Heat shock proteins and neuroprotection in cerebral ischemia

描述(由申请人提供)： 摘要中风是美国退伍军人的主要医疗问题。血脑屏障(BBB)破坏是脑缺血/再灌注(I/R)损伤发病机制中的灾难性事件。我们最近的研究表明，脑内皮细胞(EC)的结构变化，包括肌动蛋白聚合异常和由此导致的连接蛋白的重新分布，是导致I/R(30分钟-3小时)早期BBB泄漏的新机制；而基质金属蛋白酶(MMP2/9)激活，被认为是导致卒中后BBB破坏的主要机制，以相对延迟的方式导致BBB泄漏到更大的分子(40 KDa)。因此，恢复EC的结构/功能可能为早期BBB的I/R保护提供一种创新的治疗策略，而阻断MMP2/9可能对BBB提供相对延迟的保护。热休克蛋白27(HSP27)是小分子热休克蛋白家族的一员，在包括脑I/R损伤在内的多种中枢神经系统疾病模型中具有神经保护作用。除了其已知的蛋白质伴侣和抗凋亡功能外，HSP27还可能在某些细胞类型(如EC)中发挥强大的肌动蛋白解聚因子的作用，从而潜在地抑制肌动蛋白聚合介导的BBB破坏。此外，HSP27可能抑制核因子-kappaB的激活，核因子-kappaB是产生MMP2/9和促炎介质的中央信号分子。我们最近观察到HSP27在脑I/R后的EC中表达短暂上调，然而，HSP27在I/R后EC功能完整性中的确切作用和潜在机制仍不清楚。利用过表达HSP27的转基因小鼠和慢病毒基因转染法，我们获得了新的初步数据，表明HSP27过表达在体内外保护内皮免受I/R诱导的高通透性；HSP27抑制氧葡萄糖剥夺诱导的肌动蛋白聚合和连接蛋白在EC中的重新分布；静脉注射含有细胞可渗透转导结构域的HSP27(TAT-HSP27)可以使蛋白快速进入脑微血管，减少BBB损伤并抑制MMP2/9活性。本研究将进一步探讨HSP27对I/R脑损伤的血脑屏障保护作用并阐明其可能的机制。需要验证的中心假设是，HSP27通过稳定I/R后早期微血管内皮细胞骨架组织，抑制MMP2/9和促炎蛋白的产生，保护BBB免受I/R损伤。目标1：确定内皮靶向过表达HSP27是否足以提供对血脑屏障完整性的早期保护和对脑I/R的长期保护。目标2：验证HSP27通过双重机制保护I/R后内皮完整性的假说：稳定肌动蛋白细胞骨架和 抑制NFkB依赖的MMP2/9的产生和炎症。目的3：验证卒中后向EC传递TAT-HSP27对BBB损伤具有保护作用并改善青年和老年小鼠预后的假说。这项拟议的研究试图将HSP27开发成一种新的、临床上可行的治疗策略，以改善中风患者的中风后血脑屏障损害，阻止脑损伤的进展，并改善中风患者的长期神经功能。这项拟议研究的成功完成将有助于改善中风退伍军人的生活质量。

DESCRIPTION (provided by applicant): Abstract Stroke is a major medical concern for United States military veterans. Disruption of the blood-brain barrier (BBB) is a catastrophic event in the pathogenesis of ischemic/reperfusion (I/R) brain injury. Our recent studies suggested that structural alterations in brain endothelial cells (EC), including abnormal actin polymerization and the resulting redistribution of junctional proteins, is a novel mechanism responsible for early BBB leakage after I/R (30 min-3h); while matrix metalloproteinase (MMP) 2/9 activation, a predominant mechanism thought to contribute to post-stroke BBB disruption, contribute to the BBB leakage to larger molecules (>40kDa) in a relatively delayed manner. Therefore, restoring EC structure/function may offer an innovative therapeutic strategy for early BBB protection against I/R, while blocking MMP2/9 may provide a relatively delayed protection to BBB. Heat shock protein 27 (HSP27), a member of the small heat shock protein family, confers neuroprotection in several models of CNS diseases, including I/R brain injury. In addition to its well-known protein chaperone and anti-apoptotic functions, HSP27 may act as a potent actin depolymerization factor in certain cell types such as EC, thus potentially inhibiting actin polymerization-mediated BBB disruption. Further, HSP27 may inhibit the activation of NF-kappaB, a central signaling molecule for the production of MMP2/9 and pro-inflammatory mediators. We recently observed that HSP27 is transiently upregulated in brain EC after I/R. However, the precise role of HSP27 in the functional integrity of EC following I/R and the underlying mechanism remain unknown. Using transgenic mice overexpressing HSP27 and the lentiviral gene-transfection approach, we have obtained novel pilot data showing that HSP27 overexpression protects the endothelium from I/R-induced hyperpermeability in vitro and in vivo; that HSP27 inhibited oxygen glucose deprivation-induced actin polymerization and redistribution of junctional proteins in EC; and that intravenous administration of HSP27 containing a cell permeable transduction domain (TAT-HSP27) enabled rapid delivery of the protein into brain micro-vasculatures, reduced BBB damage and inhibited MMP2/9 activity after I/R. This proposal will further explore the BBB protective effect of HSP27 on I/R brain injury and elucidate the underlying mechanisms. The central hypothesis to be tested is that HSP27 protects BBB against I/R injury by stabilizing EC cytoskeletal organization in microvasculature early after I/R and inhibiting the production of MMP2/9 as well as pro-inflammatory proteins. The following specific aims are proposed: Aim 1: To determine whether endothelial targeted overexpression of HSP27 is sufficient to provide early protection on BBB integrity and to confer long term protection against cerebral I/R. Aim 2: Test the hypothesis that HSP27 protects endothelial integrity following I/R by dualistic mechanisms: stabilizing the actin cytoskeleton and inhibiting NFkB-dependent MMP2/9 production and inflammation. Aim 3: Test the hypothesis that post-stroke delivery of TAT-HSP27 to EC protects against BBB damage and improves outcomes in both young adult and aged mice. The proposed study attempts to develop HSP27 into a novel, clinically feasible therapeutic strategy to ameliorate post-stroke BBB damage, brake down the progression of brain damage, and improve long-term neurological functions in stroke victims. The successful completion of this proposed study will help improve the quality of life for veterans suffering from stroke.