振荡剪切应力诱导的EPCs外泌体circ_1199在EPCs间质转化及病理性血管重构中的作用及机制

Pathological vascular remodeling is a common pathophysiological process of cardiovascular diseases, such as atherosclerosis(AS). Endothelial to mesenchymal transition (EndoMT) of endothelial progenitor cells (EPCs) may contribute to the onset and progression of pathological vascular remodeling. The oscillatory shear stress(OS) is closely related to EndoMT, which involved in AS. Emerging studies have shown that OS induces EndoMT but the underlying mechanisms are unclear. Based on previous studies and our preliminary data, here we innovatively put forward the hypothesis: under the stimuli of OS, a part of OS-sensitive EPCs secrete exosomes that play an autocrine role in communication between EPCs. Circ_1199 enriched in exosomes functions as an endogenous let-7g “sponge” to regulate the expression of let-7g target gene, HMGA2. And then the Circ_1199/let-7g/HMGA2 axis further modulate EPC EndoMT through activating the TGF-β/Smad or(and) Wnt/β-Catenin pathways. To test the hypothesis, we will apply multidisciplinary techniques, such as RNA sequencing (RNA-Seq),luciferase assay and the carotid arterial injury model and so on. These studies would have a high probability of revealing the mechanisms of EndoMT and provide new interventional strategies and targets for the pathological vascular remodeling.

病理性血管重构是动脉粥样硬化（AS）等心血管疾病的共同病理生理学过程，内皮祖细胞（EPCs）的间质转化（EndoMT）可能是病理性血管重构的普遍机制，而振荡剪切应力可诱导EndoMT并与AS进程密切相关。本课题组根据前人研究及预实验结果，创新性提出：振荡剪切应力作用下，力“敏感”性EPCs合成的外泌体以自分泌形式传递给其他EPCs，外泌体内富集的circ_1199发挥其分子“海绵”效应，吸附let-7g，从而上调let-7g靶基因HMGA2的表达，进而活化TGF-β/Smad、Wnt/β-Catenin等EndoMT相关信号通路，引发EPCs的EndoMT，促进病理性血管重构的发生。据此，本研究将从分子、细胞水平和颈动脉损伤动物模型层面对上述假设进行求证，以全新的视角阐明振荡剪切应力调控EPCs发生EndoMT的分子机制，从而为AS等病理性血管重构相关疾病探寻潜在的生物标志物和干预靶点。

病理性血管重构是动脉粥样硬化（AS）等心血管疾病的共同病理生理学过程，内皮祖细胞（EPCs）的间质转化（EndoMT）可能是病理性血管重构的普遍机制。我们前期研究表明：振荡剪切应力（OSS）可诱导EndoMT并与AS进程密切相关，但机制不明。本研究探讨了OSS诱导的EPCs外泌体circ_1199 在EPCs EndoMT中的作用及机制。结果显示：1. OSS促进EPCs外泌体释放。2.OSS外泌体被EPCs摄取后，诱导EPCs EndoMT。3.高通量测序结果显示，OSS作用前后EPCs差异表达的circRNAs有circ_1199等35种。qRT-PCR、RNA.FISH证实OSS上调EPCs内circ_1199表达。4.EPCs内过表达circ_1199诱导EPCs EndoMT。5.TargetScan和miRanda在线预测显示let-7g可与circ_1199或HMGA2配对结合。双荧光素酶报告基因检测提示let-7g对circ_1199或HMGA2具有靶向作用。LV-circ_1199下调let-7g，上调HMGA2及EndoMT相关信号分子snail及p-Smad3/Smad3表达。let-7g mimics，siHMGA2则明显下调HMGA2、snail及p-Smad3/Smad3表达。6.Let-7g mimics、siHMGA2不同程度下调α-SMA及SM22α表达。7.OSS外泌体干预后，在升高EPCs circ_1199，降低let-7g同时，增加HMGA2，snail及p-Smad3/Smad3的表达。8.OSS导致EPCs细胞骨架重排，伪足增多，且伪足周围聚集大量外泌体，上调EPCs细胞骨架相关蛋白Cortactin、Arp2和Arp3表达。siCortactin、CK636（阻断Arp2/3复合物的激活）或CytoD（抑制肌动蛋白聚合），可抑制OSS诱导的细胞骨架重排，降低OSS诱导的EPCs外泌体释放。综上所述，OSS作用下，EPCs合成外泌体增加，外泌体内富集的circ_1199发挥分子“海绵”效应，吸附let-7g，上调let-7g靶基因HMGA2的表达，进而激活EndoMT相关信号分子，引发EPCs EndoMT。本研究以全新的视角阐明了EndoMT的发生机制，为AS等病理血管重构疾病提供了生物学、力学防治策略。