CircKrt4通过外泌体介导的细胞间转运对肺血管重构的调控研究

Proliferation of pulmonary artery smooth muscle cells (PASMCs) which leading to pulmonary vascular remodeling is the main pathological process of pulmonary arterial hypertension (PAH), the mechanism is unknown. Exosomes shuttle play an important role in cell signal transmission. We hypothesized that the interaction of pulmonary vascular endothelial cells (PAECs) and PASMCs was through the exosome transport. Moreover, the delivery and release of exosome contents eventually lead to the proliferation of recipient PASMCs. By transmission electron microscopy, western blot and in situ hybridization we found that: 1. The secretion of exosomes was increased under the condition of PAH, and exosome inhibitor GW4869 injection significantly inhibited pulmonary vascular medial thickening; 2. The exosomes secreted by PAECs can be taken up by PASMCs and promoted their abnormal proliferation; 3. Hypoxia promoted the increase of circKrt4 expression and its abundant enrichment in exosomes; 4. CircKrt4 in PAECs mediated hypoxia-induced abnormal proliferation of PASMCs via the exosome transport and release, the relative targets and molecular networks need to be further determined. This application will first investigate the role of exosomes and their contents, circKrt4 in PAH, which possess the original innovation and provide a new theoretical basis and breakthrough point for the pathogenesis research and the treatment of PAH.

肺动脉平滑肌细胞(PASMCs)增殖导致肺血管重构是肺动脉高压(PAH)主要病理过程，机制未明。外泌体转运在细胞间信号传递中发挥重要作用。我们假设肺动脉内皮细胞(PAECs)与PASMCs相互作用通过外泌体转运实现。PAECs外泌体内容物转运及释放最终导致受体PASMCs增殖。通过透射电镜、免疫印迹、原位杂交等方法得到以下结果：1.PAH中外泌体分泌增加，外泌体抑制剂GW4869注射能够缓解肺血管重构；2. PAECs分泌的外泌体可被PASMCs摄取并促进PASMCs增殖；3. 缺氧促进PAECs外泌体内circKrt4富集；4. PAECs中circKrt4可通过外泌体转运释放介导缺氧诱导的PASMCs异常增殖，调控靶点及分子网络有待确定。本申请将首次探讨外泌体及其内容物circKrt4在PAH中的作用，具有一定的原始创新性，为PAH发病机理和治疗研究提供新的理论依据与突破点。

肺血管重构是肺动脉高压(PH)主要病理过程，机制未明。环状非编码RNA在细胞增殖、代谢等生物过程中发挥重要作用。本研究通过RNA测序、透射电镜、免疫印迹、免疫组化及原位杂交等方法得到以下结果：1. PH中外泌体分泌增加，外泌体抑制剂GW4869注射能够缓解肺血管重构；2. 低氧条件下，CircKrt4在肺组织和血浆中表达上调；3. 在细胞核中，circKrt4通过与转录激活蛋白Pura α (Pura)相互作用诱导EndoMT，促进N-cadherin基因激活；4. 在细胞质中，增加的circKrt4通过抑制线粒体结合型甘油激酶(Glpk)的细胞质-线粒体穿梭导致线粒体功能障碍；5. 有趣的是，circKrt4被鉴定为超级增强子相关的circRNA，它被转录因子CCAAT增强子结合蛋白α (CEBPA)转录激活；6. 此外，我们发现RNA结合基序蛋白25 (RBM25)通过增加Krt4基因的反向剪接来调节circKrt4环化。本申请首次探讨外泌体及circKrt4在PH中的作用，为PH发病机理和治疗研究提供新的理论依据与突破点。