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Growing evidence suggests that the immune system plays a critical role in all the stages of atherogenesis, from lesion formation to plaque rupture.1 The inflammatory component of atherothrombosis is a topic of intensive research, since it is still unclear whether inflammatory markers measurable in peripheral blood can be useful tools for risk assessment in the general population; it is also unknown whether therapeutic strategies leading to a decrease of these inflammatory markers can modify cardiovascular risk.w1
INFLAMMATORY MECHANISMS IN ATHEROSCLEROSIS
Early in the atherogenesis process, resident or circulating leucocytes bind to the site of a developing lesion in response to oxidised low density lipoprotein cholesterol (LDL-C), injury, or infection. Proinflammatory cytokines play a central regulatory role in this early stage of atherogenesis, since they induce the migration of these inflammatory cells to the subendothelial space, both by acting directly on these leucocytes and by upregulating the expression of several adhesion molecules (such as vascular cells adhesion molecules, intercellular adhesion molecules and selectins) which participate in leucocyte adhesion, rolling and subendothelial migration (fig 1).w2
As these monocytes accumulate in the subendothelial space, they continue to ingest chemically modified lipids and lipoproteins, they become macrophages and finally develop into foam cells, and initiate fatty streaks. At the same time, other inflammatory cells, such as activated T cells and mast cells, also attach themselves to the endothelium, contributing to the formation of the atheromatous lesion, which consists of a lipid pool covered by a fibrous cap.w3 During the whole process, smooth muscle cells (SMCs) secrete chemoattracting factors that recruit additional monocytes.w4 Local stimulation of SMCs in the artery wall can amplify the inflammatory response and promote a local procoagulant effect.w3 The activation of all these immune cells and SMCs leads to the release of additional mediators, including cytokines, chemokines and growth factors, molecules which lead to further immune activation and trigger further steps in atherogenesis.w5
Interleukin-6 (IL-6), interleukin 1b (IL-1b) and tumour necrosis factor α (TNFα) are the principal pro-atherogenic cytokines,1 which are also produced in tissues other than the vascular wall and immune system, such as adipose tissue, myocardium, intestine, etc.1 They upregulate the expression of adhesion molecules on vascular endothelium,w2 depress nitric oxide synthesisw6 and promote the subendothelial migration of leucocytes. Further to their local regulatory role at a vascular level, these cytokines induce the liver-derived synthesis of acute phase proteins, such as fibrinogen, plasminogen, C-reactive protein (CRP) and serum amyloid α (SAA), which amplify inflammatory and pro-coagulant responses.w7 w8
Although the role of inflammation is critical in all the stages of atherogenesis, from plaque formation to plaque rupture and the development of acute coronary syndromes (ACS), it is unclear whether measurement of circulating levels of inflammatory molecules can be useful in risk assessment or even in the design of therapeutic approaches against the development and/or progression of coronary atherosclerosis. In this review we examine the clinical importance of the most widely known inflammatory markers, in the assessment of cardiovascular risk.
PROINFLAMMATORY CYTOKINES AS PREDICTORS FOR CARDIOVASCULAR DISEASE
Initial evidence suggested that IL-6 may be a better predictor of coronary artery disease than CRP,w9 w10 while its effect on the risk for stroke was particularly strong (relative risk (RR) 3.7, 95% confidence interval (CI) 1.67 to 8.21). However, it seems that IL-6 is also elevated in the presence of subclinical atherosclerosis, and it is hard to distinguish between patients with advanced and subclinical atherosclerosis.w11 The association between subclinical disease and IL-6 is consistent with data from the Health ABC Study, which showed that both IL-6 and TNFα are higher in older adults with subclinical cardiovascular disease.w12
Furthermore, in a study by Ridker et alw13 it was found that apparently healthy subjects with IL-6 at the highest quartile had relative risk for future myocardial infarction, in a 6 year follow up period, 2.3 times higher than those in the lowest quartile (95% CI 1.3 to 4.3), with an increase of risk by 38% for each quartile increase in IL-6.
Although TNFα is directly implicated in atherogenesis, it has not been frequently measured in epidemiologic studies. In the Health ABC Study, it was moderately correlated with IL-6 and weakly correlated with CRP,w12 while a stronger relationship between TNFα and coronary heart disease (CHD) than with either IL-6 or CRP has been reported.w9 Additionally, in a nested case–control study, Ridker et alw14 reported a multivariable-adjusted relative risk of recurrent coronary events of 2.5 (95% CI 1.3 to 5.1) among men whose TNFα levels exceeded the 95th centile, as compared with men with lower levels. However, TNFα has a limited half-life and is difficult to measure in large-scale epidemiologic studies,w15 and this is the main reason for the observed lack of data from prospective studies about the predictive role of TNFα in clinical practice.
Alternatively, the soluble forms of TNF receptors 1 (sTNF-R1) and 2 (sTNF-R2) levels can be measured; they may be of greater significance than direct measurement of TNFα, and they can be measured with greater sensitivity and reliability.w15 The soluble TNF receptors may attenuate the bioactivity of TNFα but may also serve as slow-release reservoirs and promote inflammation in the absence of free TNF ligand.w16 Only a few studies have examined the relationship between levels of sTNF-R1/sTNFR2 and the risk of CHD.2w9 w17 However, recent evidence suggested that measurement of these receptors is a rather weak approach to predicting cardiovascular risk compared to classic inflammatory markers, such as CRP or IL-6.w18
Another cytokine with a potential interest for future clinical research, but with limited clinical data currently available, is IL-10. IL-10 is an anti-inflammatory cytokine that inhibits the production of a variety of inflammatory cytokines such as IL-2, TNFα and IFNγ, and it is strongly associated with better prognosis in patients with ACS.w10 w20 However, IL-10 has not been evaluated in any epidemiologic study, and its clinical use is still obscure.
ACUTE PHASE PROTEINS
C-reactive protein (CRP)
CRP is a widely known acute phase protein, produced by the liver in response to proinflammatory cytokines, and especially IL-6, TNFα and IL-1b.w8 Although it is a non-specific inflammatory marker, it appears to be a stronger predictor of cardiovascular risk compared to most of the other known circulating inflammatory molecules.w21
At a clinical level, measurement of CRP in healthy individuals predicts the future development of coronary artery disease (tables 1 and 2). However, in the Thrombogenic Risk Factor (THROMBO) study, CRP was not a predictor of risk after adjustment for important predictors of prognosis, such as left ventricular ejection fraction and the presence of pulmonary congestion.w22 The potential association between CRP and cardiovascular prognosis was first illustrated in patients presenting with ACS.w23 The Thrombolysis In Myocardial Infarction (TIMI) investigators have since shown that the increased risk associated with high CRP values may be evident as early as 14 days after presentation with an ACS.w24 The CAPTUREw25 trial investigators found that, although only troponin T was predictive in the initial 72 h period, both CRP and troponin T were independent predictors of risk at 6 months, while the FRISCw26 investigators reported that the risk associated with elevated CRP values at the time of index event continues to increase for several years. In each of the above studies, the predictive value of CRP was independent of, and additive to, troponin levels. Most importantly, CRP has been found to have prognostic value among patients without evidence of myocyte necrosis; specifically, even among patients with negative troponin T, an elevated CRP is predictive of future adverse events.w24–26
Measuring and evaluating CRP
On the basis of all these data, the US Centers for Disease Control and Prevention and the American Heart Association issued guidelines in 2003 for the use of high sensitivity CRP (hsCRP) in clinical practice (table 3).3 Subjects are defined as low risk if CRP is <1.0 mg/litre, average risk if CRP is 1.0–3.0 mg/litre, and high risk if CRP is >3.0 mg/litre; however, high CRP values may be associated with an inflammatory disease, and this possibility should be taken into account when evaluating CRP values.
CRP is easily and inexpensively measured, and standardised high sensitivity assays are commercially available.w27 w28 These assays provide similar results in fresh or frozen plasma, while the predictive value of hsCRP measurement may be further improved by repeating several serial measurements.w29
Further to its role as an acute phase protein, fibrinogen plays a pivotal role in coagulation mechanisms, as the substrate for thrombin, while there is evidence that it may be involved in multiple mechanisms at all stages of the atherothrombotic process.w30
At a clinical level, the first meta-analysis4 examining the role of plasma fibrinogen as a risk factor for CHD in 4018 subjects, showed a risk of 1.8 for the top third (>350 mg/dl) versus the bottom third (<250 mg/dl), a finding confirmed in another meta-analysis in 7213 cases with CHD (table 1).5
Although the direct involvement of acute phase proteins in the progression of coronary atherosclerosis needs to be documented, it is likely that acute phase proteins may be markers reflecting the levels of proinflammatory cytokines, in a more reliable way than the direct measurement of these cytokines in plasma, since they are less variable throughout the day.
Serum amyloid α (SAA)
SAA is an amphipathic, α-helical apolipoprotein that is transported in the circulation primarily in association with high density lipoprotein (HDL),w31 and like CRP and fibrinogen, is an acute phase protein. SAA can induce the expression of proteinases thought to degrade extracellular matrix,w32 which might be important during tissue injury. In several observational and prospective studies, the risk of cardiovascular disease associated with SAA changed in parallel with that seen with CRP,2w33 although the absolute level of risk was generally smaller. Moreover, elevated plasma SAA is observed in the presence of risk factors such as obesity,w34 insulin resistance,w35 the metabolic syndrome,w36 and diabetes.w35 A recent meta-analysis6 suggests that subjects with SAA at higher tertile have a significant increase in cardiovascular risk up to 1.49 (95% CI 1.37 to1.62), although its use in clinical practice is still controversial. Although additional studies are needed, these observations raise the possibility that SAA may serve as a marker for an increased risk of cardiovascular disease in humans.
SOLUBLE FORMS OF ADHESION MOLECULES
Cellular adhesion molecules are molecules expressed on the surface of cells that mediate the adhesion of the cell to other cells or to the extracellular matrix.w2 A number of adhesion molecules expressed mainly on endothelial cells, leucocytes and platelets are actively involved in atherogenesis (table 4). The expression of these molecules is upregulated on the surface of these cells under the stimulatory effect of proinflammatory cytokines,w2 and they play a key role in atherogenesis (fig 1).
Adhesion molecules and risk prediction in coronary atherosclerosis
Since the expression of both acute phase proteins and adhesion molecules is largely regulated by proinflammatory cytokines, it is expected that the soluble forms of adhesion molecules (such as sICAM-l) correlate with acute phase reactants like CRP, and they may provide similar predictive information to CRP in settings of primary prevention.w37 In large prospective studies, sICAM-l, but not sVCAM-1, has been consistently related to incident coronary artery disease (CAD), at least in healthy populations (table 1).w37–40 Similarly, data from the Women’s Health Study indicated an association between sICAM-1 values and a combined cardiovascular end point,2 although after controlling for classical risk factors and other inflammatory biomarkers such as CRP, IL-6, and SAA, this association lost significance. Therefore, sICAM-l appears to be a general marker of pro-inflammatory status, and it is expressed in healthy individuals under stable clinical conditions.
Several studies have assessed the prognostic role of soluble adhesion molecules in a setting of secondary prevention. In the AtheroGene study, a prospective cohort of patients with established CAD, sVCAM-1 was identified as a strong independent predictor of future fatal cardiovascular events, independently of most potential confounders, especially other inflammatory markers including hsCRP.w41 In the Bezafibrate Infarction Prevention (BIP) study based on patients with CAD, baseline sICAM-1 concentration was an independent predictor for future coronary eventsw42 and stroke.w43 Similarly, in CAD patients and high-risk subjects (for example, diabetic patients), sVCAM-1 appears to be a strong predictor of cardiovascular mortality.w44
Only few data are available about the relationship between selectin levels and extent of atherosclerosis or their use for cardiovascular risk assessment. The first evidence, summarised in a recent meta-analysis,7 suggests that soluble selectin values may be predictors of cardiovascular disease in healthy populations; however, these data are based on small numbers of patients, and further studies are needed to establish their role in risk assessment in the general population.
OTHER NEW INFLAMMATORY MARKERS
CD40 ligand is a transmembrane protein related to TNFα.w45 A variety of cells involved in atherosclerosis, such as endothelial cells, smooth muscle cells, macrophages, T lymphocytes and platelets, express CD40L.w46 Evidence suggests that sCD40L is a strong predictor for clinical outcome in patients with ACS,w20 w47 w48 while high sCD40L was correlated with late restenosis after coronary angioplasty.w49 Although the first results from risk assessment studies were promising, there is still lack of sufficient data about the clinical importance of sCD40L as a predictive marker for coronary atherosclerosis, and further studies are needed to establish its role in the clinical setting.
Lipoprotein-associated phospholipase A2 (Lp-PLA2)
Lp-PLA2 is an enzyme distinct from secretory PLA2 and is transported primarily in LDL.w50 Lp-PLA2 is secreted by cells of the monocyte–macrophage series, T lymphocytes and mast cells,w51 and hydrolyses oxidised phospholipids, generating lysophosphatidylcholine which upregulates adhesion molecule expression.w51 Recent evidencew52 suggests that Lp-PLA2 is associated with coronary endothelial dysfunction, implying that it could be used as a predictor for cardiovascular risk.
Clinical evidence suggests that elevated Lp-PLA2 values are associated with increased risk of CHD and stroke, independently of lipid values (table 5).8,9w50 This association was shown in men enrolled in the West of Scotland Coronary Prevention Study (WOSCOPS), among whom Lp-PLA2 values in the highest quintile were independently associated with a twofold increase in risk compared with those in the lowest quintile.8 In the Women’s Health Study, the baseline Lp-PLA2 value was significantly higher in women who had a cardiovascular event during a mean 3 year follow up (1.20 mg/litre vs 1.05 mg/litre in controls); however, after adjustment for other cardiovascular risk factors, the relative risk in the highest versus the lowest quartile of Lp-PLA2 was not statistically significant.9 Recent evidence also suggests that Lp-PLA2 and CRP have additive effects to increase risk for cardiovascular events. In the ARIC studyw53 which included 12 819 men and women free of CHD at baseline, both Lp-PLA2 and CRP were associated with increased risk for CHD over a 6 year follow up period. In the presence of low LDL-C, CHD risk was three times greater for individuals with both Lp-PLA2 and hsCRP in the highest versus the lowest category.10w28
Inflammatory markers in atherosclerosis: key points
Inflammation is a key element of atherogenesis
Proinflammatory cytokines, adhesion molecules and acute phase proteins are the main inflammatory markers measured in humans
C-reactive protein is the strongest inflammatory predictor for atherosclerosis
Fibrinogen and serum amyloid α have been associated with atherosclerosis, but their predictive role is still controversial
Among adhesion molecules, the soluble form of sICAM-1 is a predictor of cardiovascular risk in healthy individuals, and sVCAM-1 in high-risk patients
Lp-PLA2 and sCD40L are new promising inflammatory markers with predictive value in cardiovascular disease, but their clinical usefulness is still under investigation
MPO is an abundant enzyme stored in the primary granules of neutrophils and monocytes. MPO has been demonstrated in human atherosclerotic lesions and has been implicated as a catalyst for LDL oxidation, leading to increased uptake and foam cell formation,w21 and may also contribute to endothelial dysfunction. MPO values may be elevated among individuals with CAD.w53 Increasing concentrations of leucocyte-MPO and blood-MPO were significant predictors of the risk for CAD.11 After adjustment for white blood cell count and Framingham risk score, individuals in the highest quartile of blood-MPO had a 20-fold higher risk of CAD than individuals in the lowest quartile.w53 However, further to these initial reports, there is a lack of prospective studies examining its long-term predictive value in cardiovascular risk.
The role of inflammation in atherogenesis is now well established. Although a large variety of inflammatory markers participating at several stages of atherogenesis can be measured in plasma or serum, only some of them seem to be clinically important (table 6). The acute phase proteins such as CRP and fibrinogen as well as SAA are the most well studied inflammatory markers, which can be used for cardiovascular risk assessment in the general population. Among the soluble forms of adhesion molecules, sICAM-1 is a promising inflammatory marker which can predict cardiovascular risk in healthy individuals, while measurement of sVCAM-1 is more useful for risk assessment in high-risk subjects and in patients with advanced atherosclerosis. Newer inflammatory markers such as sCD40L, Lp-PLA2, or MPO also seem promising for the prediction of cardiovascular risk, but further studies are needed to establish their role in cardiovascular disease.
Additional references appear on the Heart website— http://heart.bmj.com/supplemental
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