for oxidative stress, markers of plaque erosion and thrombosis, lipid‐associated markers, markers of endothelial dysfunction, metabolic markers, markers of neovascularization, and genetic markers. The last six biomarker categories are not treated in the presented chapter but only listed in Table 1.1. As mentioned earlier, some of these markers may indeed reflect the natural history of atherosclerotic plaque growth and may not be directly related to an increased risk of cardiovascular events. The best outcomes may be achieved by a panel of markers that will capture all of the different processes involved in plaque progression and plaque rupture, and that will enable clinicians to quantify an individual patient’s true cardiovascular risk. In all likelihood, a combination of genetic (representing heredity) and serum markers (representing the net interaction between heredity and environment) will ultimately be the ones that should be utilized in primary prevention. Finally, different non‐invasive and invasive imaging techniques may be coupled with biomarkers detection to increase the specificity, sensitivity, and overall predictive value of each potential diagnostic technique.
Markers of inflammation
Markers of inflammation include C‐reactive protein (CRP), inflammatory cytokines soluble CD40L (sCD40L), soluble vascular adhesion molecules (sVCAM), and tumour necrosis factor (TNF).
C‐reactive protein is a circulating pentraxin that plays a major role in the human innate immune response [184] and provides a stable plasma biomarker for low‐grade systemic inflammation. C‐reactive protein is produced predominantly in the liver as part of the acute phase response. However, CRP is also expressed in smooth muscle cells within diseased atherosclerotic arteries [185] and has been implicated in multiple aspects of atherogenesis and plaque vulnerability, including expression of adhesion molecules, induction of nitric oxide, altered complement function, and inhibition of intrinsic fibrinolysis [186], CRP is considered to be an independent predictor of unfavourable cardiovascular events in patients with atherosclerotic disease. Beyond CRP’s ability to predict risk among both primary and secondary prevention patients, interest in it has increased with the recognition that statin‐induced reduction of CRP is associated with less progression in adverse cardiovascular events that is independent of the lipid‐associated changes [187] and that the efficacy of statin therapy may be related to the underlying level of vascular inflammation as detected by hs‐CRP. Among patients with stable angina and established CAD, plasma levels of hs‐CRP have consistently been shown associated with recurrent risk of cardiovascular events [188, 189]. Similarly, during acute coronary ischemia, levels of hs‐CRP are predictive of high vascular risk even if troponin levels are non‐detectable, suggesting that inflammation is associated with plaque vulnerability even in the absence of detectable myocardial necrosis [190, 191]. Despite these data, the most relevant use of hs‐CRP remains in the setting of primary prevention. To date, over two dozen large‐scale prospective studies have shown baseline levels of hs‐CRP to independently predict future myocardial infarction, stroke, cardiovascular death, and incident peripheral arterial disease [192,193]. Moreover, eight major prospective studies have had adequate power to evaluate hs‐CRP after adjustment for all Framingham covariates, and all have confirmed the independence of hs‐CRP [194]. The association of plaque morphology prone to rupture with hs‐CRP has been also shown in the preliminary study [195]. Subsequently, a number of drug approaches to the inflammatory cardiovascular residual risk have been guided by knowledge of the inflammatory basis of cardiovascular disease. Recently, the CANTOS trial with the anti‐IL‐1‐beta monoclonal antibody canakinumab, demonstrated the reduction of hs‐CRP by 35% and reduced the composite of nonfatal‐MI, nonfatal stroke or cardiovascular death (hazard ratio (HR) =0.85, 95%CI: 0.74–0.98) [196]. Additionally, canakinumab reduced MACE rates to a similar extent in patients with or without diabetes197 and was associated with a significant reduction in deaths from lung cancer [198].
Colchicine, another anti‐inflammatory drug widely used for gout and pericarditis, has been examined to have a beneficial effect on cardiovascular disease, which showed the reduction of ischemic cardiovascular events in patients with MI [199], whereas a further trial investigating the efficacy of another anti‐inflammatory drug of methotrexate, most commonly used for arthritis, on patients with coronary artery disease was stopped given that it did not result in the lowering of IL‐1beta, IL‐6 or hs‐CRP compared to placebo and was associated with raised liver enzymes, reduction in leukocytes and hematocrit and a higher incidence of non‐basal cell skin cancer compared to placebo [200]. Thus, given unclear the anti‐inflammatory protective mechanism further investigation is required to better understand the association between inflammation and atherosclerotic disease.
Cellular adhesion molecules can be considered potential markers of vulnerability since such molecules are activated by inflammatory cytokines and then released by the endothelium.201 These molecules represent the one available marker to assess endothelial activation and vascular inflammation. The Physicians’ Health Study evaluated more than 14 000 healthy subjects and demonstrated ICAM‐1 expression positive correlation with cardiovascular risk and showed that subjects in the higher quartile of ICAM‐1 expression showed 1.8 times higher risk compared to subjects in the lower quartile [202]. Furthermore, soluble ICAM‐1 and VCAM‐1 levels showed a positive correlation with atherosclerosis disease burden [203]. IL‐6 is expressed during the early phases of inflammation and it is the principle stimuli for CRP liver production. In addition, CD 40 ligand, a molecule expressed on cellular membrane, is a TNF‐α homologue which stimulates activated macrophages proteolytic substances production.204 CD40 and CD40L have been found on platelets and several other cell types in functional‐bound and soluble (sCD40L) forms. Although many platelet‐derived factors have been identified, recent evidence suggests that CD40L is actively involved in the pathogenesis of acute coronary syndrome (ACS). CD40L drives the inflammatory response through the interaction between CD40L on activated platelets and the CD40 receptor on endothelial cells. Such interactions facilitate increased expression of adhesion molecules on the surface of endothelial cells and release of various stimulatory chemokines. These events, in turn, facilitate activation of circulating monocytes as a trigger of atherosclerosis. Beyond known proinflammatory and thrombotic properties of CD40L, experimental evidence suggests that CD40L‐induced platelet activation leads to the production of reactive oxygen and nitrogen species, which are able to prevent endothelial cell migration and angiogenesis [205]. As a consequence of inhibiting endothelial cell recovery, the risk of subsequent coronary events may be greater. Clinical studies have supported the involvement of CD40L in ACS and the prognostic value in ACS populations. Levels of sCD40L have been shown to be an independent predictor of adverse cardiovascular events after ACS [206] with increased levels portending a worse prognosis [207]. Importantly, specific therapeutic strategies have shown to be beneficial in reducing risk associated with sCD40L [208]. IL‐18 is a pro‐inflammatory cytokine mostly produced by monocytes and macrophages, and it acts synergistically with IL‐12 [176]. Both these interleukines are expressed in the atherosclerotic plaque and they stimulate IFN‐γ induction which, on its turn, inhibits collagen synthesis, preventing a thick fibrous cap formation and facilitating plaque destabilization. Mallat et al. [209] examined 40 stable and unstable atherosclerotic plaques obtained from patients undergoing carotid endarterectomy and they highlighted how IL‐18 expression was higher in macrophages and endothelial cells extracted from unstable rather than stable lesions and it correlated with clinical (symptomatic plaques) and pathological (ulceration) signs of vulnerability (Figures 1.7 and 1.8).
Pregnancy Associated Plasma Protein‐A (PAPP‐A), is a high‐molecular‐weight, zinc‐binding metalloproteinase, typically measured in women’s blood during pregnancy and later found in macrophages and smooth muscle cells inside unstable coronary atherosclerotic plaques. This protease cleaves the bond between Insulin Like Growth Factor‐1 (IGF‐1) and its specific inhibitor (IGFBP‐4 e IGFBP‐5), increasing free IGF‐1 levels. IGF‐1 is important for monocytes‐macrophages chemotaxis and activation in the atherosclerotic lesion, with consequent pro‐inflammatory cytokine and proteolytic enzyme release, and stimulates endothelial
