PROJECT SUMMARY Considerable data now suggests that inflammatory processes play a critical role in the pathogenesis of diabetic retinopathy. Leukocytes in particular appear to play a major role in the diabetes-induced degeneration of retinal capillaries (which sets up the conditions for eventual development of retinal ischemia, release of vaso- proliferative factors like VEGF, and ultimately, retinal neovascularization). How leukocytes mediate this capillary cell damage in diabetes is not known. Neutrophils contain large quantities of proteases, which they use to kill foreign invaders in the body. Neutrophils are known to release neutrophil elastase (NE) as a part of their response to injury, but failure to regulate their levels can result in tissue injury. We present evidence that neutrophil elastase plays and important role in the endothelial damage and cytotoxicity in diabetes, and postulate that the diabetes-induced induction of retinal inflammation and the vascular damage that is characteristic of early diabetic retinopathy are secondary to neutrophils via transport of NE in extracellular vesicles to endothelial cells, where the NE cleaves Gasdermin D (GSDMD) to cause cytotoxic pores in the endothelial cell membranes. We propose 3 specific aims: Aim 1. To investigate the effect of pharmacologic inhibition of NE on early stages of diabetic retinopathy. We will use structurally NE inhibitors, and will administer the therapies systemically as well as via eyedrops. Aim 2. To investigate mechanism(s) by which NE increases death of retinal endothelial cells in diabetes. Aim 3. Investigate the role of GSDMD in the pathogenesis of diabetic retinopathy. These studies will be conducted initially using GSDMD-/- mice. These studies will be conducted in vivo using pharmacological means to inhibit NE in diabetes and using mice genetically deficient in GSDMD. This proposal is novel because it focuses on (i) the role of a neutrophil protease in the pathogenesis of the retinopathy, and (ii) toxicity to retinal endothelial cells as a result of transfer of the protease from neutrophils to the endothelial cells via extracellular vesicles. This area is new and has not been previously been studied with respect to diabetic retinopathy. The insights learned from these studies can lead to development of novel and effective therapies that inhibit the development of diabetic retinopathy by targeting a protease secreted by neutrophils or the subsequent cleavage of GSDMD.