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，病死率高，病死率低。 治疗选择。在这种翻译PPG(TPPG)的第一周期中进行的研究发现 氧化还原失衡在激活的肌成纤维细胞(myo-FBS)中的关键作用，它可能推动疾病的进展 IPF。在肺纤维化的动物模型中，靶向活性氧物种(ROS)生成酶， NADPH氧化酶-4(NOX4)，通过遗传方法或通过药物方法(GKT137831，待测试 在IIb期临床试验中，见项目1)预防纤维化。UAB-tPPG调查人员最近的研究表明 发现了一种以有氧糖酵解(Agly)升高为特征的myo-FBS的代谢重新编程，以及 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 (转化生长因子-β-1)，诱导产生三羧酸循环代谢物，包括琥珀酸和富马酸， 它们可以稳定/激活缺氧诱导因子1(HIF-1)(7-9)。我们的初步研究表明 在NOX4基因缺失的胎牛中，转化生长因子-NOX1诱导的缺氧诱导因子-1的激活受到抑制。 此外，我们已经建立了一种新的3D-椭圆形组织培养系统，它提供了一种补充 以患者特有的方式测试myo-Fb侵袭性和筛选抗纤维化化合物的方法。 激活的巨噬细胞(见项目3)和B淋巴细胞(见项目4)的旁分泌效应在 调节Fb和myo-FBS表型；然而，NOX酶的作用和代谢潜力 这些细胞类型对myo-fbs的重新编程还不是很清楚。 在这个项目中要检验的假设是NOX4通过代谢介导纤维化效应 重新编程涉及线粒体功能障碍和产生TCA循环代谢物以授予 对myo-FBS具有侵袭性和抗凋亡表型，从而阻碍纤维化的消退。具体目标 要检测的是：(1)确定NOX4的表达和/或FBS的侵袭能力 预测IPF的严重程度和/或进展；并表征对抗-HBs反应的异质性 纤维化药物；(2)确定NOX4代谢重编myo-FBS以诱导 凋亡抵抗和侵袭；并确定这些促纤维化的myo-Fb表型是否 被激活的巨噬细胞和/或B细胞调节；以及(3)确定 肝纤维化动物模型中NOX4的遗传/药物抑制是通过逆转促纤维化作用介导的 新陈代谢计划。 这些研究的完成将：(A)将NOX4的生物学与代谢重新编程联系起来；(B) 为中间代谢在决定纤维化基因表达和促纤维化中的作用提供新的见解。 纤维细胞表型；以及(C)利用新的3D-椭圆形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.