Mechanisms of Neurovascular Injury in Cerebral Ischemia

描述(申请人提供)：内皮细胞(EC)是血脑屏障(BBB)的主要细胞成分。脑缺血/再灌注(I/R)后早期发生的内皮损伤或功能障碍是卒中后血脑屏障破坏和神经血管损伤发展的重要因素。阐明I/R后EC完整性和功能受损的机制是确定新的EC保护策略的关键一步，这些策略可能减少进行性脑损伤，改善卒中后的长期神经预后。血脑屏障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，I/R后EC的结构变化不仅是早期(30min-3h)BBB渗漏到小分子的关键，而且使微血管对基质金属蛋白酶(MMPs)介导的继发性BBB破坏和渗漏到大分子敏感。I/R发作在EC启动氧化应激介导的炎症过程，促进外周免疫细胞(包括中性粒细胞和单核细胞)的募集和渗透。由此产生的免疫细胞、促炎介质和中性粒细胞衍生的蛋白酶的聚集进一步促进了血脑屏障的破坏和脑梗塞的进展。因此，恢复EC结构的同时阻断微血管的炎症反应可能为脑I/R损伤的脑保护提供一种统一的、创新的治疗策略。过氧化还蛋白4(Prx4)是抗氧化酶(Prx1-6)家族中的一员。我们最近发现Prx4仅在脑内皮细胞中表达，并且在I/R后其水平一过性升高。然而，Prx4在I/R后血管完整性中的功能作用及其潜在的作用机制在中枢神经系统中尚未被探索。这一提议将检验内皮Prx4通过双重机制保护I/R神经血管损伤的总体假设：1)Prx4通过稳定EC的细胞骨架组织来防止最初的BBB破坏 一条独特的信号通路；以及2)Prx4抑制EC在微血管系统中启动的炎症。利用在内皮细胞中选择性高表达Prx4的转基因小鼠，我们已经获得了支持这一假说的关键证据。我们提出了三个特定的目标来验证以下工作假说：1)Prx4的内皮靶向性过表达足以保护I/R后的BBB损伤并改善长期预后。2)Prx4分别通过调节ROCK/MCL/Actin信号通路和ASK1/p38信号通路来抑制ECs最初诱导的BBB破坏和炎症反应。3)给予细胞膜通透性的TAT-Prx4可保护中风小鼠血脑屏障的完整性，并改善预后。综上所述，本研究旨在探讨Prx4在脑I/R中新的BBB保护作用及其机制。该研究的积极结果将有助于开发一种针对中风及其破坏性神经血管后遗症的新的临床相关治疗策略。

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.