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Statins as Protective Agents for Aortic Endothelial Cells Robert Kreisberg West Liberty State College Our Facilities WARNING!! Experimental Design To determine whether statins inhibit IL-8 and VCAM production in human aortic endothelial cells (HAEC) in response to C-reactive protein (CRP), TNFa, and ox-LDL. We will establish whether statins protect cultured HAEC from injury induced by: C-reactive protein (CRP) TNFα (10 ng/mL) ox-LDL (100 ug/ml) Injury will be monitored by determining the levels of Il-8 and VCAM-1 in HAEC by immunoblotting, ELISA, and RTqPCR Monocytes Role in the development of atherosclerotic plaques (accumulation of lipids and fibrous elements in the arteries) Adhere to arterial walls, migrate through the layer of endothelial cells and differentiate into macrophages Receptors on cell surface bind lipoprotein particles, internalize them and accumulate lipid droplets, “foamy” appearance Foamy macrophages secrete: proteolytic enzymes, degrade ECM---resulting in formation of plaques reactive oxygen species (ROS) and Cytokines (TNFα, CRP, IL-6) recruit more inflammatory cells into the newly formed plaques increase lipid migration into the formed plaque C-reactive protein (CRP) An acute phase response protein Induces production of IL-8, TNFα, IL-6, and IL-1. Increased levels are associated with higher risk of coronary heart disease Cell Adhesion Molecules Hold tissues together Used by leukocytes to interact with tissue cells Increase the strength of the functional interactions between cells of the immune system May be constitutively expressed or expressed only in response to increases in local concentration of cytokines Vascular Cell Adhesion Molecule (VCAM) Member of the immunoglobulin superfamily Expressed on vascular endothelial cells Little or no expression in resting human endothelium Cell surface expression enhanced in inflamed tissue Function is to increase migration of monocytes, neutrophils, lymphocytes into inflamed tissue TNFα Secreted by activated macrophages Acts locally and systemically Induces expression of VCAM Acts on macrophages and endothelial cells to induce production of chemokines resulting in an influx of neutrophils) Activates macrophages and neutrophils Increased phagocytosis Increased release of lytic enzymes into tissue spaces Causes influx of eosinophils, basophils, neutrophils, monocytes, and mast cells Participate in clearance of antigen and tissue healing IL-8 (Neutrophil Activating Factor) Produced by neutrophils and endothelial cells Chemoattractant Synergizes with IFN , TNF , GM-CSF and G-CSF Enhances cytotoxic functions of neutrophils Enhances antibody-dependent cytotoxicity Neutrophil recruitment across the endothelium (concentration gradient) Statins Introduced in 1982 (Merck Laboratories) Inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase Lower serum cholesterol Successfully treat atherosclerosis and hypercholesterolemia Attenuation of chronic inflammation Cholesterol Synthesis Acetyl-CoA Numerous Rxns Site of Restriction Numerous Rxns Cholesterol Treatment Assay: Confluent cultures of HAEC CRP (50μg/ml) Lovastatin (0.1 to 50μmole/L) Mevalonate (200μM) X X X X Time span: 15min Ox-LDL (100μg/ml) X TNFα (10ng/ml) X X X X X X 24hr. X Cells treated with TNF At each time point: Supernatant IL-8 ELISA Cell lysate mRNA prep for RT-qPCR Cell lysate VCAM immunoblotting L og fold c hang e from z ero tim e point RT-qPCR 10.00 8.00 6.00 4.00 IL -8 2.00 VC AM 0.00 -2.00 -4.00 15 minutes 30 minutes 60 minutes 4 hours 8 hours IL-8 ELISA 2500 2000 Supernatant % increase Cellular Lysate % increase 1500 1000 500 m 15 in m i 30 n m 60 in m in 4 hr s 8 hr 24 s hr s 0 0 Percent (%) Increase The Expression of Il-8 from TNF-a Treated Human Aortic Endothelial Cells Time Summary T.C. facilities are up-and –running Technician/students are trained in aseptic tissueculture technique TNFα stimulates IL-8 and VCAM mRNA production TNFα stimulates the production of IL-8 protein Production of VCAM protein between 8-24 hours has not been detected Concerns $$$$ The Gang: Cassie, Amanda, Katie, and King Kreisberg