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: 10528244
• 负责人: ANA RODRIGUEZ
• 金额: $ 9.79万
• 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/10528244
• 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处将有助于重要的翻译应用 对于各种疾病，内皮完整性受到损害。作为第一个模型，我们选择了大脑 疟疾（CM），它仍然是疟疾的最致命表现。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。 尽管抗疟疾药从血液中清除寄生虫，但它们对商业没有特定的影响。 我们发现恶性疟原虫-IRBC诱导的人脑微血管内皮细胞的破坏 血管紧张素（ANG）II接收器的激活阻止了小鼠中CM的连接和发育 类型2（AT2）。 ANG II只是G蛋白偶联受体AT2的偏置激动剂，因为它不会激活G- 通过受体的蛋白质。我们发现了AT2（EA）的实际内源性激动剂，该激活GαS 并防止破坏内皮完整性。基于计算机建模和体外实验，我们 还鉴定了一个AT2特异性的非肽受体激动剂（SNPA），该非肽受体激动剂（SNPA）也激活Gα。 我们假设AT2的特定细胞内信号通路的激活保护人脑和 恶性疟原虫引起的视网膜内皮细胞诱导的内皮间接头破坏，从而维持 内皮功能，减少/预防水肿和出血，从而减少CM和相关视网膜病。 我们将首先确定哪种AT2介导的细胞内信号通路是保护内皮的关键 完整性，通过测试4种不同作用化合物：ANG II，EA，SNPA和非特异性激动剂C21，在AT2-中 转染的细胞以及人和鼠脑内皮细胞中的细胞和细胞内信号传导 分子在屏障完整性中很重要。 siRNA在中的药物方法和针对抑制AT2 原发性人（脑，视网膜）和小鼠（野生型和AT2缺乏）内皮细胞将确定最多 在脑内皮保护方面有效的激动剂。我们将确定激动剂相互作用的特定影响 在内皮激活，连接完整性和选定的第二个使者上使用AT2。最后，我们将测试 用不同激动剂治疗如何影响野生型和相关视网膜病的结果 AT2缺乏小鼠，对视网膜的分析提供了脑微血管功能投资的范围。 该项目的主要目标是识别铅化合物，该化合物可以刺激特定的细胞内信号传导 在AT2受体中介导内皮完整性的基本保护。我们的实验将奠定基础 为了开发小分子药物，将在临床试验中立即测试以治疗 商业危及生命的病理，并在其他出血疾病中可能采用未来的应用。 该建议是根据美国 - 爱尔兰研发合作计划提交的，该计划的重点是 开发新的治疗方法。由于爱尔兰 资金机构已经在NIH的积极资助决定中提出了有条件的财政支持。