Knockdown of lung phosphodiesterase 2A attenuates alveolar inflammation and protein leak in a two-hit mouse model of acute lung injury

Phosphodiesterase 2A (PDE2A) is stimulated by cGMP to hydrolyze cAMP, a potent endothelial barrier-protective molecule. We previously found that lung PDE2A contributed to a mouse model of ventilator-induced lung injury (VILI). The purpose of the present study was to determine the contribution of PDE2A in a two-hit mouse model of 1-day intratracheal (IT) LPS followed by 4 h of 20 ml/kg tidal volume ventilation. Compared with IT water controls, LPS alone (3.75 μg/g body wt) increased lung PDE2A mRNA and protein expression by 6 h with a persistent increase in protein through day 4 before decreasing to control levels on days 6 and 10. Similar to the PDE2A time course, the peak in bronchoalveolar lavage (BAL) neutrophils, lactate dehydrogenase (LDH), and protein concentration also occurred on day 4 post-LPS. IT LPS (1 day) and VILI caused a threefold increase in lung PDE2A and inducible nitric oxide synthase (iNOS) and a 24-fold increase in BAL neutrophilia. Compared with a control adenovirus, PDE2A knockdown with an adenovirus expressing a short hairpin RNA administered IT 3 days before LPS/VILI effectively decreased lung PDE2A expression and significantly attenuated BAL neutrophilia, LDH, protein, and chemokine levels. PDE2A knockdown also reduced lung iNOS expression by 53%, increased lung cAMP by nearly twofold, and improved survival from 47 to 100%. We conclude that in a mouse model of LPS/VILI, a synergistic increase in lung PDE2A expression increased lung iNOS and alveolar inflammation and contributed significantly to the ensuing acute lung injury.     

N-acetylcysteine pretreatment attenuates paraquat-induced lung leak in rats

We investigated the effects of N-acetylcysteine (NAC) pretreatment on paraquat-induced lung inflammation and leak. We found that administering a single intravenous dose (60 mg/kg) of paraquat rapidly (2 h) increased lung leak, lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels, and lung myeloperoxidase (MPO) activity in rats. Rats pretreated with NAC (150 mg/kg, intraperitoneally) had increased lung tissue glutathione (GSH + GSSG) levels compared to saline-pretreated rats. In addition, rats pretreated with NAC and then given paraquat 2.5 h later had decreased lung leak compared to saline-pretreated rats given paraquat. In contrast, NAC pretreated rats given paraquat had the same lung lavage CINC levels and lung tissue MPO activity as saline-pretreated rats given paraquat. Our results indicate that paraquat causes an oxidative injury which may be decreased by the GSH-increasing or other properties of NAC.     

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.     

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.     

Inhaled nitric oxide attenuates acute lung injury via inhibition of nuclear factor-kappa B and inflammation.

The effect of inhaled nitric oxide (NO) on inflammatory process in acute lung injury (ALI) is unclear. The aims of this study were to 1) examine whether inhaled NO affects the biochemical lung injury parameters and cellular inflammatory responses and 2) determine the effect of inhaled NO on the activation of nuclear factor-kappa B (NF-kappa B) in lipopolysaccharide (LPS)-induced ALI. Compared with saline controls, rabbits treated intravenously with LPS showed increases in total protein and lactate dehydrogenase in the bronchoalveolar lavage (BAL) fluid, indicating ALI. LPS-treated animals with NO inhalation (LPS-NO) showed significant decreases in these parameters. Neutrophil numbers in the BAL fluid, the activity of reactive oxygen species in BAL cells, and the levels of interleukin (IL)-1 beta and IL-8 in alveolar macrophages were increased in LPS-treated animals. In contrast, neutrophil numbers and these cellular activities were substantially decreased in LPS-NO animals, compared with LPS-treated animals. NF-kappa B activation in alveolar macrophages from LPS-treated animals was also markedly increased, whereas this activity was effectively blocked in LPS-NO animals. These results suggest that inhaled NO attenuates LPS-induced ALI and pulmonary inflammation. This attenuation may be associated with the inhibition of NF-kappa B activation.     

Fullerene derivative attenuates ischemia-reperfusion-induced lung injury

Reactive oxygen species are the major contributing factors to lung ischemia-reperfusion (IR) injury. In this study, we tested whether a water soluble antioxidant fullerene derivative [C(60)(ONO(2))(7 +/- 2)] attenuates IR lung injury. Young Wistar rats were divided into two groups: control and C(60)(ONO(2))(7 +/- 2). Under ventilation with 95% air-5% CO(2) gas mixture and a 2.5 cm H(2)O end-expiratory pressure, the isolated lungs were perfused with a physiological solution. The experimental protocol included three periods: baseline (10 min), ischemia (45 min) and reperfusion (60 min, ventilated with 95% O(2)-5% CO(2) gas mixture). Before and after ischemia, we measured pulmonary arterial pressure (Ppa), pulmonary venous pressure and lung weight (W). Then, pulmonary capillary pressure and filtration coefficient (K(fc)) were calculated. Ischemia caused increases in Ppa, W and K(fc) in the control group. For most cases, the above ischemia-induced increases were attenuated by the C(60)(ONO(2))(7 +/- 2) pretreatment. Our results suggest that the antioxidant C(60)(ONO(2))(7 +/- 2) attenuates IR-induced lung injury.     

Cytokine-mediated inflammation in acute lung injury

Clinical acute lung injury (ALI) is a major cause of acute respiratory failure in critically ill patients. There is considerable experimental and clinical evidence that pro- and anti-inflammatory cytokines play a major role in the pathogenesis of inflammatory-induced lung injury from sepsis, pneumonia, aspiration, and shock. A recent multi-center clinical trial found that a lung-protective ventilatory strategy reduces mortality by 22% in patients with ALI. Interestingly, this protective ventilatory strategy was associated with a marked reduction in the number of neutrophils and the concentration of pro-inflammatory cytokines released into the airspaces of the injured lung. Further research is needed to establish the contribution of cytokines to both the pathogenesis and resolution of ALI.     

Sildenafil attenuates pulmonary inflammation and fibrin deposition, mortality and right ventricular hypertrophy in neonatal hyperoxic lung injury

Background

Phosphodiesterase-5 inhibition with sildenafil has been used to treat severe pulmonary hypertension and bronchopulmonary dysplasia (BPD), a chronic lung disease in very preterm infants who were mechanically ventilated for respiratory distress syndrome.

Methods

Sildenafil treatment was investigated in 2 models of experimental BPD: a lethal neonatal model, in which rat pups were continuously exposed to hyperoxia and treated daily with sildenafil (50–150 mg/kg body weight/day; injected subcutaneously) and a neonatal lung injury-recovery model in which rat pups were exposed to hyperoxia for 9 days, followed by 9 days of recovery in room air and started sildenafil treatment on day 6 of hyperoxia exposure. Parameters investigated include survival, histopathology, fibrin deposition, alveolar vascular leakage, right ventricular hypertrophy, and differential mRNA expression in lung and heart tissue.

Results

Prophylactic treatment with an optimal dose of sildenafil (2 × 50 mg/kg/day) significantly increased lung cGMP levels, prolonged median survival, reduced fibrin deposition, total protein content in bronchoalveolar lavage fluid, inflammation and septum thickness. Treatment with sildenafil partially corrected the differential mRNA expression of amphiregulin, plasminogen activator inhibitor-1, fibroblast growth factor receptor-4 and vascular endothelial growth factor receptor-2 in the lung and of brain and c-type natriuretic peptides and the natriuretic peptide receptors NPR-A, -B, and -C in the right ventricle. In the lethal and injury-recovery model we demonstrated improved alveolarization and angiogenesis by attenuating mean linear intercept and arteriolar wall thickness and increasing pulmonary blood vessel density, and right ventricular hypertrophy (RVH).

Conclusion

Sildenafil treatment, started simultaneously with exposure to hyperoxia after birth, prolongs survival, increases pulmonary cGMP levels, reduces the pulmonary inflammatory response, fibrin deposition and RVH, and stimulates alveolarization. Initiation of sildenafil treatment after hyperoxic lung injury and continued during room air recovery improves alveolarization and restores pulmonary angiogenesis and RVH in experimental BPD.     

Hypoxia decreases lung neprilysin expression and increases pulmonary vascular leak

Although prior studies suggest that hypoxia may increase pulmonary vascular permeability, the mechanisms responsible for that effect remain uncertain. Neprilysin (neutral endopeptidase) is a cell surface metallopeptidase that degrades several vasoactive peptides including substance P and bradykinin. We hypothesized that hypoxia could reduce lung neprilysin expression, leading to increased vascular leak. Weanling rats were exposed to normobaric hypoxia (inspired O(2) fraction = 0.1). Lung neprilysin activity was significantly decreased after 24 and 48 h of hypoxia (P < 0.006). The decrease in enzyme activity was associated with decreased lung neprilysin protein content and decreased lung neprilysin mRNA expression. Immunohistochemistry showed a predominantly perivascular distribution of neprilysin, with clear reductions in neprilysin immunoreactivity after exposure to hypoxia. Exposure to hypoxia for 24 h also caused marked increases in vascular leak (P = 0.008), which were reversed by the administration of recombinant neprilysin. The hypoxia-induced increase in leak was also reversed by substance P and bradykinin receptor antagonists. We conclude that in young rats hypoxia decreases lung neprilysin expression, which contributes to increased pulmonary vascular leak via substance P and bradykinin receptors.     

Cannabinoid receptor 1 antagonist genistein attenuates marijuana-induced vascular inflammation

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Plant recombinant erythropoietin attenuates inflammatory kidney cell injury

Human erythropoietin (EPO) is a pleiotropic cytokine with remarkable tissue-protective activities in addition to its well-established role in red blood cell production. Unfortunately, conventional mammalian cell cultures are unlikely to meet the anticipated market demands for recombinant EPO because of limited capacity and high production costs. Plant expression systems may address these limitations to enable practical, cost-effective delivery of EPO in tissue injury prevention therapeutics. In this study, we produced human EPO in tobacco and demonstrated that plant-derived EPO had tissue-protective activity. Our results indicated that targeting to the endoplasmic reticulum (ER) provided the highest accumulation levels of EPO, with a yield approaching 0.05% of total soluble protein in tobacco leaves. The codon optimization of the human EPO gene for plant expression had no clear advantage; furthermore, the human EPO signal peptide performed better than a tobacco signal peptide. In addition, we found that glycosylation was essential for the stability of plant recombinant EPO, whereas the presence of an elastin-like polypeptide fusion had a limited positive impact on the level of EPO accumulation. Confocal microscopy showed that apoplast and ER-targeted EPO were correctly localized, and N -glycan analysis demonstrated that complex plant glycans existed on apoplast-targeted EPO, but not on ER-targeted EPO. Importantly, plant-derived EPO had enhanced receptor-binding affinity and was able to protect kidney epithelial cells from cytokine-induced death in vitro . These findings demonstrate that tobacco plants may be an attractive alternative for the production of large amounts of biologically active EPO.     

TREM-1 deficiency attenuates the inflammatory responses in LPS-induced murine endometritis

Endometritis, which is usually caused by bacterial infection, is characterized by high levels of pro-inflammatory cytokines and a high infertility rate. Triggering receptor expressed on myeloid cells-1 (TREM-1) has been recognized as a potent amplifier of inflammatory reactions. Studies have demonstrated reduced inflammatory responses and mortality rates of animals with bacterial infection due to the blocking of TREM-1 expression. However, whether TREM-1 deficiency could alleviate the inflammatory reaction in bacterial endometritis is still unclear. Here, TREM-1 knock-out (Trem-1^−/−) mice were used to inhibit TREM-1 signalling to evaluate its role in inflammatory reactions after a highly pathogenic LPS infection in mice uteri. The results demonstrated that TREM-1 deficiency attenuated the inflammation in mice uteri; markedly reduced the number of polymorphonuclear neutrophils; and suppressed interleukin-1β (IL-1β), IL-6, and tumour necrosis factor-α (TNF-α) concentrations in serum as well as their production in inflamed uteri after LPS stimulation. Our results illustrate an anticipated pathogenic impact of TREM-1 on endometritis during LPS infection and indicate that blocking of TREM-1 in LPS-induced endometritis holds considerable promise for blunting excessive inflammation.     

IQGAP1 is necessary for pulmonary vascular barrier protection in murine acute lung injury and pneumonia

We recently reported that integrin α(v)β(3) is necessary for vascular barrier protection in mouse models of acute lung injury and peritonitis. Here, we used mass spectrometric sequencing of integrin complexes to isolate the novel β(3)-integrin binding partner IQGAP1. Like integrin β(3), IQGAP1 localized to the endothelial cell-cell junction after sphingosine-1-phosphate (S1P) treatment, and IQGAP1 knockdown prevented cortical actin formation and barrier enhancement in response to S1P. Furthermore, knockdown of IQGAP1 prevented localization of integrin α(v)β(3) to the cell-cell junction. Similar to β(3)-null animals, IQGAP1-null mice had increased pulmonary vascular leak compared with wild-type controls 3 days after intratracheal LPS. In an Escherichia coli pneumonia model, IQGAP1 knockout mice had increased lung weights, lung water, and lung extravascular plasma equivalents of (125)I-labeled albumin compared with wild-type controls. Taken together, these experiments indicate that IQGAP1 is necessary for S1P-mediated vascular barrier protection during acute lung injury and is required for junctional localization of the barrier-protective integrin α(v)β(3).     

Linking lung function and inflammatory responses in ventilator-induced lung injury

Despite decades of research, the mechanisms of ventilator-induced lung injury are poorly understood. We used strain-dependent responses to mechanical ventilation in mice to identify associations between mechanical and inflammatory responses in the lung. BALB/c, C57BL/6, and 129/Sv mice were ventilated using a protective [low tidal volume and moderate positive end-expiratory pressure (PEEP) and recruitment maneuvers] or injurious (high tidal volume and zero PEEP) ventilation strategy. Lung mechanics and lung volume were monitored using the forced oscillation technique and plethysmography, respectively. Inflammation was assessed by measuring numbers of inflammatory cells, cytokine (IL-6, IL-1β, and TNF-α) levels, and protein content of the BAL. Principal components factor analysis was used to identify independent associations between lung function and inflammation. Mechanical and inflammatory responses in the lung were dependent on ventilation strategy and mouse strain. Three factors were identified linking 1) pulmonary edema, protein leak, and macrophages, 2) atelectasis, IL-6, and TNF-α, and 3) IL-1β and neutrophils, which were independent of responses in lung mechanics. This approach has allowed us to identify specific inflammatory responses that are independently associated with overstretch of the lung parenchyma and loss of lung volume. These data provide critical insight into the mechanical responses in the lung that drive local inflammation in ventilator-induced lung injury and the basis for future mechanistic studies in this field.     

Verapamil attenuates lung vascular responses to endotoxin in sheep

Experiments were conducted on five chronically instrumented unanesthetized sheep to determine the effects of verapamil, a calcium channel inhibitor, on the pulmonary hemodynamic and microvascular permeability responses to endotoxemia. Paired control endotoxemia experiments (E) and endotoxemia with verapamil treatment (30-60 micrograms.kg-1.min-1) experiments (V + E) were conducted on each sheep in random order. In the V + E experiments sheep were pretreated with a continuous intravenous infusion of verapamil 1.5-2.0 h before endotoxin infusion (1.0 microgram/kg, given over 15 min). Verapamil significantly increased base-line pulmonary arterial pressure, left atrial pressure, lung lymph flow rate, and circulating blood leukocyte levels and significantly decreased base-line cardiac output. During the endotoxin response, verapamil significantly attenuated both phase I pulmonary arterial hypertension and phase II lung lymph flow rate compared with control endotoxin experiments. The results indicate that verapamil attenuates both the pulmonary hemodynamic and increased lung microvascular permeability response to endotoxin in sheep. In a series of in vitro experiments, verapamil was found to be a potent inhibitor of phorbol myristate acetate-induced superoxide production in isolated sheep granulocytes. These data suggest that the beneficial in vivo effects of verapamil during endotoxemia may in part be due to its inhibition of increased free cytosol calcium concentration and/or inhibition of toxic O2 metabolite production.     

Paeonol attenuates airway inflammation and hyperresponsiveness in a murine model of ovalbumin-induced asthma

Paeonol, the main active component isolated from Moutan Cortex, possesses extensive pharmacological activities such as anti-inflammatory, anti-allergic, and immunoregulatory effects. In the present study, we examined the effects of paeonol on airway inflammation and hyperresponsiveness in a mouse model of allergic asthma. BALB/c mice sensitized and challenged with ovalbumin were administered paeonol intragastrically at a dose of 100?mg/kg daily. Paeonol significantly suppressed ovalbumin-induced airway hyperresponsiveness to acetylcholine chloride. Paeonol administration significantly inhibited the total inflammatory cell and eosinophil count in bronchoalveolar lavage fluid. Treatment with paeonol significantly enhanced IFN-γ levels and decreased interleukin-4 and interleukin-13 levels in bronchoalveolar lavage fluid and total immunoglobulin E levels in serum. Histological examination of lung tissue demonstrated that paeonol significantly attenuated allergen-induced lung eosinophilic inflammation and mucus-producing goblet cells in the airway. These data suggest that paeonol exhibits anti-inflammatory activity in allergic mice and may possess new therapeutic potential for the treatment of allergic bronchial asthma.