Comparison of pulmonary and systemic NO- and PGI_{2}-dependent endothelial function in diabetic mice

Diabetes increases the risk of pulmonary hypertension and is associated with alterations in pulmonary vascular function. Still, it is not clear whether alterations in the phenotype of pulmonary endothelium induced by diabetes are distinct, as compared to peripheral endothelium. In the present work, we characterized differences between diabetic complications in the lung and aorta in db/db mice with advanced diabetes. Male, 20-week-old db/db mice displayed increased HbA1c and glucose concentration compatible with advanced diabetes. Diabetic lungs had signs of mild fibrosis, and pulmonary endothelium displayed significantly ultrastructural changes. In the isolated, perfused lung from db/db mice, filtration coefficient (Kf,c) and contractile response to TXA2 analogue were enhanced, while endothelial NO-dependent modulation of pulmonary response to hypoxic ventilation and cumulative production of NO2− were impaired, with no changes in immunostaining for eNOS expression. In turn, 6-keto-PGF1α release from the isolated lung from db/db mice was increased, as well as immunostaining of thrombomodulin (CD141). In contrast to the lung, NO-dependent, acetylcholine-induced vasodilation, ionophore-stimulated NO2− generation, and production of 6-keto-PGF1α were all impaired in aortic rings from db/db mice. Although eNOS immunostaining was not changed, that of CD141 was clearly lowered. Interestingly, diabetes-induced nitration of proteins in aorta was higher than that in the lungs. In summary, diabetes induced marked ultrastructural changes in pulmonary endothelium that were associated with the increased permeability of pulmonary microcirculation, impaired NO-dependent vascular function, with compensatory increase in PGI2 production, and increased CD141 expression. In contrast, endothelial dysfunction in the aorta was featured by impaired NO-, PGI2-dependent function and diminished CD141 expression

Time Response of Oxidative/Nitrosative Stress and Inflammation in LPS-Induced Endotoxaemia—A Comparative Study of Mice and Rats

Sepsis is a severe and multifactorial disease with a high mortality rate. It represents a strong inflammatory response to an infection and is associated with vascular inflammation and oxidative/nitrosative stress. Here, we studied the underlying time responses in the widely used lipopolysaccharide (LPS)-induced endotoxaemia model in mice and rats. LPS (10 mg/kg; from Salmonella Typhosa) was intraperitoneally injected into mice and rats. Animals of every species were divided into five groups and sacrificed at specific points in time (0, 3, 6, 9, 12 h). White blood cells (WBC) decreased significantly in both species after 3 h and partially recovered with time, whereas platelet decrease did not recover. Oxidative burst and iNOS-derived nitrosyl-iron hemoglobin (HbNO) increased with time (maxima at 9 or 12 h). Immune cell infiltration (CD68 and F4/80 content) showed an increase with time, which was supported by increased vascular mRNA expression of VCAM-1, P-selectin, IL-6 and TNF-α. We characterized the time responses of vascular inflammation and oxidative/nitrosative stress in LPS-induced endotoxaemic mice and rats. The results of this study will help to interpret and compare data from different animal species in LPS-induced endotoxaemia models for the identification of new drug targets

Formation of 2-nitrophenol from salicylaldehyde as a suitable test for low peroxynitrite fluxes

There has been some dispute regarding reaction products formed at physiological peroxynitrite fluxes in the nanomolar range with phenolic molecules, when used to predict the behavior of protein-bound aromatic amino acids like tyrosine. Previous data showed that at nanomolar fluxes of peroxynitrite, nitration of these phenolic compounds was outcompeted by dimerization (e.g. biphenols or dityrosine). Using 3-morpholino sydnonimine (Sin-1), we created low fluxes of peroxynitrite in our reaction set-up to demonstrate that salicylaldehyde displays unique features in the detection of physiological fluxes of peroxynitrite, yielding detectable nitration but only minor dimerization products. By means of HPLC analysis and detection at 380 nm we could identify the expected nitration products 3- and 5-nitrosalicylaldehyde, but also novel nitrated products. Using mass spectrometry, we also identified 2-nitrophenol and a not fully characterized nitrated dimerization product. The formation of 2-nitrophenol could proceed either by primary generation of a phenoxy radical, followed by addition of the NO2-radical to the various resonance structures, or by addition of the peroxynitrite anion to the polarized carbonyl group with subsequent fragmentation of the adduct (as seen with carbon dioxide). Interestingly, we observed almost no 3- and 5-nitrosalicylic acid products and only minor dimerization reaction. Our results disagree with the previous general assumption that nitration of low molecular weight phenolic compounds is always outcompeted by dimerization at nanomolar peroxynitrite fluxes and highlight unique features of salicylaldehyde as a probe for physiological concentrations of peroxynitrite

Influence of rosuvastatin treatment on cerebral inflammation and nitro-oxidative stress in experimental lung injury in pigs

Background!#!Many patients with acute respiratory distress syndrome (ARDS) suffer from cognitive impairment after hospital discharge. Different mechanisms have been implicated as potential causes for this impairment, inter alia cerebral inflammation. A class of drugs with antioxidant and anti-inflammatory properties are β-HMG-CoA-reductase inhibitors ('statins'). We hypothesized that treatment with rosuvastatin attenuates cerebral cytokine mRNA expression and nitro-oxidative stress in an animal model of acute lung injury.!##!Methods!#!After approval of the institutional and state animal care committee, we performed this prospective randomized controlled animal study in accordance with the international guidelines for the care and use of laboratory animals. Thirty-two healthy male pigs were randomized to one of four groups: lung injury by central venous injection of oleic acid (n = 8), statin treatment before and directly after lung injury (n = 8), statin treatment after lung injury (n = 8), or ventilation-only controls (n = 8). About 18 h after lung injury and standardized treatment, the animals were euthanised, and the brains and lungs were collected for further examinations. We determined histologic lung injury and cerebral and pulmonal cytokine and 3-nitrotyrosine production.!##!Results!#!We found a significant increase in hippocampal IL-6 mRNA after lung injury (p < 0.05). Treatment with rosuvastatin before and after induction of lung injury led to a significant reduction of hippocampal IL-6 mRNA (p < 0.05). Cerebral 3-nitrotyrosine was significantly higher in lung-injured animals compared with all other groups (p < 0.05 vs. animals treated with rosuvastatin after lung injury induction; p < 0.001 vs. all other groups). 3-Nitrotyrosine was also increased in the lungs of the lung-injured pigs compared to all other groups (p < 0.05 each).!##!Conclusions!#!Our findings highlight cerebral cytokine production and nitro-oxidative stress within the first day after induction of lung injury. The treatment with rosuvastatin reduced IL-6 mRNA and 3-nitrotyrosine concentration in the brains of the animals. In earlier trials, statin treatment did not reduce mortality in ARDS patients but seemed to improve quality of life in ARDS survivors. Whether this is attributable to better cognitive function because of reduced nitro-oxidative stress and inflammation remains to be elucidated

Comparison of Pulmonary and Systemic NO- and PGI 2

Diabetes increases the risk of pulmonary hypertension and is associated with alterations in pulmonary vascular function. Still, it is not clear whether alterations in the phenotype of pulmonary endothelium induced by diabetes are distinct, as compared to peripheral endothelium. In the present work, we characterized differences between diabetic complications in the lung and aorta in db/db mice with advanced diabetes. Male, 20-week-old db/db mice displayed increased HbA1c and glucose concentration compatible with advanced diabetes. Diabetic lungs had signs of mild fibrosis, and pulmonary endothelium displayed significantly ultrastructural changes. In the isolated, perfused lung from db/db mice, filtration coefficient (Kf,c) and contractile response to TXA2 analogue were enhanced, while endothelial NO-dependent modulation of pulmonary response to hypoxic ventilation and cumulative production of NO2− were impaired, with no changes in immunostaining for eNOS expression. In turn, 6-keto-PGF1α release from the isolated lung from db/db mice was increased, as well as immunostaining of thrombomodulin (CD141). In contrast to the lung, NO-dependent, acetylcholine-induced vasodilation, ionophore-stimulated NO2− generation, and production of 6-keto-PGF1α were all impaired in aortic rings from db/db mice. Although eNOS immunostaining was not changed, that of CD141 was clearly lowered. Interestingly, diabetes-induced nitration of proteins in aorta was higher than that in the lungs. In summary, diabetes induced marked ultrastructural changes in pulmonary endothelium that were associated with the increased permeability of pulmonary microcirculation, impaired NO-dependent vascular function, with compensatory increase in PGI2 production, and increased CD141 expression. In contrast, endothelial dysfunction in the aorta was featured by impaired NO-, PGI2-dependent function and diminished CD141 expression

Pentaerythritol Tetranitrate In Vivo Treatment Improves Oxidative Stress and Vascular Dysfunction by Suppression of Endothelin-1 Signaling in Monocrotaline-Induced Pulmonary Hypertension

Objective. Oxidative stress and endothelial dysfunction contribute to pulmonary arterial hypertension (PAH). The role of the nitrovasodilator pentaerythritol tetranitrate (PETN) on endothelial function and oxidative stress in PAH has not yet been defined. Methods and Results. PAH was induced by monocrotaline (MCT, i.v.) in Wistar rats. Low (30 mg/kg; MCT30), middle (40 mg/kg; MCT40), or high (60 mg/kg; MCT60) dose of MCT for 14, 28, and 42 d was used. MCT induced endothelial dysfunction, pulmonary vascular wall thickening, and fibrosis, as well as protein tyrosine nitration. Pulmonary arterial pressure and heart/body and lung/body weight ratio were increased in MCT40 rats (28 d) and reduced by oral PETN (10 mg/kg, 24 d) therapy. Oxidative stress in the vascular wall, in the heart, and in whole blood as well as vascular endothelin-1 signaling was increased in MCT40-treated rats and normalized by PETN therapy, likely by upregulation of heme oxygenase-1 (HO-1). PETN therapy improved endothelium-dependent relaxation in pulmonary arteries and inhibited endothelin-1-induced oxidative burst in whole blood and the expression of adhesion molecule (ICAM-1) in endothelial cells. Conclusion. MCT-induced PAH impairs endothelial function (aorta and pulmonary arteries) and increases oxidative stress whereas PETN markedly attenuates these adverse effects. Thus, PETN therapy improves pulmonary hypertension beyond its known cardiac preload reducing ability