Saenz-Pipaon Goren, Wacker Bradley K, Bi Lianxiang, Stamatikos Alexis, Dichek David A
Department of Medicine, Division of Cardiology, University of Washington, Seattle, WA 98195, USA.
Department of Food, Nutrition, and Packaging Sciences, Clemson University, Clemson, SC 29634, USA.
Biology (Basel). 2024 Nov 24;13(12):965. doi: 10.3390/biology13120965.
Atherosclerosis is caused by the accumulation of cholesterol within intimal smooth muscle cells (SMCs) and macrophages. However, the transporter ATP-binding cassette subfamily A, member 1 (ABCA1), can remove cholesterol from these intimal, cells reducing atherosclerosis. Antagomir-mediated inhibition of miR-33a-5p, a microRNA that represses ABCA1 translation, promotes ABCA1-dependent cholesterol efflux and may impede atherosclerosis development. In our previous work, transducing cultured endothelial cells (ECs) with a helper-dependent adenoviral vector (HDAd) that expresses X-motif-tagged anti-miR-33a-5p enhanced antagomir packaging into EC-derived exosomes, which delivered the antagomir to cultured SMCs and macrophages. In this present study, we tested whether in vivo transduction of rabbit carotid artery endothelium can deliver an X-motif-tagged anti-miR-33a-5p to subendothelial cells. Rabbit carotid endothelial cells were transduced in vivo with an HDAd expressing anti-miR-33a-5p either with or without the X-motif ( = 11 arteries per vector). Contralateral carotids received HDAd that express scrambled oligonucleotides. Three days after transduction, the antagomir-without the X-motif-was detected in the intima but not in the media of transduced carotids ( = 0.062). The X-motif antagomir was detected in 82% of the intimal extracts (9 out of 11 carotids) and 27% of medial samples (3 out of 11 carotids, = 0.031). However, the X-motif did not significantly enhance antagomir delivery to the media ( = 0.214 vs. non-X-motif antagomir). Expression of the antagomirs-with and without the X-motif-was sub-stoichiometric in ECs and SMCs. No antagomir-related changes in miR-33a-5p or ABCA1 expressions were detected. Despite its potential as a therapeutic strategy, our exosome-targeted gene transfer system requires further improvements to enhance antagomir expression and delivery to the subendothelial cells.
动脉粥样硬化是由胆固醇在血管内膜平滑肌细胞（SMC）和巨噬细胞内蓄积所致。然而，转运蛋白ATP结合盒A亚家族成员1（ABCA1）可从这些内膜细胞中清除胆固醇，从而减轻动脉粥样硬化。抗miR-33a-5p（一种抑制ABCA1翻译的微小RNA）通过拮抗剂介导的抑制作用可促进ABCA1依赖的胆固醇外流，并可能阻碍动脉粥样硬化的发展。在我们之前的研究中，用表达X基序标记的抗miR-33a-5p的辅助依赖腺病毒载体（HDAd）转导培养的内皮细胞（EC），可增强拮抗剂包装到EC来源的外泌体中，这些外泌体将拮抗剂递送至培养的SMC和巨噬细胞。在本研究中，我们测试了兔颈动脉内皮细胞的体内转导是否能将X基序标记的抗miR-33a-5p递送至内皮下细胞。将表达抗miR-33a-5p（有或无X基序）的HDAd体内转导兔颈动脉内皮细胞（每个载体n = 11条动脉）。对侧颈动脉接受表达乱序寡核苷酸的HDAd。转导后三天，在转导颈动脉的内膜中检测到无X基序的拮抗剂，但在中膜中未检测到（P = 0.062）。在82%的内膜提取物（11条颈动脉中的9条）和27%的中膜样本（11条颈动脉中的3条，P = 0.031）中检测到X基序拮抗剂。然而，X基序并未显著增强拮抗剂向中膜的递送（与无X基序拮抗剂相比，P = 0.214）。有和无X基序的拮抗剂在EC和SMC中的表达均低于化学计量。未检测到拮抗剂相关的miR-33a-5p或ABCA1表达变化。尽管其作为一种治疗策略具有潜力，但我们的外泌体靶向基因转移系统需要进一步改进，以增强拮抗剂的表达并递送至内皮下细胞。
