Transcript Experimental Design To determine whether statins inhibit IL

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)

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
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
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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

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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