Structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells. II. Evidence for a pentasaccharide sequence that contains a 3-O-sulfate group.

Abstract

Abstract. Earlier work from our laboratory demonstrated that heparin inhibited the proliferation of vascular smooth muscle cells in vivo and in vitro. Both anticoagulant and non-anticoagulant heparin species were equally effective as antiproliferative agents. Previous structure-function studies indicated that hexasaccharide and larger fragments retained antiproliferative activity, whereas tetra-and disaccharides were inactive. These experiments also suggested that both Nand O-sulfates of heparin were necessary for growth inhibitory capacity. In this paper, we have further analyzed the structural determinants of the antiproliferative activity of heparin. These experiments were done using synthetically prepared and therefore chemically defined heparin oligosaccharides. We present evidence that a pentasaccharide fragment retains antiproliferative activity, and that the 3-O-sulfate on the internal glucosamine residue is critical for growth inhibitory capacity of the pentasaccharide. We also show that heparins obtained from different manufacturers differ significantly in their ability to suppress smooth muscle cell proliferation.T HE proliferation of vascular smooth muscle cells (SMC) ~ after endothelial injury is thought to be a key step in the pathogenesis of arteriosclerosis (Ross and Glomset, 1973). Although much attention has been focused on factors that stimulate SMC proliferation (Schwartz et al., 1982), very little is known about the mechanisms involved in maintaining these cells in a quiescent growth state in healthy blood vessels, and in re-establishing the quiescent growth state of SMC after their proliferative response to intimal injury.Earlier work from our laboratory demonstrated that heparin inhibited the proliferation of SMC in vivo and in vitro (Clowes and Karnovsky, 1977;Guyton et al., 1980;Hoover et al., 1980; Castellot et al., 1981). Both anticoagulant and non-anticoagulant heparin species were equally effective as SMC antiproliferative agents. These studies suggested both a pharmacological basis for the use ofhepafin after vessel injury, and a possible physiological role for heparin-like species as regulators of SMC growth in the vasculature.Previous structure-function studies (Castellot et al., 1984) indicated that hexasaccharide and larger fragments retained antiproliferative activity, whereas tetra-and disaccharides were inactive against SMC growth. These experiments also indicated that both the N-and O-sulfates of heparin were necessary for antiproliferative activity, although the N-sulfates could be replaced with acetyl groups without significant loss of activity. Both the hexasaccharide fragment and the Ndesulfated, re-N-acetylated heparin are devoid of anticoagulant activity (Kazatchkine et al., 1981;Oosta et al., 1981), thus providing a chemical basis for the difference between the antiproliferative and anticoagulant activities of this glycosaminoglycan.In this paper, we have further analyzed the structural determinants of the antiproliferative effect of heparin. These experiments were ...

Heparin and hormonal regulation of mRNA synthesis and abundance of autocrine growth factors: relevance to clonal growth of tumors.

“…Nevertheless, the scarcity of glycosaminoglycan reference standards forces investigators to use the commercial heparins of weaker and variable specific activity. These findings are analogous to those reported by Castellot et al (5) in their structure-function analysis of heparin effects on inhibition of growth of smooth muscle cells. The data shown are from Sigma's bovine lung-derived heparin, lot 53F-0532.…”

Trans-repressor activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription.

“…The results of the binding experiments support the conclusions from the transient transfection studies and provide evidence for a possible mechanism of action in which nuclear GAG act as a direct competitive inhibitor of DNA thereby blocking the trans-activation response. Castellot et al (1986) reported the inhibition of SMC proliferation by GAG required the presence of a 3-0-sulfated pentasaccharide. Similarly, Resink et al (1989) found that pentosan polysulfate and heparin were competitive for the same cellular binding site and that both were inhibitory to vascular SMC proliferation .…”

Effect of dalteparin and radiation on survival and thromboembolic events in glioblastoma multiforme: a phase II ECOG trial

“…Mechanistically, the potential anti-neoplastic properties of LMWH include anti-angiogenisis [2,3], effects on cellular matrix [4,5], and reduced cell proliferation [6]. In this regard, it is of interest to note that based on preclinical studies, anti-angiogensis agents can putatively serve as radio-sensitizers [7,8].…”