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Background: Endothelial cells depend on glycolysis for much of their energy production. Impaired endothelial glycolysis has been associated with various vascular pathobiologies, including impaired angiogenesis and atherogenesis. IFN-γ (interferon-γ)–producing CD4 + and CD8 + T lymphocytes have been identified as the predominant pathological cell subsets in human atherosclerotic plaques. Although the immunologic consequences of these cells have been extensively evaluated, their IFN-γ–mediated metabolic effects on endothelial cells remain unknown. The purpose of this study was to determine the metabolic consequences of the T-lymphocyte cytokine, IFN-γ, on human coronary artery endothelial cells. Methods: The metabolic effects of IFN-γ on primary human coronary artery endothelial cells were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux analyses and molecular mechanistic studies. Cellular phenotypic correlations were made by measuring altered endothelial intracellular cGMP content, wound-healing capacity, and adhesion molecule expression. Results: IFN-γ exposure inhibited basal glycolysis of quiescent primary human coronary artery endothelial cells by 20% through the global transcriptional suppression of glycolytic enzymes resulting from decreased basal HIF1α (hypoxia-inducible factor 1α) nuclear availability in normoxia. The decrease in HIF1α activity was a consequence of IFN-γ–induced tryptophan catabolism resulting in ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF1β sequestration by the kynurenine-activated AHR (aryl hydrocarbon receptor). In addition, IFN-γ resulted in a 23% depletion of intracellular nicotinamide adenine dinucleotide in human coronary artery endothelial cells. This altered glucose metabolism was met with concomitant activation of fatty acid oxidation, which augmented its contribution to intracellular ATP balance by >20%. These metabolic derangements were associated with adverse endothelial phenotypic changes, including decreased basal intracellular cGMP, impaired endothelial migration, and a switch to a proinflammatory state. Conclusions: IFN-γ impairs endothelial glucose metabolism by altered tryptophan catabolism destabilizing HIF1, depletes nicotinamide adenine dinucleotide, and results in a metabolic shift toward increased fatty acid oxidation. This work suggests a novel mechanistic basis for pathological T lymphocyte–endothelial interactions in atherosclerosis mediated by IFN-γ, linking endothelial glucose, tryptophan, and fatty acid metabolism with the nicotinamide adenine dinucleotide balance and ATP generation and their adverse endothelial functional consequences.

Introduction: Advanced-stage pulmonary arterial hypertension (asPAH) is characterized by endothelial dysfunction and fibrotic remodeling of pulmonary arterioles that promotes irreversible right heart failure. Thus, identifying mechanisms that regulate vascular fibrosis in early PAH (esPAH) may have translational importance. Hypothesis: We hypothesized that profibrotic molecular pathways differentiate esPAH from asPAH. Methods: To test this hypothesis, male Sprague-Dawley rats were administered one dose of monocrotaline (60 mg/kg; day 0) to induce inflammatory PAH. Cardiac catheterization and pulmonary artery endothelial cell (PAEC) transcriptomic analyses were performed on days 15 and 21 for esPAH and asPAH, respectively. Results: The esPAH profile included: right ventricular-pulmonary arterial (RV-PA) uncoupling (1.13 ± 0.05 vs. 0.90 ± 0.06, RV end-systolic elastance/PA elastance, P=0.02, N=6) and increased indexed pulmonary vascular resistance (50 ± 8 vs. 213 ± 29 mmHg*min*g -1 *mL -1 , Pin silico . Pulmonary endothelial expression of Csk protein by immunofluorescence correlated with vascular collagen by picrosirius red stain (r=+0.87, P=0.006, N=4). To validate these findings in vitro , human PAECs (HPAEC) were treated with an inflammatory stimulus of lipopolysaccaride (0.03 mg/mL) + interferon-γ (50 ng/mL) + interleukin-1β (50 ng/mL), or vehicle control (V). Compared to V-control, inflammation increased Csk protein and mRNA expression by 2.3- and 2.0-fold, respectively (P-4 , N=4) vs. V-treated cells. Overexpression of Csk by adenovirus transfection attenuated inflammation-mediated hydroxyproline accumulation by 84% (P=1.3x10 -5 , N=3). Conclusions: These data suggest that impaired Csk may underlie HPAEC fibrosis in esPAH, which, in turn, may have potential therapeutic implications for the prevention of fibrotic vascular remodeling and PAH progression.

Introduction: Endothelial cells depend on glycolysis for much of energy production. Deranged endothelial glycolysis has been associated with various vascular pathobiologies. Interferon-gamma (IFN-γ) producing T-cells have been identified as the predominant pathologic cell subset in human atherosclerotic plaque. While the immunological consequences of these cells have been evaluated, their metabolic effects on endothelial cells remain unknown. The objective of this study was to determine the metabolic consequences of IFN-γ on human coronary artery endothelial cells (HCAEC). Hypothesis: We hypothesized that IFN-γ inhibits endothelial glucose metabolism and results in pathologic phenotypic changes in HCAEC. Methods: The metabolic effects of IFN-γ on primary HCAEC were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux and molecular mechanistic studies. Relevant cellular phenotype was assessed by measuring endothelial intracellular cGMP content and wound healing capacity. Results: IFN-γ inhibited glycolysis of HCAEC by 20 % ( p = 0.006) via global transcriptional suppression of glycolytic enzymes resulting from decreased basal nuclear hypoxia inducible factor 1α (HIF1α) availability. HIF1α destabilization (FC 0.72; p = 0.0039) was mediated by the sequestration of its binding partner HIF1β by aryl hydrocarbon receptor via a kynurenine surge (FC 6.24; p < 0.0001) resulting from IFN-γ-induced tryptophan degradation. Endothelial fatty acid oxidation (FAO) increased by 19% ( p = 0.014) with its inhibition leading to a 23% ATP deficit ( p < 0.0001). These metabolic derangements were associated with pathologic endothelial phenotypic changes, including decreased intracellular cGMP by 29 % ( p = 0.025) and impaired wound healing capacity by 38 % ( p = 0.0098). Conclusions: IFN-γ impairs endothelial glucose metabolism via tryptophan catabolism destabilizing HIF1α and results in a metabolic shift toward increased FAO. These findings suggest a novel mechanistic basis for pathologic T-lymphocyte-endothelial interaction in coronary artery disease mediated by IFN-γ, linking endothelial glucose, amino acid, and fatty acid metabolism and their adverse endothelial functional consequences.

Background— The molecular mechanism(s) regulating hypoxia-induced vascular fibrosis are unresolved. Hyperaldosteronism correlates positively with vascular remodeling in pulmonary arterial hypertension, suggesting that aldosterone may contribute to the pulmonary vasculopathy of hypoxia. The hypoxia-sensitive transcription factors c-Fos/c-Jun regulate steroidogenic acute regulatory protein (StAR), which facilitates the rate-limiting step of aldosterone steroidogenesis. We hypothesized that c-Fos/c-Jun upregulation by hypoxia activates StAR-dependent aldosterone synthesis in human pulmonary artery endothelial cells (HPAECs) to promote vascular fibrosis in pulmonary arterial hypertension. Methods and Results— Patients with pulmonary arterial hypertension, rats with Sugen/hypoxia–pulmonary arterial hypertension, and mice exposed to chronic hypoxia expressed increased StAR in remodeled pulmonary arterioles, providing a basis for investigating hypoxia–StAR signaling in HPAECs. Hypoxia (2.0% FiO 2 ) increased aldosterone levels selectively in HPAECs, which was confirmed by liquid chromatography–mass spectrometry. Increased aldosterone by hypoxia resulted from enhanced c-Fos/c-Jun binding to the proximal activator protein-1 site of the StAR promoter in HPAECs, which increased StAR expression and activity. In HPAECs transfected with StAR–small interfering RNA or treated with the activator protein-1 inhibitor SR-11302 [3-methyl-7-(4-methylphenyl)-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid], hypoxia failed to increase aldosterone, confirming that aldosterone biosynthesis required StAR activation by c-Fos/c-Jun. The functional consequences of aldosterone were confirmed by pharmacological inhibition of the mineralocorticoid receptor with spironolactone or eplerenone, which attenuated hypoxia-induced upregulation of the fibrogenic protein connective tissue growth factor and collagen III in vitro and decreased pulmonary vascular fibrosis to improve pulmonary hypertension in vivo. Conclusion— Our findings identify autonomous aldosterone synthesis in HPAECs attributable to hypoxia-mediated upregulation of StAR as a novel molecular mechanism that promotes pulmonary vascular remodeling and fibrosis.