Endothelial Heparan Sulfate Proteoglycan. I. Inhibitory Effects on Smooth Muscle Cell Proliferation

Abstract

Proliferation of smooth muscle cells is an important component of pulmonary arterial morphogenesis, both during normal development and pathologic remodeling. However, little is known of the factors that regulate smooth muscle proliferation in these vessels. To investigate the hypothesis that factors produced by endothelial cells may regulate smooth muscle cell growth, we studied the effects of culture medium conditioned by fetal bovine pulmonary arterial endothelium on proliferation of smooth muscle cells in culture. This conditioned medium contains an inhibitor of smooth muscle proliferation that is degraded by nitrous acid, heparinase, and heparitinase, but resists degradation by protease, boiling, and chondroitin ABC lyase, indicating that the inhibitor is structurally similar to heparin. Inhibitor release occurs in both growing and confluent endothelial cell cultures and in the presence and absence of serum. A growth-inhibiting proteoglycan purified to homogeneity from endothelial cell-conditioned medium has physicochemical characteristics similar to those of the prototypic basement membrane heparan sulfate proteoglycan of the Englebreth-Holm-Swarm tumor: an overall size of approximately 10(6) D, heparan sulfate chains of 60,000 D, and a buoyant density of 1.33 g/ml. Antibody raised against the tumor basement proteoglycan recognizes this endothelial heparan sulfate proteoglycan, and Western blotting after SDS-PAGE demonstrates that the core proteins of both proteoglycans migrate as a doublet at apparent molecular weights of 450,000 and 360,000 D. Heparan sulfate glycosaminoglycan prepared from purified medium proteoglycan is a potent inhibitor of smooth muscle cell growth, exhibiting activity approximately 1,000 times greater than that of heparin. These results indicate that endothelial cells cultured from fetal bovine pulmonary arteries produce a basement membrane heparan sulfate proteoglycan that is a potent inhibitor of smooth muscle proliferation. This proteoglycan may mediate endothelial regulation of smooth muscle growth during development or pathologic pulmonary arterial remodeling.

Perlecan regulates Oct-1 gene expression in vascular smooth muscle cells.

“…Perlecan is widely expressed in the basement membranes of adult tissues and in all vascular structures (Couchman, 1987;Murdoch et al, 1994) and has been directly implicated as a potent endothelial cell-derived inhibitor of SMC replication (Benitz et al, 1990). Our data indicate that at least some of the gene-regulating activity of perlecan resides in its heparan sulfate side chains, consistent with a large body of evidence implicating heparin-like molecules in the regulation of various SMC functions (Clowes and Karnovsky, 1977;Castellot et al, 1981;Majack and Bornstein, 1984;Majack and Clowes, 1984;Marcum and Rosenberg, 1984;Majack et al, 1985;Fritze et al, 1985;Benitz et al, 1990;Campbell et al, 1992;Pukac et al, 1992;Au et al, 1993).…”

“…The protein core consists of five distinct molecular domains, including a perlecan-specific domain containing heparan sulfate attachment sites and domains homologous with the low-density lipoprotein receptor, the laminin A globular region, immunoglobulin repeats, and epidermal growth factor-like repeats (Noonan et al, 1991;Noonan and Hassell, 1993;lozzo et al, 1994). Perlecan is widely expressed in the basement membranes of adult tissues and in all vascular structures (Couchman, 1987;Murdoch et al, 1994) and has been directly implicated as a potent endothelial cell-derived inhibitor of SMC replication (Benitz et al, 1990). Our data indicate that at least some of the gene-regulating activity of perlecan resides in its heparan sulfate side chains, consistent with a large body of evidence implicating heparin-like molecules in the regulation of various SMC functions (Clowes and Karnovsky, 1977;Castellot et al, 1981;Majack and Bornstein, 1984;Majack and Clowes, 1984;Marcum and Rosenberg, 1984;Majack et al, 1985;Fritze et al, 1985;Benitz et al, 1990;Campbell et al, 1992;Pukac et al, 1992;Au et al, 1993).…”

“…Endogenous HSPGs of several types are produced by both endothelial cells and SMCs (Castellot et al, 1981;Marcum and Rosenberg, 1984;Fritze et al, 1985;Benitz et al, 1990;Jarvelainen et al, 1991;Cizmeci-Smith et al, 1992;Bernfield et al, 1993), and administration of soluble heparin after experimental balloon catheter injury to the rat carotid artery in vivo inhibits SMC replication, migration, and neointimal formation and alters the composition of the ECM surrounding SMCs (Clowes and Karnovsky, 1977;Snow et al, 1990;Au et al, 1993). In addition, culturing SMCs on a heparan sulfate-rich glycosaminoglycan extract inhibited SMC phenotypic modulation in a heparinase-sensitive manner (Campbell et al, 1992).…”

The cell cycle: A critical therapeutic target to prevent vascular proliferative disease

“…In vivo, perlecan has been shown to inhibit thrombosis and intimal hyperplasia after arterial injury (153). Several studies have shown that endothelialderived perlecan is a potent inhibitor of SMC proliferation in vitro (149,154,155). Perlecan binds to heparin-binding mitogens such as FGF-2, leading to inactivation of FGF-2, a potent stimulatory growth factor, due to its heparin-binding property.…”

Heparin and its derivatives in the treatment of arterial thrombosis: a review

“…This action is multifactorial, including an inhibition of mitogens in plasma, inhibition of excretion of platelet-derived growth factor (PDGF), interference with thrombin mitogenic activity, deliberation of cell-bound thrombospondin (free thrombospondin is unable to interact with PDGF) (Majack, 1985(Majack, , 1986(Majack, , 1988, inhibition of DNA synthesis in smooth muscle cells by intracellular heparin (Reilly et al, 1986;Wright et al, 1989), heparin-dependent inhibition of VSMC protein synthesis Cochran et al, 1985) and protecting of heparan sulphate from biodegradation by heparinase released from platelets (Fritz et al, 1985;Castellot et al, 1987;Wright et al, 1989). In the last case, the following events have been observed: (1) both heparin and heparan sulphate inhibit VSMC growth in a similar fashion (Castellot et al, 1981(Castellot et al, , 1987Benitz et al, 1990), endothelial cells synthesize heparan sulphate in the same manner as smooth muscle cells; (2) smooth muscle cells synthesize heparan sulphate more during continuous growth than during growth on exponential cell-lines (Fritz et al, 1985); (3) media containing heparan sulphate (those fixed by heparinase from flavobacteria) increase the sensitivity of VSMC's to various mitogens and growth stimulators (Castellot et al, 1981). Heparinase itself can be released by internal platelet and monocyte activation (Oldberg et al, 1980;Castellot et al, 1982;Wright et al, 1989).…”