SHAPE Society

1. Editorial Slides (B)
VP Watch – September 18, 2002 – Volume 1, Issue 37
Yet Another Reason to Eat Less Fat…
Provided by:
Mark Rekhter, Ph.D.
Pfizer Global Research and Development, Ann Arbor Labs

2. How Does Lipid Lowering Work?
Clinical trials have established that lipid lowering by statins
reduces acute coronary events.
Lipid lowering may do it via “stabilization” of plaques in a
functional manner, specifically by reducing vascular inflammation1
.
Oxidative stress (directly or through LDL modification) induces
endothelial cell expression of vascular cell adhesion molecule 1
(VCAM-1)2
and monocyte chemoattractant protein 1 (MCP-1)3
and
reduces levels of atheroprotective NO4
, leading to monocyte
recruitment and macrophage accumulation.
Can lipid lowering work through decreasing oxidative stress5
and
improving endothelial functions related to inflammatory cell
accumulation?

3. As reported in VP Watch of this week, Aikawa
et al6
. showed that dietary lipid lowering in a rabbit
model of atherosclerosis can reduce oxidative stress
and endothelial cell “activation” in vivo.
Thirty rabbits consumed an atherogenic diet for 4
months to create atheroma. Balloon injury of the
thoracic aortas was performed 1 week after initiation of
the diet. Fifteen rabbits euthanized at 4 months
constituted the BaselineBaseline group. Five animals continued
to consume atherogenic diet for additional 16 months
(High group). The remaining animals consumed a chow
diet with no added cholesterol and fat for 16 months
(Low group).

4. Lipid Accumulation and Oxidation
In hypercholesterolemic rabbits, apoB-100, a major
component of LDL and VLDL particles, accumulated in
atheroma and co-localized with oxLDL epitopes (detected by
the antibody against MDA-LDL).
Lipid lowering reduced the amount of immunoreactive
apoB-100 and LDL epitopes
Plasma levels of autoantibodies (IgG) against oxLDL
epitopes (MDA-LDL) in the Baseline and High groups
significantly exceeded those of the Low group.

5. Endothelial “Activation”
Cholesterol feeding was associated with increased VCAM-1
expression by endothelial cells overlaying atherosclerotic
lesions. Lipid lowering reduced VCAM-1 expression.
MCP-1 localized in endothelial cells, smooth muscle cells
and macrophages in atheroma of hypercholesterolemic
rabbits. Lipid lowering lead to significant reduction in MCP-1
expression.
Endothelial cells after lipid lowering exhibitied more
“normal” ultrastructure.

6. Lipid lowering reduced oxLDL
accumulation and VCAM-1
expression in rabbit atheroma.
A, OxLDL epitopes (MDA-lysine)
accumulated in the aortic intima
beneath VCAM-1–positive ECs in
hypercholesterolemic rabbits fed the
atherogenic diet for 4 months
(Baseline, top panels) or 20 months
(High, middle panels). Bottom panels,
oxLDL epitopes and VCAM-1 were
barely detectable in the intima of
rabbit aorta after 16 months of lipid
lowering (Low), whereas CD31, an EC
marker, indicated an intact monolayer.
Scale bar, 50 µmol/L.
B, Data for VCAM-1 are reported as
percentage of CD31-positive
endothelium also bearing VCAM-1
measured by computer-assisted color
image analysis. Bars represent SEM.

7. Endothelial NO Synthase (eNOS) Production
Endothelial cells in rabbit atheroma showed
less eNOS immunoreactivity than those in
normal aortas.
Low group rabbits showed more eNOS
positivity than hypercholesterolemic rabbits.

8. Reactive oxygen species (ROS) production
ROS production (detected as Tiron-inducible
lucigenin chemiluminescence) in freshly
isolated aortic segments from the Baseline- and
High-group rabbits exceeded that of aortas from
age-matched normal rabbits.
After 16 months of dietary lipid lowering, ROS
production decreased to levels similar to those
of the normal rabbits.

9. ROS production detected by lucigenin
chemiluminescence and NBT reducing
activity assay and plasma levels of
autoantibody against MDA-LDL.
A, Production of ROS including O2
-
(detected by lucigenin
chemiluminescence) in fresh aortic segments of high
cholesterol–fed rabbits from Baseline (n=6) and High
(n=5) groups significantly exceeded that of aortas of age-
matched normal rabbits (n=4). After 16 months of lipid
lowering (Low, n= 7), ROS production decreased to levels
similar to those of the normal rabbits. Bars, SEM.
B, Top panel, Aortic ECs of the Baseline group showed
intense reaction with NBT. Spindle-shaped cells (probably
SMCs) also stained blue with NBT. Middle panel, Cluster
of circular cells (likely macrophages) in the deeper intima
of the baseline lesion also stained blue with NBT. Bottom,
NBT staining was greatly reduced by cotreatment with the
cell-permeant superoxide scavenger Tiron (10 mmol/L).
Scale bar, 50 µmol/L.
C, Plasma levels of autoantibodies against oxLDL
epitopes (MDA-LDL) were measured by
chemiluminescence immunoassay. Levels of anti-MDA-
LDL IgG in the Baseline and High groups significantly
exceeded those of the Low group. Bars, SEM.

10. Conclusions:
Dietary lipid lowering can reduce oxidative
stress and endothelial “activation” in vivo.
These mechanisms may contribute to
improvement in endothelial function and plaque
stabilization observed clinically.

11. Questions
What is the mechanism of oxidative stress reduction
by dietary lipid lowering?
Is inhibition of ROS production a root cause of
endothelial “pacifying” or lipid lowering has multiple
independent effects on ROS, eNOS, MCP-1, etc.?
If lipid lowering is sufficient for restoration of
endothelial function and plaque stabilization, why
aggressive lipid lowering fails to eradicate heart attack?
Why anti-oxidants are not blockbusters yet?
What new drugs do we need: better lipid lowering
drugs, anti-oxidants or direct “endothelial pacifiers”?

12. References
1. Libby P, Ridker PM, Maseri A: Inflammation and atherosclerosis. Circulation 2002, 105:1135-1143.
2. Marui N, Offermann MK, Swerlick R, Kunsch C, Rosen CA, Ahmad M, Alexander RW, Medford RM:
Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated
through an antioxidant-sensitive mechanism in human vascular endothelial cells. J.Clin.Invest.
1993, 92:1866-1874.
3. Cushing SD, Berliner JA, Valente AJ, Territo MC, Navab M, Parhami F, Gerrity R, Schwartz CJ,
Fogelman AM: Minimally modified low density lipoprotein induces monocyte chemotactic
protein 1 in human endothelial cells and smooth muscle cells. Proceedings of the National
Academy of Sciences of the United States of America 1990, 87:5134-5138.
4. Espey MG, Miranda KM, Thomas DD, Xavier S, Citrin D, Vitek MP, Wink DA: A chemical
perspective on the interplay between NO, reactive oxygen species, and reactive nitrogen oxide
species. Annals of the New York Academy of Sciences 2002, 962:195-206.
5. Ohara Y, Peterson TE, Sayegh HS, Subramanian RR, Wilcox JN, Harrison DG: Dietary correction
of hypercholesterolemia in the rabbit normalizes endothelial superoxide anion production.
Circulation 1995, 92:898-903.
6. Aikawa M, Sugiyama S, Hill CC, Voglic SJ, Rabkin E, Fukumoto Y, Schoen FJ, Witztum JL, Libby P: