Neutrophil elastase and Gasdermin D in diabetic retinopathy

项目总结 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。尤其是白细胞似乎在糖尿病引起的视网膜变性中起主要作用。 毛细血管(它为最终发展为视网膜缺血，血管释放- 增殖因子，如血管内皮生长因子，最终，视网膜新生血管)。白细胞是如何调节这一过程的 糖尿病对毛细血管细胞的损伤尚不清楚。 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。 已知中性粒细胞会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 调节它们的水平会导致组织损伤。我们提出了中性粒细胞弹性蛋白酶发挥作用的证据 在糖尿病血管内皮细胞损伤和细胞毒性中的重要作用，并假设糖尿病诱导 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 中性粒细胞通过细胞外小泡向内皮细胞运输去甲肾上腺素，去甲肾上腺素在血管内皮细胞裂解 Gasdermin D(GSDMD)可引起内皮细胞膜上的细胞毒性毛孔。 我们提出了三个具体目标： 目的1.探讨去甲肾上腺素的药理抑制作用对早期糖尿病视网膜病变的影响。我们 将使用结构上的去甲肾上腺素抑制剂，并将系统地给予治疗以及通过滴眼液。 目的2.探讨去甲肾上腺素(NE)增加糖尿病视网膜内皮细胞死亡的机制(S)。 目的3.探讨GSDMD在糖尿病视网膜病变发病机制中的作用。这些研究将是 最初使用GSDMD-/-小鼠进行。 这些研究将在体内进行，使用药物手段抑制糖尿病患者的去甲肾上腺素，并使用小鼠 GSDMD存在遗传缺陷。这个提议很新颖，因为它侧重于(I)中性粒细胞的作用 蛋白水解酶在视网膜病变发病机制中的作用，以及(Ii)转移对视网膜内皮细胞的毒性 通过胞外小泡将中性粒细胞中的蛋白水解酶转移到内皮细胞。这个地区是新的，也不是 之前已经被研究过关于糖尿病视网膜病变。从这些研究中学到的见解可以 导致开发新的有效的治疗方法，通过以下方式抑制糖尿病视网膜病变的发展 靶向中性粒细胞分泌的蛋白水解酶或随后切割的GSDMD。

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.