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

• 批准号: 9323226
• 负责人: Reiko Matsui
• 金额: $ 8.23万
• 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: 2016-08-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9323226
• 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

 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.

 描述（由申请方提供）：缺血诱导的血管生成反应因老化而减弱。这与血管内皮生长因子（VEGF）和缺氧诱导因子（HIF）-1活性降低有关。低水平的氧化剂对于通过氧化翻译后修饰抑制生理信号传导是必不可少的。由于细胞内谷胱甘肽（GSH）丰富，蛋白巯基上的GSH加合物（S-谷胱甘肽化）通常在轻度氧化应激下形成，并被小的细胞溶质巯基转移酶（也称为谷氧还蛋白-1（Glutaroxin））逆转。我们发现，在中年小鼠股动脉结扎后，Glycoprotein转基因（TG）小鼠表现出血流恢复受损。相比之下，Glycoprotein敲除（KO）小鼠改善了后肢缺血后的血流恢复。我们的初步数据表明，在Glyphoid KO小鼠的缺血肌肉中，HIF-1α和VEGF的蛋白表达较高，并且Glyphoid KO肌肉中HIF-1α的可逆巯基修饰增加。HIF-1α可通过氧依赖性降解结构域中Cys 533处的S-亚硝基化（R-SNO）进行修饰，其通过防止降解稳定HIF-1α并促进其活化。R-SNO加合物与丰富的GSH迅速反应，成为更稳定的修饰型GSH加合物（R-SSG），其将因缺乏Glucose而增加。因此，我们假设Glucose KO可能增加HIF-1α-GSH加合物，导致HIF-1α的稳定和活化，并增加VEGF的产生。具体目的1是在体外检测Glycoprotein对HIF-1α活性和VEGF产生的调节。为此目的，1）将在C2 C12肌肉细胞系中评估Glrx敲减或过表达对HIF-1α稳定性、活性和VEGF产生的影响。2)将通过生物素转换试验和质谱法检测HIF-1α上的GSH加合物。3)将测试Cys突变体HIF-1α是否缺乏Glycoprotein抑制作用。具体目的二是研究局部抑制Gln对小鼠后肢缺血的影响。将在肢体肌肉中注射表达短发夹RNA以沉默Glrx表达的腺相关病毒（腺相关病毒），并在年轻和中年（8个月大）野生型小鼠中测试后肢缺血血运重建。已知中年小鼠后肢缺血恢复受损。AAV介导的基因递送提供持久的表达，并且可以提供治疗潜力。我们的研究结果将揭示GSH加合物增加、氧化修饰、促进缺血再血管化的新概念和机制。这些研究可以扩展到产生组织特异性Glucose KO小鼠，并应用于其他衰老中的缺血模型。