Therapeutic Targeting of the Class IB PI3-Kinase Gamma for Treatment of Acute Ischemic Stroke

描述(由申请人提供)：针对急性缺血性中风的治疗靶向IB PI3-激酶伽玛。重组tPA仍然是FDA批准的唯一和最有益的已证实的治疗缺血性中风的干预措施。然而，tPA的临床应用受到限制，因为它的治疗窗口很窄，而且出血转化(HT)的风险增加。在这项建议中，我们假设AS605240单独或与tPA联合治疗抑制PI3Kg可以保护缺血和tPA诱导的脑损伤和HT，并增加tPA治疗缺血性中风的治疗窗口。我们这一想法的分子基础是，PI3Kg的基因缺失不仅可以减少卒中所致的血脑屏障破坏和组织梗塞，而且几乎完全阻断了卒中后6小时给予tPA所致的脑出血。我们的数据是有希望的，提示PI3Kg在tPA诱导的高血压和缺血性卒中后的脑损伤中起着关键作用。此外，我们的试点数据显示，用AS605240抑制PI3Kg可显著防止脑损伤，并减少大鼠栓塞性中风后缺血性脑中的纤维蛋白沉积/血栓形成。这些令人兴奋的初步数据表明，PI3Kg可能是治疗急性缺血性卒中的一个有前途的治疗靶点。在此应用程序中，我们建议开发一种 通过靶向PI3Kg和AS605240治疗急性缺血性中风的新疗法，并研究AS605240介导的有益效果的分子机制。我们现在提出三个目标来研究这种新的中风疗法。在目标1中，我们将确定AS605240单独和联合tPA在大鼠局灶性栓塞性卒中后6小时治疗的效果。我们将确定AS605240单独以及与tPA联合治疗急性卒中的最佳剂量和延长的治疗时间窗口。脑梗塞、神经功能障碍、脑出血和动物死亡率将在中风后72小时进行检查。在目标2中，我们将确定AS605240单独或与tPA联合治疗急性卒中的有益效果的机制。血脑屏障损伤将通过关键血脑屏障蛋白的时空分析以及与脑血管上核因子-κB、CD147、MMP9和/或MMP3激活/表达变化的潜在相关性来评估。通过分析脑血管壁及其周围的纤维蛋白沉积和血栓前因子(PAI-1、组织因子-TF)在缺血脑中的表达，来评估中风后缺血脑中的血栓形成。在目标3中，我们将评估AS605240单独以及在卒中栓塞术后6小时联合tPA治疗的长期结果。动物将在5周内接受一系列行为测试。最后取脑测量病变大小、纤维蛋白沉积/血栓形成、脑血管密度/效力及其与促血管生成因子(VEGF、Ang-1)和促血栓形成因子(PAI-1、组织因子-TF)表达的相关性。如果成功，这些拟议的实验将为IB类PI3Kg信号如何在缺血和tPA诱导的高血压和脑损伤中发挥作用提供新的见解，并最终可能产生治疗急性缺血性中风的新疗法，并将tPA对缺血性神经血管损伤的不利影响降至最低，从而改善缺血性中风后的长期预后。

DESCRIPTION (provided by applicant): Therapeutic targeting of the class IB PI3-kinase gamma for treatment of acute ischemic stroke. Recombinant tPA remains the only FDA approved and the most beneficial proven intervention for treatment of ischemic stroke. However, clinical use of tPA is limited due to its narrow therapeutic window and increased risk of hemorrhagic transformation (HT). In this proposal, we hypothesize that therapeutic inhibition of PI3Kg with AS605240, alone or in combination with tPA, protects against the ischemia- and tPA-induced brain injury and HT and increases therapeutic window for tPA treatment of ischemic stroke. The molecular basis for our idea is that genetic deletion of PI3Kg not only reduces stroke-induced BBB disruption and tissue infarction but also almost completely blocked cerebral hemorrhage induced by tPA administered at 6 hours after stroke. Our data are promising and suggest a critical role of PI3Kg in tPA-induced HT and brain injury after ischemic stroke. Further, our pilot data show that inhibition of PI3Kg with AS605240 significantly protects against brain injury and reduces fibrin deposition/thrombosis in the ischemic brain after embolic stroke in rats. These exciting preliminary data suggest that PI3Kg may represent a promising therapeutic target for treating acute ischemic stroke. In this application, we propose to develop a new stroke therapy by therapeutic targeting of PI3Kg with AS605240 for treatment of acute ischemic stroke and to investigate molecular mechanisms underlying the AS605240-mediated beneficial effects. We now propose three aims to investigate this new stroke therapy. In Aim 1, we will determine the effects of AS605240, alone and in combination with tPA, treated at 6 hrs after focal embolic stroke in rats. We will identify the optimal dosage and the extended therapeutic time window for AS605240 alone and in combination with tPA for treatment of acute stroke. Brain infarction, neurological deficits, brain hemorrhage, and animal mortality will be examined 72h after stroke. In Aim 2, we will define the mechanisms that underlie the beneficial effects of AS605240 alone or in combination with tPA in the treatment of acute stroke. BBB damage will be assessed by the spatial and temporal analysis of key BBB proteins and potential correlation with changes in activation/expression of NF-κB, CD147, MMP-9 and/or MMP-3 on cerebral vessels. Thrombus formation in the ischemic brain after stroke will be assessed by analyzing fibrin deposition within and around the cerebral vessel wall and the expression of prothrombotic factors (PAI-1, tissue factor-TF) in the ischemic brain. In Aim 3, we will assess long term outcomes of AS605240 alone and in combination with tPA treated at 6 hrs after embolic stroke. Animals will be followed over 5 weeks with a battery of behavioral tests. At the end, brains are removed for measurements of lesion size, fibrin deposition/thrombosis, cerebral vascular density/potency, and their correlation with the expression of proangiogenic factors (VEGF, Ang-1) and prothrombotic factors (PAI-1, tissue factor-TF). If successful, these proposed experiments should provide new insights of how the class IB PI3Kg signaling contributes to the ischemia- and tPA-induced HT and brain injury and may ultimately yield new therapies to treat acute ischemic stroke and to minimize the adverse effect of tPA on ischemic neurovascular damage, leading to improved long term outcomes after ischemic stroke.