Autophagy maintains vascular function through a novel glycolysis-linked pathway regulating eNOS.

摘要 一份报告表明，内皮细胞(EC)自噬在老年人中受到损害。我们证明了基因 抑制内皮细胞自噬否定切应力诱导的内皮型一氧化氮合酶的激活 也没有一代人。目前尚不清楚内皮细胞中自噬的干扰是否具有功能相关性 在体内，被抑制的EC自噬不影响任何世代的机制尚不清楚。在……里面 目的1我们将检验条件删除成年小鼠内皮细胞自噬(IecAtg3KO)的假设 (小鼠)出现老年小鼠的动脉功能障碍。此外，基于令人信服的初步 数据，我们将检验这样的假设，即抑制突变小鼠和老年小鼠的EC自噬会唤起P53- 介导阻断糖酵解，导致细胞外三磷酸腺苷通过P2Y1-R和蛋白激酶Cδ减少信号转导 内皮型一氧化氮合酶，导致动脉功能障碍。目标2将探索这一新途径的翻译潜力 人类老龄化的背景。在永生化的人动脉内皮细胞(HAECs)中，我们假设 自噬抑制可阻止切应力诱导的嘌呤能信号传导至eNOS。接下来，这条路径将是 评价老年(60岁)和成人(18-30岁)受试者手术前后的动脉内皮细胞 在两组中，有节奏的握力运动都能提高臂动脉的切变率。ECS将用于 量化EC自噬、eNOS激活和NO生成。重要的是，药理学和遗传学 恢复嘌呤能介导的eNOS信号的方法将用于这些遗传和 人内皮细胞中与衰老相关的自噬抑制。Aim 3将使用成年和老年突变小鼠 确定运动训练是否能减缓与衰老相关的EC自噬功能的下降，以及是否 训练诱导的血管改善需要完整的自噬。为了评估翻译潜力，我们 将辨别老年受试者的单肢有节奏的握力训练是否足以 提高基础和剪切诱导的EC自噬启动、eNOS激活和NO生成与 对侧久坐肢体。这项工作的结果具有巨大的潜力来揭示一种新的治疗方法 老年人口血管功能恢复/维持的目标和途径。

SUMMARY One report indicates endothelial cell (EC) autophagy is compromised in aged humans. We showed that genetic repression of autophagy in ECs negates shear-stress induced EC nitric oxide (NO) synthase (eNOS) activation and NO generation. It is unknown whether disruption of autophagy specifically in ECs has functional relevance in vivo, and the mechanism whereby repressed EC autophagy compromises NO generation is not known. In Aim 1 we will test the hypothesis that conditional deletion of autophagy in ECs from adult mice (iecAtg3KO mice) phenocopies arterial dysfunction that is present in old mice. Further, based on compelling preliminary data, we will test the hypothesis that repressed EC autophagy in mutant mice and old mice evokes a p53- mediated block in glycolysis, leading to decreased signaling by extracellular ATP via the P2Y1-R and PKCδ to eNOS, resulting in arterial dysfunction. Aim 2 will explore the translational potential of this novel pathway in the context of human aging. In immortalized human arterial endothelial cells (HAECs) we hypothesize that genetic autophagy suppression prevents shear-stress induced purinergic signaling to eNOS. Next, this pathway will be evaluated in primary arterial ECs obtained from old (> 60 y) and adult (18-30 y) subjects before and following rhythmic handgrip exercise that elevates brachial artery shear-rate similarly in both groups. ECs will be used to quantify EC autophagy, eNOS activation, and NO generation. Importantly, pharmacological and genetic approaches that restore purinergic mediated signaling to eNOS will be used in these models of genetic and aging-associated autophagy repression in human ECs. Aim 3 will use adult and aged mutant mice to determine whether exercise-training attenuates the aging-associated decline in EC autophagy, and whether intact autophagy is required for training-induced vascular improvements. To evaluate translational potential, we will discern whether one-limb rhythmic handgrip exercise training by old (> 60 y) human subjects is sufficient to elevate basal and shear-induced EC autophagy initiation, eNOS activation, and NO generation vs. the contralateral sedentary limb. Results from this work have tremendous potential to reveal a new therapeutic target and approach for restoring / maintaining vascular function in the aging population.