Abstract 235: Hypoxia Enhanced Delivery of Mitochondria-Targeted Catalase Protects Choroid Retinal Microvascular Endothelial Cells from Oxidative Stress
Loss of pericytes is a critical event early in the progression of microvascular dysfunction in diabetic retinopathy. Pericyte loss may be linked to high glucose mediated reactive oxygen species generation, blocking N-cadherin trafficking to the endothelial cell surface preventing pericyte recruitment and vessel stabilization. Hydrogen peroxide has been identified as a major free radical produced during high glucose exposure in endothelial cells. The goal of this research is to determine if tissue-specific hypoxia-regulated expression of a mitochondria-targeted catalase can prevent or limit RF/6A microvascular endothelial cell apoptosis and decrease vascular permeability by limiting cellular oxidative stress. For the development of tissue-specific and hypoxia-enhanced expression vectors, promoters were constructed with nine tandem combinations of HREs. This 9x HRE oligomer enhancer was inserted together into a pGL3 firefly luciferase plasmid with the Tie2(short) promoter for endothelial-specific expression. The 9xHRE-Tie2(sh) promoter construct was highly selective for RF/6A cells producing a basal amount of mitochondria-targeted catalase equivalent to the Tie2(short) promoter alone. In response to hypoxia (pO2=1%), the 9xHRE-Tie2(short) promoter showed a 21-fold hypoxia-inducible activation similar in strength to the CMV promoter, measured by dual luciferase assay. The hybrid promoters were incorporated into a replication deficient AAV delivery system for apoptosis and cell culture based endothelial permeability assays. In preliminary assays using RF/6A microvascular endothelial cells, apoptosis was reduced by 58% and permeability was reduced by 46%. The results suggest that mitochondria-targeted catalase protects RF/6A microvascular endothelial cells from apoptosis and reduces endothelial permeability in a high-glucose, low-oxygen environment.