Oladosu Olanrewaju, Chin Emma, Barksdale Christian, Powell Rhonda R, Bruce Terri, Stamatikos Alexis
Department of Food, Nutrition, and Packaging Sciences, Clemson University, Clemson, SC 29634, USA.
Clemson Light Imaging Facility, Clemson University, Clemson, SC 29634, USA.
Pathophysiology. 2024 Feb 28;31(1):117-126. doi: 10.3390/pathophysiology31010009.
Atherosclerosis is caused by cholesterol accumulation within arteries. The intima is where atherosclerotic plaque accumulates and where lipid-laden foam cells reside. Intimal foam cells comprise of both monocyte-derived macrophages and macrophage-like cells (MLC) of vascular smooth muscle cell (VSMC) origin. Foam cells can remove cholesterol via apoAI-mediated cholesterol efflux and this process is regulated by the transporter ABCA1. The microRNA miR-33a-5p is thought to be atherogenic via silencing ABCA1 which promotes cholesterol retention and data has shown inhibiting miR-33a-5p in macrophages may be atheroprotective via enhancing apoAI-mediated cholesterol efflux. However, it is not entirely elucidated whether precisely inhibiting miR-33a-5p in MLC also increases ABCA1-dependent cholesterol efflux. Therefore, the purpose of this work is to test the hypothesis that inhibition of miR-33a-5p in cultured MLC enhances apoAI-mediated cholesterol efflux. In our study, we utilized the VSMC line MOVAS cells in our experiments, and cholesterol-loaded MOVAS cells to convert this cell line into MLC. Inhibition of miR-33a-5p was accomplished by transducing cells with a lentivirus that expresses an antagomiR directed at miR-33a-5p. Expression of miR-33a-5p was analyzed by qRT-PCR, ABCA1 protein expression was assessed via immunoblotting, and apoAI-mediated cholesterol efflux was measured using cholesterol efflux assays. In our results, we demonstrated that lentiviral vector-mediated knockdown of miR-33a-5p resulted in decreasing expression of this microRNA in cultured MLC. Moreover, reduction of miR-33a-5p in cultured MLC resulted in de-repression of ABCA1 expression, which caused ABCA1 protein upregulation in cultured MLC. Additionally, this increase in ABCA1 protein expression resulted in enhancing ABCA1-dependent cholesterol efflux through increasing apoAI-mediated cholesterol efflux in cultured MLC. From these findings, we conclude that inhibiting miR-33a-5p in MLC may protect against atherosclerosis by promoting ABCA1-dependent cholesterol efflux.
动脉粥样硬化是由动脉内胆固醇积累引起的。内膜是动脉粥样硬化斑块积累的部位，也是富含脂质的泡沫细胞所在之处。内膜泡沫细胞包括单核细胞衍生的巨噬细胞和血管平滑肌细胞（VSMC）来源的巨噬细胞样细胞（MLC）。泡沫细胞可通过载脂蛋白AI（apoAI）介导的胆固醇流出清除胆固醇，这一过程由转运蛋白ABCA1调节。微小RNA miR-33a-5p被认为通过沉默ABCA1而具有致动脉粥样硬化作用，ABCA1沉默会促进胆固醇潴留，并且数据表明在巨噬细胞中抑制miR-33a-5p可能通过增强apoAI介导的胆固醇流出而具有抗动脉粥样硬化保护作用。然而，MLC中精确抑制miR-33a-5p是否也会增加ABCA1依赖性胆固醇流出尚未完全阐明。因此，本研究的目的是验证在培养的MLC中抑制miR-33a-5p可增强apoAI介导的胆固醇流出这一假说。在我们的研究中，实验使用了VSMC系MOVAS细胞，并将加载胆固醇的MOVAS细胞转化为MLC。通过用表达针对miR-33a-5p的抗miR的慢病毒转导细胞来实现对miR-33a-5p的抑制。通过qRT-PCR分析miR-33a-5p的表达，通过免疫印迹评估ABCA1蛋白表达，并使用胆固醇流出测定法测量apoAI介导的胆固醇流出。在我们的结果中，我们证明慢病毒载体介导的miR-33a-5p敲低导致培养的MLC中该微小RNA的表达降低。此外，培养的MLC中miR-33a-5p的减少导致ABCA1表达去抑制，从而使培养的MLC中ABCA1蛋白上调。此外，ABCA1蛋白表达的这种增加通过增加培养的MLC中apoAI介导的胆固醇流出而导致ABCA1依赖性胆固醇流出增强。从这些发现中，我们得出结论，在MLC中抑制miR-33a-5p可能通过促进ABCA1依赖性胆固醇流出而预防动脉粥样硬化。
