Endothelial-Pericyte Crosstalk in Diabetic Stroke

项目摘要 组织纤溶酶原激活剂（tPA）仍然是FDA批准的唯一治疗急性缺血性卒中的药物。 不幸的是，三分之一到一半的大脑血管再通成功的患者没有 良好的临床效果。这部分是由于不完全的微血管再灌注，称为局灶性“无复流”， 在糖尿病中风中尤为普遍。目前的建议将确定内皮周细胞的作用， 串扰，介导的作用，内皮类花生酸环氧二十碳三烯酸（ESTA）对周细胞， 小鼠糖尿病卒中后微血管无复流。我们将检验内皮源性内皮素 局部缺血后，部分通过保护周细胞免受缺血性损伤来保持脑中的毛细血管灌注， 糖尿病降低微血管内皮细胞的增殖，从而导致更严重的周细胞损伤和毛细血管损伤 糖尿病中风后我们还将确定是否Eclampin通过抑制G蛋白偶联受体39来保护周细胞 （GPR39）。在患有和不患有2型糖尿病（T2 D）的小鼠中诱导中风，并且在患有或不患有2型糖尿病（T2 D）的小鼠中诱导较高或较低的内皮细胞密度。 EETs（由于EETs降解酶可溶性环氧化物水解酶，sEH， 在内皮中）使用管腔内闭塞细丝或原位凝血酶注射来闭塞中间 大脑动脉（MCA）。使用光学微血管造影术（OMAG）和双光子显微镜进行活体成像 (2PM)将用于评估周细胞形态、BBB完整性、毛细血管血流量以及血小板和白细胞 在MCA闭塞后，在体内实时观察梗塞周围区域内的形态学和动力学。小鼠将被 中风后存活4天，以评估细胞和组织损伤（H&E，EM，IHC），并存活28天，以评估 长期功能缺陷（神经认知和躯体感觉）。我们建议保留内皮细胞- 周细胞E3/GPR 39信号传导保护周细胞，防止无复流并减少脑组织损伤， 神经行为功能缺陷增强内皮细胞的内皮细胞，无论是在体外还是在转基因小鼠中 高内皮素也将增强tPA疗效，减少卒中后tPA相关出血 在糖尿病小鼠中。

Project Summary Tissue plasminogen activator (tPA) remains the only FDA-approved therapy for acute ischemic stroke. Unfortunately, one-third to half of patients with successful recanalization of large cerebral vessels do not have good clinical outcome. This is in part due to incomplete microvascular reperfusion termed focal “no-reflow”, which is particularly prevalent in diabetic stroke. The current proposal will determine the role of endothelial-pericyte crosstalk, mediated by the action of the endothelial eicosanoids epoxyeicosatrienoates (EETs) on pericytes, in microvascular no-reflow after diabetic stroke in mice. We will test the hypothesis that endothelial-derived EETs preserve capillary perfusion in brain after ischemia, in part by protecting pericytes from ischemic injury, and that diabetes reduces microvascular endothelial EETs; thus, leading to greater pericyte injury and capillary damage after diabetic stroke. We will also determine if EETs protect pericytes by inhibiting G-protein coupled receptor 39 (GPR39). Stroke is induced in mice with and without type 2 diabetic (T2D), and with higher or lower endothelial EETs (due to transgenic overexpression or deletion of EETs-degrading enzyme soluble epoxide hydrolase, sEH, in endothelium) using an intraluminal occlusive filament or in-situ thrombin injection to occlude the middle cerebral artery (MCA). Intra-vital imaging using optical microangiography (OMAG) and two-photon microscopy (2PM) will be used to assess pericyte morphology, BBB integrity, capillary blood flow, and platelet and leukocyte morphology and dynamics within the peri-infarct region in-vivo, in real-time after MCA occlusion. Mice will be survived for 4 days after stroke to assess cellular and tissue damage (H&E, EM, IHC), and for 28 days to assess long-term functional deficit (neurocognitive and somatosensory). We propose that preserving endothelial- pericyte EETs/GPR39 signaling protects pericytes, prevents no-reflow and reduces brain tissue damage and neurobehavioral functional deficit. Enhancing endothelial EETs, either pharmacologically or in transgenic mice with higher endothelial EETs, will also enhance tPA efficacy and reduce tPA-associated hemorrhage after stroke in diabetic mice.