Limiting Overdistention or Collapse When Mechanically Ventilating Injured Lungs: A Randomized Study in a Porcine Model.

Rationale: It is unknown whether preventing overdistention or collapse is more important when titrating positive end-expiratory pressure (PEEP) in acute respiratory distress syndrome (ARDS). Objectives: To compare PEEP targeting minimal overdistention or minimal collapse or using a compromise between collapse and overdistention in a randomized trial and to assess the impact on respiratory mechanics, gas exchange, inflammation, and hemodynamics. Methods: In a porcine model of ARDS, lung collapse and overdistention were estimated using electrical impedance tomography during a decremental PEEP titration. Pigs were randomized to three groups and ventilated for 12 hours: PEEP set at ⩽3% of overdistention (low overdistention), ⩽3% of collapse (low collapse), and the crossing point of collapse and overdistention. Measurements and Main Results: Thirty-six pigs (12 per group) were included. Median (interquartile range) values of PEEP were 7 (6-8), 11 (10-11), and 15 (12-16) cm H2O in the three groups (P < 0.001). With low overdistension, 6 (50%) pigs died, whereas survival was 100% in both other groups. Cause of death was hemodynamic in nature, with high transpulmonary vascular gradient and high epinephrine requirements. Compared with the other groups, pigs surviving with low overdistension had worse respiratory mechanics and gas exchange during the entire protocol. Minimal differences existed between crossing-point and low-collapse animals in physiological parameters, but postmortem alveolar density was more homogeneous in the crossing-point group. Inflammatory markers were not significantly different. Conclusions: PEEP to minimize overdistention resulted in high mortality in an animal model of ARDS. Minimizing collapse or choosing a compromise between collapse and overdistention may result in less lung injury, with potential benefits of the compromise approach.

Therapeutic Characteristics of Alveolar-like Macrophages in Mouse Models of Hyperoxia and LPS-Induced Lung Inflammation.

Acute respiratory distress syndrome (ARDS) is a severe lung disease of high mortality (30-50%). Patients require lifesaving supplemental oxygen therapy; however, hyperoxia can induce pulmonary inflammation and cellular damage. Although alveolar macrophages (AMs) are essential for lung immune homeostasis, they become compromised during inflammatory lung injury. To combat this, stem cell-derived alveolar-like macrophages (ALMs) are a prospective therapeutic for lung diseases like ARDS. Using in vitro and in vivo approaches, we investigated the impact of hyperoxia on murine ALMs during acute inflammation. In vitro, ALMs retained their viability, growth and antimicrobial abilities when cultured at 60% O2,while they die at 90% O2. In contrast, ALMs instilled in mouse lungs remained viable during exposure of mice to 90% O2. The ability of the delivered ALMs to phagocytose Pseudomonas aeruginosa was not impaired by exposure to 60 or 90% O2. Furthermore, ALMs remained immunologically stable in a murine model of LPS-induced lung inflammation when exposed to 60 and 90% O2 and effectively attenuated the accumulation of CD11b+ inflammatory cells in the airways. These results support the potential use of ALMs in ARDS patients receiving supplemental oxygen therapy.

Preservation of recipient plasma sphingosine-1-phosphate levels reduces transfusion-related acute lung injury.

Cold-stored (CS) platelets are once again being reintroduced for clinical use. Transfused CS platelets offer benefits over room temperature-stored (RTS) platelets such as increased hemostatic effects and prolongation of shelf-life. Despite these advantages little is known about their association with transfusion-related acute lung injury (TRALI). TRALI is associated with prolonged storage of RTS platelets and has a mortality of >15%. Determining the safety of CS platelets is important considering their proposed use in TRALI-vulnerable populations with inflammation such as surgical patients or patients with trauma. Donor platelet-derived ceramide causes TRALI, whereas donor platelet sphingosine-1-phosphate (S1P) is barrier protective. Females have higher plasma levels of S1P than males. Cold temperatures increase S1P levels in cells. Therefore, we hypothesized that female (donors or recipients) and/or CS platelets would decrease TRALI. To test this, we compared how male and female donor and recipient allogeneic platelet transfusions of CS (4°C) versus RTS (23°C) platelets stored for 5 days influence murine TRALI. Transfusion of CS platelets significantly reduced recipient lung tissue wet-to-dry ratios, bronchoalveolar lavage total protein, lung tissue myeloperoxidase enzyme activity, histological lung injury scores, and increased plasma sphingosine-1-phosphate (S1P) levels compared with RTS platelet transfusions. Female as opposed to male recipients had less TRALI and higher plasma S1P levels. Female donor mouse platelets had higher S1P levels than males. Mouse and human CS platelets had increased S1P levels compared with RTS platelets. Higher recipient plasma S1P levels appear protective considering females, and males receiving platelets from females or male CS platelets had less TRALI.NEW & NOTEWORTHY Transfusion-related acute lung injury (TRALI) though relatively rare represents a severe lung injury. The sphingolipid sphingosine-1-phosphate (S1P) regulates the severity of platelet-mediated TRALI. Female platelet transfusion recipient plasmas or stored platelets from female donors have higher S1P levels than males, which reduces TRALI. Cold storage of murine platelets preserves platelet-S1P, which reduces TRALI in platelet-transfused recipients.

Low foraging rates drive large insectivorous bats away from urban areas.

Urbanization has significant impacts on wildlife and ecosystems and acts as an environmental filter excluding certain species from local ecological communities. Specifically, it may be challenging for some animals to find enough food in urban environments to achieve a positive energy balance. Because urban environments favor small-sized bats with low energy requirements, we hypothesized that common noctules (Nyctalus noctula) acquire food at a slower rate and rely less on conspecifics to find prey in urban than in rural environments due to a low food abundance and predictable distribution of insects in urban environments. To address this, we estimated prey sizes and measured prey capture rates, foraging efforts, and the presence of conspecifics during hunting of 22 common noctule bats equipped with sensor loggers in an urban and rural environment. Even though common noctule bats hunted similar-sized prey in both environments, urban bats captured prey at a lower rate (mean: 2.4 vs. 6.3 prey attacks/min), and a lower total amount of prey (mean: 179 vs. 377 prey attacks/foraging bout) than conspecifics from rural environments. Consequently, the energy expended to capture prey was higher for common noctules in urban than in rural environments. In line with our prediction, urban bats relied less on group hunting, likely because group hunting was unnecessary in an environment where the spatial distribution of prey insects is predictable, for example, in parks or around floodlights. While acknowledging the limitations of a small sample size and low number of spatial replicates, our study suggests that scarce food resources may make urban habitats unfavorable for large bat species with higher energy requirements compared to smaller bat species. In conclusion, a lower food intake may displace larger species from urban areas making habitats with high insect biomass production key for protecting large bat species in urban environments.

Extracellular vesicles: effectors of transfusion-related acute lung injury.

Respiratory transfusion reactions represent some of the most severe adverse reactions related to receiving blood products. Of those, transfusion-related acute lung injury (TRALI) is associated with elevated morbidity and mortality. TRALI is characterized by severe lung injury associated with inflammation, pulmonary neutrophil infiltration, lung barrier leak, and increased interstitial and airspace edema that cause respiratory failure. Presently, there are few means of detecting TRALI beyond clinical definitions based on physical examination and vital signs or preventing/treating TRALI beyond supportive care with oxygen and positive pressure ventilation. Mechanistically, TRALI is thought to be mediated by the culmination of two successive proinflammatory hits, which typically comprise a recipient factor (1st hit-e.g., systemic inflammatory conditions) and a donor factor (2nd hit-e.g., blood products containing pathogenic antibodies or bioactive lipids). An emerging concept in TRALI research is the contribution of extracellular vesicles (EVs) in mediating the first and/or second hit in TRALI. EVs are small, subcellular, membrane-bound vesicles that circulate in donor and recipient blood. Injurious EVs may be released by immune or vascular cells during inflammation, by infectious bacteria, or in blood products during storage, and can target the lung upon systemic dissemination. This review assesses emerging concepts such as how EVs: 1) mediate TRALI, 2) represent targets for therapeutic intervention to prevent or treat TRALI, and 3) serve as biochemical biomarkers facilitating TRALI diagnosis and detection in at-risk patients.

Aberrant lung lipids cause respiratory impairment in a Mecp2-deficient mouse model of Rett syndrome.

Severe respiratory impairment is a prominent feature of Rett syndrome, an X-linked disorder caused by mutations in methyl CpG-binding protein 2 (MECP2). Despite MECP2's ubiquitous expression, respiratory anomalies are attributed to neuronal dysfunction. Here, we show that neutral lipids accumulate in mouse Mecp2-mutant lungs, whereas surfactant phospholipids decrease. Conditional deletion of Mecp2 from lipid-producing alveolar epithelial 2 (AE2) cells causes aberrant lung lipids and respiratory symptoms, whereas deletion of Mecp2 from hindbrain neurons results in distinct respiratory abnormalities. Single-cell RNA sequencing of AE2 cells suggests lipid production and storage increase at the expense of phospholipid synthesis. Lipid production enzymes are confirmed as direct targets of MECP2-directed nuclear receptor co-repressor 1/2 transcriptional repression. Remarkably, lipid-lowering fluvastatin improves respiratory anomalies in Mecp2-mutant mice. These data implicate autonomous pulmonary loss of MECP2 in respiratory symptoms for the first time and have immediate impacts on patient care.

Autophagy Is Impaired in Fetal Hypoplastic Lungs and Rescued by Administration of Amniotic Fluid Stem Cell Extracellular Vesicles.

Rationale: Pulmonary hypoplasia secondary to congenital diaphragmatic hernia is characterized by reduced branching morphogenesis, which is responsible for poor clinical outcomes. Administration of amniotic fluid stem cell extracellular vesicles (AFSC-EVs) rescues branching morphogenesis in rodent fetal models of pulmonary hypoplasia. Herein, we hypothesized that AFSC-EVs exert their regenerative potential by affecting autophagy, a process required for normal lung development. Objectives: To evaluate autophagy in hypoplastic lungs throughout gestation and establish whether AFSC-EV administration improves branching morphogenesis through autophagy-mediated mechanisms. Methods: EVs were isolated from c-kit+ AFSC-conditioned medium by ultracentrifugation and characterized for size, morphology, and EV markers. Branching morphogenesis was inhibited in rat fetuses by nitrofen administration to dams and in human fetal lung explants by blocking RAC1 activity with NSC23766. The expression of autophagy activators (BECN1 and ATG5) and adaptor (SQSTM1/p62) was analyzed in vitro (rat and human fetal lung explants) and in vivo (rat fetal lungs). Mechanistic studies on rat fetal primary lung epithelial cells were conducted using inhibitors for microRNA-17 and -20a contained in the AFSC-EV cargo and known to regulate autophagy. Measurements and Main Results: Rat and human models of fetal pulmonary hypoplasia showed reduced autophagy mainly at pseudoglandular and canalicular stages. AFSC-EV administration restored autophagy in both pulmonary hypoplasia models by transferring miR-17∼92 cluster members contained in the EV cargo. Conclusions: AFSC-EV treatment rescues branching morphogenesis partly by restoring autophagy through microRNA cargo transfer. This study enhances our understanding of pulmonary hypoplasia pathogenesis and creates new opportunities for fetal therapeutic intervention in congenital diaphragmatic hernia babies.

Impact of Reverse Triggering Dyssynchrony during Lung-Protective Ventilation on Diaphragm Function: An Experimental Model.

Rationale: Reverse triggering dyssynchrony (RT) is a patient-ventilator interaction where a respiratory muscle contraction is triggered by a passive mechanical insufflation. Its impact on diaphragm structure and function is unknown. Objectives: To establish an animal model of RT with lung injury receiving lung-protective ventilation and to assess its impact on the structure and function of the diaphragm. Methods: Lung injury was induced by surfactant depletion and high-stress ventilation in 32 ventilated pigs. Animals were allocated to receive passive mechanical ventilation (Vt: 10 ml/kg; respiratory rate [RR]: 30-35 breaths/min; n = 8) or a more lung-protective strategy (Vt: 6-8 ml/kg; n = 24) with adjustments in RR to facilitate the occurrence of RT for 3 hours. Diaphragm function (transdiaphragmatic pressure [Pdi] during phrenic nerve stimulation [force/frequency curve]) and structure (biopsies) were assessed. The impact of RT on diaphragm function was analyzed according to the breathing effort assessed by the pressure-time product. Measurements and Main Results: Compared with passive ventilation, the protective ventilation group with RT received significantly lower Vt (7 vs. 10 ml/kg) and higher RR (45 vs. 31 breaths/min). An entrainment pattern of 1:1 was the most frequently occurring in 83% of the animals. Breathing effort induced by RT was highly variable across animals. RT with the lowest tercile of breathing effort was associated with 23% higher twitch Pdi compared with passive ventilation, whereas RT with high breathing effort was associated with a 10% lower twitch Pdi and a higher proportion of abnormal muscle fibers. Conclusions: In a reproducible animal model of RT with variable levels of breathing effort and entrainment patterns, RT with high effort is associated with impaired diaphragm function, whereas RT with low effort is associated with preserved diaphragm force.

Therapeutic stem cell-derived alveolar-like macrophages display bactericidal effects and resolve Pseudomonas aeruginosa-induced lung injury.

Bacterial lung infections lead to greater than 4 million deaths per year with antibiotic treatments driving an increase in antibiotic resistance and a need to establish new therapeutic approaches. Recently, we have generated mouse and rat stem cell-derived alveolar-like macrophages (ALMs), which like primary alveolar macrophages (1'AMs), phagocytose bacteria and promote airway repair. Our aim was to further characterize ALMs and determine their bactericidal capabilities. The characterization of ALMs showed that they share known 1'AM cell surface markers, but unlike 1'AMs are highly proliferative in vitro. ALMs effectively phagocytose and kill laboratory strains of P. aeruginosa (P.A.), E. coli (E.C.) and S. aureus, and clinical strains of P.A. In vivo, ALMs remain viable, adapt additional features of native 1'AMs, but proliferation is reduced. Mouse ALMs phagocytose P.A. and E.C. and rat ALMs phagocytose and kill P.A. within the lung 24 h post-instillation. In a pre-clinical model of P.A.-induced lung injury, rat ALM administration mitigated weight loss and resolved lung injury observed seven days post-instillation. Collectively, ALMs attenuate pulmonary bacterial infections and promote airway repair. ALMs could be utilized as an alternative or adjuvant therapy where current treatments are ineffective against antibiotic-resistant bacteria or to enhance routine antibiotic delivery.

Repeated endo-tracheal tube disconnection generates pulmonary edema in a model of volume overload: an experimental study.

BACKGROUND: An abrupt lung deflation in rodents results in lung injury through vascular mechanisms. Ventilator disconnections during endo-tracheal suctioning in humans often cause cardio-respiratory instability. Whether repeated disconnections or lung deflations cause lung injury or oedema is not known and was tested here in a porcine large animal model. METHODS: Yorkshire pigs (~ 12 weeks) were studied in three series. First, we compared PEEP abruptly deflated from 26 cmH2O or from PEEP 5 cmH2O to zero. Second, pigs were randomly crossed over to receive rapid versus gradual PEEP removal from 20 cmH2O. Third, pigs with relative volume overload, were ventilated with PEEP 15 cmH2O and randomized to repeated ETT disconnections (15 s every 15 min) or no disconnection for 3 h. Hemodynamics, pulmonary variables were monitored, and lung histology and bronchoalveolar lavage studied. RESULTS: As compared to PEEP 5 cmH2O, abrupt deflation from PEEP 26 cmH2O increased PVR, lowered oxygenation, and increased lung wet-to-dry ratio. From PEEP 20 cmH2O, gradual versus abrupt deflation mitigated the changes in oxygenation and vascular resistance. From PEEP 15, repeated disconnections in presence of fluid loading led to reduced compliance, lower oxygenation, higher pulmonary artery pressure, higher lung wet-to-dry ratio, higher lung injury score and increased oedema on morphometry, compared to no disconnects. CONCLUSION: Single abrupt deflation from high PEEP, and repeated short deflations from moderate PEEP cause pulmonary oedema, impaired oxygenation, and increased PVR, in this large animal model, thus replicating our previous finding from rodents. Rapid deflation may thus be a clinically relevant cause of impaired lung function, which may be attenuated by gradual pressure release.

Positive End-Expiratory Pressure, Pleural Pressure, and Regional Compliance during Pronation: An Experimental Study.

Rationale: The physiological basis of lung protection and the impact of positive end-expiratory pressure (PEEP) during pronation in acute respiratory distress syndrome are not fully elucidated. Objectives: To compare pleural pressure (Ppl) gradient, ventilation distribution, and regional compliance between dependent and nondependent lungs, and investigate the effect of PEEP during supination and pronation. Methods: We used a two-hit model of lung injury (saline lavage and high-volume ventilation) in 14 mechanically ventilated pigs and studied supine and prone positions. Global and regional lung mechanics including Ppl and distribution of ventilation (electrical impedance tomography) were analyzed across PEEP steps from 20 to 3 cm H2O. Two pigs underwent computed tomography scans: tidal recruitment and hyperinflation were calculated. Measurements and Main Results: Pronation improved oxygenation, increased Ppl, thus decreasing transpulmonary pressure for any PEEP, and reduced the dorsal-ventral pleural pressure gradient at PEEP < 10 cm H2O. The distribution of ventilation was homogenized between dependent and nondependent while prone and was less dependent on the PEEP level than while supine. The highest regional compliance was achieved at different PEEP levels in dependent and nondependent regions in supine position (15 and 8 cm H2O), but for similar values in prone position (13 and 12 cm H2O). Tidal recruitment was more evenly distributed (dependent and nondependent), hyperinflation lower, and lungs cephalocaudally longer in the prone position. Conclusions: In this lung injury model, pronation reduces the vertical pleural pressure gradient and homogenizes regional ventilation and compliance between the dependent and nondependent regions. Homogenization is much less dependent on the PEEP level in prone than in supine positon.

Role of Positive End-Expiratory Pressure and Regional Transpulmonary Pressure in Asymmetrical Lung Injury.

Rationale: Asymmetrical lung injury is a frequent clinical presentation. Regional distribution of Vt and positive end-expiratory pressure (PEEP) could result in hyperinflation of the less-injured lung. The validity of esophageal pressure (Pes) is unknown.Objectives: To compare, in asymmetrical lung injury, Pes with directly measured pleural pressures (Ppl) of both sides and investigate how PEEP impacts ventilation distribution and the regional driving transpulmonary pressure (inspiratory - expiratory).Methods: Fourteen mechanically ventilated pigs with lung injury were studied. One lung was blocked while the contralateral one underwent surfactant lavage and injurious ventilation. Airway pressure and Pes were measured, as was Ppl in the dorsal and ventral pleural space adjacent to each lung. Distribution of ventilation was assessed by electrical impedance tomography. PEEP was studied through decremental steps.Measurements and Results: Ventral and dorsal Ppl were similar between the injured and the noninjured lung across all PEEP levels. Dorsal Ppl and Pes were similar. The driving transpulmonary pressure was similar in the two lungs. Vt distribution between lungs was different at zero end-expiratory pressure (≈70% of Vt going in noninjured lung) owing to different respiratory system compliance (8.3 ml/cm H2O noninjured lung vs. 3.7 ml/cm H2O injured lung). PEEP at 10 cm H2O with transpulmonary pressure around zero homogenized Vt distribution opening the lungs. PEEP ≥16 cm H2O equalized distribution of Vt but with overdistension for both lungs.Conclusions: Despite asymmetrical lung injury, Ppl between injured and noninjured lungs is equalized and esophageal pressure is a reliable estimate of dorsal Ppl. Driving transpulmonary pressure is similar for both lungs. Vt distribution results from regional respiratory system compliance. Moderate PEEP homogenizes Vt distribution between lungs without generating hyperinflation.

Hyperpolarized 129Xe magnetic resonance spectroscopy in a rat model of bronchopulmonary dysplasia.

Premature infants often require mechanical ventilation and oxygen therapy, which can result in bronchopulmonary dysplasia (BPD), characterized by developmental arrest and impaired lung function. Conventional clinical methods for assessing the prenatal lung are not adequate for the detection and assessment of long-term health risks in infants with BPD, highlighting the need for a noninvasive tool for the characterization of lung microstructure and function. Theoretical diffusion models, like the model of xenon exchange (MOXE), interrogate alveolar gas exchange by predicting the uptake of inert hyperpolarized (HP) 129Xe gas measured with HP 129Xe magnetic resonance spectroscopy (MRS). To investigate HP 129Xe MRS as a tool for noninvasive characterization of pulmonary microstructural and functional changes in vivo, HP 129Xe gas exchange data were acquired in an oxygen exposure rat model of BPD that recapitulates the fewer and larger distal airways and pulmonary vascular stunting characteristics of BPD. Gas exchange parameters from MOXE, including airspace mean chord length (Lm), apparent hematocrit in the pulmonary capillaries (HCT), and pulmonary capillary transit time (tx), were compared with airspace mean axis length and area density (MAL and ρA) and percentage area of tissue and air (PTA and PAA) from histology. Lm was significantly larger in the exposed rats (P = 0.003) and correlated with MAL, ρA, PTA, and PAA (0.59<|ρ|<0.66 and P < 0.05). Observed increase in HCT (P = 0.012) and changes in tx are also discussed. These findings support the use of HP 129Xe MRS for detecting fewer, enlarged distal airways in this rat model of BPD, and potentially in humans.

Fast detection of FOXF1 variants in patients with alveolar capillary dysplasia with misalignment of pulmonary veins using targeted sequencing.

BACKGROUND: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a lethal congenital lung disorder associated with heterozygous variants in the FOXF1 gene or its regulatory region. Patients with ACD/MPV unnecessarily undergo invasive and expensive treatments while awaiting a diagnosis. The aim of this study was to reduce the time to diagnose ACD/MPV by developing a targeted next-generation sequencing (NGS) panel that detects FOXF1 variants. METHODS: A FOXF1-targeted NGS panel was developed for detection of mutations and large genomic alterations and used for retrospective testing of ACD/MPV patients and controls. Results were confirmed with Sanger sequencing and SNP array analysis. RESULTS: Each amplicon of the FOXF1-targeted NGS panel was efficiently sequenced using DNA isolated from blood or cell lines of 15 ACD/MPV patients and 8 controls. Moreover, testing of ACD/MPV patients revealed six novel and six previously described pathogenic or likely pathogenic FOXF1 alterations. CONCLUSION: We successfully designed a fast and reliable targeted genetic test to detect variants in the FOXF1 gene and its regulatory region in one run. This relatively noninvasive test potentially prevents unnecessary suffering for patients and reduces the use of futile and expensive treatments like extra-corporeal membrane oxygenation. IMPACT: FOXF1-targeted NGS potentially prevents ACD/MPV patients from unnecessary suffering and expensive treatments. FOXF1-targeted NGS potentially reduces the number of misdiagnosis in ACD/MPV patients. Retrospective testing of ACD/MPV patients using FOXF1-targeted NGS revealed six novel pathogenic or likely pathogenic variants.

Embryonic-Derived Myb- Macrophages Enhance Bacterial Clearance and Improve Survival in Rat Sepsis.

Peritoneal resident macrophages play a key role in combating sepsis in the peritoneal cavity. We sought to determine if peritoneal transplantation of embryonic Myb- "peritoneal-like" macrophages attenuate abdominal fecal sepsis. Directed differentiation of rodent pluripotent stem cells (PSCs) was used in factor-defined media to produce embryonic-derived large "peritoneal-like" macrophages (Ed-LPM) that expressed peritoneal macrophage markers and demonstrated phagocytic capacity. Preclinical in vivo studies determined Ed-LPM efficacy in rodent abdominal fecal sepsis with or without Meropenem. Ex vivo studies explored the mechanism and effects of Ed-LPM on host immune cell number and function, including phagocytosis, reactive oxygen species (ROS) production, efferocytosis and apoptosis. Ed-LPM reduced sepsis severity by decreasing bacterial load in the liver, spleen and lungs. Ed-LPM therapy significantly improved animal survival by ~30% and reduced systemic bacterial burden to levels comparable to Meropenem therapy. Ed-LPM therapy decreased peritoneal TNFα while increasing IL-10 concentrations. Ed-LPMs enhanced peritoneal macrophage phagocytosis of bacteria, increased macrophage production of ROS and restored homeostasis via apoptosis and efferocytosis-induced clearance of neutrophils. In conclusion, Ed-LPM reduced systemic sepsis severity, improved survival and reduced bacterial load by enhancing peritoneal macrophage bacterial phagocytosis and killing and clearance of intra-peritoneal neutrophils. Macrophage therapy may be a potential strategy to address sepsis.

Hyperpolarized 129 Xe imaging of embryonic stem cell-derived alveolar-like macrophages in rat lungs: proof-of-concept study using superparamagnetic iron oxide nanoparticles.

PURPOSE: To measure regional changes in hyperpolarized 129 Xe MRI signal and apparent transverse relaxation ( T2∗ ) because of instillation of SPION-labeled alveolar-like macrophages (ALMs) in the lungs of rats and compare to histology. METHODS: MRI was performed in 6 healthy mechanically ventilated rats before instillation, as well as 5 min and 1 h after instillation of 4 million SPION-labeled ALMs into either the left or right lung. T2∗ maps were calculated from 2D multi-echo data at each time point and changes in T2∗ were measured and compared to control rats receiving 4 million unlabeled ALMs. Histology of the ex vivo lungs was used to compare the regional MRI findings with the locations of the SPION-labeled ALMs. RESULTS: Regions of signal loss were observed immediately after instillation of unlabeled and SPION-labeled ALMs and persisted at least 1 h in the case of the SPION-labeled ALMs. This was reflected in the measurements of T2∗ . One hour after the instillation of SPION-labeled ALMs, the T2∗ decreased to 54.0 ± 7.0% of the baseline, compared to a full recovery to baseline after the instillation of unlabeled ALMs. Histology confirmed the co-localization of SPION-labeled ALMs with regions of signal loss and T2∗ decreases for each rat. CONCLUSION: Hyperpolarized 129 Xe MRI can detect the presence of SPION-labeled ALMs in the airways 1 h after instillation. This approach is promising for targeting and tracking of stem cells for the treatment of lung disease.

Acid Sphingomyelinase Inhibition Attenuates Cell Death in Mechanically Ventilated Newborn Rat Lung.

RATIONALE: Premature infants subjected to mechanical ventilation (MV) are prone to lung injury that may result in bronchopulmonary dysplasia. MV causes epithelial cell death and halts alveolar development. The exact mechanism of MV-induced epithelial cell death is unknown. OBJECTIVES: To determine the contribution of autophagy to MV-induced epithelial cell death in newborn rat lungs. METHODS: Newborn rat lungs and fetal rat lung epithelial (FRLE) cells were exposed to MV and cyclic stretch, respectively, and were then analyzed by immunoblotting and mass spectrometry for autophagy, apoptosis, and bioactive sphingolipids. MEASUREMENTS AND MAIN RESULTS: Both MV and stretch first induce autophagy (ATG 5-12 [autophagy related 5-12] and LC3B-II [microtubule-associated proteins 1A/1B light chain 3B-II] formation) followed by extrinsic apoptosis (cleaved CASP8/3 [caspase-8/3] and PARP [poly(ADP-ribose) polymerase] formation). Stretch-induced apoptosis was attenuated by inhibiting autophagy. Coimmunoprecipitation revealed that stretch promoted an interaction between LC3B and the FAS (first apoptosis signal) cell death receptor in FRLE cells. Ceramide levels, in particular C16 ceramide, were rapidly elevated in response to ventilation and stretch, and C16 ceramide treatment of FRLE cells induced autophagy and apoptosis in a temporal pattern similar to that seen with MV and stretch. SMPD1 (sphingomyelin phosphodiesterase 1) was activated by ventilation and stretch, and its inhibition prevented ceramide production, LC3B-II formation, LC3B/first apoptosis signal interaction, caspase-3 activation, and, ultimately, FLRE cell death. SMPD1 inhibition also attenuated ventilation-induced autophagy and apoptosis in newborn rats. CONCLUSIONS: Ventilation-induced ceramides promote autophagy-mediated cell death, and identifies SMPD1 as a potential therapeutic target for the treatment of ventilation-induced lung injury in newborns.

Abrupt Deflation after Sustained Inflation Causes Lung Injury.

RATIONALE: Ventilator management in acute respiratory distress syndrome usually focuses on setting parameters, but events occurring at ventilator disconnection are not well understood. OBJECTIVES: To determine if abrupt deflation after sustained inflation causes lung injury. METHODS: Male Sprague-Dawley rats were ventilated (low Vt, 6 ml/kg) and randomized to control (n = 6; positive end-expiratory pressure [PEEP], 3 cm H2O; 100 min) or intervention (n = 6; PEEP, 3-11 cm H2O over 70 min; abrupt deflation to zero PEEP; ventilation for 30 min). Lung function and injury was assessed, scanning electron microscopy performed, and microvascular leak timed by Evans blue dye (n = 4/group at 0, 2, 5, 10, and 20 min after deflation). Hemodynamic assessment included systemic arterial pressure (n = 6), echocardiography (n = 4), and right (n = 6) and left ventricular pressures (n = 6). MEASUREMENTS AND MAIN RESULTS: Abrupt deflation after sustained inflation (vs. control) caused acute lung dysfunction (compliance 0.48 ± 1.0 vs. 0.82 ± 0.2 m/cm H2O, oxygen saturation as measured by pulse oximetry 67 ± 23.5 vs. 91 ± 4.4%; P < 0.05) and injury (wet/dry ratio 6.1 ± 0.6 vs. 4.6 ± 0.4; P < 0.01). Vascular leak was absent before deflation and maximal 5-10 minutes thereafter; injury was predominantly endothelial. At deflation, left ventricular preload, systemic blood pressure, and left ventricular end-diastolic pressure increased precipitously in proportion to the degree of injury. Injury caused later right ventricular failure. Sodium nitroprusside prevented the increase in systemic blood pressure and left ventricular end-diastolic pressure associated with deflation, and prevented injury. Injury did not occur with gradual deflation. CONCLUSIONS: Abrupt deflation after sustained inflation can cause acute lung injury. It seems to be mediated by acute left ventricular decompensation (caused by increased left ventricular preload and afterload) that elevates pulmonary microvascular pressure; this directly injures the endothelium and causes edema, which is potentiated by the surge in pulmonary perfusion.

Autophagy and the unfolded protein response promote profibrotic effects of TGF-ß1 in human lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease in adults with limited treatment options. Autophagy and the unfolded protein response (UPR), fundamental processes induced by cell stress, are dysregulated in lung fibroblasts and epithelial cells from humans with IPF. Human primary cultured lung parenchymal and airway fibroblasts from non-IPF and IPF donors were stimulated with transforming growth factor-ß1 (TGF-ß1) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3ßII and Atg5-12), UPR (BIP, IRE1α, and cleaved XBP1), and fibrosis (collagen 1α2 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3ßII) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-ß1-induced collagen 1α2 and fibronectin protein production was significantly higher in IPF lung fibroblasts compared with lung and airway fibroblasts from non-IPF donors. TGF-ß1 induced the accumulation of LC3ßII in parallel with collagen 1α2 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-ß1-induced collagen and fibronectin biosynthesis was significantly reduced by inhibiting autophagy flux in fibroblasts from the lungs of non-IPF and IPF donors. Conversely, only in lung fibroblasts from IPF donors did TGF-ß1 induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-ß1-induced collagen 1α2 and fibronectin biosynthesis in IPF lung fibroblasts but not those from non-IPF donors. The IRE1 arm of the UPR response is uniquely induced by TGF-ß1 in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these cells.

Mechanical Ventilation Induces Desensitization of Lung Axl Tyrosine Kinase Receptors.

BACKGROUND: Lower tidal volumes are increasingly used in acute respiratory distress syndrome, but mortality has changed little in the last 20 yr. Therefore, in addition to ventilator settings, it is important to target molecular mediators of injury. Sepsis and other inflammatory states increase circulating concentrations of Gas6, a ligand for the antiinflammatory receptor Axl, and of a soluble decoy form of Axl. We investigated the effects of lung stretch on Axl signaling. METHODS: We used a mouse model of early injury from high tidal volume and assessed the effects of inhibiting Axl on in vivo lung injury (using an antagonist R428, n = 4/group). We further determined the effects of stretch on Axl activation using in vitro lung endothelial cells. RESULTS: High tidal volume caused mild injury (compliance decreased 6%) as intended, and shedding of the Axl receptor (soluble Axl in bronchoalveolar fluid increased 77%). The Axl antagonist R428 blocked the principal downstream Axl target (suppressor of cytokine signaling 3 [SOCS3]) but did not worsen lung physiology or inflammation. Cyclic stretch in vitro caused Axl to become insensitive to activation by its agonist, Gas6. Finally, in vitro Axl responses were rescued by blocking stretch-activated calcium channels (using guanidinium chloride [GdCl3]), and the calcium ionophore ionomycin replicated the effect of stretch. CONCLUSIONS: These data suggest that lung endothelial cell overdistention activates ion channels, and the resultant influx of Ca inactivates Axl. Downstream inactivation of Axl by stretch was not anticipated; preventing this would be required to exploit Axl receptors in reducing lung injury.