Statins as Protective Agents for Aortic Endothelial Cells Robert Kreisberg West Liberty State College

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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) C-reactive protein (CRP) TNFα (10 ng/mL) TNFα (10 ng/mL) ox-LDL (100 ug/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 RT- qPCR Injury will be monitored by determining the levels of Il-8 and VCAM-1 in HAEC by immunoblotting, ELISA, and RT- qPCR

Monocytes Role in the development of atherosclerotic plaques (accumulation of lipids and fibrous elements in the arteries) 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 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 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 proteolytic enzymes, degrade ECM---resulting in formation of plaques reactive oxygen species (ROS) and Cytokines (TNFα, CRP, IL-6) reactive oxygen species (ROS) and Cytokines (TNFα, CRP, IL-6) recruit more inflammatory cells into the newly formed plaques recruit more inflammatory cells into the newly formed plaques increase lipid migration into the formed plaque increase lipid migration into the formed plaque

C-reactive protein (CRP) An acute phase response protein An acute phase response protein Induces production of IL-8, TNFα, IL-6, and IL-1. Induces production of IL-8, TNFα, IL-6, and IL-1. Increased levels are associated with higher risk of coronary heart disease Increased levels are associated with higher risk of coronary heart disease

Cell Adhesion Molecules Cell Adhesion Molecules Hold tissues together Hold tissues together Used by leukocytes to interact with tissue cells Used by leukocytes to interact with tissue cells Increase the strength of the functional interactions between cells of the immune system 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 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 Member of the immunoglobulin superfamily Expressed on vascular endothelial cells Expressed on vascular endothelial cells Little or no expression in resting human endothelium Little or no expression in resting human endothelium Cell surface expression enhanced in inflamed tissue Cell surface expression enhanced in inflamed tissue Function is to increase migration of monocytes, neutrophils, lymphocytes into inflamed tissue Function is to increase migration of monocytes, neutrophils, lymphocytes into inflamed tissue

TNFα Secreted by activated macrophages Secreted by activated macrophages Acts locally and systemically Acts locally and systemically Induces expression of VCAM Induces expression of VCAM Acts on macrophages and endothelial cells to induce production of chemokines resulting in an influx of neutrophils) Acts on macrophages and endothelial cells to induce production of chemokines resulting in an influx of neutrophils) Activates macrophages and neutrophils Activates macrophages and neutrophils Increased phagocytosis Increased phagocytosis Increased release of lytic enzymes into tissue spaces Increased release of lytic enzymes into tissue spaces Causes influx of eosinophils, basophils, neutrophils, monocytes, and mast cells Causes influx of eosinophils, basophils, neutrophils, monocytes, and mast cells Participate in clearance of antigen and tissue healing Participate in clearance of antigen and tissue healing

IL-8 (Neutrophil Activating Factor) Produced by neutrophils and endothelial cells Produced by neutrophils and endothelial cells Chemoattractant Chemoattractant Synergizes with IFN, TNF, GM-CSF and G-CSF Synergizes with IFN, TNF, GM-CSF and G-CSF Enhances cytotoxic functions of neutrophils Enhances cytotoxic functions of neutrophils Enhances antibody-dependent cytotoxicity Enhances antibody-dependent cytotoxicity Neutrophil recruitment across the endothelium (concentration gradient) Neutrophil recruitment across the endothelium (concentration gradient)

Statins Introduced in 1982 (Merck Laboratories) Introduced in 1982 (Merck Laboratories) Inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase Inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase Lower serum cholesterol Lower serum cholesterol Successfully treat atherosclerosis and hypercholesterolemia Successfully treat atherosclerosis and hypercholesterolemia Attenuation of chronic inflammation Attenuation of chronic inflammation

Cholesterol Synthesis Acetyl-CoA Numerous Rxns Cholesterol Site of Restriction

Treatment Assay: Confluent cultures of HAEC CRP (50μg/ml)Ox-LDL (100μg/ml)TNFα (10ng/ml) Lovastatin (0.1 to 50μmole/L) XXXXXX Mevalonate (200μM) XXXXXX Time span : 15min24hr.

Cells treated with TNF

Cell lysate mRNA prep for RT-qPCR At each time point: Supernatant IL-8 ELISA Cell lysate VCAM immunoblotting

RT-qPCR

IL-8 ELISA

Summary T.C. facilities are up-and –running T.C. facilities are up-and –running Technician/students are trained in aseptic tissue- culture technique Technician/students are trained in aseptic tissue- culture technique TNFα stimulates IL-8 and VCAM mRNA production TNFα stimulates IL-8 and VCAM mRNA production TNFα stimulates the production of IL-8 protein TNFα stimulates the production of IL-8 protein Production of VCAM protein between 8-24 hours has not been detected Production of VCAM protein between 8-24 hours has not been detected

Concerns $$$$

The Gang: Cassie, Amanda, Katie, and King Kreisberg