Inflammatory Cytokines and Atherosclerotic Plaque Progression. Therapeutic Implications

A subgroup of patients with larger plaques have an increased population of CD4⁺ T cells. The expression of class II antigens on neighbouring cells provided evidence for the functional activity of these lymphocytes in the arterial plaque [46, 47]. Several hypotheses have been presented, including the possibility that the lipids inside the plaque could represent an autoimmune stimulus. The results of the cardiovascular inflammation reduction trial (CIRT) showed that a low dose of methotrexate did not reduce circulatory levels of several pro-atherogenesis cytokines and there was not a reduction of cardiovascular events in the treated group [48]. Inflammatory and immune responses can operate to promote disease, or to promote resolution or repair of lesions. B1 lymphocytes exert an anti atherogenesis effect, but B2 lymphocytes tend to aggravate atherogenesis. Interleukins have different actions on inflammation and atherosclerosis progression; IL10 and IL16 seem to have a protective effect [49, 50].

Epidemiological studies have found an association between several conditions characterized by local or systemic infections and inflammation (chronic pulmonary infections, dental infections, urinary tract infections, herpes virus infections) and increased systemic levels of inflammatory cytokines associated with atherosclerosis [51‐55]. This finding supports the possibility that ‘distant’ stimuli might have a local effect on aged-related disorders, an effect defined by Libby et al.’s ‘echo effect’ [56••]. A systemic effect is also possible. In patients with human immunodeficiency virus (HIV), a link between chronic infection, immune deregulation and age-related diseases, including cardiovascular events, is possible. Microbial communities in the mouth have been shown to cause infectious diseases such as gingivitis and to be associated with increased prevalence of systemic disorders such as atherosclerosis and cancer. Cytomegalovirus infection is related to high levels of circulating inflammatory cytokines and to increased prevalence of classic age-related diseases, including atherosclerosis [57, 58].

Inflammatory cytokines can exert their action locally, stimulating cell growth, apoptosis and reduced removal of necrotic tissue; conceptually, it is possible that elevated levels of inflammatory cytokines might lead to a de-regulation of the immune system through a systemic effect. Both the innate and the adaptive arm of the immune system undergo marked changes with age, a phenomenon to which has been given the unspecific definition as ‘the process of immune-senescence’. With ageing, functioning of the adaptive immune system generally declines. Immuno-senescence is characterized by a thymic involution with lower production of naïve T-cells, and less sophisticated T-cell repertoire [59].

Despite this logical deterioration of the immune system, the innate immune system seems hyper-active in association with several degenerative diseases, as evidenced by increased serum levels of inflammatory cytokines such as IL-6, TNF-α and acute phase proteins, in patients with diffuse atherosclerosis [60•, 61]. It is possible that systemic chronic inflammation deregulates the central immune system, with the activation of an abnormal immunological response (Fig. 2). Chronic inflammation may represent a general stimulus for a deranged hematopoietic stem cell, with the possibility of proliferation of selected clones of genetically modified cells. Many of the elderly patients will host hematopoietic cell clones for prolonged periods of times without developing any clinical problem [62••, 63, 64]. This condition has been named clonal hematopoiesis of indeterminate potential (CHIP). An initial observation, however, highlighted a significant increased risk from cardiovascular events in patients who carried CHIP involving approximately 20% of their circulating blood cells.

The topographic localization of the atherosclerotic plaque suggests a potential role for hemodynamic forces in the development and distribution of atherosclerosis [65, 66]. Flow separation, with the simultaneous occurrence of areas of high and low shear stress, has been shown to be present at the level of areas predisposed to develop the atherosclerotic plaque, such as the origin or the bifurcation of the carotid and coronary arteries, or arterial segments with a sharp curvature [67]. Several studies have correlated abnormal flow dynamics to increased production of growth factors, which could explain the initial abnormal proliferation of smooth muscle cells and endothelial cells, leading to myointimal hyperplasia [68, 69] (Fig. 3).

The atherosclerotic plaque occurs preferentially in regions of disturbed flow, with significant stress to the wall. Under long-term pulsatile radial pressure, elastin and collagen can lose their structural integrity. Deterioration of collagen and elastin per se activate a significant inflammatory reaction [70].

High levels of shear stress induce the release of inflammatory mediators from endothelial cells [71, 72]. PDGF, b-FGF and VEGF production is also increased in areas of high shear stress and high radial pressure. Several in vitro and in vivo studies have shown that high shear stress has a protective effect on atherosclerosis formation [73, 74]. In conditions of high shear stress, despite the increased presence of mitogens such as PDGF, bFGF and VEGF, endothelial and smooth muscle cells tend to have a limited proliferation rate. The atherosclerotic plaques form and progress in areas of low shear stress. However, atherosclerosis does not form in the venous system, where the blood flows at low velocity with consequent low shear stress. In the venous flow, the concentration of growth factors is much reduced. In vitro studies show that endothelial and smooth muscle cells, not exposed to any shear stress, have a much higher proliferation rate in the presence of the same concentrations of growth factors, compared with cells subjected to shear stress [75, 76]. Thus, the simultaneous presence of low and high shear stress, as present in turbulence, facilitates the action of growth factors; growth factors production is increased in areas of high shear stress, and they can act, through a paracrine mechanism, in the neighbour cells, located in the low shear stress zone, where they can easily proliferate.

Endothelial and smooth muscle cells situated in areas of low shear stress, in the context of a turbulence zone, can easily proliferate. A similar action mechanism is associated with the increased production of IL-1, IL-2, IL-6 and TNF alfa in areas of high shear stress; these cytokines may exert their inflammatory action on the neighbour cells, subjected to a low shear stress. The inflammatory action may promote proliferation of the endothelial and smooth muscle cell, and may facilitate LDL transport, through increased permeability of the endothelial cells and differentiation of smooth muscle cells in monocytes and macrophages involved in LDL transport. The involvement of the inflammasome complex may lead to oxidation of LDL. The transport of LDL in a zone of low shear stress is facilitated by the accumulation in this zone of LDL, in a general condition of turbulence [69, 77‐80].

Ultrasound studies demonstrated the changes of a plaque from a homogenous structure to a complex heterogeneous structure, and vice-versa [81, 82]. In completely occluded arteries, the plaque always appeared homogenous during ultrasound. Pathology showed that the ‘heterogeneous’ structure was often correlated to the presence of intraplaque hemorrhage and/or a large soft lipidic core [82‐84]. These observations testify to the conceptual possibility that the inflammatory reaction precedes the progression from stable to unstable plaque. On the other hand, with time, an initial ‘heterogenous’, vulnerable plaque, related often to intraplaque hemorrhage, can transform into a homogenous, stable plaque, if for several reasons (mainly slow progression) a valid balance is established between the internal pressure of the plaque and a thick fibrous cap [85•].

In vivo and in vitro experiments have shown that high shear stress per se prevents occlusive changes in the artery. The atherosclerotic plaque forms preferentially in areas of low shear stress in the context of disturbed flow. However, once the atherosclerotic plaque has formed, high levels of shear stress, favoured by the increased flow velocity in the narrowed lumen, can directly determine a trauma to the endothelium with desquamation and exposure of the content of the plaque to the blood and its coagulation factors [22, 86]. In the case of recently developed plaque, there is the possibility that the content of the plaque is composed mainly of hemorrhage and a lipid rich core, which, for their intrinsic thrombogenic nature, can determine platelet adhesion and production of inflammatory cytokines [22, 87•, 88]. In this condition, the production of inflammatory cytokines represents a secondary event and not a causal factor. The inflammatory cascade contributes to the severity of the local and systemic clinical picture. Alternatively, if the content of the plaque is formed mainly by fibrosis and collagen, as it is commonly found in slowly formed plaques, the desquamation of the endothelium determines exposure to the blood and its coagulation factors of a material less thrombogenic; in this condition, there is mainly aggregation of platelets, forming a white thrombus with minimal inflammatory reaction. Ultrastructural studies showed increased content of proteoglycan and glycosaminoglycans, hyaluronic acid and hyaluronan receptor CD44 in chronic lesions complicated by superficial erosion [31, 32, 89‐91]. Eroded chronic plaques have few inflammatory cells and many smooth muscle cells, whereas vulnerable plaques (presumably formed in a shorter time), have many mononuclear cells (macrophages and monocytes) and few smooth muscle cells. The fibrous cap shows various characteristics: in the eroded chronic plaque, the fibrous cap is thick, whereas in the unstable, acutely formed plaque, the fibrous plaque is thin. The type of thrombus associated with superficial erosion differs from the thrombus formed in the case of an unstable plaque rupture. Thrombus aspirated by percutaneous coronary techniques shows different patterns. Thrombi complicating eroded chronic lesions have a white color and they are rich in platelets, whereas thrombi complicating ruptured unstable plaques are predominantly red and rich in fibrin. The thrombi overlying ruptured unstable plaques are much richer in myeloperoxidase-positive inflammatory cells [31, 32, 89, 90, 91, 92•]. These findings suggest marked differences in the pathophysiology of erosion of chronic stable plaques from rupture of unstable plaques with thin fibrous cap. The clinical picture will depend on the characteristics of the plaque itself. In the PROSPECT (Providing Regional Observations to Study Predictors of Events in the Coronary Tree) study, intravascular ultrasound techniques were used to evaluate the character of tissue in coronary atherosclerotic plaques [84]. Serial intravascular imaging studies suggested that the morphology of human coronary atheromata evolves in time, from an unstable to a stable morphology, and vice-versa.