Data from the 4S, CARE and LIPID trials have shown that lipid lowering therapy with statins in patients with known and established CAD is associated with a significant reduction in overall mortality (8%-30%) and coronary heart disease (CHD) related deaths (20%-42%), and the major ischemic events of angina, MI, stroke, and death (24%-34%). Additionally, there is reduced need for invasive revascularization, such as angioplasty or bypass surgery (24%-37%).

Prevention, regression and stabilization are three potential mechanisms by which statins may produce their benefit. Evidence from angiographic studies support the notion that statins may prevent the formation of new atherosclerotic plaques. Statins may induce regression of pre-established atherosclerotic disease, and there is some evidence of minor degrees of regression. By changing the composition of the plaque, statins render it more stable, making it more quiescent and less prone to rupture and produce thrombosis. Plaque stabilization is gaining the most notoriety in terms of the potential mechanism of action by which statins produce their clinical benefit.

A new paradigm

Trivial reductions in the magnitude of stenosis reduction has been seen in serial coronary angiographic studies using quantitative techniques in patients given either placebo or statins. This reduction ranges from 0.69% to 1.5% +/-4.0 in five studies. In sharp contrast, there is a greater and disproportionate reduction in clinical events in these studies, ranging from 39% to 89%. This disparity has given rise to the new paradigm that statins may be working by mechanisms other than a direct influence on the severity of stenosis, perhaps through plaque stabilization, making them less prone to result in ACS. By decreasing the frequency and severity of the complications of atherosclerosis, i.e., plaque rupture, thrombosis, and vasoconstriction, a decrease in lethal clinical events would be expected.

The presence of a large, amorphous lipid rich core, the thinning of the fibrous cap, and the presence of inflammation due to cellular infiltration, usually under sites of a thinning fibrous cap determine the vulnerability of a plaque to rupture, cause thrombosis, and lead to clinical events. The cellular infiltration tends to be foam cells and inflammatory cells, primarily macrophages derived from mononuclear cells that are recruited into the plaque in a dynamic fashion. Features that confer plaque instability and promote clinical events include:

• Increased lipid content
• Reduced collagen content in a thinned fibrous cap
• Increased inflammatory cell infiltration
• Increased expression of matrix-degrading metalloproteinases (MMP)
• Reduced expression of tissue inhibitor of MMP (TIMP)
• Reduced smooth muscle cell (SMC) content and function

Promotion of plaque stability to reduce clinical events requires:

• Reduced lipid content
• Increased collagen content in fibrous cap
• Reduced inflammation
• Reduced MMP
• Reduced TIMP
• Increased SMC content and function to synthesize collagen and other matrix components that form the protective fibrous cap

The first evidence that diet-induced lipid lowering could alter the composition of atherosclerotic plaques without changing its size or stenosis severity came from Armstrong and colleagues in the 1970s who reduced the lipid content of lipid-rich atherosclerotic plaques when switching monkeys from a high cholesterol to a low cholesterol diet.

More recently Libby and colleagues have shown in hypercholesterolemic rabbits fed high cholesterol diets that there is marked infiltration of macrophages accompanied by expression of MMP co-localizing with the macrophages. When the rabbits are placed on a low cholesterol diet for 8 or 16 months there is a rapid disappearance of the macrophages accompanied by rapid disappearance of MMP immunoreactivity in atherosclerotic plaques. Libby's laboratory has also shown that using a statin, such as cerivistatin, can also reduce the tissue factor content of the plaques.

Plaque stabilization in humans

Results from a study in humans by Shah in collaboration with a Swedish group support the concept that lipid lowering therapy with pravastatin changes human carotid plaque composition favoring plaque stability. This supports the concept of plaque stabilization as a potential mechanism for the beneficial effects of pravastatin and possibly other statins.

To determine whether treatment with pravastatin would result in a change in the composition of carotid atherosclerotic plaque they studied 24 consecutive patients with greater than 70% angiographically-confirmed internal carotid stenosis scheduled for carotid endarterectomy 3 months following diagnosis of carotid disease. During the 3-month period the patients were allocated to either pravastatin (40 mg daily) or no lipid lowering medication. At the time of endarterectomy carotid plaques were removed and subjected to detailed analysis.

The plaques were analyzed for 1) lipid content, 2) degree of oxidized LDL, 3) incidence of SMC apoptosis, 4) macrophage, T-cell, SMC immunoreactivity, 5) collagen content, 6) MMP and TIMP immunoreactivity, and 7) Nf-kB immunoreactivity as measure of oxidant stress.

At baseline, the total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides were comparable in both groups. After three months of pravastatin, there was a statistically significant reduction in total cholesterol, LDL and triglycerides (P<0.05, respectively), and a slight increase in HDL ((P<0.05). In the carotid plaques, pravastatin significantly reduced the lipid content from about 25% of the plaque area to about 9% (P<0.05) and reduced oxidized LDL content from about 22% to about12-13% (P<0.001).

Associated with the lipid depletion there was evidence of an anti-inflammatory effect with pravastatin as measured by T-cell reduction (24% to 10%; P<0.05) and macrophage reduction (25% to 15%; P<0.05). Further, pravastatin reduced cell death, primarily SMC, from about 32% to 18% (P<0.05). A trend towards a reduction in the pro-inflammatory adhesion cell molecules VCAM-1 and ICAM-1 and in Nf-kB, was seen in the pravastatin-treated group, although this did not reach statistical significance.

Evidence of matrix-degrading MMP-2 immunoreactivity was seen in the pravastatin-treated group (about 8% to 3%; P=0.03), which was accompanied by a significant increase in TIMP-1 immunoreactivity (3% to 8.5%; P=0.02). Collagen content also increased with pravastatin from about 7% in the control group to 12% (P=0.02).

Study with recombinant HDL

Shah's laboratory is investigating the use of recombinant HDL to alter plaque composition. In Apo E knock-out mice fed a high cholesterol diet treated with either buffer, liposomes, or a recombinant HDL containing a mutant of human Apo A-1 (Apo A-1_Milano), there is a marked reduction in the lipid content of the plaques 48 hours after a single dose (400 mg/kg) of human recombinant Apo A-1_Milano. This suggests the possibility that change in the lipid content of atherosclerotic lesions could be accomplished as rapidly as 48 hours after injection of recombinant HDL and raises the very tantalizing possibility that rapid stabilization of atherosclerotic lesions could be achieved with therapeutic administration of HDL or Apo A-1_Milano.

Macrophage immunoreactivity is also significantly reduced in the mice receiving recombinant HDL containing Apo A-1_Milano. These observations, coupled with the clinical observations, strongly support the concept that it is possible to achieve changes in plaque composition that can favor stability of atherosclerosis, namely depletion of lipids, oxidized lipids, and inflammatory cells, reduction in matrix degrading activity, and increased collagen content.

Q: Is the anti-inflammatory effect of statins due to lipid lowering or is it an independent effect?

A: Presently, the overwhelming evidence suggests that lipid lowering is the dominant, if not only, mechanism, because most of the changes in plaque composition occur around the time that lipid lowering has already occurred. This question will be difficult to answer definitively in humans as another technique would be needed that could monitor plaque composition at multiple time points and its relation to anti-inflammatory effects. In the animal model there is some data to suggest that anti-inflammatory effects are dissociated from lipid-lowering effects.

Q: Do the findings from the carotid arteries apply to coronary arteries?

A: It is likely that what occurs in one area of the circulation applies to other areas of the circulation. However, it is difficult to be absolutely certain. Coronary plaques are more difficult to assess in this manner, so we are limited to using extra-coronary sites to gain some insights. The findings are consistent with other clinical observations.

Q: How low should LDL be lowered?

A: This is still an area of controversy. There are some clinical trials that suggest that lowering LDL below 120-125, once that level is achieved, does not produce further benefit. Other studies suggest there is a continuous relationship between further lowering of LDL and greater benefit. For example, in the Post-CABG clinical trial, the greatest benefit in preserving saphenous vein graft patency was achieved when the LDL was lowered below 90 mg/dl. So, it is still not clear how low LDL should be reduced, as it is only one of the risk factors. There are patients who develop MI and stroke who have perfectly normal LDL levels. Thus, there may be a threshold below which little additional benefit is seen. Whether that level is 125 or 100 is still subject to question.

Q: Is there any clinical data for the Apo A-1_Milano in ischemic heart disease?

A: No. The natural carriers of this mutation are resistant to atherosclerosis, despite having low HDL and high triglycerides, which is a very promising insight. In 2001, our group will begin human studies with recombinant HDL containing Apo A-1_Milano. It will be a year or so before we have clinical results to know how and whether this will be beneficial in humans.