Li Min, Riddle Suzette, Zhang Hui, D'Alessandro Angelo, Flockton Amanda, Serkova Natalie J, Hansen Kirk C, Moldovan Radu, McKeon B Alexandre, Frid Maria, Kumar Sushil, Li Hong, Liu Hongbing, Caánovas Angela, Medrano Juan F, Thomas Milton G, Iloska Dijana, Plecitá-Hlavatá Lydie, Ježek Petr, Pullamsetti Soni, Fini Mehdi A, El Kasmi Karim C, Zhang QingHong, Stenmark Kurt R
From Cardiovascular Pulmonary Research Laboratories, Department of Pediatrics and Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO (M.L., S.R., H.Z., A.F., B.A.M., M.F., S.K., M.A.F., K.R.S.); Department of Biochemistry and Molecular Genetics and Biological Mass Spectrometry Shared Resource (A.D., K.C.H.), Department of Anesthesiology (N.J.S.), Advanced Light Microscopy Core Facility (R.M.), Department of Dermatology (H.L., H.L., Q.Z.), and Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition (K.C.E.K.), University of Colorado, Denver; Department of Mitochondrial Physiology, Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic (L.P.-H., P.J.); Department of Lung Development and Remodeling, Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany (D.I., S.P.); Center for Genetic Improvement of Livestock, Department of Animal Bioscience, University of Guelph, Guelph, ON, Canada (A.C.); Department of Animal Science, University of California-Davis, Davis (J.F.M.); and Department of Animal Science, Colorado State University, Fort Collins (M.G.T.).
Circulation. 2016 Oct 11;134(15):1105-1121. doi: 10.1161/CIRCULATIONAHA.116.023171. Epub 2016 Aug 25.
Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells, including fibroblasts, in pulmonary hypertension (PH). Here, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and proinflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional corepressor C-terminal binding protein 1 (CtBP1).
RNA sequencing, quantitative polymerase chain reaction,C-nuclear magnetic resonance, fluorescence-lifetime imaging, mass spectrometry-based metabolomics, and tracing experiments with U-C-glucose were used to assess glycolytic reprogramming and to measure the NADH/NAD ratio in bovine and human adventitial fibroblasts and mouse lung tissues. Immunohistochemistry was used to assess CtBP1 expression in the whole-lung tissues. CtBP1 siRNA and the pharmacological inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were used to abrogate CtBP1 activity in cells and hypoxic mice.
We found that adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with idiopathic pulmonary arterial hypertension (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with idiopathic pulmonary arterial hypertension and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB or genetically blocking CtBP1 with siRNA upregulated the cyclin-dependent genes (p15 and p21) and proapoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. Chromatin immunoprecipitation analysis demonstrated that CtBP1 directly binds the HMOX1 promoter. Treatment of hypoxic mice with MTOB decreased glycolysis and expression of inflammatory genes, attenuated proliferation, and suppressed macrophage numbers and remodeling in the distal pulmonary vasculature.
CtBP1 is a critical factor linking changes in cell metabolism to cell phenotype in hypoxic and other forms of PH and a therapeutic target.
有研究表明，代谢变化有助于肺动脉高压（PH）中血管壁细胞（包括成纤维细胞）的异常表型形成。在此，我们检验以下假设：向有氧糖酵解的代谢重编程是高血压血管壁中成纤维细胞的关键适应性变化，其通过涉及烟酰胺腺嘌呤二核苷酸（NADH）敏感性转录共抑制因子C末端结合蛋白1（CtBP1）活性增加的机制驱动增殖和促炎激活。
采用RNA测序、定量聚合酶链反应、碳核磁共振、荧光寿命成像、基于质谱的代谢组学以及用U-¹³C-葡萄糖进行示踪实验，以评估糖酵解重编程并测量牛和人外膜成纤维细胞以及小鼠肺组织中的NADH/ NAD比值。采用免疫组织化学评估全肺组织中CtBP1的表达。使用CtBP1小干扰RNA（siRNA）和药理抑制剂4-甲硫基-2-氧代丁酸（MTOB）消除细胞和低氧小鼠中的CtBP1活性。
我们发现，来自严重低氧诱导的PH小牛和特发性肺动脉高压患者（PH-成纤维细胞）的外膜成纤维细胞在常氧培养条件下表现出有氧糖酵解，同时游离NADH和NADH/ NAD比值增加。在特发性肺动脉高压患者、患PH的动物以及培养的PH-成纤维细胞的肺外膜成纤维细胞中，NADH传感器CtBP1的表达在体内和体外均增加。用MTOB进行药理作用降低NADH或用siRNA进行基因阻断CtBP1可上调细胞周期蛋白依赖性基因（p15和p21）和促凋亡调节因子（NOXA和PERP），减弱增殖，纠正PH-成纤维细胞的糖酵解重编程表型，并增强抗炎基因血红素加氧酶1（HMOX1）的转录。染色质免疫沉淀分析表明，CtBP1直接结合HMOX1启动子。用MTOB处理低氧小鼠可降低糖酵解和炎症基因的表达，减弱增殖，并抑制远端肺血管中的巨噬细胞数量和重塑。
CtBP1是将低氧及其他形式的PH中细胞代谢变化与细胞表型联系起来的关键因素，也是一个治疗靶点。
