Cardiac fibrosis in mice with hypertrophic cardiomyopathy is
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Transcriptional regulation of EndMT in cardiac fibrosis: Role
of MRTF-A and ATF3
Journal: Canadian Journal of Physiology and Pharmacology
Manuscript ID cjpp-2016-0634.R1
Manuscript Type: Review
Date Submitted by the Author: 21-Jan-2017
Complete List of Authors: Sharma, Vibhuti; Post Graduate Institute of Medical Education and Research, Department of Histopathology Dogra, Nilambra; Post Graduate Institute of Medical Education and Research, Department of Experimental Medicine and Biotechnology Saikia, Uma; Post Graduate Institute of Medical Education and Research, Department of Histopathology
Khullar, Madhu; Department of Experimental Medicine and Biotechnology
Is the invited manuscript for consideration in a Special
Issue?: IACS Sherbrooke 2016 special issue Part 2
Keyword: Cardiac fibrosis, diabetes, EndMT, MRTF-A, ATF3
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Title: Transcriptional regulation of EndMT in cardiac fibrosis:
Role of MRTF-A and ATF3
Authors: Vibhuti Sharma1, Nilambra Dogra2, Uma Nahar Saikia1, Madhu
Khullar2
1 Department of Histopathology, Post Graduate Institute of Medical Education
and Research (PGIMER), Chandigarh, India-160012.
2 Department of Experimental Medicine and Biotechnology, Post Graduate
Institute of Medical Education and Research (PGIMER), Chandigarh, India-
160012.
Corresponding Author:
Prof. Madhu Khullar
Department of Experimental Medicine and Biotechnology
Post Graduate Institute of Medical Education and Research (PGIMER),
Chandigarh, India-160012.
Email: [email protected]
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Key Words: Cardiac fibrosis, diabetes, EndMT, MRTF-A, ATF3
ABSTRACT
The etiology of cardiac fibrogenesis is quite diverse but a common feature is
the presence of activated fibroblasts. Experimental evidence suggests that a
subset of cardiac fibroblasts is derived via transition of vascular endothelial
cells into fibroblasts by endothelial to mesenchymal transition (EndMT). During
EndMT, endothelial cells lose their endothelial characteristics and acquire a
mesenchymal phenotype. Molecular mechanisms and the transcriptional
mediators controlling EndMT in heart during development or disease remain
relatively undefined. Myocardin related transcription factor-A (MRTF-A)
facilitates serum response factor (SRF) in the transcription of cytoskeletal
genes during fibrosis, therefore its specific role in cardiac EndMT might be of
importance.
Activating transcription factor 3 (ATF3) activation during cardiac EndMT is
speculated, since it responds to TGF-β stimulus and controls the expression of
primary EMT markers snail, slug and twist.
Although the role of TGF-β has been established in EndMT mediated cardiac
fibrosis, targeting of TGF-β ligand has not proven to be a viable anti-fibrotic
strategy due to its wide functional importance. Thus, targeting downstream
transcriptional mediators may provide useful therapeutic approach in
attenuating cardiac fibrosis.
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Here, we discuss some of the potential transcription factors that may regulate
EndMT mediated cardiac fibrosis and their implication in type-II diabetes.
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Introduction
Fibrosis is the response of an injured tissue to repair itself, which involves
enhanced fibroblast activity leading to increased deposition of extracellular matrix
(ECM) proteins at the damaged site. Fibrosis, though an adaptive and reparative
process initially, on persistence can lead to organ dysfunction. Different
pathological conditions such as myocardial infarction, pressure or volume
overload, diabetes etc. are known to promote cardiac fibrosis, leading to adverse
remodeling of the heart architecture, resulting in impaired ventricular dysfunction
and heart failure (Berk et al. 2007; Russo & Frangogiannis 2016; Sutton &
Sharpe 2000). The pathological features and signaling events associated with
myocardial fibrosis are similar to those occurring in kidneys, liver or lungs (M.
Zeisberg & Kalluri, 2013). The extra-cellular matrix in the heart is composed of an
intricate network of collagen fibers, fibroblasts and smooth muscle cells that
provide support to cardiomyocytes (Rienks, Papageorgiou, Frangogiannis, &
Heymans, 2014). Fibroblasts are the major collagen producing cells and their
activity is required for maintaining a balanced collagen homeostasis in the heart.
Initially, fibroblast to myofibroblast differentiation serves to preserve cardiac
contractility, however, sustained stimulation under pathological conditions, results
in excessive deposition of collagen and other matrix proteins. These changes in
ECM induce processes such as inflammation and cytoskeletal reorganization,
promoting cardiac remodeling and ventricular dysfunction, leading to heart
failure. In fact, cardiac fibrosis is a major cause of cardiac morbidity and mortality
in end stage heart failure, irrespective of its etiology (Ichiki et al., 2014). Several
growth factors and cytokines such as TGF-β, TNF-α, Endothelin-I and
Angiotensin-II are known to influence collagen synthesis by stimulating
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conversion of fibroblasts to myofibroblasts that actively secrete extracellular
matrix components (Dobaczewski, Chen, & Frangogiannis, 2011; Nishida et al.,
2007; Porter & Turner, 2009). Although resident cardiac fibroblasts are the major
players in cardiac fibrosis, recent studies suggest that endothelial cells
transitioning into fibroblasts and myofibroblasts (EndMT) may also contribute to
cardiac fibrosis (E. M. Zeisberg et al., 2007). In the present review, we discuss
EndMT mediated cardiac fibrosis and its transcriptional regulation.
Cardiac Fibrosis
Fibrosis is a process of excessive fibroblast activity and deposition of
extracellular matrix proteins leading to remodeling of the organ architecture and
impaired function (M. Zeisberg & Kalluri, 2013). The extracellular matrix protein,
especially collagen, is principally involved in the morphological and structural
changes. Out of the five different forms of collagens found in extracellular
matrix of the heart, Type I and III are the most abundant forms (Bishop &
Laurent, 1995). Type I collagen molecules form thick fibres that provide
structural support and Type III collagen molecules form a fine network of fibrils
and are found in the blood vessels (Weber, Sun, Tyagi, & Cleutjens, 1994). An
imbalance of collagen metabolism involving its synthesis and degradation is
responsible for structural remodeling of a tissue. Collagen turnover rate is
tissue specific and varies with different organs with age, mechanical or
chemical stimuli. In the normal human heart the exact turnover rate of collagen
has not been determined so far. In rat models of left coronary artery ligation the
rate of collagen degradation is found to be more than its synthesis at the
myocardial infarct site (Wei, Chow, Shum, Qin, & Sanderson, 1999).
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Activated fibroblasts or myofibroblasts secrete fibrillar collagens type I and type
III. They are also responsible for α-smooth muscle actin gene expression as
well as ECM proteins (Leslie, Taatjes, Schwarz, & others, 1991). Physiological
activity of fibroblasts is required during the wound healing process which
becomes uncontrolled in a fibrotic disease causing excessive deposition of
collagen and other matrix proteins in the affected tissue.
On the basis of experiments on animal models of left ventricular pressure
overloading and acute myocardial infarction (MI), the process of fibrogenesis is
classified as of two types i.e. reactive interstitial fibrosis and replacement
fibrosis (Anderson, Sutton, & Lie, 1979; Weber, Pick, Jalil, Janicki, & Carroll,
1989). Reactive interstitial fibrosis occurs initially as an adaptive response to an
increase in the pressure overload in heart and is accompanied by
cardiomyocyte hypertrophy and necrosis (Isoyama & Nitta-Komatsubara,
2002). Animal models of acute MI on the other hand, show replacement fibrosis
as a result of tissue injury, myocyte death and inflammation (Hasenfuss, 1998).
Pathologically the replacement fibrosis is associated with loss of
cardiomyocytes whereas, reactive interstitial fibrosis leads to cardiomyocyte
hypertrophy and occurs in the perivascular regions, spreading towards the
interstitium (Anderson et al., 1979; Weber et al., 1989).
It is speculated that there exists heterogeneity among the fibroblasts that
contribute to fibrosis and their origin is thought to occur from different sources.
The valvular fibroblasts are derived from cardiac endothelium by EndMT during
embryonic development of heart (de Lange et al., 2004), whereas the interstitial
fibroblasts are derived from mesenchymal cells and increase in number by
proliferation of resident fibroblast cells (Weber, 1997).
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The different origins of activated fibroblasts or myofibroblasts in a fibrous
cardiac tissue are still being investigated. Recent studies have shown that
recapitulation of molecular pathways associated with embryonic development
occurs under the influence of growth factors and cytokines during pathological
fibrosis in the heart contributing to phenotypic transition of endothelial cells into
fibroblasts, a process termed as endothelial mesenchymal transition (EndMT)
(E. M. Zeisberg et al., 2007).
Endothelial to Mesenchymal Transition (EndMT) and Cardiac Fibrosis
Endothelial cells lining the blood vessels in the heart exhibit phenotypic
variability and plasticity depending upon their origin and response to
stimulation. While this cellular plasticity facilitates vessel remodeling during
angiogenesis, it also contributes to fibrosis via endothelial to mesenchymal
transition in the heart. During EndMT, endothelial cells delaminate from a
polarized cell layer, lose their cellular junctions, acquire migratory capacity and
penetrate the underlying tissue. This process is similar to epithelial
mesenchymal transition (EMT) which is a well studied phenomenon during
embryonic development as well as in cancer progression.
EndMT is classified as type II EMT wherein endothelial cells transition to
fibroblasts. EMT occuring during embryonic development and connective tissue
formation is classified as type I and type III EMT occurs during cancer and
metastasis (Carew, Wang, & Kantharidis, 2012).
Although the process of EndMT is very much similar to EMT, still there are
certain pathophysiological differences between the two processes. EMT occurs
extensively during gastrulation to form neural crest, heart and musculoskeletal
system, whereas EndMT is responsible for the formation of myocardial valves,
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septa and aotic intima during heart development. EMT changes are mainly
related to tumorigenesis, however, EndMT is not seen in such cases. The
cellular mediators such as TGF-β, Wnt, Notch, MMPs, FGF-2 play a common
role in both transitioning processes. The major difference lies in the change in
expression of different cellular markers during the transitioning event (Carew et
al., 2012) (Table1).
The role of endothelial to mesenchymal transition contributing to the origin of
activated fibroblasts or myofibroblasts has recently gained importance. The
origin of fibroblasts in cardiac fibrosis has been studied by fate mapping
experiments that are done to trace the lineage of any cell in the body. Zeisberg
et al. for the first time demonstrated the origin of cardiac fibroblast cells from
endothelial cells in an adult tissue via EndMT. They used transgenic mice
expressing green fluorescent protein (GFP) under the control of fibroblast
specific protein 1 (FSP1) gene promoter and the cells of endothelial origin were
marked by lacZ reporter gene expression. Cardiac fibrosis was experimentally
induced in these mice by aortic banding causing pressure overload in the heart.
LacZ positivity was observed throughout the fibrotic tissue in banded hearts
compared to the normal hearts where it was restricted only to the endothelial
cells associated with blood vessels. The lacZ positive cells also showed co-
expression of β-gal and FSP1 confirming their mesenchymal origin.This data
was further supported by in vitro studies performed in human coronary artery
endothelial cells where the authors conclusively demonstrated that cardiac
fibrosis was associated with the emergence of fibroblasts originating from
endothelial cells. They also showed that recombinant human bone
morphogenic protein 7 (hBMP7) - an antagonist in the TGF-β fibrotic pathway,
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allowed preservation of the endothelial cell phenotype, and that its systemic
administration significantly inhibited EndMT and the progression of cardiac
fibrosis in mouse models of pressure overload and chronic allograft rejection.
(E. M. Zeisberg et al., 2007).
Owing to the lack of cell specific markers differentiating fibroblasts from
myofibroblasts, the exact cellular precursor that gives rise to cardiac
myofibroblasts following an injury still remains a controversial topic. α-SMA
which is generally considered to be a myofibroblast marker is also expressed in
smooth muscle cells. More recently periostin has emerged as a marker protein
exclusively expressed by myofibroblasts as determined by RNAseq analysis
and periostin lineage-tracing has been used to identify the cellular source of
myofibroblasts in an injured heart. Contrary to the earlier findings where about
70% of cardiac myofibroblasts were found to originate via EndMT in a pressure
overload model (E. M. Zeisberg et al., 2007), Kanisicak et al. found negligible
contribution of cardiac endothelial cells or smooth muscle cells towards
myofibroblasts generation in the infarct regions of adult mouse hearts with
pressure overload or MI. They demonstrated that majority of cardiac
myofibroblasts were derived from Tcf21- expressing tissue resident fibroblasts
in the injured heart (Kanisicak et al., 2016). Therefore, identification of specific
myofibroblasts marker still remain a challenging task and further studies are
required to investigate the generation of cardiac myofibroblasts during
pathological conditions.
Diabetes associated cardiac fibrosis and EndMT
Type-II diabetes mellitus is a global pandemic that is associated with a greater
risk of developing heart failure (Olokoba, Obateru, & Olokoba, 2012). Cardiac
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fibrosis is a prominent feature observed in diabetic patients and is one of the
major predisposing factors for development of heart failure in diabetic patients
(Regan et al., 1977). In a diabetic heart, fibrosis is an end stage phenomenon
and is a result of several pathological conditions such as hyperglycemia,
ischemia, stretch, inflammation, oxidative stress, advanced glycation end
products and several neurohormonal mediators (Creemers & Pinto, 2010; Pitt &
Zannad, 2012). Diabetes induced increased extra cellular matrix (ECM) protein
deposition, interstitial fibrosis and myocyte hypertrophy results in ventricular
dysfunction and eventually leads to heart failure in diabetic patients. In general,
diabetic patients develop left ventricular hypertrophy, narrowing of intramural
coronary arteries and thickening of arteriolar wall due to excessive fibrosis
accompanied by accumulation of muco-polysaccharides and elastic tissue
remodeling (Fischer, Barner, & Larose, 1984; GREGOR, WIDIMSKY,
ROSTLAPIL, CERVENKA, & VISEK, 1984).
Angiotensin II has been shown to have potential role in the development of
cardiac fibrosis in diabetic rats (Singh, Le, Khode, Baker, & Kumar, 2008). It
has been suggested that Angiotensin II may also be mediating EndMT changes
in diabetic hearts. Tang et al reported that diabetic rats treated with angiotensin
II receptor blocker Irbesartan, showed diminished EndMT features (Tang et al.,
2013).The role of EndMT in cardiac fibrosis in diabetic hearts have also been
observed in type- I diabetes model of endothelial cell–specific ET-1 knockout
mice (Widyantoro et al., 2010). Persistent high levels of endothelin-1 (ET-1) - a
known vasoconstrictor in the plasma of diabetic patients is shown to be
associated with the development of cardiac fibrosis and experimental studies
have reported the beneficial effect of endothelin receptor antagonist in
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attenuating diabetes mellitus–induced myocardial dysfunction. To identify the
molecular mechanism linking ET-1 to diabetic cardiomyopathy, Widyantoro et
al. developed type-I diabetes model of endothelial cell–specific ET-1 knockout
mice. They showed that diabetes mellitus–induced cardiac fibrosis was
associated with the emergence of fibroblasts from endothelial cells. Further,
this EndMT appeared to be mediated by ET-1 as its targeted gene silencing
inhibited high glucose induced EndMT in cultured endothelial cells (Schneider
et al., 2002; Widyantoro et al., 2010).
In a study carried out by our group on streptozotocin treated rat model of type-II
diabetes, we observed EndMT changes in cardiac vessels of diabetic rats
(manuscript in preparation). We observed a significant decrease in the
epithelial marker protein VE-cadherin and increase in mesenchymal marker α-
SMA specifically in the vascular endothelium of diabetic rats, suggesting
EndMT. We also observed co-expression of endothelial and fibroblast marker
proteins CD-31 and α-SMA in the endothelial lining of cardiac vessels, further
supporting occurrence of EndMT in diabetic hearts. Similar changes have been
reported in diabetic nephropathy where about 35% of fibroblasts have been
shown to be derived via EndMT from microvascular endothelial cells (E. M.
Zeisberg, Potenta, Sugimoto, Zeisberg, & Kalluri, 2008).
Thus, our results further support the occurrence of EndMT mediated cardiac
fibrosis in diabetes.
Transcription factors involved in EMT/EndMT and cardiac fibrosis
A number of transcription factors have been shown to activate EMT during
development and disease. Majority of the recognized inducers of EMT are
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implicated in the loss of cell adhesion and act as E-cadherin repressors while
some of them affect extracellular matrix remodeling and cytoskeletal
reorganization. E-cadherin is a calcium dependent cell-cell adhesion
glycoprotein required for establishment of apical-basal polarity in epithelial cells
(Wheelock, Shintani, Maeda, Fukumoto, & Johnson, 2008). It is a component of
adherens junctions and also regulates the formation of tight junctions and
desmosomes. The junctional protein E-cadherin is degraded during EMT
causing transcriptional activation of catenin proteins (β-catenin, p120) and is
accompanied by increased expression of neural cadherin (N-cadherin)
(Wheelock et al., 2008). N-cadherin interacts with catenins and receptor
tyrosine kinases (PDGF, FGFRs) to form cytoskeletal connections (Wheelock
et al., 2008). Transcription factors SNAIL,TWIST and ZEB are master
regulators known to drive EMT and are activated early in the process (Peinado,
Olmeda, & Cano, 2007).
SNAIL transcription factors: SNAIL1 and SNAIL 2 (SLUG) are zinc- finger
containing transcription factors. They repress epithelial genes by binding to
E-box DNA sequences through their carboxy-terminal zinc-finger domains.
Snail proteins play an important role during embryonic development facilitating
cell migration required for the formation of different tissues and is thus often
referred to as mesodermal determinant gene. Almost all known EMT triggers
such as epidermal growth factor (EGF), fibroblast growth factor (FGF), bone
morphogenetic proteins (BMPs), transforming growth factor-β (TGF-β) induce
expression of snail genes. The major function of snail proteins is to regulate cell
movement, which in a pathological state such as cancer becomes fatal
favouring tumour progression and metastasis (Jin et al., 2010). EMT associated
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with organ fibrosis and wound healing also involves activation of snail genes.
Snail 1 expression is observed in mesothelial cells of patients with renal fibrosis
and also in cultured human mesothelial cells upon TGF- β treatment (Yáñez-Mó
et al., 2003). Snail 1 is also activated in lens epithelial cells undergoing EMT
(Saika et al., 2004). SNAIL 1 represses E-cadherin expression and induces
EMT when overexpressed in epithelial cells (Batlle et al., 2000).While TGF-β
signals have been shown to induce the expression of SNAIL during EMT of
mammary epithelial cells (Shirakihara, Saitoh, & Miyazono, 2007), the causal
relationship between TGF-β-induced SNAIL expression and EMT has not yet
been fully elucidated.
Lee et al. investigated role of SNAIL in cardiac fibrosis in a mouse model of
ischemia reperfusion injury. They showed that SNAIL expression was
significantly increased in the endocardial and myocardial regions at the site of
tissue injury. Hypoxia-reoxygenation in cultured endothelial cells, cardiac
fibroblasts or cardiomyocytes induced SNAIL expression, specifically in the
endothelial cells. Further, overexpression of Snail gene in endothelial cells
caused EndMT and stimulated conversion of cardiac fibroblasts to activated
myofibroblasts through connective tissue growth factor (CTGF) secretion (Lee
et al., 2013). Thus, these authors reported that paracrine signalling via CTGF
was important player in trans-differentiation of cardiac fibroblasts to
myofibroblasts and showed CTGF as a downstream factor of Snail gene
expression responsible for cross-talk between endothelial and fibroblast cells
during cardiac fibrosis (Lee et al., 2013).
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Basic Helix-Loop-Helix transcription factors: TWIST 1, TWIST 2, E12, E47
and ID proteins belonging to this class of transcription factors are known to play
key role in EMT by repressing E-cadherin expression (Peinado et al., 2007).
Scleraxis, a basic Helix-Loop-Helix transcription factor, has been shown to
regulate collagen gene expression in the cardiac fibroblasts and in the fibrotic
scars following myocardial infarction (Espira et al., 2009). It has been also
shown to regulate cardiac fibroblast to myofibroblast transition. A number of
matrix proteins have been identified as its direct transcriptional targets; for
example, Scleraxis was shown to control the transcription of vimentin, MMP2,
fibronectin1, snail, twist and fibrillar collagens in primary cardiac fibroblasts and
myofibroblasts. Scleraxis was found to directly bind to α-SMA promoter and
activate assembly of stress fibers which is a characteristic feature of
myofibroblasts (Bagchi et al., 2016). Levay et al reported that Scleraxis was
required for remodeling heart valve structures as Scleraxis knockout mice
showed thickened heart valves and fibrous annulus of atrio-ventricular canal.
Both these structures are known to be dependent on epicardial EMT. Further,
scleraxis deficient mice showed persistent increased expression of MSX1 and
SNAI1, mesenchymal markers, suggesting a potential role in EMT (Levay et al.,
2008). However, its role in EndMT associated cardiac fibrosis remains to be
examined.
Transcription factor 21 (TCF21) is another bHLH transcription factor expressed
during epicardial development (Tandon, Miteva, Kuchenbrod, Cristea, &
Conlon, 2013). Lineage tracing experiments have revealed that TCF21 is
required for epicardial EMT in the developing heart and its expression was
found to be induced in a subset of epicardial cells prior to the onset of EMT
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process, indicating its early involvement in the formation of cardiac fibroblasts
from epicardial cells (Acharya et al., 2012). Although, TCF21 activity is known
to regulate EMT during cardiac valve remodeling at the time of embryonic heart
development, its role in cardiac EMT during pathological states and valve
disease is yet to be ascertained.
ZEB transcription factors: ZEB 1, ZEB 2 are zinc-finger-homeodomain
proteins and act as transcriptional activators or repressors by binding to E-box
regulatory sequences located in the promoters of various genes. ZEB factors
are implicated in EMT in several tumour types and are essentially identified as
regulators of cell polarity and junctional proteins such as CRB3, PATJ, LGL2,
occludin and claudin-7 (Günzel & Alan, 2013; Lamouille, Xu, & Derynck, 2014;
Moreno-Bueno, Portillo, & Cano, 2008). Zeb factors are also reported to
repress transcription of type II collagen gene (Col2a1) during chondrogenesis
(Murray, Precht, Balakir, & Horton, 2000) and type I collagen gene (Col1a2)
expression in osteoblasts(Terraz, Toman, Delauche, Ronco, & Rossert, 2001).
During EndMT mesenchymal cells are formed that express α-SMA and serum
response factor (SRF) activity is required for α-SMA transcription. SRF activity
is facilitated by interaction with ZEB1 and SMAD3 (Nishimura et al., 2006). Zeb
binding sites have also been identified in TGF-β/BMP promoters, however their
role in TGF-β /BMP signaling is not well defined. Zeb1 is reported to repress E-
cadherin expression by recruiting chromatin remodeling protein BRG1
(Spaderna et al. 2008; Xu et al. 2009). Although, SNAIL1 activity is required for
the expression of the ZEB genes(Thiery, Acloque, Huang, & Nieto, 2009), loss
of E-cadherin expression in TGF-β induced EMT is found to be independent of
SNAIL activity and dependent on ZEB factors(Kato et al., 2007).
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Loss of E-cadherin alone is not sufficient to initiate EMT(Thiery, 2002) and
additional transcription factors belonging to GATA, FOX and SOX families of
transcription factors also contribute in inducing EMT either by regulating the
epithelial gene expression or by synergizing with other factors(Bresnick, Lee,
Fujiwara, Johnson, & Keles, 2010; Eijkelenboom & Burgering, 2013; Kondoh &
Kamachi, 2010). Previous studies suggest that transcription factors playing a
crucial role in EMT are also implicated in EndMT-mediated cardiac fibrosis. It is
of great interest to identify transcription factors specifically activated in heart
following an injury or in response to a pathological condition, triggering cardiac
EndMT and fibrogenesis.
Myocardin Related Transcription Factor-A (MRTF-A)
MRTF-A is a transcriptional co-activator of serum response factor (SRF) that
synergistically activates transcription of genes encoding cardiac and smooth
muscle cell restricted cytoskeletal and contractile proteins in multiple cell
lineages such as undifferentiated embryonic stem cells and fibroblasts (Wang
et al. 2002). MRTF-A is sequestered in the cytoplasm forming a stable complex
with monomeric G-actin (Miralles, Posern, Zaromytidou, & Treisman, 2003). G-
actin monomers control the nuclear-cytoplasmic shuttling of MRTF-A and its
nuclear activity, which in turn controls the activity of SRF. Actin polymerization
leads to release of MRTF-A and its nuclear translocation eliciting SRF-directed
target gene activation (Miralles et al., 2003).
It has been shown that MRTF-A activates fibrotic gene program that includes
genes involved in extracellular matrix production and smooth muscle cell
differentiation in the heart and also promotes the conversion of cardiac
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fibroblasts to myofibroblasts following myocardial infarction (MI) (Small et al.,
2010).
Falling concentrations of cytoplasmic G-actin owing to the assembly of F-actin
filaments cause MRTF-A to associate with SRF in the nucleus, activating the
transcription of contractile genes and fibrosis associated proteins including
collagen1A2 (Parmacek, 2010) (Figure 1). TGF-β signaling through Smad
pathway activation is another mechanism of MRTF-A mediated transcriptional
activation. In the nucleus, Smad proteins associate with MRTF-A and the
complex binds to regulatory elements controlling transcription of fibrosis
associated genes (Parmacek, 2010).
MRTF-A is expected to contribute to EndMT changes occurring during
pathological fibrotic remodeling of heart, since it involves extensive actin
dynamics and activation of cytoskeletal genes.
We examined cardiac tissue sections of Streptozotocin treated type-II diabetic
rats and observed nuclear localization of transcription factor MRTF-A
specifically in cardiac endothelial cells indicating its activation in these cells in
response to a diabetic milieu.
We also examined cardiac autopsy tissues of subjects having a history of long
standing type-II diabetes. We observed MRTF-A nuclear localization in cardiac
endothelium similar to the changes seen in diabetic rats. MRTF-A activation
was further confirmed by observing its target gene collagen expression in
diabetic hearts. Trichrome staining in the myocardial sections of diabetic rats
and diabetic archived autopsy tissues revealed extensive deposition of collagen
around the blood vessels and also in the interstitium suggesting a role of
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MRTF-A in diabetes induced pathological fibrotic remodeling of the heart
(results communicated). Our results are supported by a previous study related
to scleroderma related fibrosis, which has reported nuclear localization of
MRTF-A in scleroderma tissues (Shiwen et al., 2015).
To our knowledge, this is the first study examining the expression of MRTF-A in
diabetic heart tissues and shows that MRTF-A is localized in the nuclei of
cardiac endothelial cells of diabetic hearts.
Activating Transcription Factor 3 (ATF3)
ATF3 is a member of ATF/CREB family of transcription factors which regulate
gene expression by binding to a common DNA sequence motif (TGACGTCA).
It has been characterized as an immediate-early stress response gene. ATF3 is
a Smad4 dependent TGF-β regulated gene. In cultured cells, ATF3 is induced
by a variety of signals, including cytokines, genotoxic agents and cell death
inducing agents (Hai & Hartman, 2001). The role of ATF3 in TGF-β induced
epithelial to mesenchymal transition (EMT) has been shown in breast cancer
cells (Yin et al., 2010). ATF3 is also induced in pancreatic β-cells by cytokines,
elevated glucose or free fatty acids and is expressed in the islets of type- I and
type-II diabetic patients, where it is known to induce β-cell apoptosis (Hartman
et al., 2004). ATF3 was shown to repress Insulin Receptor Substrate 2 (IRS2)
gene - a prosurvival factor in pancreatic β-cells and its proapoptotic function
has been reported (Zmuda et al., 2010). ATF3 is also identified as an essential
transcription factor activated in response to endoplasmic reticulum (ER) stress
occurring due to unfolded protein response (Brooks et al., 2014). ATF3
maintains a balance between proliferative and apoptotic signals during the
development of cancer. Functionally, ectopic expression of ATF3 leads to
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morphological changes and alterations of markers consistent with EMT in
MCF10CA1a human breast cancer cells. Overexpression of ATF3 promoted
metastasis in these cells by up regulating fibronectin-1, TWIST1 and Slug
transcripts (Yin et al., 2010), which are key regulators of cell-cell or cell-
extracellular matrix interaction.
In the heart, ATF3 is induced by myocardial ischemia and myocardial ischemia
coupled with reperfusion. Studies in transgenic mice demonstrated that cardiac
specific expression of ATF3 caused atrial enlargement and cardiomyocyte
hypertrophy (Okamoto et al., 2001). In the heart, hypoxia,
ischemia/reperfusion, hypertrophy, pressure overload, heart failure, diabetes
and drug-induced insults can result in activation of ER stress (Eizirik, Cardozo,
& Cnop, 2008; Toth et al., 2007). ER stress which is known to trigger EndMT in
renal tubular cells in experimental models of nephropathy (Han et al., 2008;
Pallet et al., 2008), is also speculated to be involved in cardiac EndMT through
ATF3 activation.
We examined the cardiac expression of ATF3 in the ventricular tissue sections
of Streptozotocin treated diabetic rats, as well as in the subjects (cardiac
autopsy tissue) having prolonged type-II diabetes by immunohistochemistry.
ATF3 expression was specifically observed in the endothelial lining of small
blood vessels (capillaries) in these tissue sections. To explore the functional
relevance of ATF3 activation with EndMT in the vascular endothelium of
diabetic hearts, we measured ATF3 expression in cardiac microvascular
endothelial cells (CMVECs) after high glucose treatment (HG, 30mM). ATF3
induction and intra nuclear localization was observed in these cells following
HG treatment (manuscript in preparation). A luciferase reporter assay
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confirmed activation of Slug promoter in CMVECs in response to HG treatment.
Slug is a well-established EMT/EndMT marker and in a previous report it has
been shown as an ATF3 target gene (Yin et al., 2010). Further, overexpression
of ATF3 caused upregulation of Slug gene expression, indicating its role in HG
induced EndMT in these cells. Our results suggest that ATF3 may have a role
in HG induced cardiac EndMT.
Therapeutics Targeting EndMT mediated Cardiac Fibrosis
TGF-β1, a profibrotic cytokine is one of the several factors shown to induce
EndMT mediated cardiac fibrosis (M. Zeisberg & Kalluri, 2013). Current
therapeutic approaches explore targeting of TGF-β induced EndMT pathway for
inhibiting/attenuating cardiac fibrosis. A synthetic small molecular inhibitor of
TGF-β-receptor I (TβRI) kinase (SB431542) has been shown to effectively
block TGF-β2-induced cardiac EndMT (Ghosh, Nagpal, Covington, Michaels, &
Vaughan, 2012). Similarly, targeting the epigenetic regulator Acetyltransferase
p300 (ATp300), a coactivator of profibrotic signaling which is significantly
elevated during EndMT is also being explored (Ghosh et al., 2012). Potential
involvement of other components of TGF-β signaling or EMT-related
transcription factors in regulating EndMT-mediated cardiac fibrosis is currently
being investigated. Recently, it has been found that dipeptidyl peptidase 4
(DPP-4) inhibitor- Linagliptin inhibits the endothelial-mesenchymal transition
(EndMT) and ameliorates diabetic kidney fibrosis via the induction of miR-29
(Shi, Kanasaki, & Koya, 2016). However, the substantial contribution of EndMT
and underlying molecular basis of this process in pathological fibrosis of the
heart is still under investigation. Zeisberg, Kalluri et al. have also shown that
systemic administration of recombinant human BMP-7 (rhBMP-7) attenuated
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EndMT both in vitro and in vivo and can be used to inhibit EndMT mediated
cardiac fibrosis. In a mouse model of chronic heart rejection rhBMP-7
administration reduced fibrosis by 50%compared to the vehicle treated mice.
They also showed substantial inhibition of TGF-β induced EndMT by rhBMP-7
treatment in human coronary endothelial cells (HCECs) (E. M. Zeisberg et al.,
2007). Effect of exogenous recombinant BMP-7 supplementation has also been
shown to effectively ameliorate endothelial to mesenchymal transition in rat
model of endocardial fibroelastosis (Xu et al., 2015). Xu et al. observed that
aberrant DNA promoter methylation and inhibition of Ras-GTP activity
enhanced EndMT and cardiac fibrosis, suggesting epigenetic mechanisms may
regulate EndMT and cardiac fibrosis.
The underlying mechanism by which BMP-7 exhibits anti-fibrotic effects is by
inhibiting TGF-β induced EMT (M. Zeisberg, Shah, & Kalluri, 2005). BMP-7
induces ID proteins that promote E-cadherin expression and maintain epithelial
phenotype (Kowanetz, Valcourt, Bergström, Heldin, & Moustakas, 2004). BMP-
7 also induces SMAD7 which blocks the Smad dependent TGF- β signalling
pathway (Benchabane & Wrana, 2003). During EMT an intricate networking
occurs between BMP-7 and TGF-β to decide the fate of cell towards either
epithelial or mesenchymal type.
microRNAs in EndMT: Further, the role of microRNAs in the EndMT
progression is increasingly being recognized. Ghosh et al reported differential
expression of several microRNAS in mouse cardiac endothelial cells during
EndMT. Micro RNAs such as miR-125b, Let-7c, Let-7g, miR-21, miR-30b and
miR-195 were significantly elevated and miR-122a, miR-127, miR-196, and
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miR-375 were found to be significantly downregulated during EndMT (Ghosh et
al., 2012). Constitutive expression of miR-31 has been shown to positively
regulate TGF-β induced EndMT, actin remodeling and MRTF-A activation by
targeting VAV3 guanine nucleotide exchange factor (Katsura et al., 2016).
Cardiac muscle enriched microRNA miR-486 promotes (PI3K)–AKT signalling
and its transcription is directly controlled by SRF and MRTF-A (Small et al.,
2010). Elevated levels of miR-494 are found in mouse hearts after ischemia-
reperfusion injury and cardiac-specific overexpression of mir-494 in transgenic
mice subjected to ischemia-reperfusion, improved recovery of cardiac tissue by
suppressing cardiomyocyte apoptosis (X. Wang et al., 2010). ATF3 has been
identified as a target of miR-494 and release of miR-494 in urine has been
associated with inhibition of ATF3 and activation of inflammatory cytokine gene
expression post I/R induced kidney injury (Lan et al., 2012). Thus, its role in
inflammatory pathways operating in cardiac pathologies is also speculated and
targeting these microRNAs offers a potential therapeutic potential for mitigating
cardiac fibrosis.
Conclusion and perspective
Cardiac fibrosis, irrespective of its etiology, is one of the major factors
contributing to heart failure. Cardiac fibroblasts are the major players
contributing to cardiac fibrosis, however, their source and molecular
mechanisms leading to their increased proliferation are not well delineated.
Recent literature suggests that endothelial to mesenchymal transition (EndMT)
may be an important contributor to the cardiac fibrosis in diseased states. A few
reports, on the other hand, suggest that mesenchymal transitions do not
significantly contribute to the pool of activated fibroblasts in heart. Therefore,
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further investigations are required to determine the extent to which EndMT
contributes to fibrosis in the heart and the stimuli that trigger EndMT in an
injured or diseased heart need to be ascertained. Various stimuli such as
inflammation, hyperglycemia, hyperlipidemia can trigger EndMT by activating
cellular pathways facilitating this transition. Growth factors and signaling
pathways that govern EMT are also involved in EndMT. However, compared to
EMT, less is known about the transcriptional mediators regulating EndMT in the
embryonic heart and cardiac fibrosis and is focus of several recent studies.
Transcriptional activation of MRTF-A and ATF3 in the endothelium of diabetic
hearts is an important observation to further understand EndMT in context of
cardiac fibrosis and may hold a key to develop improved therapies. Thus,
research in the area of EndMT holds potential to reveal new perspectives that
can contribute to the development of possible therapeutic interventions to
suppress cardiac fibrosis.
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Figure 1: Mechanism of MRTF-A action: MRTF-A is sequestered in the cytoplasm bound to G-
actin monomers. Rho activation in response to mechanical or chemical stimuli causes
F-actin assembly dissociating MRTF-A from G-actin and its intra-nuclear localization
where it activates cytoskeletal and fibrotic genes by associating with Serum Response
Factor (SRF).
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