Markers of inflammation

Several inflammatory factors involved in the cascade described earlier have been studied in clinical trials in the last decade. These include the proinflammatory cytokines interleukin (IL)-1, IL-6, tumor necrosis factor a), catalysts of the inflammatory reaction (CD40 ligand), cellular adhesion molecules (intercellular cell adhesion molecule [ICAM]-1, vascular cell adhesion molecule [VCAM]-1, selectins), and acute phase reactants (fibrinogen, white blood count, C-reactive protein [CRP], serum amyloid A).

C-reactive protein

CRP is the best studied of the inflammatory markers in cardiovascular disease. It is an acute-phase protein that has been shown to be a marker of systemic inflammation, elevated in response to acute injury, infection, and other inflammatory stimuli [9]. Hepatic production is directly related to IL-6 stimulation and, unlike other acute phase reactants, its levels remain stable over long periods of time in the absence of new stimuli [10]. Traditional CRP assays with limits of quantification of 3 to 8 mg/L lack adequate sensitivity to detect levels required for atherosclerotic risk prediction. The development of a standardized high-sensitivity CRP (hsCRP) assay has improved precision at low concentrations of CRP that permits its use in cardiovascular risk assessment.

In several studies of patients who have ACS (Chimeric c7E3 Antiplatelet Therapy in Unstable angina REfactory to standard treatment, Thrombolysis In Myocardial Infarction [TIMI]-11a, and FRagmin during Instability in Coronary artery disease [FRISC] trials), elevated hsCRP at hospital admission independently predicted increased mortality [11-13]. In a Global Use of Strategies to open Occluded arteries IV substudy, hsCRP elevation during the acute stage of unstable CAD was associated with an increased 30-day mortality independent of troponin levels; there was no association with recurrent MI [14]. However, in stable post-MI patients, elevated hsCRP predicted a significantly higher risk for recurrent nonfatal MI or fatal coronary events (75% higher in the highest versus lowest quintile of hsCRP), suggesting that it is not merely a marker for the extent of myocardial damage [15].

In the European Concerted Action on Thrombosis and Disabilities Angina Pectoris Study, 2121 patients admitted with angina were followed over a 2-year period. A plasma concentration of CRP greater than 3.6 mg/L at study entry was associated with a 45% increase in the relative risk for nonfatal MI or sudden cardiac death (95% confidence intervale [CI], 1.15-1.83) [16]. However, the usefulness of measuring hsCRP in patients who have ACS is limited because there is already sufficient evidence warranting maximal lipid-lowering, antiplatelet, and other cardioprotective drug therapies in these patients, so that measurement of hsCRP does not provide incremental information.

The setting in which hsCRP may be most useful is primary prevention. A population-based cross-sectional study in Great Britain showed that the prevalence of CAD increased 1.5 fold (95% CI, 1.25-1.92) for each doubling of hsCRP among men aged 50 to 69 years [17]. In the Multiple Risk Factor Intervention Trial cohort of middle-aged men who had traditional high cardiovascular risk factors, CRP was elevated more in smokers versus nonsmokers. Over 17 years of follow-up, an elevated baseline CRP was associated with a 2.8-fold increased risk for coronary heart disease mortality (95% CI, 1.4-5.4) [18].

Several prospective studies of hsCRP in apparently healthy individuals have also shown that elevated baseline levels of hsCRP are correlated with higher risk for future cardiovascular morbidity and mortality after adjustment for potential confounders. The Physicians Health Study, a prospective, nested case-control study of men who did not have prior history of CAD and had low rates of cigarette use, showed that men in the highest quartile of hsCRP (R2.1 mg/L) had a significant 2.9-fold increase in risk for MI that was independent of smoking status, lipid levels, and other traditional risk factors for CAD [19]. This finding was confirmed in the Monitoring Trends and Determinants of Cardiovascular Disease (MONICA)-Augsburg prospective study in Europe that followed 936 healthy, middle-aged men over 8 years and noted a 19% increase in risk for future coronary event for each standard deviation increase in baseline hsCRP after adjustment for multiple risk factors [20].

This relationship also bears out in women. In a prospective nested case-control study involving postmenopausal women enrolled in the Women's Health Study, Ridker and colleagues [21] showed hsCRP to be the most powerful predictor of cardiovascular risk compared with other inflammatory markers, baseline lipid levels, and homocysteine. Women in the highest quartile had a relative risk of 4.4 (95% CI, 2.2-8.9, P < .001) compared with those in the lowest quartile. Addition of hsCRP to cholesterol measurement increased the area under the receiver operating characteristic (ROC) curve from 0.59 to 0.66 (P < .001). Furthermore, in women who had LDL levels less than 130 mg/dL (the target level recommended for primary prevention by the National Cholesterol Education Program), those who had elevated baseline CRP were still at increased risk for future events with a 3.1 relative risk in the highest quartile compared with the lowest (95% CI, 1.7-11.3, P = .002) after adjustment for traditional risk factors and high-density lipoprotein (HDL) cholesterol levels.

Prospective data show that hsCRP levels minimally correlate with lipid levels and are a stronger predictor of risk than LDL cholesterol (area under the ROC curve 0.64 versus 0.60) [22]. Therefore, the role of CRP is adjunctive to lipid screening and the additive predictive value of hsCRP to the Framingham 10-year risk score, LDL, total, and HDL cholesterol measurements has been demonstrated in several studies [23,24]. However, there is not yet a consensus that the degree of hsCRP elevation correlates with atherosclerotic burden.

Tataru and colleagues [25] showed that in survivors of MI, there was a significant association of hsCRP level with angiographically detected degree of coronary artery stenosis. In addition, patients who had coronary disease who had sonographi-cally detectable peripheral arterial disease had even higher levels of hsCRP compared with those who had CAD alone. In a small Denmark study of 269 patients referred for elective coronary angi-ography, hsCRP levels were significantly higher in patients who had coronary stenoses than those who did not, but no difference in hsCRP levels were found comparing groups with single, 2-, or 3-vessel disease [26]. In a nested case-control sub-study of the Prospective Army Coronary Calcium trial that looked at healthy men between the age of 40 and 45 years who did not have coronary disease, the prevalence of coronary artery calcium as assessed by electron-beam CT was similar across all hsCRP quartiles [27]. Therefore, although hsCRP has good risk assessment value, there is no definitive evidence supporting its role in selecting patients for coronary angiography.

The role of hsCRP in predicting CAD has been established. What remains uncertain at this time is whether CRP should be a target of therapy or used to guide therapy. In vitro and in vivo studies show that hsCRP contributes to plaque development by increasing monocyte adherence, inducing expression of cell surface adhesion molecules [28], and increasing LDL scavenger cell uptake of cholesterol [29]. Furthermore, high CRP is associated with decreased nitric oxide availability impacting vasomotor activity [30] and increased monocyte production of tissue factor [31]. CRP is also shown to activate complement and neutrophils [32] and decrease fibrinolytic capacity [33], thereby contributing to plaque instability and thrombus formation.

In the Physicians Health Study, use of aspirin was associated with a statistically significant, 55.7% risk reduction for first MI among men who had hsCRP levels in the highest quartile compared with a nonsignificant 13.9% reduction in those who had hsCRP levels in the lowest quartile [19]. No follow-up levels were drawn to deduce whether aspirin directly lowered hsCRP levels.

The anti-inflammatory effects of statins are still unclear; experimental evidence posits statin-mediated reduction in macrophage activation, antiproliferative effects on smooth muscle cells, improvements in endothelial function and vasomo-tion, and antithrombotic effects [34,35]. At 5-year follow-up of stable post-MI patients randomized in the Cholesterol and Recurrent Events trial, mean hsCRP levels decreased by 37.8% in those randomized to pravastatin therapy, whereas levels increased in the placebo patients [36]. This reduction in hsCRP was independent of the magnitude of changes in lipid levels. Furthermore, patients randomized to pravastatin therapy had a twofold reduction in risk for recurrent MI or fatal coronary event in the elevated hsCRP group compared with the group that did not have elevated hsCRP, even though baseline lipid levels were identical in both groups [15].

More recently, PRavastatin Or atorVastatin Evaluation and Infection Therapy (PROVE-IT) trial demonstrated that intensive statin therapy that lowered hsCRP levels to a mean of less than 2 mg/L resulted in a decreased risk for recurrent MI

or death from coronary causes, irrespective of the degree of LDL lowering [37]. In the PRavastatin Inflammation CRP Evaluation (PRINCE) study, pravastatin was shown to also reduce hsCRP levels in patients who did not have prior history of cardiovascular disease [38]. There is not yet evidence linking hsCRP reduction to a reduction in cardiovascular events in the primary prevention setting.

The Centers for Disease Control and Prevention/American Heart Association consensus statement advocates use of hsCRP for risk assessment in patients who are at intermediate risk for cardiovascular events (10% to 20% 10-year risk of coronary event) [39]. However, there is no prospective randomized clinical trial yet examining the benefits or harm of screening with hsCRP. Presently the guidelines rate a hsCRP level less than 1 mg/L as normal, 1 to 3 mg/L intermediate, and more than 3 mg/L as high. A level greater than 10 mg/L indicates a noncardiovascular source of inflammation and should prompt a search for a source of infection or other inflammation. For patients who have ACS, hsCRP may provide some prognostic information, and a cutoff level of more than 10 mg/L is most predictive of risk for adverse outcomes.

Interleukin-6

IL-6 is an inflammatory cytokine that induces hepatic synthesis of all the acute phase proteins and is the primary determinant of CRP levels. The presence of IL-6-expressing macrophages in coronary atherosclerotic plaques [40] suggests that increased IL-6 levels are not just a marker of inflammation but also play a direct role in increasing plaque vulnerability to fissuring and thrombosis. In addition to its proinflammatory effects, it also has procoagulant effects by modulating fi-brinogen synthesis, enhancing platelet adhesion, and impairing endothelial vasodilation. High IL-6 levels in healthy men correlated with increased risk for future MI independently of hsCRP [41]. In patients who had ACS, IL-6 elevation (>5 ng/mL) was associated with 3.5-fold higher 1-year mortality than levels less than 5 ng/mL, independent of troponin and hsCRP [42]. Furthermore, in this study it appeared that patients who had inflammation as determined by IL-6 levels greater than 5 ng/mL had a greater response to an invasive versus conservative strategy than patients who had IL-6 levels less than 5 ng/mL.

Lipoprotein-associatedphospholipase A2

Lipoprotein-associated phospholipase A2 (Lp-PLA2), previously described as platelet-activating factor acetylhydrolase, is a novel marker whose role in atherosclerosis has been heavily debated. Initially considered atheroprotective because of its ability to degrade platelet-activating factor, this enzyme has since been discovered to cleave oxidized phosphatidylcholine into lysophosphati-dylcholine and oxidized free fatty acids which promote the inflammatory process of atherosclerosis. Lp-PLA2 is produced by macrophages and T lymphocytes and can be detected in human atherosclerotic lesions [43]. In humans, Lp-PLA2 is predominantly bound to LDL cholesterol particles and is activated once LDL particles undergo oxidative damage [44].

The West of Scotland Coronary Prevention Study first demonstrated that baseline Lp-PLA2 elevation in hyperlipidemic men predicted risk for coronary events independently of other inflammatory markers, such as CRP and fibrinogen. There was a 60% statistically significant increase in risk between the highest and lowest quintile of Lp-PLA2 [45]. However, in a lower risk population of women (in a nested case-control analysis of the Women's Health Study), the predictive ability of Lp-PLA2 was not statistically significant after adjustment for traditional risk factors [46].

The Atherosclerosis Risk in Communities study enrolled men and women who had a wide range of LDL levels and found that in patients who had LDL levels below 130 mg/dL, Lp-PLA2 was significantly and independently associated with CAD [47]. This finding suggests a role for Lp-PLA2, similar to CRP, in identifying high-risk patients who may benefit from drug therapy and are not targeted for statin use under the current Adult Treatment Panel (ATP) III guidelines. In the most recent study by Brilakis and colleagues [48], Lp-PLA2 levels correlated with extent of angiographic CAD; however, this was not independently predictive after adjusting for CRP, lipid status, and other traditional risk factors. Inhibition of Lp-PLA2 in animal models has been shown to be effective in reversing atherosclerosis in animal models [49], and phase II trials of SB-480848, a specific Lp-PLA2 inhibitor, as a potential treatment of atherosclerosis are currently underway.

CD40 ligand

CD40 ligand (CD40L) is a transmembrane protein expressed on CD4+ T cells, macrophages, and activated platelets. Its interaction with the CD40 receptor on endothelial cells, smooth muscle cells, and phagocytes has been shown to stimulate expression of proinflammatory cytokines, cellular adhesion molecules, matrix metal-loproteinases, and procoagulant tissue factor [50]. Furthermore, CD40 ligand has a KGD sequence that is a known binding motif for platelet integrin alip3. T cells expressing CD40L are found in atherosclerotic plaques [51], and disruption of the CD40-CD40L interaction in vitro diminishes ath-eroma formation and promotes stabilization of the established plaque [52].

Biologically active soluble CD40L (sCD40L) released from stimulated lymphocytes can be measured in plasma. Healthy women who have high levels of sCD40L have been shown to be at increased risk for cardiovascular events [53]. In patients who have ACS, sCD40L elevation (>5 mg/L) correlated with increased 6-month mortality (l8.6% versus 7.1%, P < .001) [54]. Treatment of these patients with abciximab before coronary angioplasty reduced risk for death or nonfatal MI (hazard ratio 0.12; 95% CI, 0.080.49; P<.001). However, patients who had lower levels of sCD40L did not experience the same treatment benefit. Among troponin-negative patients, those who had elevated sCD40L had an increased risk for cardiac events (13.6%) similar to that of patients who were troponin-positive (14%). Treatment of these patients with abcixi-mab also reduced risk for cardiac events (5.5% versus 13.6%, P = .03).

Selectins

Selectins (P-selectin, E-selectin) and cellular adhesion molecules (VCAM-1, ICAM-1) mediate the initial leukocyte rolling along the endothelium and attachment/subintimal transmigration, respectively, that are the initial steps of atherosclerosis. Immunohistochemical studies have demonstrated expression of these molecules, stimulated by oxidized LDL, in the endothelium overlying the atherosclerotic plaque. Circulating levels of VCAM-1, ICAM-1, and P- and E-selectin have been detected in plasma and are thought to arise from proteolytic cleavage from endothelial cells, particularly during inflammatory conditions [55]. In a nested case-control analysis of the Physicians Health Study, risk for future MI was 60% higher in patients who had soluble ICAM-1 elevation, and this increased risk was independent of smoking status or lipid levels [56]. Elevated baseline soluble

P-selectin levels in healthy women in the Women's Health Study were also associated with increased cardiovascular risk [57]. The levels of ICAM-1 and E-selectin, but not VCAM-1, have been shown to correlate with atherosclerotic burden as measured by carotid ultrasound, suggesting the usefulness of circulating adhesion molecules as indicators of subclinical disease [58]. Despite these intriguing data, the challenges of sample handling (samples are unstable unless frozen) currently limit the clinical usefulness of these markers.

Serum amyloid A

Like CRP, serum amyloid A (SAA) is an acute-phase protein that is synthesized in the liver in response to stress, injury, or inflammation. During the acute phase, SAA becomes the predominant apolipoprotein on HDL cholesterol, thus altering HDL-mediated cholesterol delivery to cells [59]. In the Women's Ischemia Syndrome Evaluation study of women referred for coronary angiogra-phy for suspected myocardial ischemia, SAA level correlated with 3-year risk for cardiovascular events and with angiographic severity of CAD [60].

Interleukin-10

IL-10 is a cytokine whose function is to limit the inflammatory reaction once the pathogen is eliminated. Its effects on macrophages include down-regulation of proinflammatory cytokine production and adhesion molecule expression. It also inhibits synthesis of matrix metalloproteinases implicated in plaque destabilization [61]. Overexpression of human IL-10 compensates for the rapid atherosclerosis of LDL-receptor knockout mice [62]. A small study in the United Kingdom showed higher IL-10 levels in patients who had stable versus unstable coronary syndrome [63].

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