Transforming growth factor-beta 1 and ascorbate regulate proliferation of cultured smooth muscle cells by independent mechanisms.

We previously reported that ascorbate (vitamin C) can regulate the growth of cultured vascular smooth muscle cells (VSMC) directly as well as by altering the properties of extracellular matrix (ECM) [Mol Cell Cardiol 1997;29:3293-303]. In the present study we compared the effects of ascorbate and transforming growth factor-beta 1 (TGF-beta1) on VSMC growth in order to determine whether their actions were mediated by similar mechanisms. When VSMC proliferation was stimulated by fetal bovine serum, the addition of TGF-beta1 (20 ng/ml) or ascorbate (1 mM) to the cell culture medium inhibited the cellular incorporation of [3H]thymidine by 19 and 59%, respectively, and by 85% when added together. The cell growth inhibitory effects of TGF-beta1 and ascorbate were partially mediated by changing the growth-regulatory properties of the ECM produced by the cells. Thus, VSMC grew more slowly on ECM deposited by VSMC under treatment with 20 ng/ml TGF-beta1 or 1 mM ascorbate (52 and 46% inhibition, respectively) than on control ECM, and their combination had an additional inhibitory effect (84%). Anti-TGF-beta1 neutralizing antibodies prevented the direct and ECM-mediated effects of TGF-beta1 on VSMC growth, but did not alter the effects of ascorbate. When ECM was pre-incubated with increasing concentrations of TGF-beta1, the growth rate of freshly plated VSMC gradually decreased, indicating that ECM-bound TGF-beta1 retained its biological activity. Comparison of the patterns of TGF-betal binding to ECM produced by VSMC in the presence or absence of ascorbate revealed no significant differences. Extraction of ECM-bound TGF-beta1 by incubation of exposed ECM with plasmin did not affect the ECM-mediated inhibitory effect of ascorbate, as the rate of proliferation of secondary VSMC cultures grown on ascorbate-dependent and independent matrices treated with plasmin were equally increased. These results suggest that the amount of ECM-bound TGF-beta1 was not altered by ascorbate. The secretion of TGF-beta1 into the cell culture medium by VSMC also did not depend on the ascorbate supply. Finally, addition of heparin to the VSMC culture medium during ECM production abolished the ECM-mediated growth inhibitory effects of ascorbate, but did not affect the action of TGF-beta1. Our data demonstrate that the growth inhibitory effects of ascorbate on cultured VSMC are independent of the action of TGF-beta1, and the effects of these two compounds on VSMC growth are additive.