Neutrophil elastase and Gasdermin D in diabetic retinopathy

项目摘要 大量数据表明炎症过程在糖尿病的发病机制中起着关键作用， 视网膜病变特别是白细胞似乎在糖尿病诱导的视网膜变性中起主要作用。 毛细血管（其为视网膜缺血的最终发展、血管紧张素的释放、血管紧张素的释放和视网膜的缺血性病变的形成提供了条件， 增殖因子，如VEGF，以及最终的视网膜新生血管形成）。白细胞如何介导这一过程 糖尿病中的毛细血管细胞损伤是未知的。 中性粒细胞含有大量的蛋白酶，它们用来杀死体内的外来入侵者。 已知中性粒细胞释放中性粒细胞弹性蛋白酶（NE）作为其对损伤的反应的一部分，但不能释放中性粒细胞弹性蛋白酶。 调节它们的水平会导致组织损伤。我们提出的证据表明，中性粒细胞弹性蛋白酶发挥作用， 在糖尿病的内皮损伤和细胞毒性中起重要作用，并推测糖尿病诱导的 诱导视网膜炎症和血管损伤是早期糖尿病视网膜病变的特征， 继发于中性粒细胞，通过细胞外囊泡中的NE转运至内皮细胞，在内皮细胞中NE裂解 Gasdermin D（GSDMD）在内皮细胞膜中引起细胞毒性孔。 我们提出三个具体目标： 目标1。探讨药物抑制去甲肾上腺素对早期糖尿病视网膜病变的影响。我们 将使用结构上的NE抑制剂，并且将全身性地以及通过滴眼剂施用治疗。 目标2.研究NE增加糖尿病视网膜内皮细胞死亡的机制。 目标3。探讨GSDMD在糖尿病视网膜病变发病机制中的作用。这些研究报告将 最初使用GSDMD-/-小鼠进行。 这些研究将在体内进行，使用药理学手段抑制糖尿病中的NE，并使用小鼠 GSDMD基因缺陷这个建议是新颖的，因为它集中在（i）中性粒细胞的作用， 蛋白酶在视网膜病变的发病机制，和（ii）毒性视网膜内皮细胞作为转移的结果 蛋白酶从中性粒细胞通过细胞外囊泡到达内皮细胞。这是一个新的领域， 以前曾研究过糖尿病视网膜病变。从这些研究中获得的见解可以 从而导致开发新的和有效的疗法，其通过以下方式抑制糖尿病视网膜病变的发展： 靶向嗜中性粒细胞分泌的蛋白酶或随后的GSDMD裂解。

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.