Regulation of ischemic limb vascularization by glutaredoxin-1

严重肢体缺血是糖尿病和脑缺血损害的主要并发症之一。 代谢紊乱导致的血管重建仍然知之甚少。升高的氧化剂可能是 有害的，但越来越多的证据支持这样的观点，即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 形成稳定的蛋白质谷胱甘肽加合物(或S-谷胱甘肽基化)，改变蛋白质功能。GSH加合物是 被一种胞浆酶谷氧还蛋白-1(Glrx)逆转，这种酶在糖尿病时可能会增加。Glrx 通过减少GSH加合物来调节氧化还原信号。我们发现Glrx过表达转基因 小鼠在股动脉结扎后血流恢复受损(Murdoch 2014)。相比之下，我们的 新的研究表明，Glrx基因敲除(KO)小鼠改善了后肢血流的恢复 脑缺血与谷胱甘肽加合物增加及缺氧诱导因子-1和血管内皮生长因子蛋白水平升高相关 肌肉缺血症。缺氧诱导因子-1的活性通常在蛋白水平调节，而S-亚硝化(R-NO) 据报道，在氧依赖降解结构域中的Cys533通过阻止抑制HIF-1来稳定HIF-1 退化。R-SNO与丰富的GSH反应生成更稳定的GSH加合物(R-SSG)， 将由Glrx监管。我们的研究表明，在Glrx KO小鼠中，HIF-1-GSH加合物增加 稳定和激活缺氧诱导因子-1，增加血管内皮生长因子的产生 肢体缺血。C2C12肌肉细胞显示Glrx基因敲除后诱导HIF-1蛋白和 增加血管生成因子的表达，包括血管内皮生长因子和前列腺素C-1，这也诱导 血管内皮生长因子与肌肉血管生成我的假设是骨骼肌中的Glrx协调抗- 血管生成功能，同时抑制Glrx增加GSH加合物，促进缺血肢体 部分通过增加肌肉中的缺氧诱导因子-1、和血管生成因子进行血管重建。自糖尿病以来 与HIF-1稳定性受损和PGC-1水平降低有关，Glrx的抑制作用可能会增加 低氧诱导因子-1和/或前列腺素C-1水平和改善糖尿病患者的肢体血管形成。 我将研究Glrx对骨骼肌细胞血管生成因子的调节(目标1)，研究 肌肉特异性Glrx在缺血肢体血运重建中的体内过表达(AIM 2)及其影响 腺相关病毒转导shRNA对缺血肢体Glrx基因缺失或抑制的影响 饮食诱导的糖尿病小鼠的血管重建(目标3)。这些研究将阐明有益的和 可逆性GSH加合物在缺血肌肉血管生成中的机制作用 糖尿病肢体缺血的治疗靶点。

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.