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.
Diabetes causes limb ischemia which is due in part to impaired angiogenesis, the formation of new blood vessels. We propose that angiogenesis is impaired because the enzyme (glutaredoxin) which removes oxidative changes in proteins is up-regulated and reduce angiogenic factors. This study will not only help explain why limb blood flow recovery is impaired in diabetes, but also provide insight into the therapeutic mechanism to improve diabetic vascular disease.
