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

使用 AT2 受体激动剂针对脑型疟疾期间受损的脑内皮屏障功能

• 批准号: 10312024
• 负责人: ANA RODRIGUEZ
• 金额: $ 43.55万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-15 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10312024
• 关键词: AGTR2 gene; Adherence; Affect; Agonist; Angiotensin II; Angiotensin Receptor; Antimalarials; Apoptosis; Biological; Blood; Blood - brain barrier anatomy; Blood Vessels; Blood flow; Blood-Retinal Barrier; Brain; Caliber; Cells; Cerebral Malaria; Clinical Treatment; Clinical Trials; Correlation Studies; Dependovirus; Development; Disease; Edema; Electrical Resistance; Embryo; Endothelial Cells; Endothelium; Erythrocytes; Financial Support; Fingerprint; Foundations; Funding; Funding Agency; Future; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Genetic Models; Goals; Hemorrhage; Human; In Vitro; Intercellular Junctions; Intervention; Investigation; Ireland; Ischemia; Knockout Mice; Lead; Leukocytes; Life; Ligands; Malaria; Maps; Measures; Mediating; Modeling; Movement; Mus; Outcome; Parasites; Pathogenesis; Pathology; Pathway interactions; Patients; Permeability; Pharmaceutical Preparations; Pharmacology; Phosphorylation; Plasmodium falciparum; Pre-Clinical Model; Proteins; Proteomics; Publications; Reporting; Retina; Retinal Diseases; Retinal Hemorrhage; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; Stroke; Syndrome; Testing; Therapeutic; Tight Junctions; Tissues; Trees; Type 2 Angiotensin II Receptor; United States National Institutes of Health; Virus Diseases; Wild Type Mouse; Work; base; beta catenin; blood-brain barrier disruption; brain endothelial cell; capillary bed; cross reactivity; electric impedance; endothelial repair; experimental study; in silico; in vitro Model; in vivo; kidney cell; monocyte; monolayer; neurovascular unit; novel therapeutic intervention; overexpression; prevent; prognostic; programs; receptor; receptor expression; repair function; research and development; response; small molecule; therapeutic target; tool; translational applications; treatment comparison

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.

概括 加强内皮细胞的细胞间连接将促进重要的转化应用 用于内皮完整性受损的多种疾病。作为第一个模型，我们选择了大脑 疟疾（CM），仍然是疟疾最致命的表现形式。它是由恶性疟原虫引起的 受感染的红细胞 (iRBC) 粘附在宿主脑内皮细胞上并损害血脑屏障。 虽然抗疟疾药物可以清除血液中的寄生虫，但它们对 CM 没有特效。 我们发现恶性疟原虫-iRBC 诱导人脑微血管内皮细胞破坏 血管紧张素 (Ang) II 受体的激活可阻止小鼠 CM 的连接和发展 2 型（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 通过受体的蛋白质。我们发现了 AT2 真正的内源性激动剂 (EA)，它可以激活 Gαs 并防止内皮完整性受到破坏。基于计算机模拟和体外实验，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 特定细胞内信号通路的激活可以保护人类大脑和 来自恶性疟原虫的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对于保护内皮细胞至关重要。 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 AT2- 转染细胞以及人和鼠脑内皮细胞中的细胞并量化细胞内信号传导 对屏障完整性很重要的分子。 siRNA 对 AT2 的药理学方法和靶向抑制 原代人类（大脑、视网膜）和小鼠（野生型和 AT2 缺陷型）内皮细胞将识别最 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 AT2 对内皮激活、连接完整性和选定的第二信使的影响。最后我们来测试一下 不同激动剂的治疗如何影响野生型和相关视网膜病变的 CM 和相关视网膜病变的结果 AT2 缺陷小鼠，视网膜分析为研究大脑微血管功能提供了空间。 该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 AT2 受体介导内皮完整性的重要保护。我们的实验将奠定基础 开发一种小分子药物，并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-爱尔兰研发合作伙伴计划提交的，该计划的重点是 开发新的治疗方法。仅美国部分需要资金，因为爱尔兰 资助机构已经承诺以 NIH 积极的资助决定为条件提供财政支持。