Role of Endothelial to Mesenchymal Transition in the Pathogenesis of the Vascular Alterations in Systemic Sclerosis

1. Introduction

Scleroderma or Systemic Sclerosis (SSc) is an autoimmune disease of unknown etiology characterized by progressive fibrosis of skin and multiple internal organs and prominent and often severe alterations in the microvasculature [1]. Although SSc is the third most common systemic inflammatory autoimmune disease and has the highest case-specific mortality among this group of idiopathic disorders, there is currently no effective disease-modifying therapy for SSc. Therefore, there is an urgent unmet need for the development of effective disease-modifying therapies to improve the devastating health consequences and high mortality caused by the disease. The cells responsible for the severe fibroproliferative process in SSc are activated myofibroblasts, a unique population of mesenchymal cells displaying unique biological functions including increased production of fibrillar type l and type lll collagens, initiation of expression of α-smooth muscle actin (α-SMA), a molecular marker of activated myofibroblasts, and reduction in the expression of genes encoding extracellular matrix (ECM)-degradative enzymes. The accumulation of myofibroblasts in affected tissues and the persistence of their elevated biosynthetic functions are crucial determinants of the extent and rate of progression of the fibrotic process in SSc, and of its clinical course, response to therapy, prognosis, and overall mortality. The origins of the myofibroblasts responsible for the exaggerated and uncontrolled production of collagen and other ECM proteins in SSc have not been completely elucidated. Extensive research studies have shown that these cells originate from several sources, including expansion of resident tissue fibroblasts and migration and tissue accumulation of bone marrow-derived circulating fibrocytes, or from epithelial cells which have undergone epithelial to mesenchymal transition (EMT). More recent studies, however, have demonstrated that endothelial cells are also capable of undergoing endothelial to mesenchymal transition (EndoMT) and that this transition might be an important source of the mesenchymal cells participating in the fibroproliferative vasculopathy and the fibrotic process in SSc. Thus, this novel mechanism of generation of activated myofibroblasts may represent an important and currently unexplored target for the development of disease-modifying therapeutic interventions for this currently incurable disease.

2. SSc Pathogenesis: Overview

The pathogenesis of SSc is extremely complex, and despite numerous studies that examined several aspects of its intricate picture, the exact mechanisms involved are not well understood. However, it is apparent that the clinical and pathologic manifestations of the disease are the result of three distinct processes: (1) fibroproliferative vascular lesions of small arteries and arterioles, (2) excessive and often progressive deposition of collagen and other ECM macromolecules in skin and various internal organs, and (3) alterations of humoral and cellular immunity characterized by innate immunity alterations, involvement of macrophages and T- and B-lymphocytes, and the production of numerous disease-specific autoantibodies [2–4]. It has not been established which of these processes is of primary importance or how they are temporally related during the development and progression of the disease. However, numerous recent studies have clarified some of the early events in SSc pathogenesis [5–11]. A current hypothesis for SSc pathogenesis posits that there is a sequence of pathogenetic events initiated by unknown etiologic factors in a genetically receptive host which trigger microvascular injury characterized by structural and functional endothelial cell abnormalities. The endothelial cell abnormalities result in the increased production and release of numerous and potent mediators including cytokines, chemokines, polypeptide growth factors, and various other substances such as nitric oxide, prostaglandins, reactive oxygen species (ROS), and thrombogenic and pro-coagulant activities or in the reduction of important compounds such as prostacyclin. The endothelial cell dysfunction triggers the chemokine and cytokine-mediated attraction of specific inflammatory cellular elements from the bloodstream and bone marrow and their transmigration into the surrounding tissues. These events lead to the establishment of a chronic inflammatory process with participation of macrophages and T- and B-lymphocytes, with further production and secretion of cytokines and growth factors that induce the tissue accumulation of activated myofibroblasts, the effector cells responsible for the fibrotic process [12–14]. This sequence of events, diagrammatically illustrated in Figure 1, results in the development of a severe and often progressive fibroproliferative vasculopathy and in the exaggerated and widespread accumulation of fibrotic tissue in the skin and multiple internal organs, which are the most salient characteristics of the disease.

The purpose of this review is to discuss recent studies that have substantially advanced the current understanding of SSc pathogenesis regarding the endothelial cell and vascular abnormalities and the role of endothelial to mesenchymal transition (EndoMT) in the pathogenesis of this currently incurable disease. However, the genetic, innate, and acquired immunological abnormalities and the cytokine, chemokine, and growth factor abnormalities, all of which play an extremely important role in SSc pathogenesis, will not be reviewed here owing to the availability of numerous outstanding reviews about these topics that have been recently published [15–22].

3. Vascular Abnormalities in SSc

Vascular dysfunction is one of the earliest and most noticeable manifestations of SSc as indicated by the occurrence of Raynaud phenomenon, nailfold capillary microvascular alterations, and digital ulcers almost universally in SSc patients often preceding the appearance of clinical evidence of tissue fibrosis [23, 24]. Furthermore, there is a remarkable microvascular fibroproliferative vasculopathy present in essentially all SSc affected organs that is responsible for the most important symptoms and clinical manifestations of SSc and often leads to serious and even fatal complications. Although the effects of vascular abnormalities and dysfunction in patients with SSc are most dramatic when they involve the pulmonary and renal arterioles, causing renal crisis [25, 26] and pulmonary artery hypertension [27–29], respectively, there are numerous other important clinical manifestations of the disease that are caused or mediated by the prominent fibroproliferative vasculopathy. These include capillary rarefaction and capillary loop dilation in the nailfold capillaries [30, 31], cutaneous and mucosal telangiectasias [32–34], erectile dysfunction resulting from alterations in penile blood flow [35–37], and cardiac dysfunction including nonartherosclerotic myocardial infarcts [38, 39], gastric antral vascular ectasia [40–42], central retinal artery occlusion [43, 44], and involvement of larger vessels [45, 46]. Histopathologically, the affected vessels display marked narrowing or even complete occlusion of the vessel lumen with remarkable accumulation of mesenchymal cells and fibrous tissue in the subendothelial compartment and associated endothelial cell abnormalities, which include swelling and apoptotic changes, as well as thickening of the basement membrane. Occasionally, endothelial cell detachment and intravascular platelet thrombi are found. On transmission, electron microscopy universal morphological changes of endothelial cells and basement membrane duplication and lamellation are characteristic alterations. The histopathological changes in the microvasculature of several affected organs are illustrated in Figure 2.

4. Mechanisms of Vascular and Endothelial Cell Injury in SSc

The initial events responsible for the vascular and endothelial cell injury and their subsequent activation are not known although numerous putative etiologic factors have been suggested. Some of these include exogenous chemical substances, vasculotropic viral pathogens, antiendothelial cell antibodies, cellular products from inflammatory cells, tissue hypoxia, or ROS generated during episodes of ischemia/reperfusion [8–10, 47–52]. The injured/activated endothelial cells may undergo apoptosis or may detach from the vascular endothelium, leaving a denuded vascular lumen which triggers the release of endothelial cell precursors from the bone marrow in attempts to repair the endothelial lining defects. Supporting this notion are the observations of increased numbers of circulating endothelial cells and endothelial cell precursors in SSc patients [53–55]. The activation of endothelial cells also induces the expression of cell adhesion molecules such as ICAM, VCAM-1, and E-selectin [56, 57]. The induced expression of cell adhesion molecules by the endothelial cells leads to recruitment and activation of chronic inflammatory cells, including T- and B-lymphocytes and profibrotic macrophage populations and their accumulation in the perivascular tissue and in the interstitium of parenchymal organs. The activated chronic inflammatory cells are responsible for the increased production of transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF), and other profibrotic polypeptide growth factors which together with the mediators released by the endothelial cells, such as endothelin-1, induce subsequent pathogenetic events leading to the severe tissue fibrosis and fibroproliferative vasculopathy characteristic of the disease [1–11]. Besides the endothelial cell abnormalities, other vascular alterations include increased proliferation of smooth muscle cells in the medial layer of affected vessels, marked accumulation of fibrotic tissue in the subendothelial compartment, and initiation of platelet aggregation and intravascular thrombosis, eventually causing microvascular occlusion [58, 59]. These multiple events result in tissue hypoxia which can cause activation of hypoxia-dependent profibrogenic processes, including further increases in production of TGF-β and interstitial collagens as well as other ECM macromolecules [60, 61]. In addition to the structural vascular changes described above, there are also functional vascular alterations which include a reduction in endothelium dependent vasodilator molecules and dysfunction of the neurovascular and neuroendothelial control of vasodilation [62–65], as well as a relative deficiency of vasodilator molecules such as prostacyclin and nitric oxide.

The injured or cytokine/growth factor-activated endothelial cells also produce increased amounts of the potent profibrotic and vasoconstrictor polypeptide, endothelin-1 [66, 67], and numerous other vasoactive and prothrombogenic compounds that are capable of directly stimulating various target cells such as vascular smooth muscle cells and fibroblasts [8–10, 66, 67]. The important role of endothelin-1 in the development of SSc-associated tissue fibrosis and fibroproliferative vasculopathy has received increasing attention recently. Indeed, elevated levels of endothelin-1 have been found in plasma and bronchoalveolar lavage of SSc patients [68–70] and correlate with clinical parameters and subsets of the disease [71, 72]. Numerous studies have demonstrated that endothelin-1 is a potent inducer of proliferation and ECM production by fibroblastic cells [73–76]. The exaggerated vasoconstrictor response to the increased endothelin levels causes vascular hypoxia and further endothelial injury, thus establishing and maintaining a vicious cycle of endothelial injury and fibrosis. The chronic inflammatory cells accumulated in the perivascular environment also participate in the maintenance of a powerful profibrotic cycle since the numerous cytokines, chemokines, and growth factors they produce can in turn induce further activation of the endothelial cells and their production of profibrotic mediators [67]. The mutual interaction between inflammatory and endothelial cells has been validated by a recent study describing the upregulation of endothelin-1 and TGF-β in human microvascular endothelial cells induced by interferon-γ, one of the potent cytokines released by the infiltrating inflammatory cells [77]. Additional alterations which contribute to the severe vascular dysfunction and rarefaction in SSc are the result of disordered angiogenesis [78–84] and impaired differentiation of bone marrow stem cells into endothelial cells [85].

5. Endothelial to Mesenchymal Transition (EndoMT) in the Pathogenesis of SSc

One of the most characteristic histopathologic alterations in SSc is a severe fibroproliferative vasculopathy affecting the microvasculature as well as some larger vessels [86]. The proliferative vasculopathy of SSc has two distinct components. The first one is a marked proliferation of smooth muscle cells in the media of medium size and small size arterioles, a process which plays a crucial role in SSc-associated pulmonary hypertension. The second component is most prominent in the small arterioles of parenchymal organs, such as the lungs and kidneys, and is characterized by the subendothelial accumulation of activated fibroblasts or myofibroblasts and the production of abundant fibrotic tissue. The origin of mesenchymal cells responsible for the fibrotic microvascular occlusion in SSc is not known, but recent studies have suggested that at least some of these cells may result from EndoMT, that is, the transdifferentiation of endothelial cells into subintimal fibroblasts induced by locally-secreted cytokines and growth factors. During EndoMT, endothelial cells lose their specific endothelial cell markers, such as vascular endothelial cadherin (VE cadherin) and von Willebrand factor, and acquire a mesenchymal or myofibroblastic phenotype initiating expression of α-SMA, vimentin, and type I collagen. In addition, these cells become motile and are capable of migrating into surrounding tissues. EndoMT has been described as an important process during cardiac valve and pulmonary artery embryonic development [87–89]. More recently, EndoMT has emerged as a possible mechanism in the pathogenesis of tissue fibrosis in various diseases, including diabetic nephropathy, cardiac fibrosis, intestinal fibrosis, portal hypertension, and pulmonary hypertension [90–100]. Although there is some experimental evidence supporting the participation of EndoMT in SSc, further studies will be required to conclusively demonstrate that EndoMT plays a role in the pathogenesis of SSc-associated fibroproliferative vasculopathy and progressive tissue fibrosis. A firm demonstration of the occurrence of EndoMT in SSc and a further understanding of the molecular mechanisms involved may lead to the pharmacologic modulation or abrogation of this pathway in SSc.

6. Molecular Mechanisms of EndoMT

The molecular mechanisms involved in the EndoMT process have not been fully elucidated, and despite the remarkable importance of this process to normal development and to various pathologic conditions including SSc, only a few studies have examined the molecular changes and the regulatory events occurring in endothelial cells during their transdifferentiation into mesenchymal cells or myofibroblasts. However, substantial recent evidence has accumulated demonstrating the crucial role of TGF-β signaling [101–104] in the initiation of EndoMT during normal development as well as in various diseases.

7. Conclusions and Future Directions

Scleroderma or Systemic Sclerosis (SSc) is a systemic autoimmune disease of unknown etiology characterized by progressive fibrosis of skin and multiple internal organs and severe alterations in the microvasculature [1]. SSc is the third most common systemic inflammatory autoimmune disease and has the highest case-specific mortality among this group of idiopathic disorders. Whereas remarkable therapeutic advances have recently been accomplished for Rheumatoid Arthritis and Systemic Lupus Erythematosus, there is currently no effective disease-modifying therapy for SSc. Therefore, there is an urgent unmet need for the development of effective disease-modifying therapies to improve the devastating health consequences and high mortality caused by the disease. The effector cells ultimately responsible for the severe fibroproliferative process in SSc are activated myofibroblasts. These cells display very active protein synthesis producing increased amounts of ECM proteins and acquiring a motile and contractile phenotype expressing a high level of α-SMA [12–14]. Although it is widely recognized that there are numerous inflammatory and immunological events in the pathogenesis of SSc, myofibroblasts have been recognized as the crucial determinant of the fibrotic process in SSc and other fibrotic disorders [6, 11–13, 201–203]. Furthermore, their accumulation in affected tissues and the persistence of their elevated biosynthetic functions are the primary determinants of the extent and severity of the clinical manifestations in SSc, and of its clinical course, response to therapy, prognosis, and overall mortality. Thus, activated myofibroblasts have become an important target for SSc disease-modifying therapeutic approaches [204–206].

Extensive research studies have shown that these cells originate from several sources [94, 207], including expansion and phenotypic activation of resident tissue fibroblasts and migration and tissue accumulation of bone marrow-derived circulating fibrocytes, or from epithelial cells which have undergone EMT. More recent studies, however, have demonstrated that endothelial cells are also capable of undergoing a phenotypic change to activated mesenchymal cells in a complex process known as EndoMT. Although there are very few studies that have examined the possible participation of EndoMT in the initiation and progression of the fibrotic and fibroproliferative processes in SSc, it is expected that given its potential importance in the pathogenesis of this currently incurable disease this area of investigation may attract further scientific attention.

Despite the relatively recent research interest in the role of EndoMT in the SSc pathogenesis, important components of the complex pathway of TGF-β-induced EndoMT and the molecular mechanisms involved in the generation of activated tissue myofibroblasts have already been identified. These observations suggest that targeting components of these pathways may be a feasible therapeutic goal to modify crucial steps in the development of SSc fibroproliferative vasculopathy [208]. Furthermore, the important role that miRNAs have been shown to play in the regulation of gene expression has clearly opened the possibility of developing a novel therapeutic approach for SSc by targeting these extremely versatile noncoding RNA species. Obviously, subsequent preclinical studies employing suitable animal models will be required to further support the potential therapeutic role of EndoMT and/or miRNA modulation for the fibrosis and proliferative vasculopathy of SSc.