Cell surface thrombospondin is functionally essential for vascular smooth muscle cell proliferation

Abstract

Abstract. Thrombospondin (TS) is an extracellular glycoprotein whose synthesis and secretion by vascular smooth muscle cells (SMC) is regulated by plateletderived growth factor. We have used a panel of five monoclonal antibodies against TS to determine an essential role for thrombospondin in the proliferation of cultured rat aortic SMC. All five monoclonal antibodies inhibited SMC growth in 3-d and extended cell number assays; the growth inhibition was specific for anti-TS IgG. The effects of one antibody (134.6) were examined in detail and were found to be reversable and dose dependent. Cells treated with D4.6 at 50 Ixg/ml (which resulted in a >60% reduction in cell number at day 8) were morphologically identical to control cells. D4.6-treated SMC were analyzed by flow cytofluorimetry and were found to be arrested in the G1 phase of the cell cycle. To determine a possible cellular site of action of TS in cell growth, SMC were examined by immunofluorescence using a polyclonal antibody against TS. TS was observed diffusely bound to the cell surface of serum-or platelet-derived growth factor-treated cells. The binding of TS to SMC was abolished in the presence of heparin, which prevents the binding of TS to cell surfaces and inhibits the growth of SMC. Monoclonal antibody D4.6, like hepatin, largely abolished cell surface staining of TS but had no detectable effect on the cellular distribution of fibronectin. These results were corroborated by metabolic labeling experiments. We conclude that cell surface-associated TS is functionally essential for the proliferation of vascular SMC, and that this requirement is temporally located in the G~ phase of the cell cycle. Agents that perturb the interaction of TS with the SMC surface, such as heparin, may inhibit SMC proliferation in this manner.

Colocalization of thrombospondin and syndecan during murine development

“…Such a role may also apply to developing smooth muscle, which is positive for thrombospondin in the bladder, gastrointestinal tract, and some (but not all) blood vessels. Thrombospondin might also function in regulating the growth of smooth muscle cells, as suggested by Majack et al (1986Majack et al ( , 1988 based on observations in vitro.…”

“…Together, these effects may account for the increased cord formation by BAEC observed in the presence of anti-thrombospondin antibodies (Iruela-Arispe et al, 1991). In contrast to its effects on BAEC, thrombospondin promotes the growth of rat aortic smooth muscle cells (Majack et al, 1986(Majack et al, , 1987(Majack et al, , 1988.…”

Cell biology of arterial proteoglycans.

“…It remains to be determined whether thrombospondin associates with the surface of smooth muscle cells as it does with endothelial cells. Majack et al 166 made the interesting suggestion that surface thrombospondin, presumably bound to a membrane form of HSPG, interact with certain proteases to facilitate the matrix degradation and remodeling that accompanies cell proliferation. Heparin, which inhibits SMC proliferation, also releases thrombospondin from the cell surface.…”

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

“…Heparin, as well as heparan sulphate, blocks cellular multiplication at the G0 and G1 stage Wright et al, 1989). 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).…”