[Show abstract][Hide abstract] ABSTRACT: mTORC1 activation occurs frequently in cancers, yet clinical efficacy of rapalogs is limited due to the associated activation of upstream survival pathways. An alternative approach is to inhibit downstream of mTORC1; therefore, acquired resistance to fludarabine (Flu), a purine analog and anti-metabolite chemotherapy, active agent for chronic lymphocytic leukemia (CLL) was investigated. Elevated phospho-p70S6K (T389), an mTORC1 activation marker, predicted Flu resistance in a panel of B-cell lines, isogenic Flu-resistant (FluR) derivatives, and primary human CLL cells. Consistent with the anabolic role of mTORC1, FluR cells had higher rates of glycolysis and oxidative phosphorylation than Flu-sensitive (FluS) cells. Rapalogs (everolimus, rapamycin), induced moderate cell death in FluR and primary CLL cells, and everolimus significantly inhibited glycolysis and oxidative phosphorylation in FluR cells. Strikingly, the higher oxidative phosphorylation in FluR cells was not coupled to higher ATP synthesis. Instead it contributed primarily to an essential, dihydroorotate dehydrogenase (DHODH) catalyzed, step in de novo pyrimidine biosynthesis. mTORC1 promotes pyrimidine biosynthesis by p70S6 kinase-mediated phosphorylation of CAD (Ser1859) and favors S-phase cell cycle progression. We found increased phospho-CAD (S1859) and higher S-phase population in FluR cells. Pharmacological inhibition of de novo pyrimidine biosynthesis using N-phosphonacetyl-Laspartate (PALA) and leflunomide, RNAi-mediated knockdown of p70S6K, and inhibition of mitochondrial respiration were selectively cytotoxic to FluR, but not FluS cells. These results reveal a novel link between mTORC1-mediated metabolic reprogramming and Flu resistance identifying mitochondrial respiration and de novo pyrimidine biosynthesis as potential therapeutic targets. Implications: This study provides the first evidence for mTORC1/p70S6K-dependent regulation of pyrimidine biosynthesis in a relevant disease setting.
Molecular Cancer Research 07/2014; · 4.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The early biological impact of short-term mechanical ventilation on healthy lungs is unknown. The authors aimed to characterize the immediate tidal volume (VT)-related changes on lung injury biomarkers in patients with healthy lungs and low risk of pulmonary complications.
Twenty-eight healthy patients for knee replacement surgery were prospectively randomized to volume-controlled ventilation with VT 6 (VT6) or 10 (VT10) ml/kg predicted body weight. General anesthesia and other ventilatory parameters (positive end-expiratory pressure, 5 cm H2O, FIO2, 0.5, respiratory rate titrated for normocapnia) were managed similarly in the two groups. Exhaled breath condensate and blood samples were collected for nitrite, nitrate, tumor necrosis factor-α, interleukins-1β, -6, -8, -10, -11, neutrophil elastase, and Clara Cell protein 16 measurements, at the onset of ventilation and 60 min later.
No significant differences in biomarkers were detected between the VT groups at any time. The coefficient of variation of exhaled breath condensate nitrite and nitrate decreased in the VT6 but increased in the VT10 group after 60-min ventilation. Sixty-minute ventilation significantly increased plasma neutrophil elastase levels in the VT6 (35.2 ± 30.4 vs. 56.4 ± 51.7 ng/ml, P = 0.008) and Clara Cell protein 16 levels in the VT10 group (16.4 ± 8.8 vs. 18.7 ± 9.5 ng/ml, P = 0.015). Exhaled breath condensate nitrite correlated with plateau pressure (r = 0.27, P = 0.042) and plasma neutrophil elastase (r = 0.44, P = 0.001). Plasma Clara Cell protein 16 correlated with compliance (r = 0.34, P = 0.014).
No tidal volume-related changes were observed in the selected lung injury biomarkers of patients with healthy lungs after 60-min ventilation. Plasma neutrophil elastase and plasma Clara Cell protein 16 might indicate atelectrauma and lung distention, respectively.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Hypobaric hypoxia occurs during ascent to higher altitude. In this observational study of healthy lowland dwellers, intracellular red-cell forms of nitric oxide, S-nitrosohemoglobin, and iron nitrosyl hemoglobin increased strikingly during ascent to 5050 m.
New England Journal of Medicine 11/2011; 365(20):1942-4. · 54.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pulmonary arterioles respond to hypoxia with constriction that raises vascular resistance and pulmonary artery blood pressure. The response is sustained indefinitely by the chronic hypoxia of high-altitude residence among highlanders of European and Andean descent, but not Tibetans. The objective of this study was to identify the consequences of lifelong hypoxia exposure for the pulmonary vasculature among Amhara high-altitude natives from Ethiopia.
A three-way static group comparison tested for the effect of Amhara ancestry and high residence altitude on pulmonary hemodynamics measured using echocardiography in samples of 76 healthy adult Amhara lifelong residents at 3700 m, 54 Amhara lifelong residents at 1200 m, and 46 U.S. low-altitude residents at 282 m.
Amhara at 3700 m had average Doppler-estimated pulmonary artery systolic pressure (tricuspid regurgitant gradient) of 27.9 ± 8.4 (SD) mm Hg as compared with 21.9 ± 4.0 among Amhara at low altitude and 16.5 ± 3.6 in the U.S. low-altitude reference sample. However, there was no residence altitude effect on pulmonary blood flow or vascular resistance. Amhara ancestry was associated with greater pulmonary artery systolic pressure and pulmonary blood flow, yet lower pulmonary vascular resistance.
The Amhara at 3700 m had elevated pulmonary artery pressure, but without the elevated pulmonary vascular resistance characteristic of the classic model of the response to long-term hypoxia by the pulmonary vasculature. The elevated pressure among Amhara may be a consequence of high pulmonary blood flow regardless of altitude and represent a newly identified pattern of response.
American Journal of Human Biology 03/2011; 23(2):168-76. · 2.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
American Journal Of Pathology 03/2010; 176(3):1130-8. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The regulation of nitric oxide synthase 2 (NOS2) in airway epithelial cells plays a key role in the innate host response to a wide variety of microbial agents and also participates in the generation of pathologic airway inflammation. Among the important signalling cascades that direct NOS2 gene expression are nuclear factor kappaB (NFkappaB) and interferon-gamma (IFNgamma)/signal transducer and activator of transcription 1 (STAT-1). Previous studies suggest activator protein-1 (AP-1), in particular c-Fos component of AP-1, influences NOS2 expression. We investigated the effect of c-Fos modulation using RNA interference siRNA on NOS2 gene expression. A549 cells stably transfected with a plasmid overexpressing a c-Fos siRNA construct (FOSi) resulted in a decrease of NOS2 protein inducibility by IFN gamma. In contrast, classical IFN gamma inducible signal transduction pathways interferon regulated factor-1 (IRF-1) and pSTAT-1 were activated at a similar magnitude in FOSi and control cells. DNA-protein binding assays showed that c-Fos binding was present in wild type cells, but reduced in FOSi clones. FOSi clones had activation of NFkappaB detectable by DNA-protein binding assays, which may have contributed to a decrease of NOS2 expression. Overall, these studies indicate that c-Fos is a requisite and specific component for inducible NOS2 expression.
[Show abstract][Hide abstract] ABSTRACT: Nasal polyposis is characterized by impaired regulation of nasal tissue growth and is associated with chronic inflammation, sinus infections, and low levels of nitric oxide (NO). Based on its critical role in mediating cell growth and antimicrobial function, decrease of NO levels has been implicated in the pathogenesis of nasal polyposis.
We sought to evaluate mechanisms for the low NO level in polyposis, including factors regulating NO synthase (NOS) expression and activity and NO consumptive processes in nasal epithelial cells and nasal lavage fluid.
Eighteen patients with nasal polyposis and 8 healthy control subjects were studied. Nasal brushings, nasal lavage fluid, and nasal biopsy specimens were collected and analyzed.
NO metabolite levels (nitrite and nitrate) in nasal lavage fluid from patients with polyps were less than those in control subjects, but activation of signal transduction and inducer of transcription 1, which regulates inducible NOS gene expression and protein expression, was present at higher levels in polyp than in healthy control tissue. Levels of arginine, methylarginine, and endogenous NOS inhibitors were similar between polyp and control tissue. In contrast, superoxide dismutase activity of polyp tissues was lower than that seen in control tissue and associated with increased nitrotyrosine, a biomarker of oxidant consumptive products of NO.
Taken together, these data suggest that the nasal polyp environment is characterized by abnormalities in NO metabolism that might predispose to altered regulation of tissue growth and infection.
Identification of NO metabolic abnormalities might lead to novel treatments for sinonasal polyposis targeted against the pathways identified within this study.
The Journal of allergy and clinical immunology 01/2008; 120(6):1346-53. · 12.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The low barometric pressure at high altitude causes lower arterial oxygen content among Tibetan highlanders, who maintain normal levels of oxygen use as indicated by basal and maximal oxygen consumption levels that are consistent with sea level predictions. This study tested the hypothesis that Tibetans resident at 4,200 m offset physiological hypoxia and achieve normal oxygen delivery by means of higher blood flow enabled by higher levels of bioactive forms of NO, the main endothelial factor regulating blood flow and vascular resistance. The natural experimental study design compared Tibetans at 4,200 m and U.S. residents at 206 m. Eighty-eight Tibetan and 50 U.S. resident volunteers (18-56 years of age, healthy, nonsmoking, nonhypertensive, not pregnant, with normal pulmonary function) participated. Forearm blood flow, an indicator of systemic blood flow, was measured noninvasively by using plethysmography at rest, after breathing supplemental oxygen, and after exercise. The Tibetans had more than double the forearm blood flow of low-altitude residents, resulting in greater than sea level oxygen delivery to tissues. In comparison to sea level controls, Tibetans had >10-fold-higher circulating concentrations of bioactive NO products, including plasma and red blood cell nitrate and nitroso proteins and plasma nitrite, but lower concentrations of iron nitrosyl complexes (HbFeIINO) in red blood cells. This suggests that NO production is increased and that metabolic pathways controlling formation of NO products are regulated differently among Tibetans. These findings shift attention from the traditional focus on pulmonary and hematological systems to vascular factors contributing to adaptation to high-altitude hypoxia.
Proceedings of the National Academy of Sciences 11/2007; 104(45):17593-8. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Idiopathic pulmonary arterial hypertension (IPAH) is pathogenetically related to low levels of the vasodilator nitric oxide (NO). Because NO regulates cellular respiration and mitochondrial biogenesis, we hypothesized that abnormalities of bioenergetics may be present in IPAH. Evaluation of pulmonary artery endothelial cells from IPAH and control lungs in vitro revealed that oxygen consumption of IPAH cells was decreased, especially in state 3 respiration with substrates glutamate-malate or succinate, and this decrease paralleled reduction in Complex IV activity and IPAH cellular NO synthesis. IPAH pulmonary artery endothelial cells had decreased mitochondrial dehydrogenase activity and lowered mitochondrial numbers per cell and mitochondrial DNA content, all of which increased after exposure to NO donors. Although IPAH/pulmonary artery endothelial cells' ATP content was similar to control under normoxia, cellular ATP did not change significantly in IPAH cells under hypoxia, whereas ATP decreased 35% in control cells, identifying a greater dependence on cellular respiration for energy in control cells. Evidence that glucose metabolism was subserving the primary role for energy requirements of IPAH cells was provided by the approximately 3-fold greater glycolytic rate of IPAH cells. Positron emission tomography scan with [18F]fluoro-deoxy-D-glucose performed on IPAH patients and healthy controls revealed significantly higher uptake in IPAH lungs as compared with controls, confirming that the glycolytic rate was increased in vivo. Thus, there are substantial changes in bioenergetics of IPAH endothelial cells, which may have consequences for pulmonary hypertensive responses and potentially in development of novel imaging modalities for diagnosis and evaluation of treatment.
Proceedings of the National Academy of Sciences 02/2007; 104(4):1342-7. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Viral infections produce severe respiratory morbidity in children with cystic fibrosis (CF). CF cells are more susceptible to virus in part because of impaired airway epithelial activation of signal transducer and activator of transcription 1 (Stat1). As Stat1 is a fundamental regulator of antiviral defenses, we hypothesized that there may be multiple alterations in the antiviral defense of CF epithelium compared with normal (NL). To obtain a comprehensive view of mucosal host responses to influenza and characterize the difference between CF and NL responses to influenza, gene expression profiles of primary human airway epithelial cells (HAEC) were evaluated using an interferon (IFN)-stimulated genes/AU/double-stranded RNA (dsRNA) microarray or quantitative real-time polymerase chain reaction (PCR) following influenza A infection. Gene expression was significantly modified by influenza in NL (228 genes) and CF (101 genes), with a similar pattern of gene response but with overall less numbers of responsive genes in CF (p < 0.05). Moreover, CF cells had less IFN-related antiviral gene induction at 24 h but greater inflammatory cytokine gene induction at 1 h after infection. Taken together, the lesser antiviral and greater early inflammatory response likely contribute to the severe respiratory illness of CF patients with viral infections.
Journal of Interferon & Cytokine Research 10/2006; 26(9):609-27. · 3.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen species and reactive nitrogen species produced by epithelial and inflammatory cells are key mediators of the chronic airway inflammation of asthma. Detection of 3-nitrotyrosine in the asthmatic lung confirms the presence of increased reactive oxygen and nitrogen species, but the lack of identification of modified proteins has hindered an understanding of the potential mechanistic contributions of nitration/oxidation to airway inflammation. In this study, we applied a proteomic approach, using nitrotyrosine as a marker, to evaluate the oxidation of proteins in the allergen-induced murine model of asthma. Over 30 different proteins were targets of nitration following allergen challenge, including the antioxidant enzyme catalase. Oxidative modification and loss of catalase enzyme function were seen in this model. Subsequent investigation of human bronchoalveolar lavage fluid revealed that catalase activity was reduced in asthma by up to 50% relative to healthy controls. Analysis of catalase isolated from asthmatic airway epithelial cells revealed increased amounts of several protein oxidation markers, including chloro- and nitrotyrosine, linking oxidative modification to the reduced activity in vivo. Parallel in vitro studies using reactive chlorinating species revealed that catalase inactivation is accompanied by the oxidation of a specific cysteine (Cys(377)). Taken together, these studies provide evidence of multiple ongoing and profound oxidative reactions in asthmatic airways, with one early downstream consequence being catalase inactivation. Loss of catalase activity likely amplifies oxidative stress, contributing to the chronic inflammatory state of the asthmatic airway.
The Journal of Immunology 06/2006; 176(9):5587-97. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A cellular prooxidant state promotes cells to neoplastic growth, in part because of modification of proteins and their functions. Reactive nitrogen species formed from nitric oxide (NO) or its metabolites, can lead to protein tyrosine nitration, which is elevated in lung cancer.
To determine the alteration in these NO derivatives and the role they may play in contributing to lung carcinogenesis.
We analyzed levels of NO, nitrite (NO2-), nitrate (NO3-), and the location of the protein nitration and identified the proteins that are modified.
Although exhaled NO and NO2- were increased, endothelial NO synthase or inducible NO synthase expression was similar in the tumor and tumor-free regions. However, immunohistochemistry showed that nitrotyrosine was increased in the tumor relative to non-tumor-bearing sections. We used proteomics to identify the modified proteins (two-dimensional polyacrylamide gel electrophoresis; mass spectrometry). Both the degree of nitration and the protein nitration profile were altered. We identified more than 25 nitrated proteins, including metabolic enzymes, structural proteins, and proteins involved in prevention of oxidative damage. Alterations of the biology of NO metabolites and nitration of proteins may contribute to the mutagenic processes and promote carcinogenesis.
This study provides evidence in favor of a role for reactive nitrogen and oxygen species in lung cancer.
American Journal of Respiratory and Critical Care Medicine 10/2005; 172(5):597-605. · 11.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Airway hyperresponsiveness and remodeling are defining features of asthma. We hypothesized that impaired superoxide dismutase (SOD) antioxidant defense is a primary event in the pathophysiology of hyperresponsiveness and remodeling that induces apoptosis and shedding of airway epithelial cells. Mechanisms leading to apoptosis were studied in vivo and in vitro. Asthmatic lungs had increased apoptotic epithelial cells compared to controls as determined by terminal dUTP nick-end labeling-positive cells. Apoptosis was confirmed by the finding that caspase-9 and -3 and poly (ADP-ribose) polymerase were cleaved. On the basis that SOD inactivation triggers cell death and low SOD levels occur in asthma, we tested whether SOD inactivation plays a role in airway epithelial cell death. SOD inhibition increased cell death and cleavage/activation of caspases in bronchial epithelial cells in vitro. Furthermore, oxidation and nitration of MnSOD were identified in the asthmatic airway, correlating with physiological parameters of asthma severity. These findings link oxidative and nitrative stress to loss of SOD activity and downstream events that typify asthma, including apoptosis and shedding of the airway epithelium and hyperresponsiveness.
American Journal Of Pathology 04/2005; 166(3):663-74. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pulmonary arterial hypertension (PAH), a fatal disease of unknown etiology characterized by impaired regulation of pulmonary hemodynamics and vascular growth, is associated with low levels of pulmonary nitric oxide (NO). Based upon its critical role in mediating vasodilation and cell growth, decrease of NO has been implicated in the pathogenesis of PAH. We evaluated mechanisms for low NO and pulmonary hypertension, including NO synthases (NOS) and factors regulating NOS activity, i.e. the substrate arginine, arginase expression and activity, and endogenous inhibitors of NOS in patients with PAH and healthy controls. PAH lungs had normal NOS I-III expression, but substrate arginine levels were inversely related to pulmonary artery pressures. Activity of arginase, an enzyme that regulates NO biosynthesis through effects on arginine, was higher in PAH serum than in controls, with high-level arginase expression localized by immunostaining to pulmonary endothelial cells. Further, pulmonary artery endothelial cells derived from PAH lung had higher arginase II expression and produced lower NO than control cells in vitro. Thus, substrate availability affects NOS activity and vasodilation, implicating arginase II and alterations in arginine metabolic pathways in the pathophysiology of PAH.
The FASEB Journal 12/2004; 18(14):1746-8. · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Decreased production of vasodilator substances such as nitric oxide (NO) has been proposed as important in development of pulmonary arterial hypertension (PAH). We hypothesize that NO measured over time serves as a non invasive marker of severity of PAH and response to therapy. We prospectively and serially measured exhaled NO and carbon monoxide (CO), a vasodilator and anti-inflammatory product of heme oxygenases, in 17 PAH patients in conjunction with hemodynamic parameters over 2 years. Although pulmonary artery pressures and NO were similar in all patients at entry to the study, NO increased in the 12 individuals who survived to complete the study, and correlated with change in pulmonary artery pressures. In contrast, CO did not change or correlate with hemodynamic parameters. Investigation of NO-oxidant reaction products in PAH in comparison to controls suggests that NO synthesis is impaired in the lung and that reactive oxygen species may be involved in the pathophysiology of pulmonary hypertension. Endogenous NO is inversely related to pulmonary artery pressure in PAH, with successful therapy of PAH associated with increase in NO.
Free Radical Biology and Medicine 11/2004; 37(7):1010-7. · 5.27 Impact Factor