The Warburg Effect and Diabetic Retinopathy - Project Summary/Abstract Retinal neovascularization (RNV) is a debilitating complication of advanced diabetic retinopathy, which despite the use of anti-VEGF and laser treatments continues to cause blindness. Less is known as to why RNV develops only after patients have had diabetes for decades. Although endothelial cell (EC) angiogenic activation is a hallmark of this transition, how ECs adapt their metabolism to sustain such activation remains a significant gap in our knowledge. Our long-term goal is to determine the bioenergetic mechanisms of RNV. The Warburg effect is a metabolic shift from mitochondrial oxidative phosphorylation (OxPhos) to hyperglycolysis that was first found in cancer cells. This metabolic shift not only produces ATP faster than OxPhos, albeit less efficiently, but also provides precursors required for lipid, protein, and nucleotide synthesis during cell proliferation. Recently the Warburg effect was rediscovered as a key contributor in various endothelial-related diseases; however, its role in diabetic retinopathy is not well-defined. In this application, the overall objective(s) are to define the role of the Warburg effect in diabetic retinopathy and to identify its underlying mechanisms. Here, we propose that multiple hits are needed to cooperatively alter EC metabolism to fulfill biosynthetic demands of transforming a quiescent EC into an angiogenic cell. Tissue hypoxia is the most common risk factor associated with advanced diabetic retinopathy. Thus, our central hypothesis is that diabetes primes quiescent ECs to be angiogenic (first hit) and that hypoxia (second hit) is necessary for angiogenic switch via the metabolic adaptation of the Warburg effect. Aim1 will test the hypothesis that persistent activation of the energy sensor, AMP-activated protein kinase (AMPK) sustains the Warburg effect to mediate EC angiogenic activation. Our approach is to use a two-hit model of diabetes and hypoxia in AMPKα1 endothelial-specific conditional knockout (AMPKα1End-/-) mice and in AMPKα1 silenced human retinal ECs (HRECs) to achieve this aim. We will also use vitreous samples from patients with proliferative diabetic retinopathy to test the correlation between the development of RNV and the Warburg effect- associated metabolites. Aim2 will investigate the role of endoplasmic reticulum (ER) stress in mediating the Warburg effect-induced EC angiogenic activation. We hypothesize that activation of Inositol-requiring enzyme (IRE)1, a unique ER-stress sensor protein with kinase and RNase activities, is a key mediator for the Warburg effect-induced EC angiogenic activation. We will use CRISPR/Cas9 to test the effect of inhibiting downstream signaling of IRE1 kinase and RNase activities on the Warburg effect-induced EC angiogenic activation. We will also test our hypothesis in vivo using a two-hit model of hypoxia and diabetes and selective pharmacological inhibitors. Overall, this new-investigator initiated R01 capitalizes on the interdisciplinary expertise of a biochemist, a mitochondrial biologist, an ER biologist, and a clinician to use a novel two-hit model of advanced diabetic retinopathy to gain mechanistic insights into the bioenergetic basis of RNV. Understanding the role of the Warburg effect will reveal novel targets in the treatment of diabetic retinopathy.
