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      Heparin Fails to Inhibit the Proliferation of Human Vascular Smooth Muscle Cells in the Presence of Human Serum

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          The proliferation of vascular smooth muscle cells (VSMC) plays a significant part in both the developing atherosclerotic lesion and in restenosis. Heparin has been widely reported to inhibit the growth of VSMC in culture and intimal VSMC in some animal models of vascular hyperplasia. Clinical trials with heparin, however, have failed to inhibit restenosis following angioplasty. Bovine serum is normally used as a growth supplement in in vitro VSMC growth assays. We have compared the effects of human serum with those of bovine serum on the cellular response to heparin in human VSMC culture. While heparin inhibited the proliferation of human VSMC in the presence of bovine serum, it was totally ineffective in the presence of human serum. These observations were consistent over a wide range of serum and VSMC samples. Experiments utilizing neutralizing antibodies to a number of growth factors showed that cells in either serum were similarly dependent on platelet-derived growth factor for proliferation. In contrast, proliferation in the presence of bovine serum was shown to be dependent on extracellular basic fibroblast growth factor, whereas that in human serum was not. Direct binding of [<sup>3</sup>H]-heparin to VSMC was significantly reduced in the presence of human serum compared with bovine serum, and the former contained twice the concentration of heparin-binding factors of the latter. Removal of heparin-binding factors from either serum type significantly reduced the proliferation potential. Fractionation of heparin-binding factors from human serum showed that the major growth-promoting activity, together with heparin resistance, was contained within a fraction excluded by a 100,000 molecular weight membrane. We conclude that the mechanism of resistance to heparin in human serum is likely to be due to a combination of differential growth factor binding and interference with heparin interaction with cellular receptors by a high molecular weight heparin-binding factor. This phenomenon may significantly contribute to the lack of success of heparin as an antirestenotic agent in clinical trials.

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          Most cited references 5

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          Heparin selectively inhibits a protein kinase C-dependent mechanism of cell cycle progression in calf aortic smooth muscle cells [published erratum appears in J Cell Biol 1990 Mar;110(3):863]

          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.
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            Entry and distribution of fluorescent antiproliferative heparin derivatives into rat vascular smooth muscle cells: comparison between heparin-sensitive and heparin-resistant cultures.

             T Bârzu,  M. Pascal,  M Maman (1996)
            We studied the binding and entry of fluorescein (FITC)-labeled heparin derivatives into rat aortic smooth muscle cells (SMC) by confocal microscopy. FITC-labeled heparin fractions or FITC-labeled SR 80037A, a potent antiproliferative heparin derivative (Bârzu et al., Eur. J. Pharmacol., 219:225-233 1992), were prepared and their antiproliferative activity was confirmed. By incubating SMC with FITC-labeled heparins, a specific cell-associated fluorescence was found. Cellular fluorescence was mostly located around the nucleus and at the level of cell contacts or cell adhesion. The fluorescence was displaced neither by chasing with excess of unlabeled heparins nor by washing with 1 M NaCl, which proved that labeled heparins had been internalized by SMC. Kinetics of internalization of FITC-heparins suggested receptor-mediated endocytosis of heparins by SMC. Double labeling of SMC with biotinylated Concanavalin A and FITC-SR 80037A also indicated that heparin derivative enters the endocytic pathway. The process was accelerated when serum was present in the incubation medium. Treatment of cells with chloroquine (50 microM) induced accumulation of FITC-SR 80037A in the late endosomes, around the nucleus. No fluorescence labeling could be evidenced inside the nucleus. Neither electron microscopy nor cell fractionation experiments performed with SMC previously incubated with [3H]-heparin were able to ascertain nuclear uptake of heparin, as proposed by other workers (Busch et al., Cell Biol., 116:31-42; 1992; Sing et al., Drug Dev. Res., 29:129-136 1993). The cell-associated fluorescence was very weak in SMC resistant to the antiproliferative activity of heparin, selected by long-term heparin treatment (HT-SMC) as previously shown [Bârzu et al., J. Cell. Physiol., 160:239-248, 1994]. The HT-SMC differed from control SMC with regard to expression of extracellular matrix proteins. These cells exhibited very low expression of fibronectin and prevalent expression of laminin and synthesized less cell-associated glycosaminoglycans. From our results, the following conclusions can be drawn: (1) the antiproliferative heparins are bound and internalized by SMC without being taken up into the nucleus; (2) there is a correlation between the binding and/or the internalization process and the sensitivity of SMC to the antiproliferative activity of heparins; and (3) selection of heparin-resistant SMC by long treatment with heparin results in particular growth pattern of SMC (absence of focal overgrowth), associated with changes in the expression of the extracellular matrix components (fibronectin, laminin, and cell-bound glycosaminoglycans).
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              Protein Kinase C α Expression Is Required for Heparin Inhibition of Rat Smooth Muscle Cell Proliferationin Vitroandin Vivo


                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                December 1998
                23 September 2008
                : 35
                : 6
                : 449-460
                Co-operative Research Centre for Cardiac Technology and CSIRO Molecular Science, North Ryde, N.S.W., Australia
                25616 J Vasc Res 1998;35:449–460
                © 1998 S. Karger AG, Basel

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                Page count
                Figures: 5, Tables: 3, References: 50, Pages: 12
                Research Paper


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