Abstract

Perlecan is a large heparan sulfate proteoglycan (HSPG), which is a major component of the vessel wall. In relation to vascular biology, perlecan has been shown to be a potent inhibitor of smooth muscle cell (SMC) activity. In vivo experiments in animal models of arterial injury have shown that perlecan may inhibit thrombosis and intimal hyperplasia. On the other hand, perlecan has been shown to have opposing effects on endothelial cells (ECs), where it promotes in vitro and in vivo angiogenesis and plays an important role in mediating tumor growth. These diverse biological effects, or “the perlecan paradox”, are discussed in this review paper. The properties of perlecan including inhibition of SMC activity and thrombosis while enhancing EC proliferation are ideal for the prevention of in-stent restenosis. Perlecan's pro-angiogenic effects may be used for the treatment of various ischemic diseases such as intractable coronary artery disease and peripheral vascular disease.

1. Introduction

Proteoglycans (PGs) are ubiquitous macromolecules that consist of a core protein, to which one or more glycosaminoglycans (chondroitin sulfate, dermatan sulfate, heparan sulfate or keratin sulfate) are covalently bound [1]. These complex molecules influence many arterial properties, such as viscoelasticity, permeability, lipid metabolism, hemostasis, thrombosis and extracellular matrix (ECM) assembly [2–4]. A specific class of PGs, known as heparan sulfate proteoglycan (HSPG), is emerging as key molecules governing crucial events in embryonic development, inflammation, wound repair, and cancer [5]. A variety of growth factors bind to HSPG, including members of the fibroblast growth factor (FGF) family [6], vascular endothelial growth factor (VEGF) [7], heparin-binding epidermal growth factor (EGF) [8] and cytokines, such as transforming growth factor-β (TGF-β) [9]. A common feature of the heparin-binding growth factors family members is their high affinity towards HSPGs and especially perlecan. The interaction between FGF-2 and HSPG/perlecan enhances high affinity binding to the FGF cell surface tyrosine kinase receptor, and is essential for mediating receptor phosphorylation and receptor-mediated signal transduction [6,10].

Vascular endothelial cells (ECs) synthesize HSPGs, including perlecan [11], members of the syndecan family of transmembrane PGs [12,13] and the cell surface-associated PG, glypican-1 [12]. ECs also constitutively synthesize and secrete the large aggregating chondroitin sulfate PG, versican [14], and the small leucine-rich chondroitin/dermatan sulfate PG, biglycan [15,16]. In addition, the expression of another small leucine-rich chondroitin/dermatan sulfate PG, decorin, is induced during formation of neovessels both in vitro and in vivo [17,18].

2. Perlecan

Perlecan is a large (467 kDa) HSPG that is expressed in most ECM and basement membranes [19,20]. It is the major extracellular HPSG in blood vessel ECM. The core protein consists of five distinct domains with homologies to molecules involved in cell proliferation, lipoprotein uptake and cell adhesion (Fig. 1). Each of these domains has exhibited one or more binding sites for a number of ligands including basement membrane components [19–22], cell adhesion molecules [23] and growth factors [23,25]. The structural characteristics of the complete human perlecan gene and its promoter have been fully described [26]. Deletion of the perlecan gene causes embryonic lethality with severe cephalic, myocardial and cartilage abnormalities, including transposition of the great vessels [27–29]. Perlecan-deficient embryos demonstrate abnormally abundant mesenchymal cells expressing smooth muscle cell (SMC)-specific alpha-actin isoform in the left ventricular outflow tract, which can lead to varying levels of outflow tract obstruction [29].

Fig. 1

Structure of perlecan.

Fig. 1

Structure of perlecan.

In relation to cardiovascular biology, perlecan has been identified in the atherosclerotic intimal lesions of apoE−/− and LDLR−/− mice [30] and may contribute to the retention of lipoproteins at the earliest stage of atherosclerosis. Perlecan is associated with intermediate and advanced lesions of hypercholesterolemic nonhuman primates and in cultures of medial SMC from human atherosclerotic tissue [31–33]. The exact role of perlecan in the arterial wall is still unclear. Perlecan has a module domain that shares homology with the binding domain of the LDL receptor, suggesting that perlecan could potentially bind lipoproteins [34,35].

A different type of a membranal HSPG that has been studied more extensively is syndecan. The regulation of the interaction between heparin-binding growth factors and their associated receptors by perlecan may have many similarities to syndecan. All heparin binding growth factors such as FGF, VEGF and heparin-binding EGF are highly regulated by syndecan, reflecting the ability of syndecan and possibly other HSPGs to contain specific binding sites within the architecture of the heparan sulfate chains [36]. Specifically with FGF, syndecan has been shown to bind not only the growth factor but also the receptor forming a high affinity tri-molecular complex that is necessary for receptor activation, phosphorylation and subsequent signal transduction [37]. Perlecan has also been shown to induce high affinity binding of FGF-2 both to cells deficient in heparan sulfate and to soluble FGF receptors, thus promoting angiogenesis and tumor growth [25]. On the other hand, perlecan also appears to inhibit SMC proliferation in cell culture [38].

3. Effects of perlecan on smooth muscle cells

3.1. Adhesion and migration

Perlecan interferes with the adhesion of SMC to fibronectin, an interstitial ECM glycoprotein that accumulates around migrating and proliferating cells in the arterial media and intima [39,40]. HSPGs also mediate a potent inhibitory signal for migration of SMC [41].

3.2. Proliferation

Endogenous perlecan gene expression in aortic SMC is largely limited to non-replicating cells. Perlecan mRNA in cultured SMC is significantly higher in non-replicating (serum-starved) cultures compared to replicating cultures [42]. The data suggest that the expression of perlecan by vascular SMC is regulated by cellular growth state. In vascular SMC, perlecan inhibits the expression of Oct-1, a member of a family of transcription factors involved in cell growth processes. However, when cells are exposed to other components of the ECM such as laminin, type IV collagen or fibronectin, Oct-1 is readily detectable. This suggests that perlecan plays a biologically important role in negatively regulating SMC at the transcriptional level [43]. It has been known for many years that heparan sulfate glycosaminoglycans (HSGAGs) are potent inhibitors of SMC proliferation [44,45] independent of its anticoagulant property [45,46].

Several studies have shown that endothelial-derived perlecan is a potent inhibitor of SMC proliferation in vitro [13,47,48]. Perlecan binds to heparin-binding mitogens, such as FGF-2 and prevents them from stimulating SMCs [48,49]. It appears that PGs with larger heparan sulfate chains (such as perlecan) are more potent inhibitors of FGF-2 binding than the PGs with smaller heparin sulfate chains [48]. An additional mechanism of this antiproliferative effect may be related to apolipoprotein E [35], which was specifically found to induce the expression of perlecan in SMCs. The induction of perlecan by apolipoprotein E was correlated with the inhibition of SMC proliferation. Recently, Garl et al. [50] showed that perlecan-induced suppression of SMC proliferation is mediated through increased activity of the tumor suppressor PTEN, which is an important inhibitor of growth factor receptor- and integrin-stimulated signaling, thus promoting cell cycle arrest, decreased cell migration and apoptosis. Perlecan may also interact with non-heparin binding mitogens such as thrombin and platelet-derived growth factor (PDGF). Rauch et al. [51] demonstrated that both thrombin and factor Xa increase SMC proliferation by transactivation of FGF receptor-1. This activation was inhibited by heparin and by anti-FGF-2 antibodies. Gohring et al. [52] mapped the perlecan binding site of PDGF suggesting that perlecan serves as a storage site of PDGF, a non-heparin binding growth factor.

3.3. The role of perlecan in maintaining cell quiescence

Recently, Pillarisetti et al. have suggested that perlecan may be required to maintain SMC quiescence [53]. This is based on the observation that serum starvation induces perlecan expression by SMC. Strategies to block perlecan by anti-perlecan antibodies or deoxyribozyme transfection increased DNA synthesis despite serum-free conditions. Furthermore, antiproliferative agents such as apoE, cGMP and nitric oxide have no effect on SMC growth when perlecan is blocked further suggesting the important role of perlecan as a master switch regulating SMC quiescence [53].

4. Role of perlecan after arterial injury

Due to perlecan's inhibitory effects on SMC, its role in arterial repair after injury is interesting. Unfortunately, there are few studies on this relationship in vivo. In an experimental carotid artery injury in rats, mRNA expression of several HSPG including perlecan, syndecan and ryudocan are increased beginning only at 1–2 weeks after injury in both the intima and media [54]. Perlecan expression was not affected by heparin. Using the same rat model, Kinsella et al. [55] showed by immunohistochemistry and in situ hybridization that perlecan expression was low in uninjured arteries and up to 7 days after injury. By 2 weeks after injury, perlecan expression was increased significantly and remained highly expressed in advanced lesions, 35–42 days after injury. Ex vivo, arterial explants were treated by heparin lyase to remove any HSPG effects. The authors showed that the responsiveness to PDGF could have been restored by the removal of endogenous HSPG from the neo intimal ECM. In a different in vivo experimental study by Bingley et al. [56], a mixture of HSPG (including perlecan) that was extracted from normal rabbit aorta was applied as peri-adventitial gel to rabbit carotid arteries undergoing balloon injury. Arterial HSPG mixture reduced neointimal formation by 35% at 14 days after injury, whereas the low-molecular-weight heparin enoxaparin was ineffective. Most recently, Tran et al. have analyzed transgenic mice with heparan sulfate-deficient perlecan with respect to vascular phenotype and intimal lesion formation. SMCs obtained from the transgenic animals showed increased proliferation and reduced ECM binding capacity of FGF-2. In vivo, transgenic animals had an augmented intimal hyperplasia after flow cessation of the carotid artery. The authors suggest the importance of the heparan sulfate side chains in contributing to SMC growth control by the ability of these side chains to sequestrate heparin-binding growth factors such as FGF-2 [57].

5. Anti-thrombotic effects of perlecan

In a study by Nugent et al. [38], perlecan-deficient EC were generated by using an antisense vector targeting the domain III of perlecan. First, perlecan-deficient cells showed a reduced ability to inhibit the binding of FGF-2 to its cell surface receptor. Second, these cells were seeded adjacent to porcine carotid arteries subjected to deep injury. In contrast to perlecan-expressing cells that completely prevented occlusive thrombosis, injured arterial segments containing perlecan-deficient cells had a 23% thrombotic occlusion rate. Furthermore, HSPG produced by EC may exert an anticoagulant effect by local binding of anti-thrombin III [46]. In contrast, HajMohammadi et al. [58] found no prothrombotic state in transgenic mice lacking HS 3-O-sulfotransferase-1. These animals have reduced HS in either endothelial cells or in the subendothelial space. However, it is possible that luminal heparan sulfates exerts an anticoagulant effect [59].

6. Effects of perlecan on endothelial cells

6.1. Endothelial cell proliferation

FGF-2, VEGF and hepatocyte growth factor/scatter factor (HGF) are all heparin-binding growth factors and important regulators of EC growth and the angiogenic process under both physiological and pathological conditions [60,61]. FGF-2 can interact with the heparan sulfate chains of proteoglycans on the EC cell surface [6,62]. When complexed with heparan sulfate chains, FGF-2 is protected from proteolytic degradation [63,64] and maintains a capacity for long-term stimulation of EC inducing proliferation, migration [65] and increased plasminogen activator activity [62,65]. The ECM is a second source of perlecan, which may also act as a physiologic buffer whereby there is FGF binding when concentrations are high and releasing it later for interaction with its receptor. Several studies have identified a crucial role for heparin-like molecules in the formation of distinct FGF-2-heparin complexes that are essential for FGF binding and activation of its tyrosine kinase receptor [6,66].

6.2. Angiogenesis

Perlecan, by inducing high affinity binding of FGF-2 to heparin sulfate deficient cells or to soluble FGF receptors, possesses angiogenic properties and promotes tumor angiogenesis [25,67]. Increased perlecan levels are detected in breast carcinomas [68,69] and in metastatic melanomas [70] that correlates with a more aggressive phenotype [71]. It has also been shown that perlecan suppression can inhibit tumor angiogenesis. Antisense targeting of perlecan inhibited FGF-2 function in human melanoma cells [72] and blocked tumor growth and angiogenesis in vivo [24]. Adatia et al. [73] were successful in the suppression of the invasive behavior of melanoma cells by stable expression of antisense perlecan cDNA. It should be stated that although perlecan has been correlated with tumor growth, several other studies have shown the opposite effects. Human fibrosarcoma cells that were transfected with anti-sense perlecan cDNA grew faster, formed larger colonies on agar gel and induced faster formation of subcutaneous tumors in nude mice than the wild-type cells [74]. Recently, perlecan was shown to increase the activity of an important tumor suppressor PTEN further suggesting the complexity of the effects of perlecan on tumor growth [50].

VEGF is an endothelial cell-specific angiogenic growth factor, which stimulates EC proliferation, increases endothelial permeability and acts as an endothelial “survival factor” [75,76]. VEGF can also interact with the heparan sulfate chains of proteoglycans on the EC surface [77]. VEGF has been shown to stimulate the development of collateral arteries in animal models of peripheral [78–82] and in human peripheral vascular disease [83,84]. HGF is another heparin-binding growth factor (60–65 kDa) that has mitogenic, angiogenic, antiapoptotic and antifibrotic effects in a variety of cell types, including hepatocytes, epithelial cells and EC [85,86]. Recently, it has been shown that HGF is upregulated in a murine model of critical limb ischemia and its expression is regulated by FGF-2 [87]. Although VEGF and HGF bind HSPG, few in vitro and no in vivo studies have specifically addressed perlecan–VEGF or perlecan–HGF interactions. In an in vitro study, transfection of an immortalized cell line, derived from a human Kaposi's sarcoma, with an antisense perlecan construct significantly reduced migration and proliferation in response to VEGF and HGF compared to controls [88]. In fact, the in vivo angiogenic effects of perlecan outside of tumor angiogenesis have received little attention to date. The exception is one study using the rabbit ear crush model of angiogenesis, which showed that perlecan encapsulated in alginate beads bound FGF-2 and promoted angiogenesis [25].

7. The “perlecan paradox”

Perlecan has a number of complex biologic effects that depend on cell type and the environment (Table 1). In SMCs, perlecan has been shown to be a potent inhibitor of adhesion, migration and proliferation, and may represent a novel strategy to inhibit intimal hyperplasia after arterial injury. However, the mechanism(s) by which perlecan inhibits SMCs has not been fully explored. In contrast to its inhibitory effect on SMCs, perlecan appears to be a potent pro-angiogenic agent. This duality of function depending on cell type may seem paradoxical. There are several possible explanations. These differences may be due to the HSGAG side chains that directly affect cell signaling or through interactions with heparin-binding growth factors, particularly FGF-2 and its receptor. For example, COP-1, a member of the CCN family of heparin-regulated genes, is specifically induced by heparin in SMCs, but not in ECs, and has anti-proliferative activity [89]. The activity of FGF is modulated by HSGAG, found both in the ECM and on the cell surface. Differences in FGF2-mediated proliferation in SMCs and ECs appear to be related to the distinct cell surface HSGAG of the two cell types [90]. Recently, it has been reported that heparin inhibition of EC proliferation and organization requires a specific chain length and molecular size [91].

Table 1

Biological effects of perlecan

Effect Reference 
Smooth muscle cells 
Interference with adhesion to fibronectin [39] 
Inhibition of migration [41] 
Inhibition of Oct-1 expression [43] 
Inhibition of proliferation by EC-derived perlecan [45–47] 
Binding to FGF-2/interference with FGF receptor binding [48,49] 
Inhibition of apoE binding to SMC [35] 
Increased activity of tumor suppressor PTEN [50] 
Arterial injury 
Restoration of PDGF activity after removal of perlecan from intimal ECM [53] 
Reduced neo-intimal formation by adventitial delivery of perlecan [38] 
Augmented intimal hyperplasia in transgenic mice lacking HS of perlecan [57] 
Thrombosis 
Perlecan-expressive cells prevented occlusive thrombosis after injury [38] 
Perlecan binds to anti-thrombin III [46] 
Angiogenesis 
Perlecan is a crucial cofactor for FGF-2/VEGF binding [25] 
FGF-2/perlecan complexes are resistant to proteolytic degradation [57,58] 
Long-term stimulation of EC proliferation and migration [65] 
Perlecan increases EC plasminogen activator activity [62,65] 
Perlecan induces in vivo angiogenesis [25] 
Perlecan is involved in tumor growth and is correlated with aggressiveness [70–73] 
Perlecan antisense causes proliferation of fibrosarcoma cells [74] 
Potential effects 
Effects on other factors such as PDGF, TGF-β, INF-γ [34,52,53] 
Effect Reference 
Smooth muscle cells 
Interference with adhesion to fibronectin [39] 
Inhibition of migration [41] 
Inhibition of Oct-1 expression [43] 
Inhibition of proliferation by EC-derived perlecan [45–47] 
Binding to FGF-2/interference with FGF receptor binding [48,49] 
Inhibition of apoE binding to SMC [35] 
Increased activity of tumor suppressor PTEN [50] 
Arterial injury 
Restoration of PDGF activity after removal of perlecan from intimal ECM [53] 
Reduced neo-intimal formation by adventitial delivery of perlecan [38] 
Augmented intimal hyperplasia in transgenic mice lacking HS of perlecan [57] 
Thrombosis 
Perlecan-expressive cells prevented occlusive thrombosis after injury [38] 
Perlecan binds to anti-thrombin III [46] 
Angiogenesis 
Perlecan is a crucial cofactor for FGF-2/VEGF binding [25] 
FGF-2/perlecan complexes are resistant to proteolytic degradation [57,58] 
Long-term stimulation of EC proliferation and migration [65] 
Perlecan increases EC plasminogen activator activity [62,65] 
Perlecan induces in vivo angiogenesis [25] 
Perlecan is involved in tumor growth and is correlated with aggressiveness [70–73] 
Perlecan antisense causes proliferation of fibrosarcoma cells [74] 
Potential effects 
Effects on other factors such as PDGF, TGF-β, INF-γ [34,52,53] 

Differences in the ECM of SMC and EC also profoundly affect cellular behavior. The sequestering of heparin-binding growth factors such as FGF by perlecan in the SMC ECM may be an important factor in inhibiting SMC proliferation. Perlecan may behave differently in the presence of other matrix compounds such as fibronectin [39]. Thus, the bioactivity of HSPG such as perlecan is dependent on its cell origin, subtle changes in structure (including secondary interactions the size of the heparin side chains and its glycosolation status), the concentration and binding kinetics of the growth factor and the expression of a specific receptor and its isoforms [92,93].

This discussion of the “perlecan paradox” has focused on the heparan binding sites of perlecan that interact particularly with FGF-2. However, within the enormous core protein of perlecan, there are a number of additional binding sites, which have received scant attention. For example, several growth factors and cytokines bind to various domains of the perlecan protein core including FGF-7 [94], PDGF AA and BB [53], FGF-BP [95], HGF, interleukins, TGF-β, and IFN-γ [34]. The physiologic consequences of the interactions between these mediators of cell growth and the perlecan core protein merit further investigation. Fig. 2 illustrates a proposed mechanism for the differential effects of perlecan on SMC and EC. Similar to syndecan, in ECs, perlecan is essential for the formation of high affinity tri-molecular complex in order to activate the receptor. In SMCs, cell surface or extracellular matrix perlecan side chains may be different in length or conformation so perlecan traps FGF molecules thus inhibiting FGF receptor binding. This suggested mechanism is probably more validated in ECs and only speculative regarding SMCs. However, other mechanisms may explain this duality of action. The sequence and the differential sulfation patterns of the heparan sulfate side chains may also be an important determinant of perlecan's specifications in different cell types [96]. Moreover, it has been also suggested that perlecan in the vessel wall binds FGF-2 and serves as a reservoir of growth factors that then can be released by several enzyme such as stromelysin and collagenase [97]. These properties may also be different between ECs and SMCs.

Fig. 2

A proposed mechanism for the differential effects of perlecan on SMC and EC. FGF interaction with its cell surface receptor serves as an example. In ECs, perlecan is essential for the formation of high affinity tri-molecular complex in order to activate/phosphorylate the receptor (Panel A). In SMCs, cell surface or extracellular matrix perlecan side chains may be different in length or conformation so perlecan traps FGF molecules inhibiting FGF receptor binding (Panel B).

Fig. 2

A proposed mechanism for the differential effects of perlecan on SMC and EC. FGF interaction with its cell surface receptor serves as an example. In ECs, perlecan is essential for the formation of high affinity tri-molecular complex in order to activate/phosphorylate the receptor (Panel A). In SMCs, cell surface or extracellular matrix perlecan side chains may be different in length or conformation so perlecan traps FGF molecules inhibiting FGF receptor binding (Panel B).

8. Conclusions and clinical implications

We still lack the exact mechanism by which perlecan acts differently on two important vascular cell types, SMC and EC. These opposing effects merit further studies. These diverse biological effects of perlecan are ideal for the prevention of in-stent restenosis. Perlecan inhibits SMC proliferation and arterial thrombosis while enhancing EC proliferation, which may promote rapid healing after stenting. However, in vivo studies of perlecan administration have been hampered by lack of availability of purified or partially purified perlecan. Therefore, additional studies are required to fully explore the role of perlecan in arterial repair, especially after stenting. The role of perlecan in other vasculopathies such as vein graft repair, and transplant vasculopathy also merit further experimental attention. On the other hand, perlecan's pro-angiogenic effects and the augmentation of the effects of heparin-binding growth factors, such as FGF and VEGF, may serve as a new modality for the induction of angiogenesis in ischemic conditions such as ischemic heart disease and peripheral vascular diseases.

Acknowledgements

Dr. A. Segev is a Research Fellow of the Heart and Stroke Foundation of Canada.

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Author notes

Time for primary review 29 days