Endothelial-to-Mesenchymal Transition: Role in Cardiac Fibrosis

Abstract

Endothelial-to-mesenchymal transition (EndMT) is a complex biological process by which endothelial cells lose their endothelial cell characteristics and acquire mesenchymal cell properties under certain physiological or pathological conditions. Recently, it has been found that EndMT plays an important role in the occurrence and development of fibrotic cardiovascular diseases. In this review, we first summarize the main induction pathways involved in EndMT process. In addition, we discuss the role of EndMT in fibrotic cardiovascular diseases and its potential implication in new therapeutic interventions.

Introduction

Endothelial cells (ECs) and mesenchymal cells are two different cell types, although both are derived from mesoderm. They are distinct in cell morphology, cell phenotype, physiological function, and gene sequence. Under some physiological and pathological conditions including fibrosis, embryonic development, wound repair, inflammation and tumor, the phenotype of endothelial cells may switch to mesenchymal cells, termed epithelial-to mesenchymal transition (EndMT). EndMT was first discovered by Johannes holtfreter in the study of early embryonic development and differentiation in 1939. ¹ During the process of EndMT, endothelial cells lose some of the characteristics of endothelial cells, and obtained several characteristics of mesenchymal cells, including the loss of tight connection, the enhancement of motility, and increased extracellular matrix protein secretion (Figure 1). ²

Figure 1.

Phenotypic modifications occur in the progression of EndMT which is controlled by several pathological factors and signaling pathways.

Cardiac fibrosis is a pathological manifestation in response to stress and injury by various cardiovascular diseases such as cardiomyopathy, coronary heart disease, hypertension, and heart failure. In recent years, several publications have described the critical role of EndMT in the pathogenesis and progression of cardiac fibrosis in the tissues from patients with cardiovascular diseases. Promoted by various pathological factors, myofibroblasts derived from endothelial cells through EndMT process secrete excessive extracellular matrix to the tissues surrounding the heart and blood vessels, which cause excessive deposition of collagen. The accumulation of collagen damages myocardial structure and results in ventricular remodeling and cardiac fibrosis. ³ Long-term cardiac fibrosis further leads to the ventricular dysfunction, cardiac microcirculation blocking, decreased cardiac compliance and cardiac contractility. ⁴ It has been shown that EndMT promotes the development of interstitial and perivascular fibrosis in acute myocardial infarction (AMI). ⁵ The expression levels of EndMT-related genes including Snail, Slug, and Twist are also dramatically increased in the left ventricular myocardial tissues from patients with end-stage of cardiac failure. ⁶

Signaling Pathways Involved in EndMT

The molecular mechanisms of EndMT in cardiovascular diseases are mainly controlled by the following signaling pathways and pathological factors (Figure 2).

Figure 2.

Overview of various signaling pathways and pathological factors involved in EndMT process. In Smad dependent TGF-β signaling pathway, TGF-β I/II receptor complex is activated upon TGF-β binding on the cell membrane. Smad2 and Smad3 proteins are in turn phosphorylated and the Smad2/Smad3/Smad4 complex is formed by the recruitment of Smad4 to the activated Smad2 and Smad3. Then entire complex then translocates to the nucleus and binds to Smad binding elements (SBE), inducing the expression of profibrotic extracellular matrix (ECM) and transcription factors such as Snail, Slug and Twist1. Smad6/Smad7 inhibits TGF-β-induced signaling transduction by negative regulation on the activated TGF-β receptors and/or Smad complex. TGF-β induces Smad2/3-independent pathways mainly by the mitogen activated protein kinase (MAPK) family including extracellular signal- regulated kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinases (JNK) and other kinases such as phosphatidylinositol 3-kinase (PI3K). Beside TGF-β induced EndMT process, Notch, Wnt, hypoxia and reactive oxygen species (ROS) are also involved in the regulation of EndMT process. The downstream effectors of Notch and Wnt pathways, NICD and catenin, are important modulators of EndMT. HIF-1 mediates the activation of Snail1 in hypoxia induced EndMT. ROS accumulation induces the activation of NOX1/4 oxidases which subsequently promotes the activation of NF-κB. The consequences of these pathways are either decreased expression of endothelial cell specific markers or increased expression of mesenchymal cell specific marker which induced by EndMT responsive transcription factors including Snail and Twist.

TGF-β Signaling Pathway

In mammalians, the TGF-β superfamily mainly comprise three subtypes of TGF-β (TGF-β1-3) and six subtypes of bone morphogenetic proteins (BMP2-7). ⁷ The ligands induce the downstream signaling through the heterodimer receptors complex formed by two type I TGF-β receptors (ALK5 and ALK2) and two type II TGF-β receptors (TGF-βR2) with intrinsic serine/threonine kinase activity. ^{8 -10} TGF-β induced signaling cascade is the most important regulatory pathway in the initiation and progression of EndMT. ¹⁰ It participates in the regulation of myocardial fibrosis by activating both Smad-dependent and Smad-independent signaling pathways. ¹¹ These downstream processes are mediated by numerous transcriptional regulators such as Snail family of zinc finger transcription factors Snail, Slug and Twist, as well as Zeb transcriptional factors Zeb1 and Zeb2. ^{11 -14} The transcription factors Snail, Slug, and Twist are activated to promote the occurrence of EndMT. Snail and Slug proteins inhibit the expression of endothelial cell specific-expressed adhesion proteins including endothelial cadherin, VE-cadherin by directly binding to their promoter element, leading to the reduction of endothelial cell junction. ^{15 -19} Snail also up-regulates the expression of RhoA and vimentin, which promote the degradation of endothelial basement membrane and rearrangement of the cytoskeleton by increased expression of MMPs. ^{19 -21} Twist promotes EndMT by down-regulating the expression of VE-cadherin through negatively regulation of transcription via the basic-helix-loop-helix. ^{21 -23}

In Smad-dependent signaling pathway, upon the binding of TGF-β to TGF-βRI and TGF-βRII complex on the endothelial cell membrane, TGF-βRI is phosphorylated by TGF-βRII kinase. The intracellular effector proteins, Smad2 and Smad3, are subsequently recruited to the docking site in the glycine–serine-rich domain (GS domain) of TGF-βRI. The specific Serine residues in the c-terminal domain of Smads are phosphorylated and the activated Smad2/3 forms a heteromeric complex with Smad4. The entire Smad complex then transfers to the nucleus and binds Smad binding elements (SBE), promoting the transcription of EndMT related genes. ²⁴

Smads also directly interact with the Snail1 promoter to induce transcription of EndMT-related genes and inhibit the expression of VE-cadherin and occluding. ^17,25 This interaction also indirectly affects the expression of other factors including Zeb transcription factor and high mobility group factor HGMA2, regulating the expression of Snail, Slug and Twist. ^{26 -28} In addition, TGF-β-mediated signaling is antagonized by inhibitory Smad proteins (Smad6/7) which block Smad2/3 recruitment to TGF-βRI and the formation of Smad2/3/4 complex. ^{29 -31}

In Smad-independent pathways, TGF-β participates in the EndMT process by activating mitogen-activated protein kinase (MAPK) ERK, JNK, p38, small G proteins (Ras, RhoA, Rac1, CDC42, mTOR), as well as NF-κB. Wylie-Sears et al revealed that TGF-β1 induces EndMT by ERK phosphorylation in sheep heart valve endothelial cells. ³² The EndMT process is suppressed after applying ERK phosphorylation inhibitors. Montorfano et al. demonstrated that the EndMT would be induced in endothelial cells treated with H₂O₂ through TGF-β/p38 MAPK signaling pathway. ³³ TGF-β induces EndMT process through phosphatidylinositol 3-kinase (PI3 K)/Akt signaling pathway by activating autoreceptor EGF and PDGF receptors. ^34,35

Further studies found that TGFβ1-Smad2/3 signaling pathway mediates the occurrence of EndMT and further promotes cardiac fibrosis. Lee et al. found that in the mice model of cardiac fibrosis induced by myocardial infarction, the expression of Snail is increased significantly in EndMT-derived cells. ³⁶ The up-regulation of Snail subsequently promotes the release of connective tissue growth factor (CTGF) which induces the transformation of cardiac fibroblasts into myofibroblasts by paracrine and ultimately promotes myocardial fibrosis. ^25,37 Moreover, current research has found that inhibition of EndMT reduces myocardial fibrosis. In Zeisberg’s study, myocardial fibrosis was much reduced in mice treated with human recombinant BMP protein 7, rhBMP7, which inhibit the EndMT process by blocking the activation of the TGFβ1-Smad2/3 signaling pathway. ³² Jeong et al. showed that connective tissue growth factor 5 (CCN5) inhibits myocardial fibrosis in pressure overload mice model by inhibiting EndMT. ³⁸ Similar results were observed by in vitro investigations, CCN5 inhibits TGFβ2-induced EndMT in human coronary artery endothelial cells.

In addition, BMPs negatively regulate EndMT by activating downstream molecules Smad1, 5 and 8. Smad1, 5 and 8, together with Smad4 translocating to the nucleus where the Smad complex induces the activation of BMP related genes. Several studies have described the critical role of BMPs (in particular BMP7) in cardiac fibrosis by inhibiting pathological EndMT. ^6,32

Notch Signaling Pathway

The Notch receptor is a single transmembrane receptor which is divided into three parts: the extracellular region, the transmembrane region and the intracellular region (NICD). After activation of Notch heterodimer formed by different receptors (Notch1-4) on the cell membrane, the cleaved soluble NICD translocate into the nucleus where it binds to different transcriptional factors and regulates target gene expression. ³⁹

The Notch pathway is key signal in myocardial fibrosis by promoting the expression of transcription factors Snail, Slug, and Zeb1 in the EndMT process. The interaction of Notch and Slug is crucial for inhibiting the activation of VE-cadherin and β-catenin. Notch overexpression leads to decreased expression of VE-cadherin in the endothelial cells, and EndMT process is subsequently initiated. In the study of human umbilical vein endothelial cells (HUVECs), it was found that Notch signaling plays an important role in the development of EndMT induced by TGF-β. ²⁸ Similar phenomenon was found in the study of human glomerular endothelial cells. After blocking Notch pathway by its antagonist, this process of EndMT was inhibited. ⁴⁰

Wnt Signaling Pathway

The classical Wnt/β-catenin signaling pathway has been shown as a stimulus of promoting EndMT process. β-catenin is the most important downstream effector in the Wnt signaling pathway by which induces the activation of Wnt-related target genes. Under unstimulated conditions, β-catenin maintains at a low level as the protein is degraded by the degradation complex formed by Glycogen synthase kinase 3β (GSK-3β), adenomatous polyposis coli (APC) and Axin. Upon activation, Wnt protein interacts with the receptor Frizzled and co-receptors LRP5/LRP6 at the cell membrane and inhibits the activity of the degradation complex. Thereby β-catenin accumulates in the cytoplasm and subsequently transfer to the nucleus, where it binds the transcription factor T-cell factor (TCF) and lymphoid enhancing factor (LEF), inducing the expression of target genes such as Snail and Slug. ^40,41 In contrast, EndMT is strongly inhibited by Wnt7a mediated Wnt signaling. Also, EndMT is suppressed by the dominant negative effect of endothelial-specific β-catenin mutants, suggesting the key role of Wnt signaling in EndMT. ^8,42,43 Originated from endothelial cells, a significant proportion of myofibroblasts in fibrotic myocardium are derived from EndMT through β-catenin dependent Wnt signaling pathway in the mouse model of myocardial infarction. ⁴⁰ This is confirmed by other subsequent reports that the accumulation of β-catenin in the nucleus causes the decreased expression of VE-cadherin expression and increased expression of fibronectin. It was also shown that the accumulation of β-catenin in the nucleus plays a key role in the formation of cardiac scars through EndMT process after cardiac ischemic injury. ⁴⁴

Oxidative Stress

Oxidative stress refers to the process by which large amount of reactive oxygen species (ROS) and reactive nitrogen are generated under the pathological stimulation. Oxidative stress has been recently recognized as one of the important mechanisms to promote the development of cardiac fibrosis. In the process of heart failure, ROS are mainly derived from dysfunctional mitochondria, activated NAPDH oxidase (NOX) and overload intracellular Ca²⁺. Among the NOX protein family, NOX2 and NOX4 have been proved to be most relevant in the physiological and pathological functions in vascular endothelial cells. The two NOX isoforms are highly expressed in the vascular endothelial cells and the function of NOX4 depends on its expression level since NOX4 is constitutively active without any other adaptor protein. Both in vivo and in vitro evaluations suggested that NOX4-induced ROS generation promoted endothelial cell fibrosis through EndMT by activating important profibrotic signaling pathway, TGF-β/Smad2/3. ⁴⁵ TGF-β can also induce the protein level of NOX4, leading to the induction of EndMT. The EndMT process is enhanced in NOX2 specific overexpressed endothelial cells from transgenic mice. The EndMT related gene expression is also significantly increased in cultured NOX2 overexpressed human aortic endothelial cells, indicating the important role of NOX2 in EndMT. ⁴⁶ ROS accumulation induces the secretion of inflammatory factors such as MCP1, NF-κB in inflammatory cells and promotes the occurrence of fibrosis. The expression of fibrogenic proteins, including CTGF, MMP2 and MMP9, are consequently up-regulated upon the ROS generation. Treating with potent ROS scavenger compound, N-acetyl-L-cysteine in HUVECs, the EndMT process induced by endotoxin is blocked.

Hypoxia Signal Pathway

The crosstalk of hypoxia and other signaling pathways have been described as a potent inducer of various EndMT-related systemic diseases including cardiac fibrosis. Numerous investigations suggested that hypoxia inducible factor-1α (HIF-1α) is the key molecule in regulation of EndMT process in diverse hypoxia model, both in vitro and in vivo. Under physiological conditions, HIF-1α is catalyzed and degraded by prolyl hydroxylase (such as PHD2, PHD3). In contrast, the degradation is inhibited in the various diseases. As a transcription factor, HIF-1α directly interacts with the hypoxic response element promoter to regulate the expression of EndMT-related genes, such as Snail, Slug and Twist. ^47,48 In addition, HIF-1α induces the expression of interstitial markers, such as vimentin, N-cadherin, which is essential for HDAC3-mediated histone methyltransferase complex formation. ⁴⁹ Furthermore, it is shown that hypoxia also induces the EndMT process through TGF-β/Smad signaling pathways in the cardiac remodeling model. The investigations in different in vivo experimental animal models revealed the effect of hypoxia induced EndMT associated with typical changes of cell characteristics in pulmonary artery hypertension. Enhanced Smad1/5 and Smad2 phosphorylation was observed and the effects are inhibited by specifically blocking ALK1 or ALK5. HIF-1α and TGF-β/Smad signaling pathways synergistically involve in hypoxia-induced EndMT by direct Snail induction and autocrine stimulation of TGF-β in ventricular cardiomyocytes. ⁴⁸

MicroRNAs Regulate EndMT

Micro RNAs (miRNAs) are a class of non-coding regulatory RNAs with the length of approximately 20 to 25 nucleotides. Through binding with the complementary sequence of the target gene mRNA, miRNAs regulate the expression of proteins at the epigenetic level. miRNAs are involved in processes such as cell proliferation, death, and differentiation. In recent years, it has been found that miRNAs are critical in regulating the occurrence and development of cardiovascular diseases such as cardiac fibrosis, myocardial hypertrophy, heart failure, and pulmonary hypertension by regulating the EndMT process. Several studies had proved that miRNAs can modulate EndMT process by directly targeting the key components of different EndMT signaling pathways. It has been demonstrated that miRNAs modulate the expression of mRNA transcripts in multiple TGF-β mediated signaling pathways including TGF-β/Smad signaling cascade.

Kumarswamy et al. found that miR-21 promotes the endogenous generation of TGF-β induced HUVECs by regulating PTEN/Akt pathway. ¹¹ The EndMT process is inhibited by reversing the TGF-β1-induced miR-21upregulation. ⁵⁰ In addition, the TGF-β2-induced repression of EndMT responsive gene is reverted by inhibition of miR-21. The above results suggest miR-21 is a positive regulator of EndMT. Other miRNAs which facilitate EndMT are miR-27b, miR-2 and miR-20. Preclusion of miR-27b suppresses EndMT by inhibition of EndMT responsive gene expression. ⁵¹ Knocking down the expression of the miR-2 gene in rat endothelial cells reduce the expression of collagen and fibronectin, thereby delaying the progression of myocardial fibrosis. Similarly, miR-20 downregulates ALK5 and TGF-βRII, and inhibits TGF-β-induced HUVECs from EndMT. ⁵² Extensive studies reveal that miR-18a-5p and miR-532 are negative regulators of EndMT during cardiac fibrosis and myocardial infarction. miR-18a-5p inhibits EndMT by regulating the expression of Notch2. An investigation in diabetic mice indicated that miR-18a-5p suppresses EndMT in cardiac fibrosis. High glucose-induced EndMT were suppressed by miR-18a-5p overexpression. The expression of the mesenchymal markers S100A4, Vimentin, and Fibronectin was reduced and the expression of CD31 was upregulated. ⁵³ Furthermore, inhibiting miR-532 enhanced TGF-β2-induced EndMT in the myocardial infarction model. Increased expression of Collagen-3, Snail and ACTA2 and decreased expression of CD31 and vWF were observed. ⁵⁴

Many studies support that expression level of miRNAs in endothelial cells is altered under pathological conditions and the change of miRNAs expression promotes the pathogenesis of human diseases by facilitating EndMT process. For example, during the pathological occurrence of EndMT, the expression levels of miRLet-7 decreased significantly under diabetic condition, leading to the diabetic kidney fibrosis. In addition, The peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) have been showed the capability of inhibiting diabetic kidney fibrosis by inhibiting EndMT through restoring the expression level of miRLet-7. ⁵⁵

EndMT in Cardiovascular Diseases

EndMT is a key mechanism in generating diverse cells with migratory and invasive abilities, which involves numerous physiological and pathological events. ^{56 -58} After cardiac cushion formation, endothelial cells of outflow tract under transformation into mesenchymal cells via EndMT to generate the primordia of the valves and membranous septa. ^58,59 During development, most fibroblasts in the interventricular septum are originated from endocardial cells via EndMT. ^60,61 Despite necessary in cardio-genesis, EndMT is kept low level in postnatal and adult heart to tissue homeostasis. ^{62 -64}

However, pressure overload-induced cardiac fibroblasts also transdifferentiate from endothelial cells, which share similarity to that of formation of the atrioventricular cushion in the embryonic heart. ³² EndMT is able to co-express with osteogenic markers in a mouse model of aortic valve calcification and human aortic valves obtained from patients with calcific aortic valve disease. EndMT precedes VEC osteogenesis and EndMT-derived eVICs probably populate the valve leaflet and differentiate into osteoblastic cells (oVICs), contributing to the pathological remodeling observed in CAVD. ⁶⁵ Diabetes mellitus induces cardiomyopathy via EndMT-meditated cardiac fibrosis. ⁶⁶ In hearts of patients with stage 3-4 chronic kidney disease (CKD) and end stage renal disease (ESRD), microvascular is reduced and fibroblasts derived from EndMT was 17% higher than that in preserved renal function patients. ⁶⁷ EndMT deteriorates fibrosis in the endocardial fibroelastosis (EFE) diseases, and the redundant EFE fibroblasts are proposed to be derived from postnatal endocardial cells via EndMT caused by dysregulated TGF-β/BMP signals. ⁶⁸ In pressure-overload hypertrophied rabbit hearts, EndMT-induced fibrosis mediates excessive deposition of collagen, which further reduces myocardial diastolic stiffness and heart failure. ⁶⁹

EndMT induced fibrosis also usually leads to excessive deposition of extracellular matrix. In an aortic banding mice model (pressure overload and chronic allograft rejection), approximately 30% EndMT derived fibroblasts present in the fibrotic myocardium. Cardiac fibrosis induces a progressive stiffening of the ventricular walls, and it decreases extent of microvasculature and contractility. These abnormalities ultimately disrupt myocardial structures and cardiac conductance. ³²

Prospect

Endothelial-to-Mesenchymal Transition (EndMT) is an evolutionarily highly conserved biological process involving a cellular transition from an endothelial to a mesenchymal phenotype, which plays a key role in development, chronic inflammation, tissue reconstruction, cancer metastasis and various fibrotic diseases. ^{70 -73} Endothelial cells are the main components of the inner wall of the heart, blood vessels, and lymphatics. They play an important role in the physiological function of cardiovascular system, and endothelial dysfunction is involved in a variety of acute and chronic cardiovascular diseases. EndMT regulates the transition of endothelial cells into mesenchymal cells, and is accompanied by changes in the expression of various transcription factors and cytokines, which plays a key physiological and pathological role in the development of vascular endothelial injury, vascular remodeling, and myocardial fibrosis disease (Figure 3). It was estimated that approximately 30% fibroblasts are derived from EndMT of endothelial cells, and the occurrence of EndMT is reduced when the degree of myocardial fibrosis is low. ⁷⁴ Although various stimuli, such as inflammatory cytokines, growth factors, proteases and hypoxia, can induce EndMT, their effects in various disease models may differ. In addition, mesenchymal-to-endothelial transition (MEndoT) is involved in the regulation of angiogenesis after acute myocardial ischemic injury. ⁷⁵ It is well known that myocardial fibrosis is the result of persistent myocardial fibers and/or repeated exacerbation of myocardial ischemia and hypoxia, whose pathological morphology is mainly manifested by excessive deposition of collagen fibers in myocardial extracellular matrix and changes in myocardial structure. It results in decreased microcirculation, dysfunction of systolic and diastolic function, progressive development of heart failure, arrhythmia and sudden cardiac death. Abnormal EndMT has been proved to be the key mechanism of myocardial fibrosis in a variety of rodent animal models, which suggested that EndMT may be an important target for the prevention and treatment of myocardial fibrosis. ⁶⁸ More importantly, although extensive scientific investigations conclusively revealed the critical role of EndMT in human cardiovascular disorders, it is believed that a large number of unresolved questions still need to be identified in future studies including the exploration of initiate pathological event in EndMT responsive gene expression and modification and the subsequent pathophysiological processes of myocardial fibrosis. The contribution of syringed crosstalk between different signaling pathways and pathological factors in EndMT also needs to be addressed. The clinical identifications of both positive and negative regulators in EndMT mediated fibrotic cardiovascular diseases which associate with losing endothelial cell phenotype and adopting mesenchymal cell characteristics can be considered as attractive prospective biomarkers in clinical diagnostic. The property of endothelial cells located in different tissues and organs is varying due to distinct surrounding physiological environment. Therefore, the EndMT process in these endothelial cells might not be respond to identical outside pathophysiological stimulations through the same signaling pathways. It is still needed to explore the role of EndMT induced by different signal networks in different disease states such as atherosclerosis, cardiac fibrosis and pulmonary artery hypertension. Given that EndMT is the central mediating process involved in multiple cardiovascular diseases, the discovery of key target molecules that regulate the process of EndMT and the mechanism of EndMT/MEndoT interaction regulation will provide new targets in prevention and treatment of fibrotic cardiovascular diseases caused by EndMT.

Figure 3.

The transition process of endothelial cells differentiation to cardiac fibroblasts contributes to the development of cardiac fibrosis.