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

描述（由申请方提供）：有明确证据表明，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知之甚少。此外，虽然有令人信服的证据表明，氧化磷脂（oxPL）在动脉粥样硬化形成过程中的内皮细胞和单核细胞/巨噬细胞的活化中发挥关键作用，几乎没有什么是已知的oxPL在控制血管SMC的表型转换的作用。本提案的重点是检验以下假设：氧化的1-棕榈酰-2-花生四烯酸酰-sn-甘油-3-磷酸胆碱（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，并且这些化合物的作用至少部分通过G/C阻遏物/TCE结合蛋白KLF 4和ERK介导。依赖于ELK 1的磷酸化。为了支持这一假设，我们发现oxPAPC、POVPC和PGPC深刻地抑制了迄今为止测试的所有SMC分化标志物基因的表达，包括在培养的SMC中以及在体内颈动脉中的SM α-肌动蛋白、SM肌球蛋白重链（MHC）和心肌蛋白。相比之下，oxPL增加KLF 4的表达，KLF 4是我们先前已经显示的基因，可以显著抑制有效的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，我们目前的初步数据表明，POVPC诱导的抑制SMC分化标记基因在培养的SMC和体内可以抑制siRNA诱导的抑制KLF 4。目的1将确定oxPL抑制SMC分化标记基因如SM α-肌动蛋白、SM MHC和SM 22 a表达的机制，并将包括研究KLF 4和磷酸化-ELK 1抑制CArG-SRF-心肌蛋白依赖性转录的作用。目的2将确定oxPLs调节SMC表型转换的作用和机制，在体内血管损伤和/或实验性动脉粥样硬化使用一种新的pluronic凝胶系统在我们的实验室开发的独特的SMC启动子报告基因转基因小鼠，和条件KLF 4/ApoE基因敲除小鼠相结合。总之，研究将提供关于oxPL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，并可能导致开发用于抑制动脉粥样硬化病变形成、进展和/或斑块破裂的新疗法。

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.