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1.  In vivo hypoxic preconditioning protects from warm liver ischemic/reperfusion injury through the adenosine A2B receptor 
Transplantation  2012;94(9):894-902.
BACKGROUND
Liver ischemia(I)/reperfusion(R) injury(I) is a known risk factor for the postoperative outcome of patients undergoing liver surgery/transplantation. Attempts to protect from organ damage require multidisciplinary strategies and are of emerging interest in view of patients with higher age and ASA-status. Ischemic preconditioning has been successfully applied to prevent from IRI during liver resections/transplantation. Since even short periods of ischemia during preconditioning inevitably lead to hypoxia and formation of anti-inflammatory/ cytoprotective acting adenosine, we reasoned that short non-ischemic hypoxia also protects against hepatic IRI.
METHODS
Mice underwent hypoxic preconditioning(HPC) by breathing 10%-oxygen for 10 minutes, followed by 10 minutes of 21%-oxygen prior to left-liver-lobe-ischemia(45 min) and reperfusion(4 hrs). The interactions of hypoxia->adenosine->adenosine-receptors were tested by pharmacologic antagonism at adenosine receptor(AR) sites in wild type mice and in mice with genetic deletions at the A1-;A2A-;A2B- and A3-ARs. Hepatocellular damage, inflammation and metabolic effects were quantified by enzyme activities, cytokines, liver-myeloperoxidase(MPO), blood adenosine and tissue-adenosinemonophosphate(AMP), respectively.
RESULTS
Hepatoprotection by HPC was significant in wild type and A1-, A2A-and A3 AR-knock-out mice as quantified by lower ALT serum activities, cytokine levels, histological damage-scores, tissue-myeloperoxidase-concentrations and as well as preserved AMP-concentrations. Protection by HPC was blunted in mice pretreated with the A2B-AR-antagonist MRS1754 or in A2B-AR“knock-outs”.
CONCLUSION
Because liver protective effects of HPC are negated when the A2B receptor is non-functional, the "hypoxia->adenosine->A2B receptor" pathway plays a critical role in the prevention of warm ischemia reperfusion injury in vivo. Hypoxic activation of this pathway warrants use of selective A2B-AR-agonists or even intermittent hypoxia (e.g. in deceased organ donors) to protect from liver ischemia/reperfusion injury.
doi:10.1097/TP.0b013e31826a9a46
PMCID: PMC3491139  PMID: 23073466
hypoxia; murine liver ischemia; preconditioning; hepatoprotection
2.  Oxygenation Inhibits the Physiological Tissue-Protecting Mechanism and Thereby Exacerbates Acute Inflammatory Lung Injury 
PLoS Biology  2005;3(6):e174.
Acute respiratory distress syndrome (ARDS) usually requires symptomatic supportive therapy by intubation and mechanical ventilation with the supplemental use of high oxygen concentrations. Although oxygen therapy represents a life-saving measure, the recent discovery of a critical tissue-protecting mechanism predicts that administration of oxygen to ARDS patients with uncontrolled pulmonary inflammation also may have dangerous side effects. Oxygenation may weaken the local tissue hypoxia-driven and adenosine A2A receptor (A2AR)-mediated anti-inflammatory mechanism and thereby further exacerbate lung injury. Here we report experiments with wild-type and adenosine A2AR-deficient mice that confirm the predicted effects of oxygen. These results also suggest the possibility of iatrogenic exacerbation of acute lung injury upon oxygen administration due to the oxygenation-associated elimination of A2AR-mediated lung tissue-protecting pathway. We show that this potential complication of clinically widely used oxygenation procedures could be completely prevented by intratracheal injection of a selective A2AR agonist to compensate for the oxygenation-related loss of the lung tissue-protecting endogenous adenosine. The identification of a major iatrogenic complication of oxygen therapy in conditions of acute lung inflammation attracts attention to the need for clinical and epidemiological studies of ARDS patients who require oxygen therapy. It is proposed that oxygen therapy in patients with ARDS and other causes of lung inflammation should be combined with anti-inflammatory measures, e.g., with inhalative application of A2AR agonists. The reported observations may also answer the long-standing question as to why the lungs are the most susceptible to inflammatory injury and why lung failure usually precedes multiple organ failure.
A mouse model suggests that oxygen therapy may exacerbate lung injury by weakening the anti-inflammatory mechanisms driven by hypoxia.
doi:10.1371/journal.pbio.0030174
PMCID: PMC1088279  PMID: 15857155
3.  Hypoxia-preconditioned mesenchymal stem cells attenuate bleomycin-induced pulmonary fibrosis 
Introduction
Idiopathic pulmonary fibrosis is a progressive diffuse parenchymal lung disorder of unknown etiology. Mesenchymal stem cell (MSC)-based therapy is a novel approach with great therapeutic potential for the treatment of lung diseases. Despite demonstration of MSC grafting, the populations of engrafted MSCs have been shown to decrease dramatically 24 hours post-transplantation due to exposure to harsh microenvironments. Hypoxia is known to induce expression of cytoprotective genes and also secretion of anti-inflammatory, anti-apoptotic and anti-fibrotic factors. Hypoxic preconditioning is thought to enhance the therapeutic potency and duration of survival of engrafted MSCs. In this work, we aimed to prolong the duration of survival of engrafted MSCs and to enhance the effectiveness of idiopathic pulmonary fibrosis transplantation therapy by the use of hypoxia-preconditioned MSCs.
Methods
Hypoxic preconditioning was achieved in MSCs under an optimal hypoxic environment. The expression levels of cytoprotective factors and their biological effects on damaged alveolar epithelial cells or transforming growth factor-beta 1-treated fibroblast cells were studied in co-culture experiments in vitro. Furthermore, hypoxia-preconditioned MSCs (HP-MSCs) were intratracheally instilled into bleomycin-induced pulmonary fibrosis mice at day 3, and lung functions, cellular, molecular and pathological changes were assessed at 7 and 21 days after bleomycin administration.
Results
The expression of genes for pro-survival, anti-apoptotic, anti-oxidant and growth factors was upregulated in MSCs under hypoxic conditions. In transforming growth factor-beta 1-treated MRC-5 fibroblast cells, hypoxia-preconditioned MSCs attenuated extracellular matrix production through paracrine effects. The pulmonary respiratory functions significantly improved for up to 18 days of hypoxia-preconditioned MSC treatment. Expression of inflammatory factors and fibrotic factor were all downregulated in the lung tissues of the hypoxia-preconditioned MSC-treated mice. Histopathologic examination observed a significant amelioration of the lung fibrosis. Several LacZ-labeled MSCs were observed within the lungs in the hypoxia-preconditioned MSC treatment groups at day 21, but no signals were detected in the normoxic MSC group. Our data further demonstrated that upregulation of hepatocyte growth factor possibly played an important role in mediating the therapeutic effects of transplanted hypoxia-preconditioned MSCs.
Conclusion
Transplantation of hypoxia-preconditioned MSCs exerted better therapeutic effects in bleomycin-induced pulmonary fibrotic mice and enhanced the survival rate of engrafted MSCs, partially due to the upregulation of hepatocyte growth factor.
doi:10.1186/s13287-015-0081-6
PMCID: PMC4487587  PMID: 25986930
4.  Monocytes and tissue factor promote thrombosis in a murine model of oxygen deprivation. 
Journal of Clinical Investigation  1997;99(7):1729-1738.
Clinical conditions associated with local or systemic hypoxemia can lead to prothrombotic diatheses. This study was undertaken to establish a model of whole-animal hypoxia wherein oxygen deprivation by itself would be sufficient to trigger tissue thrombosis. Furthermore, this model was used to test the hypothesis that hypoxia-induced mononuclear phagocyte (MP) recruitment and tissue factor (TF) expression may trigger the local deposition of fibrin which occurs in response to oxygen deprivation. Using an environmental chamber in which inhaled oxygen tension was lowered to 6%, hypoxic induction of thrombosis was demonstrated in murine pulmonary vasculature by 8 h based upon: (a) immunohistologic evidence of fibrin formation in hypoxic lung tissue using an antifibrin antibody, confirmed by 22.5-nm strand periodicity by electron microscopy; (b) immunoblots revealing fibrin gamma-gamma chain dimers in lungs from hypoxic but not normoxic mice or hypoxic mice treated with hirudin; (c) accelerated deposition of 125I-fibrin/fibrinogen and 111In-labeled platelets in the lung tissue of hypoxic compared with normoxic animals; (d) reduction of tissue 125I-fibrin/fibrinogen accumulation in animals which had either been treated with hirudin or depleted of platelets before hypoxic exposure. Because immunohistochemical analysis of hypoxic pulmonary tissue revealed strong MP staining for TF, confirmed by increased TF RNA in hypoxic lungs, and because 111In-labeled murine MPs accumulated in hypoxic pulmonary tissue, we evaluated whether recruited MPs might be responsible for initiation of hypoxia-induced thrombosis. This hypothesis was supported by several lines of evidence: (a) MP depletion before hypoxia reduced thrombosis, as measured by reduced 125I-fibrin/fibrinogen deposition and reduced accumulation of cross-linked fibrin by immunoblot; (b) isolated murine MPs demonstrated increased TF immunostaining when exposed to hypoxia; and (c) administration of an anti-rabbit TF antibody that cross-reacts with murine TF decreased 125I-fibrin/fibrinogen accumulation and cross-linked fibrin accumulation in response to hypoxia in vivo. In summary, these studies using a novel in vivo model suggest that MP accumulation and TF expression may promote hypoxia-induced thrombosis.
PMCID: PMC507994  PMID: 9120018
5.  Adenosine promotes vascular barrier function in hyperoxic lung injury 
Physiological Reports  2014;2(9):e12155.
Abstract
Hyperoxic lung injury is characterized by cellular damage from high oxygen concentrations that lead to an inflammatory response in the lung with cellular infiltration and pulmonary edema. Adenosine is a signaling molecule that is generated extracellularly by CD73 in response to injury. Extracellular adenosine signals through cell surface receptors and has been found to be elevated and plays a protective role in acute injury situations. In particular, ADORA2B activation is protective in acute lung injury. However, little is known about the role of adenosine signaling in hyperoxic lung injury. We hypothesized that hyperoxia‐induced lung injury leads to CD73‐mediated increases in extracellular adenosine, which is protective through ADORA2B signaling pathways. To test this hypothesis, we exposed C57BL6, CD73−/−, and Adora2B−/− mice to 95% oxygen or room air and examined markers of pulmonary inflammation, edema, and monitored lung histology. Hyperoxic exposure caused pulmonary inflammation and edema in association with elevations in lung adenosine levels. Loss of CD73‐mediated extracellular adenosine production exacerbated pulmonary edema without affecting inflammatory cell counts. Furthermore, loss of the ADORA2B had similar results with worsening of pulmonary edema following hyperoxia exposure without affecting inflammatory cell infiltration. This loss of barrier function correlated with a decrease in occludin in pulmonary vasculature in CD73−/− and Adora2B−/− mice following hyperoxia exposure. These results demonstrate that exposure to a hyperoxic environment causes lung injury associated with an increase in adenosine concentration, and elevated adenosine levels protect vascular barrier function in hyperoxic lung injury through the ADORA2B‐dependent regulation of occludin.
e12155
Hyperoxic lung injury is characterized by cellular damage from high oxygen concentrations that lead to an inflammatory response in the lung with cellular infiltration and pulmonary edema, and extracellular adenosine has been found to be elevated and plays a protective role in acute injury situations; however, little is known about the role of adenosine signaling in hyperoxic lung injury. We hypothesized that hyperoxia‐induced lung injury leads to CD73‐mediated increases in extracellular adenosine, which is protective through ADORA2B signaling pathways. Our results demonstrate that exposure to a hyperoxic environment causes lung injury associated with an increase in adenosine concentration, and elevated adenosine levels protect vascular barrier function in hyperoxic lung injury through the ADORA2B‐dependent regulation of occludin.
doi:10.14814/phy2.12155
PMCID: PMC4270235  PMID: 25263205
Adenosine; hyperoxic lung injury; vascular barrier function
6.  Crucial Role for Ecto-5′-Nucleotidase (CD73) in Vascular Leakage during Hypoxia 
The Journal of Experimental Medicine  2004;200(11):1395-1405.
Extracellular adenosine has been widely implicated in adaptive responses to hypoxia. The generation of extracellular adenosine involves phosphohydrolysis of adenine nucleotide intermediates, and is regulated by the terminal enzymatic step catalyzed by ecto-5′-nucleotidase (CD73). Guided by previous work indicating that hypoxia-induced vascular leakage is, at least in part, controlled by adenosine, we generated mice with a targeted disruption of the third coding exon of Cd73 to test the hypothesis that CD73-generated extracellular adenosine functions in an innate protective pathway for hypoxia-induced vascular leakage. Cd73−/− mice bred and gained weight normally, and appeared to have an intact immune system. However, vascular leakage was significantly increased in multiple organs, and after subjection to normobaric hypoxia (8% O2), Cd73−/− mice manifested fulminant vascular leakage, particularly prevalent in the lung. Histological examination of lungs from hypoxic Cd73−/− mice revealed perivascular interstitial edema associated with inflammatory infiltrates surrounding larger pulmonary vessels. Vascular leakage secondary to hypoxia was reversed in part by adenosine receptor agonists or reconstitution with soluble 5′-nucleotidase. Together, our studies identify CD73 as a critical mediator of vascular leakage in vivo.
doi:10.1084/jem.20040915
PMCID: PMC1237012  PMID: 15583013
adenosine; inflammation; edema; endothelium; knockout
7.  Crucial Role for Ecto-5′-Nucleotidase (CD73) in Vascular Leakage during Hypoxia 
The Journal of experimental medicine  2004;200(11):1395-1405.
Extracellular adenosine has been widely implicated in adaptive responses to hypoxia. The generation of extracellular adenosine involves phosphohydrolysis of adenine nucleotide intermediates, and is regulated by the terminal enzymatic step catalyzed by ecto-5′-nucleotidase (CD73). Guided by previous work indicating that hypoxia-induced vascular leakage is, at least in part, controlled by adenosine, we generated mice with a targeted disruption of the third coding exon of Cd73 to test the hypothesis that CD73-generated extracellular adenosine functions in an innate protective pathway for hypoxia-induced vascular leakage. Cd73−/− mice bred and gained weight normally, and appeared to have an intact immune system. However, vascular leakage was significantly increased in multiple organs, and after subjection to normobaric hypoxia (8% O2), Cd73−/− mice manifested fulminant vascular leakage, particularly prevalent in the lung. Histological examination of lungs from hypoxic Cd73−/− mice revealed perivascular interstitial edema associated with inflammatory infiltrates surrounding larger pulmonary vessels. Vascular leakage secondary to hypoxia was reversed in part by adenosine receptor agonists or reconstitution with soluble 5′-nucleotidase. Together, our studies identify CD73 as a critical mediator of vascular leakage in vivo.
doi:10.1084/jem.20040915
PMCID: PMC1237012  PMID: 15583013
adenosine; inflammation; edema; endothelium; knockout; ANOVA, analysis of variance; APCP, α,β -methylene ADP; E-Ado, etheno-adenosine; E-AMP, etheno-AMP; ES, embryonic stem; NECA, 5′-(N-ethylcarboxamido)-adenosine; 5′-NT, 5′-nucleotidase
8.  Hostile, Hypoxia-A2-Adenosinergic Tumor Biology as the Next Barrier to the Tumor Immunologists 
Cancer immunology research  2014;2(7):598-605.
The hypoxia-driven and A2A or A2B adenosine receptors (A2AR/A2BR)-mediated (“Hypoxia-A2-Adenosinergic”) and T cell autonomous immunosuppression was first recognized as critical and non-redundant in protection of normal tissues from inflammatory damage and autoimmunity. However, this immunosuppressive mechanism is high-jacked by bacteria and tumors to misguidedly protect pathogens and cancerous tissues. The inhibitors of Hypoxia-A2-Adenosinergic pathway represent the conceptually novel type of immunological co-adjuvants to be combined with cancer vaccines, adoptive cell transfer and/or blockade of immunological negative regulators in order to further prolong survival and minimize side effects. In support of this approach are preclinical studies and findings that some human cancers are resistant to chemotherapies and immunotherapies due to the tumor-generated extracellular adenosine and intracellular cAMP-elevating A2AR and A2BR on anti-tumor T and NK cells. Among co-adjuvants are i) antagonists of A2AR/A2BR; ii) extracellular adenosine-degrading drugs; iii) inhibitors of adenosine generation by CD39/CD73 ecto-enzymes and iv) inhibitors of the hypoxia-HIF-1 alpha signaling. It is emphasized that even after the multi-combinatorial blockade of immunological negative regulators the anti-tumor T and NK cells would be still vulnerable to inhibition by hypoxia and A2AR and A2BR. The advantage of combining these co-adjuvants with the blockade of the CTLA4-A and/or PD-1 is in expectations of additive or even synergistic effects of targeting both immunological and physiological tumor-protecting mechanisms. Yet to be tested is the potential capacity of co-adjuvants to minimize the side effects of blockade of CTLA-4 and/or PD1 by decreasing the dose of blocking antibodies or by eliminating the need in dual blockade.
doi:10.1158/2326-6066.CIR-14-0075
PMCID: PMC4331061  PMID: 24990240
9.  Adenosine and inosine exert cytoprotective effects in an in vitro model of liver ischemia-reperfusion injury 
Liver ischemia represents a common clinical problem. In the present study, using an in vitro model of hepatic ischemia-reperfusion injury, we evaluated the potential cytoprotective effect of the purine metabolites, such as adenosine and inosine, and studied the mode of their pharmacological actions. The human hepatocellular carcinoma-derived cell line HepG2 was subjected to combined oxygen-glucose deprivation (COGD; 0-14-24 h), followed by re-oxygenation (0-4-24 h). Adenosine or inosine (300–1,000 μM) were applied in pretreatment. Cell viability and cytotoxicity were measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and lactate dehydrogenase methods, respectively. The results showed that both adenosine and inosine exerted cytoprotective effects, and these effects were not related to receptor-mediated actions, since they were not prevented by selective adenosine receptor antagonists. On the other hand, the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA, 10 μM) markedly and almost fully reversed the protective effect of adenosine during COGD, while it did not influence the cytoprotective effect of inosine in the same assay conditions. These results suggest that the cytoprotective effects are related to intracellular actions, and, in the case of adenosine also involve intracellular conversion to inosine. The likely interpretation of these findings is that inosine serves as an alternative source of energy to produce ATP during hypoxic conditions. The protective effects are also partially dependent on adenosine kinase, as the inhibitor 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-d]pyrimidine, 2HCl (ABT 702, 30 μM) significantly reversed the protective effect of both adenosine and inosine during hypoxia and re-oxygenation. Collectively, the current results support the view that during hypoxia, adenosine and inosine exert cytoprotective effects via receptor-independent, intracellular modes of action, which, in part, depend on the restoration of cellular bioenergetics. The present study supports the view that testing of inosine for protection against various forms of warm and cold liver ischemia is relevant.
doi:10.3892/ijmm.2012.1203
PMCID: PMC3981016  PMID: 23232950
adenosine; inosine; cytoprotection; liver; ischemia-reperfusion; hepatocytes
10.  Cysteinyl Leukotriene Signaling Aggravates Myocardial Hypoxia in Experimental Atherosclerotic Heart Disease 
PLoS ONE  2012;7(7):e41786.
Background
Cysteinyl-leukotrienes (cys-LT) are powerful spasmogenic and immune modulating lipid mediators involved in inflammatory diseases, in particular asthma. Here, we investigated whether cys-LT signaling, in the context of atherosclerotic heart disease, compromises the myocardial microcirculation and its response to hypoxic stress. To this end, we examined Apoe−/− mice fed a hypercholesterolemic diet and analysed the expression of key enzymes of the cys-LT pathway and their receptors (CysLT1/CysLT2) in normal and hypoxic myocardium as well as the potential contribution of cys-LT signaling to the acute myocardial response to hypoxia.
Methods and principal findings
Myocardial biopsies from Apoe−/− mice demonstrated signs of chronic inflammation with fibrosis, increased apoptosis and expression of IL-6, as compared to biopsies from C57BL/6J control mice. In addition, we found increased leukotriene C4 synthase (LTC4S) and CysLT1 expression in the myocardium of Apoe−/− mice. Acute bouts of hypoxia further induced LTC4S expression, increased LTC4S enzyme activity and CysLT1 expression, and were associated with increased extension of hypoxic areas within the myocardium. Inhibition of cys-LT signaling by treatment with montelukast, a selective CysLT1 receptor antagonist, during acute bouts of hypoxic stress reduced myocardial hypoxic areas in Apoe−/− mice to levels equal to those observed under normoxic conditions. In human heart biopsies from 14 patients with chronic coronary artery disease mRNA expression levels of LTC4S and CysLT1 were increased in chronic ischemic compared to non-ischemic myocardium, constituting a molecular basis for increased cys-LT signaling.
Conclusion
Our results suggest that CysLT1 antagonists may have protective effects on the hypoxic heart, and improve the oxygen supply to areas of myocardial ischemia, for instance during episodes of sleep apnea.
doi:10.1371/journal.pone.0041786
PMCID: PMC3404957  PMID: 22848603
11.  Endothelial PAS Domain Protein 1 Activates the Inflammatory Response in the Intestinal Epithelium to Promote Colitis in Mice 
Gastroenterology  2013;145(4):831-841.
Background & Aims
Hypoxic inflammation (decreased oxygen tension at sites of inflammation) is a feature of inflammatory bowel disease (IBD). The hypoxia response is mediated by the transcription factors hypoxia-inducible factor (HIF)1α and endothelial PAS domain protein 1 (EPAS1 or HIF2α), which are induced in intestinal tissues of patients with IBD. HIF1α limits intestinal barrier dysfunction, but the role of EPAS1 has not been assessed under conditions of hypoxic inflammation or in models of IBD.
Methods
Acute colitis was induced by administration of Citrobacter rodentium ordextran sulfate sodium (DSS) to transgenic hypoxia reporter mice (ODD-Luc), mice with conditional overexpression of Epas1 (Epas1LSL/LSL), mice with intestinal epithelium-specific deletion of Epas1 (Epas1ΔIE), or wild-type littermates (controls). Colon tissues from these mice and from patients with ulcerative colitis (UC) or Crohn's disease (CD) were assessed by histologic and immunoblot analyses, immunohistochemistry, and quantitative PCR.
Results
Levels of hypoxia and EPAS1 were increased in colon tissues of mice following induction of colitis and patients with UC or CD, compared with controls. Epas1ΔIE mice had attenuated colonic inflammation and were protected from DSS-induced colitis. Intestine-specific overexpression of EPAS1, but not HIF-1α, led to spontaneous colitis, increased susceptibility to induction of colitis by C rodentium or DSS, and reduced survival times compared with controls. Disruption of intestinal epithelial EPAS1 attenuated the inflammatory response following administration of DSS or C rodentium, whereas intestine-specific overexpression of EPAS1 increased this response. We found EPAS1 to be a positive regulator of tumor necrosis factor (TNF)α production by the intestinal epithelium. Blocking TNFα completely reduced hypoxia-induced intestinal inflammation. We found EPAS1 to be a positive regulator of tumor necrosis factor (TNF)α production by the intestinal epithelium. Blocking TNFα completely reduced hypoxia-induced intestinal inflammation.
Conclusions
EPAS1 is a transcription factor that activates mediators of inflammation, such as TNFα, in the intestinal epithelium and promotes development of colitis in mice.
doi:10.1053/j.gastro.2013.07.010
PMCID: PMC3799890  PMID: 23860500
mouse model; CD; UC; oxygen
12.  Hepatic oxygen and glucose metabolism in the fetal lamb. Response to hypoxia. 
Journal of Clinical Investigation  1983;71(5):1047-1061.
Although the fetal liver is an active metabolic organ, its oxygen and glucose requirements have not previously been described. We measured hepatic blood flows and the oxygen and glucose differences across the liver in 12 late gestation fetal lambs in utero. Four animals were studied at least 1 wk postsurgically and again 2-5 d later to assess daily variations in hepatic blood flow and metabolism (group I). A second group of eight animals was studied 3-5 d postsurgically during a control period and during acute fetal hypoxia (group II). Under control conditions total hepatic blood flow averaged 400 ml/min per 100 g in both groups, and 75-80% was of umbilical origin. Liver blood flow and oxygen consumption were usually similar during repeated measurements, but in one animal varied considerably. During periods of normoxia, oxygen consumption for both the right and left lobes of liver was 4 ml/min per 100 g. Oxygen consumption of the whole liver accounted for 20% of total fetal oxygen consumption. This was achieved with oxygen extraction of 10-15%, so that hepatic venous blood was well oxygenated and provided an important source of oxygen for other fetal tissues. Under control conditions we could demonstrate no net hepatic uptake or release of glucose suggesting that the liver ultimately utilizes another carbon source to support its oxidative metabolism. During acute hypoxia total liver blood flow and its umbilical venous contribution both fell by 20%. Blood flow to the right lobe of the liver fell twice as much as that to the left lobe. Hepatic oxygen consumption was linearly related to oxygen delivery during the control and hypoxic periods. Consequently, right hepatic oxygen uptake fell by 45% whereas left hepatic oxygen uptake was unchanged, suggesting a functional difference between the lobes. During hypoxia glucose was released from both liver lobes; 6 mg/min per 100 g for the right lobe and 9 mg/min per 100 g for the left lobe. Total hepatic release of glucose was estimated to nearly equal umbilical uptake, so that 45% of the glucose available to fetal tissues was of hepatic origin. We conclude that the fetal liver responds to acute hypoxia by reducing its own oxygen consumption and releasing glucose to facilitate anaerobic metabolism.
PMCID: PMC436966  PMID: 6682864
13.  Adenosine signaling in normal and sickle erythrocytes and beyond 
Microbes and infection / Institut Pasteur  2012;14(10):10.1016/j.micinf.2012.05.005.
Sickle cell disease (SCD) is a debilitating hemolytic genetic disorder with high morbidity and mortality affecting millions of individuals worldwide. Although SCD was discovered more than a century ago, no effective mechanism-based prevention and treatment are available due to poorly understood molecular basis of sickling, the fundamental pathogenic process of the disease. SCD patients constantly face hypoxia. One of the best-known signaling molecules to be induced under hypoxic conditions is adenosine. Recent studies demonstrate that hypoxia-mediated elevated adenosine signaling plays an important role in normal erythrocyte physiology. In contrast, elevated adenosine signaling contributes to sickling and multiple life threatening complications including tissue damage, pulmonary dysfunction and priapism. Here, we summarize recent research on the role of adenosine signaling in normal and sickle erythrocytes, progression of the disease and therapeutic implications.
In normal erythrocytes, both genetic and pharmacological studies demonstrate that adenosine can enhance 2,3-bisphosphoglycerate (2,3-BPG) production via A2B receptor (ADORA2B) activation, suggesting that elevated adenosine has an unrecognized role in normal erythrocytes to promote O2 release and prevent acute ischemic tissue injury. However, in sickle erythrocytes, the beneficial role of excessive adenosine-mediated 2,3-BPG induction becomes detrimental by promoting deoxygenation, polymerization of sickle hemoglobin and subsequent sickling. Additionally, adenosine signaling via the A2A receptor (ADORA2A) on invariant natural killer T (iNKT) cells inhibits iNKT cell activation and attenuates pulmonary dysfunction in SCD mice. Finally, elevated adenosine coupled with ADORA2BR activation is responsible for priapism, a dangerous complication seen in SCD.
Overall, the research reviewed here reveals a differential role of elevated adenosine in normal erythrocytes, sickle erythrocytes, iNK cells and progression of disease. Thus, adenosine signaling represents a potentially important therapeutic target for the treatment and prevention of disease.
doi:10.1016/j.micinf.2012.05.005
PMCID: PMC3842013  PMID: 22634345
sickle cell disease; malaria; adenosine; adenosine A2B receptor; 2,3-diphosphoglycerate; adenosine deaminase
14.  HYPOXIA INDUCED CHANGES IN EXPRESSION OF PROTEINS INVOLVED IN IRON UPTAKE AND STORAGE IN CULTURED LENS EPITHELIAL CELLS 
Experimental eye research  2014;125:135-141.
Hypoxia inducible factor (HIF) regulates expression of over 60 genes by binding to hypoxia response elements (HRE) located upstream of the transcriptional start sites. Many genes encoding proteins involved in iron transport and homeostasis are regulated by HIF. Expression of iron handling proteins can also be translationally regulated by binding of iron regulatory protein (IRP) to iron responsive elements (IREs) on the mRNA of ferritin chains and transferrin receptor (TfR). Lens epithelial cells (LEC) function in a low oxygen environment. This increases the risk of iron catalyzed formation of reactive oxygen species (ROS) and oxidative cell damage. We examined changes in expression of ferritin (iron storage protein) and Tf/TfR1 (iron uptake proteins) in LEC cultured under hypoxic conditions. Ferritin consists of 24 subunits of two types, heavy (H-chain) and light (L-chain) assembled in a cell specific ratio. Real-time PCR showed that 24 h exposure to hypoxia lowered transcription of both ferritin chains by over 50% when compared with normoxic LEC. However it increased the level of ferritin chain proteins (20% average). We previously found that 6 h exposure of LEC to hypoxia increased the concentration of cytosolic iron which would stimulate translation of ferritin chains. This elevated ferritin concentration increased the iron storage capacity of LEC. Hypoxic LEC labeled with 59FeTf incorporated 70% more iron into ferritin after 6 h as compared to normoxic LEC. Exposure of LEC to hypoxia for 24 h reduced the concentration of TfR1 in cell lysates. As a result, hypoxic LEC internalized less Tf at this later time point. Incorporation of 59Fe into ferritin of hypoxic LEC after 24 h did not differ from that of normoxic LEC due to lower 59FeTf uptake. This study showed that hypoxia acutely increased iron storage capacity and lowered iron uptake due to changes in expression of iron handling proteins. These changes may better protect LEC against oxidative stress by limiting iron-catalyzed ROS formation in the low oxygen environment in which the lens resides.
doi:10.1016/j.exer.2014.05.010
PMCID: PMC4154372  PMID: 24877740
lens; iron; iron proteins; hypoxia
15.  Hypoxic Preconditioning Results in Increased Motility and Improved Therapeutic Potential of Human Mesenchymal Stem Cells 
Stem cells (Dayton, Ohio)  2008;26(8):2173-2182.
Mesenchymal stem cells (MSC) are adult multipotent cells found in bone marrow, adipose tissue, and other adult tissues. MSC have been shown to improve regeneration of injured tissues in vivo, but the mechanisms remain unclear. Typically, MSC are cultured under ambient, or normoxic, conditions (21% oxygen). However, the physiological niches for MSC in the bone marrow and other sites have much lower oxygen tension. When used as a therapeutic tool to repair tissue injuries, MSC cultured in standard conditions must adapt from 21% oxygen in culture to less than 1% oxygen in the ischemic tissue. We therefore examined the effects of preculturing human bone marrow-derived MSC in hypoxic conditions (1%–3% oxygen) to elucidate the best conditions that enhance their tissue regenerative potential. We demonstrated that MSC cultured in hypoxia activate the Akt signaling pathway while maintaining their viability and cell cycle rates. We also showed that MSC cultured in hypoxia induced expression of cMet, the major receptor for hepatocyte growth factor (HGF), and enhanced cMet signaling. MSC cultured in hypoxic conditions increased their migration rates. Since migration and HGF responsiveness are thought to be key mediators of MSC recruitment and/or activation in vivo, we next examined the tissue regenerative potential of MSC cultured under hypoxic conditions, using a murine hind limb ischemia model. We showed that local expression of HGF is increased in ischemic muscle in this model. Intra-arterial injection of MSC cultured in either normoxic or hypoxic conditions 24 hours after surgical induction of hind limb ischemia enhanced revascularization compared with saline controls. However, restoration of blood flow was observed significantly earlier in mice that had been injected with hypoxic preconditioned MSC. Collectively, these data suggest that preculturing MSC under hypoxic conditions prior to transplantation improves their tissue regenerative potential.
doi:10.1634/stemcells.2007-1104
PMCID: PMC3017477  PMID: 18511601
Immune-deficient mice; Human stem cells; Mesenchymal stem cells; Hypoxia; Transplantation; Tissue repair
16.  Effects of Hypoxia Exposure on Hepatic Cytochrome P450 1A (CYP1A) Expression in Atlantic Croaker: Molecular Mechanisms of CYP1A Down-Regulation 
PLoS ONE  2012;7(7):e40825.
Hypoxia-inducible factor-α (HIF-α) and cytochrome P450 1A (CYP1A) are biomarkers of environmental exposure to hypoxia and organic xenobiotic chemicals that act through the aryl hydrocarbon receptor, respectively. Many aquatic environments heavily contaminated with organic chemicals, such as harbors, are also hypoxic. Recently, we and other scientists reported HIF-α genes are upregulated by hypoxia exposure in aquatic organisms, but the molecular mechanisms of hypoxia regulation of CYP1A expression have not been investigated in teleost fishes. As a first step in understanding the molecular mechanisms of hypoxia modulation of CYP1A expression in fish, we characterized CYP1A cDNA from croaker liver. Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 2 to 4 weeks) caused significant decreases in hepatic CYP1A mRNA and protein levels compared to CYP1A levels in fish held in normoxic conditions. In vivo studies showed that the nitric oxide (NO)-donor, S-nitroso-N-acetyl-DL-penicillamine, significantly decreased CYP1A expression in croaker livers, whereas the competitive inhibitor of NO synthase (NOS), Nω-nitro-L-arginine methyl ester, restored CYP1A mRNA and protein levels in hypoxia-exposed (1.7 mg DO/L for 4 weeks) fish. In vivo hypoxia exposure also markedly increased interleukin-1β (IL-1β, a cytokine), HIF-2α mRNA and endothelial NOS (eNOS) protein levels in croaker livers. Pharmacological treatment with vitamin E, an antioxidant, lowered the IL-1β, HIF-2α mRNA and eNOS protein levels in hypoxia-exposed fish and completely reversed the down-regulation of hepatic CYP1A mRNA and protein levels in response to hypoxia exposure. These results suggest that hypoxia-induced down-regulation of CYP1A is due to alterations of NO and oxidant status, and cellular IL-1β and HIF-α levels. Moreover, the present study provides the first evidence of a role for antioxidants in hepatic eNOS and IL-1β regulation in aquatic vertebrates during hypoxic stress.
doi:10.1371/journal.pone.0040825
PMCID: PMC3397942  PMID: 22815834
17.  ERYTHROPOIETIN: A MODEL SYSTEM FOR STUDYING OXYGEN-DEPENDENT GENE REGULATION 
The Journal of experimental biology  1998;201(Pt 8):1197-1201.
Summary
The physiological regulation of the red cell mass depends upon enhanced transcription of the erythropoietin (Epo) gene in response to hypoxia. Studies of Epo gene expression have been useful in investigating the mechanism by which cells and tissues sense hypoxia and respond with biologically appropriate alterations in gene expression. It is likely that oxygen sensing involves a heme protein in which cobalt and nickel can substitute for iron in the porphyrin ring. Indirect evidence suggests that the sensor is present in all cells and is a multi-subunit assembly containing an NAD(P)H oxidase capable of generating peroxide and reactive oxygen intermediates, which serve as signaling molecules. The up-regulation of Epo gene transcription by hypoxia is mediated by at least two known DNA-binding transcription factors, hypoxia-inducible factor 1 (HIF-1) and hepatic nuclear factor 4 (HNF-4), which bind to cognate response elements in a critical 3′ enhancer approximately 50 bp in length. HIF-1 binding is induced by hypoxia as well as by cobalt. The activation of HIF-1 by hypoxia depends upon the selective protection of its α subunit from ubiquitin-dependent proteolysis by means of a mechanism that involves redox chemistry and perhaps phosphorylation. HNF-4 is an orphan nuclear receptor that is constitutively expressed in kidney and liver and which cooperates with HIF-1 to give maximal hypoxic induction. In hypoxic cells, p300 or a related family member forms a macromolecular assembly with HIF-1 and HNF-4, enabling transduction from the Epo 3′ enhancer to the apparatus on the promoter responsible for the initiation of transcription.
PMCID: PMC3044471  PMID: 9510530
erythropoietin; hypoxia; gene regulation; oxygen sensing; HIF-1; HNF-4; p300
18.  Renal Cell Protection of Erythropoietin beyond Correcting The Anemia in Chronic Kidney Disease Patients 
Cell Journal (Yakhteh)  2013;15(4):378-380.
Currently many patients with chronic renal failure have profited from the use of erythropoietin to correct anemia (1,2). In chronic kidney disease, anemia is believed to be a surrogate index for tissue hypoxia that continues preexisting renal tissue injury (1-3). Erythropoietin is an essential glycoprotein that accelerates red blood cell maturation from erythroid progenitors and facilitates erythropoiesis. It is a 30.4 kD glycoprotein and class I cytokine containing 165 amino acids (3,4). Approximately 90% of systemic erythropoietin in adults is produced by peritubular interstitial fibroblasts in the renal cortex and outer medulla of the kidney (3-5). A feedback mechanism involving oxygen delivery to the tissues seems to regulate erythropoietin production. Hypoxia-inducible factor regulates transcription of the erythropoietin gene in the kidney, which determines erythropoietin synthesis (3-5). Erythropoietin is an essential glycoprotein that accelerates red blood cell maturation from erythroid progenitors and mediates erythropoiesis in the bone marrow (4-6). Kidney fibrosis is the last common pathway in chronic renal failure irrespective of the initial etiology (5,6). Constant inflammatory cell infiltration and pericyte-myofibroblast transition lead to renal fibrosis and insufficiency which result in decreased production of erythropoietin (4-7). Thus far, therapeutic efforts to treat patients with chronic renal failure by administering erythropoietin have been made only to correct anemia and putative hypoxic tissue damage. The introduction of recombinant human erythropoietin has marked a significant advance in the management of anemia associated with chronic renal failure (6-9). With an increasing number of patients with chronic renal failure receiving erythropoietin treatment, emerging evidence suggests that erythropoietin not only has an erythropoietic function, but also has renoprotective potential. In fact, in recent years, the additional non-erythropoietic tissue/ organ protective efficacy of erythropoietin has become evident, especially in the kidneys (5-12). Various investigations have shown the kidney protective property of erythropoietin in acute kidney injury. In a study to evaluate the ameliorative effects of erythropoietin on renal tubular cells, we studied 40 male rats. We found that erythropoietin was able to prevent the increase in serum creatinine and blood urea nitrogen. Furthermore, co-administration of gentamicin and erythropoietin effectively reduced kidney tissue damage compared to the control group. However, the protective properties of erythropoietin were also evident in our study. When the drug was applied after gentamicin- induced tubular damage we were able to show that the drug was still effective after tissue injury onset. This indicates that erythropoietin may have curative effects in addition to its preventive properties (13). Thus, erythropoietin is a promising kidney protective agent to prevent, ameliorate or attenuate tubular damage induced by gentamicin or other nephrotoxic agents that act in a similar manner to this drug (14-17). Recent studies have elucidated the cellular mechanism involved in kidney erythropoietin production and the consequent events that lead to kidney fibrosis, showing that they are closely related to each other (18-20). In contrast to previous findings, fibroblasts originating from damaged renal tubular epithelial cells do not have an important role in kidney fibrosis, but renal erythropoietin- producing cells, stemming from neural crests, have been shown to trans-differentiate into myofibroblasts after long-term exposure to inflammatory situations related to kidney fibrosis. In fact, almost all myofibroblasts expressing α-smooth muscle actin originate from renal erythropoietin-producing cells, which are naturally peritubular interstitial fibroblastic cells expressing neural cell marker genes but not α-smooth muscle actin. Macrophages and myofibroblasts are responsible for fibrosis in the renal tissue. Macrophages could be differentiated to phenotype M1 (classically activated) or M2 (wound healing) according to the distinctive cytokine production and behavior that follows different routes of activation (6,8,21,22). While erythropoietin can disengage macrophages by stopping the activity of NF-κB, it is possible that one of the mechanisms explaining the antifibrotic effects of erythropoietin in chronic kidney disease is in vivo macrophage regulation (20-25). These important findings stipulate the missing link in chronic renal failure between anemia and kidney fibrosis (6,8,21,22). In patients with chronic kidney disease, anemia due to reduced erythropoietin production eventually appears (1,4,5). Recombinant human erythropoietin has been used for more than 20 years in chronic kidney disease to recompense for reduced endogenous erythropoietin production (1,4,5,25). Recent investigations have pointed out that erythropoietin administration improves kidney functions in chronic kidney disease either directly or indirectly (17-24). The therapeutic benefits of erythropoietin beyond the correction of anemia are still questioned. However, it is notable that various pieces of evidence simply reflect the pleiotropic effects of erythropoietinon on the central nervous, cardiovascular system and on the kidney (18,20,25). In brief, clinical evidence shows the kidney protective potential of erythropoietin in patients with chronic renal failure, however, additional clinical investigations are crucial to outline when to start erythropoietin treatment and what is the optimal erythropoietin dosage for slowing disease progression in patients with chronic renal failure. The application of erythropoietin treatment for renoprotection may need to be earlier than that for erythropoiesis, while it is possible that the erythropoietin attenuation of renal fibrosis through macrophage regulation and endothelial cell protection operates through other unidentified mechanisms. While agents restoring the initial function of renal erythropoietin-producing cells could delay kidney fibrosis, further laboratory studies are necessary to clarify the cellular target of erythropoietin in the kidney and for developing a novel erythropoietin derivative or mimetic for kidney protection.
PMCID: PMC3866543  PMID: 24381864
Erythropoietin; Erythropoiesis
19.  Mutation of von Hippel–Lindau Tumour Suppressor and Human Cardiopulmonary Physiology 
PLoS Medicine  2006;3(7):e290.
Background
The von Hippel–Lindau tumour suppressor protein–hypoxia-inducible factor (VHL–HIF) pathway has attracted widespread medical interest as a transcriptional system controlling cellular responses to hypoxia, yet insights into its role in systemic human physiology remain limited. Chuvash polycythaemia has recently been defined as a new form of VHL-associated disease, distinct from the classical VHL-associated inherited cancer syndrome, in which germline homozygosity for a hypomorphic VHL allele causes a generalised abnormality in VHL–HIF signalling. Affected individuals thus provide a unique opportunity to explore the integrative physiology of this signalling pathway. This study investigated patients with Chuvash polycythaemia in order to analyse the role of the VHL–HIF pathway in systemic human cardiopulmonary physiology.
Methods and Findings
Twelve participants, three with Chuvash polycythaemia and nine controls, were studied at baseline and during hypoxia. Participants breathed through a mouthpiece, and pulmonary ventilation was measured while pulmonary vascular tone was assessed echocardiographically. Individuals with Chuvash polycythaemia were found to have striking abnormalities in respiratory and pulmonary vascular regulation. Basal ventilation and pulmonary vascular tone were elevated, and ventilatory, pulmonary vasoconstrictive, and heart rate responses to acute hypoxia were greatly increased.
Conclusions
The features observed in this small group of patients with Chuvash polycythaemia are highly characteristic of those associated with acclimatisation to the hypoxia of high altitude. More generally, the phenotype associated with Chuvash polycythaemia demonstrates that VHL plays a major role in the underlying calibration and homeostasis of the respiratory and cardiovascular systems, most likely through its central role in the regulation of HIF.
Editors' Summary
Background.
Human cells (like those of other multicellular animals) use oxygen to provide the energy needed for daily life. Having not enough oxygen is a problem, but having too much is also dangerous because it damages proteins, DNA, and other large molecules that keep cells functioning. Consequently, the physiological systems—including the heart, lungs, and circulation—work together to balance oxygen supply and demand throughout the body. When oxygen is limiting (a condition called hypoxia), as happens at high altitudes, the cellular oxygen supply is maintained by increasing the heart rate, increasing the speed and depth of breathing (hyperventilation), constricting the blood vessels in the lung (pulmonary vasoconstriction), and increasing the number of oxygen-carrying cells in the blood. All these physiological changes increase the amount of oxygen that can be absorbed from the air, but how they are regulated is poorly understood. By contrast, researchers know quite a bit about how individual cells respond to hypoxia. When oxygen is limited, a protein called hypoxia-inducible factor (or HIF) activates a number of target proteins that help the cell get enough oxygen (for example, proteins that stimulate the growth of new blood vessels). When there is plenty of oxygen, another protein, called von Hippel–Lindau tumor suppressor (abbreviated VHL), rapidly destroys HIF. Recently, researchers discovered that a genetic condition called Chuvash polycythaemia, characterised by the overproduction of red blood cells, is caused by a specific defect in VHL that reduces its ability to destroy HIF. As a result, the expression of certain HIF target proteins is increased even when oxygen levels are normal.
Why Was This Study Done?
Chuvash polycythaemia is very rare, and so far little is known about how this genetic abnormality affects the physiology and long-term health of patients. By studying heart and lung function in patients with Chuvash polycythaemia, the researchers involved in this study hoped to discover more about the health consequences of the condition and to find out whether the VHL–HIF system controls systemic responses to hypoxia as well as cellular responses.
What Did the Researchers Do and Find?
The researchers recruited and studied three patients with Chuvash polycythaemia, and, as controls for the comparison, several normal individuals and patients with an unrelated form of polycythaemia. They then measured how the lungs and hearts of these people reacted to mild hypoxia (similar to that experienced on commercial air flights) and moderate hypoxia (equiv alent to being on the top of an Alpine peak). They found that patients with Chuvash polycythaemia naturally breathe slightly quicker and deeper than normal individuals, and that their breathing rate increased dramatically and abnormally when oxygen was reduced. They also found that at normal oxygen levels the pulmonary blood vessels of these patients were more constricted than those of control individuals, and that they reacted more extremely to hypoxia. Similarly, the normal heart rate of the patients was slightly higher than that of the controls and increased much more in response to mild hypoxia.
What Do These Findings Mean?
The physiological differences measured by the researchers between Chuvash polycythaemia patients and control individuals are similar to the adaptations seen in people traveling to high altitudes where oxygen is limited. Thus, the VHL–HIF proteins may regulate the response to different oxygen concentrations both in individual cells and at the systemic level, although more physiological studies are needed to confirm this. Because the pulmonary blood vessels of patients with Chuvash polycythaemia are always abnormally constricted, and even more so when oxygen is limited, these people should avoid living at high altitude and should minimise air travel, suggest the researchers. The increased blood pressure in their lungs (pulmonary hypertension) could conceivably cause heart failure under such circumstances. Finally, this study has implications for the development of drugs directed at the VHL–HIF system. Agents are currently being designed to promote the development of new blood vessels after strokes or heart attacks by preventing the destruction of HIF, but based on the findings here such agents might have undesirable physiological affects. Conversely, HIF inhibitors (which act as anti-cancer reagents by increasing hypoxia in the centre of tumors and so inhibiting their growth) might be useful in the treatment of pulmonary hypertension.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030290.
• Online Mendelian Inheritance in Man page on Chuvash polycythaemia
• Information from the VHL Family Alliance on von Hippel–Lindau disease, including information on Chuvash polycythaemia
• Wikipedia page on polycythaemia and von Hippel–Lindau disease (note: Wikipedia is a free online encyclopaedia that anyone can edit)
Physiological study of patients with Chuvash polycythemia (caused by mutation of VHL) reveals characteristics similar to those associated with acclimatization to the hypoxia of high altitude.
doi:10.1371/journal.pmed.0030290
PMCID: PMC1479389  PMID: 16768548
20.  Dietary nitrate increases arginine availability and protects mitochondrial complex I and energetics in the hypoxic rat heart 
The Journal of Physiology  2014;592(21):4715-4731.
Key points
Exposure to environmental hypoxia, at high altitude or in a chamber, impairs cardiac energetics and alters mitochondrial function.
Inorganic nitrate, a ubiquitous dietary constituent, improves mitochondrial efficiency, lowering the oxygen cost of exercise, whilst elevated circulating nitrogen oxide levels in high-altitude natives enhances blood flow.
Here we report that dietary nitrate supplementation prevents hypoxia-induced changes in cardiac mitochondrial function and energetics, whilst ameliorating oxidative stress, suggesting improved tissue oxygenation.
Furthermore, nitrate supplementation suppresses cardiac arginase expression and increases tissue l-arginine levels under both hypoxic and normoxic conditions, underpinning a novel mechanism to enhance the availability of nitric oxide.
Nitrate supplementation may thus be of benefit to individuals exposed to hypobaric hypoxia at altitude or in patients with diseases characterised by tissue hypoxia and energetic impairment, such as heart failure and chronic obstructive pulmonary disease, or in the critically ill.
Abstract
Hypoxic exposure is associated with impaired cardiac energetics in humans and altered mitochondrial function, with suppressed complex I-supported respiration, in rat heart. This response might limit reactive oxygen species generation, but at the cost of impaired electron transport chain (ETC) activity. Dietary nitrate supplementation improves mitochondrial efficiency and can promote tissue oxygenation by enhancing blood flow. We therefore hypothesised that ETC dysfunction, impaired energetics and oxidative damage in the hearts of rats exposed to chronic hypoxia could be alleviated by sustained administration of a moderate dose of dietary nitrate. Male Wistar rats (n = 40) were given water supplemented with 0.7 mmol l−1 NaCl (as control) or 0.7 mmol l−1 NaNO3, elevating plasma nitrate levels by 80%, and were exposed to 13% O2 (hypoxia) or normoxia (n = 10 per group) for 14 days. Respiration rates, ETC protein levels, mitochondrial density, ATP content and protein carbonylation were measured in cardiac muscle. Complex I respiration rates and protein levels were 33% lower in hypoxic/NaCl rats compared with normoxic/NaCl controls. Protein carbonylation was 65% higher in hearts of hypoxic rats compared with controls, indicating increased oxidative stress, whilst ATP levels were 62% lower. Respiration rates, complex I protein and activity, protein carbonylation and ATP levels were all fully protected in the hearts of nitrate-supplemented hypoxic rats. Both in normoxia and hypoxia, dietary nitrate suppressed cardiac arginase expression and activity and markedly elevated cardiac l-arginine concentrations, unmasking a novel mechanism of action by which nitrate enhances tissue NO bioavailability. Dietary nitrate therefore alleviates metabolic abnormalities in the hypoxic heart, improving myocardial energetics.
doi:10.1113/jphysiol.2014.275263
PMCID: PMC4253472  PMID: 25172947
21.  Emerging evidence of the physiological role of hypoxia in mammary development and lactation 
Hypoxia is a physiological or pathological condition of a deficiency of oxygen supply in the body as a whole or within a tissue. During hypoxia, tissues undergo a series of physiological responses to defend themselves against a low oxygen supply, including increased angiogenesis, erythropoiesis, and glucose uptake. The effects of hypoxia are mainly mediated by hypoxia-inducible factor 1 (HIF-1), which is a heterodimeric transcription factor consisting of α and β subunits. HIF-1β is constantly expressed, whereas HIF-1α is degraded under normal oxygen conditions. Hypoxia stabilizes HIF-1α and the HIF complex, and HIF then translocates into the nucleus to initiate the expression of target genes. Hypoxia has been extensively studied for its role in promoting tumor progression, and emerging evidence also indicates that hypoxia may play important roles in physiological processes, including mammary development and lactation. The mammary gland exhibits an increasing metabolic rate from pregnancy to lactation to support mammary growth, lactogenesis, and lactation. This process requires increasing amounts of oxygen consumption and results in localized chronic hypoxia as confirmed by the binding of the hypoxia marker pimonidazole HCl in mouse mammary gland. We hypothesized that this hypoxic condition promotes mammary development and lactation, a hypothesis that is supported by the following several lines of evidence: i) Mice with an HIF-1α deletion selective for the mammary gland have impaired mammary differentiation and lipid secretion, resulting in lactation failure and striking changes in milk compositions; ii) We recently observed that hypoxia significantly induces HIF-1α-dependent glucose uptake and GLUT1 expression in mammary epithelial cells, which may be responsible for the dramatic increases in glucose uptake and GLUT1 expression in the mammary gland during the transition period from late pregnancy to early lactation; and iii) Hypoxia and HIF-1α increase the phosphorylation of signal transducers and activators of transcription 5a (STAT5a) in mammary epithelial cells, whereas STAT5 phosphorylation plays important roles in the regulation of milk protein gene expression and mammary development. Based on these observations, hypoxia effects emerge as a new frontier for studying the regulation of mammary development and lactation.
doi:10.1186/2049-1891-5-9
PMCID: PMC3929241  PMID: 24444333
Glucose transporter; Hypoxia; Hypoxia inducible factor; Lactation; Mammary development; Metabolism
22.  Hypoxia-inducible Factor-dependent Production of Profibrotic Mediators by Hypoxic Kupffer Cells 
Aim
Liver fibrosis develops when chronic liver injury stimulates cells in the liver to produce mediators that activate hepatic stellate cells and stimulate them to secrete collagen. Recent studies suggest that the hypoxia-regulated transcription factor, hypoxia-inducible factor-1α, is essential for upregulation of profibrotic mediators, such as platelet-derived growth factor, in the liver during the development of liver fibrosis. What remains unknown, however, is the cell type-specific regulation of profibrotic mediators by hypoxia-inducible factors. Accordingly, in the present study the hypothesis was tested that hypoxia-inducible factors regulate production of profibrotic mediators by hypoxic Kupffer cells.
Methods
Kupffer cells were isolated from Control mice and hypoxia-inducible factor-1β-Deficient mice and exposed to room air or 1% oxygen (i.e., hypoxia). Levels of profibrotic mediators were quantified by real-time PCR.
Results
Exposure of Kupffer cells isolated from Control mice to 1% oxygen activated hypoxia-inducible factor-1α, and increased mRNA levels of platelet-derived growth factor-B, vascular endothelial growth factor, Angiopoietin-1, and monocyte chemotactic protein-1. Upregulation of all of these mediators by hypoxia was prevented in Kupffer cells isolated from hypoxia-inducible factor-1β-Deficient mice.
Conclusion
Results from these studies suggest that hypoxia-inducible factors are critical regulators of profibrotic mediator production by hypoxic Kupffer cells.
doi:10.1111/j.1872-034X.2010.00635.x
PMCID: PMC2886188  PMID: 20412331
Angiogenesis; Fibrosis; Hypoxia; Hypoxia-inducible Factors; Kupffer cells; Liver
23.  In vivo T Cell Activation in Lymphoid Tissues is Inhibited in the Oxygen-Poor Microenvironment 
Activation of immune cells is under control of immunological and physiological regulatory mechanisms to ensure adequate destruction of pathogens with the minimum collateral damage to “innocent” bystander cells. The concept of physiological negative regulation of immune response has been advocated based on the finding of the critical immunoregulatory role of extracellular adenosine. Local tissue oxygen tension was proposed to function as one of such physiological regulatory mechanisms of immune responses. In the current study, we utilized in vivo marker of local tissue hypoxia pimonidazole hydrochloride (Hypoxyprobe-1) in the flowcytometric analysis of oxygen levels to which lymphocytes are exposed in vivo. The level of exposure to hypoxia in vivo was low in B cells and the levels increased in the following order: T cells < NKT cells < NK cells. The thymus was more hypoxic than the spleen and lymph nodes, suggesting the variation in the degree of oxygenation among lymphoid organs and cell types in normal mice. Based on in vitro studies, tissue hypoxia has been assumed to be suppressive to T cell activation in vivo, but there was no direct evidence demonstrating that T cells exposed to hypoxic environment in vivo are less activated. We tested whether the state of activation of T cells in vivo changes due to their exposure to hypoxic tissue microenvironments. The parallel analysis of more hypoxic and less hypoxic T cells in the same mouse revealed that the degree of T cell activation was significantly stronger in better-oxygenated T cells. These observations suggest that the extent of T cell activation in vivo is dependent on their localization and is decreased in environment with low oxygen tension.
doi:10.3389/fimmu.2011.00027
PMCID: PMC3342240  PMID: 22566817
T cell; oxygen; hypoxia; hyperoxia; Hypoxyprobe-1; cytometry; tumor
24.  Loss of von Hippel-Lindau Protein (VHL) Increases Systemic Cholesterol Levels through Targeting Hypoxia-Inducible Factor 2α and Regulation of Bile Acid Homeostasis 
Molecular and Cellular Biology  2014;34(7):1208-1220.
Cholesterol synthesis is a highly oxygen-dependent process. Paradoxically, hypoxia is correlated with an increase in cellular and systemic cholesterol levels and risk of cardiovascular diseases. The mechanism for the increase in cholesterol during hypoxia is unclear. Hypoxia signaling is mediated through hypoxia-inducible factor 1α (HIF-1α) and HIF-2α. The present study demonstrates that activation of HIF signaling in the liver increases hepatic and systemic cholesterol levels due to a decrease in the expression of cholesterol hydroxylase CYP7A1 and other enzymes involved in bile acid synthesis. Specifically, activation of hepatic HIF-2α (but not HIF-1α) led to hypercholesterolemia. HIF-2α repressed the circadian expression of Rev-erbα, resulting in increased expression of E4BP4, a negative regulator of Cyp7a1. To understand if HIF-mediated decrease in bile acid synthesis is a physiologically relevant pathway by which hypoxia maintains or increases systemic cholesterol levels, two hypoxic mouse models were assessed, an acute lung injury model and mice exposed to 10% O2 for 3 weeks. In both models, cholesterol levels increased with a concomitant decrease in expression of genes involved in bile acid synthesis. The present study demonstrates that hypoxic activation of hepatic HIF-2α leads to an adaptive increase in cholesterol levels through inhibition of bile acid synthesis.
doi:10.1128/MCB.01441-13
PMCID: PMC3993569  PMID: 24421394
25.  Physiological control of NKT cell-dependent hepatitis induction by extracellular adenosine 
European journal of immunology  2014;44(4):1119-1129.
Summary
Extracellular adenosine regulates inflammatory responses via A2A adenosine receptor (A2AR). A2AR-deficiency results in much exaggerated acute hepatitis, indicating non-redundancy of adenosine-A2AR pathway in inhibitory mechanisms of immune activation. To identify a critical target of immunoregulatory effect of extracellular adenosine, we focused on NKT cells, which play an indispensable role in hepatitis. A2AR agonist abolished NKT cell-dependent induction of acute hepatitis by Con A or α-galactosylceramide (α-GalCer), corresponding to down-regulation of activation markers and cytokines in NKT cells and of NK cell co-activation. These results show that A2AR signaling can down-regulate NKT cell activation and suppress NKT cell-triggered inflammatory responses. Next, we hypothesized that NKT cells might be under physiological control of the adenosine-A2AR pathway. Indeed, both Con A and α-GalCer induced more severe hepatitis in A2AR−/− mice than in wild-type controls. Transfer of A2AR−/− NKT cells into A2AR-expressing recipients resulted in exaggeration of Con A-induced liver damage, suggesting that NKT cell activation is controlled by endogenous adenosine via A2AR, and this physiological regulatory mechanism of NKT cells is critical in the control of tissue-damaging inflammation. The current study suggests the possibility to manipulate NKT cell activity in inflammatory disorders through intervention to the adenosine-A2AR pathway.
doi:10.1002/eji.201343866
PMCID: PMC4482763  PMID: 24448964
NKT cell; adenosine; A2A adenosine receptor; hepatitis; immunoregulation

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