Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Corepressor C-Terminal Binding Protein-1

Abstract

Background Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells including fibroblasts in pulmonary hypertension (PH). Herein, 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 pro-inflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional co-repressor C-terminal binding protein 1 (CtBP1). Methods RNA-Sequencing, qPCR, 13C-NMR, fluorescence-lifetime imaging, mass spectrometry-based metabolomics and tracing experiments with U-13C-glucose were used to assess glycolytic reprogramming and to measure NADH/NAD+ ratio in bovine and human adventitial fibroblasts, and mouse lung tissues. Immunohistochemistry was utilized to assess CtBP1 expression in the whole lung tissues. CtBP1 siRNA and the pharmacologic inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were utilized to abrogate CtBP1 activity in cells and hypoxic mice. Results We found adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with IPAH (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 IPAH and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB, or genetically blocking CtBP1 using siRNA, upregulated the cyclin-dependent genes (p15 and p21) and pro-apoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. ChIP 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. Conclusions CtBP1 is a critical factor linking changes in cell metabolism to cell phenotype in hypoxic and other forms of PH and a therapeutic target.

Mechanisms contributing to persistently activated cell phenotypes in pulmonary hypertension

“…Increased CtBP activity enhances cell proliferation and apoptosis resistance by decreasing expression of cell cycle inhibitors such as p15 and p21 and pro‐apoptosis genes such as NOXA and PERP (Li et al . ). Importantly, normalizing metabolic activity via metabolic inhibitors such as 2‐deoxyglucose (2DG) or directly reducing CtBP1 expression reduces PH fibroblast proliferation and apoptosis resistance (Li et al .…”

“…Importantly, normalizing metabolic activity via metabolic inhibitors such as 2‐deoxyglucose (2DG) or directly reducing CtBP1 expression reduces PH fibroblast proliferation and apoptosis resistance (Li et al . ).…”

“…These activated phenotypes are likely due to a persistently high expression of genes such as CCND1 , CCNA2 (hyper‐proliferation), BCL2 , BCL2L1 and Survivin (apoptosis‐resistance), CCL2 , CXCL12 , GM‐CSF , IL6 and VCAM1 (pro‐inflammation), epidermal growth factor receptor (EGFR) ligands ( AREG , EREG , TGFA ), fibroblast growth factor receptor (FGFR) ligands ( FGF7 ) and MET ligand ( HGF ) (Li et al . ; Pullamsetti et al . , )(authors unpublished observations).…”

SNP detection using RNA-sequences of candidate genes associated with puberty in cattle

“…Six tissues were collected and included from each steer: left ventricle (heart), right ventricle (heart), pulmonary artery, aorta, longissimus dorsi muscle, and lung Li et al, 2016).…”

“…Fibroblasts from the pulmonary artery were harvested and cultured, and transcriptome analysis was performed using RNA from these cells (Li et al, 2016).…”