Role of statins on modulation of inflammation and cellular adhesion biomarkers in vivo and in vitro

Abstract

The inflammatory process plays a key role in the genesis and development of atherosclerosis, and endothelial dysfunction is considered one of the early stages of atherogenesis. Through the inhibition of hydroxyl-methyl glutaryl CoA reductase (HMGCR), statins reduce cholesterol biosynthesis and the formation of isoprenoids, which are intermediate products of cholesterol synthesis that are important in post-transcriptional modification of small GTPases, which are involved in endothelial dysfunction and vascular inflammation. This mechanism is also known as pleiotropic effects of statins. In order to elucidate the molecular mechanisms by which statins can modulate inflammatory biomarkers and cellular adhesion, we will study the protein and mRNA expression, as well as mRNA stability of pro-inflammatory markers and adhesion molecules in response to simvastatin and ezetimibe. The effects of simvastatin and ezetimibe (a non-statin cholesterol-lowering drug) on mRNA expression of inflammatory biomarkers (eNOS, TNF±, IL-6, NFkB, IL-1b, IL-8, MMP-9 and MCP-1) and adhesion molecules (E-selectin, P-selectin, VCAM-1, ICAM-1, PSGL1, L-selectin, VLA-4, LFA-1 and Mac-1) will be evaluated by real time PCR, using in vitro models of endothelial cells (HUVEC) and monocytes (THP-1), as well as peripheral blood mononuclear cells (PBMC) from individuals with hypercholesterolemia (HC) under cholesterol-lowering therapy as a in vivo model. The expression of proteins will be evaluated by Western blotting and flow cytometry. The effect of these drugs on the mRNA stability will be also evaluated by real time PCR. The effect of lipid-lowering drugs on activity of NFkB, transcription factor that modulate the expression of inflammatory proteins, will be evaluated by Electrophoretic Mobility Shift Assay (EMSA) that enables to study DNA-protein interactions. To study the participation of eNOS in modulating the effects of simvastatin on inflammatory and adhesion biomarkers, it will be performed gene silencing by RNA interference (RNAi) to generate eNOS deficient HUVEC cells. The expression profile of the micro-RNAs miR-221 e miR-222 will also be evaluated in HUVEC cells by real time PCR. The results of this study help to clarify the molecular mechanisms by which statins may regulate the inflammatory response and improve endothelial dysfunction. (AU)