Abstract 884: Expression of Anti-mir-33a-5p in Cultured Endothelial Cells Increases Macrophage and Smooth Muscle Cell Cholesterol Efflux by Exosome-mediated Transfer of Anti-mir-33a-5p

Abstract

Background: The microRNA miR-33a-5p impairs apoAI-mediated cholesterol efflux by silencing ABCA1 expression. Systemic injection of antagomirs to miR-33a-5p reduces atherosclerosis but can cause hepatic steatosis and hypertriglyceridemia. In contrast, local delivery of miR-33a-5p antagomirs to lipid-laden intimal cells [smooth muscle cells (SMC) and macrophages (Mϕ)] could potentially treat atherosclerosis without causing metabolic abnormalities. Helper-dependent adenoviral vectors (HDAd) provide durable transgene expression in endothelial cells (EC) in vivo; however, HDAd does not cross an intact large vessel EC layer and therefore cannot deliver antagomirs directly to SMC and Mϕ. We hypothesized that transducing EC with an HDAd expressing anti-miR-33a-5p (HDAdAnti) could result in packaging of anti-miR-33a-5p into EC-derived exosomes that, in turn, deliver anti-miR-33a-5p to neighboring SMC and Mϕ. Inhibition of miR-33a-5p in SMC and Mϕ would upregulate ABCA1, and enhance apoAI-mediated cholesterol efflux. Methods: We transduced cultured EC with either HDAdAnti or a control HDAd expressing a “scrambled” shRNA (HDAdScr), harvested conditioned medium (CM) from the EC, and treated SMC and Mϕ (some of which were cholesterol-loaded) with the CM. We measured levels of anti-miR-33a-5p and miR-33a-5p, ABCA1 protein, and apoAI-mediated cholesterol efflux in the treated SMC and Mϕ. Results: Exosomes containing anti-miR-33a-5p were present only in CM from HDAdAnti-transduced EC. Compared to Mϕ incubated with CM from EC transduced with HDAdScr, Mϕ incubated with CM from HDAdAnti-transduced EC had 80% lower levels of miR-33a-5p, a 2.2-fold increase in ABCA1 protein, and a 1.6-fold increase in apoAI-mediated cholesterol efflux (all P<0.001). Similar results were obtained with SMC: 65% lower levels of miR-33a-5p expression, 1.6-fold increase in ABCA1 protein, and 1.4-fold increase in apoAI-mediated cholesterol efflux (all P≤0.01). Conclusions: EC transduced with HDAdAnti release exosomes containing anti-miR-33a-5p. These exosomes transfer anti-miR-33a-5p to SMC and Mϕ, increasing cholesterol efflux. Application of this approach in vivo may accelerate vessel wall cholesterol efflux, preventing and reversing atherosclerosis.