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.

PUBLIC HEALTH RELEVANCE: VISN-19, a diagnosis database query performed by Dr. Larry Meyer, ACOS for Research, Salt Lake City VA, identified 151 Veterans with corneal transplants, 4,873 veterans with macular degeneration, and 4,282 veterans with diabetic retinopathy. There are approximately 220,000 Veterans in VISN-19 and about 24 million living Veterans. By extrapolation, this would indicate that nationally, there are 467,127 Veterans with diabetic retinopathy. Of the more than 1 million Veterans with low vision (defined as less than 20/70), about a quarter are due to diabetic retinopathy. The VA spends >$43,000 per low vision case for rehabilitation and >$30,000 per patient-year on anti-VEGF therapy for ocular retinopathies. Determining whether improving retinal blood vessel stability can prevent and rescue structural and functional changes of the retina in diabetic retinopathy would be the first approach to supply a positive mediator, breaking the mold of the current destructive treatments (laser ablation of retina or painful intravitreal injections) used in diabetic retinopathy