Matina Kardara

Inhaled activated protein C protects mice from ventilator-induced lung injury

IntroductionActivated Protein C (APC), an endogenous anticoagulant, improves tissue microperfusion and endothelial cell survival in systemic inflammatory states such as sepsis, but intravenous administration may cause severe bleeding. We have thus addressed the role of APC delivered locally by inhalation in preventing acute lung injury from alveolar overdistention and the subsequent ventilator-induced lung injury (VILI). We also assessed the effects of APC on the activation status of Extracellular- Regulated Kinase 1/2 (ERK) pathway, which has been shown to be involved in regulating pulmonary responses to mechanical stretch.MethodsInhaled APC (12.5 μg drotrecogin-α × 4 doses) or saline was given to tracheotomized C57/Bl6 mice starting 20 min prior to initiation of injurious mechanical ventilation with tidal volume 25 mL/Kg for 4 hours and then hourly thereafter; control groups receiving inhaled saline were ventilated with 8 mL/Kg for 30 min or 4 hr. We measured lung function (respiratory system elastance H), arterial blood gases, surrogates of vascular leak (broncho-alveolar lavage (BAL) total protein and angiotensin-converting enzyme (ACE)-activity), and parameters of inflammation (BAL neutrophils and lung tissue myeloperoxidase (MPO) activity). Morphological alterations induced by mechanical ventilation were examined in hematoxylin-eosin lung tissue sections. The activation status of ERK was probed in lung tissue homogenates by immunoblotting and in paraffin sections by immunohistochemistry. The effect of APC on ERK signaling downstream of the thrombin receptor was tested on A549 human lung epithelial cells by immunoblotting. Statistical analyses were performed using ANOVA with appropriate post-hoc testing.ResultsIn mice subjected to VILI without APC, we observed hypoxemia, increased respiratory system elastance and inflammation, assessed by BAL neutrophil counts and tissue MPO activity. BAL total protein levels and ACE activity were also elevated by VILI, indicating compromise of the alveolo-capillary barrier. In addition to preserving lung function, inhaled APC prevented endothelial barrier disruption and attenuated hypoxemia and the inflammatory response. Mechanistically, we found a strong activation of ERK in lung tissues by VILI, which was prevented by APC, suggestive of pathogenetic involvement of the Mitogen-Activated Kinase pathway. In cultured human lung epithelial cells challenged by thrombin, APC abrogated the activation of ERK and its downstream effector, cytosolic Phospholipase A2.ConclusionsTopical application of APC by inhalation may effectively reduce lung injury induced by mechanical ventilation in mice.

Role of caveolin-1 expression in the pathogenesis of pulmonary edema in ventilator-induced lung injury

Caveolin-1 is a key regulator of pulmonary endothelial barrier function. Here, we tested the hypothesis that caveolin-1 expression is required for ventilator-induced lung injury (VILI). Caveolin-1 gene-disrupted (Cav-1−/−) and age-, sex-, and strain-matched wild-type (WT) control mice were ventilated using two protocols: volume-controlled with protective (8 mL/kg) versus injurious (21 mL/Kg) tidal volume for up to 6 hours; and pressure-controlled with protective (airway pressure = 12 cm H2O) versus injurious (30 cm H2O) ventilation to induce lung injury. Lung microvascular permeability (whole-lung 125I-albumin accumulation, lung capillary filtration coefficient [Kf, c]) and inflammatory markers (bronchoalveolar lavage [BAL] cytokine levels and neutrophil counts) were measured. We also evaluated histologic sections from lungs, and the time course of Src kinase activation and caveolin-1 phosphorylation. VILI induced a 1.7-fold increase in lung 125I-albumin accumulation, fourfold increase in Kf, c’ significantly increased levels of cytokines CXCL1 and interleukin-6, and promoted BAL neutrophilia in WT mice. Lung injury by these criteria was significantly reduced in Cav-1−/− mice but fully restored by i.v. injection of liposome/Cav-1 cDNA complexes that rescued expression of Cav-1 in lung microvessels. As thrombin is known to play a significant role in mediating stretch-induced vascular injury, we observed in cultured mouse lung microvascular endothelial cells (MLECs) thrombin-induced albumin hyperpermeability and phosphorylation of p44/42 MAP kinase in WT but not in Cav-1−/− MLECs. Thus, caveolin-1 expression is required for mechanical stretch-induced lung inflammation and endothelial hyperpermeability in vitro and in vivo.

Early serum levels of soluble triggering receptor expressed on myeloid cells–1 in septic patients: Correlation with monocyte gene expression☆

PURPOSE To define early kinetics of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) and of TREM-1 monocyte gene expression in critically ill patients with sepsis. METHODS Blood was sampled at regular time intervals from 105 patients with sepsis. Concentrations of tumour necrosis factor α (TNFα), interleukin (IL)-6, IL-8 and IL-10 and IL-12p70 and sTREM-1 were measured by an enzyme immunoassay. Blood mononuclear cells were isolated on day 0 from 20 patients and 10 healthy volunteers; RNA was extracted and gene expression of TREM-1 and TNFα were assessed by reverse transcriptase polymerase chain reaction. RESULTS Early serum concentrations of sTREM-1 were greater among patients with severe sepsis/shock than among patients with sepsis; those of TNFα, IL-6, IL-8 and IL-10 were pronounced among patients with septic shock. Gene transcripts of TNFα were lower among patients with severe sepsis/shock than among patients with sepsis; that was not the case for TREM-1. Early serum levels of sTREM-1 greater than 180 pg/mL were predictors of shorter duration of mechanical ventilation. CONCLUSIONS Although serum levels of sTREM-1 are increased early upon advent of severe sepsis/shock, gene expression of TREM-1 on monocytes in severe sepsis/shock is not increased. These findings add considerably to our knowledge on the pathophysiology of sepsis.

Metformin attenuates ventilator-induced lung injury

IntroductionDiabetic patients may develop acute lung injury less often than non-diabetics; a fact that could be partially ascribed to the usage of antidiabetic drugs, including metformin. Metformin exhibits pleiotropic properties which make it potentially beneficial against lung injury. We hypothesized that pretreatment with metformin preserves alveolar capillary permeability and, thus, prevents ventilator-induced lung injury.MethodsTwenty-four rabbits were randomly assigned to pretreatment with metformin (250 mg/Kg body weight/day per os) or no medication for two days. Explanted lungs were perfused at constant flow rate (300 mL/min) and ventilated with injurious (peak airway pressure 23 cmH2O, tidal volume ≈17 mL/Kg) or protective (peak airway pressure 11 cmH2O, tidal volume ≈7 mL/Kg) settings for 1 hour. Alveolar capillary permeability was assessed by ultrafiltration coefficient, total protein concentration in bronchoalveolar lavage fluid (BALF) and angiotensin-converting enzyme (ACE) activity in BALF.ResultsHigh-pressure ventilation of the ex-vivo lung preparation resulted in increased microvascular permeability, edema formation and microhemorrhage compared to protective ventilation. Compared to no medication, pretreatment with metformin was associated with a 2.9-fold reduction in ultrafiltration coefficient, a 2.5-fold reduction in pulmonary edema formation, lower protein concentration in BALF, lower ACE activity in BALF, and fewer histological lesions upon challenge of the lung preparation with injurious ventilation. In contrast, no differences regarding pulmonary artery pressure and BALF total cell number were noted. Administration of metformin did not impact on outcomes of lungs subjected to protective ventilation.ConclusionsPretreatment with metformin preserves alveolar capillary permeability and, thus, decreases the severity of ventilator-induced lung injury in this model.

Acute effects of smoke exposure on airway and systemic inflammation in forest firefighters

Introduction The aim of this study was to assess respiratory health and airway and systemic inflammation in professional forest firefighters post firefighting. Methods A total of 60 firefighters who participated in forest firefighting operations in Greece during 2008 were included in the study. A questionnaire consisting of symptoms and exposure, pulmonary function, atopy, bronchial hyperresponsiveness, and markers of inflammation in induced sputum, serum, and bronchoalveolar lavage (BAL) fluid was assessed. Results A measurable eosinophilic and neutrophilic inflammation was shown to be induced in the bronchial airways after acute exposure during forest firefighting. This was associated with increased respiratory symptoms from the upper and lower respiratory tract and pulmonary function impairment. Additionally, a measurable systemic inflammatory response was demonstrated. This study showed that acute exposure during forest firefighting significantly augments the intensity of airway and systemic inflammation in relation to the baseline inflammatory background due to chronic exposure. Conclusion The repeated acute exposures during firefighting augment the burden of chronic airway and systemic inflammation and may eventually lead to allergic sensitization of the airways and increased incidence of rhinitis and asthma after prolonged exposure.