Ischemia-induced angiogenic responses are blunted by aging. This is associated with decreased vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1 activity. Low levels of oxidants are essential to transduce physiological signaling through oxidative post-translational modifications. Because of the abundance of cellular glutathione (GSH), GSH adducts on protein thiols (S-glutathionylation) are commonly formed under mild oxidative stress, and reversed by a small cytosolic thioltransferase, also known as glutaredoxin-1 (Glrx). We found that Glrx transgenic (TG) mice showed impaired blood flow recovery after femoral artery ligation in middle-aged mice. In contrast, Glrx knockout (KO) mice improved blood flow recovery after hindlimb ischemia. Our preliminary data indicate higher protein expression of HIF-1? and VEGF in ischemic muscles of Glrx KO mice, and increased reversible thiol modification of HIF-1? in Glrx KO muscle. HIF-1? can be modified by S-nitrosylation (R-SNO) at Cys533 in the oxygen-dependent degradation domain which stabilizes HIF-1? by preventing degradation and promotes its activation. R-SNO adducts react rapidly with abundant GSH to become the more stable modification GSH adducts (R-SSG) which would be increased by lack of Glrx. Therefore, we hypothesize that Glrx KO may increase HIF-1?-GSH adducts resulting in stabilization and activation of HIF-1? and increased VEGF production.
Specific aim 1 is to examine regulation of HIF-1? activity and VEGF production by Glrx in vitro. For this aim, 1) the effects of Glrx knockdown or overexpression on HIF-1? stability, activity, and VEGF production will be assessed in C2C12 muscle cell line. 2) GSH adducts on HIF-1? will be detected by the biotin switch assay and mass spectrometry. 3) Cys mutant HIF-1? will be tested for lack of the effect of Glrx inhibition.
Specific aim 2 is to sudy the effect of local inhibition of Glrx on hindlimb ischemia in mice. Adeno-associated virus (AAV) expressing short hairpin RNA to silence Glrx expression will be injected in limb muscle, and hindlimb ischemia revascularization will be tested in young and middle-aged (8 month old) wild type mice. Middle-aged mice are known that hindlimb ischemia recovery is impaired. AAV-mediated gene delivery gives long lasting expression, and may provide therapeutic potential. Our findings will reveal the novel concept and mechanism that increased GSH adducts, oxidative modification, improve ischemic revascularization. These studies can be extended to generating tissue-specific Glrx KO mice and to apply to other ischemic models in aging.
The elderly have poor blood flow recovery from ischemic limbs which is associated with decreased level of angiogenic factors to stimulate new vascular formation. We propose that inhibiting an enzyme (glutaredoxin) can activate one of the factors and promote ischemic limb revascularization. This study will not only analyze the mechanism to improve blood flow recovery, but also explore the therapeutic use to improve ischemic disease.
| Anathy, Vikas; Lahue, Karolyn G; Chapman, David G et al. (2018) Reducing protein oxidation reverses lung fibrosis. Nat Med 24:1128-1135 |
| Matsui, Reiko; Watanabe, Yosuke; Murdoch, Colin E (2017) Redox regulation of ischemic limb neovascularization - What we have learned from animal studies. Redox Biol 12:1011-1019 |
