Annexin A2 as a cerebrovascular therapy in traumatic brain injury

 描述(申请人提供)：脑血管后遗症是创伤性脑损伤(TBI)的重要组成部分。在急性期，血脑屏障(BBB)的破坏会导致脑水肿。在延迟期，持续的微血管功能障碍损害了血管生成和神经生成。因此，任何治疗脑外伤的尝试都必须考虑到随着时间的推移这些不断演变的脑血管机制。我们的总体假设是：(I)在急性期，重组Annexin A2结合内皮细胞膜F-肌动蛋白，从而保护内皮细胞膜的完整性，从而防止脑损伤引起的血脑屏障破坏；(Ii)在延迟期，Annexin A2通过促进血管生成促进重塑，同时增加血管衍生营养因子的分泌，促进内源性神经发生和神经修复。目的1：探讨膜联蛋白A2在脑外伤后早期脑血管保护中的作用机制。我们将在活体脑损伤模型和体外血管内皮细胞/星形胶质细胞共培养中测试血脑屏障通透性的时间分布及其与连接蛋白表达和脑水肿的相关性。变性的RA2或药物抑制剂将被用来测试RA2在与F-肌动蛋白结合、F-肌动蛋白应激纤维形成和通透性调节的信号通路中的特定作用。目的：探讨膜联蛋白A2在脑外伤后脑血管重塑中的作用机制。我们将通过免疫组织化学方法检测血管生成的时间分布。原位纤溶酶活性分析将检测血管纤溶活性；基因芯片将检测血管内皮细胞衍生的血管生成/抗血管生成以及分离的脑微血管片段中营养因子的表达谱。颅脑损伤后将进行长达三个月的长期神经功能检查。在血管内皮细胞培养中，我们将通过体外血管生成试验来测试RA2在血管生成中的作用和机制。目的：探讨膜联蛋白A2促进血管源性营养因子表达促进内源性神经发生和神经修复的机制。基因芯片将检测分离的微血管片段中神经营养因子的表达，免疫组织化学、RT-PCR和Western blotts将研究脑损伤后海马神经元的变性、不同脑区的神经再生以及脑白质随时间的损伤和修复。

 DESCRIPTION (provided by applicant): Cerebrovascular sequelae comprise a critical part of traumatic brain injury (TBI). In the acute phase, damage to the blood-brain barrier (BBB) leads to cerebral edema. In the delayed phase, persisting dysfunction in microvessels impair angiogenesis and neurogenesis. Hence, any attempt to treat TBI must take into account these evolving cerebrovascular mechanisms over time. Our overall hypothesis states that (i) in the acute phase, recombinant annexin A2 binds endothelial cell membrane F-actin that preserves endothelial integrity thus protecting against TBI-induced BBB disruption; and (ii) in the delayed phase, annexin A2 promotes remodeling with enhanced angiogenesis, and concurrently increases vascular-derived trophic factor secretion for promoting endogenous neurogenesis and neurorepair. Aim 1: Investigate mechanisms of annexin A2 in early cerebrovascular protection after TBI. We will test temporal profile of BBB permeability and correlate with junction protein expression, and brain edema in vivo TBI model and in vitro endothelial/astrocytes co-cultures. Denatured rA2 or pharmacological inhibitors will be applied to test specific roles of rA2 in binding to F-actin, F-actin stress fiber formation, and permeability modulated signaling pathways. Aim 2: Investigate mechanisms of annexin A2 in cerebrovascular remodeling after TBI. We will test temporal profile of angiogenesis by immunohistochemistry. In situ plasmin activity assay will examine vascular fibrinolytic activity; mRNA microarrays will examine expression profiles of vascular endothelium derived angiogenic/anti-angiogenic, and trophic factors in isolated cerebral microvascular fragments. Long-term neurological function will be examined for up to three months post-TBI. In endothelial cultures, we will test rA2 effects and mechanisms in angiogenesis with in vitro angiogenic assays. Aim 3: Investigate mechanisms of annexin A2 in promoting vascular-derived trophic factor expression for enhancing endogenous neurogenesis and neurorepair. mRNA microarray will examine expression of neuronal trophic factors in isolated microvascular fragments, immunohistochemistry, RT-PCR and western blots will investigate hippocampus neuron degeneration, neurogenesis in different brain areas, and white matter injury and repair over time after TBI.