- Email: [email protected]
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Abstracts / Nitric Oxide 31 (2013) S13–S48
to generate new cardiac muscle. We investigated the effects of hypoxia and nitrite on zebrafish heart regeneration and found that low concentrations (10 lM) of nitrite reduced scar tissue at 5 and 10 days post cryoinjury. Exposure to nitrite under normoxia or to hypoxia alone had no positive effects on regeneration. However simultaneous exposure to nitrite and hypoxia resulted in an increase of cell proliferation showing a positive interaction of hypoxia and nitrite. A specific increase in proliferating cardiomyocytes is detectable at 5 days after cryoinjury and amputation. These studies show that physiological levels of nitrite improve cardiomyocyte proliferation and cardiac regeneration following cardiac injury in the zebrafish. Disclosure: Nothing to disclose.
mediated through the G protein beta 2 receptor (Gb2), as nitriteinduced phosphorylation of Drp1 was attenuated in the presence of the selective Gb2 inhibitor, ICI 118,551. Conclusions: These data are the first to elucidate a mechanism by which nitrite confers ischemic resilience priorto I/R and are the first to report normoxic NO2 dependent activation of PKA and regulation of mitochondrial dynamics. Disclosure: This work was supported by the Institute of Transfusion Medicine and the Hemophilia Center of Western Pennsylvania, the NIH (1R01HL096973 to SS; R01NS065789 to CTC) and by the American Heart Association (09SDG2150066 to SS). http://dx.doi.org/10.1016/j.niox.2013.02.045
http://dx.doi.org/10.1016/j.niox.2013.02.044
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Nitrite activates protein kinase A in normoxia to promote mitochondrial fusion and confer delayed tolerance to ischemia/ reperfusion
Nitric oxide may modulate matrix metalloproteinase-2 during renovascular hypertension
a
b
c
b
Christelle Kamga , Li Mo , Ruben K. Dagda , Lisa Geary , Catherine Corey b, Sergey Zharikov b, Claudette St Croix d, Donna Beer-Stolz d, Nicholas Khoo e, Sruti Shiva e a University of Pittsburgh, Pittsburgh, PA, United States b University of Pittsburgh, Vascular Medicine Institute, Pittsburgh, PA, United States c University of Pittsburgh, Department of Pathology, Pittsburgh, PA, United States d University of Pittsburgh, Center for Biologic Imaging, Pittsburgh, PA, United States e University of Pittsburgh, Department of Pharmacology and Chemical Biology, Pittsburgh, PA, United States 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 nitriteinduced 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 lM; 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/R initiated 5 min–72 h later. Mechanistically, nitrite-mediated cytoprotection was dependent on increased activity of protein kinase A (PKA; 157.8 ± 23.8% of control), which propagated mitochondrial fusion through the phosphorylation and inhibition of dynamin related protein 1 (Drp1). This morphological change in mitochondrial phenotype led to a functional change characterized by a slight increase in mitochondrial superoxide generation (157.2 ± 16.3% of control). We show that this superoxide generated is responsible for the oxidation and subsequent activation of the cardioprotective mediator AMP kinase (214.7 ± 28.8% of control), as scavenging of reactive oxygen species by the mitochondrially targeted scavenger mitoTEMPO significantly abrogated AMPK activation and cytoprotection after H/R. Ongoing studies are investigating the mechanism by which nitrite modulates PKA activity. Preliminary data suggest that nitrite signaling may be
Danielle Guimaraes a, Jefferson Amaral a, Elen Rizzi a, Lucas Cesar Pinheiro a, Raquel Fernanda Gerlach b, Jose Eduardo Tanus-Santos a a FMRP/University of Sao Paulo, Ribeirao Preto, Brazil b FORP/University of Sao Paulo, Ribeirao Preto, Brazil Decreased nitric oxide (NO) bioavailability and up-regulated MMP activity contribute to hypertension-induced cardiovascular hypertrophy. However, it is uncertain whether NO affects MMP activity. Dietary nitrite is an alternative source of NO and may exert antioxidant effects, thus affecting up-regulated MMP hypertension. We evaluated whether sodium nitrite treatment reduces cardiac MMP in 2-kidney, 1-clip (2K1C) hypertension. Sham and 2K1C rats were treated with vehicle or sodium nitrite (15 mg/kg orally) for 4 weeks. Systolic blood pressure was monitored weekly. Plasma nitrite concentrations were analyzed by chemiluminescence. Cardiac MMP level/activity were determined by gelatin and in situ zymography. Sodium nitrite treatment exerted antihypertensive effects in 2K1C rats (P < 0.05). We found lower plasma nitrite concentrations in 2K1C rats when compared with the Sham group (P < 0.05), and nitrite treatment restored these levels in 2K1C rats. 2K1C hypertension increased cardiac MMP-2 levels and MMP activity (P < 0.05). Sodium nitrite treatment reduced hypertension-induced increases in both MMP levels and activity (P < 0.05). Our results suggest that sodium nitrite treatment prevents 2K1C hypertension-induced MMP up-regulation, and therefore may prevent cardiac remodeling associated with hypertension. Supported by: FAPESP, CNPq. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2013.02.046
P45 Enhanced nitrite reaction rates of MP4OX, a novel ischemic rescue therapy Scott D. Olsen, Ashok Malavalli, Kim Vandegriff Sangart Inc., San Diego, CA, United States Background: Hemoglobin-based oxygen carriers have been associated with vasoconstriction, attributed primarily to binding of nitric oxide (NO) as a heme ligand. MP4OX, an ischemic rescue therapy, designed with high O2 affinity using poly (ethylene glycol) (PEG)
Abstracts / Nitric Oxide 31 (2013) S13–S48
conjugation chemistry, has been observed not to cause vasoconstriction and to preserve microvascular blood flow, despite reducing perivascular levels of NO [1]. Recent evidence shows that reduction of nitrite to NO by deoxyhemoglobin has the ability to vasodilate blood vessels [2]. It also has been reported that the oxidative reaction of oxyhemoglobin with nitrite may facilitate the release of NO from heme [3]. We hypothesized that MP4OX may have enhanced reaction rates with nitrite and regeneration of NO, which may contribute to its lack of systemic vasoconstriction. Materials and methods: MP4OX was prepared by reacting human stroma-free hemoglobin (SFH) with 2-iminothiolane and maleimide-activated PEG to produce a PEG-hemoglobin conjugate, with approximately 8 PEGs/Hb. Rates of the deoxyHb/nitrite reaction were measured by mixing deoxygenated SFH or MP4OX anaerobically with a 10-fold excess of sodium nitrite in a sealed cuvette in the presence of sodium dithionite. The reaction was monitored spectrophotometrically, and the spectra were deconvoluted using parent spectra for deoxyhemoglobin, iron-nitrosyl-hemoglobin (HbNO), and methemoglobin. Rates of the oxyHb/nitrite reaction were measured by mixing oxygenated SFH or MP4 with a 20-fold excess of nitrite. These spectra were deconvoluted using parent spectra for oxyhemoglobin, methemoglobin, and nitrite-bound methemoglobin. Results: Time courses for both deoxy-and oxyHb reactions showed autocatalytic properties that deviated from pseudo first-order kinetics. Therefore, the maximum reaction rates were used for comparison purposes. Fully deoxygenated SFH and MP4OX (i.e., MP4DX) reduced nitrite to NO with maximum rates of 0.022 and 0.206 lM/s, respectively, showing a nearly 10-fold higher rate for MP4DX compared to deoxySFH. For the oxyHb/nitrite reaction, SFH and MP4OX showed maximum rates of 0.55 and 0.92 lM/s, respectively, a nearly 2-fold enhancement for MP4OX over SFH. Conclusions: Our results demonstrate that deoxygenated MP4OX is more effective at reducing nitrite to NO than unmodified Hb. In addition, oxygenated MP4OX reacts more rapidly with nitrite, which may then accelerate the release of NO from heme sites. Taken together, these enhanced nitrite reaction rates for MP4 may compensate for NO scavenging and explain, at least in part, why MP4OX does not induce vasoconstriction in vivo. The increased reaction rates of MP4OX appear to be due to R-state stabilization due to the PEG conjugation. Further studies are required to understand the extent to which MP4OX’s interactions with nitrite contribute to its ability to improve perfusion and oxygenation of ischemic tissues. Disclosure: This work was funded in part by Sangart Inc. and under US Army Contract No. W81XWH1020111 to Sangart. References [1] Tsai et al., Blood 108 (2006) 3603. [2] Cosby et al., Nat. Med. 9 (2003) 1498. [3] Grubina et al., J. Biol. Chem. 282 (2007) 17. http://dx.doi.org/10.1016/j.niox.2013.02.047
P46 Low sodium nitrite (but not nitrate) concentrations attenuate MMP-9 production by endothelial cells Cesar Arruda Meschiari a, Raquel Fernanda Gerlach b, Jose Eduardo Tanus-Santos a a Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil b Dental School of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil
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Reduced nitric oxide (NO) bioavailability and imbalanced matrix metalloproteinase (MMP) activity have important roles in the pathogenesis of cardiovascular diseases, and some NO donors were shown to downregulate MMP expression in some cell types. However, while nitrite and nitrate can be recycled back to NO by non enzymatic and enzymatic mechanisms as an alternative to NO formation from L-arginine, no previous study has examined whether sodium nitrite can downregulate stimulated MMP release by endothelial cells. Objective: We examined the effect of sodium nitrite and sodium nitrate on MMP-9 production by endothelial cells. Methods: Human umbilical vein endothelial cells were cultured in a modified DMEM (iron(III) nitrate is replaced by iron(III) sulfate) and treated for 24 h with 10 nM phorbol myristate acetate (PMA; a MMP-9 inducer) and sodium nitrite or sodium nitrate, or their vehicles. Conditioned medium were analyzed by gelatin zimography to assess MMP-9 activity. Results: PMA increased MMP-9 activity from 0.02 ± 0.01 arbitrary units (AU) to 1.14 ± 0.34 AU (P < 0.05). While low nitrite concentrations (0.2 or 2 lM) attenuated the increases in MMP-9 activity induced by PMA (0.77 ± 0.12 AU or 0.71 ± 0.14 AU, respectively; both P < 0.05 vs. PMA), high nitrite concentrations (10 or 20 lM) had no effects on PMA-induced increases in MMP-9 activity (0.88 ± 0.14 AU or 1.18 ± 0.36 AU, respectively; both P > 0.05 vs. PMA). Both low (10 or 100 lM) or high (200 or 400 lM) nitrate concentrations had no effects on PMA-induced increases in MMP-9 activity PMA (P > 0.05 vs. PMA). Conclusion: Our results suggest that low nitrite (but not nitrate) concentrations attenuate MMP-9 production by endothelial cells. Financial support: FAPESP, CNPq, FAEPA. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2013.02.048
P47 AMPK activation by nitrite and metformin increases glut-4 mediated glucose uptake and normalizes pulmonary venous hypertension in a rat model of severe metabolic syndrome Yen Chun Lai a, Diana M. Tabima a,b, Jeff B. Baust a, John J. Dube c, Alexander Chacon c, Juan C. Alvarez-Perez d, Bret H. Goodpaster c, Adolfo Garcia-Ocaña d, Stevan P. Tofovic a,e, Ana L. Mora a,e, Mark T. Gladwin a,e a University of Pittsburgh, Vascular Medicine Institute, Pittsburgh, PA, United States b University de Los Andes, Department of Biomedical Engineering, Bogota-Colombia, Colombia c University of Pittsburgh, Division of Endocrinology and Metabolism, Department of Medicine, Pittsburgh, PA, United States d Diabetes, Obesity and Metabolism Institute, The Mount Sinai Medical Center, New York City, NY, United States e University of Pittsburgh, Pulmonary, Allergy and Critical Care Medicine, Pittsburgh, PA, United States Metabolic syndrome is associated with decreased bioavailability of nitric oxide (NO) and has recently been defined as a risk factor for the development of pulmonary venous hypertension in both humans and animal models. Nitrite, an endogenous circulating oxidation product of NO, has been recognized as an important signaling molecule that regulates blood flow and blood pressure, and exhibits therapeutic efficacy in pre-clinical models of pulmonary hyperten sion (PH). Nitrite NO2 is formed from the reduction of nitrate NO3 by oral commensal bacteria and nitrate has recently been reported to improve metabolic syndrome in the eNOS knock out
Abstracts / Nitric Oxide 31 (2013) S13–S48
to generate new cardiac muscle. We investigated the effects of hypoxia and nitrite on zebrafish heart regeneration and found that low concentrations (10 lM) of nitrite reduced scar tissue at 5 and 10 days post cryoinjury. Exposure to nitrite under normoxia or to hypoxia alone had no positive effects on regeneration. However simultaneous exposure to nitrite and hypoxia resulted in an increase of cell proliferation showing a positive interaction of hypoxia and nitrite. A specific increase in proliferating cardiomyocytes is detectable at 5 days after cryoinjury and amputation. These studies show that physiological levels of nitrite improve cardiomyocyte proliferation and cardiac regeneration following cardiac injury in the zebrafish. Disclosure: Nothing to disclose.
mediated through the G protein beta 2 receptor (Gb2), as nitriteinduced phosphorylation of Drp1 was attenuated in the presence of the selective Gb2 inhibitor, ICI 118,551. Conclusions: These data are the first to elucidate a mechanism by which nitrite confers ischemic resilience priorto I/R and are the first to report normoxic NO2 dependent activation of PKA and regulation of mitochondrial dynamics. Disclosure: This work was supported by the Institute of Transfusion Medicine and the Hemophilia Center of Western Pennsylvania, the NIH (1R01HL096973 to SS; R01NS065789 to CTC) and by the American Heart Association (09SDG2150066 to SS). http://dx.doi.org/10.1016/j.niox.2013.02.045
http://dx.doi.org/10.1016/j.niox.2013.02.044
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Nitrite activates protein kinase A in normoxia to promote mitochondrial fusion and confer delayed tolerance to ischemia/ reperfusion
Nitric oxide may modulate matrix metalloproteinase-2 during renovascular hypertension
a
b
c
b
Christelle Kamga , Li Mo , Ruben K. Dagda , Lisa Geary , Catherine Corey b, Sergey Zharikov b, Claudette St Croix d, Donna Beer-Stolz d, Nicholas Khoo e, Sruti Shiva e a University of Pittsburgh, Pittsburgh, PA, United States b University of Pittsburgh, Vascular Medicine Institute, Pittsburgh, PA, United States c University of Pittsburgh, Department of Pathology, Pittsburgh, PA, United States d University of Pittsburgh, Center for Biologic Imaging, Pittsburgh, PA, United States e University of Pittsburgh, Department of Pharmacology and Chemical Biology, Pittsburgh, PA, United States 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 nitriteinduced 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 lM; 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/R initiated 5 min–72 h later. Mechanistically, nitrite-mediated cytoprotection was dependent on increased activity of protein kinase A (PKA; 157.8 ± 23.8% of control), which propagated mitochondrial fusion through the phosphorylation and inhibition of dynamin related protein 1 (Drp1). This morphological change in mitochondrial phenotype led to a functional change characterized by a slight increase in mitochondrial superoxide generation (157.2 ± 16.3% of control). We show that this superoxide generated is responsible for the oxidation and subsequent activation of the cardioprotective mediator AMP kinase (214.7 ± 28.8% of control), as scavenging of reactive oxygen species by the mitochondrially targeted scavenger mitoTEMPO significantly abrogated AMPK activation and cytoprotection after H/R. Ongoing studies are investigating the mechanism by which nitrite modulates PKA activity. Preliminary data suggest that nitrite signaling may be
Danielle Guimaraes a, Jefferson Amaral a, Elen Rizzi a, Lucas Cesar Pinheiro a, Raquel Fernanda Gerlach b, Jose Eduardo Tanus-Santos a a FMRP/University of Sao Paulo, Ribeirao Preto, Brazil b FORP/University of Sao Paulo, Ribeirao Preto, Brazil Decreased nitric oxide (NO) bioavailability and up-regulated MMP activity contribute to hypertension-induced cardiovascular hypertrophy. However, it is uncertain whether NO affects MMP activity. Dietary nitrite is an alternative source of NO and may exert antioxidant effects, thus affecting up-regulated MMP hypertension. We evaluated whether sodium nitrite treatment reduces cardiac MMP in 2-kidney, 1-clip (2K1C) hypertension. Sham and 2K1C rats were treated with vehicle or sodium nitrite (15 mg/kg orally) for 4 weeks. Systolic blood pressure was monitored weekly. Plasma nitrite concentrations were analyzed by chemiluminescence. Cardiac MMP level/activity were determined by gelatin and in situ zymography. Sodium nitrite treatment exerted antihypertensive effects in 2K1C rats (P < 0.05). We found lower plasma nitrite concentrations in 2K1C rats when compared with the Sham group (P < 0.05), and nitrite treatment restored these levels in 2K1C rats. 2K1C hypertension increased cardiac MMP-2 levels and MMP activity (P < 0.05). Sodium nitrite treatment reduced hypertension-induced increases in both MMP levels and activity (P < 0.05). Our results suggest that sodium nitrite treatment prevents 2K1C hypertension-induced MMP up-regulation, and therefore may prevent cardiac remodeling associated with hypertension. Supported by: FAPESP, CNPq. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2013.02.046
P45 Enhanced nitrite reaction rates of MP4OX, a novel ischemic rescue therapy Scott D. Olsen, Ashok Malavalli, Kim Vandegriff Sangart Inc., San Diego, CA, United States Background: Hemoglobin-based oxygen carriers have been associated with vasoconstriction, attributed primarily to binding of nitric oxide (NO) as a heme ligand. MP4OX, an ischemic rescue therapy, designed with high O2 affinity using poly (ethylene glycol) (PEG)
Abstracts / Nitric Oxide 31 (2013) S13–S48
conjugation chemistry, has been observed not to cause vasoconstriction and to preserve microvascular blood flow, despite reducing perivascular levels of NO [1]. Recent evidence shows that reduction of nitrite to NO by deoxyhemoglobin has the ability to vasodilate blood vessels [2]. It also has been reported that the oxidative reaction of oxyhemoglobin with nitrite may facilitate the release of NO from heme [3]. We hypothesized that MP4OX may have enhanced reaction rates with nitrite and regeneration of NO, which may contribute to its lack of systemic vasoconstriction. Materials and methods: MP4OX was prepared by reacting human stroma-free hemoglobin (SFH) with 2-iminothiolane and maleimide-activated PEG to produce a PEG-hemoglobin conjugate, with approximately 8 PEGs/Hb. Rates of the deoxyHb/nitrite reaction were measured by mixing deoxygenated SFH or MP4OX anaerobically with a 10-fold excess of sodium nitrite in a sealed cuvette in the presence of sodium dithionite. The reaction was monitored spectrophotometrically, and the spectra were deconvoluted using parent spectra for deoxyhemoglobin, iron-nitrosyl-hemoglobin (HbNO), and methemoglobin. Rates of the oxyHb/nitrite reaction were measured by mixing oxygenated SFH or MP4 with a 20-fold excess of nitrite. These spectra were deconvoluted using parent spectra for oxyhemoglobin, methemoglobin, and nitrite-bound methemoglobin. Results: Time courses for both deoxy-and oxyHb reactions showed autocatalytic properties that deviated from pseudo first-order kinetics. Therefore, the maximum reaction rates were used for comparison purposes. Fully deoxygenated SFH and MP4OX (i.e., MP4DX) reduced nitrite to NO with maximum rates of 0.022 and 0.206 lM/s, respectively, showing a nearly 10-fold higher rate for MP4DX compared to deoxySFH. For the oxyHb/nitrite reaction, SFH and MP4OX showed maximum rates of 0.55 and 0.92 lM/s, respectively, a nearly 2-fold enhancement for MP4OX over SFH. Conclusions: Our results demonstrate that deoxygenated MP4OX is more effective at reducing nitrite to NO than unmodified Hb. In addition, oxygenated MP4OX reacts more rapidly with nitrite, which may then accelerate the release of NO from heme sites. Taken together, these enhanced nitrite reaction rates for MP4 may compensate for NO scavenging and explain, at least in part, why MP4OX does not induce vasoconstriction in vivo. The increased reaction rates of MP4OX appear to be due to R-state stabilization due to the PEG conjugation. Further studies are required to understand the extent to which MP4OX’s interactions with nitrite contribute to its ability to improve perfusion and oxygenation of ischemic tissues. Disclosure: This work was funded in part by Sangart Inc. and under US Army Contract No. W81XWH1020111 to Sangart. References [1] Tsai et al., Blood 108 (2006) 3603. [2] Cosby et al., Nat. Med. 9 (2003) 1498. [3] Grubina et al., J. Biol. Chem. 282 (2007) 17. http://dx.doi.org/10.1016/j.niox.2013.02.047
P46 Low sodium nitrite (but not nitrate) concentrations attenuate MMP-9 production by endothelial cells Cesar Arruda Meschiari a, Raquel Fernanda Gerlach b, Jose Eduardo Tanus-Santos a a Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil b Dental School of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil
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Reduced nitric oxide (NO) bioavailability and imbalanced matrix metalloproteinase (MMP) activity have important roles in the pathogenesis of cardiovascular diseases, and some NO donors were shown to downregulate MMP expression in some cell types. However, while nitrite and nitrate can be recycled back to NO by non enzymatic and enzymatic mechanisms as an alternative to NO formation from L-arginine, no previous study has examined whether sodium nitrite can downregulate stimulated MMP release by endothelial cells. Objective: We examined the effect of sodium nitrite and sodium nitrate on MMP-9 production by endothelial cells. Methods: Human umbilical vein endothelial cells were cultured in a modified DMEM (iron(III) nitrate is replaced by iron(III) sulfate) and treated for 24 h with 10 nM phorbol myristate acetate (PMA; a MMP-9 inducer) and sodium nitrite or sodium nitrate, or their vehicles. Conditioned medium were analyzed by gelatin zimography to assess MMP-9 activity. Results: PMA increased MMP-9 activity from 0.02 ± 0.01 arbitrary units (AU) to 1.14 ± 0.34 AU (P < 0.05). While low nitrite concentrations (0.2 or 2 lM) attenuated the increases in MMP-9 activity induced by PMA (0.77 ± 0.12 AU or 0.71 ± 0.14 AU, respectively; both P < 0.05 vs. PMA), high nitrite concentrations (10 or 20 lM) had no effects on PMA-induced increases in MMP-9 activity (0.88 ± 0.14 AU or 1.18 ± 0.36 AU, respectively; both P > 0.05 vs. PMA). Both low (10 or 100 lM) or high (200 or 400 lM) nitrate concentrations had no effects on PMA-induced increases in MMP-9 activity PMA (P > 0.05 vs. PMA). Conclusion: Our results suggest that low nitrite (but not nitrate) concentrations attenuate MMP-9 production by endothelial cells. Financial support: FAPESP, CNPq, FAEPA. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2013.02.048
P47 AMPK activation by nitrite and metformin increases glut-4 mediated glucose uptake and normalizes pulmonary venous hypertension in a rat model of severe metabolic syndrome Yen Chun Lai a, Diana M. Tabima a,b, Jeff B. Baust a, John J. Dube c, Alexander Chacon c, Juan C. Alvarez-Perez d, Bret H. Goodpaster c, Adolfo Garcia-Ocaña d, Stevan P. Tofovic a,e, Ana L. Mora a,e, Mark T. Gladwin a,e a University of Pittsburgh, Vascular Medicine Institute, Pittsburgh, PA, United States b University de Los Andes, Department of Biomedical Engineering, Bogota-Colombia, Colombia c University of Pittsburgh, Division of Endocrinology and Metabolism, Department of Medicine, Pittsburgh, PA, United States d Diabetes, Obesity and Metabolism Institute, The Mount Sinai Medical Center, New York City, NY, United States e University of Pittsburgh, Pulmonary, Allergy and Critical Care Medicine, Pittsburgh, PA, United States Metabolic syndrome is associated with decreased bioavailability of nitric oxide (NO) and has recently been defined as a risk factor for the development of pulmonary venous hypertension in both humans and animal models. Nitrite, an endogenous circulating oxidation product of NO, has been recognized as an important signaling molecule that regulates blood flow and blood pressure, and exhibits therapeutic efficacy in pre-clinical models of pulmonary hyperten sion (PH). Nitrite NO2 is formed from the reduction of nitrate NO3 by oral commensal bacteria and nitrate has recently been reported to improve metabolic syndrome in the eNOS knock out