The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325027, China; The Affiliated Kangning Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325007, China; School of Mental Health, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China.
Institute of Comparative Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China.
Biochim Biophys Acta Gen Subj. 2019 Jun;1863(6):1079-1087. doi: 10.1016/j.bbagen.2019.04.001. Epub 2019 Apr 4.
The endothelial cell (EC) barrier disruption has been implicated in vascular leakage and pulmonary edema. Many reports have shown that the EC barrier dysfunction is regulated by the sphingosine-1-phophate (S1P)/S1P receptor-1 (S1PR1) axis. Identifying downstream effectors for the S1P/S1PR1 axis in pulmonary vasculature has been limited by mixed populations in vitro cultures that do not retain physiological EC phenotype and complex of tedious proteomics. In this study, we used a combination of in vivo biotinylation and liquid chromatograph tandem mass spectrometry on three mouse models of S1pr1 expression, namely normal, knockout (KO) and high, to identify EC membrane proteins whose cell-surface expression is S1pr1-dependent. EC-specific KO of S1pr1 caused severe pulmonary vascular disruption and reduction of many membrane proteins on ECs. Using the MaxQuant software we were able to identify novel membrane targets of S1pr1, for instance, Cd105 and Plvap, by comparison with their membrane expressions among the three EC model systems. Moreover, regulation of Cd105 and Plvap by S1pr1 were validated with Western blot and immunostaining in vivo and in vitro. Our data suggest that S1pr1 dictates cell-surface localization of several apical membrane proteins in ECs. Our results are insightful for development of novel therapeutics to specifically target EC barrier function.
内皮细胞（EC）屏障的破坏与血管渗漏和肺水肿有关。许多报道表明，EC 屏障功能障碍受鞘氨醇-1-磷酸（S1P）/S1P 受体-1（S1PR1）轴的调节。由于体外培养的细胞群体混杂，不能保留生理 EC 表型和繁琐的蛋白质组学，因此，确定肺血管中 S1P/S1PR1 轴的下游效应物受到限制。在这项研究中，我们使用了三种 S1pr1 表达的小鼠模型的体内生物素化和液相色谱串联质谱联用的方法，鉴定了 S1pr1 依赖性细胞表面表达的 EC 膜蛋白。S1pr1 的 EC 特异性敲除导致严重的肺血管破坏和许多 EC 上的膜蛋白减少。通过与三种 EC 模型系统中的膜表达进行比较，我们使用 MaxQuant 软件能够鉴定 S1pr1 的新的膜靶标，例如 Cd105 和 Plvap。此外，我们通过体内和体外实验验证了 S1pr1 对 Cd105 和 Plvap 的调节作用。我们的数据表明，S1pr1 决定了 EC 中几个顶膜蛋白的细胞表面定位。我们的研究结果为开发针对 EC 屏障功能的新型治疗方法提供了有价值的见解。
