American Journal of Physiology-Lung Cellular and Molecular Physiology

A key feature of bronchopulmonary dysplasia (BPD) is impaired alveolar septation. In later live, BPD survivors are more susceptible to childhood respiratory problems and have reduced respiratory function as adults. Chymotrypsin-like elastase 1 (CELA1) is a serine protease expressed in AT2 cells that mediates emphysema progression in adult mouse models. CELA1 binds and cleaves tropoelastin in response to strain. Its expression is developmentally regulated. Using the mouse hyperoxia model of impaired alveolar development we hypothesized a role for CELA1 in impaired alveolar development (IAD). In C57BL6 mouse pup lungs exposed to 80% oxygen for 14 days Cela1 mRNA increased 1.9-fold (p<0.05) and protein 2.6-fold (p<0.01). Protein levels normalized after 14 days in room air. Analysis of an existing single cell mRNA-seq dataset showed Cela1 mRNA in AT2 cells, alveolar macrophages and interstitial macrophages. The fraction of cells with Cela1 mRNA increased with hyperoxia. By flow cytometry the only Cela1-specific difference in immune cell populations was a 2-fold increase in lung eosinophils in room air (p<0.05). After 14 days of exposure to 80% oxygen Cela1-/- mice had better alveolarization with an average mean linear intercept of 80 m compared to 111m (p<0.001). Treatment of hyperoxia-exposed pups with subcutaneous anti-Cela1 KF4 antibody offered similar protection compared to IgG (59 m vs. 67 m, p<0.001).Human BPD specimens demonstrated CELA1 in AT2 cells and myeloid cells. These data indicate that hyperoxia-induced increases in CELA1 are partially responsible for IAD and suggest a potential role in premature neonates exposed to high FiO2.

Inhalation exposure to ozone (O3) causes adverse respiratory health effects that result from airway inflammation, a complex response mediated by changes to airway cellular transcriptional programs. These programs may be regulated in part by a subset of microRNAs transferred between cells (e.g. epithelial cells and macrophages) via extracellular vesicles (EV miRNA). To explore this, we exposed female C57BL/6J mice to filtered air (FA), 1, or 2 ppm O3 by inhalation and collected bronchoalveolar lavage fluid (BALF) 21 hours later for markers of airway inflammation, EVs, and EV miRNA. Both concentrations of O3 significantly increased markers of inflammation (neutrophils and total protein) and the number of EVs in the BALF. Using high-throughput small RNA sequencing, we identified several differentially expressed (DE) BALF EV miRNAs after 1 ppm (16 DE miRNAs) and 2 ppm (99 DE miRNAs) O3 versus FA exposure. O3 concentration response patterns in EV miRNA expression were apparent, particularly for the two most highly expressed (miR-2137 and miR-126-3p) and lowly expressed (miR-378-3p and miR-351-5p) miRNAs. Integrative analysis of EV miRNA expression and airway cellular mRNA expression identified EV miR-22-3p as a candidate regulator of transcriptomic responses to O3 in airway macrophages. In contrast, we did not identify candidate miRNA regulators of mRNA expression data from conducting airways (predominantly composed of epithelial cells). In summary, our data show that O3 exposure alters EV release and EV miRNA expression, suggesting that further investigation of EVs may provide insight into their effects on airway macrophage function and other mechanisms of O3-induced respiratory inflammation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

RationaleIn patients with severe lung injury, extracorporeal life support can enable lung rest, but the optimal strategy is unknown. ObjectiveTo compare liquid lung rest versus standard gas rest ventilation in a porcine model of acute lung injury and extracorporeal life support. MethodsTwenty neonatal pigs with lung injury after systemic injection of oleic acid were placed on extracorporeal life support. Pigs were ventilated with standard rest settings (peak inspiratory pressure: 20 cmH2O; end expiratory pressure: 10 cmH2O; rate: 10 breaths/minute) and randomized to receive no additional therapy (gas ventilation) or 5 mL/kg perfluorooctylbromide instilled endotracheally (liquid lung rest). The study continued for 4 hours with hourly pulmonary compliance measurements. After euthanasia, lung samples were taken for histologic analysis. Inflammation was quantified via blood and lung tissue cytokines. Measurements and Main ResultsAll pigs achieved significant lung injury; static compliance decreased by a mean of 54%. After 4 hours of lung rest, static compliance was higher with liquid lung rest compared to gas ventilation (0.88{+/-}0.05 mL/kg/cmH2O versus 0.57{+/-}0.05, p<0.001). Histology revealed 2.1-fold greater airspace in dependent lung regions with liquid lung rest (p<0.001) and a reduced airspace heterogeneity index (LLR: 0.071{+/-}0.004, gas: 0.095{+/-}0.010, p=0.04), suggesting more uniform alveolar recruitment. Cytokine analysis demonstrated a 3.7-fold decrease in tissue interleukin-10 levels with liquid lung rest (p=0.02). ConclusionsLiquid lung rest improved pulmonary compliance, achieved better alveolar recruitment, and decreased tissue interleukin-10 levels compared to standard gas ventilation. This strategy may enhance lung recovery in acute lung injury requiring extracorporeal life support.

Acute hypoxemic respiratory failure presents therapeutic challenges due to ventilation/perfusion mismatch and shunt. The goal of management strategies is to improve arterial oxygenation, however each management strategy presents risk to patients from iatrogenic injury. Intravenous oxygen therapeutics present an appealing option to improve arterial oxygenation without these risks. We used a two-hit murine model of acute lung injury to evaluate the effect of intravenous dodecafluoropentane (NanO2) on oxygen saturation and bronchoalveolar lavage cell count and protein. Mice were given intratracheal lipopolysaccharide and 20 hours later were intubated and ventilated with high tidal volumes. NanO2 was given by bolus injection at the initiation of mechanical ventilation and again at 2 hours, while oxygen saturation was measured every 15 minutes. At the conclusion of the experiment (4 hours), a bronchoalveolar lavage was performed. There was no difference in mean O2 saturation at time zero, however the difference between the mean O2 saturation immediately prior to injection and the mean first O2 saturation after injection in the control saline group were 91% and 83%, mean difference -7.5%; whereas mean O2 saturation in the NanO2 treated group rose from 89% to 91%, mean difference +2.5%, net difference 10% [95% CI: 2.7,17.3], p=0.01). There was a statistically significant difference in cell count, but not protein, on the bronchoalveolar lavage analysis. These data show that NanO2 rapidly improves oxygen saturation in a two-hit model of acute lung injury, and shows potential as an intravenous oxygen therapeutic in the management in acute hypoxemic respiratory failure.

Obstructive sleep apnea and its characteristic intermittent hypoxia (IH) are widely recognized as significant contributors to various pulmonary diseases, including asthma, pulmonary arterial hypertension, fibrosis, and chronic obstructive pulmonary disease. While single-cell RNA sequencing (scRNA-seq) has provided valuable insights into cell-type-specific responses to IH, previous studies have primarily focused on post-hypoxic recovery states, leaving immediate molecular responses during active IH exposure unexplored. To address this critical knowledge gap, we investigated real-time transcriptional responses to IH at single-cell resolution in lung tissue using male mice (n=3/group) exposed to either normoxia or IH (30 cycles/h, nadir 6% O2, 12 h/day) for 14 days, with tissue collection during active IH exposure. Our analysis revealed pronounced cell-type-specific transcriptional reprogramming, particularly in airway smooth muscle cells (ASMC), arterial endothelial cells (AEC), and lymphatic endothelial cells (LEC). These changes were characterized by enrichment in pathways related to epithelial-to-mesenchymal transition (ASMC, LEC), myogenesis (ASMC), and antioxidant defenses (AEC, LEC). Most cell types demonstrated substantial upregulation of genes encoding mitochondrial complex I-IV proteins and TCA cycle enzymes accompanied by a decreased expression of genes encoded by mitochondrial DNA that was markedly present in LEC, AEC, and cells of the alveolar-capillary unit, revealing a mito-nuclear imbalance. These findings provide novel insights into the immediate cellular responses to IH, showing previously uncharacterized metabolic reorganization that may underlie the development of IH-related pulmonary complications. This improved understanding of early molecular events during active IH exposure advances our knowledge of sleep apnea-related lung pathologies and may inform future therapeutic strategies.

Regional pulmonary perfusion (Q) has been investigated using blood volume (Fb) imaging as an easier-to-measure surrogate. However, it is unclear if changing pulmonary conditions could affect their relationship. We hypothesized that vascular changes in early acute respiratory distress syndrome (ARDS) affect Q and Fb differently. Five sheep were anesthetized and received protective mechanical ventilation for 20 hours while endotoxin was continuously infused. Using dynamic 18F-FDG and 13NN Positron Emission Tomography (PET), regional Fb and Q were analysed in 30 regions of interest (ROIs) and normalized by tissue content (Fbn and Qn, respectively). After 20 hours, the animals lung injury showed characteristics of early ARDS, including gas exchange and lung mechanics. PET images of Fbn and Qn showed substantial differences between baseline and lung injury. Lung injury caused a significant change in the Fbn-Qn relationship compared to baseline (p<0.001). The best models at baseline and lung injury were Fbn=0.32+0.690Qn and Fbn=1.684Qn-0.538Qn2, respectively. Early ARDS changed the relationship between Fb and Q from linear to curvilinear. Effects of endotoxin exposure on the vasoactive blood flow regulation were most likely the key factor for this change limiting the quantitative accuracy of Fb imaging as a surrogate for regional Q.

RationalePulmonary angiogenesis is a key driver of alveolarization. Our prior studies showed that nuclear factor kappa-B (NF{kappa}B) promotes pulmonary angiogenesis during early alveolarization. However, the mechanisms regulating temporal-specific NF{kappa}B activation in the pulmonary vasculature are unknown. ObjectivesTo identify mechanisms that activate pro-angiogenic NF{kappa}B signaling in the developing pulmonary vasculature. MethodsProteomic analysis of the lung secretome was performed using 2D-DIGE. NF{kappa}B activation and angiogenic function was assessed in primary pulmonary endothelial cells (PEC) and TGFBI-regulated genes identified using RNA-sequencing. Alveolarization and pulmonary angiogenesis was assessed in WT and TGFBI null mice exposed to normoxia or hyperoxia. Lung TGFBI expression was determined in premature lambs supported by invasive and noninvasive respiratory support. Measurements and Main ResultsSecreted factors from the early alveolar, but not the late alveolar or adult lung, promoted proliferation and migration in quiescent, adult PEC. Proteomic analysis identified transforming growth factor beta-induced protein (TGFBI) as a protein highly expressed by myofibroblasts in the early alveolar lung that promoted PEC migration by activating NF{kappa}B via v{beta}3 integrins. RNA-sequencing identified Csf3 as a TGFBI-regulated gene that enhances nitric oxide production in PEC. Loss of TGFBI in mice exaggerated the impaired pulmonary angiogenesis induced by chronic hyperoxia, and TGFBI expression was disrupted in premature lambs with impaired alveolarization. ConclusionsOur studies identify TGFBI as a developmentally-regulated protein that promotes NF{kappa}B-mediated angiogenesis during early alveolarization by enhancing nitric oxide production. We speculate that dysregulation of TGFBI expression may contribute to diseases marked by impaired alveolar and vascular growth.

Alveolarization is dependent on myo-, matrix- and lipo- fibroblast functions by interstitial PDGFRa+ fibroblasts. While these fibroblasts are derived from GLI and PDGFRa expressing fibroblasts, the transcriptional control of their functional specification remains unknown. Perinatally, the transcription factor GATA6 is upregulated in PDGFRa+ fibroblasts. To study the role of GATA6 during fibroblast differentiation, we generated PDGFRaCreER/GATA6flx/flx mice and deleted GATA6 in the perinatal period and in adult mice prior to left lobe pneumonectomy. Loss of GATA6 in the PDGFRa+-fibroblasts impaired alveolarization, and extracellular matrix deposition, in association with increased TCF21 expression and lipofibroblast differentiation. Loss of GATA6 in PDGFRa+ fibroblasts resulted in loss of alveolar type 1 (AT1) cells and gain of transitional alveolar type 2 (AT2) cells. Loss of GATA6 was associated with reduced WNT signaling. Restoration of WNT signaling in GATA6 deficient alveolar lung organoids restored AT2 and AT1 cell differentiation. GATA6 induces matrix fibroblast functions and represses lipofibroblast functions, serving as key regulator of fibroblast differentiation during alveolarization and regeneration. Present findings link matrix fibroblast functions with the ability of transitional AT2 cells to differentiate into AT1 cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/494950v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@47375forg.highwire.dtl.DTLVardef@184630borg.highwire.dtl.DTLVardef@8285b3org.highwire.dtl.DTLVardef@117e47a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO C_FIG

RationaleThe role of MUC5B mucin expression in IPF pathogenesis is unknown. Bleomycin-exposed rodent models do not exhibit sustained fibrosis or airway remodeling. Unlike mice, ferrets have human-like distribution of MUC5B expressing cell types and natively express the risk-conferring variant that induces high MUC5B expression in humans. We hypothesized that ferrets would consequently exhibit aberrant repair to propagate fibrosis similar to human IPF. MethodsBleomycin (5U/kg) or saline-control was micro-sprayed intratracheally then wild-type ferrets were evaluated through 22 wks. Clinical phenotype was assessed with lung function. Fibrosis was assessed with {micro}CT imaging and comparative histology with Ashcroft scoring. Airway remodeling was assessed with histology and quantitative immunofluorescence. ResultsBleomycin ferrets exhibited sustained restrictive physiology including decreased inspiratory capacity, decreased compliance, and shifted Pressure-Volume loops through 22 wks. Volumetric {micro}CT analysis revealed increased opacification of the lung bleomycin-ferrets. Histology showed extensive fibrotic injury that matured over time and MUC5B-positive cystic structures in the distal lung suggestive of honeycombing. Bleomycin ferrets had increased proportion of small airways that were double-positive for CCSP and alpha-tubulin compared to controls, indicating an aberrant proximalization repair phenotype. Notably, this aberrant repair was associated with extent of fibrotic injury at the airway level. ConclusionsBleomycin-exposed ferrets exhibit sustained fibrosis through 22 wks and have pathologic features of IPF not found in rodents. Ferrets exhibited proximalization of the distal airways and other pathologic features characteristic of human IPF. MUC5B expression through native cell types may play a key role in promoting airway remodeling and lung injury in IPF.

RationaleExposure to humidifier disinfectants has been linked to an array of pulmonary disorders and diminished lung functionality particularly reduced Forced Vital Capacity (FVC). ObjectivesThis investigation sought to identify diagnostic biomarkers for early detection of children at elevated risk of developing chronic respiratory conditions following such exposure. MethodsOur research employed a comprehensive multi-omics strategy analyzing 70 pediatric patients alongside 10 controls, seamlessly integrating clinical assessments with transcriptomics, methylomics, proteomics, and metabolomics data. The analytical framework utilized a sophisticated combination of Non-negative Matrix Factorization (NMF), Multi-Omics Factor Analysis (MOFA), and advanced machine learning algorithms. Measurements and Main ResultsNMF clustering uncovered distinctive protein expression patterns associated with integrin-mediated signaling pathways and immune response mechanisms. Complementarily, MOFA identified latent factors correlating with lung function metrics, highlighting critical molecular pathways involved in integrin cell surface interactions and lipid metabolism regulation. Machine learning-based analysis facilitated the development of a multi-marker panel-comprising IGHV2-70, LysoPC (16:0), and hexadecyl ferulate-which achieved 81.46% accuracy in identifying pulmonary dysfunction cohort. ConclusionsThese findings suggest that alterations in integrin-related signaling networks and dysregulation of lipid metabolism play pivotal roles in mediating the long-term pulmonary consequences of humidifier disinfectant exposure. The proposed multi-marker panel offers significant potential for enhanced risk stratification and timely therapeutic intervention. At a Glance CommentaryO_ST_ABSScientific Knowledge on the SubjectC_ST_ABSExtensive epidemiological evidence has established the causal relationship between humidifier disinfectant exposure and pulmonary dysfunction; however, clinically validated biomarkers for predicting chronic lung disease progression remain limited. Pediatric populations demonstrate unique pathophysiological mechanisms distinct from adults, highlighting the critical necessity for biomarker identification grounded in comprehensive molecular understanding. Despite advances in omics technologies, recent investigations have encountered significant obstacles in achieving deeper mechanistic insights, predominantly attributable to methodological constraints in harmonizing clinical phenotypes with high-dimensional molecular datasets. What This Study Adds to the FieldThis investigation elucidates the fundamental contributions of integrin-mediated signaling cascades and lipid metabolic networks to persistent pulmonary dysfunction following humidifier disinfectant exposure. Our analyses revealed coordinated regulation of integrin signaling pathways and immune response networks through NMF clustering, indicating dynamic temporal evolution of inflammatory responses during chronic disease progression, with temporally distinct molecular signatures identified across discrete observation intervals. Multi-omics factor analysis (MOFA) corroborated integrin pathway dysregulation while additionally uncovering systematic suppression of lipid metabolic processes. Furthermore, machine learning algorithms enabled development of a robust three-component biomarker panel--encompassing IGHV2-70, LysoPC (16:0), and hexadecyl ferulate--demonstrating 81.46% classification accuracy for pulmonary dysfunction phenotypes. Collectively, these findings substantially advance mechanistic understanding of chronic lung injury in vulnerable pediatric cohorts and identify clinically relevant biomarkers with translational potential for risk stratification and therapeutic targeting in clinical practice.

As an intrinsically mechanosensitive organ, the lung experiences a range of mechanical forces. Chronic obstructive pulmonary disease (COPD) involves abnormal macroscopic cellular and extracellular matrix (ECM) changes that impact mechanical properties of the lung. Mechanosensitive Piezo1/2 channels are expressed in the lung including on airway smooth muscle cells (ASM) that mediate cellular responses to stretch and ECM biomechanics. The expression and roles of Piezos in COPD lung ASM are not known. We hypothesized that Piezo expression and activation are altered in COPD lung ASM influencing ECM regulation. Distribution of Piezo proteins in ASM and epithelium of small airways of COPD stage II and IV vs. non-COPD controls was assessed using immunohistochemistry and ImageJ (n=10-17/group). Isolated ASM cells from control (n=6) vs. COPD stage II and IV patients (n=3 each stage) were exposed to stretch or the Piezo1 agonist Yoda1 followed by measurement of ECM gene and protein expression. Less Piezo2 staining was observed in COPD IV patients compared to controls, with lesser area and intensity of staining in the epithelial layer, and lower intensity of staining in ASM and small airways as a whole. Fura-2-based imaging of ASM Ca2+ showed lower influx after Yoda1 exposure in COPD II compared to control and COPD IV. Gene expression of Piezo1 increased upon stretching in controls but not in COPD ASM, while Piezo2 protein expression decreased with stretching in all groups. Yoda1 treatment resulted in decreased collagen1, fibulin1 and periostin gene and collagen 1 and periostin protein expression in ASM. Overall, these results support a role for Piezo activation in abnormal ECM-ASM cell crosstalk in COPD.

Precision-cut lung slices (PCLS) are increasingly utilized for ex vivo disease modeling, but a high-resolution characterization of cellular phenotype stability in PCLS has not been reported. Comparing the single-cell transcriptomic profile of human PCLS after five days of culture to freshly isolated human lung tissue, we found striking changes in endothelial cell and alveolar epithelial cell programs, reflecting both injury and pathways activated in static culture, while immune cell frequencies and programs remained largely intact and similar to the native lung. These cellular dynamics should be considered when utilizing PCLS as a model of the human lung.

The lungs mechanosensitive immune response, which occurs when pulmonary alveoli are overstretched, is a major impediment to ventilation therapy for hypoxemic respiratory failure. The cause is not known. We tested the hypothesis that alveolar stretch causes stretch of alveolar macrophages (AMs), leading to the immune response. In lungs viewed by optical imaging, sessile AMs expressed gap junctional protein connexin-43 (Cx43), and they communicated with the alveolar epithelium through gap junctions. Alveolar hyperinflation increased Ca2+ in the AMs but did not stretch the AMs. The Ca2+ response, and concomitant TNF secretion by AMs were blocked in mice with AM-specific deletion of Cx43. The AM responses, as also lung injury due to mechanical ventilation at high tidal volume, were inhibited by AM-specific delivery of lipid nanoparticles containing Xestospongin C, which blocked the induced Ca2+ increases. We conclude, Cx43- and Ca2+-dependent AM-epithelial interactions determine the lungs mechanosensitive immunity, providing a basis for therapy for ventilator- induced lung injury.

Prolonged exposure to hypoxia causes structural remodeling and sustained contraction of the pulmonary vasculature, resulting in the development of pulmonary hypertension. Both pulmonary arterial smooth muscle cell (PASMC) proliferation and migration contribute to the vascular remodeling. We previously showed that the protein expression of aquaporin 1 (AQP1), a membrane water channel protein, is elevated in PASMCs during following in vivo or in vitro exposure to hypoxia. Studies in other cell types suggest that AQP1 is a direct transcriptional target of hypoxia inducible factor (HIF)-1. Moreover, we and others have shown that an increase in intracellular calcium concentration ([Ca2+]i) is a hallmark of hypoxic exposure in PASMCs. Thus, we wanted to determine whether HIF regulates AQP1 in PASMCs and, if so, whether the process occurred via transcriptional regulation or was Ca2+-dependent. PASMCs were exposed to hypoxia, incubated with DMOG, which inhibits HIF protein degradation or infected with constitutively active forms of HIF-1 or HIF-2. Hypoxia, DMOG and HIF1/2 produced a time-dependent increase in AQP1 protein, but not mRNA. Interestingly, incubation with increasing HIF1/2a levels and DMOG increased [Ca2+]i in PASMCs, and this elevation was prevented by the voltage-gated Ca2+ channel inhibitor, verapamil (VER) and nonselective cation channel inhibitor SKF96365 (SKF). VER and SKF also blocked upregulation of AQP1 protein by DMOG or HIF1/2, but had no effect on expression of GLUT1, a canonical HIF transcriptional target. Silencing of AQP1 abrogated increases in PASMC migration and proliferation induced by HIF1/2, suggesting induction of AQP1 protein by HIF1/2 has a functional outcome in these cells. Thus, our results show that contrary to reports in other cell types, in PASMCs, AQP1 does not appear to be a direct target for HIF transcriptional regulation. Instead, AQP1 protein may be upregulated by a mechanism involving HIF-dependent increases in [Ca2+]i.

We assessed the mechanisms by which non-encapsulated heme, released in the plasma of mice post exposure to chlorine (Cl2) gas, resulted in the initiation and propagation of acute lung injury. We exposed adult C57BL/6 male and female to Cl2 (500 ppm for 30 min) in environmental chambers and returned them to room air and injected them intramuscularly with a single dose of human hemopexin (hHPX; 5 {micro}g/ g BW), the most efficient scavenger of heme, 30-60 min post exposure. Concentrations of hHPX in plasma of air and Cl2 exposed mice were 9081{+/-}900 vs. 1879{+/-} 293 at 6 h and 2966{+/-}463 vs. 1555{+/-}250 at 50 h post injection (ng/ml; X{+/-}1 SEM=3; p<0.01). Cl2 exposed mice developed progressive acute lung injury post exposure characterized by increased concentrations of plasma heme, marked inflammatory response, respiratory acidosis and increased concentrations of plasma proteins in the alveolar space. Injection of hHPX decreased the onset of acute lung injury at 24 h post exposure; mean survival, for the saline and hHPX groups were 40 vs. 80% (P<0.001) at 15 d post exposure. Non-supervised global proteomics analysis of mouse lungs at 24 h post exposure, revealed the upregulation of 92 and downregulation of 145 lung proteins. Injection of hHPX at one h post exposure moderated the Cl2 induced changes in eighty-three of these 237 lung proteins. System biology analysis of the global proteomics data showed that hHPX reversed changes in mitochondrial dysfunction and elF2 and integrin signaling. Western blot analysis of lung tissue showed significant increase of phosphorylated elF2 at 24 h post exposure in vehicle treated mice but normal levels in those injected with hHPX. Similarly, RT-PCR analysis of lung tissue showed that hHPX reversed the onset of mtDNA lesions. A form of recombinant human hemopexin generated in tobacco plants was equally effective in reversing acute lung and mtDNA injury. The results of this study offer new insights as to the mechanisms by which exposure to Cl2 results in acute lung injury and to the therapeutic effects of hemopexin.

Acute respiratory distress syndrome (ARDS) is a highly lethal condition that impairs lung function and causes respiratory failure. Mechanical ventilation maintains gas exchange in patients with ARDS, but exposes lung cells to physical forces that exacerbate lung injury. Our data demonstrate that mTOR complex 1 (mTORC1) is a mechanosensor in lung epithelial cells and that activation of this pathway during mechanical ventilation exacerbates lung injury. We found that mTORC1 is activated in lung epithelial cells following volutrauma and atelectrauma in mice and humanized in vitro models of the lung microenvironment. mTORC1 is also activated in lung tissue of mechanically ventilated patients with ARDS. Deletion of Tsc2, a negative regulator of mTORC1, in epithelial cells exacerbates physiologic lung dysfunction during mechanical ventilation. Conversely, treatment with rapamycin at the time mechanical ventilation is initiated prevents physiologic lung injury (i.e. decreased compliance) without altering lung inflammation or barrier permeability. mTORC1 inhibition mitigates physiologic lung injury by preventing surfactant dysfunction during mechanical ventilation. Our data demonstrate that in contrast to canonical mTORC1 activation under favorable growth conditions, activation of mTORC1 during mechanical ventilation exacerbates lung injury and inhibition of this pathway may be a novel therapeutic target to mitigate ventilator induced lung injury during ARDS.

Vaping of flavored liquids has been touted as safe alternative to traditional cigarette smoking with decreased health risks. The popularity of vaping has dramatically increased over the last decade, particularly among teenagers who incorporate vaping into their daily life as a social activity. Despite widespread and increasing adoption of vaping among young adults there is little information on long term consequences of vaping and potential health risks. This study demonstrates Vaping-Induced Pulmonary Injury (VAPI) using commercial JUUL pens with flavored vape juice using an inhalation exposure murine model. Profound pathological changes to upper airway, lung tissue architecture, and cellular structure are evident within 9 weeks of exposure. Marked histologic changes include increased parenchyma tissue density, cellular infiltrates proximal to airway passages, alveolar rarefaction, increased collagen deposition, and bronchial thickening with elastin fiber disruption. Transcriptional reprogramming includes significant changes to gene families coding for xenobiotic response, glycerolipid metabolic processes, and oxidative stress. Cardiac contractile performance for systemic output is moderately but significantly impaired, and the shows severe pulmonary side structural remodeling with chamber enlargement. This VAPI model with pulmonary circuit failure demonstrates mechanistic underpinnings of vaping-related pathologic injury.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive and excessive accumulation of myofibroblasts and extracellular matrix in the lung. Connective-tissue growth factor (CTGF) is known to exacerbate pulmonary fibrosis in radiation-induced lung fibrosis, and in this study, we show the upregulation of CTGF from a rat lung fibrosis model induced by adenovirus vector encoding active TGF-β1 (AdTGF-β1), and also in patients with IPF. The expression of CTGF was upregulated in vascular smooth muscle cells cultured from fibrotic lungs on days 7 or 14 as well as endothelial cells sorted from fibrotic lungs on day 14 or 28 respectively. These findings suggest the role of different cells in maintaining the fibrotic phenotype during fibrogenesis. Treatment of fibroblasts with recombinant CTGF along with TGF-β increases pro-fibrotic markers in fibroblasts, confirming the synergistic effect of recombinant CTGF with TGF-β in inducing pulmonary fibrosis. Also, fibrotic extracellular matrix upregulated the expression of CTGF, as compared to normal extracellular matrix, suggesting that not only profibrotic mediators but also a profibrotic environment contributes to fibrogenesis. We also showed that pamrevlumab, a CTGF inhibitory antibody, partially attenuates fibrosis in the model. These results suggest that pamrevlumab could be an option for the treatment of pulmonary fibrosis.Competing Interest StatementT. Yanagihara was funded by the Uehara Memorial Foundation Research Fellowship and Mitacs Canada, and the research institute of St Joseph Hospital, Hamilton, ON, Canada (Post-doctoral Fellowship Award). K. Ask reports grants and personal fees from Boehringer Ingelheim, grants from Canadian Pulmonary Fibrosis Foundation, Synairgen, Alkermes, GlaxoSmitheKline, Pharmaxis, Unity, Avalyn, Canadian Institutes of Health Research, Ceapro, Pieris, outside the submitted work. M. Kolb reports grants from the Canadian Institute for Health Research and grants/ personal fees from Roche, Boehringer Ingelheim, Prometic, Respivert, Alkermes, and Pharmaxis and personal fees from Genoa. K.E. Lipson is an employee and shareholder of FibroGen, Inc. J. Tikkanen reports personal fees from CSL Behring, outside the submitted work. S.G. Chong, M. Gholiof, Q. Zhou, C. Scallan, C. Upagupta, and S. Keshavjee report no conflict of interest.View Full Text

Chronic obstructive pulmonary disease (COPD) is a devastating lung disease, characterized by a progressive decline in lung function, alveolar loss (emphysema), and airflow limitation due to excessive mucus secretion (chronic bronchitis), that can occur even after the injurious agent is removed. It is slated to rise to the 3rd leading cause of death due to chronic disease by 2030 globally, and the 4th leading cause of death due to chronic disease in the USA. While there is substantial evidence indicating loss of E-cadherin in the lung epithelium of patients with COPD, it is not known if this is causal to the disease. We investigated if loss of E-cadherin can result in lung disease using in both in vitro models of primary, differentiated human cells and in mouse models. Using a cell type-specific promoter using Cre/LoxP mice system to knock-out E- cadherin in ciliated and alveolar epithelial cell (Type 1 and Type 2) populations in adult mouse models, we determined that loss of E-cadherin caused airspace enlargement, as well as increased airway hyperresponsiveness indicating that it does have a causative role in causing COPD. Strategies to upregulate CDH1 (encodes for E-cadherin) in CHBEs and cigarette-smoke injured NHBEs can rescue the dysfunctional epithelium.

Synthetic textiles shed fibers that accumulate indoors and this results in continuous exposure when indoors. High exposure to microplastic fibers in nylon flock workers has been linked to the development of airway and interstitial lung disease, but the exact health effects of microplastic fibers on the lungs are unknown. Here we determined effects of polyester and nylon textile microplastic fibers on airway and alveolar epithelial cells using human and murine lung organoids. We observed that particularly nylon microfibers had a negative impact on the growth and development of airway organoids. We demonstrated that this effect was mediated by components leaking from nylon. Moreover, our data suggested that microplastic textile fibers may especially harm the developing airways or airways undergoing repair. Our results call for a need to assess exposure and inhalation levels in indoor environments to accurately determine the actual risk of these fibers to human health. TeaserAirborne fibers shed from synthetic textiles, in particular nylon, can inhibit repair of the cells coating the airways