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: 9040025
• 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/9040025
• 关键词: ANGPT1 gene; Ablation; Address; Adhesions; Adverse effects; Age; Age-Months; Antibodies; Apoptosis; Binding; Biological Assay; Biological Response Modifier Therapy; Blindness; Blood Vessels; Blood capillaries; Blood-Retinal Barrier; Bone Growth; CASP3 gene; 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; 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; cytokine; database query; density; diabetic; experience; improved; in vivo; indexing; inflammatory marker; innovation; insight; intravitreal injection; laser photocoagulation; neurovascular; novel; novel therapeutic intervention; novel therapeutics; 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）： 高血糖引起的残留血管丧失和血管功能障碍是糖尿病性视网膜病（DR）的主要病理生理机制。高血糖导致周细胞的丧失，这为促进细胞存活，成熟和屏障完整性提供了营养信号。在长期高血糖症中，血管变得过度耐受并增加炎症性细胞因子的产生，并经历了原始疗法丧失，导致残留缺氧。 DR中丢失的关键周细胞衍生的营养信号是Angiopietin-1（Ang1）， 结合了内皮表面受体TIE2。这分别通过PI3K/AKT和血管内皮（VE） - 钙粘着蛋白促进内皮生存率，并提高渗透率。直到最近，将ANG1用作治疗靶点的使用一直受到其不溶性和聚集的阻碍。开发了一种新颖，稳定，可溶性和更多潜在版本，软骨寡核基质蛋白ANG1（COMP-ANG1）以克服ANG1的局限性。具有COMP-ANG1的血管成熟可缓解糖尿病性肾病，促进中风后神经元的生长，并在缺血性损伤后恢复骨骼生长。 Comp-Ang1在糖尿病性视网膜病变中的影响尚未研究。该项目将确定同时使用新型蛋白质Comp-Ang1的玻璃体内递送生长内皮细胞（OEC）是否可以再生在晚期糖尿病性视网膜病中丢失的视网膜血管树。我们假设Comp-Ang1将挽救糖尿病性视网膜病变的视网膜神经血管结构和功能性缺陷。我们的假设将在以下特定目的中进行检验：目标1。确定通过AAV2玻璃体内递送的Comp-Ang1递送的动力学，安全性和有效性。小鼠将通过玻璃体内注射AAV2表达Comp-Ang1或对照进行监测，并进行表达并进行测定以进行炎症标记以及功能和结构变化。我们将执行OCT（光学相干断层扫描）来监测体内结构效应，分析TNF-€，VEGF和IL-1的存在（感染标记）和评估凋亡（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。 AIM 2。确定Comp-Ang1的组成表达是否可以增强OEC整合到糖尿病性视网膜中，并由糖尿病性视网膜病变引起的反向损害。用COMP-ANG1或对照处理的糖尿病小鼠将对OEC进行玻璃体内注射，以确定Comp-Ang1是否可以增加OEC整合并反向容器损失。该目标将确定Comp-Ang1 Plus OEC治疗是否可以通过促进血管再生来逆转小鼠的晚期糖尿病性视网膜病变。具体而言，我们将确定构型COMP-ANG1表达是否可以增强OEC整合并恢复老年糖尿病小鼠的残留脉管系统。这将是再生医学的协同生物学和细胞疗法的重大进步。患有晚期糖尿病性视网膜病的小鼠（6个月大）将接受AAV2.comp-ang1，aAV2.GFP或PBS治疗。两周后，将从健康的小鼠中收获OEC，用PKH染料标记为红色，并通过玻璃体内注射给药。在四个时间点（72小时至4个月），我们将恢复鼠标视网膜，并确定AAV2.comp-ang1改善了以下结构和功能指标相对于控制：1）船舶能力以及残留的结构和物理完整性，2）将OEC的整合到现有的血管床中，3）增加血管密度（CD31），4）牛群（CD31），4）牛群的变化（CD31），5）均变化。 5）炎症标记。