An antiproliferative heparan sulfate species produced by postconfluent smooth muscle cells.

Abstract

Heparan sulfate was isolated from the cell surface, cell pellet, and culture medium of exponentially growing as well as postconfluent bovine aortic smooth muscle cells (SMCs). After chromatography on DEAE-Sephadex and Sepharose 4B, the various mucopolysaccharides were examined for their ability to cause growth inhibition in a SMC bioassay. The heparan sulfate isolated from the surface of postconfluent SMCs possessed approximately eight times the antiproliferative potency per cell of the heparan sulfate obtained from the surface of exponentially growing SMCs. Heparan sulfate isolated from other fractions of exponentially growing or postconfluent SMCs possesses little growth inhibitory activity. The difference in the antiproliferative activities of heparan sulfate obtained from the surface of SMCs in the two growth states could not be attributed to the synthesis of a greater mass of mucopolysaccharide by postconfluent SMCs. Indeed, heparan sulfate isolated from the surface of the postconfluent SMCs exhibits a specific antiproliferative activity which is 13-fold greater than mucopolysaccharide obtained from the surface of exponentially growing SMCs and more than 40-fold greater than commercially available heparin. In addition, exponentially growing SMCs did not exhibit an enhanced ability to degrade the complex carbohydrate. Furthermore, other investigations indicate that the small amount of growth inhibitory activity intrinsic to heparan sulfate isolated from the surface of exponentially growing SMCs is due to residual, biologically active, mucopolysaccharide produced by the primary postconfluent SMCs from which the exponentially growing SMCs were derived. These studies suggest that bovine aortic SMCs are capable of controlling their own growth by the synthesis of a specific form of heparan sulfate with antiproliferative potency.Most connective tissues contain glycosaminoglycans as major components of their extracellular matrix. These high molecular weight, negatively charged sulfated mucopolysaccharides have been implicated in determining certain general overall properties of tissues such as hydration, elasticity, and permeability (1). It has also been suggested that glycosaminoglycans that are known to be associated with the surface of the cell could be involved in basic cellular functions, such as adhesion and motility (2, 3). However, little is known about whether specific structural elements on the glycosaminoglycans are responsible for the biologic properties of these components.Several investigators have demonstrated that the mucopolysaccharide, heparin, must satisfy unique structure-function relationships to interact with its protein co-factor, antithrombin, to alter allosterically the conformation of this protease inhibitor, and thereby accelerate neutralization of coagulation system enzymes (4). Our laboratory has recently shown that anticoagulantly active heparan sulfate molecules are present on the surface of endothelial cells and are, in part, responsible for maintaining the nonthrombogenic p...

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).…”

“…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).…”

Fibronectin stimulates macrophage uptake of low density lipoprotein-heparin-collagen complexes.

“…4950 These structural differences are related to functional properties, such as the ability to inhibit smooth muscle cell proliferation. 49 - 50 In addition, there may be focal differences in the nature of the heparan sulfates in the arterial wall, such as the increased charge of heparan sulfate found in an area of pigeon aorta predisposed to lesion development. 51 Highly charged heparan sulfate proteoglycan may play a role similar to the Hep Fn used in this study.…”

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

“…The presence of sulphate is necessary for this process, since de-sulphated heparin loses its inhibitory properties for VSMC (Castellot et al, 1984). Other glycosaminoglycans, such as dermatan sulphate, hyaluronic acid and chondroitin sulphate do not inhibit VSMC proliferation with the same strength as heparan sulphate (Castellot et al, 1981;Fritz et al, 1985).…”

“…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).…”