CHARACTERIZATION OF RAT AORTIC SMOOTH-MUSCLE CELLS RESISTANT TO THE ANTIPROLIFERATIVE ACTIVITY OF HEPARIN FOLLOWING LONG-TERM HEPARIN TREATMENT

Vascular smooth muscle cells (SMC) do not represent a homogeneous popu lation (Schwartz et al., 1990, Am. J. Pathol. 136:1417-1428). Cellular clones resistant to the antiproliferative activity of heparin were is olated from rat aortic SMC cultures (Pukac et al., 1990, Cell Regul., 1:435-443; San Antonio et al., 1993, Arterioscler. Thromb., 73:748-757 ) and from explant of human arterial restenotic lesions (Chan et al., 1993, Lancet, 341:341-342). We have shown in the present study that lo ng-term treatment (growth medium supplemented with 200 mu g/ml heparin , from the second to the tenth passage) of rat aortic SMC, without cel l cloning, resulted in a significant loss of sensitivity to the growth inhibition by heparin and its derivatives. The heparin resistance was stable after growing cells for two passages in heparin-free medium, s uggesting the selection of a particular phenotype. We tried to charact erize these cells and to determine the causes of the resistance to the growth inhibition by heparin. Heparin-treated SMC (HT-SMC) were small er than their control culture at the same passage, expressed less alph a-SM actin, and did not overgrow after reaching confluence. As in the heparin-resistant clones (San Antonio et al., 1993, Cell Regul., 1:435 -443) expression of alpha-SM actin could be increased in HT-SMC by hep arin addition before Western blotting. Heparin resistance was associat ed with a tenfold decrease in [H-3]-heparin binding capacity (B-max = 1.9 x 10(6) sites per cell) compared to control cultures (B-max = 1.7 x 10(7) sites per cell), which was irreversible after growing the cell s for two additional passages in heparin-free medium. We also investig ated protein kinase C (PKC) in HT-SMC in terms of both enzymatic activ ity and protein expression (evaluated by [H-3]-staurosporine and [H-3] -phorbol-12,13-dibutyrate binding). We found that HT-SMC had only half the PKC activity and expression as control SMC. Therefore, long-term treatment of rat aortic SMC with heparin allowed the selection of a le ss differentiated subpopulation of cells, exhibiting low sensitivity t o the growth inhibition by heparin, which could be related to the low capacity of binding heparin and to a lower PKC activity and/or express ion. (C) 1994 Wiley-Liss, Inc.