BACKGROUND Endothelial cells (ECs) use glycolysis to produce energy. In preclinical models of peripheral arterial disease, further activation of EC glycolysis was ineffective or deleterious in promoting hypoxia-dependent angiogenesis, whereas pentose phosphate pathway activation was effective. Hexosamine biosynthesis pathway, pentose phosphate pathway, and glycolysis are closely linked. Glucosamine directly activates hexosamine biosynthesis pathway. METHODS Hind-limb ischemia in endothelial nitric oxide synthase knockout (eNOS-/-) and BALB/c mice was used. Glucosamine (600 μg/g per day) was injected intraperitoneally. Blood flow recovery was assessed using laser Doppler perfusion imaging and angiogenesis was studied by CD31 immunostaining. In vitro, human umbilical vein ECs and mouse microvascular ECs with glucosamine, L-glucose, or vascular endothelial growth factor (VEGF165a) were tested under hypoxia and serum starvation. Cell Counting Kit-8, tube formation, intracellular reactive oxygen species, electric cell-substrate impedance sensing, and fluorescein isothiocyanate dextran permeability were assessed. Glycolysis and oxidative phosphorylation were assessed by seahorse assay. Gene expression was assessed using RNA sequencing, real-time quantitative polymerase chain reaction, and Western blot. Human muscle biopsies from patients with peripheral arterial disease were assessed for EC O-GlcNAcylation before and after supervised exercise versus standard medical care. RESULTS On day 3 after hind-limb ischemia, glucosamine-treated versus control eNOS-/- mice had less necrosis (n=4 or 5 per group). Beginning on day 7 after hind-limb ischemia, glucosamine-treated versus control BALB/c mice had higher blood flow, which persisted to day 21, when ischemic muscles showed greater CD31 staining per muscle fiber (n=8 per group). In vitro, glucosamine versus L-glucose ECs showed improved survival (n=6 per group) and tube formation (n=6 per group). RNA sequencing of glucosamine versus L-glucose ECs showed increased amino acid metabolism (n=3 per group). That resulted in increased oxidative phosphorylation (n=8-12 per group) and serine biosynthesis pathway without an increase in glycolysis or pentose phosphate pathway genes (n=6 per group). This was associated with better barrier function (n=6-8 per group) and less reactive oxygen species (n=7 or 8 per group) compared with activating glycolysis by VEGF165a. These effects were mediated by activating transcription factor 4, a driver of exercise-induced angiogenesis. In muscle biopsies from humans with peripheral arterial disease, EC/O-GlcNAcylation was increased by 12 weeks of supervised exercise versus standard medical care (n=6 per group). CONCLUSIONS In cells, mice, and humans, activation of hexosamine biosynthesis pathway by glucosamine in peripheral arterial disease induces an "exercise-like" angiogenesis and offers a promising novel therapeutic pathway to treat this challenging disorder.

中文翻译：

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葡萄糖胺介导的己糖胺生物合成途径激活使用 ATF4 促进 PAD 中的“运动样”血管生成和灌注恢复。


背景 内皮细胞 （EC） 利用糖酵解产生能量。在外周动脉疾病的临床前模型中，进一步激活 EC 糖酵解在促进缺氧依赖性血管生成方面无效或有害，而磷酸戊糖途径激活是有效的。己糖胺生物合成途径、磷酸戊糖途径和糖酵解密切相关。葡萄糖胺直接激活己糖胺生物合成途径。方法 采用内皮一氧化氮合酶敲除 （eNOS-/-） 和 BALB/c 小鼠后肢缺血。葡萄糖胺 （600 μg/g 每天） 腹膜内注射。使用激光多普勒灌注成像评估血流恢复，并通过 CD31 免疫染色研究血管生成。在体外，在缺氧和血清饥饿下测试人脐静脉 ECs 和具有葡萄糖胺、L-葡萄糖或血管内皮生长因子 （VEGF165a） 的小鼠微血管 ECS。评估细胞计数试剂盒 8、试管形成、细胞内活性氧、电细胞-底物阻抗感应和异硫氰酸荧光素葡聚糖通透性。通过 seahorse 测定评估糖酵解和氧化磷酸化。使用 RNA 测序、实时定量聚合酶链反应和 Western blot 评估基因表达。与标准医疗护理相比，评估外周动脉疾病患者在监督运动前后的人体肌肉活检的 EC O-GlcNAcylation。结果 在后肢缺血后第 3 天，葡萄糖胺处理与对照 eNOS-/- 小鼠坏死较少 （每组 n=4 或 5）。 从后肢缺血后第 7 天开始，葡萄糖胺处理与对照 BALB/c 小鼠的血流量较高，这种情况持续到第 21 天，此时缺血肌肉显示每根肌纤维的 CD31 染色更大 （每组 n=8）。在体外，葡萄糖胺与 L-葡萄糖 ECs 相比，存活率 （每组 n=6） 和管形成 （每组 n=6） 得到改善。葡萄糖胺与 L-葡萄糖 ECs 的 RNA 测序显示氨基酸代谢增加 （每组 n=3）。这导致氧化磷酸化增加 （每组 n=8-12） 和丝氨酸生物合成途径，而糖酵解或磷酸戊糖途径基因 （每组 n=6） 增加。与通过 VEGF165a 激活糖酵解相比，这与更好的屏障功能 （每组 n=6-8） 和更少的活性氧 （每组 n=7 或 8） 相关。这些作用是通过激活转录因子 4 介导的，转录因子 4 是运动诱导的血管生成的驱动因素。在外周动脉疾病患者的肌肉活检中，与标准医疗护理相比，12 周的监督运动增加了 EC/O-GlcNAcylation （每组 n=6）。结论在细胞、小鼠和人类中，葡萄糖胺在外周动脉疾病中激活己糖胺生物合成途径会诱导 “运动样” 血管生成，并为治疗这种具有挑战性的疾病提供了一种有前途的新型治疗途径。