Inhaled nitric oxide attenuates pulmonary inflammation and fibrin deposition and prolongs survival in neonatal hyperoxic lung injury

Administration of inhaled nitric oxide (iNO) is a potential therapeutic strategy to prevent bronchopulmonary dysplasia (BPD) in premature newborns with respiratory distress syndrome. We evaluated this approach in a rat model, in which premature pups were exposed to room air, hyperoxia, or a combination of hyperoxia and NO (8.5 and 17 ppm). We investigated the anti-inflammatory effects of prolonged iNO therapy by studying survival, histopathology, fibrin deposition, and differential mRNA expression (real-time RT-PCR) of key genes involved in the development of BPD. iNO therapy prolonged median survival 1.5 days (P = 0.0003), reduced fibrin deposition in a dosage-dependent way up to 4.3-fold (P < 0.001), improved alveolar development by reducing septal thickness, and reduced the influx of leukocytes. Analysis of mRNA expression revealed an iNO-induced downregulation of genes involved in inflammation (IL-6, cytokine-induced neutrophilic chemoattractant-1, and amphiregulin), coagulation, fibrinolysis (plasminogen activator inhibitor 1 and urokinase-type plasminogen activator receptor), cell cycle regulation (p21), and an upregulation of fibroblast growth factor receptor-4 (alveolar formation). We conclude that iNO therapy improves lung pathology and prolongs survival by reducing septum thickness, inhibiting inflammation, and reducing alveolar fibrin deposition in premature rat pups with neonatal hyperoxic lung injury.     

Suppression of nuclear factor-kappa B activity by nitric oxide and hyperoxia in oxygen-resistant cells

Inhaled nitric oxide (iNO) is used clinically to treat pulmonary hypertension in newborns, often in conjunction with hyperoxia (NO/O2). Prolonged exposure to NO/O2 causes synergistic lung injury and death of lung epithelial cells. To explore the mechanisms involved, oxygen-resistant HeLa-80 cells were exposed to NO +/- O2. Exposure to NO and O2 induced a synergistic cytotoxicity, accompanied with apoptotic characteristics, including elevated caspase-3-like activity, Annexin V incorporation, and nuclear condensation. This apoptosis was associated with a synergistic suppression of NF-kappaB activity. Cells lacking functional NF-kappaB p65 subunit were more sensitive to NO/O2 than their wild type counterparts. This injury was partially rescued by transfection with a p65 expression construct, suggesting an inverse relationship between NF-kappaB and susceptibility to the cytotoxicity of NO/O2. Despite the reduced NF-kappaB activity in cells exposed to NO +/- O2, IkappaBalpha was degraded, suggesting that pathways regulating the steady-state levels of IkappaB were not involved. However, exposure to NO/O2 caused a marked reduction in nuclear localization and an increase in protein carbonyl formation of NF-kappaB p65 subunit. These results suggest that NO/O2-induced apoptosis occurs by suppressing NF-kappaB activity.     

Hydrogen inhalation reduced epithelial apoptosis in ventilator-induced lung injury via a mechanism involving nuclear factor-kappa B activation

Entry of enveloped viruses into cells is initiated by binding of their envelope glycoproteins (Envs) to cell surface-associated receptors. The Crimean-Congo hemorrhagic fever virus (CCHFV) has two Envs, Gn and Gc, with poorly understood role in binding to susceptible cells. We expressed codon optimized Gn and Gc, and identified independently folded soluble Env fragments, one of which (Gc residues 180–300) bound CCHFV susceptible cells supposedly by interacting with a putative receptor. This receptor binding domain (RBD) was used to identify its interacting partner by coimmunoprecipitation and mass spectrometry. Thus we identified the human cell surface nucleolin as a putative CCHFV entry factor. Nucleolin was expressed on all susceptible cells tested but not on the surface of cells resistant to CCHFV infection. Further studies are needed to explore the nucleolin function as a plausible CCHFV receptor and the molecular mechanisms of the Gc-nucleolin interactions. The identification of the CCHFV RBD and its binding partner could provide novel targets for therapy and tools for prevention as well as more complete understanding of the mechanisms of CCHFV entry and pathogenesis.     

Hydrogen inhalation reduced epithelial apoptosis in ventilator-induced lung injury via a mechanism involving nuclear factor-kappa B activation

We recently demonstrated the inhalation of hydrogen gas, a novel medical therapeutic gas, ameliorates ventilator-induced lung injury (VILI); however, the molecular mechanisms by which hydrogen ameliorates VILI remain unclear. Therefore, we investigated whether inhaled hydrogen gas modulates the nuclear factor-kappa B (NFκB) signaling pathway. VILI was generated in male C57BL6 mice by performing a tracheostomy and placing the mice on a mechanical ventilator (tidal volume of 30 ml/kg or 10 ml/kg without positive end-expiratory pressure). The ventilator delivered either 2% nitrogen or 2% hydrogen in balanced air. NFκB activation, as indicated by NFκB DNA binding, was detected by electrophoretic mobility shift assays and enzyme-linked immunosorbent assay. Hydrogen gas inhalation increased NFκB DNA binding after 1 h of ventilation and decreased NFκB DNA binding after 2 h of ventilation, as compared with controls. The early activation of NFκB during hydrogen treatment was correlated with elevated levels of the antiapoptotic protein Bcl-2 and decreased levels of Bax. Hydrogen inhalation increased oxygen tension, decreased lung edema, and decreased the expression of proinflammatory mediators. Chemical inhibition of early NFκB activation using SN50 reversed these protective effects. NFκB activation and an associated increase in the expression of Bcl-2 may contribute, in part, to the cytoprotective effects of hydrogen against apoptotic and inflammatory signaling pathway activation during VILI.     

Inhibition of neuronal nitric oxide synthase by 7-nitroindazole attenuates acute lung injury in an ovine model

Nitric oxide (NO) has been shown to play a major role in acute lung injury (ALI) after smoke inhalation. In the present study, we developed an ovine sepsis model, created by exposing sheep to smoke inhalation followed by instillation of bacteria into the airway, that mimics human sepsis and pneumonia. We hypothesized that the inhibition of neuronal NO synthase (nNOS) might be beneficial in treating ALI associated with this model. Female sheep (n = 26) were surgically prepared for the study and given a tracheostomy. This was followed by insufflation of 48 breaths of cotton smoke (40 degrees C) into the airway of each animal and subsequent instillation of live Pseudomonas aeruginosa [5 x 10(11) colony forming units (CFU)] into each sheep's lung. All sheep were mechanically ventilated using 100% O2. Continuous infusion of 7-nitroindazole (7-NI), an nNOS inhibitor, NG-monomethyl-l-arginine (l-NMMA), a nonspecific NOS inhibitor, or aminoguanidine (AG), an inducible NOS inhibitor, was started 1 h after insult. The administration of 7-NI improved pulmonary gas exchange (PaO2/FiO2; where PaO2 is arterial PO2 and FiO2 is fractional inspired oxygen concentration) and pulmonary shunt fraction and attenuated the increase in lung wet-to-dry weight ratio seen in the nontreated sheep. Histologically, 7-NI prevented airway obstruction. The increase in airway blood flow after injury in the nontreated group was significantly inhibited by 7-NI. The increase in plasma concentration of nitrate and nitrite (NOx) was inhibited by 7-NI as well. Posttreatment with l-NMMA improved the pulmonary gas exchange, but AG did not. The results of the present study show that nNOS may be involved in the pathogenesis of ALI after smoke inhalation injury followed by bacterial instillation in the airway.     

Curcumin attenuates ovalbumin-induced airway inflammation by regulating nitric oxide

Curcumin has been strongly implicated as an anti-inflammatory agent, but the precise mechanisms of its action are largely unknown. In this study, we show that curcumin contributes to anti-inflammatory activity in the murine asthma model and lung epithelial cell A549 through suppression of nitric oxide (NO). To address this problem, curcumin was injected into the peritoneum of ovalbumin (OVA)-sensitized mice before the last allergen challenge. OVA challenge resulted in activation of the production of inducible nitric oxide (iNOS) in lung tissue, inflammatory cytokines, recruitment of eosinophils to lung airways, and airway hyper-responsiveness to inhaled methacholine. These effects of ovalbumin challenge were all inhibited by pretreatment of mice with curcumin. Furthermore, supplementation with curcumin in the A549 human airway epithelial cells decreased iNOS and NO production induced by IFN-γ. These findings show that curcumin may be useful as an adjuvant therapy for airway inflammation through suppression of iNOS and NO.     

Chronic inhibition of nuclear factor kappa B attenuates aldosterone/salt-induced renal injury

AimsRecent studies suggested that nuclear factor kappa B (NF-κB) plays a key role in the pathogenesis of renal injury. This study investigated whether NF-κB inhibition attenuates progressive renal damage in aldosterone/salt-induced renal injury and its mechanisms.Main methodsAdult male rats were uninephrectomized and treated with one of the following for 4 weeks: vehicle (0.5% ethanol, subcutaneously); vehicle/1% NaCl (1% NaCl in drinking solution); aldosterone/1% NaCl (1% NaCl in drinking solution and aldosterone, 0.75 μg/h, subcutaneously); or aldosterone/1%NaCl + pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB (100 mg/kg/day, by gavage). The activity of NF-κB was measured by EMSA and immunohistochemistry, CTGF and ICAM-1 were measured by Western blot and real-time PCR, and TGF-β and CTGF were measured by immunohistochemistry.Key findingsRats that received aldosterone/1% NaCl exhibited hypertension and severe renal injury. Renal cortical mRNA levels of CTGF, TGF-β, ICAM-1 and collagen IV, protein expression of CTGF and ICAM-1, and NF-κB–DNA binding activity were significantly upregulated in rats that received aldosterone/1% NaCl. Treatment with PDTC significantly decreased the percentage of cells positive for CTGF and TGF-β; mRNA levels of CTGF, TGF-β, ICAM-1 and collagen IV, and protein levels of CTGF and ICAM-1 were also inhibited by PDTC.SignificanceThese data suggest that the NF-κB signal pathway plays a role in the progression of aldosterone/salt-induced renal injury.     

Depletion of tissue plasminogen activator attenuates lung ischemia-reperfusion injury via inhibition of neutrophil extravasation

Ischemia-reperfusion (IR) injury following lung transplantation remains a major source of early morbidity and mortality. Histologically, this inflammatory process is characterized by neutrophil infiltration and activation. We previously reported that lung IR injury was significantly attenuated in plasminogen activator inhibitor-1-deficient mice. In this study, we explored the potential role of tissue plasminogen activator (tPA) in a mouse lung IR injury model. As a result, tPA knockout (KO) mice were significantly protected from lung IR injury through several mechanisms. At the cellular level, tPA KO specifically blocked neutrophil extravasation into the interstitium, and abundant homotypic neutrophil aggregation (HNA) was detected in the lung microvasculature of tPA KO mice after IR. At the molecular level, inhibition of neutrophil extravasation was associated with reduced expression of platelet endothelial cell adhesion molecule-1 mediated through the tPA/ LDL receptor-related protein/NF-κB signaling pathway, whereas increased P-selectin triggered HNA. At the functional level, tPA KO mice incurred significantly decreased vascular permeability and improved lung function following IR. Protection from lung IR injury in tPA KO mice occurs through a fibrinolysis-independent mechanism. These results suggest that tPA could serve as an important therapeutic target for the prevention and treatment of acute IR injury after lung transplantation.     

Proinflammatory role of inducible nitric oxide synthase in acute hyperoxic lung injury

BackgroundHyperoxic exposures are often found in clinical settings of respiratory insufficient patients, although oxygen therapy (>50% O₂) can result in the development of acute hyperoxic lung injury within a few days. Upon hyperoxic exposure, the inducible nitric oxide synthase (iNOS) is activated by a variety of proinflammatory cytokines both in vitro and in vivo. In the present study, we used a murine hyperoxic model to evaluate the effects of iNOS deficiency on the inflammatory response.MethodsWild-type and iNOS-deficient mice were exposed to normoxia, 60% O₂ or >95% O₂ for 72 h.ResultsExposure to >95% O₂ resulted in an increased iNOS mRNA and protein expression in the lungs from wild-type mice. No significant effects of iNOS deficiency on cell differential in bronchoalveolar lavage fluid were observed. However, hyperoxia induced a significant increase in total cell count, protein concentration, LDH activity, lipid peroxidation, and TNF-α concentration in the bronchoalveolar lavage fluid compared to iNOS knockout mice. Moreover, binding activity of NF-κB and AP-1 appeared to be higher in wild-type than in iNOS-deficient mice.ConclusionTaken together, our results provide evidence to suggest that iNOS plays a proinflammatory role in acute hyperoxic lung injury.     

Dual role of inducible nitric oxide synthase in acute asbestos-induced lung injury

Reactive oxygen and nitrogen species have been implicated in the pathogenesis of asbestos fibers-associated pulmonary diseases. By comparing the responses of inducible nitric oxide synthase (iNOS) knockout and wild-type mice we investigated the consequences of iNOS expression for the development of the inflammatory response and tissue injury upon intratracheal instillation of asbestos fibers. Exposure to asbestos fibers resulted in an increased iNOS mRNA and protein expression in the lungs from wild-type mice. Moreover, iNOS knockout mice exhibited an exceeded pulmonary expression and production of TNF-alpha as well as a higher influx of neutrophils into the alveolar space than wild-type mice. In contrast, iNOS knockout animals displayed an attenuated oxidant-related tissue injury reflected in a decrease in protein leakage and LDH release into the alveolar space as well as weaker nitrotyrosine staining of lung tissue compared to wild-type mice. Data presented here indicate that iNOS-derived NO exerts a dichotomous role in acute asbestos-induced lung injury in that iNOS deficiency resulted in an exacerbated inflammatory response but improved oxidant-promoted lung tissue damage.     

Inhalation of an endothelin receptor A antagonist attenuates pulmonary inflammation in experimental acute lung injury

We recently demonstrated that inhalation of the endothelin receptor A (ETA) antagonist LU 135252 improved arterial oxygenation and reduced pulmonary artery pressure in experimental acute lung injury (ALI). In this study we analyzed potential immune modulatory effects of inhaled LU 135252 in experimental ALI. ALI was induced by repeated lung lavage in intubated (100% O2) and anesthetized piglets. Animals were randomly assigned to inhale either nebulized LU 135252 (0.3 mg.kg-1, ALI + LU group, n = 8) or saline buffer (ALI control group, n = 16), both for 30 min. Surviving animals were sacrificed 6 h after induction of ALI, and lung tissue specimens were obtained from all animals for histology and immunhistochemistry. Induction of ALI significantly decreased arterial oxygenation in all animals. Inhalation of LU 135252 significantly reduced mortality and induced significant and sustained increase in Pao2 (316 +/- 47 mm Hg vs. control 53 +/- 3 mm Hg, p < 0.001). We measured a significant reduction in the number of pulmonary leukocyte L1 antigen-positive cells in ALI + LU animals (8% +/- 1% positive cells vs. control 12% +/- 2% positive cells, p < 0.05). The number of CD3-positive cells was not altered by treatment with LU 135252. Pulmonary tissue concentration of IL-6 was significantly suppressed by LU 135252 inhalation (4 +/- 1 pg.100 mg-1 wet weight vs. control 7 +/- 1 pg.100 mg-1 wet weight, p < 0.05). Concentrations of TNF-alpha, IL-1beta, and ET-1 in pulmonary tissue were not influenced by inhalation of LU 135252. In conclusion, we demonstrated that inhalation of LU 135252 not only improves mortality and gas exchange, but also blunts the local immune response in experimental ALI.     

Catalase protects cardiomyocytes via its inhibition of nitric oxide synthesis.

Nitric oxide (NO) has been reported to play an important role as an effector molecule in cytokine signal transduction in cardiomyocytes. A treatment of neonatal rat ventricular cardiomyocytes with interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) induces apoptosis via an NO-dependent pathway. However, cardiomyocytes were more resistant to NO-dependent cell death in the presence of catalase, while producing inducible nitric oxide synthase. This paper reports that catalase stimulates the NF-kappaB-binding affinity. However, the NO synthase activity is abolished by the addition of catalase, suggesting that H(2)O(2) is involved in NO synthesis in a posttranslation state. The catalase-induced inhibition of NO was partially but significantly reversed by H(4)B, an important cofactor of NO synthesis. Treatment of myocytes with IL-1beta, TNF-alpha, and IFN-gamma induced a significant increase in the formation of peroxynitrite, and a pretreatment with catalase was found to quench the production of peroxynitrite. This paper shows that the catalase activity was significantly down-regulated by H(4)B in a concentration-dependent manner. The treatment of H(4)B induced reactive oxygen species (ROS) release in cardiac cell system. These results suggest that catalase interferes with NO and peroxynitrite production as well as with the related apoptosis of cardiomyocytes. This study also shows that the catalase-induced inhibition of NO release may be reversed by H(4)B by the release of ROS.     

Inhaled nitric oxide attenuates pulmonary hypertension and improves lung growth in infant rats after neonatal treatment with a VEGF receptor inhibitor

VEGF plays a critical role during lung development and is decreased in human infants with bronchopulmonary dysplasia. Inhibition of VEGF receptors in the newborn rat decreases vascular growth and alveolarization and causes pulmonary hypertension (PH). Nitric oxide (NO) is a downstream mediator of VEGF, but whether the effects of impaired VEGF signaling are due to decreased NO production is unknown. Therefore, we sought to determine whether impaired VEGF signaling downregulates endothelial NO synthase (eNOS) expression in the developing lung and whether inhaled NO (iNO) decreases PH and improves lung growth after VEGF inhibition. Newborn rats received a single dose of SU-5416 (a VEGF receptor inhibitor) or vehicle by subcutaneous injection and were killed up to 3 wk of age for assessments of right ventricular hypertrophy (RVH), radial alveolar counts (RAC), lung eNOS protein, and NOx production in isolated perfused lungs (IPL). Neonatal treatment with SU-5416 increased RVH in infant rats and reduced RAC. Compared with controls, SU-5416 reduced lung eNOS protein expression by 89% at 5 days (P < 0.01). IPL studies from day 14 rats demonstrated increased baseline pulmonary artery pressure and lower perfusate NOx concentration after SU-5416 treatment. Importantly, iNO treatment prevented the increase in RVH and improved RAC after SU-5416 treatment. We conclude that treatment of neonatal rats with SU-5416 downregulates lung eNOS expression and that iNO therapy decreases PH and improves lung growth after SU-5416 treatment. We speculate that decreased NO production contributes to PH and decreases distal lung growth caused by impaired VEGF signaling.     

Simvastatin attenuates vascular leak and inflammation in murine inflammatory lung injury

Therapies to limit the life-threatening vascular leak observed in patients with acute lung injury (ALI) are currently lacking. We explored the effect of simvastatin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor that mediates endothelial cell barrier protection in vitro, in a murine inflammatory model of ALI. C57BL/6J mice were treated with simvastatin (5 or 20 mg/kg body wt via intraperitoneal injection) 24 h before and again concomitantly with intratracheally administered LPS (2 microg/g body wt). Inflammatory indexes [bronchoalveolar lavage (BAL) myeloperoxidase activity and total neutrophil counts assessed at 24 h with histological confirmation] were markedly increased after LPS alone but significantly reduced in mice that also received simvastatin (20 mg/kg; approximately 35-60% reduction). Simvastatin also decreased BAL albumin (approximately 50% reduction) and Evans blue albumin dye extravasation into lung tissue (100%) consistent with barrier protection. Finally, the sustained nature of simvastatin-mediated lung protection was assessed by analysis of simvastatin-induced gene expression (Affymetrix platform). LPS-mediated lung gene expression was significantly modulated by simvastatin within a number of gene ontologies (e.g., inflammation and immune response, NF-kappaB regulation) and with respect to individual genes implicated in the development or severity of ALI (e.g., IL-6, Toll-like receptor 4). Together, these findings confirm significant protection by simvastatin on LPS-induced lung vascular leak and inflammation and implicate a potential role for statins in the management of ALI.