Systemic markers of inflammation – are they useful predictive tools in coronary artery disease?

[BvU1]Our understanding of the mechanisms underlying atherosclerotic disorders has evolved beyond the view of a progressive collection of lipids and cellular debris in the vascular wall. Current evidence implicates endothelial dysfunction, oxidative stress and inflammation as important pathogenic mechanisms in atherogenesis and plaque destabilization, ultimately leading to acute coronary syndromes (ACS) and other thrombotic complications of atherosclerosis 1, 2. The production of inflammatory mediators such as adhesion molecules and cytokines in the vascular wall may lead to enhanced proliferation of vascular smooth muscle cells, increased lipid accumulation and prominent infiltration of inflammatory cells (i.e. activated monocytes/macrophages and T-cells), which again will result in further enhancement of the vascular inflammation, representing a vicious circle in atherogenesis. Inflammatory processes are not only involved in plaque growth, but seem also to play a critical role in plaque rupture by promoting platelet activation, apoptosis and production of matrix-degrading enzymes and reactive oxygen species (ROS) 2. While not necessarily the primary event, inflammation and oxidative stress may represent common final pathways to various stimuli in atherogenesis. In fact, it seems that “traditional” risk factors for cardiovascular disease such as hyperlipidemia, hypertension and diabetes at least partly may mediate their atherogenic effects through inflammatory mechanisms 1, 2.

Predictive value of inflammatory markers in CAD

In consideration of the important role of inflammation in plaque progression and stability, recent work has focused on whether plasma markers of inflammation can non-invasively diagnose and prognosticate coronary artery disease (CAD). Numerous studies have shown that inflammatory markers can help in identifying patients with stable CAD and ACS, as well as being predictive for development of CAD in high-risk patients 3, 4. Moreover, several inflammatory markers improve risk stratification in CAD patients, being independent prognostic markers for cardiovascular events 3, 4.

Of these markers, C-reactive peptide (CRP) has been the most widely studied. CRP has proven remarkably robust as a marker of cardiovascular risk and gives predictive value beyond that of traditional risk factors in CAD patients 5. Several large scale studies have shown baseline levels of CRP to independently predict future myocardial infarction, stroke and cardiovascular death in apparently healthy individuals 3–5. Moreover, among patients with stable angina and established CAD, plasma levels of CRP have consistently been shown associated with risk of cardiovascular events 3–5. Similarly, in patients with ACS, levels of CRP are predictive of high coronary risk even in those with non-detectable troponin levels 6. Finally, measuring CRP in CAD patients may be useful in monitoring responses to various interventions such as statin therapy. A recent sub-study of the Aggrastat-to-Zocor Trial showed that the achieved CRP levels at 30 days and 4 months after ACS are independently associated with long-term survival 7.

Other markers of the inflammatory cascade have also been found useful in predicting risk for vascular events in different populations 3, 4. In the Women's Health Study several inflammatory markers such as interleukin (IL)-6, serum amyloid A and CRP were all found to predict cardiovascular events in apparently healthy postmenopausal women 8. IL-6, which is a typical upstream mediator of CRP, is also predictive of recurrent vascular instability. Thus, elevated levels of IL-6 are associated with an adverse in-hospital prognosis among patients with ACS 9. The clinical usefulness of measuring IL-6 in ACS was also demonstrated in a sub-study of the FRISC-II trial showing that IL-6 may improve the ability to identify high-risk patients who benefit from an early invasive strategy 10. Interestingly, inflammatory mediators directly derived from activated platelets, such as soluble CD40 ligand (sCD40L), marker of platelet-mediated inflammation, have been identified as powerful markers of thrombotic activation in ACS 11. In a CAPTURE sub-study sCD40L did not only identify those patients with ACS who were at the highest risk for ischemic events, but also predicted which patients would derive major benefit from anti-platelet treatment with the GP IIb/IIIa receptor antagonist abciximab 12. Finally, recent studies have suggested that measurements of anti-inflammatory mediators may be useful in predicting vascular risk. Thus, elevated levels of IL-10 were associated with an improved outcome in patients with ACS, independently of elevated troponin levels 13. The beneficial effect of IL-10 was limited to patients with elevated CRP levels, further supporting the concept that the balance between pro- and anti-inflammatory mediators is a major determinant of a patient's outcome in ACS.

Endothelial dysfunction – an early and reliable marker for presence and progression of atherosclerosis

The endothelium regulates vasomotor tone, growth of vascular smooth muscle cells, and local inflammation by elaborating a number of paracrine factors 14. Moreover, endothelial cells control fibrinolysis by producing tissue plasminogen activator and plasminogen activator inhibitor 1 and is the source of both pro-coagulant (e.g., von Willebrand factor [vWF]) and anti-thrombotic (e.g., thrombomodulin [TM]) mediators. Under normal conditions the endothelium maintains a vasodilatory, anti-thrombotic, and anti-inflammatory state. However, both classic and recently recognized cardiovascular risk factors are associated with a shift to a vasoconstricted, pro-thrombotic and inflammatory condition 1, 2. These profound changes in endothelial phenotype seem to be of importance in all stages of atherosclerosis, suggesting that endothelial dysfunction could provide pivotal information as both a diagnostic and prognostic tool in patients at risk in CAD 15. Particular attention has been drawn towards the possibility of using plasma proteins as markers of endothelial cell dysfunction in this setting. Increased expression of adhesion molecules (e.g., intercellular adhesion molecule-1 [ICAM-1], vascular-cell adhesion molecule-1 [VCAM-1], P-selectin and E-selectin) on the endothelium is an important pathogenic event in both early and late atherogenesis 4, and there are several studies on the prognostic value of measuring the soluble forms of these adhesion molecules in plasma. Thus, in a nested case-control study in healthy men enrolled in the Physicians' Health Study, the authors reported a significant association between increasing concentration of sICAM-1 and risk of future myocardial infarction 16. However, other studies have failed to demonstrate that adhesion molecules are useful predictors in CAD, possibly reflecting that some of these mediators are derived from several cellular sources (e.g., platelets, leukocytes and macrophages) and may not necessarily be markers of endothelial dysfunction. Moreover, although sE-selectin is selectively derived from endothelial cells, its role as a prognostic marker in CAD is controversial, underscoring the problems of finding useful and reliable soluble markers of endothelial cell activation in atherosclerotic disorders.

Endothelial cell-derived coagulation factors such as vWF and TM may represent more specific markers for endothelial dysfunction 17. Both vWF and TM have been identified as predictors of adverse events among subjects with peripheral or coronary atherosclerosis, and TM has also been found to predict a fatal outcome in patients who already had a stroke 18, 19. Interestingly, these mediators provide the same prognostic information although they may have opposite effects in atherothrombotic disease. Thus, while vWF promotes atherosclerosis and thrombosis, TM seems to have potent anti-thrombotic and anti-inflammatory effects, possibly playing a protective role in vascular disease. This may seem contradictory, but could merely underscore that they both reflect endothelial cell activation and dysfunction.

Is oxidative stress of questionable importance in CAD?

Various risk factors for atherosclerosis have in common the enhanced generation of ROS leading to oxidative stress if not balanced by appropriate levels of anti-oxidants. These elementary molecules are thought to be harmful to the vasculature, leading to pathological processes such as apoptosis and inflammation 20. Moreover, enhanced oxidative stress has been implicated in pro-atherogenic modification of proteins, including lipoproteins such as LDL. According to “the oxidation hypothesis” of atherosclerosis, LDL, retained in the intima, undergoes oxidative modification, and this oxidized form of LDL (oxLDL) is regarded as a major inflammatory stimulus within the vessel wall 21.

In the present issue of Scand Cardiovasc Journal, Ruef and co-workers compare the levels of several markers of endothelial dysfunction, oxidative stress and inflammation in patients with stable and instable CAD 22. Their main findings were increased levels of several mediators associated with endothelial dysfunction (e.g., TM and vWF) and inflammation (CRP) in CAD patients, also correlated to severity of the disease. However, their results regarding oxidative stress were somewhat more conflicting. Thus, while plasma levels of superoxide dismutase (SOD) and myeloperoxidase (MPO) were elevated in these patients, no significant changes were observed for important markers of oxidative stress such as glutathione, lipid peroxides and 8-isoprostane. The authors conclude that besides SOD and MPO, markers of oxidative stress fail to be useful predictive markers of disease in CAD patients. Although some previous studies have shown that biomarkers for oxidative stress may correlate to CAD 23, 24, several other studies including the paper of Ruef et al. have failed to do so, possibly suggesting that the importance of oxidative stress in atherogenesis may be exaggerated. Indeed, despite extensive studies in both observational and randomized trials, the weight of evidence points to little or no benefit from antioxidant therapy in CAD. Hence, initial trials of antioxidant vitamins, mostly using vitamin C and vitamin E, have mainly given negative results including the HOPE trial and Heart Protection Study 25, 26.

However, one may argue that these studies do not necessarily exclude a role for oxidative stress in atherogenesis and plaque destabilization, but that they merely underscore the difficulties in performing such studies aiming to modulate a delicate balance in the anti- and pro-oxidant equilibrium. Moreover, the tools for measuring oxidative stress in vivo may also be insufficient. Thus several biomarkers including SOD are not useful during longitudinal testing in vivo, and they poorly identify patients who could benefit from an anti-oxidative treatment strategy. Moreover, while the intracellular balance between reduced and oxidized glutathione seems to be a major marker for intracellular oxidative stress, the value of measuring total glutathione in circulation is rather questionable.

Novel markers for oxidative stress as useful predictors of vascular events

Several “new” mediators reflecting oxidative stress have been found to serve as useful prognostic markers in CAD. First, recent studies measuring oxLDL in peripheral circulation have showed a close association between circulating levels of oxLDL and severity of disease in patients with ACS 27, 28. Moreover, another recent study showed that the circulating levels of oxLDL were strongly associated with angiographically documented coronary CAD 29. Second, the leukocyte-derived lipoprotein-associated A₂ (Lp-PLA₂) has also been found to have predictive value in CAD 30. Although, the exact role of Lp-PLA₂ still has to be determined, several studies have suggested that it accounts for much of the anti-atherogenic role of HDL by promoting resistance against oxidative stress 30. Interestingly, in the nested case-control study of hyperlipidemic patients from the West of Scotland Coronary Prevention Study (WOSCOPS) elevated Lp-PLA₂ was found to be an independent predictor of death, myocardial infarction, and revascularization in men 31. Also, in the MONICA study, Lp-PLA₂ levels also predicted vascular events in patients with hypercholesterolemia at 14-year follow-up 32. Finally, MPO, another enzyme involved in oxidative stress has recently been found to add to traditional risk markers in predicting CAD 33. MPO is produced in activated phagocytes generating ROS through a reaction with hydrogen peroxide, inducing oxidative damage during atherogenesis. Accumulating evidence suggest that MPO may play a pathogenic role in plaque vulnerability, and several studies have suggested that MPO levels may provide useful diagnostic and prognostic data in both stable and unstable CAD 34, 35. Ruef and co-workers also identified MPO as the most useful marker for oxidative stress in CAD patient in their study, further supporting that MPO together with oxLDL and Lp-PLA₂ could represent interesting new markers for oxidative stress in CAD 22. However, the usefulness of these markers may also be due to their ability to reflect other important atherogenic processes such as lipoprotein dysregulation and enhanced inflammation.

Future considerations

Useful biomarkers should in general provide independent diagnostic and prognostic value by reflecting the underlying disease. Clinical application also requires that besides being predictive of disease, the marker should substantively add to traditional risk factors. The clinical utility of measuring CRP has been well established, and the Centers of Disease Control and Prevention and the American Heart Association have endorsed the use of CRP as an adjunct to global risk prediction, particularly among patients at intermediate risk 36. The same guidelines also suggested use of CRP in risk stratification of patients with ACS and for secondary prevention. For other markers of inflammation and endothelial dysfunction, testing in multiple prospective cohorts is required before they may become useful tools in clinical medicine. As shown in the present as well as in other studies, markers of oxidative stress are at time being of very limited relevance in CAD. However, as oxidative stress, endothelial dysfunction and inflammation are different aspects of a single, shared pathogenic pathway in CAD, stable biomarkers reflecting all this pathogenic processes could represent potential future biomarkers in CAD.