Previous work from our laboratory has demonstrated that heparin specifically inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. In this paper, we examine the binding and mode of internalization of heparin by smooth muscle cells. For these studies, radiolabeled and fluoresceinated (FITC) heparin probes were synthesized that retained their antiproliferative capacity. Binding of 3H-heparin to these cells occurs via specific, high-affinity binding sites (Kd = 10(-9) M, 100,000 binding sites per cell). Approximately 80% of the heparin bound to the cell surface was shed into the culture medium within 2 hr. The heparin that was left on the cell surface was internalized with biphasic kinetics. Approximately 50% of the bound material was internalized within 2 hr. After this initial rapid uptake, the rate slowed substantially, with the remaining heparin requiring 1-2 days to be internalized. Binding and uptake of FITC heparin was monitored using video image intensification fluorescence microscopy. When smooth muscle cells were exposed to FITC heparin at 4 degrees C, a diffuse surface staining pattern was observed. After warming the cells to 37 degrees C, intensely fluorescent vesicles were seen superimposed over the diffuse surface staining within 2 min. After 15 min at 37 degrees C, numerous large punctate vesicles were seen inside the cell. After 2 hr these vesicles had concentrated in the perinuclear region. This pattern of uptake, when considered along with the presence of specific, high-affinity binding sites and the initial rapid uptake of 3H-heparin, suggests that heparin enters smooth muscle cells by both receptor-mediated and other endocytic pathways.

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Heparin is a potent inhibitor of vascular smooth muscle cell (VSMC) growth. In this paper we show that heparin suppressed the induction of c-fos and c-myc mRNA in rat and calf VSMC. This effect of heparin is closely associated with its growth-inhibitory activity, as shown by isolating and characterizing a strain of rat VSMC that was resistant to heparin's antiproliferative effect; heparin did not suppress c-fos mRNA induction in these cells. Moreover, neither a nonantiproliferative heparin fragment or other glycosaminoglycans that lack growth-inhibitory activity repressed c-fos or c-myc mRNA levels. The effect of heparin on c-fos mRNA induction was selective for specific mitogens, as heparin inhibited c-fos mRNA induction in phorbol 12-myristate 13-acetate (TPA) stimulated but not epidermal growth factor (EGF) stimulated VSMC. The effect of heparin on gene expression is independent of ongoing protein synthesis, and inhibition of c-fos mRNA is at the transcriptional level. These results suggest that heparin may selectively inhibit a protein kinase C-dependent pathway for protooncogene induction and that this may be one mechanism used by heparin to inhibit cell proliferation.

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Heparin is a complex glycosaminoglycan that inhibits the proliferation of several cell types in culture and in vivo. To begin to define the mechanism(s) by which heparin exerts its antiproliferative effects, we asked whether heparin interferes with the expression of the growth factor-inducible protooncogenes c-fos and c-myc. We show that heparin suppressed the induction of c-os and c-myc mRNA by serum in murine (BALB/c) 3T3 fibroblasts. Using purified mitogens, we further show that suppression was most marked when protooncogene expression was induced by phorbol 12-myristate 13-acetate, an activator of protein kinase C. By contrast, there was little or no suppression when the cells were stimulated by epidermal growth factor, which, in these cells, utilizes a protein kinase C-independent pathway for the induction of gene expression. Heparin also inhibited the change in cell morphology induced by the phorbol ester but had no effect on the morphological change induced by epidermal growth factor and agents that raise intracellular cAMP. Heparin did not inhibit intracellular protein kinase C activity, phorbol ester-induced down-regulation of protein kinase C, or phosphorylation of the 80-kDa intracellular protein kinase C substrate. These results suggest that heparin inhibits a protein kinase C-dependent pathway for cell proliferation and suppresses the induction of c-fos and c-myc mRNA at a site distal to activation of the kinase.The proliferation of higher eukaryotic cells is regulated primarily by extracellular factors that provide growthstimulatory and growth-inhibitory signals (1). Over the last decade, a number of growth-promoting factors have been described. Considerable progress has been made in understanding the signal transduction mechanisms utilized by various growth factors and in identifying nuclear events that are regulated by growth factors. Among the nuclear events is