Statins as Statins as Protective Protective Agents for Agents for

Aortic Aortic Endothelial Endothelial

CellsCellsRobert KreisbergRobert Kreisberg

West Liberty State CollegeWest Liberty State College

Our FacilitiesOur Facilities

WARNING!!

Experimental Experimental DesignDesign

To determine whether statins To determine whether statins inhibit IL-8 and VCAM production inhibit IL-8 and VCAM production in human aortic endothelial cells in human aortic endothelial cells (HAEC) in response to C-reactive (HAEC) in response to C-reactive protein (CRP), TNFa, and ox-LDL.protein (CRP), TNFa, and ox-LDL.

We will establish whether We will establish whether statins protect cultured statins protect cultured

HAECHAEC from injury induced from injury induced by: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 Injury will be monitored by determining the levels of Il-determining the levels of Il-8 and VCAM-1 in HAEC by 8 and VCAM-1 in HAEC by immunoblotting, ELISA, immunoblotting, ELISA,

and RT-qPCRand RT-qPCR

MonocytesMonocytes

Role in the development of atherosclerotic Role in the development of atherosclerotic plaques (accumulation of lipids and plaques (accumulation of lipids and fibrous elements in the arteries)fibrous elements in the arteries)

Adhere to arterial walls, migrate Adhere to arterial walls, migrate through the layer of endothelial cells through the layer of endothelial cells and differentiate into macrophagesand differentiate into macrophages

Receptors on cell surface bind lipoprotein Receptors on cell surface bind lipoprotein particles, internalize them and accumulate particles, internalize them and accumulate lipid droplets, “foamy” appearancelipid droplets, “foamy” appearance

Foamy macrophages Foamy macrophages secrete:secrete:

proteolytic enzymes, degrade ECM---proteolytic enzymes, degrade ECM---resulting in formation of plaquesresulting in formation of plaques

reactive oxygen species (ROS) and reactive oxygen species (ROS) and Cytokines (TNFα, CRP, IL-6)Cytokines (TNFα, CRP, IL-6)

recruit more inflammatory cells into the recruit more inflammatory cells into the newly formed plaquesnewly formed plaques

increase lipid migration into the formed increase lipid migration into the formed plaqueplaque

C-reactive protein (CRP)C-reactive protein (CRP)

An acute phase response proteinAn 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 Increased levels are associated with higher risk of coronary heart diseasecoronary heart disease

Cell Adhesion Molecules Cell Adhesion Molecules

Hold tissues togetherHold tissues together Used by leukocytes to interact with tissue Used by leukocytes to interact with tissue

cellscells Increase the strength of the functional Increase the strength of the functional

interactions between cells of the immune systeminteractions between cells of the immune system

May be constitutively expressed or expressed only in May be constitutively expressed or expressed only in response to increases in local concentration of cytokines response to increases in local concentration of cytokines

Vascular Cell Adhesion Vascular Cell Adhesion Molecule (VCAM)Molecule (VCAM)

Member of the immunoglobulin superfamilyMember of the immunoglobulin superfamily

Expressed on vascular endothelial cellsExpressed on vascular endothelial cells

Little or no expression in resting human endotheliumLittle 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, Function is to increase migration of monocytes, neutrophils, lymphocytes into inflamed tissue lymphocytes into inflamed tissue

TNFαTNFα Secreted by activated macrophagesSecreted by activated macrophages

Acts locally and systemicallyActs locally and systemically

Induces expression of VCAMInduces expression of VCAM Acts on macrophages and endothelial cells to induce Acts on macrophages and endothelial cells to induce

production of chemokines resulting in an influx of neutrophils)production of chemokines resulting in an influx of neutrophils)

Activates macrophages and neutrophilsActivates macrophages and neutrophils Increased phagocytosisIncreased phagocytosis Increased release of lytic enzymes into tissue spacesIncreased release of lytic enzymes into tissue spaces

Causes influx of eosinophils, basophils, neutrophils, monocytes, and mast cellsCauses 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 IL-8 (Neutrophil Activating Factor)Factor)

Produced by neutrophils and Produced by neutrophils and endothelial cellsendothelial cells

ChemoattractantChemoattractant Synergizes with IFN , TNF , GM-CSF Synergizes with IFN , TNF , GM-CSF

and G-CSFand G-CSF Enhances cytotoxic functions of Enhances cytotoxic functions of

neutrophilsneutrophils Enhances antibody-dependent Enhances antibody-dependent

cytotoxicitycytotoxicity Neutrophil recruitment across the Neutrophil recruitment across the

endothelium (concentration gradient)endothelium (concentration gradient)

StatinsStatins

Introduced in 1982 (Merck Introduced in 1982 (Merck Laboratories)Laboratories)

Inhibit 3-hydroxy-3-methylglutaryl Inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductasecoenzyme A reductase

Lower serum cholesterolLower serum cholesterol Successfully treat atherosclerosis Successfully treat atherosclerosis

and hypercholesterolemiaand hypercholesterolemia Attenuation of chronic inflammationAttenuation of chronic inflammation

Cholesterol SynthesisCholesterol SynthesisAcetyl-CoA

Numerous Rxns

Numerous Rxns

Cholesterol

Site of Restriction

Treatment Assay: Treatment Assay: Confluent cultures of HAECConfluent cultures of HAEC

CRP (50μg/ml) Ox-LDL (100μg/ml) TNFα (10ng/ml)

Lovastatin(0.1 to 50μmole/L)

X X X X X X

Mevalonate (200μM)

X X X X X X

Time span: 15min 24hr.

Cells treated with TNFCells treated with TNF

Cell lysate mRNA prep for RT-qPCR

At each time point:At each time point:

Supernatant IL-8 ELISA

Cell lysate VCAM immunoblotting

RT-qPCRRT-qPCR

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IL-8 ELISAIL-8 ELISAThe Expression of Il-8 from TNF-a

Treated Human Aortic Endothelial Cells

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SummarySummary

T.C. facilities are up-and –runningT.C. facilities are up-and –running Technician/students are trained in Technician/students are trained in

aseptic tissue-culture techniqueaseptic tissue-culture technique TNFα stimulates IL-8 and VCAM mRNA TNFα stimulates IL-8 and VCAM mRNA

productionproduction TNFα stimulates the production of IL-8 TNFα stimulates the production of IL-8

proteinprotein Production of VCAM protein between 8-Production of VCAM protein between 8-

24 hours has not been detected 24 hours has not been detected

ConcernsConcerns

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