Molecular and single-cells analyses of atherosclerotic lesions in murine knockout models Atherosclerosis is a chronic disease initiated by the accumulation of materials such as lipids, resulting in the recruitment of monocytes/lymphocytes into arterial plaques and their adhesion to activated endothelial cells. Although it has been established that leukocyte recruitment and expression of pro-inflammatory cytokines characterize early atherosclerotic lesions many aspects of plaque formation and evolution remain unsolved. Peroxisome Proliferator-Activated Receptor gamma (PPARgamma), a regulator of lipid metabolism (uptake and efflux) in macrophages has been implicated also in the regulation of inflammatory gene expression. The goal of this project is to gain a comprehensive view on the molecular events governing the atherosclerotic plaques formation and evolution, focusing on target genes, PPARgamma-induced, involved in monocyte trafficking and accumulation, such as CCR2 and CX3CR1, within the plaque. The Apoe KO mouse model represent an invaluable tool to study the pathogenesis of atherosclerotic lesions. Thus, the use of a selected genetically defined population (inbred strain C57BL/6J) of mice will allow: i) to fix the genetic contribution (animals with identical genetic background); ii) to perform the molecular analyses of the disease. The Apoe-/-/Ppargfx/fx/Ccr2-/- and the Apoe-/-/Ppargfx/fx/Cx3cr1-/- mouse models, kept onto C57BL/6J inbred genetic background, will be produced to analyze the process of monocyte/macrophage recruitment in the early phases of atherogenesis. The in vivo study will be combined with ex vivo manipulations and with in vitro experiments to investigate: a) the transcription factors involved in the control of the PPARgamma expression and activity levels; b) the molecular mechanisms involved in monocyte recruitment, focusing on the role of chemokine (C-C motif) receptor 2 (CCR2) and chemokine (CX3-C motif) receptor 1 (CX3CR1) in modulating the inflammatory elements of the atherosclerotic lesion phenotypes. The genes, potentially involved in monocyte/macrophage switching will be investigated on Laser-Capture Microdissected single-cells by quantitative Real-Time PCR (qRT-PCR). In addition, extracellular nucleotide metabolism in hearts of all knockouts and controls will be studied by evaluation of e5N and eNTPD activities and in Langendorff heart perfusion system that will include infusion of nucleotides and measurement its metabolism by high performance liquid chromatography. This will consent to identify shifts in expression pattern and function of extracellular nucleotide metabolism related to genetic manipulations and atherosclerosis development. The results obtained in this study will allow to generate novelty regarding the biological function of PPARgamma and other regulative genes important to maintain the homeostasis of the vascular wall and that are involved in the atherosclerotic lesion formation.
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