Variation in Early Management Practices in Moderate-to-Severe ARDS in the United States: The Severe ARDS: Generating Evidence Study.

BACKGROUND: Although specific interventions previously demonstrated benefit in patients with ARDS, use of these interventions is inconsistent, and patient mortality remains high. The impact of variability in center management practices on ARDS mortality rates remains unknown. RESEARCH QUESTION: What is the impact of treatment variability on mortality in patients with moderate to severe ARDS in the United States? STUDY DESIGN AND METHODS: We conducted a multicenter, observational cohort study of mechanically ventilated adults with ARDS and Pao2 to Fio2 ratio of ≤ 150 with positive end-expiratory pressure of ≥ 5 cm H2O, who were admitted to 29 US centers between October 1, 2016, and April 30, 2017. The primary outcome was 28-day in-hospital mortality. Center variation in ventilator management, adjunctive therapy use, and mortality also were assessed. RESULTS: A total of 2,466 patients were enrolled. Median baseline Pao2 to Fio2 ratio was 105 (interquartile range, 78.0-129.0). In-hospital 28-day mortality was 40.7%. Initial adherence to lung protective ventilation (LPV; tidal volume, ≤ 6.5 mL/kg predicted body weight; plateau pressure, or when unavailable, peak inspiratory pressure, ≤ 30 mm H2O) was 31.4% and varied between centers (0%-65%), as did rates of adjunctive therapy use (27.1%-96.4%), methods used (neuromuscular blockade, prone positioning, systemic steroids, pulmonary vasodilators, and extracorporeal support), and mortality (16.7%-73.3%). Center standardized mortality ratios (SMRs), calculated using baseline patient-level characteristics to derive expected mortality rate, ranged from 0.33 to 1.98. Of the treatment-level factors explored, only center adherence to early LPV was correlated with SMR. INTERPRETATION: Substantial center-to-center variability exists in ARDS management, suggesting that further opportunities for improving ARDS outcomes exist. Early adherence to LPV was associated with lower center mortality and may be a surrogate for overall quality of care processes. Future collaboration is needed to identify additional treatment-level factors influencing center-level outcomes. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT03021824; URL: www.clinicaltrials.gov.

Claudin-18-mediated YAP activity regulates lung stem and progenitor cell homeostasis and tumorigenesis.

Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18-/- AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18-/- mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.

Region-specific role for Pten in maintenance of epithelial phenotype and integrity.

Previous studies have demonstrated resistance to naphthalene-induced injury in proximal airways of mice with lung epithelial-specific deletion of the tumor-suppressor gene Pten, attributed to increased proliferation of airway progenitors. We tested effects of Pten loss following bleomycin injury, a model typically used to study distal lung epithelial injury, in conditional PtenSFTPC-cre knockout mice. Pten-deficient airway epithelium exhibited marked hyperplasia, particularly in small bronchioles and at bronchoalveolar duct junctions, with reduced E-cadherin and ß-catenin expression between cells toward the luminal aspect of the hyperplastic epithelium. Bronchiolar epithelial and alveolar epithelial type II (AT2) cells in PtenSFTPC-cre mice showed decreased expression of epithelial markers and increased expression of mesenchymal markers, suggesting at least partial epithelial-mesenchymal transition at baseline. Surprisingly, and in contrast to previous studies, mutant mice were exquisitely sensitive to bleomycin, manifesting rapid weight loss, respiratory distress, increased early mortality (by day 5), and reduced dynamic lung compliance. This was accompanied by sloughing of the hyperplastic airway epithelium with occlusion of small bronchioles by cellular debris, without evidence of increased parenchymal lung injury. Increased airway epithelial cell apoptosis due to loss of antioxidant defenses, reflected by decreased expression of superoxide dismutase 3, in combination with deficient intercellular adhesion, likely predisposed to airway sloughing in knockout mice. These findings demonstrate an important role for Pten in maintenance of airway epithelial phenotype integrity and indicate that responses to Pten deletion in respiratory epithelium following acute lung injury are highly context-dependent and region-specific.

Knockout Mice Reveal a Major Role for Alveolar Epithelial Type I Cells in Alveolar Fluid Clearance.

Active ion transport by basolateral Na-K-ATPase (Na pump) creates an Na(+) gradient that drives fluid absorption across lung alveolar epithelium. The α1 and ß1 subunits are the most highly expressed Na pump subunits in alveolar epithelial cells (AEC). The specific contribution of the ß1 subunit and the relative contributions of alveolar epithelial type II (AT2) versus type I (AT1) cells to alveolar fluid clearance (AFC) were investigated using two cell type-specific mouse knockout lines in which the ß1 subunit was knocked out in either AT1 cells or both AT1 and AT2 cells. AFC was markedly decreased in both knockout lines, revealing, we believe for the first time, that AT1 cells play a major role in AFC and providing insights into AEC-specific roles in alveolar homeostasis. AEC monolayers derived from knockout mice demonstrated decreased short-circuit current and active Na(+) absorption, consistent with in vivo observations. Neither hyperoxia nor ventilator-induced lung injury increased wet-to-dry lung weight ratios in knockout lungs relative to control lungs. Knockout mice showed increases in Na pump ß3 subunit expression and ß2-adrenergic receptor expression. These results demonstrate a crucial role for the Na pump ß1 subunit in alveolar ion and fluid transport and indicate that both AT1 and AT2 cells make major contributions to these processes and to AFC. Furthermore, they support the feasibility of a general approach to altering alveolar epithelial function in a cell-specific manner that allows direct insights into AT1 versus AT2 cell-specific roles in the lung.

Combinations of differentiation markers distinguish subpopulations of alveolar epithelial cells in adult lung.

Distal lung epithelium is maintained by proliferation of alveolar type II (AT2) cells and, for some daughter AT2 cells, transdifferentiation into alveolar type I (AT1) cells. We investigated if subpopulations of alveolar epithelial cells (AEC) exist that represent various stages in transdifferentiation from AT2 to AT1 cell phenotypes in normal adult lung and if they can be identified using combinations of cell-specific markers. Immunofluorescence microscopy showed that, in distal rat and mouse lungs, ∼ 20-30% of NKX2.1(+) (or thyroid transcription factor 1(+)) cells did not colocalize with pro-surfactant protein C (pro-SP-C), a highly specific AT2 cell marker. In distal rat lung, NKX2.1(+) cells coexpressed either pro-SP-C or the AT1 cell marker homeodomain only protein x (HOPX). Not all HOPX(+) cells colocalize with the AT1 cell marker aquaporin 5 (AQP5), and some AQP5(+) cells were NKX2.1(+). HOPX was expressed earlier than AQP5 during transdifferentiation in rat AEC primary culture, with robust expression of both by day 7. We speculate that NKX2.1 and pro-SP-C colocalize in AT2 cells, NKX2.1 and HOPX or AQP5 colocalize in intermediate or transitional cells, and HOPX and AQP5 are expressed without NKX2.1 in AT1 cells. These findings suggest marked heterogeneity among cells previously identified as exclusively AT1 or AT2 cells, implying the presence of subpopulations of intermediate or transitional AEC in normal adult lung.

Knockout mice reveal key roles for claudin 18 in alveolar barrier properties and fluid homeostasis.

Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that regulate paracellular permeability to ions and solutes. Claudin 18, a member of the large claudin family, is highly expressed in lung alveolar epithelium. To elucidate the role of claudin 18 in alveolar epithelial barrier function, we generated claudin 18 knockout (C18 KO) mice. C18 KO mice exhibited increased solute permeability and alveolar fluid clearance (AFC) compared with wild-type control mice. Increased AFC in C18 KO mice was associated with increased ß-adrenergic receptor signaling together with activation of cystic fibrosis transmembrane conductance regulator, higher epithelial sodium channel, and Na-K-ATPase (Na pump) activity and increased Na-K-ATPase ß1 subunit expression. Consistent with in vivo findings, C18 KO alveolar epithelial cell (AEC) monolayers exhibited lower transepithelial electrical resistance and increased solute and ion permeability with unchanged ion selectivity. Claudin 3 and claudin 4 expression was markedly increased in C18 KO mice, whereas claudin 5 expression was unchanged and occludin significantly decreased. Microarray analysis revealed changes in cytoskeleton-associated gene expression in C18 KO mice, consistent with observed F-actin cytoskeletal rearrangement in AEC monolayers. These findings demonstrate a crucial nonredundant role for claudin 18 in the regulation of alveolar epithelial TJ composition and permeability properties. Increased AFC in C18 KO mice identifies a role for claudin 18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties that may, at least in part, compensate for increased permeability.

Interactions between ß-catenin and transforming growth factor-ß signaling pathways mediate epithelial-mesenchymal transition and are dependent on the transcriptional co-activator cAMP-response element-binding protein (CREB)-binding protein (CBP).

Interactions between transforming growth factor-ß (TGF-ß) and Wnt are crucial to many biological processes, although specific targets, rationale for divergent outcomes (differentiation versus block of epithelial proliferation versus epithelial-mesenchymal transition (EMT)) and precise mechanisms in many cases remain unknown. We investigated ß-catenin-dependent and transforming growth factor-ß1 (TGF-ß1) interactions in pulmonary alveolar epithelial cells (AEC) in the context of EMT and pulmonary fibrosis. We previously demonstrated that ICG-001, a small molecule specific inhibitor of the ß-catenin/CBP (but not ß-catenin/p300) interaction, ameliorates and reverses pulmonary fibrosis and inhibits TGF-ß1-mediated α-smooth muscle actin (α-SMA) and collagen induction in AEC. We now demonstrate that TGF-ß1 induces LEF/TCF TOPFLASH reporter activation and nuclear ß-catenin accumulation, while LiCl augments TGF-ß-induced α-SMA expression, further confirming co-operation between ß-catenin- and TGF-ß-dependent signaling pathways. Inhibition and knockdown of Smad3, knockdown of ß-catenin and overexpression of ICAT abrogated effects of TGF-ß1 on α-SMA transcription/expression, indicating a requirement for ß-catenin in these Smad3-dependent effects. Following TGF-ß treatment, co-immunoprecipitation demonstrated direct interaction between endogenous Smad3 and ß-catenin, while chromatin immunoprecipitation (ChIP)-re-ChIP identified spatial and temporal regulation of α-SMA via complex formation among Smad3, ß-catenin, and CBP. ICG-001 inhibited α-SMA expression/transcription in response to TGF-ß as well as α-SMA promoter occupancy by ß-catenin and CBP, demonstrating a previously unknown requisite TGF-ß1/ß-catenin/CBP-mediated pro-EMT signaling pathway. Clinical relevance was shown by ß-catenin/Smad3 co-localization and CBP expression in AEC of IPF patients. These findings suggest a new therapeutic approach to pulmonary fibrosis by specifically uncoupling CBP/catenin-dependent signaling downstream of TGF-ß.

Retention of human bone marrow-derived cells in murine lungs following bleomycin-induced lung injury.

We studied the capacity of adult human bone marrow-derived cells (BMDC) to incorporate into distal lung of immunodeficient mice following lung injury. Immunodeficient NOD/SCID and NOD/SCID/beta(2) microglobulin (beta(2)M)(null) mice were administered bleomycin (bleo) or saline intranasally. One, 2, 3 and 4 days after bleo or saline, human BMDC labeled with CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) were infused intravenously. Retention of CMFDA(+) cells was maximal when delivered 4 days after bleo treatment. Seven days after bleo, <0.005% of enzymatically dispersed lung cells from NOD/SCID mice were CMFDA(+), which increased 10- to 100-fold in NOD/SCID/beta(2)M(null) mice. Preincubation of BMDC with Diprotin A, a reversible inhibitor of CD26 peptidase activity that enhances the stromal-derived factor-1 (SDF-1/CXCL12)/CXCR4 axis, resulted in a 30% increase in the percentage of CMFDA(+) cells retained in the lung. These data indicate that human BMDC can be identified in lungs of mice following injury, albeit at low levels, and this may be modestly enhanced by manipulation of the SDF-1/CXCR4 axis. Given the overall low number of human cells detected, methods to increase homing and retention of adult BMDC, and consideration of other stem cell populations, will likely be required to facilitate engraftment in the treatment of lung injury.

Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-beta1: potential role in idiopathic pulmonary fibrosis.

The hallmark of idiopathic pulmonary fibrosis (IPF) is the myofibroblast, the cellular origin of which in the lung is unknown. We hypothesized that alveolar epithelial cells (AECs) may serve as a source of myofibroblasts through epithelial-mesenchymal transition (EMT). Effects of chronic exposure to transforming growth factor (TGF)-beta1 on the phenotype of isolated rat AECs in primary culture and a rat type II cell line (RLE-6TN) were evaluated. Additionally, tissue samples from patients with IPF were evaluated for cells co-expressing epithelial (thyroid transcription factor (TTF)-1 and pro-surfactant protein-B (pro-SP-B), and mesenchymal (alpha-smooth muscle actin (alpha-SMA)) markers. RLE-6TN cells exposed to TGF-beta1 for 6 days demonstrated increased expression of mesenchymal cell markers and a fibroblast-like morphology, an effect augmented by tumor necrosis factor-alpha (TNF-alpha). Exposure of rat AECs to TGF-beta1 (100 pmol/L) resulted in increased expression of alpha-SMA, type I collagen, vimentin, and desmin, with concurrent transition to a fibroblast-like morphology and decreased expression of TTF-1, aquaporin-5 (AQP5), zonula occludens-1 (ZO-1), and cytokeratins. Cells co-expressing epithelial markers and alpha-SMA were abundant in lung tissue from IPF patients. These results suggest that AECs undergo EMT when chronically exposed to TGF-beta1, raising the possibility that epithelial cells may serve as a novel source of myofibroblasts in IPF.

Effects of transdifferentiation and EGF on claudin isoform expression in alveolar epithelial cells.

Rat alveolar epithelial type II cells grown on polycarbonate filters form high-resistance monolayers and concurrently acquire many phenotypic properties of type I cells. Treatment with EGF has previously been shown to increase transepithelial resistance across alveolar epithelial cell (AEC) monolayers. We investigated changes in claudin expression in primary cultured AEC during transdifferentiation to the type I cell-like phenotype (days 0, 1, and 8), and on day 5 in culture +/- EGF (10 ng/ml) from day 0 or day 4. Claudins 4 and 7 were increased, whereas claudins 3 and 5 were decreased, on later compared with earlier days in culture. Exposure to EGF led to increases in claudins 4 and 7 and decreases in claudins 3 and 5. Claudin 1 was only faintly detectable in freshly isolated type II cells and remained unchanged over time in culture and after exposure to EGF. These results suggest that increases in transepithelial resistance accompanying AEC transdifferentiation and/or EGF exposure are mediated, at least in part, by changes in the pattern of expression of specific claudin isoforms.

Subunit-specific coordinate upregulation of sodium pump activity in alveolar epithelial cells by lentivirus-mediated gene transfer.

Resolution of alveolar edema depends on active ion transport by sodium pumps located on the basolateral surface of alveolar epithelial cells (AECs), suggesting that upregulation of sodium pump activity may facilitate clearance of edema fluid. We have investigated the use of lentiviral vectors to augment sodium pump activity via gene transfer of sodium pump subunits to AECs. Full-length cDNA for the alpha(1) or beta(1) subunit of rat Na(+),K(+)-ATPase was cloned into the lentiviral vector pRRLsin.hCMV.IRES.EGFP. Rat AECs in primary culture were transduced on day 4 with lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein G. Transduction with lentiviral vectors encoding either alpha(1) subunit (Lenti-alpha(1)-EGFP) or beta(1) subunit (Lenti-beta(1)-EGFP) led to dose-dependent increases in mRNA and protein for the corresponding subunit. Transduction with Lenti-beta(1)-EGFP was accompanied by coordinate upregulation of endogenous alpha(1) expression, whereas endogenous beta(1) expression was unchanged after transduction with Lenti-alpha(1)-EGFP. Consistent with these findings, transduction with Lenti-beta(1)-EGFP, but not Lenti-alpha(1)-EGFP, led to augmentation of sodium pump activity as a result of increases in Na(+),K(+)-ATPase holoenzyme. Sodium pump alpha(2) subunit and sodium channel protein did not change after Lenti-beta(1)-EGFP transduction. These results demonstrate that overall sodium pump activity can be efficiently upregulated in AECs specifically via gene transfer of the sodium pump beta(1) subunit and support the feasibility of lentivirus-mediated gene transfer to augment alveolar fluid clearance.

Alveolar epithelial type I cells express beta2-adrenergic receptors and G-protein receptor kinase 2.

Beta2-Adrenergic agonists stimulate alveolar epithelial sodium (Na(+)) transport and lung fluid clearance. Alveolar type II (AT2) cells have been reported to express beta2-adrenergic receptors (beta2AR). Given the large surface area covered by alveolar type I (AT1) cells and their potential role in alveolar fluid removal, we were interested in learning if AT1 cells express beta2AR as well. Because beta2AR is potentially susceptible to desensitization by G-protein-coupled receptor kinase 2 (GRK2), we also undertook localization of GRK2. beta2AR and GRK2 expression was evaluated in whole lung, isolated alveolar epithelial cells (AECs), and AECs in primary culture, and was localized to specific AEC phenotypes by immunofluorescence techniques. beta2AR is highly expressed in AT1 cells. beta2AR mRNA increases with time in culture as AT2 cells transdifferentiate towards the AT1 cell phenotype. Immunoreactive GRK2 is seen in both AT1 and AT2 cells in similar amounts. These data suggest that both AT1 and AT2 cells may contribute to the increased alveolar Na(+) and water clearance observed after exposure to beta2 adrenergic agents. Both cell types also express GRK2, suggesting that both may undergo desensitization of beta2AR with subsequent decline in the stimulatory effects of beta2-adrenergic agonists over time.

Foxj1 is required for apical localization of ezrin in airway epithelial cells.

Establishment and maintenance of epithelial cell polarity depend on cytoskeletal organization and protein trafficking to polarized cortical membranes. ERM (ezrin, radixin, moesin) family members link polarized proteins with cytoskeletal actin. Although ERMs are often considered to be functionally similar, we found that, in airway epithelial cells, apical localization of ERMs depend on cell differentiation and is independently regulated. Moesin was present in the apical membrane of all undifferentiated epithelial cells. However, in differentiated cells, ezrin and moesin were selectively localized to apical membranes of ciliated airway cells and were absent from secretory cells. To identify regulatory proteins required for selective ERM trafficking, we evaluated airway epithelial cells lacking Foxj1, an F-box factor that directs programs required for cilia formation at the apical membrane. Interestingly, Foxj1 expression was also required for localization of apical ezrin, but not moesin. Additionally, membrane-cytoskeletal and threonine-phosphorylated ezrin were decreased in Foxj1-null cells, consistent with absent apical ezrin. Although apical moesin expression was present in null cells, it could not compensate for ezrin because ERM-associated EBP50 and the beta2 adrenergic receptor failed to localize apically in the absence of Foxj1. These findings indicate that Foxj1 regulates ERM proteins differentially to selectively direct the apical localization of ezrin for the organization of multi-protein complexes in apical membranes of airway epithelial cells.

Identification of three genes of known function expressed by alveolar epithelial type I cells.

To identify genes of known function expressed by type I (AT1) cells, changes in gene expression during transdifferentiation of alveolar epithelial cells (AEC) in primary culture from type II (AT2) to type I-like cell phenotype were evaluated. Total RNA from AEC on Day 0 or Day 8 was hybridized to a rat microarray for screening. Eight upregulated genes on Day 8 were selected for further investigation. Northern analysis confirmed upregulation of three of these genes, PAI-1, P2X4, and P15INK4B. The corresponding proteins were evaluated in cultured AEC and results correlated with expression in AT1 cells. In AEC monolayers, all three proteins increased between Day 1 and Day 8. In mixed populations of freshly isolated rat lung cells, concurrent labeling with the AT1 cell-specific antibody, VIIIB2, localized these proteins to AT1 cells. In whole lung, all three proteins were detected in alveolar epithelium in a location consistent with expression in AT1 cells. Identification of novel AT1 cell genes of known function suggests an active role for AT1 cells in alveolar homeostasis. Furthermore, expression of these gene products in AT1-like cells, in freshly isolated AT1 cells, and AT1 cells in whole lung indicates that AT1-like cells reflect many of the properties of AT1 cells in situ.

Na transport proteins are expressed by rat alveolar epithelial type I cells.

Despite a presumptive role for type I (AT1) cells in alveolar epithelial transport, specific Na transporters have not previously been localized to these cells. To evaluate expression of Na transporters in AT1 cells, double labeling immunofluorescence microscopy was utilized in whole lung and in cytocentrifuged preparations of partially purified alveolar epithelial cells (AEC). Expression of Na pump subunit isoforms and the alpha-subunit of the rat (r) epithelial Na channel (alpha-ENaC) was evaluated in isolated AT1 cells identified by their immunoreactivity with AT1 cell-specific antibody markers (VIIIB2 and/or anti-aquaporin-5) and lack of reactivity with antibodies specific for AT2 cells (anti-surfactant protein A) or leukocytes (anti-leukocyte common antigen). Expression of the Na pump alpha(1)-subunit in AEC was assessed in situ. Na pump subunit isoform and alpha-rENaC expression was also evaluated by RT-PCR in highly purified (approximately 95%) AT1 cell preparations. Labeling of isolated AT1 cells with anti-alpha(1) and anti-beta(1) Na pump subunit and anti-alpha-rENaC antibodies was detected, while reactivity with anti-alpha(2) Na pump subunit antibody was absent. AT1 cells in situ were reactive with anti-alpha(1) Na pump subunit antibody. Na pump alpha(1)- and beta(1)- (but not alpha(2)-) subunits and alpha-rENaC were detected in highly purified AT1 cells by RT-PCR. These data demonstrate that AT1 cells express Na pump and Na channel proteins, supporting a role for AT1 cells in active transalveolar epithelial Na transport.