PUTATIVE HEPARIN RECEPTOR IN SMOOTH MUSCLE CELLS

描述（改编自研究者摘要）：血管过度生长 平滑肌细胞是血管疾病晚期的标志， 最终导致受影响的动脉阻塞。肝素已经 显示在体外和在选定体内动物中减缓平滑肌细胞生长 和人类研究。肝素对血管平滑肌细胞的作用已被证实， 可以用包括肝素受体在内的各种理论来解释， 肝素与生长因子的相互作用和肝素的内吞作用。数据显示 肝素治疗导致活性MAPK水平降低。这 研究计划的目的是：检验肝素改变行为的假设 敏感的血管平滑肌细胞， 通过肝素与一种 细胞膜上的肝素结合位点。这项工作将涉及培养 血清饥饿然后激活的猪主动脉平滑肌细胞 佛波醇肉豆蔻酸酯或血小板衍生生长因子。肝素诱导cGMP升高的能力 浓度和降低MAPK活性将使用Western blot监测。 来自用或不用肝素或cGMP处理的细胞的蛋白质印迹 类似物将使用市售的ELISA测定法监测细胞内 cGMP水平。针对假定的肝素受体的抗体， PI的实验室，并已被证明作为肝素激动剂，在以前的 研究将比较肝素诱导cGMP的能力 增大cGMP引起MKP-1合成增加的能力，如图所示。 在PI的肝素实验室中，也将由Western 印迹最后，PI实验室中用于部分 将改进推定的肝素受体的纯化。所得 将来将使用受体制剂来获得克隆序列 假定受体并允许进一步表征 受体-配体相互作用的机制。这项工作将共同确认 假定的肝素受体在肝素诱导的变化中的重要性 血管平滑肌细胞，并提供了一个显着的进步， 了解肝素如何改变敏感的VSMC。

DESCRIPTION (Adapted from Investigator's Abstract): Over growth of vascular smooth muscle cells is a hallmark of the late stages of vascular disease and contributes to the eventual blocking of affected arteries. Heparin has been shown to slow smooth muscle cell growth in vitro and in selected in vivo animal and human studies. Heparin effects on vascular smooth muscle cells have been explained by a variety of theories of theories including heparin receptors, heparin interaction with growth factors, and endocytosis of heparin. Data show that heparin treatment resulted in decreased levels of active MAPK. This research proposal aims: to test hypothesis that heparin modifies the behavior of sensitive vascular smooth muscle cells by causing an increase in intracellular cyclic GMP levels through the interaction of heparin with a heparin-binding site on the cell membrane. This work will involve cultured porcine aortic smooth muscle cells that are serum starved and then activated with phorbol myristate or PDGF. The ability of heparin to induce rises in cGMP concentration and decrease MAPK activity will be monitored using western blotting of proteins from cells treated with or without heparin or cGMP analogs. Commercially available ELISA assays will be used to monitor cellular cGMP levels. antibodies to a putative heparin receptor that were developed in the PI's laboratory and have been shown to act as heparin agonists in previous studies will be compared with heparin for their ability to induce cGMP increases. The ability of cGMP to cause increases in MKP-1 synthesis, as shown in the PI's laboratory for heparin, will also be determined by western blotting. Finally, the current protocol used in the PI's laboratory for partial purification of the putative heparin receptor will be improved. the resultant receptor preparations will be used in the future to obtain sequence for cloning the putative receptor and to allow further characterization of characterization of the receptor-ligand interactions. Together this work will confirm the importance of the putative heparin receptor in heparin-induced changes in vascular smooth muscle cells and provide a significant advancement in understanding of how heparin alters sensitive VSMC.