精胺富集的内皮细胞外囊泡在吸烟所致肺动脉高压发病机制中的作用

A variety of pathogenic factors such as cigarette smoke can trigger pulmonary endothelial cell damage and smooth muscle cell proliferation and lead to the development of pulmonary hypertension. In the past, smoking was thought to induce pulmonary emphysema and hypoxemia, resulting in pulmonary vascular injury and remodeling, and eventually pulmonary hypertension. However, recent studies have shown that smoking can directly cause PH through other effects than hypoxia. But, the molecular mechanism by which smoking directly induces the development of pulmonary hypertension is unclear. Preliminary experiments of the applicant show that smoking induced an increase of endothelial extracellular vesicle (eEVs) secretion and an elevation of spermine carried by eEVs in the blood plasma of rats. In addition, pulmonary hypertension was induced in rats by injecting exogenous eEVs through tail vein. Considering that spermine is a potent agonist of calcium-sensing receptor (CaSR), and the pivotal role of CaSR in hypoxia and monocrotaline-induced pulmonary hypertension as recently revealed from several laboratories including ours, the applicant hypothesis that CaSR in pulmonary artery smooth muscle cells may be activated by increased spermine on eEVs, and this causing contraction and proliferation of pulmonary artery smooth muscle cells, eventually inducing the development of pulmonary hypertension. The successful implementation of this project is expected to provide a new direction for the prevention and treatment of smoking-induced pulmonary hypertension.

以往认为吸烟通过引起慢性阻塞性肺疾病，造成低氧血症，进而能引发肺血管损伤和重构，最终导致肺动脉高压（PH）的发生。但新的研究进展表明，吸烟也可以通过缺氧以外的其他作用直接引起PH。然而，吸烟直接诱导PH发生发展的分子机制却不清楚。申请人预实验发现，吸烟诱导大鼠血浆中内皮细胞外囊泡（eEVs）分泌增多且其携带的精胺增加。通过尾静脉注射外源性eEVs能够诱导大鼠PH发生。考虑到精胺是钙敏感受体（CaSR）的强效激动剂，而申请人前期研究已发现CaSR介导野百合碱和缺氧导致的PH。以此为基础，申请人推测精胺富集的eEVs可能激活肺动脉平滑肌细胞的CaSR，引起肺动脉平滑肌细胞收缩、增殖，最终引起PH。本项目的顺利实施有望为吸烟诱导的PH的防治提供新的探索方向。

本研究旨在确定内皮细胞外囊泡（eEV）的富集代谢物是否以及如何对香烟烟雾引起的内皮细胞直接损伤和肺动脉高压的发展至关重要。来自吸烟人类受试者的内皮细胞外囊泡的代谢组学筛选显示精胺显着升高，精胺是一种多胺代谢物，是钙敏感受体（CaSR）的强效激动剂。钙敏感受体的抑制剂Calhex231或CaSR敲低可减轻香烟烟雾引起的大鼠肺动脉高压，而无肺气肿变化或慢性低氧血症。香烟烟雾暴露会增加精胺阳性eEV的产生及其精胺含量增加。免疫荧光染色和免疫金电子显微镜结果显示精胺不仅在eEV内的富集，还在eEV膜外表面富集。抑制精胺合成、精胺的类似物—N1-丹磺酰精胺、Calhex231或CaSR敲低可显着抑制eEV诱导的细胞内钙离子浓度升高、肺动脉收缩和平滑肌细胞增殖。免疫组织化学染色的共聚焦成像表明，香烟烟雾暴露大鼠中，精胺阳性eEV从肺动脉内皮迁移到肺动脉平滑肌细胞。抑制精胺合成或敲除CaSR抑制eEV引起的肺动脉高压。香烟烟雾通过诱导精胺富集的eEV产生增加，从而激活CaSR并诱导平滑肌细胞收缩和增殖，最终直接导致肺动脉高压的发展。