Transforming growth factor beta 1 inhibits the proliferative effect of insulin on human infragenicular vascular smooth muscle cells.

Abstract

The distribution of atherosclerotic arterial disease in diabetes mellitus characteristically involves the infragenicular arterial tree including the anterior tibial, posterior tibial, and peroneal arteries. The proliferation of vascular smooth muscle cell (VSMC) is essential in the development of the atherosclerotic lesion. It has long been held that insulin plays a causative role in the formation of the atherosclerotic lesion in diabetes. We studied the role played by insulin in the proliferation of these cells in culture and the interaction of insulin with transforming growth factor beta 1 (TGF beta 1), a factor known for its possible inhibitory effects. We have grown and characterized a line of VSMC harvested from atherosclerotic infragenicular arteries of human subjects undergoing below-knee amputation. The cultures were defined as being of VSMC origin by immunohistochemical staining with alpha-smooth muscle actin. Confluent cultures of passages 4 through 7 were seeded into six well plates at a density of 5000 cells/well. After serum deprivation the cells were exposed to insulin (100 ng/ml) alone or in combination with TGF beta 1 (6 ng/ml). Our findings indicate that a 48-hour incubation with insulin augments the proliferation of human infragenicular VSMC, producing a 207% increase in cell number when compared with control cells (11,328 +/- 686, n = 56 vs 3682 +/- 182, n = 87; p < 0.0001). The addition of TGF beta 1 in combination with insulin abolished the accelerated growth rate seen in test groups treated with insulin alone (3614 +/- 247, n = 32 vs 11,328 +/- 686, n = 56; p < 0.0001). These results strongly suggest that insulin is a potent stimulant of human infragenicular VSMC proliferation. The mitogenic effect of insulin is inhibited by TGF beta 1, producing proliferation rates comparable to those observed in control cells incubated with serum-free media.