Synergistic OEC-biologic Use for Diabetic Retinal Regeneration

描述（由申请人提供）： 糖尿病视网膜病变（DR）的主要病理生理机制是高血糖引起的视网膜血管损伤和血管功能障碍。高血压导致周细胞的损失，周细胞向内皮提供营养信号，促进细胞存活、成熟和屏障完整性。在长时间的高血糖期间，血管变得高渗透性并增加炎性细胞因子的产生，并经历内皮损失，导致视网膜缺氧。在DR中丢失的一个关键的周细胞来源的营养信号是血管生成素-1（Ang 1）， 其结合内皮表面受体Tie 2。这分别促进内皮细胞存活并降低通过PI 3 K/Akt和血管内皮（VE）-钙粘蛋白的渗透性。直到最近，Ang 1作为治疗靶点的使用一直受到其不溶性和聚集性的阻碍。开发了一种新的、稳定的、可溶的和更有效的Ang 1版本，软骨寡基质蛋白Ang 1（COMP-Ang 1），以克服Ang 1的局限性。COMP-Ang 1的血管成熟可减轻糖尿病肾病，促进中风后神经元生长，并恢复缺血性损伤后的骨生长。COMP-Ang 1在糖尿病视网膜病变中的作用尚未研究。该项目将确定同时玻璃体内递送生长内皮细胞（OEC）与一种新型蛋白COMP-Ang 1是否可以再生在晚期糖尿病视网膜病变中丢失的视网膜血管树。我们假设COMP-Ang 1将挽救糖尿病视网膜病变中的视网膜神经血管结构和功能缺陷。我们的假设将在以下具体目标中得到检验：目标1。确定通过AAV 2玻璃体内递送的COMP-Angl递送的动力学、安全性和功效。将用玻璃体内注射表达COMP-Ang 1的AAV 2或对照处理小鼠，并监测表达和测定炎性标志物以及功能和结构变化。我们将进行OCT（光学相干断层扫描）以监测体内结构效应，分析TNF-α、VEGF和IL-1（炎症标志物）的存在，并评估细胞凋亡（TUNEL染色; caspase 3）。我们将使用RT-PCR、原位杂交和Western印迹评估表达动力学。目标二。确定COMP-Ang 1的组成型表达是否可以增强OEC整合到糖尿病视网膜中并逆转糖尿病视网膜病变引起的损伤。用COMP-Ang 1或对照治疗的糖尿病小鼠将被给予OEC的玻璃体内注射以确定COMP-Ang 1是否可以增加OEC整合和逆转血管损失。该目的将确定COMP-Ang 1加OEC治疗是否可以通过促进血管再生来逆转小鼠中的晚期糖尿病视网膜病变。具体来说，我们将确定组成性COMP-Ang 1表达是否可以增强老年糖尿病小鼠OEC整合和恢复视网膜血管。这将是再生医学协同生物和细胞治疗的重大进展。将用AAV 2. COMP-Ang 1、AAV2.GFP或PBS治疗患有晚期糖尿病视网膜病变（6月龄）的小鼠。两周后，将从健康小鼠收获OEC，用PKH染料标记红色，并通过玻璃体内注射施用。在四个时间点（72小时至4个月），我们将收获小鼠视网膜并确定AAV 2. COMP-Ang 1相对于对照是否改善以下结构和功能指数：1）血管能力和视网膜结构和生理完整性，2）OEC整合到现有血管床中，3）增加血管密度（CD 31），4）视网膜氧合的变化，5）血视网膜屏障功能障碍，和5）炎性标志物。

DESCRIPTION (provided by applicant): Hyperglycemia-induced loss of retinal blood vessels and vascular dysfunction is a primary pathophysiologic mechanism of diabetic retinopathy (DR). Hyperglycemia results in loss of pericytes, which provide trophic signals to the endothelium promoting cell survival, maturation, and barrier integrity. During prolonged hyperglycemia, blood vessels become hyperpermeable and increase inflammatory cytokine production, and experience endothelial loss, resulting in retinal hypoxia. A key pericyte-derived trophic signal that is lost in DR is angiopoietin-1 (Ang1), which binds the endothelial surface receptor Tie2. This promotes endothelial survival and decreases permeability through PI3K/Akt and vascular endothelial (VE)-cadherin, respectively. Until recently, use of Ang1 as a therapeutic target has been hindered by its insolubility and aggregation. A novel, stable, soluble and more potent version of Ang1, cartilage oligo matrix protein Ang1 (COMP-Ang1) was developed to overcome the limitations of Ang1. Vascular maturation with COMP-Ang1 mitigates diabetic nephropathy, promotes neuron growth after a stroke, and restores bone growth after ischemic injury. The effects of COMP-Ang1 in diabetic retinopathy have yet to be studied. This project will determine whether concurrent intravitreal delivery of outgrowth endothelial cells (OEC) with a novel protein, COMP-Ang1, can regenerate the retinal vascular tree that is lost in advanced diabetic retinopathy. We hypothesize that COMP-Ang1 will rescue the retinal neurovascular structural and functional deficits in diabetic retinopathy. Our hypothesis will be tested in the following specific aims: Aim 1. Determine the kinetics, safety, and efficacy of COMP-Ang1 delivery via AAV2 intravitreal delivery. Mice will be treated with an intravitreal injection of AAV2 expressing COMP-Ang1 or control and monitored for expression and assayed for inflammatory markers as well as functional and structural changes. We will perform OCT (optical coherence tomography) to monitor in vivo structural effects, analyze presence of TNF-¿, VEGF, and IL-1 (markers of inflammation), and assess apoptosis (TUNEL staining; caspase 3). We will assess expression kinetics using RT-PCR, in situ hybridization, and Western blot. Aim 2. Determine whether constitutive expression of COMP-Ang1 can enhance OEC integration into the diabetic retina and reverse damage caused by diabetic retinopathy. Diabetic mice, treated with COMP-Ang1 or control will be given intravitreal injections of OECs to determine if COMP-Ang1 can increase OEC integration and reverse vessel loss. This aim will determine whether COMP- Ang1 plus OEC therapy could reverse advanced diabetic retinopathy in mice by promoting vascular regeneration. Specifically we will determine whether constitutive COMP-Ang1 expression can enhance OEC integration and restore retinal vasculature in aged diabetic mice. This would be a significant advance in synergistic biologic and cell therapy for regenerative medicine. Mice with advanced diabetic retinopathy (6 months of age) will be treated with AAV2.COMP-Ang1, AAV2.GFP, or PBS. Two weeks later, OECs will be harvested from healthy mice, labeled red with PKH dye, and administered by intravitreal injection. At four time points (72 hours to 4 months) we will harvest the mouse retinas and determine whether AAV2.COMP-Ang1 improves the following structural and functional indices relative to control: 1) Vessel competence and retinal structural and physiological integrity, 2) Integration of OECs into existing vascular beds, 3) Increase vascular density (CD31), 4) Changes in retinal oxygenation, 5) blood retinal barrier dysfunction, and 5) Inflammatory markers.