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.
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