Restoration of autophagy in endothelial cells from patients with diabetes mellitus improves nitric oxide signaling.
Restoration of autophagy in endothelial cells from patients with diabetes mellitus improves nitric oxide signaling.
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DOI：
10.1016/j.atherosclerosis.2016.01.043
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发表时间：
2016-04
期刊：
影响因子：
5.3
通讯作者：
Vita JA
中科院分区：
文献类型：
--
作者：
Fetterman JL;Holbrook M;Flint N;Feng B;Bretón-Romero R;Linder EA;Berk BD;Duess MA;Farb MG;Gokce N;Shirihai OS;Hamburg NM;Vita JA
内皮功能障碍是糖尿病心血管疾病的重要因素。自噬是从细胞中清除受损蛋白质和细胞器的多步骤机制。在糖尿病条件下，不充分的自噬促进非血管组织中的细胞功能障碍和胰岛素抵抗。我们假设受损的自噬有助于糖尿病的内皮功能障碍。我们测量了自噬标记物和内皮型一氧化氮合酶（eNOS）激活新鲜分离的内皮细胞从糖尿病受试者（n=45）和非糖尿病对照组（n=41）。p62水平在来自糖尿病患者的细胞中更高（34.2±3.6对20.0±1.6，P=0.001），表明自噬通量降低。巴弗洛霉素抑制胰岛素诱导的eNOS激活（−21±5% vs. 64± 22%，P=0.003），证实了完整的自噬是eNOS信号传导所必需的。在糖尿病患者的内皮细胞中，亚精胺激活自噬恢复eNOS激活，表明受损的自噬有助于内皮功能障碍（P=0.01）。自噬起始的指标，包括LC 3结合的斑点和beclin 1表达的数量在糖尿病患者和对照组中相似，而自噬终末相指标，溶酶体蛋白Lamp 2a，在糖尿病患者中较高。在糖尿病条件下的内皮细胞中，亚精胺对eNOS激活的有益作用被自噬抑制剂巴弗洛霉素或3-甲基腺嘌呤阻断。用巴弗洛霉素阻断终末阶段的自噬增加了糖尿病患者细胞中的p62（P=0.01），但程度低于对照组细胞（P=0.04），这表明自噬清除正在进行，但不充分。自噬不足导致糖尿病患者内皮功能障碍，可能成为糖尿病血管疾病治疗的靶点。
Endothelial dysfunction contributes to cardiovascular disease in diabetes mellitus. Autophagy is a multistep mechanism for removal of damaged proteins and organelles from the cell. Under diabetic conditions, inadequate autophagy promotes cellular dysfunction and insulin resistance in non-vascular tissue. We hypothesized that impaired autophagy contributes to endothelial dysfunction in diabetes mellitus. We measured autophagy markers and endothelial nitric oxide synthase (eNOS) activation in freshly isolated endothelial cells from diabetic subjects (n=45) and non-diabetic controls (n=41). p62 levels were higher in cells from diabetics (34.2±3.6 vs. 20.0±1.6, P=0.001), indicating reduced autophagic flux. Bafilomycin inhibited insulin-induced activation of eNOS (−21±5% vs. 64±22%, P=0.003) in cells from controls, confirming that intact autophagy is necessary for eNOS signaling. In endothelial cells from diabetics, activation of autophagy with spermidine restored eNOS activation, suggesting that impaired autophagy contributes to endothelial dysfunction (P=0.01). Indicators of autophagy initiation including the number of LC3-bound puncta and beclin 1 expression were similar in diabetics and controls, whereas an autophagy terminal phase indicator, the lysosomal protein Lamp2a, was higher in diabetics. In endothelial cells under diabetic conditions, the beneficial effect of spermidine on eNOS activation was blocked by autophagy inhibitors bafilomycin or 3-methyladenine. Blocking the terminal stage of autophagy with bafilomycin increased p62 (P=0.01) in cells from diabetics to a lesser extent than in cells from controls (P=0.04), suggesting ongoing, but inadequate autophagic clearance. Inadequate autophagy contributes to endothelial dysfunction in patients with diabetes and may be a target for therapy of diabetic vascular disease.