**8. Efferocytosis**

The process in which apoptotic cells are cleared is called efferocytosis. While mainly managed by macrophages, vascular smooth muscle cells and neighboring cells may also have efferocytotic roles. As discussed above, apoptosis is a significant phenomenon that defines the progression of a plaque: EC, VSMC, and foamy macrophages all undergo apoptosis fueled by the growing plaque's chemokine environment. Tabas et al. have concluded that clearance of such apoptotic cells is the real issue within an atherosclerotic

plaque than apoptosis itself [10]. Martinet et al. have shown that efferocytosis is reduced by approximately 20-fold in a plaque relative to normal [30,31]. Interestingly, macrophages that become foam cells do not unload the engulfed ox-LDL to HDLs due to their defective efferocytosis, suppressing reverse cholesterol pathways' normal functioning. Defective efferocytosis also triggers macrophages to secrete pro-inflammatory signals such as TGFβ or IL-10. When efferocytosis is not complete, macrophages undergo cell membrane lysis spilling out necrotic chemicals such as proteases, thrombogenic tissue factors, and angiogenesis-promoting cytokines, creating more pro-inflammatory pathways (Figure 2). It is also thought that phenotype switching of macrophages from anti-inflammatory M2 to pro-inflammatory M1 has a role in diminishing the efficiency of efferocytosis [32].

Several classes of cellular molecules highly regulate efferocytosis: "find me" ligands that recruit phagocytes to the site of apoptosis, bridging molecules that link phagocytes to their targets, and "eat me" ligands on the apoptotic cell surface. These "eat me" ligands on cell surfaces bind and activate engulfment receptors on phagocytes. A counter molecule class called "don't eat me" ligand is present on viable cells but is downregulated in apoptotic cells. Studies have shown that ox-LDL in plaques induces auto-antibody generation within macrophages and other phagocytes. These autoantibodies mask the cell-surface "eat me" ligands on the dying cell. Ox-LDL also seems to act as a competitive inhibitor of scavenger receptors, making them less efficient in clearing apoptotic cells, as shown in (Figure 3). Calreticulin (Calr) is one of the key "eat me" ligands that binds to LDL Receptor-Related Protein (LRP1) on phagocytic cells and induces engulfment. Carriers of a risk allele at chromosome 9p21 are shown to express less Calr due to an inherited defect in TGF-β signaling, resulting in a more extensive lesion formation. Plaques of such mice models (that lack one of the 9p21 candidate genes) have been shown to exhibit plaque destabilizing features. VSMCs deficient in Calr have been shown to resist phagocytosis in vitro, induce pro-inflammatory foam-cell phenotype on cocultured macrophages, and suppress reverse cholesterol transport. It was shown that an exogenous introduction of Calr reversed these effects in-vitro [32].

**Figure 3.** RvD1 prevents the nuclear location of 5-LOX, increasing the production of pro-resolving LXA4 and reducing the production of pro-inflammatory LTA4 in response to stress signals.

As mentioned above, the "don't eat me" ligands maintain the balance of efferocytosis and protect healthy cells from being phagocytosed. CD47 is one such ligand on healthy cells that interacts with the alpha receptor on phagocytes, shutting off the efferocytotic pathways within the phagocytes. TNF-α weakens the downregulation of CD47 in atherosclerotic plaque cells and renders them resistant to efferocytosis. CD47 blocking antibodies have shown to have beneficial effects in mouse models by preventing atherosclerotic progression, regressing the necrotic core, and preventing the plaque from rupturing [32].

A few other molecules have been implicated in the failure of efferocytosis in atherosclerosis, including Milk fat globule epidermal growth factor 8 (Mfge8) and Mer receptor tyrosine kinase (Mertk). Mfge8 is a bridging molecule between αvβ3 integrin on the macrophages and externalized phosphatidyl serine on the apoptotic body [14]. This molecule seems to be expressed less in atherosclerotic plaque. In mouse models that were created with both LDLR−/<sup>−</sup>, and transplanted Mfge8−/− bone marrow had advanced atherosclerosis with larger necrotic core and systemic inflammation. It is thought that Mfge8 might also have a role in reverse cholesterol transport by binding transglutaminase 2 [32].

Mouse models with both LDLR−/<sup>−</sup>, and transplanted Mertk−/− bone marrow showed similar plaque properties to those with absent Mfge8 and LDLR. Furthermore, a mouse with a defective kinase form of Mertk resulted in more plaque necrosis than those found in ApoE−/<sup>−</sup>. Metalloproteinases, generally found in abundance in proinflammatory settings, cleave Mertk into a soluble inactive form. This inactive molecule provides a decoy receptor to Growth Arrest Specific 6 and leads to competitive inhibition of efferocytosis [32].

Weissman and colleagues have found that cancer cells upregulate "don't eat me" ligands to evade phagocytosis. Antibodies and decoy molecules that inhibit such processes and restore normal phagocytosis have been developed and are under study. If such treatments prove effective, they will have immense potential in treating atherosclerosis. Similarly, antibodies to TNF-α have been shown to reduce the expression of CD47. Anti-TNF-α antibodies are used in patients with rheumatological conditions. Such patients appear to be protected from myocardial infarction or adverse cardiovascular effects. Mouse treated with a combination of anti-CD47 and anti-TNF-α antibodies showed a better reduction in atherosclerosis than anti-CD47 alone. Given that there is a genetic susceptibility in reducing the efficiency of efferocytosis through the example of Calr expression, a genotype-driven therapy will most benefit such individuals [32].
