Synergistic OEC-biologic Use for Diabetic Retinal Regeneration

OEC-生物制剂协同用于糖尿病视网膜再生

• 批准号: 8820208
• 负责人: BALAMURALI K AMBATI
• 金额: --
• 依托单位: VA SALT LAKE CITY HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-11-01 至 2016-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8820208
• 关键词: Ablation; Address; Adhesions; Adverse effects; Age; Age-Months; Angiopoietin-1; Antibodies; Apoptosis; Binding; Biological Assay; Biological Response Modifier Therapy; Blindness; Blood Vessels; Blood capillaries; Blood-Retinal Barrier; Bone Growth; Cartilage; Cell Survival; Cell Therapy; Cicatrix; Cities; Competence; Dependovirus; Diabetes Mellitus; Diabetic Nephropathy; Diabetic Retinopathy; Diabetic mouse; Diagnosis; Disease; Dyes; Endothelial Cells; Endothelium; Event; Extravasation; Functional disorder; Growth; Harvest; Heart Diseases; Hour; Human; Hyperglycemia; Hypoxia; In Situ Hybridization; In Situ Nick-End Labeling; Inflammation; Inflammatory; Injury; Interleukin-1; Ischemia; Keratoplasty; Kinetics; Label; Lasers; Lead; Leukocytes; Leukostasis; Life; Macular degeneration; Mediator of activation protein; Molds; Monitor; Mus; Natural regeneration; Neurons; Oligonucleotides; Optical Coherence Tomography; PECAM1 gene; Pain; Patients; Pericytes; Peripheral Vascular Diseases; Permeability; Phenotype; Physiological; Population; Prevalence; Production; Proteins; Regenerative Medicine; Rehabilitation therapy; Relative (related person); Research; Resistance; Retina; Retinal; Retinal Diseases; Reverse Transcriptase Polymerase Chain Reaction; Safety; Serotyping; Signal Transduction; Sodium Chloride; Source; Staining method; Stains; Stem cells; Stroke; Supporting Cell; Surface; TIE-2 Receptor; TNF gene; Testing; Time; Trees; Vascular Diseases; Vascular Endothelial Growth Factors; Veterans; Vision; Visual Acuity; Visual impairment; Western Blotting; Work; aged; cadherin 5; capillary; caspase-3; cytokine; database query; density; diabetic; experience; improved; in vivo; indexing; inflammatory marker; innovation; insight; intravitreal injection; laser photocoagulation; novel; novel therapeutic intervention; prevent; public health relevance; retina blood vessel structure; retinal damage; retinal regeneration; therapeutic target; vascular bed

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.

描述(由申请人提供)： 高血糖引起的视网膜血管丢失和血管功能障碍是糖尿病视网膜病变(DR)的主要病理生理机制。高血糖导致周细胞丢失，周细胞为内皮提供营养信号，促进细胞存活、成熟和屏障完整性。在长期的高血糖中，血管变得高通透性，增加炎性细胞因子的产生，并经历内皮细胞的丢失，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(Ang1)， 它与内皮细胞表面受体Tie2结合。这通过PI3K/Akt和血管内皮细胞(VE)-钙粘蛋白分别促进内皮细胞存活和降低通透性。直到最近，Ang1作为治疗靶点的使用一直受到其不溶性和聚集性的阻碍。为了克服Ang1的局限性，开发了一种新的、稳定的、可溶的、功能更强的Ang1版本-软骨寡聚基质蛋白Ang1(comp-Ang1)。Comp-Ang1的血管成熟可减轻糖尿病肾病，促进中风后神经元生长，并恢复缺血损伤后的骨生长。Comp-Ang1在糖尿病视网膜病变中的作用还有待研究。该项目将确定同时在玻璃体内输送生长内皮细胞(OEC)和一种新的蛋白质comp-Ang1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-Ang1将挽救糖尿病视网膜病变的视网膜神经血管结构和功能缺陷。我们的假设将在以下特定目标中得到验证：目的1.确定AAV2玻璃体内注射COMP-Ang1的动力学、安全性和有效性。小鼠将接受玻璃体内注射表达comp-Ang1或对照的AAV2，并监测其表达，分析炎症标志物以及功能和结构变化。我们将进行光学相干断层扫描(OCT)来监测体内的结构效应，分析肿瘤坏死因子、血管内皮生长因子和IL-1(炎症标志物)的存在，并评估细胞凋亡(TUNEL染色；caspase3)。我们将使用RT-PCR、原位杂交和Western印迹来评估表达动力学。目的2.确定COMP-Ang1的组成性表达是否能促进OEC整合到糖尿病视网膜，逆转糖尿病视网膜病变造成的损伤。接受Comp-Ang1治疗的糖尿病小鼠或对照组将被给予玻璃体内注射OECs，以确定Comp-Ang1是否可以增加OEC的整合和逆转血管丢失。这一目标将决定COMP-Ang1联合OEC治疗是否可以通过促进血管再生来逆转小鼠晚期糖尿病视网膜病变。具体地说，我们将确定结构性comp-Ang1表达是否可以促进OEC整合，并恢复老年糖尿病小鼠的视网膜血管系统。这将是再生医学生物和细胞协同疗法的重大进步。患有晚期糖尿病视网膜病变(6个月龄)的小鼠将接受AAV2.COMP-Ang1、AAV2.GFP或PBS的治疗。两周后，将从健康小鼠身上获取嗅鞘细胞，用PKH染料标记为红色，并通过玻璃体内注射给药。在4个时间点(72小时至4个月)，我们将采集小鼠视网膜，并确定AAV2是否为AAV2。COMP-Ang1可改善与对照组相关的以下结构和功能指标：1)血管活性和视网膜结构和生理完整性，2)嗅鞘细胞与现有血管床整合，3)增加血管密度(CD31)，4)视网膜氧合变化，5)血视网膜屏障功能障碍，5)炎症标志物。