Hypoxia inducible-factor1alpha regulates the metabolic shift of pulmonary hypertensive endothelial cells.

Severe pulmonary hypertension is irreversible and often fatal. Abnormal proliferation and resistance to apoptosis of endothelial cells (ECs) and hypertrophy of smooth muscle cells in this disease are linked to decreased mitochondria and preferential energy generation by glycolysis. We hypothesized this metabolic shift of pulmonary hypertensive ECs is due to greater hypoxia inducible-factor1alpha (HIF-1alpha) expression caused by low levels of nitric oxide combined with low superoxide dismutase activity. We show that cultured ECs from patients with idiopathic pulmonary arterial hypertension (IPAH-ECs) have greater HIF-1alpha expression and transcriptional activity than controls under normoxia or hypoxia, and pulmonary arteries from affected patients have increased expression of HIF-1alpha and its target carbonic anhydrase IX. Decreased expression of manganese superoxide dismutase (MnSOD) in IPAH-ECs paralleled increased HIF-1alpha levels and small interfering (SI) RNA knockdown of MnSOD, but not of the copper-zinc SOD, increased HIF-1 protein expression and hypoxia response element (HRE)-driven luciferase activity in normoxic ECs. MnSOD siRNA also reduced nitric oxide production in supernatants of IPAH-ECs. Conversely, low levels of a nitric oxide donor reduced HIF-1alpha expression in normoxic IPAH-ECs. Finally, mitochondria numbers increased in IPAH-ECs with knockdown of HIF-1alpha. These findings indicate that alterations of nitric oxide and MnSOD contribute to pathological HIF-1alpha expression and account for lower numbers of mitochondria in IPAH-ECs.

Integrative proteomics and phosphoproteomics in pulmonary arterial hypertension.

Pulmonary arterial endothelial cells (PAEC) are mechanistically linked to origins of pulmonary arterial hypertension (PAH). Here, global proteomics and phosphoproteomics of PAEC from PAH (n = 4) and healthy lungs (n = 5) were performed using LC-MS/MS to confirm known pathways and identify new areas of investigation in PAH. Among PAH and control cells, 170 proteins and 240 phosphopeptides were differentially expressed; of these, 45 proteins and 18 phosphopeptides were located in the mitochondria. Pathologic pathways were identified with integrative bioinformatics and human protein-protein interactome network analyses, then confirmed with targeted proteomics in PAH PAEC and non-targeted metabolomics and targeted high-performance liquid chromatography of metabolites in plasma from PAH patients (n = 30) and healthy controls (n = 12). Dysregulated pathways in PAH include accelerated one carbon metabolism, abnormal tricarboxylic acid (TCA) cycle flux and glutamate metabolism, dysfunctional arginine and nitric oxide pathways, and increased oxidative stress. Functional studies in cells confirmed abnormalities in glucose metabolism, mitochondrial oxygen consumption, and production of reactive oxygen species in PAH. Altogether, the findings indicate that PAH is typified by changes in metabolic pathways that are primarily found in mitochondria.

Arginine metabolic endotypes related to asthma severity.

AIMS: Arginine metabolism via inducible nitric oxide synthase (iNOS) and arginase 2 (ARG2) is higher in asthmatics than in healthy individuals. We hypothesized that a sub-phenotype of asthma might be defined by the magnitude of arginine metabolism categorized on the basis of high and low fraction of exhaled nitric oxide (FENO). METHODS: To test this hypothesis, asthmatics (n = 52) were compared to healthy controls (n = 51) for levels of FENO, serum arginase activity, and airway epithelial expression of iNOS and ARG2 proteins, in relation to clinical parameters of asthma inflammation and airway reactivity. In parallel, bronchial epithelial cells were evaluated for metabolic effects of iNOS and ARG2 expression in vitro. RESULTS: Asthmatics with high FENO (≥ 35 ppb; 44% of asthmatics) had higher expression of iNOS (P = 0.04) and ARG2 (P = 0.05) in the airway, indicating FENO is a marker of the high arginine metabolic endotype. High FENO asthmatics had the lowest FEV1% (P < 0.001), FEV1/FVC (P = 0.0002) and PC20 (P < 0.001) as compared to low FENO asthmatics or healthy controls. Low FENO asthmatics had near normal iNOS and ARG2 expression (both P > 0.05), and significantly higher PC20 (P < 0.001) as compared to high FENO asthmatics. In vitro studies to evaluate metabolic effects showed that iNOS overexpression and iNOS+ARG2 co-expression in a human bronchial epithelial cell line led to greater reliance on glycolysis with higher rate of pyruvate going to lactate. CONCLUSIONS: The high FENO phenotype represents a large portion of the asthma population, and is typified by greater arginine metabolism and more severe and reactive asthma.

A novel method for pulmonary research: assessment of bioenergetic function at the air-liquid interface.

Air-liquid interface cell culture is an organotypic model for study of differentiated functional airway epithelium in vitro. Dysregulation of cellular energy metabolism and mitochondrial function have been suggested to contribute to airway diseases. However, there is currently no established method to determine oxygen consumption and glycolysis in airway epithelium in air-liquid interface. In order to study metabolism in differentiated airway epithelial cells, we engineered an insert for the Seahorse XF24 Analyzer that enabled the measure of respiration by oxygen consumption rate (OCR) and glycolysis by extracellular acidification rate (ECAR). Oxidative metabolism and glycolysis in airway epithelial cells cultured on the inserts were successfully measured. The inserts did not affect the measures of OCR or ECAR. Cells under media with apical and basolateral feeding had less oxidative metabolism as compared to cells on the inserts at air-interface with basolateral feeding. The design of inserts that can be used in the measure of bioenergetics in small numbers of cells in an organotypic state may be useful for evaluation of new drugs and metabolic mechanisms that underlie airway diseases.