Isolation of vascular smooth muscle cell cultures with altered responsiveness to the antiproliferative effect of heparin.

Smooth muscle cell (SMC) hyperplasia in the arterial wall is an important component of both atherogenesis and post-vascular surgical restenosis. One naturally-occurring group of molecules which can suppress SMC proliferation in animal models and in cell culture systems are the complex carbohydrates of the heparan sulfate class, including heparin. In this communication, we have used retrovirus vectors to introduce several oncogenes into SMC: SV40 Large T antigen (SVLT), polyoma virus Large T antigen (PyLT), v-myc, and adenovirus E1a. We analyzed a total of 11 cultures. A combination of Western blot analysis, immunoprecipitation, and indirect immunofluorescence confirmed the expression of the infected oncogenic protein in each culture we isolated. All four oncogenes permitted the maintenance of a normal SMC phenotype, as assessed by the general morphology of cells in the light microscope and the presence of SMC-specific alpha-actin in an immunofluorescence assay. Doubling times in infected cells ranged from 20 to 33 hr, and final cell densities in infected cultures ranged from 4 x 10(4) to 5 x 10(5) cells per cm2. By comparison, the parent line had a doubling time of 30 hr and reached a final cell density of 1 x 10(5) cells per cm2. Despite the differences sometimes observed in these proliferation parameters, neither one was strongly correlated with heparin responsiveness. PyLT, v-myc, and E1a all produced SMC cultures or lines which retained sensitivity to the antiproliferative activity of heparin (ED50 = 50 micrograms/ml). In contrast, SVLT expression yielded SMC lines which were highly resistant to heparin (ED50 > 300 micrograms/ml). These results suggest that altered responsiveness to heparin is dependent upon which oncogenic protein is being expressed in the cells. The availability of cloned, immortal SMC lines with a wide range of heparin responsiveness should aid in the understanding of the cellular and molecular mechanism of action of this potentially important growth regulator and therapeutic agent.

Heparin binding, internalization, and metabolism in vascular smooth muscle cells: I. Upregulation of heparin binding correlates with antiproliferative activity.

Vascular smooth muscle cell (SMC) hyperplasia is an important component in the pathogenesis of arteriosclerotic lesions and is responsible for the failure of many vascular surgical procedures. SMC proliferation is inhibited by the glycosaminoglycan heparin; however, the precise mechanisms of action are still not understood. One important question in this regard is whether binding, internalization, and metabolism of heparin are necessary for the antiproliferative activity. In this study, we have analyzed SMC rendered resistant to the antiproliferative effect of heparin by drug selection and retroviral infection of SMC. We first examined the ability of heparin to bind to SMC. Experiments using [3H]heparin indicate the presence of saturable, heparin-displaceable, protease-sensitive binding sites on both sensitive and resistant SMC. The affinity of heparin binding does not correlate with the antiproliferative response. Using fluorescent and radiolabeled heparin probes, we observed that early heparin internalization kinetics in both sensitive and resistant SMC is similar, indicating that resistance to heparin is not due to changes in the ability of cells to take up heparin. In contrast, we observed during the continuous incubation with heparin that binding to resistant SMC is rapidly downregulated, whereas sensitive cells continue to bind and internalize heparin. These results suggest that upregulation of heparin binding to the SMC surface is required for an antiproliferative response. In an accompanying paper (Letourneur et al. [1995] J. Cell Physiol., 165:687-695, this issue), we describe the degradation and secretion of internalized heparin in both sensitive and resistant SMC.

Heparin binding, internalization, and metabolism in vascular smooth muscle cells: II. Degradation and secretion in sensitive and resistant cells.

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 al. [1995] J. Cell Physiol., 165:676-686), 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. [1995] 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.

Heparin inhibits c-fos and c-myc mRNA expression in vascular smooth muscle cells.

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.

Heparin selectively inhibits a protein kinase C-dependent mechanism of cell cycle progression in calf aortic smooth muscle cells.

The proliferation of arterial smooth muscle cells (SMCs) plays a critical role in the pathogenesis of arteriosclerosis. Previous studies have indicated that the glycosaminoglycan heparin specifically inhibited the growth of vascular SMCs in vivo and in culture, although the precise mechanism(s) of action have not been elucidated. In this study, we have examined the ability of specific mitogens (PDGF, EGF, heparin-binding growth factors, phorbol esters, and insulin) to stimulate SMC proliferation. Our results indicate that SMCs derived from different species and vascular sources respond differently to these growth factors. We next examined the ability of heparin to inhibit the proliferative responses to these mitogens. In calf aortic SMCs, heparin inhibits a protein kinase C-dependent pathway for mitogenesis. Detailed cell cycle analysis revealed several new features of the effects of heparin on SMCs. For example, heparin has two effects on the Go----S transition: it delays entry into S phase and also reduces the number of cells entering the cycle from Go. Using two separate experimental approaches, we found that heparin must be present during the last 4 h before S phase, suggesting a mid-to-late G1 heparin block. In addition, our data indicate that heparin-treated SMCs, while initially blocked in mid-to-late G1, slowly move back into a quiescent growth state in the continued presence of heparin. These results suggest that heparin may have multiple targets for its antiproliferative effect.

Binding and internalization of heparin by vascular smooth muscle cells.

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.