Targeting the compromised brain endothelial barrier function during cerebral malaria with AT2 receptor agonists

摘要 加强内皮细胞的细胞间连接将促进重要的翻译应用 用于内皮完整性受损的各种疾病。作为第一个模型，我们选择了大脑 疟疾(CM)，它仍然是疟疾最致命的表现形式。它是由恶性疟原虫引起的 感染红细胞(IRBC)附着在宿主脑内皮细胞上，破坏血脑屏障。 虽然抗疟疾药物清除了血液中的寄生虫，但它们对CM没有特殊效果。 我们发现恶性疟原虫-IRBC诱导的人脑微血管内皮细胞破裂 血管紧张素II受体的激活阻止了小鼠心肌的连接和发育 类型2(AT2)。Ang II只是G蛋白偶联受体AT2的偏向激动剂，因为它不激活G-蛋白偶联受体AT2。 蛋白质通过受体。我们已经发现了真正的AT_2(EA)内源性激动剂，它激活G-αS 并防止内皮细胞完整性的破坏。基于计算机模拟和体外实验，我们 我们还发现了第一个AT2特异性非肽受体激动剂(SNPA)，它也能激活GαS。 我们假设AT2的特定细胞内信号通路的激活保护人类的大脑和 恶性疟原虫视网膜内皮细胞诱导内皮细胞间连接的破坏，从而维持 内皮功能，减少/预防水肿和出血，从而减少CM和相关的视网膜病变。 我们将首先确定哪些AT2介导的细胞内信号通路是保护内皮细胞的关键 完整性，通过测试4种不同作用的化合物：Ang II，EA，SNPA和AT2-2中的非特异性激动剂C21 转染人和小鼠脑内皮细胞及细胞内信号的定量 对屏障完整性很重要的分子。小干扰RNA对血管紧张素Ⅱ受体2的靶向抑制及药理学研究 原代人类(脑、视网膜)和小鼠(野生型和AT2缺乏)内皮细胞将识别最多 有效的脑内皮细胞保护激动剂。我们将确定激动剂相互作用的具体效果。 AT2对内皮激活、连接完整性和选定的第二信使起作用。最后，我们将测试 不同激动剂的治疗如何影响野生和大鼠CM和相关视网膜病变的结局 AT2基因缺陷的小鼠，借此对视网膜的分析为研究大脑微血管功能提供了范围。 这个项目的主要目标是确定一种能够刺激特定细胞内信号传递的先导化合物。 AT2受体介导内皮完整性的基本保护。我们的实验将奠定基础 开发一种小分子药物，立即在临床试验中进行测试，用于治疗 CM的危及生命的病理学，以及未来可能在其他出血性疾病中的应用。 这项提案是根据美国-爱尔兰研发伙伴计划提交的，该计划的重点是 开发新的治疗方法。只为美国部分申请资金，因为爱尔兰 资助机构已经承诺提供财政支持，条件是NIH做出积极的资助决定。

Summary Strengthening of inter-cellular junctions of endothelial cells would facilitate important translational applications for a variety of diseases where endothelial integrity is compromised. As a first model, we have chosen cerebral malaria (CM), which remains the deadliest manifestation of malaria. It is caused by Plasmodium falciparum infected erythrocytes (iRBC) adhering to host brain endothelial cells and compromising the blood brain barrier. While anti-malarial drugs clear parasites from the blood, they do not have specific effects against CM. We have found that P. falciparum-iRBC-induced disruption of human brain microvascular endothelial cell junctions and development of CM in mice was prevented by the activation of the angiotensin (Ang) II receptor type 2 (AT2). Ang II is only a biased agonist of the G-protein coupled receptor AT2, since it does not activate G- proteins via the receptor. We have discovered the real endogenous agonist for AT2 (EA), which activates Gαs and protects against disruption of endothelial integrity. Based on in silico modelling and in vitro experiments, we have also identified a first AT2-specific non-peptidic receptor agonist (SNPA) that also activates Gαs. We hypothesize that activation of specific intracellular signaling pathways of AT2 protects human brain and retinal endothelial cells from P. falciparum-induced disruption of inter-endothelial junctions, thus maintaining endothelial function, reducing/preventing edema and hemorrhages and thus CM and related retinopathy. We will first identify which AT2-mediated intracellular signaling pathways are key in the protection of endothelial integrity, by testing 4 differently acting compounds: Ang II, EA, SNPA and the non-specific agonist C21, in AT2- transfected cells and in human and murine brain endothelial cells and quantifying intracellular signaling molecules important in barrier integrity. A pharmacological approach and targeted inhibition of AT2 by siRNA in primary human (brain, retina) and mouse (wild-type and AT2-deficient) endothelial cells will identify the most efficient agonist in brain endothelial protection. We will determine the specific effects of the agonists interacting with AT2 on endothelial activation, junction integrity, and on selected second messengers. Finally, we will test how treatment with the different agonists affects the outcome of CM and related retinopathy in wild-type and AT2-deficient mice, whereby analysis of the retina offers scope for investigation of brain microvascular function. The main goal of this project is to identify a lead compound, which can stimulate specific intracellular signaling at the AT2 receptor to mediate essential protection of endothelial integrity. Our experiments will lay the foundation for the development of a small molecule drug to be immediately tested in clinical trials for the treatment of the life-threatening pathology of CM, with possible future applications in other hemorrhagic diseases. This proposal is submitted under the US-Ireland R&D Partnership Programme, which is focused on the development of new therapeutic approaches. Funding is requested only for the US component, since the Irish funding agencies have already committed their financial support conditional on a positive NIH funding decision.