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中风的药物。 不幸的是，成功再通大脑血管的患者中，有三分之一到一半没有 临床效果良好。这在一定程度上是由于被称为局灶性“无复流”的不完全微血管再灌注所致。 在糖尿病中风中尤其普遍。目前的建议将确定内皮细胞-周细胞的作用 串扰，由内皮二十烷基类环氧二十碳三烯酸酯(EETs)对周细胞的作用而介导，在 糖尿病小鼠卒中后微血管无复流。我们将检验这样一种假设：内皮来源的EETs 保护脑缺血后的毛细血管灌流，部分是通过保护周细胞免受缺血损伤，并且 糖尿病使微血管内皮EETs减少，从而导致更大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EET是否通过抑制G蛋白偶联受体39来保护周细胞 (GPR39)。患有和不患有2型糖尿病(T2D)以及内皮细胞水平较高或较低的小鼠均可诱发中风 EETs(由于EETs降解酶可溶性环氧化物水解酶，sEH， 在内皮中)使用闭塞细丝或原位凝血酶注射来闭塞中段 大脑动脉(MCA)。利用光学微血管造影(OMAG)和双光子显微镜进行活体成像 (下午2：00)将用于评估周细胞形态、血脑屏障完整性、毛细血管血流以及血小板和白细胞。 大脑中动脉闭塞后，在体脑梗塞周围区域的形态和动态变化。老鼠会成为 卒中后存活4天以评估细胞和组织损伤(H&E、EM、IHC)，并持续28天评估 长期功能缺陷(神经认知和躯体感觉)。我们建议保存内皮细胞- 周细胞EETS/GPR39信号通路保护周细胞，防止无复流，减少脑组织损伤 神经行为功能缺陷。在药物或转基因小鼠中增强内皮EETs 具有较高的内皮EETs，也将增强tPA疗效并减少卒中后tPA相关出血 在糖尿病小鼠身上。

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.