**9. Resolvins**

### *9.1. Resolvin D Series (RvD)*

RvD1 binds to two receptors, ChemR23 and BLT1, and increases macrophage phagocytosis and PMN apoptosis, respectively. RvD1 also upregulates anti-inflammatory IL-10 and downregulates pro-inflammatory LTB4 [20]. Overexpression of the enzyme 15- Lipoxygenase (15-LOX) reduced atherosclerotic plaques in rabbits [8]. Lack of resolution of inflammation and the increased ratio of LTB4 to RvD1 have been implicated as the real culprit behind what starts as a host-beneficial process to life-threatening events [33,34]. Since SPMs act in their local environments, the microclimate of the area of tissue necrosis is vital in determining their viability and action; thus, a 5-LOX closer to the cell periphery can interact with 12/15 LOX to produce RvD1 from DHA. Furthermore, RvD1 prevents the nuclear location of 5-LOX, increasing the production of pro-resolving LXA4 and reducing the production of LTA4 (Figure 3) [35]. Liquid Chromatography tandem Mass Spectrometry of human carotid plaques revealed LXA4 and RvD1 as the major SPMs, requiring 5-LOX. Furthermore, these pro-resolving molecules were much less in vulnerable plaques than the stable ones. However, the intermediates such as 5-HEPE, 15-HEPE, 17, and 14-HDHA through the actions of 5/15/12-LOX were high in vulnerable plaques, indicating that the enzymes (5-LOX and 15-LOX) themselves were bioactive even in vulnerable plaques [33]. One mechanism by which there could be a reduction in RvD1 was proposed to be the relocation of 5-LOX to the nucleus via the persistent activation of Ca2+/Calmodulin Dependent Protein Kinase II (CAMKII) caused by the oxidative stress from substances such as 7-ketocholesterol (7-KC) found in the atherosclerotic plaque. This was proven by the reduction in the nuclear localization of 5-LOX once CAMKII expression was suppressed in human macrophages [35]. Inhibition of CAMKII also prevented the reduction of RvD1 production by 7-KC [33]. RvD1 also blocked the synthesis of LTB4 from AA. Such pathways were mediated by the receptors formyl peptide receptor 2/ lipoxin A4 receptor (FPR2/ALX), as both a receptor blocking antibody, as well as an antagonist, blocked this reduction of LTB4 by RvD1 [35]. Nuclear localization of 5-LOX produces LTA4, which is then transformed to LTB4 through the action of LTA4 hydrolase. This nuclear localization is brought about by the phosphorylation of LOX-5 at Ser271 by p38MAPK-activated protein kinase 2 (MK2). RvD1 blocks this phosphorylation through the receptors FPR2/ALX and G-protein-coupled receptor (GPCR) Gi (and GPR32 in humans). LXA4, which shares the same receptors, also has been found to reduce the phosphorylation. RvD1 could not block the synthesis of LTB4 when incubated with large quantities of LTA4, demonstrating that RvD1 cannot alter the pathway once LTA4 is formed [35]. Fredman et al. showed that the decrease in RvD1 was associated with the progression of the atherosclerotic plaque in the aortic arch of Ldlr −/− mice fed with a western diet for 8 or 17 weeks. Analysis of the lipid mediators in early vs. late plaques showed an approximately 87-fold decrease in RvD1 in advanced plaques but no significant change in LTB4 [33]. An increase in the lesional RvD1 levels was noticed when RvD1 was administered within the physiologic range. This increase resulted in the reduction of LTB4 levels, showing that RvD1 could have facilitated this reduction by preventing the nuclear localization of 5-LOX. This simultaneous increase in RvD1 (through external administration) and reduction in LTB4 (through RvD1 mediated reduction in the nuclear localization of 5-LOX) resulted in the ratio of RvD1:LTB4 being reverted to its early plaque levels. In addition, RvD1 increased other p SPMs, reduced oxidized CAMKII, reduced oxidative stress in the plaques, enhanced efferocytosis of macrophages, reduced the size of necrotic cores, and reduced the levels of collagenase and MMP9 without a concomitant reduction in the number of macrophages or VSMCs, resulting in a thickened fibrous cap. All these effects contribute to the stability of the plaque and in slowing its progression to an advanced type [33]. RvD1 was shown to act on human PMN, in-vitro, through a GPCR receptor that was inhibited by Pertussis toxin (PTX) and reduced their actin polymerization. They also blocked β2 integrin molecules on human PMN that were regulated by LTB4. Enhancement of phagocytosis by macrophages was also observed through the interaction of the receptors ALX, GPR32, and RvD1 [36]. RvD1 was also found to limit monocyte adhesion, reactive oxygen species (ROS), and pro-inflammatory cytokine production in VSMCs derived from the saphenous vein in vitro and in rabbit arteries that underwent balloon angioplasty [20]. It was found to alter the cytoskeletal properties of arterial smooth muscle cells (ASMC) in rats, thereby inhibiting their migration. Furthermore, it reduces their proliferation, oxidative stress, and translocation of p65, a molecule vital in NF-κB stimulation, which is implicated widely in the inflammation processes. All these beneficial effects were observed without damaging the viability of ASMCs [18]. Interestingly, a positive effect of RvD1 on reducing neutrophil infiltration comes from the analysis done by Recchiuti et al. They found that RvD1, possibly through its upregulation of certain micro RNAs (miRNAs) in humans, brought about a reduction in the resolution interval by ~4 h. These miRNAs were found to target immune-competent proteins such as the NF-κB pathway and 5-LOX (in the leukotriene pathway). By blocking these pathways, the concentration of pro-inflammatory mediators is reduced [37].

Akagi et al. demonstrated that pretreatment of ASMCsin vitro with DHA-derived SPMs, RvD2, and maresin-1(MaR1), impaired their migration towards PDGF in a dosedependent manner by 74% and 80%, respectively at a 100 nM concentration [38]. They also showed that GPCR could have mediated this response since the reduction in migration was attenuated in the presence of PTX, which inhibits GPCR proteins [20,36,38]. Of the many immunologically active cytokines, TNF α induces the NFκB pathway by nuclear translocation of p65 (Figure 4). This pathway results in the transcription of many pro-

inflammatory cytokines such as TNF<sup>α</sup>, Interleukin (IL)-1, IL-6, and IL-8. An in vitro study showed that RvD2 or MaR1 treatment of mouse ASMCs reduced p65 nuclear translocation by 24% and 28%, respectively, at a concentration of 500 nM. At the same concentration, such treatment also has reduced TNF-α induced superoxide production by 46% and 53%, respectively [38]. Furthermore, RvD2 reduced the cultured VSMCs production of VCAM-1 and ICAM-1 induced by TNF-α [20]. Such effects of these two SPMs demonstrate their anti-inflammatory and pro-resolving characteristics in their local environment. This proresolving action reduced the neo-intimal hyperplasia (neointima: media area ratio), which has been proven to be a result of chronic inflammation, by 67% and 71% by RvD2 and MaR1, respectively. The same study also showed a decrease in migration of neutrophils and monocytes to the area of injury, achieved by the suppression of MCP-1 expression by activated VSMCs and an increase in the M2 phenotype of macrophages which promoted resolution of the ongoing inflammation [38].

**Figure 4.** TNFα induces the NFκB pathway by nuclear translocation of p65. This pathway results in the transcription of many pro-inflammatory cytokines such as TNF<sup>α</sup>, Interleukin (IL)-1, IL-6, and IL-8. RvD2 or MaR1 treatment reduces p65 nuclear translocation, demonstrating their anti-inflammatory and pro-resolving characteristics in their local environment.

Aspirin-triggered RvD1 (AT-RvD1) has been shown to activate the nuclear factor erythropoietin 2 related factor 2 (Nrf2), increasing the expression of genes such as heme oxygenase-1 (HMOX1), and NAD(P)H quinone oxidoreductase 1 (NQO-1), aiding in combating oxidative stress in mice lung injuries [39].

### *9.2. Resovlin E Series (RvE)*

Resolvin E (RvE) series (E1 and E2) molecules are produced from EPA through the action of 5-lipoxygenase (5-LOX) [40]. In addition, RvE1 receptors have been found to be GPCRs [41].

RvE series mediate the resolution of inflammation through the following means:


receptor, BLT1. RvE1 binds BLT1 and ChemR23 equally, but RvE2 is a weak agonist of the ChemR23 receptor [42]. Platelet aggregation is one of the hallmarks of acute inflammation, brought about in part by ADP, which activates other platelets and leukocytes through intracellular signaling pathways. These pathways ultimately end with activation of platelet receptor GP IIb/IIIa, and granule secretion. RvE1 has been shown to regulate an ADP-mediated pathway that results in P-selectin surface mobilization through the ChemR23 receptor [43].

These SPMs have proven to be strong local modulators of acute inflammation through these actions. One of the major risks in atherosclerosis—thrombus formation- is avoided by preventing platelet aggregation. GP IIb/IIIa is a receptor for fibrinogen that is activated by ADP within platelets. RvE1, at concentrations of ~100 nM, has reduced fibrinogen binding to platelets. The lack of complete blockage of ADP stimulation of platelets by RvE1 is beneficiary as platelet aggregation, and thrombus formation are required for hemostasis [43].
