Neurorestorative therapy of stroke with HUCBC in type two diabetic mice

描述(由申请人提供)：糖尿病(DM)导致经历缺血性中风的风险增加3-4倍。高血糖和糖尿病引发一系列事件，导致血管内皮细胞功能障碍、血管通透性增加和缺血性中风后恢复不良。糖尿病动物卒中后脑白质(WM)损伤较非糖尿病动物严重。糖尿病患者和非糖尿病患者对卒中治疗的反应也不同。在非糖尿病人群中对中风的有效治疗不一定会转移到糖尿病人群中，这促使需要开发专门设计的治疗方法，以减少糖尿病人群中中风后的神经功能障碍。我们的初步数据显示，T2 DM显著降低了小鼠循环和缺血脑中microRNA-126(miR-126)和Angiopoietin-1(Ang1)的表达。人脐血细胞(HUCBC)对T2 DM小鼠卒中的治疗作用于卒中后3天开始，显著促进神经功能的恢复，并增加缺血脑组织miR-126和Ang1的表达。因此，基于我们稳健的初步数据，我们建议将HUCBCs用于T2 DM小鼠的卒中治疗，并探讨通过包裹在外切体/微囊泡(EMVS)中的miR-126进行细胞间通讯在介导HUCBCs对缺血性卒中的治疗益处中的作用。这项申请包括三个目标。目的1验证miR-126是否介导HUCBC治疗T2 DM小鼠卒中后的神经修复作用。我们假设miR-126介导了HUCBC治疗诱导的血管完整性、轴突生长和WM重塑，并改善了T2 DM小鼠卒中后的功能结局。目的2将检测人脐血细胞产生的miR-126是否通过EMVS转移到脑内皮细胞(BECs)和实质细胞。我们推测HUCBCs分泌含有miR-126的EMVs，由BEC和实质细胞摄取。目的3研究miR-126调节Ang1是否促进HUCBC诱导的T2 DM小鼠卒中后的神经修复作用。我们假设：1)HUCBC治疗T2 DM小鼠卒中可提高缺血脑组织Ang-1信号活性；2)miR-126调控Ang1表达，从而调控血管重塑、轴突生长及少突胶质细胞的存活和分化；3)用Ang1模拟肽恢复Ang1将挽救T2 DM小鼠卒中后HUCBC中miR-126基因敲除的神经修复作用。在这一应用中，我们首次提出，人脐血细胞产生包裹在EMVS中的miR-126有助于其强大的治疗恢复作用，并且miR-126及其对Ang-1的调节介导了HUCBC诱导的神经血管和WM重建，从而促进T2 DM小鼠中风功能的恢复。这项建议具有很高的临床相关性，如果成功，将对糖尿病患者的治疗产生重大影响，可能还会影响所有中风患者的治疗。

DESCRIPTION (provided by applicant): Diabetes mellitus (DM) leads to a 3-4 fold higher risk of experiencing ischemic stroke. Hyperglycemia and diabetes instigate a cascade of events leading to vascular endothelial cell dysfunction, increased vascular permeability and poor recovery after ischemic stroke. Diabetic animal's exhibit more severely injured white matter (WM) than non-DM animals after stroke. There is also a differential response to treatment of stroke between DM and non-DM subjects. Effective therapy of stroke in the non-DM population may not necessarily transfer to the DM population, prompting the need to develop therapeutic approaches specifically designed to reduce neurological deficits after stroke in the DM population. Our preliminary data show that T2DM significantly decreases microRNA-126 (miR-126) and Angiopoietin-1 (Ang1) expression in the circulation and in the ischemic brain of mice. Human umbilical cord blood cell (HUCBC) treatment of stroke in T2DM mice starting at 3 days after stroke significantly improves recovery of neurological function as well as increases miR-126 and Ang1 expression in the ischemic brain. Therefore, based on our robust preliminary data, we propose to use HUCBCs for the treatment of stroke in the T2DM mice and to investigate the role of intercellular communication via miR-126 encapsulated within Exosomes/Microvesicles (EMVs) in mediating the therapeutic benefit on HUCBCs for ischemic stroke. This application includes three Aims. Aim 1 will test if miR-126 mediates HUCBC treatment induced neurorestorative effects after stroke in T2DM mice. We hypothesize that miR-126 mediates HUCBC treatment-induced vascular integrity, axonal outgrowth, and WM remodeling, and improves functional outcome after stroke in T2DM mice. Aim 2 will test whether miR-126 generated by HUCBCs is transferred to brain endothelial cells (BECs) and parenchymal cells via EMVs. We hypothesize that HUCBCs secrete EMVs containing miR-126 which are taken up by BECs and parenchymal cells. Aim 3 will investigate whether miR-126 regulation of Ang1 promotes the HUCBC-induced neurorestorative effects after stroke in T2DM mice. We hypothesize that: 1) HUCBC treatment of stroke in T2DM mice increases Ang-1 signaling activity in the ischemic brain; 2) miR-126 regulates Ang1 expression and thereby regulates vascular remodeling, axonal outgrowth and oligodendrocyte survival and differentiation; 3) Restoration of Ang1 with an Ang1 mimetic peptide will rescue the neurorestorative effects of knockdown of miR-126 in HUCBC after stroke in T2DM mice. In this application, we are the first to propose that, generation of miR-126 encapsulated in EMVs by HUCBCs contributes to its robust therapeutic restorative effects and that miR-126 and its regulation of Ang-1 mediate HUCBC-induced neurovascular and WM remodeling, and thereby improve stroke functional recovery in T2DM mice. This proposal is highly clinically relevant and if successful, will significantly impact the treatment of diabetic and possibly all stroke patients