ISOLATION OF VASCULAR SMOOTH-MUSCLE CELL-CULTURES WITH ALTERED RESPONSIVENESS TO THE ANTIPROLIFERATIVE EFFECT OF HEPARIN

Smooth muscle cell (SMC) hyperplasia in the arterial wall is an import ant component of both atherogenesis and post-vascular surgical resteno sis. One naturally-occurring group of molecules which can suppress SMC proliferation in animal models and in cell culture systems are the co mplex carbohydrates of the heparan sulfate class, including heparin. I n this communication, we have used retrovirus vectors to introduce sev eral oncogenes into SMC: SV40. Large T antigen (SVLT), polyoma virus L arge T antigen (PyLT), v-myc, and adenovirus E1a. We analyzed a total of 11 cultures. A combination of Western blot analysis, immunoprecipit ation, and indirect immunofluorescence confirmed the expression of the infected oncogenic protein in each culture we isolated. All four onco genes permitted the maintenance of a normal SMC phenotype, as assessed by the general morphology of cells in the light microscope and the pr esence of SMC-specific alpha-actin in an immunofluorescence assay. Dou bling times in infected cells ranged from 20 to 33 hr, and final cell densities in infected cultures ranged from 4 x 10(4) to 5 x 10(5) cell s per cm(2). By comparison, the parent line had a doubling time of 30 hr and reached a final cell density of 1 x 10(5) cells per cm(2). Desp ite the differences sometimes observed in these proliferation paramete rs, neither one was strongly correlated with heparin responsiveness. P yLT, v-myc, and E1a all produced SMC cultures or lines which retained sensitivity to the antiproliferative activity of heparin (ED(50) = 50 mu g/ml). In contrast, SVLT expression yielded SMC lines which were hi ghly resistant to heparin (ED(50) > 300 mu g/ml). These results sugges t that altered responsiveness to heparin is dependent upon which oncog enic protein is being expressed in the cells. The availability of clon ed, immortal SMC lines with a wide range of heparin responsiveness sho uld aid in the understanding of the cellular and molecular mechanism o f action of this potentially important growth regulator and therapeuti c agent. (C) 1996 Wiley-Liss, Inc.