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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