miR-33a Expression Attenuates ABCA1-Dependent Cholesterol Efflux and Promotes Macrophage-Like Cell Transdifferentiation in Cultured Vascular Smooth Muscle Cells

Abstract

Recent evidence suggests that the majority of cholesterol-laden cells found in atherosclerotic lesions are vascular smooth muscle cells (VSMC) that have transdifferentiated into macrophage-like cells (MLC). Furthermore, cholesterol-laden MLC of VSMC origin have demonstrated impaired ABCA1-dependent cholesterol efflux, but it is poorly understood why this occurs. A possible mechanism which may at least partially be attributed to cholesterol-laden MLC demonstrating attenuated ABCA1-dependent cholesterol efflux is a miR-33a expression, as a primary function of this microRNA is to silence ABCA1 expression, but this has yet to be rigorously investigated. Therefore, the VSMC line MOVAS cells were used to generate miR-33a knockout (KO) MOVAS cells, and we used KO and wild-type (WT) MOVAS cells to delineate any possible proatherogenic role of miR-33a expression in VSMC. When WT and KO MOVAS cells were cholesterol-loaded to convert into MLC, this resulted in the WT MOVAS cells to exhibit impaired ABCA1-dependent cholesterol efflux. In the cholesterol-loaded WT MOVAS MLC, we also observed a delayed restoration of the VSMC phenotype when these cells were exposed to the ABCA1 cholesterol acceptor, apoAI. These results imply that miR-33a expression in VSMC drives atherosclerosis by triggering MLC transdifferentiation via attenuated ABCA1-dependent cholesterol efflux.

Inhibition of miR-33a-5p in Macrophage-like Cells In Vitro Promotes apoAI-Mediated Cholesterol Efflux

“…However, a recent report in our laboratory also showed that ablation of the miR-33a gene in the immortalized VSMC line MOVAS cells did not enhance apoAI-mediated cholesterol efflux, even though we did detect increased ABCA1 protein expression from miR-33a ablation [25]. When we MβCD:Chol-loaded miR-33a deficient MOVAS cells to induce MLC trans-differentiation though, we did observe increases in both ABCA1 protein expression and enhanced apoAI-mediated cholesterol efflux [25]. Since miR-33a deletion results in absent expression of both miR-33a-5p and miR-33a-3p, we cannot confidently infer from our previous published results whether the increase in ABCA1-dependent cholesterol efflux observed in MLC is actually due to miR-33a-5p deficiency, which led to us to performing this study.…”

“…We previously reported that inhibiting miR-33a-5p expression in primary VSMC does result in increasing ABCA1 protein expression and enhancing apoAI-mediated cholesterol efflux [48]. However, a recent report in our laboratory also showed that ablation of the miR-33a gene in the immortalized VSMC line MOVAS cells did not enhance apoAI-mediated cholesterol efflux, even though we did detect increased ABCA1 protein expression from miR-33a ablation [25]. When we MβCD:Chol-loaded miR-33a deficient MOVAS cells to induce MLC trans-differentiation though, we did observe increases in both ABCA1 protein expression and enhanced apoAI-mediated cholesterol efflux [25].…”

“…Western blotting was performed as follows [ 25 ]. Briefly, transduced MOVAS MLC were washed with PBS and cell lysates collected with RIPA lysis buffer which contained protease inhibitors (VWR Life Science).…”

“…Much of the published work involving miR-33a-5p inhibition has involved cultured macrophages and we and others have shown that inhibiting miR-33a-5p in these cells results in increasing ABCA1-dependent cholesterol efflux [ 21 , 22 , 23 , 24 ]. A recent report from our laboratory has also shown that miR-33a deficient cultured MLC exhibit increased ABCA1-dependent cholesterol efflux [ 25 ]. While our results infer that inhibiting miR-33a-5p in MLC may be atheroprotective, the proof-of-principle approach we utilized also resulted in ablating expression of the other miR-33a mature strand, miR-33a-3p, and evidence implies this mature strand may be capable of silencing ABCA1 expression, too [ 26 ].…”

Dissecting the Impact of Vascular Smooth Muscle Cell ABCA1 versus ABCG1 Expression on Cholesterol Efflux and Macrophage-like Cell Transdifferentiation: The Role of SR-BI

“…The primary purpose of this work was to attempt to uncover the atheroprotective roles of ABCA1 and ABCG1 expression in cultured VSMC. For our study, we utilized the well-characterized immortalized mouse aortic smooth muscle cell line MOVAS cells [29][30][31] for the generation of ABCA1 knockout (A1-KO) MOVAS cells, ABCG1 knockout (G1-KO) MOVAS cells, and ABCA1/ABCG1 double-knockout (DKO) MOVAS cells to compare to parental wild-type (WT) MOVAS cells. Using these cells, we tested the hypothesis that ABC transporter expression restores the VSMC phenotype in MLC by preserving apoAI/HDLmediated cholesterol efflux.…”