PUTATIVE HEPARIN RECEPTOR IN SMOOTH MUSCLE CELLS

平滑肌细胞的增殖是动脉粥样硬化的主要事件 动脉粥样硬化的纤维增生阶段。 这种扩散是 可能由血管化学变化引起，包括 生长因子增加，抑制因子相对减少， 分子。 肝素已被证明会干扰血清和生长 因子刺激平滑肌增殖在体内和体外。 然而，肝素抑制平滑肌的机制 扩散并不明确。 本提案的目标是 调查肝素通过特定肝素发挥作用的假设 平滑肌细胞上的结合位点（受体）。 的抗体 阻断肝素与猪主动脉内皮细胞的结合， 也能阻断肝素与平滑肌细胞的结合。 在 此外，抗体免疫沉淀平滑肌细胞蛋白 类似于细胞表面的肝素结合蛋白， 内皮细胞 这些抗体将被用来解决这个问题 这些抗体识别的假定受体是否发挥作用 在肝素信号传导中的作用。 具体而言，将评估抗体 因为它们能够模拟肝素作用并阻断肝素信号传导。 将对每项实验的肝素和对照样品进行分析。 测定 将包括测量胸苷掺入和细胞增殖， 对血清和生长因子刺激的反应。 如果，作为初步的 结果表明，抗体模拟肝素， 基于受体活性的肝素激动剂。 阻断抗体 肝素信号传导被认为是拮抗剂。 任一类型的 活动将提供一个系统来研究信号通路。 的 抗体也将被用来研究肝素块在 平滑肌细胞蛋白激酶C信号通路。 佛波醇酯 (and其他测定中的血清）将用于刺激信号传导 通路 抗体干扰肝素作用的能力，或 模拟肝素效应的方法将通过测量丝裂原活化 蛋白激酶活性 对于这些实验，激酶和 磷酸酪氨酸特异性抗体将用于 免疫沉淀和蛋白质印迹实验。 C-fos和c-Myc 在活化后，还将通过北方印迹来测量转录 蛋白激酶C通路的一个重要组成部分。 从这些数据中获得的数据 实验将支持或反驳肝素信号传导 蛋白激酶C途径是通过 被抗体识别的假定肝素受体。 这是一 了解平滑肌细胞生长调节重要一步 血管系统 这样的知识可能会导致开发治疗 基于控制平滑肌细胞增殖的策略， 血管疾病

Proliferation of smooth muscle cells is a primary event in the fibroproliferative stage of atherosclerosis. This proliferation is probably induced by changes in the blood vessel chemistry which include increases in growth factors and relative decreases in inhibitory molecules. Heparin has been shown to interfere with serum and growth factor stimulated smooth muscle proliferation both in vivo and in vitro. However, the mechanism by which heparin inhibits smooth muscle proliferation is not clear. The goal of the present proposal is to investigate the hypothesis that heparin acts through specific heparin binding sites (receptors) on the smooth muscle cells. Antibodies which block the binding of heparin to porcine aortic endothelial cells have been found to block heparin binding to smooth muscle cells as well. In addition, the antibodies immunoprecipitate a smooth muscle cell protein similar to the cell surface heparin binding protein precipitated from endothelial cells. These antibodies will be used to address the question of whether the putative receptor recognized by these antibodies plays a role in heparin signaling. Specifically, the antibodies will be assessed for their ability to mimic heparin actions and to block heparin signaling. Heparin and control samples will be analyzed for each experiment. Assays will include measuring thymidine incorporation and cell proliferation in response to serum and growth factor stimulation. If, as preliminary results suggest, the antibodies mimic heparin they will be considered heparin agonists for receptor based activity. Antibodies which block heparin signaling would be considered antagonists. Either type of activity would provide a system to investigate the signaling pathway. The antibodies will also be used to investigate the heparin block in the smooth muscle cell protein kinase C signaling pathway. Phorbol esters (and serum in other assays) will be used to stimulate the signaling pathway. The ability of antibodies to interfere with heparin effects or to mimic heparin effects will be assessed by measuring mitogen activated protein kinase activity. For these experiments, kinase and phosphotyrosine specific antibodies will be employed in immunoprecipitation and western blotting experiments. C-fos and c-myc transcription will also be measured by northern blotting after activation of the protein kinase C pathway. Together the data obtained from these experiments will support or refute the hypothesis that heparin signaling in proliferation and the protein kinase C pathway is mediated through the putative heparin receptor recognized by the antibodies. This is an important step in understanding smooth muscle cell growth regulation in the vasculature. Such knowledge may lead to developing treatment strategies based on the control of smooth muscle cell proliferation in vascular disease.