内皮源性GAPDH在缺氧性肺动脉高压血管重构中的作用及机制

This project is based on the recently discovered that hypoxia induced increasing GAPDH secretion by pulmonary artery endothelial cells and caused the serum GAPDH concentration in pulmonary hypertension rats to rise, to investigate the role of endothelial cell derived GAPDH in vascular remodeling of hypoxic pulmonary hypertension; Applicants according to a possible target protein that is exogenous pulmonary artery smooth muscle cell membrane protein interaction with GAPDH by protein mass screening and immunoprecipitation, initially selected two target proteins of HSP70, CD64 as a candidate, so as to confirm the interaction between GAPDH and target protein. In addition, the project will use integrated cells, isolated vascular strips and animal experiment system, further explore the signaling pathway of endothelial GAPDH with pulmonary artery smooth muscle cell membrane HSP70,CD64interactions, elucidate the molecular mechanism of endothelial GAPDH in vascular remodeling in pulmonary hypertension.

本项目拟在申请人新近发现的缺氧诱导肺动脉内皮细胞分泌GAPDH及肺动脉高压大鼠血清GAPDH含量增加的基础之上，探讨内皮源性GAPDH在缺氧性肺动脉高压血管重构中的作用；申请人根据蛋白质质谱及免疫共沉淀的进一步筛选外源性GAPDH与肺动脉平滑肌细胞膜蛋白相互作用的可能靶蛋白，初步选定两种靶蛋白HSP70、CD64作为候选，从而全面确认GAPDH与靶蛋白的相互作用，此外，本项目还将综合运用细胞、离体血管条和整体动物等系列实验，进一步深入探讨内皮源性GAPDH与肺动脉平滑肌细胞膜HSP70、CD64相互作用的信号通路，阐明内皮源性GAPDH在肺动脉高压血管重构中的分子机制。

近些年的研究发现细胞外 GAPDH 可通过与细胞膜上的靶蛋白相互作用等多种机制影响细胞功能。研究中我们发现，缺氧诱导肺动脉内皮细胞 GAPDH 蛋白表达增高，同时肺动脉内皮细胞向细胞外分泌的 GAPDH 也显著增加，而缺氧时平滑肌细胞GAPDH表达减少且几乎不分泌GAPDH。本项目旨在探讨内皮源性GAPDH在缺氧性肺动脉高压血管重构中的作用。根据蛋白质质谱分析外源性GAPDH与肺动脉平滑肌细胞膜钙敏感受体(CaSR)有相互作用，用免疫荧光共定位及免疫功沉淀进一步确认了GAPDH与CaSR的相互作用。在平滑肌细胞实验中发现GAPDH作用于CaSR导致细胞内钙信号改变并且导致细胞增殖。本项目发现内皮源性GAPDH与在肺动脉平滑肌细胞上CaSR相互作用参与了肺动脉高压血管重构的发生发展。