A Putative Heparin Receptor in Smooth Muscle Cells

描述（由申请方提供）：晚期血管疾病的特征是炎症变化，包括血管平滑肌细胞过度生长，最终导致受影响动脉阻塞。肝素是最有名的抗凝剂，也已被证明在体外和选定的体内研究中减缓平滑肌细胞生长。肝素对血管平滑肌细胞的作用已经通过几种理论来解释，包括肝素与生长因子的相互作用、肝素受体和肝素的内吞作用以改变转录。PI的研究小组先前确定了血管细胞上的假定肝素受体和抗受体抗体，其模拟肝素在阻断血管平滑肌细胞中的DNA合成和降低ERK活化中的功能。PI实验室的数据表明，在肝素诱导的ERK活性和DNA合成降低中，该受体和信号转导涉及cGMP产生、cGMP敏感性蛋白激酶（PKG）的激活和特异性双功能磷酸酶MKP 1的合成。拟议的研究将扩展初步结果，表明肝素治疗也会降低Raf活性，并将证实肝素通过受体和PKG发挥作用，导致活性降低。确认肝素通过PKG途径对平滑肌细胞增殖的影响将涉及使用cGMP模拟物和PKG抑制剂评价肝素对细胞周期蛋白/细胞周期蛋白激酶抑制剂水平和细胞周期蛋白D（G1期细胞周期蛋白）合成的影响。PI实验室的其他结果证明了活性JNK和p38应激活化蛋白激酶亚组的细胞骨架定位以及肝素降低JNK和p38活化的能力，如通过蛋白质印迹法评估的。拟议的研究将确定肝素是否也会导致细胞骨架相关JNK和p38以及细胞质中发现的效应物的活性降低，或者肝素的作用是否仅限于MKP 1所在的细胞核。为了进一步评价肝素处理通过PKG激活cGMP信号传导的程度，将评价已知的PKG靶点（如VASP和PAK 1），以确定它们是否响应于肝素处理而特异性磷酸化，因为它们是在NO或PKG酶的人工激活剂增加后，并将评价基因表达中共有一组变化的可能性。PI实验室获得的初步结果表明，肝素处理后PKG水平降低，表明PKG降解可能由肝素信号传导和PKG活化诱导。这种情况发生的可能性将涉及使用蛋白酶体抑制剂和检测与PKG相关的泛素的方法。最后，这项研究将继续工作，旨在分离肝素受体，并获得序列信息，将有助于基因鉴定。总之，拟议的研究将证实肝素受体在肝素诱导的血管平滑肌细胞变化中的重要性，并将为理解肝素如何改变敏感的血管平滑肌细胞提供重大进展。血管中最复杂的信息系统之一是控制细胞生长和分裂的系统，细胞生长和分裂既可以导致损伤修复，也可以在过度活跃时导致血管疾病。拟议的研究继续PI实验室的调查模式，重点是了解导致细胞生长的信息传递通常被关闭的方式，重点是抗凝剂分子肝素。在分子水平上更好地理解这些负性途径，包括肝素开启的途径，以及已知参与降低血管张力的蛋白激酶G，将有助于设计改善血管疾病的治疗方法。

DESCRIPTION (provided by applicant): Late stage vascular disease is characterized by inflammatory changes including the overgrowth of vascular smooth muscle cells which eventually contribute to blocking of affected arteries. Heparin is best known as an anticoagulant that has also been shown to slow smooth muscle cell growth in vitro and in selected in vivo studies. Heparin effects on vascular smooth muscle cells have been explained by several theories including heparin interactions with growth factors, heparin receptors and endocytosis of heparin to alter transcription. The PI's research group previously identified a putative heparin receptor on vascular cells and anti-receptor antibodies that mimic heparin functions in blocking DNA synthesis and decreasing ERK activation in vascular smooth muscle cells. Data from the PI's laboratory implicate this receptor and signal transduction involving cGMP production, the activation of cGMP sensitive protein kinase (PKG) and synthesis of a specific dual function phosphatase, MKP1, in heparin induced decreases in ERK activity and DNA synthesis. The proposed research will expand on preliminary results indicating Raf activity is also decreased in response to heparin treatment and will confirm that heparin works through the receptor and PKG to cause this decreased activity. Confirmation that the effects of heparin on smooth muscle cell proliferation occur through the PKG pathway will involve evaluating heparin effects on cyclin/cyclin kinase inhibitor level and cyclin D (G1 phase cyclin) synthesis using cGMP mimics and PKG inhibitors. Additional results from the PI's laboratory demonstrate cytoskeletal localization of a subset of active JNK and p38 stress activated protein kinase enzymes and the ability of heparin to decrease activation of JNK and p38 as assessed by western blotting. Proposed research will determine whether heparin also results in decreased activity of the cytoskeletal associated JNK and p38 and effectors found in the cytoplasm, or whether heparin effects are restricted to the nucleus where the MKP1 resides. To further evaluate the extent to which heparin treatment activates cGMP signaling through PKG, known PKG targets such as VASP and PAK1 will be evaluated to determine whether they are specifically phosphorylated in response to heparin treatment as they are after increases in NO or artificial activators of the PKG enzyme and the possibility of a shared set of changes in gene expression will be evaluated. Preliminary results obtained in the PI's laboratory indicate that PKG levels decrease after heparin treatment suggesting that PKG degradation might be induced by heparin signaling and PKG activation. The possibility that this occurs will involve use of proteasome inhibitors and methods to detect ubiquitin linked to PKG. Finally, this study will continue work aimed at isolating the heparin receptor and obtaining sequence information that will facilitate gene identification. Together, the proposed studies will confirm the importance of the heparin receptor in heparin induced changes in vascular smooth muscle cells and will provide a significant advancement in understanding of how heparin alters sensitive vascular smooth muscle cells. One of the most complicated informational systems in blood vessels is that which controls cell growth and division that can lead both to damage repair, and when over active, to vascular disease. The proposed research continues a pattern of investigation in the PI's laboratory focused on understanding the way in which the information transfer leading to cell growth is normally turned off with a focus on the anti-coagulant molecule heparin. A better understanding of these negative pathways at the molecular level, including the pathway turned on by heparin and following through protein kinase G already known for its involvement in reducing blood vessel tension, will be helpful in designing improved treatments for vascular disease.