Smooth muscle cell (SMC) proliferation plays a critical role in several pathological states, including atherosclerosis and hypertension. Heparin suppresses SMC proliferation in vivo and in culture, but the mechanism of action is still poorly understood. In an accompanying article in this issue (Letourneur et at. 119951 J. Cell Physiol., 765676-6861, we observed that heparin binding was up-regulated in heparin-sensitive SMC but was rapidly down-regulated in heparin-resistant SMC continuously exposed to heparin. In this communication, we examine the degradation and secretion of internalized heparin in sensitive and resistant SMC, using gel filtration chromatography to analyze heparin degradation products. Pulse-chase experiments using radiolabeled heparin indicate that sensitive and resistant SMC secrete heparin during the first few hours after exposure. Experiments in which cells are continuously exposed to heparin indicate that degradation and secretion occur in both sensitive and resistant SMC for approximately 5-8 hr. After that time, however, binding and internalization in resistant SMC rapidly decrease and degradation and secretion stop. In contrast, heparin binding and uptake continue in sensitive SMC; degradation and secretion also continue. Chloroquine prevents degradation in both sensitive and resistant SMC, suggesting that catabolism occurs in the lysosomal compartment. The results presented in this and the accompanying article (Letourneur et al. [19951 J. Cell. Physiol., 165:676-686) suggest that heparin acts to upregulate its receptors, and that increased binding of heparin is required for the antiproliferative response. Degradation and secretion kinetics parallel the internalization kinetics and appear to be strongly linked to the binding process. o 1995 WiIey-Liss, Inc.
The proliferation of vascular smooth muscle cells (SMC) is a critical event in early atherogenesis (Ross, 19861, and is the cause of failure in up to 30% of vascular surgical procedures. Much research has been aimed at understanding the mechanisms regulating SMC growth. Previous studies demonstrated that heparin, a complex glycosaminoglycan composed of repeating sulfated sugar units, could inhibit the proliferation of SMC in vivo (Clowes and Karnovsky, 1977; Guyton e t al., 1980) and in cell culture (Hoover e t al., 1980;Castellot et al., 1981 Castellot et al., , 1986a)). The mechanism of action is not well understood. One reason for this is that until very recently, heparin-resistant SMC cultures were not available for more rigorous testing of putative antiproliferative mechanisms. The isolation of several different heparin-resistant SMC cultures (Pukac et al., 1990; San Antonio e t al., 1993; Caleb et al., manuscript in preparation) now permits the re-examination of the important question of the relationship between heparin binding, internalization, metabolism, and the antiproliferative response. In a n accompanying article (Letourneur e t al., 1995), 0 1995 WILEY-LISS. INC.
we found that continuous exposure of heparin to resistant SMC resulted in a rapid down-regulation of binding. In contrast, heparin binding to sensitive SMC actually increased with time. Once bound to the SMC surface, heparin is internalized via receptor-mediated endocytosis in both sensitive (Castellot et al., 1985a; Letourneur e t al., 1995) and resistant SMC (Letourneur et al., 1995). In this communication, we complete our examination of the fate of heparin in SMC by analyzing the degradation and secretion of this glycosaminoglycan in sensitive and resistant SMC cultures. These experiments have been carried out under conditions which closely resemble the conditions used to ob-