Invest. 1992. 90:136-141.)

No 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.

We studied in vitro the effects heparin on the growth of rat aortic smooth muscle cells. Measurements of growth were monitored by [3H]thymidine uptake and changes in cell number over a period of 3 days. Our results show that heparin-highly anticoagulant or nonanticoagulant-significantly inhibits growth of smooth muscle cells. We also show that this is a highly specfic interaction with regard to molecule and cell type: i.e., other polyanions, except for a low molecular weight dextran sulfate, do not have the same effect on growth, and not all cells are inhibited by heparin; e.g., endothelial cell growth actually is enhanced. After removing antithrombin from our media, we carried out experiments which show that heparin is effective even though thrombin, a potent mitogenic agent, is still present and active. We also found that passing the platelet extract over a heparin column did not remove all of the motogenic activity of the platelet preparation. Both experiments indicate an inhibitory role for the heparin molecule, per se. Our results support the findings of a recent paper (Guyton et al., 1980) showing that heparin can limit the size of myointimal plaques in rats after carotid injuries by inhibiting smooth muscle cell proliferation.

Since recent studies indicate that cyclosporine (CsA) disrupts endothelial integrity and that injured endothelial cells release excess endothelin, we examined endothelin's role in acute cyclosporine nephrotoxicity. Following CsA (20 mg/kg i.v.), rabbit anti-porcine endothelin (aE) serum was continuously infused into a first order branch of the main renal artery in Munich-Wistar rats whereupon the hemodynamics of glomeruli not infused with aE as well as those infused with aE within the same kidney were simultaneously assessed by micropuncture techniques. In CsA treated kidneys, in glomeruli not infused with aE, single nephron GFR (SNGFR) and glomerular plasma flow rate (QA) fell profoundly (on average by 42 and 48%, respectively) below the baseline values in association with lower glomerular capillary pressure and elevated afferent arteriolar resistance. By contrast, in glomeruli infused with aE within the same CsA treated kidneys, this vasoconstrictive pattern was markedly attenuated: SNGFR was, on average, only 19% lower than baseline and values for QA as well as other parameters determining glomerular filtration were at or near the levels observed before administration of CsA. In another group of rats (N = 6) an identical dose of CsA was given to measure the circulating level of endothelin. In these CsA treated rats, endothelin level (measured by radioimmunoassay) was elevated at 41.7 +/- 14.7 pg/ml, contrasting the value of less than 2 pg/ml uniformly observed in identically instrumented normal rats not given CsA (N = 5). Thus, cyclosporine is a potential inducer for endothelin release and endothelin appears to have a pivotal role in pathophysiology of cyclosporine-induced acute renal vasoconstriction and glomerular dysfunction.