NICOTINE AND COTININE STIMULATE SECRETION OF BASIC FIBROBLAST GROWTH-FACTOR AND AFFECT EXPRESSION OF MATRIX METALLOPROTEINASES IN CULTURED HUMAN SMOOTH-MUSCLE CELLS

Purpose: We have recently shown that nicotine and its metabolite cotin ine are mitogenic for smooth muscle cells in vitro. In the present stu dy, we examined the effect of nicotine and cotinine on the production of growth factors and the expression of matrix metalloproteinases in s mooth muscle cells. Methods: Smooth muscle cells were harvested from h uman arteries and grown in culture. Subconfluent cultures were incubat ed for 24 hours in M199 containing 0.1% fetal bovine serum with or wit hout nicotine or cotinine at concentrations ranging from 10(-9) mol/L to 10(-6) mol/L. The supernatants and cell lysates were assayed by enz yme-linked immunosorbent assay for basic fibroblast growth factor (bFG F), tumor necrosis factor alpha (TNF-alpha), platelet-derived growth f actor AB (PDGF-AB), and transforming growth factor beta (TGF-beta). Ma trix metalloproteinase expression was determined in subconfluent cultu res incubated in albumin with or without nicotine or cotinine at 10(-8 ) mol/L and 10(-7) mol/L for 6, 12, 18, 24 and 36 hours. Northern blot analyses were performed with human cDNA probes for collagenase-1, str omelysin-1, gelatinase A, gelatinase B, and triose phosphate isomerase . Blots were quantified by phosphor-imaging techniques. Results: Both nicotine and cotinine stimulated the production and secretion of bFGF in a dose-dependent manner. PDGP, TNF-alpha, and TGP-beta secretions w ere not significantly affected by nicotine or cotinine. Collagenase wa s up-regulated by nicotine at 18 and 24 hours (4.5-fold to 5.8-fold) a nd by cotinine at 18 hours (from 5.0-fold to 29-fold). Stromelysin-1 w as up-regulated by nicotine and cotinine at 12 and 18 hours (1.5-fold to 7.0-fold). Gelatinase A generally peaked at 12 hours and was up-reg ulated by both agents (2.0-fold to 6.5-fold). Conclusion: Nicotine and cotinine enhanced the production of bFGF, a major mitogen for smooth muscle cells, and up-regulated the expression of several matrix metall oproteinases that are critical in cell migration. These data demonstra te mechanisms by which smoking may contribute to the development of in timal hyperplasia, atherosclerosis, and aneurysms.