THE	ROLE	OF	THE	INTERLEUKIN-1	FAMILY	IN	THE	DEVELOPMENT	OF	DIABETIC	
RETINOPATHY	
	
By		
	
Derrick	J.	Feenstra	
	
	
	
	
	
	
	
	
	
	
	
	
	
	
A	DISSERTATION	
	
Submitted	to	
Michigan	State	University	
in	partial	fulfillment	of	the	requirements	
for	the	degree	of	
	
Physiology	–	Doctor	of	Philosophy	
	
2017	
	

	

	
ABSTRACT	
	
THE	ROLE	OF	THE	INTERLEUKIN-1	FAMILY	IN	THE	DEVELOPMENT	OF	DIABETIC	
RETINOPATHY	
	
By		
	
Derrick	J.	Feenstra	
	
	

Diabetic	retinopathy	is	one	of	the	most	prominent	complications	of	diabetes	

with	approximately	67%	of	diabetic	patients	experiencing	some	form	of	retinopathy	
(1).		Currently	there	is	no	cure	for	the	disease,	and	in	order	to	develop	more	reliable	
therapies	 a	 better	 understanding	 of	 mechanisms	 leading	 to	 disease	 onset	 and	
progression	 are	 crucial.	 The	 aim	 of	 this	 dissertation	 was	 to	 identify	 underlying	
mechanisms	promoting	diabetic	retinopathy.	Since	there	is	a	growing	consent	that	
chronic	 retinal	 inflammation	 might	 be	 causing	 and	 driving	 the	 progression	 of	
diabetic	 retinopathy	 the	 research	 was	 specifically	 focused	 on	 identifying	 potential	
mediators	 and	 cell	 types	 participating	 in	 the	 inflammation,	 and	 testing	 whether	
targeting	 specific	 inflammatory	 pathways	 will	 be	 a	 valid	 therapeutic	 strategy	 to	
treat	diabetic	retinopathy.	
First,	in	order	to	understand	the	role	of	inflammation	in	diabetic	retinopathy	
we	 examined	 the	 caspase-1/IL-1β	 pathway.	 We	 demonstrated	 that	 caspase-1	
activity	is	increased	in	the	retinas	of	streptozotocin	(STZ)-induced	diabetic	mice	at	
10	 and	 20	 weeks	 of	 diabetes.	 	 Interestingly,	 elevated	 caspase-1	 activity	 was	
prevented	in	diabetic	IL-1R1-/-	mice	at	20	weeks	indicating	that	sustained	caspase-1	
activity	 is	 dependent	 on	 feedback	 through	 IL-1R1.	 	 The	 same	 phenomena	 was	
observed	 in	 Müller	 cells	 in	 vitro.	 Furthermore,	 we	 identified	 Receptor	 Interacting	

	

	
Protein-2	(RIP2)	as	a	central	regulator	of	caspase-1	activity	induced	by	either	high	
glucose	or	IL-1β.		It	was	further	identified	that	this	caspase-1	activity	leads	to	Müller	
cell	death	both	in	vitro	and	in	vivo.		
To	 confirm	 that	 the	 activation	 of	 the	 caspase-1/IL-1β	 pathway	 is	 indeed	
responsible	 for	 retinal	 pathologies	 associated	 with	 retinopathy	 we	 used	 the	
galactosemic	 mouse	 model,	 another	 model	 of	 retinopathy	 leading	 to	 the	 same	
vascular	pathologies	seen	in	the	STZ	diabetic	mouse	model.		Knockout	of	caspase-1	
prevented	the	formation	of	acellular	capillaries	in	galactosemic	mice.	When	Müller	
cells	were	treated	with	elevated	galactose	levels,	caspase-1	was	activated	and	led	to	
cell	 death.	 Interestingly,	 mediators	 associated	 with	 caspase-1	 activation	 such	 as	
Thioredoxin	 Interacting	 Protein	 (TXNIP)	 and	 oxidative	 stress	 were	not	 induced	 by	
galactosemia	as	they	are	in	hyperglycemic	conditions.		
Finally,	to	identify	potential	roles	of	other	IL-1	family	members	we	began	to	
examine	the	role	of	IL-1α	in	the	activation	of	the	caspase-1/IL-1β	pathway.	Our	data	
suggest	that	IL-1α	contributes	to	caspase-1	activity	and	Müller	cell	activation	since	
treatment	 with	 an	 IL-1α	 neutralizing	 antibody	 inhibited	 high	 glucose	 induced	
caspase-1	activity.	Furthermore,	we	observed	that	IL-1α	appeared	to	translocate	in	
to	the	nucleus	under	high	glucose	conditions	in	Müller	cells	in	vitro.	
Collectively,	these	findings	indicate	that	caspase-1	activation	and	subsequent	
IL-1β	 production	 are	 crucial	 for	 the	 development	 and	 progression	 of	 diabetic	
retinopathy.	 Müller	 cells	 are	 a	 prominent	 site	 of	 active	 caspase-1	 in	 the	 diabetic	
retina.	 It	 seems	 that	 targeting	 the	 caspase-1/IL-1β	 pathway	 might	 be	 a	 potential	
new	strategy	to	develop	therapies	to	treat	diabetic	retinopathy.		
	

	

	
ACKNOWLEDGEMENTS	
	
I	 would	 like	 to	 express	 my	 sincere	 gratitude	 for	 my	 mentor,	 Dr.	 Susanne	
Mohr,	 as	 without	 her	 guidance	 and	 support	 none	 of	 this	 work	 would	 have	 been	
possible.		Throughout	my	training	she	was	a	constant	source	of	encouragement	and	
was	always	dedicated	to	ensuring	that	I	achieved	my	goals.	I	will	always	appreciate	
the	 support	 she	 gave	 me,	 not	 only	 in	 lab	 so	 that	 I	 could	 learn	 the	 technical	 skills	
required	to	be	a	successful	scientist,	but	also	outside	of	lab	so	that	I	found	myself	in	
a	position	to	put	those	technical	skills	into	practice	when	I	completed	my	training.	
Something	 I	 will	 always	 be	 thankful	 for	 is	 that	 Dr.	 Mohr	 always	 encouraged	 me	 to	
apply,	participate	and	make	the	most	of	every	opportunity.	By	doing	so,	I	was	able	to	
become	 a	 more	 well-rounded	 scientist	 and	 also	 have	 opportunities	 that	 I	 would	
have	never	imagined	and	experiences	I	will	never	forget.	Following	her	advice	I	was	
fortunate	enough	to	attend	and	present	at	numerous	international	conferences,	and	
it	was	at	these	conferences	that	she	always	made	it	a	top	priority	to	introduce	me	to	
some	 of	 the	 leading	 researchers	 in	 our	 field	 and	 include	 me	 at	 dinners	 and	 social	
events	 so	 that	 I	 could	 make	 contacts	 that	 would	 be	 crucial	 for	 success	 at	 the	 next	
level.	She	also	encouraged	me	to	apply	to	the	Fundamental	Issues	in	Vision	Research	
course,	a	special	topics	course	held	at	the	Marine	Biology	Lab	in	Woods	Hold,	MA.		
This	 was	 an	 amazing	 experience	 where	 I	 was	 able	 to	 spend	 two	 weeks	 learning	
about	 vision	 science	 from	 leaders	 in	 the	 field	 and	 also	 meets	 other	 aspiring	
scientists	with	similar	research	interests.		Additionally,	Dr.	Mohr	always	encouraged	
me	 to	 seek	 out	 an	 opportunity	 to	 research	 abroad,	 and	 it	 was	 through	 this	
iv	

	
encouragement	 that	 I	 found	 a	 one-year	 internship	 at	 the	 pharmaceutical	 company	
Hoffmann	La-Roche	in	Basel,	Switzerland.	This	gave	me	my	first	taste	of	what	it	is	
like	to	work	in	industry	rather	than	academia,	and	also	gave	me	the	opportunity	to	
live	 in	 a	 new	 country	 for	 a	 year,	 an	 experience	 that	 provided	 many	 learning	
opportunities	in	itself.	
	

Finally,	 in	 addition	 to	 her	 support	 and	 encouragement,	 Dr.	 Mohr	 always	

made	 sure	 that	 the	 lab	 had	 fun	 together.	 Although	 Dr.	 Mohr	 always	 pushed	 me	 in	
lab,	it	is	the	things	we	did	outside	of	the	lab	that	I	will	remember	most,	whether	it	be	
Segway	 tours	 or	 Giants	 games	 in	 San	 Francisco,	 white	 water	 rafting	 in	 Colorado,	
snorkeling	in	Hawaii,	or	seeing	castles	in	Germany.		
	

I	 would	 also	 like	 to	 thank	 the	 other	 members	 of	 the	 lab,	 most	 specifically	

Brandon	 Coughlin,	 Dr.	 Prathiba	 Jayaguru	 and	 Barbara	 Christian	 for	 their	 help	 and	
support	 in	 my	 experiments.	 	 Also,	 Dr.	 Lou	 Glazer	 has	 contributed	 greatly	 to	 help	
understand	diabetic	retinopathy	at	a	clinical	level.	Finally,	I	would	like	to	thank	my	
entire	family	who	has	provided	immense	support	outside	of	lab.			
	
	

	

v	

	
TABLE	OF	CONTENTS	

	
LIST	OF	TABLES	....................................................................................................................	viii	
LIST	OF	FIGURES	.....................................................................................................................	ix	
KEY	TO	ABBREVIATIONS	.....................................................................................................	xi	
Chapter	1.	Introduction	........................................................................................................	1	
1.1	Diabetes	Mellitus	.......................................................................................................................	1	
1.2	Diabetic	Retinopathy	................................................................................................................	2	
1.2.1-	Introduction	to	Diabetic	Retinopathy	.......................................................................................	2	
1.2.2	Non-proliferative	Diabetic	Retinopathy	....................................................................................	3	
1.2.3	Proliferative	Diabetic	Retinopathy	..............................................................................................	5	
1.2.4	Diabetic	Macular	Edema	...................................................................................................................	7	
1.2.5	Current	Therapies	for	Diabetic	Retinopathy	.........................................................................	10	
1.3	Retina	Structure	.......................................................................................................................	11	
1.4	Müller	Cells	................................................................................................................................	17	
1.4.1	Müller	Cells	in	the	Healthy	Retina	..............................................................................................	17	
1.4.2	Müller	Cells	in	Diabetic	Retinopathy	.........................................................................................	20	
1.5	Caspase	Family	.........................................................................................................................	22	
1.5.1	Caspase	Enzymes-	Functions	and	Outcomes	.........................................................................	22	
1.5.2	Caspase-1	..............................................................................................................................................	23	
1.5.3	Caspase-1	in	Disease	........................................................................................................................	25	
1.6	The	Interleukin-1	Family	and	Signaling	..........................................................................	25	
1.6.1	The	Interleukin-1	Family	...............................................................................................................	25	
1.6.2	IL-1β,	IL-1β	Receptor	Signaling,	and	Regulation	.................................................................	26	
1.6.3	Autoinflammatory	Disease	............................................................................................................	27	
1.7	Objectives	of	the	Dissertation	.............................................................................................	28	
Chapter	 2.	 Interleukin-1	 Receptor-Dependent	 and	 Independent	 Caspase-1	
Activation	in	Diabetic	Retinopathy	.................................................................................	30	
2.1	Introduction	..............................................................................................................................	30	
2.2	Research	Design	And	Methods	............................................................................................	32	
2.3	Results	.........................................................................................................................................	36	
2.3.1	Inhibition	of	Diabetic	Retinopathy	in	STZ	mice	by	Caspase-1	Knockout.	.................	36	
2.3.2	 Sustained	 Caspase-1	 Activity	 in	 STZ	 Mice	 Dependent	 on	 Feedback	 Through	 the	
IL-1	Receptor.	.................................................................................................................................................	37	
2.3.3	 Inhibition	 of	 Caspase-1	 Activity	 in	 Human	 Müller	 cells	 by	 IL-1	 Receptor	
Antagonist.	.......................................................................................................................................................	37	
2.3.4	Glucose	Consumption	in	Müller	Cells	Exposed	to	Hyperglycemic	Conditions.	......	38	
2.3.5	RIP2	Mediated	Caspase-1	Activation	by	Hyperglycemia.	................................................	39	
2.3.6	Inhibition	of	IL-β-Induced	Caspase-1	Activity	by	RIP2	Knock	Down.	........................	40	
2.3.7	 Hyperglycemia-Induced	 Müller	 Cell	 Death	 Mediated	 by	 Caspase-1/IL-1β/IL-1R1	
Feedback	Signaling.	......................................................................................................................................	40	
2.3.8	Diabetic	Caspase-1-/-	Mice	Are	Protected	from	Müller	Cell	Loss.	.................................	41	
2.4	Discussion	..................................................................................................................................	53	

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Chapter	3.	Modes	of	Retinal	Cell	Death	in	Diabetic	Retinopathy	..........................	58	
3.1	Abstract	.......................................................................................................................................	58	
3.2	Introduction	..............................................................................................................................	59	
3.3.	Apoptosis:	Extrinsic	Versus	Intrinsic	..............................................................................	61	
3.4	Necrosis:	A	Regulated	Pathway	to	Cell	Death?	..............................................................	66	
3.5	Autophagic	Cell	Death	............................................................................................................	67	
3.6	Pyroptosis:	Inflammation	Driven	Cell	Death	.................................................................	69	
3.7	Conclusion	..................................................................................................................................	75	
Chapter	4.	Caspase-1	Activation	in	Galactosemia	......................................................	78	
4.1	Introduction	..............................................................................................................................	78	
4.2	Materials	and	Methods	..........................................................................................................	81	
4.3	Results	.........................................................................................................................................	83	
4.3.1	Galactosemia	Increases	Caspase-1	Activity	in	Müller	Cells	.............................................	83	
4.3.2	High	Glucose,	But	Not	High	Galactose	Generates	Reactive	Oxygen	Species	............	84	
4.3.3	Glucose	Specific	Increases	in	TXNIP	..........................................................................................	84	
4.3.4	 Caspase-1	 Inhibition	 Prevents	 Galactosemia-Induced	 Müller	 Cell	 Death	 and	
Acellular	Capillary	Formation	.................................................................................................................	85	
4.4	Conclusion	..................................................................................................................................	92	

Chapter	 5.	 The	 Role	 of	 Interleukin-1α	 in	 Caspase-1	 Activation	 and	 Cell	 Death	
in	Müller	Cells	.........................................................................................................................	95	
5.1	Introduction	..............................................................................................................................	95	
5.2	Materials	and	Methods	..........................................................................................................	96	
5.3	Results	.........................................................................................................................................	98	
5.4	Conclusion	...............................................................................................................................	102	
Chapter	6.	Summary	and	Future	Outlook	..................................................................	103	
6.1	Summary	of	data	regarding	caspase-1/IL-1β	feedback	signaling	in	the	retina	of	
diabetic	mice	.................................................................................................................................	103	
6.2	 Summary	 of	 data	 regarding	 the	 role	 of	 IL-1α	 in	 hyperglycemia-induced	
caspase-1	activation	in	Müller	cells	......................................................................................	106	
6.3	Implications	of	the	data	collected	during	this	thesis	project	to	the	field	of	Müller	
cells		and	diabetic	retinopathy	................................................................................................	107	
6.4	Conclusion	...............................................................................................................................	115	

Chapter	7.	Internship	Experience	at	Hoffmann-La	Roche	....................................	117	
REFERENCES	........................................................................................................................	119	
	
	
	
	
	

	

vii	

	
LIST	OF	TABLES	
	
Table	1:	Summary	of	Animal	Data.	..................................................................................	52	
Table	2.	Characteristics	of	modes	of	cell	death	and	potential	retinal	cell	types	
undergoing	cell	death	in	diabetic	retinopathy.	..........................................................	77	
	
	

	

viii	

	
LIST	OF	FIGURES	
	
Figure	 1.	 Fluorescein	 angiography	 of	 patients	 with	 non-proliferative	 diabetic	
retinopathy.	..............................................................................................................................	4	
Figure	 2.	 Fluorescein	 angiography	 of	 patients	 with	 proliferative	 diabetic	
retinopathy	with	neovascularization	of	the	disc	and	elsewhere.	...........................	6	
Figure	3.	OCT	of	patients	with	DME	..................................................................................	9	
Figure	4.		Anatomy	of	the	Eye	and	Retina.	....................................................................	12	
Figure	5.	Fundus	and	OCT	of	Healthy	Retina	...............................................................	16	
Figure	6.	Retinal	capillary	degeneration	in	non-diabetic	and	diabetic	wild	type	
and	caspase-1	knockout	mice.	..........................................................................................	42	
Figure	7.	Caspase-1	activity	pattern	in	the	retinas	of	non-diabetic	and	diabetic	
wild	type	and	IL-1R1-/-mice.	..............................................................................................	43	
Figure	 8.	 Inhibition	 of	 hyperglycemia-and	 IL-β-induced	 caspase-1	 activity	 in	
human	Müller	cells	by	IL-1	receptor	antagonist.	.......................................................	44	
Figure	9.	Glucose	consumption	normalized	over	time	in	Müller	cells.	...............	45	
Figure	 10.	 RIP2	 mediated	 caspase-1	 activity	 and	 IL-β	 production	 in	 Müller	
cells.	...........................................................................................................................................	46	
Figure	11:	IL-1β-induced	caspase-1	activity	mediated	by	RIP2.	...........................	48	
Figure	 12:	 Inhibition	 of	 hyperglycemia-induced	 Müller	 cell	 death	 by	 IL-1ra,	
YVAD-fmk,	and	RIP2siRNA.	................................................................................................	49	
Figure	13:	Caspase-1	dependent	Müller	cell	death	in	retinas	of	diabetic	mice.
	.....................................................................................................................................................	50	
Figure	 14:	 Proposed	 mechanism	 of	 sustained	 caspase-1	 activation	 via	
feedback	signaling	in	Müller	cells.	..................................................................................	51	
Figure	15:	IL-1R1	Dependent	Müller	cell	death	in	diabetes.	..................................	73	
Figure	16.	Scheme	of	pyroptosis	in	Müller	cells.	........................................................	74	
Figure	17.	High	Glucose	and	Galactose-Induced	Caspase-1	Activity.	..................	86	
Figure	18.		ROS	induced	by	high	glucose	but	not	high	galactose.	..........................	87	
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Figure	19.		TXNIP	protein	induction	is	glucose	specific.	(Cont.	on	next	page)	.	88	
Figure	20.		High	Galactose	leads	to	caspase-1	dependent	Müller	cell	death.	....	90	
Figure	 21.	 Acellular	 capillary	 formation	 in	 non-diabetic	 and	 galactosemic	
mice.	...........................................................................................................................................	91	
Figure	 22.	 IL-1α	 blockage	 decreases	 caspase-1	 activity	 in	 Müller	 Cells	 Under	
High	Glucose	Conditions.	....................................................................................................	99	
Figure	23.	High	glucose-induced	Müller	cell	death	inhibited	by	anti-IL-1α.	.	100	
Figure	24.	Nuclear	Accumulation	of	IL-1α	In	Müller	Cells	Under	High	Glucose	
Conditions.	............................................................................................................................	101	
Figure	 25:	 Schematic	 outlining	 the	 caspase-1/IL-1β	 feedback	 signaling	 the	
diabetic	mouse.	...................................................................................................................	105	
Figure	26:	Timeline	of	Diabetic	Retinopathy	In	Mice	and	Rats.	.........................	114	
	
	

	

x	

	
KEY	TO	ABBREVIATIONS	
	
AGE	-	Advanced	Glycation	End	Product	
AIF-Apoptosis-Inducing	Factor	
AIM2-	Absent	In	Melanoma-2	
AMD	-	Age	Related	Macular	Degeneration	
BID	-	BH3-Interacting	Domain	
CAPS	-	Cryopyrin-Associated	Periodic	Syndromes	
CARD	-	Caspase	Activation	and	Recruitment	Domain	
Casp-/-	-	Caspase-1	Knockout	
CCL2	-	Chemokine	Ligand-2	
CNV	-	Choroidal	Neovascularization	
COX2	-	Cyclooxygenase-2	
DAMP	-	Danger	Associated	Molecular	Pattern	
DCCT	-	Diabetes	Control	and	Complications	Trial	
DED	-	Death	Effector	Domain	
DIRA	-	Deficiency	of	IL-1ra	
DISC	-	Death-Inducing	Signaling	Complex	
DME	-	Diabetic	Macular	Edema	
DMEM	-	Dulbecco's	Modified	Eagle's	Medium	
ETDRS	-	Early	Treatment	Diabetic	Retinopathy	Study	
FBS	-	Fetal	Bovine	Serum	
FMF	-	Familial	Mediterranean	Fever	
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GABA	-	γ-aminobutryic	acid		
GAPDH	-	Glyceraldehyde-3-Phosphate	Dehydrogenase	
GFAP	-	Glial	Fibrillary	Acidic	Protein	
GHb	-	Glycated	Hemoglobin	
GLAST	-	Glutamate	Aspartate	Transporter	
H2DCFDA	-	2’-7’-dichlorofluroescin	diacetate	
HIF-1	-	Hypoxia	Inducible	Factor-1	
hMC	-	Human	Müller	Cell	
ICE	-	Interleukin-1	Converting	Enzyme	
IFNγ	-	Interferon-γ	
IKKβ	-	Inhibitor	of	NF-κB	Kinase	b		
IL	-	Interleukin	
ILM	-	Inner	Limiting	Membrane	
INL	-	Inner	Nuclear	Layer	
iNOS	-	Inducible	Nitric	Oxide	Synthase	
IRAK	-	IL-1R-Associated	Kinase	
MYD88	-	Myeloid	Differentiation	Primary	Response	Protein	88	
NLRP	-	Nod	Like	Receptor	Family	Pyrin	Domain	Containing	
OCT	-		Optical	Coherence	Tomography	
PAMP	-	Pathogen	Associated	Molecular	Pattern	
PEDF	-	Pigment	Epithelium-Derived	Factor	
PGE2	-	Prostaglandin	E2	
PI	-	Propidium	Iodide	
xii	

	
PRR	-	Pattern	Recognition	Receptor	
PS	-	Penicillin/Streptomycin	
PTP	-	Permeability	Transition	Pore	
RIP2	-	Receptor	Interacting	Protein-2	
ROS	-	Reactive	Oxygen	Species	
RPE	-	Retinal	Pigment	Epithelium	
STZ	-	Streptozotocin	
TH2	-	T	Helper	Type-2	
TIR	-	Toll/IL-1	receptor	
TNFα	-	Tumor	Necrosis	Factor-α	
TRAIL	-	TNF-Related	Apoptosis	Inducing	Ligand	
TUNEL	-	Terminal	dUTP	Nick	End	Labeling	
TXNIP	-	Thioredoxin	Interacting	Protein	
VEGF	-	Vascular	Endothelial	Growth	Factor	
WT	-	Wild	Type	
	
	
	

xiii	

	
Chapter	1.	Introduction	
1.1	Diabetes	Mellitus	
Diabetes	mellitus	is	a	disease	characterized	by	elevated	fasting	blood	glucose	
(Fasting	 blood	 glucose	 ≥	 126	 mg/dl)	 (2).	 	 Although	 there	 are	 numerous	 types	 of	
diabetes,	the	two	most	common	types	of	diabetes	are	Type	1	and	Type	2	diabetes.	In	
patients	with	Type	1	diabetes,	also	known	as	insulin-dependent	or	juvenile	diabetes,	
there	is	an	autoimmune	destruction	of	the	pancreatic	beta	cells,	the	cell	type	that	is	
responsible	 for	 producing	 insulin,	 which	 renders	 the	 body	 unable	 to	 produce	 the	
insulin	required	to	stimulate	glucose	uptake	from	the	blood	stream.		Patients	with	
Type	2	diabetes	have	a	similar	increase	in	blood	glucose	but	by	an	entirely	different	
mechanism.	 	 Type	 2	 diabetes,	 also	 known	 as	 insulin-independent	 diabetes,	 is	 the	
most	common	form	of	diabetes	and	results	from	improper	insulin	signaling.		Unlike	
patients	with	Type	1	diabetes,	these	patients	are	able	to	produce	insulin,	however	
the	 insulin	 cannot	 act	 on	 its	 receptor	 to	 trigger	 glucose	 uptake	 from	 the	 blood	
stream	 into	 target	 tissues	 such	 as	 muscle	 and	 adipose	 tissue,	 a	 process	 known	 as	
insulin	resistance.	
Diabetes	is	the	first	non-contagious	disease	labeled	an	epidemic	by	the	World	
Health	 Organization.	 The	 number	 of	 people	 affected	 by	 the	 disease	 is	 staggering;	
approximately	29	million	people	in	the	United	States	have	diabetes,	a	number	that	
accounts	for	9.3%	of	the	total	population.	To	make	matters	worse,	86	million	people	
in	 the	 United	 States	 have	 pre-diabetes,	 meaning	 they	 have	 either	 elevated	 blood	
glucose	 levels	 or	 impaired	 glucose	 tolerance,	 which	 puts	 them	 at	 severe	 risk	 of	
developing	 diabetes	 in	 the	 future	 (2).	 According	 to	 the	 American	 Diabetes	
1	

	
Association	 the	 disease	 has	 cost	 the	 United	 States	 a	 total	 of	 $245	 billion	 in	 2012.		
These	costs	are	a	result	of	both	the	reduced	productivity	of	the	afflicted	individuals	
in	 the	 work	 force,	 and	 also	 direct	 medical	 costs	 from	 the	 many	 complications	 that	
arise	with	the	disease.		Some	of	the	common	complications	of	diabetes	include	skin	
ulcers,	impaired	wound	healing,	nephropathy,	neuropathy,	and	retinopathy.	
	
1.2	Diabetic	Retinopathy	
1.2.1-	Introduction	to	Diabetic	Retinopathy	
Diabetic	 retinopathy	 is	 one	 of	 leading	 causes	 of	 acquired	 blindness	 in	
working	age	adults.		As	the	prevalence	of	diabetes	increases	so	does	the	incidence	of	
diabetic	 complications	 such	 as	 diabetic	 retinopathy.	 Diabetic	 retinopathy	 is	a	 sight	
threatening	 disease	 that	 occurs	 in	 patients	 with	 both	 Type	 1	 and	 Type	 2	 diabetes.		
Duration	 of	 diabetes	 seems	 to	 be	 the	 strongest	 predictor	 of	 the	 incidence	 of	
retinopathy	 as	 approximately	 8%	 of	 diabetic	 patients	 had	 retinopathy	 at	 3	 years,	
25%	by	5	years,	60%	by	10	years	and	80%	by	15	years.		
	Another	predictor	of	disease	progression	is	glycemic	control.		In	the	Diabetes	
Control	and	Complications	Trial	(DCCT)	a	total	of	1,441	patients	who	had	either	no	
retinopathy	(primary	prevention	cohort)	or	minimal-to-moderate	non-proliferative	
DR	 (secondary	 progression	 cohort)	 were	 treated	 by	 either	 conventional	 treatment	
using	 one	 or	 two	 daily	 injections	 of	 insulin	 or	 by	 intensive	 diabetes	 management	
with	 three	 or	 more	 daily	 insulin	 injections	 or	 a	 continuous	 subcutaneous	 insulin	
infusion.	 	 During	 the	 first	 36	 months,	 incidence	 of	 progression	 of	 retinopathy	 was	
similar	 between	 the	 two	 treatments	 in	 the	 primary	 prevention	 cohort.	 	 After	 36	
2	

	
months	however,	there	was	a	substantial	decrease	in	incidence	of	retinopathy	in	the	
intensive	 treatment	 group	 compared	 to	 the	 conventional	 treatment	 group.	 	 In	 the	
secondary	 progression	 cohort,	 the	 intensive	 treatment	 group	 had	 higher	 initial	
incidence	 during	 the	 first	 year,	 however	 by	 36	 months	 the	 intensive	 group	 had	
lower	 risks	 of	 progression.	 	 Together,	 this	 indicated	 that	 glycemic	 control	 was	
important	in	both	the	prevention	and	intervention	of	diabetic	retinopathy.	
1.2.2	Non-proliferative	Diabetic	Retinopathy	
The	initial	stage	of	diabetic	retinopathy	is	referred	to	as	non-proliferative,	or	
background,	 diabetic	 retinopathy	 (Figure	 1).	 The	 retinal	 changes	 associated	 with	
non-proliferative	 diabetic	 retinopathy	 include	 microaneurysms,	 lipid	 exudates,	
microhemorrhages,	 cotton-wool	 spots,	 and	 thickening	 of	 the	 basement	
membrane(3).	 These	 retinal	 changes	 are	 reversible	 and	 the	 defects	 in	 a	 patients	
vision	 are	 minimal(4).	 The	 associated	 changes	 in	 vision	 of	 patients	 with	 nonproliferative	 diabetic	 retinopathy	 include	 decreased	 contrast	 sensitivity	 unless	 the	
patient	begins	to	show	signs	of	edema,	which	can	lead	to	decreased	visual	acuity(3).	
	

	

3	

	
Figure	 1.	 Fluorescein	 angiography	 of	 patients	 with	 non-proliferative	 diabetic	
retinopathy.		
A)	Early	frame	fluorescein	angiogram	from	a	non-proliferative	diabetic	retinopathy	
patient	 showing	 microaneurysms.	 B)	 Mid	 and	 C)	 late	 frame	 non-proliferative	
angiogram	from	the	same	patient	showing	DME	and	leakage	from	microaneurysms.		

	
	

	

4	

	
1.2.3	Proliferative	Diabetic	Retinopathy	
As	 the	 disease	 progresses	 patients	 can	 develop	 more	 severe	 visionthreatening	 pathologies.	 	 These	 changes	 are	 commonly	 a	 result	 in	 the	 disease	
progressing	 from	 non-proliferative	 to	 proliferative	 diabetic	 retinopathy,	 where	
there	is	abnormal	growth	of	new	blood	vessels.		These	new	blood	vessels	can	lead	to	
leakage	 of	 blood	 into	 the	 retinal	 tissue	 or	 vitreous	 fluid	 which	 blocks	 light	 from	
passing	through	the	fluid	to	activate	photoreceptors	in	the	back	of	the	retina	(Figure	
2)	Additionally,	these	blood	vessels	can	lead	to	tractional-detachment	of	the	retina	
which	can	result	in	permanent	damage	if	not	treated(3,4).	
	

	

5	

	
Figure	 2.	 Fluorescein	 angiography	 of	 patients	 with	 proliferative	 diabetic	
retinopathy	with	neovascularization	of	the	disc	and	elsewhere.		
A)	Early	stage,	imaged	for	disc.	B)	Early	stage,	imaged	for	macula.	C)	Mid	stage	D)	
Late	stage.	

	
	

	

6	

	
1.2.4	Diabetic	Macular	Edema	
In	 addition	 to	 the	 non-proliferative	 and	 proliferative	 stages	 of	 diabetic	
retinopathy,	diabetic	patients	are	also	at	risk	of	developing	diabetic	macular	edema	
(DME).	 	 DME	 is	 the	 most	 common	 cause	 of	 vision	 loss	 in	 patients	 with	 diabetic	
retinopathy	 and	 can	 occur	 in	 either	 the	 non-proliferative	 or	 proliferative	 stage	 of	
diabetic	 retinopathy.	 DME	 arises	 from	 the	 breakdown	 of	 the	 inner	 blood	 retinal	
barrier	(primarily	from	endothelial	cells)	rather	than	the	outer	blood	retinal	barrier	
(primarily	 from	 retinal	 pigment	 epithelium(RPE))	 and	 is	 caused	 by	 the	
redistribution	 of	 tight	 junction	 proteins	 such	 as	 claudin	 and	 occludin	 leading	 to	
leakage	of	fluid	from	the	vasculature	into	the	retina(5,6).		In	a	healthy	retina,	excess	
fluid	 is	 removed	 primarily	 by	 the	 RPE	 pump,	 where	 RPE	 transport	 potassium	 and	
chloride	ions	out	of	the	extracellular	space	allowing	for	passive	removal	of	water	via	
aquaporin-1	 channels(7,8).	 In	 addition	 to	 the	 RPE	 pump,	 Müller	 cells	 also	 play	 a	
major	 role	 in	 the	 removal	 of	 fluid	 from	 the	 retina.	 	 Müller	 cells	 act	 as	 potassium	
shuttle	by	taking	up	potassium	from	the	extracellular	fluid	through	Kir2.1	potassium	
channels	 and	 depositing	 the	 potassium	 into	 the	 vasculature	 using	 Kir4.1	 channels	
that	are	found	on	the	Müller	cell	processes	that	encompass	the	blood	vessels(9,10).		
This	 leads	 to	 osmotic	 fluid	 removal	 through	 aquaporin-4(8,10–12).	 In	 diabetes,	
Müller	cells	have	been	shown	to	downregulate	the	Kir4.1	channels,	but	not	Kir2.1,	
leading	

to	

continued	

potassium	

uptake	

with	

no	

release	

into	

the	

microvasculature(13–15).	 This	 leads	 to	 subsequent	 swelling	 of	 Müller	 cells	
contributing	to	Müller	cell	dysfunction	and	decreased	fluid	removal	contributing	to	
DME.	DME	leads	to	thickening	of	the	macula	due	to	fluid	accumulation	and	can	be	
7	

	
observed	by	optical	coherence	tomography	(OCT)	(Figure	3).	The	thickening	of	the	
macula	 due	 to	 fluid	 accumulation	 typically	 leads	 to	 disruption	 of	 the	 retinal	
structure	and	changes	in	visual	acuity.	
	

	

8	

	
Figure	3.	OCT	of	patients	with	DME	
(A)	 Line	 scan	 OCT	 from	 patient	 with	 DME	 with	 visible	 fluid	 accumulation	 in	 the	
macula.	(B)	Map	scan	from	patient	with	DME	showing	increased	macular	thickening	
due	to	fluid	accumulation.	

	
9	

	
1.2.5	Current	Therapies	for	Diabetic	Retinopathy	
There	 are	 currently	 two	 main	 therapies	 for	 the	 treatment	 of	 diabetic	
retinopathy.	 	 The	 first	 are	 agents	 that	 target	 VEGF	 (vascular	 endothelial	 growth	
factor)	and	are	typically	used	in	the	treatment	of	proliferative	diabetic	retinopathy	
and	DME.	Elevated	levels	of	VEGF	are	found	in	the	vitreous	of	patients	with	diabetic	
retinopathy	and	DME.	VEGF	is	a	growth	factor	that	promotes	neovascularization	and	
also	 increases	 vascular	 permeability	 by	 increasing	 occludin	 phosphorylation	 and	
decreasing	 occludin	 levels	 in	 endothelial	 cells	 in	 the	 inner	 retinal	 vasculature.	
Therefore,	 neutralizing	 VEGF	 using	 specific	 antibodies	 has	 been	 successful	 in	
correcting	vascular	leakage	and	neovascularization.	Unfortunately,	not	every	patient	
responds	 to	 this	 treatment	 and	 patients	 that	 do	 require	 monthly	 injections	 of	 the	
drug	to	keep	symptoms	from	reoccurring.	Overall,	this	treatment	strategy	is	a	huge	
burden	to	the	patient..	
	

The	 second	 therapy	 is	 laser	 focal	 photocoagulation.	 The	 Early	 Treatment	

Diabetic	 Retinopathy	 Study	 (ETDRS)	 was	 the	 first	 large	 scale	 study	 examining	 the	
effects	 of	 using	 a	 argon	 blue-green	 or	 green	 laser	 to	 inflict	 50-200μM	 moderate	
intensity	burns	in	a	grid	pattern	in	the	macula	of	the	retina.		In	this	study	the	laser	
burns	 were	 focused	 on	 microaneurysms,	 intraretinal	 microvascular	 abnormalities	
and	 other	 potential	 leakage	 or	 neovascular	 sites.	 It	 was	 observed	 that	 laser	
photocoagulation	had	a	led	to	a	significant	improvement	in	visual	acuity	in	treated	
eyes	 compared	 to	 untreated	 eyes	 3	 years	 later.	 Since	 this	 study,	 laser	
photocoagulation	is	still	a	common	treatment	for	proliferative	diabetic	retinopathy,	
however,	 the	 methods	 have	 changed	 for	 focal	 laser	 treatment	 for	 DME.	 	 Currently,	
10	

	
burns	are	smaller	(50	μM)	and	have	a	lighter	intensity	(using	yellow	and	green,	but	
no	 longer	 blue-green	 laser)	 to	 prevent	 scar	 expansion.	 Although,	 the	 exact	
mechanism	 by	 which	 focal	 and	 panretinal	 laser	 work	 is	 unclear,	 there	 have	 been	
numerous	proposed	mechanisms.		The	most	common	proposed	mechanism	is	that	
the	burns	destroy	the	photoreceptors	in	the	peripheral	retina	leaving	more	oxygen	
remaining	healthy	tissue.		This	eliminates	areas	of	hypoxia,	which	is	one	of	the	main	
drivers	 of	 VEGF	 production.	 Additionally,	 photocoagulation	 destroys	 the	 leakage	
sources	such	as	microaneurysms	and	leaky	vessels.		Other	studies	indicate	that	laser	
photocoagulation	actually	improves	the	RPE	pumping	of	ions	and	water	out	of	the	
retina	and	into	the	choroid.			
	

In	sum,	the	lack	of	available	therapies	points	to	the	need	for	identifying	new	

targets	involved	in	the	progression	of	diabetic	retinopathy.		In	order	to	identify	new	
targets	there	needs	to	be	increased	understanding	of	how	the	retina	changes	during	
diabetes.		
	
1.3	Retina	Structure	
The	retina	is	a	highly	organized	tissue	consisting	of	various	layers	located	in	
the	back	of	the	eye	(Figure	4A).	The	cross-sectional	layers	of	the	retina	from	inner	
most	layer	to	the	outer	most	layer	are	ordered	as	follows	(Figure	4B),	the	(1)	Inner	
Limiting	Membrane	(ILM),	which	forms	the	boundary	between	the	neural	retina	and	
the	vitreous	humor,	a	clear	jelly-like	fluid	that	fills	the	eye.	The	ILM	is	made	up	of	
the	end	feet	of	Müller	cells,	which	are	then	coated	in	a	mucopolysaccharide	on	the	
surface	in	order	to	form	a	true	boundary	between	the		
11	

	
Figure	4.		Anatomy	of	the	Eye	and	Retina.		
A)	 Anatomy	 of	 the	 eye	 showing	 location	 of	 the	 retina.	 B)	 Illustration	 of	 the	 cross	
section	 of	 the	 retina	 showing	 each	 retinal	 cell	 type.	 *This	 figure	 is	 used	 with	
permission	from	webvision.utah.med.edu.	
A	

	
B	

	
12	

	
retina	 and	 vitreous	 humor(16,17).	 	 The	 (2)	 nerve	 fiber	 layer	 consists	 of	 the	 axons	
from	 the	 ganglion	 cells	 that	 run	 across	 the	 inner	 surface	 of	 the	 retina,	 just	 behind	
the	 ILM,	 and	 converge	 at	 the	 optic	 nerve	 to	 where	 they	 eventually	 return	
information	to	the	brain	for	processing.	They	are	supported	by	astrocytes,	one	of	the	
three	 glia	 cell	 types	 in	 the	 retina.	 The	 (3)	 ganglion	 cell	 layer	 synapses	 in	 the	 (4)	
inner	 plexiform	 with	 the	 bipolar	 cells	 and	 amacrine	 cells.	 	 The	 (5)	 inner	 nuclear	
layer	(INL)	consists	of	the	cell	bodies	of	amacrine	cells,	bipolar	cells,	horizontal	cells,	
microglia,	 and	 Müller	 cells,	 another	 glia	 cell	 type.	 The	 bipolar	 cells	 serve	 as	 a	
“bridge”	 to	 pass	 the	 signal	 from	 photoreceptor	 cells	 in	 the	 outer	 retina	 to	 the	
ganglion	cells	in	the	inner	retina.		The	(6)	outer	plexiform	layer	is	where	the	bipolar	
cells	 and	 horizontal	 cells	 synapse	 with	 the	 photoreceptor	 inner	 segments.	 	 Just	
behind	 this	 synapse	 is	 the	 (7)	 outer	 nuclear	 layer,	 which	 contains	 the	 highly	
organized	 nuclei	 of	 the	 photoreceptors,	 which	 are	 connected	 to	 the	 photoreceptor	
outer	 segments,	 which	 is	 the	 starting	 location	 of	 the	 visual	 cycle	 and	 signal	
transduction.	 	 The	 outer	 segment	 of	 the	 photoreceptors	 makes	 up	 the	 (8)	
photoreceptor	layer.		Just	behind	the	photoreceptors	is	the	final	layer	of	the	retina,	
which	is	the	(9)	retinal	pigment	epithelium	cell	layer.			
	

The	retina	is	unique	in	that	it	has	two	blood	supplies.	The	first	blood	

supply	 comes	 from	 the	 central	 retinal	 artery	 and	 branches	 after	 it	 enters	 through	
the	optic	nerve	(Figure	5).		These	blood	vessels	are	located	within	the	retinal	layers	
and	can	easily	be	seen	in	a	fundus	image.	The	blood	supply	from	the	central	retinal	
artery	 is	 responsible	 for	 supplying	 nutrients	 and	 oxygen	 to	 the	 inner	 retina.	 	 The	
second	 blood	 supply	 is	 the	 choroid.	 	 The	 choroid	 lies	 just	 behind	 the	 retina	 and	
13	

	
supplies	 oxygen	 and	 nutrients	 specifically	 to	 the	 RPE	 and	 photoreceptors	 that	 are	
located	in	the	outer	retina,	one	of	the	most	metabolically	active	sites	in	the	body.		By	
having	their	own	blood	supply	just	posterior	to	the	retina	photoreceptors	are	very	
well	 suited	 to	 cope	 with	 the	 high	 metabolic	 demand.	 	 Interestingly,	 there	 are	
speculations	that	this	unique	design	of	the	retina	(having	photoreceptors	in	the	back	
instead	 on	 the	 front	 of	 the	 eye	 and	 retina)	 evolved	 exactly	 to	 guarantee	 the	 high	
metabolic	demand	could	be	supported.	This	is	also	the	reason	that	the	macula	can	
survive	 as	 an	 avascular	 region;	 since	 it	 is	 a	 region	 that	 consists	 of	 only	 the	
photoreceptor	 cell	 layer	 that	 receives	 is	 nutrients	 solely	 from	 the	 choroid	 located	
behind	the	retina	and	doesn’t	depend	on	blood	vessels	within	the	retinal	tissue	that	
originate	from	the	central	retinal	artery.	
Looking	 at	 a	 fundus	 of	 the	 retina	 it	 becomes	 apparent	 that	 the	 retina	 is	 a	
tissue	 that	 is	 not	 only	 highly	 organized	 in	 the	 cross-sectional	 layers,	 but	 also	
organized	 spatially	 from	 the	 anterior	 view.	 	 The	 area	 in	 the	 center	 of	 the	 retina	 is	
known	as	the	macula,	which	is	then	further	divided	into	the	fovea,	parafovea	and	the	
perifovea	(Figure	5B).		This	region	of	the	retina	is	responsible	for	the	highest	visual	
acuity	due	to	the	high	concentration	of	cone	photoreceptors.	Furthermore,	this	is	an	
avascular	region	of	the	retina	and	also	the	thinnest	layer	of	the	retina	as	it	contains	
only	 cone	 photoreceptors	 and	 end	 feet	 of	 Müller	 cells.	 Therefore,	 each	 photon	 of	
light	can	pass	directly	through	the	fovea	to	activate	the	photoreceptors	with	limited	
interference	 from	 the	 other	 retinal	 cell	 layers.	 The	 area	 outside	 of	 the	 macula	 is	
known	 as	 the	 peripheral	 retina.	 The	 peripheral	 retina	 is	 a	 highly	 vascular	 region	

14	

	
responsible	 for	 contrast	 sensitivity	 due	 to	 the	 increased	 concentration	 of	 rod	
photoreceptors.		
	

	

15	

	
Figure	5.	Fundus	and	OCT	of	Healthy	Retina	
A)	Fundus	image	of	retina	showing	central	retinal	artery	entering	retina	through	the	
optic	disc.	The	fovea	(shown	by	arrow)	can	also	be	seen	in	the	center	of	the	retina.		
B)	 OCT	 showing	 the	 cross	 sectional	 layers	 of	 the	 same	 retina	 with	 arrow	 showing	
foveal	 pit	 that	 contains	 cone	 photoreceptors.	 *This	 figure	 is	 used	 with	 permission	
from	webvision.med.utah.edu.	

	
	

	

16	

	
1.4	Müller	Cells	
1.4.1	Müller	Cells	in	the	Healthy	Retina	
Müller	cells	are	the	principle	glia	of	the	retina.	They	are	the	only	cells	to	span	
the	entire	width	of	the	retina	and	have	intimate	contact	with	both	the	retinal	blood	
vessels	 and	 retinal	 neurons	 (Figure	 4B).	 Because	 of	 this	 arrangement,	 Müller	 cells	
have	a	variety	of	important	functions	in	the	healthy	retina.		Functions	of	Müller	cells	
can	 be	 divided	 into	 3	 major	 categories:	 (1)	 Uptake	 and	 recycling	 of	
neurotransmitters,	 retinoic	 acid	 compounds,	 and	 ions	 (such	 as	 potassium	 K+),	 (2)	
control	 of	 metabolism	 and	 supply	 of	 nutrients	 for	 the	 retina,	 and	 (3)	 regulation	 of	
blood	flow	and	maintenance	of	the	blood	retinal	barrier.		
The	extensive	contact	of	Müller	cells	with	retinal	neurons	allows	Müller	cells	
to	actively	participate	in	proper	neurotransmission.	They	rapidly	take	up	and	clear	
glutamate	 and	 γ-aminobutryic	 acid	 (GABA)	 in	 the	 inner	 plexiform	 layer(18–21).	
Studies	 have	 shown	 that	 Müller	 cells	 take	 up	 extracellular	 glutamate	 through	 the	
Glutamate	Aspartate	Transporter	(GLAST)	and	indicate	that	glutamate	removal	and	
prevention	 of	 neurotoxicity	 in	 the	 retina	 is	 achieved	 primarily	 by	 this	
mechanism(22,23).	 Once	 taken	 up,	 glutamate	 is	 converted	 to	 glutamine	 by	
glutamine	 synthetase	 and	 released	 back	 to	 neurons	 for	 re-synthesis	 of	 glutamate	
and	GABA(24).	This	process	provides	substrate	for	neurotransmitter	synthesis	and	
also	 prevents	 glutamate	 toxicity.	 Müller	 cells	 further	 maintain	 proper	 retinal	
function	 by	 participating	 in	 a	 process	 known	 as	 “potassium	 spatial	 buffering”,	 a	
process	that	redistributes	and	normalizes	K+	in	the	surrounding	microenvironment	
to	avoid	prolonged	accumulation	of	K+(25).	It	has	been	shown	that	Müller	cells	can	
17	

	
take	 up	 K+	 from	 the	 inner	 and	 outer	 plexiform	 layers	 where	 neuronal	 synapses	
occur	 and	 release	 the	 K+	 into	 the	 vitreous	 humor	 in	 an	 effort	 to	 redistribute	 K+	
ions(26).	 This	 process	 is	 also	 involved	 in	 retinal	 fluid	 removal.	 Müller	 cells	 act	 as	
potassium	shuttle	by	taking	up	potassium	from	the	extracellular	fluid	through	Kir2.1	
potassium	channels	and	depositing	the	potassium	into	the	vasculature	using	Kir4.1	
channels	 that	 are	 found	 on	 the	 Müller	 cell	 processes	 that	 encompass	 the	 blood	
vessels(9,10).	This	leads	to	osmotic	fluid	removal	through	aquaporin-4(8,10–12).	
In	addition	to	regulating	neurotransmitters	and	ion	levels	within	the	retina,	
Müller	cells	also	participate	in	the	retinoid	cycle	with	cone	photoreceptors	by	taking	
up	 all-trans	 retinol	 from	 the	 subretinal	 space(27–30).	 During	 the	 visual	 cycle,	
photons	of	light	lead	to	isomerization	of	11-cis	retinal	to	all-trans	retinal	in	the	rod	
and	 cone	 photoreceptors.	 	 Once	 isomerized,	 all-trans	 retinal	 is	 expelled	 from	 the	
opsin	protein	to	be	reduced	by	retinol	dehydrogenases	to	all-trans	retinol(31).	The	
all-trans	retinol	from	the	cones	is	then	released	into	the	extracellular	space	where	it	
is	 taken	 up	 by	 Müller	 cells,	 isomerized	 back	 to	 11-cis	 retinol	 by	 all-trans	 retinol	
isomerase,	and	released	back	to	the	extracellular	space	to	be	taken	up	by	the	cone	
photoreceptors	where	it	can	finally	be	oxidized	from	11-cis	retinol	back	to	original	
11-cis	retinal	to	restart	the	visual	cycle(27–29,32).	
Müller	cells	seem	a	primary	site	of	nutrient	storage	for	the	retina.	It	has	been	
shown	 that	 ATP	 production	 in	 Müller	 cells	 drastically	 declines	 when	 glycolysis	 is	
inhibited.	 However,	 ATP	 levels	 remained	 equal	 in	 aerobic	 versus	 anaerobic	
conditions	 as	 long	 as	 glucose	 was	 provided,	 indicating	 that	 Müller	 cells	 live	
primarily	 from	 glycolysis	 rather	 than	 oxidative	 phosphorylation(33).	 This	 is	
18	

	
important	 as	 it	 spares	 oxygen	 for	 retinal	 neurons	 and	 other	 cell	 types	 that	 use	
oxidative	 phosphorylation	 for	 ATP	 production.	 Furthermore,	 Müller	 cells	 are	 the	
primary	 site	 of	 glycogen	 storage	 in	 the	 retina(33,34).	 When	 nutrient	 supplies	 are	
low	 Müller	 cells	 can	 utilize	 this	 glycogen	 storage	 to	 provide	 metabolites	 for	 other	
cell	types.	Furthermore,	the	large	amounts	of	lactate	they	produce	via	glycolysis	and	
irreversible	 conversion	 of	 pyruvate	 to	 lactate	 due	 to	 a	 specific	 lactate	
dehydrogenase	 isoform	 can	 be	 transported	 to	 photoreceptors	 to	 be	 used	 as	 a	
potential	 alternative	 source	 of	 energy	 in	 case	 of	 need(33,35,36).	 Interestingly,	
studies	 suggest	 that	 the	 metabolism	 of	 glucose	 and	 glycogen	 by	 Müller	 cells	 is	
regulated	 by	 light	 being	 absorbed	 by	 the	 photoreceptors(24).	 This	 means	 that	 as	
photoreceptors	absorb	light,	the	Müller	cells	respond	by	metabolizing	more	glucose	
in	 order	 to	 provide	 more	 lactate	 for	 photoreceptors	 as	 needed,	 indicating	 that	
Müller	cells	and	photoreceptors	are	tightly	coupled	in	their	respective	functions	by	
metabolism.	 In	 addition	 to	 providing	 lactate	 as	 a	 fuel	 source	 for	 photoreceptors,	
Müller	cells	can	also	regulate	nutrient	supplies	to	the	retina	via	regulation	of	retinal	
blood	 flow.	 In	 a	 healthy	 retina,	 increased	 light	 stimulation	 results	 in	 increased	
retinal	 blood	 flow,	 which	 is	 required	 to	 supply	 the	 activated	 neurons	 with	 oxygen	
and	 other	 nutrients,	 a	 process	 termed	 neurovascular	 coupling.	 Müller	 cells	 play	 a	
crucial	 role	 in	 neurovascular	 coupling	 as	 they	 release	 metabolites	 controlling	
vasoconstriction	and	vasodilation	of	retinal	blood	vessels(37,38).	
One	 of	 the	 most	 important	 functions	 of	 Müller	 cells	 is	 their	 regulation	 of	
retinal	 blood	 flow	 and	 contribution	 to	 the	 blood	 retinal	 barrier.	 The	 blood	 retinal	
barrier	 is	 essential	 for	 preventing	 leakage	 of	 blood	 and	 other	 potentially	 harmful	
19	

	
stimuli	such	as	pathogens	from	entering	the	retinal	tissue.		It	has	been	shown	that	
Müller	 cells	 induce	 blood-barrier	 properties	 in	 retinal	 endothelial	 cells(39,40).	
Studies	 using	 conditional	 ablation	 of	 Müller	 cells	 showed	 severe	 blood	 retinal	
barrier	 breakdown(41).	 The	 exact	 mechanism	 of	 how	 Müller	 cells	 maintain	 the	
blood	retinal	barrier	is	debated	but	includes	the	secretion	of	factors	such	as	pigment	
epithelium-derived	factor	(PEDF)	and	thrombospondin-1	which	are	anti-angiogenic	
and	increase	the	tightness	of	the	endothelial	barrier(42,43).	
It	 is	 clear	 that	 Müller	 cells	 are	 an	 integral	 part	 of	 a	 healthy	 and	 well	
functioning	retina.	Any	disturbance	to	these	cells	certainly	affects	cellular	cross-talk	
within	the	retina	and	its	proper	function.	However,	despite	their	importance	Müller	
cells	 are	 still	 an	 under-studied	 cell	 type	 in	 the	 context	 of	 diseases	 such	 as	 diabetic	
retinopathy.	The	following	aims	to	provide	an	overview	about	the	effects	of	diabetes	
on	Müller	cells	and	the	role	Müller	cells	play	in	pathological	events	in	the	diabetic	
retina.	
	
1.4.2	Müller	Cells	in	Diabetic	Retinopathy	
	

Functional	changes	that	have	been	determined	in	Müller	cells	begin	early	in	

the	disease,	with	significant	decreases	in	glutamate	transport	via	GLAST	beginning	
after	 just	 4	 weeks	 of	 diabetes	 in	 rats(44).	 This	 is	 consistent	 with	 reports	 showing	
significantly	increased	glutamate	accumulation	in	the	retinas	of	diabetic	rats(45,46).	
Furthermore,	 these	 studies	 have	 shown	 that	 there	 is	 decreased	 glutamine	
synthetase	 activity	 and	 a	 subsequent	 decrease	 in	 the	 conversion	 of	 glutamate	 to	
glutamine	necessary	for	neurotransmitter	regeneration(45,46).	These	results	are	in	
20	

	
line	 with	 reports	 demonstrating	 glutamate	 increases	 to	 a	 potentially	 neurotoxic	
level	in	the	vitreous	of	diabetic	patients(47).	However,	in	neurological	diseases	such	
as	 stroke,	 therapies	 targeting	 glutamate	 increase	 have	 been	 ineffective	 indicating	
that	 increased	 glutamate	 levels	 might	 not	 play	 a	 pathophysiological	 role(48,49).	
Whether	 increased	 glutamate	 levels	 actually	 cause	 neurotoxicity	 over	 time	 in	
diabetic	retinopathy	has	yet	to	be	determined.	
It	 seems	 that	 Müller	 cells	 not	 only	 contribute	 to	 glutamate	 toxicity	 directly	 by	
decreased	 glutamate	 uptake,	 but	 Müller	 cells	 also	 contribute	 indirectly	 via	
decreased	 K+	 uptake	 during	 the	 progression	 of	 diabetic	 retinopathy.	 There	 is	
decreased	K+	conductance	on	the	plasma	membrane	of	Müller	cells	isolated	from	rat	
retinas	after	4	months	of	experimental	diabetes(15).	Redistribution	of	the	Kir4.1	K+	
channel	 has	 been	 identified	 as	 the	 mechanism	 of	 decreased	 K+	 conductance(15).	
This	decrease	in	K+	conductance	was	also	observed	in	Müller	cells	of	patients	with	
proliferative	 diabetic	 retinopathy(50).	 Alteration	 of	 the	 Kir4.1	 K+	 channel	
localization	 in	 Müller	 cells	 in	 the	 diabetic	 retina	 has	 been	 attributed	 to	 the	
accumulation	 of	 advanced	 glycation	 end	 products	 (AGEs)(51).	 Together,	 this	 can	
lead	 to	 an	 imbalance	 in	 K+	 concentrations	 and	 altered	 K+	 homeostasis	 leading	 to	
neuronal	 excitation	 and	 subsequent	 glutamate	 toxicity.	 	 The	 contribution	 of	 K+	
spatial	buffering	in	the	context	of	DME	has	been	discussed	in	section	1.2.4.	
In	addition	to	the	defects	in	neurotransmitter	clearance	and	K+	homeostasis	
during	 diabetic	 retinopathy,	 Müller	 cells	 become	 activated	 as	 shown	 by	 increased	
expression	 of	 glial	 fibrillary	 acidic	 protein	 (GFAP),	 a	 common	 marker	 of	 reactive	
gliosis(45,52,53).	 	 Upon	 activation,	 Müller	 cells	 have	 been	 shown	 to	 secrete	 a	
21	

	
number	 of	 pro-inflammatory	 cytokines	 and	 pro-angiogeneic	 growth	 factors	
including	 vascular	 endothelial	 growth	 factor	 (VEGF),	 pigment	 epithelium-derived	
factor	 (PEDF),	 interleukin-1β	 (IL-β),	 interleukin-6	 (IL-6),	 tumor	 necrosis	 factor-α	
(TNF-α),	 chemokine	 ligand-2	 (CCL2),	 prostaglandin	 E2	 (PGE2),	 inducible	 nitric	
oxide	synthase	(iNOS)	and	cyclooxygenase-2	(COX2)(54–60).		
One	 specific	 pro-inflammatory	 pathway	 seems	 to	 stand	 out	 when	 talking	
about	 Müller	 cells	 and	 their	 role	 in	 inflammatory	 events.	 Once	 stimulated,	 Müller	
cells	 predominantly	 activate	 the	 caspase-1/interleukin-1beta	 (IL-1β)	 pathway(61–
63)	 leading	 to	 speculations	 that	 IL-1β	 might	 not	 only	 be	 produced	 during	
inflammatory	events	but	also	for	physiological	events	such	as	regulation	of	glucose	
consumption.	
	
1.5	Caspase	Family	
1.5.1	Caspase	Enzymes-	Functions	and	Outcomes	
Caspases	 are	 a	 family	 of	 enzymes	 responsible	 for	 regulating	 inflammation	
and	 cell	 death	 in	 both	 normal	 and	 disease	 states.	 These	 endoproteases	 share	 a	
common	 mechanism	 of	 action	 in	 which	 they	 recognize	 their	 specific	 substrate	 and	
use	 a	 catalytic	 cysteine	 residue	 in	 their	 active	 site	 to	 cleave	 the	 substrate	 after	 an	
aspartic	acid	residue.		It	is	this	“C”ysteine-mediated	cleavage	after	an	“As”partic	acid	
residue	 from	 which	 the	 “Cas”pase	 name	 is	 derived.	 Caspases	 have	 been	 classified	
into	two	main	categories,	the	pro-inflammatory	caspases	that	regulate	inflammation	
or	 pro-apoptotic	 caspases	 that	 initiate	 and	 carry	 out	 cell	 death.	 The	 proinflammatory	 caspases	 include	 caspase-1,	 -4,	 -5,	 -11,	 and	 -12(64).	 	 The	 pro22	

	
apoptotic	 caspases	 can	 be	 further	 divided	 into	 subgroups	 including	 initiator	
caspases	(caspase-8	and	-9)	or	executioner	caspases	(caspase-3,	-6,	and	-7)(64).		
Caspases	 are	 typically	 produced	 as	 an	 inactive	 pro-caspase	 that	 requires	
dimerization	 and	 autocatalytic	 cleavage	 of	 their	 pro-domain	 before	 they	 can	 carry	
out	their	own	enzymatic	function.	The	mechanism	of	dimerization	and	activation	is	
dependent	 on	 the	 specific	 caspase	 in	 question,	 however	 the	 binding	 of	 an	 adaptor	
protein	 to	 the	 pro-domain	 of	 the	 procaspase,	 allowing	 for	 autocatalytic	 cleavage	
between	the	pro-	and	active-domain	on	the	caspase	to	produce	the	active	caspase,	
typically	 facilitates	 activation.	 This	 mechanism	 is	 known	 as	 the	 proximity	 induced	
mechanism	of	caspase	activation(65).		
In	 the	 pro-apoptotic	 caspases	 the	 pro-domain	 contains	 a	 death	 effector	
domain	 (DED)	 to	 facilitate	 dimerization	 and	 activation.	 Activation	 of	 an	 initiator	
caspase	 via	 the	 DED	 typically	 leads	 to	 a	 cascade	 of	 caspase	 activation	 where	 one	
caspase	cleaves	and	activates	the	next,	leading	to	eventual	cleavage	and	activation	of	
executioner	 caspases	 resulting	 in	 cell	 death.	 With	 the	 pro-inflammatory	 caspases	
the	adaptor	protein	typically	binds	the	caspase	activation	and	recruitment	domain	
(CARD)	rather	than	a	DED,	leading	to	dimerization	and	cleavage	in	order	to	activate	
the	 caspase.	 	 Furthermore,	 where	 as	 activation	 of	 pro-apoptotic	 caspases	 leads	 to	
activation	 of	 other	 caspases,	 activation	 of	 pro-inflammatory	 caspases	 typically	
results	in	activation	of	pro-inflammatory	cytokines.		
1.5.2	Caspase-1	
Caspase-1	 is	 arguably	 the	 most	 well	 studied	 member	 of	 the	 caspase	 family.		
Formerly	 known	 as	 interleukin-1-converting	 enzyme	 (ICE),	 the	 most	 prominent	
23	

	
action	 of	 caspase-1	 is	 its	 ability	 cleave	 and	 activate	 interleukin-1β,	 a	
proinflammatory	 cytokine	 involved	 in	 innate	 immunity(66,67).	 Caspase-1	 itself	 is	
produced	as	a	45	kDa	inactive	zymogen,	and	is	cleaved	at	two	locations	resulting	in	
three	 separate	 subunits-	 the	 CARD	 domain	 required	 for	 activation,	 with	 the	
remaining	 two	 subunits	 being	 10	 (p10)	 and	 20	 (p20)	 kDA	 polypeptides(68–71).		
Following	 cleavage	 of	 multiples	 caspases,	 two	 p10	 subunits	 and	 two	 p20	 subunits	
come	 together	 to	 form	 heterotetramer	 capable	 of	 cleaving	 various	
substrates(69,71,72).		Caspase-1	is	in	the	group	of	proinflammatory	caspases	rather	
than	 the	 pro-apoptotic	 group,	 and	 thus	 contains	 a	 CARD	 required	 for	 recruitment	
and	activation	rather	that	a	DED.		The	activation	of	caspase-1	is	typically	facilitated	
by	 a	 molecular	 platform	 termed	 the	 inflammasome.	 The	 inflammasome	 platform	
usually	 contains	 a	 pattern	 recognition	 receptor	 (PRR),	 which	 is	 responsible	 for	
sensing	 various	 harmful	 stimuli,	 either	 pathogen	 associated	 molecular	 patterns	
(PAMPs)	or	damage	associated	molecular	patterns	(DAMPs).	There	are	a	number	of	
known	 activators	 of	 caspase-1	 including	 NLR	 family	 pyrin	 domain	 containing	 1	
(NLRP1),	 NLRP3,	 NLRP6,	 NLRP7,	 NLR	 family	 CARD	 domain	 containing	 protein	 4	
(NLRC4),	 absent	 in	 melanoma	 2	 (AIM2),	 RIG-1-like	 receptors,	 and	 receptor	
interacting	protein-2	(RIP2)(73,74).		
In	 addition	 to	 its	 primary	 function	 in	 IL-1β	 production,	 caspase-1	 has	 been	
shown	 to	 be	 a	 relatively	 promiscuous	 enzyme	 that	 is	 able	 to	 cleave	 a	 variety	 of	
substrates.	 	 Caspase-1	 can	 cleave	 and	 activate	 IL-18,	 a	 cytokine	 that	 can	 stimulate	
inflammation	 via	 induction	 of	 tumor	 necrosis	 factor-α(TNFα)	 and	 interferon-γ	
(IFNγ)(75–78).	Furthermore,	caspase-1	can	be	linked	to	adaptive	immunity	due	to	
24	

	
it’s	 role	 in	 activation	 of	 IL-33	 which	 modulates	 T	 helper	 type	 2	 (TH2)	 immune	
response(74,79).	Recent	studies	have	shown	that	there	may	be	at	least	121	caspase1	substrates,	many	of	them	related	to	cell	death	independent	of	IL-1β	action(73).	
1.5.3	Caspase-1	in	Disease	
Caspase-1	 has	 been	 implicated	 in	 a	 number	 of	 diseases	 involving	
inflammation.		These	disease	range	from	inflammatory	disease	such	as	septic	shock,	
inflammatory	 bowel	 disease,	 rheumatoid	 and	 osteoarthritis,	 to	 neurodegenerative	
diseases	such	as	Huntington’s	disease,	amyotrophic	lateral	sclerosis	and	Alzheimer’s	
disease,	and	also	diseases	of	the	eye	including	diabetic	retinopathy	and	age	related	
macular	degeneration	(AMD)(62,66,80–88).	The	harmful	effects	of	caspase-1	can	be	
mediated	by	cleavage	of	its	vast	number	of	substrates,	although	IL-1β	and	IL-18	are	
the	 most	 prominent	 effectors.	 The	 beneficial	 effects	 of	 caspase-1	 inhibition	 have	
been	 implicated	 in	 a	 number	 of	 diseases	 ranging	 from	 rheumatoid	 arthritis,	
epilepsy,	and	diabetic	retinopathy(88–90).		
	
1.6	The	Interleukin-1	Family	and	Signaling	
1.6.1	The	Interleukin-1	Family	
Interleukin-1	 (IL-1)	 was	 the	 first	 interleukin	 to	 be	 identified	 and	 the	 IL-1	
family	has	since	grown	to	a	total	of	11	members.		These	11	family	members	include	
IL-1α,	 IL-1β,	 IL-1	 receptor	 antagonist	 (IL-1Ra),	 IL-18,	 IL-33,	 and	 IL-1F5-F10.	 	 The	
effects	 of	 these	 family	 members	 have	 increased	 in	 complexity	 with	 recent	 studies.		
Some	of	these	family	members	seem	to	be	inflammatory	by	acting	as	a	Type	1	IL-1	
receptor	(IL-1R1)	agonist	(IL-1α,	IL-1β,	IL-18,	IL-1F6,	IL-1F8,	IL-1F9,	IL-1F11),	some	
25	

	
can	 be	 suppressors	 of	 inflammation	 by	 acting	 as	 an	 IL-1R1	 antagonist	 (IL-1Ra,	 IL1F10),	and	some	have	even	been	shown	to	be	anti-inflammatory	in	nature	(IL-1F5,	
IL-1F7)(91–93).	 Furthermore,	 IL-1α,	 IL-33,	 and	 IL-1F7	 have	 been	 found	 in	 the	
nucleus	and	exert	their	effects	by	acting	as	a	nuclear	factor	by	binding	to	DNA	and	
influencing	 gene	 transcription(91,94–97).	 Interestingly,	 although	 the	 majority	 of	
these	 IL-1	 family	 members	 act	 as	 extracellular	 cytokines,	 only	 IL-1ra	 has	 a	
traditional	 signal	 peptide	 allowing	 it	 to	 be	 secreted	 by	 the	 classical	 secretory	
pathway	

involving	

the	

endoplasmic	

reticulum	

and	

Golgi	

complex	

mechanism(93,97).	 	 The	 exact	 mechanism	 by	 which	 the	 other	 IL-1	 members	 are	
secreted	is	currently	unknown.	
1.6.2	IL-1β ,	IL-1β 	Receptor	Signaling,	and	Regulation	
IL-1β	is	arguably	the	most	widely	researched	member	of	the	IL-1	family.	This	proinflammatory	 cytokine	 plays	 a	 crucial	 role	 in	 innate	 immunity,	 and	 is	 typically	
produced	 by	 monocytes,	 macrophages	 or	 microglia	 in	 response	 to	 DAMPs	 or	
PAMPs(92).	 IL-1β	 is	 produced	 as	 a	 biologically	 inactive	 31	 kDa	 precursor	 (pro-IL1β)	 and	 requires	 cleavage	 by	 caspase-1	 to	 produce	 the	 17	 kDa	 active	 form	 that	 is	
secreted	 and	 involved	 in	 inflammation	 and	 cell	 death	 in	 innate	 immunity(68,98–
100).	IL-1β	signaling	is	facilitated	by	ligand	binding	to	IL-1R1	on	the	cell	membrane.	
Much	 like	 Toll	 Like	 receptors	 (TLRs),	 IL-1R1	 contains	 an	 extracellular	
immunoglobulin	domain	to	which	IL-1β	binds.	Upon	binding,	IL-1R1	recruits	the	IL1	 receptor	 accessory	 protein	 (IL-1RAcP)	 that	 is	 necessary	 for	 signal	 transduction.		
Both	IL-1R1	and	IL-1RAcP	have	intracellular	Toll/IL-1	receptor	(TIR)	domains	that	
upon	 the	 formation	 of	 the	 heterodimer,	 come	 together	 and	 recruit	 myeloid	
26	

	
differentiation	primary	response	protein	88	(MYD88)	leading	to	phosphorylation	of	
IL-1R-associated	kinases	(IRAKs)	and	inhibitor	of	NF-κB	kinase	β	(IKKβ)	resulting	in	
activation	 of	 NF-κB	 and	 eventual	 gene	 transcription.	 Due	 to	 the	 severity	 of	 IL-1	
signaling,	 this	 pathway	 is	 tightly	 controlled	 by	 a	 number	 of	 redundant	 negative	
regulators.	The	first	level	of	regulation	comes	from	within	the	IL-1	family	itself,	IL1ra.		IL-1ra	can	bind	to	IL-1R1	and	prevent	recruitment	of	the	crucial	IL-1RAcP,	thus	
occupying	 IL-1R1	 and	 preventing	 signaling(93,101).	 	 Another	 level	 of	 regulation	
comes	from	the	Type	2	IL-1	receptor	(IL-1R2)	that	serves	as	a	decoy	receptor.		IL1R2	is	able	to	bind	IL-1β	but	the	receptor	lacks	the	cytoplasmic	TIR	domain	and	is	
therefore	unable	to	signal.		In	addition	to	serving	as	an	IL-1β	trap,	IL-1R2	provides	
further	sequestration	of	signaling	since	it	recruits	the	IL-1RAcP	after	it	binds	IL-1β,	
leaving	less	IL-1RAcP	to	dimerize	with	IL-1R1.			
1.6.3	Autoinflammatory	Disease	
IL-1β	 plays	 such	 a	 significant	 role	 in	 some	 inflammatory	 diseases	 that	 there	 is	 an	
entire	 class	 of	 syndromes	 that	 are	 characterized	 by	 aberrant	 IL-1β	 signaling.		
Termed	 “Autoinflammatory”	 diseases,	 these	 diseases	 involve	 increased	 IL-1β	
signaling,	 although	 it	 can	 come	 from	 either	 uncontrolled	 caspase-1	 activation	 and	
IL-1β	 secretion	 as	 in	 the	 case	 of	 familial	 Mediterranean	 fever	 (FMF),	 cryopyrinassociated	 periodic	 syndroms	 (CAPSs),	 and	 Muckle-Wells	 syndrome,	 or	 decreased	
regulation	 of	 the	 IL-1β	 signaling	 as	 in	 the	 case	 of	 deficiency	 of	 IL-1ra	
(DIRA)(97,102–104).	 One	 common	 characteristic	 of	 these	 autoinflammatory	
diseases	is	their	general	responsiveness	to	IL-1β	blockade,	whether	it	be	through	an	
IL-1	receptor	antagonist	or	an	IL-1β	neutralizing	antibody(91,105–107).	
27	

	
1.7	Objectives	of	the	Dissertation	
In	 the	 last	 few	 years,	 it	 has	 become	 clear	 that	 diabetic	 retinopathy	 has	
multiple	 features	 of	 a	 chronic	 inflammatory	 disease.	 Interestingly,	 preliminary	
studies	 have	 indicated	 that	 the	 pro-inflammatory	 caspase-1/IL-1β	 pathway	 is	
activated	 in	 diabetic	 retinopathy.	 Since	 autoinflammatory	 diseases	 are	 defined	 by	
caspase-1	 activation	 and	 IL-1β	 production	 it	 was	 an	 intriguing	 idea	 to	 investigate	
whether	 diabetic	 retinopathy	 has	 some	 features	 of	 an	 autoinflammatory	 disease.	
Supporting	such	an	idea	was	the	fact	that	Müller	cells	seem	to	preferentially	activate	
this	pathway	once	stimulated	and	could	serve	as	a	main	source	for	active	caspase-1	
and	IL-1β.			
	
Therefore,	the	working	hypothesis	investigated	in	this	dissertation	is	as	follows:	
	
Hypothesis:	 We	 hypothesize	 that	 hyperglycemia	 induces	 caspase-1	 activity	
and	IL-1β	production	in	retinal	Müller	cells	in	vitro	and	retinal	tissue	in	vivo.	
We	 postulate	 that	 caspase-1	 is	 sustained	 by	 an	 IL-1β-induced	 feedback	
mechanism	that	requires	signaling	via	a	functional	IL-1	receptor	as	it	has	been	
discussed	 for	 autoinflammatory	 diseases.	 	 We	 further	 hypothesize	 that	
intervening	in	the	caspase-1/IL-1β	pathway	will	prevent	diabetic	retinopathy.	
	
The	 goal	 of	 this	 study	 was	 to	 identify	 mechanisms	 involved	 in	 the	 activation	 of	 the	
caspase-1/IL-1β	 pathway	 and	 potential	 feedback	 signaling	 involved	 in	 sustained	
inflammation.	 Furthermore,	 we	 aimed	 to	 identify	 the	 consequence	 of	 prolonged	
28	

	
caspase-1	 activation	 in	 the	 fate	 of	 Müller	 cells	 and	 also	 in	 the	 development	 and	
progression	 of	 diabetic	 retinopathy	 in	 order	 to	 potentially	 identify	 new	 therapeutic	
targets	in	disease	treatment.			
	

	

29	

	
	

Chapter	2.	Interleukin-1	Receptor-Dependent	and	Independent	Caspase-1	
Activation	in	Diabetic	Retinopathy	

2.1	Introduction	
Diabetes	 leads	 to	 many	 complications,	 one	 of	 them	 being	 diabetic	 retinopathy.	
Diabetic	 retinopathy	 is	 characterized	 by	 microaneurysms,	 vascular	 leakage,	
neovascularization	and/or	macular	edema	within	the	retina	and	ultimately	leads	to	
vision	 loss(108).	 Diabetic	 retinopathy	 was	 once	 thought	 of	 as	 a	 purely	
microvascular	 disease,	 however	 more	 recent	 studies	 indicate	 that	 the	 disease	 may	
actually	 originate	 within	 the	 retinal	 tissue.	 Sustained	 low-grade	 retinal	 tissue	
inflammation	 has	 been	 discussed	 as	 the	 cause	 for	 retinal	 cell	 dysfunction	 and	 cell	
death	 leading	 to	 subsequent	 microvascular	 changes(109,110).	 Despite	 increasing	
evidence	 that	 chronic	 inflammation	 and	 cell	 death	 contribute	 to	 development	 and	
progression	 of	 diabetic	 retinopathy,	 there	 have	 been	 few	 treatments	 aimed	 at	
preventing	 these	 events.	 In	 order	 to	 develop	 such	 treatments,	 a	 better	
understanding	 of	 mechanisms	 underlying	 initiation	 and	 maintenance	 of	 chronic	
inflammation	is	crucial.	
Interleukin-1β	 (IL-1β)	 has	 emerged	 as	 one	 of	 the	 prominent	 pro-inflammatory	
cytokines	 associated	 with	 diabetic	 retinopathy(111–115).	 Levels	 of	 IL-1β	 are	
elevated	 in	 the	 vitreous	 and	 serum	 of	 patients	 with	 proliferative	 diabetic	
retinopathy	 compared	 to	 healthy	 individuals(113).	 Caspase-1,	 originally	 named	
Interleukin-1β	 Converting	 Enzyme	 (ICE),	 is	 the	 enzyme	 responsible	 for	 producing	
IL-1β	by	converting	pro-IL-1β	into	its	active	form,	which	in	turn	exerts	its	effects	via	
the	 Type	 1	 receptor	 (IL-1R1).	 Our	 previous	 work	 has	 indicated	 that	 caspase-1	 is	
30	

	
activated	 throughout	 the	 progression	 of	 diabetic	 retinopathy	 in	 STZ	 and	
galactosemic	mice(62,88,116).	Caspase-1	activity	was	also	elevated	in	retinal	tissue	
samples	 from	 diabetic	 donors(62,117).	 Müller	 cells	 are	 one	 of	 the	 cell	 types	
identified	as	being	a	source	of	active	caspase-1	and	IL-1β	production.		
	

Typically,	 caspase-1	 activation	 occurs	 in	 response	 to	 a	 bacterial	 infection.	

This	mechanism	is	well	established	and	usually	involves	sensing	of	the	pathogen	via	
the	Nlrp3	inflammasome(118).	In	the	diabetic	retina,	caspase-1	activation	is	unique	
in	that	it	occurs	in	response	to	elevated	glucose	levels	rather	than	a	pathogen.	Very	
little	 is	 known	 about	 caspase-1	 activation	 in	 so	 called	 “sterile	 inflammation.”	
Alternative	 pathways	 that	 are	 independent	 of	 the	 Nlrp3	 inflammasome	 have	 been	
suggested	to	activate	caspase-1	in	pathogen	free	conditions.	For	example,	Receptor	
Interacting	 Protein	 2	 (RIP2)	 can	 act	 as	 a	 potential	 activator	 of	 caspase-1	 in	 sterile	
inflammation(119–123).	In	Huntington’s	disease,	which	is	considered	to	be	a	sterile	
inflammatory	 disease,	 RIP2-mediated	 caspase-1	 activation	 leads	 to	 chronic	 tissue	
inflammation	 and	 cell	 death(82).	 To	 date,	 the	 mechanism	 by	 which	 caspase-1	
activation	is	initiated,	and	more	importantly	sustained,	in	the	retina	and	retinal	cells	
under	diabetic	conditions	is	not	known.	
Therefore,	 this	 study	 aimed	 to	 identify	 mechanisms	 of	 how	 caspase-1	 activity	 is	
induced	 and	 maintained	 in	 hyperglycemic	 conditions	 in	vitro	 and	 in	vivo	 and	 how	
this	 process	 contributes	 to	 the	 sustained	 low-grade	 inflammation	 seen	 in	 diabetic	
retinopathy.		
	

31	

	
2.2	Research	Design	And	Methods	
Antibodies	 and	 Reagents.	 Caspase-1	 antibody	 was	 from	 Invitrogen	 (Burlington,	
Ontario).	 Goat	 Anti-Rabbit	 IgG	 conjugated	 to	 horseradish	 peroxidase	 (HRP)	 and	 420%	 Gradient	 Tris-SDS-PAGE	 gels	 were	 from	 BioRad	 (Hercules,	 CA).	 TRIzol,	 7amino-4-trifluoro-methylcoumarin	(AFC)	and	glucose	assay	kit	were	from	Sigma	(St.	
Louis,	 MO).	 GHb	 kits	 were	 from	 Glyc-Affin	 (Rockford,	 IL).	 Caspase-1	 substrate,	
YVAD-fmk	 and	 elastase	 Calbiochem	 (San	 Diego,	 CA).	 Human	 IL-1β,	 IL-1	 receptor	
antagonist	 (IL-1ra),	 and	 high	 sensitivity	 IL-1β	 ELISA	 was	 from	 R&D	 Systems	
(Minneapolis,	MN).	Rabbit	polyclonal	anti-RIP2	antibody	(ab8428)	was	from	Abcam	
(Cambridge,	 MA).	 ON-TARGETplus	SMARTpool	 Human	 RIPK2	 and	 ON-TARGETplus	
Control	 siRNA	 Non-Targeting	 siRNA	 #1	 were	 purchased	 from	 Thermo	 Scientific	
Dharmacon.	Amaxa	Cell	Line	Nucleofector	Kit	L	was	purchased	from	Lonza	(Basel,	
Switzerland).	 Mouse	 anti-Glutamine	 synthetase	 (#610517,	 clone	 6)	 was	 obtained	
from	Transduction	Laboratories.		
Animal	Models.	Caspase-1	knockout	mice	(Casp1-/-)	(gift	from	Dr.	T.	McCormick,	Case	
Western	Reserve	University),	and	IL-1	receptor	knockout	mice	(IL-1R1-/-)	(Jackson	
Laboratories;	 strain	 name:	 B6.129S7-Il1r1tm1jm)	 (both	 in	 a	 C57BL/6	 background)	
mice	 were	 bred	 using	 homozygous	 breeding	 pairs.	 Recent	 studies	 have	
demonstrated	 that	 these	 caspase-1	 null	 mice	 (derived	 from	 the	 original	 strain	
described	 by	 Li	 et	 al	 (66))	 lack	 both	 caspase-1	 and	 caspase-11	 (Casp1-/-	 Casp11-/-)	
(124,125).	Male	mice	(Wild-type	(WT)	C57BL/6,	Casp1-/-,	and	IL-1R1-/-)	weighing	20	
g	 were	 randomly	 assigned	 to	 be	 either	 diabetic	 or	 non-diabetic	 controls.	
Streptozotocin	(STZ)	injections	(60mg/kg	body	wt	i.p.	on	5	consecutive	days)	were	
32	

	
utilized	 to	 induce	 diabetes	 as	 previously	 described	 (62).	 Diabetic	 animals	 were	
maintained	with	insulin	injections	(0.1-0.2	units	of	NPH	insulin	subcutaneously)	as	
needed.	Animals	had	free	access	to	food	and	water	and	were	maintained	under	a	12	
h	 on/12	 h	 off	 light	 cycle.	 Treatment	 of	 animals	 conforms	 to	 the	 Association	 for	
Research	 in	 Vision	 and	 Ophthalmology	 Resolution	 on	 Treatment	 of	 Animals	 in	
Research.	 GHb	 levels	 were	 measured	 at	 the	 end	 of	 the	 each	 study	 to	 determine	
severity	of	diabetes	(Table	1).		
Histologic	assessment	of	retinal	vascular	pathology.	Isolated	retinas	were	washed	in	
running	 water	 overnight	 and	 then	 digested	 in	 elastase	 solution	 (0.4U/ml	 in	
100mmol/L	 sodium	 phosphate	 buffer,	 150	 mmol/L	 sodium	 chloride,	 5	 mmol/L	
EDTA)	 for	 30-45	 min	 at	 37°C.	 The	 retinal	 tissue	 was	 transferred	 into	 100mmol/L	
Tris-Hydrochloric	Acid	buffer	(pH	8.5)	and	left	overnight	at	RT.	The	cleaned	vessel	
network	 was	 dried	 onto	 a	 mounting	 slide,	 stained	 with	 hematoxylin	 and	 PAS,	
dehydrated	 and	 covered	 with	 coverslip.	 Numbers	 of	 acellular	 capillaries	 were	
counted	in	8	areas	per	retina	(20X	magnification),	averaged,	and	reported	per	mm2	
retina	as	previously	described(126).	
Tissue	 Culture.	 Human	 retinal	 Müller	 cells	 (hMC):	 	 Handling	 of	 human	 tissue	
conformed	 to	 the	 tenets	 of	 Declaration	 of	 Helsinki	 for	 research	 involving	 human	
tissue.	Human	Müller	cells	were	isolated	from	retinal	tissue	of	healthy	donors	with	
no	 history	 of	 diabetes	 and	 chronic	 inflammatory	 diseases	 as	 previously	
described(127,128).		
Treatment:	 hMC	 (1x106)	 were	 treated	 with	 either	 5	 mmol/L	 glucose	 DMEM	 or	 25	
mmol/L	 glucose	 DMEM	 supplemented	 with	 2%	 FBS,	 1%	 P/S	 for	 either	 48	 or	 96	
33	

	
hours.	 Cells	 treated	 with	 5	 mmol/L	 glucose	 medium	 served	 as	 controls.	 For	 IL-1β	
studies,	 recombinant	 human	 IL-1β	(1-5ng/ml)	 was	 used.	 For	 IL-1ra	 studies,	
following	pretreatment	with	100ng/ml	IL-1ra	for	1	hour,	hMC	were	incubated	in	25	
mmol/L	 glucose	 DMEM	 for	 48	 or	 96	 hours,	 or	 in	 5	 mmol/L	 glucose	 DMEM	 plus	
recombinant	human	IL-1β	(2ng/ml)	for	24	hours.		
siRNA	 Electroporation:	 hMCs	 were	 electroporated	 with	 either	 siRNA	 against	 RIP2	
(50	 nmol)	 or	 scramble	 RNA	 control	 (50	 nmol)	 using	 a	 Nucleofector	 II	 device	 from	
Amaxa	Biosystems	(Cologne,	Germany).		
Preparation	 of	 cytosolic	 lysates.	 Following	 treatment,	 hMC	 were	 lysed	 in	 100μl	 of	
lysate	buffer	[CHAPS	Buffer	(100mM	HEPES,	pH	7.5	containing	10%	sucrose,	0.1%	
CHAPS),	 1mmol/L	 EDTA,	 1	 mmol/L	 PMSF	 and	 leupeptin	 (10μg/ml)]	 as	 described	
previously(126).		
Caspase-1	 Activity	 Assay.	 Caspase-1	 activities	 were	 measured	 as	 described	
previously	(62,117,126–129).	Briefly,	equal	amounts	of	sample	protein	(15	μg)	were	
incubated	 in	 the	 presence	 of	 the	 specific	 caspase-1	 substrate	 (YVAD-AFC;	
2.5μmol/L)	 for	 1	 hour	 at	 32°C.	 AFC	 fluorescence	 was	 detected	 by	 a	 Tecan	 Spectra	
FluorPlus	fluorescence	plate	reader	(excitation:	400	nm,	emission:	510	nm).	Release	
of	 AFC	 by	 active	 caspase-1	 was	 calculated	 against	 an	 AFC	 standard	 curve	 and	
expressed	as	pmol	AFC/mg	protein/min.		
Cytokine	Assays.	 Medium	 (150µl)	 from	 hMC	 treated	 with	 5	 mmol/L	 or	 25	 mmol/L	
glucose	 containing	 medium	 was	 added	 to	 pre-coated	 96	 well	 plates.	 IL-1β	 ELISAs	
were	performed	according	to	the	manufacturer’s	directions.	Levels	of	cytokine	were	
normalized	to	mg	of	total	protein.		
34	

	
Glucose	Consumption	Assay.	hMC	(1x106)	were	cultured	with	either	5	mmol/L	or	25	
mmol/L	 glucose	 media	 for	 96	 hours.	 Media	 was	 changed	 at	 48	 hours.	 After	
collection	 of	 media	 at	 48	 and	 96	 hours,	 glucose	 concentration	 in	 media	 was	
measured	using	glucose	assay	kits	according	to	manufacturer’s	directions.	
Western	Blot	Analysis.	 Proteins	 (40μg)	 were	 separated	 in	 4-20%	 SDS	 gradient	 gels	
and	blotted	on	nitrocellulose	membrane.	Membranes	were	incubated	with	primary	
antibody	 against	 RIP2	 (1:1000	 dilution	 in	 PBS/0.05%	 Tween	 20)	 overnight	 at	 4°C	
followed	by	incubation	with	secondary	antibody	(1:5,000	dilution)	for	1	hour	at	RT	
and	 developed	 using	 LICOR	 Biosciences	 Odyssey	 Imaging	 System	 (Lincoln,	 NE).	
Membranes	were	re-probed	for	β-actin	and	relative	densities	of	RIP2/β-actin	were	
calculated.	
Müller	 Cell	 Death	 Detection.	 In	 vivo:	 Animals	 (see	 above)	 were	 sacrified	 after	 6	
months	 of	 diabetes.	 Eyes	 were	 fixed	 in	 formalin,	 embedded	 in	 paraffin,	 sectioned	
(10μM),	and	blocked	with	mouse	on	mouse	(M.O.M.)	Ig	blocking	solution	for	1	hour	
at	 RT.	 Slides	 were	 incubated	 with	 primary	 anti-Glutamine	 synthetase	 antibody	
(1:1000)	for	1	hour	at	RT.	Following	staining	with	secondary	antibody,	slides	were	
treated	with	Fluresceine	Avidin	DCS	for	10	min	in	dark	and	cover-slipped	with	DAPI	
containing	 mounting	 media.	 Blinded	 samples	 were	 visualized	 using	 scanning	 laser	
confocal	microscopy	(LSM	510;	Carl	Zeiss	Meditec,	Göttingen,	Germany)	and	a	water	
objective	 (63X	 Plan-Neofluor;	 Carl	 Zeiss	 Meditec).	 The	 number	 of	 Müller	 cells	 (GS	
positive	 cells)	 per	 standard	 retinal	 area	 (143μm	 x	 143μm)	 was	 established	 from	
eight	 independent	 areas	 per	 section.	 Five	 individual	 sections	 per	 experimental	
group	 were	 counted	 and	 the	 average	 number	 of	 Müller	 cells	 was	 determined	 and	
35	

	
graphed.	 In	 vitro:	 Following	 treatment,	 cells	 were	 suspended	 and	 100μL	 of	 cell	
suspension	was	mixed	with	100μL	of	trypan	blue	solution.	Cell	death	was	quantified	
using	hemocytometer.	
Statistical	 Analysis.	 Analysis	 of	 data	 was	 performed	 using	 the	 Anova	 One-Way	
(correlated	 samples,	 p<0.05)	 test	 followed	 by	 Tukey’s	 post	 analysis	 or	 KruskalWallis	 test	 (ordinal	 data;	 p<0.05)	 followed	 by	 Dunn’s	 post	 analysis	 to	 determine	
statistical	 significance	 among	 groups.	 For	 details	 in	 statistical	 analysis	 see	
VasserStats	 Statistical	 Computation	 Web	 Site.	 All	 data	 are	 presented	 as	 mean	 ±	
SDEV.	
	
2.3	Results	
2.3.1	Inhibition	of	Diabetic	Retinopathy	in	STZ	mice	by	Caspase-1	Knockout.		
We	 have	 previously	 shown	 that	 diabetes	 leads	 to	 sustained	 caspase-1	
activation	 throughout	 the	 development	 of	 diabetic	 retinopathy(126).	 To	
demonstrate	 that	 this	 activation	 of	 caspase-1	 is	 necessary	 for	 the	 development	 of	
diabetic	 retinopathy,	 a	 long-term	 study	 using	 4	 experimental	 groups	 non-diabetic	
WT,	diabetic	WT,	non-diabetic	Casp1-/-	and	diabetic	Casp1-/-	mice	was	initiated.	After	
6	 months	 of	 diabetes,	 there	 was	 a	 significant	 increase	 in	 the	 number	 of	 acellular	
capillaries/mm2	 retina	 in	 diabetic	 WT	 mice	 (12.2	 ±	 1.6)	 compared	 to	 non-diabetic	
WT	mice	(5.2	±	0.8).	Caspase-1	knockout	prevented	diabetes-induced	formation	of	
acellular	capillaries	indicating	that	activation	of	caspase-1	is	indeed	crucial	for	the	
development	 of	 diabetic	 retinopathy	 (Figure	 6).	 Supplemental	 Table	 1	 shows	 that	
inhibition	of	diabetic	retinopathy	was	not	due	to	changes	in	severity	of	diabetes.		
36	

	
2.3.2	Sustained	Caspase-1	Activity	in	STZ	Mice	Dependent	on	Feedback	Through	
the	IL-1	Receptor.		
Caspase-1	activation	leads	to	IL-1β	production.	Reports	in	the	literature	have	
suggested	that	IL-β	itself	can	promote	activation	of	caspase-1	via	signaling	through	
the	IL-1R1	receptor(130,131).	Since	sustained	caspase-1	activation	is	important	for	
the	 development	 of	 diabetic	 retinopathy	 we	 tested	 whether	 intervention	 in	 the	
downstream	 signaling	 of	 the	 caspase-1/IL-1β/IL-1R1	 signaling	 pathway	 using	 IL1R1	 knockout	 mice	 would	 impact	 caspase-1	 activation	 throughout	 disease	
progression.	 After	 10	 weeks	 of	 diabetes,	 caspase-1	 activity	 was	 significantly	
increased	in	both	diabetic	WT	and	diabetic	IL-1R1-/-	mice	by	59.6	±	15.3%	and	33.2	
±	 9.8%	 respectively	 compared	 to	 non-diabetic	 mice	 (Figure	 7A).	 At	 20	 weeks	 of	
diabetes,	caspase-1	activity	was	increased	by	30.2	±	3.8%	in	the	retinas	of	diabetic	
WT	 mice	 compared	 to	 non-diabetic	 WT	 mice.	 However,	 there	 was	 no	 significant	
increase	in	caspase-1	activity	in	retinas	of	diabetic	IL-1R1-/-	mice	compared	to	nondiabetic	 IL-1R1-/-	 or	 WT	 mice	 indicating	 that	 caspase-1	 activation	 progresses	 from	
an	IL-1R1	independent	mechanism	at	10	weeks	of	diabetes	to	an	IL-1R1	dependent	
mechanism	 at	 20	 weeks	 of	 diabetes	 (Figure	 7B).	 	 These	 data	 are	 consistent	 with	 a	
caspase-1/IL-1β/IL-1R1	feedback	signaling	mechanism.	
2.3.3	 Inhibition	 of	 Caspase-1	 Activity	 in	 Human	 Müller	 cells	 by	 IL-1	 Receptor	
Antagonist.		
Previously	we	have	shown	that	hyperglycemia	induces	caspase-1	activation	
and	 IL-1β	 production	 in	 human	 Müller	 cells(54,62,88).	 Therefore,	 these	 cells	 were	
used	 to	 explore	 mechanisms	 of	 caspase-1/IL-1β/IL-1R1	 feedback	 signaling	 under	
37	

	
hyperglycemic	 conditions.	 To	 determine	 whether	 caspase-1	 activation	 progresses	
from	an	IL-1R1	independent	mechanism	to	an	IL-1R1	dependent	mechanism	in	this	
cellular	 model,	 human	 Müller	 cells	 (hMCs)	 were	 treated	 with	 5	 mmol/L	 or	 25	
mmol/L	glucose	media	in	the	presence	or	absence	of	an	IL-1	receptor	antagonist	for	
48	 or	 96	 hours.	 At	 48	 hours,	 caspase-1	 activity	 was	 increased	 by	 32.5	 ±	 3.6%	 in	
hyperglycemic	conditions	compared	to	normal.	IL-1ra	treatment	had	no	significant	
effect	on	caspase-1	activity	(Figure	8A).	At	96	hours,	treatment	with	IL-1ra	led	to	a	
97.7	 ±	 5.3%	 reduction	 in	 caspase-1	 activity	 in	 cells	 incubated	 in	 hyperglycemic	
conditions	 indicative	 of	 a	 caspase-1/IL-1β/IL-1R1	 feedback	 signaling	 (Figure	 8B).	
To	 further	 confirm	 the	 concept	 of	 caspase-1/IL-1β/IL-1R1	 feedback	 signaling,	 we	
tested	 whether	 IL-1β	 alone	 can	 induce	 caspase-1	 activity	 in	 hMCs.	 hMCs	 were	
treated	with	increasing	concentrations	of	IL-1β	(0.5-2	ng/mL)	in	5	mmol/L	glucose	
conditions	 for	 24	 hours.	 IL-1β	 induced	 caspase-1	 activation	 in	 a	 concentration	
dependent	 fashion	 demonstrating	 that	 IL-1β	 is	 capable	 of	 inducing	 caspase-1	
activity	(Figure	8C).	Treatment	with	IL-1ra	led	to	an	87.2	±	3.4%	inhibition	of	IL-1βinduced	 caspase-1	 activity	 demonstrating	 that	 the	 effects	 seen	 by	 IL-1β	 were	
mediated	by	the	IL-1	receptor	(Figure	8D).	
2.3.4	Glucose	Consumption	in	Müller	Cells	Exposed	to	Hyperglycemic	Conditions.		
In	order	to	understand	how	sustained	caspase-1	activity	becomes	dependent	
on	feedback	signaling	through	the	IL-1	receptor	despite	the	constant	presence	of	a	
high	 glucose	 stimulus,	 we	 determined	 how	 much	 glucose	 Müller	 cells	 consume	
when	exposed	to	prolonged	periods	of	hyperglycemia.	Over	the	first	48	hours,	hMCs	
cultured	 in	 25	 mmol/L	 glucose	 consumed	 nearly	 twice	 as	 much	 glucose	 (6.3	 ±	 0.4	
38	

	
mmol/L)	 as	 cells	 cultured	 5	 mmol/L	 glucose	 (3.3	 ±	 0.4	 mmol/L).	 Interestingly,	
between	 48	 and	 96	 hours	 there	 was	 no	 significant	 difference	 in	 glucose	
consumption	 between	 Müller	 cells	 cultured	 in	 25	 mmol/L	 glucose	 (3.5	 ±	 1.0	
mmol/L)	compared	to	cells	cultured	in	5	mmol/L	glucose	(3.2	±0.5	mmol/L)	(Figure	
9).	 These	 data	 indicate	 that	 glucose	 consumption	 is	 drastically	 increased	 at	 early	
time	 points	 at	 which	 treatment	 with	 IL-1ra	 is	 ineffective.	 At	 later	 time	 points	
however,	 glucose	 consumption	 decreases	 to	 normal	 levels	 and	 chronic	 caspase-1	
activation	becomes	less	dependent	on	hyperglycemia	and	mostly	dependent	on	IL1β,	 which	 can	 directly	 activate	 caspase-1	 as	 we	 have	 shown	 above,	 explaining	 the	
effectiveness	of	IL-1ra	treatment	in	hyperglycemic	conditions.			
2.3.5	RIP2	Mediated	Caspase-1	Activation	by	Hyperglycemia.		
Our	 results	 so	 far	 indicate	 that	 caspase-1	 activation	 in	 Müller	 cells	 is	
seemingly	 maintained	 by	 two	 stimuli.	 Initially,	 activation	 of	 caspase-1	 is	
predominantly	 driven	 by	 hyperglycemia.	 Once	 activated,	 caspase-1	 activity	 is	
sustained	 by	 feedback	 signaling	 of	 IL-1β	 through	 the	 IL-1	 receptor.	 Therefore,	 we	
were	 interested	 whether	 both	 stimuli	 activate	 caspase-1	 by	 the	 same	 mechanism.	
Several	 known	 activators	 of	 caspase-1	 were	 tested;	 only	 RIP2	 played	 a	 significant	
role	 in	 hyperglycemia-induced	 caspase-1	 activation.	 As	 shown	 in	 Figure	 10A,	 RIP2	
protein	is	significantly	upregulated	in	hMCs	cultured	in	25	mmol/L	glucose	(77.6	±	
25.5%)	compared	to	control.	To	confirm	that	increased	RIP2	levels	are	responsible	
for	 increased	 hyperglycemia-induced	 caspase-1	 activity,	 siRNA	 against	 RIP2	 was	
used.	 Caspase-1	 activity	 was	 significantly	 increased	 in	 hMCs	 transfected	 with	
scramble	 siRNA	 or	 left	 non-transfected	 in	 hyperglycemic	 conditions	 compared	 to	
39	

	
control.	Knock	down	of	RIP2	attenuated	high	glucose-induced	caspase-1	activity	by	
85.8	 ±	 1.8%	 demonstrating	 that	 RIP2	 is	 necessary	 for	 hyperglycemia-induced	
caspase-1	activity	(Figure	10B).	Consistently,	knock	down	of	RIP2	also	prevented	IL1β	production	under	these	conditions	(Figure	10C).		
2.3.6	Inhibition	of	IL-β -Induced	Caspase-1	Activity	by	RIP2	Knock	Down.		
Since	 RIP2	 upregulation	 was	 crucial	 for	 hyperglycemia-driven	 caspase-1	
activation,	 we	 further	 investigated	 the	 role	 of	 RIP2	 in	 IL-1β-induced	 caspase-1	
activation.	 Treatment	 of	 hMCs	 with	 IL-1β	 in	 5	 mmol/L	 glucose	 media	 led	 to	 a	
2.38±0.6	 fold	 increase	 in	 RIP2	 protein	 levels	 (Figure	 11A).	 siRNA	 against	 RIP2	
prevented	 IL-1β-induced	 caspase-1	 activation	 as	 seen	 in	 Figure	 11B.	 RIP2	 is	 not	
only	 involved	 in	 hyperglycemia-induced	 caspase-1	 activation	 but	 also	 promotes	
caspase-1	 activation	 by	 IL-1β.	 Together,	 this	 indicates	 that	 RIP2	 is	 a	 central	
regulator	of	the	caspase-1/IL-1β/IL-1R1	feedback	signaling	that	controls	Müller	cell	
function	and	viability.		
2.3.7	 Hyperglycemia-Induced	 Müller	 Cell	 Death	 Mediated	 by	 Caspase-1/IL1β /IL-1R1	Feedback	Signaling.		
We	have	previously	shown	that	prolonged	exposure	to	hyperglycemia	causes	
Müller	 cell	 death.	 To	 determine	 the	 role	 of	 caspase-1/IL-1β/IL-1R1	 pathway	 and	
feedback	 signaling	 in	 Müller	 cell	 death,	 hMCs	 were	 treated	 with	 5	 mmol/L	 or	 25	
mmol/L	glucose	media	in	the	presence	or	absence	of	RIP2	siRNA	or	an	IL-1ra	for	96	
hours.	 Upstream	 inhibition	 of	 the	 caspase-1/IL-1β	 signaling	 by	 RIP2	 knock	 down	
prevented	 hyperglycemia	 mediated	 cell	 death	 of	 Müller	 cells	 (Figure	 12A).	
Additionally,	 IL-1ra	 treatment	 prevented	 hyperglycemia-induced	 cell	 death	
40	

	
demonstrating	 that	 feedback	 signaling	 affects	 proper	 Müller	 cell	 function	 and	
viability	(Figure	12B).	These	data	indicate	that	interference	in	caspase-1/IL-1β/IL1R1	 feedback	 signaling	 at	 any	 point	 of	 the	 pathway	 is	 beneficial	 for	 Müller	 cell	
survival.	
2.3.8	Diabetic	Caspase-1-/-	Mice	Are	Protected	from	Müller	Cell	Loss.		
We	 have	 demonstrated	 that	 hyperglycemia-induced	 sustained	 caspase-1	
activation	 is	 detrimental	 for	 Müller	 survival	 in	vitro.	 To	 confirm	 the	 importance	 of	
caspase-1	 activation	 on	 Müller	 cell	 viability	 in	 vivo,	 numbers	 of	 Müller	 cell	 were	
counted	in	retinal	sections	of	non-diabetic	or	STZ-diabetic	WT	and	non-diabetic	or	
STZ-diabetic	 caspase-1-/-	 mice.	 Diabetes	 caused	 a	 12.2	 ±	 2.0%	 loss	 of	 Müller	 cells	
after	 6	 months	 of	 diabetes.	 Knock	 out	 of	 caspase-1	 prevented	 diabetes-induced	
Müller	cell	loss	indicating	that	activation	of	caspase-1	is	detrimental	for	Müller	cell	
survival	not	only	in	vitro	but	more	importantly	in	vivo	(Figure	13).	
	

	

41	

	
Figure	6.	Retinal	capillary	degeneration	in	non-diabetic	and	diabetic	wild	type	
and	caspase-1	knockout	mice.		
Retinas	 of	 non-diabetic	 (n=6)	 and	 diabetic	 (n=8)	 wild	 type	 mice,	 and	 non-diabetic	
(n=6)	 and	 diabetic	 (n=5)	 casp1-/-	 mice	 were	 isolated	 at	 7	 months	 of	 diabetes.	
Number	of	acellular	capillaries	was	determined	and	expressed	as	mean	±	SDEV.	

	
42	

	
Figure	7.	Caspase-1	activity	pattern	in	the	retinas	of	non-diabetic	and	diabetic	
wild	type	and	IL-1R1-/-mice.		
Retinas	 of	 non-diabetic	 (n=10)	 and	 diabetic	 (n=10)	 wild	 type	 and	 non-diabetic	
(n=12)	 and	 diabetic	 (n=10)	 IL-1R1-/-	 mice	 were	 isolated	 at	 10	 weeks	 (A)	 and	 20	
weeks	(B).	Caspase-1	activity	was	measured	and	expressed	as	mean	±	SDEV	with	(*)	
=	p<	0.05	compared	to	non-diabetic	animals.		

	
	

	

43	

	
Figure	 8.	 Inhibition	 of	 hyperglycemia-and	 IL-β-induced	 caspase-1	 activity	 in	
human	Müller	cells	by	IL-1	receptor	antagonist.			
hMCs	 were	 cultured	 in	 either	 5	 mmol/L	 or	 25	 mmol/L	 glucose	 media	 in	 the	
presence	or	absence	of	100ng/ml	IL-1ra	for	(A)	48	or	(B)	96	hours.	(C)	hMCs	were	
treated	 in	 5	 mmol/L	 glucose	 or	 5	 mmol/L	 glucose	 +	 IL-1β	 (0.5,	 1,	 or	 2ng/ml)	
containing	media	for	24hrs.	(D)	hMCs	were	treated	with	IL-1ra	(100ng/ml)	in	either	
5	mmol/L	glucose	or	5	mmol/L	glucose	+	IL-1β	(2ng/ml)	media	for	24hrs.	Caspase1	 activity	 was	 measured	 and	 expressed	 as	 mean	 ±	 SDEV	 with	 (*)	 =	 p<	 0.05	
compared	to	control	and	(#)	=	P<0.01	compared	to	25	mmol/L	glucose.	
	

	
	

	

44	

	
Figure	9.	Glucose	consumption	normalized	over	time	in	Müller	cells.	
hMCs	were	incubated	in	either	5	mmol/l	glucose	or	25	mmol/l	glucose	for	eitehr	48	
or	96	hours.		Glucose	consumption	was	measured	and	expressed	as	mean	±	SDEV,	
n=5,	p<0.05.	

	
	

	

45	

	
Figure	 10.	 RIP2	 mediated	 caspase-1	 activity	 and	 IL-β	 production	 in	 Müller	
cells.	
(A)	 hMCs	were	incubated	in	either	5	mmol/L	glucose	or	25	mmol/L	glucose	media	
for	 48	 hours.	 RIP2	 protein	 levels	 were	 determined	 by	 Western	 Blot	 analysis,	
normalized	 to	 β-actin,	 and	 expressed	 as	 mean	 ±	 SDEV	 (n=5)	 with	 (*)	 =	 p<	 0.05	
compared	to	5	mmol/L	glucose.	(B)	 hMCs	transfected	with	either	scramble	RNA	or	
siRNA	 were	 incubated	 in	 5	 mmol/L	 glucose	 or	 25	 mmol/L	 glucose	 media	 for	 48	
hours.	 Caspase-1	 activity	 was	 assessed	 and	 expressed	 as	 mean	 ±	 SDEV	 (n=5)	 with	
(*)	 =	 p<	 0.01	 compared	 to	 5	 mmol/L	 glucose	 and	 (#)	 =	 p<0.01	 compared	 to	 25	
mmol/L	 glucose.	 hMCs	 without	 transfection	 served	 as	 controls.	 (C)	 IL-1β	 release	
into	media	was	measured	using	ELISA	assays.		IL-1β	levels	are	expressed	as	mean	±	
SDEV	 (n=3)	 with	 (*)	 =	 p<	 0.05	 compared	 to	 5	 mmol/L	 glucose	 and	 (#)	 =	 p<0.05	
compared	to	25	mmol/L	glucose.		
	

	

46	

	
Figure	10	cont.	
RIP2 (62 kDa)

RIP2 (62 kDa)

β-Actin (42 kDa)

β-Actin (42 kDa)

A

B

*

2.0

20.0

Caspase-1 Activity
(pmol AFC/mg/min)

Relative Density
(RIP2/b -Actin)

2.5

1.5
1.0
0.5
0.0

*

5 mmol/l Glucose
25 mmol/l Glucose

15.0

10.0

5.0

0.0

25

tr
an
sf
ec
Sc
tio
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A
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o
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an
sf
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ra
n
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bl
e
R
N
A
si
R
N
A

5

N
o

Glucose (mmol/l)

*

C

IL-1b Release
(pg/ml/mg protein)

10.0
8.0

*

5 mmol/l Glucose
25 mmol/l Glucose

*

6.0
4.0
2.0

N
o

tr
an
sf
ec
Sc
tio
ra
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R
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R
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A
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A

0.0

	
	

	

47	

	
Figure	11:	IL-1β-induced	caspase-1	activity	mediated	by	RIP2.			
(A)	 hMCs	were	incubated	in	either	5	mmol/L	glucose	or	5	mmol/L	glucose	+	IL-1β	
(2ng/ml)	 for	 24	 hours.	 RIP2	 protein	 levels	 were	 determined	 by	 Western	 Blot	
analysis,	normalized	to	β-actin,	and	expressed	as	mean	±	SDEV	(n=5)	with	(*)	=	p<	
0.05	 compared	 to	 5	 mmol/L	 glucose.	 (B)	 hMCs	 transfected	 with	 either	 scramble	
RNA	 or	 siRNA	 were	 incubated	 in	 5	 mmol/L	 glucose	 or	 5	 mmol/L	 glucose	 +	 IL-1β	
(2ng/ml)	 for	 24	 hours.	 Caspase-1	 activity	 was	 assessed	 and	 expressed	 as	 mean	 ±	
SDEV	 (n=5)	 with	 (*)	 =	 p<	 0.01	 compared	 to	 5	 mmol/L	 glucose	 and	 (#)	 =	 p<0.01	
compared	to	25	mmol/L	glucose.		
RIP2 (62 kDa)
A

β-Actin (42 kDa)

Relative Density
(RIP2/b -Actin)

5.0

*

4.0
3.0
2.0
1.0
0.0

Glucose (mmol/l)

5

5

IL-1b (ng/ml)

-

2

B

Caspase-1 Activity
(pmol AFC/mg/min)

25.0
20.0

*

5 mmol/l Glucose

*

5 mmol/l Glucose
+ 2ng/ml IL-1b

15.0
10.0
5.0

N
o

tr

an
sf
ec
Sc
tio
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R
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A
N
o
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m
bl
e
R
N
A
si
R
N
A

0.0

	

48	

	

	
Figure	 12:	 Inhibition	 of	 hyperglycemia-induced	 Müller	 cell	 death	 by	 IL-1ra,	
YVAD-fmk,	and	RIP2siRNA.		
hMCs	 were	 cultured	 in	 either	 5	 mmol/L	 or	 25	 mmol/L	 glucose	 media	 in	 the	
presence	or	absence	of	(A)	RIP2	siRNA	or	(B)	100ng/ml	IL-1ra	for	96	hours.	At	96	
hours,	 cell	 death	 was	 determined	 using	 the	 Trypan	 Blue	 Exclusion	 assay	 and	
expressed	as	mean	±	SDEV	(n=5)	with	(*)	=	p<0.01	compared	to	5	mmol/L	glucose,	
(#)	=	P<0.01	compared	to	25	mmol/L	glucose.		

	

49	

	

	
Figure	13:	Caspase-1	dependent	Müller	cell	death	in	retinas	of	diabetic	mice.		
Eyes	 of	 STZ-diabetic	 wt	 (n=10)	 and	 non-diabetic	 wt	 (n=10),	 STZ-diabetic	 cas-1-/-	
(n=5)	and	non-diabetic	cas-1-/-	(n=5)	mice	were	isolated	after	7	months	duration	of	
diabetes.	Number	of	Müller	cells	is	presented	as	mean	±	SDEV	with	(#)	=	p<0.02.	

	
	

	

50	

	
Figure	 14:	 Proposed	 mechanism	 of	 sustained	 caspase-1	 activation	 via	
feedback	signaling	in	Müller	cells.	

	
	

	

51	

	
Table	1:	Summary	of	Animal	Data.		
Severity	 of	 diabetes	 was	 estimated	 by	 measuring	 non-enzymatically	 glycated	
hemoglobin	 (GHb)	 levels	 using	 affinity	 chromatography	 (Glyc-Affin;	 Pierce,	
Rockford,	 IL).	 (*)	 =	 p<	 0.05	 significantly	 different	 from	 normal	 wild	 type	 animals	
compared	to	diabetic	wild	type	animals	and	normal	Cas-1	-/-	(A)	or	IL-1R1	-/-	(B)	
animals	compared	to	diabetic	Cas-1	(A)	or	IL-1R1	-/-	(B)	animals	.		
	

	
	

	

52	

	
2.4	Discussion	
The	 importance	 of	 inflammation	 in	 the	 progression	 of	 diabetic	 retinopathy	 has	
become	 increasingly	 apparent.	 A	 variety	 of	 pro-inflammatory	 cytokines	 have	 been	
identified	in	the	vitreous	of	patients	compared	to	healthy	individuals,	among	them	
IL-1β(111–115,132).	 Our	 previous	 studies	 have	 shown	 that	 caspase-1	 activity	 is	
consistently	increased	in	the	retinas	of	diabetic	animals	and	patients(62,133).	This	
new	 study	 provides	 novel	 mechanistic	 insights	 into	 the	 process	 of	 prolonged	
caspase-1	 activation	 and	 IL-β	 production	 in	 diabetic	 retinopathy.	 First,	 using	
caspase-1-/-	 mice	 we	 demonstrated	 that	 caspase-1	 activation	 is	 indeed	 crucial	 for	
the	 development	 of	 diabetic	 retinopathy.	 Furthermore,	 using	 IL-1R1-/-	 mice	 we	
identified	 that	 diabetes-induced	 caspase-1	 activity	 progresses	 from	 an	 IL-1R1	
independent	 mechanism	 to	 an	 IL-1R1	 dependent	 mechanism	 throughout	 disease	
progression.	 This	 is	 consistent	 with	 our	 previous	 observation	 that	 knock	 down	 of	
the	IL-1R1	receptor	prevented	the	development	of	diabetic	retinopathy(133).	These	
data	 provide	 for	 the	 first	 time	 an	 indication	 of	 a	 caspase-1/IL-1β/IL-1R1	 feedback	
signaling	mechanism	that	keeps	caspase-1	active	in	the	diabetic	retina.	Using	Müller	
cells	known	to	produce	active	caspase-1	and	IL-1β	under	hyperglycemic	conditions	
we	 confirmed	 that	 prolonged	 exposure	 to	 hyperglycemia	 leads	 to	 caspase-1/IL1β/IL-1R1	feedback	signaling	causing	sustained	caspase-1	activity.	We	were	able	to	
show	that	hyperglycemia	initiates	caspase-1	activity	and	IL-1β	continues	to	promote	
caspase-1	activation	despite	the	fact	that	initial	increased	glucose	consumption	has	
been	normalized.	In	addition,	activation	of	caspase-1	by	both	stimuli,	hyperglycemia	
and	IL-1β,	was	mediated	by	RIP2	as	determined	by	RIP2	knockdown	experiments.	
53	

	
Finally,	 we	 determined	 that	 activation	 of	 the	 caspase-1/IL-1β/IL-1R1	 feedback	 is	
detrimental	leading	to	Müller	cells	death.	Hyperglycemia	is	not	only	detrimental	for	
Müller	cells	in	vitro	but	most	importantly	in	vivo.	We	confirmed	that	Müller	cell	loss	
in	vivo	 is	 dependent	 on	 activation	 of	 caspase-1.	 Taken	 together,	 this	 indicates	 that	
hyperglycemia	 induces	 caspase-1	 activation	 and	 IL-1β	 production	 initiating	 a	
seemingly	 IL-1β	 driven	 caspase-1/IL-1β/IL-1R1	 feedback	 cycle	 that	 is	 detrimental	
to	 the	 viability	 of	 retinal	 cells	 such	 as	 Müller	 cells	 and	 promotes	 the	 development	
and	progression	of	diabetic	retinopathy	(Figure	14).	
The	 idea	 of	 a	 caspase-1/IL-1β/IL-1R1	 feedback	 signaling	 is	 intriguing	 for	 several	
reasons.	 Although	 our	 study	 used	 Müller	 cells	 to	 demonstrate	 hyperglycemiainduced	 caspase-1/IL-1β/IL-1R1	 feedback	 signaling,	 other	 retinal	 cells	 might	 be	
capable	 of	 producing	 similar	 feedback	 cycles	 leading	 to	 sustained	 caspase-1	
activation	 and	 prolonged	 IL-1β	 production.	 Several	 retinal	 cell	 types	 such	 as	
astrocytes	 and	 microglia	 have	 been	 identified	 to	 produce	 IL-1β	 when	 exposed	 to	
elevated	 glucose	 levels(134–136).	 This	 leaves	 the	 strong	 possibility	 that	 IL-1β	
produced	 by	 one	 retinal	 cell	 type	 feeds	 into	 IL-1β	 feedback	 signaling	 of	 another	
retinal	 cells	 type	 even	 further	 augmenting	 IL-1β	 production.	 Despite	 our	 in	 vitro	
studies	showing	that	autocrine	activation	is	sufficient	to	induce	caspase-1/IL-1β/IL1R1	feedback	signaling	the	combination	of	several	IL-1β-producing	cells	amplifying	
feedback	signaling	might	actually	be	necessary	to	reach	high	enough	IL-1β	levels	for	
IL-1β	to	drive	caspase-1	activation	via	the	IL-1R1	in	the	diabetic	retina.	This	could	
explain	 the	 long	 duration	 of	 diabetes	 required	 for	 caspase-1	 activity	 to	 become	

54	

	
dependent	 on	 IL-1β	 feedback	 in	 vivo.	 However,	 more	 studies	 need	 to	 be	 done	 to	
positively	 confirm	 this	 concept	 of	 caspase-1/IL-1β/IL-1R1	 feedback	 signaling	 in	
other	retinal	cell	types.	Future	studies	will	also	have	to	identify	how	other	cytokines	
such	 as	 TNFα	 that	 have	 been	 associated	 with	 the	 development	 of	 diabetic	
retinopathy	 influence	 the	 regulation	 of	 the	 caspase-1/IL-β/IL-1R1	 feedback	
signaling.	
Looking	deeper	into	the	switch	of	caspase-1	activation	from	IL-1R1	independent	to	
IL-1R1	 dependent	 mechanisms	 despite	 the	 presence	 of	 an	 obvious	 hyperglycemic	
environment	 we	 examined	 glucose	 consumption	 in	 Müller	 cells.	 Müller	 cells	 are	
unique	in	their	metabolism	since	they	derive	their	energy	primarily	from	glycolysis	
and	 are	 known	 to	 take	 up	 large	 amounts	 of	 glucose(33).	 Although	 hyperglycemia	
initially	 drives	 caspase-1	 activation,	 over	 time	 glucose	 consumption	 surprisingly	
decreased	 and	 eliminated	 glucose	 as	 a	 mediator	 of	 prolonged	 caspase-1	 activity.	
This	 indicates	 that	 Müller	 cells	 may	 be	 capable	 of	 regulating	 glucose	 consumption	
overtime	to	avoid	excessive	caspase-1	activation	by	hyperglycemia.	However,	cells	
are	unable	to	compensate	for	the	increased	levels	of	IL-1β,	which	itself	is	capable	of	
driving	 caspase-1	 activation.	 This	 leads	 to	 caspase-1	 activation	 being	 driven	 solely	
by	 IL-1β	 feedback	 signaling,	 which	 explains	 the	 effectiveness	 of	 IL-1ra	 treatment	
since	 glucose	 is	 playing	 a	 minor	 role	 in	 caspase-1	 activation	 once	 the	 feedback	
signaling	is	set	in	motion.	How	much	glucose	is	actually	needed	to	trigger	caspase-1	
activation	and	to	support	ongoing	caspase-1/IL-1β/IL-1R1	feedback	signaling	has	to	
be	 determined	 in	 more	 detail	 in	 future	 studies.	 Interestingly,	 although	 caspase-1	
activation	 during	 this	 feedback	 signaling	 is	 mediated	 by	 two	 distinct	 stimuli,	
55	

	
hyperglycemia	 and	 IL-1β,	 both	 pathways	 are	 controlled	 by	 one	 regulator,	 RIP2.	
RIP2,	 a	 62	 kDa	 CARD	 domain	 containing	 protein,	 can	 act	 as	 a	 scaffold	 protein	
capable	

of	

binding	

and	

activating	

pro-caspase-1	

via	

CARD-CARD	

interaction(82,119,120).	 Aberrant	 RIP2	 activity	 has	 been	 implicated	 as	 a	 driver	 of	
inflammation	 in	 diseases	 such	 as	 Huntington’s	 Disease,	 where	 it	 causes	 increased	
caspase-1	 activation	 and	 IL-1β	 production,	 ultimately	 leading	 to	 neuronal	 cell	
death(82).	 For	 the	 first	 time	 this	 study	 shows	 that	 RIP2	 plays	 a	 major	 role	 in	
hyperglycemia-mediated	caspase-1	activation	and	cell	death	in	retinal	Müller	cells.	
This	 is	 interesting	 since	 the	 more	 prominent	 mechanisms	 of	 caspase-1	 activation	
seem	 to	 be	 inflammasome-mediated	 mechanisms	 involving	 NLRP3(118).	 NLRP3	
upregulation	has	been	shown	in	some	retinal	cells(137)	but	was	not	observed	in	our	
studies	in	Müller	cells	(data	not	shown).		
Prolonged	activation	of	caspase-1	and	IL-1β	production	causes	Müller	cell	death	in	
vitro	 and	 in	 vivo.	 Müller	 cell	 death	 in	 diabetic	 retinopathy	 was	 first	 identified	 in	
1980,	 however,	 few	 studies	 have	 looked	 at	 actual	 mechanisms	 underlying	 Müller	
cell	 death(54,133,138–140).	 Intervention	 in	 the	 caspase-1/IL-1β/IL-1R1	 feedback	
signaling	by	inhibition	of	caspase-1	as	shown	in	this	study	or	by	the	IL-1	receptor	as	
we	 previously	 reported	 prevents	 Müller	 cell	 death	 in	 diabetic	 retinopathy	
demonstrating	 the	 importance	 of	 this	 signaling	 pathway	 to	 Müller	 cell	
viability(140).	The	protective	effect	of	caspase-1	inhibition	on	Müller	cell	viability	is	
not	 surprising.	 	 Increasing	 evidence	 shows	 that	 there	 are	 a	 number	 of	 modes	 of	
programmed	 cell	 death	 in	 addition	 to	 the	 classical	 apoptosis(141,142).	 One	 of	 the	
more	 recently	 identified	 types	 of	 cell	 death,	 termed	 pyroptosis,	 is	 an	 inherently	
56	

	
inflammatory	 based	 cell	 death	 defined	 as	 being	 caspase-1	 dependent	 and	
preventable	 by	 inhibition	 of	 caspase-1(142).	 This	 current	 study	 demonstrates	 that	
hyperglycemia-induced	Müller	cell	death	fulfills	all	criteria	for	pyroptosis	and	links	
the	 pro-inflammatory	 function	 of	 caspase-1	 and	 IL-1β	 production	 to	 cell	 death.	
Excessive	IL-1β	production	by	Müller	cells	might	not	only	affect	viability	of	Müller	
cells	 but	 also	 affect	 viability	 of	 surrounding	 retinal	 cells	 as	 we	 have	 previously	
shown	for	endothelial	cells(143).	Furthermore,	Müller	cell	death	preceded	acellular	
capillary	formation	suggesting	a	potential	role	for	Müller	cells	in	the	progression	of	
diabetic	retinopathy.	
In	this	study	we	have	outlined	a	mechanism	of	prolonged	caspase-1	activation	that	
contributes	 to	 chronic	 inflammatory	 events	 in	 the	 diabetic	 retina	 and	 while	 set	 in	
motion	 by	 hyperglycemia	 seemingly	 becomes	 independent	 of	 its	 original	
hyperglycemic	 insult.	 	 If	 confirmed	 in	 diabetic	 patients	 it	 could	 potentially	 explain	
why	 diabetic	 retinopathy	 still	 progresses	 despite	 good	 control	 of	 blood	 glucose	
levels.	 It	 also	 might	 open	 new	 venues	 for	 treatment	 of	 diabetic	 retinopathy.	
Currently	 the	 most	 common	 therapy	 seeks	 to	 inhibit	 VEGF,	 a	 growth	 factor	 that	
promotes	increased	vascular	permeability	and	neovascularization(144–148).	While	
these	 drugs	 have	 been	 successful	 in	 some	 patients,	 they	 do	 not	 provide	 reliable	
benefits	for	all	patients.	Our	data	suggest	that	treatment	of	inflammatory	events	are	
potentially	 more	 pressing	 than	 inhibition	 of	 growth	 factors	 such	 as	 VEGF.	 Several	
studies	have	shown	that	IL-1β	can	lead	to	increased	production	of	VEGF(149–151).	
Inhibition	of	caspase-1/IL-1β	signaling	may	provide	a	therapeutic	target	upstream	
of	VEGF,	without	inhibiting	VEGF	actions	directly.	
57	

	

	
Chapter	3.	Modes	of	Retinal	Cell	Death	in	Diabetic	Retinopathy	
	
This		chapter	is	a	modified	version	of	an	article	published	in	Journal	of	Clinical	and	
Experimental	Ophthalmology	
Authors:	Derrick	J.	Feenstra,	E.	Chepchumba	Yego,	and	Susanne	Mohr	
	
3.1	Abstract	
Cell	 death	 seems	 to	 be	 a	 prominent	 feature	 in	 the	 progression	 of	 diabetic	
retinopathy.	Several	retinal	cell	types	have	been	identified	to	undergo	cell	death	in	a	
diabetic	 environment.	 Most	 emphasis	 has	 been	 directed	 towards	 identifying	
apoptosis	in	the	diabetic	retina.	However,	new	 research	has	established	that	there	
are	 multiple	 forms	 of	 cell	 death.	 This	 review	 discusses	 the	 different	 modes	 of	 cell	
death	 and	 attempts	 to	 classify	 cell	 death	 of	 retinal	 cells	 known	 to	 die	 in	 diabetic	
retinopathy.	Special	emphasis	is	given	to	apoptosis,	necrosis,	autophagic	cell	death,	
and	pyroptosis.	It	seems	that	different	retinal	cell	types	are	dying	by	diverse	types	of	
cell	 death.	 Whereas	 endothelial	 cells	 predominantly	 undergo	 apoptosis,	 pericytes	
might	 die	 by	 apoptosis	 as	 well	 as	 necrosis.	 On	 the	 other	 hand,	 Müller	 cells	 are	
suggested	to	die	by	a	pyroptotic	mechanism.	Diabetes	leads	to	significant	Müller	cell	
loss	at	7	months	duration	of	diabetes	in	retinas	of	diabetic	mice	compared	to	nondiabetic,	 which	 is	 prevented	 by	 the	 inhibition	 of	 the	 caspase-1/IL-1β	 (interleukin1beta)	 pathway	 using	 the	 IL-1	 receptor	 knockout	 mouse.	 Since	 pyroptosis	 is	
characterized	 by	 the	 activation	 of	 the	 caspase-1/IL-1β	 pathway	 subsequently	
leading	 to	 cell	 death,	 Müller	 cells	 seem	 to	 be	 a	 prime	 candidate	 for	 this	 form	 of	
58	

	
inflammation-driven	 cell	 death.	 Considering	 that	 diabetic	 retinopathy	 is	 now	
discussed	 to	 potentially	 be	 a	 chronic	 inflammatory	 disease,	 pyroptotic	 cell	 death	
might	play	an	important	role	in	disease	progression.	Understanding	mechanisms	of	
cell	 death	 will	 lead	 to	 a	 more	 targeted	 approach	 in	 the	 development	 of	 new	
therapies	to	treat	diabetic	retinopathy.		
	
3.2	Introduction	
The	 most	 common	 features	 of	 diabetic	 retinopathy	 are	 alterations	 to	 the	
retinal	 microvasculature	 leading	 to	 microaneurysms,	 macular	 edema,	 leakage	 of	
blood	 into	 the	 retinal	 tissue	 and	 vitreous,	 and	 eventual	 blindness(3,108).	
Endothelial	 cells,	 which	 line	 the	 microvasculature	 and	 provide	 the	 blood-retinal	
barrier,	 have	 long	 been	 regarded	 as	 a	 scapegoat	 for	 explaining	 changes	 in	 the	
increased	vascular	permeability	in	the	course	of	diabetic	retinopathy.	However,	the	
blood-retinal	 barrier	 function	 of	 the	 endothelial	 cells	 is	 supported	 by	 surrounding	
cells,	 such	 as	 Müller	 cells,	 pericytes,	 and	 astrocytes(39).	 Since	 the	 blood-retinal	
barrier	 depends	 so	 heavily	 on	 this	 interdependent	 microenvironment	 where	 the	
function	 of	 one	 cell	 type	 depends	 on	 support	 from	 other	 cell	 types,	 any	 cellular	
injury	and	cell	loss	will	have	vast	effects	on	proper	retinal	barrier	function	and	for	
that	matter	any	retinal	function(143,152,153).		
Indeed,	 loss	 of	 retinal	 cells	 seems	 to	 be	 a	 prominent	 feature	 of	 diabetic	
retinopathy.	Diabetes-induced	cell	death	has	been	observed	in	numerous	retinal	cell	
types	 such	 as	 endothelial	 cells(143,154–156),	 pericytes(156–158),	 neural	 retinal	
cells	 such	 as	 ganglion	 cells(159–161),	 and	 retinal	 glial	 cells	 such	 as	 Müller	 cells,	
59	

	
astrocytes,	and	microglia(5,52,88,135,162–166).	Endothelial	cell	death	and	pericyte	
loss	have	long	been	assumed	to	play	an	important	role	in	the	loss	of	proper	bloodretinal	 barrier	 function(152,156,167,168).	 Despite	 increasing	 efforts	 to	
demonstrate	 retinal	 cell	 death	 in	 diabetic	 retinopathy,	 mechanisms	 leading	 to	 cell	
death	by	diabetes	are	only	poorly	understood	to	date.	Identifying	potential	modes	of	
cell	death	is	complicated	by	the	fact	that	for	some	forms	of	cell	death,	the	pathways	
and	markers	are	poorly	understood	and	are	still	being	discovered.	In	recent	years,	
existing	types	of	cell	death	(apoptosis	and	necrosis)	have	been	re-classified	and	new	
subtypes	of	cell	death	have	been	added.		
According	to	the	most	recent	cell	death	nomenclature	paper	published	by	the	
Nomenclature	 Committee	 on	 Cell	 Death	 (NCCD)	 there	 are	 now	 13	 subroutines	 of	
regulated	 cell	 death	 identified(142).	 These	 include	 anoikis,	 autophagic	 cell	 death,	
caspase-dependent	 intrinsic	 apoptosis,	 caspase-independent	 intrinsic	 apoptosis,	
cornification,	entosis,	extrinisic	apoptosis	by	death	receptors,	extrinsic	apoptosis	by	
dependence	 receptors,	 mitotic	 catastrophy,	 regulated	 necrosis,	 netosis,	
parthanatosis,	 and	 pyroptosis.	 Each	 type	 of	 cell	 death	 has	 different,	 and	 often	 not	
fully	defined,	characteristics	and	markers	leading	to	increased	complexity	in	correct	
identification	 of	 cell	 death	 mechanisms	 both	 in	vitro	 and	 more	 importantly	 in	vivo.	
Apoptosis	is	the	most	studied	type	of	cell	death	in	diabetic	retinopathy.	It	has	welldefined	features	and	is	easily	detectable	with	established	techniques,	such	as	TUNEL	
(Terminal	dUTP	Nick	End	Labeling)	assay.	However,	some	of	the	cell	death	types	are	
far	 more	 difficult	 to	 detect	 due	 to	 the	 lack	 of	 established	 markers	 and	 techniques	
available.	
60	

	
In	 order	 to	 establish	 mechanisms	 underlying	 the	 development	 of	 diabetic	
retinopathy	and	to	determine	whether	cell	death	is	crucial	for	the	progression	of	the	
disease,	a	better	understanding	of	potential	types	of	cell	death	in	the	diabetic	retina	
must	 be	 achieved.	 This	 will	 then	 allow	 for	 more	 targeted	 therapies	 to	 combat	 cell	
death	in	diabetic	retinopathy.		This	review	will	provide	an	overview	of	the	various	
retinal	cell	types	undergoing	cell	death	in	diabetic	retinopathy	and	attempt	to	assign	
cell	death	classification	to	these	dying	cells.	
	
3.3.	Apoptosis:	Extrinsic	Versus	Intrinsic	
	

The	 most	 well	 defined	 form	 of	 cell	 death	 is	 apoptosis.	 Apoptosis,	 originally	

introduced	by	Kerr	et	al.	in	1972,	is	a	term	that	describes	a	form	of	programmed	cell	
death	resulting	in	cytoplasmic	and	nuclear	condensation,	a	specific	pattern	of	DNA	
fragmentation,	 and	 eventual	 demise	 of	 the	 cell	 into	 apoptotic	 bodies	 to	 be	
phagocytosed	 by	 surrounding	 cells	 with	 very	 little	 inflammation	 involved	 in	 the	
process(169).	The	common	theme	in	identification	of	apoptotic	cell	death	is	the	use	
of	 TUNEL	 staining	 or	 other	 methods	 that	 are	 aimed	 to	 specifically	 detect	 the	
apoptotic	DNA	laddering	pattern(170–172).	However,	due	to	recent	advancements	
in	 cell	 death	 studies	 and	 changes	 in	 cell	 death	 nomenclature,	 classification	 of	
apoptosis	is	not	as	simple	as	detection	of	specific	DNA	fragmentation.		
	

Apoptosis	 can	 be	 divided	 into	 various	 subcategories	 according	 to	 both	 the	

stimuli	and	the	pathways	leading	to	execution	of	cell	death,	and	should	therefore	be	
a	 term	 used	 with	 caution.	 	 ‘Extrinisic	 Apoptosis’	 for	 example	 is	 used	 to	 define	 cell	
death	 induced	 by	 binding	 of	 lethal	 ligands	 including	 FAS/CD95	 ligand,	 tumor	
61	

	
necrosis	factor	α	(TNFα),	or	TNF-related	apoptosis	inducing	ligand	(TRAIL)	to	their	
respective	 death	 receptor(142,173).	 	 Upon	 binding	 of	 these	 ligands	 to	 the	 death	
receptor,	 the	 “death	 domain”	 of	 the	 receptor	 recruits	 the	 assembly	 of	 the	 “deathinducing	 signaling	 complex”	 (DISC),	 a	 platform	 of	 various	 proteins.	 The	 DISC	 can	
differ	depending	on	the	death	receptor	involved	but	typically	results	in	activation	of	
caspase-8(142,174–176).	 	 Depending	 on	 cell	 type,	 active	 caspase-8	 initiates	 one	 of	
two	distinct	pathways.	First,	active	caspase-8	can	directly	cleave	caspase-3,	known	
as	 an	 executioner	 caspase	 in	 the	 apoptotic	 process(177).	 	 Alternatively,	 active	
caspase-8	 can	 cleave	 BH3-interacting	 domain	 death	 agonist	 (BID)	 creating	
truncated	 BID	 (tBID).	 tBID	 then	 binds	 Bcl-2	 allowing	 BAX	 	 to	 form	 a	 pore	 in	 the	
outer	membrane	of	the	mitochondria	enabling	the	release	of	cytochrome	c	into	the	
cytosol.	 	 This	 triggers	 formation	 of	 the	 canonical	 ‘apoptosome’	 via	 assembly	 of	
APAF1	with	pro-caspase-9,	cytochrome	c,	and	dATP	leading	to	caspase-9	activation,	
which	in	turn	activates	caspase-3(142,178).		
	

Another	 type	 of	 apoptosis,	 ‘intrinsic	 apoptosis’	 is	 similar	 to	 extrinsic	

apoptosis	 in	 that	 there	 is	 eventual	 activation	 of	 caspase-3	 as	 the	 executioner	
caspase.	 However,	 rather	 than	 an	 extrinsic	 ligand	 binding	 to	 a	 death	 receptor,	
apoptosis	is	triggered	by	intracellular	stress	such	as	DNA	damage,	oxidative	stress,	
or	 excitotoxicity(142).	 	 Regardless	 of	 the	 intracellular	 stress	 that	 initiates	 intrinsic	
apoptosis,	 the	 intrinsic	 and	 extrinsic	 pathways	 converge	 at	 the	 mitochondria.	
Increased	pore	formation	by	either	bak	or	bax,	or	pore	formation	by	a	multi-protein	
complex	 termed	 the	 permeability	 transition	 pore	 (PTP)	 promotes	 the	 release	 of	
proteins	such	as	cytochrome	c,	apoptosis-inducing	factor	(AIF),	and	endonuclease	G	
62	

	
(ENDOG)	 from	 the	 mitochondria	 into	 the	 cytosol(142,179,180).	 In	 addition,	
alterations	of	the	respiratory	chain	lead	to	increased	reactive	oxygen	species	(ROS)	
production(142).	As	described	above,	apoptosome	formation	induces	caspase-9	and	
subsequent	 caspase-3	 activation.	 Activation	 of	 caspse-3	 initiates	 events	 that	 are	
responsible	 for	 the	 specific	 DNA	 cleavage	 pattern	 seen	 in	 apoptotic	 cell	 death.	 In	
contrast,	 AIF	 and	 ENDOG	 can	 translocate	 to	 the	 nucleus	 leading	 to	 DNA	
fragmentation	that	is	independent	of	caspase	activation(178,181–186).	In	this	case,	
apoptotic	cell	death	occurs	even	in	the	absence	of	active	caspases	or	when	caspases	
are	 pharmacologically	 inhibited.	 This	 allows	 for	 even	 further	 classification	 of	
intrinsic	 apoptosis	 into	 caspase-dependent	 and	 caspase-independent	 intrinsic	
apoptosis(142).	 Therefore,	 observation	 of	 DNA	 fragmentation	 alone	 by	 TUNEL	
staining	is	not	sufficient	to	distinguish	between	the	different	types	of	apoptosis.	
	

Much	of	the	research	in	diabetes-induced	retinal	cell	death	has	been	focused	

on	 identifying	 apoptosis	 using	 the	 TUNEL	 assay	 as	 the	 method	 of	 choice.	 Some	
TUNEL	based	studies	were	supported	by	additional	data	identifying	active	caspase3.	 	 TUNEL	 staining	 has	 identified	 increased	 endothelial	 cell	 apoptosis	 in	 retinas	 of	
diabetic	and	galactose	fed	rats,	compared	to	control	animals(156,187,188).	A	similar	
increase	 in	 TUNEL	 staining	 was	 seen	 in	 retinal	 endothelial	 cells	 of	 diabetic	
mice(189).	A	recent	study	confirmed	these	results	in	the	retinas	of	human	subjects	
with	diabetic	retinopathy	compared	to	those	without(190).	TUNEL	staining	has	also	
been	used	in	studies	showing	that	neutrophils	from	diabetic	rats,	when	co-cultured	
with	human	endothelial	cells,	led	to	increased	endothelial	cell	apoptosis	indicating	
that	other	cells	types	when	exposed	to	hyperglycemia	induce	endothelial	cell	death	
63	

	
via	an	apoptotic	pathway(191).		In	other	studies,	propidium	iodide	(PI),	which	when	
injected	 intravenously	 will	 fluoresce	 after	 leakage	 through	 injured	 cell	 membrane	
and	 bind	 to	 DNA	 or	 RNA,	 has	 been	 used	 to	 detect	 endothelial	 cell	 apoptosis	 in	
diabetic	rats(154).	However,	PI	staining	does	not	allow	for	a	discrimination	between	
cells	undergoing	apoptosis	or	necrosis(154).	While	much	of	the	apoptosis	research	
has	 used	 DNA	 fragmentation	 alone,	 some	 more	 detailed	 studies	 have	 shown	 that	
high	 glucose	 leads	 to	 cytochrome	 c	 release	 and	 changes	 in	 mitochondrial	
morphology	 in	 endothelial	 cells	 indicating	 a	 mitochondria-mediated	 apoptotic	
mechanism(192).	 This	 is	 further	 supported	 by	 a	 study	 that	 demonstrated	 that	
overexpression	 of	 bcl2,	 an	 anti-apoptotic	 member	 of	 the	 bcl2	 family,	 prevented	
capillary	degeneration	in	diabetic	mice(193).	Other	studies	have	demonstrated	that	
hyperglycemia	can	initiate	pro-apoptotic	pathways	in	endothelial	cells	by	measuring	
caspase-8	and	caspase-3	activity	indicating	the	caspase	dependency	of	the	apoptotic	
process(143,189,194,195).	 Although	 these	 studies	 provide	 good	 evidence	 that	
caspase-dependent	 apoptosis	 is	 the	 predominant	 type	 of	 cell	 death	 for	 endothelial	
cells	 when	 exposed	 to	 a	 hyperglycemic	 environment,	 more	 studies	 are	 needed	 to	
determine	 whether	 apoptotic	 cell	 death	 in	 endothelial	 cells	 is	 mediated	 by	 an	
intrinsic	or	extrinsic	mechanism	in	diabetic	retinopathy.	
Apoptosis	 has	 also	 been	 suggested	 as	 the	 type	 of	 cell	 death	 in	 pericytes	
during	 the	 progression	 of	 diabetic	 retinopathy.	 TUNEL	 staining	 demonstrated	
increased	 pericyte	 apoptosis	 in	 retinas	 of	 diabetic	 and	 galactose	 fed	 rodents	
compared	 to	 control	 animals(156,187,196).	 Increased	 pericyte	 apoptosis	 has	 also	
been	shown	in	retinal	tissue	of	diabetic	patients	compared	to	non-diabetic	patients,	
64	

	
again	using	TUNEL	staining(157,158).	In	addition,	increased	caspase-8	and	caspase3	activity	is	seen	in	rat	retinal	pericytes	in	high	glucose	conditions(197).		Similarly	
to	 endothelial	 cells,	 mitochondria	 of	 retinal	 pericytes	 display	 significant	
fragmentation	and	metabolic	dysregulation	and	this	has	been	directly	implicated	in	
accelerated	 apoptosis	 in	 retinal	 pericytes	 in	 diabetic	 retinopathy(198).	 These	
studies	all	indicate	a	similar	caspase-dependent	apoptotic	mechanism	for	pericytes	
as	seen	for	endothelial	cells.		
	

Additionally,	TUNEL	staining	has	been	used	to	identify	apoptosis	in	a	variety	

of	 retinal	 cell	 types,	 although	 numbers	 of	 these	 studies	 are	 limited.	 Increased	
apoptosis	in	neural	retinal	cells	such	as	ganglion	cells	of	diabetic	rats	compared	to	
non-diabetic	rats	has	been	detected(135,159,160,188,196).	Amacrine	cells	have	also	
been	shown	to	undergo	apoptosis,	as	characterized	by	TUNEL	staining	and	staining	
for	active	caspase-3,	using	the	Ins2Akita	mouse	model(199).		It	has	been	suggested	
that	 there	 is	 selective	 S-cone	 loss	 as	 identified	 by	 TUNEL	 staining	 in	 diabetic	
retinopathy(200).	 	 However,	 more	 detailed	 studies	 are	 needed	 to	 further	 confirm	
the	 aforementioned	 mechanisms	 and	 to	 allow	 for	 a	 distinct	 classification	 of	
apoptosis	in	these	cells.	Although	numerous	studies	have	established	that	apoptosis	
of	several	cell	types	occurs	in	the	diabetic	retina,	the	next	important	step	will	be	to	
identify	 the	 link	 between	 increased	 glucose	 levels	 and	 the	 initiation	 of	 caspasedependent	apoptosis.	
	

65	

	
3.4	Necrosis:	A	Regulated	Pathway	to	Cell	Death?	
Historically,	necrosis	was	considered	the	type	of	cell	death	“on	the	other	end”	of	the	
cell	death	spectrum.	Necrosis	has	been	a	term	used	for	‘accidental	cell	death’	rather	
than	‘programmed	cell	death,’	which	was	reserved	for	apoptosis.		It	was	defined	in	
the	classification	of	cell	death	article	published	by	the	NCCD	in	2005,	as	cell	death	
with	 no	 apparent	 signs	 of	 apoptosis	 or	 autophagy(201).	 The	 morphological	
appearance	of	cells	undergoing	necrosis	were	described	as	having	features	such	as	
cytoplasmic	 swelling,	 mechanical	 rupture	 of	 the	 plasma	 membrane,	 dilation	 of	
cytoplasmic	 organelles,	 and	 chromatin	 condensation(201).	 The	 understanding	 of	
pathways	 leading	 to	 necrosis	 in	 vivo	 was	 vague	 at	 best.	 Due	 to	 the	 lack	 of	 a	 clear	
mechanism	 for	 necrosis,	 new	 terms	 describing	 “necrosis-like”	 cell	 death	 were	
introduced.	One	of	these	new	terms	was	“apoptonecrosis”	where	apoptosis	evolves	
into	necrosis,	although	use	of	this	term	was	discouraged	to	avoid	further	confusion	
until	pathways	involved	in	this	process	were	fully	identified(201).	However,	out	of	
this	 research,	 the	 picture	 of	 “regulated	 necrosis”	 and	 it’s	 importance	 in	 various	
physiological	 and	 pathological	 settings	 evolved(142,202).	 Triggers	 for	 regulated	
necrosis	include	excitotoxicity,	DNA	damage	resulting	in	DNA	alkylation,	and	ligands	
such	 as	 TNF	 and	 FasL	 binding	 to	 their	 respective	 death	 receptors(142,203–207).	
These	 triggers	 initiate	 ubiquitination	 of	 receptor	 interacting	 kinase-1	 (RIPK1)	 and	
subsequent	 activation	 of	 RIP3.	 	 Whereas	 RIP3	 would	 activate	 pro-caspase-8	 in	
apoptotic	 conditions,	 in	 experimental	 or	 pathological	 settings	 where	 caspase-8	 is	
absent	 RIP3	 can	 lead	 directly	 to	 execution	 of	 regulated	 necrosis(142,203–205).	
Crucial	 characteristics	 of	 regulated	 necrosis	 include	 death	 receptor	 signaling,	
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absence	 of	 caspase	 activity,	 and	 RIP1	 and	 /or	 RIP3	 activation.	 Activation	 of	
pathways	 in	 regulated	 necrosis	 still	 lead	 to	 the	 classical	 morphological	 features	
associated	 with	 necrosis(142).	 All	 these	 new	 studies	 indicate	 that	 the	 process	 of	
necrotic	 cell	 death	 can	 be	 regulated	 depending	 on	 microenvironment	 rather	 than	
being	a	random	event	as	previously	assumed.		
Necrosis	 has	 been	 implicated	 in	 the	 process	 of	 diabetic	 retinopathy.	
Increased	 necrotic	 cell	 death	 of	 pericytes	 has	 been	 observed	 in	 the	 retinas	 of	
diabetic	 rats	 and	 humans	 using	 light	 and	 electron	 microscopy(208–210).	 This	
particular	 pericyte	 cell	 death	 was	 later	 described	 as	 “selective	 necrosis”(138).		
Reasoning	for	this	designation	was	most	likely	due	to	the	assumption	that	this	cell	
death	 caused	 by	 diabetic	 conditions	 was	 accidental.	 Although	 the	 newer	 studies	
claim	apoptosis	as	the	major	type	of	cell	death	for	pericytes	in	diabetic	retinopathy,	
one	 cannot	 exclude	 that	 some	 pericytes	 might	 undergo	 cell	 death	 via	 regulated	
necrosis	 depending	 on	 microenvironment	 and	 the	 progression	 of	 the	 disease.		
Further	clarification	of	the	definition	for	necrosis	and	the	pathways	involved	may	be	
necessary	to	better	understand	and	identify	this	process	in	the	diabetic	retina.		
	
3.5	Autophagic	Cell	Death	
Autophagic	cell	death	may	be	the	most	puzzling	type	of	cell	death	identified	
to	date.	It	is	currently	defined	by	the	NCCD	as	“a	type	of	cell	death	that	occurs	in	the	
absence	 of	 chromatin	 condensation	 but	 accompanied	 by	 massive	 autophagic	
vacuolization	of	the	cytoplasm”(141).	The	first	study	demonstrating	that	autophagic	
cell	 death	 exists	 in	 vivo	 showed	 that	 knockdown	 of	 key	 genes	 required	 for	
67	

	
autophagy	 reduced	 cell	 death	 in	 Drosophila	 melanogaster(211).	 Autophagic	 cell	
death	has	also	been	identified	in	cancer	cells	exposed	to	chemotherapeutic	agents	in	
vitro(212,213).	Cells	dying	by	autophagic	cell	death	have	very	little	association	with	
phagocytes,	contrary	to	cells	dying	by	apoptosis	which	are	eventually	removed	via	
phagocytosis(141).	In	order	to	determine	autophagic	cell	death,	cell	death	must	be	
prevented	 by	 inhibition	 of	 the	 autophagic	 pathway	 either	 by	 chemicals	 or	
knockdown	 of	 essential	 autophagic	 proteins(141,142).	 Detection	 of	 common	
markers	 used	 to	 observe	 increases	 or	 decreases	 in	 autophagy,	 such	 as	 LC3	
(microtubule-associated	 protein	 1	 light	 chain	 3)	 or	 ATG	 (autophagy)	 family	
members,	are	not	sufficient	to	indicate	autophagic	cell	death.		
Autophagy	 is	 the	 process	 of	 removing	 unwanted	 or	 damaged	 cellular	
material	 or	 organelles	 by	 packaging	 these	 materials	 into	 autophagosomes,	 which	
are	 then	 targeted	 for	 degradation.	 In	 most	 physiological	 settings,	 autophagy	 is	
considered	a	beneficial	and	a	pro-survival	mechanism	used	by	the	cell	and	inhibition	
of	 autophagy	 can	 actually	 lead	 to	 increased	 apoptosis(214–218).	 Therefore,	 an	
increase	 in	 autophagic	 flux	 does	 not	 always	 imply	 autophagic	 cell	 death.	 	 For	
example,	 if	 cell	 death	 occurs	 with	 increased	 markers	 of	 autophagy	 but	 cannot	 be	
blocked	 by	 autophagy	 inhibition,	 it	 is	 not	 indicative	 of	 autophagic	 cell	 death.	 	 To	
classify	 cells	 as	 dying	 by	 autophagic	 cell	 death,	 inhibition	 of	 proteins	 within	 the	
autophagic	pathway	must	promote	cell	survival.	
Whether	 autophagic	 cell	 death	 is	 occurring	 in	 the	 progression	 of	 diabetic	
retinopathy	 has	 not	 been	 determined	 to	 date.	 A	 recent	 study	 indicated	 increased	
autophagy	 by	 measuring	 levels	 of	 ATG5,	 but	 whether	 this	 ultimately	 leads	 to	
68	

	
autophagic	 cell	 death	 in	 retinal	 cells	 during	 diabetic	 retinopathy	 has	 not	 been	
addressed(219).	
	
3.6	Pyroptosis:	Inflammation	Driven	Cell	Death	
An	 emerging	 type	 of	 cell	 death	 that	 is	 attracting	 increasing	 attention	 is	
‘pyroptosis.’	Pyroptosis	is	an	inherently	inflammatory-mediated	form	of	cell	death,	
defined	 as	 being	 caspase-1-dependent(73,142,220).	 	 During	 pyroptosis,	 there	 is	
assembly	 of	 a	 multiprotein	 platform	 allowing	 for	 induced	 proximity-mediated	
activation	 of	 caspase-1.	 Active	 caspase-1	 then	 cleaves	 the	 pro-inflammatory	
cytokines	IL-1β	and	IL-18	from	their	inactive	precursors	to	their	biologically	active	
forms(73,75,76,220).		The	multiprotein	platform	allowing	for	caspase-1	activation	is	
termed	either	the	inflammasome	or	pyroptosome.	Inflammasomes	are	comprised	of	
the	 ASC	 (Apoptosis-associated	 Speck-like	 protein	 containing	 a	 CARD)	 adaptor	
protein	 and	 a	 cytosolic	 sensor	 of	 either	 DAMPS	 (Danger	 Associated	 Molecular	
Patterns)	 or	 PAMPS	 (Pathogen	 Associated	 Molecular	 Patterns)	 such	 as	 a	 NLRs	
(NOD-like	receptors)	or	AIM2	(absent	in	melanoma	2)(221–225).	The	pyroptosome	
is	an	assembly	of	ASC	dimers	that	can	directly	activate	caspase-1(226).	Prevention	
of	pyroptosis	is	accomplished	by	inhibition	of	caspase-1	either	by	pharmacological	
intervention	or	caspase-1	knockout	in	animal	models.	Although	it	is	now	very	well	
established	that	initiation	of	pyroptosis	is	caspase-1	and	IL-1β	driven,	the	execution	
phase	 of	 pyroptosis	 is	 not	 yet	 completely	 understood.	 It	 has	 been	 shown	 that	
pyroptosis	 shares	 traits	 with	 both	 apoptosis	 and	 necrosis	 in	 the	 execution	
phase(141,227).	 Execution	 of	 pyroptotic	 cell	 death	 might	 depend	 on	 cell	 type,	
69	

	
microenvironment,	 and	 stimulus.	 Different	 pathways	 might	 be	 involved	 in	 the	
execution	 of	 pyroptosis	 bringing	 into	 question	 whether	 TUNEL	 staining	 is	 actually	
able	to	identify	all	pyroptotic	cells.		
This	inflammatory-mediated	process	of	cell	death	is	particularly	intriguing	in	
the	context	of	diabetic	retinopathy.		A	disease	which	was	originally	thought	of	as	a	
purely	 microvascular	 disease,	 diabetic	 retinopathy	 is	 now	 being	 viewed	 as	 a	
potential	 chronic	 inflammatory	 disease	 leading	 to	 changes	 in	 the	 retinal	
microvasculature(3,110,228).	 	 Studies	 have	 demonstrated	 that	 diabetes	 leads	 to	
activation	 of	 caspase-1	 and	 IL-1β	 production	 in	 the	 retinas	 of	 diabetic	 and	
galactosemic	mice	as	well	as	diabetic	rats(135,229,230).	Active	caspase-1	and	IL-1β	
was	 also	 detected	 in	 retinal	 tissue	 of	 diabetic	 patients(88,162)	 and	 the	 vitreous	 of	
patients	 with	 proliferative	 diabetic	 retinopathy(113,231).	 Inhibition	 of	 the	
caspaspe-1/IL-β	 signaling	 pathway	 prevented	 the	 development	 of	 diabetic	
retinopathy	in	diabetic	and	galactosemic	animals	indicating	that	this	inflammatory	
pathway	is	important	for	disease	development,	potentially	via	pyroptotic	cell	death	
of	retinal	cells	that	are	crucial	for	proper	retinal	function(88,135).	
When	 looking	 at	 specific	 cell	 types	 undergoing	 pyroptotic	 cell	 death	 in	 the	
course	of	diabetic	retinopathy,	retinal	glial	cells	stand	out.		It	has	been	shown	that	
caspase-1	activity	and	IL-1β	production	is	increased	in	vitro	in	Müller	cells	following	
exposure	 to	 hyperglycemic	 conditions	 and	 cells	 die	 as	 a	 consequence(116,232).	
Inhibition	 of	 the	 caspase-1	 pathway	 prevented	 Müller	 cell	 death	 under	 these	
conditions.	In	Müller	cells	and	microglia,	it	has	been	shown	that	use	of	minocycline,	
a	 drug	 that	 decreases	 caspase-1/IL-1β	 signaling,	 is	 able	 to	 prevent	 cell	
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death(88,135).	These	in	vitro	studies	are	an	indication	that	glial	cells	might	respond	
to	chronically	elevated	glucose	levels	by	undergoing	pyroptotic	cell	death.	
Since	 execution	 of	 pyropototic	 cell	 death	 lacks	 specific	 markers,	 identifying	
retinal	cells	dying	by	pyroptosis	in	vivo	is	a	difficult	task.	Studies	using	EM	show	that	
there	is	Müller	cell	death	occurring	in	diabetic	retinopathy(138).	Dying	Müller	cells	
are	 described	 as	 being	 hypertrophic	 consistent	 with	 the	 notion	 that	 during	
pyroptosis,	 cells	 swell	 rather	 than	 shrink	 as	 observed	 in	 apoptotic	 cell	 death(53).	
Other	 studies	 indicate	 that	 there	 is	 simply	 hypertrophy	 and	 glial	 dysfunction	
associated	 with	 the	 disease(135).	 	 A	 previous	 study	 by	 us	 has	 shown	 that	 GAPDH	
(glyceraldehyde-3-phosphate	dehydrogenase)	accumulates	in	the	nucleus	of	Müller	
cells	in	the	retinas	of	diabetic	rats(163).	Nuclear	accumulation	of	GAPDH	has	been	
closely	associated	with	cell	death	induction(233–235).	Interestingly,	hyperglycemiainduced	nuclear	accumulation	of	GAPDH	was	mediated	by	activation	of	the	caspase1/IL-1β	 pathway(54,139).	 	 Whether	 caspase-1/IL-1β-mediated	 GAPDH	 nuclear	
accumulation	is	part	of	the	pyroptotic	pathway	in	general	has	yet	to	be	determined.	
As	our	results	indicate	(Figure	15),	diabetes	leads	to	Müller	cell	loss	in	the	retinas	of	
diabetic	 mice.	 Due	 to	 the	 lack	 of	 specific	 markers	 for	 pyroptotic	 cell	 death,	 Müller	
cells	 were	 stained	 against	 glutamine	 synthetase	 and	 CRALPB	 and	 counted.	 To	
confirm	that	Müller	cell	loss	was	dependent	on	the	activation	of	the	caspase-1/IL-1β	
pathway,	 IL-1	 receptor	 knockout	 mice	 were	 made	 diabetic	 and	 Müller	 cells	 were	
counted	 in	 retinas	 of	 non-diabetic	 and	 diabetic	 IL-1	 receptor	 knockout	 mice.	
Inhibition	 of	 the	 caspase-1/IL-1β	 pathway	 prevented	 diabetes-induced	 Müller	 cell	
loss.	 These	 studies	 are	 the	 first	 to	 clearly	 demonstrate	 Müller	 cell	 loss	 in	 diabetes	
71	

	
and	to	suggest	that	cell	death	might	occur	via	a	pyroptotic	mechanism.	Based	on	our	
studies,	 we	 suggest	 that	 hyperglycemia	 leads	 to	 activation	 of	 caspase-1	 and	
subsequent	production	of	IL-1β	leading	to	Müller	cells	death	via	pyroptosis	(Figure	
16).	 Since	 glial	 cells	 in	 general	 respond	 to	 hyperglycemia	 by	 producing	 proinflammatory	cytokines,	future	studies	need	to	determine	whether	all	glial	cell	types	
are	able	to	undergo	pyroptotic	cell	death.	
	

	

72	

	
Figure	15:	IL-1R1	Dependent	Müller	cell	death	in	diabetes.		
Diabetic	wild	type	(gHb=	11.0	±	1.8)	and	IL-1R1-/-	(gHb=	12.1	±	0.4)	were	sacrificed	
after	7	months	of	diabetes	along	with	age	matched	normal	controls	(wild	type	gHb=	
3.8	±	0.55,	IL-1R1-/-	gHb=	3.2	±	0.2).	Animals	were	sacrified	and	eyes	were	isolated	
and	fixed	in	formalin.	Retinal	sections	were	processed	for	both	glutamine	synthase,	
CRALBP,	 and	 DAPI	 staining	 and	 blinded	 samples	 were	 visualized	 using	 confocal	
microscopy	 Z-sections.	 The	 number	 of	 Müller	 cells	 was	 determined	 by	 counting	
three	 independent	 areas	 per	 retinal	 section	 and	 three	 retinal	 sections	 per	 animal.		
The	number	of	Müller	cells	per	mm2	retina	is	expressed	as	mean	±	STDV	(n=10	per	
group).	

	

	

	
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Figure	16.	Scheme	of	pyroptosis	in	Müller	cells.	
	

Hyperglycemia

	

	

Caspase-1/IL-1β
Signaling
• Rapid caspase-1 activation
• IL-1β Secretion
• Cell swelling

	

	

Müller Cell
PYROPTOSIS
	

	

74	

	
3.7	Conclusion	
	

In	conclusion,	cell	death	seems	to	be	a	prominent	feature	in	the	progression	

of	 diabetic	 retinopathy.	 Several	 retinal	 cell	 types	 have	 been	 shown	 to	 undergo	
various	 forms	 of	 cell	 death	 (Table	 2).	 A	 lot	 of	 emphasis	 has	 been	 given	 to	 study	
apoptosis	of	retinal	cells	during	the	progression	of	the	disease.	Based	on	the	studies	
available,	 it	 is	 fair	 to	 say	 that	 endothelial	 cells	 are	 predominantly	 dying	 by	 an	
apoptotic	process,	however	the	type	of	apoptosis	has	yet	to	be	determined.	Several	
studies	 point	 to	 a	 caspase-dependent	 mechanism	 involving	 mitochondrial	 damage	
but	 more	 studies	 are	 needed	 to	 fully	 determine	 an	 intrinsic	 or	 extrinsic	 apoptotic	
pathway.	 Other	 cell	 types	 suggested	 to	 undergo	 apoptosis	 are	 ganglion	 cells,	
amacrine	cells,	and	S-cones.	However,	due	to	the	limited	number	of	studies	on	cell	
death	 in	 these	 cell	 types,	 a	 conclusion	 as	 to	 whether	 these	 cells	 are	 truly	 dying	 of	
apoptosis	 cannot	 be	 made	 at	 this	 time.	 The	 picture	 is	 not	 clear	 for	 pericytes;	 both	
apoptosis	as	well	as	necrosis	have	been	suggested	as	modes	of	cell	death.	Given	the	
new	research	on	necrosis	that	indicates	necrosis	can	also	be	a	regulated	process	like	
apoptosis,	 more	 studies	 are	 needed	 to	 determine	 precise	 mechanisms	 of	 pericyte	
cell	 death	 in	 diabetic	 retinopathy.	 The	 type	 of	 cell	 death	 might	 depend	 on	 the	
phenotype	 of	 pericytes	 and	 the	 microenvironment	 surrounding	 these	 cells.	
Inflammation-driven	pyroptosis	is	an	emerging	form	of	cell	death	that	is	receiving	a	
lot	of	attention	right	now.	This	type	of	cell	death	is	intriguing	to	study	in	the	context	
of	 diabetic	 retinopathy	 since	 new	 understandings	 of	 the	 disease	 suggest	 that	
diabetic	 retinopathy	 is	 a	 chronic	 inflammatory	 disease.	 Müller	 cells	 are	 prime	
candidates	for	this	form	of	cell	death	due	to	the	fact	that	diabetes-induced	cell	death	
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is	 dependent	 on	 the	 activation	 of	 the	 caspase-1/IL-1β	 pathway.	 More	 studies	 are	
needed	to	fully	understand	the	mechanism	underlying	the	process	of	pyroptosis	and	
to	 determine	 whether	 glial	 cells	 including	 macro	 and	 micro	 glial	 cells	 are	
undergoing	 pyroptosis	 in	 diabetes.	 	 Loss	 of	 other	 retinal	 cell	 types	 such	 as	
astrocytes	has	been	reported	but	identification	of	the	type	of	cell	death	has	not	been	
made(166).	
Other	 types	 of	 cell	 death	 such	 as	 anoikis,	 entosis,	 parthanatos,	 netosis,	
cornification,	 and	 mitotic	 catastrophe,	 have	 yet	 to	 be	 identified	 in	 the	 course	 of	
diabetic	 retinopathy	 but	 cannot	 be	 excluded	 from	 the	 process	 of	 disease	
development.		Further	development	of	tools	capable	of	assessing	these	modes	of	cell	
death	 is	 needed	 to	 determine	 whether	 these	 cell	 death	 modalities	 are	 present	 in	
diabetic	retinopathy.	
A	better	understanding	of	how	retinal	cells	are	dying	during	the	development	
and	progression	of	diabetic	retinopathy	will	allow	for	a	more	targeted	approach	to	
intervene	 in	 this	 process.	 Although	 the	 general	 consensus	 is	 that	 inhibition	 of	 cell	
death	 is	 beneficial	 for	 disease	 prognosis,	 timing	 at	 which	 intervention	 should	 be	
started	 and	 targeting	 of	 specific	 retinal	 cell	 types	 will	 be	 crucial	 for	 a	 successful	
outcome	of	treatments	aiming	to	inhibit	cell	death.		Therefore,	the	more	knowledge	
that	 is	 attained	 on	 which	 cell	 types	 undergo	 what	 type	 of	 cell	 death,	 the	 more	
therapeutic	strategies	can	be	developed.	
	

	

76	

	
Table	2.	Characteristics	of	modes	of	cell	death	and	potential	retinal	cell	types	
undergoing	cell	death	in	diabetic	retinopathy.		
Retinal	cell	types	were	assigned	into	a	cell	death	category	based	on	identification	of	
at	least	two	characteristics	of	the	respective	mode	of	cell	death.	Abbreviations:	AIF,	
apoptosis-inducing	 factor;	 ENDOG,	 endonuclease	 G;	 TNF,	 tumor	 necrosis	 factor;	
LC3/Atg8,	microtubule-associated	protein	1	light	chain	3;	SQSTM1,	sequestosome	1;	
DAMP,	danger	associated	molecular	pattern;	PAMP,	pathogen	associated	molecular	
pattern.	
	
Cell	Death	Characteristics	

Retinal	Cell	Types	

• Triggered	by	binding	of	lethal	ligands	(FAS/CD95,	TNF,	TRAIL)	to	
respective	death	receptor	
• Caspase-8	activation	leading	to	eventual	caspase-9	and	caspase-3	
activation	as	the	executioner	caspase		
• DNA	fragmentation	
	

•
•
•
•
•
	

Endothelial	cells	
Pericyte	
Ganglion	cells	
Amacrine	
S-cones	

• Triggered	 by	 intracellular	 stress	 (DNA	 damage,	 oxidative	 stress,	
excitoxicity)	
• Increased	mitochondrial	outer	membrane	permeability	
• Release	of	proteins	from	mitochondria	(cytochrome	c)	
• AIF	and	ENDOG	nuclear	translocation	
• DNA	fragmentation	
• Caspase-9	 activation	 leading	 to	 caspase-3	 activation	 as	 the	
executioner	caspase		
• Triggered	 by	 excitotoxicity,	 DNA	 damage,	 or	 binding	 of	 lethal	
ligands	(TNF,	FasL)	
• Ubiquitination	of	RIP1	and	subsequent	RIP3	activation	
• Cell	death	even	in	the	absence	of	caspase	activity	

•
•
•
•
•
	

Endothelial	cells	
Pericytes	
Ganglion	cells	
Amacrine	
S-cones	

• Presence	of	autophagy	markers	(lipidation	of	LC3/Atg8)	
• Increased	degradation	of	autophagic	substrates	(SQSTM1)	
• Prevented	by	inhibition	of	autophagy	
	

• ?	

• Triggered	by	either	DAMPs	or	PAMPs	
• Assembly	of	either	inflammasome	or	pyroptosome	complex	
• Caspase-1	activation	and	subsequent	IL-1β	or	IL-18		

• Müller	cells	

	
Extrinsic	
apoptosis	

Intrinsic	
apoptosis	
	

Necrosis	
	

• Pericytes	

	
	
Autophagic	
cell	death	
	
	
Pyroptosis	

77	

	
Chapter	4.	Caspase-1	Activation	in	Galactosemia	
	
4.1	Introduction	
Diabetic	 retinopathy	 is	 the	 leading	 cause	 of	 acquired	 blindness	 in	 adults.		
Currently	there	is	no	cure	for	the	disease	and	the	few	treatments	that	are	available	
have	limited	success,	work	on	a	case-by-case	basis,	and	are	both	a	burden	to	the	life	
quality	of	the	diabetic	patient	and	a	financial	burden	to	the	health	care	system.	The	
most	common	therapies	are	designed	to	treat	the	vascular	changes	seen	in	diabetic	
retinopathy.		Research	has	shown	however,	that	inflammation	plays	a	key	role	in	the	
development	of	diabetic	retinopathy	and	may	preclude	the	vascular	changes	seen	in	
disease	progression	(61,62,111–113,115,189).		Recently,	a	study	identified	at	least	
19	 cytokines	 are	 elevated	 in	 the	 vitreous	 of	 patients	 with	 proliferative	 diabetic	
retinopathy	 compared	 to	 healthy	 individuals	 (132).	 Therefore,	 understanding	
inflammation	 in	 the	 progression	 of	 diabetic	 retinopathy	 may	 help	 to	 identify	 new	
targets	for	therapies.	
One	 of	 the	 major	 concerns	 in	 diabetic	 retinopathy	 research	 is	 the	 lack	
disease-relevant	 of	 animal	 models.	 	 Mice	 have	 an	 inherently	 different	 retinal	
structure	 as	 they	 have	 no	 macula,	 and	 disease	 pathology	 doesn’t	 progress	 to	
proliferative	diabetic	retinopathy	as	it	does	in	humans.		One	interesting	model	is	the	
galactosemia	model.		The	galactosemia	model	was	first	identified	in	a	study	by	the	
Kern	 lab	 and	 showed	 that	 dogs	 fed	 a	 30%	 D-galactose	 diet	 showed	 retinal	
pathologies	 similar	 to	 those	 in	 the	 diabetic	 retina	 including	 microaneurysms,	
hemorrhages,	 acellular	 capillaries,	 vascular	 lesions	 and	 pericyte	 dropout(236).	
78	

	
Interestingly,	these	pathologies	developed	even	though	the	animals	lacked	many	of	
the	 systemic	 abnormalities	 typically	 associated	 with	 diabetes	 as	 they	 had	 normal	
blood	glucose,	free	fatty	acids	and	serum	insulin(236).		Since	then,	the	galactosemia	
model	has	been	used	extensively	as	a	tool	to	study	retinal	diabetic-like	retinopathy	
without	systemic	diabetes-related	changes(237–244).		Mice	and	rats	fed	galactoseenriched	 diet	 show	 similar	 retinal	 pathologies	 to	 dogs	 including	 increased	 retinal	
capillary	 width	 and	 microaneurysms	 at	 8	 months	 of	 age,	 and	 increased	 retinal	
microaneurysms,	 acellular	 capillary	 formation,	 pericyte	 ghosts	 and	 capillary	
basement	 membrane	 thickening	 as	 duration	 of	 galactosemia	 is	 extended(244–
246,246–251).	Previous	reports	show	that	there	is	chronic,	low-grade	inflammation	
in	 the	 retinas	 of	 galactosemic	 mice(61,62,248).	 Several	 studies	 indicate	 there	 may	
be	multiple	sources	contributing	to	this	inflammation,	however	all	reports	conclude	
that	reducing	this	inflammation	leads	to	protection	from	disease	development	and	
may	 be	 a	 useful	 strategy	 in	 the	 treatment	 of	 diabetic	 retinopathy.	 	 Studies	 using	
galactosemic	CD18	and	ICAM-1	deficient	mice	to	inhibit	leukocyte	adherence	show	
there	 is	 decreased	 retinal	 inflammation	 which	 results	 in	 decreased	 breakdown	 of	
blood-retina	 barrier,	 decreased	 endothelial	 cell	 death	 and	 decreased	 formation	 of	
acellular	capillaries(248).	Previously,	we	have	reported	increased	caspase-1	activity	
and	IL-1β	in	the	retinas	of	galactosemic	mice(61,62).		Furthermore,	administration	
of	 the	 drug	 minocycline	 inhibited	 caspase-1	 activity	 and	 IL-1β	 production	 in	 the	
retinas	 of	 galactosemic	 mice	 and	 this	 resulted	 in	 decreased	 formation	 of	 acellular	
capillaries(61).	 In	 addition	 to	 increased	 inflammation,	 studies	 have	 shown	
abnormalities	in	metabolism	in	the	retinas	of	galactosemic	mice	and	this	may	be	a	
79	

	
driver	 of	 inflammation(247,252–258).	 Interestingly,	 these	 studies	 show	 there	 are	
key	 differences	 between	 the	 experimental-diabetes	 and	 galactosemia	 models	 with	
regards	 to	 metabolic	 abnormalities.	 	 In	 diabetic	 mice	 there	 were	 increases	 in	 both	
systemic	and	retinal	oxidative	stress,	however	in	galactosemic	mice	oxidative	stress	
increased	 only	 in	 the	 retinas,	 but	 was	 normal	 in	 the	 plasma(252).	 	 Furthermore,	
there	 are	 differential	 effects	 of	 antioxidants	 in	 the	 retinas	 of	 these	 two	
models(252,256).		
Caspase-1,	formerly	known	as	Interleukin-1β	Converting	Enzyme	(ICE),	is	an	
enzyme	produced	as	an	inactive	zymogen	that	can	be	activated	to	convert	inactive	
pro-IL-1β	into	biologically	active	IL-1β	(75,259–262).	Caspase-1	activation	has	been	
linked	to	increased	production	of	reactive	oxygen	species	(ROS)	and	upregulation	of	
TXNIP,	 a	 protein	 which	 can	 bind	 and	 inhibit	 thioredoxin	 leading	 to	 ROS	
generation(137,263).	 Studies	 show	 that	 TXNIP	 plays	 a	 significant	 role	 in	 diabetic	
retinopathy	 and	 that	 inhibition	 of	 TXNIP	 prevents	 diabetes-induced	 inflammation,	
gliosis,	pericyte	loss	and	ganglion	cell	injury(137,197,264).	Although	many	studies	
have	 focused	 on	 the	 differences	 in	 metabolism,	 inflammation	 and	 retinopathy	
pathology	 between	 high	 glucose	 and	 high	 galactose	 models,	 relatively	 few	 studies	
have	been	performed	examining	the	effects	of	high	galactose	at	the	cellular	level.		In	
the	present	study,	we	examine	differences	between	hyperglycemia	and	galactosemia	
and	how	they	affect	oxidative	stress	and	caspase-1	activity	in	the	retina	and	retinal	
Müller	 cells.	 Furthermore,	 we	 explore	 whether	 potential	 differences	 in	 caspase-1	
activity	can	be	linked	to	differences	in	oxidative	stress	between	hyperglycemic	and	
galactosemic	conditions.	
80	

	
4.2	Materials	and	Methods	
Materials:	Caspase-1	substrate	YVAD-AFC	and	caspase-1	inhibitor	YVAD-FMK	were	
purchased	 from	 Calbiochem	 (San	 Diego,	 CA).	 Mouse	 anti-TXNIP	 antibody	 was	
purchased	from	MBL	International	(Woburn,	MA).	Gradient-Tris-SDS-PAGE	(4-20%)	
gels	 were	 from	 BioRad	 (Hercules,	 CA).	 7-amino-4-trifluoro-methylcoumarin	 (AFC)	
standards	came	from	Sigma	(St.	Louis,	MO).			
Animal	Models:	Male	mice	(C57BL/6)	weighing	20	g	were	randomly	assigned	to	be	
made	 diabetic,	 galactosemic,	 or	 left	 as	 normal	 controls.	 Diabetes	 was	 induced	 by	
streptozotocin	 injections	 (60	 mg/kg	 body	 wt	 i.p.	 on	 5	 consecutive	 days)	 as	
described	 previously	 (13).	 Insulin	 was	 given	 to	 diabetic	 animals	 as	 needed	 to	
achieve	 slow	 weight	 gain	 without	 preventing	 hyperglycemia	 and	 glycosuria	 (0.1–	
0.2	units	of	NPH	insulin	subcutaneously,	two	to	three	times	a	week).	Galactosemia	
was	induced	by	feeding	normal	mice	a	diet	enriched	with	30%	galactose.	
Tissue	 Culture:	 The	 transformed	 rat	 retinal	 Müller	 cell	 line	 (rMC-1)	 has	 been	
established	 previously	 as	 a	 relevant	 cell	 type	 for	 retinal	 Müller	 cell	 studies	
(54,62,163,265).	 rMC-1	 cells	 were	 maintained	 in	 normal	 (5mmol/L)	 glucose	
Dulbecco’s	Modified	Eagle’s	Medium	(DMEM)	supplemented	with	10%	fetal	bovine	
serum	(FBS)	and	1%	penicillin/streptomycin	(PS)	in	a	humidified	incubator	kept	at	
37	degrees	Celsius	with	5%	CO2.	
High	Glucose	and	Galactose	Treatment:	For	high	glucose	studies	rMC-1	were	treated	
in	DMEM	supplemented	with	glucose	for	final	glucose	concentration	of	25	mmol/L.		
For	 high	 galactose	 treatment	 rMC-1	 were	 treated	 in	 DMEM	 containing	 standard	 5	
mmol/L	 glucose	 and	 supplemented	 with	 20	 mmol/L	 galactose.	 	 Studies	 using	 the	
81	

	
caspase-1	 inhibitor	 (YVAD-fmk;	 100μM)	 were	 performed	 as	 described	 in	 previous	
studies(54).	
Preparation	of	Cytosolic	Lysates:	Following	respective	treatments,	rMC-1	were	lysed	
in	 cytosolic	 lysate	 buffer	 [CHAPS	 Buffer	 (100mM	 HEPES,	 pH	 7.5	 containing	 10%	
sucrose,	0.1%	CHAPS),	1	mmol/L	EDTA,	1	mmol/L	PMSF	and	leupeptin	(10μg/ml)	
as	described	previously	(54,88).	
Caspase-1	 Activity	 assays:	 Caspase-1	 activity	 was	 measured	 as	 described	
previously(54,62,88,117,139,163).	Equal	amounts	of	sample	(15	μg)	were	incubated	
in	 the	 presence	 of	 the	 caspase-1	 specific	 substrate	 (YVAD-AFC;	 2.5	 μmol/L)	 for	 1	
hour	at	32	degrees	C.		AFC	fluorescence	was	detected	by	a	Tecan	Spectra	FluorPlus	
fluorescence	 plate	 reader	 (excitation:	 400	 nm,	 emission:	 510	 nm).	 The	 release	 of	
AFC	 after	 cleavage	 by	 active	 caspase-1	 was	 calculated	 against	 AFC	 standard	 curve	
and	expressed	as	pmol	AFC/mg	protein/min.	
Western	Blot	Analysis:	Protein	(40ug)	was	separated	in	4-20%	SDS	gradient	gels	and	
blotted	 on	 nitrocellulose	 membrane.	 	 Membranes	 were	 incubated	 against	 TXNIP	
(1:1000	 dilution	 in	 PBS/0.05%	 Tween	 20)	 overnight	 at	 4	 degrees	 C	 followed	 by	
incubation	with	anti-mouse	secondary	antibody	(1:5,000	dilution)	for	1	hour	at	RT.		
Membranes	 were	 imaged	 using	 LICOR	 Biosciences	 Odyssey	 Imaging	 System.		
Membranes	 were	 reprobed	 using	 anti-β-actin	 antibody	 and	 normalized	 by	 the	
relative	density	of	TXNIP/β-actin.	
ROS	Measurement:		ROS	was	measured	using	cell	permeable	2’-7’-dichlorofluroescin	
diacetate	 (H2DCFDA)	 (Invitrogen,	 Burlington,	 Ontario).	 	 rMC-1	 were	 plated	 on	
Costar	96	well	plates,	black	with	a	clear	bottom	(Corning,	NY)	at	100,000	cells	per	
82	

	
well.		After	treatment,	media	was	removed,	and	100	μl	of	10	μM	H2DCFDA	diluted	in	
serum	free	media	was	added	to	each	well	for	15	minutes,	after	which	H2DCFDA	was	
aspirated	 out	 of	 well	 and	 replaced	 with	 serum	 free	 media.	 	 ROS	 was	 measured	 on	
TECAN	Infinite	m1000	plate	reader	at	an	excitation	of	494	nm	and	emission	of	525	
nm.	 	 Following	 measurement	 of	 ROS,	 fluorescence	 was	 normalized	 using	 cell	
permeable	Hoechst	33342	nucleic	acid	stain	and	normalized	to	a	standard	curve	to	
determine	cell	number.		
Trypan	 Blue	 Cell	 Death	 Assay:	 Cell	 death	 was	 determined	 as	 described	
previously(44,54,88,163,266).	 	 Briefly,	 following	 treatment	 rMC-1	 samples	 were	
masked	and	assessed	for	trypan	blue	inclusion	indicating	cell	death.	Cell	death	was	
quantified	 and	 expressed	 as	 fold	 change	 increase	 compared	 to	 cells	 cultured	 in	
normal	glucose.	
Statistical	 Analysis:	 Analysis	 of	 data	 was	 performed	 using	 the	 One-Way	 Anova	
(correlated	 samples;	 p<0.05)	 test	 followed	 by	 Tukey’s	 post	 analysis	 or	 KruskalWallis	 test	 (ordinal	 data;	 p<0.05)	 followed	 by	 Dunn’s	 post	 analysis	 to	 determine	
statistical	 significance	 between	 groups.	 For	 details	 in	 statistical	 analysis	 see	
VassarStats,	 Statistical	 computational	 website.	 	 All	 data	 are	 presented	 as	 mean	
±SDEV.	
	
4.3	Results	
4.3.1	Galactosemia	Increases	Caspase-1	Activity	in	Müller	Cells	
	

It	 is	 known	 that	 there	 is	 chronic	 low-grade	 inflammation	 in	 the	 retinas	 of	

galactosemic	 mice.	 	 Previously	 we	 have	 shown	 that	 there	 is	 increased	 caspase-1	
83	

	
activity	in	the	retinas	of	hyperglycemic	and	galactosemic	mice	and	that	inhibition	of	
caspase-1	 prevents	 the	 formation	 of	 acellular	 capillaries	 in	 these	 animals.	 	 To	
identify	 the	 cellular	 source	 of	 this	 caspase-1	 we	 treated	 rMC-1	 cells	 with	 either	 5	
mmol/L	 glucose,	 25	 mmol/L	 glucose	 (high	 glucose)	 or	 5	 mmol/L	 glucose	 and	 20	
mmol/L	glucose	(high	galactose).			We	found	that	treatment	with	either	high	glucose	
or	 high	 galactose	 led	 to	 a	 significant	 increase	 in	 caspase-1	 activity	 in	 rMC-1	 cells	
(Figure	17).	
4.3.2	High	Glucose,	But	Not	High	Galactose	Generates	Reactive	Oxygen	Species	
	

Caspase-1	 activity	 has	 been	 linked	 to	 mitochondrial	 dysfunction	 and	

increases	in	ROS.		In	order	to	determine	whether	the	observed	increases	in	caspase1	activity	were	due	to	generation	of	ROS	we	treated	cells	measure	ROS	production	in	
rMC-1	cells	over	the	course	of	96	hours.		Interestingly,	we	observed	increased	ROS	
production	in	rMC-1	cells	treated	with	high	glucose,	however	high	galactose	had	no	
effect	on	ROS	production	(Figure	18A,B).			
4.3.3	Glucose	Specific	Increases	in	TXNIP		
	

TXNIP	 is	 one	 of	 the	 primary	 targets	 of	 studies	 involving	 increased	 ROS	

accumulation.		Interestingly,	numerous	reports	have	shown	that	TXNIP	also	plays	a	
role	in	caspase-1	activition,	and	TXNIP	is	shown	to	be	increased	in	studies	relating	
to	 diabetic	 retinopathy.	 We	 observed	 that	 there	 are	 increased	 protein	 levels	 of	
TXNIP	 in	 rMC-1	 cells	 treated	 with	 high	 glucose	 for	 24	 hours	 (Figure	 19A).	
Interestingly,	in	the	same	manner	that	ROS	generation	was	specific	to	glucose	and	
not	 galactose,	 there	 was	 no	 increase	 in	 TXNIP	 protein	 in	 rMC-1	 cells	 treated	 with	
high	galactose	(Figure	19B).		Interestingly,	we	observed	two	bands	in	our	Western	
84	

	
Blot	 analysis	 of	 TXNIP,	 the	 lower	 band	 was	 consistent	 the	 molecular	 weight	 of	
TXNIP,	 however	 the	 upper	 band	 was	 found	 around	 90	 kDA.	 More	 interestingly,	 in	
high	glucose	treated	samples;	the	upper	band	appeared	to	decrease	in	intensity	as	
the	 lower	 band	 increased.	 	 Next,	 we	 measured	 TXNIP	 levels	 in	 rMC-1	 over	 the	
course	of	96	hours	to	determine	whether	TXNIP	was	increased	at	all	time	points	of	
increased	ROS	production.		We	observed	significant	increases	in	TXNIP	levels	at	24,	
48,	72	and	96	hours	of	high	glucose	treatment	(Figure	19C)			
4.3.4	Caspase-1	Inhibition	Prevents	Galactosemia-Induced	Müller	Cell	Death	and	
Acellular	Capillary	Formation	
Previously	 we	 have	 shown	 that	 inhibition	 of	 caspase-1	 can	 prevent	 Müller	
cell	 death	 and	 acellular	 capillary	 formation	 in	 diabetic	 animals.	 To	 determine	
whether	 galactosemia	 can	 also	 lead	 to	 Müller	 cell	 death	 in	 a	 caspase-1	 dependent	
manner	 we	 measured	 cell	 death	 in	 rMC-1	 treated	 with	 high	 galactose	 in	 the	
presence	or	absence	of	a	caspase-1	inhibitor.		We	found	that	high	galactose	led	to	a	
significant	 increase	 in	 cell	 death,	 and	 that	 treatment	 with	 a	 caspase-1	 inhibitor	
significantly	 decreased	 cell	 death	 (Figure	 20).	 To	 examine	 whether	 inhibition	 of	
caspase-1	 affected	 disease	 progression	 in	 vivo,	 acellular	 capillary	 formation	 was	
counted	 in	 either	 non-diabetic	 or	 galactosemic	 mice.	 	 We	 observed	 increased	
acellular	capillary	formation	in	galactosemic	animals,	however	inhibition	of	caspase1	 prevented	 increased	 acellular	 capillary	 formation	 in	 galactosemic	 cas-1-/-	 mice	
(Figure	21).	
	

	

85	

	
Figure	17.	High	Glucose	and	Galactose-Induced	Caspase-1	Activity.	
rMC-1	were	treated	with	either	normal	or	high	galactose	for	24	hours	and	measured	
for	caspase-1	activity.		Data	expressed	as	mean	±	SDEV.	n=5,	p<0.05.	

	
	

	

86	

	
Figure	18.		ROS	induced	by	high	glucose	but	not	high	galactose.	
rMC-1	were	treated	with	either	normal	glucose,	A)	high	glucose,	or	B)	high	galactose	
for	 up	 to	 72	 hours.	 	 ROS	 production	 was	 measured	 and	 expressed	 as	 mean	 AFU	 ±	
SDEV.	n=5,	p<0.05.	

A

5 mmol/l Glucose
25 mmol/l Glucose

*

AFU/100k Cells

1,200.0
1,000.0

*

800.0
600.0
400.0
200.0
0.0

0hr	

12hr	

0

24	hr	

12

24

36	hr	

48	hr	

36

48

72	hr	

72

Hours

B

5 mmol/l Glucose
5 mmol/l Glucose+ 20
mmol/l Galactose

350.0

AFU/100k Cells

300.0
250.0
200.0
150.0
100.0
50.0
0.0

1	

2	

0

24

3	

48

Hours

	

	

87	

4	

72

	

	
Figure	19.		TXNIP	protein	induction	is	glucose	specific.	(Cont.	on	next	page)	
rMC-1	 were	 treated	 with	 either	 normal	 glucose,	 A)	 high	 galactose,	 or	 B)	 high	
glucose.	TXNIP	protein	levels	were	determined	by	Western	Blot	analysis.	C)	protein	
levels	 were	 determined	 by	 Western	 Blot	 analysis,	 normalized	 to	 β-actin,	 and	
expressed	as	mean	±	SDEV	(n=5)	with	(*)	=	p<	0.05	compared	to	5	mmol/L	glucose.	

	
	

	
88	

	
Figure	19	cont..			

C
TXNIP (50 kDa)
Actin (42 kDa)

TXNIP:Actin ratio

0.120

*

0.100

*

0.080

5 mmol/l Glucose
25 mmol/l Glucose

*

0.060

*

0.040
0.020
0.000

24

48

72
Hours

	

	

89	

96

	

	
Figure	20.		High	Galactose	leads	to	caspase-1	dependent	Müller	cell	death.	
rMC-1	were	treated	with	normal	or	high	galactose	in	the	presence	or	absence	of	a	
caspase-1	 specific	 inhibitor	 (YVAD-FMK).	 	 Cell	 death	 was	 measured	 using	 trypan	
blue	exclusion	method.	

Cell Death (Fold Change)

1.8

*

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

	

5mM	

25mM	Gal	

25mM	Gal+	Inhibitor	

Glucose (mmol/L)

5

5

5

Galactose (mmol/L)
Caspase-1 Inhibitor (µM)

-

20

-

-

20
100

	

90	

	

	
Figure	 21.	 Acellular	 capillary	 formation	 in	 non-diabetic	 and	 galactosemic	
mice.	
Retinas	 of	 non-diabetic	 (n=6)	 and	 galactosemic	 (n=10)	 wild	 type	 mice,	 and	 nondiabetic	 (n=6)	 and	 galactosemic	 (n=6)	 casp1-/-	 mice	 were	 isolated	 at	 7	 months	 of	
diabetes.	Number	of	acellular	capillaries	was	determined	and	expressed	as	mean	±	
SDEV.	

Non-Diabetic
Galactosemic
18.0

*

Acellular Capillaries
(number/mm2)

16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0

WT	

Cas-1-/-	

Wild Type
	

Cas-1-/-

	

91	

	

	
4.4	Conclusion	
Previous	 studies	 have	 shown	 that	 the	 galactosemia	 model	 induces	 diabeticlike	 retinopathy	 with	 similar	 retinal	 pathologies	 to	 experimental	 diabetes	 in	 vivo.		
Relatively	few	studies	have	been	performed	examining	the	effects	of	high	galactose	
at	the	cellular	level.		It	is	known	that	diabetes	leads	to	increased	oxidative	stress	and	
inflammation,	and	that	one	cellular	source	of	this	is	Müller	cells.	Studies	have	linked	
increased	oxidative	stress	and	inflammation	in	high	glucose	to	increases	in	TXNIP.	
Thioredoxin,	 along	 with	 glutathione	 and	 catalase,	 represents	 one	 of	 the	 three	
primary	 antioxidant	 pathways	 used	 to	 eliminate	 ROS	 from	 the	 environment(267).	
TXNIP	functions	as	negative	regulator	of	thioredoxin	and	inhibits	it	from	scavenging	
ROS,	leading	to	ROS	accumulation.	In	the	present	study,	we	compared	the	effects	of	
high	 glucose	 and	 high	 galactose	 on	 Müller	 cells	 specifically	 on	 oxidative	 stress,	
TXNIP	regulation,	caspase-1	activity	and	Müller	cell	death.			
First,	 reports	 indicate	 that	 high	 glucose	 leads	 to	 increased	 caspase-1	
activation	and	IL-1β	production	in	Müller	cells(62,88,137).		We	observed	that	there	
was	increased	caspase-1	activity	in	rMC-1	cells	treated	with	either	high	galactose	or	
high	 glucose.	 This	 indicates	 that	 some	 of	 the	 retinal	 pathologies	 observed	 in	
galactosemic	 mice,	 such	 as	 increased	 inflammation	 may	 be	 due	 to	 increased	
caspase-1	activity	and	subsequent	IL-1β	production	by	Müller	cells.	
Next,	 we	 examined	 differences	 in	 ROS	 production	 in	 rMC-1	 under	 high	
glucose	or	high	galactose	conditions.	Studies	indicate	that	there	are	abnormalities	in	
retinal	 metabolism	 and	 oxidative	 stress	 in	 galactosemic	 mice,	 however	 there	 are	
significant	 differences	 in	 retinal	 metabolism	 and	 antioxidant	 response	 between	
92	

	
diabetic	 and	 galactosemic	 mice.	 We	 observed	 there	 is	 significantly	 increased	 ROS	
production	 in	 rMC-1	 beginning	 at	 36	 hours	 of	 high	 glucose	 treatment	 and	 that	 it	
increases	progressively	at	all	time	points	to	96	hours.		Interestingly,	there	were	no	
significant	difference	in	ROS	production	in	rMC-1	treated	with	high	galactose	at	any	
time	 point	 between	 24	 and	 96	 hours.	 	 This	 indicates	 that	 caspase-1	 activity	 may	
actually	 be	 independent	 of	 ROS	 production	 in	 galactosemic	 conditions.		
Furthermore,	 under	 high	 glucose	 conditions,	 increases	 in	 caspase-1	 activity	 are	
detectable	 as	 early	 as	 24	 hours,	 which	 is	 prior	 to	 increased	 ROS	 production,	
indicating	that	caspase-1	activity	may	be	independent	of	ROS	production	under	high	
glucose	conditions	as	well,	at	least	initially.	
Since	increased	caspase-1	activity	and	oxidative	stress	have	both	been	linked	
to	 increases	 in	 TXNIP	 expression	 we	 determined	 if	 there	 are	 similar	 changes	 in	
TXNIP	protein	levels.	Interestingly,	when	comparing	TXNIP	protein	levels	in	either	
high	 glucose	 to	 high	 galactose	 we	 observed	 similar	 differences	 between	 the	 two	
models	to	what	was	observed	in	ROS	production.		There	is	increased	TXNIP	protein	
levels	 in	 rMC-1	 under	 high	 glucose	 conditions	 and	 it	 is	 observed	 at	 all	 time	 points	
between	24	and	96	hours.	Consistent	with	TXNIP’s	function	as	a	negative	regulator	
of	thioredoxin,	TXNIP	protein	levels	increase	just	before	the	accumulation	of	ROS	in	
rMC-1	 cells.	 There	 was	 no	 observed	 increase	 in	 TXNIP	 levels	 in	 the	 high	 galactose	
samples.		Interestingly,	in	western	blot	images	analysis	we	observed	two	bands,	one	
at	55	kDa	molecular	weight	corresponding	to	TXNIP,	and	another	near	95	kDa.		In	
the	high	glucose	treated	samples,	as	there	was	increased	intensity	of	the	lower	band	
for	TXNIP,	there	was	a	decrease	in	the	upper	band	as	if	it	was	a	bound	form	that	was	
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decreasing	 in	 high	 glucose	 conditions.	 	 This	 change	 in	 the	 upper	 band	 was	 not	
observed	and	remained	consistent	in	high	galactose	treated	samples.		
Previously,	 we	 have	 found	 that	 inhibition	 of	 caspase-1	 prevents	 Müller	 cell	
death	 in	 rMC-1	 treated	 with	 high	 glucose(88).	 Numerous	 studies	 in	 other	 systems	
have	 also	 shown	 that	 caspase-1	 activity	 contributes	 to	 pyroptotic	 cell	 death(268–
270).		Interestingly,	we	observed	that	just	as	there	is	increased	caspase-1	activity	in	
rMC-1	treated	with	high	galactose,	there	is	also	increased	cell	death	after	prolonged	
exposure.		Inhibition	of	caspase-1	with	a	caspase-1	inhibitor	(YVAD-fmk)	prevented	
this	 cell	 death.	 	 The	 importance	 of	 caspase-1	 to	 the	 progression	 of	 retinopathy	 in	
galactosemic	 mice	 was	 illustrated	 in	 cas-1-/-	 mice	 fed	 a	 galactose	 enriched	 diet.		
Retinopathy	 progression	 was	 prevented	 in	 these	 animals	 indicating	 that	 the	
caspase-1	 pathway	 is	 activated	 not	 only	 in	 the	 STZ	 diabetic	 model	 as	 reported	
previously,	but	also	in	the	galactosemic	model	of	retinopathy.		
Collectively	this	indicates	that	there	are	many	similarities	and	differences	in	
rMC-1	treated	with	either	high	glucose	or	high	galactose.		While	both	treatments	led	
to	significantly	increased	inflammation	as	observed	by	increased	caspase-1	activity,	
only	 high	 glucose	 led	 to	 increased	 TXNIP	 levels	 and	 ROS	 accumulation.	 	 This	
indicates	that	caspase-1	may	be	activated	independently	of	oxidative	stress	in	rMC1.	 	 Furthermore,	 both	 models	 lead	 to	 a	 similar	 eventual	 out	 come	 of	 caspase-1dependent	cell	death.	

	

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Chapter	5.	The	Role	of	Interleukin-1α	in	Caspase-1	Activation	and	Cell	Death	
in	Müller	Cells	
5.1	Introduction	
Previously	we	have	demonstrated	that	IL-1β	plays	a	crucial	role	in	sustained	
caspase-1	activity	and	cell	death	in	Müller	cells	exposed	to	high	glucose	conditions	
and	 also	 in	 the	 progression	 of	 diabetic	 retinopathy.	 Therefore,	 it	 was	 important	 to	
examine	the	role	of	another	IL-1	family	member,	IL-1α,	in	these	events.			
There	are	a	number	similarities	and	differences	between	IL-1α	and	IL-1β.	In	
terms	 of	 similarities,	 IL-1α	 and	 IL-1β	 are	 both	 synthesized	 as	 a	 31	 kDa	 precursor	
(pro-form)	that	can	be	cleaved	into	17	kDa	mature	forms.	Additionally,	both	of	these	
cytokines	 function	 as	 ligands	 of	 IL-1R1	 and	 exert	 similar	 inflammatory	
responses(271).		
There	are	many	differences	between	IL-1α	and	IL-1β	however,	for	example	
they	 have	 unique	 amino	 acid	 sequences	 and	 have	 very	 different	
bioavailability(99,272).	Whereas	IL-1β	is	only	synthesized	and	secreted	after	a	cell	
encounters	 harmful	 stimuli,	 IL-1α	 is	 constitutively	 expressed	 in	 many	 cell	 types	
even	 under	 normal	 conditions(273).	 	 Furthermore,	 IL-1β	 is	 a	 secreted	 cytokine,	
whereas	 IL-1α	 can	 be	 secreted	 or	 membrane	 bound(274–278).	 	 Another	 key	
difference	 is	 that	 unlike	 IL-1β	 that	 is	 cleaved	 by	 caspase-1	 and	 requires	 this	
processing	to	be	biologically	active,	IL-1α	is	cleaved	instead	by	calpain,	however	it	
does	not	require	cleavage	in	order	to	be	active(271).	Both	the	precursor	and	cleaved	

95	

	
forms	 of	 IL-1α	 are	 agonists	 of	 IL-1R1	 and	 produce	 identical	 biological	
responses(271,279).	
In	addition	to	its	functions	as	a	proinflammatory	cytokine,	IL-1α	is	unique	in	
that	 it	 has	 a	 nuclear	 localization	 signal	 (NLS)	 on	 its	 N-terminal	 pro-piece	
(NTP)(280,281).	 This	 allows	 either	 the	 uncleaved	 pro-IL-1α	 or	 the	 cleaved	 IL-1αNTP	 to	 translocate	 to	 the	 nucleus.	 The	 exact	 function	 of	 IL-1α	 in	 the	 nucleus	 is	
largely	 unknown,	 however	 studies	 indicate	 that	 it	 can	 bind	 to	 the	 chromatin	 and	
directly	regulate	transcription	of	proinflammatory	genes(282–284).	
	
5.2	Materials	and	Methods	
Tissue	 Culture.	 Human	 retinal	 Müller	 cells	 (hMC):	 	 Handling	 of	 human	 tissue	
conformed	 to	 the	 tenets	 of	 Declaration	 of	 Helsinki	 for	 research	 involving	 human	
tissue.	Human	Müller	cells	were	isolated	from	retinal	tissue	of	healthy	donors	with	
no	 history	 of	 diabetes	 and	 chronic	 inflammatory	 diseases	 as	 previously	
described(127,128).		
Treatment:	 hMC	 (1x106)	 were	 treated	 with	 either	 5	 mmol/L	 glucose	 DMEM	 or	 25	
mmol/L	 glucose	 DMEM	 supplemented	 with	 2%	 FBS,	 1%	 P/S	 for	 either	 48	 or	 96	
hours.	 Cells	 treated	 with	 5	 mmol/L	 glucose	 medium	 served	 as	 controls.	 For	 IL-1β	
studies,	 recombinant	 human	 IL-1β	(1-5ng/ml)	 was	 used.	 For	 IL-1ra	 studies,	
following	pretreatment	with	100ng/ml	IL-1ra	for	1	hour,	hMC	were	incubated	in	25	
mmol/L	 glucose	 DMEM	 for	 48	 or	 96	 hours,	 or	 in	 5	 mmol/L	 glucose	 DMEM	 plus	
recombinant	human	IL-1β	(2ng/ml)	for	24	hours.		

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Caspase-1	 Activity	 Assay.	 Caspase-1	 activities	 were	 measured	 as	 described	
previously	(62,117,126–129).	Briefly,	equal	amounts	of	sample	protein	(15	μg)	were	
incubated	 in	 the	 presence	 of	 the	 specific	 caspase-1	 substrate	 (YVAD-AFC;	
2.5μmol/L)	 for	 1	 hour	 at	 32°C.	 AFC	 fluorescence	 was	 detected	 by	 a	 Tecan	 Spectra	
FluorPlus	fluorescence	plate	reader	(excitation:	400	nm,	emission:	510	nm).	Release	
of	 AFC	 by	 active	 caspase-1	 was	 calculated	 against	 an	 AFC	 standard	 curve	 and	
expressed	as	pmol	AFC/mg	protein/min.		
Müller	Cell	Death	In	vitro:	Following	treatment,	cells	were	suspended	and	100μL	of	
cell	 suspension	 was	 mixed	 with	 100μL	 of	 trypan	 blue	 solution.	 Cell	 death	 was	
quantified	using	hemocytometer.	
Immunofluorescence.	 Cells	 were	 plated	 on	 cover	 slips	 in	 a	 6	 well	 plate	 with	 35k	
cells/well.		After	treatment	cells	were	washed,	permeabilized	and	stained	for	IL-1α	
using	 anti-IL-1α	 antibody	 (Abcam,	 ab9875),	 and	 nuclei	 was	 stained	 for	 DAPI.		
Following	staining,	samples	were	imaged	using	confocal	microscope.	
Statistical	 Analysis.	 Analysis	 of	 data	 was	 performed	 using	 the	 Anova	 One-Way	
(correlated	 samples,	 p<0.05)	 test	 followed	 by	 Tukey’s	 post	 analysis	 or	 KruskalWallis	 test	 (ordinal	 data;	 p<0.05)	 followed	 by	 Dunn’s	 post	 analysis	 to	 determine	
statistical	 significance	 among	 groups.	 For	 details	 in	 statistical	 analysis	 see	
VasserStats	 Statistical	 Computation	 Web	 Site.	 All	 data	 are	 presented	 as	 mean	 ±	
SDEV.	
	

97	

	
5.3	Results	
In	order	to	examine	determine	if	IL-1α	plays	a	role	in	high	glucose-induced	
caspase-1	activity	in	Müller	cells,	we	first	treated	hMCs	with	either	normal	or	high	
glucose	for	96	hours	and	used	an	anti-IL-1α	antibody	to	block	the	effects	of	IL-1α.		
We	 observed	 that	 treatment	 with	 anti-IL-1α	 prevented	 high	 glucose	 induced	
caspase-1	activity	at	96	hours,	similar	to	what	we	previously	observed	using	an	IL-1	
receptor	antagonist	(Figure	22).		To	determine	whether	this	blockage	of	caspase-1	
activity	 prevented	 subsequent	 Müller	 cell	 death	 we	 measured	 cell	 death	 in	 the	
aforementioned	 experiment.	 	 Interestingly,	 we	 saw	 that	 anti-IL-1α	 also	 prevented	
high	glucose	induced	Müller	cell	death	(Figure	23).	
Next,	since	IL-1α	has	a	nuclear	localization	sequence	we	determined	whether	
there	could	be	any	nuclear	translocation	in	Müller	cells.		Here	we	treated	hMCs	with	
normal	 or	 high	 glucose	 and	 used	 immunofluorescence	 to	 examine	 cellular	
localization	 (Figure	 24A).	 	 Interestingly,	 we	 observed	 an	 increase	 in	 the	 cells	
positive	for	nuclear	IL-1α	after	96	hours	of	high	glucose	treatment	(Figure	24B).	
	
	

	

98	

	
Figure	 22.	 IL-1α	 blockage	 decreases	 caspase-1	 activity	 in	 Müller	 Cells	 Under	
High	Glucose	Conditions.	
Caspase-1	activity	after	treatment	with	5mmol/l	or	25	mmol/l	glucose	in	the	
presence	or	absence	of	anti-IL-1α.	N=5,	p<0.05.	
50.0

*

Caspase-1 Activity
(pmol AFC/mg/min)

45.0
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0

	

96/5	

96/5+10ug/ml	an/-IL-1a	

96/25	

96/25+10ug/ml	an/-IL-1a	

Glucose (mmol/l)

5

5

25

25

Anti-IL-1α (ug/ml)

-

10

-

10

	

99	

	

	
Figure	23.	High	glucose-induced	Müller	cell	death	inhibited	by	anti-IL-1α.	
Müller	cell	death	measured	by	Trypan	Blue	exclusion	after	treatment	with	5mmol/l	
or	25	mmol/l	glucose	in	the	presence	or	absence	of	anti-IL-1α.	N=5,	p<0.05.	
25.0	

*

Cell Death (%)

20.0	
15.0	
10.0	
5.0	
0.0	

	

96/5	

96/5+10ug/ml	an1-IL-1a	

96/25	

96/25+an1-IL-1a	

Glucose (mmol/l)

5

5

25

25

Anti-IL-1α (ug/ml)

-

10

-

10

	

100	

	

	
	
Figure	24.	Nuclear	Accumulation	of	IL-1α	In	Müller	Cells	Under	High	Glucose	
Conditions.	
(A)	 Immunofluroescence	 staining	 of	 Müller	 cells	 for	 IL-1α	 (red)	 or	 DAPI	 (blue).		
Cells	were	treated	for	48	hours-	5mM	glucose	(upper	left),	48	hours-	25mM	glucose	
(upper	 right),	 96	 hours-	 5mmol/l	 glucose	 (lower	 left),	 or	 96	 hours-	 25	 mmol/l	
glucose	 (lower	 right).	 	 (B)	 Cells	 positive	 for	 nuclear	 IL-1α	 were	 counted	 and	
expressed	as	mean	±	SDEV.	n=5,	p<0.05.	

A

B
Percent of Cells with IL-1α
in the Nucleus

60.00

#
*

50.00

5mM Glucose
25mM Glucose

40.00

30.00

20.00

10.00

0.00
1	

48 Hr

2	

96 Hr

101	

	

	

	
5.4	Conclusion	
In	previous	chapters,	we	examined	the	caspase-1/IL-1β/IL-1R1	pathway	and	
its	 role	 in	 sustained	 caspase-1	 activation	 in	 Müller	 cells	 under	 high	 glucose	
conditions.	 	 In	 this	 study	 we	 examined	 another	 IL-1	 family	 member,	 IL-1α,	 and	 its	
potential	 role	 caspase-1	 activation.	 	 Interestingly,	 we	 found	 that	 blockage	 of	 IL-1α	
prevents	 sustained	 caspase-1	 activity	 and	 subsequent	 cell	 death,	 similar	 to	
treatment	 with	 an	 IL-1	 receptor	 antagonist.	 	 Collectively,	 this	 indicates	 that	 IL-1α	
may	 act	 in	 combination	 with	 IL-1β	 in	 driving	 caspase-1	 activation	 through	
activation	of	IL-1R1.	Furthermore,	we	have	shown	that	high	glucose	induces	nuclear	
translocation	 of	 IL-1α.	 	 It	 remains	 to	 be	 determined	 the	 exact	 role	 of	 IL-1α	 in	 the	
nucleus	of	Müller	cells	under	these	conditions,	however	it	appears	to	be	localized	at	
focal	points,	consistent	with	previous	reports	of	IL-1α	binding	to	specific	locations	
on	the	nuclear	chromatin(283).			
	

	

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Chapter	6.	Summary	and	Future	Outlook	
6.1	 Summary	 of	 data	 regarding	 caspase-1/IL-1β	 feedback	 signaling	 in	 the	
retina	of	diabetic	mice	
The	 importance	 of	 inflammation	 in	 the	 progression	 of	 diabetic	 retinopathy	
has	 become	 increasingly	 apparent.	 A	 variety	 of	 pro-inflammatory	 cytokines	 have	
been	 identified	 in	 the	 vitreous	 of	 patients	 compared	 to	 healthy	 individuals,	 among	
them	IL-1β.	Our	previous	studies	have	shown	that	caspase-1	activity	is	consistently	
increased	in	the	retinas	of	diabetic	animals	and	patients.	New	data	from	this	thesis	
project	provide	novel	mechanistic	insights	into	the	process	of	prolonged	caspase-1	
activation	and	IL-β	production	in	diabetic	retinopathy.	Our	results	indicate:		
	
•

Using	caspase-1-/-	mice	we	demonstrated	that	caspase-1	activation	is	indeed	
crucial	for	the	development	of	diabetic	retinopathy.	

•

Furthermore,	 using	 IL-1R1-/-	 mice	 we	 identified	 that	 diabetes-induced	
caspase-1	activity	progresses	from	an	IL-1R1	independent	mechanism	to	an	
IL-1R1	dependent	mechanism	throughout	disease	progression.	

•

These	 data	 provide	 for	 the	 first	 time	 an	 indication	 of	 a	 caspase-1/IL-1β/IL1R1	 feedback	 signaling	 mechanism	 that	 keeps	 caspase-1	 active	 in	 the	
diabetic	retina.	

•

Using	 Müller	 cells	 known	 to	 produce	 active	 caspase-1	 and	 IL-1β	 under	
hyperglycemic	 conditions	 we	 confirmed	 that	 prolonged	 exposure	 to	
hyperglycemia	 leads	 to	 caspase-1/IL-1β/IL-1R1	 feedback	 signaling	 causing	
sustained	caspase-1	activity.	
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•

Hyperglycemia	 increased	 RIP2	 protein	 levels	 in	 Müller	 cells	 after	 24	 hours.	
Most	 importantly,	 siRNA	 against	 RIP2	 decreases	 caspase-1	 activation	 and	
subsequent	IL-1β	secretion	under	hyperglycemic	conditions.	

•

Consistent	with	a	decrease	in	caspase-1	activation	and	IL-1β	secretion,	siRNA	
against	 RIP2	 prevented	 hyperglycemia-induced	 Müller	 cell	 death	 after	 96	
hours	of	hyperglycemia	exposure.	

•

Interestingly,	 IL-1β	 also	 increased	 RIP2	 protein	 levels.	 siRNA	 against	 RIP2	
also	attenuates	caspase-1	activity	induced	by	exogenous	IL-1β	indicating	that	
RIP2	 is	 not	 only	 activating	 caspase-1	 under	 hyperglycemic	 conditions,	 but	
also	by	IL-1β	feedback	signaling.		

•

Diabetic	and	galactosemic	caspase-1-/-	mice	were	protected	from	Müller	cell	
loss	 and	 the	 formation	 of	 acellular	 capillaries,	 confirming	 that	 caspase-1	
activation	plays	a	significant	role	in	Müller	cell	loss	in	vivo	and	is	crucial	for	
disease	progression.	

	
Future	 outlook:	 Future	 studies	 are	 requires	 to	 determine	 whether	 interfering	 in	
caspase-1	activation	by	targeting	RIP2	represents	a	potential	therapeutic	target	for	
treatment	of	diabetic	retinopathy.	In	addition,	it	would	be	valuable	to	test	drugs	that	
target	 caspase-1	 or	 IL-1β	 directly	 which	 are	 already	 on	 the	 market	 for	 other	
diseases	in	the	context	of	diabetic	retinopathy.	This	would	increase	possibilities	of	
drug	choices	dramatically.	
	

	

104	

	
Figure	 25:	 Schematic	 outlining	 the	 caspase-1/IL-1β	 feedback	 signaling	 the	
diabetic	mouse.	

Hyperglycemia	

STZ	or	
Müller	Cells	
Galactosemic	
Mouse	Model	

RIP2	

IL-1R1	

Ac)ve	Caspase-1	
Pro-IL-1β	

Ac)ve	IL-1β	

Ac)ve	IL-1β	

Feedback	Signaling	

Diabe)c	
Re)nopathy	

	
	

	

105	

	
6.2	 Summary	 of	 data	 regarding	 the	 role	 of	 IL-1α	 in	 hyperglycemia-induced	
caspase-1	activation	in	Müller	cells	
Hyperglycemia	 leads	 to	 sustained	 activation	 of	 caspase-1	 and	 IL-1β	 production	 in	
the	 retinas	 of	 diabetic	 animals	 and	 patients	 in	 vivo	 and	 in	 Müller	 cells	 in	 vitro.	
Interference	 in	 this	 signaling	 by	 knockout	 of	 caspase-1	 or	 the	 IL-1R1	 prevents	
diabetic	 retinopathy	 indicating	 that	 the	 IL-1	 pathway	 is	 important	 for	 the	
development	 of	 the	 disease.	 The	 IL-1	 family	 is	 comprised	 of	 multiple	 members	
including	 IL-1α.	 This	 study	 was	 focused	 on	 identifying	 the	 role	 of	 IL-1α	 in	
hyperglycemia-induced	chronic	inflammation	in	human	Müller	cells.	Data	show	that:	
	
•

Hyperglycemia	led	to	a	decrease	in	cytosolic	IL-1α	in	Müller	cells	at	24	hours.		

•

Treatment	with	anti-IL-1α	inhibited	hyperglycemia-induced	chronic	caspase1	 activity	 and	 subsequent	 cell	 death	 in	 Müller	 cells	 suggesting	 that	 IL-1α	
plays	a	role	in	caspase-1/IL-1β	inflammatory	feedback	signaling.		

•

Cytosolic	 localization	 of	 IL-1α	 became	 increasingly	 localized	 to	 the	
cytoskeleton	during	high	glucose	treatment	compared	to	control.			

•

There	was	a	significant	increase	in	nuclear	IL-1α	at	96	hours	indicating	that	
IL-1α	 might	 function	 on	 a	 transcriptional	 level	 to	 sustain	 inflammation	 at	
prolonged	high	glucose	exposure.		

•

Nuclear	 IL-1α	 appeared	 punctated	 rather	 than	 diffusely	 spread	 throughout	
the	nucleus.		

Future	 outlook:	 This	 study	 has	 shown	 that	 IL-1α	 seems	 to	 participate	 in	
hyperglycemia-induced	 chronic	 caspase-1/IL-1β/IL-1R1-mediated	 inflammation.	
106	

	
Further	 studies	 are	 necessary	 to	 identify	 the	 role	 of	 IL-1α	 in	 the	 development	 of	
diabetic	 retinopathy	 and	 whether	 IL-1α	 can	 be	 targeted	 for	 drug	 development.		
Furthermore,	the	role	of	IL-1α	in	the	nucleus	remains	to	be	examined.	
	
6.3	Implications	of	the	data	collected	during	this	thesis	project	to	the	field	of	
Müller	cells		and	diabetic	retinopathy		
	
Hyperglycemia	promotes	release	of	(1)	growth	factors,	such	as	vascular	endothelial	
growth	 factor	 (VEGF)	 and	 pigment	 epithelium-derived	 factor	 (PEDF),	 and	 (2)	
cytokines	 and	 chemokines	 including	 interleukin-1β	 (IL-β),	 interleukin-6	 (IL-6),	
tumor	necrosis	factor-α	(TNF-α),	and	chemokine	ligand-2	(CCL2)(54–60,285)	(61–
63)	 from	 Müller	 cells.	 In	 vitro	 studies	 have	 provided	 ample	 evidence	 that	 Müller	
cells	are	a	potential	source	for	growth	factors	and	cytokines	when	stimulated	with	
elevated	glucose	levels.	Considering	that	most	of	the	growth	factors,	cytokines,	and	
chemokines	released	by	Müller	cells	have	been	identified	in	the	vitreous	of	diabetic	
patients	 it	 is	 fair	 to	 assume	 that	 Müller	 cells	 contribute	 to	 the	 overall	 synthesis	 of	
these	factors	in	vivo(231,286–288).		
How	 much	 Müller	 cell	 derived	 growth	 factors	 really	 contribute	 to	 the	
pathology	 of	 diabetic	 retinopathy	 in	 vivo	 is	 still	 not	 fully	 understood.	 The	 first	
studies	to	understand	the	contribution	and	effect	of	Müller	cell	derived	VEGF	to	the	
development	and	progression	of	diabetic	retinopathy	were	done	by	the	group	of	Y.Z.	
Le.	This	group	disrupted	VEGF	in	Müller	cells	with	an	inducible	Cre/lox	system	and	
examined	 diabetes-induced	 retinal	 inflammation	 and	 vascular	 leakage	 in	 these	
107	

	
conditional	 VEGF	 knockout	 (KO)	 mice.	 The	 diabetic	 conditional	 VEGF	 KO	 mice	
exhibited	 an	 overall	 decrease	 in	 parameters	 associated	 with	 the	 pathology	 of	
diabetic	 retinopathy	 such	 as	 leukostasis,	 expression	 of	 inflammatory	 biomarkers,	
depletion	 of	 tight	 junction	 proteins,	 numbers	 of	 acellular	 capillaries,	 and	 vascular	
leakage	compared	to	diabetic	control	mice(59,289,290).	Additional	studies	focusing	
on	altering	known	regulators	of	VEGF	production	such	as	HIF-1	(hypoxia	inducible	
factor	1)(291)	and	the	Wnt	signaling	pathway(292)	specifically	in	Müller	cells	have	
supported	the	notion	that	Müller	cell	derived	VEGF	is	actually	a	major	component	in	
the	process	of	retinal	angiogenesis		and	pathology	in	diabetic	retinopathy.	Besides	
VEGF,	Müller	cell	derived	PEDF	has	also	been	suggested	to	have	its	part	in	diabetesinduced	retinal	angiogenesis(42).	Taken	together,	it	seems	that	Müller	cell	derived	
growth	 factors	 contribute	 heavily	 to	 pathological	 vascular	 events	 in	 diabetic	
retinopathy.		
Although	Müller	cell	derived	VEGF	contributes	to	detrimental	effects	on	the	
microvasculature	in	the	diabetic	retina,	the	intent	of	such	growth	factor	production	
by	 Müller	 cells	 in	 the	 first	 place	 might	 have	 been	 to	 protect	 itself	 and	 the	 retinal	
neurons	 from	 a	 diabetic	 insult.	 This	 idea	 is	 supported	 by	 a	 study	 using	 mice	 that	
carry	 a	 disrupted	 VEGFR2	 specifically	 in	 Müller	 cells.	 Loss	 of	 VEGFR2	 caused	 a	
gradual	 reduction	 in	 Müller	 glial	 density,	 decreased	 of	 scotopic	 and	 photopic	
electroretinography	 amplitudes,	 and	 accelerated	 loss	 of	 photoreceptors,	 ganglion	
cells,	and	inner	nuclear	layer	neurons	in	the	diabetic	retina(293).	More	studies	are	
needed	to	fully	explore	and	understand	the	beneficial	effects	of	Müller	cell	derived	
growth	 factors	 on	 Müller	 cells	 itself	 and	 retinal	 neurons	 in	 the	 context	 of	 disease.	
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This	 is	 especially	 important	 since	 long-term	 anti-VEGF	 treatment	 might	 hamper	
functional	 integrity	 of	 Müller	 cells	 and	 neurons	 causing	 unexpected	 additional	
problems	in	treating	diabetic	retinopathy.	
Besides	 growth	 factors,	 Müller	 cells	 release	 a	 variety	 of	 cytokines	 and	
chemokines	under	hyperglycemic	conditions.	For	example,	Müller	cells	are	a	major	
source	 of	 retinal	 interleukin-1beta	 (IL-1β)	 production	 as	 discussed	 in	 this	
dissertation(62,126,128,294,295).	 Caspase-1,	 originally	 named	 interleukin-1β	
converting	enzyme	(ICE),	produces	the	active	cytokines	IL-1β	and	IL-18	by	cleavage	
of	their	inactive	proform(259,260,296,297).	In	Müller	cells,	hyperglycemia	strongly	
induces	 the	 activation	 of	 the	 caspase-1/IL-1β	 signaling	 pathway	 as	 we	 have	
previously	shown(62,128).	Increased	caspase-1	activation	and	elevated	IL-1β	levels	
have	 also	 been	 identified	 in	 the	 retinas	 of	 diabetic	 mice	 and	 retinal	 tissue	 and	
vitreous	 fluid	 of	 diabetic	 patients(62,113,134,295,298).	 We	 have	 identified	 that	
targeting	this	pathway	by	knocking	down	caspase-1	or	the	IL-1	receptor	(IL-1R1)	or	
by	 pharmacological	 intervention	 protects	 against	 the	 development	 of	 diabetic	
retinopathy	 in	 diabetic	 rats	 and	 mice(126,135).	 Prolonged	 IL-1β	 production	 by	
Müller	 cells	 has	 been	 shown	 to	 affect	 endothelial	 cell	 viability	 in	 a	 paracrine	
fashion(295).	 Endothelial	 cells	 are	 extremely	 susceptible	 to	 IL-1β	 and	 rapidly	
progress	 to	 cell	 death	 in	 response	 to	 this	 pro-inflammatory	 cytokine(295).	
Endothelial	 cell	 death	 is	 detectable	 in	 the	 retinal	 microvasculature	 of	 diabetic	
animals	 and	 isolated	 retinal	 blood	 vessels	 of	 diabetic	 donors	 and	 has	 been	
associated	with	the	formation	of	acellular	capillaries,	a	hallmark	of	retinal	pathology	
in	diabetic	retinopathy(156).	Besides	IL-1β,	Müller	cells	produce	other	well-known	
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pro-inflammatory	 cytokines	 such	 as	 tumor	 necrosis	 factor	 alpha	 (TNFα)	 and	
interleukin-6	 (IL-6)(126,128,135,140,162,299,300).	 Anti-TNFα	 therapy	 has	 been	
proposed	

as	

a	

strategy	

to	

treat	

diabetic	

retinopathy	

in	

diabetic	

animals(111,112,115,189).	 Detrimental	 effects	 of	 IL-6	 have	 been	 associated	 with	
vascular	 dysfunction	 and	 promotion	 of	 angiogenesis(301–303)	 which	 is	 why	 IL-6	
recently	 has	 become	 a	 new	 therapeutical	 target	 of	 interest	 to	 prevent	 diabetesinduced	 vascular	 damage.	 The	 production	 and	 release	 of	 pro-inflammatory	
cytokines	 by	 Müller	 cells	 strongly	 contributes	 to	 the	 chronic	 inflammatory	
environment	detected	in	the	diabetic	retina	that	over	time	promotes	drop-out	of	a	
retinal	cells.	
From	 a	 vascular	 perspective,	 other	 cytokines	 such	 as	 IL-6	 has	 been	 solely	
associated	with	detrimental	effects(301–303).	However,	we	have	previously	shown	
that	 IL-6	 prevents	 hyperglycemia-induced	 Müller	 cell	 dysfunction	 and	 loss	 clearly	
supporting	a	beneficial	and	protective	nature	of	IL-6(128).	This	observation	is	well	
in	line	with	reports	that	in	the	retina	IL-6	is	an	important	cytokine	responsible	for	
maintaining	 proper	 neuronal	 function	 as	 well	 as	 stimulating	 neuroprotective	
effects(128,304–307).	 Treatment	 with	 IL-6	 has	 been	 shown	 to	 protect	 retinal	
ganglion	 cells	 from	 pressure-induced	 cell	 death(304).	 Additionally,	 in	 an	
experimental	model	of	retinal	detachment,	genetic	ablation	or	neutralization	of	IL-6	
led	 to	 a	 significant	 increase	 in	 photoreceptor	 cell	 death.	 However,	 treatment	 with	
exogenous	 IL-6	 resulted	 in	 a	 significant	 increase	 in	 photoreceptor	 density	 in	 the	
outer	nuclear	layer(307).	These	different	effects	of	IL-6	can	potentially	be	attributed	
to	 the	 two	 distinct	 signaling	 pathways	 IL-6	 acts	 through.	 Classical	 IL-6	 signaling	 -	
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thought	 to	 be	 the	 anti-inflammatory	 and	 protective	 pathway	 -	 is	 mediated	 by	 the	
membrane-bound	form	of	the	IL-6	receptor	(IL-6R)	and	the	ubiquitously	expressed	
glycoprotein	130	(gp130).	Only	cells	such	as	Müller	cells	(but	not	endothelial	cells)	
that	express	IL-6R	are	able	to	signal	through	classical	IL-6	signaling.	Conversely,	IL6	trans-signaling,	which	is	mediated	by	binding	of	IL-6	to	the	soluble	form	of	the	IL6	receptor	(sIL-6R)	and	gp130,	is	thought	to	be	the	more	pro-inflammatory	and	proangiogenic	 pathway(128,302,305,308–318).	 In	 diabetic	 patients,	 correlations	
between	 increased	 levels	 of	 IL-6	 and	 the	 development	 of	 complications	 in	 the	 eye	
have	been	made(319–324).	However,	whether	IL-6	levels	are	increased	in	diabetes	
as	 an	 attempt	 to	 protect	 from	 a	 pro-inflammatory	 environment	 or	 whether	 high	
levels	of	IL-6	synergistically	exaggerate	diabetes-induced	inflammation	has	yet	to	be	
determined.	
Whether	 Müller	 cells	 die	 in	 diabetic	 retinopathy	 has	 long	 been	 a	 matter	 of	
debate.	It	is	easy	to	see	that	Müller	cells	are	“sturdy”	cells	taking	into	account	how	
well	 equipped	 these	 cells	 are	 to	 produce	 fair	 amounts	 of	 protective	 factors	 that	
shield	 them	 at	 least	 in	 the	 beginning	 from	 a	 chronic	 diabetic	 insult	 as	 discussed	
above.	However,	newer	studies	indicate	that	over	time	Müller	cells	actually	do	begin	
to	die	the	longer	diabetic	retinopathy	progresses.	Frequency	of	Müller	cell	death	in	
the	 diabetic	 retina	 rapidly	 accelerates	 when	 protective	 growth	 factors	 are	
blunted(293).	
Better	understanding	of	types	of	cell	deaths	has	furthered	studies	to	look	for	
mechanisms	 other	 than	 apoptosis	 by	 which	 Müller	 cells	 can	 die	 in	 a	 diabetic	
environment.	 In	 this	 dissertation	 (Chapter	 3)	 we	 have	 identified	 one	 particular	
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mechanism	 of	 cell	 death	 that	 stands	 out	 and	 can	 explain	 histological	 features	
described	 for	 Müller	 cells	 in	 the	 diabetic	 retina.	 Pyroptosis	 is	 an	 inherently	
inflammatory	 mediated	 mechanism	 of	 cell	 death,	 defined	 as	 being	 caspase-1dependent(73,142,220).	 Müller	 cells	 show	 increased	 caspase-1	 activity	 and	 IL-1β	
production	 following	 exposure	 to	 hyperglycemic	 conditions	 and	 cells	 die	 as	 a	
consequence(116,232).	 Although	 it	 is	 now	 very	 well	 established	 that	 initiation	 of	
pyroptosis	 is	 caspase-1	 and	 IL-1β	 driven,	 the	 execution	 phase	 of	 pyroptosis	 is	 not	
yet	 completely	 understood.	 It	 has	 been	 shown	 that	 pyroptosis	 shares	 traits	 with	
both	 apoptosis	 and	 necrosis	 in	 the	 execution	 phase(141,227).	 Since	 execution	 of	
pyropototic	 cell	 death	 lacks	 specific	 marker,	 identifying	 retinal	 cells	 dying	 by	
pyroptosis	in	vivo	is	a	difficult	task.	Markers	such	as	TUNEL	staining	used	to	detect	
apoptotic	 cell	 death	 may	 not	 adequately	 detect	 pyroptosis.	 Therefore,	 we	 have	
performed	a	study	actually	counting	Müller	cells	in	the	healthy	and	diabetic	retina	
and	 determined	 roughly	 15%	 cell	 death	 at	 7	 months	 of	 diabetes(140).	 Even	 more	
important,	 inhibition	 of	 the	 caspase-1/IL-1β	 pathway	 inhibited	 diabetes-induced	
Müller	cell	death	in	vivo	as	we	had	previously	shown	in	vitro(126,128,140).	Several	
other	 studies	 are	 in	 line	 with	 our	 observation	 that	 Müller	 cells	 die	 in	 a	
hyperglycemic	 environment.	 The	 first	 study	 to	 describe	 dying	 Müller	 cells	 in	
diabetic	 retinopathy	 was	 done	 using	 EM	 analysis(138).	 Dying	 Müller	 cells	 are	
described	as	being	hypertrophic	consistent	with	the	notion	that	during	pyroptosis,	
cells	 swell	 rather	 than	 shrink	 as	 observed	 in	 apoptotic	 cell	 death(53).	 To	 collect	
more	 evidence	 for	 Müller	 cells	 death	 in	 the	 diabetic	 retina	 we	 looked	 at	 earlier	
markers	 of	 cell	 death	 and	 we	 have	 identified	 that	 GAPDH	 (glyceraldehyde-3112	

	
phosphate	dehydrogenase)	accumulates	in	the	nucleus	of	Müller	cells	in	the	retinas	
of	 diabetic	 rats(163).	 Nuclear	 accumulation	 of	 GAPDH	 has	 been	 closely	 associated	
with	 cell	 death	 induction(233–235,325).	 Consistent	 with	 our	 finding	 that	 Müller	
cells	 die	 by	 pyroptotic	 cell	 death,	 hyperglycemia-induced	 nuclear	 accumulation	 of	
GAPDH	 depends	 on	 the	 activation	 of	 the	 caspase-1/IL-1β	 pathway(54,139).	 The	
consequences	of	dying	Müller	cells	are	multi	faceted.	On	the	bad	side	–	Müller	cell	
death	 will	 promote	 loss	 of	 retinal	 blood	 barrier	 integrity,	 increased	 vascular	
permeability,	and	loss	of	neuroprotection	affecting	both	neurons	and	vascular	cells.	
Loss	of	Müller	cells	in	diabetes	has	also	been	associated	with	aneurysm	formation,	a	
clinical	 characteristic	 of	 diabetic	 retinopathy(138).	 However,	 one	 can	 also	 argue	
that	 on	 the	 good	 side	 –	 removal	 of	 activated	 and	 pro-inflammatory	 Müller	 cells	
might	 be	 a	 “shut	 off”	 mechanism	 to	 deal	 with	 an	 increasing	 inflammatory	
environment	in	the	diabetic	retina.	A	lot	more	studies	are	needed	to	determine	the	
full	pathway	of	Müller	cells	death	and	to	identify	whether	all	Müller	cells	are	equally	
affected	by	hyperglycemia.	
	

	

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Figure	26:	Timeline	of	Diabetic	Retinopathy	In	Mice	and	Rats.	
Timeline illustrating course caspase-1 activation, cytokine secretion, Müller cell
death initiation and execution in comparison to other prominent events
associated with diabetic retinopathy in retinas of STZ diabetic mice and rats.
	
	

	
	

	

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6.4	Conclusion	
Müller	 cells	 are	 a	 major	 component	 of	 a	 healthy	 retinal	 environment.	 Once	
chronic	 hyperglycemia	 disturbs	 their	 environment,	 Müller	 cells	 become	
dysfunctional	 and	 start	 activating	 pathways	 to	 counter-regulate	 and	 “repair”	 the	
environment.	
In	 order	 to	 do	 so,	 Müller	 cells	 release	 a	 large	 variety	 of	 growth	 factors	 and	
cytokines	in	a	diabetic	environment.	Most	of	the	research	to	date	has	focused	on	the	
detrimental	effects	the	release	of	these	growth	factors	and	cytokines	causes	to	the	
retina.	When	taking	a	closer	look	most	of	these	effects	are	associated	with	vascular	
dysfunction	and	angiogenesis.	On	the	other	hand,	it	seems	that	production	of	these	
growth	 factors	 and	 cytokines	 by	 Müller	 cells	 are	 primarily	 intended	 to	 protect	
Müller	 cells	 and	 consequently	 retinal	 neurons	 from	 diabetic	 insult	 and	 might	 only	
secondarily	 turn	 into	 the	 damaging	 components	 observed	 in	 diabetic	 retinopathy.	
Very	 few	 studies	 have	 started	 to	 consider	 the	 protective	 nature	 of	 Müller	 cell	
derived	 growth	 factors	 and	 cytokines	 in	 regards	 to	 the	 integrity	 of	 glia	 cells	 and	
neurons.	 A	 lot	 more	 studies	 are	 needed	 to	 understand	 the	 nature	 of	 Müller	 cells	
derived	 growth	 factors	 and	 cytokines.	 For	 a	 successful	 development	 of	 a	 new	
therapy	targeting	these	factors	both	detrimental	as	well	as	beneficial	effects	need	to	
be	considered.		
Understanding	 Müller	 cell	 functions	 within	 the	 retina	 and	 restoring	 such	
function	 in	 diabetic	 retinopathy	 should	 become	 a	 cornerstone	 for	 developing	
effective	therapies	to	treat	diabetic	retinopathy.	Some	approaches	have	been	tested	
to	 increase	 Müller	 cell	 function	 by	 stimulating	 the	 beta-adrenergic	
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pathway(326,327).	 Whether	 these	 studies	 materialize	 into	 effective	 therapy	
strategies	has	to	be	seen	in	the	future.	
	
	

	

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Chapter	7.	Internship	Experience	at	Hoffmann-La	Roche	
	
I	spent	the	final	year	of	the	graduate	program	working	at	Hoffmann	La-Roche	
at	 their	 campus	 in	 Basel,	 Switzerland.	 	 I	 was	 accepted	 for	 a	 one	 year	 internship	 to	
the	 Roche	 internship	 for	 Scientific	 Exchange	 (RiSE)	 program	 and	 my	 appointment	
was	 in	 the	 ophthalmology	 department	 of	 pharmaceutical	 Research	 and	 Early	
Development	(pRED).		The	RiSE	program	is	designed	to	give	graduate	students	an	
opportunity	 to	 work	 at	 a	 pharmaceutical	 company	 with	 the	 goal	 of	 learning	 new	
techniques	and	learn	what	it	is	like	to	work	in	industry.			
	

The	 ophthalmology	 department	 at	 Hoffmann	 La-Roche	 focuses	 on	

translational	research	with	a	goal	of	identifying	new	therapeutic	targets	in	a	number	
of	 vision	 related	 diseases.	 My	 experience	 in	 the	 field	 of	 diabetic	 retinopathy	 and	
inflammation	was	extremely	useful	and	allowed	me	to	be	a	valuable	member	to	the	
research	 team.	 	 I	 was	 given	 my	 own	 research	 project	 and	 it	 allowed	 me	 to	 lead	 a	
project	and	design	the	experiments	necessary	to	complete	the	project.		Additionally,	
I	 worked	 with	 many	 of	 the	 other	 team	 members	 and	 it	 allowed	 me	 to	 collaborate	
with	 a	 team	 on	 portfolio	 related	 projects.	 	 During	 this	 work	 I	 learned	 many	 new	
techniques	 and	 how	 to	 incorporate	 them	 into	 research	 projects.	 	 One	 of	 the	 new	
techniques	that	I	have	used	in	the	internship	is	RNA	sequencing	and	how	to	analyze	
the	data	obtained.		This	technique	has	been	useful	in	helping	to	identify	new	targets	
for	 potential	 therapies	 in	 a	 number	 of	 ocular	 diseases.	 In	 addition	 to	 RNA	
sequencing,	 I	 have	 also	 become	 more	 familiar	 with	 confocal	 and	 two	 photon	
microscopy	 and	 luminex	 magnetic	 bead	 cytokine	 assays.	 	 I	 have	 learned	 to	 use	 a	
117	

	
number	 of	 new	 devices	 including	 the	 Roche	 MagnaPure	 for	 RNA	 extraction	 and	
purification,	Heidelberg	for	Optical	Coherence	Tomography	(OCT),	and	the	Phoenix	
Micron	 IV	 for	 fundus	 imaging	 and	 laser	 photocoagulation.	 	 Furthermore,	 I	 have	
increased	 my	 skills	 in	 vitro	 by	 continuing	 to	 work	 with	 the	 STZ	 diabetic	 mouse	
model	 and	 have	 begun	 to	 work	 on	 the	 Choroidal	 Neovascularization	 (CNV)	 mouse	
model.		
	

Collectively,	the	experience	of	the	RiSE	internship	was	extremely	valuable	and	

further	confirmed	that	I	would	like	to	pursue	a	career	at	a	pharmaceutical	company.		
	
	

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REFERENCES		

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