Role of Oxidized Phospholipids in Phenotypic Switching of Smooth Muscle Cells (SM

描述(由申请人提供)：有明确的证据表明，血管平滑肌细胞(SMC)的表型转换在动脉粥样硬化性疾病的发展和斑块破裂/血栓形成等终末期临床后果中起着关键作用。然而，在动脉粥样硬化形成中调节SMC表型转换的机制和因素却知之甚少。此外，尽管有令人信服的证据表明氧化磷脂(Oxpls)在动脉粥样硬化形成过程中对内皮细胞和单核/巨噬细胞的激活起关键作用，但关于oxpls在控制血管SMC表型转换中的作用几乎一无所知。这一建议的重点是验证氧化1-棕榈酰基-2-花生烯酰-sn-甘油-3-磷酰胆碱(OxPAPC)及其oxPL组分POVPC和PGPC在调节与实验性血管损伤/动脉粥样硬化相关的SMC表型转换中起关键作用的假说，以及这些化合物的作用至少部分是通过G/C抑制物/TCE结合蛋白KLF4和ELK1的ERK依赖的磷酸化来调节的。为了支持这一假说，我们发现oxPAPC、POVPC和PGPC能显著抑制所有SMC分化标志物基因的表达，包括SM a-肌动蛋白、SM肌球蛋白重链(MHC)和Myocardin在培养的SMC以及体内颈动脉中的表达。相反，oxPLs增加了KLF4的表达，我们之前已经证明KLF4基因可以显著抑制强大的SMC选择性SRF共激活因子myocardin(和myocardin样因子)的表达，并诱导与染色质凝聚和转录沉默相关的SMC标记基因位点的组蛋白修饰。此外，我们提供的初步数据表明，POVPC诱导的SMC分化标志基因在培养的SMC和体内的抑制可以被siRNA诱导的KLF4抑制所抑制。目的1探讨oxPLs抑制SMC分化标志物基因如SM a-肌动蛋白、SM MHC和SM22a表达的机制，以及KLF4和磷酸化ELK1对Carg-SRF-myocardin依赖转录的抑制作用。目的2利用我们实验室开发的Pluronic凝胶系统，结合独特的SMC启动子-报告基因转基因小鼠和条件性KLF4/ApoE基因敲除小鼠，确定oxPLs在体内调节SMC表型转换以应对血管损伤和/或实验性动脉粥样硬化的作用和机制。综上所述，这些研究将提供关于oxpls在动脉粥样硬化形成中促进SMC表型转换的细胞和分子机制的新见解，并可能导致抑制动脉粥样硬化病变形成、进展和/或斑块破裂的新疗法的开发。

DESCRIPTION (provided by applicant): There is clear evidence that phenotypic switching of the vascular smooth muscle cell (SMC) plays a critical role in development of atherosclerotic disease, and end stage clinical consequences such as plaque rupture/thrombosis. However, the mechanisms and factors that regulate SMC phenotypic switching in atherogenesis are poorly understood. In addition, although there is compelling evidence that oxidized phospholipids (oxPLs) play a critical role in activation of endothelial cells and monocytes/macrophages during atherogenesis, virtually nothing is known regarding the role of oxPLs in control of phenotypic switching of vascular SMC. The focus of this proposal is to test the hypothesis that oxidized 1-palmitoyl-2-arachidonoyl-sn- glycero-3-phosphorylcholine (OxPAPC) and its oxPL components POVPC and PGPC play a key role in regulating SMC phenotypic switching associated with experimental vascular injury/atherogenesis, and that the effects of these compounds are mediated at least in part through the G/C repressor/TCE binding protein KLF4 and ERK-dependent phosphorylation of ELK1. In support of this hypothesis, we found that oxPAPC, POVPC, and PGPC profoundly suppressed expression of all SMC differentiation marker genes tested to date including SM a-actin, SM myosin heavy chain (MHC), and myocardin in cultured SMC, as well as in carotid arteries in vivo. In contrast, oxPLs increased expression of KLF4, a gene we have previously shown can markedly suppress expression of the potent SMC selective SRF co-activator myocardin (and myocardin like factors), and induce histone modifications of SMC marker gene loci associated with chromatin condensation and transcriptional silencing. In addition, we present preliminary data showing that POVPC-induced suppression of SMC differentiation marker genes in cultured SMC and in vivo can be inhibited by siRNA-induced suppression of KLF4. Aim 1 will determine mechanisms by which oxPLs suppress expression of SMC differentiation marker genes such as SM a-actin, SM MHC, and SM22a and will include investigation of the role of inhibition of CArG-SRF-myocardin dependent transcription by KLF4 and phospho-ELK1. Aim 2 will determine the role and mechanisms by which oxPLs regulate SMC phenotypic switching in vivo in response to vascular injury and/or experimental atherosclerosis using a novel pluronic gel system developed in our labs in combination with unique SMC promoter-reporter transgenic mice, and conditional KLF4/ApoE knockout mice. Taken together, studies will provide novel insights regarding cellular and molecular mechanisms whereby oxPLs contribute to phenotypic switching of SMC in atherogenesis, and may lead to development of novel therapies for inhibiting atherosclerotic lesion formation, progression, and/or plaque rupture.