Regulation of brain endothelial integrity by angiotensin receptors as treatment for cerebral malaria

概括。脑型疟疾（CM）仍然是疟疾中毒性最强、最致命的表现形式。它是 由粘附在宿主脑内皮细胞上的恶性疟原虫感染的红细胞（iRBC）引起 损害血脑屏障。虽然现有的抗疟疾药物可以有效清除寄生虫 从血液来看，它们对 CM 没有特异性作用。 我们发现恶性疟原虫-iRBC 会诱导人脑微血管内皮细胞的破坏 通过激活血管紧张素 (Ang) II 受体 2 型 (AT2) 来阻止连接，从而实现 保护内皮完整性。我们还发现 AT2 激动剂对实验性 CM 具有保护作用， 缺乏 AT2 受体的小鼠更容易患上这种综合征。然而，在过去的几年里，我们 通过识别额外的受体（Mas 和 MrgD）扩展了肾素-血管紧张素系统 由类似于或等于 AT2 激动剂的激动剂激活，例如 C21、Ang-(1-7) 和 Ang-(1-9)。我们还可以 表明这些化合物可以防止恶性疟原虫引起的内皮完整性破坏。 因此，我们假设肾素有益臂的一个、两个或全部三个受体的激活 血管紧张素系统可保护脑内皮细胞免受寄生虫引起的屏障功能破坏。 为了确定哪些受体对于保护内皮完整性至关重要，我们将首先量化 受体转染的 HEK293 细胞以及人和小鼠脑内皮细胞中的 Ang-(1-9)、Ang-(1-7) 和 C21 细胞内信号分子，已知受激动剂调节，并且在细胞内信号分子中很重要 维持内皮屏障的完整性。针对不同血管紧张素受体的受体阻滞剂将 测试了它们阻断由保护性血管紧张肽/类似物诱导的细胞内信号传导的能力。 原代内皮细胞也将被针对 AT2 和/或 Mas 和/或 MrgD 的 siRNA 靶向，以鉴定 负责信号传导作用的受体。最后，从小鼠体内分离出原代脑内皮细胞 缺乏一种、两种或三种受体的基因将被用作遗传模型来证实从 药理学和siRNA实验。然后我们将识别参与保护的受体 由恶性疟原虫诱导的脑内皮细胞的 Ang-(1-9)、Ang-(1-7) 和 C21 介导 扰乱。我们将使用受体拮抗剂和 siRNA 的药理学方法来对抗 受体以确定它们对内皮激活和连接完整性以及选定的第二个的影响 使者。最后，我们将测试 Ang-(1-9)、Ang-(1-7) 和 C21 治疗如何影响 CM 的结果 在野生型和遗传缺陷小鼠中，三种受体之一以及双重和三重敲除。 该项目的主要目标是鉴定介导内皮保护的血管紧张素受体 CM 期间的完整性。我们的结果将为开发针对该受体的激动剂奠定基础 作为 CM 和潜在的脑内皮完整性受损的其他疾病的辅助治疗。

Summary. Cerebral malaria (CM) remains the most virulent and 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 available anti-malarial drugs are effective at clearing 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 was prevented by the activation of the angiotensin (Ang) II receptor type 2 (AT2), achieving protection of endothelial integrity. We also found that AT2 agonists are protective against experimental CM, and mice deficient in the AT2 receptor are more susceptible to this syndrome. However, over the last years, we have expanded the renin-angiotensin system by identifying additional receptors (Mas and MrgD) that are activated by agonists similar or equal to AT2 agonists, such as C21, Ang-(1-7) and Ang-(1-9). We could also show that these compounds are protective against P. falciparum-induced disruption of endothelial integrity. Thus, we hypothesize that activation of one, two, or all three receptors of the beneficial arm of the renin- angiotensin system protects brain endothelial cells from parasite-induced disruption of their barrier function. To identify which receptor(s) are key in the protection of endothelial integrity, we will first quantify the effect of Ang-(1-9), Ang-(1-7) and C21 in receptor-transfected HEK293 cells and in human and murine brain endothelial cells on intracellular signaling molecules, known to be regulated by the agonists and important in the maintenance of endothelial barrier integrity. Receptor blockers targeting different angiotensin receptors will be tested for their ability to block the intracellular signaling induced by the protective Ang peptides/analogues. Primary endothelial cells will be also targeted with siRNA against AT2 and/or Mas and/or MrgD to identify the responsible receptor(s) for the signaling effects. Finally, primary brain endothelial cells isolated from mice deficient in one, two or three receptors will be used as a genetic model to confirm the conclusions made from the pharmacological and siRNA experiments. We will then identify the receptors involved in the protection mediated by Ang-(1-9), Ang-(1-7), and C21 of brain endothelial cells from P. falciparum-induced endothelial disruption. We will use a pharmacological approach with the receptor antagonists and siRNA against the receptors to determine their effects on endothelial activation and junction integrity and on selected second messengers. Finally, we will test how treatment with Ang-(1-9), Ang-(1-7) and C21 affects the outcome of CM in wild-type and genetically deficient mice in one of the three receptors as well as double and triple knockouts. The main goal of this project is to identify the angiotensin receptor(s) that mediate protection of endothelial integrity during CM. Our results will lay the foundation for the development of agonists against this receptor(s) as adjunct therapy for CM and potentially for other diseases where brain endothelial integrity is compromised.