The	
  mRNA	
  prepared	
  from	
  pulmonary	
  vein	
  samples	
  were	
  of	
  insufficient	
  quality	
  for	
  analysis	
  
for	
  all	
  horses	
  after	
  the	
  first	
  exercise	
  period	
  and	
  for	
  2	
  horses	
  after	
  the	
  second	
  exercise	
  
period;	
  therefore,	
  mRNA	
  samples	
  for	
  4	
  horses	
  obtained	
  after	
  the	
  second	
  exercise	
  period	
  
were	
  analyzed	
  via	
  PCR	
  assay	
  for	
  determination	
  of	
  gene	
  expression.	
  Results	
  of	
  initial	
  
analysis	
  indicated	
  exercise	
  of	
  horses	
  had	
  an	
  effect	
  on	
  gene	
  expression	
  in	
  pulmonary	
  vein	
  
samples,	
  but	
  the	
  interaction	
  of	
  the	
  variables	
  pulmonary	
  wedge	
  resection	
  site	
  (caudodorsal	
  
vs	
  cranioventral)	
  X	
  exercise	
  was	
  not	
  significant.	
  Therefore,	
  mean	
  values	
  for	
  gene	
  
expression	
  in	
  pulmonary	
  vein	
  samples	
  obtained	
  from	
  the	
  caudodorsal	
  regions	
  of	
  lungs	
  and	
  
for	
  those	
  obtained	
  from	
  the	
  cranioventral	
  regions	
  of	
  lungs	
  were	
  used	
  for	
  analysis.	
  Exercise	
  
of	
  horses	
  significantly	
  decreased	
  expression	
  of	
  5	
  of	
  the	
  10	
  genes	
  evaluated	
  (MMP-­‐2	
  [P	
  =	
  
0.017],	
  MMP-­‐9	
  [P	
  =	
  0.035],	
  TIMP-­‐2	
  [P	
  =	
  .039],	
  TGF-­‐β	
  [P	
  =	
  0.003],	
  and	
  VEGF	
  [P	
  =	
  0.007];	
  
(Figure	
  15).	
  Gene	
  expression	
  did	
  not	
  significantly	
  change	
  after	
  exercise	
  for	
  TIMP-­‐1	
  (P	
  =	
  
0.270),	
  collagen	
  type	
  I	
  (P	
  =	
  0.130),	
  tenascin-­‐C	
  (P	
  =	
  0.659),	
  ET-­‐1	
  (P	
  =	
  0.077),	
  and	
  PDGF	
  (P	
  =	
  
0.119).	
  	
  	
  
The	
  only	
  gene	
  with	
  differential	
  expression	
  between	
  pulmonary	
  vein	
  samples	
  obtained	
  from	
  
caudodorsal	
  regions	
  of	
  lungs	
  and	
  those	
  obtained	
  from	
  cranioventral	
  regions	
  of	
  lungs	
  was	
  
tenascin-­‐C.	
  The	
  mRNA	
  expression	
  of	
  tenascin-­‐C	
  was	
  approximately	
  four	
  times	
  as	
  greater	
  in	
  
pulmonary	
  vein	
  samples	
  obtained	
  from	
  cranioventral	
  regions	
  of	
  lungs	
  as	
  it	
  was	
  in	
  samples	
  
obtained	
  from	
  caudodorsal	
  regions	
  of	
  lungs;	
  these	
  gene	
  expression	
  values	
  were	
  
significantly	
  (P	
  =	
  0.033)	
  different.	
  However,	
  tenascin-­‐C	
  expression	
  in	
  each	
  of	
  those	
  lung	
  
regions	
  did	
  not	
  significantly	
  change	
  after	
  exercise.	
  	
  

	
  
121	
  

Protein	
  expression	
  in	
  lung	
  samples	
  was	
  determined	
  via	
  immunohistochemical	
  methods	
  for	
  
all	
  6	
  horses	
  that	
  completed	
  the	
  study;	
  results	
  indicated	
  exercise	
  had	
  no	
  effect	
  on	
  protein	
  
expression	
  of	
  tenascin-­‐C,	
  TGF-­‐β,	
  or	
  VEGF	
  in	
  tissue	
  samples	
  obtained	
  from	
  caudodorsal	
  or	
  
cranioventral	
  regions	
  of	
  lungs.	
  The	
  percentage	
  of	
  collagen	
  in	
  tissue	
  samples	
  obtained	
  from	
  
caudodorsal	
  regions	
  of	
  lungs	
  was	
  significantly	
  (P	
  <0.05)	
  higher	
  than	
  that	
  in	
  tissue	
  samples	
  
obtained	
  from	
  cranioventral	
  regions	
  of	
  lungs,	
  although	
  the	
  percentage	
  of	
  collagen	
  was	
  not	
  
significantly	
  different	
  in	
  lung	
  samples	
  obtained	
  before	
  and	
  after	
  exercise	
  (Figure	
  16).	
  	
  
	
  
Discussion	
  
Venous	
  remodeling	
  is	
  important	
  in	
  the	
  pathogenesis	
  of	
  EIPH.	
  Alterations	
  in	
  mRNA	
  
expression	
  are	
  expected	
  to	
  precede	
  structural	
  changes	
  in	
  vasculature.	
  Therefore,	
  the	
  
purpose	
  of	
  this	
  study	
  was	
  to	
  determine	
  whether	
  2	
  weeks	
  of	
  intense	
  exercise	
  would	
  affect	
  
mRNA	
  and	
  protein	
  expression	
  of	
  mediators	
  of	
  pulmonary	
  intralobular	
  vein	
  remodeling	
  in	
  a	
  
manner	
  expected	
  to	
  favor	
  vascular	
  remodeling	
  in	
  caudodorsal	
  but	
  not	
  cranioventral	
  
regions	
  of	
  lungs	
  of	
  horses.	
  
	
  	
  

The	
  thoracoscopic	
  technique	
  used	
  to	
  obtain	
  lung	
  samples	
  from	
  standing	
  horses	
  in	
  

this	
  study	
  was	
  previously	
  reported	
  (115)	
  and	
  validated	
  by	
  personnel	
  in	
  our	
  laboratory.	
  No	
  
intraoperative	
  complications	
  were	
  detected,	
  and	
  horses	
  had	
  no	
  substantial	
  problems	
  
attributable	
  to	
  the	
  surgery.	
  The	
  endoscopic	
  device	
  used	
  to	
  obtain	
  lung	
  samples	
  resulted	
  in	
  
collection	
  of	
  an	
  adequate	
  amount	
  of	
  tissue	
  for	
  harvest	
  of	
  veins	
  and	
  preparation	
  of	
  mRNA.	
  
To	
  reduce	
  the	
  effects	
  of	
  surrounding	
  tissues	
  on	
  results	
  for	
  pulmonary	
  veins,	
  a	
  
microdissection	
  technique	
  was	
  used	
  to	
  ensure	
  that	
  only	
  the	
  cells	
  of	
  interest	
  (intralobular	
  
venous	
  wall	
  cells)	
  were	
  isolated	
  and	
  assayed.	
  

	
  
122	
  

The	
  markers	
  of	
  venous	
  remodeling	
  evaluated	
  in	
  the	
  present	
  study	
  were	
  selected	
  on	
  
the	
  basis	
  of	
  studies	
  conducted	
  with	
  animals	
  of	
  other	
  species,	
  because	
  such	
  information	
  was	
  
not	
  available	
  for	
  horses,	
  to	
  the	
  authors’	
  knowledge.	
  The	
  activities	
  of	
  MMP-­‐2	
  and	
  MMP-­‐9,	
  
which	
  have	
  predominantly	
  proteolytic	
  actions,	
  are	
  regulated	
  by	
  TIMP-­‐1	
  and	
  TIMP-­‐2	
  (142);	
  
these	
  factors	
  regulate	
  the	
  protein	
  content	
  of	
  extracellular	
  matrix.	
  In	
  general,	
  hypertension	
  
results	
  in	
  increased	
  expression	
  of	
  MMP-­‐2	
  and	
  MMP-­‐9	
  mRNA	
  or	
  protein	
  (109,	
  225)	
  and	
  
decreased	
  (29,	
  235)	
  or	
  no	
  change	
  (23)	
  in	
  TIMP	
  expression.	
  	
  	
  
Results	
  of	
  other	
  studies	
  indicate	
  collagen	
  content	
  is	
  increased	
  in	
  severely	
  affected	
  
regions	
  of	
  lungs	
  of	
  horses	
  with	
  EIPH	
  (38)	
  and	
  in	
  walls	
  of	
  remodeled	
  veins	
  in	
  humans	
  (25)	
  
and	
  rabbits	
  (235).	
  Tenascin-­‐C	
  (an	
  extracellular	
  matrix	
  protein)	
  expression	
  is	
  upregulated	
  
by	
  MMPs	
  (89)	
  and	
  PDGF	
  (223)	
  and	
  is	
  expressed	
  during	
  venous	
  remodeling	
  (2,	
  222).	
  
Endothelin-­‐1	
  causes	
  vasoconstriction	
  in	
  vivo	
  (136)	
  and	
  	
  has	
  been	
  implicated	
  in	
  pulmonary	
  
(204)	
  	
  and	
  systemic	
  (224)	
  venous	
  remodeling.	
  Platelet-­‐derived	
  growth	
  factor	
  is	
  a	
  potent	
  
mitogen	
  of	
  connective	
  tissue	
  cells	
  (65)	
  and	
  is	
  associated	
  with	
  venous	
  remodeling	
  in	
  pigs	
  
(48).	
  The	
  cytokine	
  TGF-­‐β	
  is	
  important	
  in	
  various	
  developmental	
  and	
  pathological	
  processes	
  
(161)	
  and	
  has	
  been	
  implicated	
  in	
  vein	
  graft	
  remodeling	
  (85).	
  Vascular	
  endothelial	
  growth	
  
factor	
  is	
  also	
  a	
  mitogen	
  that	
  is	
  produced	
  by	
  vascular	
  endothelial	
  cells(46);	
  that	
  cytokine	
  has	
  
a	
  role	
  in	
  formation	
  of	
  neointima	
  in	
  remodeled	
  blood	
  vessels	
  (156,	
  241).	
  	
  
We	
  expected	
  that	
  expression	
  of	
  the	
  genes	
  evaluated	
  in	
  this	
  study	
  (except	
  TIMPs)	
  
would	
  increase	
  in	
  pulmonary	
  veins	
  of	
  caudodorsal	
  regions	
  of	
  lungs	
  after	
  exercise	
  of	
  horses.	
  
Results	
  of	
  this	
  study	
  indicated	
  that	
  mean	
  expression	
  values	
  of	
  all	
  genes	
  evaluated	
  
decreased	
  in	
  pulmonary	
  veins	
  after	
  exercise;	
  these	
  findings	
  were	
  significant	
  for	
  MMP-­‐2,	
  
MMP-­‐9,	
  TIMP-­‐2,	
  TGF-­‐β,	
  and	
  VEGF.	
  Because	
  the	
  collection	
  site	
  X	
  treatment	
  interaction	
  was	
  

	
  
123	
  

not	
  significant,	
  decreases	
  in	
  expression	
  were	
  attributed	
  to	
  causes	
  other	
  than	
  lung	
  region.	
  
Although	
  expression	
  of	
  tenascin-­‐C	
  mRNA	
  was	
  not	
  increased	
  after	
  exercise,	
  tenascin-­‐C	
  
mRNA	
  expression	
  was	
  higher	
  in	
  pulmonary	
  veins	
  in	
  cranioventral	
  regions	
  of	
  lungs	
  versus	
  
those	
  in	
  caudodorsal	
  regions	
  of	
  lungs.	
  
	
  	
  

The	
  main	
  advantage	
  of	
  qRT-­‐PCR	
  assays	
  for	
  determination	
  of	
  gene	
  expression	
  in	
  

pulmonary	
  veins	
  is	
  that	
  the	
  technique	
  has	
  high	
  sensitivity;	
  therefore,	
  mRNA	
  expression	
  can	
  
be	
  determined	
  for	
  small	
  amounts	
  of	
  tissue.	
  Furthermore,	
  expression	
  of	
  multiple	
  genes	
  can	
  
be	
  evaluated	
  for	
  a	
  tissue	
  sample	
  via	
  that	
  technique.	
  Data	
  regarding	
  expression	
  of	
  mRNA	
  are	
  
commonly	
  used	
  to	
  infer	
  other	
  information	
  about	
  molecular	
  pathways	
  in	
  cells,	
  including	
  
information	
  regarding	
  protein	
  expression.	
  However,	
  because	
  of	
  translational	
  and	
  
posttranslational	
  control	
  mechanisms,	
  such	
  inferences	
  may	
  not	
  be	
  correct	
  (186).	
  For	
  
example,	
  results	
  of	
  another	
  study	
  indicate	
  differential	
  mRNA	
  and	
  protein	
  expression	
  of	
  
MMP-­‐2,	
  MMP-­‐9,	
  and	
  TIMP-­‐1	
  (111).	
  Because	
  of	
  this	
  possibility,	
  we	
  determined	
  vascular	
  
expression	
  of	
  TGF-­‐β,	
  VEGF,	
  and	
  tenascin-­‐C	
  via	
  immunohistochemical	
  methods.	
  Unlike	
  the	
  
results	
  for	
  gene	
  expression,	
  no	
  significant	
  decrease	
  in	
  protein	
  expression	
  was	
  detected	
  by	
  
use	
  of	
  that	
  semiquantitative	
  method	
  in	
  the	
  present	
  study.	
  Immunohistochemistry	
  was	
  used	
  
rather	
  than	
  quantitative	
  techniques	
  (such	
  as	
  Western	
  blot	
  analysis)	
  because	
  an	
  insufficient	
  
amount	
  of	
  protein	
  would	
  have	
  been	
  obtained	
  from	
  the	
  microdissected	
  veins	
  for	
  
performance	
  of	
  such	
  assays.	
  	
  	
  
Analysis	
  was	
  performed	
  for	
  determination	
  of	
  the	
  effects	
  of	
  exercise	
  on	
  collagen	
  
content	
  of	
  lung	
  samples	
  in	
  this	
  study	
  because	
  results	
  of	
  another	
  study	
  (38)	
  indicate	
  the	
  
amount	
  of	
  collagen	
  in	
  EIPH-­‐affected	
  lung	
  tissue	
  is	
  higher	
  than	
  that	
  in	
  unaffected	
  lung	
  tissue.	
  
Results	
  of	
  the	
  present	
  study	
  indicated	
  that	
  exercise	
  did	
  not	
  have	
  a	
  significant	
  effect	
  on	
  

	
  
124	
  

collagen	
  content	
  of	
  lung	
  samples.	
  However,	
  collagen	
  content	
  was	
  significantly	
  different	
  in	
  
tissue	
  samples	
  obtained	
  from	
  caudodorsal	
  and	
  cranioventral	
  regions	
  of	
  lungs.	
  Although	
  
areas	
  of	
  slides	
  with	
  pleural	
  tissue	
  were	
  excluded	
  from	
  analysis,	
  that	
  finding	
  was	
  likely	
  
attributable	
  to	
  anatomic	
  differences	
  between	
  caudodorsal	
  and	
  cranioventral	
  regions	
  of	
  
lungs.	
  Also,	
  expression	
  of	
  collagen	
  type	
  I	
  mRNA	
  was	
  not	
  affected	
  by	
  exercise	
  of	
  horses.	
  
Similar	
  morphometric	
  analysis	
  for	
  the	
  proteins	
  evaluated	
  via	
  immunohistochemical	
  
methods	
  (TGF-­‐β,	
  VEGF	
  and	
  tenascin-­‐C)	
  was	
  not	
  performed	
  because	
  differences	
  in	
  
expression	
  of	
  those	
  proteins	
  were	
  not	
  detected	
  via	
  routine	
  microscopy.	
  	
  
Interactions	
  among	
  mediators	
  of	
  venous	
  remodeling	
  are	
  complex	
  and	
  affected	
  by	
  
the	
  type	
  and	
  severity	
  of	
  a	
  stimulus	
  and	
  the	
  timing	
  of	
  tissue	
  sample	
  collection.	
  For	
  example,	
  
during	
  development	
  of	
  TGF-­‐β–mediated	
  intimal	
  hyperplasia	
  in	
  vein	
  grafts	
  in	
  rabbits,	
  
activities	
  of	
  MMP-­‐2	
  and	
  MMP-­‐9	
  concurrently	
  decrease	
  (85).	
  Results	
  of	
  another	
  study	
  
indicate	
  there	
  is	
  a	
  temporal	
  pattern	
  of	
  MMP-­‐2	
  and	
  MMP-­‐9	
  expression	
  during	
  venous	
  
remodeling,	
  with	
  an	
  initial	
  increase	
  in	
  expression	
  followed	
  by	
  a	
  decrease	
  in	
  expression	
  to	
  
undetectable	
  levels	
  (195).	
  The	
  significant	
  decrease	
  in	
  expression	
  of	
  MMP-­‐2	
  and	
  MMP-­‐9	
  
mRNA	
  detected	
  in	
  the	
  present	
  study	
  after	
  exercise	
  of	
  horses	
  may	
  have	
  been	
  attributable	
  to	
  
a	
  period	
  of	
  blood	
  vessel	
  remodeling	
  during	
  which	
  those	
  substances	
  had	
  low	
  expression.	
  
	
  	
  

A	
  limitation	
  of	
  the	
  present	
  study	
  was	
  the	
  fact	
  that	
  lung	
  samples	
  were	
  evaluated	
  for	
  

only	
  one	
  time	
  after	
  exercise	
  of	
  horses.	
  Results	
  of	
  another	
  study	
  in	
  which	
  gene	
  expression	
  in	
  
autologous	
  vein	
  grafts	
  was	
  evaluated	
  via	
  high	
  throughput	
  microarray	
  analysis	
  (92)	
  indicate	
  
expression	
  of	
  TIMP-­‐1	
  and	
  VEGF	
  mRNA	
  is	
  increased	
  only	
  on	
  day	
  1	
  after	
  graft	
  implantation,	
  
and	
  not	
  on	
  days	
  7,	
  14,	
  or	
  30	
  after	
  graft	
  implantation;	
  results	
  of	
  that	
  study	
  also	
  indicate	
  
collagen	
  expression	
  is	
  decreased	
  on	
  days	
  1	
  and	
  7,	
  and	
  increased	
  on	
  days	
  14	
  and	
  30	
  after	
  

	
  
125	
  

graft	
  implantation.	
  Because	
  data	
  have	
  not	
  been	
  published	
  regarding	
  gene	
  expression	
  in	
  
equine	
  pulmonary	
  veins,	
  to	
  the	
  authors’	
  knowledge,	
  the	
  timing	
  of	
  lung	
  sample	
  collection	
  
and	
  the	
  duration	
  of	
  exercise	
  of	
  horses	
  in	
  this	
  study	
  were	
  selected	
  on	
  the	
  basis	
  of	
  other	
  
information.	
  Continuous	
  hypertension	
  causes	
  substantial	
  structural	
  alterations	
  in	
  the	
  
tunica	
  media	
  and	
  adventitia	
  of	
  pulmonary	
  veins	
  in	
  sheep	
  after	
  only	
  4	
  days	
  (86);	
  therefore,	
  
we	
  predicted	
  that	
  alterations	
  in	
  gene	
  expression	
  (which	
  should	
  precede	
  structural	
  
alterations)	
  in	
  vein	
  walls	
  of	
  horses	
  in	
  the	
  present	
  study	
  would	
  be	
  detectable	
  2	
  weeks	
  after	
  
the	
  end	
  of	
  a	
  6-­‐session	
  intense	
  exercise	
  period.	
  Because	
  results	
  of	
  this	
  study	
  indicated	
  
mRNA	
  expression	
  of	
  various	
  MMPs	
  and	
  growth	
  factors	
  was	
  significantly	
  different	
  after	
  
exercise	
  versus	
  gene	
  expression	
  before	
  exercise,	
  that	
  duration	
  and	
  intensity	
  of	
  exercise	
  for	
  
horses	
  seemed	
  to	
  be	
  adequate	
  to	
  cause	
  changes	
  in	
  gene	
  expression.	
  	
  
The	
  high-­‐intensity	
  exercise	
  protocol	
  used	
  in	
  the	
  present	
  study	
  was	
  intended	
  to	
  simulate	
  
race	
  training	
  (after	
  horses	
  underwent	
  2	
  weeks	
  of	
  low-­‐intensity	
  conditioning	
  exercise).	
  The	
  
intensity	
  of	
  exercise	
  was	
  expected	
  to	
  be	
  an	
  adequate	
  stimulus	
  for	
  evaluation	
  of	
  changes	
  in	
  
gene	
  expression	
  in	
  lungs	
  of	
  horses.	
  Each	
  horse	
  exercised	
  at	
  a	
  speed	
  corresponding	
  to	
  a	
  
heart	
  rate	
  of	
  120%	
  of	
  the	
  HRmax	
  value.	
  The	
  variable	
  HRmax	
  is	
  a	
  reproducible	
  measurement	
  
for	
  exercising	
  horses	
  (45),	
  and	
  horses	
  require	
  maximum	
  effort	
  to	
  maintain	
  a	
  position	
  on	
  a	
  
treadmill	
  at	
  a	
  speed	
  corresponding	
  to	
  120%	
  	
  of	
  HRmax.	
  Furthermore,	
  33.3%	
  of	
  horses	
  in	
  this	
  
study	
  had	
  EIPH	
  (as	
  diagnosed	
  via	
  respiratory	
  tract	
  endoscopy);	
  this	
  finding	
  suggested	
  that	
  
the	
  exercise	
  was	
  of	
  adequate	
  intensity.	
  	
  
There	
  was	
  a	
  12-­‐month	
  period	
  between	
  collection	
  of	
  pre-­‐	
  and	
  postexercise	
  lung	
  
samples	
  in	
  this	
  study.	
  This	
  period	
  allowed	
  healing	
  of	
  surgical	
  sites	
  after	
  the	
  first	
  procedure.	
  
Ageing	
  of	
  animals	
  is	
  associated	
  with	
  remodeling	
  of	
  blood	
  vessel	
  walls	
  (66)	
  (particularly	
  

	
  
126	
  

arterial	
  walls	
  (110));	
  however,	
  such	
  findings	
  have	
  only	
  been	
  detected	
  for	
  very	
  young	
  and	
  
very	
  old	
  animals	
  (110)	
  and	
  humans	
  (104,	
  155).	
  Therefore,	
  it	
  was	
  unlikely	
  that	
  ageing	
  
during	
  the	
  12-­‐month	
  period	
  affected	
  blood	
  vessel	
  wall	
  characteristics	
  in	
  horses	
  in	
  the	
  
present	
  study.	
  
The	
  role	
  of	
  venous	
  remodeling	
  in	
  the	
  pathogenesis	
  of	
  EIPH	
  is	
  not	
  known,	
  to	
  the	
  
authors’	
  knowledge.	
  However,	
  the	
  distribution	
  of	
  venous	
  remodeling	
  in	
  lungs	
  of	
  horses	
  
with	
  EIPH	
  (lesions	
  are	
  colocalized	
  with	
  hemosiderin	
  in	
  caudodorsal	
  regions	
  of	
  lungs	
  of	
  
affected	
  horses	
  (38))	
  suggests	
  that	
  it	
  is	
  important	
  in	
  the	
  pathogenesis	
  of	
  EIPH.	
  Because	
  
high	
  intravascular	
  pressures	
  induce	
  remodeling	
  in	
  systemic	
  (2,	
  28,	
  29,	
  64,	
  112,	
  180,	
  235)	
  
and	
  pulmonary	
  (25,	
  86)	
  veins,	
  we	
  propose	
  that	
  intermittent	
  periods	
  of	
  high	
  pressures	
  in	
  
the	
  pulmonary	
  circulation	
  during	
  exercise	
  cause	
  remodeling	
  of	
  pulmonary	
  veins	
  in	
  
caudodorsal	
  regions	
  of	
  lungs	
  of	
  horses.	
  Such	
  venous	
  remodeling	
  may	
  result	
  in	
  high	
  
pulmonary	
  capillary	
  pressures	
  in	
  affected	
  regions	
  of	
  lungs	
  and	
  an	
  increased	
  risk	
  of	
  
capillary	
  rupture	
  and	
  hemorrhage	
  and	
  development	
  of	
  EIPH.	
  	
  
	
  

Results	
  of	
  the	
  present	
  study	
  did	
  not	
  support	
  the	
  hypothesis	
  that	
  2	
  

weeks	
  of	
  intense	
  exercise	
  would	
  cause	
  alterations	
  in	
  gene	
  and	
  protein	
  expression	
  in	
  
pulmonary	
  veins	
  in	
  a	
  manner	
  expected	
  to	
  favor	
  venous	
  remodeling.	
  However,	
  few	
  data	
  
regarding	
  timing	
  of	
  expression	
  of	
  genes	
  during	
  vascular	
  remodeling	
  in	
  horses	
  have	
  been	
  
published.	
  Further	
  studies	
  are	
  warranted	
  to	
  determine	
  the	
  mechanisms	
  and	
  timing	
  of	
  
venous	
  remodeling	
  in	
  horses	
  with	
  EIPH.	
  	
   	
  
	
  

	
  

	
  
127	
  

	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
APPENDIX	
  

	
  
128	
  

	
  

	
  
	
  
Figure	
  15:	
  Mean	
  ±	
  SEM	
  fold	
  changes	
  in	
  mRNA	
  expression	
  of	
  10	
  genes	
  in	
  pulmonary	
  vein	
  
samples	
  of	
  4	
  horses	
  after	
  a	
  2-­‐week	
  period	
  of	
  intense	
  exercise	
  versus	
  expression	
  before	
  
exercise.	
  *Expression	
  is	
  significantly	
  (P	
  <	
  0.05)	
  different	
  between	
  pulmonary	
  vein	
  samples	
  
collected	
  before	
  and	
  after	
  exercise.	
  	
  
	
  
	
  
	
  
	
  
	
  

	
  

	
  
129	
  

	
  
	
  
Figure	
  16:	
  Least	
  square	
  mean	
  ±	
  SEM	
  	
  percentage	
  of	
  collagen	
  in	
  samples	
  of	
  cranioventral	
  	
  
(CV)	
  and	
  caudodorsal	
  (CD)	
  regions	
  of	
  lung	
  of	
  6	
  horses	
  before	
  (black	
  bars)	
  and	
  after	
  (grey	
  	
  
bars)	
  a	
  2-­‐week	
  period	
  of	
  intense	
  exercise.	
  Bars	
  indicate	
  no	
  significant	
  (P	
  <	
  0.05)	
  differences	
  
between	
  lung	
  samples	
  obtained	
  before	
  and	
  after	
  exercise	
  within	
  a	
  region.	
  *Mean	
  value	
  for	
  
pre-­‐	
  and	
  postexercise	
  tissue	
  samples	
  obtained	
  from	
  caudodorsal	
  regions	
  of	
  lungs	
  are	
  
significantly	
  (P	
  <	
  0.05)	
  higher	
  than	
  those	
  obtained	
  from	
  cranioventral	
  regions	
  of	
  lungs.	
  	
  
	
  
	
  

	
  

	
  
130	
  

	
  
	
  
	
  
	
  
GenBank	
  
accession	
  
No.	
  

Forward	
  primer	
  
(5’–3’)	
  

Reverse	
  primer	
  (5’–
3’)	
  

TCCGAGTCTGGAGT
GATGTGA	
  
NM-­‐
GCAAGGAGTACTCT
001111302	
   GCCTGTA	
  
NM_00108 GCCAGGGCTTCACC
2515	
  
AAGA	
  
CTGACAAGGACATC
AJ010315	
  
GAGTTCATCTA	
  
XM001914 GAGCCCAGAGCAGA
PDGF	
  
920	
  
TGCAA	
  
NM-­‐
GGAATGGCTGTCCT
TGF-­‐β	
  
001081849	
   TTGATGTCA	
  
CGACATCATCTGGG
ET-­‐1	
   AY730629	
  
TCAACACT	
  
Collag
CGGACAGCCTGGAC
en	
  
AF034691	
  
TCC	
  
type	
  I	
  
NM_00108 GCAAATGTGAATGC
VEGF	
  
1821	
  
AGACCAAAGAA	
  
Tenas
GTGGAGTATTTCAT
AY246747	
  
cin-­‐C	
  
CCGTGTGTTTG	
  
Beta-­‐ NM_00108 GGGACCTGACGGAC
actin	
  
1838	
  
TACCT	
  
NM_00108 CGCCTGAGATTGAA
B2M	
  
2502	
  
ATTGATTTGCT	
  

GATCATGATGTCAGC
CTCTCCAT	
  
CCAGAGGCGCCCATC
A	
  
CAGTGTCACTCTGCA
GTTTGC	
  

Gene	
  
MMP-­‐
2	
  
MMP-­‐
9	
  
TIMP-­‐
1	
  
TIMP-­‐
2	
  

EF-­‐1	
  

AJ010314	
  

AY237113	
  

Probe	
  

CTTCTTGCTCTGACC
CACGAT	
  
CGAAGGCCCTCCATT
GC	
  
GGATCGCTTGGACCT
GGAA	
  

CCCACTACGGT
TTTCT	
  
CTGCGGCCCTC
TCTG	
  
ATGCTCAGTGT
TTCCC	
  
ACGGCTCCCTC
CTCG	
  
ACAGCAGCCCA
CTTGC	
  
CTGCCGCACGA
CTCC	
  
CCGAGCACATT
GTTCC	
  

CAGCAAATTTCTCAT
CATAGCCATAAGAC	
  

CCTCCTGGACC
TCCCG	
  

GCTTTCTCCGCTCTG
AGCAA	
  
GCCACCCTGGCACTG
A	
  
CCGTGGTGGTGAAGC
TGTAG	
  
GACCAGTCCTTGCTG
AAAGACA	
  

CCACAGGGATT
TTC	
  
CCATCCCGGAG
AACAA	
  
TCCGTGAGGAT
CTTCA	
  
ACCGGTCGACT
TTCAT	
  

CCACCAACTCGTCCA GACAGTACCGATACC
ACTGATAAG	
  
ACCAATTTTG	
  

CCCTTGCGTCT
GCCCC	
  

GCGAGACCCCGCACA	
  

	
  
Table	
  2	
  Primers	
  and	
  probes	
  used	
  for	
  detection	
  of	
  various	
  genes	
  in	
  pulmonary	
  vein	
  samples	
  
of	
  horses	
  via	
  qRT-­‐PCR	
  assay.	
  B2M	
  =	
  Beta-­‐2-­‐microglobulin.	
  EF-­‐1	
  =	
  Elongation	
  factor-­‐1	
  alpha.	
  

	
  
131	
  

CHAPTER	
  5	
  
	
  
Conclusions	
  and	
  directions	
  for	
  future	
  studies	
  
	
  
	
  
Recent	
  descriptions	
  of	
  EIPH	
  pathology	
  have	
  highlighted	
  deficiencies	
  in	
  the	
  capillary-­‐stress	
  
failure	
  theory	
  of	
  EIPH	
  pathogenesis,	
  which	
  until	
  recently	
  provided	
  the	
  most	
  plausible,	
  
albeit	
  incomplete,	
  explanation	
  of	
  disease	
  mechanisms.	
  	
  
	
  

Pulmonary	
  capillary	
  stress	
  failure	
  secondary	
  to	
  exercise-­‐associated	
  pulmonary	
  

circulation	
  pressure	
  elevations	
  explains	
  neither	
  the	
  predilection	
  for	
  caudodorsal	
  lung	
  of	
  
EIPH	
  pathology,	
  the	
  distribution	
  of	
  which	
  matches	
  exactly	
  that	
  of	
  pulmonary	
  blood	
  flow	
  
during	
  exercise,	
  nor	
  does	
  it	
  account	
  for	
  the	
  extensive	
  venous	
  remodeling	
  of	
  intralobular	
  
pulmonary	
  veins	
  in	
  caudodorsal	
  lung.	
  Assuming	
  however	
  that	
  stress	
  failure	
  is	
  a	
  component	
  
of	
  EIPH	
  pathogenesis,	
  and	
  evidence	
  exists	
  to	
  suggest	
  that	
  it	
  is,	
  the	
  factors	
  that	
  determine	
  
capillary	
  pressure,	
  and	
  therefore	
  pulmonary	
  capillary	
  rupture,	
  merit	
  consideration.	
  	
  
Resistance	
  to	
  flow	
  in	
  a	
  vessel	
  is	
  strongly	
  influenced	
  by	
  vessel	
  diameter,	
  which	
  in	
  
turn	
  is	
  a	
  function	
  of	
  a	
  combination	
  of	
  the	
  passive,	
  mechanical	
  characteristics	
  of	
  the	
  vessel	
  
wall	
  (i.e.	
  its	
  ability	
  to	
  resist	
  stretch)	
  and	
  the	
  degree	
  of	
  contraction	
  of	
  circumferential	
  
vascular	
  smooth	
  muscle	
  (i.e.	
  vessel	
  tone),	
  which	
  is	
  determined	
  by	
  neural,	
  humoral	
  and	
  local	
  
factors.	
  	
  
	
  

Capillary	
  pressure	
  is	
  immediately	
  influenced	
  by	
  the	
  resistance	
  to	
  blood	
  flow	
  in	
  the	
  

segments	
  supplying	
  and	
  draining	
  that	
  capillary.	
  Decreased	
  arterial	
  resistance	
  to	
  flow	
  and	
  
increased	
  venous	
  resistance	
  to	
  flow	
  are	
  both	
  conditions	
  under	
  which	
  intervening	
  
capillaries	
  will	
  be	
  exposed	
  to	
  higher	
  pressures.	
  	
  
	
  

While	
  there	
  is	
  evidence	
  that	
  regional	
  heterogeneity	
  in	
  the	
  reactivity	
  of	
  large	
  

pulmonary	
  arteries	
  in	
  the	
  horse	
  lung	
  exists,	
  whether	
  a	
  similar	
  pattern	
  exists	
  in	
  the	
  small	
  
	
  
132	
  

arteries	
  and	
  veins,	
  that	
  are	
  almost	
  immediately	
  up-­‐	
  and	
  down-­‐stream	
  from	
  the	
  pulmonary	
  
capillaries	
  had	
  not	
  been	
  investigated.	
  I	
  hypothesized	
  therefore	
  that	
  regional	
  differences	
  do	
  
exist,	
  in	
  either	
  the	
  mechanical	
  characteristics,	
  and/or	
  in	
  the	
  reactivity	
  profile	
  of	
  these	
  
vessels,	
  and	
  that	
  these	
  differences	
  would	
  provide	
  some	
  evidence	
  that	
  capillary	
  pressures	
  in	
  
caudodorsal	
  lung	
  could	
  exceed	
  those	
  in	
  cranioventral	
  lung	
  during	
  exercise.	
  Furthermore,	
  if	
  
those	
  regional	
  differences	
  predict	
  the	
  transmission	
  of	
  higher	
  pressures	
  to	
  pulmonary	
  veins	
  
in	
  the	
  caudodorsal	
  lung,	
  then	
  hemodynamic	
  stimuli	
  in	
  that	
  region,	
  while	
  transient,	
  would	
  
be	
  enough	
  to	
  initiate	
  pressure-­‐mediated	
  venous	
  remodeling.	
  Remodeling	
  would	
  reduce	
  
venous	
  compliance,	
  and	
  further	
  exacerbate	
  pulmonary	
  capillary	
  failure.	
  	
  
Accordingly,	
  my	
  overarching	
  hypothesis	
  for	
  EIPH	
  pathogenesis	
  was	
  as	
  follows:	
  	
  

	
  

During	
  intense	
  exercise	
  horses	
  experience	
  elevations	
  in	
  cardiac	
  output	
  that	
  result	
  in	
  
elevated	
  pulmonary	
  artery,	
  left	
  atrial	
  and	
  pulmonary	
  capillary	
  pressures.	
  In	
  caudodorsal	
  
regions	
  of	
  lung	
  that	
  already	
  experience	
  highest	
  flow,	
  regional	
  differences	
  in	
  determinants	
  
of	
  arterial	
  and	
  venous	
  vessel	
  diameter	
  promote	
  even	
  higher	
  pulmonary	
  capillary	
  pressures.	
  
Pulmonary	
  capillary	
  breaking	
  strength	
  is	
  exceeded	
  resulting	
  in	
  stress	
  failure	
  of	
  some	
  
capillaries,	
  and	
  extravasation	
  of	
  red	
  cells	
  and	
  airway	
  hemorrhage.	
  During	
  both	
  training	
  and	
  
racing,	
  repeated	
  episodes	
  of	
  high	
  pulmonary	
  blood	
  flow	
  and	
  pressures,	
  particularly	
  in	
  the	
  
highest	
  flow	
  regions	
  within	
  caudodorsal	
  lung,	
  provide	
  adequate	
  hemodynamic	
  stimuli	
  to	
  
result	
  in	
  pulmonary	
  venous	
  remodeling.	
  Remodeled	
  pulmonary	
  veins	
  are	
  less	
  compliant	
  
than	
  normal	
  veins	
  and	
  failure	
  of	
  these	
  vessels	
  to	
  distend	
  normally	
  further	
  increases	
  
pulmonary	
  capillary	
  pressures.	
  Those	
  capillaries	
  that	
  are	
  drained	
  by	
  remodeled	
  veins	
  are	
  
even	
  more	
  susceptible	
  to	
  rupture	
  during	
  exercise	
  as	
  venous	
  wall	
  compliance	
  is	
  reduced	
  and	
  
in	
  some	
  cases,	
  venous	
  luminal	
  area	
  is	
  diminished.	
  With	
  each	
  exercise	
  bout	
  the	
  injurious	
  

	
  
133	
  

cycle	
  is	
  repeated	
  and	
  compounded,	
  ultimately	
  resulting	
  in	
  clinically	
  detectable	
  hemorrhage,	
  
significant	
  pulmonary	
  pathology,	
  and	
  potentially	
  impaired	
  performance.	
  
	
  

In	
  the	
  studies	
  outlined	
  in	
  this	
  dissertation	
  I	
  determined	
  that	
  regional	
  differences	
  in	
  

the	
  mechanical	
  characteristics	
  of	
  both	
  arteries	
  and	
  veins	
  in	
  control,	
  unraced	
  horses	
  exist.	
  
Specifically,	
  caudodorsal	
  arteries	
  are	
  stiffer	
  than	
  arteries	
  from	
  cranioventral	
  lung,	
  and	
  the	
  
converse	
  is	
  true	
  of	
  veins.	
  	
  These	
  differences	
  do	
  not	
  necessarily	
  predict	
  that	
  during	
  exercise,	
  
caudodorsal	
  pulmonary	
  capillaries	
  and	
  as	
  a	
  result,	
  veins	
  will	
  be	
  exposed	
  to	
  higher	
  
pressures	
  than	
  those	
  in	
  cranioventral	
  regions.	
  In	
  fact,	
  less	
  distensible	
  caudodorsal	
  arteries	
  
may	
  even	
  protect	
  caudodorsal	
  capillaries	
  somewhat	
  from	
  transmission	
  of	
  the	
  highest	
  
arterial	
  pressures	
  during	
  exercise,	
  and	
  more	
  compliant	
  veins	
  in	
  this	
  region	
  also	
  provide	
  a	
  
degree	
  of	
  capillary	
  protection.	
  	
  Of	
  particular	
  interest	
  in	
  this	
  study	
  however	
  was	
  the	
  
observation	
  that	
  pulmonary	
  veins	
  from	
  caudodorsal	
  lung	
  of	
  horses	
  that	
  had	
  raced,	
  but	
  had	
  
neither	
  a	
  clinical	
  history,	
  nor	
  pathologic	
  evidence	
  of	
  severe	
  EIPH,	
  became	
  significantly	
  
stiffer	
  than	
  those	
  veins	
  from	
  control,	
  unraced	
  horses.	
  Although	
  structural	
  components	
  of	
  
the	
  study	
  vessels	
  were	
  not	
  evaluated	
  specifically,	
  it	
  is	
  most	
  likely	
  that	
  collagen	
  deposition	
  
in	
  vein	
  walls	
  such	
  as	
  has	
  been	
  reported	
  in	
  other	
  studies	
  contributed	
  to	
  the	
  increase	
  in	
  
elastic	
  modulus	
  of	
  these	
  vessels.	
  These	
  are	
  the	
  first	
  data	
  to	
  demonstrate	
  possible	
  
physiologic	
  ramifications	
  of	
  venous	
  remodeling	
  in	
  EIPH,	
  namely	
  reduced	
  venous	
  
compliance.	
  	
  
	
  

While	
  these	
  data	
  support	
  the	
  contention	
  that	
  venous	
  remodeling	
  is	
  an	
  early	
  event	
  in	
  

EIPH	
  development,	
  a	
  two-­‐week	
  intermittent	
  exercise	
  stimuls	
  was	
  insufficient	
  to	
  induce	
  
changes	
  in	
  vein	
  wall	
  mRNA	
  that	
  would	
  support	
  initiation	
  of	
  remodeling	
  at	
  that	
  stage.	
  	
  

	
  
134	
  

	
  

The	
  autonomic	
  control	
  of	
  small	
  pulmonary	
  arteries	
  and	
  veins	
  also	
  demonstrates	
  a	
  

regionally	
  heterogeneous	
  pattern.	
  In	
  the	
  absence	
  of	
  any	
  α-­‐adrenoreceptor	
  mediated	
  
vasoconstriction,	
  β-­‐adrenergic	
  activity	
  is	
  expected	
  to	
  predominate	
  in	
  the	
  high	
  sympathetic	
  
outflow	
  conditions	
  experienced	
  during	
  exercise.	
  A	
  β-­‐adrenergic	
  agonist	
  failed	
  to	
  cause	
  
relaxation	
  of	
  precontracted	
  pulmonary	
  veins,	
  and	
  relaxed	
  pulmonary	
  arteries	
  from	
  
caudodorsal	
  lung	
  to	
  a	
  greater	
  degree	
  than	
  those	
  in	
  cranioventral	
  lung,	
  a	
  combination	
  that	
  
in	
  an	
  in	
  vivo	
  setting	
  could	
  act	
  to	
  cause	
  greater	
  capillary	
  pressures	
  in	
  caudodorsal	
  lung.	
  A	
  
muscarinic	
  agonist	
  caused	
  pulmonary	
  veins	
  and	
  caudodorsal	
  arteries	
  (mediated	
  by	
  both	
  
nitric	
  oxide	
  and	
  prostanoid	
  release)	
  to	
  relax,	
  whereas	
  caudodorsal	
  arteries	
  contracted.	
  In	
  
the	
  exercising	
  horse	
  and	
  in	
  the	
  absence	
  of	
  parasympathetic	
  input,	
  the	
  inverse	
  of	
  this	
  
pattern	
  could	
  serve	
  to	
  deliver	
  the	
  highest	
  pulmonary	
  capillary	
  and	
  venous	
  pressures	
  to	
  
caudodorsal	
  regions.	
  	
  
	
  

In	
  summary,	
  regional	
  difference	
  in	
  autonomic	
  control	
  of	
  small	
  pulmonary	
  arteries	
  

and	
  veins	
  support	
  the	
  theory	
  that	
  the	
  highest	
  pulmonary	
  capillary	
  and	
  venous	
  pressures	
  
occur	
  in	
  the	
  caudodorsal	
  lung	
  during	
  exercise.	
  Mechanical	
  characteristics	
  of	
  the	
  same	
  
vessels	
  appear	
  to	
  reflect	
  this	
  regional	
  pattern	
  and	
  in	
  fact,	
  vessel	
  wall	
  structure	
  may	
  “offset”	
  
these	
  projected	
  pressure	
  differences	
  somewhat.	
  Probably	
  as	
  a	
  result	
  of	
  vessel	
  
heterogeneity,	
  venous	
  remodeling	
  in	
  caudodorsal	
  regions	
  in	
  response	
  to	
  hemodynamic	
  
stimuli	
  associated	
  with	
  racing	
  reduces	
  venous	
  compliance	
  in	
  a	
  region-­‐dependent	
  manner,	
  
even	
  before	
  the	
  development	
  of	
  severe	
  pathology.	
  	
  
With	
  regard	
  to	
  future	
  directions	
  for	
  study,	
  at	
  this	
  time	
  I	
  consider	
  a	
  more	
  detailed	
  
characterization	
  of	
  the	
  reactivity	
  profiles	
  of	
  small	
  pulmonary	
  arteries	
  and	
  veins	
  of	
  the	
  
horse,	
  in	
  particular	
  responses	
  to	
  naturally	
  occurring	
  agonists	
  such	
  as	
  endothelin,	
  and	
  

	
  
135	
  

serotonin	
  a	
  priority.	
  This	
  approach	
  will	
  add	
  depth	
  to	
  current	
  understanding	
  of	
  in	
  vivo	
  
control	
  of	
  vessel	
  tone,	
  and	
  provide	
  direction	
  for	
  future	
  investigations	
  into	
  
pharmacotherapeutic	
  interventions	
  in	
  EIPH.	
  Although	
  furosemide	
  did	
  not	
  have	
  a	
  clinically	
  
relevant	
  effect	
  on	
  pulmonary	
  venous	
  tone,	
  testing	
  this	
  drug	
  on	
  larger	
  pulmonary	
  vessels,	
  
and	
  preliminary	
  investigations	
  into	
  other	
  venous	
  specific	
  vasoactive	
  agents,	
  for	
  example,	
  C-­‐
type	
  natriuretic	
  peptide	
  (209)	
  is	
  also	
  warranted.	
  Also,	
  detailed,	
  morphometric	
  
characterization	
  of	
  the	
  changes	
  in	
  vessel	
  wall	
  structure	
  that	
  result	
  in	
  altered	
  wall	
  
mechanical	
  properties	
  will	
  provide	
  another	
  layer	
  of	
  understanding	
  of	
  the	
  venous	
  
remodeling	
  process.	
  If	
  this	
  information	
  were	
  coupled	
  with	
  a	
  detailed	
  training/racing	
  
history	
  it	
  would	
  permit	
  elucidation	
  of	
  the	
  exact	
  nature	
  of	
  the	
  stimulus	
  required	
  for	
  
mild/moderate/severe	
  remodeling,	
  and	
  enable	
  exploration	
  of	
  non-­‐pharmacologic	
  
approaches	
  to	
  EIPH	
  management	
  along	
  the	
  lines	
  of	
  training	
  modifications	
  etc.	
  	
  
	
  

It	
  is	
  noteworthy	
  that	
  regional	
  differences	
  such	
  as	
  are	
  described	
  in	
  these	
  studies	
  have	
  

not	
  been	
  reported	
  in	
  the	
  pulmonary	
  microvasculature	
  of	
  other	
  species.	
  Whether	
  these	
  
differences	
  occur	
  in	
  other	
  species	
  certainly	
  merits	
  further	
  investigation	
  at	
  this	
  time.	
  I	
  
consider	
  it	
  unlikely	
  that	
  these	
  observations	
  are	
  unique	
  to	
  the	
  horse,	
  and	
  should	
  they	
  occur	
  
across	
  other	
  mammalian	
  species,	
  they	
  have	
  potential	
  for	
  application	
  in	
  a	
  wide	
  range	
  of	
  
contexts,	
  in	
  particular	
  in	
  the	
  study	
  of	
  pulmonary	
  vascular	
  pathology	
  including	
  pulmonary	
  
hypertension,	
  pulmonary	
  veno-­‐occlusive	
  disease	
  and	
  left	
  heart	
  failure,	
  all	
  of	
  which	
  are	
  
conditions	
  associated	
  with	
  high	
  morbidity	
  rates	
  (25,	
  122).	
  Indeed,	
  until	
  recently,	
  
pulmonary	
  veins	
  were	
  viewed	
  by	
  most	
  as	
  mere	
  “conduit	
  vessels”	
  and	
  as	
  a	
  result,	
  are	
  
relatively	
  understudied	
  (50).	
  	
  

	
  
136	
  

In	
  conclusion,	
  these	
  data	
  further	
  cement	
  the	
  role	
  of	
  the	
  pulmonary	
  vein	
  as	
  a	
  pivotal	
  
component	
  of	
  EIPH	
  pathology	
  and	
  shed	
  new	
  light	
  on	
  features	
  of	
  the	
  equine	
  pulmonary	
  
circulation	
  that	
  place	
  the	
  exercising	
  horse	
  at	
  risk	
  of	
  caudodorsal	
  capillary	
  rupture,	
  and	
  
EIPH.	
  Thus,	
  these	
  investigations	
  provide	
  evidence	
  that	
  support	
  a	
  new,	
  comprehensive	
  
theory	
  of	
  EIPH	
  pathogenesis,	
  a	
  framework	
  for	
  future	
  study	
  of	
  EIPH,	
  and	
  novel	
  contributions	
  
to	
  pulmonary	
  vascular	
  biology	
  literature.	
  	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
137	
  

	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
BIBLIOGRAPHY	
  

	
  
138	
  

	
  

BIBLIOGRAPHY	
  
	
  
	
  
1.	
  
General	
  stud	
  book,	
  containing	
  pedigrees	
  of	
  race	
  horses,	
  &c.,	
  from	
  the	
  earliest	
  accounts.	
  
Lond:	
  C.	
  &	
  J.	
  Weatherby,	
  1858,	
  p.	
  18v.	
  
2.	
  
Abeles	
  D,	
  Kwei	
  S,	
  Stavrakis	
  G,	
  Zhang	
  Y,	
  Wang	
  ET,	
  and	
  Garcia-­‐Cardena	
  G.	
  Gene	
  
expression	
  changes	
  evoked	
  in	
  a	
  venous	
  segment	
  exposed	
  to	
  arterial	
  flow.	
  J	
  Vasc	
  Surg	
  44:	
  
863-­‐870,	
  2006.	
  
3.	
  
Aitsebaomo	
  J,	
  Portbury	
  AL,	
  Schisler	
  JC,	
  and	
  Patterson	
  C.	
  Brothers	
  and	
  sisters:	
  
molecular	
  insights	
  into	
  arterial-­‐venous	
  heterogeneity.	
  Circ	
  Res	
  103:	
  929-­‐939,	
  2008.	
  
4.	
  
Akbar	
  SJ,	
  Derksen	
  FJ,	
  Billah	
  AM,	
  and	
  Werney	
  U.	
  Exercise	
  induced	
  pulmonary	
  
haemorrhage	
  in	
  racing	
  camels.	
  Vet	
  Rec	
  135:	
  624-­‐625,	
  1994.	
  
5.	
  
al-­‐Tinawi	
  A,	
  Clough	
  AV,	
  Harder	
  DR,	
  Linehan	
  JH,	
  Rickaby	
  DA,	
  and	
  Dawson	
  CA.	
  
Distensibility	
  of	
  small	
  veins	
  of	
  the	
  dog	
  lung.	
  Journal	
  of	
  applied	
  physiology	
  73:	
  2158-­‐2165,	
  
1992.	
  
6.	
  
Allen	
  D,	
  Jr.,	
  Clark	
  ES,	
  Moore	
  JN,	
  and	
  Prasse	
  KW.	
  Evaluation	
  of	
  equine	
  digital	
  
Starling	
  forces	
  and	
  hemodynamics	
  during	
  early	
  laminitis.	
  Am	
  J	
  Vet	
  Res	
  51:	
  1930-­‐1934,	
  
1990.	
  
7.	
  
Angus	
  JA,	
  and	
  Wright	
  CE.	
  Techniques	
  to	
  study	
  the	
  pharmacodynamics	
  of	
  isolated	
  
large	
  and	
  small	
  blood	
  vessels.	
  Journal	
  of	
  pharmacological	
  and	
  toxicological	
  methods	
  44:	
  
395-­‐407,	
  2000.	
  
8.	
  
Araya	
  O,	
  Palma	
  P,	
  Salvi	
  M,	
  Bustamante	
  H,	
  and	
  Vits	
  L.	
  Endoscopic	
  determination	
  
of	
  exercise-­‐induced	
  pulmonary	
  haemorrhage	
  in	
  Chilean	
  Criollo	
  horses.	
  Vet	
  J	
  169:	
  311-­‐313,	
  
2005.	
  
9.	
  
Banks	
  J,	
  Booth	
  FV,	
  MacKay	
  EH,	
  Rajagopalan	
  B,	
  and	
  Lee	
  GD.	
  The	
  physcial	
  
properties	
  of	
  human	
  pulmonary	
  arteries	
  and	
  veins.	
  Clinical	
  science	
  and	
  molecular	
  medicine	
  
55:	
  477-­‐484,	
  1978.	
  
10.	
   Barman	
  SA,	
  Senteno	
  E,	
  Smith	
  S,	
  and	
  Taylor	
  AE.	
  Acetylcholine's	
  effect	
  on	
  vascular	
  
resistance	
  and	
  compliance	
  in	
  the	
  pulmonary	
  circulation.	
  Journal	
  of	
  applied	
  physiology	
  67:	
  
1495-­‐1503,	
  1989.	
  
11.	
   Barnes	
  PJ,	
  and	
  Liu	
  SF.	
  Regulation	
  of	
  pulmonary	
  vascular	
  tone.	
  Pharmacological	
  
reviews	
  47:	
  87-­‐131,	
  1995.	
  
12.	
   Bashey	
  RI,	
  Cox	
  R,	
  McCann	
  J,	
  and	
  Jimenez	
  SA.	
  Changes	
  in	
  collagen	
  biosynthesis,	
  
types,	
  and	
  mechanics	
  of	
  aorta	
  in	
  hypertensive	
  rats.	
  J	
  Lab	
  Clin	
  Med	
  113:	
  604-­‐611,	
  1989.	
  

	
  
139	
  

13.	
   Benamou	
  AE,	
  Marlin	
  DJ,	
  and	
  Lekeux	
  P.	
  Equine	
  pulmonary	
  and	
  systemic	
  
haemodynamic	
  responses	
  to	
  endothelin-­‐1	
  and	
  a	
  selective	
  ET(A)	
  receptor	
  antagonist.	
  
Equine	
  veterinary	
  journal	
  33:	
  337-­‐344,	
  2001.	
  
14.	
   Berceli	
  SA,	
  Jiang	
  Z,	
  Klingman	
  NV,	
  Pfahnl	
  CL,	
  Abouhamze	
  ZS,	
  Frase	
  CD,	
  Schultz	
  
GS,	
  and	
  Ozaki	
  CK.	
  Differential	
  expression	
  and	
  activity	
  of	
  matrix	
  metalloproteinases	
  during	
  
flow-­‐modulated	
  vein	
  graft	
  remodeling.	
  J	
  Vasc	
  Surg	
  39:	
  1084-­‐1090,	
  2004.	
  
15.	
   Bernard	
  SL,	
  Glenny	
  RW,	
  Erickson	
  HH,	
  Fedde	
  MR,	
  Polissar	
  N,	
  Basaraba	
  RJ,	
  and	
  
Hlastala	
  MP.	
  Minimal	
  redistribution	
  of	
  pulmonary	
  blood	
  flow	
  with	
  exercise	
  in	
  racehorses.	
  J	
  
Appl	
  Physiol	
  81:	
  1062-­‐1070,	
  1996.	
  
16.	
   Birks	
  EK,	
  Mathieu-­‐Costello	
  O,	
  Fu	
  Z,	
  Tyler	
  WS,	
  and	
  West	
  JB.	
  Comparative	
  aspects	
  
of	
  the	
  strength	
  of	
  pulmonary	
  capillaries	
  in	
  rabbit,	
  dog,	
  and	
  horse.	
  Respir	
  Physiol	
  97:	
  235-­‐
246,	
  1994.	
  
17.	
   Birks	
  EK,	
  Mathieu-­‐Costello	
  O,	
  Fu	
  Z,	
  Tyler	
  WS,	
  and	
  West	
  JB.	
  Very	
  high	
  pressures	
  
are	
  required	
  to	
  cause	
  stress	
  failure	
  of	
  pulmonary	
  capillaries	
  in	
  thoroughbred	
  racehorses.	
  J	
  
Appl	
  Physiol	
  82:	
  1584-­‐1592,	
  1997.	
  
18.	
   Birks	
  EK,	
  Shuler	
  KM,	
  Soma	
  LR,	
  Martin	
  BB,	
  Marconato	
  L,	
  Del	
  Piero	
  F,	
  Teleis	
  DC,	
  
Schar	
  D,	
  Hessinger	
  AE,	
  and	
  Uboh	
  CE.	
  EIPH:	
  postrace	
  endoscopic	
  evaluation	
  of	
  
Standardbreds	
  and	
  Thoroughbreds.	
  Equine	
  Vet	
  J	
  Suppl	
  375-­‐378,	
  2002.	
  
19.	
   Bronquard	
  C,	
  Maupoil	
  V,	
  Arbeille	
  B,	
  Fetissof	
  F,	
  Findlay	
  I,	
  Cosnay	
  P,	
  and	
  Freslon	
  
JL.	
  Contractile	
  and	
  relaxant	
  properties	
  of	
  rat-­‐isolated	
  pulmonary	
  veins	
  related	
  to	
  
localization	
  and	
  histology.	
  Fundamental	
  &	
  clinical	
  pharmacology	
  21:	
  55-­‐65,	
  2007.	
  
20.	
   Burton	
  AC.	
  Relation	
  of	
  structure	
  to	
  function	
  of	
  the	
  tissues	
  of	
  the	
  wall	
  of	
  blood	
  
vessels.	
  Physiol	
  Rev	
  34:	
  619-­‐642,	
  1954.	
  
21.	
   Buus	
  CL,	
  Pourageaud	
  F,	
  Fazzi	
  GE,	
  Janssen	
  G,	
  Mulvany	
  MJ,	
  and	
  De	
  Mey	
  JG.	
  
Smooth	
  muscle	
  cell	
  changes	
  during	
  flow-­‐related	
  remodeling	
  of	
  rat	
  mesenteric	
  resistance	
  
arteries.	
  Circ	
  Res	
  89:	
  180-­‐186,	
  2001.	
  
22.	
   Caro	
  CG,	
  and	
  Saffman	
  PG.	
  Extensibility	
  of	
  Blood	
  Vessels	
  in	
  Isolated	
  Rabbit	
  Lungs.	
  J	
  
Physiol	
  178:	
  193-­‐210,	
  1965.	
  
23.	
   Castro	
  MM,	
  Rizzi	
  E,	
  Prado	
  CM,	
  Rossi	
  MA,	
  Tanus-­‐Santos	
  JE,	
  and	
  Gerlach	
  RF.	
  
Imbalance	
  between	
  matrix	
  metalloproteinases	
  and	
  tissue	
  inhibitor	
  of	
  metalloproteinases	
  in	
  
hypertensive	
  vascular	
  remodeling.	
  Matrix	
  Biol	
  29:	
  194-­‐201,	
  2010.	
  
24.	
   Chay	
  S,	
  Woods	
  WE,	
  Rowse	
  K,	
  Nugent	
  TE,	
  Blake	
  JW,	
  and	
  Tobin	
  T.	
  The	
  
pharmacology	
  of	
  furosemide	
  in	
  the	
  horse.	
  V.	
  Pharmacokinetics	
  and	
  blood	
  levels	
  of	
  
furosemide	
  after	
  intravenous	
  administration.	
  Drug	
  Metab	
  Dispos	
  11:	
  226-­‐231,	
  1983.	
  

	
  
140	
  

25.	
   Chazova	
  I,	
  Robbins	
  I,	
  Loyd	
  J,	
  Newman	
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role	
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  Gravity	
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but	
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57.	
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determinant	
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remodeling.	
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68.	
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  Sex	
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pulmonary	
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69.	
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  O'Callaghan	
  PA,	
  McCaffrey	
  JP,	
  
Morley	
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  Slocombe	
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  AF.	
  Tracheobronchoscopic	
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induced	
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70.	
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  Morley	
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  Brown	
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  Dredge	
  AE,	
  O'Callaghan	
  PA,	
  
McCaffrey	
  JP,	
  Slocombe	
  RE,	
  and	
  Clarke	
  AE.	
  Association	
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pulmonary	
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71.	
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  KH.	
  Fluid	
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  the	
  
haemodynamic	
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  of	
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72.	
   Hinchcliff	
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plasma	
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73.	
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  Use	
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pulmonary	
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74.	
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exercise-­‐induced	
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235:	
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75.	
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  Jackson	
  MA,	
  Brown	
  JA,	
  Dredge	
  AF,	
  O'Callaghan	
  PA,	
  
McCaffrey	
  JP,	
  Slocombe	
  RF,	
  and	
  Clarke	
  AF.	
  Risk	
  factors	
  for	
  exercise-­‐induced	
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haemorrhage	
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76.	
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  Bernard	
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  Erickson	
  HH,	
  Fedde	
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  Gaughan	
  EM,	
  McMurphy	
  R,	
  
Emery	
  MJ,	
  Polissar	
  N,	
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  Pulmonary	
  blood	
  flow	
  distribution	
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horses	
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144	
  

77.	
   Hlastala	
  MP,	
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  RW.	
  Vascular	
  structure	
  determines	
  pulmonary	
  blood	
  flow	
  
distribution.	
  News	
  in	
  physiological	
  sciences	
  :	
  an	
  international	
  journal	
  of	
  physiology	
  produced	
  
jointly	
  by	
  the	
  International	
  Union	
  of	
  Physiological	
  Sciences	
  and	
  the	
  American	
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Society	
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78.	
   Holcomb	
  BW,	
  Jr.,	
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  JE,	
  Ely	
  EW,	
  Johnson	
  J,	
  and	
  Robbins	
  IM.	
  Pulmonary	
  veno-­‐
occlusive	
  disease:	
  a	
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  Chest	
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79.	
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  Spragg	
  RG,	
  Martin	
  TR,	
  and	
  West	
  JB.	
  
Intense	
  exercise	
  impairs	
  the	
  integrity	
  of	
  the	
  pulmonary	
  blood-­‐gas	
  barrier	
  in	
  elite	
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Am	
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80.	
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  the	
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pulmonary	
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  Journal	
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81.	
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  B,	
  Dorfmuller	
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  D,	
  Humbert	
  M,	
  and	
  Montani	
  D.	
  
Pulmonary	
  veno-­‐occlusive	
  disease:	
  advances	
  in	
  clinical	
  management	
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Expert	
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82.	
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  JM,	
  Bethea	
  B,	
  Liu	
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  PP,	
  Stacher	
  E,	
  Gandjeva	
  MR,	
  
Parish	
  E,	
  Perez	
  M,	
  Smith	
  L,	
  Graham	
  BB,	
  Kuebler	
  WM,	
  and	
  Tuder	
  RM.	
  Pulmonary	
  veins	
  
in	
  the	
  normal	
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  and	
  pulmonary	
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  due	
  to	
  left	
  heart	
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  Am	
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  Physiol	
  Lung	
  
Cell	
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83.	
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  Autonomic	
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  of	
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circulation.	
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84.	
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  Raj	
  JU.	
  Heterogeneity	
  in	
  prostacyclin	
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  thromboxane	
  
synthesis	
  in	
  ovine	
  pulmonary	
  vascular	
  tree:	
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22:	
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  1996.	
  
85.	
   Jiang	
  Z,	
  Tao	
  M,	
  Omalley	
  KA,	
  Wang	
  D,	
  Ozaki	
  CK,	
  and	
  Berceli	
  SA.	
  Established	
  
neointimal	
  hyperplasia	
  in	
  vein	
  grafts	
  expands	
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  progressive	
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Am	
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86.	
   Johnson	
  JE,	
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  Pulmonary	
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  and	
  bronchial	
  vessels	
  
undergo	
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  in	
  sustained	
  pulmonary	
  hypertension	
  induced	
  by	
  continuous	
  air	
  
embolization	
  into	
  sheep.	
  Exp	
  Lung	
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  1997.	
  
87.	
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  LR,	
  Parker	
  JL,	
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  MH.	
  Chronic	
  exercise	
  training	
  improves	
  ACh-­‐
induced	
  vasorelaxation	
  in	
  pulmonary	
  arteries	
  of	
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  Journal	
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451,	
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88.	
   Jones	
  JH,	
  Smith	
  BL,	
  Birks	
  EK,	
  Pascoe	
  JR,	
  and	
  Hughes	
  TR.	
  LEFT	
  ATRIAL	
  AND	
  
PULMONARY	
  ARTERIAL	
  PRESSURES	
  IN	
  EXERCISING	
  HORSES.	
  Faseb	
  Journal	
  6:	
  A2020-­‐
A2020,	
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145	
  

89.	
   Jones	
  PL,	
  Crack	
  J,	
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  Regulation	
  of	
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  a	
  vascular	
  smooth	
  
muscle	
  cell	
  survival	
  factor	
  that	
  interacts	
  with	
  the	
  alpha	
  v	
  beta	
  3	
  integrin	
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epidermal	
  growth	
  factor	
  receptor	
  phosphorylation	
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  J	
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1997.	
  
90.	
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  Bing	
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  Jianguang	
  Q,	
  and	
  Chaoshu	
  T.	
  Effect	
  of	
  L-­‐arginine	
  
on	
  collagen	
  of	
  high	
  flow-­‐induced	
  pulmonary	
  arterial	
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  Circulation	
  journal	
  :	
  
official	
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91.	
   Kadowitz	
  PJ,	
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  Hyman	
  AL.	
  Effect	
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  stimulation	
  on	
  
pulmonary	
  vascular	
  resistance	
  in	
  the	
  dog.	
  Circ	
  Res	
  32:	
  221-­‐227,	
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92.	
   Kalish	
  JA,	
  Willis	
  DJ,	
  Li	
  C,	
  Link	
  JJ,	
  Deutsch	
  ER,	
  Contreras	
  MA,	
  Quist	
  WC,	
  and	
  
Logerfo	
  FW.	
  Temporal	
  genomics	
  of	
  vein	
  bypass	
  grafting	
  through	
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microarray	
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93.	
   Kane	
  DW,	
  Tesauro	
  T,	
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  JH.	
  Adrenergic	
  modulation	
  of	
  the	
  pulmonary	
  
circulation	
  during	
  strenuous	
  exercise	
  in	
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  Am	
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94.	
   Karau	
  KL,	
  Johnson	
  RH,	
  Molthen	
  RC,	
  Dhyani	
  AH,	
  Haworth	
  ST,	
  Hanger	
  CC,	
  Roerig	
  
DL,	
  and	
  Dawson	
  CA.	
  Microfocal	
  X-­‐ray	
  CT	
  imaging	
  and	
  pulmonary	
  arterial	
  distensibility	
  in	
  
excised	
  rat	
  lungs.	
  Am	
  J	
  Physiol	
  Heart	
  Circ	
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95.	
   Kay	
  JM.	
  Comparative	
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  features	
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  the	
  pulmonary	
  vasculature	
  in	
  
mammals.	
  Am	
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  Dis	
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  1983.	
  
96.	
   Kedzierski	
  RM,	
  and	
  Yanagisawa	
  M.	
  Endothelin	
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  the	
  double-­‐edged	
  sword	
  in	
  
health	
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  Annual	
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  2001.	
  
97.	
   Keen	
  JA,	
  Hillier	
  C,	
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  Bunton	
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  JE.	
  Evaluation	
  of	
  equine	
  
laminar	
  vein	
  function:	
  harvesting,	
  dissection	
  and	
  the	
  use	
  of	
  functional	
  methods	
  to	
  
distinguish	
  between	
  veins	
  and	
  arteries.	
  Journal	
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  and	
  toxicological	
  
methods	
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  2008.	
  
98.	
   Kelly	
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  Hall	
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  Haworth	
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  Relationship	
  between	
  structural	
  
remodeling	
  and	
  reactivity	
  in	
  pulmonary	
  resistance	
  arteries	
  from	
  hypertensive	
  piglets.	
  
Pediatric	
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  58:	
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  2005.	
  
99.	
   Kemp	
  BK,	
  Smolich	
  JJ,	
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  Cocks	
  TM.	
  Evidence	
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  specific	
  regional	
  patterns	
  of	
  
responses	
  to	
  different	
  vasoconstrictors	
  and	
  vasodilators	
  in	
  sheep	
  isolated	
  pulmonary	
  
arteries	
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  Br	
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100.	
   Kindig	
  CA,	
  McDonough	
  P,	
  Finley	
  MR,	
  Behnke	
  BJ,	
  Richardson	
  TE,	
  Marlin	
  DJ,	
  
Erickson	
  HH,	
  and	
  Poole	
  DC.	
  NO	
  inhalation	
  reduces	
  pulmonary	
  arterial	
  pressure	
  but	
  not	
  
hemorrhage	
  in	
  maximally	
  exercising	
  horses.	
  Journal	
  of	
  applied	
  physiology	
  91:	
  2674-­‐2678,	
  
2001.	
  

	
  
146	
  

101.	
   Kobs	
  RW,	
  Muvarak	
  NE,	
  Eickhoff	
  JC,	
  and	
  Chesler	
  NC.	
  Linked	
  mechanical	
  and	
  
biological	
  aspects	
  of	
  remodeling	
  in	
  mouse	
  pulmonary	
  arteries	
  with	
  hypoxia-­‐induced	
  
hypertension.	
  Am	
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  Physiol	
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  2005.	
  
102.	
   Krenz	
  GS,	
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  Dawson	
  CA.	
  Flow	
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  pressure	
  distributions	
  in	
  vascular	
  networks	
  
consisting	
  of	
  distensible	
  vessels.	
  Am	
  J	
  Physiol	
  Heart	
  Circ	
  Physiol	
  284:	
  H2192-­‐2203,	
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103.	
   Kuhl	
  PG,	
  Bolds	
  JM,	
  Loyd	
  JE,	
  Snapper	
  JR,	
  and	
  FitzGerald	
  GA.	
  Thromboxane	
  
receptor-­‐mediated	
  bronchial	
  and	
  hemodynamic	
  responses	
  in	
  ovine	
  endotoxemia.	
  Am	
  J	
  
Physiol	
  254:	
  R310-­‐319,	
  1988.	
  
104.	
   Lakatta	
  EG.	
  Age-­‐associated	
  cardiovascular	
  changes	
  in	
  health:	
  impact	
  on	
  
cardiovascular	
  disease	
  in	
  older	
  persons.	
  Heart	
  failure	
  reviews	
  7:	
  29-­‐49,	
  2002.	
  
105.	
   Lambers	
  C,	
  Roth	
  M,	
  Zhong	
  J,	
  Campregher	
  C,	
  Binder	
  P,	
  Burian	
  B,	
  Petkov	
  V,	
  and	
  
Block	
  LH.	
  The	
  interaction	
  of	
  endothelin-­‐1	
  and	
  TGF-­‐beta1	
  mediates	
  vascular	
  cell	
  
remodeling.	
  PLoS	
  One	
  8:	
  e73399,	
  2013.	
  
106.	
   Langsetmo	
  I,	
  Meyer	
  MR,	
  and	
  Erickson	
  HH.	
  Relationship	
  of	
  pulmonary	
  arterial	
  
pressure	
  to	
  pulmonary	
  haemorrhage	
  in	
  exercising	
  horses.	
  Equine	
  veterinary	
  journal	
  32:	
  
379-­‐384,	
  2000.	
  
107.	
   Lapointe	
  JM,	
  Vrins	
  A,	
  and	
  McCarvill	
  E.	
  A	
  survey	
  of	
  exercise-­‐induced	
  pulmonary	
  
haemorrhage	
  in	
  Quebec	
  standardbred	
  racehorses.	
  Equine	
  veterinary	
  journal	
  26:	
  482-­‐485,	
  
1994.	
  
108.	
   Leach	
  RM,	
  Twort	
  CH,	
  Cameron	
  IR,	
  and	
  Ward	
  JP.	
  A	
  comparison	
  of	
  the	
  
pharmacological	
  and	
  mechanical	
  properties	
  in	
  vitro	
  of	
  large	
  and	
  small	
  pulmonary	
  arteries	
  
of	
  the	
  rat.	
  Clin	
  Sci	
  (Lond)	
  82:	
  55-­‐62,	
  1992.	
  
109.	
   Lehoux	
  S,	
  Lemarie	
  CA,	
  Esposito	
  B,	
  Lijnen	
  HR,	
  and	
  Tedgui	
  A.	
  Pressure-­‐induced	
  
matrix	
  metalloproteinase-­‐9	
  contributes	
  to	
  early	
  hypertensive	
  remodeling.	
  Circulation	
  109:	
  
1041-­‐1047,	
  2004.	
  
110.	
   Li	
  Z,	
  Froehlich	
  J,	
  Galis	
  ZS,	
  and	
  Lakatta	
  EG.	
  Increased	
  expression	
  of	
  matrix	
  
metalloproteinase-­‐2	
  in	
  the	
  thickened	
  intima	
  of	
  aged	
  rats.	
  Hypertension	
  33:	
  116-­‐123,	
  1999.	
  
111.	
   Lichtinghagen	
  R,	
  Musholt	
  PB,	
  Lein	
  M,	
  Romer	
  A,	
  Rudolph	
  B,	
  Kristiansen	
  G,	
  
Hauptmann	
  S,	
  Schnorr	
  D,	
  Loening	
  SA,	
  and	
  Jung	
  K.	
  Different	
  mRNA	
  and	
  protein	
  
expression	
  of	
  matrix	
  metalloproteinases	
  2	
  and	
  9	
  and	
  tissue	
  inhibitor	
  of	
  metalloproteinases	
  
1	
  in	
  benign	
  and	
  malignant	
  prostate	
  tissue.	
  Eur	
  Urol	
  42:	
  398-­‐406,	
  2002.	
  
112.	
   Liu	
  SQ,	
  and	
  Fung	
  YC.	
  Changes	
  in	
  the	
  organization	
  of	
  the	
  smooth	
  muscle	
  cells	
  in	
  rat	
  
vein	
  grafts.	
  Ann	
  Biomed	
  Eng	
  26:	
  86-­‐95,	
  1998.	
  
113.	
   Livak	
  KJ,	
  and	
  Schmittgen	
  TD.	
  Analysis	
  of	
  relative	
  gene	
  expression	
  data	
  using	
  real-­‐
time	
  quantitative	
  PCR	
  and	
  the	
  2(-­‐Delta	
  Delta	
  C(T))	
  Method.	
  Methods	
  25:	
  402-­‐408,	
  2001.	
  

	
  
147	
  

114.	
   Lovering	
  AT,	
  Romer	
  LM,	
  Haverkamp	
  HC,	
  Pegelow	
  DF,	
  Hokanson	
  JS,	
  and	
  
Eldridge	
  MW.	
  Intrapulmonary	
  shunting	
  and	
  pulmonary	
  gas	
  exchange	
  during	
  normoxic	
  and	
  
hypoxic	
  exercise	
  in	
  healthy	
  humans.	
  Journal	
  of	
  applied	
  physiology	
  104:	
  1418-­‐1425,	
  2008.	
  
115.	
   Lugo	
  J,	
  Stick	
  JA,	
  Peroni	
  J,	
  Harkema	
  JR,	
  Derksen	
  FJ,	
  and	
  Robinson	
  NE.	
  Safety	
  and	
  
efficacy	
  of	
  a	
  technique	
  for	
  thoracoscopically	
  guided	
  pulmonary	
  wedge	
  resection	
  in	
  horses.	
  
Am	
  J	
  Vet	
  Res	
  63:	
  1232-­‐1240,	
  2002.	
  
116.	
   Lundergan	
  CF,	
  Fitzpatrick	
  TM,	
  Rose	
  JC,	
  Ramwell	
  PW,	
  and	
  Kot	
  PA.	
  Effect	
  of	
  
cyclooxygenase	
  inhibition	
  on	
  the	
  pulmonary	
  vasodilator	
  response	
  to	
  furosemide.	
  J	
  
Pharmacol	
  Exp	
  Ther	
  246:	
  102-­‐106,	
  1988.	
  
117.	
   MacEachern	
  KE,	
  Smith	
  GL,	
  and	
  Nolan	
  AM.	
  Characteristics	
  of	
  the	
  in	
  vitro	
  hypoxic	
  
pulmonary	
  vasoconstrictor	
  response	
  in	
  isolated	
  equine	
  and	
  bovine	
  pulmonary	
  arterial	
  
rings.	
  Veterinary	
  anaesthesia	
  and	
  analgesia	
  31:	
  239-­‐249,	
  2004.	
  
118.	
   Machado	
  RD,	
  Aldred	
  MA,	
  James	
  V,	
  Harrison	
  RE,	
  Patel	
  B,	
  Schwalbe	
  EC,	
  Gruenig	
  
E,	
  Janssen	
  B,	
  Koehler	
  R,	
  Seeger	
  W,	
  Eickelberg	
  O,	
  Olschewski	
  H,	
  Elliott	
  CG,	
  Glissmeyer	
  
E,	
  Carlquist	
  J,	
  Kim	
  M,	
  Torbicki	
  A,	
  Fijalkowska	
  A,	
  Szewczyk	
  G,	
  Parma	
  J,	
  Abramowicz	
  MJ,	
  
Galie	
  N,	
  Morisaki	
  H,	
  Kyotani	
  S,	
  Nakanishi	
  N,	
  Morisaki	
  T,	
  Humbert	
  M,	
  Simonneau	
  G,	
  
Sitbon	
  O,	
  Soubrier	
  F,	
  Coulet	
  F,	
  Morrell	
  NW,	
  and	
  Trembath	
  RC.	
  Mutations	
  of	
  the	
  TGF-­‐
beta	
  type	
  II	
  receptor	
  BMPR2	
  in	
  pulmonary	
  arterial	
  hypertension.	
  Human	
  mutation	
  27:	
  121-­‐
132,	
  2006.	
  
119.	
   Mackay	
  EH,	
  Banks	
  J,	
  Sykes	
  B,	
  and	
  Lee	
  G.	
  Structural	
  basis	
  for	
  the	
  changing	
  physical	
  
properties	
  of	
  human	
  pulmonary	
  vessels	
  with	
  age.	
  Thorax	
  33:	
  335-­‐344,	
  1978.	
  
120.	
   Malmstrom	
  J,	
  Lindberg	
  H,	
  Lindberg	
  C,	
  Bratt	
  C,	
  Wieslander	
  E,	
  Delander	
  EL,	
  
Sarnstrand	
  B,	
  Burns	
  JS,	
  Mose-­‐Larsen	
  P,	
  Fey	
  S,	
  and	
  Marko-­‐Varga	
  G.	
  Transforming	
  
growth	
  factor-­‐beta	
  1	
  specifically	
  induce	
  proteins	
  involved	
  in	
  the	
  myofibroblast	
  contractile	
  
apparatus.	
  Molecular	
  &	
  cellular	
  proteomics	
  :	
  MCP	
  3:	
  466-­‐477,	
  2004.	
  
121.	
   Maloney	
  JE,	
  Rooholamini	
  SA,	
  and	
  Wexler	
  L.	
  Pressure-­‐diameter	
  relations	
  of	
  small	
  
blood	
  vessels	
  in	
  isolated	
  dog	
  lung.	
  Microvascular	
  research	
  2:	
  1-­‐12,	
  1970.	
  
122.	
   Mandel	
  J,	
  Mark	
  EJ,	
  and	
  Hales	
  CA.	
  Pulmonary	
  veno-­‐occlusive	
  disease.	
  Am	
  J	
  Respir	
  
Crit	
  Care	
  Med	
  162:	
  1964-­‐1973,	
  2000.	
  
123.	
   Manohar	
  M.	
  Furosemide	
  attenuates	
  the	
  exercise-­‐induced	
  increase	
  in	
  pulmonary	
  
artery	
  wedge	
  pressure	
  in	
  horses.	
  Am	
  J	
  Vet	
  Res	
  54:	
  952-­‐958,	
  1993.	
  
124.	
   Manohar	
  M.	
  Pulmonary	
  artery	
  wedge	
  pressure	
  increases	
  with	
  high-­‐intensity	
  
exercise	
  in	
  horses.	
  Am	
  J	
  Vet	
  Res	
  54:	
  142-­‐146,	
  1993.	
  
125.	
   Manohar	
  M,	
  and	
  Goetz	
  TE.	
  Intrapulmonary	
  arteriovenous	
  shunts	
  of	
  >15	
  microm	
  in	
  
diameter	
  probably	
  do	
  not	
  contribute	
  to	
  arterial	
  hypoxemia	
  in	
  maximally	
  exercising	
  
Thoroughbred	
  horses.	
  Journal	
  of	
  applied	
  physiology	
  99:	
  224-­‐229,	
  2005.	
  

	
  
148	
  

126.	
   Manohar	
  M,	
  and	
  Goetz	
  TE.	
  L-­‐NAME	
  does	
  not	
  affect	
  exercise-­‐induced	
  pulmonary	
  
hypertension	
  in	
  thoroughbred	
  horses.	
  Journal	
  of	
  applied	
  physiology	
  84:	
  1902-­‐1908,	
  1998.	
  
127.	
   Manohar	
  M,	
  and	
  Goetz	
  TE.	
  Pulmonary	
  vascular	
  pressures	
  of	
  exercising	
  
thoroughbred	
  horses	
  with	
  and	
  without	
  endoscopic	
  evidence	
  of	
  EIPH.	
  J	
  Appl	
  Physiol	
  81:	
  
1589-­‐1593,	
  1996.	
  
128.	
   Manohar	
  M,	
  and	
  Goetz	
  TE.	
  Pulmonary	
  vascular	
  resistance	
  of	
  horses	
  decreases	
  with	
  
moderate	
  exercise	
  and	
  remains	
  unchanged	
  as	
  workload	
  is	
  increased	
  to	
  maximal	
  exercise.	
  
Equine	
  Vet	
  J	
  Suppl	
  117-­‐121,	
  1999.	
  
129.	
   Manohar	
  M,	
  Goetz	
  TE,	
  Sullivan	
  E,	
  and	
  Griffin	
  R.	
  Pulmonary	
  vascular	
  pressures	
  of	
  
strenuously	
  exercising	
  Thoroughbreds	
  after	
  administration	
  of	
  varying	
  doses	
  of	
  frusemide.	
  
Equine	
  veterinary	
  journal	
  29:	
  298-­‐304,	
  1997.	
  
130.	
   Markham	
  G.	
  Markham's	
  masterpiece	
  containing	
  all	
  knowledge	
  belonging	
  to	
  the	
  
smith,	
  farrier,	
  or	
  horse-­‐leech,	
  touching	
  the	
  curing	
  of	
  all	
  diseases	
  in	
  horses	
  ...	
  being	
  divided	
  into	
  
two	
  books,	
  the	
  first	
  containing	
  all	
  cures	
  physical,	
  the	
  second	
  all	
  belonging	
  to	
  chirurgery.	
  
London:	
  Printed	
  at	
  London	
  by	
  Edward	
  Okes,	
  and	
  are	
  to	
  be	
  sold	
  by	
  Thomas	
  Passenger	
  ...	
  
1668,	
  p.	
  13	
  ,	
  591,	
  523	
  p.	
  
131.	
   McKane	
  SA,	
  Canfield	
  PJ,	
  and	
  Rose	
  RJ.	
  Equine	
  bronchoalveolar	
  lavage	
  cytology:	
  
survey	
  of	
  thoroughbred	
  racehorses	
  in	
  training.	
  Australian	
  veterinary	
  journal	
  70:	
  401-­‐404,	
  
1993.	
  
132.	
   McLaughlin	
  RF,	
  Tyler	
  WS,	
  and	
  Canada	
  RO.	
  A	
  study	
  of	
  the	
  subgross	
  pulmonary	
  
anatomy	
  in	
  various	
  mammals.	
  American	
  Journal	
  of	
  Anatomy	
  108:	
  149-­‐165,	
  1961.	
  
133.	
   Merkus	
  D,	
  de	
  Beer	
  VJ,	
  Houweling	
  B,	
  and	
  Duncker	
  DJ.	
  Control	
  of	
  pulmonary	
  
vascular	
  tone	
  during	
  exercise	
  in	
  health	
  and	
  pulmonary	
  hypertension.	
  Pharmacology	
  &	
  
therapeutics	
  119:	
  242-­‐263,	
  2008.	
  
134.	
   Michel	
  RP.	
  Arteries	
  and	
  veins	
  of	
  the	
  normal	
  dog	
  lung:	
  qualitative	
  and	
  quantitative	
  
structural	
  differences.	
  The	
  American	
  journal	
  of	
  anatomy	
  164:	
  227-­‐241,	
  1982.	
  
135.	
   Mills	
  PC,	
  Marlin	
  DJ,	
  Demoncheaux	
  E,	
  Scott	
  C,	
  Casas	
  I,	
  Smith	
  NC,	
  and	
  
Higenbottam	
  T.	
  Nitric	
  oxide	
  and	
  exercise	
  in	
  the	
  horse.	
  J	
  Physiol	
  495	
  (	
  Pt	
  3):	
  863-­‐874,	
  1996.	
  
136.	
   Miyauchi	
  T,	
  and	
  Masaki	
  T.	
  Pathophysiology	
  of	
  endothelin	
  in	
  the	
  cardiovascular	
  
system.	
  Annu	
  Rev	
  Physiol	
  61:	
  391-­‐415,	
  1999.	
  
137.	
   Moses	
  VS,	
  and	
  Bertone	
  AL.	
  Nonsteroidal	
  anti-­‐inflammatory	
  drugs.	
  The	
  Veterinary	
  
clinics	
  of	
  North	
  America	
  Equine	
  practice	
  18:	
  21-­‐37,	
  v,	
  2002.	
  
138.	
   Mulvany	
  MJ,	
  and	
  Halpern	
  W.	
  Contractile	
  properties	
  of	
  small	
  arterial	
  resistance	
  
vessels	
  in	
  spontaneously	
  hypertensive	
  and	
  normotensive	
  rats.	
  Circ	
  Res	
  41:	
  19-­‐26,	
  1977.	
  

	
  
149	
  

139.	
   Mulvany	
  MJ,	
  and	
  Halpern	
  W.	
  Mechanical	
  properties	
  of	
  vascular	
  smooth	
  muscle	
  
cells	
  in	
  situ.	
  Nature	
  260:	
  617-­‐619,	
  1976.	
  
140.	
   Murray	
  PA,	
  Lodato	
  RF,	
  and	
  Michael	
  JR.	
  Neural	
  antagonists	
  modulate	
  pulmonary	
  
vascular	
  pressure-­‐flow	
  plots	
  in	
  conscious	
  dogs.	
  Journal	
  of	
  applied	
  physiology	
  60:	
  1900-­‐
1907,	
  1986.	
  
141.	
   Naeije	
  R,	
  and	
  Chesler	
  N.	
  Pulmonary	
  circulation	
  at	
  exercise.	
  Comprehensive	
  
Physiology	
  2:	
  711-­‐741,	
  2012.	
  
142.	
   Nagase	
  H,	
  Visse	
  R,	
  and	
  Murphy	
  G.	
  Structure	
  and	
  function	
  of	
  matrix	
  
metalloproteinases	
  and	
  TIMPs.	
  Cardiovasc	
  Res	
  69:	
  562-­‐573,	
  2006.	
  
143.	
   Nandiwada	
  PA,	
  Hyman	
  AL,	
  and	
  Kadowitz	
  PJ.	
  Pulmonary	
  vasodilator	
  responses	
  to	
  
vagal	
  stimulation	
  and	
  acetylcholine	
  in	
  the	
  cat.	
  Circ	
  Res	
  53:	
  86-­‐95,	
  1983.	
  
144.	
   Newman	
  JH,	
  Butka	
  BJ,	
  and	
  Brigham	
  KL.	
  Thromboxane	
  A2	
  and	
  prostacyclin	
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  not	
  
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150	
  

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carotid	
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and	
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responses	
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Cool	
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Expression	
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stress)-­‐mediated	
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  and	
  Tuder	
  RM.	
  Vascular	
  endothelial	
  growth	
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the	
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220.	
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  CR.	
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and	
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221.	
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  JP,	
  Druml	
  T,	
  and	
  Brem	
  G.	
  Identification	
  
of	
  genetic	
  variation	
  on	
  the	
  horse	
  y	
  chromosome	
  and	
  the	
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  of	
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  lineages	
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modern	
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222.	
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  Sharifi	
  BG.	
  Arterialization	
  of	
  human	
  
vein	
  grafts	
  is	
  associated	
  with	
  tenascin-­‐C	
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223.	
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  H,	
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  Wilcox	
  JN.	
  Adventitial	
  
remodeling	
  after	
  angioplasty	
  is	
  associated	
  with	
  expression	
  of	
  tenascin	
  mRNA	
  by	
  adventitial	
  
myofibroblasts.	
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224.	
   Wan	
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  Yim	
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  JL,	
  Shukla	
  N,	
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  GD,	
  Smith	
  FC,	
  Dashwood	
  MR,	
  and	
  
Jeremy	
  JY.	
  The	
  endothelin	
  1A	
  receptor	
  antagonist	
  BSF	
  302146	
  is	
  a	
  potent	
  inhibitor	
  of	
  

	
  
156	
  

neointimal	
  and	
  medial	
  thickening	
  in	
  porcine	
  saphenous	
  vein-­‐carotid	
  artery	
  interposition	
  
grafts.	
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225.	
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  B,	
  Pervaiz	
  MH,	
  Watson	
  RE,	
  and	
  Fink	
  
GD.	
  Morphological	
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  biochemical	
  characterization	
  of	
  remodeling	
  in	
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  vena	
  cava	
  
of	
  DOCA-­‐salt	
  hypertensive	
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226.	
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  Epistaxis	
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exercise-­‐induced	
  pulmonary	
  haemorrhage	
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South	
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227.	
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  P,	
  Shoshani	
  O,	
  Hirschhorn	
  G,	
  
and	
  Margulis	
  A.	
  Pulmonary	
  oedema	
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  haemoptysis	
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Bmj	
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228.	
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  J,	
  Stolle	
  V,	
  Sasse	
  P,	
  Roll	
  W,	
  
Breuer	
  J,	
  and	
  Fleischmann	
  BK.	
  beta(2)-­‐adrenoceptor	
  antagonist	
  ICI	
  118,551	
  decreases	
  
pulmonary	
  vascular	
  tone	
  in	
  mice	
  via	
  a	
  G(i/o)	
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  oxide-­‐coupled	
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Hypertension	
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229.	
   West	
  JB,	
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  O.	
  Stress	
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  capillaries	
  as	
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mechanism	
  for	
  exercise	
  induced	
  pulmonary	
  haemorrhage	
  in	
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  horse.	
  Equine	
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journal	
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230.	
   West	
  JB,	
  Mathieu-­‐Costello	
  O,	
  Jones	
  JH,	
  Birks	
  EK,	
  Logemann	
  RB,	
  Pascoe	
  JR,	
  and	
  
Tyler	
  WS.	
  Stress	
  failure	
  of	
  pulmonary	
  capillaries	
  in	
  racehorses	
  with	
  exercise-­‐induced	
  
pulmonary	
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231.	
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  KJ,	
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  H,	
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  RR,	
  Steel	
  CM,	
  and	
  
Robinson	
  NE.	
  Regional	
  pulmonary	
  veno-­‐occlusion:	
  a	
  newly	
  identified	
  lesion	
  of	
  equine	
  
exercise-­‐induced	
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232.	
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  KJ,	
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  Defeijter-­‐Rupp	
  HL,	
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  Robinson	
  NE.	
  Repeated	
  blood	
  
instillation	
  into	
  the	
  airway	
  of	
  the	
  horse	
  does	
  not	
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veterinary	
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233.	
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  NE,	
  Defeijter-­‐Rupp	
  H,	
  Millerick-­‐May	
  M,	
  Stack	
  A,	
  
Hauptman	
  J,	
  and	
  Derksen	
  FJ.	
  Distribution	
  of	
  venous	
  remodeling	
  in	
  exercise-­‐induced	
  
pulmonary	
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  of	
  horses	
  follows	
  reported	
  blood	
  flow	
  distribution	
  in	
  the	
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lung.	
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234.	
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  RA,	
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  RL,	
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  and	
  Hoch	
  JR.	
  Transforming	
  
growth	
  factor-­‐beta1	
  antisense	
  treatment	
  of	
  rat	
  vein	
  grafts	
  reduces	
  the	
  accumulation	
  of	
  
collagen	
  and	
  increases	
  the	
  accumulation	
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157	
  

235.	
   Wong	
  AP,	
  Nili	
  N,	
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  ZS,	
  Qiang	
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  Leong-­‐Poi	
  H,	
  Jaffe	
  R,	
  Raanani	
  E,	
  Connelly	
  
PW,	
  Sparkes	
  JD,	
  and	
  Strauss	
  BH.	
  Expansive	
  remodeling	
  in	
  venous	
  bypass	
  grafts:	
  novel	
  
implications	
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236.	
   Wong	
  MS,	
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  PM.	
  COX-­‐mediated	
  endothelium-­‐dependent	
  
contractions:	
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  past	
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  Inflammatory	
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nasal	
  discharge	
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  respiratory	
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  in	
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  Equine	
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journal	
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238.	
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  Longitudinal	
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resistance	
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  high	
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1987.	
  
239.	
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  Heterogeneity	
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independent	
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  large	
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  porcine	
  pulmonary	
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  Pulmonary	
  
pharmacology	
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240.	
   Zhang	
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  Li	
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  Chen	
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  Q.	
  Comparison	
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  patency	
  
between	
  off-­‐pump	
  and	
  on-­‐pump	
  coronary	
  artery	
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  an	
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analysis.	
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241.	
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  L,	
  Tellides	
  G,	
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Bender	
  JR,	
  and	
  Sadeghi	
  MM.	
  VEGF	
  blockade	
  inhibits	
  lymphocyte	
  recruitment	
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ameliorates	
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242.	
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  AA,	
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  MJ.	
  Length-­‐tension	
  relationships	
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small	
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  the	
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  Am	
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Heart	
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158