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疾病的发展和终末期临床后果（如斑块破裂/血栓形成）中起着关键作用。然而，在动脉粥样硬化发生过程中调节SMC表型转换的机制和因素尚不清楚。此外，尽管有令人信服的证据表明，氧化磷脂（oxPLs）在动脉粥样硬化过程中对内皮细胞和单核/巨噬细胞的激活起着关键作用，但oxPLs在控制血管SMC表型转换中的作用几乎一无所知。本研究的重点是验证氧化1-棕榈酰-2-花生四烯酰基-sn-甘油-3-磷酸胆碱（OxPAPC）及其oxPL成分POVPC和PGPC在调节与实验性血管损伤/动脉粥样硬化相关的SMC表型转换中发挥关键作用的假设，以及这些化合物的作用至少部分通过G/C抑制物/TCE结合蛋白KLF4和erk依赖性ELK1磷酸化介导。为了支持这一假设，我们发现oxPAPC、POVPC和PGPC在体外培养的SMC和颈动脉中深刻地抑制了迄今为止测试的所有SMC分化标记基因的表达，包括SM a-actin、SM肌球蛋白重链（MHC）和心肌素。相反，oxPLs增加了KLF4的表达，该基因可以显著抑制SMC选择性SRF共激活因子心肌素（和心肌素样因子）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显示，povpc诱导的SMC分化标记基因在培养SMC和体内的抑制可以被sirna诱导的KLF4抑制所抑制。目的1将确定oxPLs抑制SMC分化标记基因（如SM a-actin， SM MHC和SM22a）表达的机制，并将包括KLF4和phospho-ELK1抑制carg - srf -心肌素依赖转录的作用。目的2将利用我们实验室开发的一种新型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KLF4/ApoE敲除小鼠，确定oxPLs在血管损伤和/或实验性动脉粥样硬化反应中调节SMC表型转换的作用和机制。综上所述，这些研究将提供关于细胞和分子机制的新见解，其中oxPLs有助于动脉粥样硬化发生中SMC的表型转换，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发展。

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.