Annexin A2 as a cerebrovascular therapy in traumatic brain injury

 描述（由申请方提供）：脑血管后遗症是创伤性脑损伤（TBI）的关键部分。在急性期，血脑屏障（BBB）的损伤导致脑水肿。在延迟期，微血管的持续功能障碍损害血管生成和神经发生。因此，任何治疗TBI的尝试都必须考虑到这些随时间推移而演变的脑血管机制。我们的总体假设表明：（i）在急性期，重组膜联蛋白A2结合内皮细胞膜F-肌动蛋白，保持内皮完整性，从而保护免受TBI诱导的BBB破坏;（ii）在延迟期，膜联蛋白A2促进重塑，增强血管生成，同时增加血管源性营养因子分泌，促进内源性神经发生和神经修复。目的1：探讨膜联蛋白A2在颅脑损伤后早期脑血管保护中的作用机制。我们将在体内TBI模型和体外内皮细胞/星形胶质细胞共培养物中测试BBB渗透性的时间曲线，并与连接蛋白表达和脑水肿相关。变性rA 2或药理学抑制剂将用于测试rA 2在与F-肌动蛋白结合、F-肌动蛋白应力纤维形成和渗透性调节信号传导途径中的特定作用。目的2：探讨膜联蛋白A2在颅脑损伤后脑血管重构中的作用机制。我们将通过免疫组织化学检测血管生成的时间分布。原位纤溶酶活性测定将检查血管纤溶活性; mRNA微阵列将检查分离的脑微血管片段中血管内皮衍生的血管生成/抗血管生成因子和营养因子的表达谱。TBI后将检查长期神经功能长达三个月。在内皮细胞培养中，我们将测试rA 2在血管生成中的作用和机制与体外血管生成试验。目标3：研究膜联蛋白A2促进血管源性营养因子表达以增强内源性神经发生和神经修复的机制。mRNA微阵列将检测分离的微血管片段中神经元营养因子的表达，免疫组织化学、RT-PCR和蛋白质印迹将研究TBI后海马神经元变性、不同脑区的神经发生以及白色物质损伤和修复。

 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.