Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China.
Key Laboratory of Cardiovascular & Cerebrovascular Medicine, School of Pharmacy, Nanjing Medical University, Nanjing, 211166, China.
Theranostics. 2019 Aug 14;9(20):5937-5955. doi: 10.7150/thno.34168. eCollection 2019.
Prolonged occlusion of multiple microvessels causes microvascular injury. G protein-coupled receptor 124 (GPR124) has been reported to be required for maintaining central nervous system (CNS) angiogenesis and blood-brain barrier integrity. However, the molecular mechanisms by which GPR124 regulates pericytes during ischemia have remained elusive. : A microsphere embolism-induced ischemia model was used to evaluate the expression of GPR124 following microsphere embolism. Immunocytochemistry and stochastic optical reconstruction microscopy imaging were used to assess the expression and distribution of GPR124 in human brain vascular pericytes (HBVPs) and after the treatment with 3-morpholino-sydnonimine (SIN-1) or oxygen-glucose deprivation (OGD). The effect of GPR124 knockdown or overexpression on HBVP migration was analyzed in vitro using wound healing assays and a microfluidic device. GPR124 loss-of-function studies were performed in HBVPs and HEK293 cells using CRISPR-Cas9-mediated gene deletion. Time-lapse imaging was used to assess dynamic changes in the formation of filopodia in an individual cell. Finally, to explore the functional domains required for GPR124 activity, deletion mutants were constructed for each of the N-terminal domains. : GPR124 expression was increased in pericytes following microsphere embolism. Morphological analysis showed localization of GPR124 to focal adhesions where GPR124 bound directly to the actin binding protein vinculin and upregulated Cdc42. SIN-1 or OGD treatment redistributed GPR124 to the leading edges of HBVPs where GPR124 signaling was required for pericyte filopodia formation and directional migration. Partial deletion of GPR124 domains decreased SIN-1-induced filopodia formation and cell migration. : Taken together, our results provide the first evidence for a role of GPR124 in pericyte migration under ischemic conditions and suggest that GPR124 was essential for Cdc42 activation and filopodia formation.
多种微血管的长时间阻塞会导致微血管损伤。已经报道 G 蛋白偶联受体 124（GPR124）对于维持中枢神经系统（CNS）血管生成和血脑屏障完整性是必需的。然而，GPR124 在缺血期间调节周细胞的分子机制仍然难以捉摸。
使用微球栓塞诱导的缺血模型来评估微球栓塞后 GPR124 的表达。免疫细胞化学和随机光学重建显微镜成像用于评估 GPR124 在人脑血管周细胞（HBVPs）中的表达和分布，以及在用 3-吗啉代- sydnonimine（SIN-1）或氧葡萄糖剥夺（OGD）处理后的表达和分布。使用划痕愈合测定和微流控装置分析 GPR124 敲低或过表达对 HBVP 迁移的影响。使用 CRISPR-Cas9 介导的基因缺失在 HBVPs 和 HEK293 细胞中进行 GPR124 功能丧失研究。使用延时成像来评估单个细胞中丝状伪足形成的动态变化。最后，为了探索 GPR124 活性所需的功能域，构建了每个 N 端结构域的缺失突变体。
GPR124 在微球栓塞后周细胞中的表达增加。形态分析表明，GPR124 定位于焦点黏附处，GPR124 直接与肌动蛋白结合蛋白 vinculin 结合，并上调 Cdc42。SIN-1 或 OGD 处理将 GPR124 重定位到 HBVPs 的前缘，GPR124 信号对于周细胞丝状伪足形成和定向迁移是必需的。GPR124 结构域的部分缺失减少了 SIN-1 诱导的丝状伪足形成和细胞迁移。
综上所述，我们的研究结果首次提供了 GPR124 在缺血条件下调节周细胞迁移的作用证据，并表明 GPR124 对于 Cdc42 的激活和丝状伪足的形成是必需的。
