Regulation of ischemic limb vascularization by glutaredoxin-1

谷氧还蛋白-1对缺血肢体血管化的调节

• 批准号: 9277579
• 负责人: Reiko Matsui
• 金额: $ 41.13万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9277579
• 关键词: Angiogenic Factor; Blood Vessels; Blood flow; Complications of Diabetes Mellitus; Creatine Kinase; Data; Dependovirus; Diabetes Mellitus; Diabetic Angiopathies; Diabetic mouse; Diet; Down-Regulation; Doxycycline; Enzymes; Fatty acid glycerol esters; Gene Deletion; Genes; Glucose; Glutathione; Grx1 protein; Hindlimb; Hypoxia; Impairment; Injectable; Ischemia; Knock-out; Knockout Mice; Ligation; Limb structure; Metabolic Diseases; Mus; Muscle; Muscle Cells; Muscle Fibers; Outcome; Oxidants; Oxidation-Reduction; Oxygen; Physiological; Post-Translational Protein Processing; Production; Proteins; Recovery; Regulation; Reporting; Response Elements; Role; SKIL gene; Signal Transduction; Skeletal Muscle; Sucrose; Sulfhydryl Compounds; Testing; Tetanus Helper Peptide; Tetracyclines; Therapeutic; Transgenic Mice; Up-Regulation; Vascular Endothelial Growth Factors; Vascularization; adduct; angiogenesis; diabetic; femoral artery; glutaredoxin; hypoxia inducible factor 1; improved; in vivo; insight; knock-down; overexpression; prevent; promoter; protein function; small hairpin RNA; therapeutic target

Critical limb ischemia is one of the major complications of diabetes and impairment of ischemic revascularization by metabolic disorders is still poorly understood. Elevated oxidants may be deleterious, but growing evidence supports the notion that physiological levels of oxidants are essential to promote ischemic angiogenesis. Protein thiols react with oxidants and cellular glutathione (GSH) forms stable protein GSH adducts (or S-glutathionylation) that alter protein function. GSH adducts are reversed by a cytosolic enzyme, glutaredoxin-1 (Glrx), which may be increased in diabetes. Glrx modulates redox signaling by reducing GSH adducts. We found that Glrx overexpressing transgenic mice have impaired blood flow recovery after femoral artery ligation (Murdoch 2014). In contrast, our new studies indicate that Glrx knockout (KO) mice have improved blood flow recovery after hindlimb ischemia in association with increased GSH adducts and higher protein levels of HIF-1and VEGF in ischemic muscles. HIF-1 activity is usually regulated at the protein level, and S-nitrosylation (R-SNO) at Cys533 in the oxygen-dependent degradation domain is reported to stabilize HIF-1 by preventing degradation. R-SNO reacts with abundant GSH to form the more stable GSH adduct (R-SSG) which would be regulated by Glrx. Our studies indicate that in Glrx KO mice HIF-1-GSH adducts increase resulting in stabilization and activation of HIF-1 which can increase VEGF production in ischemic limbs. C2C12 muscle cells show that Glrx knockdown induces HIF-1protein and increasesexpression of angiogenic factors including VEGF as well as PGC-1which also induces VEGF and muscle angiogenesis. My hypothesis is that Glrx in skeletal muscle orchestrates anti- angiogenic function, while inhibiting Glrx increases GSH adducts and promotes ischemic limb revascularization in part through increasing HIF-1 and angiogenic factors in muscle. Since diabetes is associated with impaired HIF-1 stability and lower levels of PGC-1, inhibition of Glrx could increase HIF-1 and/or PGC-1 levels and improve limb vascularization in diabetes. I will examine Glrx regulation on angiogenic factors in skeletal muscle cells (aim 1), study the effects of muscle-specific Glrx overexpression in vivo on ischemic limb revascularization (aim 2), and the effects of Glrx gene deletion or inhibition by adeno-associated virus delivery of shRNA on ischemic limb revascularization in diet-induced diabetic mice (aim 3). These studies will elucidate the beneficial and mechanistic role of reversible GSH adducts in ischemic muscle angiogenesis, and provide a potential therapeutic target for ischemic limbs in diabetes.

严重肢体缺血是糖尿病的主要并发症之一， 对代谢紊乱引起的血管重建仍然知之甚少。氧化剂的升高可能是 有害的，但越来越多的证据支持的概念，生理水平的氧化剂是必不可少的 以促进缺血性血管生成。蛋白质硫醇与氧化剂和细胞谷胱甘肽（GSH）反应 形成稳定的蛋白质GSH加合物（或S-谷胱甘肽化），改变蛋白质功能。谷胱甘肽加合物是 由胞质酶谷氧还蛋白-1（Glutaredoxin-1，Glutaredoxin-1，Glutaredoxin-1）逆转，Glutaredoxin-1在糖尿病中可能增加。格莱什 通过减少GSH加合物来调节氧化还原信号。我们发现Glycoprotein过表达的转基因 小鼠在股动脉结扎后血流恢复受损（Murdoch 2014）。相比之下，我们 新的研究表明，Glycoprotein敲除（KO）小鼠在后肢损伤后改善了血流恢复。 缺血与GSH加合物增加和HIF-1受体和VEGF蛋白水平升高相关， 缺血性肌肉HIF-1 α的活性通常在蛋白质水平调节，S-亚硝基化（R-SNO） 据报道，氧依赖性降解结构域中的Cys 533处通过阻止HIF-1 α的表达来稳定HIF-1 α。 降解R-SNO与丰富的GSH反应形成更稳定的GSH加合物（R-SSG）， 将由Glass监管。我们的研究表明，在Glyphoid KO小鼠中，HIF-1 α-GSH加合物增加， 导致HIF-1 α的稳定和活化，这可以增加VEGF的产生， 肢体缺血C2 C12肌细胞显示Glycine敲低诱导HIF-1受体蛋白， 增加血管生成因子包括VEGF和PGC-1的表达， VEGF和肌肉血管生成。我的假设是骨骼肌中的谷胱甘肽协调了抗- 血管生成功能，同时抑制Glycine增加GSH加合物，促进缺血肢体 部分通过增加肌肉中的HIF-1 α和血管生成因子来实现血管重建。由于糖尿病 与HIF-1 β稳定性受损和PGC-1 β水平降低相关，Gly的抑制可增加 HIF-1 α和/或PGC-1 β水平并改善糖尿病患者的肢体血管形成。 我将研究Glycoprotein对骨骼肌细胞中血管生成因子的调节（目的1），研究Glycoprotein对骨骼肌细胞中血管生成因子的影响。 体内肌肉特异性Glycine过表达对缺血性肢体血管重建的影响（目的2）， 腺相关病毒介导的shRNA对缺血肢体Gly蛋白基因缺失或抑制的影响 在饮食诱导的糖尿病小鼠中进行血管再生（aim 3）。这些研究将阐明有益的和 可逆GSH加合物在缺血性肌肉血管生成中的机制作用，并提供了一个潜在的 糖尿病肢体缺血的治疗靶点。