Transcriptional regulation of EndMT in cardiac fibrosis: Role of … · EndMT mediated cardiac fibrosis and its transcriptional regulation. Cardiac Fibrosis Fibrosis is a process

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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

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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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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