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

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?

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).

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.

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.

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.

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.

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.

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.

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.

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”?

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:

Lipid Lowering Reduces Oxidative Stress and Endothelial Cell Activation in Rabbit Atheroma .

Circulation 2002, 106:1390-1396.