Activation of calpains mediates early lung neutrophilic inflammation in ventilator-induced lung injury

Lung inflammatory responses in the absence of infection are considered to be one of primary mechanisms of ventilator-induced lung injury. Here, we determined the role of calpain in the pathogenesis of lung inflammation attributable to mechanical ventilation. Male C57BL/6J mice were subjected to high (28 ml/kg) tidal volume ventilation for 2 h in the absence and presence of calpain inhibitor I (10 mg/kg). To address the isoform-specific functions of calpain 1 and calpain 2 during mechanical ventilation, we utilized a liposome-based delivery system to introduce small interfering RNAs targeting each isoform in pulmonary vasculature in vivo. Mechanical ventilation with high tidal volume induced rapid (within minutes) and persistent calpain activation and lung inflammation as evidenced by neutrophil recruitment, production of TNF-α and IL-6, pulmonary vascular hyperpermeability, and lung edema formation. Pharmaceutical calpain inhibition significantly attenuated these inflammatory responses caused by lung hyperinflation. Depletion of calpain 1 or calpain 2 had a protective effect against ventilator-induced lung inflammatory responses. Inhibition of calpain activity by means of siRNA silencing or pharmacological inhibition also reduced endothelial nitric oxide (NO) synthase (NOS-3)-mediated NO production and subsequent ICAM-1 phosphorylation following high tidal volume ventilation. These results suggest that calpain activation mediates early lung inflammation during ventilator-induced lung injury via NOS-3/NO-dependent ICAM-1 phosphorylation and neutrophil recruitment. Inhibition of calpain activation may therefore provide a novel and promising strategy for the prevention and treatment of ventilator-induced lung injury.     

Inhibition of matrix metalloproteinase-9 prevents neutrophilic inflammation in ventilator-induced lung injury

Neutrophils are considered to play a central role in ventilator-induced lung injury (VILI). However, the pulmonary consequences of neutrophil accumulation have not been fully elucidated. Matrix metalloproteinase-9 (MMP-9) had been postulated to participate in neutrophil transmigration. The purpose of this study was to investigate the role of MMP-9 in the neutrophilic inflammation of VILI. Male Sprague-Dawley rats were divided into three groups: 1) low tidal volume (LVT), 7 ml/kg of tidal volume (VT); 2) high tidal volume (HVT), 30 ml/kg of VT; and 3) HVT with MMP inhibitor (HVT+MMPI). As a MMPI, CMT-3 was administered daily from 3 days before mechanical ventilation. Degree of VILI was assessed by wet-to-dry weight ratio and acute lung injury (ALI) scores. Neutrophilic inflammation was determined from the neutrophil count in the lung tissue and myeloperoxidase (MPO) activity in the bronchoalveolar lavage fluid (BALF). MMP-9 expression and activity were examined by immunohistochemical staining and gelatinase zymography, respectively. The wet-to-dry weight ratio, ALI score, neutrophil infiltration, and MPO activity were increased significantly in the HVT group. However, in the HVT+MMPI group, pretreatment with MMPI decreased significantly the degree of VILI, as well as neutrophil infiltration and MPO activity. These changes correlated significantly with MMP-9 immunoreactivity and MMP-9 activity. Most outcomes were significantly worse in the HVT+MMPI group compared with the LVT group. In conclusion, VILI mediated by neutrophilic inflammation is closely related to MMP-9 expression and activity. The inhibition of MMP-9 protects against the development of VILI through the downregulation of neutrophil-mediated inflammation.     

Angiotensin-converting enzyme inhibitor captopril attenuates ventilator-induced lung injury in rats.

We hypothesized that lung inflammation and parenchymal apoptosis in ventilator-induced lung injury (VILI) are related to ANG II and assessed the ability of the angiotensin-converting enzyme inhibitor captopril to attenuate VILI in rats. Adult male Sprague-Dawley rats were randomized to receive two ventilation strategies for 2 h: 1) tidal volume of 40 ml/kg, respiratory rate of 25 breaths/min, and inspiratory O2 fraction of 0.21 [high-volume, 0 positive end-expiratory pressure (HVZP) group] and 2) injection of captopril (100 mg/kg ip) 30 min before HVZP ventilation (HVZP+CAP group). Another group, which did not receive ventilation, served as the control. Mean arterial pressure was significantly lower in the HVZP+CAP group than in the HVZP group at 2 h of ventilation. Total protein levels were significantly higher in bronchoalveolar lavage fluid (BALF) recovered from HVZP-ventilated rats than from controls. BALF macrophage inflammatory protein-2 and lung ANG II were significantly higher in the HVZP group than in the control and HVZP+CAP groups. Lung ANG II levels correlated positively with BALF protein and macrophage inflammatory protein-2. The number of apoptotic airway and alveolar wall cells was significantly higher in the HVZP and HVZP+CAP groups than in the control group and significantly lower in the HVZP+CAP group than in the HVZP group. These results suggest that the efficiency of captopril to attenuate VILI is related to reduction of inflammatory cytokines and inhibition of apoptosis and indicate that VILI is partly mediated by the local angiotensin system.     

Activation of c-Src tyrosine kinase mediated the degradation of occludin in ventilator-induced lung injury

Background

Ventilator-induced lung injury (VILI) is characterized by increased alveolar permeability, pulmonary edema. The tyrosine kinase, c-Src, is involved in VILI but its role has not been fully elucidated. This study examined the relationship between c-Src activation and occludin levels in VILI both in vitro and in vivo.

Methods

For the in vivo study, Wistar rats were randomly divided into five groups: control (group C); normal tidal volume (group M); normal tidal volume + c-Src inhibitor (PP2) (group M + P); high tidal volume (group H); and high tidal volume + c-Src inhibitor (PP2) (group H + P). Rats in all groups but group C underwent mechanical ventilation for 4 h. For the in vitro study, MLE-12 cells pretreated with PP2 and siRNA underwent cyclic stretching at 8% or 20% for 0, 1, 2 and 4 h. The expressions of occludin, c-Src, and p-c-Src were analyzed by western blotting, hematoxylin and eosin (HE) staining, and immunofluorescence.

Results

For the in vivo study, rats in group H showed decreased occludin expression and activated c-Src compared with group C. HE staining and lung injury score showed more severe lung injury and alveolar edema in group H compared with group M and group C. Group H + P had less pulmonary edema induced by the high tidal volume ventilation. For the in vitro study, occludin expression decreased and c-Src activation increased as indicated by the phosphorylation of c-Src over time. Consistently, PP2 could restore occludin levels.

Conclusions

Mechanical ventilation can activate c-Src by phosphorylation and increase the degradation of occludin. c-Src inhibitor can ameliorate barrier function and lung injury by up-regulating occludin.     

Congenital NOS2 deficiency prevents impairment of hypoxic pulmonary vasoconstriction in murine ventilator-induced lung injury

Hypoxic pulmonary vasoconstriction (HPV) preserves systemic arterial oxygenation during lung injury by diverting blood flow away from poorly ventilated lung regions. Ventilator-induced lung injury (VILI) is characterized by pulmonary inflammation, lung edema, and impaired HPV leading to systemic hypoxemia. Studying mice congenitally deficient in inducible nitric oxide synthase (NOS2) and wild-type mice treated with a selective NOS2 inhibitor, L-N(6)-(1-iminoethyl)lysine (L-NIL), we investigated the contribution of NOS2 to the impairment of HPV in anesthetized mice subjected to 6 h of either high tidal volume (HV(T)) or low tidal volume (LV(T)) ventilation. HPV was estimated by measuring the changes of left lung pulmonary vascular resistance (LPVR) in response to left mainstem bronchus occlusion (LMBO). LMBO increased the LPVR similarly in wild-type, NOS2(-/-), and wild-type mice treated with L-NIL 30 min before commencing 6 h of LV(T) ventilation (96% +/- 30%, 103% +/- 33%, and 80% +/- 16%, respectively, means +/- SD). HPV was impaired in wild-type mice subjected to 6 h of HV(T) ventilation (23% +/- 16%). In contrast, HPV was preserved after 6 h of HV(T) ventilation in NOS2(-/-) and wild-type mice treated with L-NIL either 30 min before or 6 h after commencing HV(T) ventilation (66% +/- 22%, 82% +/- 29%, and 85% +/- 16%, respectively). After 6 h of HV(T) ventilation and LMBO, systemic arterial oxygen tension was higher in NOS2(-/-) than in wild-type mice (192 +/- 11 vs. 171 +/- 17 mmHg; P < 0.05). We conclude that either congenital NOS2 deficiency or selective inhibition of NOS2 protects mice from the impairment of HPV occurring after 6 h of HV(T) ventilation.     

Intracellular regulation of neuronal nicotinic cholinergic receptors

Effects of substances affecting intracellular secondary messengers on the membrane currents evoked by ionophoretic application of acetylcholine (ACh currents) and on the excitatory postsynaptic currents (EPSC) evoked by single stimuli applied to preganglionic nerve fibres, were studied in neurones of the rat isolated superior cervical ganglion. Forskolin, the protein kinase A activator, and isobutyl-methyxanthine, the phosphodiesterase inhibitor, decreased the ACh currents. Neither forskolin nor isobutyl-methylxanthine affected the EPSC amplitude or the EPSC decay time constant. Phorbol ester, the protein kinase C activator, decreased the ACh current but did not affect either EPSC amplitude or the EPSC decay time constant. Thapsigargin, the intracellular calcium releaser, decreased the ACh current and the EPSC amplitude but did not affect the EPSC decay time constant. The data obtained suggest that nicotinic acetylcholine receptors (nAChRs) of ganglion neurones are not modulated through the pathways involving protein kinase A or protein kinase C. The nAChRs sensitivity to both exogenous and nerve-released acetylcholine is reduced by intracellular calcium without affecting kinetics of their ionic channels.     

Anti-inflammatory effects of clarithromycin in ventilator-induced lung injury

Background

Mechanical ventilation can promote lung injury by triggering a pro-inflammatory response. Macrolides may exert some immunomodulatory effects and have shown significant benefits over other antibiotics in ventilated patients. We hypothesized that macrolides could decrease ventilator-induced lung injury.

Methods

Adult mice were treated with vehicle, clarithromycin or levofloxacin, and randomized to receive mechanical ventilation with low (12 cmH₂O, PEEP 2 cmH₂O) or high (20 cmH₂O, ZEEP) inspiratory pressures for 150 minutes. Histological lung injury, neutrophil infiltration, inflammatory mediators (NFκB activation, Cxcl2, IL-10) and levels of adhesion molecules (E-selectin, ICAM) and proteases (MMP-9 and MMP-2) were analyzed.

Results

There were no differences among groups after low-pressure ventilation. Clarithromycin significantly decreased lung injury score and neutrophil count, compared to vehicle or levofloxacin, after high-pressure ventilation. Cxcl2 expression and MMP-2 and MMP-9 levels increased and IL-10 decreased after injurious ventilation, with no significant differences among treatment groups. Both clarithromycin and levofloxacin dampened the increase in NFκB activation observed in non-treated animals submitted to injurious ventilation. E-selectin levels increased after high pressure ventilation in vehicle- and levofloxacin-treated mice, but not in those receiving clarithromycin.

Conclusions

Clarithromycin ameliorates ventilator-induced lung injury and decreases neutrophil recruitment into the alveolar spaces. This could explain the advantages of macrolides in patients with acute lung injury and mechanical ventilation.     

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.     

Activation of skeletal muscle nicotinic acetylcholine receptors

Summary and Conclusions Work over the past ten years has greatly increased our understanding of both the structure and function of the muscle nicotinic acetylcholine receptor. There is a strongly supported general picture of how the receptor functions: agonist binds rapidly to sites of low affinity and channel opening occurs at a rate comparable to the agonist dissociation rate. Channel closing is slow, so the channel has a high probability of being open if both agonist-binding sites are occupied by ACh. Results of expression studies have shown that each subunit can influence AChR activation and have given a structural basis for the major physiological change known for muscle AChR, the developmental change in AChR activation. These general statements notwithstanding, there are still major areas of uncertainty which limit our understanding. We have emphasized these areas of uncertainty in this review, to indicate what needs to be done.First, the quantitative estimates of rate constants are not as strongly supported as they should be. The major reasons are twofold—uncertainties about the interpretation of components in the kinetic data and difficulties of resolving brief events. As a result, any inferences about the functional consequences of structural alterations must remain tenuous.Second, the functional behavior of individual AChRs is not as well understood as it should be. The kinetic behavior of an individual receptor clearly can be complex (section II). In addition, there is evidence that superimposed on this complexity there may be stable and kinetically distinguishable populations of receptors (section III). Until the basis for the kinetically defined populations is clarified, kinetic parameters for receptors of defined structure cannot be unambiguously obtained.Finally, it is not surprising that the studies of AChR of altered structure have not given definitive results. Two reasons should be apparent from the preceding points: there is not a fully supported approach for kinetic analysis, and the normal population may not be clearly defined. An additional complication is also emerging, in that the available data support the idea that specific residues distributed over all subunits may influence AChR activation. This possibility renders the task of analysis that much more difficult.The muscle nicotinic AChR has served as a prototype for the family of transmitter-gated membrane channels, which includes the muscle and neuronal nicotinic receptors, the GABA_A, the glycine and possibly the non-NMDA excitatory amino acid receptor (Stroud et al., 1990). It is interesting to note that the functional properties of the GABA_A receptor, probably the best-studied of the other members of the family are rather similar. In particular, opentime and burst durations show multiple components interpreted as reflecting openings of singly and doubly liganded receptors (Mathers & Wang, 1988; Macdonald et al., 1989), the distribution of gaps indicates a relatively complex gating scheme (Twyman et al., 1990; Weiss & Magleby, 1989), and multiple kinetic modes are likely to exist (Newland et al., 1991). The situation with regards to the effects of GABA_A receptor subunit stoichiometry is more complex than for muscle AChR (e.g., Luddens & Wisden, 1991), perhaps similar to that found for neuronal nicotinic AChR (Papke et al., 1989; Luetje et al., 1990; Luetje & Patrick, 1991). Overall, it appears that the unresolved questions about the muscle nicotinic AChR are not indications that this is an exceptionally complicated transmitter-gated channel. Rather, it appears to be a relatively straightforward member of the family, and the lessons we learn from studying it are likely to be directly applicable to other receptors.We thank many friends for discussion, including Tony Auerbach, Paul Brehm, Jim Dilger, Meyer Jackson, and Chuck Stevens who told us about data before publication. Research in the authors' laboratories is supported by grants from the NIH (CL and JHS) and the AHA (CL).     

Lung-derived soluble mediators are pathogenic in ventilator-induced lung injury

Ventilator-induced lung injury (VILI) due to high tidal volume (V(T)) is associated with increased levels of circulating factors that may contribute to, or be markers of, injury. This study investigated if exclusively lung-derived circulating factors produced during high V(T) ventilation can cause or worsen VILI. In isolated perfused mouse lungs, recirculation of perfusate worsened injury (compliance impairment, microvascular permeability, edema) induced by high V(T). Perfusate collected from lungs ventilated with high V(T) and used to perfuse lungs ventilated with low V(T) caused similar compliance impairment and permeability and caused a dose-dependent decrease in transepithelial electrical resistance (TER) across rat distal lung epithelial monolayers. Circulating soluble factors derived from the isolated lung thus contributed to VILI and had deleterious effects on the lung epithelial barrier. These data demonstrate transferability of an injury initially caused exclusively by mechanical ventilation and provides novel evidence for the biotrauma hypothesis in VILI. Mediators of the TER decrease were heat-sensitive, transferable via Folch extraction, and (following ultrafiltration, 3 kDa) comprised both smaller and larger molecules. Although several classes of candidate mediators, including protein cytokines (e.g., tumor necrosis factor-α, interleukin-6, macrophage inflammation protein-1α) and lipids (e.g., eicosanoids, ceramides, sphingolipids), have been implicated in VILI, only prostanoids accumulated in the perfusate in a pattern consistent with a pathogenic role, yet cyclooxygenase inhibition did not protect against injury. Although no single class of factor appears solely responsible for the decrease in barrier function, the current data implicate lipid-soluble protein-bound molecules as not just markers but pathogenic mediators in VILI.     

Activation of lung vagal sensory receptors by circulatory endotoxin in rats

Although endotoxin is known to induce various pulmonary responses that are linked to the function of lung vagal sensory receptors, its effects on these pulmonary receptors are still not clear. This study investigated the effects of circulatory endotoxin on the afferent activity of lung vagal sensory receptors in rats. We recorded afferent activity arising from vagal pulmonary C fibers (CFs), rapidly adapting receptors (RARs), tonic pulmonary stretch receptors (T-PSRs), and phasic pulmonary stretch receptors (P-PSRs) in 64 anesthetized, paralyzed, and artificially ventilated rats. Intravenous injection of endotoxin (50 mg/kg; lipopolysaccharide) stimulated 7 of the 8 CFs, 8 of the 8 RARs, and 4 of the 8 T-PSRs studied, while having no effect on the 8 P-PSRs tested. The stimulation started 3-16 min after endotoxin injection and lasted until the end of the 90-min observation period. The evoked discharge of either CFs or RARs was not in phase with the ventilatory cycle, whereas that of T-PSRs showed a respiratory modulation. Injection of a saline vehicle caused no significant change in the discharge of these receptors. Additionally, endotoxin significantly produced an increase in total lung resistance, and decreases in dynamic lung compliance and arterial blood pressure. Our results demonstrate that a majority of lung vagal sensory receptors are activated following intravenous injection of endotoxin, and support the notion that these pulmonary receptors may function as an important afferent system during endotoxemia.     

Activation of Src-dependent Smad3 signaling mediates the neutrophilic inflammation and oxidative stress in hyperoxia-augmented ventilator-induced lung injury

Background

Mechanical ventilation and concomitant administration of hyperoxia in patients with acute respiratory distress syndrome can damage the alveolar epithelial and capillary endothelial barrier by producing inflammatory cytokines and reactive oxygen species. The Src tyrosine kinase and Smad3 are crucial inflammatory regulators used for ventilator-induced lung injury (VILI). The mechanisms regulating interactions between high-tidal-volume mechanical ventilation, hyperoxia, and acute lung injury (ALI) are unclear. We hypothesized that high-tidal-volume mechanical stretches and hyperoxia augment lung inflammation through upregulation of the Src and Smad3 pathways.

Methods

Wild-type or Src-deficient C57BL/6 mice, aged between 6 and 8 weeks, were exposed to high-tidal-volume (30 mL/kg) ventilation with room air or hyperoxia for 1–4 h after 2-mg/kg Smad3 inhibitor (SIS3) administration. Nonventilated mice were used as control subjects.

Results

We observed that the addition of hyperoxia to high-tidal-volume mechanical ventilation further induced microvascular permeability, neutrophil infiltration, macrophage inflammatory protein-2 and matrix metalloproteinase-9 (MMP-9) production, malondialdehyde, nicotinamide adenine dinucleotide phosphate oxidase activity, MMP-9 mRNA expression, hypoxemia, and Src and Smad3 activation (P < 0.05). Hyperoxia-induced augmentation of VILI was attenuated in Src-deficient mice and mice with pharmacological inhibition of Smad3 activity by SIS3 (P < 0.05). Mechanical ventilation of Src-deficient mice with hyperoxia further reduced the activation of Smad3.

Conclusions

Our data suggest that hyperoxia-increased high-tidal-volume ventilation-induced ALI partially depends on the Src and Smad3 pathways.     

Lack of matrix metalloproteinase-9 worsens ventilator-induced lung injury

Matrix metalloproteinase-9 (MMP-9) is released by neutrophils at the sites of acute inflammation. This enzyme modulates matrix turnover and inflammatory response, and its activity has been found to be increased after ventilator-induced lung injury. To clarify the role of MMP-9, mice lacking this enzyme and their wild-type counterparts were ventilated for 2 h with high- or low-peak inspiratory pressures (25 and 15 cmH2O, respectively). Lung injury was evaluated by gas exchange, respiratory mechanics, wet-to-dry weight ratio, and histological analysis. The activity of MMP-9 and levels of IL-1beta, IL-4, and macrophage inflammatory protein (MIP-2) were measured in lung tissue and bronchoalveolar lavage fluid (BALF). Cell count and myeloperoxidase activity were measured in BALF. There were no differences between wild-type and Mmp9-/- animals after low-pressure ventilation. After high-pressure ventilation, wild-type mice exhibited an increase in MMP-9 in tissue and BALF. Mice lacking MMP-9 developed more severe lung injury than wild-type mice, in terms of impaired oxygenation and lung mechanics, and higher damage in the histological study. These effects correlated with an increase in both cell count and myeloperoxidase activity in the BALF, suggesting an increased neutrophilic influx in response to ventilation. An increase in IL-1beta and IL-4 in the BALF only in knockout mice could be responsible for the differences. There were no differences between genotypes in MMP-2, MMP-8, or tissue inhibitors of metalloproteinases. These results show that MMP-9 protects against ventilator-induced lung injury by decreasing alveolar neutrophilic infiltration, probably by modulation of the cytokine response in the air spaces.     

Soluble guanylyl cyclase contributes to ventilator-induced lung injury in mice

High tidal volume (HV(T)) ventilation causes pulmonary endothelial barrier dysfunction. HV(T) ventilation also increases lung nitric oxide (NO) and cGMP. NO contributes to HV(T) lung injury, but the role of cGMP is unknown. In the current study, ventilation of isolated C57BL/6 mouse lungs increased perfusate cGMP as a function of V(T). Ventilation with 20 ml/kg V(T) for 80 min increased the filtration coefficient (K(f)), an index of vascular permeability. The increased cGMP and K(f) caused by HV(T) were attenuated by nitric oxide synthase (NOS) inhibition and in lungs from endothelial NOS knockout mice. Inhibition of soluble guanylyl cyclase (sGC) in wild-type lungs (10 muM ODQ) also blocked cGMP generation and inhibited the increase in K(f), suggesting an injurious role for sGC-derived cGMP. sGC inhibition also attenuated lung Evans blue dye albumin extravasation and wet-to-dry weight ratio in an anesthetized mouse model of HV(T) injury. Additional activation of sGC (1.5 muM BAY 41-2272) in isolated lungs at 40 min increased cGMP production and K(f) in lungs ventilated with 15 ml/kg V(T). HV(T) endothelial barrier dysfunction was attenuated with a nonspecific phosphodiesterase (PDE) inhibitor (100 muM IBMX) as well as an inhibitor (10 muM BAY 60-7550) specific for the cGMP-stimulated PDE2A. Concordantly, we found a V(T)-dependent increase in lung cAMP hydrolytic activity and PDE2A protein expression with a decrease in lung cAMP concentration that was blocked by BAY 60-7550. We conclude that HV(T)-induced endothelial barrier dysfunction resulted from a simultaneous increase in NO/sGC-derived cGMP and PDE2A expression causing decreased cAMP.     

Effects of melatonin in an experimental model of ventilator-induced lung injury

Melatonin is a free radical scavenger and a broad-spectrum antioxidant and has well-documented immunomodulatory effects. We studied the effects of this hormone on lung damage, oxidative stress, and inflammation in a model of ventilator-induced lung injury (VILI), using 8- to 12-wk-old Swiss mice (n = 48). Animals were randomized into three experimental groups: control (not ventilated); low-pressure ventilation [peak inspiratory pressure 15 cmH(2)O, positive end-expiratory pressure (PEEP) 2 cmH(2)O], and high-pressure ventilation (peak inspiratory pressure 25 cmH(2)O, PEEP 0 cmH(2)O). Each group was divided into two subgroups: eight animals were treated with melatonin (10 mg/kg ip, 30 min before the onset of ventilation) and the remaining eight with vehicle. After 2 h of ventilation, lung injury was evaluated by gas exchange, wet-to-dry weight ratio, and histological analysis. Levels of malondialdehyde, glutathione peroxidase, interleukins IL-1beta, IL-6, TNF-alpha, and IL-10, and matrix metalloproteinases 2 and 9 in lung tissue were measured as indicators of oxidation status, pro-/anti-inflammatory cytokines, and matrix turnover, respectively. Ventilation with high pressures induced severe lung damage and release of TNF-alpha, IL-6, and matrix metalloproteinase-9. Treatment with melatonin improved oxygenation and decreased histological lung injury but significantly increased oxidative stress quantified by malondialdehyde levels. There were no differences in TNF-alpha, IL-1beta, IL-6, or matrix metalloproteinases caused by melatonin treatment, but IL-10 levels were significantly higher in treated animals. These results suggest that melatonin decreases VILI by increasing the anti-inflammatory response despite an unexpected increase in oxidative stress.     

Pulmonary microRNA profiling in a mouse model of ventilator-induced lung injury

The aim of this study was to investigate the changes induced by high tidal volume ventilation (HVTV) in pulmonary expression of micro-RNAs (miRNAs) and identify potential target genes and corresponding miRNA-gene networks. Using a real-time RT-PCR-based array in RNA samples from lungs of mice subjected to HVTV for 1 or 4 h and control mice, we identified 65 miRNAs whose expression changed more than twofold upon HVTV. An inflammatory and a TGF-β-signaling miRNA-gene network were identified by in silico pathway analysis being at highest statistical significance (P = 10(-43) and P = 10(-28), respectively). In the inflammatory network, IL-6 and SOCS-1, regulated by miRNAs let-7 and miR-155, respectively, appeared as central nodes. In TGF-β-signaling network, SMAD-4, regulated by miR-146, appeared as a central node. The contribution of miRNAs to the development of lung injury was evaluated in mice subjected to HVTV treated with a precursor or antagonist of miR-21, a miRNA highly upregulated by HVTV. Lung compliance was preserved only in mice treated with anti-miR-21 but not in mice treated with pre-miR-21 or negative-control miRNA. Both alveolar-arterial oxygen difference and protein levels in bronchoalveolar lavage were lower in mice treated with anti-miR-21 than in mice treated with pre-miR-21 or negative-control miRNA (D(A-a): 66 ± 27 vs. 131 ± 22, 144 ± 10 mmHg, respectively, P < 0.001; protein concentration: 1.1 ± 0.2 vs. 2.3 ± 1, 2.1 ± 0.4 mg/ml, respectively, P < 0.01). Our results show that HVTV induces changes in miRNA expression in mouse lungs. Modulation of miRNA expression can affect the development of HVTV-induced lung injury.     

Acute lung inflammation and ventilator-induced lung injury caused by ATP via the P2Y receptors: an experimental study

Background

Many studies associated the main polyphenolic constituent of green tea, (-)-Epigallocatechin-3-gallate (EGCG), with inhibition of cancers, invasion and metastasis. To date, most of the studies have focused on the effect of EGCG on cell proliferation or death. Since cell migration is an important mechanism involved in tumor invasion, the aim of the present work was to target another approach of the therapeutic effect of EGCG, by investigating its effect on the cell migratory behavior.

Methods

The effect of EGCG (at concentrations lower than 10 μg/ml) on the migration speed of invasive cells was assessed by using 2D and 3D models of cell culture. We also studied the effects of EGCG on proteinases expression by RT-PCR analysis. By immunocytochemistry, we analyzed alterations of vimentin organization in presence of different concentrations of EGCG.

Results

We observed that EGCG had an inhibitory effect of cell migration in 2D and 3D cell culture models. EGCG also inhibited MMP-2 mRNA and protein expression and altered the intermediate filaments of vimentin.

Conclusion

Taken together, our results demonstrate that EGCG is able to inhibit the migration of bronchial tumor cells and could therefore be an attractive candidate to treat tumor invasion and cell migration.     

Inflammation and matrix remodeling during repair of ventilator-induced lung injury

High-pressure ventilation triggers different inflammatory and matrix remodeling responses within the lung. Although some of them may cause injury, the involvement of these mediators in repair is largely unknown. To identify mechanisms of repair after ventilator-induced lung injury (VILI), mice were randomly assigned to baseline conditions (no ventilation), injury [90 min of high-pressure ventilation without positive end-expiratory pressure (PEEP)], repair (injury followed by 4 h of low-pressure ventilation with PEEP), and ventilated controls (low-pressure ventilation with PEEP for 90 and 330 min). Histological injury and lung permeability increased during injury, but were partially reverted in the repair group. This was accompanied by a proinflammatory response, together with increases in TNF-α and IFN-γ, which returned to baseline during repair, and a decrease in IL-10. However, macrophage inflammatory protein-2 (MIP-2) and matrix metalloproteinases (MMP)-2 and -9 increased after injury and persisted in being elevated during repair. Mortality in the repair phase was 50%. Survivors showed increased cell proliferation, lower levels of collagen, and higher levels of MIP-2 and MMP-2. Pan-MMP or specific MMP-2 inhibition (but not MIP-2, TNF-α, or IL-4 inhibition) delayed epithelial repair in an in vitro wound model using murine or human alveolar cells cultured in the presence of bronchoalveolar lavage fluid from mice during the repair phase or from patients with acute respiratory distress syndrome, respectively. Similarly, MMP inhibition with doxycycline impaired lung repair after VILI in vivo. In conclusion, VILI can be reverted by normalizing ventilation pressures. An adequate inflammatory response and extracellular matrix remodeling are essential for recovery. MMP-2 could play a key role in epithelial repair after VILI and acute respiratory distress syndrome.