A Putative Heparin Receptor in Smooth Muscle Cells

描述(申请人提供)：晚期血管疾病的特征是炎性变化，包括血管平滑肌细胞过度生长，最终导致受影响的动脉阻塞。肝素最为人所知的是一种抗凝剂，在体外和选定的体内研究中，也被证明可以减缓血管细胞的生长。肝素对血管平滑肌细胞的作用可以用多种理论来解释，包括肝素与生长因子的相互作用、肝素受体和肝素内吞改变转录。PI的研究小组之前发现了血管细胞上可能的肝素受体和抗受体抗体，这种抗体模拟肝素在阻断血管平滑肌细胞DNA合成和减少ERK激活方面的功能。来自PI实验室的数据表明，这种受体和信号转导涉及cGMP的产生、cGMP敏感蛋白激酶(PKG)的激活和肝素中特殊的双功能磷酸酶mkp1的合成，导致ERK活性和DNA合成减少。这项拟议的研究将扩展初步结果，表明Raf活性也因肝素治疗而降低，并将证实肝素通过受体和PKG工作，导致这种活性降低。要证实肝素对平滑肌细胞增殖的影响是通过PKG途径实现的，需要使用cGMP模拟物和PKG抑制剂来评估肝素对细胞周期蛋白/细胞周期蛋白激酶抑制物水平和细胞周期蛋白D(G1期细胞周期蛋白)合成的影响。PI实验室的其他结果表明，JNK和p38应激激活的蛋白激酶酶的一部分在细胞骨架中定位，并通过Western blotting评估肝素降低JNK和p38的活性的能力。拟议的研究将确定肝素是否也会导致细胞质中与细胞骨架相关的JNK和p38以及效应器的活性降低，或者肝素的作用是否仅限于mkp1所在的细胞核。为了进一步评估肝素处理通过PKG激活cGMP信号的程度，将评估已知的PKG靶标，如Vasp和PAK1，以确定它们是否在肝素处理后被特异性磷酸化，因为它们是在增加PKG酶的NO或人工激活剂后发生的，并将评估一组共同的基因表达变化的可能性。PI实验室获得的初步结果表明，肝素处理后PKG水平下降，这表明PKG的降解可能是由肝素信号和PKG激活诱导的。发生这种情况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，这项研究将继续致力于分离肝素受体，并获得有助于基因鉴定的序列信息。总之，拟议的研究将证实肝素受体在肝素引起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，这可能会导致损伤修复，当过度活跃时，会导致血管疾病。这项拟议的研究延续了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，重点是抗凝血剂分子肝素。在分子水平上更好地了解这些负面途径，包括肝素启动的途径和通过蛋白激酶G的途径，将有助于设计改进的血管疾病治疗方法。

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.