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

总结 加强内皮细胞的细胞间连接将促进重要的翻译应用 用于多种内皮完整性受损的疾病。作为第一个模型，我们选择了大脑 疟疾（CM），这仍然是疟疾最致命的表现。它是由恶性疟原虫引起的 感染的红细胞（iRBC）粘附于宿主脑内皮细胞并损害血脑屏障。 虽然抗疟疾药物可以清除血液中的寄生虫，但它们对CM没有特异性作用。 我们发现恶性疟原虫-iRBC诱导人脑微血管内皮细胞的破坏 血管紧张素（Ang）II受体的激活阻止了小鼠CM的连接和发展 2型（AT 2）。Ang II只是G蛋白偶联受体AT 2的偏向性激动剂，因为它不激活G蛋白偶联受体AT 2。 蛋白质通过受体我们发现了真实的内源性AT 2激动剂EA，它激活Gαs 并防止内皮完整性的破坏。基于计算机模拟和体外实验，我们 还鉴定了第一种AT 2特异性非肽受体激动剂（SNPA），它也能激活Gαs。 我们推测，激活特定的细胞内信号通路的AT 2保护人脑， 视网膜内皮细胞从恶性疟原虫诱导破坏内皮细胞间连接，从而维持 内皮功能，减少/预防水肿和水肿，从而减少/预防CM和相关的视网膜病变。 我们将首先确定哪些AT 2介导的细胞内信号通路在保护内皮细胞中起关键作用。 完整性，通过测试4种不同作用的化合物：Ang II，EA，SNPA和非特异性激动剂C21，在AT 2- 转染的细胞和人和小鼠脑内皮细胞中的细胞内信号传导，并定量细胞内信号传导 分子在屏障完整性中的重要性。一种药理学方法和通过siRNA靶向抑制AT 2， 原代人（脑、视网膜）和小鼠（野生型和AT 2缺陷型）内皮细胞将鉴定最多的 脑内皮保护的有效激动剂。我们将确定激动剂相互作用的具体效果 AT 2对内皮活化、连接完整性和选定的第二信使的影响。最后，我们将测试 不同激动剂治疗如何影响野生型CM和相关视网膜病变的结局， AT 2缺陷小鼠，从而分析视网膜提供了研究脑微血管功能的范围。 本项目的主要目标是确定一种先导化合物，它可以刺激特定的细胞内信号传导 在AT 2受体介导的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，未来可能应用于其他出血性疾病。 该提案是根据美国-爱尔兰研发伙伴关系计划提交的，该计划的重点是 开发新的治疗方法。由于爱尔兰仅为美国部分申请资金 资助机构已经承诺在NIH做出积极资助决定的条件下提供财政支持。

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.