REGULATION OF ALVEOLAR EPITHELIAL CELL SURVIVAL BY
ANGIOTENSIN1-7 AND MAS
By
Xu Gao

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Physiology
2011

ABSTRACT
REGULATION OF ALVEOLAR EPITHELIAL CELL SURVIVAL BY ANGIOTENSIN II
AND MAS
By
Xu Gao
Apoptosis of alveolar epithelial cells (AECs) is believed to be a critical event in the
pathogenesis of lung fibrosis. It was previously shown that apoptosis of AECs requires
autocrine generation of angiotensin (ANG) II. ANG II can also be degraded to ANG1-7 by
angiotensin converting enzyme-2 (ACE-2). ACE2 was recently found to be protective but
significantly downregulated in experimental lung fibrosis and patients with IPF. In other
organ systems, ANG1-7 has been found to inhibit the actions of ANGII through the ANG17 receptor mas. Therefore, it was hypothesized that ANG1-7 in the lungs might antagonize
the actions of ANG II in the regulation of AEC apoptosis. To test this theory, the AEC cell
line MLE-12 and primary cultures of human AECs were stimulated by the profibrotic
apoptosis inducers ANG II or bleomycin (BLEO). Caspase-3 activation and nuclear
fragmentation were used as markers of apoptosis and were measured along with JNK
phosphorylation. Exposure to ANG II or BLEO induced caspase-3 activation, nuclear
fragmentation, and JNK phosphorylation in cultured AECs. Pretreatment with ANG1-7, at
a concentration of 0.1µM, prevented JNK phosphorylation and apoptosis. In addition,
pretreatment with A779, a specific blocker of the ANG 1-7 receptor mas, successfully
prevented ANG1-7 induced blockade of JNK phosphorylation and nuclear fragmentation.
These data indicate that ANG1-7 prevents AEC apoptosis, and its actions are mediated
through the ANG1-7 mas receptor.

Copyright by
XU GAO
2010

To Mom and Dad for their unconditional care, love, and support.

iv	
  

ACKNOWLEDGEMENTS
First and foremost, I would like to thank my mentor, Dr. Bruce D. Uhal, who was a
source of guidance and support through my undergraduate education. I would not have been able
to do this without his encouragement and effort. I also wish to thank and acknowledge the
individuals involved in my Thesis Guidance Committee: Dr. Gloria I. Perez and Dr. Arthur J.
Weber, for contributing their expertise and ideas. I would also like to thank Amal Abdul-Hafez,
Trang Dang, and Chris Plasecki for their help and friendship in the laboratory. Finally, I would
like to thank my family and friends for their motivational support through it all.

v	
  

TABLE OF CONTENTS

LIST OF FIGURES ................................................................................................................... viii
LIST OF ABBREVIATIONS ...................................................................................................... x
CHAPTER 1 – GENERAL INTRODUCTION ......................................................................... 1
Idiopathic Pulmonary Fibrosis ........................................................................................... 2
Epidemiology ................................................................................................................ 2
Clinical Features ........................................................................................................... 3
Histological Features .................................................................................................... 4
Genetic Factors ............................................................................................................. 5
Therapeutic Options ...................................................................................................... 6
Pathogenesis of Fibrosis ...................................................................................................... 8
Alveolar Epithelial Cells ............................................................................................... 8
Coagulation and Fibrogenesis ..................................................................................... 10
Profibrotic Cytokines .................................................................................................. 11
Myofibroblasts ............................................................................................................ 12
Basement Membrane Disruption and ECM Remodeling ........................................... 15
Apoptosis in Idiopathic Pulmonary Fibrosis ................................................................... 19
Apoptosis in General................................................................................................... 19
Morphological and Biochemical Changes............................................................ 19
Caspases ............................................................................................................... 20
Mechanisms of Apoptosis ...................................................................................... 21
The Extrinsic Pathway .................................................................................... 22
The Intrinsic Pathway ..................................................................................... 23
Caspase Substrates .......................................................................................... 24
Other Pathways ............................................................................................... 25
Alveolar Epithelial Cell Apoptosis in IPF .................................................................. 27
Evidence of Apoptosis ........................................................................................... 27
Induction of Pulmonary Fibrosis .......................................................................... 28
Mechanism of Alveolar Epithelial Apoptosis ........................................................ 28
p21 and p21 Upregulation ............................................................................... 28
Fas-FasL Pathway Activation ......................................................................... 29
TGF-β Induction of Apoptosis ........................................................................ 30
Oxidative Stress .............................................................................................. 30
Alveolar Epithelial-Fibroblast Crosstalk ........................................................ 31
The Renin Angiotensin System ......................................................................................... 33
General Ras ................................................................................................................. 33
Local Ras .................................................................................................................... 34
Bleomycin-Induced Experimental Pulmonary Fibrosis Model ..................................... 36
Pulmonary Angiotensin System in IPF ............................................................................ 38
General Ras ................................................................................................................. 36
Local Ras .................................................................................................................... 37

vi	
  

CHAPTER 2 – ANGIOTENSIN 1-7 REGULATES ALVEOLAR EPITHELIAL
CELL SURVIVAL THROUGH THE RECEPTOR MAS ..................................................... 42
Abstract ............................................................................................................................... 43
Introduction ........................................................................................................................ 44
Methods ............................................................................................................................... 54
Results ................................................................................................................................. 56
Discussion............................................................................................................................ 66
REFERENCES ............................................................................................................................ 69

vii	
  

LIST OF FIGURES
Figure 1.1. Pathogenesis of idiopathic pulmonary fibrosis [Figure 7.1 modified from
(Selman et al., 2001) ................................................................................................................... 17
Figure 1.2. Induction of apoptosis [Figure 11.9C.1 modified from (Siegel and Lenardo,
2002)] .......................................................................................................................................... 26
Figure 1.3. The Renin Angiotensin System (RAS) ................................................................. 35
Figure 2.1. Quantitation of ISEL-positive nuclei in alveolar (A) and airway (B) epithelial
cells after Bleo, CAPTO, and ZVAD-fmk administration ...................................................... 47
Figure 2.2. Quantitation of collagen by PR staining of alveolar (A) and peribronchial (B)
lung parenchyma.) ....................................................................................................................... 48
Figure 2.3. Dose-dependent induction of nuclear fragmentation by BLEO in primary rat
AECs and blockade by inhibitors of caspases, endonucleases, ANG-converting enzyme
(ACE), and ANG-receptor interaction ...................................................................................... 49
Figure 2.4. Blockade of Bleo-induced apoptosis in primary AECs by selective caspase or
ANG receptor blockers ............................................................................................................... 50
Figure 2.5. ANGII induces activation of caspase-9 ................................................................ 51
Figure 2.6. Pathways for the formation of ANG 1-7 .............................................................. 52
Figure 2.7. Summary of Earlier Published Data ...................................................................... 53
Figure 2.8. ANG1-7 inhibits ANG II induced JNK phosphorylation through the mas
receptor ......................................................................................................................................... 59
Figure 2.9. ANG1-7 inhibits BLEO induced JNK phosphorylation through the mas
receptor ......................................................................................................................................... 60
Figure 2.10. ANG1-7 inhibits BLEO induced caspase-3 activation of MLE-12 cells ......... 61
Figure 2.11. ANG1-7 inhibits BLEO induced caspase-9 activation of primary AECs
through the mas receptor ............................................................................................................ 62
Figure 2.12. ANG1-7 inhibits bleomycin-induced nuclear fragmentation through the mas
receptor in MLE-12 cells ............................................................................................................ 63
Figure 2.13. ANG1-7 inhibits bleomycin-induced nuclear fragmentation through the mas
receptor in primary human AECs .............................................................................................. 64

viii	
  

Figure 2.14. Summary ................................................................................................................ 65

ix	
  

LIST OF ABBREVIATIONS

ACE

Angiotensin-Converting Enzyme

ACE-2

Angiotensin Converting Enzyme-2

AEC

alveolar epithelial cell

AGT

Angiotensinogen

Ang 1-7

Angiotensin 1-7

Ang II

Angiotensin II

Ang III

Angiotensin III

Ang IV

Angiotensin IV

AT1

Type 1 Angiotensin receptor

AT2

Type 2 Angiotensin receptor

ATI

Type I alveolar epithelial cell

ATII

Type II alveolar epithelial cell

ECM

extracellular matrix

EMT

epithelial-mesenchymal transition

HIF-1

Hypoxia Inducible Factor -1

IL

Interleukin

ILD

Interstitial Lung Disease

IPF

Idiopathic Pulmonary Fibrosis

JNK

c-Jun N-terminal kinase

MAPK

mitogen activated protein kinase

NFkB

nuclear factor-kappa B

x	
  

PI3K

Phosphoinositide 3-kinase

RAS

Renin Angiotensin System

ROS

Reactive Oxygen Species

RT-PCR

Reverse Transcriptase-Polymerase Chain Reaction

siRNA

Small interfering RNA

SNP

Single Nucleotide Polymorphism

SP-C

surfactant protein C

TGF-b1

Transforming Growth Factor-beta 1

TNF-a

tumor necrosis factor-alpha

xi	
  

Chapter 1

GENERAL INTRODUCTON

1	
  

IDIOPATHIC PULMONARY FIBROSIS
Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive fibrotic lung
disease resulting from injury to the lung and an ensuing fibrotic response that leads to the
thickening of the alveolar walls and the obliteration of the alveolar space (Fonseca et al.,
2000). The American thoracic Society and the European Respiratory Society recently
issued a consensus statement that defined IPF as a distinctive type of chronic fibrosing
interstitial pneumonia of unknown etiology limited to the lung and associated with the
histology of unusual interstitial pneumonia (American Thoracic Society, European
Respiratory Society, 2002). IPF is classified as a specific presentation of idiopathic
interstitial pneumonia, which is in turn a type of interstitial lung disease (American
Thoracic Society, European Respiratory Society, 2002).

Epidemiology
IPF is the most common of the idiopathic interstitial pneumonias and is associated
with the worst prognosis (American Thoracic Society, 2002). Deaths from IPF are
estimated to be nearly 14 times greater than those from asbestos (Wells and Mannino,
1996). Differences in disease definition, classifications, diagnostic approach, and
epidemiological design of the studies greatly influence the findings. In the Unite States, a
recent study estimated prevalence to range from 14.0 to 42.7 cases per 100,000 population,
depending on the criteria used for diagnosis (Raghu et al., 2006). This is higher than the
previously reported prevalence of 3 to 6 per 100,000 persons (Cherniack et al., 1991). The
median survival rate of IPF is estimated to be from 3 to 5 years (Selman et al., 2001).

2	
  

Respiratory failure is the most frequent cause of death, and has been reported to account
for over 80% of all fatalities (Martinet et al., 2005).
Two thirds of IPF patients are older than 60 years of age with a mean age of 66 at
diagnosis (Johnston et al., 1997). The incidence, prevalence, and death rate of IPF increase
with age (Coultas et al., 1994). IPF is reported to be more common in males than in females
(Hubbard 1996). Some studies also suggest that the risk of developing IPF increases with
environmental or occupational exposure to dusts, organic solvents, or urban pollution
(Iwai, 1994). Other risk factors associated with pulmonary fibrosis include smoking,
gastro-esophageal reflux disease, commonly prescribed drugs, diabetes mellitus, infectious
agents, and genetic factors (Zisman et al., 2005).

Clinical Features
The clinical features for IPF are not specific and can also occur in various other
pulmonary disorders. The symptoms usually vary with the extent of lung damage, the rate
of disease progression, and the development of complications, such as lung infections or
cor pulmonale. IPF usually presents with inspiratory crackles, dyspnoea on exertion, and
dry, non-productive cough. Common symptoms also include weight loss and fatigue. In
more than 80% of patients, bi-basilar, end-expiratory rales are found upon physical
examination (American Thoracic Society, 2000). In addition, up to half of all patients
develop digital clubbing where ends of the fingers become thick or club-shaped (Johnston
et al., 1997). At late stages of the disease, patients also experience shortness of breath at
rest as well as cyanosis of the lips and fingers. Pulmonary hypertension may also present
late in the course (Panos et al., 1990).

3	
  

There are currently no laboratory tests specific for the diagnosis of IPF. Laboratory
evaluations are done to rule out alternative causes of interstitial lung disease such as
sarcoidosis or connective tissue disease-pulmonary fibrosis. Pulmonary function tests show
restrictive impairment, reduced diffusing capacity for carbon monoxide, and arterial
hypoxemia exaggerated or elicited by exercise (Selman et al., 2001). Greater than 90% of
patients have abnormal chest radiographs that show diffuse bilateral interstitial or
reticulonodular infiltrates in the basilar and subpleural regions of the lung (Kazerooni et
al., 1997; Wells et al., 1993). High-resolution computerized tomography scanning shows
variable

but

limited

ground-glass

opacity

subpleural

honeycombing.

Extensive

honeycombing, septal thickening, and a lack of ground glass opacities are usually
indications of a poor prognosis.
The diagnosis of idiopathic pulmonary fibrosis must include: 1) a compatible
clinical history; 2) the exclusion of other known causes of interstitial lung disease (such as
drug injuries, environmental exposures, or collagen vascular disease); 3) a surgical lung
biopsy showing usual interstitial pneumonia histologic pattern (American Thoracic Society,
2000).

Histological Features
The histological features of IPF are described as usually interstitial pneumonia
(UIP), which can be seen in other diseases as well. The histological hallmark of usual
interstitial pneumonia is heterogeneous appearance of normal-appearing lung alternating
with areas of peripheral fibrosis, interstitial inflammation, and honeycomb changes, on low
magnification (American Thoracic Society, 2000). The inflammatory component is

4	
  

typically light with mostly lymphocytes and plasma cells. At higher-power magnification,
the border between the fibrotic and normal lung, called the fibroblastic foci, can be seen
with accumulations of dense, relatively acellular, collagen bundles. Fibroblastic foci are the
regions of fibroblastic proliferation and represent the primary site of ongoing injury.
Fibroblastic foci are rich in extracellular matrix and mesenchymal cells with cell
phenotypes ranging from proliferating fibroblasts to fully differentiated smooth muscle
cells. The most abundant cell type is the myofibroblast, which contribute to active
contraction, distorted architecture and excess collagen deposition (Kuhn & McDonald
1991). Although not pathognomonic, the number of fibroblastic foci may be an important
prognostic factor and correlate with a worse prognosis (Nicholson et al., 2002, King et al.,
2001).

Genetic factors
Evidence suggests that genetic factors may play an important role in the
pathogenesis of IPF. It has been estimated that up to 4% of IPF is familial and appears to
be inherited as an autosomal dominant trait (Lawson & Lloyd, 2006). The familial form of
IPF has been reported in monozygotic twins raised in different environments, in genetically
related members of several families, in consecutive generations in the same families, and in
family members separated at an early age (Steele et al., 2005). Genetic studies found two
mutations of the SP-C gene to be associated with familial lung fibrosis (Thomas et al.,
2002). These mutations lead to misfolding of the protein resulting in abnormal surfactant
production and injury in type II alveolar epithelial cells (Thomas et al., 2002). Angiotensin
II is a growth factor that plays a key role in the physiopathology of IPF. A nucleotide

5	
  

substitution of an adenine instead of a guanine (G-6A) in the proximal promoter region of
the angiotensin II precursor, angiotensinogen, has been associated with an increased gene
transcription rate. A recent case-control study found that the distribution of G-6A
genotypes and alleles did not significantly differ between cases and controls (Molina et al.,
2008). The G-6A polymorphism of the angiotensinogen gene was found to be associated
with idiopathic pulmonary fibrosis progression but not with disease predisposition (Molina
et al., 2008). The G-6A polymorphism of the angiotensinogen gene could have a predictive
significance in idiopathic pulmonary fibrosis patients.

Therapeutic options
The two main types of therapeutic agents used to treat IPF are the anti-inflammatory
medications and the anti-fibrotic agents. Most therapies are, however, largely ineffective
with limited evidence to suggest that any one treatment improves survival or disease
progression. The conventional management of IPF with anti-inflammatory medications is
based on the concept that pulmonary fibrosis is an inflammatory disorder and the
suppression of inflammation prevents progression to fibrosis. These therapies continue to
be used despite the lack of evidence of inflammation in the pathogenesis of IPF (Raghu et
al., 2004). Clinical studies indicate that response to steroids is poor in IPF and the use of
other immunosuppressive or aggressive cytotoxic agents only offer a marginal benefit at
best (King et al., 2001). Recently, more anti-fibrotic agents are being used in the treatment
of IPF based on the concept that the disease is a fibrotic condition with a lack of significant
inflammatory component (Selman et al., 2004). A meta-analysis of the combined results of
multiple studies showed that interferon-γ1b (IFN-γ1b) therapy may be efficacious in

6	
  

improving survival and is associated with reduced mortality in patients with IPF (Bajwa et
al., 2005). However, this benefit of IFN-γ1b therapy was not seen in patients with more
severe disease (Martinez et al., 2005).
Currently, the only option for patients with end stage pulmonary fibrosis and those
who fail to respond to medical treatment is lung transplantation. Patients are referred for
transplantation when progressive symptoms have a vital capacity less than 60-70% and a
corrected diffusion capacity below 50-60% of predicted (The American Society for
transplant physicians, 1998). The five-year survival rate for patients following transplant is
estimate to be 50-60% (American Thoracic Society, 2000). A recent study estimated that
lung transplantation reduces the risk of death by 75% (Thabut et al., 2003).
To develop new therapies, recent studies have focused on targeting angiotensin II
(ANG II) and its role in the pathogenesis of pulmonary fibrosis. These studies showed that
inhibition of the RAS using angiotensin converting enzyme inhibitors (ACEi’s),
angiotensin receptor blockers or receptor deletion was able to prevent experimental lung
fibrosis (Wang et al., 2000b, Marshall et al., 2004, Uhal et al., 2007a, Molteni et al., 2000,
Molina-Molina et al., 2006) and improve lung function in IPF patients (Woo et al., 2003).

7	
  

PATHOGENESIS OF FIBROGENESIS
Until recently, pulmonary fibrosis has been considered to be an inflammatory
disorder where lower respiratory tract inflammation leads to derangements of the alveoli
resulting in scaring of the lung parenchyma (Gallin et al., 1992). Inflammation results in
the loss of functional alveolar-capillary unit, the accumulation of collagen, and the
formation of the honeycomb lung. Recent studies, along with the limited success of antiinflammatory treatments, suggest the existence of alternative hypothesis regarding the
pathogenesis of IPF. The current hypothesis is that IPF results from alveolar epithelial
injury and abnormal wound repair (Selman et al., 2001). This theory is supported by
evidence from human lung biopsies where nascent fibrotic foci were colocalized with
unrepaired or abnormal epithelia (Uhal et al., 1998).

Alveolar Epithelial Cells
The alveolus is the basic functional unit of the lung where gas exchange occurs
between the air and the capillaries. The site of gas exchange is composed of the capillary
endothelium and alveolar epithelium separated by a thin interstitium composed of
connective tissue, ECM and a few fibroblasts (Fonseca et al., 2000). The alveolar
epithelium is composed of two types of alveolar epithelial cells (AECs) that are
morphologically and functionally distinct. The first is the thin and elongated type I (AT I)
cells that are terminally differentiated and cover 95% of the alveolar surface to facilitate
gas exchange. AT I cells are metabolically active and express cell surface receptors for a
variety of substances, including extracellular matrix (ECM) proteins, growth factors, and
cytokines. The second are type II (AT II) alveolar epithelial that reside in alveolar corners

8	
  

and are cuboidal in shape with rounded nuclei. AT II cells secrete surfactant, facilitate
trans-epithelial movement of water, function as antigen presenting cells and serve as the
progenitor of both types of epithelial cells (Uhal, 1997). AT II cells are important in the
maintenance and repair of the alveolar epithelium by replacing lost cells and restoring
normal tissue architecture and lung function. The regeneration of a continuous epithelium
is vital in maintaining barrier function and in limiting airway hyper-activity. This process
is most critical in response to lung injury and other insults. Epithelial injury and blunted
epithelial repair are sufficient to promote pulmonary fibrotic processes (Adamson et al.,
1988).
One important step in the healing process is the rapid re-epithelialization of the
denuded area through epithelial cell migration, proliferation and differentiation. The
inhibition of alveolar epithelial repair in animal models increases the severity of
subsequent fibrosis (Haschek and Witschi, 1979), suggesting that the efficient repopulation
of the denuded alveolar basement membrane acts to suppress subsequent fibroblast
proliferation and extracellular matrix accumulation. IPF alveolar epithelium show
significant loss of AT I cells, hyperplasia of AT II cells and altered expression of adhesion
molecules and MHC antigens (Kasper, 1996). AT II cells in IPF have decreased capacity to
restore damaged AT I cells, resulting in epithelial cuboidalization and the presence of
transitional reactive phenotypes (Kasper, 1996), abnormalities in pulmonary surfactant
(McCormack et al., 1991), and alveolar collapse (Burkhardt, 1989).
The epithelial cells overlying fibroblastic foci are found to be hyper- and dysplastic,
with abnormal morphology and gene expression patterns (Kasper et al., 1996). These cells
secrete a variety of profibrotic cytokines and participate in a bidirectional communication

9	
  

network

with

neighboring

fibroblasts

where

each

cell

type

influences

the

proliferation/survival of the other. AEC apoptosis was also detected adjacent to
myofibroblasts-containing fibroblastic foci, the presumed primary sites of epithelial injury
in IPF. Ongoing apoptosis is believed to be a key component in the progression of IPF and
appears to be essential for the development of transforming growth factor-beta (TGF-β)
induced lung fibrosis (Lee et al., 2004; Uhal et al., 1998).

Coagulation and fibrinolysis
The disruption of blood vessels and extravasation of blood into the wound after
tissue injury reestablishes hemostasis while also providing a provisional extracellular
matrix in which the repair process can begin. AT II cells and macrophages convert the
leaked in fibrinogen into fibrin, which then becomes scaffold for fibroblast migration
(Gross et al., 1991). The efficient and orderly resolution of the fibrin matrix will
reconstitute a normal alveolar space. Continued deposition of fibroblasts and dysregulated
coagulation, however, will lead to destruction of the alveolar space and contribute to
pulmonary fibrosis. Whether fibrin is deposited or resorbed in the alveolar space is critical
in the development of fibrosis and is determined by the balance of procoagulatory,
fibrinolytic, and antifibrinolytic activities in the lungs. Fibrin removal is slowed in IPF
lungs as evidenced by increased procoagulant and anti-fibrinolytic activities found in
bronchoalveolar lavage fluid from IPF patients (Gunter et al., 2000).
During wound repair, the fibrinolytic process is required to cleave a path for
epithelial cell migration. The plasminogen activation system mediates this process and is
crucial in tissue remodeling. Plasminogen, when activated to plasmin by tissue-type

10	
  

plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA), is the primary
fibrinolytic enzyme responsible for degrading fibrin clots and promoting wound
reepithelialization. The overexpression of uPA has been shown to decrease fibrosis in a
murine model, indicating a protective role of uPA in the development of pulmonary
fibrosis (Sisson et al., 2002). Plasminogen activator inhibitors (PAIs) negatively regulate
plasminogen. Animal models have shown that the overexpression of PAIs inhibits plasmin
activity and promotes the formation of fibrosis while the lack of PAIs increase plasmin
activity and prevents fibrosis (Eitzman et al., 1996). Alveolar epithelial cells synthesize
uPA and its receptor uPAR as well as PAI-1 (Hasegawa et al., 1997; Simon et al., 1992).
PAI-1 is strongly induced by TGF-β and may play a role in TGF-β induced fibrosis.
In normal repair, epithelial cells are able to dissolve the fibrin barrier and migrate
throughout the denuded wound surface. The proximity of uPA and its receptor greatly
increases the rate of plasminogen activation on the alveolar epithelium and the clearance of
fibrin from the alveolar spaces (Hattori et al., 1989). Alveolar epithelial cells in the IPF
lung, however, seem to play a role in increasing procoagulant and antifibrinolytic
activities. BAL fluid from IPF patients was found to contain significantly more PAIs but
the same amount of uPA as compared to normal individuals (Kotani et al., 1995). This
suggests that the IPF alveolar microenvironment prefers a pro-coagulant and antifibrinolytic state that promotes ECM accumulation and inhibits alveolar reepithelialization.

Profibrotic Cytokines
Alveolar epithelial cells in IPF also express several cytokines and growth factors
that promote fibroblast migration and proliferation as well as extracellular matrix

11	
  

accumulation. Damaged AECs produce platelet-derived growth factor (PDGF) (Antoniades
et al., 1990), tumor necrosis factor alpha (TNF-α) (Kapanci et al., 1995), endothelin-1
(Giaid et al., 1993), and are the primary producers of TGF-β (Khalil et al., 1996). TGF-β
has profound effects on epithelial cells and fibroblasts and is a central regulator of
pulmonary fibrosis. TGF-β promotes epithelial cell apoptosis (Hagimoto et al., 2002),
epithelial-to-mesenchymal transition (EMT) (Kim et al., 2006), epithelial cell migration
(Yu et al.,

2008), collagen synthesis, fibroblast proliferation and transformation into

myofibroblasts (Scotton and Chambers, 2007). Studies with animal models found that
overexpression of TGF-β leads to extensive and progressive fibrosis with limited
inflammation (Sime et al., 1997). PDGF released by epithelial cells is a potent mitogen and
chemoattractant for fibroblasts. PDGF mRNA and protein were found to be upregulated in
the epithelial cells of IPF patients (Antoniades et al., 1990). Hyperplastic AT II cells in
pulmonary fibrosis synthesize TNF-α, which promotes DNA synthesis and proliferation of
fibroblasts (Battegay et al., 1995). Endothelin-1 also plays a role in stimulating fibroblast
proliferation and transdifferentiation into myofibroblasts (Shahar et al., 1999). Together,
these cytokines released by AECs after injury promote proliferation, migration, and matrix
deposition by fibroblasts.
Myofibroblasts
Fibroblasts are highly versatile and are able to interconvert between several distinct
but related cell types. This phenotypic plasticity of fibroblasts is crucial to tissue repair
after injury. After lung injury, fibroblasts are responsible for secreting ECM proteins to
provide a tissue scaffold for epithelial cell migration during repair. Additionally, the
dissolution of this scaffold along with apoptosis of fibroblasts and myofibroblasts are

12	
  

critical for restoration of normal tissue structure (Desmouliere et al., 1995; Lorena et al,
2002).
In IPF, AECs lose their ability to provide normal and homeostatic fibroblastsuppressive functions and instead initiate the pathologic process by producing most of the
factors that induce the fibroblast phenotypic changes. In the fibroblastic foci of the injured
lung, fibroblasts continually modify their interactions with the microenvironment. They
first become migratory, then proliferative, and finally become profibrotic and secret
abundant ECM (Kuhn et al., 1989). Activation of fibroblasts by the secreted factors
produce myofibroblasts that express contractile proteins and have ultrastructural features
that are intermediate between fibroblasts and smooth muscle cells (Gabbiani et al., 1971).
The contractile function of myofibroblasts is important in the re-epithelialization process
by bringing wound margins closer together. Myofibroblasts also have high synthetic
capacity for ECM proteins (Ignotz and Massague, 1986), growth factors/cytokine (Finlay et
al., 2000), growth factor receptors (Thannickal et al., 1998), integrins (Heino et a. 1989),
and oxidants (Thannickal et al 1989).
In addition to sending activation signals to mesenchymal cells, AECs themselves
can also undergo phenotypic transition to fully differentiated mesenchymal cells such as
fibroblasts and myofibroblasts (Zavadil et al., 2005). This process is called epithelial to
mesenchymal transition (EMT). Epithelial cells undergoing EMT are able to lose polarity,
disassemble cell adhesion systems, produce cell-motility machinery, and move from one
location to another (Zavadil et al., 2005). EMT plays a pivotal role in cellular
transdifferentiation during embryonic morphogenesis and tumor progression. In the adult,
injury to epithelial cells induces EMT and contributes to fibrosis in a number of organs

13	
  

(Iwano et al., 2002; Saika et al., 2004).

EMT has been investigated as a mechanism

underlying fibrosis in renal and lens epithelium. Injured renal tubular epithelial cells during
kidney fibrosis migrate through damaged basement membranes into the interstitium and
fully transdifferentiate into fibroblasts and myofibroblasts (Fan et al., 1999; Kalluri et al.,
2003). About 36% of new fibroblasts come from EMT of local epithelium in kidney
fibrosis (Iwano et al., 2002).
The stimulatory input of epidermal growth factor (EGF), hepatocyte growth factor
(HGF), fibroblast growth factors (FGF), and especially TGF-β factors stimulate epithelial
cells to lose polarity, express basement membrane-degrading matrix metalloproteinases
(MMPs), undergo cytoskeletal rearrangements, and express machinery necessary for
motility, leading to migration and complete transition to a mesenchymal phenotype
(Grunert et al., 2003; Kalluri et al., 2003; Savagner et al., 2001). It was recently
demonstrated in vitro that TGF-β1 induces EMT in AECs (Willis et al., 2005).
In addition to the highly synthetic phenotype of fibroblasts in IPF, there is also an
imbalance in the production of MMPs and tissue inhibitors of metalloproteinases (TIMPs)
that control MMP activity (Ramos et al., 2001; Selman et al., 2000). MMPs are a family of
highly regulated zinc-dependent peptidases and have been implicated in the remodeling of
ECM and cell migration (Nagase and Woessner, 1999). TIMP-2 is expressed by
myofibroblasts within the fibrotic foci and may induce mesenchymal cell proliferation.
This in part explains the survival of mesenchymal cells in the fibroblast foci in IPF, in
contrast to the expected cell death as observed in normal wound healing (Desmouliere et
al., 1995). In addition, TIMP-2 has been found to contribute to the irreversible structural
remodeling in IPF (Fukuda et al., 1998).

14	
  

Myofibroblasts have also been shown to secrete angiotensin peptides that may
induce apoptosis of adjacent alveolar epithelial cells (Uhal et al., 1998). The signaling
crosstalk between AECs and fibroblasts is critical in determining whether fibrosis occurs
after lung injury. The various roles and phenotypes of fibroblasts and myofibroblasts make
them key effector cells in the pathogenesis of fibrosis.

Basement Membrane Disruption and ECM Remodeling
The basement membrane plays an important role in maintaining the integrity and
differentiation of the alveolar epithelium. The basement membrane is a complex structure
composed of type IV collagen, laminin, entactin, fibronectin, and heparin sulfatechondroitin proteoglycans (Yurchenco and Schittny, 1990). The disruption of the basement
membrane contributes to the pathogenesis of fibrosis. MMP proteins produced by the
myofibroblasts in IPF have been shown to degrade different components of the basement
membrane, type IV collagen in particular (Segura et al., 2000). In addition, MMP proteins
in IPF have been found to coincide in areas with denuded alveolar basement membrane
(Hayashi et al., 1996; Selman et al 2000). These findings suggest that myofibroblasts play
a role in the degradation of the basement membrane to facilitate their migration into the
alveolar spaces. This disrupted basement membrane may also contribute to the failure of an
orderly repair of damaged AT I cells.
Fibroblasts and myofibroblasts also play a central role in the synthesis, deposition,
and remodeling of the ECM. Increase in the deposition of ECM, including fibrillar
collagens, fibronectin, elastic fibers, and proteoglycans, results in the aberrant tissue
remodeling observed in IPF (Selman et al., 1996). This remodeling eventually leads to

15	
  

extensive structural disorganization in the lung microenvironments and progressive loss of
the alveolar-capillary units.

16	
  

Figure 1.1.
Pathogenesis of idiopathic pulmonary fibrosis [Figure 7.1 modified from (Selman et
al., 2001)]. Alveolar epithelial cell damage (top left) induces an antifibrinolytic
environment in the alveolar spaces, which then enhances wound clot formation. Alveolar
epithelial cells also secret growth factors that induce migration and proliferation of
fibroblasts and differentiation into myofibroblasts (bottom left). Myofibroblasts secret
extracellular matrix proteins, mainly collagens. Neovascularization is induced by
angiogenic factors TIMP-2, FGF-2, and VEGF (bottom right). An imbalance between

17	
  

interstitial collagenases and tissue inhibitors of matrix metalloproteinases (MMPs)
provokes the progressive deposit of extracellular matrix (top right). Myofibroblasts also
produce angiotensin II, which provokes alveolar epithelial cell death, further impairing reepithelialisation. PDGF: platelet-derived growth factor; TGF: transforming growth factor;
TNF: tumour necrosis factor; PAI: plasminogen activator inhibitor; TIMP: tissue inhibitors
of metalloproteinases; VEGF: vascular endothelial growth factor; FGF: fibroblast growth
factor.

18	
  

APOPTOSIS IN IDIOPATHIC PULMONARY FIBROSIS
Apoptosis is programmed cell death that is a critical physiological process in the
development and maintenance of tissue homeostasis. Apoptosis is important in almost all
cell types to provide a balance between cellular proliferation and turnover. In IPF,
apoptosis affects the development of fibrosis through both increased apoptosis of epithelial
cells resulting in inefficient reepithelialization and resistance to apoptosis of fibroblasts
resulting in increased fibrosis (Antoniou et al., 2007).

Apoptosis in General
Morphological and Biochemical Changes
The cell undergoes a highly regulated series of characteristic morphological and
biochemical changes during apoptosis that distinguish it from normal and necrotic cells.
The first morphological changes during apoptosis are cytoplasmic shrinking, loss of cellcell contacts and active membrane blebbing (Allen et al., 1997). In addition, the cytoplasm
and nuclear chromatins condense and fragment into membrane-bound vesicles.
Phosphatidylserine (PS) externalizes at the intact cell surface and promotes signal
recognition by phagocytic cells. Endogenous DNase degrades chromosomal DNA and
cleaves the internucleosomal regions into double stranded DNA fragments. Another marker
of apoptosis is the activation of cytosolic caspases. Caspases are cysteine proteases that
cleave and degrade a specific subset of cellular proteins in cells undergoing apoptosis. The
entire process of apoptosis occurs in a predictable sequence that concludes with the
engulfment of apoptotic bodies by other cells.

19	
  

Caspases
Caspases are a family of aspartate-specific cysteine proteases that are present in all
animal cells as inactive zymogens. In apoptosis, caspases function as both effectors in cell
disassembly and as initiators in initiating this disassembly in response to proapoptotic
signals. Initiator caspases are the first to be activated in a death pathway. They then
activate effector caspases through a caspase cascade. Proteolytic processing at conserved
aspartic acid (Asp) residues triggers caspases to assume active states. During activation, the
zymogen pro-proteins are cleaved at a site known as the linker region to generate the large
and small subunits of the active enzymes. One small and one large subunit make up one
catalytic unit of the active caspase enzyme. Each catalytic unit contains one active site and
a dimer of identical catalytic units make up one active caspase enzyme. The N-terminal
prodomain regulates activation of the proenzyme and is highly variable in sequence and
length. Caspases are unusually highly specific and recognize the Asp residues they cleave
within tetrapeptide motifs (Nicholson and Thornberry, 1997). This strict specificity ensures
that caspases are signaling proteases intended for specific protein cleavage and not for
protein degradation.
Caspase proteins collaborate in proteolytic cascades where caspases activate
themselves and each other. Caspase enzymes cleave their substrates at Asp residues, and
are also activated by proteolytic processing at Asp residues. Activated upstream initiator
caspases then activate downstream effector caspases. The N-terminal domains of proform
initiator caspases are large and function as protein interaction modules that allow them to
interact with various proteins that trigger caspase activation. The proforms of downstream

20	
  

effector caspases contain only short N-terminal prodomains that appear to serve no
function. The effector caspases depend mostly on initiator capases for their proteolytic
processing and activation. Consequently, the sequence of the cleavage sites of zymogen
effector caspases generally matches the preferred tetrapeptide specificities of the initiator
caspases. Additionally, cleavage sites of proteins that were identified as caspase substrates
that undergo processing during apoptosis also match with the preferred specificities of the
effector caspases (Thornberry et al., 1997). Most caspases in mammals and higher
eukaryotes are directly involved in cell death except for a few, such as caspase-1, 4, and 5
that are involved in processing of pro-inflammatory cytokines in humans. The tetrapeptide
specificities of these cytokine-processing proteases do not match the cleavage sites of most
of the proteins known to undergo cleavage during apoptosis, but instead they match with
the sequences of the cleavage sites within pro-cytokines (Salvesen and Dixit, 1997).
Initiator capases can by activated by a variety of mechanisms to initiate the
apoptotic cascade. The underlying biochemical mechanisms of initiator caspase activation
are all remarkably similar and can be explain by the induced proximity model (Salvesen
and Dixit, 1999). The zymogen forms of unprocessed caspases are not completely inactive
but rather possess weak protease activities that, in some cases are at approximately 1% of
the level of the fully active enzymes. Protein interactions can bring the zymogens close
together and allow them to trans-process each other to produce fully active proteases.

Mechanisms of Apoptosis
Apoptosis can be induced by various physiological stimuli such as deformation of
the cell membrane, growth factor withdrawal, inflammatory cytokines, osmotic shock or

21	
  

UV irradiation, as well as a number of molecules and agents. When stimulated, the cells
undergo the activation of pro-apoptotic signaling pathways that transduce a signal to the
interior of the cell and activate the apoptotic machinery.
The mechanism of apoptosis begins with the initiation phase, during which various
apoptotic stimuli lead to activation of caspases, followed by the execution phase where the
activated caspases induce cell death. The caspase cascade can be activated by many
pathways, two of which have been studied in detail. Both of these pathways lead to the
activation of the signaling cascade that results in the cleavage of inactive pro-caspase
molecules into active caspase proteins.

The Extrinsic Pathway/Receptor-mediated Pathway
The first is the extrinsic or death receptor pathway that involves the activation of
death receptors present in the cell membrane, such as Fas and tumor necrosis factor (TNF)
receptors I and II (Figure 1.2). Death receptors are protein interaction modules that consist
of a compact bundle of six α-helices (Huang et al., 1996). Death receptors bind to each
other with specificity directed by differences in their surface residues. Fas receptor can be
activated by either the Fas ligand (FasL) on the surface of cytotoxic lymphocytes, or by a
soluble form of FasL (sFasL) that can be cleaved from cell membranes by MMP-7 and
MMP-3 (Matsuno et al., 2001; Powell et. al 1999). Upon ligation to FasL, the Fas receptor
forms microaggregates at the cell surface. This allows the adaptor molecule, Fas-associated
protein with death domain (FADD), to be recruited to the cytosolic tail of the Fas receptor.
FADD contains a death effector domain (DED) at its amino-terminus and a Death Domain
(DD) at its carboxy-terminus. Caspase-8 contains two DEDs at its amino-terminus. FADD

22	
  

binds Fas receptor and at least three molecules of procaspase-8 through homotypic
interactions. Within this death inducing signaling complex (DISC), procaspase-8 is able to
undergo activation by autocatalytic cleavage via the induced proximity mechanism. During
activation, the N-terminal prodomain of procaspase-8 is cleaved off and the active protease
is released into the cytosol. The active caspase-8 then cleaves and activates downstream
caspases-3, 6, and 7 as well as the pro-death protein Bid. The Bid cleavage fragment then
targets the mitochondria and leads to a mitochondrial amplification of the caspase pathway.

The Intrinsic Pathway/Mitochondrial Pathway
The intrinsic pathway responds to ionizing radiations, chemotherapeutic drugs,
mitochondrial damage, and environmental cues. Death triggers increase mitochondrial
permeability, leading to the release of cytochrome c and the subsequent recruitment and
activation of caspase-9 (Zou et al., 1999). The release of cytochrome c triggers the
formation of the apoptosome complex. The apoptotic protease-activating factor 1 (Apaf1)
is the main component of the apoptosome. Apf-1 is a multidomain protein that consists of
three functional regions: an N-terminal caspase-recruitment domain (CARD); a nucleotidebinding and oligomerization domain (NOD); and a string of WD40 repeats in the Cterminal of the protein. In the absence of an apoptotic signal, Apaf-1 exists in a monomeric
form. Cytochrome c binding allows the compact Apaf-1 molecule to stretch out and
polymerize upon biding ATP (Acehan et al., 2002). Studies of the apoptosome through
electron cryomicroscopy reveal it as a seven-spoked wheel with the caspase-9 recruitment
domain, CARD of Apaf-1, in the central hub (Renatus et al., 2001). Caspase-9 is activated
through dimerization of monomers within the apoptosome. In contrast to the activation of

23	
  

caspase-8, the N-terminal prodomains of caspase-9 is not cleaved and the caspase-9
enzyme must remain bound to Apaf-1 to be fully active (Rodriguez et al., 1999). Activated
caspase-9 is then released from the complex to cleave and activate downstream caspases-3,
6, and 7 (Budihardjo et al., 1999). Members of the B-cell lymphoma (Bcl)-2 family
proteins, including the anti-apoptotic Bcl-2/Bcl-xL and the pro-apoptotic Bax/Bid, regulate
the release of cytochrome c from the mitochondria and act to suppress or promote
apoptosis. Activated Bax/Bid migrate from the cytosol to the mitochondria to initiate
release of cytochrome c from the mitochondria and promote apoptosis. Bcl-2/Bcl-xL are
primarily localized in the outer member of the mitochondrial and are able to inhibit the
release of cytochrome c to suppress apoptosis. The balance of anti-apoptotic proteins and
pro-apoptotic proteins determines the release of cytochrome c and activation of
downstream caspases and therefore the cell’s susceptibility to apoptotic stimuli. In
addition, both Bcl-2/Bcl-xL and Bax/Bid are transcriptional targets of the tumor suppressor
protein p53, which induces cell cycle arrest and apoptosis in response to DNA damage
(Earnshaw et al., 1999)

Caspase Substrates
Activation of caspases leads to downstream cleavage of key structural components
of the cytoskeleton and nucleus as well as proteins involved in the apoptosis signaling
pathway. Pro-and anti-apoptotic regulatory proteins are among the first to be cleaved by
activated caspases. Caspase-3 cleavage of Bcl-2/Bcl-xL inactivates the anti-apoptotic
properties of these proteins by producing a pro-apoptotic product that increases
mitochondrial permeability and release of cytochrome c (Li et al., 1998). Caspase-8 can

24	
  

also induce cytochrome c release by cleaving pro-apoptotic Bid (Lou et.al, 1994). These
cleavage events allow the extrinsic pathway to communicate with and utilize the
mitochondrial pathway to amplify apoptotic signals.

Other Pathways
The MAPK pathway allows signals to be transmitted from the cell membrane to the
nucleus in response to various stimuli. The MAPK pathway also induces the
phosphorylation of intracellular substrates such as protein kinases and transcription factors
to regulate cellular functions such as cell growth, differentiation, and apoptosis (Kyriakis
and Avruch, 1996). The MAPK family of kinases include: the extracellular signal-regulated
kinases (ERKs), ERK-1 (p44 MAPK) and ERK-2 (p42 MAPK); stress-activated protein
kinases (SAPKs), also referred to as c-Jun, NH2-terminal kinases (JNKs), which include
p54 SAPK (SAPKα/,β, JNK2) and p45 SAPK (SAPKγ, JNK1); and the p38 kinases (α, β, γ,
and δ) (Widmann et al., 1999). Activation of the SAPK/JNKs and p38 kinases play a role in
apoptosis and activation of the ERK1/ERK2 pathways are involved in cell survival
(Earnshaw et al., 1999).

25	
  

Figure 1.2.
Induction of apoptosis [Figure 11.9C.1 modified from (Siegel and Lenardo, 2002)]

26	
  

Alveolar Epithelial Cell Apoptosis in IPF
Alveolar epithelial cell injury and death have consistently been found in human IPF.
The survival and recovery of epithelial cells is key to normal repair of damaged epithelia.
Fibrosis occurs through the pathological repair process of tissue remodeling. Often, the
degree of remodeling is closely associated with the patient’s prognosis. Hence, a greater
understanding of epithelial cell apoptosis may lead to the development of effective
strategies for treatment.

Evidence of Apoptosis
Alveolar epithelial cell injury is a characteristic feature of IPF. In the normal lung,
ATI cells makeup 40% of the AEC population and cover 95% of the alveolar surface
(Mason and Shannon, 1997). The number of AT1 cells are markedly decreased in the IPF
lung after injury and cell death. Recently, increased apoptosis of ATII cells has been found
in areas of IPF that do not have established apoptosis (Barbas-Filho et al., 2001), and in
epithelial cells that overlay myofibroblasts (Uhal et al., 1998). Additionally, increased
expression of proapoptotic proteins and decreased expression of anti-apoptotic proteins has
also been found in the IPF lung (Plataki et al., 2005). Experiments using caspase inhibitors
also demonstrate the involvement of apoptosis in fibrosis. The tripeptide broad-spectrum
caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD.fmk),
inhibits the intracellular activation of caspase-like proteases in vivo , and attenuates
bleomycin-induced pulmonary fibrosis in mice (Kuwano et al., 2001; Wang et al., 2000).
Thus, there is growing evidence that subpopulations of AECs in IPF are undergoing

27	
  

apoptosis in association with the dysfunctional repair of the damaged alveolar epithelium.

Induction of Pulmonary Fibrosis
Studies of animal models have provided further understanding of whether excessive
AEC apoptosis is sufficient to lead to fibrogenesis. The induction of AEC apoptosis using
aerosolized anti-Fas antibody was found to associate with the development of pulmonary
fibrosis in mice (Hagimoto et al., 1997). Targeted transgenic overexpression of bioactive
TGF-β1 to the murine lung produces a transient wave of epithelial apoptosis that is
followed by mononuclear-rich inflammation, tissue fibrosis, myofibroblast hyperplasia, and
honeycombing (Lee et al., 2004). Furthermore, a null mutation of early growth response
gene (EGF)-1 or caspase inhibition can rescue the fibrotic phenotype (Lee et al., 2004).

Mechanism of Alveolar Epithelial Apoptosis
p53 and p21 upregulation
Lung tissues from patients with IPF reveal upregulation of p53 and p21 in lung
epithelial cells (Kuwano et al., 1996). In normal cells, p53 responds to DNA damage and
mediates cell cycle arrest, DNA repair, and apoptosis. p53 expression is upregulated in
response to a variety of stresses. ATII cell apoptosis is associated with upregulation of p53
and p21 expression in diffuse alveolar damage (Guinee et al., 1996). ATII cells of patients
with IPF also express high levels of p53 in association with DNA strand breaks (Kuwano et
al., 1996; Plataki el al., 2005;). p53 and p21 are also overexpressed in AECs treated with
bleomycin (Mishra et al., 2000; Kuwano et al., 2000). p21 is a critical downstream effecter
in the p53 pathway of growth control in mammalian cells and is induced after exposure to

28	
  

DNA-damaging agents (el-Deiry et al., 1993). p21 enhances cell survival by promoting
DNA repair or by modifying cell death caused by exposure to hyperoxia (O’Reilly et al.,
2001). Transfer of the p21 gene through adenovirus to epithelial cells attenuates
bleomycin-induced pulmonary fibrosis in mice (Inoshima et al., 2004). p21 also regulates
activation of caspase-3. The formation of a procaspase-3-p21 complex is essential for cell
survival (Suzuki et al., 1998; Suzuki et al., 1999), suggesting that p21 may act as a key
regulator of DNA replication and repair after lung damage.

Fas-FasL Pathway Activation
Fas is expressed on the luminal surface of a subset of AT II cells (Fine et al., 1997).
In patients with acute lung injury, alveolar epithelial damage is in part associated with the
local upregulation of the Fas-FasL pathway and subsequent activation of the apoptotic
cascade in epithelial cells (Albertine et al., 2002). In lung tissues of IPF patients, FasL
mRNA and protein expression are upregulated in infiltrating inflammatory cells and Fas
protein is overexpressed in the epithelial cells (Kuwano et al., 1999). BAL fluid obtained
from patients with IPF or ARDS could induce apoptosis on small airway epithelial cells
through the Fas-FasL pathway (Matute-Bello et al.,1999; Hagimoto et al., 2002).
Bleomycin is used to induce pulmonary fibrosis in animal models of lung injury and
fibrosis. FasL mRNA in this model is upregulated in infiltrating lymphocytes. In addition,
excessive apoptosis is detected in the bronchiolar and alveolar epithelial cells where Fas
protein expression is upregulated (Hagimoto et al., 1997) Neutralization of FasL prevents
the development of fibrosis in this model (Kuwano et al., 1999). These findings indicate
that the Fas-mediated apoptotic pathway is essential in the bleomycin-induced pulmonary

29	
  

fibrosis model. Furthermore, the inhibition of caspases may provide a new approach to
treating pulmonary fibrosis.

TGF-β Induction of Apoptosis
TGF-β is a strong chemotactic attractant for monocytes and macrophages as well as
a potent promoter of ECM production. There is a consistent increase in TGF-β production
in epithelial cells and macrophages in lung tissues of IPF patients (Khalil et al., 1991) and
in bleomycin-induced pulmonary fibrosis in rodents (Raghow et al., 1989). Gene transfer of
a TGF antagonist reduces the fibrotic response to bleomycin (Kolb et al., 2001). TGF-β1
directly induces epithelial cell apoptosis via Fas and caspase-3 activation, and by enhancer
the FasL-Fas interaction (Hagimoto et al., 2002). The Bax-mediated, Bid-activated pathway
is involved in the pathogenesis of pulmonary fibrosis. TGF-β1 significantly stimulates Bax
and Bid expression and causes the release of MMP-12 and tissue inhibitor of
matalloproease-1 in mice (Kang et al., 2007). Bid-deficient mice are protected from
developing fibrosis after TGF-β1 activation, indicating that Bid is required for AEC
apoptosis and bleomycin-induced fibrosis in mice (Budinger, 2006). In an experiment using
mice, TGF-β1 overexpression in lung epithelial cells induced apoptosis and fibrosis. This
induction was attenuated when a caspase inhibitor was administered from day 0 but not
from day 5 after TGF-β1 overexpression (Lee et al., 2004), indicating that TGF-β1-induced
epithelial cell apoptosis is a critical early event in pulmonary fibrosis.

Oxidative Stress

30	
  

Oxidative stress has been proposed to significantly contribute to epithelial cell
damage in IPF (Kuwano et al., 2003). A number of studies demonstrate the increase of
oxidative stress in IPF. Patients with IPF exhibit an increase in the spontaneous production
of oxidants in the alveolar epithelial lining fluid (Saleh et al., 1997) as well as a significant
reduction in antioxidant capacity in the plasma and BAL fluid (Rahman et al., 1999).
Apoptosis plays a key role in hyperoxic lung injury (Albertine et al., 2002). Hyperoxia can
affect both ATI and endothelial cells as well as induce DNA damage in ATII cells (Roper
et al., 2004). Hyperoxia amplifies ventilator-induced cytokine production, neutrophil
influx, and apoptosis through activation of the JNK and ERK pathway (Ward et al., 1983).
It was traditionally thought that alveolar inflammatory cells produce the reactive oxygen
species (ROS) that induce AEC injury/apoptosis in IPF (Cantin et al., 1987; Stausz et al.,
1990). However, recent studies suggest that structural cells of the lung, particularly
activated myofibroblasts, produce sufficient amounts of extracellular ROS to induce
injury/apoptosis of adjacent epithelial cells. Bleomycin induces AEC apoptosis and fibrosis
through an increase of ROS (Wallach-Dayan et al., 2006).

Alveolar Epithelium-Fibroblast Crosstalk
Severe injury to the lung epithelial cells followed by insufficient repair disturbs
normal epithelial-fibroblast interactions. The lack of reepithelialization after damage
allows fibroblasts to proliferate and eventually resulting in pulmonary fibrosis. Epithelial
cells control fibroblasts through the release of cytokines. At the same time, fibroblasts can
also affect alveolar epithelial cells by releasing factors that disrupt their normal repair.

31	
  

Abnormal fibroblasts isolated from fibrotic human lungs produce factors that can induce
apoptosis and necrosis of AECs in vitro (Uhal et al., 1995).

32	
  

THE RENIN ANGIOTENSIN SYSTEM
General RAS
The traditional view of the renin angiotensin system (RAS) characterizes it as an
endocrine system with important roles in the regulation of fluid homeostasis, electrolyte
metabolism and blood pressure. Angiotensinogen (AGT), an α-glycoprotein, is released
from the liver and cleaved by renin in the circulation to form the decapeptide,
Angiotensinogen I (ANG I). Renin is an aspartyl protease secreted by the granular cells in
the juxtaglomerular apparatus of the kidney in response to decreased renal perfusion and
plasma sodium concentration. ANG I is activated by angiotensin converting enzyme (ACE)
to

form

the

octapeptide

angiotensin

II

(ANG

II).

ACE,

a

membrane-bound

metalloproteinase, is primarily expressed in high concentrations on the surface of
endothelial cells in the pulmonary capillaries. Alternative pathways involving enzymes
other than renin and ACE have also been shown to generate ANG I and ANG II. For
example, ANG II may be generated by chymase released from mast cells in the vascular
tissue under certain pathological conditions. ANG II is considered the main effector
peptide of the RAS and exerts its actions through specific cell surface angiotensin
receptors.
The main receptors of ANG II are AT 1 and AT2, both belong to a superfamily of
seven transmembran G-protein couple receptors. The AT1 receptor mediates most of the
well-known actions of ANG II, such as vasoconstriction, sodium retention, aldosterone
release, and cell growth and proliferation, etc. Actions mediated by the AT2 receptor are
less well characterized but appear to counteract AT1 effects by promoting vasodilation, cell
differentiation, and inhibiton of cell growth and apoptosis (de Gasparo et al., 2000).

33	
  

In additional to ANG I and ANG II, several functional peptides can be generated
through alternative cleavage pathways. These include angiotensin III (ANG III),
angiotensin IV (ANG IV), and angiotensin 1-7 (ANG 1-7). ANG III is generated from
ANG II by aminopeptidase A and plays a similar role as ANG II. Cleavage of ANG III by
aminopeptidase M generates ANG IV, which is involved in the vascular inflammatory
response (Ruiz-Ortega, 2007). ANG 1-7 heptapeptide is generated through cleavage of
ANG II by angiotensin-converting enzyme 2 (ACE2). ANG 1-7 was found to have actions
opposing those of ANG II and could act as a part of a counterbalancing mechanism in
controlling the actions of ANG II in specific tissues (Ferrario et al., 2006).

Local RAS
Recent evidence of RAS components expression in most organs and tissues suggests
the existence of a “local” RAS system that responds to physiological or pathophysiological
stimuli in an autocrine, panracrine and/or intracrine manner. This local system contains all
the components necessary to produce ANG II and other angiotensin peptides along with
their respective receptors, as well as renin/prorenin receptors. Most, if not all of the renin
found in local RAS is derived from renal renin. Local RAS systems may operate
independently of or interact with “circulating RAS” to exert novel and tissue specific
actions. These include cellular actions such as regulation of cell growth, differentiation,
proliferation and apoptosis, reactive oxygen species (ROS) generation, hormonal secretion,
tissue inflammation, and fibrosis (Leung et al., 2007; Montani & Van Vliet 2004).

34	
  

Figure 1.3.
The Renin Angiotensin System (RAS). The diagram illustrates the enzymatic cleavage of
angiotensinogen protein into angiotensin I (ANG I), angiotensin II (ANG II), angiotensin
1-9 (ANG 1-9), angiotensin 1-7 (ANG 1-7). Also shown are the known receptors for
angiotensin peptides: AT1 (angiotensin receptor 1), AT2 (angiotensin receptor 2), Mas.
ACE: angiotensin converting enzyme; ACE-2: angiotensin converting enzyme-2; aa: amino
acid.
35	
  

BLEOMYCIN-INDUCED EXPERIMENTAL PULMONARY FIBROSIS MODEL

The bleomycin-induced experimental pulmonary fibrosis model is one of many
animal models of pulmonary fibrosis that have been developed to address the pathogenesis
of pulmonary fibrosis. Bleomycin is an antineoplastic antibiotic that was isolated from a
strain of Strptomyces verticillus (Umezawa et al., 1966). Bleomycin is often used to treat
squamous cell carcinomas and various lymphomas (Ichikawa et al., 1967, 1969; Yogoda et
al., 1972; Blum et al., 1973). Instead of being just a single peptide, bleomycin consists of a
family of complex glycopeptides with different amine groups (Umezawa et al., 1966, 1967;
Umezawa 1973, 1974). The mechanisms of the antineoplastic actions of bleomycin are cell
cycle-dependent and complex. Bleomycin can intercalate between DNA base pairs, leading
to DNA unwinding and impaired protein synthesis. Bleomycin also reduces molecular
oxygen to superoxide and hydroxyl radicals, producing radical oxygen species that then
attack DNA and cause strand cleavage (Sausville et al., 1978).
Treatment with bleomycin has very little side effects on bone marrow (Kimura et
al., 1972; Boggs et al., 1974) and immunocompetence (Dlugi et al., 1974; Lehane et al.,
1975). Despite these advantages, bleomycin has severe toxic side effects on the lung and
skin organ systems (Thrall and Scalise, 1995). After parenteral administration, bleomycin
concentrations are highest in the lungs and skin, likely due to the lack of a hydrolase,
which inactivates bleomycin (Umezawa et al., 1972).
The bleomycin-induced pulmonary fibrosis animal model is currently the most
commonly used model in studying the pathogenesis of fibrotic lung disease. Pulmonary
fibrosis induced by bleomycin is dependent on dosage and administration route, most
common of which are parenteral and intratracheal. Various animals, such as mice and rats,

36	
  

have been used in models of bleomycin-induced pulmonary fibrosis (Thrall and Scalise).
The development of lung injury in these models is similar regardless of species and route
of administration. The lung injury has three stages (Thrall and Scalise, 1995): (1) the acute
inflammatory stage where the inflammatory mediator systems are activated and pulmonary
edema appears; (2) The subacute stage where collagen is synthesized and the level of net
lung collagen elevates; (3) the chronic stage that is dominated by the metabolism of
connective tissue towards reepithelialization. The bleomycin animal models are valuable
tools in studying the pathogenesis of fibrotic lung disease.

37	
  

PULMONARY ANGIOTENSIN SYSTEM IN IDIOPATHIC PULMONARY
FIBROSIS
Recent evidence suggests the existence of a local intrinsic RAS system in the distal
lung parenchymal that plays a central role in the signaling of apoptosis in alveolar
epithelial cells (Wang et al., 1999). ANG II in the lung, derived from its precursor AGT,
drives fibrosis through upregulation of collagen gene expression in lung fibroblasts
(Marshall et al., 2004; Marshall et al., 2000), induction of apoptosis in alveolar epithelial
cells (Li et al., 2003), and other profibrotic actions (Marshal, 2003).
AGT in the local system is synthesized within the lung tissue itself by alveolar
epithelial cells and myofibroblasts. Primary cultures of human or rat AECs can synthesize
and secrete AGT, which is converted to ANG II when undergoing apoptosis induced by Fas
ligand (Wang et al., 1999), TNF-α (Wang et al., 2000), amiodarone (Bargout et al., 2000)
and bleomycin (Li et al., 2003). Myofibroblasts isolated from lungs of IPF patients were
shown to synthesize AGT mRNA and protein constitutively (Wang et al., 1999).
Intratracheal administration of antisense oligonucleotides against AGT mRNA prevented
bleomycin-induced lung fibrosis in rats without affecting circulating levels of ANGEL
protein, suggesting that local pulmonary synthesis of ANG II from the precursor AGT
within the lung is required for lung fibrogenesis (Li et al., 2007).
The local angiotensin systems often utilize components of endocrine RAS system in
its pathways. For example, synthesis of local ANG II in the heart and vessel walls reply on
the uptake of circulating renin through either diffusion into the interstitial space or through
binding to prorenin receptors (Re, 2004). Renin mRNA has not been detected in the lungs.
Other proteases in the lung, such as cathepsin D (Cat D), are capable of cleaving AGT to

38	
  

ANG I. Cat D expression is upregulated in the fibrotic lungs of both rat and human. Cat D
is also overexpressed in the lungs of patients with pulmonary fibrosis (Kasper et al., 1996),
and is induced in lung cells during apoptosis (Kasper et al., 1999). It is possible that
production of ANG I from AGT in the lungs is mediated by Cat D.
The decapeptide ANG I is inactive until it is cleaved into ANG II by the dipeptidyl
carboxypeptidase ACE. ANG II is thought to be responsible for most of the physiological
and pathophysiological effects of the RAS. ACE in the pulmonary vascular endothelium is
primarily responsible for the conversion of ANG I to ANG II in the circulation (Oparil et
al., 1971). ACE2, the first known homologue of ACE, was recently discovered to be
another regulator of the RAS system (Donoghue et al., 2000). ACE2 was first described for
its ability to cleave ANG I into ANG 1-9, a peptide with no known function (Donoghue et
al., 2000). It was later found to also degrade ANG II to the biologically active peptide,
ANG 1-7 (Vickers et al., 2002). In vitro studies demonstrate that the catalytic efficiency of
ACE2 for ANG II is 400 times greater than for ANG I (Vickers et al., 2002), suggesting
that ACE2 is primarily involved in the conversion of ANG II to ANG 1-7. ACE2
expression has been identified in the heart, kidney, testis, gastrointestinal tract, brain, and
lung (Hammer et al., 2002). Despite its similarities to ACE, the in vitro enzymatic
activities of ACE2 is unaffected by ACE inhibitors (Donoghue et al., 2000; Tipnis et al.,
2000).
Both receptors of ANG II, AT1 and AT2, are found to be expressed in the lung. In
the human lung, AT1 receptor mRNA and proteins were found on vascular smooth muscle
cells, macrophages and in the stroma underlying the airway epithelium (Bullock et al.,
2001). AT2 receptor RNA and protein were highly expressed on the bronchial epithelial

39	
  

cell brush border, endothelial cells, and underlying mucous glands of the epithelium
(Bullock et al., 2001). A study of the rat lung also found AT1 to be expressed on alveolar
macrophages, alveolar type II cells, vascular smooth muscle cells, endothelial cells and
fibroblasts of the rat lung (Otsuka et al., 2004). The AT1 expression in the lung was
significantly upregulated after induction of fibrosis (Otsuka et al., 2004), suggesting that
ANG II promotes lung fibrosis via the AT1 receptor.
Although AGT in the lung can be synthesized by myofibroblasts, they have a
limited ability to convert the AGT to ANG II (Wang et al., 1999). Alveolar epithelial cells,
on the other hand, are capable of proteolytically processing AGT to ANG II as well as
undergoing apoptosis in response to the ANG II produced (Wang et al., 1999). Thus, it is
theorized that the death of AECs adjacent to underlying myofibroblasts in the fibrotic lung
is due to production of AGT by the myofibroblasts and its subsequent conversion to ANG
II by the epithelial cells.
The local RAS system can also induce apoptosis of AECs without myofibroblasts.
Purified ANG II was shown to induce apoptosis of AECs in primary culture through the
AT1 receptor (Papp et al., 2002). The induction of AEC apoptosis by Fas ligand (Wang et
al., 1999), TNF-α (Wang et al., 2000), amiodarone (Bargout et al., 2000), and bleomycin
(Li et al., 2003) were blocked by AGT antisense mRNA oligonucleotides, ACE inhibitors,
and ANG II receptor antagonists, indicating that AEC apoptosis as induced by these agents
require angiotensin synthesis de novo. The inhibition of bleomycin-induced AEC apoptosis
with either an ACE inhibitor or ANG receptor antagonist was observed both in vitro and in
vivo, indicating that ANG II may be required for apoptosis in both cultured cells and the
intact lung.

40	
  

Angiotensin system antagonists have been shown to attenuate lung fibrosis in
experimental models. Applications of ACE inhibitors, which presumably prevent ANG II
production, have been shown to block pulmonary fibrosis in animal models induced by
various agents. Treatment with the ACE inhibitor captopril, for example, effectively
inhibited monocrotaline (Molteni et al., 1985) as well as γ irradiation-induced lung fibrosis
in rats (Ward et al., 1990). Captopril was also shown to inhibit the proliferation of human
lung fibroblasts in vitro (Nguyen et al., 1994). The specific AT1 receptor antagonist
L158809 and the nonthiol ACE inhibitor enalapril were also shown to have similar effects
on radiation-induced pulmonary fibrosis in rats (Molteni et al., 2000). Captopril was also
found to prevent collagen deposition on bleomycin-treated rats (Wang et al., 2000).
Recently, it was shown that captopril and AT1 selective antagonist losartan block the
amiodarone induced pulmonary fibrosis in rats (Uhal et al., 2002). Together, these results
suggest that ANG II plays an important role in lung fibrosis through the AT1-receptor.
The ACE inhibitor captopril as well as caspase inhibitors have been found to block
pulmonary fibrosis through the inhibition of apoptosis. Caspases play key roles in
apoptosis signaling. Application of the broad-spectrum caspase inhibitor ZVADfmk, or
captopril both attenuated epithelial apoptosis and collagen deposition in intratracheally
administered bleomycin-induced pulmonary fibrosis in rats (Wang et al., 2000). Delivery of
the same caspase inhibitor through inhalation also inhibited bleomycin-induced in mice
(Kuwano et al., 2001). These results indicate that both ANG II and epithelial apoptosis are
required for lung fibrogenesis.

41	
  

Chapter 2

ANGIOTENSIN 1-7 REGULATES ALVEOLAR EPITHELIAL CELL SURVIVAL
THROUGH THE RECEPTOR MAS

42	
  

ABSTRACT
Apoptosis of alveolar epithelial cells (AECs) is believed to be a critical event in the
pathogenesis of lung fibrosis. It was previously shown that apoptosis of AECs requires
autocrine generation of angiotensin (ANG) II. ANG II can also be degraded to ANG1-7 by
angiotensin converting enzyme-2 (ACE-2). ACE2 was recently found to be protective but
significantly downregulated in experimental lung fibrosis and patients with IPF. In other
organ systems, ANG1-7 has been found to inhibit the actions of ANGII through the ANG17 receptor mas. Therefore, it was hypothesized that ANG1-7 in the lungs might antagonize
the actions of ANG II in the regulation of AEC apoptosis. To test this theory, the AEC cell
line MLE-12 and primary cultures of human AECs were stimulated by the profibrotic
apoptosis inducers ANG II or bleomycin (BLEO). Caspase-3 activation and nuclear
fragmentation were used as markers of apoptosis and were measured along with JNK
phosphorylation. Exposure to ANG II or BLEO induced caspase-3 activation, nuclear
fragmentation, and JNK phosphorylation in cultured AECs. Pretreatment with ANG1-7, at
a concentration of 0.1µM, prevented JNK phosphorylation and apoptosis. In addition,
pretreatment with A779, a specific blocker of the ANG 1-7 receptor mas, successfully
prevented ANG1-7 induced blockade of JNK phosphorylation and nuclear fragmentation.
These data indicate that ANG1-7 prevents AEC apoptosis, and its actions are mediated
through the ANG1-7 mas receptor.

43	
  

INTRODUCTION
Idiopathic pulmonary fibrosis is a pathological condition resulting from lung injury
and an ensuing fibrotic response that leads to thickening of the alveolar walls and the
obliteration of the alveolar space (Fonseca et al., 2000). The etiology of the disease is
unknown. Current evolving hypothesis about pathogenesis is that IPF results from the
epithelial injury and a failure of reepithelialization (Selman et al., 2001). Alveolar
epithelial cell apoptosis is a critical event in the pathogenesis of pulmonary fibrosis and the
regulation of AEC apoptosis is of great interest and potential clinical importance. The
bleomycin animal models are valuable tools in studying the pathogenesis of fibrotic lung
disease. Recent studies in our lab and other labs using the bleomycin-induced models in
rats and mice suggested a role of epithelial apoptosis as the profibrotic event in fibrosis.
First, apoptosis of AECs was found in both patients with IPF (Uhal, et al., 198) and animal
models (Hagimoto et al., 1997). Second, induction of apoptosis in the epithelium is
sufficient to produce a fibrotic response (Hagimoto et al., 1997). And lastly, several labs
have shown that the blockade of apoptosis could prevent the fibrotic response (Wang et al.,
2000; Kuwano et al., 2001).
Recent work from this lab demonstrated that the exposure of cultured AECs to Fas
ligand (Wang et al, 1999), tumor necrosis factor-α (Wang et al., 2000) or bleomycin (Li et
al., 2003) a induce expression of angiotensinogen mRNA and protein and its cleavage to
the peptide Angiotensin II. They also showed that bleomycin induces fibrosis through
induction of alveolar epithelial cell apoptosis (Wang et al., 2000). They found that the ACE
inhibitor captopril and the broad-spectrum caspase inhibitor ZVAD-fmk were able to
reduce bleomycin-induced apoptosis (Figure 2.1). Moreover, captopril and ZVAD-fmk also

44	
  

prevented the bleomycin-induced accumulation of collagen (Figure 2.2). Together, these
results indicate that the induction of apoptosis is required for bleomycin-induced fibrosis
and that ANG II and caspases play an important role in this pathway.
In another study, our lab demonstrated that bleomycin-induced apoptosis of alveolar
epithelial cells requires ANG II synthesis de novo. Li et al. showed that bleomycin-induced
nuclei fragmentation was blocked by ZVAD-fmk, captopril and the nonselective ANG
receptor antagonist saralasin (Figure 2.3). In addition, they also demonstrated that
bleomycin-induced apoptosis of primary AECs was blocked by the caspase-3-selective
blocker DEVD-fmk and the AT1 receptor selective blocker losartan (Figure 2.4), suggesting
that AT1 receptor mediates bleomycin-induced apoptosis as it does ANGII-induced AEC
apoptosis. Together, these data indicates that the production of ANGII is a necessary event
in bleomycin-induced AEC apoptosis.
Bleomycin induces alveolar cell apoptosis through the JNK-dependent activation of
the mitochondrial death pathway (Lee et al., 2005). Bleomycin-induced Bax activation was
inhibited by the expression of a dominant negative JNK in MLE-12 cells (Lee et al., 2005).
Additionally, recent evidence show that both bleomycin and ANGII induces caspase 9
activation, suggesting that ANG II, just like bleomycin, also induces apoptosis through the
mitochondrial death pathway requiring the activation of phosphorylation of JNK (Figure
2.5).
Angiotensin converting enzyme 2(ACE-2) degrades ANG II into the heptapeptide
ANG 1-7 (Figure 2.6). In other organ systems, ANG1-7 was found to prevent some of the
actions of ANGII through the ANG1-7 receptor mas (Gava et al., 2009; Lara et al., 2010;
Tallant et al, 2005). Precisely which lung cell types express active mas signaling is

45	
  

currently unknown. This study reports evidence that ANG1-7 in the lung acts through the
ANG1-7 receptor mas to inhibit ANGII signaling and AEC apoptosis.

46	
  

Figure 2.1.
Quantitation of ISEL-positive nuclei in alveolar (A) and airway (B)
epithelial cells after Bleo, CAPTO, and ZVAD-fmk administration. NORM,
animals exposed to vehicle only. ISEL-positive nuclei were scored as a percentage
of the total epithelial nuclei within the alveolar and airway epithelial cell
populations. See methods for details. * P < 0.01 vs. NORM. ** P < 0.01 vs. Bleo
(both by ANOVA and Student-Newman-Keuls test). (Adapted from Wang et al.,
2000)
47	
  

Figure 2.2.
Quantitation of collagen by PR staining of alveolar (A) and peribronchial (B)
lung parenchyma. Collagen was quantitated from polarized light images as the total
number of white pixels per unit area within the alveolar and peribronchial lung
parenchyma. See methods for details. * P < 0.01 vs. NORM. ** P < 0.01 vs. Bleo (both by
ANOVA and Student-Newman-Keuls test). (Adapted from Wang et al., 2000)

48	
  

Figure 2.3.
Dose-dependent induction of nuclear fragmentation by BLEO in primary rat AECs
and blockade by inhibitors of caspases, endonucleases, ANG-converting enzyme
(ACE), and ANG-receptor interaction. Rat AECs were isolated and challenged with the
indicated concentrations of Bleo on day 2 of primary culture (seematerials and methods).
Putative inhibitors were added 30 min before addition of Bleo; nuclear fragmentation was
scored as described in Fig. 1 B and materials and methods. ZVAD, N-benzylcarboxy-ValAla-Asp-[O-Me]-CH2F (60 µM); ATA, aurintricarboxylic acid (10 µM); Capto, captopril
(500 ng/ml); Saral, saralasin (50 µg/ml). Bars are the means ± SE of at least 4 observations;
*P < 0.05 vs. control (0.0 Bleo). (Adapted from Li et al., 2002)

49	
  

Figure 2.4.
Blockade of Bleo-induced apoptosis in primary AECs by selective caspase or ANG
receptor blockers. Rat AECs were isolated and challenged with 25 mU/ml Bleo alone or
in the presence of the caspase-3-selective inhibitor Asp-Glu-Val-Asp-[O-Me]-CH2F
(DEVD-fmk, 60 µM) or the ANG receptor AT1-selective antagonist losartan (Los, 10−6
M). Control cultures (Ctl) received blocker vehicles only. Nuclear fragmentation was
scored as described in Fig. 1 andmaterials and methods. Bars are means ± SE of at least 4
observations; *P < 0.05 vs. control. (Adapted from Li et al., 2002)

50	
  

Figure 2.5.
ANGII induces activation of caspase-9. Primary cultures of rat type II alveolar epithelial
cells were cultured in serum-free medium for 6 hours with or without ANGII. Cells were
harvested with NP40 buffer for Western blotting. (Xiaopeng Li, 2006)

51	
  

Figure 2.6.
Pathways for the formation of ANG 1-7. Renin cleaves angiotensinogen into the 10-aa
peptide ANG I. ANG I is then converted to angiotensin II by ACE or to ANG 1-7 by
neprilysin (NEP), prolyl-endopeptidase (POP) or thimet oliogopeptidase (TOP). ANG 1-7
can also be generated from ANG II through ACE2. ANG II can activate either the AT1
subtype of angiotensin receptor or the AT2 ANG II subtype receptor. ANG 1-7 activates the
Mas receptor.

52	
  

Figure 2.7.
Summary of Earlier Published Data. For interpretation of the references to color in this
and all other figures, the reader is referred to the electronic version of this thesis.

53	
  

METHODS
Cell culture
The mouse lung epithelial MLE-12 cell line was a gift from the laboratory of Dr.
Jeffrey Whitsett, University of Cincinnati, OH, and was grown in complete HITES medium
for MLE12 supplemented with 2% fetal bovine serum according to ATCC’s guidelines. All
cells were grown in 12-, 24-, or 6-well chambers and were analyzed at subconfluent
densities of 50-80%. 12 hours before treatment, cells were switched from growth medium
containing fetal bovine serum to serum-free medium. All subsequent incubations with
bleomycin and/or other test agents were performed in serum free medium. In all
experiments, cells were treated with inhibitors or antagonists 30 minutes prior to treatment
with bleomycin or ANGII. For exposure of extended periods, cells were exposed to test
agents as described and after one hour, culture media were replaced with new media
containing fresh A779 and ANG1-7 but not bleomycin. Due to the low biological half-lives
of the A779 and ANG1-7 peptides, they were replaced every 2 hours until cells were
harvested. Primary human alveolar epithelial cells isolated from human lung tissue were
obtained from ScienCell Research Laboratories. They were cultured in Alveolar Epithelial
Cell Medium according to ScienCell Research Laboratories guidelines.

Detection of Apoptosis
Apoptotic cells were detected by nuclear fragmentation with propidium iodide (PI)
after enzymatic digestion of ethanol-fixed cells with DNase-free RNase in PBS containing
5µg/ml PI. During fixation with 70% ethanol, detached cells were retained by
centrifugation of the 24-well or 12-well culture plates. Cells with discrete nuclear

54	
  

fragmentation containing condensed chromatin were scored as apoptotic. Apoptotic cells
were scored over four separate microscopic fields from each of four culture wells per
treatment group. The active forms of caspase-3 and caspase-9 were detected by Western
blotting using antibodies specific for the active cleaved forms.

Western Blotting
For caspase-3 and caspase-9 detections, cells were harvested and lysed in an NP40based lysis buffer containing protease inhibitors. For detection of phosphor-proteins, cells
were harvested and lysed with a modified lysis buffer containing 50mM HEPES, 150mM
NaCl, 10% glycerol, 1% Triton X-100, 1uM EGTA, 1.5mM MgCl2, phosphatase inhibitor
cocktail (Phospho Stop, Roche, Nutley, NJ) and the protease inhibitor cocktail (Complete
Mini, Roche, Nutley, NJ). Proteins were separated on polyacrylamide gels and transferred
to PVDF membranes. Immunoreactive bands were visualized by ECL detection systems
(Thermo Scientific, Rockford, IL).

55	
  

RESULTS
Earlier results from this lab, as summarized in figure 2.7, proposes that pulmonary
fibrosis induced by bleomycin begins with an increased production of ANG II. ANG II then
induces JNK phosphorylation and downstream activation of caspase 9 and 3. The signaling
eventually leads to apoptosis of the alveolar epithelial cells followed by fibrosis. In this
study, we wanted to investigate how Angiotensin 1-7 fits in to this pathway and what role it
plays in the regulation of AEC apoptosis.
To begin examining the hypothesis that ANG1-7 acts to oppose the actions of ANG
II, we assessed the effect of ANG II on JNK phosphorylation in the presence and absence
of purified ANG1-7 in serum-free media. JNK phosphorylation has previously been shown
to be required for apoptosis in alveolar epithelial cells (Lee et al., 2005). Figure 2.8 shows
that 5 minutes of exposure to ANG II (10-7 M) also induces JNK phosphorylation. 30
minute pre-incubation with ANG1-7 (10

-7

M) prevented the ANG II-induced increase in

JNK phosphorylation, indicating that ANG1-7 is able to block the actions of ANG II at
equimolar concentrations. Moreover, incubation with the specific mas receptor blocker
A779 blocked the effects of ANG1-7. This indicates that ANG1-7 is able to signal through
its receptor mas to prevent ANGII induced JNK phosphorylation.
In a previous study, it was shown that bleomycin-induced apoptosis of AECs
requires autocrine synthesis of ANGII and its subsequent binding to ANG receptor AT1 (Li
et al., 2003). Given that ANG1-7 inhibited ANG II induced JNK phosphorylation (Figure
2.8), we theorized that it may also block the JNK phosphorylation that is required in
bleomycin-stimulated JNK phosphorylation. A 30-minute pretreatment with ANG1-7 (10

-7

M) prevented the increase in p-JNK observed 5 minutes after exposure of MLE-12 cells to

56	
  

purified bleomycin (10

-7

M) (Figure 2.8). We then tested bleomycin-induced JNK

phosphorylation again with a 30-minute pretreatment of the mas antagonist A-779 (D-Ala7Ang1-7, 10

-7

M). The A779 pretreatment prevented the pJNK inhibition by ANG1-7

(Figure 2.9), suggesting that ANG1-7 prevents bleomycin-induced JNK phosphorylation
through the receptor mas.
Since

ANG1-7

prevented

both

ANG

II-

and

bleomycin-induced

JNK

phosphorylation, and JNK phosphorylation is a required event in bleomycin-induced
apoptosis, we then examined if ANG1-7 might also prevent AEC apoptosis. In Figure 2.10,
preincubation with ANG1-7 blocked the bleomycin-induced activation of caspase-3 in
MLE-12 cells. Figure 2.11 shows that ANG1-7 could also prevent bleomycin-induced
caspase-9 activation in primary cultures of human alveolar epithelial cells and that the mas
antagonist A779 prevents the action of ANG1-7, confirming that bleomycin induces apoptosis
through the mitochondrial pathway as previously established. Furthermore, Figure 2.12 shows
that ANG1-7 prevented the bleomycin-induced increase in nuclear fragmentation detected
in MLE-12 cells after 12 hours exposure to bleomycin (p<0.01). Preincubation with the
mas antagonist A779 prevented the effect exerted by ANG1-7. It was previously
demonstrated that bleomycin-induced AEC apoptosis involves the release of cytochrome c,
indicating that apoptosis is occurring versus necrosis. In Figure 2.13A, MLE-12 cells were
treated with 25mU/ml bleomycin for 20 hours and stained with active caspase-3 antibodies.
The results further confirm the activities of apoptosis. Figure 2.13 also shows that ANG1-7
could also prevent bleomycin-induced nuclear fragmentation in primary cultures of human
alveolar epithelial cells (p<0.01). As seen in MLE-12 cells, the blockade of nuclear
fragmentation by ANG1-7 was prevented by A779.

57	
  

Both nuclear fragmentation and

caspase-3 activation are markers of apoptosis. Figure 2.14 provides a brief summary of the
results of this study and related earlier works, along with topics for future research.

58	
  

Figure 2.8.
ANG1-7 inhibits ANG II induced JNK phosphorylation through the mas receptor.
MLE-12 cells in serum free conditions were preincubated with specific mas antagonist
A779 10

-7

M for 30 minutes followed by incubation in 10

-7

M ANG1-7 for an additional

30 minutes. Afterwards, cells were treated with 5mU/ml ANG II for 10 minutes and
harvested for Western blotting.

59	
  

Figure 2.9.
ANG1-7 inhibits BLEO induced JNK phosphorylation through the mas receptor. A:
MLE-12 cells in serum free conditions were preincubated with specific mas antagonist
-7

-7

A779 10 M for 30 minutes followed by incubation in 10 M ANG1-7 for an additional
30 minutes. Afterwards, cells were treated with 5mU/ml bleomycin (BLEO) for 5 minutes
and harvested for Western blotting. B: Ratio of the densitometric quantification of the
Western blot performed in Panel A and the Western blot of total JNK of the same
conditions (data not shown). Bars are the means. *: p<0.05 vs. CTRL and **: p<0.01 vs
CTRL by ANOVA and Student-Newman-Keul’s post-hoc analysis.

60	
  

Figure 2.10.
ANG1-7 inhibits BLEO induced caspase-3 activation of MLE-12 cells. MLE-12 cells in
serum free conditions were preincubated in 10

-7

M ANG1-7 for 30 minutes. Afterwards,

cells were treated with 5mU/ml bleomycin (BLEO) for 2 hours and harvested for Western
blotting.

61	
  

Figure 2.11.
ANG1-7 inhibits BLEO induced caspase-9 activation of primary AECs through the
mas receptor. Primary cultures of human alveolar epithelial cells were preincubated with
the specific mas antagonist A779 (10

-7

M) for 30 minutes followed by ANG1-7 (10

-7

M)

for an additional 30 minutes. Thereafter the cells were exposed to bleomycin (BLEO,
5mU/ml) for an additional 6 hours.

62	
  

Figure 2.12.
ANG1-7 inhibits bleomycin-induced nuclear fragmentation through the mas receptor
in MLE-12 cells. A: Primary cultures of AECs fixed in 70% ethanol and stained with
propidium iodide. Arrow points to cells that exhibit chromatin condensation and nuclear
fragmentation. B. MLE-12 cells in serum free conditions were preincubated with specific
mas antagonist A779 10

-7

M for 30 minutes followed by incubation in 10

-7

M ANG1-7 for

an additional 30 minutes. Afterwards, cells were treated with 5mU/ml bleomycin (BLEO)
for 12 hours. A779 and ANG1-7 were replaced every 2 hours. Cells were then harvested for
detection of nuclear fragmentation. Absolute value for basal (CTRL) nuclear fragmentation
was 3.30 ± 0.25%. Bars represent the means ± S.E.M. of four separate cell cultures. **:
p<0.001 vs. CTRL by ANOVA and Student-Newman-Keul’s post-hoc analysis.

63	
  

Figure 2.13.
ANG1-7 inhibits bleomycin-induced nuclear fragmentation through the mas receptor
in primary human AECs. A. Cells treated with 25 mU/ml bleomycin for 20 hrs stained
with PI or with active caspase-3 antibody. Arrows indicate fragmented nuclei with
condensed chromatin (bright fluorescence). B. Primary human alveolar epithelial cells in
-7
serum free conditions were preincubated with specific mas antagonist A779 10 M for 30
minutes followed by incubation in 10
-7

-7

M ANG1-7 for an additional 30 minutes.

Afterwards, cells were treated 10 with 5mU/ml bleomycin (BLEO) for 12 hours. A779
and ANG1-7 were replaced every 2 hours. Cells were then harvested for detection of
nuclear fragmentation. Absolute value for basal (CTRL) nuclear fragmentation was 3.11 ±
0.21%. Bars represent the means ± S.E.M. of four separate cell cultures. **: p<0.001 vs.
CTRL by ANOVA and Student-Newman-Keul’s post-hoc analysis.
64	
  

Figure 2.14.
Summary. The autocrine production of ANGII is required for AEC apoptosis and can be
stimulated by bleomycin or the endogenous proapoptotic inducers Fas ligand or TNF-α.
Apoptosis signaling proceeds through the ANG receptor AT1 and JNK phosphorylation
(pJNK). ANG 1-7 inhibits JNK phosphorylation and apoptosis through binding to the
specific ANG 1-7 receptor mas. The mechanisms of how activation of mas leads to
inhibition of JNK phosphorylation are currently unknown and may be topics for future
inquiry.

65	
  

DISCUSSION
Recent studies have established a new regulatory axis in the renin-angiotensin
system (RAS). Much attention has been paid to studying the role of the ACE-2/ANG17/mas axis in organ injury and fibrogenesis. The heptapeptide ANG1-7, has been shown to
oppose the vasoconstrictive, proliferative, and profibrotic actions of ANG II in a number of
tissues (Ferrario et al., 2005; Ferrario 2010). ANG1-7 can be generated through cleavage
by a variety of enzymes including neprilysin, prolyl-endopeptidase, and thimet
oliogopeptidase (Gallager and Tallant, 2004). The primary pathway of ANG 1-7 synthesis
is through ACE2 degradation of ANG II, in which ACE2 removes the ANG II C-terminal
phenylalanine to produce ANG1-7 (Li et al., 2008). Figure 2.6 illustrates the cleavage
pathway of AGT into ANG 1-7. Studies of the adult mouse lung indicate that the type II
pneumocytes are the only cells to express ACE2 in the lungs (Wiener et al., 2007).
The heptapeptide ANG1-7 can bind to the receptor mas, a seven trans-membrane
protein with domains containing sequences characteristic of G protein-coupled receptors
encoded by the oncogene of the same name (Young et al., 1988). Some suggested that
ANG1-7 may also bind to the AT1 receptor (Zisman, 2005). Binding data, however, reveal
low affinity of ANG1-7 for AT1 or AT2 receptors (Fontes et al., 1994). Currently there is
very little data regarding the functions of ANG1-7 in apoptosis and its role in the
regulation of organ systems. In the liver, ACE-2 and ANG1-7 may be protective against the
development of liver fibrosis (Warner et al., 2007), the molecular and cellular mechanisms
of which are still unclear.
Evidence from numerous cell systems demonstrates the ability of ANG1-7 to inhibit
the effects of ANG II. For example, in the heart, ANG 1-7 prevents the development of

66	
  

cardiac fibrosis induced by ANG II infusion (Grobe et al., 2007), and attenuates the ANG
II-stimulated production of endothelin-1 and leukaemia inhibitory factor in cardiac
fibroblasts (Iwata et al., 2005). In the rat proximal tubular cells, ANG 1-7 inhibited ANG
II-induced phosphorylation of MAPKs including p38, ERK1/2, and JNK while also
partially blocking activation of TGF-β1 promoted by ANG II (Su et al., 2006). Also in the
kidney, ANG1-7 was found to reverse ANG II-induced stimulated Na+-ATPase (Lara et al.,
2010). In all of these systems, ANG 1-7 blockade of ANG II actions was inhibited by a
knockdown of mas or the application of the specific mas receptor blocker A779.
Consistent with these results from other experimental systems, Figure 2.8
demonstrates the ability of ANG 1-7 to prevent JNK phosphorylation stimulated by ANG II
in alveolar epithelial cells. In addition, Figure 2.12 indicates that ANG 1-7 also prevents
apoptosis induced by bleomycin. The finding that bleomycin activates caspase-9 (Figure
2.11) confirms that bleomycin also induces apoptosis through the mitochondrial pathway
and that this induction is prevented by A1-7, whose effects, in turn, were blocked by A779.
Together, our data reinforces the previously established concept that autocrine production
of ANG II and its binding to the AT1 receptor is required in bleomycin-induced apoptosis
of alveolar epithelial cells. As seen in Figure 2.12 and 2.13, the mas antagonist A779 was
able to block the actions of ANG1-7 in both the MLE cell line and primary human AECs.
This suggests that in alveolar epithelial cells, the ability of ANG1-7 to inhibit ANG II
actions is mediated through the mas receptor, just as in other cell types studied thus far.
It is still unclear as to how intracellular signaling through the mas receptor can
counteract the effects of ANG II. Evidence indicate that factors such as Akt
phosphorylation, protein kinase C (PKC) activation and MAPK inhibition may play a role

67	
  

in this pathway. In adult ventricular myocytes and endothelial cells, ANG 1-7 causes NO
synthase phosphorylation and release through the phosphatidylinositol 3-kinase (PI3K)protein kinase B (Akt)– dependent pathway (Sampaio et al., 2007; Dias-Peixoto et al.,
2008). This was effectively prevented by the mas antagonist A779 or knockout of the mas
gene. Moreover, it was found in rat extracardiac tissues that ANG 1-7 induces the in vivo
phosphorylation of Akt by phosphorylating at threonine 308 and serine 473 (Muñoz et al.,
2010). Studies with isolated perfused rat atria also showed ANG 1-7 to be exerting its
effects through the mas/ PI3K/Akt pathway (Shah et al., 2010). In addition, they also found
ANG 1-7 to activate the Na+/H+ exchanger-1 and CaMKII through the mas receptor.
However, little is known about how these mechanisms interact to antagonize the signaling
of ANG II in the cell. It was previously shown that PKC expression is required for ANG IIstimulated AEC apoptosis in primary cultures (Papp et al., 2002). Recent evidence reveals
that in renal tubule cells, ANG1-7 signals via the mas receptor to inhibit ANG II-stimulated
Na+-ATPase activity by reversing PKC activation through a Gs/PKA pathway (Lara et al.,
2010). Future studies focusing on the cAMP/PKA pathways in AECs might further
elucidate the mechanism by which ANG1-7 inhibits ANG II signaling.
In summary, this study demonstrated that ANG1-7 acts through the receptor mas to
block phosphorylation of JNK stimulated by either ANG II or bleomycin. ANG 1-7
treatment also inhibited bleomycin-induced apoptosis of alveolar epithelial cells, in
agreement with the previously established requirement of JNK phosphorylation in AEC
apoptosis. Together, the data demonstrates that ANG 1-7, produced from ANG II by ACE2,
acts through the mas receptor to play a key role in the regulation of alveolar epithelial cell
survival by preventing both JNK phosphorylation and subsequent apoptosis.

68	
  

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