1. B. Mechanisms of disease
Evolution of atherosclerotic plaque
This schematic represents the evolution of atherosclerotic plaque as described by Peter Libby, MD.4 In the atherosclerotic plaque, a cholesterol-rich lipid core forms within the intimal layer and is infiltrated with cells, such as macrophages, macrophage-derived foam cells (laden with lipid drops), and smooth muscle cells:
Plaque formation begins with accumulation of lipoprotein particles in the intima. These particles aggregate and are susceptible to oxidation and glycation.
Oxidative stress can induce local cytokines.
Cytokines promote expression of adhesion molecules, which enable leukocytes to adhere to the endothelium. Cytokines also induce chemoattractant molecules (such as monocyte chemoattractant protein [MCP-1]) that facilitate migration of leukocytes.
On entering the media, monocytes interact with stimuli (such as macrophage-colony stimulating factor [M-CSF]) that promote expression of scavenger receptors.
Scavenger receptors mediate uptake of modified lipid particles leading to formation of foam cells. The macrophage-derived foam cells are a storehouse of atherogenic mediators and effector molecules, such as cytokines, superoxide anion, and matrix metalloproteinases.
Smooth muscle cells migrate from the media to the intima and proliferate.
Smooth muscle cell accumulation leads to buildup of extracellular matrix in plaque and the fatty streak evolves to fatty fibrous lesions.
Fibrosis continues in later stages, sometimes along with smooth muscle cell death. This leads to a relatively cell-free thin fibrous capsule surrounding a lipid-rich core. If plaque ruptures, the lesion’s thrombogenic core is exposed to blood in the lumen of the artery. Platelet adhesion and activation initiates formation of a thrombus.

2. Key pleiotropic effects of statins
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The statins are extremely complex drugs and exhibit a wide variety of vascular effects that appear to be independent of their lipid-modifying properties.5
Among these pleiotropic effects are beneficial effects on endothelial function and blood flow, reduction in LDL-C oxidation, antithrombotic effects including improved fibrinolytic balance, and enhanced stability of atherosclerotic plaques. Statins have also been shown to inhibit vascular smooth muscle cell proliferation and platelet aggregation, and reduce vascular inflammation.
Although the list of these cellular effects continues to grow, it remains to be determined which effects, if any, account for the clinical benefits of statin therapy in cardiovascular disease.

3. Potential markers of vascular inflammation and their sources
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There are several potential sources of inflammatory markers as well as the cytokines that promote their production.6 Some of the most widely measured and discussed inflammatory markers are derived from the liver, including C-reactive protein, fibrinogen, and serum amyloid A. Their production is stimulated by systemic cytokines, such as interleukin-1β and interleukin-6, and tumor necrosis factor α.
Cytokines are produced at several sites outside the liver, such as the heart, vessel walls, macrophages, and adipose tissue.
Macrophages are a second source of inflammatory markers. One example is secretory phospholipase A2; levels are elevated in acute and inflammatory states and may predict events in patients with coronary disease. Macrophages also produce lipoprotein-associated phospholipase A2, which is an independent marker of cardiovascular risk.
The heart secretes troponin T and I and creatine kinase MB in response to injury.
The atherosclerotic vessel wall itself is a source of inflammatory markers. Examples include soluble adhesion molecules, such as intercellular adhesion molecule I (ICAM-1), vascular-cell adhesion moledule 1 (VCAM-1), E-selectin, and P-selectin. Cytokines upregulate and express these cell-surface molecules.

4. Statins blunt markers of coagulation, systemic inflammation, and soluble vascular cell adhesion
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To gain more insight into the non-lipid effects of statin therapy, Bickel et al examined the impact of statins on laboratory markers of coagulation, inflammation, and soluble cell adhesion.7 These effects were studied in 950 hospitalized patients with angiographically proven coronary artery disease.7 The group included 277 patients who were treated with statins and 677 patients who did not receive statin therapy.
The two groups had similar levels of total cholesterol, LDL-C, HDL-C, and triglycerides because lipids were lowered as a result of treatment for the patients taking statins.
The statin-treated group, however, had significantly lower levels of markers of inflammation, coagulation, and vascular cell adhesion, including von Willebrand factor (vWF), leukocyte count, high sensitive C-reactive protein, interleukin-6, and soluble P-selectin.
These results indicate that additional non-lipid lowing mechanisms might contribute to the beneficial effects of statin therapy.

5. Cholesterol ↓ rapidly improves vascular function post ACS
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Cholesterol lowering reduces coronary events. One mechanism could be improvement of endothelial function. This study shows that reduction in cholesterol by early use of statins can result in a rapid improvement of endothelial function in patients with an acute coronary syndrome.8
Patients with acute MI or unstable angina and total cholesterol levels at admission ≥5.2 mmol/L or LDL ≥3.4 mmol/L were randomized to placebo (n = 30) or pravastatin 40 mg daily (n = 30) for 6 weeks.
Total cholesterol and LDL-C levels were similar at admission and before randomization in both groups. With pravastatin, total cholesterol decreased by 23% and LDL-C was reduced by 33% at 6 weeks.
Statin therapy produced a substantial increase in flow-mediated vasodilation from about 5% to 7%, representing a 42% relative increase. Thus, initiation of statin therapy early after acute coronary syndromes rapidly lowers lipid levels and improves endothelial function after 6 weeks of therapy.

6. Beneficial effects of statins on endothelial function and BP
in normocholesterolemic SHR
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Wassmann and colleagues studied the potential beneficial effects of statins that are independent of cholesterol lowering and appear to involve direct effects on the vascular wall.9 The study was conducted in normocholesterolemic spontaneously hypertensive rats (SHR).
Before treatment, blood pressure was similar in both SHR groups and pathologically elevated. Treatment with atorvastatin led to a significant reduction in blood pressure.
The reduction in blood pressure was related to a statin-induced improvement in vasorelaxation and reduction in vasoconstriction. The effects of atorvastatin on vasodilation and vasoconstriction were assessed in isolated aortic rings.
Atorvastatin caused a marked increase in endothelial cell-dependent relaxation (top left). The response was determined using increasing concentrations of carbachol (a cholinergic agonist). The force of contraction of phenylephrine-induced vasoconstriction with carbachol 100 mmol/L was 14% with atorvastatin compared with 32% for the control (P < 0.05).
Atorvastatin caused a nearly 50% decrease in angiotensin-II (Ang-II) induced vasoconstriction (bottom left). The force of contraction for control of KCl-induced vasoconstriction was 4.4% with atorvastatin vs 8.2% with the control (P < 0.05).
Further investigations of the mechanisms involved are shown on the following slides.

7. Statins downregulate AT1 receptors and reduce ROS production
in normocholesterolemic SHR
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Statin therapy in SHR decreased angiotensin II (Ang II) induced vasoconstriction and vascular reactive oxygen species (ROS) production.9 Because both effects are mediated through angiotensin type-1 (AT1) receptor activation, it was reasonable to assume that the receptor was influenced by atorvastatin.
AT1 receptor expression was downregulated in SHR treated with atorvastatin to 44% of the control level (P < 0.05). This was measured by analysis of vascular AT1 receptor mRNA.
The decrease in vascular responsiveness to Ang II in the statin-treated group could also lead to a lower level of free radicals in the vessel wall, which could have improved vascular function.
Atorvastatin decreased superoxide production in the vessel wall significantly to 62% of the control level (P < 0.05). This was determined by lucigenin chemiluminescence assays in isolated aortic segments.
Atorvastatin reduced NAD(P)H oxidase expression to 63% of the control level (P < 0.05). The effect of atorvastatin on vascular expression of NAD(P)H oxidase was assessed by quantifying p22phox, the essential subunit of the enzyme, in amplified DNA fragments from the vessel wall.
Clinical implications: Because the AT1 receptor is implicated in atherosclerosis, downregulation of AT1 receptor gene expression and reduction of oxidative stress may contribute to clinical benefits of statin therapy that extend beyond cholesterol lowering. The decreased expression of p22phox associated with atorvastatin therapy may be related to a reduction in oxidative stress.

8. Statins upregulate ecNOS expression and increase ecNOS activity in normocholesterolemic SHR
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Wassmann and colleagues also measured expression of endothelial cell nitric oxide synthase (ecNOS) in the aortic tissue of the atorvastatin and placebo-treated SHR.9 Expression of ecNOS was upregulated in the statin-treated SHR to 138% of control levels (P < 0.05 vs controls).
Further analysis showed that ecNOS activity was increased 2-fold in the atorvastatin-treated group (P < 0.05 vs control). The increase in ecNOS activity with atorvastatin may result in greater production of nitric oxide. The enhanced bioavailability of nitric oxide also contributes to improvement of endothelial function.

9. Statins decrease LOX-1 expression
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Accumulating evidence shows that oxidized LDL (ox-LDL) is a key component in atherogenesis.10 Several studies have shown that the receptor for ox-LDL, LOX-1, mediates the uptake of ox-LDL by endothelial cells. Expression of the LOX-1 gene is upregulated by ox-LDL, Ang II, inflammatory cytokines, and shear stress. LOX-1 is upregulated in atherosclerotic tissues.
The present study shows that two different widely used statins inhibit the expression of LOX-1, and uptake of ox-LDL, in human cultured coronary artery endothelial cells (HCAECs).
To examine the effects of statins on LOX-1 expression, HCAECs were pretreated with simvastatin or atorvastatin (1 and 10 µM) before exposure to ox-LDL. Both statins markedly decreased ox-LDL-induced upregulation of LOX-1 protein in a dose-dependent manner. In a parallel study, both statins had similar effects on LOX-1 mRNA.
Clinical implications: Statins may directly protect vascular endothelium against the adverse effect of ox-LDL. These findings further support the clinically beneficial effect of statins in patients with cardiovascular disease who do not have high cholesterol levels.

10. Oxidized LDL levels are positively related to severity of acute coronary syndromes
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There is increasing evidence that acute coronary syndromes are related to recent activation of the immunomediated inflammatory process associated with atherosclerotic plaques. Thus, increased ox-LDL levels could play a role in acute coronary syndromes.
Ehara and colleagues measured ox-LDL levels in patients with acute myocardial infarction (n = 35), unstable angina pectoris (n = 45), or stable angina pectoris (n = 45) and in 46 control subjects.11
Levels of ox-LDL were markedly higher in acute MI patients than in patients with either unstable angina (P < ) or stable angina (P < ), or in controls (P < ).
As shown in the study, ox-LDL levels are directly correlated with the severity of acute coronary syndromes. More severe lesions also contained a much higher percentage of ox-LDL-positive macrophages. These observations suggest that increased levels of ox-LDL relate to plaque stability in human coronary atherosclerotic lesions.

11. Potential interaction of LOX-1 and Ang II in endothelial cells
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As we have seen, LDL-mediated AT1 receptor upregulation leads to production of ox-LDL. Ox-LDL, in turn, upregulates its own receptor (LOX-1), which leads to activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kB (NF-kB).12 NF-kB is an important transcription factor in the expression of genes for many cytokines, enzymes, and adhesion molecules.13
Ox-LDL also increases expression of monocyte chemoattractant protein-1 (MCP-1), which plays a crucial role in monocyte adhesion to endothelial cells.14
It has been hypothesized that the adverse effects of ox-LDL on the vascular wall may be mediated by LOX-1.12,14

12. Hypertension and hypercholesterolemia: Interactions and potential mechanisms
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Hypertension and hypercholesterolemia, the major risk factors for atherosclerotic disease, frequently are present in the same patient. Interactions between dyslipidemia and activation of the neurohormonal systems, such as the renin-angiotensin system (RAS) may not only explain their frequent coexistence, but may also be important in the pathogenesis of atherosclerosis.15
Data from clinical studies suggest that hyperlipidemia enhances RAS activity. For example, blood pressure response to mental stress is much greater in hypertensive subjects than in those with normal blood pressure.
Lipoprotein-neurohormonal interactions may adversely affect vascular structure and reactivity. Experimental studies have shown that vascular RAS may be involved via LDL-mediated AT1-receptor upregulation, which leads to production of ox-LDL. Adverse effects of ox-LDL on the vascular wall may be mediated by the LOX-1 receptor.
These findings extend our understanding of the interplay among risk factors to synergistically increase cardiovascular risk.