Redox Regulation of Metabolic Reprogramming in Activated Myofibroblasts

摘要 特发性肺纤维化（IPF）是一种慢性、进行性肺部疾病，死亡率高， 治疗选择在该平移PPG（tPPG）的周期I期间进行的研究发现了 活化的肌成纤维细胞（myo-Fbs）中氧化还原失衡的关键作用，可能会推动疾病进展， IPF。在肺纤维化的动物模型中，靶向活性氧（ROS）生成酶， NADPH氧化酶-4（NOX 4），通过遗传方法或药理学方法（GKT 137831，待测 在IIb期临床试验中，参见项目1）防止纤维化。UAB-tPPG研究者最近的研究表明， 发现了肌纤维蛋白酶的代谢重编程，其特征在于有氧糖酵解（aGLY）升高， 线粒体功能障碍我们的数据表明，促纤维化细胞因子转化生长因子β1 (TGF-β1），诱导三羧酸（TCA）循环代谢产物的产生，包括琥珀酸盐和富马酸盐， 已知其稳定/激活缺氧诱导因子1 β（HIF-1 β）（7-9）。我们的初步研究显示 NOX 4基因缺失（NOX 4-/-）的Fbs抑制TGF-β1诱导的HIF-1 α活化。 此外，我们还建立了一种新的三维球体组织培养系统， 测试myo-Fb侵袭性和以患者特异性方式筛选抗纤维化化合物的方法。 活化的巨噬细胞（见项目3）和B淋巴细胞（见项目4）的旁分泌作用是至关重要的， 调节Fb和myo-Fbs表型;然而，NOX酶的作用和代谢的潜力， 这些细胞类型对myo-Fbs的重编程还不清楚。 在本项目中待检验的假设是NOX 4通过代谢途径介导纤维化效应。 涉及线粒体功能障碍和TCA循环代谢产物产生的重编程， 对myo-Fbs具有侵袭性和抗纤维化表型，从而阻碍纤维化消退。具体目标 （1）确定Fbs的N 0X 4表达和/或侵袭能力是否与N 0X 4表达和/或侵袭能力相关。 预测IPF的严重程度和/或进展;并表征抗- （2）确定NOX 4代谢重编程myo-Fbs以诱导 细胞凋亡抗性和侵袭;并确定这些促纤维化myo-Fb表型是否 由活化的巨噬细胞和/或B细胞调节;和（3）确定是否有保护作用， 纤维化动物模型中的遗传/药理学NOX 4抑制是通过逆转促纤维化 代谢程序 这些研究的完成将：（a）将NOX 4的生物学与代谢重编程联系起来;（B） 提供了新的见解，中间代谢的作用，在确定纤维化基因的表达和促纤维化， 纤维化细胞表型;和（c）利用新的3D球体组织培养模型来研究细胞侵袭， 表型多细胞疾病过程，并评估对特定药物治疗的反应性。

ABSTRACT Idiopathic pulmonary fibrosis (IPF) is chronic, progressive lung disorder with high mortality, and limited therapeutic options. Studies performed during Cycle I of this translational PPG (tPPG) have uncovered a critical role for redox imbalance in activated myofibroblasts (myo-Fbs) that may drive disease progression in IPF. In animal models of lung fibrosis, targeting the reactive oxygen species (ROS)-generating enzyme, NADPH oxidase-4 (NOX4), by genetic approaches or by a pharmacologic approach (GKT137831, to be tested in Phase IIb clinical trial, see Project 1) protects from fibrosis. Recent studies by UAB-tPPG investigators have discovered a metabolic reprogramming of myo-Fbs characterized by elevated aerobic glycolysis (aGLY), and mitochondrial dysfunction. Our data demonstrate that the pro-fibrotic cytokine, transforming growth factor-β1 (TGF-β1), induces generation of tricarboxylic acid (TCA) cycle metabolites, including succinate and fumarate, which are known to stabilize/activate hypoxia-inducible factor 1 (HIF-1) (7-9). Our preliminary studies show that TGF-β1-induced activation of HIF-1 is inhibited in Fbs with genetic deletion in NOX4 (NOX4-/-). Additionally, we have established a novel 3D-spheroid tissue culture system that provides a complementary approach to test for myo-Fb invasiveness and screen for anti-fibrotic compounds in a patient-specific manner. Paracrine effects of activated macrophages (see Project 3) and B-lymphocytes (see Project 4) are critical in regulating Fb and myo-Fbs phenotypes; however, the roles of NOX enzymes and potential for metabolic reprogramming of myo-Fbs by these cell types are not well understood. The hypothesis to be tested in this project is that NOX4 mediates fibrogenic effects by metabolic reprogramming that involves mitochondrial dysfunction and generation of TCA cycle metabolites to confer an invasive and apoptosis-resistant phenotype to myo-Fbs, thus, impeding fibrosis resolution. The specific aims to be tested are: (1) to determine whether the NOX4 expression and/or the invasive capacity of Fbs is predictive of severity and/or progression of IPF; and to characterize the heterogeneity in responses to anti- fibrotic drugs; (2) to determine the mechanisms by which NOX4 metabolically reprograms myo-Fbs to induce apoptosis resistance and invasion; and determine whether these pro-fibrotic myo-Fb phenotypes are modulated by activated macrophages and/or B-cells; and (3) to determine whether the protective effect of genetic/pharmacologic NOX4 inhibition in animal models of fibrosis is mediated by reversal of pro-fibrotic metabolic programs. These completion of these studies will: (a) link the biology of NOX4 with metabolic reprogramming; (b) provide new insights into the role of intermediary metabolism in determining fibrotic gene expression and pro- fibrotic cellular phenotypes; and (c) utilize novel 3D-spheroid tissue culture models to study cell invasion, to phenotype multi-cellular disease processes, and to assess responsiveness to specific drug therapies.