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Regulation of ischemic vascularization by glutaredoxin-1 by aging

谷氧还蛋白-1通过衰老调节缺血性血管形成

基本信息

批准号：
9480179
负责人：
Reiko Matsui
金额：
$ 7.15万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9480179
关键词：
Adult Aging Angiogenic Factor Biological Assay Biotin Blood Vessels Blood flow Caenorhabditis elegans Cell Line Data Dependovirus Disease Elderly Endothelial Cells Enzymes Gene Delivery Genetic Transcription Glutathione Grx1 protein Hindlimb Hypoxia Hypoxia Inducible Factor Impairment In Vitro Injectable Ischemia Knock-out Knockout Mice Ligation Limb structure Longevity Mass Spectrum Analysis Mediating Modeling Modification Mus Muscle Muscle Cells Muscle Fibers Myocardial Infarction Outcome Oxidants Oxidative Stress Oxygen Physiological Post-Translational Protein Processing Production Proteins Recovery Regulation SKIL gene Signal Transduction Skeletal Muscle Sulfhydryl Compounds Testing Therapeutic Therapeutic Uses Tissues Transgenic Mice Vascular Endothelial Growth Factors Vascularization Wild Type Mouse adduct aged cell motility femoral artery glutaredoxin hypoxia inducible factor 1 improved in vivo knock-down middle age mutant novel novel therapeutic intervention overexpression prevent protein expression public health relevance response small hairpin RNA therapeutic target thioltransferase

项目摘要

DESCRIPTION (provided by applicant): 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-1α and VEGF in ischemic muscles of Glrx KO mice, and increased reversible thiol modification of HIF-1α in Glrx KO muscle. HIF-1α can be modified by S-nitrosylation (R-SNO) at Cys533 in the oxygen-dependent degradation domain which stabilizes HIF-1α 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-1α-GSH adducts resulting in stabilization and activation of HIF-1α and increased VEGF production. Specific aim 1 is to examine regulation of HIF-1α activity and VEGF production by Glrx in vitro. For this aim, 1) the effects of Glrx knockdown or overexpression on HIF-1α stability, activity, and VEGF production will be assessed in C2C12 muscle cell line. 2) GSH adducts on HIF-1α will be detected by the biotin switch assay and mass spectrometry. 3) Cys mutant HIF-1α 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.