Basement Membrane Extracellular Matrix Proteins in Pulmonary Vascular and Right Ventricular Remodeling in Pulmonary Hypertension

Abstract

The extracellular matrix (ECM), a highly organized network of structural and nonstructural proteins, plays a pivotal role in cellular and tissue homeostasis. Changes in the ECM are critical for normal tissue repair, whereas dysregulation contributes to aberrant tissue remodeling. Pulmonary arterial hypertension is a severe disorder of the pulmonary vasculature characterized by pathologic remodeling of the pulmonary vasculature and right ventricle, increased production and deposition of structural and nonstructural proteins, and altered expression of ECM growth factors and proteases. Furthermore, ECM remodeling plays a significant role in disease progression, as several dynamic changes in its composition, quantity, and organization are documented in both humans and animal models of disease. These ECM changes impact vascular cell biology and affect proliferation of resident cells. Furthermore, ECM components determine the tissue architecture of the pulmonary and myocardial vasculature as well as the myocardium itself and provide mechanical stability crucial for tissue homeostasis. However, little is known about the basement membrane (BM), a specialized, self-assembled conglomerate of ECM proteins, during remodeling. In the vasculature, the BM is in close physical association with the vascular endothelium and smooth muscle cells. While in the myocardium, each cardiomyocyte is enclosed by a BM that serves as the interface between cardiomyocytes and the surrounding interstitial matrix. In this review, we provide a brief overview on the current state of knowledge of the BM and its ECM composition and their impact on pulmonary vascular remodeling and right ventricle dysfunction and failure in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a severe disorder of the pulmonary vasculature characterized by increased vasoconstriction and progressive remodeling of the pulmonary arteries (PAs). Pulmonary vascular remodeling results in increased pulmonary vascular resistance, increased right ventricular (RV) afterload, progressive RV remodeling and dysfunction, and, ultimately, death owing to RV failure (1). In PAH, the vascular remodeling involves the three-layered architecture of the arteries. Increased accumulation of ECM proteins is thought to be a key pathologic change across the vascular wall in PH (2). In the intima, there is neointimal remodeling owing to endothelial and smooth muscle cell proliferation accompanied by neointimal fibrosis. The media is the most predominant site of smooth muscle cell hypertrophy and hyperplasia. There, remodeling leads to increased medial wall thickness in small PAs and abnormal muscularization of more distal PAs (<50 μm in diameter). Throughout this process, there is aberrant expansion and accumulation of the ECM with increased collagen deposition and elastin, leading to vascular fibrosis that further contributes to the adverse remodeling (3–6). Intriguingly, several dynamic changes in the vascular ECM composition, amount, and organization are increasingly implicated as active drivers of pathogenesis in both humans and animal models of the disease (5–11). Certainly, dysregulated ECM deposition activates transcriptional coregulators, triggering proliferation of endothelial and vascular smooth muscle cells and adventitial fibroblasts in PH (5, 10). Patients with congenital heart defect and pulmonary vascular disease evidence degradation of elastin in PA. These findings are supported by hypoxia and monocrotaline (MCT) rat models of PH, which documented increased activity of elastase in PA (6, 11). The emerging role of the basement membrane (BM) and associated molecules on the vascular endothelium and its implications on lung vascular remodeling in PH have been documented in a recent review (12). Pathogenic processes implicated in ECM remodeling include vascular injury, abnormal growth factor expression, proinflammation factors, and/or hypoxia exposure (2). In addition, ECM-associated proteins play a crucial role in PA remodeling and consequently affect RV structure and function (13). Despite this knowledge, relatively little is known about the dynamics of BM changes and its ECM-associated proteins in PAH. Here, we will review the current state of knowledge regarding the BM and its ECM composition and its impact on pulmonary vascular and RV remodeling in PAH.

BM and Its ECM-associated Proteins in PAH

ECM is a highly organized extensive molecule network that plays a key role in maintaining tissue architecture and homeostasis by providing structural support for the cells composing the vessel wall. ECM also enables inter- and intracellular communication and regulates cell proliferation, migration, and apoptosis (14). The composition of the PA-ECM is dynamic, consisting predominantly of collagens and numerous other proteins contributing to specialized components of the ECM structure. The PA-ECM includes the BM, elastins, laminins, collagen type IV, fibronectin, tenascin C, proteoglycan, and various growth factors and proteases (15, 16). In addition to several ECM molecules, proteoglycans consist of core proteins richly decorated with glycosaminoglycan side chains (17, 18). Along with maintaining tissue architecture, ECM component glycosaminoglycans, such as hyaluronan (HA), are involved in mediating a variety of physiological and pathological processes (19, 20). Normally, ECM integrity and turnover are regulated by a balance between specific and unique proteolytic enzymes, such as ADAMs (a disintegrin and metalloproteases) and MMPs (matrix metalloproteinases). The activity of these enzymes is regulated, in part, by their endogenous inhibitors, such as TIMPs (tissue inhibitors of metalloproteinases). Both the expression and activity of MMPs and TIMPs are abnormally regulated during disease, as evident from numerous animal models of PAH (2). Additionally, turnover of BM components can release both proteins and/or peptides that alter cellular behavior within the microenvironment of the vessel wall. For example, the degradation of BMs by MMP-2 can promote the production of tenascin-C, an ECM component that amplifies the smooth muscle cell proliferative response (2). In PAH, an imbalance in the proteolytic enzymes and their endogenous tissue inhibitors leads to increased collagen accumulation, collagen cross-linkage, and elastin breakdown in the perivascular and intravascular compartments of the PA followed by disruption of the pulmonary vasculature structural integrity (2).

BMs are thin layers of specialized noncellular dense ECM proteins in close association with the cellular tissue components. They act as regulators of cellular behavior, adhesion, migration, differentiation, molecule transport, and cell signaling (21, 22). BMs are usually found in the basolateral aspect of vascular endothelial and epithelial cells (23) or surround pericytes and smooth muscle cells (24). At least 20 different ECM proteins have been identified from BMs, and every single protein contributes to BM assembly, stability, and function. BMs are predominantly composed of non–fiber-forming collagen, collagen type IV, which is believed to form a meshwork to which various other components can attach, including laminin, fibronectin, proteoglycans, perlecan, and entactin/nidogen, to produce highly organized BM structures and perform various functions (25). Additional BM components include agrin, fibulins, collagen type XV, and collagen type XVIII (21) (Figure 1). Beyond structural support, growing evidence indicates that BM proteins can function as a signaling platform during development and in mature tissues by sequestering a number of growth factors (12, 26, 27). Key components of the BM, proteoglycans perlecan, agrin, and collagen type XVIII, actively modulate cellular processes by tethering growth factors such as VEGF (vascular endothelial growth factor) (28), TGF-β (transforming growth factor-β) (29), and FGF (fibroblast growth factor) (30) through binding interactions with their heparan sulfate glycosaminoglycan chains (26). Furthermore, these growth factors act to maintain cell survival, migration, and proliferation (12). BM glycoproteins laminin and fibronectin are assumed to modulate cell proliferation, migration, and differentiation (31–33). Modifications in the BM composition, including its amount and distribution, have been demonstrated to dramatically affect the function of cells, tissues, and organs (31, 34, 35).

Figure 1.

Schematic illustration of the proposed changes in the basement membrane (BM) extracellular matrix (ECM) proteins in pulmonary arterial hypertension (PAH)-associated pulmonary vasculature compared with the normal pulmonary vasculature. Structure of arterial wall in normal (left) and PAH (right). The vessel wall has a three-layered architecture: intima (endothelial cells and BM), media (smooth muscle cells and ECM components), and adventitia (adventitial fibroblasts). (A) Schematic illustration of the normal vascular BM matrix. The major components of BM include collagen type IV, laminins, perlecan, nidogen, agrin, versican, and collagen type XVIII. The structure of the BM consists of independent networks of laminin and collagen type IV, which interact with cell surface receptors integrin and dystroglycan. The self-assembled collagen type IV network is recruited together with other BM-associated proteins, perlecan, agrin, nidogen, versican, and collagen type XVIII, to the developing laminin network. (B) In PH, the structure, quantity, and composition of the BM ECM matrix is altered. Based on the current knowledge, collagen IV, Col18al, endostatin, TSP1, perlecan, versican, and agrin levels are increased. Remodeling of the ECM within the arterial wall is characterized by increased deposition of elastin, tenascin C, fibronectin, hyperplasia of smooth muscle cells, and fibrillar collagens, which are secreted mainly by activated smooth muscle cells and fibroblasts. Not drawn to scale for illustrative purposes. Col18al = collagen type XVIII, α1; MMPs = matrix metalloproteinases; TSP1 = thrombospondin 1.

Several previous studies using animal models of PH and human PAH tissues document increased deposition of ECM proteins of the BM in remodeled vessels, mainly collagen, elastin, fibronectin, and tenascin-C (2, 6, 8, 9, 15, 36, 37). Aberrant collagen and elastin expression have been observed in proximal and distal vessels in both early and end-stage disease (10, 38). Increased fragmentation of the elastin lamina has been observed in the PA of both experimental PH and tissues from children with PAH associated with congenital heart disease (39–41). Several studies suggest that oxidative stress from increased production of superoxide radicals and other reactive oxygen species (42, 43) may contribute to the pathogenesis of PH. SOD3 (superoxide dismutase 3), a potent antioxidant enzyme, is tightly bound to the EC membrane through its heparan-binding domain. This provides affinity of the protein for BM-associated ECM molecules such as heparan sulfate proteoglycans (44). Animal studies confirmed overexpression of SOD3 attenuates hypoxia-induced PAH in mice (45) and MCT-induced PAH in rats (46). Thus, the association of BM heparan sulfate proteoglycans with SOD3 plays a potential protective role during the development of PAH. Moreover, abnormal expression and breakdown of HA has been observed in the rat model of MCT-induced PH. Fragmentation of HA occurs during disease progression associated with enhanced hyaluronidase activity, which may provide the inflammatory and proliferative stimulus to drive pathological vascular remodeling (47). A significant perivascular increase of HA within plexiform lesions has been observed in lungs of patients with idiopathic PAH (IPAH) (20, 48). Recently, Jandl and colleagues documented major alterations in the structure and composition of BM components such as laminin and collagen type IV contents in patients with IPAH-derived pulmonary arterial endothelial cells, which can disturb crucial endothelial cell function and integrity. This may contribute to PAH pathogenesis and provides evidence that BM imbalance could lead to the loss of homeostasis and abnormal cell behavior in disease pathogenesis (49). We and others have previously demonstrated increased circulating levels of endostatin, a proteolytic fragment of the BM protein Col18a1 (collagen type XVIII, α1) in PAH (50–54). Importantly, endostatin levels were associated with adverse hemodynamics and a strong predictor of mortality in PAH (50). This suggests that markers of BM dynamics, such as endostatin, may be linked to disease severity. Similarly, Hoffmann and colleagues demonstrated significant increased expression of BM vascular collagens, including network-forming collagen, Col4a5 (collagen type IV α5 chain), fibril-associated collagen, Col14a1 (collagen type XIV α1 chain), and the endostatin-producing collagen, Col18a1, in the intima and media of diseased PAs from patients with IPAH (53). Endostatin may contribute to the development of PH as a result of impaired angiogenesis (55). These studies emphasize the importance of BM components and their potential role in the pathophysiology of PAH (50, 53, 55).

RV ECM in PAH

Increased PA pressure and pulmonary vascular resistance lead to increased workload on the RV. Initially, the RV compensates through hypertrophy. RV morphologic changes are accompanied by extensive structural and ECM remodeling at the organ, tissue, and fiber levels, eventually leading to RV failure and death (56–60). Although remodeling of the pulmonary vasculature is the main cause of PAH, the degree of RV dysfunction is a key determinant of morbidity and mortality. RV hypertrophy followed by failure remains the primary cause of death in patients with PAH (61). RV remodeling is often characterized by extensive cardiac fibrosis and aberrant accumulation of cardiac ECM proteins (57–59). Any changes in these key structural components of RV during remodeling can disrupt RV function in several ways (57–59). Specifically, marked alterations in ECM proteins of the RV matrix are observed in patients with PH and are thought to contribute to increased morbidity and mortality (62).

In the RV, ECM is a highly organized network surrounding the cardiac cells that not only provides structural support to the cardiomyocytes and vessels but also plays an important role in the regulation of myocardial contraction and relaxation by maintaining ventricular geometry. ECM is crucial for efficient cardiac function via maintenance of proper alignment of myocyte and stabilization of appropriate tissue tensile modulus, compliance, and blood flow regulation during contraction. Thus, the ECM is an essential network required to maintain the normal pump function, the integrity of the cardiac structure, and the elasticity of the heart tissue providing mechanical stiffness (63). Alterations in the ECM homeostasis are notable in various cardiac pathologies, including hypertensive cardiac hypertrophy, myocardial infarction, and dilated cardiomyopathy, contributing to structural and functional abnormalities that cause progressive cardiac remodeling and dysfunction (64). ECM also controls the function of resident cardiac cells, such as fibroblasts, myocytes, pericytes, and endothelial and smooth muscle cells, impacting cell migration, adhesion, proliferation, differentiation, and survival, that is crucial for preserving cellular homeostasis (65, 66). These functions are mediated through interactions of ECM-associated proteins with integrins and other cardiomyocyte cell receptors (65, 66).

Normally, ECM homeostasis relies on a tightly controlled balance between the function of MMPs and TIMPs, which collectively regulate ECM-associated proteins in the process of cardiac remodeling (67–69). Historically, TIMPs control MMPs involved in dysregulated ECM remodeling (70–72). Also, higher levels of MMP, a marker of ECM breakdown, and TIMP have been observed in patients with PH compared with healthy controls, resulting in increased collagen turnover and ECM remodeling (73).

In numerous experimental models of myocardial hypertrophy and failure, an increase of RV ECM protein expression has been described, including MMP expression and altered collagen expression that contribute to adverse ventricular remodeling (74–78). The deposition of ECM is well recognized to increase ventricular stiffness and lead to the development of heart failure (79, 80). Among the ECM-affiliated proteins, the MMP family is an important contributor to PH-induced RV remodeling (77). MMPs associated with myocardial remodeling include gelatinases (e.g., MMP-2 and MMP-9), collagenases (e.g., MMP-1 and MMP-13), stromelysin (e.g., MMP-3), and the membrane-type MMP (e.g., MMP-14) (81). In the heart, MMPs are mainly expressed by cardiomyocytes and fibroblasts, which degrade ECM collagens, such as collagen types I and III (82–84). Most inactive forms of MMPs are stored extracellularly and bound to various components of the ECM. Upon stimulation, activated MMPs degrade ECM-associated proteins, including laminin, fibronectin, collagens, proteoglycan, and gelatin. Therefore, MMPs have the potential to alter tissue architecture and regulate cellular function in the heart, thus contributing to cardiovascular physiology and pathophysiology (68). Similarly, the family of ADAMTS proteinases, an additional group of extracellular metalloproteinases, also plays a major role in the degradation of ECM anchoring proteins along with strictly regulating normal physiological conditions (85). Hence, their capability to degrade ECM-associated proteins, proteoglycans, and procollagens suggests that they may influence cardiomyocyte–ECM integrity and function during the progression of RV failure (81, 82, 85–87). Thus, the loss of ECM integrity may be a major contributor to ventricular stiffness, dilatation, and cardiac dysfunction (88).

Classification of Cardiac ECM: An Overview

The cardiac ECM is essentially classified into structural and nonstructural proteins (Table 1). The structural proteins are composed of mostly fibrillar molecules, such as collagen type I and III. Nonstructural proteins of the ECM encompass a variety of nonfibrillar proteins, including glycoproteins (e.g., fibronectins, elastin, laminins, etc.), proteoglycans, nonproteoglycans, glycosaminoglycans, BM proteoglycans, and BM (pericellular) proteins. These separate cardiomyocytes from the surrounding interstitial matrix (e.g., fibronectin, laminin, collagen type IV) (89–95). Additionally, the cardiac ECM serves as a reservoir for a diverse assortment of cytokines, growth factors, ECM receptors, and proteases (MMPs and TIMPs), which are stored in the normal matrix but can be activated following injury (87, 96, 97). The maintenance of cardiac structural integrity requires a balance between ECM-associated protein synthesis and degradation. Among the ECM components, MMPs are the most important enzymatic determinant of ECM degradation and include MMP-2, MMP-9, and MMP-13 as major subtypes within the heart. The proteolytic activity of these MMPs is predominantly controlled by TIMPs, among which TIMP 2, TIMP 3, and TIMP4 are normally expressed in the heart (96). Additionally, family members of the ADAMS and ADAMTS are also implicated in the development and remodeling of the heart (98, 99).

Table 1.

Classification of the Cardiac ECM-associated Proteins

	Classification	ECM-associated Proteins
Glycoproteins	Structural proteins	Collagen type I, III, V
	Nonstructural proteins	Collagen type IV, VI
	Matricellular proteins	Tenascin, thrombospondins, SPARC, periostin osteopontin, members of the CCN family
	Basement membrane proteins	Fibronectin, laminin, collagen type IV
	Matricryptins	Endostatin, tumstatin
	Others	Elastin, fibronectin, laminin, fibrillin
Proteoglycans	ECM-associated proteoglycans	Aggrecan, versican, perlecan, nidogen
	Basement membrane–associated proteoglycans	Heparin sulfate (agrin, perlecan), collagen type XVIII, nidogen
	Cell surface–associated proteoglycans	Syndecan, Glypican
	Glycosaminoglycans	Hyaluronate, chondrotin sulfate, heparin sulfate, dermatan sulfate
	Proteases and their inhibitors	MMPs, TIMPs, ADAMS, ADAMTS
	Small leucine rich–associated proteoglycans	Biglycan, podocan, asporin, fibromodulin, decorin

Definition of abbreviations: ADAMS = a disintegrin and metalloproteinase with thrombospondin motifs; CCN = Cyr61 (cysteine-rich 61)/βIG-M1/Cef10/CCN1, CTGF (connective tissue growth factor)/Hcs-24(hypertrophic chondrocyte-specific gene 24)/βIG-M2/Fisp-12/HBGF-0.8/CCN2, and Nov (nephroblastoma overexpressed)/CCN3; ECM = extracellular matrix; MMP = matrix metalloproteinase; SPARC = secreted protein acidic and rich in cysteine; TIMP = tissue inhibitor of metalloproteinases.

Cardiac ECM also includes matricryptins, proteolytic fragments derived from ECM degradation by MMPs, which contain a biologically active cryptic site. Endostatin and tumstatin are derived fragments of BM protein collagens XVIII and IV, respectively (100). In the normal heart, nonstructural ECM proteins also involve rarely expressed proteins often referred to as matricellular proteins, which are markedly upregulated after injury and have an ability to bind, activate, and modulate the function of various molecules, including growth factors, cytokines, proteases, cell membrane receptors, and other ECM-associated proteins (101). In the RV, the upregulation of matricellular proteins such as thrombospondins, osteopontin, SPARC (secreted protein acidic and rich in cysteine), tenascin C and N, members of CCN family, and periostin has been implicated in RV remodeling (101–103) (Table 2).

Table 2.

Function and Expression/Involvement of BM-associated ECM Proteins in the Heart

BM-associated ECM Protein	Parent Protein	Function	Expression/Involvement in Heart Disease
Endostatin	Proteolytic cleavage product of collagen type XVIII α1 chain (50)	•Shows antiangiogenic activity (50) •Maintains integrity and stability of BM (12)	•Circulating elevated endostatin levels in patients with PAH are associated with worsened cardiac function and poor survival (50) •Acts as a positive regulator of thrombospondin 1 and is associated with PAH severity (51) •Stimulates proliferation, migration, and wound healing in rat cardiac fibroblasts (177, 178) •In patients with chronic heart failure and coronary heart disease, increased circulating levels may predict adverse outcomes (172, 179) •Rise in endostatin may reflect adverse cardiac remodeling (180)
Endorepellin	Cleavage fragment of perlecan from its C-terminal domain by proteolytic enzyme cathepsin L (12, 181, 182)	•Antiangiogenic, antiapoptotic, and profibrogenic in function (12) •Maintains integrity and stability of BM (12)	•Upregulated levels of cathepsin L have been reported in rats exposed to hypoxia and in the endothelium of patients with IPAH with BMPR2 mutations (183) •Marked expression in ischemic reperfusion injury under mild hypothermic conditions in pigs may contribute to an increased structural remodeling of the myocardial BM with potential functional impairment (184)
Arresten	Cleavage product of collagen type IV α1 chain by MT1-MMP and MT2-MMP (177)	•Antiangiogenic and proapoptotic in function (185–186) •Maintains integrity and stability of BM (12) •Antiangiogenic action by inhibiting proliferation and migration, promoting apoptosis in endothelial cells (186), and suppressing the migration and invasion of tumor cells (187)	•Increased expression in ischemic reperfusion injury under mild hypothermic conditions in pigs may contribute to an increased structural remodeling of the myocardial BM with potential functional impairment (184)
Canstatin	Fragment derived from α2 chain of collagen type IV by MT1-MMP and MT2-MMP (177)	•Potent antiangiogenic factor (188) •Maintains integrity and stability of BM (12)	•Increased degradation by cathepsin S at the site of infarcted area after myocardial infarction may be involved in the pathogenesis of myocardial infarction through increased turnover (188) •Stimulates the migration of rat cardiac fibroblasts through the increased expression of MMP2 (189) •Inhibits hypoxia-induced apoptosis in H9c2 cardiomyoblasts (190)
Tumstatin	Cleavage fragment of collagen type IV α3 chain (191)	•Exhibits both antiangiogenetic and antitumor properties (191) •Maintains integrity and stability of BM (12)	•Increased expression following cardiac pressure overload stimulates the proliferation and migration of cardiac fibroblasts and may play a profibrotic role (191)
Thrombospondin 1	N/A	•Shows potent antiangiogenic activity (192) •Stabilizes the ECM (193)	•Increased protein expression stimulates pulmonary endothelial cell apoptosis, inhibits migration and proliferation, and conversely promotes smooth muscle cell proliferation (51) •Increased expression promotes fibrosis and diminished cardiac function in the diabetic pressure overloaded heart (194) •Elevated plasma levels in PH cohort correlate with increased mortality (195)
Thrombospondin 2	N/A	•Preserves the integrity of the cardiac ECM (196, 197)	•Overexpression leads to progression of heart failure (196) •Increased expression reflects disease severity and predicts adverse outcomes (198)

Definition of abbreviations: BM = basement membrane; ECM = extracellular matrix; IPAH = idiopathic PAH; N/A  =  not applicable; PAH  =  pulmonary arterial hypertension; PH  =  pulmonary hypertension.

In PAH, an increased deposition of ECM proteins, primarily structural collagens, is observed in the RV (104–106). Structural ECM proteins and their contribution to disease pathogenesis are the most commonly researched; however, they are not the main focus of this article and are reviewed elsewhere (2, 49, 62, 79, 107–112). The contribution of other nonstructural ECM-associated proteins in RV remodeling remains to be determined and may shed light on the role of the BM in adaptive and maladaptive RV remodeling in disease. Thus, the current discussion is focused primarily on nonstructural BM proteins of the ECM that have been implicated in RV remodeling.

RV BM-associated Proteins in PAH

The RV BM is a well-organized layered assembly of specialized ECM proteins that lies on the external surface of the sarcolemma, encasing the cardiomyocytes and serving as the interface between these cells and the surrounding interstitial matrix (94, 113, 114). BM plays a key role in tissue function by facilitating cell-to-cell communication and organization (87). BM components serve as anchors for cells, essential for cell alignment, which is necessary for the productive contraction allowing for cell-to-cell electrical communication and adequate pumping (87).

Cardiac BM consists primarily of collagen type IV, laminin, fibronectin, entactins (nidogens), chondroitin sulfate proteoglycans, and proteoglycans, including perlecan, collagen type XVIII, and agrin (21, 113, 115–117). It serves as a layer of supportive material for cardiac myocytes. Additionally, the less abundant proteins that reside in the BMs are collagen types V, VII, X, and XIV (118). Moreover, each component performs distinct biological functions via cell surface receptors (mostly integrins) and nonintegrin receptors, such as cell development, proliferation, differentiation, growth, and migration (119, 120). Likewise, the major constituent of BM, collagen type IV, is thought to form a mesh-like structure to which many other components, such as laminin, fibronectin, perlecan, and nidogen, can attach to produce the mature BM structure required to maintain proper endothelial cell function (121). The assembly of BM-associated proteins, particularly laminin, collagen type IV, fibronectin, and entactin play an important structural role by anchoring cells to the interstitial matrix, which provides mechanical stability to the BM (31, 122) (Figure 2).

Figure 2.

Schematic illustration of the components of the BM ECM proteins in the healthy heart (A) and proposed changes in these components in RV remodeling associated with PAH (B). In PAH, potential changes in collagen IV, perlecan, versican, nidogen, aggrecan, and agrin may lead to disease progression and RV remodeling. The increased levels of Col18al, endostatin, and TSP1 may adversely influence RV remodeling and are associated with RV dysfunction and failure. Not drawn to scale for illustrative purpose (169). BM = basement membrane; ECM = extracellular matrix; LV = left ventricle; PAH = pulmonary arterial hypertension; RV = right ventricle.

In the RV, the BM actively influences the formation of sarcomere and remodeling via its interaction with integrins, which serve as receptors on the plasma membrane and perform multiple myocardial functions (114, 123). Thus, the critical roles of several main ECM-associated proteins found in the BM of the RV are discussed below.

Collagen Type IV, A Network-Forming Collagen in the BM

Collagen type IV is the most abundant and essential network-forming component of the BM that surrounds the cardiomyocytes and usually forms a close molecular association with laminin (66, 124). The major structural component of the BM collagen type IV consists of a triple helical structure with six types of α chains (i.e., α1–α6) (117, 125), among which the α1 and α2 chains are ubiquitously expressed (126, 127). Collagen type IV self assembles into a covalently stabilized network and provides the main scaffolding upon which the laminin network and other BM components, such as nidogen and heparan sulfate proteoglycan perlecan (124), adhere, thus forming a highly organized supramolecular architecture. The combined network plays an important role in the maintenance of BM structural integrity. This network-forming collagen possesses several binding sites that can interact with cellular receptors and other BM components and serves as a storage reservoir of growth factors. Thus, the BM structure contributes to cell function (128, 129).

Aberrant expression of collagen type IV has been linked to disease in transgenic animals and human. Mice deficient in Col4a1/Col4a2 (collagen type IV α1 chain/collagen type IV α2 chain) are embryonically lethal at E10.5 to E11.5 because of aberrant cardiogenesis associated with structural deficiencies in the BM (124). Structural defects of BM are not observed before E9.5, strongly suggesting that collagen type IV is not required during the initial assembly of BM proteins but is necessary for enhancing BM structural stability and function under conditions of increasing mechanical stress (124). Additionally, in rat models, increased levels of collagen type IV were observed in the remodeled RV associated with the early phases of mild hypoxia-induced RV hypertrophy (130). In humans, patients with hypertrophic cardiomyopathy exhibit significantly increased serum levels of collagen type IV, which correlates with systolic and diastolic dysfunction (131). Aberrant collagen type IV expression or mutations lead to pathological changes in the composition and structural integrity of the BM, which are common features of matrix disorders in the Goodpasture’s and Alport syndromes (132). These observations highlight an important role of collagen type IV in BM integrity in the heart.

Laminin Is One of the Major Components in the BM

Laminin is a large multidomain glycoprotein, found in the BM matrix of cardiac myocytes (133). Laminins are essential components of the BM, usually composed of three subunits of α chain, β chain, and γ chain that are organized in a three-dimensional intertwined structure (133). Among the five α isoforms, laminin α1 is involved in BM assembly and architecture and plays a significant role in cell migration through interactions with integrins α3β1 and α6β1 (134). One unique feature of all laminin α chains is that they contain a large C-terminal globular domain that is responsible for cell surface binding to integrin and α-dystroglycan (135–137). Additionally, laminin–integrin interactions via this globular domain activate signals, which are important in cellular functions (137). Furthermore, laminin polymers form independent networks and interact with collagen type IV networks via the proteoglycans nidogen and perlecan for enhanced BM stability (95). Laminins are involved in many other cell–matrix interactions and functions by anchoring cells to ECM that are important during embryonic development, tissue homeostasis, and remodeling (115).

Although there are inadequate studies on the direct involvement of laminin to RV remodeling in PAH, laminin isoforms appear to contribute to other aspects of cardiovascular remodeling. Laminin α4 chain, a laminin isoform in the heart sarcolemma is essential in maintaining cardiac structure and function (138). Disruptions in the laminin α4 chain can lead to cardiac hypertrophy and dysfunction (138). This results in apoptosis, as the laminin isoform serves as both a signaling and a structural molecule (138, 139). Thus, laminins are critical to the general scaffolding of BMs. Upcoming studies exploring the role of laminin and its isoforms may provide additional insight into their specific roles in RV adaptation and failure.

Fibronectin Is a Noncollagenous Glycoprotein in the BM

Fibronectin is another important dimeric component of the BM, which has been critically involved in several biological processes, including the regulation of cell growth, migration, wound healing, and cell adhesion (140). Embryos derived from mice lacking the fibronectin gene exhibit several defects of cardiac and vascular tissues and die early in embryonic development (141). Fibronectin serves as a bridge between cardiac myocytes and the interstitial collagen network and was shown to be essential for the development of heart and cardiac tissue repair (75, 142). Fibronectin is required for normal collagen organization and deposition by fibroblasts, and, thus, fibronectin interacts with interstitial collagen to form a fibrillar component of the ECM. Very few studies have explored the importance of fibronectin in PH. Data from chronic hypoxia–induced PH in the rat demonstrates increased expression of fibronectin in the remodeled RV (143). Increased expression of fibronectin in the hypertrophic RV may promote the aberrant accumulation of collagen, which in turn may lead to increased cardiac stiffness, RV dysfunction, and failure (140, 143). These studies indicate that the cell adhesion glycoprotein fibronectin may be an important factor in RV structure and function and could contribute to RV remodeling in response to PH.

Abnormal production, accumulation, and degradation of ECM proteins may lead to deposition of ECM components in the tissues and structural variations such as BM thickening during the development of cardiovascular diseases (142). Thus, the organization of this cell attachment glycoprotein network is crucial for the maintenance of appropriate and adequate cardiac function (144). Hence, fibronectin is an important BM component for the proper functioning of the myocardium, and further investigations will be required to examine more detailed involvement of BM fibronectin in RV remodeling and PAH pathogenesis.

Perlecan and Agrin Are Heparan Sulfate Proteoglycans in the BM

Perlecan is an important heparan sulfate proteoglycan found in the BM (145, 146), and agrin is another multidomain proteoglycan associated with BM (146). Perlecan is one of the critical components for the BM assembly. Its ability to interact with a variety of BM proteins such as laminin, collagen type IV, and entactin/nidogen in vitro highlights its crucial involvement in BM assembly and its role as an essential scaffold in stabilizing BM structures (95, 147). Also, perlecan recruits growth factors and has the ability to bind to α-dystroglycan and cell adhesion molecules such as integrins (145). Likewise, agrin binds integrins, α-dystroglycan, and laminins, which may enable increased laminin binding to cell surfaces and impact myocardial stability (95).

High levels of perlecan during embryonic development suggest vital functions in other extracellular matrices as well (148). During postimplantation stages, perlecan is detected in blood vessel walls and in the developing embryonic heart, suggesting its important role in heart development (148). Perlecan-deficient mice reveal a crucial role of perlecan in cardiac development. Heart defects have been described in these mice, including hemopericardium and an arrest of heart function (149). During heart development, perlecan acts as an adhesive substrate for cardiomyocytes and an important component of BM integrity (145). Thus, during early embryonic development and in the adult heart after injury, perlecan plays a vital role in maintaining BM assembly and physical integrity (145). Also, perlecan and agrin have been shown to bind various growth factors, including PDGF-B (platelet-derived growth factor subunit B) and FGF2 (fibroblast growth factor 2), and serve as important modulators of growth factor signaling. They may either sequester growth factors and inhibit downstream signaling or present the ligand to facilitate receptor activation (150). Whether perlecan is involved in RV remodeling in PAH remains unknown.

Nidogen Is a BM Glycoprotein

Nidogen, a small ubiquitous protein in the BM with three globular domains, functions as a bridge to connect collagen type IV and laminins (116, 151). It serves a critical coordinating role in BM assembly. Nidogen–laminin interactions are essential and play a key role in BM stabilization, mainly during late embryogenesis and again in adult life in the setting of increased mechanical stress (151). Experimental studies with nidogen knockout mice reveal heart defects that are lethal at birth (151). Any changes and/or the removal of nidogen might promote BM disintegration. Thus, there is a need for further studies on nidogen to understand its expression and involvement in RV remodeling in the setting of PAH.

Collagen Type XVIII Is a Collagen and Heparan Sulfate Proteoglycan in the BM

Collagen type XVIII, a member of the multiplexin family, possesses features of both collagen and heparin sulfate proteoglycan in the BM. It is cleaved by various proteases, including elastase, MMPs, and cathepsins (152–157). During cardiac development, collagen type XVIII is expressed in the endocardial and myocardial BM during early atrioventricular cushion development, later becoming localized to the atrioventricular and semilunar valves (158). Structurally, collagen type XVIII contains 10 collagenous domains interspersed in 11 noncollagenous domains (159). These domains are flanked by an N-terminal NC11 domain and a C-terminal NC1 domain. The C-terminal noncollagenous domain of collagen type XVIII can undergo proteolytic cleavage to produce the biologically active angiostatic peptide known as endostatin (160).

Endostatin exhibits potent antiangiogenic activity, preventing neovascularization in vitro and in vivo (50, 161). This is mediated by restricting proliferation, migration, and enhancement of apoptosis of endothelial cells (162) and is important in the growth and spreading of malignant diseases (158, 163–165). Increased levels of endostatin were observed in certain cancers such as lung cancer and in some chronic inflammatory diseases, including chronic kidney disease, diabetic retinopathy, and systemic sclerosis (166–168). We have previously reported increased serum endostatin is linked to disease severity in PAH and adverse hemodynamics. In the preclinical Sugen-hypoxia PAH model, increased RV expression of endostatin/Col18a1 is an early event during RV remodeling. In human PAH, increased serum endostatin is associated with pathologic RV remodeling as assessed by cardiac magnetic resonance (169). Furthermore, higher levels of circulating endostatin predict mortality, suggesting that endostatin serves as a prognostic biomarker in PAH (50, 52–54).

Serum endostatin has been associated with left ventricular dysfunction and increased heart failure risk (170). Higher levels of endostatin have been reported in patients with systemic hypertension and associated with worsened endothelial function, higher left ventricular mass, and increased urinary albumin/creatinine ratio (171). Moreover, increased expression of endostatin in the circulation, pericardial space, and myocardial tissue has been related to reduce collateral circulation in ischemic heart disease (172–174). In both models of pressure-overload–induced cardiac hypertrophy and chronic hypoxemia–induced PH, cardiac expression of endostatin is increased (55, 175, 176). It remains to be determined whether endostatin plays a direct role in the development of RV dysfunction and failure or serves as an indicator of adverse BM ECM remodeling in PAH. Further studies that will investigate the role of endostatin in the development of RV remodeling are, therefore, warranted.

Conclusions

In summary, PAH is characterized by profound pulmonary and RV remodeling that leads to increased afterload on the RV and subsequent RV remodeling and failure. Vascular remodeling is associated with increased deposition of ECM components, including BM-associated ECM proteins, elastins, laminins, collagen type IV, fibronectin, tenascin C, proteoglycan, and various growth factors and proteases that intensify the proliferative response of smooth muscle cells. ECM not only determines the tissue architecture of the lung vasculature and the myocardium but also provides mechanical stability. It is now clear that the ECM is a highly organized network and a crucial component for maintaining proper pulmonary vasculature and cardiac tissue homeostasis. Among the ECM components, the BM, composed of specialized ECM proteins, is recognized to likely contribute to both pulmonary vascular and RV remodeling in PAH. Likewise, PAH is associated with elevated circulating levels of matricellular proteins/peptides (thrombospondin-1/endostatin), which have been linked to adverse hemodynamics and outcomes. Although the BM and its associated ECM proteins have emerged as important players in vascular remodeling, their role in RV remodeling in PAH remains to be further explored. Specifically, further investigations are needed to understand and identify whether alterations in BM protein expression and functions play a causal role in the development of RV dysfunction in PAH. Such investigations may eventually lead to the development of innovative prognostic and therapeutic approaches that can restore or preserve pulmonary vasculature and RV homeostasis as well as improve patient outcomes in PAH.

Future Perspectives

Changes in the composition and organization of ECM components, including BM-associated ECM proteins, are linked to disease severity and adverse hemodynamics in PH. Therefore, targeting BM ECM proteins could potentially lead to the development of innovative therapeutic and prognostic approaches in PAH. Further translational research should help identify critical changes in BM ECM proteins responsible for RV remodeling and failure in PAH.