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Statins as Protective Agents for Aortic Endothelial Cells

Statins as Protective Agents for Aortic Endothelial Cells. Robert Kreisberg West Liberty State College. Our Facilities. WARNING!!. Experimental Design.

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Statins as Protective Agents for Aortic Endothelial Cells

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

  2. Our Facilities WARNING!!

  3. 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.

  4. 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)

  5. Injury will be monitored by determining the levels of Il-8 and VCAM-1 in HAEC by immunoblotting, ELISA, and RT-qPCR

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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)

  13. 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

  14. Cholesterol Synthesis Acetyl-CoA Numerous Rxns Site of Restriction Numerous Rxns Cholesterol

  15. Treatment Assay: Confluent cultures of HAEC Time span: 15min 24hr.

  16. Cells treated with TNF

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

  18. RT-qPCR

  19. IL-8 ELISA

  20. Summary • T.C. facilities are up-and –running • Technician/students are trained in aseptic tissue-culture 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

  21. Concerns $$$$

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

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