Abstract 262: Anti-miR33 Therapy Promotes PGC1α Expression, Mitochondrial Function and Immunometabolic Pathways in Macrophages in Atherosclerosis

Abstract

Macrophage foam cells are central to atherosclerotic plaque formation and therapies that increase macrophage cholesterol efflux remain an attractive yet elusive target. Therapeutically inhibiting microRNA-33, a critical regulator of lipid metabolism, increases circulating HDL and reduces atherosclerosis. However, the precise molecular mechanisms that regulate these beneficial effects are unknown. Thus, we sought to uncover novel gene networks regulated by miR33 that may contribute to atherosclerosis. Results: Microarray analysis of macrophages from anti-miR33 treated Ldlr-/- mice revealed several novel miR33 target genes that were de-repressed. Among these genes was PPARγ coactivator 1α (PGC1α), a crucial gene that controls cellular energy homeostasis and mitochondrial metabolism. 3’UTR luciferase assays verified that PGC1α is a specific and direct target of miR33. Macrophages transfected with miR33 mimics or anti-miR33 had decreased or increased PGC1α mRNA and protein respectively. Bioinformatic pathway analysis predicted that in addition to PGC1α, miR33 also regulates multiple mitochondrial proteins, such as pyruvate dehydrogenase kinase 4 (PDK4), solute carrier 25 A25 (SLC25A25) and Acyl-CoA Dehydrogenase (ACADSB). Anti-miR33 treatment of macrophages increases PDK4, SLC25A25 and ACADSB mRNA expression, validating these genes as miR33 targets. Seahorse XF Flux Analyzer studies show that macrophages treated with anti-miR33 have increased basal and maximal oxygen consumption rates, confirming that anti-miR33 therapy impacts positively on mitochondrial respiration and function. miR33 inhibition increased macrophage cholesterol efflux, which was abolished by oligomycin- a mitochondrial respiration inhibitor. This suggests that miR33 regulates cholesterol efflux at least in part by regulating mitochondrial function. Lastly, western-diet-fed apoE-/- mice treated with anti-miR33 injections (10mg/kg) for 8 weeks had a marked reduction in aortic sinus lesion area relative to control (p≤0.05), despite no change in HDL levels. Conclusion: Anti-miR33 therapy can positively impact on mitochondrial function, promoting macrophage cholesterol efflux and may contribute to HDL-independent anti-atherosclerotic effects.