KLF4-Dependent Regulation of SMC Differentiation and Phenotypic Switching

描述（由申请人提供）：平滑肌细胞（SMC）分化状态的改变在多种心血管疾病的发生和进展中起关键作用。该项目的长期目标是阐明在血管发育过程中控制SMC生长和分化的细胞和分子机制，以及这些控制过程如何在与血管损伤和疾病相关的SMC表型转换过程中发生改变。我们最近的研究结果表明，迷人的多能性基因krup普尔样因子4 （KLF4）是培养SMC表型转换的关键介质，以及在我们实验室产生的条件KLF4敲除（KO）小鼠颈动脉结膜损伤后的体内SMC表型转换。本课题的研究将验证KLF4在发育过程中调控血管SMC分化状态的转变，以及血管损伤或动脉粥样硬化发生后的表型转换中发挥关键作用的假设。目的1将确定KLF4在血管损伤中抑制SMC标记基因表达的机制，包括确定KLF4是否通过以下方式介导SMC基因的抑制：a)与大多数SMC启动子中发现的保守G/C抑制因子和/或TCE元件结合；B)诱导与转录沉默相关的表观遗传修饰；c)抑制强效smc选择性SRF共激活剂心肌素的表达；和/或d)诱导多能性基因。Aim 2将验证我们之前在条件KLF4敲除小鼠中观察到的SMC标记基因延迟抑制和血管损伤后病变形成加剧的假设，至少部分是由SMC中KLF4的丢失介导的，而不是其在EC或巨噬细胞中的丢失。研究将包括骨髓移植和SMC特异性条件KLF4小鼠损伤的研究。目的3是确定条件或SMC特异性条件敲除KLF4是否会改变ApoE敲除小鼠动脉粥样硬化内膜病变大小或细胞组成。目的4是确定KLF4在MMP3、MCP-1、iNOS和VCAM-1等基因激活中的作用，这些基因可能在血管损伤或实验性动脉粥样硬化的内膜病变发生和/或进展中发挥重要的功能作用。

DESCRIPTION (provided by applicant): Alterations in the differentiated state of the smooth muscle cell (SMC) play a key role in the development and progression of a variety of cardiovascular diseases. The long-term goal of this project is to elucidate cellular and molecular mechanisms that control the growth and differentiation of SMC during vascular development, and how these control processes are altered during phenotypic switching of SMC in association with vascular injury and disease. Results of our recent studies have implicated the fascinating pluripotency gene Krupple-Like Factor 4 (KLF4) as a key mediator of SMC phenotypic switching in cultured SMC, and in vivo following carotid ligation injury in conditional KLF4 knockout (KO) mice generated in our lab. Studies in this proposal will test the hypothesis that KLF4 plays a critical role in regulating transitions in the differentiated state of vascular SMC during development, as well as during phenotypic switching following vascular injury or development of atherosclerosis. Aim 1 will determine mechanisms by which KLF4 represses expression of SMC marker genes in vivo with vascular injury including determining if KLF4 mediates repression SMC genes by: a) binding to conserved G/C repressor and/or TCE elements found in most SMC promoters; b) inducing epigenetic modifications associated with transcriptional silencing; c) suppressing expression of the potent SMC-selective SRF co-activator myocardin; and/or d) inducing pluripotency genes. Aim 2 will test the hypothesis that delayed repression of SMC marker genes and exacerbated lesion formation following vascular injury observed in our previous studies in conditional KLF4 knockout mice, were mediated, at least in part, by loss of KLF4 in SMC rather than its loss in EC or macrophages. Studies will include bone marrow transfer, and SMC specific conditional KLF4 KO studies in mice injury. Aim 3 is to determine if conditional or SMC specific conditional knockout of KLF4 alters atherosclerotic intimal lesion size or cellular composition in ApoE knockout mice. Aim 4 is to determine the role of KLF4 in activation of genes such as MMP3, MCP-1, iNOS, and VCAM-1 which are likely to play an important functional role in intimal lesion development and/or progression in response to vascular injury or experimental atherosclerosis. PUBLIC HEALTH RELEVANCE: Abnormal control of the differentiated state (i.e. cell specific properties) of vascular smooth muscle cells (SMC) is known to play a critical role in a number of major diseases including atherosclerosis, asthma, hypertension, and tumor metastasis. Studies in this proposal will provide novel insights into mechanisms that control SMC differentiation in development and disease and may lead to new and more effective therapies.