Neurorestorative therapy of stroke with HUCBC in type two diabetic mice

描述（由申请人提供）：糖尿病（DM）导致发生缺血性卒中的风险增加3-4倍。高血压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卒中后恢复不良。糖尿病动物在中风后表现出比非糖尿病动物更严重的白色物质（WM）损伤。DM和非DM受试者对卒中治疗的反应也不同。在非DM人群中有效的卒中治疗可能不一定转移到DM人群，这促使需要开发专门设计用于减少DM人群卒中后神经功能缺损的治疗方法。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）和血管生成素-1（Ang 1）的表达。在中风后3天开始的T2 DM小鼠中风的人脐带血细胞（HUCBC）治疗显著改善神经功能的恢复以及增加缺血脑中的miR-126和Ang 1表达。因此，基于我们稳健的初步数据，我们建议使用HUCBCs治疗T2 DM小鼠中的中风，并研究通过外泌体/微囊泡（EMV）中封装的miR-126介导的细胞间通讯在HUCBCs治疗缺血性中风中的作用。这一应用包括三个目标。目的1将测试miR-126是否介导T2 DM小鼠中风后HUCBC治疗诱导的神经恢复作用。我们假设miR-126介导HUCBC治疗诱导的血管完整性、轴突生长和WM重塑，并改善T2 DM小鼠中风后的功能结局。目的2将测试HUCBCs产生的miR-126是否通过EMV转移到脑内皮细胞（BEC）和脑实质细胞。我们假设HUCBCs分泌含有miR-126的EMV，其被BEC和实质细胞摄取。目的3探讨miR-126对Ang 1的调控是否促进HUCBC诱导的T2 DM小鼠脑卒中后神经修复作用。我们假设：1）HUCBC治疗T2 DM小鼠中风增加缺血脑中的Ang-1信号传导活性; 2）miR-126调节Ang 1表达，从而调节血管重塑、轴突生长和少突胶质细胞存活和分化; 3）用Ang 1模拟肽恢复Ang 1将挽救T2 DM小鼠中风后HUCBC中miR-126敲低的神经恢复作用。在本申请中，我们首次提出，由HUCBCs产生包裹在EMV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HUCBCs诱导的神经血管和WM重塑，从而改善T2 DM小鼠的卒中功能恢复。这一建议具有高度的临床相关性，如果成功，将对糖尿病患者和可能所有中风患者的治疗产生重大影响

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