Abstract 9535: The Effect of Vascular Smooth Muscle Cell miR-33a Expression on apoAI-Mediated Cholesterol Efflux and Macrophage-Like Cell Transdifferentiation

Abstract

Background: ABCA1 removes cellular cholesterol by participating in apoAI-mediated cholesterol efflux. MiR-33a is an SREBP-2 intronic miRNA that silences ABCA1, which impairs ABCA1-dependent cholesterol efflux. Thus, miR-33a expression may be atherogenic and previous data suggests inhibiting miR-33a in certain cells is atheroprotective. However, current data is scant on whether miR-33a expression in vascular smooth muscle cells (VSMC) is atherogenic. Moreover, cholesterol accumulation in VSMC results in these cells to transdifferentiate into an atherogenic macrophage-like cell (MLC). Therefore, ablating miR-33a expression in VSMC may be atheroprotective via hindering cholesterol accumulation and MLC transdifferentiation in these cells by enhancing apoAI-mediated cholesterol efflux. Methods: An immortalized VSMC line MOVAS cells were used to generate miR-33a knockout (KO) MOVAS cells via using CRISPR/Cas9. We used these cells and parental wild-type (WT) MOVAS cells in our studies to assess whether apoAI-mediated cholesterol efflux is increased in MLC of VSMC origin when miR-33a expression is ablated. We also analyzed whether miR-33a ablation rapidly restores VSMC phenotype in MLC of VSMC origin when cholesterol-loaded cells were exposed to apoAI. Results: We used PCR to confirm that miR-33a was deleted in miR-33a KO MOVAS cells. We also showed that miR-33a-5p and miR-33a-3p expression was absent in miR-33a KO MOVAS cells via RT-PCR. SREBP-2 functionality remained intact in miR-33a KO MOVAS cells, as determined by upregulation of SREBP-2 target genes in serum-starved conditions. When we cholesterol-loaded miR-33a KO MOVAS cells to induce MLC transdifferentiation, we observed increased apoAI-mediated cholesterol efflux when compared to WT MOVAS cells exposed to the same conditions. Based on RT-qPCR analysis, we also observed a rapid restoration in VSMC phenotype when cholesterol-loaded miR-33a KO MOVAS cells were incubated with apoAI when compared to WT MOVAS cells treated with the same conditions. Conclusions: MiR-33a expression in VSMC appears to promote MLC transdifferentiation by impairing apoAI-mediated cholesterol efflux. Future studies are underway to determine if these effects are mediated by miR-33a-5p and/or miR-33a-3p.