Please welcome Dr. Felicita Andreotti and her colleagues from Europe to Cyberounds^®. We hope to have additional contributions from our international colleagues in the future. In this Cyberounds^®, Dr. Andreotti addresses the important emerging association between endothelial inflammation and the atherosclerotic process. It now appears that the initial endothelial disruption that leads to atherosclerotic deposits and plaque formation is secondary to activation of adhesion molecules, white blood cell attachment and transmigration. Additionally, plaque rupture and secondary vessel thrombosis, the etiology of most myocardial infarctions and many strokes, is most likely mediated by inflammatory processes. I'm sure you will enjoy learning about this exciting new development in vascular medicine.

-- Richard W. Smalling, M.D., Ph.D., Cardiovascular Editor

Atherosclerosis -- a focal obstructive lesion of the intima of large and medium-sized arteries -- tends to cause a progressive reduction of the arterial lumen and the gradual occurrence of symptoms. Often, however, even mildly obstructive lesions(1) cause sudden clinical manifestations in the form of myocardial infarction, stroke or death. These abrupt presentations are thought to arise from rupture of the lesion's luminal surface, with leakage of plaque material into the blood, precipitating occlusive thrombosis.

Pathogenesis

Atherosclerosis can be regarded as a vascular response to chronic injury.(2) In general, the response to injury evolves in consecutive phases: an initial phase of acute inflammation, followed by a phase of demolition by scavenger cells and a final phase of repair. If the initial injury is not removed these three phases (inflammation, demolition and attempts of repair) tend to overlap and the inflammatory response evolves into chronicity.(3)

The nature of the initial injury in atherogenesis is debated and may vary from subject to subject. Whatever its origin, it is thought to involve focal areas of endothelium, leading to endothelial activation.(4) This attracts monocyte-derived macrophages (scavenger cells) and lymphocytes into the subendothelial space.(4) Subsequently, smooth muscle cells migrate from the arterial media and proliferate in the intima, with concomitant production of connective tissue (repair).

Histology

The early lesions of atherosclerosis (fatty streaks) are found already in children. Some of these will evolve over the years into advanced fibro-lipid plaques.(2) Viewed from the lumen, the plaques appear undulated and raised. A single layer of endothelial cells covers multiple layers of smooth muscle cells embedded in dense connective tissue (the fibrous cap). Beneath, further smooth muscle cells, foam cells and lymphocytes surround necrotic debris and cholesterol (the lipid core).(4) Foam cells are macrophages and smooth muscle cells filled with densely packed lipid vesicles.

Figure 1. Macroscopic View of a Severe, Eccentric, Coronary Atherosclerotic Plaque.

Note the thick fibrous cap and the large crescent-shaped core.

By kind permission of Professor Eloisa Arbustini (University of Pavia, Italy).

Both the early and advanced lesions contain macrophages and T-lymphocytes, typical cells of chronic inflammation. Fatty streaks mainly consist of lipid-laden macrophages, with some T-lymphocytes and rare smooth muscle cells.(4) The advanced fibro-lipid plaques contain roughly equal numbers of smooth muscle cells, macrophages and T-lymphocytes in abundant connective tissue.(2)

Role of Macrophages and T-cells

Monocyte-derived macrophages act as scavengers to remove 'foreign' substances by phagocytosis and intracellular hydrolysis. In the subendothelial space, they produce a number of cytokines and growth factors, which contribute to the recruitment of further macrophages and to the proliferation of smooth muscle cells.(5) Macrophages and smooth muscle cells are responsible for producing connective tissue.(4) Activated macrophages also produce collagen-degrading enzymes, which may cause rupture of the fibrous cap.(6)

T-lymphocytes of the CD8 and CD4 subtypes are found in all phases of atherosclerosis. Their presence suggests an immune or autoimmune cell-mediated response and their contact with macrophages suggests a possible role of macrophages as antigen presenting cells.(2)

Molecular Mechanisms

The molecular steps involved in atherogenesis are not completely known. Here, a possible sequence of events is proposed.

Atherogenic risk factors, such as oxidized low density lipoprotein (LDL),(7) tobacco glycoprotein,(8) glycated proteins of diabetes, chlamydial or viral infections,(9) homocysteine,(10) and several cytokines(5) may induce vulnerable areas of endothelium (located at sites of augmented exposure or pressure, such as proximal segments or turbulent eddies at bends and branches) to produce inflammatory proteins, including macrophage colony stimulating factor (MCSF).

Figure 2. Cartoon of the Cellular and Molecular Mechanisms Thought to Play a Role in Atherogenesis.

A variety of toxic stimuli induce vulnerable areas of endothelium to secrete proinflammatory cytokines and growth factors (endothelial activation). Secretion of macrophage colony stimulating factor (MCSF), for example, promotes the release of interleukin(IL)-1 and monocyte chemotactic protein-1 (MCP-1) and the expression of intercellular adhesion molecule-1 (ICAM-1). These factors draw into the subendothelial space monocytes and lymphocytes from the blood and smooth muscle cells from the media. Monocyte-derived macrophages and smooth muscle cells proliferate, release futher cytokines and growth factors, ingest oxidized LDL (thereby becoming foam cells) and produce connective tissue. Degradation of plaque components by macrophage-derived enzymes generates necrotic debris and may cause plaque fissure (not shown).

MCSF induces the synthesis by endothelial cells of monocyte chemotactic protein-1, which attracts monocytes from the peripheral blood.(11) MCSF, additionally, stimulates the production of interleukin (IL)-1b and more MCSF by local endothelium and by newly recruited monocytes.(12) These two cytokines (MCSF and IL-1b) up-regulate the expression of leukocyte adhesion molecules on the endothelial surface (including the intercellular adhesion molecule-1 or ICAM-1) as well as specific integrins on monocytes, which enhance the adhesion of monocytes to the endothelium.(13) They also induce the activation and proliferation of monocyte-derived macrophages already in the subendothelium(14) and up-regulate the macrophage surface receptor for oxidized-LDL, with subsequent cholesterol uptake, foam-cell formation(15),(16) and plaque growth. These processes determine a further release of cytokines from vascular cells, including IL-6, which promotes smooth muscle cell proliferation.(17),(18),(19) Activated smooth muscle cells synthesize more IL-6, which may enter the systemic circulation and stimulate the production of C-reactive protein and fibrinogen by hepatocytes.(20)

Experimental findings in vivo and ex vivo support this chain of events: mice genetically deficient in MCSF show decreased progression of atherosclerosis,(21) while MCSF, IL-1b and IL-6 proteins and messenger RNA have been isolated from human atherosclerotic specimens.(22)

Clinical Evidence

Cross-sectional case-control studies The molecular pathways suggested in vitro may explain, at least in part, the increased circulating levels of MCSF, IL-1b, IL-6 and CRP found in patients with coronary atherosclerosis compared with healthy controls.(23),(24) In a recent report,(24) the plasma concentrations of both MCSF and IL-1b were associated with the extent of coronary artery disease at angiography. MCSF levels of patients with triple vessel disease were several fold higher than those of patients with single or two vessel disease; similarly, IL-1b levels were significantly higher in three-vessel disease than in two or single-vessel disease-patients or controls. The relation between anatomic extent of disease and MCSF and IL-1b suggests an important role of growth factors and inflammatory mediators in the progression of human atherosclerosis.

Epidemiology

Large cohort studies in patients with known ischemic heart disease have found blood levels of C-reactive protein, leucocyte count and fibrinogen to predict recurrent events,(25),(26) suggesting that inflammatory processes interacting with -- and/or, perhaps, originating from -- the atherosclerotic vessels may be related to the occlusive thrombotic complications of atherosclerosis. Inflammatory cytokines can stimulate macrophages to secrete collagen-degrading enzymes (e.g., metalloproteinases) which may initiate plaque fissure.(6) C-reactive protein can induce the expression of tissue factor (the trigger of the extrinsic coagulation pathway) on the surface of blood monocytes.(27) Fibrinogen is the precursor of fibrin, the constituent of blood clots.

Nested case-control studies from the Physicians' Health Study (a large population of initially healthy middle-aged male physicians followed for approximately 10 years) found that peripheral blood levels of soluble ICAM-1 and of C-reactive protein, measured at entry, were significantly higher in individuals who subsequently developed myocardial infarction and stroke, compared with controls.(28),(29) These inflammatory markers may, thus, indicate pre-clinical atherosclerosis.

Inflammatory Stimuli

Modified LDL

Minimally oxidized LDL has been proposed as a major toxic agent for the endothelium. LDL from the blood stream undergoes a low level of oxidation when bound to the endothelial receptors for LDL.2 Such modified LDL are internalized and transported through the endothelium and can then bind to the scavenger receptor on the surface of macrophages and to the LDL-receptor of smooth muscle cells in the subendothelial space. 'Ingestion' of LDL by these cells contributes to foam cell formation, the hallmark of early and advanced lesions.(2) Modified LDL can up-regulate the expression of MCSF and of monocyte chemotactic protein-1 by endothelial cells.(4) Lowering LDL concentrations in blood is associated with regression of atherosclerotic plaques in animals and humans and a reduced incidence of myocardial infarction and death in clinical trials.(30)

Homocysteine

This metabolite of methionine has been shown, in vitro or in vivo, to favor lipid peroxidation, foam cell formation, intimal vascular smooth muscle cell proliferation, platelet reactivity and to impair endothelial function.(31) Homocystinuric patients (who have severe hyper-homocysteinemia) die prematurely of atherothrombotic disease. Cross-sectional and prospective studies (with some exceptions) indicate that plasma total homocysteine levels >10 mmol/l are associated with - or predict the development of - coronary, cerebral and peripheral atherosclerotic disease.(32) The risk associated with hyper-homocysteinemia is graded and independent of traditional risk factors, with an estimated odds ratio for ischemic heart disease of 1.4 for every 5 mmol/l increase. Trials to investigate the possible benefits of reducing homocysteine levels with folic acid are currently being carried out.

Infectious Agents

Two Gram-negative germs (Chlamydia pneumoniae and Helicobacter pylori) and a herpes virus (cytomegalo virus) have shown a significant association with acute or stable coronary artery disease.(33) The overall odds ratio for disease (based on antibody titers) ranges from little >1 for H. pylori to » 4 for C. pneumoniae. For H. pylori, only the more virulent strains may confer increased risk.(34)

C. pneumoniae and cytomegalovirus have been isolated from atheromatous specimens and cytomegalovirus infection is associated with cardiac transplant vasculopathy.(33) The mechanism of increased risk may involve cross-reactivity, systemic or local inflammation and enhanced coagulability.(9) Because these infections are frequent in the general population (~50% in middle-aged individuals), if indeed they contribute to atherogenesis, it is likely that concomitant genetic or environmental factors are necessary for disease-onset. Two smaller trials, but not a larger one, in patients with prior acute or stable coronary syndromes have shown clinical benefit after macrolide therapy.(35) This evidence is insufficient to advocate the widespread use of antimicrobial drugs in patients with coronary artery disease.

Hypertension, Smoking and Diabetes

Raised intravascular pressure in hypertension, tobacco-related toxins and abnormally glycated proteins in diabetes may alter endothelial cell function and increase oxidative stress. Additionally, increased angiotensin II in hypertension and hyperinsulinemia in insulin-resistant states may promote vascular smooth muscle cell proliferation. Correction of these conditions can significantly reduce the risk of cardiovascular events.(36)

Conclusion

The lesions of atherosclerosis show many typical aspects of a chronic inflammatory disease. The precise stimulus triggering the early stages of this focal arterial inflammation is still elusive and may vary among individuals. Subsequent phases of atherogenesis probably involve multiple processes influenced by genetic and environmental factors. At the molecular level, crucial steps appear to involve inflammatory cytokines and growth factors, appropriate inhibition of which may ultimately lead to effective prevention.