Abstract 45: Antiatherosclerotic Effects of miR-33 Inhibition: Increased Reverse Cholesterol Transport and Alternative-Activation (M2) of Macrophages

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Plasma HDL levels have a protective role in atherosclerosis, yet clinical therapies to raise HDL and exploit its atheroprotective effects have remained elusive. Recent studies identified miR-33 as an intronic microRNA, located within the SREBF2 gene, that suppresses expression of the cholesterol transporter ABC transporter A1 (ABCA1) and lowers HDL levels. Conversely, mechanisms that inhibit miR-33 increase ABCA1 and plasma HDL, suggesting that antagonism of miR-33 may be atheroprotective. We hypothesized that systemic delivery of an oligonucleotide inhibitor of miR-33 would increase plasma HDL and promote reverse cholesterol transport (RCT), and therefore have a beneficial impact on atherosclerosis. To test this, we treated Ldlr -/- mice with established atherosclerotic plaques with anti-miR33 or a control anti-miR for 4 weeks. Treatment with anti-miR-33 increased circulating HDL levels by 37% and enhanced RCT to the plasma, liver, and feces by up to 80%. Consistent with this, anti-miR33-treated mice showed a marked reduction in plaque size and lipid content, as well as an increase in indicators of plaque stability. Laser capture microdissection of lesional CD68+ cells demonstrated that anti-miR33 oligonucleotides directly targeted the plaque macrophages, where they enhanced ABCA1 expression and cholesterol removal. Moreover, macrophages from anti-miR33-treated mice showed an enrichment in anti-inflammatory M2 markers (Arg1, Il10) and reduced expression of proinflammatory M1 markers (iNos and Tnfa). Notably, overexpression of miR-33 in pMφ in vitro decreases markers of the M2 phenotype, Arg1 and Il-4, and increases the expression of inflammatory cytokines such as Tnfa and Il-1b. In contrast, anti-miR-33 polarizes pMφ to an M2 phenotype (Arg1, Fizz1, Il-10 and Il-4), with an associated downregulation of inflammatory genes (Tnfa, Il-1b). Overall, these results indicate that anti-miR33 has multiple beneficial effects on atherosclerosis, including increasing HDL and RCT, and reducing lesional inflammation by promoting macrophage polarization to the reparative M2 state, highlighting the promise for anti-miR33 therapy for the treatment of cardiovascular disease.