Heat shock proteins in brain ischemia and stroke

描述（由申请人提供）： 中风是退伍军人中常见的疾病，治疗方法很少。脑缺血经常引起血脑屏障（BBB）的破坏，导致脑水肿和出血，这可能使已经是灾难性的疾病复杂化。该领域的最新研究表明，基质金属蛋白酶（MMPs），一个锌依赖性内肽酶家族，参与细胞外基质和BBB的分解，导致脑水肿和出血。我们实验室和我们合作者的工作集中在热休克蛋白的保护潜力上，即高度诱导的70 kD热休克蛋白（HSP 70）。HSP 70似乎具有细胞保护特性，其分子伴侣功能的性质，可能导致新生蛋白质折叠的增强和蛋白质聚集的预防。然而，相关领域的研究表明，热休克蛋白也可能参与MMPs的表达和加工。我们最近发现，热休克蛋白70的过度表达或其诱导热应激降低基质金属蛋白酶的表达在培养的星形胶质细胞在转录和翻译水平。除了阻止MMP的转录和翻译外，我们初步发现HSP 70还可以阻止pro-MMPs加工成其活性形式，这表明了额外的保护作用。在本申请中，我们建议进一步探索这些观察结果，即HSP 70通过抑制MMP的表达、翻译和加工来防止BBB破坏、水肿和出血，从而保护脑免受中风。目的1将使用脑源性内皮细胞和星形胶质细胞的细胞培养物来确定哪些细胞类型在各种刺激后产生HSP 70和MMPs，以及HSP 70是否可以使用体外模型防止BBB的破坏。目的2将确定HSP 70如何与MMPs相互作用。将研究缺乏功能性ATP酶或底物结合结构域的HSP 70突变体。目的3探讨热休克蛋白在脑卒中后血脑屏障完整性、脑水肿/出血中的意义，并应用脑卒中动物模型研究基质金属蛋白酶的表达。

DESCRIPTION (provided by applicant): Stroke is a common affliction among veterans, and treatments are few. Brain ischemia frequently causes disruption of the blood brain barrier (BBB) leading to brain edema and hemorrhage which can complicate an already catastrophic illness. Recent work in the field has implicated matrix metalloproteinases (MMPs), a family of Zn-dependent endopeptidases, in the breakdown of the extracellular matrix and BBB leading to brain edema and hemorrhage. Work by our labs and those of our collaborators' have focused on the protective potential of heat shock proteins, namely, the highly inducible 70 kD heat shock protein (HSP70). HSP70 appears to have cytoprotective properties by nature of its chaperone functions, presumably leading to enhancement of nascent protein folding and prevention of protein aggregation. However, work in related fields has shown that HSPs may also be involved in the expression and processing of MMPs. We recently showed that overexpression of HSP70 or its induction by heat stress reduced expression of MMPs in cultured astrocytes at the transcriptional and translational level. In addition to preventing MMP transcription and translation, we have preliminarily found that HSP70 can also prevent processing of pro-MMPs to their active form, suggesting an additional action of protection. In this application, we propose to further explore these observations that HSP70 protects the brain against stroke by preventing BBB disruption, edema and hemorrhage by inhibiting the expression, translation and processing of MMPs. Aim 1 will use cell cultures of brain derived endothelial cells and astrocytes to establish which cell types generate HSP70 and MMPs following various stimuli, and if HSP70 can prevent disruption of the BBB using in vitro models. Aim 2 will determine how HSP70 interacts with MMPs. HSP70 mutants lacking either functional ATPase or substrate binding domains will be studied. Aim 3 will explore the significance of HSPs on BBB integrity, brain edema/hemorrhage and MMPs will be studied using in vivo models of experimental stroke.