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1
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Gomez AP, Moreno MJ, Hernández A. Adventitial growth and lung connective tissue growth factor expression in pulmonary arterioles due to hypobaric hypoxia in broilers. Poult Sci 2020; 99:1832-1837. [PMID: 32241463 PMCID: PMC7587700 DOI: 10.3382/ps/pez157] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2018] [Accepted: 03/11/2019] [Indexed: 11/25/2022] Open
Abstract
Forty broilers maintained under natural hypobaric hypoxia (2,638 m above sea level) and 20 maintained under relative normoxia (460 m above sea level) were selected as pulmonary hypertensive (PHB) and nonpulmonary hypertensive (NPHB), to estimate the degree of the adventitial vascular thickness in lung arterioles and connective tissue growth factor (CTGF) expression in lung. In each group, the adventitial thickness (%AT) of 20 arterioles with 100 to 250 μm of external diameter was measured in lung samples of 24 and 42-day-old broilers. Also, mRNA extraction and real-time reverse transcription-PCR analysis were used to measure lung CTGF expression. The %AT was higher in PHB at 42 D as compared to NPHB at both ages and PHB at 24 D; however, the same differences were not evidenced at 24 D. In the 2 ages evaluated, differences were observed in the %AT between broilers under hypobaric hypoxia (PHB and NPHB) and under relative normoxia (P < 0.01). In broilers subjected to relative normoxia, no significant differences were found at any of the 2 ages. The expression levels of CTGF mRNA were higher in PHB compared to NPHB at the 2 ages. The %AT was higher in PHB with high levels of expression of CTGF mRNA than those NPHB with low expression of CTGF mRNA. This study showed that adventitial thickening is part of the pulmonary hypertension (PH) physiopathology in broilers exposed to hypobaric hypoxia, in which CTGF appears to be a fibrosis enhancer. Although present data suggest that adventitial engrossment could be a time-dependent process, individual susceptibility and the variable time-course of PH pathophysiology have to be considered.
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Affiliation(s)
- A P Gomez
- Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Bogotá DC 111311, Colombia
| | - M J Moreno
- Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Bogotá DC 111311, Colombia
| | - A Hernández
- Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Bogotá DC 111311, Colombia.
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2
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Malikova E, Carlström M, Kmecova Z, Marusakova M, Zsigmondova B, Krenek P, Klimas J, Henrohn D. Effects of inorganic nitrate in a rat model of monocrotaline-induced pulmonary arterial hypertension. Basic Clin Pharmacol Toxicol 2019; 126:99-109. [PMID: 31429204 DOI: 10.1111/bcpt.13309] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2019] [Accepted: 08/11/2019] [Indexed: 12/27/2022]
Abstract
The nitrate-nitrite-nitric oxide (NO) pathway represents an alternative source of NO generation, which is independent of NO synthase and potentiated by hypoxia. Augmentation of this pathway by dietary nitrate has proven favourable effects in several cardiovascular disease models. However, less is known regarding its potential value in pulmonary arterial hypertension (PAH). The aim of this study was to assess the effects of oral inorganic nitrate administration in monocrotaline (MCT)-induced PAH. Male 12-week-old Wistar rats were injected subcutaneously with monocrotaline (MCT, 60 mg/kg). Nitrate treatment (0.3 or 1 mmol/kg/d; drinking water) commenced on day 12 following the MCT injection and continued for 16 days. Nitrate administration did not attenuate right ventricular (RV) hypertrophy, increased lung weight and up-regulated mRNA expression of brain natriuretic peptide. Plasma nitrate and nitrite levels were significantly increased as well as lung nitrate level, whereas nitrite lung level was decreased following nitrate treatment (1 mmol/kg/d). MCT-induced PAH resulted in an increased MnSOD protein level, which was not observed following nitrate treatment. MCT-associated up-regulation of nNOS in the lung appeared to be dose-dependently prevented by nitrate treatment. Western blot analysis did not reveal any differences in eNOS, iNOS, XO or gp91phox expression in the lungs among the groups. In conclusion, nitrate treatment did not significantly attenuate pathological RV and lung remodelling in the rat MCT model of PAH. The suppression of MnSOD and nNOS expression by nitrate could be interpreted as reduced demand of endogenous antioxidant defence in this model.
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Affiliation(s)
- Eva Malikova
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, Bratislava, Slovak Republic
| | - Mattias Carlström
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
| | - Zuzana Kmecova
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, Bratislava, Slovak Republic
| | - Margareta Marusakova
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, Bratislava, Slovak Republic
| | - Bianka Zsigmondova
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, Bratislava, Slovak Republic
| | - Peter Krenek
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, Bratislava, Slovak Republic
| | - Jan Klimas
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, Bratislava, Slovak Republic
| | - Dan Henrohn
- Department of Medical Sciences, Uppsala University, Uppsala, Sweden
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Saito Y, Nakamura M, Eguchi K, Otsuki T. Mild Hypobaric Hypoxia Enhances Post-exercise Vascular Responses in Young Male Runners. Front Physiol 2019; 10:546. [PMID: 31178742 PMCID: PMC6543008 DOI: 10.3389/fphys.2019.00546] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2018] [Accepted: 04/17/2019] [Indexed: 12/12/2022] Open
Abstract
It has been reported that sustained post-exercise vasodilation may be linked to exercise-induced angiogenesis. The present study aimed to evaluate whether mild hypobaric hypoxia enhances the post-exercise reduction in systemic vascular resistance in young male runners. Seven male intercollegiate runners (aged 19–21 years) performed maximal incremental treadmill running under conditions of hypobaric hypoxia (corresponding to 2,200 m above sea level, hereinafter referred to as HH) and normobaric normoxia (corresponding to sea level, hereinafter referred to as NN). A third exercise test was performed under NN conditions, consisting of submaximal exercise with the same absolute exercise volume as was achieved during HH (submaximal exercise under NN conditions, hereinafter referred to as NNsubmax). Blood pressure and cardiac output (CO) were measured before and at 15, 30, and 60 (p60) minutes after exercise. Compared with NN, exercise time was shorter in HH and NNsubmax conditions (p < 0.05). Systolic blood pressure and mean blood pressure (MBP) were lower after exercise in HH conditions (p < 0.05). No condition-related differences were found in CO. Total peripheral resistance (TPR, defined as the ratio of MBP to CO) was significantly lower after exercise compared to baseline for all conditions (p < 0.05). However, the decrease in TPR was maintained longer after exercise in HH compared with NN and NNsubmax conditions (p < 0.05). At p60, TPR was lower than baseline for HH conditions (p < 0.05), whereas after exercise in NN, and NNsubmax conditions, TPR recovered to baseline by p60. Decreases in systemic vascular resistance after exercise were maintained longer under mild HH conditions compared with NN despite the lower exercise volume of the former.
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Affiliation(s)
- Yoko Saito
- Faculty of Human Sciences, Kanazawa Seiryo University, Kanazawa, Japan
| | - Mariko Nakamura
- Department of Sports Sciences, Japan Institute of Sports Sciences, Tokyo, Japan
| | - Kazumi Eguchi
- Department of Sports Sciences, Japan Institute of Sports Sciences, Tokyo, Japan
| | - Takeshi Otsuki
- Faculty of Sport and Health Sciences, Ryutsu Keizai University, Ryugasaki, Japan
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Sheak JR, Weise-Cross L, deKay RJ, Walker BR, Jernigan NL, Resta TC. Enhanced NO-dependent pulmonary vasodilation limits increased vasoconstrictor sensitivity in neonatal chronic hypoxia. Am J Physiol Heart Circ Physiol 2017; 313:H828-H838. [PMID: 28733445 DOI: 10.1152/ajpheart.00123.2017] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/24/2017] [Revised: 07/17/2017] [Accepted: 07/17/2017] [Indexed: 02/03/2023]
Abstract
Augmented vasoconstrictor reactivity is thought to play an important role in the development of chronic hypoxia (CH)-induced neonatal pulmonary hypertension. However, whether this response to CH results from pulmonary endothelial dysfunction and reduced nitric oxide (NO)-mediated vasodilation is not well understood. We hypothesized that neonatal CH enhances basal tone and pulmonary vasoconstrictor sensitivity by limiting NO-dependent pulmonary vasodilation. To test this hypothesis, we assessed the effects of the NO synthase (NOS) inhibitor Nω-nitro-l-arginine (l-NNA) on baseline pulmonary vascular resistance (PVR) and vasoconstrictor sensitivity to the thromboxane mimetic U-46619 in saline-perfused lungs (in situ) from 2-wk-old control and CH (12-day exposure, 0.5 atm) Sprague-Dawley rats. Basal tone was defined as that reversed by exogenous NO (spermine NONOate). CH neonates displayed elevated right ventricular systolic pressure (in vivo) and right ventricular hypertrophy, indicative of pulmonary hypertension. Perfused lungs from CH rats demonstrated greater baseline PVR, basal tone, and U-46619-mediated vasoconstriction compared with control rats in the absence of l-NNA. l-NNA markedly increased baseline PVR and reactivity to U-46619 in lungs from CH neonates, further augmenting vasoconstrictor sensitivity compared with control lungs. Exposure to CH also enhanced NO-dependent vasodilation to arginine vasopressin, pulmonary expression of NOS III [endothelial NOS (eNOS)], and eNOS phosphorylation at activation residue Ser1177 However, CH did not alter lung nitrotyrosine levels, a posttranslational modification reflecting [Formula: see text] scavenging of NO. We conclude that, in contrast to our hypothesis, enhanced basal tone and agonist-induced vasoconstriction after neonatal CH is limited by increased NO-dependent pulmonary vasodilation resulting from greater eNOS expression and phosphorylation at activation residue Ser1177NEW & NOTEWORTHY This research is the first to demonstrate enhanced nitric oxide-dependent vasodilation that limits increased vasoconstrictor reactivity in neonatal pulmonary hypertension. These results suggest that augmented vasoconstriction in this setting reflects changes in smooth muscle reactivity rather than a reduction in nitric oxide-dependent pulmonary vasodilation.
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Affiliation(s)
- Joshua R Sheak
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico
| | - Laura Weise-Cross
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico
| | - Ray J deKay
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico
| | - Benjimen R Walker
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico
| | - Nikki L Jernigan
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico
| | - Thomas C Resta
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico
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Ye QX, Xu LH, Shi PJ, Xia T, Fang JP. Indoleamine 2,3-dioxygenase and inducible nitric oxide synthase mediate immune tolerance induced by CTLA4Ig and anti-CD154 hematopoietic stem cell transplantation in a sensitized mouse model. Exp Ther Med 2017; 14:1884-1891. [PMID: 28962099 PMCID: PMC5609130 DOI: 10.3892/etm.2017.4722] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2015] [Accepted: 02/02/2017] [Indexed: 12/29/2022] Open
Abstract
Cytotoxic T-lymphocyte-associated protein 4 immunoglobulin (CTLA4Ig) and anti-cluster of differentiation 154 (anti-CD154) are able to block B7/CD28 and CD40/CD154 co-stimulatory signals in T cells. Additionally, they promote hematopoietic stem cell transplantation (HSCT) in sensitized recipients and are able to induce immune tolerance and complete hematopoietic reconstitution. Indoleamine 2, 3-dioxygenase (IDO) and nitric oxide (NO) have been implicated in T cell immune tolerance. The aim of the present report was to study the in vivo tolerogenic mechanisms by which CTLA4Ig and anti-CD154 induce transplantation survival in mice receiving HSCT. BALB/c mice were sensitized via splenocyte transfusion and pretreated with CTLA4Ig plus anti-CD154 on day-7. IDO and inducible nitric oxide synthase (iNOS) inhibitors were applied on days-7 to 0 and the mice were divided into 4 groups (n=10) and injected with IDO every other day. The mice were sacrificed on day 0, and splenocytes were separated to identify CD11c+ antigen-presenting cells, which were subsequently assessed for IDO expression and activity. The concentration of NO was tested using a nitrate reductase kit. Following the acceptance of allogeneic HSCT, mice were tested for homing and engraftment, as well as survival rate. Application of the IDO inhibitor increased the concentration of NO, whereas a decrease in NO resulted in increased IDO activity. Immune tolerance was abrogated in the presence of both IDO and iNOS inhibitors, whereas this effect was not observed with either compound alone. CTLA4Ig and anti-CD154 may induce immune tolerance by affecting the activity of IDO and iNOS. This tolerance was abrogated in the presence of both IDO and iNOS inhibitors. A cross-regulatory pathway was observed between the IDO and NO pathways, in which the inhibition of IDO stimulated the iNOS pathway and vice versa.
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Affiliation(s)
- Qi-Xiang Ye
- Department of Pediatrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510120, P.R. China.,Department of Hematology and Oncology, Guangzhou Women and Children's Medical Center, Guangzhou, Guangdong 510623, P.R. China
| | - Lv-Hong Xu
- Department of Pediatrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510120, P.R. China
| | - Pei-Jie Shi
- Department of Pediatrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510120, P.R. China
| | - Ting Xia
- Department of Pediatrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510120, P.R. China
| | - Jian-Pei Fang
- Department of Pediatrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510120, P.R. China
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6
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Melatonin Attenuates Pulmonary Hypertension in Chronically Hypoxic Rats. Int J Mol Sci 2017; 18:ijms18061125. [PMID: 28538666 PMCID: PMC5485949 DOI: 10.3390/ijms18061125] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2017] [Revised: 05/19/2017] [Accepted: 05/19/2017] [Indexed: 11/17/2022] Open
Abstract
Chronic hypoxia induces pulmonary hypertension and vascular remodeling, which are clinically relevant to patients with chronic obstructive pulmonary disease (COPD) associated with a decreased level of nitric oxide (NO). Oxidative stress and inflammation play important roles in the pathophysiological processes in COPD. We examined the hypothesis that daily administration of melatonin (10 mg/kg) mitigates the pulmonary hypertension and vascular remodeling in chronically hypoxic rats. The right ventricular systolic pressure (RVSP) and the thickness of pulmonary arteriolar wall were measured from normoxic control, vehicle- and melatonin-treated hypoxic rats exposed to 10% O2 for 14 days. Levels of markers for oxidative stress (malondialdhyde) and inflammation (tumor necrosis factor-α (TNFα), inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2)) and the expressions of total endothelial NO synthase (eNOS) and phosphorylated eNOS at serine1177 (ser1177) were determined in the lung tissue. We found that the RVSP and the thickness of the arteriolar wall were significantly increased in the vehicle-treated hypoxic animals with elevated levels of malondialdhyde and mRNA expressions of the inflammatory mediators, when compared with the normoxic control. In addition, the phosphorylated eNOS (ser1177) level was significantly decreased, despite an increased eNOS expression in the vehicle-treated hypoxic group. Melatonin treatment significantly attenuated the levels of RVSP, thickness of the arteriolar wall, oxidative and inflammatory markers in the hypoxic animals with a marked increase in the eNOS phosphorylation in the lung. These results suggest that melatonin attenuates pulmonary hypertension by antagonizing the oxidative injury and restoration of NO production.
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7
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Evans CE, Zhao YY. Molecular Basis of Nitrative Stress in the Pathogenesis of Pulmonary Hypertension. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2017; 967:33-45. [PMID: 29047079 DOI: 10.1007/978-3-319-63245-2_3] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Pulmonary hypertension (PH) is a lung vascular disease with marked increases in pulmonary vascular resistance and pulmonary artery pressure (>25 mmHg at rest). In PH patients, increases in pulmonary vascular resistance lead to impaired cardiac output and reduced exercise tolerance. If untreated, PH progresses to right heart failure and premature lethality. The mechanisms that control the pathogenesis of PH are incompletely understood, but evidence from human and animal studies implicate nitrative stress in the development of PH. Increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) result in nitrative stress, which in turn induces posttranslational modification of key proteins important for maintaining pulmonary vascular homeostasis. This affects their functions and thereby contributes to the pathogenesis of PH. In this chapter, molecular mechanisms underlying nitrative stress-induced PH are reviewed, molecular sources of ROS and RNS are delineated, and evidence of nitrative stress in PH patients is described. A better understanding of such mechanisms could lead to the development of novel treatments for PH.
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Affiliation(s)
- Colin E Evans
- Department of Pharmacology, University of Illinois College of Medicine, 835 South Wolcott Avenue, E403-MSB, M/C 868, Chicago, IL, 60612, USA.,Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, IL, USA.,British Heart Foundation Center of Research Excellence, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK
| | - You-Yang Zhao
- Department of Pharmacology, University of Illinois College of Medicine, 835 South Wolcott Avenue, E403-MSB, M/C 868, Chicago, IL, 60612, USA. .,Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, IL, USA.
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8
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Manukhina EB, Downey HF, Mallet RT. Role of Nitric Oxide in Cardiovascular Adaptation to Intermittent Hypoxia. Exp Biol Med (Maywood) 2016; 231:343-65. [PMID: 16565431 DOI: 10.1177/153537020623100401] [Citation(s) in RCA: 82] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Hypoxia is one of the most frequently encountered stresses in health and disease. The duration, frequency, and severity of hypoxic episodes are critical factors determining whether hypoxia is beneficial or harmful. Adaptation to intermittent hypoxia has been demonstrated to confer cardiovascular protection against more severe and sustained hypoxia, and, moreover, to protect against other stresses, including ischemia. Thus, the direct and cross protective effects of adaptation to intermittent hypoxia have been used for treatment and prevention of a variety of diseases and to increase efficiency of exercise training. Evidence is mounting that nitric oxide (NO) plays a central role in these adaptive mechanisms. NO-dependent protective mechanisms activated by intermittent hypoxia include stimulation of NO synthesis as well as restriction of NO overproduction. In addition, alternative, nonenzymic sources of NO and negative feedback of NO synthesis are important factors in optimizing NO concentrations. The adaptive enhancement of NO synthesis and/or availability activates or increases expression of other protective factors, including heat shock proteins, antioxidants and prostaglandins, making the protection more robust and sustained. Understanding the role of NO in mechanisms of adaptation to hypoxia will support development of therapies to prevent and treat hypoxic or ischemic damage to organs and cells and to increase adaptive capabilities of the organism.
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9
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Kalinowski L, Janaszak-Jasiecka A, Siekierzycka A, Bartoszewska S, Woźniak M, Lejnowski D, Collawn JF, Bartoszewski R. Posttranscriptional and transcriptional regulation of endothelial nitric-oxide synthase during hypoxia: the role of microRNAs. Cell Mol Biol Lett 2016; 21:16. [PMID: 28536619 PMCID: PMC5415778 DOI: 10.1186/s11658-016-0017-x] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2016] [Accepted: 08/18/2016] [Indexed: 02/07/2023] Open
Abstract
Understanding the cellular pathways that regulate endothelial nitric oxide (eNOS, NOS3) expression and consequently nitric oxide (NO) bioavailability during hypoxia is a necessary aspect in the development of novel treatments for cardiovascular disorders. eNOS expression and eNOS-dependent NO cellular signaling during hypoxia promote an equilibrium of transcriptional and posttranscriptional molecular mechanisms that belong to both proapoptotic and survival pathways. Furthermore, NO bioavailability results not only from eNOS levels, but also relies on the presence of eNOS substrate and cofactors, the phosphorylation status of eNOS, and the presence of reactive oxygen species (ROS) that can inactivate eNOS. Since both NOS3 levels and these signaling pathways can also be a subject of posttranscriptional modulation by microRNAs (miRNAs), this class of short noncoding RNAs contribute another level of regulation for NO bioavailability. As miRNA antagomirs or specific target protectors could be used in therapeutic approaches to regulate NO levels, either by changing NOS3 mRNA stability or through factors governing eNOS activity, it is critical to understand their role in governing eNOS activity during hypoxa. In contrast to a large number of miRNAs reported to the change eNOS expression during hypoxia, only a few miRNAs modulate eNOS activity. Furthermore, impaired miRNA biogenesis leads to NOS3 mRNA stabilization under hypoxia. Here we discuss the recent studies that define miRNAs’ role in maintaining endothelial NO bioavailability emphasizing those miRNAs that directly modulate NOS3 expression or eNOS activity.
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Affiliation(s)
- Leszek Kalinowski
- Department of Medical Laboratory Diagnostics and Central Bank of Frozen Tissues & Genetic Specimens, Medical University of Gdansk, Debinki 7, 80-211 Gdansk, Poland
| | - Anna Janaszak-Jasiecka
- Department of Biology and Pharmaceutical Botany, Medical University of Gdansk, Hallera 107, 80-416 Gdansk, Poland
| | - Anna Siekierzycka
- Department of Medical Laboratory Diagnostics and Central Bank of Frozen Tissues & Genetic Specimens, Medical University of Gdansk, Debinki 7, 80-211 Gdansk, Poland
| | - Sylwia Bartoszewska
- Department of Inorganic Chemistry, Medical University of Gdansk, Gdansk, Poland
| | - Marcin Woźniak
- Department of Medical Laboratory Diagnostics and Central Bank of Frozen Tissues & Genetic Specimens, Medical University of Gdansk, Debinki 7, 80-211 Gdansk, Poland
| | - Dawid Lejnowski
- Department of Biology and Pharmaceutical Botany, Medical University of Gdansk, Hallera 107, 80-416 Gdansk, Poland
| | - James F Collawn
- Department of Cell Biology, Developmental, and Integrative, University of Alabama at Birmingham, Birmingham, USA
| | - Rafal Bartoszewski
- Department of Biology and Pharmaceutical Botany, Medical University of Gdansk, Hallera 107, 80-416 Gdansk, Poland
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10
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Chalupsky K, Kračun D, Kanchev I, Bertram K, Görlach A. Folic Acid Promotes Recycling of Tetrahydrobiopterin and Protects Against Hypoxia-Induced Pulmonary Hypertension by Recoupling Endothelial Nitric Oxide Synthase. Antioxid Redox Signal 2015; 23:1076-91. [PMID: 26414244 PMCID: PMC4657514 DOI: 10.1089/ars.2015.6329] [Citation(s) in RCA: 61] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/12/2015] [Revised: 09/21/2015] [Accepted: 09/21/2015] [Indexed: 01/29/2023]
Abstract
AIMS Nitric oxide (NO) derived from endothelial NO synthase (eNOS) has been implicated in the adaptive response to hypoxia. An imbalance between 5,6,7,8-tetrahydrobiopterin (BH4) and 7,8-dihydrobiopterin (BH2) can result in eNOS uncoupling and the generation of superoxide instead of NO. Dihydrofolate reductase (DHFR) can recycle BH2 to BH4, leading to eNOS recoupling. However, the role of DHFR and eNOS recoupling in the response to hypoxia is not well understood. We hypothesized that increasing the capacity to recycle BH4 from BH2 would improve NO bioavailability as well as pulmonary vascular remodeling (PVR) and right ventricular hypertrophy (RVH) as indicators of pulmonary hypertension (PH) under hypoxic conditions. RESULTS In human pulmonary artery endothelial cells and murine pulmonary arteries exposed to hypoxia, eNOS was uncoupled as indicated by reduced superoxide production in the presence of the nitric oxide synthase inhibitor, L-(G)-nitro-L-arginine methyl ester (L-NAME). Concomitantly, NO levels, BH4 availability, and expression of DHFR were diminished under hypoxia. Application of folic acid (FA) restored DHFR levels, NO bioavailability, and BH4 levels under hypoxia. Importantly, FA prevented the development of hypoxia-induced PVR, right ventricular pressure increase, and RVH. INNOVATION FA-induced upregulation of DHFR recouples eNOS under hypoxia by improving BH4 recycling, thus preventing hypoxia-induced PH. CONCLUSION FA might serve as a novel therapeutic option combating PH.
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Affiliation(s)
- Karel Chalupsky
- Experimental and Molecular Pediatric Cardiology, German Heart Center Munich at the Technical University Munich, Munich, Germany
| | - Damir Kračun
- Experimental and Molecular Pediatric Cardiology, German Heart Center Munich at the Technical University Munich, Munich, Germany
| | - Ivan Kanchev
- Experimental and Molecular Pediatric Cardiology, German Heart Center Munich at the Technical University Munich, Munich, Germany
| | - Katharina Bertram
- Experimental and Molecular Pediatric Cardiology, German Heart Center Munich at the Technical University Munich, Munich, Germany
| | - Agnes Görlach
- Experimental and Molecular Pediatric Cardiology, German Heart Center Munich at the Technical University Munich, Munich, Germany
- DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany
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11
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Wandall-Frostholm C, Skaarup LM, Sadda V, Nielsen G, Hedegaard ER, Mogensen S, Köhler R, Simonsen U. Pulmonary hypertension in wild type mice and animals with genetic deficit in KCa2.3 and KCa3.1 channels. PLoS One 2014; 9:e97687. [PMID: 24858807 PMCID: PMC4032241 DOI: 10.1371/journal.pone.0097687] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2013] [Accepted: 04/22/2014] [Indexed: 11/18/2022] Open
Abstract
Objective In vascular biology, endothelial KCa2.3 and KCa3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of KCa2.3 and KCa3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of KCa2.3 and KCa3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension. Approach and Result Male wild type and KCa3.1−/−/KCa2.3T/T(+DOX) mice were exposed to chronic hypoxia for four weeks to induce pulmonary hypertension. The degree of pulmonary hypertension was evaluated by right ventricular pressure and assessment of right ventricular hypertrophy. Segments of pulmonary arteries were mounted in a wire myograph for functional studies and morphometric studies were performed on lung sections. Chronic hypoxia induced pulmonary hypertension, right ventricular hypertrophy, increased lung weight, and increased hematocrit levels in either genotype. The KCa3.1−/−/KCa2.3T/T(+DOX) mice developed structural alterations in the heart with increased right ventricular wall thickness as well as in pulmonary vessels with increased lumen size in partially- and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the KCa2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the KCa2.3 and KCa3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of KCa2.3 and KCa3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype. Conclusion Despite the deficits of the KCa2.3 and KCa3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of KCa2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a novel mechanism to counteract pulmonary hypertension and to a potential therapeutic utility of KCa2.3/KCa3.1 activators for the treatment of pulmonary hypertension.
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Affiliation(s)
| | | | - Veeranjaneyulu Sadda
- Department of Biomedicine, Aarhus University, Aarhus, Denmark
- Institute for Molecular Medicine, Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark
| | - Gorm Nielsen
- Institute for Molecular Medicine, Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark
| | | | - Susie Mogensen
- Department of Biomedicine, Aarhus University, Aarhus, Denmark
| | - Ralf Köhler
- Institute for Molecular Medicine, Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark
- Aragon Institute of Health Sciences I+CS and ARAID, Zaragoza, Spain
| | - Ulf Simonsen
- Department of Biomedicine, Aarhus University, Aarhus, Denmark
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12
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Dolkart O, E A, S S, S M, P G, Aa W. Temporal determination of lung NO system and COX-2 upregulation following ischemia-reperfusion injury. Exp Lung Res 2013; 40:22-9. [PMID: 24354410 DOI: 10.3109/01902148.2013.858196] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
BACKGROUND Pulmonary ischemia-reperfusion (IR) is a biopathological event detectable in several clinical conditions, including lung transplantation, cardiopulmonary bypass, resuscitation, and pulmonary embolism. The understanding behind the activation of various inflammatory mediators regulating the apoptotic pathways remains largely unknown. We investigated the temporal expression of endothelial nitric oxide (eNOS), inducible (iNOS), and cyclooxygenase-2 (COX-2) proteins following lung-IR injury. METHODS Lung IR was induced in anesthetized rats. One hour ischemia was performed by clamping the left hilum. eNOS, iNOS, and COX-2 levels in the bronchoalveolar lavage (BAL) were measured at different time points after restoring lung perfusion in conjunction with histological changes and cellular apoptosis. RESULTS BAL-eNOS levels were increased as early as 3 hours post IR, attaining the highest values (5.5 U/mL) at 3 hours, compared to non-IR values (2.8 U/mL). BAL-iNOS increased at 3-hour post-IR (3 U/mL). iNOS reached the highest levels at 24 hours (4.5 U/mL) as compared to nonischemic lungs (1.8 U/mL). COX-2 peaked at 12 hours (.025 U/mL) compared to 3, 24, and 48 hours. Highest apoptotic rates were detected at 12 and 48 hours following IR. CONCLUSIONS The time-associated involvement of eNOS, iNOS, and COX-2 enzymes during the evolution of IR injury may point to an early reaction of the NOSs system versus the COX-2. Similar patterns of enzymatic activity were previously shown in the context of lung IR injury. This temporal activation may indicate an involvement of eNOS in an early reparative response, and possibly the late-pathological response, mediated by the coinduction of iNOS-COX-2.
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Affiliation(s)
- Oleg Dolkart
- 1Pre-Clinical Research Laboratory & Post-Anesthesia Care Unit and
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13
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Dick AS, Ivanovska J, Kantores C, Belcastro R, Keith Tanswell A, Jankov RP. Cyclic stretch stimulates nitric oxide synthase-1-dependent peroxynitrite formation by neonatal rat pulmonary artery smooth muscle. Free Radic Biol Med 2013; 61:310-9. [PMID: 23619128 DOI: 10.1016/j.freeradbiomed.2013.04.027] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/29/2012] [Revised: 02/27/2013] [Accepted: 04/16/2013] [Indexed: 11/28/2022]
Abstract
Peroxynitrite, the reaction product of nitric oxide and superoxide, contributes to the pathogenesis of chronic pulmonary hypertension in immature animals by stimulating proliferation of pulmonary arterial smooth muscle cells (PASMCs). Pulmonary vasoconstriction, secondary to hypoxia and other stimuli, leads to enhanced pulsatile stretch of cells in the vascular wall, particularly in smooth muscle, which we hypothesized would cause increased peroxynitrite generation. Our objectives in this study were to determine whether cyclic mechanical stretch, at supraphysiologic levels, would cause increased production of reactive oxygen species (ROS), nitric oxide, and peroxynitrite in vitro. Early passage neonatal rat PASMCs were seeded and grown to subconfluence on collagen-coated elastomer-bottom plates and subjected to cyclic mechanical stretch (10% ("physiologic") or 20% ("supraphysiologic") at 0.5 Hz) for up to 24 h. Compared to nonstretched controls and to cells subjected to 10% stretch, 20% stretch increased H2O2 (stable marker of ROS) and nitrate/nitrite (stable marker of nitric oxide) in conditioned medium. These effects were accompanied by increased peroxynitrite, as evidenced by increased in situ dihydroethidium fluorescence and immunoreactive nitrotyrosine and by increased expression of nitric oxide synthase (NOS)-1 and NADPH oxidase 4 (NOX4), but not NOS-2. Stretch-induced H2O2 release and increased dihydroethidium fluorescence were prevented by pretreatment with a superoxide scavenger, nonspecific inhibitors of NADPH oxidase or NOS, or a peroxynitrite decomposition catalyst. Short-interfering RNA-mediated knockdown of NOS-1 or NOX4 attenuated increased nitric oxide and H2O2 content, respectively, in stretched-cell-conditioned medium. Knockdown of NOS-1 also attenuated increased immunoreactive nitrotyrosine content and stretch-induced proliferation, whereas knockdown of NOS-2 had no effect. We conclude that increased peroxynitrite generation by neonatal rat PASMCs subjected to supraphysiologic levels of cyclic stretch is NOS-1-dependent and that increased ROS production is predominantly mediated by NADPH oxidase, specifically NOX4.
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Affiliation(s)
- Andrew S Dick
- Physiology & Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 1X8
| | - Julijana Ivanovska
- Physiology & Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 1X8
| | - Crystal Kantores
- Physiology & Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 1X8
| | - Rosetta Belcastro
- Physiology & Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 1X8
| | - A Keith Tanswell
- Physiology & Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 1X8; Department of Physiology, Division of Neonatology, Department of Paediatrics, and Heart and Stroke Richard Lewar Centre of Excellence, University of Toronto, Toronto, ON, Canada M5S 1A8; Division of Neonatology, Department of Paediatrics, and Heart and Stroke Richard Lewar Centre of Excellence, University of Toronto, Toronto, ON, Canada M5S 1A8
| | - Robert P Jankov
- Physiology & Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 1X8; Department of Physiology, Division of Neonatology, Department of Paediatrics, and Heart and Stroke Richard Lewar Centre of Excellence, University of Toronto, Toronto, ON, Canada M5S 1A8; Division of Neonatology, Department of Paediatrics, and Heart and Stroke Richard Lewar Centre of Excellence, University of Toronto, Toronto, ON, Canada M5S 1A8; Heart and Stroke Richard Lewar Centre of Excellence, University of Toronto, Toronto, ON, Canada M5S 1A8.
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14
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Rafikov R, Rafikova O, Aggarwal S, Gross C, Sun X, Desai J, Fulton D, Black SM. Asymmetric dimethylarginine induces endothelial nitric-oxide synthase mitochondrial redistribution through the nitration-mediated activation of Akt1. J Biol Chem 2012; 288:6212-26. [PMID: 23255608 DOI: 10.1074/jbc.m112.423269] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
We have recently demonstrated that asymmetric dimethylarginine (ADMA) induces the translocation of endothelial nitric-oxide synthase (eNOS) to the mitochondrion via a mechanism that requires protein nitration. Thus, the goal of this study was elucidate how eNOS redistributes to mitochondria and to identify the nitrated protein responsible for this event. Our data indicate that exposure of pulmonary arterial endothelial cells to ADMA enhanced eNOS phosphorylation at the Akt1-dependent phosphorylation sites Ser(617) and Ser(1179). Mutation of these serine residues to alanine (S617A and S1179A) inhibited nitration-mediated eNOS translocation to the mitochondria, whereas the phosphormimic mutations (S617D and S1179D) exhibited increased mitochondrial redistribution in the absence of ADMA. The overexpression of a dominant-negative Akt1 also attenuated ADMA-mediated eNOS mitochondrial translocation. Furthermore, ADMA enhanced Akt1 nitration and increased its activity. Mass spectrometry identified a single nitration site in Akt1 located at the tyrosine residue (Tyr(350)) located within the client-binding domain. Replacement of Tyr(350) with phenylalanine abolished peroxynitrite-mediated eNOS translocation to mitochondria. We also found that in the absence of ADMA, eNOS translocation decreased mitochondrial oxygen consumption and superoxide production without altering cellular ATP level. This suggests that under physiologic conditions, eNOS translocation enhances mitochondria coupling. In conclusion, we have identified a new mechanism by which eNOS translocation to mitochondria is regulated by the phosphorylation of eNOS at Ser(617) and Ser(1179) by Akt1 and that this is enhanced when Akt1 becomes nitrated at Tyr(350).
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Affiliation(s)
- Ruslan Rafikov
- Pulmonary Disease Program, Vascular Biology Center, Georgia Health Sciences University, Augusta, Georgia 30912, USA
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15
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Lung oxidative damage by hypoxia. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2012; 2012:856918. [PMID: 22966417 PMCID: PMC3433143 DOI: 10.1155/2012/856918] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/14/2012] [Accepted: 07/11/2012] [Indexed: 12/12/2022]
Abstract
One of the most important functions of lungs is to maintain an adequate oxygenation in the organism. This organ can be affected by hypoxia facing both physiological and pathological situations. Exposure to this condition favors the increase of reactive oxygen species from mitochondria, as from NADPH oxidase, xanthine oxidase/reductase, and nitric oxide synthase enzymes, as well as establishing an inflammatory process. In lungs, hypoxia also modifies the levels of antioxidant substances causing pulmonary oxidative damage. Imbalance of redox state in lungs induced by hypoxia has been suggested as a participant in the changes observed in lung function in the hypoxic context, such as hypoxic vasoconstriction and pulmonary edema, in addition to vascular remodeling and chronic pulmonary hypertension. In this work, experimental evidence that shows the implied mechanisms in pulmonary redox state by hypoxia is reviewed. Herein, studies of cultures of different lung cells and complete isolated lung and tests conducted in vivo in the different forms of hypoxia, conducted in both animal models and humans, are described.
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16
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Abstract
It has been known for more than 60 years, and suspected for over 100, that alveolar hypoxia causes pulmonary vasoconstriction by means of mechanisms local to the lung. For the last 20 years, it has been clear that the essential sensor, transduction, and effector mechanisms responsible for hypoxic pulmonary vasoconstriction (HPV) reside in the pulmonary arterial smooth muscle cell. The main focus of this review is the cellular and molecular work performed to clarify these intrinsic mechanisms and to determine how they are facilitated and inhibited by the extrinsic influences of other cells. Because the interaction of intrinsic and extrinsic mechanisms is likely to shape expression of HPV in vivo, we relate results obtained in cells to HPV in more intact preparations, such as intact and isolated lungs and isolated pulmonary vessels. Finally, we evaluate evidence regarding the contribution of HPV to the physiological and pathophysiological processes involved in the transition from fetal to neonatal life, pulmonary gas exchange, high-altitude pulmonary edema, and pulmonary hypertension. Although understanding of HPV has advanced significantly, major areas of ignorance and uncertainty await resolution.
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Affiliation(s)
- J T Sylvester
- Division of Pulmonary & Critical Care Medicine, Department of Medicine, The Johns Hopkins University School ofMedicine, Baltimore, Maryland, USA.
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17
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Norton CE, Jernigan NL, Kanagy NL, Walker BR, Resta TC. Intermittent hypoxia augments pulmonary vascular smooth muscle reactivity to NO: regulation by reactive oxygen species. J Appl Physiol (1985) 2011; 111:980-8. [PMID: 21757577 DOI: 10.1152/japplphysiol.01286.2010] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension. IH causes oxidative stress that may limit bioavailability of the endothelium-derived vasodilator nitric oxide (NO) and thus contribute to this hypertensive response. We therefore hypothesized that increased vascular superoxide anion (O(2)(-)) generation reduces NO-dependent pulmonary vasodilation following IH. To test this hypothesis, we examined effects of the O(2)(-) scavenger tiron on vasodilatory responses to the endothelium-dependent vasodilator ionomycin and the NO donor S-nitroso-N-acetylpenicillamine in isolated lungs from hypocapnic-IH (H-IH; 3 min cycles of 5% O(2)/air flush, 7 h/day, 4 wk), eucapnic-IH (E-IH; cycles of 5% O(2), 5% CO(2)/air flush), and sham-treated (air/air cycled) rats. Next, we assessed effects of endogenous O(2)(-) on NO- and cGMP-dependent vasoreactivity and measured O(2)(-) levels using the fluorescent indicator dihydroethidium (DHE) in isolated, endothelium-disrupted small pulmonary arteries from each group. Both E-IH and H-IH augmented NO-dependent vasodilation; however, enhanced vascular smooth muscle (VSM) reactivity to NO following H-IH was masked by an effect of endogenous O(2)(-). Furthermore, H-IH and E-IH similarly increased VSM sensitivity to cGMP, but this response was independent of either O(2)(-) generation or altered arterial protein kinase G expression. Finally, both H-IH and E-IH increased arterial O(2)(-) levels, although this response was more pronounced following H-IH, and H-IH exposure resulted in greater protein tyrosine nitration indicative of increased NO scavenging by O(2)(-). We conclude that IH increases pulmonary VSM sensitivity to NO and cGMP. Furthermore, endogenous O(2)(-) limits NO-dependent vasodilation following H-IH through an apparent reduction in bioavailable NO.
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Affiliation(s)
- Charles E Norton
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131-0001, USA.
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18
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Rus A, Molina F, Peinado MA, del Moral ML. Endothelial NOS-derived nitric oxide prevents injury resulting from reoxygenation in the hypoxic lung. Free Radic Res 2011; 44:1027-35. [PMID: 20815765 DOI: 10.3109/10715762.2010.498479] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
To date, the role that NO derived from endothelial NO synthase (eNOS) plays in the development of the injuries occurring under hypoxia/reoxygenation (H/R) in the lung remains unknown and thus constitutes the subject of the present work. A follow-up study was conducted in Wistar rats submitted to H/R (hypoxia for 30 min; reoxygenation of 0 h, 48 h and 5 days), with or without prior treatment using the eNOS inhibitor L-NIO (20 mg/kg). Lipid peroxidation, apoptosis, protein nitration and NO production (NOx) were analysed. The results showed that L-NIO administration lowered NOx levels in all the experimental groups. Contrarily, the lipid peroxidation level and the percentage of apoptotic cells rose, implying that eNOS-derived NO may have a protective effect against the injuries occurring during H/R in the lung. These findings could open the possibility of future studies to design new therapies for this type of hypoxia based on NO-pharmacology.
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Affiliation(s)
- Alma Rus
- Department of Experimental Biology, University of Jaén, Spain
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19
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Rus A, Peinado MA, Castro L, Del Moral ML. Lung eNOS and iNOS are reoxygenation time-dependent upregulated after acute hypoxia. Anat Rec (Hoboken) 2010; 293:1089-98. [PMID: 20225207 DOI: 10.1002/ar.21141] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Nitric oxide plays a critical role in many physiological and physiopathological processes in the lung. Changes in the NO/NOS (Nitric Oxide/Nitric Oxide Synthase) system after hypoxia situations remain controversial in this organ, so that the aim of this work is to perform a complete study of this system in the hypoxic lung after different reoxygenation times ranging from 0 h to 5 days posthypoxia. This is a novel follow-up study carried out in Wistar rats submitted for 30 min to acute hypobaric hypoxia. We measured endothelial and inducible NOS (eNOS, iNOS) mRNA and protein expression, location, and in situ NOS activity as well as nitrated protein expression and location. In addition, NO levels were indirectly quantified (NOx) as well as the apoptosis level. Results showed an increase in eNOS mRNA, protein, activity as well as eNOS positive immunostaining at 0 h posthypoxia, coinciding with raised NOx levels. Contrary, iNOS, nitrated protein expression and apoptosis level augmented during the final reoxygenation times. The lung NO/NOS system provokes two responses to the hypoxia/reoxygenation processes: (i) eNOS is responsible of the immediate response, producing NO, which causes vasodilation and bronchodilation, and (ii) iNOS is related to the second late response, which seems to be involved in some of the deleterious consequences that hypoxia induces in the lung.
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Affiliation(s)
- Alma Rus
- Department of Experimental Biology, University of Jaén, Jaén, Spain
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20
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Rus A, Castro L, Del Moral ML, Peinado A. Inducible NOS inhibitor 1400W reduces hypoxia/re-oxygenation injury in rat lung. Redox Rep 2010; 15:169-78. [PMID: 20663293 DOI: 10.1179/174329210x12650506623609] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Nitric oxide (NO(*)) from inducible NO(*) synthase (iNOS) has been reported to either protect against, or contribute to, hypoxia/re-oxygenation lung injury. The present work aimed to clarify this double role in the hypoxic lung. With this objective, a follow-up study was made in Wistar rats submitted to hypoxia/re-oxygenation (hypoxia for 30 min; re-oxygenation of 0 h, 48 h, and 5 days), with or without prior treatment with the selective iNOS inhibitor 1400W (10 mg/kg). NO(*) levels (NOx), lipid peroxidation, apoptosis, and protein nitration were analysed. This is the first time-course study which investigates the effects of 1400W during hypoxia/re-oxygenation in the rat lung. The results showed that the administration of 1400W lowered NOx levels in all the experimental groups. In addition, lipid peroxidation, the percentage of apoptotic cells, and nitrated protein expression fell in the late post-hypoxia period (48 h and 5 days). Our results reveal that the inhibition of iNOS in the hypoxic lung reduced the damage observed before the treatment with 1400W, suggesting that iNOS-derived NO(*) may exert a negative effect on this organ during hypoxia/re-oxygenation. These findings are notable, since they indicate that any therapeutic strategy aimed at controlling excess generation of NO(*) from iNOS may be useful in alleviating NO(*)-mediated adverse effects in hypoxic lungs.
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Affiliation(s)
- Alma Rus
- Department of Experimental Biology, University of Jaén, Jaén, Spain
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21
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den Hengst WA, Gielis JF, Lin JY, Van Schil PE, De Windt LJ, Moens AL. Lung ischemia-reperfusion injury: a molecular and clinical view on a complex pathophysiological process. Am J Physiol Heart Circ Physiol 2010; 299:H1283-99. [PMID: 20833966 DOI: 10.1152/ajpheart.00251.2010] [Citation(s) in RCA: 285] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Lung ischemia-reperfusion injury remains one of the major complications after cardiac bypass surgery and lung transplantation. Due to its dual blood supply system and the availability of oxygen from alveolar ventilation, the pathogenetic mechanisms of ischemia-reperfusion injury in the lungs are more complicated than in other organs, where loss of blood flow automatically leads to hypoxia. In this review, an extensive overview is given of the molecular and cellular mechanisms that are involved in the pathogenesis of lung ischemia-reperfusion injury and the possible therapeutic strategies to reduce or prevent it. In addition, the roles of neutrophils, alveolar macrophages, cytokines, and chemokines, as well as the alterations in the cell-death related pathways, are described in detail.
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Affiliation(s)
- Willem A den Hengst
- Department of Thorax and Vascular Surgery, University of Antwerp, Antwerp, Belgium
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22
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Rus A, Molina F, Peinado MÁ, Del Moral ML. Endogenous nitric oxide can act as beneficial or deleterious in the hypoxic lung depending on the reoxygenation time. Anat Rec (Hoboken) 2010; 293:2193-201. [PMID: 20734424 DOI: 10.1002/ar.21229] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2010] [Accepted: 06/01/2010] [Indexed: 12/28/2022]
Abstract
Nitric oxide (NO) has been implicated in many pathophysiological situations in the lung, including hypoxia/reoxygenation. This work seeks to clarify the current controversy concerning the double protective/toxic role of endogenous NO under hypoxia/reoxygenation situations in the lung by using a nitric oxide synthase (NOS) inhibitor, in a novel approach to address the problems raised from assaults under such circumstances. A follow-up study was conducted in Wistar rats submitted to hypoxia/reoxygenation (hypoxia for 30 min; reoxygenation of 0 h, 48 h, and 5 days), with or without prior treatment using the nonselective NOS inhibitor L-NAME (1.5 mM, in drinking water). Lipid peroxidation, apoptosis level, protein nitration, in situ NOS activity and NO production (NOx) were analyzed. This is the first work to focus on the time-course effects of L-NAME in the adult rat lung submitted to hypoxia/reoxygenation. The results showed that after L-NAME administration, in situ NOS activity was almost completely eliminated and consequently, NOx levels fell. Lipid peroxidation and the percentage of apoptotic cells rose at the earliest reoxygenation time (0 h), but decreased in the later period (48 h and 5 days). Also nitrated protein expression decreased at 48 h and 5 days posthypoxia. These results suggest that NOS-derived NO exerts two different effects on lung hypoxia/reoxygenation injury depending on the reoxygenation time: NO has a beneficial role just after the hypoxic stimulus and a deleterious effect in the later reoxygenation times. Moreover, we propose that this dual role of NO depends directly on the producer NOS isoform.
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Affiliation(s)
- Alma Rus
- Department of Experimental Biology, University of Jaén, Jaén, Spain
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23
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Tourneux P, Markham N, Seedorf G, Balasubramaniam V, Abman SH. Inhaled nitric oxide improves lung structure and pulmonary hypertension in a model of bleomycin-induced bronchopulmonary dysplasia in neonatal rats. Am J Physiol Lung Cell Mol Physiol 2009; 297:L1103-11. [DOI: 10.1152/ajplung.00293.2009] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Whether inhaled nitric oxide (iNO) prevents the development of bronchopulmonary dysplasia (BPD) in premature infants is controversial. In adult rats, bleomycin (Bleo) induces lung fibrosis and pulmonary hypertension, but the effects of Bleo on the developing lung and iNO treatment on Bleo-induced neonatal lung injury are uncertain. Therefore, we sought to determine whether early and prolonged iNO therapy attenuates changes of pulmonary vascular and alveolar structure in a model of BPD induced by Bleo treatment of neonatal rats. Sprague-Dawley rat pups were treated with Bleo (1 mg/kg ip daily) or vehicle (controls) from day 2 to 10, followed by recovery from day 11 to 19. Treatment groups received early ( days 2–10), late ( days 11–19), or prolonged iNO therapy (10 ppm; days 2–19). We found that compared with controls, Bleo increased right ventricular hypertrophy (RVH), and pulmonary arterial wall thickness, and reduced vessel density alveolarization. In each iNO treatment group, iNO decreased RVH ( P < 0.01) and wall thickness ( P < 0.01) and restored vessel density after Bleo ( P < 0.05). iNO therapy improved alveolarization for each treatment group after Bleo; however, the values remained abnormal compared with controls. Prolonged iNO treatment had greater effects on lung structure after bleomycin than late treatment alone. We conclude that Bleo induces lung structural changes that mimic BPD in neonatal rats, and that early and prolonged iNO therapy prevents right ventricle hypertrophy and pulmonary vascular remodeling and partially improves lung structure.
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Affiliation(s)
- Pierre Tourneux
- Pediatric Heart Lung Center, Sections of Neonatology and Pulmonary Medicine, Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado; and
- Neonatal and Pediatric Intensive Care Unit, Amiens University Medical Center, and PériTox, Faculty of Medicine, Jules Verne University of Picardy, Amiens, France
| | - Neil Markham
- Pediatric Heart Lung Center, Sections of Neonatology and Pulmonary Medicine, Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado; and
| | - Gregory Seedorf
- Pediatric Heart Lung Center, Sections of Neonatology and Pulmonary Medicine, Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado; and
| | - Vivek Balasubramaniam
- Pediatric Heart Lung Center, Sections of Neonatology and Pulmonary Medicine, Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado; and
| | - Steven H. Abman
- Pediatric Heart Lung Center, Sections of Neonatology and Pulmonary Medicine, Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado; and
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Mishra OP, Ashraf QM, Delivoria-Papadopoulos M. Mechanism of increased tyrosine (Tyr(99)) phosphorylation of calmodulin during hypoxia in the cerebral cortex of newborn piglets: the role of nNOS-derived nitric oxide. Neurochem Res 2009; 35:67-75. [PMID: 19590958 DOI: 10.1007/s11064-009-0031-8] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2009] [Accepted: 06/25/2009] [Indexed: 11/28/2022]
Abstract
The present study aims to investigate the mechanism of calmodulin modification during hypoxia and tests the hypothesis that hypoxia-induced increase in Tyr(99) phosphorylation of calmodulin in the cerebral cortex of newborn piglets is mediated by NO derived from nNOS. Fifteen piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, F(i)O(2) of 0.07 for 1 h, n = 5) and hypoxic-pretreated with nNOSi (Hx-nNOSi, n = 5) groups. nNOS inhibitor I (selectivity >2,500 vs. eNOS and >500 vs. iNOS) was administered (0.4 mg/kg, I.V.) 30 min prior to hypoxia. Cortical membranes were isolated and tyrosine phosphorylation (Tyr(99) and total) of calmodulin determined by Western blot using anti-phospho-(pTyr(99))-calmodulin and anti-pTyr antibodies. Protein bands were detected by enhanced chemiluminescence, analyzed by densitometry and expressed as absorbance. The pTyr(99) calmodulin (ODxmm(2)) was 78.55 +/- 10.76 in Nx, 165.05 +/- 12.26 in Hx (P < 0.05 vs. Nx) and 96.97 +/- 13.18 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Expression of total tyrosine phosphorylated calmodulin was 69.24 +/- 13.69 in Nx, 156.17 +/- 16.34 in Hx (P < 0.05 vs. Nx) and 74.18 +/- 3.9 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). The data show that administration of nNOS inhibitor prevented the hypoxia-induced increased Tyr(99) phosphorylation of calmodulin. Total tyrosine phosphorylation of calmodulin was similar to Tyr(99) phosphorylation. We conclude that the mechanism of hypoxia-induced modification (Tyr(99) phosphorylation) of calmodulin is mediated by NO derived from nNOS. We speculate that Tyr(99) phosphorylated calmodulin, as compared to non-phosphorylated, binds with a higher affinity at the calmodulin binding site of nNOS leading to increased activation of nNOS and increased generation of NO.
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Affiliation(s)
- Om Prakash Mishra
- Department of Pediatrics, Drexel University College of Medicine and St. Christopher's Hospital for Children, Philadelphia, PA 19102, USA.
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25
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Liang Y, Fang M, Li J, Yew DT. IMMUNOHISTOCHEMICAL LOCALIZATION OF ENDOTHELIAL ISOFORM (eNOS) IN HUMAN CEREBRAL ARTERIES AND THE AORTA. Int J Neurosci 2009; 116:1403-17. [PMID: 17145676 DOI: 10.1080/00207450500514375] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
The common carotid, vertebral, posterior cerebral arteries, and the aorta were studied in the human in terms of its eNOS expression. In around 10 weeks of gestation, the developing intima began to express notable eNOS. In the adult, the positive eNOS sites were in the endothelial cells and the tunica media where the smooth muscles were. In the vessels with athrosclerotic changes, eNOS was down regulated in the endothelial layer and most of the tunica media but was significantly upregulated in the tunica media around the lesion. The protein changes are related to the onset of the athrosclerotic diseases.
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Affiliation(s)
- Yong Liang
- Institute of Cell Biology, Medical College of Zhejiang University, Hangzhou, Zhejiang, P.R., China
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26
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Pronko TP, Zinchuk VV. Effect of nebivolol on blood oxygen transport indices and endothelial dysfunction in patients with arterial hypertension. Clin Physiol Funct Imaging 2009; 29:170-6. [DOI: 10.1111/j.1475-097x.2008.00852.x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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Peyter AC, Muehlethaler V, Liaudet L, Marino M, Di Bernardo S, Diaceri G, Tolsa JF. Muscarinic receptor M1 and phosphodiesterase 1 are key determinants in pulmonary vascular dysfunction following perinatal hypoxia in mice. Am J Physiol Lung Cell Mol Physiol 2008; 295:L201-13. [PMID: 18469116 DOI: 10.1152/ajplung.00264.2007] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Perinatal adverse events such as limitation of nutrients or oxygen supply are associated with the occurrence of diseases in adulthood, like cardiovascular diseases and diabetes. We investigated the long-term effects of perinatal hypoxia on the lung circulation, with particular attention to the nitric oxide (NO)/cGMP pathway. Mice were placed under hypoxia in utero 5 days before delivery and for 5 days after birth. Pups were then bred in normoxia until adulthood. Adults born in hypoxia displayed an altered regulation of pulmonary vascular tone with higher right ventricular pressure in normoxia and increased sensitivity to acute hypoxia compared with controls. Perinatal hypoxia dramatically decreased endothelium-dependent relaxation induced by ACh in adult pulmonary arteries (PAs) but did not influence NO-mediated endothelium-independent relaxation. The M(3) muscarinic receptor was implicated in the relaxing action of ACh and M(1) muscarinic receptor (M(1)AChR) in its vasoconstrictive effects. Pirenzepine or telenzepine, two preferential inhibitors of M(1)AChR, abolished the adverse effects of perinatal hypoxia on ACh-induced relaxation. M(1)AChR mRNA expression was increased in lungs and PAs of mice born in hypoxia. The phosphodiesterase 1 (PDE1) inhibitor vinpocetine also reversed the decrease in ACh-induced relaxation following perinatal hypoxia, suggesting that M(1)AChR-mediated alteration of ACh-induced relaxation is due to the activation of calcium-dependent PDE1. Therefore, perinatal hypoxia leads to an altered pulmonary circulation in adulthood with vascular dysfunction characterized by impaired endothelium-dependent relaxation and M(1)AChR plays a predominant role. This raises the possibility that muscarinic receptors could be key determinants in pulmonary vascular diseases in relation to "perinatal imprinting."
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Affiliation(s)
- Anne-Christine Peyter
- Neonatal Research Laboratory, Department of Pediatrics, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
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Zhang HY, Han DW, Su AR, Zhang LT, Zhao ZF, Ji JQ, Li BH, Ji C. Intestinal endotoxemia plays a central role in development of hepatopulmonary syndrome in a cirrhotic rat model induced by multiple pathogenic factors. World J Gastroenterol 2007; 13:6385-95. [PMID: 18081228 PMCID: PMC4205458 DOI: 10.3748/wjg.v13.i47.6385] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To characterize the correlation between severity of hepatopulmonary syndrome (HPS) and degree of hepatic dysfunction, and to explore how intestinal endotoxemia (IETM) affects the development of HPS in cirrhotic rats.
METHODS: Male Wister rats were fed with a diet containing maize flour, lard, cholesterol, and alcohol and injected subcutaneously with CCl4 oil solution every two days for 8 wk to induce typical cirrhosis and development of HPS. The animals were also given a nitric oxide (NO) production inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME) intraperitoneally, and an iNOS inhibitor, aminoguanidine hydrochloride (AG) via gavage daily from the end of the 4th wk to the end of the 6th or 8th wk, or a HO-1 inhibitor, zinc protoporphyrin (ZnPP) intraperitoneally 12 h prior to killing. Blood, liver and lung tissues were sampled.
RESULTS: Histological deterioration of the lung paralleled to that of the liver in the cirrhotic rats. The number of pulmonary capillaries was progressively increased from 6.1 ± 1.1 (count/filed) at the 4th wk to 14.5 ± 2.4 (count/filed) at the 8th wk in the cirrhotic rats. Increased pulmonary capillaries were associated with increased blood levels of lipopolysaccharide (LPS) (0.31 ± 0.08 EU/mL vs control 0.09 ± 0.03 EU/mL), alanine transferase (ALT, 219.1 ± 17.4 U/L vs control 5.9 ± 2.2 U/L) and portal vein pressure. Compared with normal control animals, the number of total cells in bronchoalveolar lavage fluid (BALF) of the cirrhotic rats at the 8th wk was not changed, but the number of macrophages and the ratio of macrophages to total cells were increased by nearly 2-fold, protein expression of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) started to increase significantly at the 4th wk, and reached its peak at the 8th wk in the lung of cirrhotic rats. The increase of iNOS expression appeared to be quicker than that of eNOS. NO2-/NO3- was also increased, which was correlated to the increase of iNOS (r = 0.7699, P < 0.0001) and eNOS (r = 0.5829, P < 0.002). mRNA expression of eNOS and iNOS was highly consistent with their protein expression.
CONCLUSION: Progression and severity of HPS as indicated by both increased pulmonary capillaries and histological changes are closely associated with LPS levels and progression of hepatic dysfunction as indicated by increased levels of ALT and portal vein pressure. Intestinal endotoxemia plays a central role in the development of HPS in the cirrhotic rat model by inducing NO and/or CO.
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Takemori K, Kobayashi K, Sakamoto A. Expression of pulmonary vasoactive factors after sevoflurane anaesthesia in rats: a quantitative real-time polymerase chain reaction study. Br J Anaesth 2007; 100:190-4. [PMID: 18089613 DOI: 10.1093/bja/aem347] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
BACKGROUND Sevoflurane is a fluorinated volatile anaesthetic agent that lowers arterial pressure, in part by vasodilation. We previously showed, in rat lungs, that sevoflurane affected the expression of endothelin-1 (ET-1), a potent vasoconstrictor peptide. Therefore, we hypothesized that the vasodilation induced by sevoflurane involved vasodilatory and vasoconstrictor components. METHODS Rats were anaesthetized with sevoflurane 4% for 0, 2, and 6 h (n=9 each group) before death. In addition, a further group (n=9) were anaesthetized for 6 h then awoken for 2 h before death (n=9). We measured expression of mRNA encoding ET-1, nitric oxide synthase-1, 2, 3 (NOS1, 2, 3), haeme oxygenase-1, 2 (HO-1, 2), adrenomedullin (ADM), calcitonin gene-related peptide, vasoactive intestinal peptide, and prostacyclin synthase in whole lung using real-time reverse transcriptase-polymerase chain reaction. RESULTS Expressions of ET-1 and ADM were significantly increased by inhalation of sevoflurane for 2 and 6 h (P<0.05). Expression of NOS3 was significantly increased at 6 h (P<0.05). After awaking from anaesthesia, the expressions of NOS3, ET-1, and ADM returned to baseline levels. CONCLUSIONS Sevoflurane increased the expressions of ET-1, NOS3, and ADM. Our results suggest that the increased expressions of NOS3 and ADM may counteract that of ET-1 and so regulate pulmonary circulation under sevoflurane anaesthesia.
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Affiliation(s)
- K Takemori
- Department of Anaesthesiology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan.
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Snow JB, Kitzis V, Norton CE, Torres SN, Johnson KD, Kanagy NL, Walker BR, Resta TC. Differential effects of chronic hypoxia and intermittent hypocapnic and eucapnic hypoxia on pulmonary vasoreactivity. J Appl Physiol (1985) 2007; 104:110-8. [PMID: 17947499 DOI: 10.1152/japplphysiol.00698.2005] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension (PH) and right heart failure, similar to chronic sustained hypoxia (CH). Supplemental CO(2), however, attenuates hypoxic PH. We therefore hypothesized that, similar to CH, IH elicits PH and associated increases in arterial endothelial nitric oxide synthase (eNOS) expression, ionomycin-dependent vasodilation, and receptor-mediated pulmonary vasoconstriction. We further hypothesized that supplemental CO(2) inhibits these responses to IH. To test these hypotheses, we measured eNOS expression by Western blot in intrapulmonary arteries from CH (2 wk, 0.5 atm), hypocapnic IH (H-IH) (3 min cycles of 5% O(2)/air flush, 7 h/day, 2 wk), and eucapnic IH (E-IH) (3 min cycles of 5% O(2), 5% CO(2)/air flush, 7 h/day, 2 wk) rats and their respective controls. Furthermore, vasodilatory responses to the calcium ionophore ionomycin and vasoconstrictor responses to the thromboxane mimetic U-46619 were measured in isolated saline-perfused lungs from each group. Hematocrit, arterial wall thickness, and right ventricle-to-total ventricle weight ratios were additionally assessed as indexes of polycythemia, arterial remodeling, and PH, respectively. Consistent with our hypotheses, E-IH resulted in attenuated polycythemia, arterial remodeling, RV hypertrophy, and eNOS upregulation compared with H-IH. However, in contrast to CH, neither H-IH nor E-IH increased ionomycin-dependent vasodilation. Furthermore, H-IH and E-IH similarly augmented U-46619-induced pulmonary vasoconstriction but to a lesser degree than CH. We conclude that maintenance of eucapnia decreases IH-induced PH and upregulation of arterial eNOS. In contrast, increases in pulmonary vasoconstrictor reactivity following H-IH are unaltered by exposure to supplemental CO(2).
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Affiliation(s)
- Jessica B Snow
- Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131-0001, USA
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Abstract
Homeostasis in the pulmonary vasculature is maintained by the actions of vasoactive compounds, including nitric oxide (NO). NO is critical for normal development of the pulmonary vasculature and continues to mediate normal vasoregulation in adulthood. Loss of NO bioavailability is one component of the endothelial dysfunction and vascular pathology found in pulmonary hypertension (PH). A broad research effort continues to expand our understanding of the control of NO production and NO signaling and has generated novel theories on the importance of pulmonary NO production in the control of the systemic vasculature. This understanding has led to exciting developments in our ability to treat PH, including inhaled NO and phosphodiesterase inhibitors, and to several promising directions for future therapies using nitric oxide-donor compounds, stimulators of soluble guanylate cyclase, progenitor cells expressing NO synthase (NOS), and NOS gene manipulation.
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Affiliation(s)
- Matthew P Coggins
- Cardiology Division, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114, USA.
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Kirsch M, Kemp-Harper B, Weissmann N, Grimminger F, Schmidt HHHW. Sildenafil in hypoxic pulmonary hypertension potentiates a compensatory up‐regulation of NO‐cGMP signaling. FASEB J 2007; 22:30-40. [PMID: 17679609 DOI: 10.1096/fj.06-7526com] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
The availability of inhibitors of cGMP-specific phosphodiesterase 5 (PDE 5), such as sildenafil, has revolutionized the treatment of pulmonary hypertension (PH). Sildenafil may exert its protective effects in a mechanism-based fashion by targeting a pathophysiologically attenuated NO-cGMP signaling pathway. To elucidate this, we analyzed changes in the pulmonary expression and activity of key enzymes of NO-cGMP signaling as well as the functional pulmonary responses to sildenafil in the 5 or 21 day hypoxia mouse model of PH. Surprisingly, we found doubled NO synthase (NOS) II and III levels, no evidence for attenuated NO bioavailability as evidenced by the nitrosative/oxidative stress marker protein nitro tyrosine, and no changes in the expression and activity of the NO receptor, soluble guanylyl cyclase (sGC). PDE 5 was either unchanged at day 5 or, after 21 days of hypoxia, even significantly decreased along with unchanged activity. Biochemically, these changes were mirrored by increased cGMP spillover into the lung perfusate and cGMP-dependent phosphorylation of the vasodilator-stimulated phosphoprotein, VASP. Sildenafil further augmented cGMP and phospho-VASP levels in lungs of mice exposed for 5 or 21 days and decreased pulmonary arterial pressure in mice after 5 days but not 21 days of hypoxia. In conclusion, NO-cGMP signaling is compensatorily up-regulated in the hypoxic mouse model of PH, and sildenafil further augments this pathway to functionally alleviate pulmonary vasoconstriction.
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Affiliation(s)
- Mark Kirsch
- Rudolf-Buchheim-Institute of Pharmacology, Justus-Liebig-University Giessen, Germany
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Frederiksen LJ, Sullivan R, Maxwell LR, Macdonald-Goodfellow SK, Adams MA, Bennett BM, Siemens DR, Graham CH. Chemosensitization of cancer in vitro and in vivo by nitric oxide signaling. Clin Cancer Res 2007; 13:2199-206. [PMID: 17404104 DOI: 10.1158/1078-0432.ccr-06-1807] [Citation(s) in RCA: 107] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
PURPOSE Hypoxia contributes to drug resistance in solid cancers, and studies have revealed that low concentrations of nitric oxide (NO) mimetics attenuate hypoxia-induced drug resistance in tumor cells in vitro. Classic NO signaling involves activation of soluble guanylyl cyclase, generation of cyclic GMP (cGMP), and activation of cGMP-dependent protein kinase. Here, we determined whether chemosensitization by NO mimetics requires cGMP-dependent signaling and whether low concentrations of NO mimetics can chemosensitize tumors in vivo. EXPERIMENTAL DESIGN Survival of human prostate and breast cancer cells was assessed by clonogenic assays following exposure to chemotherapeutic agents. The effect of NO mimetics on tumor chemosensitivity in vivo was determined using a mouse xenograft model of human prostate cancer. Drug efflux in vitro was assessed by measuring intracellular doxorubicin-associated fluorescence. RESULTS Low concentrations of the NO mimetics glyceryl trinitrate (GTN) and isosorbide dinitrate attenuated hypoxia-induced resistance to doxorubicin and paclitaxel. Similar to hypoxia-induced drug resistance, inhibition of various components of the NO signaling pathway increased resistance to doxorubicin, whereas activation of the pathway with 8-bromo-cGMP attenuated hypoxia-induced resistance. Drug efflux was unaffected by hypoxia and inhibitors of drug efflux did not significantly attenuate hypoxia-induced chemoresistance. Compared with mice treated with doxorubicin alone, tumor growth was decreased in mice treated with doxorubicin and a transdermal GTN patch. The presence of GTN and GTN metabolites in plasma samples was confirmed by gas chromatography. CONCLUSION Tumor hypoxia induces resistance to anticancer drugs by interfering with endogenous NO signaling and reactivation of NO signaling represents a novel approach to enhance chemotherapy.
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Teshfam M, Brujeni GN, Hassanpour H. Evaluation of endothelial and inducible nitric oxide synthase mRNA expression in the lung of broiler chickens with developmental pulmonary hypertension due to cold stress. Br Poult Sci 2006; 47:223-9. [PMID: 16641034 DOI: 10.1080/00071660600611169] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
Abstract
To clarify the effect of cold-induced pulmonary hypertension on endothelial and inducible nitric oxide synthase (eNOS and iNOS) mRNA expression in the lung of broiler chickens, semi quantitative reverse transcription-PCR was performed on total RNAs isolated from lungs of the broiler chickens exposed to 5 weeks of cold stress. The eNOS gene was expressed increasingly with the increasing age during the rearing period. Comparing the treatment group with its related control group eNOS was expressed significantly only at d 21. Expression of iNOS mRNA also increased in both control and treatment groups with increasing age until d 28 and then decreased at d 35 and 42. Comparing the treatment group with its control group, iNOS mRNA level was significantly higher at 21 d of age in the cold-exposed chickens. It was concluded that, although cold exposure could significantly increase eNOS and iNOS gene expression, cold-induced pulmonary hypertension is not associated with significant variations of eNOS and iNOS expression in the lungs of broiler chickens.
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Affiliation(s)
- M Teshfam
- Department of Physiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
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35
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Maxey TS, Fernandez LG, Reece TB, Keeling WB, Kron IL, Laubach VE. Endothelial nitric oxide synthase is essential for postpneumonectomy compensatory vasodilation. Ann Thorac Surg 2006; 81:1234-8. [PMID: 16564249 DOI: 10.1016/j.athoracsur.2005.11.049] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/27/2005] [Revised: 11/18/2005] [Accepted: 11/28/2005] [Indexed: 11/16/2022]
Abstract
BACKGROUND After pneumonectomy, the remaining lung vasculature must vasodilate to compensate for increased blood volume. We hypothesized that endothelial nitric oxide synthase (eNOS) is essential for compensatory vasodilation after pneumonectomy. METHODS Adult, wild-type C57BL6 (WT) and eNOS knockout (eNOS-/-) mice underwent left pneumonectomy and recovered under normoxic conditions. Animals were lightly anesthetized at 1, 3, 7, or 14 days after pneumonectomy, and closed chest, systolic right ventricular pressure (RVP) was recorded using fine-needle cannulation. The right ventricle to left ventricle plus septum weight ratios were measured as an index of right ventricular hypertrophy. Two additional groups of mice (WT and eNOS-/-) were recovered after pneumonectomy in inhaled nitric oxide (iNO, 10 ppm), and RVP was measured on day 7. RESULTS The eNOS-/- mice had significantly higher preoperative RVP than did WT (17.1 +/- 0.4 versus 14.2 +/- 0.2 cmH2O, p = 0.001). Both groups exhibited transient periods of pulmonary hypertension after pneumonectomy. On day 1, RVP was 80% above baseline in eNOS-/- mice (30.7 +/- 0.8 cmH2O) versus 42% in WT mice (20.2 +/- 0.7 cmH2O, p = 0.0001). The RVP returned to baseline in WT mice (16.3 +/- 0.2 cmH2O) but remained significantly elevated in eNOS-/- mice (28.6 +/- 0.9 cmH2O) at day 3 and at each time thereafter (p = 0.0001). The iNO significantly reduced RVP in eNOS-/- animals to 15.2 +/- 0.3 cmH2O (p = 0.0001) while having no effect in WT animals. Right ventricular hypertrophy was not observed in any group. CONCLUSIONS Pneumonectomy results in a transient increase in RVP. Under normal circumstances, these pressures return to baseline within 3 days. The eNOS-/- mice failed to display compensatory vasodilation yet could be rescued with iNO. These results suggest that eNOS is essential for postpneumonectomy compensatory vasodilation.
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Affiliation(s)
- Thomas S Maxey
- Department of Surgery, University of South Florida, Tampa, Florida, USA
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Sehara Y, Hayashi T, Deguchi K, Nagotani S, Zhang H, Shoji M, Abe K. Distribution of inducible nitric oxide synthase and cell proliferation in rat brain after transient middle cerebral artery occlusion. Brain Res 2006; 1093:190-7. [PMID: 16701577 DOI: 10.1016/j.brainres.2006.03.092] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2005] [Revised: 03/13/2006] [Accepted: 03/16/2006] [Indexed: 11/20/2022]
Abstract
Nitric oxide (NO) can be neuroprotective or neurotoxic during cerebral ischemia, depending on the NO synthase (NOS) isoform involved. In addition to neurotoxic effect in ischemic brain, inducible NOS (iNOS) also adversely affect ischemic outcome by blocking neurogenesis. In the present study, therefore, we studied the chronological and spatial change of the distribution of iNOS and cell proliferation in subventricular zone (SVZ) after transient focal cerebral ischemia. After 90 min of transient middle cerebral artery occlusion (tMCAO), iNOS-positive cells decreased in the ischemic core at 1 to 21 days, and increased in the ipsilateral periischemic area at 1 and 3 days. 5-Bromodeoxyuridine (BrdU)-positive cells appeared in the ischemic core at 3 to 21 days, appeared in the periischemic area at 3 and 7 days, and increased in the ipsilateral SVZ at 7 days. ED-1-positive cells appeared in the ischemic core at 3 to 21 days, and some of them were double positive with BrdU or iNOS, but the majority were BrdU-negative. The present study suggests that astrocytes are born within the periischemic area at early stage after tMCAO and migrate from SVZ into periischemic area at later stage, and that time-dependent and spatial changes of iNOS expression may be involved in the proliferation and differentiation of adult neurogenesis after focal cerebral ischemia.
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Affiliation(s)
- Yoshihide Sehara
- Department of Neurology Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
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Searles CD. Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression. Am J Physiol Cell Physiol 2006; 291:C803-16. [PMID: 16738003 DOI: 10.1152/ajpcell.00457.2005] [Citation(s) in RCA: 141] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The ability of the endothelium to produce nitric oxide is essential to maintenance of vascular homeostasis; disturbance of this ability is a major contributor to the pathogenesis of vascular disease. In vivo studies have demonstrated that expression of endothelial nitric oxide synthase (eNOS) is vital to endothelial function and have led to the understanding that eNOS expression is subject to modest but significant degrees of regulation. Subsequently, numerous physiological and pathophysiological stimuli have been identified that modulate eNOS expression via mechanisms that alter steady-state eNOS mRNA levels. These mechanisms involve changes in the rate of eNOS gene transcription (transcriptional regulation) and alteration of eNOS mRNA processing and stability (posttranscriptional regulation). In cultured endothelial cells, shear stress, transforming growth factor-beta1, lysophosphatidylcholine, cell growth, oxidized linoleic acid, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, and hydrogen peroxide have been shown to increase eNOS expression. In contrast, tumor necrosis factor-alpha, hypoxia, lipopolysaccaride, thrombin, and oxidized LDL can decrease eNOS mRNA levels. For many of these stimuli, both transcriptional and posttranscriptional mechanisms contribute to regulation of eNOS expression. Recent studies have begun to further define signaling pathways responsible for changes in eNOS expression and have characterized cis- and trans-acting regulatory elements. In addition, a role has been identified for epigenetic control of eNOS mRNA levels. This review will discuss transcriptional and posttranscriptional regulation of eNOS with emphasis on the molecular mechanisms that have been identified for these processes.
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Affiliation(s)
- Charles D Searles
- Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
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Min J, Jin YM, Moon JS, Sung MS, Jo SA, Jo I. Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells. Hypertension 2006; 47:1189-96. [PMID: 16651461 DOI: 10.1161/01.hyp.0000222892.37375.4d] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (-1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(-1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36+/-0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(-1600). However, progressive 5'-deletion from -1600 to -873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element-like site, located at -924 to -921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression.
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Affiliation(s)
- Jiho Min
- Department of Biomedical Sciences, National Institute of Health, 194 Tongilo, Eunpyeong-gu, Seoul 122-701, Korea
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Dudzinski DM, Igarashi J, Greif D, Michel T. The regulation and pharmacology of endothelial nitric oxide synthase. Annu Rev Pharmacol Toxicol 2006; 46:235-76. [PMID: 16402905 DOI: 10.1146/annurev.pharmtox.44.101802.121844] [Citation(s) in RCA: 287] [Impact Index Per Article: 15.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Nitric oxide (NO) is a small, diffusible, lipophilic free radical gas that mediates significant and diverse signaling functions in nearly every organ system in the body. The endothelial isoform of nitric oxide synthase (eNOS) is a key source of NO found in the cardiovascular system. This review summarizes the pharmacology of NO and the cellular regulation of endothelial NOS (eNOS). The molecular intricacies of the chemistry of NO and the enzymology of NOSs are discussed, followed by a review of the biological activities of NO. This information is then used to develop a more global picture of the pharmacological control of NO synthesis by NOSs in both physiologic conditions and pathophysiologic states.
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Affiliation(s)
- David M Dudzinski
- Cardiovascular Division, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA
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40
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Ricart K, J Pearson R, Viera L, Cassina P, Kamaid A, Carroll SL, Estévez AG. Interactions between beta-neuregulin and neurotrophins in motor neuron apoptosis. J Neurochem 2006; 97:222-33. [PMID: 16524373 DOI: 10.1111/j.1471-4159.2006.03739.x] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Neuregulins play a major role in the formation and stabilization of neuromuscular junctions, and are produced by both motor neurons and muscle. Although the effects and mechanism of neuregulins on skeletal muscle (e.g. regulation of acetylcholine receptor expression) have been studied extensively, the effects of neuregulins on motor neurons remain unknown. We report that neuregulin-1beta (NRGbeta1) inhibited apoptosis of rat motor neurons for up to 7 days in culture by a phosphatidylinositol 3 kinase-dependent pathway and synergistically enhanced motor neuron survival promoted by glial-derived neurotrophic factor (GDNF). However, binding of neurotrophins, including brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), to the p75 neurotrophin receptor (p75NTR) abolished the neuregulin anti-apoptotic effect on motor neurons. Inhibitors of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase prevented motor neuron death caused by co-incubation of NRGbeta1 and BDNF or NGF, as well as by trophic factor deprivation. Motor neuron apoptosis resulting from both trophic factor deprivation and exposure to NRGbeta1 plus neurotrophins required the induction of neuronal nitric oxide synthase and peroxynitrite formation. Because motor neurons express both p75NTR and neuregulin erbB receptors during the period of embryonic programmed cell death, motor neuron survival may be the result of complex interactions between trophic and death factors, which may be the same molecules acting in different combinations.
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Affiliation(s)
- Karina Ricart
- Department of Physiology and Biophysics, The University of Alabama at Birmingham, Birmingham, Alabama, USA
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Ward ME, Toporsian M, Scott JA, Teoh H, Govindaraju V, Quan A, Wener AD, Wang G, Bevan SC, Newton DC, Marsden PA. Hypoxia induces a functionally significant and translationally efficient neuronal NO synthase mRNA variant. J Clin Invest 2006; 115:3128-39. [PMID: 16276418 PMCID: PMC1265848 DOI: 10.1172/jci20806] [Citation(s) in RCA: 91] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2003] [Accepted: 08/30/2005] [Indexed: 11/17/2022] Open
Abstract
We tested the hypothesis that induction of neuronal NO synthase (nNOS) impairs vascular smooth muscle contractility after hypoxia. nNOS protein was increased in aorta, mesenteric arterioles, pulmonary arteries, brain, and diaphragm from rats exposed to 8% O2 for 48 hours and in human aortic SMCs after hypoxic incubation (1% O2). Ca-dependent NO synthase activity was increased in endothelium-denuded aortic segments from hypoxia-exposed rats. N-nitro-L-arginine methyl ester enhanced the contractile responses of endothelium-denuded aortic rings and mesenteric arterioles from hypoxia-exposed but not normoxic rats (P < 0.05). The hypoxia-inducible mRNA transcript expressed by human cells was found to contain a novel 5'-untranslated region, consistent with activation of transcription in the genomic region contiguous with exon 2. Translational efficiency of this transcript is markedly increased compared with previously described human nNOS mRNAs. Transgenic mice possessing a lacZ reporter construct under control of these genomic sequences demonstrated expression of the construct after exposure to hypoxia (8% O2, 48 hours) in the aorta, mesenteric arterioles, renal papilla, and brain. These results reveal a novel human nNOS promoter that confers the ability to rapidly upregulate nNOS expression in response to hypoxia with a functionally significant effect on vascular smooth muscle contraction.
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Affiliation(s)
- Michael E Ward
- Division of Respirology, University of Toronto, Toronto, Ontario, Canada.
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42
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Jiang L, Quarck R, Janssens S, Pokreisz P, Demedts M, Delcroix M. Effect of adenovirus-mediated gene transfer of nitric oxide synthase on vascular reactivity of rat isolated pulmonary arteries. Pflugers Arch 2006; 452:213-21. [PMID: 16404609 DOI: 10.1007/s00424-005-0028-3] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2005] [Revised: 10/03/2005] [Accepted: 11/18/2005] [Indexed: 11/25/2022]
Abstract
Aerosol gene transfer of endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) to rat lungs increased NOS expression and activity, and prevented hypoxic pulmonary vasoconstriction (HPV) in vivo. Hereby, we examined the effect of eNOS and iNOS aerosol gene transfer on the endothelium-dependent relaxation (EDR) and on acute HPV in isolated rat pulmonary arteries. Changes in isometric forces were recorded in organ baths for large conduit arteries (diameter 1.8+/-0.1 mm) and in a wire myograph for small resistance arteries (258+/-35 microm). Male Wistar rats were randomly aerosolized with adenovirus (Ad) encoding beta-galactosidase (control), eNOS, or iNOS. Four days later, exhaled nitric oxide was measured, NOS expression within rat lungs was evaluated by quantitative real-time polymerase chain reaction and immunohistochemistry, vasoconstricting agonist and acetylcholine concentration response curves were generated, and the time course of HPV was recorded. Human eNOS and murine iNOS were expressed within rat lung tissue mostly in parenchyma and endothelial cells. Large arteries isolated from Ad-i, eNOS-aerosolized rats developed lower agonist-induced tension than those of control rats. The first and second contractions of the HPV were smaller in the Ad-i, eNOS-aerosolized rats. Contractions were modestly, but significantly and inversely, related to exhaled NO. Agonist- and hypoxia-induced contractions were even more reduced after eNOS aerosolization. There was no significant effect on EDR and no notable difference between small and large vessels. We conclude that adenovirus (Ad)-mediated NOS gene transfer can counteract both pharmacologically and hypoxia-induced increases in pulmonary vascular tone in isolated rat pulmonary arteries. eNOS seems as efficient as iNOS in regulating pulmonary vascular tone.
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Affiliation(s)
- Leifu Jiang
- Department of Pneumology, University Hospital Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium
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Guix FX, Uribesalgo I, Coma M, Muñoz FJ. The physiology and pathophysiology of nitric oxide in the brain. Prog Neurobiol 2005; 76:126-52. [PMID: 16115721 DOI: 10.1016/j.pneurobio.2005.06.001] [Citation(s) in RCA: 480] [Impact Index Per Article: 24.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2005] [Revised: 06/10/2005] [Accepted: 06/14/2005] [Indexed: 12/11/2022]
Abstract
Nitric oxide (NO) is a molecule with pleiotropic effects in different tissues. NO is synthesized by NO synthases (NOS), a family with four major types: endothelial, neuronal, inducible and mitochondrial. They can be found in almost all the tissues and they can even co-exist in the same tissue. NO is a well-known vasorelaxant agent, but it works as a neurotransmitter when produced by neurons and is also involved in defense functions when it is produced by immune and glial cells. NO is thermodynamically unstable and tends to react with other molecules, resulting in the oxidation, nitrosylation or nitration of proteins, with the concomitant effects on many cellular mechanisms. NO intracellular signaling involves the activation of guanylate cyclase but it also interacts with MAPKs, apoptosis-related proteins, and mitochondrial respiratory chain or anti-proliferative molecules. It also plays a role in post-translational modification of proteins and protein degradation by the proteasome. However, under pathophysiological conditions NO has damaging effects. In disorders involving oxidative stress, such as Alzheimer's disease, stroke and Parkinson's disease, NO increases cell damage through the formation of highly reactive peroxynitrite. The paradox of beneficial and damaging effects of NO will be discussed in this review.
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Affiliation(s)
- F X Guix
- Laboratori de Fisiologia Molecular, Unitat de Senyalització Cellular, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Carrer Dr. Aiguader, 80, Barcelona 08003, Spain
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Jernigan NL, Broughton BRS, Walker BR, Resta TC. Impaired NO-dependent inhibition of store- and receptor-operated calcium entry in pulmonary vascular smooth muscle after chronic hypoxia. Am J Physiol Lung Cell Mol Physiol 2005; 290:L517-25. [PMID: 16243900 DOI: 10.1152/ajplung.00308.2004] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
We have recently demonstrated that chronic hypoxia (CH) attenuates nitric oxide (NO)-mediated decreases in pulmonary vascular smooth muscle (VSM) intracellular free calcium concentration ([Ca2+]i) and promotes NO-dependent VSM Ca2+ desensitization. The objective of the current study was to identify potential mechanisms by which CH interferes with regulation of [Ca2+]i by NO. We hypothesized that CH impairs NO-mediated inhibition of store-operated (capacitative) Ca2+ entry (SOCE) or receptor-operated Ca2+ entry (ROCE) in pulmonary VSM. To test this hypothesis, we examined effects of the NO donor, spermine NONOate, on SOCE resulting from depletion of intracellular Ca2+ stores with cyclopiazonic acid, and on UTP-induced ROCE in isolated, endothelium-denuded, pressurized pulmonary arteries (213 +/- 8 microm inner diameter) from control and CH (4 wk at 0.5 atm) rats. Arteries were loaded with fura-2 AM to continuously monitor VSM [Ca2+]i. We found that the change in [Ca2+]i associated with SOCE and ROCE was significantly reduced in vessels from CH animals. Furthermore, spermine NONOate diminished SOCE and ROCE in vessels from control, but not CH animals. We conclude that NO-mediated inhibition of SOCE and ROCE is impaired after CH-induced pulmonary hypertension.
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MESH Headings
- Animals
- Bronchodilator Agents/pharmacology
- Calcium/metabolism
- Calcium Channels/drug effects
- Calcium Channels/metabolism
- Calcium-Transporting ATPases/antagonists & inhibitors
- Chronic Disease
- Endothelium, Vascular/metabolism
- Enzyme Inhibitors/pharmacology
- Hypertension, Pulmonary/metabolism
- Hypertension, Pulmonary/pathology
- Hypoxia/metabolism
- Indoles/pharmacology
- Male
- Muscle, Smooth, Vascular/cytology
- Muscle, Smooth, Vascular/drug effects
- Muscle, Smooth, Vascular/metabolism
- Nitric Oxide/pharmacology
- Nitric Oxide Donors/pharmacology
- Nitrogen Oxides/pharmacology
- Pulmonary Artery/cytology
- Pulmonary Artery/drug effects
- Pulmonary Artery/metabolism
- Rats
- Rats, Sprague-Dawley
- Spermine/analogs & derivatives
- Spermine/pharmacology
- Uridine Triphosphate/pharmacology
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Affiliation(s)
- Nikki L Jernigan
- Vascular Physiology Group, Dept. of Cell Biology and Physiology, Univ. of New Mexico Health Sciences Center, MSC 08-4750, 1 Univ. of New Mexico, Albuquerque, NM 87131-0001, USA
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Murata T, Kinoshita K, Hori M, Kuwahara M, Tsubone H, Karaki H, Ozaki H. Statin protects endothelial nitric oxide synthase activity in hypoxia-induced pulmonary hypertension. Arterioscler Thromb Vasc Biol 2005; 25:2335-42. [PMID: 16166567 DOI: 10.1161/01.atv.0000186184.33537.48] [Citation(s) in RCA: 90] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
OBJECTIVE We investigated the effects of fluvastatin on hypoxia-induced (1 to 3 weeks, 10% O2) pulmonary hypertension with focus on endothelial nitric oxide synthase (eNOS) activity. METHODS AND RESULTS Oral fluvastatin treatment (1 mg/kg daily) prevented the causing and progression of pulmonary hypertension as determined by the right ventricular pressure, right ventricular hypertrophy, and muscularization of pulmonary artery. We also revealed that fluvastatin treatments prevented the hypoxia-induced decrease in cGMP production in the rat lung and restored the endothelium-dependent relaxation in the pulmonary artery. We revealed that this beneficial effect was not dependent on the increase in eNOS mRNA or protein expression, but was dependent on the inhibition of the eNOS-tight coupling with caveolin-1, the eNOS dissociation from heat shock protein 90, and the decrease in eNOS Ser1177-phosphorylation induced by hypoxia. Furthermore, in a whole-mount immunostaining the hypoxia-induced eNOS protein condensation with caveolin-1 of pulmonary endothelial cells was restored by the fluvastatin-treatment. CONCLUSIONS These results suggest that the fluvastatin exerts beneficial effects on chronic hypoxia-induced pulmonary hypertension by protecting against the eNOS activity at the post-transcriptional level.
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Affiliation(s)
- Takahisa Murata
- Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.
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Wei IH, Huang CC, Chang HM, Tseng CY, Tu HC, Wen CY, Shieh JY. Neuronal NADPH-d/NOS expression in the nodose ganglion of severe hypoxic rats with or without mild hypoxic preconditioning. J Chem Neuroanat 2005; 29:149-56. [PMID: 15652701 DOI: 10.1016/j.jchemneu.2004.11.001] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2004] [Revised: 09/20/2004] [Accepted: 11/22/2004] [Indexed: 12/25/2022]
Abstract
This study aimed to test the hypothesis that mild hypoxic preconditioning (MHPC)-induced NOS expression would attenuate the neuropathological changes in the nodose ganglion (NG) of severe hypoxic exposure (SHE) rats. Thus, the young adult rats were caged in the altitude chamber for 4 weeks prior to SHE for 4 h to gain hypoxic preconditioning. The altitude chamber was used to set the height at the level from 5500 m (0.50 atm; pO2=79 Torr) to 10,000 m (0.27 atm; pO2=43 Torr) for MHPC and SHE, respectively. The experimental animals were allowed to survive for 0, 7, 14, 30 and 60 successive days, respectively. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry were used to detect NADPH-d/nNOS reactivity in the NG at various time points following hypoxic exposure. The present results showed that about 38% of the neurons in the NG displayed NADPH-d/nNOS positive [NADPH-d/nNOS(+)] in normoxic rats. In SHE rats, a peak in the percentage (71%) and staining intensity (230%) of NADPH-d/nNOS(+) nodose neurons at 0 day, which then gradually decreased at 7-60 days. About 25% of the nodose neurons died 60 days after SHE. However, in MHPC rats subjected to SHE, NADPH-d/nNOS(+) neurons peaked in the percentage (51%) and staining intensity (171%) at 0 day, which then decreased at 7-60 days. In addition, neuronal survival was markedly increased by MHPC. These results suggested that MHPC might have a neuroprotective effect that reduces the susceptibility of the nodose neurons to NOS mediated neuropathy subsequent to SHE.
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Affiliation(s)
- I-Hua Wei
- Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei, Taiwan
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Rodnenkov OV, Luneva OG, Ulyanova NA, Maksimov GV, Rubin AB, Orlov SN, Chazov EI. Erythrocyte membrane fluidity and haemoglobin haemoporphyrin conformation: features revealed in patients with heart failure. ACTA ACUST UNITED AC 2005; 11:209-213. [PMID: 15837166 DOI: 10.1016/j.pathophys.2004.12.001] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2004] [Revised: 12/20/2004] [Accepted: 12/21/2004] [Indexed: 10/25/2022]
Abstract
This study examined the possible involvement of abnormal erythrocyte oxygen (O(2)) transport in the pathogenesis of heart failure. Haemoglobin (Hb) haemoporphyrin conformation was assessed by Raman spectroscopy (RS) of blood samples, whereas membrane fluidity was estimated at depths of 0.6-0.8 and 2.2nm by electron-paramagnetic resonance spectroscopy of erythrocytes loaded with spin-labeled 5-doxylstearic acid and 16-doxylstearic acid, respectively. The fluidity of erythrocyte membranes from patients with heart failure was decreased in the area near the membrane surface and remained unchanged in the deeper hydrophobic membrane regions. The same differences were also detected in healthy controls subjected to chronic high-altitude hypoxia. RS demonstrated that in heart failure the total content of Hb-ligand complexes and the relative content of Hb-nitric oxide (NO) complexes with cleaved Fe(2+)-globin bond was decreased, whereas content of Hb-NO complexes with preserved Fe(2+)-globin bond was increased. We propose that this phenomenon contributes to the reduced O(2) tissue supply seen in patients with heart failure.
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Affiliation(s)
- O V Rodnenkov
- A.L. Myasnikov Institute of Clinical Cardiology, Russian Cardiological Research Complex, Moscow, Russia
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48
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Gao W, Gao Y, Zhang G, Song L, Sun B, Shi J. Hypoxia-induced expression of HIF-1alpha and its target genes in umbilical venous endothelial cells of Tibetans and immigrant Han. Comp Biochem Physiol C Toxicol Pharmacol 2005; 141:93-100. [PMID: 15970466 DOI: 10.1016/j.cca.2005.05.006] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/13/2005] [Revised: 05/07/2005] [Accepted: 05/09/2005] [Indexed: 10/25/2022]
Abstract
The better adaptation of native Tibetans to hypoxia is thought to be partly due to improved umbilical circulation, which results in reduced pre- and postnatal fatalities. We hypothesized that the difference in umbilical circulation between native Tibetans and other high-altitude inhabitants was due to differences in the expression of hypoxia-induced factor (HIF-1) and its target genes vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). We tested this hypothesis by examining the effect of hypoxia on the expression of HIF-1alpha, VEGF, and iNOS in cultured umbilical venous endothelial cells (UVECs) from native Tibetans and immigrant Hans. UVECs were collected and cultured under hypoxic (0.5% oxygen) or normoxic conditions for 2, 4, 12 and 24 h. The mRNA levels of HIF-1alpha, VEGF, endothelial nitric oxide synthase (eNOS) and iNOS and the protein level of HIF-1alpha were determined with RT-PCR and Western blot analyses, respectively. In both immigrant Han and Tibetans, HIF-1alpha mRNA was constitutively expressed under normoxic condition, and remained constant after hypoxic exposure. In contrast, HIF-1alpha protein was undetectable under normoxic condition, but underwent dynamic changes in response to hypoxia. It was induced at 4 h, peaked at 12 h, and remained elevated at 24 h. Concurrent with the induction of HIF-1alpha protein, the mRNA levels of VEGF and iNOS were also up-regulated whereas that of eNOS was down-regulated. The lack of a hypoxia-related difference in the expression of HIF-1alpha and its target genes suggests that HIF-1alpha does not play a critical role in high altitude adaptation. Alternative mechanisms may be responsible for the better adaptation of native Tibetans.
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Affiliation(s)
- Wenxiang Gao
- Department of Pathophysiology and Institute of High Altitude Medicine, Third Military Medical University, 30 Gao Tan Yan, Shapingba, Chongqing 400038, PR China
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Liu R, Evgenov OV, Ichinose F. NOS3 deficiency augments hypoxic pulmonary vasoconstriction and enhances systemic oxygenation during one-lung ventilation in mice. J Appl Physiol (1985) 2005; 98:748-52. [PMID: 15465885 DOI: 10.1152/japplphysiol.00820.2004] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Nitric oxide (NO), synthesized by NO synthases (NOS), plays a pivotal role in regulation of pulmonary vascular tone. To examine the role of endothelial NOS (NOS3) in hypoxic pulmonary vasoconstriction (HPV), we measured left lung pulmonary vascular resistance (LPVR), intrapulmonary shunting, and arterial Po2 (PaO2) before and during left mainstem bronchus occlusion (LMBO) in mice with and without a deletion of the gene encoding NOS3. The increase of LPVR induced by LMBO was greater in NOS3-deficient mice than in wild-type mice (151 ± 39% vs. 109 ± 36%, mean ± SD; P < 0.05). NOS3-deficient mice had a lower intrapulmonary shunt fraction than wild-type mice (17.1 ± 3.6% vs. 21.7 ± 2.4%, P < 0.05) during LMBO. Both real-time PaO2 monitoring with an intra-arterial probe and arterial blood-gas analysis during LMBO showed higher PaO2 in NOS3-deficient mice than in wild-type mice ( P < 0.05). Inhibition of all three NOS isoforms with Nω-nitro-l-arginine methyl ester (l-NAME) augmented the increase of LPVR induced by LMBO in wild-type mice (183 ± 67% in l-NAME treated vs. 109 ± 36% in saline treated, P < 0.01) but not in NOS3-deficient mice. Similarly, systemic oxygenation during one-lung ventilation was augmented by l-NAME in wild-type mice but not in NOS3-deficient mice. These findings indicate that NO derived from NOS3 modulates HPV in vivo and that inhibition of NOS3 improves systemic oxygenation during acute unilateral lung hypoxia.
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Affiliation(s)
- Rong Liu
- Dept. of Anesthesia and Critical Care, Massachusetts General Hospital, 55 Fruit St., Boston, MA 02114, USA
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Abstract
Normal tissue function depends on adequate supply of oxygen through blood vessels. Reduced oxygen supply (hypoxia) induces a variety of specific adaptation mechanisms in mammals that occur at the cellular, local and systemic level. These mechanisms are in part governed by the activation of the hypoxia-inducible transcription factors HIF-1 and HIF-2. Prolyl and asparaginyl hydroxylases as recently characterized oxygen sensors allow the regulation of HIFs that in turn modulate expression of hypoxically regulated genes such as VEGF. VEGF plays a key role in the formation of a functional and integrated vascular network required during physiological processes such as embryogenesis or female reproductive cycle as well as during a variety of pathological processes such tumor growth, wound healing, retinopathy and ischemic diseases (myocardial infarction, cerebral ischemia). However, other angiogenic factors, such as angiopoietins, PDGF, ephrins and erythropoietin are additionally needed to enable the formation of a functional vascular network. Many of these factors are activated during hypoxia although no HIF binding sites have yet been identified in the regulatory sequences of theses genes. Hypoxia-induced gene products that result in new vessel growth may be part of a self-regulated physiological protection mechanism preventing cell injury, especially under conditions of chronically reduced blood blow (chronic ischemia).
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Affiliation(s)
- Hugo H Marti
- Institute of Physiology and Pathophysiology, University of Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany.
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