lisa geary - Academia.edu (original) (raw)
Papers by lisa geary
Journal of Biological Chemistry, May 1, 2011
Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved fr... more Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ϳ2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-tofive-coordinate heme pocket control. We hypothesize that the sixcoordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.
Blood, May 1, 2014
• Sickle cell patients show mitochondrial dysfunction (complex V inhibition, oxidant formation), ... more • Sickle cell patients show mitochondrial dysfunction (complex V inhibition, oxidant formation), which is associated with platelet activation. • Complex V inhibition is induced by hemolysis and causes platelet activation, which is attenuated by mitochondrial therapeutics. Bioenergetic dysfunction, although central to the pathogenesis of numerous diseases, remains uncharacterized in many patient populations because of the invasiveness of obtaining tissue for mitochondrial studies. Although platelets are an accessible source of mitochondria, the role of bioenergetics in regulating platelet function remains unclear. Herein, we validate extracellular flux analysis in human platelets and use this technique to screen for mitochondrial dysfunction in sickle cell disease (SCD) patients, a population with aberrant platelet activation of an unknown mechanism and in which mitochondrial function has never been assessed. We identify a bioenergetic alteration in SCD patients characterized by deficient complex V activity, leading to decreased mitochondrial respiration, membrane hyperpolarization, and augmented oxidant production compared with healthy subjects. This dysfunction correlates with platelet activation and hemolysis in vivo and can be recapitulated in vitro by exposing healthy platelets to hemoglobin or a complex V inhibitor. Further, reproduction of this dysfunction in vitro activates healthy platelets, an effect prevented by attenuation of mitochondrial hyperpolarization or by scavenging mitochondrial oxidants. These data identify bioenergetic dysfunction in SCD patients for the first time and establish mitochondrial hyperpolarization and oxidant generation as potential pathogenic mechanism in SCD as well as a modulator of healthy platelet function.
Free Radical Biology and Medicine, Nov 1, 2012
Free Radical Biology and Medicine, Nov 1, 2012
Nitric Oxide
Background Nitrite has been demonstrated to confer potent cardioprotection in animal models of ac... more Background Nitrite has been demonstrated to confer potent cardioprotection in animal models of acute myocardial infarction. Notably, administration of exogenous nitrite or elevations in endogenous nitrite during ischemia, or hours to days prior to the ischemic episode, significantly decrease infarct size after ischemia/reperfusion (I/R). When present during ischemia, the reduction of nitrite to bioactive NO by deoxygenated heme proteins accounts for its protective effects. However, the mechanism underlying normoxic nitrite-induced ischemic tolerance remains unexplored. Methods We established an in vitro cell model of hypoxia/reoxygenation (H/R) using H9c2 cardiomyocytes. Cells were treated with nitrite (10–100 μM; 30 min; 21% O2), after which nitrite was removed, and cells incubated in normoxia for 5 min to 72 h prior to initiation of an ischemic episode (1% O2, pH 6.2) and reperfusion. Results Transient (30 min) normoxic nitrite treatment significantly attenuated cell death after H...
Journal of Biological Chemistry, 2011
Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved fr... more Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ϳ2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-tofive-coordinate heme pocket control. We hypothesize that the sixcoordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.
Free Radical Biology and Medicine, 2012
Cardiovascular Research, 2013
Methods and results Using a model of hypoxia/reoxygenation (H/R) in cultured rat H9c2 cardiomyocy... more Methods and results Using a model of hypoxia/reoxygenation (H/R) in cultured rat H9c2 cardiomyocytes, we demonstrate that a transient (30 min) normoxic nitrite treatment significantly attenuates cell death after a hypoxic episode initiated 1 h later. Mechanistically, this protection depends on the activation of protein kinase A, which phosphorylates and inhibits dynaminrelated protein 1, the predominant regulator of mitochondrial fission. This results morphologically, in the promotion of mitochondrial fusion and functionally in the augmentation of mitochondrial membrane potential and superoxide production. We identify AMP kinase (AMPK) as a downstream target of the mitochondrial reactive oxygen species (ROS) generated and show that its oxidation and subsequent phosphorylation are essential for cytoprotection, as scavenging of ROS prevents AMPK activation and inhibits nitrite-mediated protection after H/R. The protein kinase A-dependent protection mediated by nitrite is reproduced in an intact isolated rat heart model of I/R. Conclusions These data are the first to demonstrate nitrite-dependent normoxic modulation of both mitochondrial morphology and function and reveal a novel signalling pathway responsible for nitrite-mediated cardioprotection.
Blood, 2014
Key PointsSickle cell patients show mitochondrial dysfunction (complex V inhibition, oxidant form... more Key PointsSickle cell patients show mitochondrial dysfunction (complex V inhibition, oxidant formation), which is associated with platelet activation. Complex V inhibition is induced by hemolysis and causes platelet activation, which is attenuated by mitochondrial therapeutics.
Antioxidants & Redox Signaling, 2012
Aims: Once dismissed as an inert byproduct of nitric oxide (NO) auto-oxidation, nitrite (NO 2-) i... more Aims: Once dismissed as an inert byproduct of nitric oxide (NO) auto-oxidation, nitrite (NO 2-) is now accepted as an endocrine reservoir of NO that elicits biological responses in major organs. While it is known that tissue nitrite is derived from NO oxidation and the diet, little is known about how nitrite is metabolized by tissue, particularly at intermediate oxygen tensions. We investigated the rates and mechanisms of tissue nitrite metabolism over a range of oxygen concentrations. Results: We show that the rate of nitrite consumption differs in each organ. Further, oxygen regulates the rate and products of nitrite metabolism. In anoxia, nitrite is reduced to NO, with significant formation of iron-nitrosyl proteins and S-nitrosothiols. This hypoxic nitrite metabolism is mediated by different nitrite reductases in each tissue. In contrast, low concentrations (*3.5 lM) of oxygen increase the rate of nitrite consumption by shifting nitrite metabolism to oxidative pathways, yielding nitrate. While cytochrome P 450 and myoglobin contribute in the liver and heart, respectively, mitochondrial cytochrome c oxidase plays a significant role in nitrite oxidation, which is inhibited by cyanide. Using cyanide to prevent artifactual nitrite decay, we measure metabolism of oral and intraperitoneally administered nitrite in mice. Innovation: These data provide insight into the fate of nitrite in tissue, the enzymes involved in nitrite metabolism, and the role of oxygen in regulating these processes. Conclusion: We demonstrate that even at low concentrations, oxygen is a potent regulator of the rate and products of tissue nitrite metabolism. Antioxid. Redox Signal. 17, 951-961.
Free Radical Biology and Medicine, 2012
Nitrite, a dietary constituent and endogenous signaling molecule, mediates a number of physiologi... more Nitrite, a dietary constituent and endogenous signaling molecule, mediates a number of physiological responses including modulation of ischemia/reperfusion injury, glucose tolerance, and vascular remodeling. Although the exact molecular mechanisms underlying nitrite's actions are unknown, the current paradigm suggests that these effects depend on the hypoxic reduction of nitrite to nitric oxide (NO). Mitochondrial biogenesis is a fundamental mechanism of cellular adaptation and repair. However, the effect of nitrite on mitochondrial number has not been explored. Herein, we report that nitrite stimulates mitochondrial biogenesis through a mechanism distinct from that of NO. We demonstrate that nitrite significantly increases cellular mitochondrial number by augmenting the activity of adenylate kinase, resulting in AMP kinase phosphorylation, downstream activation of sirtuin-1, and deacetylation of PGC1a, the master regulator of mitochondrial biogenesis. Unlike NO, nitrite-mediated biogenesis does not require the activation of soluble guanylate cyclase and results in the synthesis of more functionally efficient mitochondria. Further, we provide evidence that nitrite mediates biogenesis in vivo. In a rat model of carotid injury, 2 weeks of continuous oral nitrite treatment postinjury prevented the hyperproliferative response of smooth muscle cells. This protection was accompanied by a nitrite-dependent upregulation of PGC1a and increased mitochondrial number in the injured artery. These data are the first to demonstrate that nitrite mediates differential signaling compared to NO. They show that nitrite is a versatile regulator of mitochondrial function and number both in vivo and in vitro and suggest that nitrite-mediated biogenesis may play a protective role in the setting of vascular injury.
Blood
5310 Once dismissed as an inert byproduct of nitric oxide (NO) auto-oxidation, nitrite (NO2−) is ... more 5310 Once dismissed as an inert byproduct of nitric oxide (NO) auto-oxidation, nitrite (NO2−) is now accepted as an endocrine reserve of NO that elicits a number of fundamental biological responses in all major organ systems. While it is known that tissue nitrite is derived from both oxidation of NO and from dietary nitrite and nitrate, much less is known about how nitrite is metabolized by tissue or about the factors that influence this metabolism. Here we investigate the rates and mechanisms by which nitrite is metabolized by tissue over a range of oxygen tensions in rats and mice. We show that the rate of nitrite metabolism differs in heart, liver, lung and brain tissue. Further, oxygen regulates the rate and products of nitrite metabolism in each of these tissues. In hypoxic tissue, nitrite is predominantly reduced to NO, with significant formation of iron-nitrosyl heme proteins and S-nitrosothiols. Interestingly, this hypoxic nitrite metabolism is mediated by different sets of ...
Free Radical Biology and Medicine, 2010
Free Radical Biology and Medicine, 2010
Free Radical Biology and Medicine, 2010
Journal of Experimental Medicine, 2009
), previously viewed as a physiologically inert metabolite and biomarker of the endogenous vaso... more ), previously viewed as a physiologically inert metabolite and biomarker of the endogenous vasodilator NO, was recently identified as an important biological NO reservoir in vasculature and tissues, where it contributes to hypoxic signaling, vasodilation, and cytoprotection after ischemia-reperfusion injury. Reduction of nitrite to NO may occur enzymatically at low pH and oxygen tension by deoxyhemoglobin, deoxymyoglobin, xanthine oxidase, mitochondrial complexes, or NO synthase (NOS). We show that nitrite treatment, in sharp contrast with the worsening effect of NOS inhibition, significantly attenuates hypothermia, mitochondrial damage, oxidative stress and dysfunction, tissue infarction, and mortality in a mouse shock model induced by a lethal tumor necrosis factor challenge. Mechanistically, nitrite-dependent protection was not associated with inhibition of mitochondrial complex I activity, as previously demonstrated for ischemiareperfusion, but was largely abolished in mice deficient for the soluble guanylate cyclase (sGC) 1 subunit, one of the principal intracellular NO receptors and signal transducers in the cardiovasculature. Nitrite could also provide protection against toxicity induced by Gram-negative lipopolysaccharide, although higher doses were required. In conclusion, we show that nitrite can protect against toxicity in shock via sGC-dependent signaling, which may include hypoxic vasodilation necessary to maintain microcirculation and organ function, and cardioprotection.
Free Radical Biology and Medicine, 2011
Free Radical Biology and Medicine, 2011
in addition, two functional polymorphisms C-1306T and C735T of MMP-2 may affect the expression an... more in addition, two functional polymorphisms C-1306T and C735T of MMP-2 may affect the expression and activity level of this enzyme. We investigated whether these polymorphisms and oxidative stress affect the circulating levels of MMP-2 in obese and healthy children and adolescents. Methods: We studied 102 obese children and adolescents and 122 healthy children and adolescents. Genomic DNA was extracted from whole blood and genotypes for C-1306T and C735T polymorphisms were determined. MMP-2 levels were measured in plasma samples by zymography and oxidative stress by TBARs (Thio-barbituric Acid Reactive Species) levels. Results: Although obese children and adolescents have increased circulating TBARs levels (P<0.05), we found no differences in MMP-2 between groups, and circulating TBARs levels were not correlated with MMP-2 in the childhood obesity (P>0.05). Interestingly, circulating TBARs levels was positively correlated with waist-to-hip ratio (r=0.38; P<0.001) and BMI (r=0.54; P<0.001) in obese children and adolescents. We found no significant difference in the genotype and allelic distribution for the two polymorphisms when obese children and adolescents were compared with control group, and there was not influence of MMP-2 genotypes on plasma MMP-2 levels in both groups (p>0.05). Conclusion: Oxidative stress levels and matrix metalloproteinase-2 (MMP-2) genetic variations do not affect plasma MMP-2 levels in obese children and adolescents. However, we found evidence indicating higher oxidative stress in obese and adolescents children, and was positively correlated with BMI and waist-to-hip ratio in obese. These findings suggest a possibly relevant pathophysiological mechanism that may be involved in the increase of cardiovascular risk associated with childhood obesity. Support: FAPESP and CNPq.
Nitric Oxide-biology and Chemistry, 2011
Journal of Biological Chemistry, May 1, 2011
Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved fr... more Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ϳ2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-tofive-coordinate heme pocket control. We hypothesize that the sixcoordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.
Blood, May 1, 2014
• Sickle cell patients show mitochondrial dysfunction (complex V inhibition, oxidant formation), ... more • Sickle cell patients show mitochondrial dysfunction (complex V inhibition, oxidant formation), which is associated with platelet activation. • Complex V inhibition is induced by hemolysis and causes platelet activation, which is attenuated by mitochondrial therapeutics. Bioenergetic dysfunction, although central to the pathogenesis of numerous diseases, remains uncharacterized in many patient populations because of the invasiveness of obtaining tissue for mitochondrial studies. Although platelets are an accessible source of mitochondria, the role of bioenergetics in regulating platelet function remains unclear. Herein, we validate extracellular flux analysis in human platelets and use this technique to screen for mitochondrial dysfunction in sickle cell disease (SCD) patients, a population with aberrant platelet activation of an unknown mechanism and in which mitochondrial function has never been assessed. We identify a bioenergetic alteration in SCD patients characterized by deficient complex V activity, leading to decreased mitochondrial respiration, membrane hyperpolarization, and augmented oxidant production compared with healthy subjects. This dysfunction correlates with platelet activation and hemolysis in vivo and can be recapitulated in vitro by exposing healthy platelets to hemoglobin or a complex V inhibitor. Further, reproduction of this dysfunction in vitro activates healthy platelets, an effect prevented by attenuation of mitochondrial hyperpolarization or by scavenging mitochondrial oxidants. These data identify bioenergetic dysfunction in SCD patients for the first time and establish mitochondrial hyperpolarization and oxidant generation as potential pathogenic mechanism in SCD as well as a modulator of healthy platelet function.
Free Radical Biology and Medicine, Nov 1, 2012
Free Radical Biology and Medicine, Nov 1, 2012
Nitric Oxide
Background Nitrite has been demonstrated to confer potent cardioprotection in animal models of ac... more Background Nitrite has been demonstrated to confer potent cardioprotection in animal models of acute myocardial infarction. Notably, administration of exogenous nitrite or elevations in endogenous nitrite during ischemia, or hours to days prior to the ischemic episode, significantly decrease infarct size after ischemia/reperfusion (I/R). When present during ischemia, the reduction of nitrite to bioactive NO by deoxygenated heme proteins accounts for its protective effects. However, the mechanism underlying normoxic nitrite-induced ischemic tolerance remains unexplored. Methods We established an in vitro cell model of hypoxia/reoxygenation (H/R) using H9c2 cardiomyocytes. Cells were treated with nitrite (10–100 μM; 30 min; 21% O2), after which nitrite was removed, and cells incubated in normoxia for 5 min to 72 h prior to initiation of an ischemic episode (1% O2, pH 6.2) and reperfusion. Results Transient (30 min) normoxic nitrite treatment significantly attenuated cell death after H...
Journal of Biological Chemistry, 2011
Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved fr... more Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ϳ2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-tofive-coordinate heme pocket control. We hypothesize that the sixcoordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.
Free Radical Biology and Medicine, 2012
Cardiovascular Research, 2013
Methods and results Using a model of hypoxia/reoxygenation (H/R) in cultured rat H9c2 cardiomyocy... more Methods and results Using a model of hypoxia/reoxygenation (H/R) in cultured rat H9c2 cardiomyocytes, we demonstrate that a transient (30 min) normoxic nitrite treatment significantly attenuates cell death after a hypoxic episode initiated 1 h later. Mechanistically, this protection depends on the activation of protein kinase A, which phosphorylates and inhibits dynaminrelated protein 1, the predominant regulator of mitochondrial fission. This results morphologically, in the promotion of mitochondrial fusion and functionally in the augmentation of mitochondrial membrane potential and superoxide production. We identify AMP kinase (AMPK) as a downstream target of the mitochondrial reactive oxygen species (ROS) generated and show that its oxidation and subsequent phosphorylation are essential for cytoprotection, as scavenging of ROS prevents AMPK activation and inhibits nitrite-mediated protection after H/R. The protein kinase A-dependent protection mediated by nitrite is reproduced in an intact isolated rat heart model of I/R. Conclusions These data are the first to demonstrate nitrite-dependent normoxic modulation of both mitochondrial morphology and function and reveal a novel signalling pathway responsible for nitrite-mediated cardioprotection.
Blood, 2014
Key PointsSickle cell patients show mitochondrial dysfunction (complex V inhibition, oxidant form... more Key PointsSickle cell patients show mitochondrial dysfunction (complex V inhibition, oxidant formation), which is associated with platelet activation. Complex V inhibition is induced by hemolysis and causes platelet activation, which is attenuated by mitochondrial therapeutics.
Antioxidants & Redox Signaling, 2012
Aims: Once dismissed as an inert byproduct of nitric oxide (NO) auto-oxidation, nitrite (NO 2-) i... more Aims: Once dismissed as an inert byproduct of nitric oxide (NO) auto-oxidation, nitrite (NO 2-) is now accepted as an endocrine reservoir of NO that elicits biological responses in major organs. While it is known that tissue nitrite is derived from NO oxidation and the diet, little is known about how nitrite is metabolized by tissue, particularly at intermediate oxygen tensions. We investigated the rates and mechanisms of tissue nitrite metabolism over a range of oxygen concentrations. Results: We show that the rate of nitrite consumption differs in each organ. Further, oxygen regulates the rate and products of nitrite metabolism. In anoxia, nitrite is reduced to NO, with significant formation of iron-nitrosyl proteins and S-nitrosothiols. This hypoxic nitrite metabolism is mediated by different nitrite reductases in each tissue. In contrast, low concentrations (*3.5 lM) of oxygen increase the rate of nitrite consumption by shifting nitrite metabolism to oxidative pathways, yielding nitrate. While cytochrome P 450 and myoglobin contribute in the liver and heart, respectively, mitochondrial cytochrome c oxidase plays a significant role in nitrite oxidation, which is inhibited by cyanide. Using cyanide to prevent artifactual nitrite decay, we measure metabolism of oral and intraperitoneally administered nitrite in mice. Innovation: These data provide insight into the fate of nitrite in tissue, the enzymes involved in nitrite metabolism, and the role of oxygen in regulating these processes. Conclusion: We demonstrate that even at low concentrations, oxygen is a potent regulator of the rate and products of tissue nitrite metabolism. Antioxid. Redox Signal. 17, 951-961.
Free Radical Biology and Medicine, 2012
Nitrite, a dietary constituent and endogenous signaling molecule, mediates a number of physiologi... more Nitrite, a dietary constituent and endogenous signaling molecule, mediates a number of physiological responses including modulation of ischemia/reperfusion injury, glucose tolerance, and vascular remodeling. Although the exact molecular mechanisms underlying nitrite's actions are unknown, the current paradigm suggests that these effects depend on the hypoxic reduction of nitrite to nitric oxide (NO). Mitochondrial biogenesis is a fundamental mechanism of cellular adaptation and repair. However, the effect of nitrite on mitochondrial number has not been explored. Herein, we report that nitrite stimulates mitochondrial biogenesis through a mechanism distinct from that of NO. We demonstrate that nitrite significantly increases cellular mitochondrial number by augmenting the activity of adenylate kinase, resulting in AMP kinase phosphorylation, downstream activation of sirtuin-1, and deacetylation of PGC1a, the master regulator of mitochondrial biogenesis. Unlike NO, nitrite-mediated biogenesis does not require the activation of soluble guanylate cyclase and results in the synthesis of more functionally efficient mitochondria. Further, we provide evidence that nitrite mediates biogenesis in vivo. In a rat model of carotid injury, 2 weeks of continuous oral nitrite treatment postinjury prevented the hyperproliferative response of smooth muscle cells. This protection was accompanied by a nitrite-dependent upregulation of PGC1a and increased mitochondrial number in the injured artery. These data are the first to demonstrate that nitrite mediates differential signaling compared to NO. They show that nitrite is a versatile regulator of mitochondrial function and number both in vivo and in vitro and suggest that nitrite-mediated biogenesis may play a protective role in the setting of vascular injury.
Blood
5310 Once dismissed as an inert byproduct of nitric oxide (NO) auto-oxidation, nitrite (NO2−) is ... more 5310 Once dismissed as an inert byproduct of nitric oxide (NO) auto-oxidation, nitrite (NO2−) is now accepted as an endocrine reserve of NO that elicits a number of fundamental biological responses in all major organ systems. While it is known that tissue nitrite is derived from both oxidation of NO and from dietary nitrite and nitrate, much less is known about how nitrite is metabolized by tissue or about the factors that influence this metabolism. Here we investigate the rates and mechanisms by which nitrite is metabolized by tissue over a range of oxygen tensions in rats and mice. We show that the rate of nitrite metabolism differs in heart, liver, lung and brain tissue. Further, oxygen regulates the rate and products of nitrite metabolism in each of these tissues. In hypoxic tissue, nitrite is predominantly reduced to NO, with significant formation of iron-nitrosyl heme proteins and S-nitrosothiols. Interestingly, this hypoxic nitrite metabolism is mediated by different sets of ...
Free Radical Biology and Medicine, 2010
Free Radical Biology and Medicine, 2010
Free Radical Biology and Medicine, 2010
Journal of Experimental Medicine, 2009
), previously viewed as a physiologically inert metabolite and biomarker of the endogenous vaso... more ), previously viewed as a physiologically inert metabolite and biomarker of the endogenous vasodilator NO, was recently identified as an important biological NO reservoir in vasculature and tissues, where it contributes to hypoxic signaling, vasodilation, and cytoprotection after ischemia-reperfusion injury. Reduction of nitrite to NO may occur enzymatically at low pH and oxygen tension by deoxyhemoglobin, deoxymyoglobin, xanthine oxidase, mitochondrial complexes, or NO synthase (NOS). We show that nitrite treatment, in sharp contrast with the worsening effect of NOS inhibition, significantly attenuates hypothermia, mitochondrial damage, oxidative stress and dysfunction, tissue infarction, and mortality in a mouse shock model induced by a lethal tumor necrosis factor challenge. Mechanistically, nitrite-dependent protection was not associated with inhibition of mitochondrial complex I activity, as previously demonstrated for ischemiareperfusion, but was largely abolished in mice deficient for the soluble guanylate cyclase (sGC) 1 subunit, one of the principal intracellular NO receptors and signal transducers in the cardiovasculature. Nitrite could also provide protection against toxicity induced by Gram-negative lipopolysaccharide, although higher doses were required. In conclusion, we show that nitrite can protect against toxicity in shock via sGC-dependent signaling, which may include hypoxic vasodilation necessary to maintain microcirculation and organ function, and cardioprotection.
Free Radical Biology and Medicine, 2011
Free Radical Biology and Medicine, 2011
in addition, two functional polymorphisms C-1306T and C735T of MMP-2 may affect the expression an... more in addition, two functional polymorphisms C-1306T and C735T of MMP-2 may affect the expression and activity level of this enzyme. We investigated whether these polymorphisms and oxidative stress affect the circulating levels of MMP-2 in obese and healthy children and adolescents. Methods: We studied 102 obese children and adolescents and 122 healthy children and adolescents. Genomic DNA was extracted from whole blood and genotypes for C-1306T and C735T polymorphisms were determined. MMP-2 levels were measured in plasma samples by zymography and oxidative stress by TBARs (Thio-barbituric Acid Reactive Species) levels. Results: Although obese children and adolescents have increased circulating TBARs levels (P<0.05), we found no differences in MMP-2 between groups, and circulating TBARs levels were not correlated with MMP-2 in the childhood obesity (P>0.05). Interestingly, circulating TBARs levels was positively correlated with waist-to-hip ratio (r=0.38; P<0.001) and BMI (r=0.54; P<0.001) in obese children and adolescents. We found no significant difference in the genotype and allelic distribution for the two polymorphisms when obese children and adolescents were compared with control group, and there was not influence of MMP-2 genotypes on plasma MMP-2 levels in both groups (p>0.05). Conclusion: Oxidative stress levels and matrix metalloproteinase-2 (MMP-2) genetic variations do not affect plasma MMP-2 levels in obese children and adolescents. However, we found evidence indicating higher oxidative stress in obese and adolescents children, and was positively correlated with BMI and waist-to-hip ratio in obese. These findings suggest a possibly relevant pathophysiological mechanism that may be involved in the increase of cardiovascular risk associated with childhood obesity. Support: FAPESP and CNPq.
Nitric Oxide-biology and Chemistry, 2011