Whereas foam cell accumulation characterizes fatty streaks, deposition of fibrous tissue defines the more advanced atherosclerotic lesion [20]. Macrophage-derived foam cells are crucial in lesion progression because they amplify the inflammatory response through the secretion of numerous growth factors and cytokines, including tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β (Fig. 2.2). In addition, T lymphocytes join macrophages in the intima and govern the adaptive immune responses. These cells, as well as ECs, secrete additional cytokines and growth factors that promote the migration and proliferation of VSMCs (Fig. 2.2). In response to inflammatory stimulation, VSMCs express specialized enzymes that can degrade elastin and collagen, allowing their penetration into the expanding lesion.

Chemokines within the plaque elicit activation of ECs and different leukocyte subsets (such as T cells), causing additional release of inflammatory cytokines and chemokines, which then promote further recruitment and activation of leukocytes, in a cycle of amplification of inflammation [3, 16]. Chemokine receptor CXCR3 interacts with its ligands (CXCL9 [MIG], CXCL10 [IP-10], and CXCL11 [I-TAC]) and promotes an inflammatory T-cell phenotype that causes macrophage activation [16]. Activation of macrophages also occurs directly by chemokines, which trigger increased release of cytokines and production of tissue factor (TF), MMPs, and ROS, and enhanced lipid loading and foam cell formation by upregulation of scavenger receptors. Altogether, these mechanisms, and the interaction between MCP-1 and CCR2, will highly alter macrophages to a pro-inflammatory, matrix-degrading, procoagulant, and proapoptotic phenotype [16].

Beyond their effects on T cells and macrophages, chemokines also interfere with VSMCs. While chemokines may promote VSMC migration into the lesion, an early event in atherogenesis, they could also transform these cells from a nonproliferative contractile phenotype, typical in healthy arteries, into actively proliferative cells (synthetic phenotype), which migrate when attracted by chemotactic agents, and increase matrix synthesis [16]. The migration of VSMCs from the vascular media to the intima is a hallmark process in intimal thickening and vascular remodeling characterizing atherosclerosis (Fig. 2.1). Recent data suggests that circulating bone marrow cells and progenitor cells present in the adventitia may also be a potential source of VSMCs in the intima [21] (Fig. 2.2).

T lymphocytes have been shown to contribute to atherogenesis and lesion development and progression [22, 23]. T cells are recruited to the forming lesion by mechanisms similar to those described for monocytes, despite using different patterns of activation, which is dependent upon recognition of cognate antigens and concomitant ligation of costimulatory receptors. Adhesion molecules (such as VCAM-1) and chemokines trigger T cells to enter the atheroma, where they react to several local molecules (peptide antigens) bound to major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs), such as oxLDL, HSP60, and microbial antigens. T-cell activation elicits distinct responses, involving T-helper-1 (Th1) cells, regulatory T cells (Treg), Th2 cells, and natural killer (NK) T cells [15, 22]. The precise role played by Th2 cells remains to be elucidated, while NK T cells recognize lipid antigens presented by CD1 molecules and trigger responses identical to those elicited by MHC-restricted T cells, including release of pro-inflammatory mediators and progression of lesion. While Th1 response, the most common in atheroma, involves induction of pro-inflammatory cytokines and promotion of lesion formation, Treg cells have an inhibitory effect on this process. In fact, Th1 cells produce IFN-γ, TNF-α, and CD40 ligand (CD40L), among other mediators, which are responsible for several responses. IFN-γ elicits activation of macrophages and endothelial cells via production of adhesion molecules, cytokines, chemokines, radicals, proteases, and coagulation factors, thus contributing to atherogenic lesion progression. TNF-α is produced by Th1 cells, but also by macrophages and NK cells, and has pro-inflammatory effects via NF-κB pathways [15, 22]. Other cytokines also play important roles in the atherogenic process [3, 16]. IL-18 receptor is expressed in endothelial and VSMCs, as well as in macrophages, and this cytokine elicits several important actions in atherogenesis, including induction of adhesion molecules (namely, VCAM-1), chemokines (i.e., IL-8), cytokines (in particular, IL-6), and several [1, 9, 13] MPPs, and, in combination with IL-12, upregulates the expression of IFN-γ in T cells, in macrophages, and in VSMCs, thus influencing important pro-inflammatory pathways involved in atherogenesis [16]. In opposition to Th1 cells, Treg cells are implicated in the maintenance of self-tolerance and regulation of autoimmunity, and the anti-inflammatory properties seem to derive from the production of transforming growth factor beta (TGF-β) and/or IL-10. T cells in atheroma display an activated/memory phenotype, and the proportion of activated T cells is particularly high in advanced lesions causing acute coronary syndrome (ACS).

The contribution of vascular dendritic cells (DCs) and mast cells to atherogenesis progression has been also suggested [24]. Although the exact role of vascular DCs remains poorly understood, they are thought to show antigen to naïve T cells accelerating lymphocyte recruitment into the atherosclerotic vessel. Mast cells seem to coordinate both the innate and acquired immunity through the activation of TLRs and cytokine release (Fig. 2.2).

Platelets also play a major role in the pathogenesis of atherosclerosis by several mechanisms, including production of inflammatory mediators (such as CD40L, myeloid-related protein-8/14 [MRP-8/14], and platelet-derived growth factor [PDGF]), and elicit leukocyte adhesion and incorporation into plaques, which is a robust example of the interplay between inflammation and thrombosis in the biology of atherothrombosis (Fig. 2.2) [20]. Platelets, as well as other cells involved in the disease (ECs and VSMCs, macrophages, and T cells), express CD40L and its receptor CD40 [25]. This pro-inflammatory cytokine plays a relevant role in this stage of atherogenesis, including stimulation of expression of adhesion molecules and of secretion of cytokines and MMPs which are involved in the degradation of extracellular matrix, as well as expression of tissue factor in ECs, VSMCs, and macrophages, which is responsible for the initiation of the coagulation cascade when exposed to factor VII. Thus, CD40L participates in the molecular events that will contribute to destabilization of plaques and increases the risk for plaque rupture and thrombosis.

Plaque rupture and the ensuing thrombosis commonly cause the most serious acute complications of atherosclerosis. Recent data suggest that inflammatory mechanisms, including infiltration and activation of leukocytes into the atherosclerotic plaque, rather than the degree of stenosis, are involved in plaque destabilization and rupture, resulting in coronary thrombosis leading to myocardial ischemia and infarction, for example (Fig. 2.1). In fact, ACS patients present typical features of an inflammatory phenotype, presenting several blood disturbances including increased contents of acute-phase reactants, cytokines, and activated T cells.

Plaque destabilization seems to occur due to the action of several types of molecules, including pro-inflammatory cytokines, chemokines, proteases, coagulation factors, vasoactive molecules, and radicals, mainly produced by activated macrophages, T cells, and mast cells located at sites of rupture (Fig. 2.2) [15, 16, 20]. These mediators are involved in several mechanisms that promote plaque activation, rupture, thrombosis, and ischemia, including inhibition of stable fibrous cap formation, degradation of collagen cap, and initiation of thrombus formation. Chemokines have been associated with the immune-mediated plaque destabilization due to several distinct mechanisms, namely, recruiting of activated leukocytes, stimulation of matrix degradation by macrophages, and induction of tissue factor in vascular ECs and VSMCs, as well as by promotion of oxidative stress and apoptosis in the plaques [16]. In addition, besides their role in thrombus formation, platelets could also have a major role on ACS, namely, due to release of pro-inflammatory mediators, including chemokines (such as CCL5, CXCL1, CXCL7, and epithelial neutrophil-activating peptide-78 [ENA-78/CXCL5]), and due to exacerbated inflammation in leukocytes and ECs [16].

The inflammatory milieu within atherosclerotic plaques stimulates the degradation of collagen fibers and inhibits the formation of new collagen, thus impairing the integrity of the interstitial collagen of the fibrous cap. In addition, activated T cells secrete IFN-γ which inhibits production of collagen by VSMCs. T lymphocytes are involved in several mechanisms that contribute to plaque destabilization, including production of IL-1 and CD40L which elicits macrophages to release interstitial collagenases, including MMPs [1, 8, 9, 13], that were detected in the shoulder region of plaques as well as in areas of foam cell accumulation, where activation and rupture of the plaque occur [22, 23]. MMPs are involved in the deregulation of extracellular matrix and intravascular thrombus formation, due to overexpression of tissue factor and activation of the coagulation cascade, thus contributing to plaque rupture [26].

Proteolytic destruction of collagen and inhibition of VSMC growth cause the reduction of cap thickness and strength, which become unable to support the hemodynamic forces and fissures, thus exposing components of the vascular matrix, including, e.g., different types of collagen, von Willebrand factor (vWF), fibronectin, laminin, fibulin, and thrombospondin (Fig. 2.2) [24]. ACS widely occurs due to physical disruption of the fibrous cap, either frank cap fracture or superficial endothelial erosion. The contact of blood with thrombogenic material (platelets and coagulation factors) initiates the formation of a thrombus, which can lead to abrupt and impressive obstruction of blood flow through the affected artery, and ischemia occurs in the end organ, such as the myocardium or the brain [20]. When the thrombus is nonocclusive or transient, the result could be clinically silent or cause symptoms typical of an ACS, including acute MI, unstable angina, or sudden death. The concentrations of circulating plasminogen activator inhibitor 1 (PAI-1) and fibrinogen usually determine the fate of a given plaque disruption [20].

Considering the role played by inflammation in all the stages of atherosclerotic disease, a variety of inflammatory mediators have been tested, in experimental and in clinical studies, as putative biomarkers of the pathogenic mechanisms that underlie the initiation and evolution of the disease, as well as predictors of risk and future events. The main question is whether plasma markers of inflammation can be noninvasively and feasibly used to improve diagnosis and prognosis of distinct forms of atherosclerosis.

The putative biomarkers of inflammation include different types of molecules, such as acute-phase reactants (namely, C-reactive protein [CRP], serum amyloid A (SAA) proteins, and fibrinogen), cytokines (including IL-1β, TNF-α, IL-6, and IFN-γ), adipokines (i.e., adiponectin), adhesion molecules (such as selectins and VCAM-1), platelet products (namely, CD40 ligand and MRP-8/14), and proteases (namely, MMP-9) [15, 16, 20]. Some of these analytes may participate in a pathogenic pathway, but not acting as an effective biomarker, while others may reflect the local inflammatory process in the artery, but do not have a causal role on the disease (thus acting as a biomarker, but not as a risk factor).

CRP has been viewed as the prototype of inflammatory markers. Accumulated evidence suggests that it could be a feasible marker of cardiovascular risk in MI patients, which presents augmented levels, and a predictor of future events in apparently healthy individuals [27, 28]. The development of a high-sensitivity assay for CRP (hsCRP) allowed accurate measurement of this molecule as a tool to enhance risk stratification. In fact, hsCRP has been shown predictive value beyond that of traditional risk factors, at least in specific populations with intermediate risk according to conventional criteria (namely, the Framingham algorithm), to whom the determination of hsCRP was already suggested (by US Centers for Disease Control and Prevention [CDC] and American Heart Association [AHA]) as a way to improve identification and stratification of risk [29]. Note that this intermediate risk group accounts for much of the burden of cardiovascular events recommending improved prediction tools. The Reynolds risk score adds hsCRP (as well as the family history of premature CAD) to traditional risk factors in a clinically useful manner [30].

Considering a successful application to identify and stratify cardiovascular risk, as well as its independence from traditional risk factors (namely, hypercholesterolemia), biomarkers of inflammation were suggested as a way to identify individuals who might benefit from therapeutic intervention, regardless of low estimates of cardiovascular risk according on traditional risk factors. The JUPITER (Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin) trial enrolled more than 17,000 individuals without known CVD, low LDL-c levels (<130 mg/dL), and hsCRP >2 mg/L in order to evaluate the effect of statin therapy in cardiovascular events and mortality [31, 32]. The results showed a major (>40 %) reduction of events and all-cause mortality, and additional analyses suggested that the benefits could be derived both from LDL reduction and improved anti-inflammatory effect viewed by hsCRP reduction. Evidence that a direct anti-inflammatory therapy positively impacts on atherosclerotic events cannot be drawn from the study, but ongoing trials (Canakinumab Anti-Inflammatory Thrombosis Outcomes Study [CANTOS] and Cardiovascular Inflammation Reduction Trial [CIRT]) could provide evidence on this, which is expected to have a major impact in the way CVD could be managed in the near future.

All these putative useful roles played by hsCRP need to be precisely defined through adequate and larger clinical trials that may test a number of targets and mediators. At the moment, doubts remain whether CRP could alone reflect the inflammatory mechanisms underlying atherosclerosis development, especially knowing the involvement of several other mediators (including a variety of cytokines), whether CRP could be viewed as a risk factor (with a causal role) or just as a surrogate marker of the process, and finally whether CRP or any other emergent biomarker of inflammation and cardiovascular risk could effectively and consistently improve clinical performance (namely, diagnosis, prediction of risk and/or of mortality, therapeutic decisions) in distinct populations.

The validation of the concept that inflammation is crucial for atherogenesis and its translation to clinical practice is an ongoing mission that deserves continuous efforts.