Cell cycle control of vascular remodeling

描述（由申请人提供）：动脉平滑肌细胞（SMC）通常以分化的静止状态存在，部分通过从内膜内皮细胞释放抗有丝分裂剂（如前列环素（PGI 2））来维持。动脉损伤，如与动脉粥样硬化和球囊血管成形术相关的损伤，损害了这些抗有丝分裂原的释放，并且（连同损伤相关的局部有丝分裂原暴露和细胞外基质的变化）导致血管SMC向增殖状态的“表型调节”。SMC增殖在血管损伤后重塑反应中的重要性已经确立，但控制静止和增殖状态的细胞周期调控机制尚未确立。我们研究了促分裂剂和PGI 2对细胞周期蛋白依赖性激酶（cdks;调节细胞周期进程的关键酶）的影响，发现这些正性和负性调节剂主要通过控制cdk抑制蛋白p27 kip 1的水平起作用。此外，这种收敛也见于Skp 2的调节，Skp 2是控制p27 kip 1降解的E3泛素连接酶。一些研究已经表征了转录后调节Skp 2的机制，但是我们发现促分裂剂和PGI 2的主要作用是对成纤维细胞和SMC中Skp 2 mRNA的表达。因此，我们提出了三个具体的目标来测试的假设，Skp 2基因是有丝分裂原和PGI 2信号在损伤诱导的血管重塑的主要焦点。在目的1中，我们将使用SMC和MEFs来表征有丝分裂原调节Skp 2基因诱导的机制。相关的信号转导级联和关键启动子元件将被确定。将探索潜在的丝裂原对Skp 2 mRNA稳定性的影响。在目标2中，我们将使用相同的系统和平行的方法来表征PGI 2拮抗Skp 2 mRNA的有丝分裂原依赖性诱导的机制。在目标3中，我们将使用基因敲除和转基因小鼠的股动脉损伤来确定PGI 2-IP-Skp 2通路对体内SMC增殖的重要性。总之，这些结果将为控制Skp 2基因表达的基本调控机制提供新的见解，并直接测试Skp 2在体内模型中对SMC增殖的重要性，该模型与心血管病理学具有明确的相关性。相关性：平滑肌细胞的异常增殖是心血管疾病的一个重要方面。这项研究将有助于识别调节平滑肌细胞增殖的基因和蛋白质。它还将测试这些分子在血管损伤的体内模型中的重要性。

DESCRIPTION (provided by applicant): Arterial smooth muscle cells (SMCs) typically exist in a differentiated, quiescent state that is maintained, in part, by the release of antimitogens, such as prostacyclin (PGI2), from intimal endothelial cells. Arterial injury, such as that associated with atherosclerosis and balloon angioplasty, impairs the release of these antimitogens and (together with injury-associated exposure to local mitogens and changes in the extracellular matrix) leads to the "phenotypic modulation" of vascular SMCs to a proliferative state. The importance of SMC proliferation in the vascular remodeling response to injury is established, but the cell cycle regulatory mechanisms that control the quiescent and proliferative states are not. We have studied the effects of mitogens and PGI2 on the cyclin-dependent kinases (cdks; the critical enzymes regulating cell cycle progression) and find that these positive and negative regulators act largely by controlling levels of the cdk inhibitory protein, p27kip1. Moreover, this convergence is also seen in the regulation of Skp2, the E3 ubiquitin ligase that controls the degradation of p27kip1. Several studies have characterized mechanisms that regulate Skp2 post-transcriptionally, but we find that the major effect of mitogens and PGI2 is on the expression of Skp2 mRNA in both fibroblasts and SMCs. We therefore propose three specific aims to test the hypothesis that the Skp2 gene is the major focal point of mitogen and PGI2 signaling during injury-induced vascular remodeling. In Aim 1, we will use SMCs and MEFs to characterize the mechanisms by which mitogens regulate induction of the Skp2 gene. Relevant signal transduction cascades and key promoter elements will be identified. Potential mitogen effects on Skp2 mRNA stability will be explored. In Aim 2, we will use the same systems and parallel approaches to characterize the mechanisms by which PGI2 antagonizes mitogen-dependent induction of Skp2 mRNA. In Aim 3, we will use femoral artery injury in knock-out and transgenic mice to determine the importance of the PGI2-IP-Skp2 pathway on SMC proliferation in vivo. Together, the results will provide new insight into the basic regulatory mechanisms controlling Skp2 gene expression and directly test the importance of Skp2 on SMC proliferation in an in vivo model with clear relevance to cardiovascular pathology. Relevance: Aberrant proliferation of smooth muscle cells is an important aspect of cardiovascular disease. This research will help to identify the genes and proteins that regulate smooth muscle cell proliferation. It will also test the importance of these molecules in an in vivo model of vascular injury.