Isolation of heparin-insensitive aortic smooth muscle cells. Growth and differentiation

Previous work has shown heparin and heparan sulfates to be potent inhibitors of vascular smooth muscle cell (VSMC) growth. This laboratory has previously isolated a VSMC line insensitive to the antiproliferative action of heparin by subjecting VSMCs that grew out from rat aortic medial explants to continuous passage in media containing heparin at 200 micrograms/mL. In the present study, we have isolated two additional heparin-resistant (HR) cell lines and have used the HR cells to investigate cellular mechanisms responsible for the potent antiproliferative activity of heparin. In contrast to normal heparin-sensitive VSMCs, the HR cells were smaller, displayed elongated processes, and possessed altered growth characteristics; however, both HR and normal cells bound and internalized comparable amounts of heparin. Immunohistochemical detection of smooth muscle cell-specific actin in growth-arrested cells showed staining of nearly all normal VSMCs and of a much smaller percentage of HR cells; heparin treatment caused a marked increase in the percentage of HR cells expressing smooth muscle cell alpha-actin, indicating that the antiproliferative and differentiation-promoting actions of heparin are independent. Proteins from control VSMCs and HR cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and heparin affinity chromatography. Several proteins were expressed preferentially by either HR cells or normal VSMCs, with the most significant difference being the secretion of a high-affinity, heparin-binding protein (M(r), 38,000) by control VSMCs but not by HR cells. We conclude that the aortic VSMC population may give rise to HR cells under selective conditions and that their unique characteristics, such as alterations in their ability to produce heparin-binding proteins, will prove useful in deciphering the cellular mechanisms involved in heparin's regulation of VSMC growth and differentiation.