Mechanisms of Neurovascular Injury in Cerebral Ischemia

描述（由申请方提供）：内皮细胞（EC）是血脑屏障（BBB）的主要细胞组分。脑缺血/再灌注（I/R）后早期发生的内皮损伤或功能障碍是脑卒中后BBB破坏和神经血管损伤发展的关键因素。阐明I/R后EC完整性和功能受损的机制是确定新的EC保护策略的重要一步，这些策略可能会减少卒中后进行性脑损伤并改善长期神经功能结局。BBB完整性的病理性破坏不可避免地与EC的结构改变有关，包括肌动蛋白细胞骨架的重组和连接蛋白的重新分布。我们最近的研究表明，EC结构的变化，I/R后，不仅是至关重要的早期（30 min-3 h）BBB泄漏到小分子，但敏感的微血管基质金属蛋白酶（MMP）介导的继发性BBB破坏和泄漏到大分子。I/R的发作在EC中启动氧化应激介导的炎症过程，其促进外周免疫细胞（包括中性粒细胞和单核细胞）的募集和浸润。由此产生的免疫细胞、促炎介质和嗜中性粒细胞衍生的蛋白酶的积累进一步促进BBB破坏和脑梗死的进展。因此，恢复EC结构，同时阻断微血管中的炎症可能为脑保护免受I/R损伤提供统一和创新的治疗策略。 过氧化物氧还蛋白4（Peroxiredoxin 4，Prx 4）是抗氧化酶家族（Prx 1 -6）中的一员。我们最近发现Prx 4仅在脑内EC中表达，并且其水平在I/R后短暂升高。然而，Prx 4在I/R后血管完整性中的功能作用及其潜在的作用机制尚未在CNS中探索。该提议将检验内皮Prx 4通过双重机制保护I/R神经血管损伤的总体假设：1）Prx 4通过以下途径稳定EC中的细胞骨架组织来防止BBB破坏的初始诱导： 一种独特的信号通路; 2）Prx 4抑制微血管中EC引发的炎症。使用转基因小鼠选择性过表达Prx 4的EC，我们已经获得了关键的证据来支持这一假设。提出了三个具体的目的来测试以下工作假设：1）Prx 4的内皮靶向过表达足以提供针对BBB损伤的保护并改善I/R后的长期结果。2)Prx 4分别通过调节ROCK/MCL/Actin信号通路和ASK 1/p38信号通路抑制BBB破坏和EC炎症反应的初始诱导。3)细胞膜可渗透的TAT-Prx 4的施用保护BBB完整性并改善中风小鼠的结果。综上所述，本研究探讨了Prx 4在脑I/R中的新的BBB保护作用和机制。这项研究的积极结果将有助于开发一种新的临床相关的治疗策略，以对抗中风及其破坏性的神经血管后遗症。

DESCRIPTION (provided by applicant): Endothelial cells (EC) are the major cellular component of the blood brain barrier (BBB). Endothelial damage or dysfunction, which occurs early after cerebral ischemic/reperfusion (I/R), contributes critically to the BBB disruption and development of neurovascular injury after stroke. Elucidation of the mechanisms by which EC integrity and function are compromised after I/R is an essential step in identifying novel EC-protecting strategies that may reduce the progressive brain damage and improve long-term neurological outcome after stroke. The pathological breach of BBB integrity is inevitably associated with structural alterations in EC, including reorganization of the actin cytoskeleton and redistribution of junctional proteins. Our recent studies revealed that the structural changes in EC after I/R not only are critical for the early (30 min-3 h) BBB leakage to smaller molecules, but sensitize microvessels to matrix metalloproteinase (MMP)-mediated secondary BBB disruption and leakage to larger molecules. An episode of I/R initiates oxidative stress-mediated inflammatory processes in EC, which facilitate the recruitment and infiltration of peripheral immune cells (including neutrophils and monocytes). The resulting accumulation of immune cells, pro-inflammatory mediators, and neutrophil-derived proteases further promote BBB disruption and the progression of brain infarct. Therefore, restoring EC structure while simultaneously blocking inflammation in microvasculature may provide a unified and innovative therapeutic strategy for brain protection against I/R injury. Peroxiredoxin 4 (Prx4) is a member in a family of antioxidant enzymes (Prx1-6). We recently discovered that Prx4 is expressed exclusively in EC in the brain, and its level is transiently elevated after I/R. However, the functional role of Prx4 in vascular integrity following I/R and its underlying mechanism of action have not been explored in CNS. This proposal will test the overarching hypothesis that endothelial Prx4 protects against I/R neurovascular injury through dual mechanisms: 1) Prx4 prevents the initial induction of BBB disruption by stabilizing cytoskeletal organization in EC via a distinctive signaling pathway; and 2) Prx4 inhibits EC-initiated inflammation in the microvasculature. Using transgenic mice overexpressing Prx4 selectively in ECs, we have obtained critical evidence to support this hypothesis. Three specific Aims are proposed to test the following working hypotheses: 1) Endothelium-targeted overexpression of Prx4 is sufficient to confer protection against BBB damage and improve long-term outcome after I/R. 2) Prx4 inhibits the initial induction of BBB disruption and the inflammatory responses in ECs via modulating the ROCK/MCL/Actin signaling pathway and the ASK1/p38 signaling pathway, respectively. 3) Administration of cell membrane-permeable TAT-Prx4 protects BBB integrity and improves outcomes in stroke mice. In sum, the proposed studies investigate novel BBB- protecting effect and mechanism of Prx4 in cerebral I/R. A positive outcome of the study will help develop a novel and clinical relevant therapeutic strategy against stroke and its devastating neurovascular sequelae.