Cell cycle control of vascular remodeling

描述(申请人提供)：动脉平滑肌细胞(SMCs)通常以分化的静止状态存在，这种状态部分是通过从内膜内皮细胞释放抗肿瘤原，如前列环素(PGI2)来维持的。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，会损害这些抗肿瘤原的释放，并(连同与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向增殖状态的表型改变。SMC增殖在损伤后血管重塑反应中的重要性已得到证实，但控制静止和增殖状态的细胞周期调节机制尚未确定。我们研究了丝裂原和前列腺素I2对细胞周期蛋白依赖性蛋白激酶(CDKs)的影响，发现这些正负调控因子主要通过控制CDK抑制蛋白p27kip1的水平发挥作用。此外，这种趋同还体现在Skp2的调节上，Skp2是控制p27kip1降解的E3泛素连接酶。一些研究已经确定了转录后调节Skp2的机制，但我们发现丝裂原和PGI2的主要作用是影响成纤维细胞和SMC中Skp2的mRNA表达。因此，我们提出了三个特定的目标来检验这一假设，即Skp2基因是损伤诱导的血管重塑过程中有丝分裂原和PGI2信号的主要焦点。在目标1中，我们将使用SMCs和MEF来表征丝裂原调节Skp2基因诱导的机制。将确定相关的信号转导级联和关键的启动子元件。我们将探讨丝裂原对Skp2基因稳定性的潜在影响。在目标2中，我们将使用相同的系统和平行的方法来表征PGI2拮抗丝裂原依赖的Skp2mRNA诱导的机制。在目的3中，我们将利用基因敲除和转基因小鼠股动脉损伤来确定PGI2-IP-Skp2通路在体内SMC增殖中的重要性。总之，这些结果将为控制Skp2基因表达的基本调控机制提供新的见解，并直接在与心血管病理明显相关的体内模型中测试Skp2对SMC增殖的重要性。相关性：血管平滑肌细胞的异常增殖是心血管疾病的一个重要方面。这项研究将有助于确定调控平滑肌细胞增殖的基因和蛋白质。它还将在血管损伤的活体模型中测试这些分子的重要性。

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.