Novel Targets and Therapeutic Interventions against Cerebral Ischemia-Reperfusion Injury

项目摘要/摘要 脑缺血再灌注(I/R)损伤是急性心肌梗死患者治疗的主要挑战。 通过静脉溶栓和血管内治疗治疗缺血性卒中。目前，还没有有效的 可用于治疗/预防脑I/R损伤的干预措施。新出现的证据表明，血栓形成和 脑部炎症反应(血栓炎症)和异常内质网应激 脑I/R引起的脑损伤是继发性脑损伤和神经功能恶化的重要原因。在这 我们希望开发一种新的基于miRNA的治疗策略来同时针对这些 脑I/R损伤不同分子机制的病理过程。最近，下降了 在患者和动物模型中，miR-30c的表达与许多病理情况有关。 在初步研究中，我们发现miR-30c在血小板、脑微血管和 正常小鼠的皮质/海马神经元，但其水平随着年龄的增长而下降。血和血中MIR-30c水平 缺血性卒中后脑组织明显减少及单次静脉注射合成激素后miR-30c升高 MIR-30c对脑I/R损伤有明显的保护作用。我们进一步表明，增加的表达式 MiR-30c的直接靶基因(包括血液和脑中的PAI-1，脑中的elF2α和Caspase-3) 静脉注射miR-30c模拟治疗可显著降低脑I/R损伤大鼠的脑损伤程度。基于这些 令人振奋的年轻成年小鼠的新发现，我们提出了创新的假设，即miR-30c作为一种 脑I/R损伤所致脑缺血后血栓炎症和内质网应激的关键调节因子 因此，靶向miR-30c是对抗脑I/R损伤的一种新的治疗方法。跟随 最新的中风治疗学术行业圆桌会议(STAIR)临床前指南，我们将测试这一点 老年雄性和雌性小鼠的假说。具体来说，我们将确定静脉注射miR-30c的有效性和安全性。 模仿为新的中风疗法(Aim1)。使用补充方法，即“函数增益”(miR- 30C模拟)和功能丧失(反义Morpholino寡核苷酸，专为与 MIR-30c对于每个单独的靶基因的3‘UTR3’UTR端的结合位点，从而起到“靶保护器”的作用)，我们将 确认PAI-1是miR-30c调节卒中后血栓炎症的关键分子靶点(目标2)， 并确定elF2、α和Caspase3是miR-30c调控卒中后神经元内质网的关键分子靶点 应激与神经细胞死亡(目标3)。这些研究的长期目标是评估靶向miR-30c是否 对于中风高危人群的男性和女性老年人群来说，这是一种可行的中风治疗选择。

Project Summary/Abstract The cerebral ischemia/reperfusion (I/R) injury is a major challenge for the treatment of patients with acute ischemic stroke by intravenous (IV) thrombolysis and endovascular therapy. Currently, there is no effective intervention available to treat/prevent cerebral I/R injury. Emerging evidence suggests that thrombotic and inflammatory responses (thrombo-inflammation) and aberrant endoplasmic reticulum (ER) stress in the brain elicited by cerebral I/R contributes importantly to secondary brain injury and neurologic deterioration. In this proposal we wish to develop a novel miRNA-based therapeutic strategy to simultaneously target these pathological processes of cerebral I/R injury through distinct molecular mechanisms. Recently, decreased expression of miR-30c has been implicated in many pathological conditions in both patients and animal models. In preliminary studies, we show that miR-30c is highly expressed in blood platelets, cerebral microvessels, and cortical/hippocampal neurons in normal mice but its levels decline with age. miR-30c levels in both blood and brain are markedly decreased after ischemic stroke and elevating miR-30c by single IV injection of synthetic miR-30c mimic significantly protects against cerebral I/R injury. We further show that the increased expressions of the direct target genes of miR-30c (including PAI-1 in both blood and brain, elF2α and caspase-3 in the brain) induced by cerebral I/R injury were significantly decreased by IV miR-30c mimic treatment. Based on these exciting new findings from young adult mice, we propose the innovative hypothesis that miR-30c functions as a critical regulator of thrombo-inflammation and ER stress in the ischemic brain elicited by cerebral I/R injury, and thus targeting miR-30c represents a novel therapeutic approach for combating cerebral I/R injury. Following updated stroke Therapy Academic Industry Roundtable (STAIR) pre-clinical guidelines, we will test this hypothesis in aged male and female mice. Specifically, we will determine the efficacy and safety of IV miR-30c mimic as novel stroke therapeutics (Aim1). Using complementary approaches, i.e. the “gain-of-function” (miR- 30c mimic) and “loss-of-function” (anti-sense Morpholino oligos that are designed to specifically compete with miR-30c for binding sites of 3’UTR of each individual target gene and thus acts as a “target protector”), we will identify PAI-1 as a key molecular target of miR-30c in regulation of post-stroke thrombo-inflammation (Aim 2), and identify elF2α and caspase 3 as key molecular targets of miR-30c in regulation of post-stroke neuronal ER stress and neuronal cell death (Aim 3). The long-term goal of these studies is to evaluate if targeting miR-30c is a viable option for stroke therapy in both male and female older populations at high risk for stroke.