3-nitrotyrosine and Pulmonary-Edema

Acute lung injury (ALI) and sepsis are major contributors to the morbidity and mortality of critically ill patients. The current study was designed further evaluate the mechanism of pulmonary vascular hyperpermeability in sheep with these injuries.. Sheep were randomized to a sham-injured control group (n=6) or ALI/sepsis group (n=7). The sheep in the ALI/sepsis group received inhalation injury followed by instillation of Pseudomonas aeruginosa into the lungs. These groups were monitored for 24 h. Additional sheep (n=16) received the injury and lung tissue was harvested at different time points to measure lung wet/dry weight ratio, vascular endothelial growth factor (VEGF) mRNA and protein expression as well as 3-nitrotyrosine protein expression in lung homogenates.. The injury induced severe deterioration in pulmonary gas exchange, increases in lung lymph flow and protein content, and lung water content (P<0.01 each). These alterations were associated with elevated lung and plasma nitrite/nitrate concentrations, increased tracheal blood flow, and enhanced VEGF mRNA and protein expression in lung tissue as well as enhanced 3-nitrotyrosine protein expression (P<0.05 each).. This study describes the time course of pulmonary microvascular hyperpermeability in a clinical relevant large animal model and may improve the experimental design of future studies.

Topics: Acute Lung Injury; Animals; Capillary Permeability; Disease Models, Animal; Female; Lung; Microcirculation; Nitric Oxide; Pneumonia; Pseudomonas aeruginosa; Pseudomonas Infections; Pulmonary Circulation; Pulmonary Edema; Pulmonary Gas Exchange; RNA, Messenger; Sepsis; Sheep; Smoke Inhalation Injury; Time Factors; Tyrosine; Vascular Endothelial Growth Factor A

The extracellular signal-regulated kinase (ERK) cascade has long been known to be central to the activation of cellular processes such as proliferation, differentiation, and oncogenic transformation. The mitogen-activated protein (MAP) serine/threonine family of protein kinases, of which ERK is a member, is activated by a mechanism that includes protein kinase cascades. Mitogen-activated protein kinases (MAPKs) are well-conserved enzymes connecting cell surface receptors to intracellular regulatory targets; they are activated in response to a wide variety of stimuli. The aim of this study was to investigate the effects of PD98059, a highly selective inhibitor of MAP/ERK kinase1 (MEK1) activation, on the development of lung inflammation and fibrosis. Lung injury was induced by intratracheal instillation of bleomycin (1 mg/kg), and PD98059 (10 mg/kg, 10% dimethyl sulfoxide, i.p.) was administrated 1 h after bleomycin instillation and daily for 7 days. PD98059 treatment shows therapeutic effects on pulmonary damage, decreasing many inflammatory and apoptotic parameters, such as (1) cytokine production; (2) IkBα degradation and NF-kB nuclear translocation; (3) iNOS expression; (4) nitrotyrosine and PAR localization; and (5) the degree of apoptosis, as evaluated by Bax and Bcl-2 balance, FAS ligand expression, and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. In particular, to assess whether PD98059 treatment influences MAPKs pathway, we have also investigated the expression of activated ERK and JNK after bleomycin-induced pulmonary fibrosis, showing that the inhibition of the cascade reduces the inflammatory processes that lead to the appearance of the fibrosis. Taken together, all our results clearly show that PD98059 reduces the lung injury and inflammation due to the intratracheal bleomycin administration in mice.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Bleomycin; Body Weight; Butadienes; Fas Ligand Protein; Flavonoids; I-kappa B Proteins; Instillation, Drug; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; Lung; Male; MAP Kinase Kinase 1; Mice; Mice, Inbred Strains; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Neutrophils; NF-KappaB Inhibitor alpha; Nitric Oxide Synthase Type II; Nitriles; Phosphorylation; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Pulmonary Edema; Pulmonary Fibrosis; Transcription Factor RelA; Tumor Necrosis Factor-alpha; Tyrosine

Acute lung injury (ALI) is a serious illness, the incidence and mortality of which are very high. Free radicals, such as hydroxyl radicals (OH) and peroxynitrite (ONOO(-)), are considered to be the final causative molecules in the pathogenesis of ALI. Hydrogen, a new antioxidant, can selectively reduce OH and ONOO(-). In the present study, we investigated the hypothesis that hydrogen inhalation could ameliorate ALI induced by intra-tracheal lipopolysaccharide (LPS, 5mg/kg body weight). Mice were randomized into three groups: sham group (physiological saline+2% hydrogen mixed gas), control group (LPS+normal air) and experiment group (LPS+2% hydrogen mixed gas). Bronchoalveolar lavage fluid (BALF) was performed to determine the total protein concentrations and pro-inflammatory cytokines. Lung tissues were assayed for oxidative stress variables, wet/dry (W/D) ratio, histological, immunohistochemistry and Western blotting examinations. Our experiments exhibited that hydrogen improved the survival rate of mice and induced a decrease in lung W/D ratio. In addition, hydrogen decreased malonaldehyde and nitrotyrosine content, inhibited myeloperoxidase and maintained superoxide dismutase activity in lung tissues and associated with a decrease in the expression of TNF-α, IL-1β, IL-6 and total protein concentrations in the BALF. Hydrogen further attenuated histopathological alterations and mitigated lung cell apoptosis. Importantly, hydrogen inhibited the activation of P-JNK, and also reversed changes in Bax, Bcl-xl and caspase-3. In conclusion, our data demonstrated that hydrogen inhalation ameliorated LPS-induced ALI and it may be exerting its protective role by preventing the activation of ROS-JNK-caspase-3 pathway.

Topics: Acute Lung Injury; Administration, Inhalation; Animals; Antioxidants; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Bronchoalveolar Lavage Fluid; Caspase 3; Cytokines; Hydrogen; Lipopolysaccharides; Male; Malondialdehyde; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Peroxidase; Pulmonary Edema; Superoxide Dismutase; Treatment Outcome; Tyrosine

We have previously implicated transient receptor potential vanilloid 4 (TRPV4) channels and alveolar macrophages in initiating the permeability increase in response to high peak inflation pressure (PIP) ventilation. Alveolar macrophages were harvested from TRPV4(-/-) and TRPV4(+/+) mice and instilled in the lungs of mice of the opposite genotype. Filtration coefficients (K(f)) measured in isolated perfused lungs after ventilation with successive 30-min periods of 9, 25, and 35 cmH(2)O PIP did not significantly increase in lungs from TRPV4(-/-) mice but increased >2.2-fold in TRPV4(+/+) lungs, TRPV4(+/+) lungs instilled with TRPV4(-/-) macrophages, and TRPV4(-/-) lungs instilled with TRPV4(+/+) macrophages after ventilation with 35 cmH(2)O PIP. Activation of TRPV4 with 4-alpha-phorbol didecanoate (4alphaPDD) significantly increased intracellular calcium, superoxide, and nitric oxide production in TRPV4(+/+) macrophages but not TRPV4(-/-) macrophages. Cross-sectional areas increased nearly 3-fold in TRPV4(+/+) macrophages compared with TRPV4(-/-) macrophages after 4alphaPDD. Immunohistochemistry staining of lung tissue for nitrotyrosine revealed increased amounts in high PIP ventilated TRPV4(+/+) lungs compared with low PIP ventilated TRPV4(+/+) or high PIP ventilated TRPV4(-/-) lungs. Thus TRPV4(+/+) macrophages restored susceptibility of TRPV4(-/-) lungs to mechanical injury. A TRPV4 agonist increased intracellular calcium and reactive oxygen and nitrogen species in harvested TRPV4(+/+) macrophages but not TRPV4(-/-) macrophages. K(f) increases correlated with tissue nitrotyrosine, a marker of peroxynitrite production.

Topics: Animals; Disease Susceptibility; Genotype; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Immunohistochemistry; In Vitro Techniques; Lung; Macrophage Activation; Macrophages, Alveolar; Mice; Mice, Knockout; Permeability; Phorbol Esters; Pulmonary Edema; Pulmonary Ventilation; Reactive Nitrogen Species; Reactive Oxygen Species; Staining and Labeling; TRPC Cation Channels; Tyrosine; Ventilator-Induced Lung Injury

In this study, we evaluated the protective effect and therapeutic potential of the prostaglandin E(2) (PGE(2)) synthetic analog 16,16-dimethyl-PGE(2) (dmPGE(2)) in the animal model of pulmonary fibrosis induced by bleomycin. Mice subjected to intratracheal administration of bleomycin (1 mg/kg) received a dmPGE(2) dose of 30 microg/kg/day by continuous subcutaneous infusion. Bronchoalveolar lavage (BAL); immunohistochemical analysis for IL-1, TNF-alpha, and nitrotyrosine; measurement of fluid content in lung; myeloperoxidase activity assay; and lung histology were performed 1 week later. Lung histology and Sircol assay for collagen deposition were performed 3 weeks after treatments. Changes of body weight and survival rate were also evaluated at 1 and 3 weeks. Compared with bleomycin-treated mice, dmPGE(2) co-treated mice exhibited a reduced degree of body weight loss and mortality rate as well as of lung damage and inflammation, as shown by the significant reduction of: (1) lung infiltration by leukocytes; (2) myeloperoxidase activity; (3) IL-1, TNF-alpha, and nitrotyrosine immunostaining; (4) lung edema; and (5) histologic evidence of lung injury and collagen deposition. In a separate set of experiments, dmPGE(2) treatment was started 3 days after bleomycin administration, and the evaluation of lung damage and inflammation was assessed 4 days later. Importantly, delayed administration of dmPGE(2) also was able to protect from inflammation and lung injury induced by bleomycin. These results, indicating that dmPGE(2) is able to prevent and to reduce bleomycin-induced lung injury through its regulatory and anti-inflammatory properties, encourage further research to find new options for the treatment of pulmonary fibrosis.

Topics: 16,16-Dimethylprostaglandin E2; Animals; Bleomycin; Body Weight; Bronchoalveolar Lavage Fluid; Collagen; Disease Models, Animal; Infusions, Subcutaneous; Interleukin-1beta; Lung; Lung Injury; Male; Mice; Peroxidase; Pneumonia; Protective Agents; Pulmonary Edema; Pulmonary Fibrosis; Time Factors; Tumor Necrosis Factor-alpha; Tyrosine

The myeloperoxidase (MPO)-hydrogen peroxide-halide system is an efficient oxygen-dependent antimicrobial component of polymorphonuclear leukocyte (PMN)-mediated host defense. However, MPO deficiency results in few clinical consequences indicating the activation of compensatory mechanisms. Here, we determined possible mechanisms protecting the host using MPO(-/-) mice challenged with live gram-negative bacterium Escherichia coli. We observed that MPO(-/-) mice unexpectedly had improved survival compared with wild-type (WT) mice within 5-12 h after intraperitoneal E. coli challenge. Lungs of MPO(-/-) mice also demonstrated lower bacterial colonization and markedly attenuated increases in microvascular permeability and edema formation after E. coli challenge compared with WT. However, PMN sequestration in lungs of both groups was similar. Basal inducible nitric oxide synthase (iNOS) expression was significantly elevated in lungs and PMNs of MPO(-/-) mice, and NO production was increased two- to sixfold compared with WT. Nitrotyrosine levels doubled in lungs of WT mice within 1 h after E. coli challenge but did not change in MPO(-/-) mice. Inhibition of iNOS in MPO(-/-) mice significantly increased lung edema and reduced their survival after E. coli challenge, but iNOS inhibitor had the opposite effect in WT mice. Thus augmented iNOS expression and NO production in MPO(-/-) mice compensate for the lack of HOCl-mediated bacterial killing, and the absence of MPO-derived oxidants mitigates E. coli sepsis-induced lung inflammation and injury.

Topics: Animals; Escherichia coli; Escherichia coli Infections; Gene Expression Regulation, Enzymologic; Lung; Lung Injury; Mice; Mice, Knockout; Neutrophils; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidants; Peroxidase; Pulmonary Edema; Sepsis; Tyrosine

Pulmonary manifestations of oxygen toxicity were studied and quantified in rats breathing >98% O(2) at 1, 1.5, 2, 2.5, and 3 ATA to test our hypothesis that different patterns of pulmonary injury would emerge, reflecting a role for central nervous system (CNS) excitation by hyperbaric oxygen. At 1.5 atmosphere absolute (ATA) and below, the well-recognized pattern of diffuse pulmonary damage developed slowly with an extensive inflammatory response and destruction of the alveolar-capillary barrier leading to edema, impaired gas exchange, respiratory failure, and death; the severity of these effects increased with time over the 56-h period of observation. At higher inspired O(2) pressures, 2-3 ATA, pulmonary injury was greatly accelerated but less inflammatory in character, and events in the brain were a prelude to a distinct lung pathology. The CNS-mediated component of this lung injury could be attenuated by selective inhibition of neuronal nitric oxide synthase (nNOS) or by unilateral transection of the vagus nerve. We propose that extrapulmonary, neurogenic events predominate in the pathogenesis of acute pulmonary oxygen toxicity in hyperbaric oxygenation, as nNOS activity drives lung injury by modulating the output of central autonomic pathways.

Topics: Animals; Behavior, Animal; Blood Gas Analysis; Body Fluids; Bronchoalveolar Lavage Fluid; Hyperoxia; L-Lactate Dehydrogenase; Lung; Lung Diseases; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Oxygen; Pneumonia; Pulmonary Edema; Rats; Rats, Sprague-Dawley; Survival Analysis; Tyrosine; Vagotomy

Human immunodeficiency virus (HIV)-1 causes lung disease by increasing the host's susceptibility to pathogens. HIV-1 also causes an increase in systemic oxidative/nitrosative stress, perhaps enhancing the deleterious effects of secondary infections. Here we examined the ability of HIV-1 proteins to increase lung oxidative/nitrosative stress after lipopolysaccharide (LPS) (endotoxin) administration in an HIV-1 transgenic mouse model. Lung oxidative/nitrosative stress biomarkers studied 3 and 6 h after LPS administration were as follows: lung edema, tissue superoxide, NO metabolites, nitrotyrosine, hydrogen peroxide, and bronchoalveolar lavage fluid (BALF) glutathione (GSH). Blood serum cytokine levels were quantified to verify immune function of our nonimmunocompromised animal model. Results indicate that 3 h after LPS administration, HIV-1 transgenic mouse lung tissue has significantly greater edema and superoxide. Furthermore, NO metabolites are significantly elevated in HIV-1 transgenic mouse BALF, lung tissue, and blood plasma compared with those of wild-type mice. HIV-1 transgenic mice also produce significantly greater lung nitrotyrosine and hydrogen peroxide than wild-type mice. In addition, HIV-1 transgenic mice produce significantly less BALF GSH than wild-type mice 3 h after LPS treatment. Without treatment, serum cytokine levels are similar for HIV-1 transgenic and wild-type mice. After treatment, serum cytokine levels are significantly elevated in both HIV-1 transgenic and wild-type mice. Therefore, HIV-1 transgenic mice have significantly greater lung oxidative/nitrosative stress after endotoxin administration than wild-type mice, independent of immune function. These results indicate that HIV-1 proteins may increase pulmonary complications subsequent to a secondary infection by altering the lung redox potential.

Topics: Animals; Cytokines; Glutathione; HIV Infections; HIV-1; Hydrogen Peroxide; Lipopolysaccharides; Lung; Mice; Mice, Transgenic; Nitrates; Nitrites; Nitrosation; Oxidative Stress; Pulmonary Edema; Tyrosine

To investigate the effects of recombinant human activated protein C (rhAPC) on pulmonary function in acute lung injury (ALI) resulting from smoke inhalation in association with a bacterial challenge.. Prospective, randomized, controlled, experimental animal study with repeated measurements.. Investigational intensive care unit at a university hospital.. Eighteen sheep (37.2 +/- 1.0 kg) were operatively prepared and randomly allocated to either the sham, control, or rhAPC group (n = 6 each). After a tracheotomy had been performed, ALI was produced in the control and rhAPC group by insufflation of 4 sets of 12 breaths of cotton smoke. Then, a 30 mL suspension of live Pseudomonas aeruginosa bacteria (containing 2-5 x 10(11) colony forming units) was instilled into the lungs according to an established protocol. The sham group received only the vehicle, i.e., 4 sets of 12 breaths of room air and instillation of 30 mL normal saline. The sheep were studied in the awake state for 24 hrs and were ventilated with 100% oxygen. RhAPC (24 mug/kg/hr) was intravenously administered. The infusion was initiated 1 hr post-injury and lasted until the end of the experiment. The animals were resuscitated with Ringer's lactate solution to maintain constant pulmonary artery occlusion pressure.. In comparison with nontreatment in controls, the infusion of rhAPC significantly attenuated the fall in Pao2/Fio2 ratio (control group values were 521 +/- 22 at baseline [BL], 72 +/- 5 at 12 hrs, and 74 +/- 7 at 24 hrs, vs. rhAPC group values of 541 +/- 12 at BL, 151 +/- 29 at 12 hours [p < .05 vs. control], and 118 +/- 20 at 24 hrs), and significantly reduced the increase in pulmonary microvascular shunt fraction (Qs/Qt; control group at BL, 0.14 +/- 0.02, and at 24 hrs, 0.65 +/- 0.08; rhAPC group at BL, 0.24 +/- 0.04, and at 24 hrs, 0.45 +/- 0.02 [p < .05 vs. control]) and the increase in peak airway pressure (mbar; control group at BL, 20 +/- 1, and at 24 hrs, 36 +/- 4; rhAPC group at BL, 21 +/- 1, and at 24 hrs, 28 +/- 2 [p < .05 vs. control]). In addition, rhAPC limited the increase in lung 3-nitrotyrosine (after 24 hrs [%]: sham, 7 +/- 2; control, 17 +/- 1; rhAPC, 12 +/- 1 [p < .05 vs. control]), a reliable indicator of tissue injury. However, rhAPC failed to prevent lung edema formation. RhAPC-treated sheep showed no difference in activated clotting time or platelet count but exhibited less fibrin degradation products (1/6 animals) than did controls (4/6 animals).. Recombinant human activated protein C attenuated ALI after smoke inhalation and bacterial challenge in sheep, without bleeding complications.

Topics: Airway Obstruction; Animals; Blood Pressure; Body Temperature; Disease Models, Animal; Female; Fibrinolytic Agents; Infusions, Intravenous; Lung; Nitrates; Nitrites; Organ Size; Prospective Studies; Protein C; Pulmonary Edema; Random Allocation; Recombinant Proteins; Respiratory Function Tests; Sepsis; Sheep; Smoke Inhalation Injury; Tyrosine

Mice deficient in inducible nitric oxide synthase (iNOS; C57Bl/6Ai-[KO]NOS2 N5) or wild-type C57Bl/6 mice were exposed to 1 part/million of ozone 8 h/night or to filtered air for three consecutive nights. Endpoints measured included lavagable total protein, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, cell content, and tyrosine nitration of whole lung proteins. Ozone exposure caused acute edema and an inflammatory response in the lungs of wild-type mice, as indicated by significant increases in lavage protein content, MIP-2 and MMP-9 content, and polymorphonuclear leukocytes. The iNOS knockout mice showed significantly greater levels of lung injury by all of these criteria than did the wild-type mice. We conclude that iNOS knockout mice are more susceptible to acute lung damage induced by exposure to ozone than are wild-type C57Bl/6 mice and that protein nitration is associated with the degree of inflammation and not dependent on iNOS-derived nitric oxide.

Topics: Acute Disease; Animals; Bronchoalveolar Lavage Fluid; Chemokine CXCL2; Chemokines; Disease Susceptibility; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Ozone; Pneumonia; Proteins; Pulmonary Edema; Tyrosine

Reactive oxygen and nitrogen species such as superoxide and nitric oxide are released into the extracellular spaces by inflammatory and airway epithelial cells. These molecules may exacerbate lung injury after influenza virus pneumonia. We hypothesized that enhanced expression of extracellular superoxide dismutase (EC SOD) in mouse airways would attenuate the pathological effects of influenza pneumonia. We compared the pathogenic effects of a nonlethal primary infection with mouse-adapted Hong Kong influenza A/68 virus in transgenic (TG) EC SOD mice versus non-TG (wild-type) littermates. Compared with wild-type mice, EC SOD TG mice showed less lung injury and inflammation as measured by significant blunting of interferon-gamma induction, reduced cell count and total protein in bronchoalveolar lavage fluid, reduced levels of lung nitrite/nitrate nitrotyrosine, and markedly reduced lung pathology. These results demonstrate that enhancing EC SOD in the conducting and distal airways of the lung minimizes influenza-induced lung injury by both ameliorating inflammation and attenuating oxidative stress.

Topics: Animals; Antioxidants; Biomarkers; Bronchoalveolar Lavage Fluid; Cytokines; Female; Gene Expression Regulation, Enzymologic; Glutathione Disulfide; Humans; Influenza A virus; Influenza, Human; Lung; Male; Mice; Mice, Transgenic; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Oxidative Stress; Pneumonia, Viral; Pulmonary Edema; RNA, Messenger; Superoxide Dismutase; Thromboxane B2; Tyrosine

Nitric oxide (NO) may either protect against or contribute to inflammatory lung injury. In this study we investigated whether inhalation of 20 ppm NO alters tyrosine nitration, and we assessed the degree of lung inflammation and edema in rats after lipopolysaccharide (LPS) instillation. The amount of nitrotyrosine relative to the total amount of tyrosine was measured in lung homogenates, and lung tissue sections were stained for nitrotyrosine and aminotyrosine (a reduced form of nitrotyrosine). Leukocytes in bronchoalveolar lavage fluid (BALF) were counted, and myeloperoxidase activity was measured in lung homogenate. Lung edema and inflammatory cell accumulation in lung tissue were estimated by extravascular lung water weight (EVLW) and extravascular dry lung weight (EVDW), respectively. LPS instillation caused increases in nitrotyrosine concentration and immunohistochemical staining of nitrotyrosine and aminotyrosine in the lungs. LPS instillation increased the BALF leukocyte count, myeloperoxidase activity in lung tissue, and both EVLW and EVDW. Inhalational exposure to 20 ppm NO induced nitrotyrosine and aminotyrosine formation only in bronchial epithelial cell surface of the lungs not instilled with LPS. NO inhalation reduced the increases in nitrotyrosine and aminotyrosine in LPS-instilled lung tissue as well as the leukocyte count in BALF and myeloperoxidase activity in lung tissue, but it did not significantly change EVLW or EVDW. Leukocyte depletion in LPS-instilled rats reduced interstitial inflammatory cells, which were stained with nitrotyrosine and aminotyrosine, and attenuated the nitrotyrosine staining of alveolar capillaries. These results suggest that inhalation of 20 ppm NO reduces leukocyte accumulation in the lungs and inhibits tyrosine nitration caused by LPS instillation.

Topics: Administration, Inhalation; Animals; Bronchoalveolar Lavage Fluid; Instillation, Drug; Leukocyte Count; Lipopolysaccharides; Lung; Male; Nitric Oxide; Peroxidase; Pulmonary Edema; Rats; Rats, Sprague-Dawley; Tyrosine