HDAC3非常规剪接通过Ⅳ型胶原蛋白水溶性A1亚型调控内皮间充质转化参与肺动脉高压的作用机制研究

Pulmonary hypertension (PH) is a common pathological status, characterized by the increased mean blood pressure in lung vascular system. It is related to endothelial cells (EC) or smooth muscle cells (SMC)-mediated pulmonary vascular remodeling. Among all cellular activities, endothelial-mesenchymal transition (EndMT) is one of the important mechanisms underlying pulmonary vascular remodeling. Our pilot studies revealed that histone deacetylase 3 (HDAC3) underwent unconventional splicing and one of the spliced products, HDAC3α was involved in embryonic cardiovascular system development. Moreover, when overexpressing spliced HDAC3α in VSMC, a novel 53kD soluble Collagen 4 A1 isoform (Col4A1s), designated as Col4A1s, was identified in the culture to induce EC to mesenchymal cell types. According to the results and literature review, we hypothesize that HDAC3α may regulate the production of Col4A1s in SMC of pulmonary artery of PH. Then Col4A1s induces EC undergoing EndMT, contributing to pulmonary arterial remolding. In the present project, we intend to decipher the mechanisms underlying HDAC3α-induced Col4A1s expression and the role of Col4A1s in EndMT of PH in vitro and in vivo. It may provide novel insights into the mechanisms underlying PH and opportunities for the future exploration of therapeutic target in clinics.

肺动脉高压（PH）是一种肺平均动脉压异常升高的病理生理状态。PH的发展与内皮细胞（EC）、平滑肌细胞（SMC）参与的肺血管重构有关，而内皮间充质转化（EndMT）是其中的重要形式之一。课题组前期研究发现，组蛋白去乙酰化酶3（HDAC3）非常规剪接产生的α亚型参与了胚胎心血管发育过程中的EndMT。同时，在过表达HDAC3α的肺动脉SMC培养基中可检测到一种水溶性IV型胶原蛋白A1（Col4A1s）亚型，后者可诱导EC发生EndMT。结合文献回顾及预实验结果，我们推测PH的原发或继发性病理刺激可能会激活SMC中HDAC3α的表达，继而调控Col4A1s的合成并通过旁分泌途径诱导EC发生EndMT，最终促进肺血管重构。在本课题中，我们将进一步在体外和体内试验中研究Col4A1s的产生机制及其对EndMT的具体调控通路，这将为PH血管重构发生机制提供新的理论基础，并为以后临床治疗PH提供新思路。

急性呼吸窘迫综合征（ARDS）是脓毒症发生发展过程中最常见、最严重的并发症之一。由脓毒症所致ARDS病死率高达70%以上。ARDS的典型特征之一为肺动脉高压（PH）。PH的发展与内皮细胞（EC）、平滑肌细胞（SMC）参与的肺血管重塑有关，而内皮间充质转化（EndMT）是重要的病理机制。课题组前期研究发现，组蛋白去乙酰化酶3（HDAC3）非常规剪接产生的α亚型参与了胚胎心血管发育过程中的EndMT。同时，在过表达HDAC3α的肺动脉SMC培养基中可检测到一种可溶性IV型胶原蛋白A1（Col4A1s）亚型，后者可诱导EC发生EndMT。本项目在前期研究的基础上，首先在脓毒症相关的ARDS小鼠模型及LPS处理的肺血管内皮细胞（HPMEC）中初步证实了EndMT、HDAC3α和Col4A1s参与脓毒症相关ARDS引起的PH。之后，设计Flag标记的Flag-Col4A1s转染质粒，以及可以拮抗Col4A1s功能的α-Flag琼脂糖磁珠，在体外实验证实了Col4A1s的分泌可以诱导HPMEC发生EndMT。Western blot和Co-IP实验发现HDAC3α通过XBP1s介导的可变剪切上调Col4A1s。通过构建过表达GFP-ITGB6、GFP-ITGB8和Flag-Col4A1s的HPMEC，采用ELISA和Co-IP技术证实了Col4A1s通过与整合素αvβ6（ITGB6）结合，激活细胞外基质中的TGFβ1前体，进而调控EndMT的发生。综上，本研究为脓毒症相关ARDS引起的肺动脉高压的发生发展的分子机制提供了新的基础证据，同时为今后临床靶向治疗脓毒症相关ARDS提供了重要的理论依据和线索。