KLF4-Dependent Regulation of SMC Differentiation and Phenotypic Switching

描述(申请人提供)：平滑肌细胞(SMC)分化状态的改变在各种心血管疾病的发生和发展中起着关键作用。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，以及这些控制过程在SMC表型转换过程中如何改变与血管损伤和疾病相关。我们最近的研究结果表明，迷人的多能性基因Krupple-like factor4(KLF4)是SMC表型转换的关键介质，在我们实验室产生的条件性KLF4基因敲除(KO)小鼠的颈动脉结扎损伤后，KLF4是SMC表型转换的关键介质。这项研究将验证KLF4在血管SMC发育过程中以及在血管损伤或动脉粥样硬化发展后的表型转换过程中，在调节血管SMC分化状态转变中发挥关键作用的假设。目的1确定KLF4在体内血管损伤时抑制SMC标志基因表达的机制，包括确定KLF4是否通过以下途径介导抑制SMC基因的表达：a)与大多数SMC启动子中发现的保守的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)抑制强大的SMC选择性SRF共激活因子myocardin的表达；和/或d)诱导多能基因。目的2将验证我们以前在条件性KLF4基因敲除小鼠中观察到的血管损伤后SMC标志基因的延迟抑制和加重病变形成的假设，至少部分是通过SMC中KLF4的丢失而不是它在EC或巨噬细胞中的丢失来调节的。研究将包括骨髓移植，以及SMC特异性KLF4 KO在小鼠损伤中的条件研究。目的3是确定条件性或SMC特异性的KLF4条件性敲除是否改变了ApoE基因敲除小鼠的动脉粥样硬化内膜病变的大小或细胞组成。目的4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血管内膜损伤发生和/或进展中发挥重要功能。 公共卫生相关性：血管平滑肌细胞(SMC)分化状态(即细胞特异性)的异常控制被认为在许多重大疾病中发挥关键作用，包括动脉粥样硬化、哮喘、高血压和肿瘤转移。这项提案中的研究将为控制SMC在发育和疾病中分化的机制提供新的见解，并可能导致新的更有效的治疗方法。

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.