Vulnerable plaques are characterized by a thin fibrous cap overlying a macrophage-rich atheromatous core. Regional macrophages overexpress matrix degrading enzymes such as matrix metalloproteinases (MMPs) and pro-thrombotic molecules including tissue factor. Both can contribute to plaque vulnerability and thrombogenecity resulting in the onset of acute coronary events. Activated smooth muscle cells (SMC) also overexpressed MMPs and tissue factor. Activated endothelial cells play an important role in monocyte recruitment into the intima and macrophage-rich atheroma formation. Their current understanding is that lipid-lowering therapy can decrease acute coronary events in patients probably by changing the nature of established plaques qualitatively or functionally, rather than decreasing stenosis. However, the precise molecular and cellular mechanisms are unknown. Potential mechanisms that may play a role in stabilizing atherosclerotic plaques in lipid lowering are shown in Figure 1.

To improve the mechanistic understanding of plaque stabilization, they tested their hypothesis that lipid lowering by diet improves inflammatory cell accumulation and vascular cell activation in rabbit atherosclerotic plaques.

Rabbit atheroma was created by balloon injury and high cholesterol feeding for four months. Fifteen animals were sacrificed at 4 months to determine the nature of baseline lesions. Five animals continued the atherogenic diet for another 16 months. The remaining animals consumed a regular chow diet with no added cholesterol or fat.

Long-term lipid lowering by diet was shown to reduce macrophage accumulation, MMP expression and activity, and in parallel increase interstitial collagen content in rabbit atheroma.

After 4 months of the atherogenic diet, rabbit aorta contained a large number of macrophages, but after 16 months of lipid lowering macrophages were nearly undetectable. In the baseline lesion, macrophages expressed high levels of MMP-1 or collagenase-1. In such lesions, collagen accumulation was seen at very low levels. However, lipid lowering reduced collagenase expression and in parallel increased collagen accumulation, a key determinant of plaque stability. The conversion from a soft plaque to a fibrous plaque was also confirmed by magnetic resonance imaging (MRI).

Smooth muscle cell activation

Several years ago, Aikawa isolated the cDNA clones encoding human smooth muscle myosin heavy chain isoforms and characterized the expression pattern. He also demonstrated that intimal smooth muscle cells in atheroma have an immature phenotype determined by decreased expression of smooth muscle myosin heavy chain isoforms.

Recently, they found that in rabbit atheroma lipid lowering promotes a more mature phenotype of smooth muscle cells as gauged by increased expression of myosin isoform and decreased expression of MMP. After 4 months of hypercholesterolemia SMC in the fibrous cap (detected by alpha-actin antibody) did not show detectable levels of SM2 expression, a specific marker for mature SMC, whereas medial SMC expressed both alpha-actin and SM2. This suggests that SMC in the fibrous cap have an immature phenotype. However, lipid lowering by diet promoted a more mature SMC in the fibrous cap as determined by increased expression of SM2.

Pathologic significance of intimal SMC maturation

After lipid lowering therapy, more mature SMC expressing SM2 showed less expression of MMP-3 and MMP-9, compared to the baseline lesion. These results suggest that lipid lowering can stabilize the plaque by not only by reducing the number of macrophages but also by promoting a more mature SMC in the intima. One candidate mechanism for the maturation of SMC in the intima is decreased expression of PDGF-ß associated with a reduced number of macrophages, since PDGF-ß is known to suppress SMC differentiation.

Reduced prothrombotic potential

Recently this group showed that dietary lipid lowering reduces tissue factor expression and activity by macrophages and SMC in rabbit atheroma. Tissue factor is a potent contributor to acute coronary events. After 4 months of the high cholesterol diet, lesional macrophages expressed tissue factor in the intima. However, after lipid lowering therapy tissue factor was nearly undetectable, associated with a reduced number of macrophages. These results suggest that lipid lowering can stabilize the plaque by decreasing proteolytic and prothrombotic macrophages. However, the mechanism by which lipid lowering decreases macrophages remains unclear.

Atherosclerotic lesions produce excess reactive oxygen species (ROS) including. is known to induce oxidative modification of LDL. A number of in vitro experiments have suggested such oxidative stress can promote endothelial cell activation or dysfunction and induce expression of cell adhesion molecules such as VCAM-1 and chemokines including MCP-1, leading to increased monocyte recruitment.

To understand the mechanisms of reduced number of macrophages in the lesion they first measured the level of ROS production using lucigenin chemiluminescence assay. Aortic rings from hypercholesterolemic rabbits elaborated high levels of ROS production, compared to normal aorta. However, lipid lowering decreased the production of ROS to levels similar to those in normal aorta.

Endothelial cell activation

Further studies in the rabbit model suggest that lipid lowering can limit endothelial cell activation and dysfunction based on the decreased expression of VCAM-1 and MCP-1 and increased expression of eNOS.

Oxidized LDL is known to induce VCAM-1 expression by endothelial cells in vitro. After 4 months of hypercholesterolemia oxidized LDL accumulated in the intima underlying endothelial cells overexpressing VCAM-1. However, after lipid lowering therapy oxidized LDL accumulation and VCAM-1 expression were nearly undetectable.

MCP-1 is a potent monocyte chemoattractant that can induce monocyte recruitment into the intima. After 4 months of hypercholesterolemia MCP-1 was detected in endothelial cells as well as SMC and macrophages. However, after lipid lowering therapy nearly no MCP-1 could be detected in the rabbit lesion.

Several in vitro studies suggested that oxidative stress can decrease the expression of endothelial nitric oxide synthase (eNOS) by endothelial cells. Decreased eNOS should promote macrophage-rich atheroma formation, since eNOS can limit the monocyte-endothelial cell interaction (Fig. 2). In rabbit atheroma after 4 months of hypercholesterolemia few if any endothelial cells stained positively for eNOS antibody. However, after 16 months of lipid lowering therapy eNOS expression substantially increased.

These results in total suggest the potential mechanisms of plaque stabilization by lipid lowering therapy to be: decreased LDL should limit oxidative modification of LDL. Decreased oxidative stress should improve endothelial cell activation or dysfunction leading to decreased monocyte recruitment and decreased accumulation of proteolytic and prothrombotic macrophages in the lesion. These results should provide further experimental validation by which lipid lowering improves clinical outcomes.