Binding and internalization of heparin by vascular smooth muscle cells

Abstract

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.

Heparin plays a key regulatory role via a p53/FAK‐dependent signaling in melanoma cell adhesion and migration

“…In fact, previous studies, performed with radioactive or fluorescent heparin derivatives showed that these compounds were bound and internalized by SMC (20), human endothelial cells (21) and rat neurons (33). Other reports show that heparin binds to specific cell surface adhesion molecules, for example, integrins to modulate the adhesive capabilities of different cell lines (34,35).…”

“…The ability of cells to internalize heparin has previously been demonstrated on other cell lines (20,21). We hypothesized therefore, that heparin is uptaken by melanoma cells to exert its influence on p53 transcription and/or stabilization.…”

Cell biology of arterial proteoglycans.

“…Smooth muscle cells possess specific high-affinity receptors for heparirVheparan sulfate and can internalize this bound glycosaminoglycan. 145 These results raise the interesting possibility that heparin/heparan sulfate and heparin/heparan sulfate fragments may suppress the growth of smooth muscle cells at specific intracellular sites. Additional studies support this possibility.…”

Inhibition of cellular proliferation after experimental balloon angioplasty by low-molecular-weight heparin.

“…43,46 Growth-arrested SMCs in cell culture are 50-100 times more sensitive to the antiproliferative effects of heparin than are exponentially growing cells in culture. 44 Thus, it seems adequate and important to start with LMWH treatment before SMCs are activated by the vessel wall injury caused by PTCA.…”