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Ischemia/reperfusion injury holds a key position in many pathological conditions such as acute kidney injury and in the transition to chronic stages of renal damage. We hypothesized that besides a reported disproportional activation of vasoconstrictor response, hypoxia/reoxygenation (H/R) adversely affects endothelial dilatory systems and impairs relaxation in renal arteries. Rat renal interlobar arteries were studied under isometric conditions. Hypoxia was induced by application of 95% N2, 5% CO2 for 60 min to the bath solution, followed by a 10 min period of reoxygenation (95% O2, 5% CO2). The effect of H/R on relaxation was assessed using various inhibitors of endothelial dilatory systems. mRNA expression of phosphodiesterase 5 (PDE5), NADPH oxidases (NOX), and nitric oxide synthase (NOS) isoforms were determined using qRT-PCR; cGMP was assayed with direct cGMP ELISA. Acetylcholine induced relaxation was impaired after H/R. Inhibition of the NOS isoforms with L-NAME, and cyclooxygenases (COXs) by indomethacin did not abolish the H/R effect. Moreover, blocking the calcium activated potassium channels KCa3.1 and KCa2.1, the main mediators of the endothelium-derived hyperpolarizing factor, with TRAM34 and UCL1684, respectively, showed similar effects in H/R and control. Arterial stiffness did not differ comparing H/R with controls, indicating no impact of H/R on passive vessel properties. Moreover, superoxide was not responsible for the observed H/R effect. Remarkably, H/R attenuated the endothelium-independent relaxation by sodium nitroprusside, suggesting endothelium-independent mechanisms of H/R action. Investigating the signaling downstream of NO revealed significantly decreased cGMP and impaired relaxation during PDE5 inhibition with sildenafil after H/R. Inhibition of PKG, the target of cGMP, did not normalize SNP-induced relaxation following H/R. However, the soluble guanylyl cyclase (sGC) inhibitor ODQ abolished the H/R effect on relaxation. The mRNA expressions of the endothelial and the inducible NOS were reduced. NOX and PDE5 mRNA were similarly expressed in H/R and control. Our results provide new evidence that impaired renal artery relaxation after H/R is due to a dysregulation of sGC leading to decreased cGMP levels. The presented mechanism might contribute to an insufficient renal reperfusion after ischemia and should be considered in its pathophysiology.
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BACKGROUND: Inorganic nitrite has shown beneficial effects in cardiovascular and metabolic diseases partly via attenuation of NADPH-oxidase (NOX)-mediated oxidative stress. However, the exact mechanisms are still unclear. Here we investigated the role of S-nitrosation or altered expression of NOX subunits, and the role of xanthine oxidoreductase (XOR) in nitrite-derived nitric oxide (NO) production. METHODS: Mouse macrophages were activated with LPS in the presence or absence of nitrite. NOX activity was measured by lucigenin-dependent chemiluminescence. Gene and protein expression of NOX2 subunits and XOR were investigated using qPCR and Western Blot. S-nitrosation of Nox2 and p22phox was studied with a Biotin Switch assay. Uric acid levels in cell culture medium were analyzed as a measure of XOR activity, and NO production was assessed by DAF-FM fluorescence. RESULTS: NOX activity in activated macrophages was significantly reduced by nitrite. Reduced NOX activity was not attributed to decreased NOX gene expression. However, protein levels of p47phox and p67phox subunits were reduced by nitrite in activated macrophages. Protein expression of Nox2 and p22phox was not influenced by this treatment and neither was their S-nitrosation status. Increased uric acid levels after nitrite and diminished NO production during XOR-inhibition with febuxostat suggest that XOR is more active during nitrite-treatment of activated macrophages and plays an important role in the bioactivation of nitrite. CONCLUSIONS: Our findings contribute to the mechanistic understanding about the therapeutic effects associated with nitrite supplementation in many diseases. We show that nitrite-mediated inhibition of NOX activity cannot be explained by S-nitrosation of the NOX enzyme, but that changes in NOX2 expression and XOR function may contribute.
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Lipopolisacáridos/farmacología , Macrófagos Peritoneales/metabolismo , NADPH Oxidasas/metabolismo , Nitritos/farmacología , Xantina Deshidrogenasa/metabolismo , Animales , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Activación de Macrófagos , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/efectos de los fármacos , Ratones , NADPH Oxidasas/genética , Óxido Nítrico/metabolismo , Nitrosación , Estrés Oxidativo , Ácido Úrico/metabolismo , Xantina Deshidrogenasa/genéticaRESUMEN
Xanthine oxidoreductase (XOR) is generally known as the final enzyme in purine metabolism and as a source of reactive oxygen species (ROS). In addition, this enzyme has been suggested to mediate nitric oxide (NO) formation via reduction of inorganic nitrate and nitrite. This NO synthase (NOS)-independent pathway for NO generation is of particular importance during certain conditions when NO bioavailability is diminished due to reduced activity of endothelial NOS (eNOS) or increased oxidative stress, including aging and cardiovascular disease. The exact interplay between NOS- and XOR-derived NO generation is not fully elucidated yet. The aim of the present study was to investigate if eNOS deficiency is associated with changes in XOR expression and activity and the possible impact on nitrite, NO and ROS homeostasis. Plasma levels of nitrate and nitrite were similar between eNOS deficient (eNOS-/-) and wildtype (wt) mice. XOR activity was upregulated in eNOS-/- compared with wt, but not in nNOS-/-, iNOS-/- or wt mice treated with the non-selective NOS inhibitor L-NAME. Following an acute dose of nitrate, plasma nitrite increased more in eNOS-/- compared with wt, and this augmented response was abolished by the selective XOR inhibitor febuxostat. Livers from eNOS-/- displayed higher nitrite reducing capacity compared with wt, and this effect was attenuated by febuxostat. Dietary supplementation with nitrate increased XOR expression and activity, but concomitantly reduced superoxide generation. The latter effect was also seen in vitro after nitrite administration. Treatment with febuxostat elevated blood pressure in eNOS-/-, but not in wt mice. A high dose of dietary nitrate reduced blood pressure in naïve eNOS-/- mice, and again this effect was abolished by febuxostat. In conclusion, eNOS deficiency is associated with an upregulation of XOR facilitating the nitrate-nitrite-NO pathway and decreasing the generation of ROS. This interplay between XOR and eNOS is proposed to play a significant role in NO homeostasis and blood pressure regulation.
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Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico/sangre , Xantina Deshidrogenasa/genética , Animales , Presión Sanguínea/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Febuxostat/farmacología , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Noqueados , NG-Nitroarginina Metil Éster/farmacología , Nitratos/sangre , Nitratos/farmacología , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/deficiencia , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/deficiencia , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/deficiencia , Nitritos/sangre , Nitritos/farmacología , Oxidación-Reducción , Transducción de Señal , Superóxidos/metabolismo , Xantina Deshidrogenasa/antagonistas & inhibidores , Xantina Deshidrogenasa/metabolismoRESUMEN
BACKGROUND: Early-life reduction in nephron number (uninephrectomy [UNX]) and chronic high salt (HS) intake increase the risk of hypertension and chronic kidney disease. Adenosine signaling via its different receptors has been implicated in modulating renal, cardiovascular, and metabolic functions as well as inflammatory processes; however, the specific role of the A3 receptor in cardiovascular diseases is not clear. In this study, gene-modified mice were used to investigate the hypothesis that lack of A3 signaling prevents the development of hypertension and attenuates renal and cardiovascular injuries following UNX in combination with HS (UNX-HS) in mice. METHODS AND RESULTS: Wild-type (A3 (+/+)) mice subjected to UNX-HS developed hypertension compared with controls (mean arterial pressure 106±3 versus 82±3 mm Hg; P<0.05) and displayed an impaired metabolic phenotype (eg, increased adiposity, reduced glucose tolerance, hyperinsulinemia). These changes were associated with both cardiac hypertrophy and fibrosis together with renal injuries and proteinuria. All of these pathological hallmarks were significantly attenuated in the A3 (-/-) mice. Mechanistically, absence of A3 receptors protected from UNX-HS-associated increase in renal NADPH oxidase activity and Nox2 expression. In addition, circulating cytokines including interleukins 1ß, 6, 12, and 10 were increased in A3 (+/+) following UNX-HS, but these cytokines were already elevated in naïve A3 (-/-) mice and did not change following UNX-HS. CONCLUSIONS: Reduction in nephron number combined with chronic HS intake is associated with oxidative stress, chronic inflammation, and development of hypertension in mice. Absence of adenosine A3 receptor signaling was strongly protective in this novel mouse model of renal and cardiovascular disease.
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Hipertensión/genética , Nefrectomía , Receptor de Adenosina A3/genética , Insuficiencia Renal Crónica/genética , Cloruro de Sodio Dietético/efectos adversos , Adiposidad/genética , Animales , Cardiomegalia/genética , Modelos Animales de Enfermedad , Femenino , Fibrosis , Intolerancia a la Glucosa/etiología , Intolerancia a la Glucosa/genética , Hiperinsulinismo/etiología , Hiperinsulinismo/genética , Hipertensión/etiología , Inflamación/etiología , Inflamación/genética , Masculino , Ratones , Ratones Noqueados , Miocardio/patología , Estrés Oxidativo/genética , Proteinuria/etiología , Proteinuria/genética , Insuficiencia Renal Crónica/etiologíaRESUMEN
Advanced age is associated with increased risk for cardiovascular disease and type 2 diabetes. A proposed central event is diminished amounts of nitric oxide (NO) due to reduced generation by endothelial NO synthase (eNOS) and increased oxidative stress. In addition, it is widely accepted that increased angiotensin II (ANG II) signaling is also implicated in the pathogenesis of endothelial dysfunction and hypertension by accelerating formation of reactive oxygen species. This study was designed to test the hypothesis that dietary nitrate supplementation could reduce blood pressure and improve glucose tolerance in aged rats, via attenuation of NADPH oxidase activity and ANG II receptor signaling. Dietary nitrate supplementation for two weeks reduced blood pressure (10-15mmHg) and improved glucose clearance in old, but not in young rats. These favorable effects were associated with increased insulin responses, reduced plasma creatinine as well as improved endothelial relaxation to acetylcholine and attenuated contractility to ANG II in resistance arteries. Mechanistically, nitrate reduced NADPH oxidase-mediated oxidative stress in the cardiovascular system and increased cGMP signaling. Finally, nitrate treatment in aged rats normalized the gene expression profile of ANG II receptors (AT1A, AT2, AT1A/AT2 ratio) in the renal and cardiovascular systems without altering plasma levels of renin or ANG II. Our results show that boosting the nitrate-nitrite-NO pathway can partly compensate for age-related disturbances in endogenous NO generation via inhibition of NADPH oxidase and modulation of ANG II receptor expression. These novel findings may have implications for nutrition-based preventive and therapeutic strategies against cardiovascular and metabolic diseases.
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Envejecimiento/metabolismo , Presión Sanguínea/efectos de los fármacos , Suplementos Dietéticos , Hipertensión/prevención & control , Nitratos/administración & dosificación , Receptores de Angiotensina/sangre , Acetilcolina/farmacología , Envejecimiento/genética , Angiotensina II/sangre , Angiotensina II/genética , Animales , GMP Cíclico/sangre , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Hipertensión/sangre , Hipertensión/genética , Hipertensión/fisiopatología , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/metabolismo , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/sangre , NADPH Oxidasas/genética , Nitratos/sangre , Óxido Nítrico Sintasa de Tipo III/sangre , Óxido Nítrico Sintasa de Tipo III/genética , Nitritos/sangre , Ratas , Ratas Sprague-Dawley , Receptores de Angiotensina/genética , Transducción de Señal , Técnicas de Cultivo de TejidosRESUMEN
BACKGROUND AND PURPOSE: NO deficiency and oxidative stress are crucially involved in the development or progression of cardiovascular disease, including hypertension and stroke. We have previously demonstrated that acute treatment with the newly discovered organic nitrate, 2-nitrate-1,3-dibuthoxypropan (NDBP), is associated with NO-like effects in the vasculature. This study aimed to further characterize the mechanism(s) and to elucidate the therapeutic potential in a model of hypertension and oxidative stress. EXPERIMENTAL APPROACH: A combination of ex vivo, in vitro and in vivo approaches was used to assess the effects of NDBP on vascular reactivity, NO release, NADPH oxidase activity and in a model of hypertension. KEY RESULTS: Ex vivo vascular studies demonstrated NDBP-mediated vasorelaxation in mesenteric resistance arteries, which was devoid of tolerance. In vitro studies using liver and kidney homogenates revealed dose-dependent and sustained NO generation by NDBP, which was attenuated by the xanthine oxidase inhibitor febuxostat. In addition, NDBP reduced NADPH oxidase activity in the liver and prevented angiotensin II-induced activation of NADPH oxidase in the kidney. In vivo studies showed that NDBP halted the progression of hypertension in mice with chronic angiotensin II infusion. This was associated with attenuated cardiac hypertrophy, and reduced NADPH oxidase-derived oxidative stress and fibrosis in the kidney and heart. CONCLUSION AND IMPLICATIONS: The novel organic nitrate NDBP halts the progression of angiotensin II-mediated hypertension. Mechanistically, our findings suggest that NDBP treatment is associated with sustained NO release and attenuated activity of NADPH oxidase, which to some extent requires functional xanthine oxidase.
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Angiotensina II/farmacología , Hipertensión/tratamiento farmacológico , Hipertensión/prevención & control , Nitratos/farmacología , Óxido Nítrico/biosíntesis , Estrés Oxidativo/efectos de los fármacos , Propano/análogos & derivados , Angiotensina II/administración & dosificación , Animales , Relación Dosis-Respuesta a Droga , Hipertensión/inducido químicamente , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Nitratos/administración & dosificación , Propano/administración & dosificación , Propano/farmacología , Ratas , Ratas WistarRESUMEN
RATIONALE: Accumulating studies suggest that nitric oxide (NO) deficiency and oxidative stress are central pathological mechanisms in type 2 diabetes (T2D). Recent findings demonstrate therapeutic effects by boosting the nitrate-nitrite-NO pathway, which is an alternative pathway for NO formation. This study aimed at investigating the acute effects of inorganic nitrate on glucose and insulin signaling in adenosine A2B receptor knockout mice (A(-/-) 2B), a genetic mouse model of impaired metabolic regulation. METHODS: Acute effects of nitrate treatment were investigated in aged wild-type (WT) and A(-/-) 2B mice. One hour after injection with nitrate (0.1 mmol/kg, i.p.) or placebo, metabolic regulation was evaluated by intraperitoneal glucose and insulin tolerance tests. NADPH oxidase-mediated superoxide production and AMPK phosphorylation were measured in livers obtained from non-treated or glucose-treated mice, with or without prior nitrate injection. Plasma was used to determine insulin resistance (HOMA-IR) and NO signaling. RESULTS: A(-/-) 2B displayed increased body weight, reduced glucose clearance, and attenuated overall insulin responses compared with age-matched WT mice. Nitrate treatment increased circulating levels of nitrate, nitrite and cGMP in the A(-/-) 2B, and improved glucose clearance. In WT mice, however, nitrate treatment did not influence glucose clearance. HOMA-IR increased following glucose injection in the A(-/-) 2B, but remained at basal levels in mice pretreated with nitrate. NADPH oxidase activity in livers from A(-/-) 2B, but not WT mice, was reduced by nitrate treatment. Livers from A(-/-) 2B displayed reduced AMPK phosphorylation compared with WT mice, and this was increased by nitrate treatment. Finally, injection with the anti-diabetic agent metformin induced similar therapeutic effects in the A(-/-) 2B as observed with nitrate. CONCLUSION: The A(-/-) 2B mouse is a genetic mouse model of metabolic syndrome. Acute treatment with nitrate improved the metabolic profile in it, at least partly via reduction in oxidative stress and improved AMPK signaling in the liver.
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AIMS/HYPOTHESIS: Adenosine is an important regulator of metabolism; however, the role of the A1 receptor during ageing and obesity is unclear. The aim of this study was to investigate the effects of A1 signalling in modulating metabolic function during ageing. METHODS: Age-matched young and aged A 1 (also known as Adora1)-knockout (A1(-/-)) and wild-type (A1(+/+)) mice were used. Metabolic regulation was evaluated by body composition, and glucose and insulin tolerance tests. Isolated islets and islet arterioles were used to detect islet endocrine and vascular function. Oxidative stress and inflammation status were measured in metabolic organs and systemically. RESULTS: Advanced age was associated with both reduced glucose clearance and insulin sensitivity, as well as increased visceral adipose tissue (VAT) in A1(+/+) compared with A1(-/-) mice. Islet morphology and insulin content were similar between genotypes, but relative changes in in vitro insulin release following glucose stimulation were reduced in aged A1(+/+) compared with A1(-/-) mice. Islet arteriolar responses to angiotensin II were stronger in aged A1(+/+) mice, this being associated with increased NADPH oxidase activity. Ageing resulted in multiple changes in A1(+/+) compared with A1(-/-) mice, including enhanced NADPH oxidase-derived O2(-) formation and NADPH oxidase isoform 2 (Nox2) protein expression in pancreas and VAT; elevated levels of circulating insulin, leptin and proinflammatory cytokines (TNF-α, IL-1ß, IL-6 and IL-12); and accumulation of CD4(+) T cells in VAT. This was associated with impaired insulin signalling in VAT from aged A1(+/+) mice. CONCLUSIONS/INTERPRETATION: These studies emphasise that A1 receptors regulate metabolism and islet endocrine and vascular functions during ageing, including via the modulation of oxidative stress and inflammatory responses, among other things.
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Inflamación/genética , Estrés Oxidativo/genética , Receptor de Adenosina A1/genética , Tejido Adiposo/metabolismo , Envejecimiento/metabolismo , Angiotensina II/farmacología , Animales , Composición Corporal/genética , Linfocitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Femenino , Intolerancia a la Glucosa/genética , Insulina/metabolismo , Resistencia a la Insulina , Islotes Pancreáticos/irrigación sanguínea , Masculino , Glicoproteínas de Membrana/metabolismo , Metabolismo/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Flujo Sanguíneo Regional/fisiología , Transducción de Señal/genéticaRESUMEN
The kidney is vulnerable to hypoxia, and substantial efforts have been made to ameliorate renal ischemic injury secondary to pathological conditions. Stimulation of the nitrate-nitrite-nitric oxide pathway is associated with renal and cardiovascular protection in disease models, but less is known about the vascular effects during renal ischemia. This study was aimed at investigating the vascular effects of nitrite in the kidney during normoxic and ischemic conditions. Using a multiwire myograph system, we assessed nitrite-mediated relaxation (10(-9)-10(-4)mol/L) in isolated and preconstricted renal interlobar arteries from C57BL/6 mice under normal conditions (pO2 13kPa; pH 7.4) and with low oxygen tension and low pH to mimic ischemia (pO2 3kPa; pH 6.6). Xanthine oxidoreductase expression was analyzed by quantitative PCR, and production of reactive nitrogen species was measured by DAF-FM DA fluorescence. During normoxia significant vasodilatation (15±3%) was observed only at the highest concentration of nitrite, which was dependent on NO-sGC-cGMP signaling. The vasodilatory responses to nitrite were greatly sensitized and enhanced during hypoxia with low pH, demonstrating significant dilatation (11±1%) already in the physiological range (10(-8)mol/L), with a maximum response of 27±2% at 10(-4) mol/L. In contrast to normoxia, and to that observed with a classical NO donor (DEA NONOate), this sensitization was independent of sGC-cGMP signaling. Moreover, inhibition of various enzymatic systems reported to reduce nitrite in other vascular beds, i.e., aldehyde oxidase (raloxifene), aldehyde dehydrogenase (cyanamide), and NO synthase (L-NAME), had no effect on the nitrite response. However, inhibition of xanthine oxidoreductase (XOR; febuxostat or allopurinol) abolished the sensitized response to nitrite during hypoxia and acidosis. In conclusion, in contrast to normoxia, nitrite exerted potent vasorelaxation during ischemic conditions already at physiological concentrations. This effect was dependent on functional XOR but independent of classical downstream signaling by sGC-cGMP.
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GMP Cíclico/metabolismo , Nitritos/farmacología , Vasodilatación , Animales , Hipoxia de la Célula , Isquemia/metabolismo , Riñón/irrigación sanguínea , Masculino , Ratones Endogámicos C57BL , Sistemas de Mensajero Secundario , Xantina Oxidasa/metabolismoRESUMEN
Oxidative stress contributes to the pathogenesis of many disorders, including diabetes and cardiovascular disease. Immune cells are major sources of superoxide (O2(â-)) as part of the innate host defense system, but exaggerated and sustained O2(â-) generation may lead to progressive inflammation and organ injuries. Previous studies have proven organ-protective effects of inorganic nitrite, a precursor of nitric oxide (NO), in conditions manifested by oxidative stress and inflammation. However, the mechanisms are still not clear. This study aimed at investigating the potential role of nitrite in modulating NADPH oxidase (NOX) activity in immune cells. Mice peritoneal macrophages or human monocytes were activated by lipopolysaccharide (LPS), with or without coincubation with nitrite. O2(â-) and peroxynitrite (ONOO(-)) formation were detected by lucigenin-based chemiluminescence and fluorescence techniques, respectively. The intracellular NO production was measured by DAF-FM DA fluorescence. NOX isoforms and inducible NO synthase (iNOS) expression were detected by qPCR. LPS increased both O2(â-) and ONOO(-) production in macrophages, which was significantly reduced by nitrite (10µmol/L). Mechanistically, the effects of nitrite are (1) linked to increased NO generation, (2) similar to that observed with the NO donor DETA-NONOate, and (3) can be abolished by the NO scavenger carboxy-PTIO or by the xanthine oxidase (XO) inhibitor febuxostat. Nox2 expression was increased in activated macrophages, but was not influenced by nitrite. However, nitrite attenuated LPS-induced upregulation of iNOS expression. Similar to that observed in mice macrophages, nitrite also reduced O2(â-) generation in LPS-activated human monocytes. In conclusion, XO-mediated reduction of nitrite attenuates NOX activity in activated macrophages, which may modulate the inflammatory response.
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Apoptosis/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Monocitos/metabolismo , NADPH Oxidasas/metabolismo , Óxido Nítrico/metabolismo , Nitritos/farmacología , Superóxidos/metabolismo , Animales , Western Blotting , Proliferación Celular , Células Cultivadas , Humanos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/patología , Ratones , Monocitos/efectos de los fármacos , Monocitos/patología , NADPH Oxidasas/genética , Estrés Oxidativo , Ácido Peroxinitroso/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Renal oxidative stress and nitric oxide (NO) deficiency are key events in hypertension. Stimulation of a nitrate-nitrite-NO pathway with dietary nitrate reduces blood pressure, but the mechanisms or target organ are not clear. We investigated the hypothesis that inorganic nitrate and nitrite attenuate reactivity of renal microcirculation and blood pressure responses to angiotensin II (ANG II) by modulating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and NO bioavailability. Nitrite in the physiological range (10(-7)-10(-5) mol/L) dilated isolated perfused renal afferent arterioles, which were associated with increased NO. Contractions to ANG II (34%) and simultaneous NO synthase inhibition (56%) were attenuated by nitrite (18% and 26%). In a model of oxidative stress (superoxide dismutase-1 knockouts), abnormal ANG II-mediated arteriolar contractions (90%) were normalized by nitrite (44%). Mechanistically, effects of nitrite were abolished by NO scavenger and xanthine oxidase inhibitor, but only partially attenuated by inhibiting soluble guanylyl cyclase. Inhibition of NADPH oxidase with apocynin attenuated ANG II-induced contractility (35%) similar to that of nitrite. In the presence of nitrite, no further effect of apocynin was observed, suggesting NADPH oxidase as a possible target. In preglomerular vascular smooth muscle cells and kidney cortex, nitrite reduced both basal and ANG II-induced NADPH oxidase activity. These effects of nitrite were also abolished by xanthine oxidase inhibition. Moreover, supplementation with dietary nitrate (10(-2) mol/L) reduced renal NADPH oxidase activity and attenuated ANG II-mediated arteriolar contractions and hypertension (99±2-146±2 mm Hg) compared with placebo (100±3-168±3 mm Hg). In conclusion, these novel findings position NADPH oxidase in the renal microvasculature as a prime target for blood pressure-lowering effects of inorganic nitrate and nitrite.
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Presión Sanguínea/efectos de los fármacos , Hipertensión/enzimología , Riñón/irrigación sanguínea , Microvasos/embriología , NADPH Oxidasas/biosíntesis , Nitratos/farmacología , Nitritos/farmacología , Animales , Modelos Animales de Enfermedad , Hipertensión/tratamiento farmacológico , Hipertensión/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Microvasos/patología , Estrés OxidativoRESUMEN
AIMS: Inorganic nitrate and nitrite from endogenous and dietary sources have emerged as alternative substrates for nitric oxide (NO) formation in addition to the classic L-arginine NO synthase (NOS)-dependent pathway. Here, we investigated a potential cross-talk between these two pathways in the regulation of vascular function. RESULTS: Long-term dietary supplementation with sodium nitrate (0.1 and 1 mmol kg(-1) day(-1)) in rats caused a reversible dose-dependent reduction in phosphorylated endothelial NOS (eNOS) (Ser1177) in aorta and a concomitant increase in phosphorylation at Thr495. Moreover, eNOS-dependent vascular responses were attenuated in vessels harvested from nitrate-treated mice or when nitrite was acutely added to control vessels. The citrulline-to-arginine ratio in plasma, as a measure of eNOS activity, was reduced in nitrate-treated rodents. Telemetry measurements revealed that a low dietary nitrate dose reduced blood pressure, whereas a higher dose was associated with a paradoxical elevation. Finally, plasma cyclic guanosine monophosphate increased in mice that were treated with a low dietary nitrate dose and decreased with a higher dose. INNOVATION AND CONCLUSIONS: These results demonstrate the existence of a cross-talk between the nitrate-nitrite-NO pathway and the NOS-dependent pathway in control of vascular NO homeostasis.
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Sistema Cardiovascular/metabolismo , Homeostasis , Redes y Vías Metabólicas , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Animales , Aorta/metabolismo , Arginina/sangre , Presión Sanguínea , Citrulina/sangre , Suplementos Dietéticos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Nitratos/química , Óxido Nítrico/química , Óxido Nítrico Sintasa de Tipo III/genética , Nitritos/química , Fosforilación , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: Genome-wide association studies identified a risk haplotype on chromosome 9p21.3 to be associated with coronary artery disease (CAD) and myocardial infarction (MI). Since this region does not contain a clear candidate gene with known pathophysiology, we performed a haplotype-specific expression study in human macrophages during pro-inflammatory stimulation to investigate the locus-dependent expression patterns in a model of atherosclerosis, the underlying cause for CAD and MI. METHODS: Blood samples were taken from 40 male stable MI patients either homozygous for 9p21.3 risk (n = 20) or non-risk haplotype (n = 20) as well as from 28 healthy male individuals (n = 14 for each haplotype). Monocytes were isolated by density gradient centrifugation followed by differentiation into macrophages via M-CSF. Macrophages were either incubated with a pro-inflammatory IFNγ-LPS cocktail or kept untreated as controls. After 24 h, RNA was isolated and applied to Affymetrix Human Exon 1.0 ST Arrays. RESULTS: Macrophages from MI patients and controls stratified for 9p21.3 haplotypes, exhibited marked differences in gene expression. Most pronounced differences were found in inflammatory mediators, like the chemokines CCL8 and CCL2 and the lectines CLEC4E and CLEC5A. Differences in expression changes could be seen most obviously during inflammatory stimulation for both, the interleukins IL12B and IL1B, and members of metallothionein gene family. CONCLUSION: These findings show that gene expression is different in 9p21.3 haplotype-stratified macrophages. While these effects are relatively small in our in vitro model of atherosclerosis, these biological effects may contribute to a long term effect in risk haplotype carriers increasing susceptibility to CAD and MI.
Asunto(s)
Cromosomas Humanos Par 9 , Enfermedad de la Arteria Coronaria/diagnóstico , Enfermedad de la Arteria Coronaria/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Macrófagos/metabolismo , Adulto , Empalme Alternativo , Estudios de Casos y Controles , Quimiocinas/metabolismo , Enfermedad de la Arteria Coronaria/metabolismo , Genotipo , Haplotipos , Homocigoto , Humanos , Inflamación , Interleucinas/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/citología , Masculino , Persona de Mediana Edad , Infarto del Miocardio/sangre , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Dilated cardiomyopathy (DCM) is a structural heart disease with strong genetic background. Monogenic forms of DCM are observed in families with mutations located mostly in genes encoding structural and sarcomeric proteins. However, strong evidence suggests that genetic factors also affect the susceptibility to idiopathic DCM. To identify risk alleles for non-familial forms of DCM, we carried out a case-control association study, genotyping 664 DCM cases and 1,874 population-based healthy controls from Germany using a 50K human cardiovascular disease bead chip covering more than 2,000 genes pre-selected for cardiovascular relevance. After quality control, 30,920 single nucleotide polymorphisms (SNP) were tested for association with the disease by logistic regression adjusted for gender, and results were genomic-control corrected. The analysis revealed a significant association between a SNP in HSPB7 gene (rs1739843, minor allele frequency 39%) and idiopathic DCM (p = 1.06 × 10â»6, ORâ = 0.67 [95% CI 0.57-0.79] for the minor allele T). Three more SNPs showed p < 2.21 × 10â»5. De novo genotyping of these four SNPs was done in three independent case-control studies of idiopathic DCM. Association between SNP rs1739843 and DCM was significant in all replication samples: Germany (n =564, n = 981 controls, p = 2.07 × 10⻳, ORâ= 0.79 [95% CI 0.67-0.92]), France 1 (n = 433 cases, n = 395 controls, p =3.73 × 10⻳, ORâ = 0.74 [95% CI 0.60-0.91]), and France 2 (n = 249 cases, n = 380 controls, p = 2.26 × 10â»4, ORâ = 0.63 [95% CI 0.50-0.81]). The combined analysis of all four studies including a total of n = 1,910 cases and n = 3,630 controls showed highly significant evidence for association between rs1739843 and idiopathic DCM (p = 5.28 × 10⻹³, OR= 0.72 [95% CI 0.65-0.78]). None of the other three SNPs showed significant results in the replication stage.This finding of the HSPB7 gene from a genetic search for idiopathic DCM using a large SNP panel underscores the influence of common polymorphisms on DCM susceptibility.