Endothelial autophagy promotes atheroprotective communication between endothelial and smooth muscle cells via exosome-mediated delivery of miR-204-5p

Background: Cellular communication among different types of vascular cells is indispensable for maintenance of vascular homeostasis and prevention of atherosclerosis (AS). However, the biological mechanism involved in cellular communication among these cells and whether this biological mechanism can be used to treat AS remain unknown. This study hypothesizes that endothelial autophagy mediates the cellular communication of vascular tissue through exosome-mediated delivery of AS-related genes.Methods: ApoE-/- mice fed with high-fat diet (HFD) were received rapamycin to activate autophagy, or were intravenously injected with adeno-associated virus (AAV) vectors carrying shRNA against Atg7 mRNA under the Tie (tyrosine kinase) promoter to specifically inhibit endothelial autophagy. miRNA microarray, in vivo and in vitro experiments and human vascular tissues/blood vessels were used to explore the molecular mechanisms of atheroprotective effect of endothelial autophagy. Exosomes were isolated and characterized. Immunofluorescence and exosomes coculture experiments were conducted to examine the role of endothelial autophagy in regulating communication between endothelial cells (ECs) and smooth muscle cells (SMCs) via exosome.Results: Endothelial autophagy was inhibited in ECs of thoracic aortas in HFD fed ApoE-/- mice. Furthermore, rapamycin alleviated HFD-triggered atherosclerotic burden and endothelial dysfunction, while endothelial-specific depletion of Atg7 aggravated atherosclerotic burden. miRNA microarray, in vivo and in vitro experiments and human vascular tissues analysis revealed that endothelial autophagy alleviated endothelial dysfunction by downregulating miR-204-5p expression by directly targeting BCL2 to regulate apoptosis in ECs. Moreover, endothelial autophagy decreased miR-204-5p expression by loading this molecule into multivesicular bodies and secreting them out of cells via exosomes, and ECs-derived exosomal miR-204-5p could be transferred to SMCs, antagonized SMCs calcification.Conclusions: Our study first revealed that endothelial autophagy was a critical regulator of atherogenesis by transferring miR-204-5p from ECs to SMCs by exosomes, and endothelial autophagy activation by rapamycin, could alleviate AS in a “one stone hit two birds” manner. Overall design: The small RNA sequencing in the exosomes of the human umbilical vein endothelial cells treated palmitic acid with/without pretreated with atg7 siRNA or rapamycin were performed on an Illumina SE50 platform

背景：不同类型血管细胞间的细胞通讯对于维持血管稳态（vascular homeostasis）、预防动脉粥样硬化（atherosclerosis, AS）不可或缺。然而，此类细胞间通讯所涉及的生物学机制，以及该机制能否用于治疗AS，目前仍不明晰。本研究提出假说：内皮自噬（endothelial autophagy）可通过外泌体（exosome）介导的动脉粥样硬化相关基因递送，介导血管组织的细胞通讯。方法：本研究对喂食高脂饮食（high-fat diet, HFD）的载脂蛋白E基因敲除（ApoE-/-）小鼠，给予雷帕霉素（rapamycin）以激活自噬，或经尾静脉注射搭载靶向Atg7 mRNA的短发夹RNA（shRNA）的腺相关病毒（adeno-associated virus, AAV）载体，该载体以Tie（酪氨酸激酶）启动子（tyrosine kinase promoter）实现内皮特异性自噬抑制。本研究采用微小RNA芯片（miRNA microarray）、体内（in vivo）与体外（in vitro）实验以及人体血管组织，探究内皮自噬发挥抗动脉粥样硬化作用的分子机制。研究人员分离并鉴定了外泌体，通过免疫荧光实验与外泌体共培养实验，考察内皮自噬是否通过外泌体调控内皮细胞（endothelial cells, ECs）与平滑肌细胞（smooth muscle cells, SMCs）间的通讯。结果：在喂食HFD的ApoE-/-小鼠的胸主动脉内皮细胞中，内皮自噬受到抑制。进一步实验显示，雷帕霉素可缓解HFD诱导的动脉粥样硬化负荷与内皮功能障碍，而内皮特异性敲除Atg7则会加重动脉粥样硬化负荷。微小RNA芯片、体内与体外实验以及人体血管组织分析结果表明，内皮自噬可通过下调miR-204-5p的表达，直接靶向BCL2基因以调控内皮细胞凋亡，从而缓解内皮功能障碍。此外，内皮自噬可将miR-204-5p装载至多囊泡体中，并通过外泌体分泌至细胞外，从而降低细胞内miR-204-5p的表达；ECs来源的外泌体miR-204-5p可被转运至SMCs，拮抗SMCs钙化。结论：本研究首次揭示，内皮自噬可通过外泌体将miR-204-5p从ECs转运至SMCs，是动脉粥样硬化发生的关键调控因子；而通过雷帕霉素激活内皮自噬，可通过"一石二鸟"的方式缓解动脉粥样硬化。总体实验设计：本研究在Illumina SE50测序平台上，对经棕榈酸（palmitic acid）处理、或预先用Atg7小干扰RNA（siRNA）/雷帕霉素处理的人脐静脉内皮细胞的外泌体进行了小RNA测序（small RNA sequencing）。