**4. VSMCs**

Within the growing stable plaque, an interaction between the T cells and macrophages secrete a wide array of chemokines and growth factors that target circulating monocytes, local endothelial cells, and smooth muscle cells. This proliferation can lead to lesion expansion. As discussed above, the stability of the plaque is derived from the presence of a thick fibrous cap produced in part by the VSMCs. Activated VSMCs switch their phenotype from contractile to synthetic and synthesize fibrotic proteins such as collagen [18]. Typically, TGF-β is a potent activator of collagen synthesis by VSMCs; however, T-cells secrete INF-γ, which inhibits collagen synthesis, especially types I and III, that are majorly found in extracellular matrices of arteries [9,19]. It has been shown that INF-γ inhibit collagen production by VSMCs even in the presence of TGF-β [19]. The source of INF-γ is T-cells, the presence of which in atherosclerotic lesions helps to tie the link between adaptive immune response to the stability of a plaque.

VSMCs also express scavenger receptors such as LOX-1 and CD-36 that internalize ox-LDL, generating foam cells out of VSMCs, as they would with macrophages. This generation of foam cells from VSMCs is most likely due to VSMCs' phenotypic change to resemble monocytes and mesenchymal stem cells. Endothelial and VSMCs, under the conditions of acute inflammation, start to produce pro-inflammatory molecules such as TNFα, IL-1β, and MCP-1, among many others, which attract neutrophils to the inflammatory site. Neutrophils, in turn, act on these cells to increase their pro-inflammatory effects, thus producing a snowball effect that prolongs the inflammatory response and intimal hyperplasia [20]. Studies have shown that a knockout of the transcription factor, Klf-4, which possibly mediates the phenotypic change in VSMCs, results in a reduction of VSMC-derived macrophage-like cells, a smaller lesion size, and increased fibrous cap thickness. These changes consequently increase plaque stability [21].

### **5. Macrophages, T Cells, Platelets**

Macrophages, classically have been categorized into M1, involved in pro-inflammatory pathways, and M2, participating in anti-inflammatory events. As noted above, monocytes recruited to the site of plaque formation are activated by toll-like receptors (TLRs), and INFγ present in the micro-environment of a growing plaque to M1 subtype [17,22,23]. These M1 type macrophages go on to secrete other pro-inflammatory cytokines such as IL-1b, IL-6, TNF, IL-12, and IL-23 along with molecules such as reactive oxygen and nitrogen species that sustain the ongoing inflammation [17]. M1 type macrophage also recruit Th1 and natural killer cells to the growing plaque further exacerbating the injury due to uncontrolled inflammation [24].

While historically, atherosclerotic plaques were characterized mainly by M1 type macrophages, recent studies have brought to fore other subtypes of macrophages, such as M4, Mox, M(Hb), Mhem. These subtypes vary in their local milieu and therefore their activation, their phenotypic markers, and in their participation or role in the pathogenesis as well as the characteristics of an atherosclerotic plaque. Recent studies with mouse models and carotid plaques have shown the presence of M2 subtype in advanced lesions [25]. However, since the cause of their presence could not be narrowed down, and since macrophage phenotypic switching based on their micro-environment is well-known, such representation is challenging and studies are still ongoing to demonstrate the presence of distinct types of macrophages and their role in plaque stability and severity [17].

The reduction in collagen levels in a plaque is partly due to decreased production mediated by T cells and increased levels of breakdown by macrophages, neutrophils, endothelial cells, and SMCs. Studies have shown that macrophages secrete several matrix metalloproteinases (MMPs), such as MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13, which are structurally identical to enzymes that degrade fibrillar collagen types I and III, proteoglycans, and elastin. Such MMPs also activate platelet aggregation and adhesion. Ox-LDL increases MMP-14 expression in endothelial cells, which in turn activates MMP-2, a potent gelatinase that acts on collagen IV, a constituent of the basement membrane. T-cells can bring such secretions by macrophages through the CD40 ligand (CD40L). The finding that the lack of MMPs resulted in increased and better-organized collagen in such plaques proved the role of MMPs in plaque stability. Elastases such as cathepsins S and K, and neutrophil elastase, have been found in plaques, which lower plaque stability possibly by degrading the extra-cellular matrix [19]. CD40L by T cells not only increases the secretion of MMPs but has also been attributed to the expression of tissue factor (TF) expression by macrophages. TF is the major activator of thrombosis when the cells encounter coagulation factors in blood. CD40L are also derived from platelets, which can cause its aggregation when the local environment is conducive. This can lead to local small arteries that feed the plaque to rupture leading to an intraplaque hemorrhage. This loop can continue whereby the exposed CD40L can activate TF in the local environment, leading to the growth of the thrombus, which in turn can cause further inflammation. While rupture of a plaque is the climax of the negative cardiac events, superficial erosion of the endothelial cells lining the plaque contributes to the weakening of its cap [19].

Not all small-scale changes that occur regularly within a plaque that result in mural thrombus formation lead to negative cardiac events, as seen in the thrombus formed within the vasculature of patients that did not die of heart conditions. These mural thrombi have platelets as its participants and platelet derived growth factor (PDGF) thus secreted might have led to the fibrosis of the plaque conferring it with stability. This 'healing' of the minor plaque ruptures lead to the 'expansive remodeling' of the artery rather than the 'constrictive remodeling' observed in an immature growing inflamed plaque [19].
