Characterization of rat aortic smooth muscle cells resistant to the antiproliferative activity of heparin following long‐term heparin treatment

Publisher Website
Google Scholar
Abstract
Vascular smooth muscle cells (SMC) do not represent a homogeneous population (Schwartz et al., 1990, Am. J. Pathol. 136:1417–1428). Cellular clones resistant to the antiproliferative activity of heparin were isolated from rat aortic SMC cultures (Pukac et al., 1990, Cell Regul., 1:435–443; San Antonio et al., 1993, Arterioscler. Thromb., 13:748–757) and from explant of human arterial restenotic lesions (Chan et al., 1993, Lancet, 341:341–342). We have shown in the present study that long‐term treatment (growth medium supplemented with 200 μg/ml heparin, from the second to the tenth passage) of rat aortic SMC, without cell 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, suggesting the selection of a particular phenotype. We tried to characterize these cells and to determine the causes of the resistance to the growth inhibition by heparin. Heparin‐treated SMC (HT‐SMC) were smaller than their control culture at the same passage, expressed less α‐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 α‐SM actin could be increased in HT‐SMC by heparin addition before Western blotting. Heparin resistance was associated with a tenfold decrease in [3H]‐heparin binding capacity (Bmax=1.9 × 106 sites per cell) compared to control cultures (Bmax = 1.7 × 107 sites per cell), which was irreversible after growing the cells for two additional passages in heparin‐free medium. We also investigated protein kinase C (PKC) in HT‐SMC in terms of both enzymatic activity and protein expression (evaluated by [3H]‐staurosporine and [3H]‐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 less differentiated subpopulation of cells, exhibiting low sensitivity to the growth inhibition by heparin, which could be related to the low capacity of binding heparin and to a lower PKC activity and/or expression.