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

总结。脑型疟疾(CM)仍然是疟疾最致命和最致命的表现形式。它是 由恶性疟原虫感染的红细胞(IRBC)黏附宿主脑内皮细胞引起 破坏血脑屏障。虽然现有的抗疟疾药物在清除寄生虫方面是有效的 从血液中看，它们对CM没有特定的作用。 我们发现恶性疟原虫-IRBC可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型受体(AT2)来阻止连接，实现 保护内皮细胞的完整性。我们还发现AT2激动剂对实验性CM有保护作用， 而AT2受体缺陷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(mA和mRgD)扩展了肾素-血管紧张素系统 被类似或等于AT2激动剂的激动剂激活，如C21、Ang-(1-7)和Ang-(1-9)。我们还可以 表明这些化合物对恶性疟原虫诱导的内皮完整性破坏具有保护作用。 因此，我们假设肾素受益臂的一个、两个或全部三个受体的激活- 血管紧张素系统保护脑内皮细胞免受寄生虫引起的屏障功能的破坏。 为了确定哪些受体(S)在保护内皮完整性中起关键作用，我们将首先量化 Ang-(1-9)、Ang-(1-7)和C21在转导受体的HEK293细胞及人和小鼠脑内皮细胞中的表达 细胞内信号分子，已知受激动剂调节，在 维持内皮细胞屏障的完整性。针对不同血管紧张素受体的受体阻滞剂将是 测试它们阻断保护性Ang多肽/类似物诱导的细胞内信号的能力。 原代内皮细胞也将被针对AT2和/或mAs和/或mrgD的siRNA靶向，以识别 负责信号效应的受体(S)。最后，从小鼠脑内皮细胞中分离出原代细胞 缺乏一个、两个或三个受体将被用作遗传模型，以证实从 药理实验和siRNA实验。然后我们将确定参与保护的受体 恶性疟原虫诱导的脑内皮细胞Ang-(1-9)、Ang-(1-7)和C21的介导 颠覆。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的影响以及对所选秒的影响 信使。最后，我们将测试Ang-(1-9)、Ang-(1-7)和C21治疗对CM结局的影响 在野生型和遗传缺陷小鼠中，三种受体中的一种以及双重和三重敲除。 该项目的主要目标是确定介导内皮保护的血管紧张素受体(S) 在CM过程中的完整性。我们的结果将为开发针对该受体的激动剂奠定基础(S) 作为CM的辅助治疗，以及可能用于脑血管内皮完整性受损的其他疾病。

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.