Dysregulated metabolism characterizes both animal and human forms of pulmonary hypertension (PH). Enzymes involved in fatty acid metabolism have previously not been assessed in human pulmonary arteries affected by pulmonary arterial hypertension (PAH), and how inhibition of fatty acid oxidation (FAO) may attenuate PH remains unclear. Fatty acid metabolism gene transcription was quantified in laser-dissected pulmonary arteries from 10 explanted lungs with advanced PAH (5 idiopathic, 5 associated with systemic sclerosis), and 5 donors without lung diseases. Effects of oxfenicine, a FAO inhibitor, on female Sugen 5416-chronic hypoxia (SuHx) rats were studied in vivo using right heart catheterization, and ex vivo using perfused lungs and pulmonary artery ring segments. The impact of pharmacologic (oxfenicine) and genetic (carnitine palmitoyltransferase 1a heterozygosity) FAO suppression was additionally probed in mouse models of Schistosoma and hypoxia-induced PH. Potential mechanisms underlying FAO-induced PH pathogenesis were examined by quantifying ATP and mitochondrial mass in oxfenicine-treated SuHx pulmonary arterial cells, and by assessing pulmonary arterial macrophage infiltration with immunohistochemistry. We found upregulated pulmonary arterial transcription of 26 and 13 FAO genes in idiopathic and systemic sclerosis-associated PAH, respectively. In addition to promoting de-remodeling of pulmonary arteries in SuHx rats, oxfenicine attenuated endothelin-1-induced vasoconstriction. FAO inhibition also conferred modest benefit in the two mouse models of PH. Oxfenicine increased mitochondrial mass in cultured rat pulmonary arterial cells, and decreased the density of perivascular macrophage infiltration in pulmonary arteries of treated SuHx rats. In summary, FAO inhibition attenuated experimental PH, and may be beneficial in human PAH.

Acute lung injury (ALI) is an inflammatory lung disease, which manifests itself in patients as acute respiratory distress syndrome (ARDS). Previous studies have implicated alveolar-epithelial succinate in ALI protection. Therefore, we hypothesized that targeting alveolar succinate dehydrogenase SDH A would result in elevated succinate levels and concomitant lung protection. Wild-type (WT) mice or transgenic mice with targeted alveolar-epithelial Sdha or hypoxia-inducible transcription factor Hif1a deletion were exposed to ALI induced by mechanical ventilation. Succinate metabolism was assessed in alveolar-epithelial via mass spectrometry as well as redox measurements and evaluation of lung injury. In WT mice, ALI induced by mechanical ventilation decreased SDHA activity and increased succinate in alveolar-epithelial. In vitro, cell-permeable succinate decreased epithelial inflammation during stretch injury. Mice with inducible alveolar-epithelial Sdha deletion (Sdha(loxp/loxp) SPC-CreER mice) revealed reduced lung inflammation, improved alveolar barrier function, and attenuated histologic injury. Consistent with a functional role of succinate to stabilize HIF, Sdha(loxp/loxp) SPC-CreER experienced enhanced Hif1a levels during hypoxia or ALI. Conversely, Hif1a(loxp/loxp) SPC-CreER showed increased inflammation with ALI induced by mechanical ventilation. Finally, wild-type mice treated with intra-tracheal dimethlysuccinate were protected during ALI. These data suggest that targeting alveolar-epithelial SDHA dampens ALI via succinate-mediated stabilization of HIF1A. Translational extensions of our studies implicate succinate treatment in attenuating alveolar inflammation in patients suffering from ARDS.

Altered metabolism in pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs) contributes to the pathology of pulmonary hypertension (PH), but changes in substrate uptake and how substrates are utilized have not been fully characterized. We hypothesized stable isotope metabolomics would identify increased glucose, glutamine and fatty acid uptake and utilization in human PASMCs and PAECs from PH versus control specimens, and that TGF-β treatment would phenocopy these metabolic changes. We used 13 C-labeled glucose, glutamine or a long-chain fatty acid mixture added to cell culture media, and mass spectrometry-based metabolomics to detect and quantify 13 C-labeled metabolites. We found PH PASMCs had increased glucose uptake and utilization by glycolysis and the pentose shunt, but no changes in glutamine or fatty acid uptake or utilization. Diseased PAECs had increased proximate glycolysis pathway intermediates, less pentose shunt flux, increased anaplerosis from glutamine, and decreased fatty acid β-oxidation. TGF-β treatment increased glycolysis in PASMCs, but did not recapitulate the PAEC disease phenotype. In TGF-β-treated PASMCs, glucose, glutamine and fatty acids all contributed carbons to the TCA cycle. In conclusion, PASMCs and PAECs collected from PH subjects have significant changes in metabolite uptake and utilization, partially recapitulated by TGF-β treatment.

in situ Smad3 expression in the lungs of patients with idiopathic PAH and control subjects, PAs were laser microdissected, followed by qPCR.Smad3 mRNA (trend, statistically not significant) and miR-130a/301b (P , 0.01) expression was higher in the PAs and plexiform lesions of patients with idiopathic PAH compared with control subjects (Figure 1E).Thus, our human data support the notion that boosted signaling of TGF-b1 and its canonical downstream effector Smad3 in human PAs is crucial for PAH development and severity.We designed this focused study to address the controversy regarding TGF-b1-mediated Smad3 signaling in PAH.We show that chronic TGF-b1 signaling in vivo leads to spontaneous PAH in mice; is associated with canonical Smad3 activation (Smad3 phosphorylation) but not Smad3 downregulation; and drives PAH by means of proliferation, inflammation, and metabolism in the lung.Our results are supported by several other important publications (2,(6)(7)(8) 10).In contrast to the study by Zabini and colleagues (9), we cannot confirm a loss of Smad3 with chronic TGF-b1 signaling in our PAH rodent models (TG-TGF-b1 mouse and SuHx rat).Zabini and colleagues focused only on total Smad3 expression and did not investigate Smad3 phosphorylation, which is the hallmark of canonical TGF-b1 activation.It is certainly possible that in PAH, downregulation of Smad3 mRNA and/or protein expression occurs in pulmonary arterial endothelial cells or other non-SMCs, such as pericytes and fibroblasts; however, experimental evidence is currently lacking, and resolving this issue would require additional studies.A better understanding of the functional antagonism of TGF-b1 and BMP2 (2) (and other BMPs) and their downstream pathways is crucial for our understanding of PAH.Emerging discoveries in this field will guide the future development of more efficient therapies for PAH, which currently is incurable.Taken together, our results indicate that chronic TGF-b1 signaling in PAH induces sustained canonical Smad3 signaling in PASMCs, and that such signals correlate with the hemodynamic and morphological PAH phenotype in rodents.

AIMS:Transforming growth factor-β (TGF-β) signalling is required for chronic hypoxia-induced pulmonary hypertension (PH). The activation of TGF-β by thrombospondin-1 (TSP-1) contributes to the pathogenesis of hypoxia-induced PH. However, neither the cellular source of pathologic TSP-1 nor the downstream signalling pathway that link activated TGF-β to PH have been determined. In this study, we hypothesized that circulating monocytes, which are recruited to become interstitial macrophages (IMs), are the major source of TSP-1 in hypoxia-exposed mice, and TSP-1 activates TGF-β with increased Rho-kinase signalling, causing vasoconstriction.METHODS AND RESULTS:Flow cytometry revealed that a specific subset of IMs is the major source of pathologic TSP-1 in hypoxia. Intravenous depletion and parabiosis experiments demonstrated that these cells are circulating prior to recruitment into the interstitium. Rho-kinase-mediated vasoconstriction was a major downstream target of active TGF-β. Thbs1 deficient bone marrow (BM) protected against hypoxic-PH by blocking TGF-β activation and Rho-kinase-mediated vasoconstriction.CONCLUSION:In hypoxia-challenged mice, BM derived and circulating monocytes are recruited to become IMs which express TSP-1, resulting in TGF-β activation and Rho-kinase-mediated vasoconstriction.

Schistosomiasis is a leading cause of pulmonary hypertension (PH) worldwide. Recent studies reveal that the type‐2 immune cytokines IL‐4 and IL‐13, as well as consequent activation of TGF‐β, are key factors in the pathogenesis of Schistosoma‐PH. Paclitaxel has been reported to act as an adjuvant for Th2 inflammation while downregulating TGF‐β activation. Moreover, paclitaxel blocks PH in monocrotaline and SU5416‐hypoxia models. We hypothesized that paclitaxel would augment Th2 inflammation while blocking TGF‐β activation and PH after schistosomiasis exposure. Wild‐type mice (C57BL6/J; 6/group) were intraperitoneally (IP) sensitized and then intravenously (IV) challenged with Schistosoma mansoni eggs. One day after IV egg challenge, the mice were treated with a single IP dose of 25 mg/kg paclitaxel or vehicle. Right ventricular (RV) catheterization was performed and granuloma volumes and vascular remodeling were quantified. Lung cytokines were quantified by ELISA and reverse transcription polymerase chain reaction, and the quantity of active TGF‐β was determined using a cell reporter line. We also investigated hypoxia‐induced PH. Paclitaxel treatment significantly protected mice from Schistosoma‐PH, with decreased RV systolic pressure (P = 0.005) and pulmonary vascular media thickness. Inflammation was significantly suppressed, contrary to our hypothesis, with decreased IL‐4 and IL‐13 levels, smaller granulomas, and less active TGF‐β following paclitaxel treatment. There was no change in IFN‐γ or FoxO1 or FoxO3 expression. Paclitaxel did not suppress chronic hypoxia‐induced PH, which is also TGF‐β‐driven but independent of type‐2 immunity. Paclitaxel protects against Schistosoma‐induced PH in mice, although by blocking proximate Th2 inflammation rather than suppressing distal TGF‐β activation.

Optimal right ventricular (RV) function in pulmonary hypertension (PH) requires structural and functional coupling between the RV cardiomyocyte and its adjacent capillary network. Prior investigations have indicated that RV vascular rarefaction occurs in PH, which could contribute to RV failure by reduced delivery of oxygen or other metabolic substrates. However, it has not been determined if rarefaction results from relative underproliferation in the setting of tissue hypertrophy or from actual loss of vessels. It is also unknown if rarefaction results in inadequate substrate delivery to the RV tissue. In the present study, PH was induced in rats by SU5416-hypoxia-normoxia exposure. The vasculature in the RV free wall was assessed using stereology. Steady-state metabolomics of the RV tissue was performed by mass spectrometry. Complementary studies were performed in hypoxia-exposed mice and rats. Rats with severe PH had evidence of RV failure by decreased cardiac output and systemic hypotension. By stereology, there was significant RV hypertrophy and increased total vascular length in the RV free wall in close proportion, with evidence of vessel proliferation but no evidence of endothelial cell apoptosis. There was a modest increase in the radius of tissue served per vessel, with decreased arterial delivery of metabolic substrates. Metabolomics revealed major metabolic alterations and metabolic reprogramming; however, metabolic substrate delivery was functionally preserved, without evidence of either tissue hypoxia or depletion of key metabolic substrates. Hypoxia-treated rats and mice had similar but milder alterations. There is significant homeostatic vascular adaptation in the right ventricle of rodents with PH.

Tobacco smoke (TS) causes chronic obstructive pulmonary disease, including chronic bronchitis, emphysema, and asthma. Rtp801, an inhibitor of mechanistic target of rapamycin, is induced by oxidative stress triggered by TS. Its up-regulation drives lung susceptibility to TS injury by enhancing inflammation and alveolar destruction. We postulated that Rtp801 is not only increased by reactive oxygen species (ROS) in TS but also instrumental in creating a feedforward process leading to amplification of endogenous ROS generation. We used cigarette smoke extract (CSE) to model the effect of TS in wild-type (Wt) and knockout (KO-Rtp801) mouse lung fibroblasts (MLF). The production of superoxide anion in KO-Rtp801 MLF was lower than that in Rtp801 Wt cells after CSE treatment, and it was inhibited in Wt MLF by silencing nicotinamide adenine dinucleotide phosphate oxidase-4 (Nox4) expression with small interfering Nox4 RNA. We observed a cytoplasmic location of ROS formation by real-time redox changes using reduction-oxidation-sensitive green fluorescent protein profluorescent probes. Both the superoxide production and the increase in the cytoplasmic redox were inhibited by apocynin. Reduction in the activity of Sod and decreases in the expression of Sod2 and Gpx1 genes were associated with Rtp801 CSE induction. The ROS produced by Nox4 in conjunction with the decrease in cellular antioxidant enzymatic defenses may account for the observed cytoplasmic redox changes and cellular damage caused by TS.

Pulmonary arterial hypertension (PAH) is an obstructive disease of the precapillary pulmonary arteries. Schistosomiasis-associated PAH shares altered vascular TGF-β signalling with idiopathic, heritable and autoimmune-associated etiologies; moreover, TGF-β blockade can prevent experimental pulmonary hypertension (PH) in pre-clinical models. TGF-β is regulated at the level of activation, but how TGF-β is activated in this disease is unknown. Here we show TGF-β activation by thrombospondin-1 (TSP-1) is both required and sufficient for the development of PH in Schistosoma-exposed mice. Following Schistosoma exposure, TSP-1 levels in the lung increase, via recruitment of circulating monocytes, while TSP-1 inhibition or knockout bone marrow prevents TGF-β activation and protects against PH development. TSP-1 blockade also prevents the PH in a second model, chronic hypoxia. Lastly, the plasma concentration of TSP-1 is significantly increased in subjects with scleroderma following PAH development. Targeting TSP-1-dependent activation of TGF-β could thus be a therapeutic approach in TGF-β-dependent vascular diseases.

When evaluating the role of redox-regulating signaling in pulmonary vascular diseases, it is intriguing to consider the modulation of key antioxidant enzymes like superoxide dismutase (SOD) because SOD isoforms are regulated by redox reactions, and, in turn, modulate downstream redox sensitive processes. The emerging field of redox biology is built upon understanding the regulation and consequences of tightly controlled and specific reduction-oxidation reactions that are critical for diverse cellular processes including cell signaling. Of relevance, both the site of production of specific reactive oxygen and nitrogen species and the site of the antioxidant defenses are highly compartmentalized within the cell. For example, superoxide is generated during oxidative phosphorylation in the mitochondria as well as by a number of enzymatic sources within the cytosol and at the cell membrane. In the pulmonary circulation, these sources include the mitochondrial electron transport chain, NADPH oxidases (NOX1-4, Duox1,2), nitric oxide synthases, and xanthine oxidase; this important topic has been thoroughly reviewed recently [1]. In parallel with these different cellular sites of superoxide production, the three SOD isoforms are also specifically localized to the cytosol (SOD1), mitochondria (SOD2) or extracellular compartment (SOD3). This chapter focuses on the role of redox mechanisms regulating SOD2 and SOD3, with an emphasis on these processes in the setting of pulmonary hypertension.

The mechanistic target of rapamycin (mTOR) is a central regulator of cellular responses to environmental stress. mTOR (and its primary complex mTORC1) is, therefore, ideally positioned to regulate lung inflammatory responses to an environmental insult, a function directly relevant to disease states such as the acute respiratory distress syndrome. Our previous work in cigarette smoke-induced emphysema identified a novel protective role of pulmonary mTORC1 signaling. However, studies of the impact of mTORC1 on the development of acute lung injury are conflicting. We hypothesized that Rtp801, an endogenous inhibitor of mTORC1, which is predominantly expressed in alveolar type II epithelial cells, is activated during endotoxin-induced lung injury and functions to suppress anti-inflammatory epithelial mTORC1 responses. We administered intratracheal lipopolysaccharide to wild-type mice and observed a significant increase in lung Rtp801 mRNA. In lipopolysaccharide-treated Rtp801(-/-) mice, epithelial mTORC1 activation significantly increased and was associated with an attenuation of lung inflammation. We reversed the anti-inflammatory phenotype of Rtp801(-/-) mice with the mTORC1 inhibitor, rapamycin, reassuring against mTORC1-independent effects of Rtp801. We confirmed the proinflammatory effects of Rtp801 by generating a transgenic Rtp801 overexpressing mouse, which displayed augmented inflammatory responses to intratracheal endotoxin. These data suggest that epithelial mTORC1 activity plays a protective role against lung injury, and its inhibition by Rtp801 exacerbates alveolar injury caused by endotoxin.

Ethnopharmacological importance: Cucurbita ficifolia is used in Mexican traditional medicine as an antidiabetic and anti-inflammatory agent and its actions can be mediated by antioxidant mechanisms. Disturbance in the homeostasis of glutathione has been implicated in the etiology and progression of diabetes mellitus and its complications.Material and methods: It was evaluated, the effect of an aqueous extract of Cucurbita ficifolia on glycemia, plasma lipid peroxidation; as well as levels of reduced (GSH) and oxidized (GSSG) glutathione and activities of enzymes involved in glutathione redox cycle: glutathione peroxidase (GPx) and glutathione reductase (GR) in liver, pancreas, kidney and heart homogenates of streptozotocin-induced diabetic mice.Results: Increased blood glucose and lipid peroxidation, together with decreased of GSH concentration, GSH/GSSG ratio and its redox potential (E-h), and enhanced activity of GPx and GR in liver, pancreas and kidney were the salient features observed in diabetic mice. Administration of the aqueous extract of Cucurbita ficifolia to diabetic mice for 30 days, used at a dose of 200 mg/kg, resulted in a significant reduction in glycemia, polydipsia, hyperphagia and plasma lipid peroxidation. Moreover, GSH was increased in liver, pancreas and kidney, and GSSG was reduced in liver, pancreas and heart, therefore GSH/GSSG ratio and its E-h were restored. Also, the activities involved in the glutathione cycle were decreased, reaching similar values to controls.Conclusions: An aqueous extract of Cucurbita ficifolia with hypoglycemic action, improve GSH redox state, increasing glutathione pool, GSH, GSH/GSSG ratio and its E-h, mechanism that can explain, at least in part, its antioxidant properties, supporting its use as an alternative treatment for the control of diabetes mellitus, and prevent the induction of complications by oxidative stress. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

We previously described a coding mutation (L60F) in the mitochondrial superoxide dismutase (SOD2) gene of the human T cell leukemia-derived cell line Jurkat. In cell extracts the L60F mutant enzyme showed unusual inhibition by thiol reagents not seen in wild-type enzyme. Here we compare the properties of purified recombinant L60F SOD2 with a previously described SOD2 mutant, I58T. Both mutant proteins display a weakened dimer-dimer interaction and thermal instability at 55 degrees C. Both I58T and L60F lose activity at 37 degrees C in the presence of 5 mM N-ethylmaleimide, whereas the wild-type SOD2 does not. Each subunit contains one exposed, reactive cysteine residue at position 196 and a second cysteine residue at 140, which is buried and unreactive in the wild-type tetramer. We propose that the mutant enzymes, which exist largely as dimers, allow both cysteine residues to react with thiol reagents. When the cysteine residue at 140 was changed to serine by site-directed mutagenesis, both double mutants I58T/C140S and L60F/C140S lost their increased thiol sensitivity. The evolutionary significance of Cys140 is discussed.

There is growing evidence of the correlation between cancer and reactive oxygen species (ROS), especially superoxide. Low expression levels of the Mn‐superoxide dismutase (SOD2) enzyme have been reported in cancer patients. Genetic variation in the regulatory regions of the SOD2 gene may increase the risk of cancer. We identified a genetic variation (G1677T, rs2Y758Y339) in the vicinity of the enhancer region located in intron 2 of the SOD2 gene that creates a potential glucocorticoid responsive element, and developed an assay to screen DNA samples of 220 individuals (73 control, 59 prostate cancer survival individuals and 88 lung cancer biopsies). There were no significant differences in the genotype frequency distribution among prostate, lung cancer and control (p = 0.074 and 0.057, respectively). However, we identified an association of T allele with a decreased risk of lung cancer (OR = 0.525, p = 0.037). The use of the G1677T polymorphism of SOD2 gene as a genetic risk marker may suggest new approaches for detection, prevention, treatment, and prognosis of cancer. Copyright © 2009 John Wiley & Sons, Ltd.

OBJETIVO: Conocer la prevalencia de las dislipidemias en una población de sujetos en apariencia sanos y su relación con la resistencia a la insulina (RI). MATERIAL Y MÉTODOS: Este es un estudio transversal que incluyó a 1 179 individuos, donadores voluntarios de 35 a 65 años. Se obtuvo el historial clínico y se realizaron examen físico, determinación del perfil de lípidos, glucemia y niveles de insulina en ayuno. RESULTADOS: La edad promedio fue de 44 ± 7 años; 836 (71%) correspondían al género masculino. La prevalencia de hipertrigliceridemia fue de 57.3%, hipoalfalipoproteinemia de 52.4% e hipercolesterolemia de 48.7 por ciento. De los sujetos con obesidad (perímetro de cintura aumentado), 36.8% tenía hipertrigliceridemia/hipoalfalipoproteinemia, 35.2% dislipidemia mixta y 33.4% hipertrigliceridemia. La prevalencia de los patrones de dislipidemias fue mayor en sujetos con RI. CONCLUSIONES: La hipertrigliceridemia e hipoalfalipoproteinemia, vinculadas con RI, son comunes en la población mexicana; empero, una considerable proporción de casos carece de diagnóstico.OBJECTIVE: Evaluate dyslipidemia prevalence and its association with insulin resistance in a cohort of apparently healthy subjects. MATERIAL AND METHODS: A cross-sectional study was conducted with 1 179 donors ages 35 to 65 years. The sample population was comprised of 71% men, with an average age of 44 ± 7. Clinical records, anthropometric data, lipid profile, fasting glycaemia, and insulin levels were obtained. RESULTS: Prevalence of hypertriglyceridemia was 57.3%, C-HDL under normal limits was 52.4%, and hypercholesterolemia was 48.7%. In addition, 36.8% of the obese individuals (as measured by waist perimeter) had hypertriglyceridemia/hypoalphalipoproteinemia, 35.2% had mixed dyslipidemia, and 33.4% had hypertriglyceridemia. Patterns of dyslipidemia were higher in subjects diagnosed with insulin resistance. CONCLUSIONS: Insulin resistance associated with hypertriglyceridemia and hypoalphalipoproteinemia was common among our studied population. However, a significant proportion of cases of apparent healthy individuals continue to go undiagnosed.