GPR4 in blood brain barrier dysfunction in brain ischemia

摘要 脑微血管内皮细胞(BMEC)，通过紧密连接和粘连连接紧密连接， 为血脑屏障(BBB)提供结构基础。在人类患者和动物模型中，血脑屏障 中断与卒中结局呈正相关。发现保护内皮细胞的新机制 屏障的完整性将为更好地针对缺血再灌注诱导的治疗提供重要的见解 神经元损伤。脑缺血的一个普遍过程是大脑pH值持续下降，这意味着 质子作为一种重要的胞外信号。在以前的研究中，最强调大脑质子信号的是 一直在研究它在神经元中的作用。相比之下，很少有研究评估酸中毒是否会改变血脑屏障。 完整性，这是导致缺血诱导的神经元损伤的重要因素之一。此应用程序将重点放在 GPR4是一种质子敏感型G蛋白偶联受体，在BMEC中大量表达。使用 结合体内和体外模型，拟议的研究将确定GPR4是否介导酸 BMEC中的信号转导，并确定其在酸化和缺血条件下的下游介体。与基因 操作和药物干预，我们将确定是否删除或抑制GPR4 尤其是内皮细胞可减轻缺血诱导的BMEC功能障碍并保护大脑 缺血诱导的神经元损伤。为了更好地理解这一机制，这项研究将分析下游 并对急性分离的BMEC的转录组变化进行无偏见的分析。最后，这项研究 将使用液-质联用方法来确定GPR4的药代动力学 脑组织和血液组织中的抑制物。一旦成功完成，这项研究将提供GPR4抑制作为一种 减轻脑缺血损伤的新神经保护方法。

ABSTRACT Brain microvascular endothelial cells (BMEC), tightly connected through tight junctions and adhesions junctions, provide the structural basis for the blood-brain barrier (BBB). In both human patients and animal models, BBB disruption exhibits positive correlation with stroke outcome. Discovering novel mechanisms to protect endothelial barrier integrity will provide important insights into better therapeutic targeting of ischemia-reperfusion-induced neuronal injury. One prevalent process in brain ischemia is the prolonged reduction of brain pH, which implicates protons as an important extracellular signal. In previous studies, most emphasis on brain proton signaling has been on its role in neurons. In contrast, few studies have assessed whether acidosis alters blood-brain barrier integrity, which is one important contributor to ischemia-induced neuronal injury. This application will focus on GPR4, a proton-sensitive G protein-coupled receptor which exhibits abundant expression in BMEC. Using a combination of in vivo and in vitro models, the proposed research will determine whether GPR4 mediates acid signaling in BMEC, and identify its downstream mediator in acidotic and ischemic conditions. With genetic manipulation and pharmacological interventions, we will determine whether deleting or inhibiting GPR4 specifically in endothelial cells attenuates ischemia-induced BMEC dysfunction and protects the brain from ischemia-induced neuronal injury. To better understand the mechanism, the research will analyze downstream signaling and perform unbiased analysis of transcriptome changes from acutely isolated BMEC. Lastly, this study will use the liquid chromatography-mass spectrometry approach to determine the pharmacokinetics of a GPR4 inhibitor in brain and blood tissue. Once successfully accomplished, the study will offer GPR4 inhibition as a novel neuroprotective approach to alleviate ischemia-induced brain injury.