Linkage of NAMPT promoter variants to eNAMPT secretion, plasma eNAMPT levels, and ARDS severity.

BACKGROUND AND OBJECTIVES: eNAMPT (extracellular nicotinamide phosphoribosyltransferase), a novel DAMP and TLR4 ligand, is a druggable ARDS therapeutic target with NAMPT promoter SNPs associated with ARDS severity. This study assesses the previously unknown influence of NAMPT promoter SNPs on NAMPT transcription, eNAMPT secretion, and ARDS severity. METHODS AND DESIGN: Human lung endothelial cells (ECs) transfected with NAMPT promoter luciferase reporters harboring SNPs G-1535A, A-1001 C, and C-948A, were exposed to LPS or LPS/18% cyclic stretch (CS) and NAMPT promoter activity, NAMPT protein expression, and secretion assessed. NAMPT genotypes and eNAMPT plasma measurements (Days 0/7) were assessed in two ARDS cohorts (DISCOVERY n = 428; ALVEOLI n = 103). RESULTS: Comparisons of minor allelic frequency (MAF) in both ARDS cohorts with the 1000 Human Genome Project revealed the G-1535A and C-948A SNPs to be significantly associated with ARDS in Blacks compared with controls and trended toward significance in non-Hispanic Whites. LPS-challenged and LPS/18% CS-challenged EC harboring the -1535G wild-type allele exhibited significantly increased NAMPT promoter activity (compared with -1535A) with the -1535G/-948A diplotype exhibiting significantly increased NAMPT promoter activity, NAMPT protein expression, and eNAMPT secretion compared with the -1535A/-948 C diplotype. Highly significant increases in Day 0 eNAMPT plasma values were observed in both DISCOVERY and ALVEOLI ARDS cohorts (compared with healthy controls). Among subjects surviving to Day 7, Day 7 eNAMPT values were significantly increased in Day 28 non-survivors versus survivors. The protective -1535A SNP allele drove -1535A/-1001A and -1535A/-948 C diplotypes that confer significantly reduced ARDS risk (compared with -1535G, -1535G/-1001 C, -1535G/-948A), particularly in Black ARDS subjects. NAMPT SNP comparisons within the two ARDS cohorts did not identify significant association with either APACHE III scores or plasma eNAMPT levels. CONCLUSION: NAMPT SNPs influence promoter activity, eNAMPT protein expression/secretion, plasma eNAMPT levels, and ARDS severity. NAMPT genotypes are a potential tool for stratification in eNAMPT-focused ARDS clinical trials.

Multiple Fibroblast Subtypes Contribute to Matrix Deposition in Pulmonary Fibrosis.

Progressive pulmonary fibrosis results from a dysfunctional tissue repair response and is characterized by fibroblast proliferation, activation, and invasion and extracellular matrix accumulation. Lung fibroblast heterogeneity is well recognized. With single-cell RNA sequencing, fibroblast subtypes have been reported by recent studies. However, the roles of fibroblast subtypes in effector functions in lung fibrosis are not well understood. In this study, we incorporated the recently published single-cell RNA-sequencing datasets on murine lung samples of fibrosis models and human lung samples of fibrotic diseases and analyzed fibroblast gene signatures. We identified and confirmed the novel fibroblast subtypes we reported recently across all samples of both mouse models and human lung fibrotic diseases, including idiopathic pulmonary fibrosis, systemic sclerosis-associated interstitial lung disease, and coronavirus disease (COVID-19). Furthermore, we identified specific cell surface proteins for each fibroblast subtype through differential gene expression analysis, which enabled us to isolate primary cells representing distinct fibroblast subtypes by flow cytometry sorting. We compared matrix production, including fibronectin, collagen, and hyaluronan, after profibrotic factor stimulation and assessed the invasive capacity of each fibroblast subtype. Our results suggest that in addition to myofibroblasts, lipofibroblasts and Ebf1+ (Ebf transcription factor 1+) fibroblasts are two important fibroblast subtypes that contribute to matrix deposition and also have enhanced invasive, proliferative, and contraction phenotypes. The histological locations of fibroblast subtypes are identified in healthy and fibrotic lungs by these cell surface proteins. This study provides new insights to inform approaches to targeting lung fibroblast subtypes to promote the development of therapeutics for lung fibrosis.

A cortactin CTTN coding SNP contributes to lung vascular permeability and inflammatory disease severity in African descent subjects.

The cortactin gene (CTTN), encoding an actin-binding protein critically involved in cytoskeletal dynamics and endothelial cell (EC) barrier integrity, contains single nucleotide polymorphisms (SNPs) associated with severe asthma in Black patients. As loss of lung EC integrity is a major driver of mortality in the Acute Respiratory Distress Syndrome (ARDS), sepsis, and the acute chest syndrome (ACS), we speculated CTTN SNPs that alter EC barrier function will associate with clinical outcomes from these types of conditions in Black patients. In case-control studies, evaluation of a nonsynonymous CTTN coding SNP Ser484Asn (rs56162978, G/A) in a severe sepsis cohort (725 Black subjects) revealed significant association with increased risk of sepsis mortality. In a separate cohort of sickle cell disease (SCD) subjects with and without ACS (177 SCD Black subjects), significantly increased risk of ACS and increased ACS severity (need for mechanical ventilation) was observed in carriers of the A allele. Human lung EC expressing the cortactin S484N transgene exhibited: (i) delayed EC barrier recovery following thrombin-induced permeability; (ii) reduced levels of critical Tyr486 cortactin phosphorylation; (iii) inhibited binding to the cytoskeletal regulator, nmMLCK; and (iv) attenuated EC barrier-promoting lamellipodia dynamics and biophysical responses. ARDS-challenged Cttn+/- heterozygous mice exhibited increased lung vascular permeability (compared to wild-type mice) which was significantly attenuated by IV delivery of liposomes encargoed with CTTN WT transgene but not by CTTN S484N transgene. In summary, these studies suggest that the CTTN S484N coding SNP contributes to severity of inflammatory injury in Black patients, potentially via delayed vascular barrier restoration.

Abnormal respiratory progenitors in fibrotic lung injury.

Recent advances in single-cell RNA sequencing (scRNA-seq) and epithelium lineage labeling have yielded identification of multiple abnormal epithelial progenitor populations during alveolar type 2 (ATII) cell differentiation into alveolar type 1 (ATI) cells during regenerative lung post-fibrotic injury. These abnormal cells include basaloid/basal-like cells, ATII transition cells, and persistent epithelial progenitors (PEPs). These cells occurred and accumulated during the regeneration of distal airway and alveoli in response to both chronic and acute pulmonary injury. Among the alveolar epithelial progenitors, PEPs express a distinct Krt8+ phenotype that is rarely found in intact alveoli. However, post-injury, the Krt8+ phenotype is seen in dysplastic epithelial cells. Fully understanding the characteristics and functions of these newly found, injury-induced abnormal behavioral epithelial progenitors and the signaling pathways regulating their phenotype could potentially point the way to unique therapeutic targets for fibrosing lung diseases. This review summarizes recent advances in understanding these epithelial progenitors as they relate to uncovering regenerative mechanisms.

Transcriptomics of bronchoalveolar lavage cells identifies new molecular endotypes of sarcoidosis.

BACKGROUND: Sarcoidosis is a multisystem granulomatous disease of unknown origin with a variable and often unpredictable course and pattern of organ involvement. In this study we sought to identify specific bronchoalveolar lavage (BAL) cell gene expression patterns indicative of distinct disease phenotypic traits. METHODS: RNA sequencing by Ion Torrent Proton was performed on BAL cells obtained from 215 well-characterised patients with pulmonary sarcoidosis enrolled in the multicentre Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study. Weighted gene co-expression network analysis and nonparametric statistics were used to analyse genome-wide BAL transcriptome. Validation of results was performed using a microarray expression dataset of an independent sarcoidosis cohort (Freiburg, Germany; n=50). RESULTS: Our supervised analysis found associations between distinct transcriptional programmes and major pulmonary phenotypic manifestations of sarcoidosis including T-helper type 1 (Th1) and Th17 pathways associated with hilar lymphadenopathy, transforming growth factor-ß1 (TGFB1) and mechanistic target of rapamycin (MTOR) signalling with parenchymal involvement, and interleukin (IL)-7 and IL-2 with airway involvement. Our unsupervised analysis revealed gene modules that uncovered four potential sarcoidosis endotypes including hilar lymphadenopathy with increased acute T-cell immune response; extraocular organ involvement with PI3K activation pathways; chronic and multiorgan disease with increased immune response pathways; and multiorgan involvement, with increased IL-1 and IL-18 immune and inflammatory responses. We validated the occurrence of these endotypes using gene expression, pulmonary function tests and cell differentials from Freiburg. CONCLUSION: Taken together, our results identify BAL gene expression programmes that characterise major pulmonary sarcoidosis phenotypes and suggest the presence of distinct disease molecular endotypes.

The acute respiratory distress syndrome biomarker pipeline: crippling gaps between discovery and clinical utility.

Recent innovations in translational research have ushered an exponential increase in the discovery of novel biomarkers, thereby elevating the hope for deeper insights into "personalized" medicine approaches to disease phenotyping and care. However, a critical gap exists between the fast pace of biomarker discovery and the successful translation to clinical use. This gap underscores the fundamental biomarker conundrum across various acute and chronic disorders: how does a biomarker address a specific unmet need? Additionally, the gap highlights the need to shift the paradigm from a focus on biomarker discovery to greater translational impact and the need for a more streamlined drug approval process. The unmet need for biomarkers in acute respiratory distress syndrome (ARDS) is for reliable and validated biomarkers that minimize heterogeneity and allow for stratification of subject selection for enrollment in clinical trials of tailored therapies. This unmet need is particularly highlighted by the ongoing SARS-CoV-2/COVID-19 pandemic. The unprecedented numbers of COVID-19-induced ARDS cases has strained health care systems across the world and exposed the need for biomarkers that would accelerate drug development and the successful phenotyping of COVID-19-infected patients at risk for development of ARDS and ARDS mortality. Accordingly, this review discusses the current state of ARDS biomarkers in the context of the drug development pipeline and highlight gaps between biomarker discovery and clinical implementation while proposing potential paths forward. We discuss potential ARDS biomarkers by category and by context of use, highlighting progress in the development continuum. We conclude by discussing challenges to successful translation of biomarker candidates to clinical impact and proposing possible novel strategies.

Direct Extracellular NAMPT Involvement in Pulmonary Hypertension and Vascular Remodeling. Transcriptional Regulation by SOX and HIF-2α.

We previously demonstrated involvement of NAMPT (nicotinamide phosphoribosyltransferase) in pulmonary arterial hypertension (PAH) and now examine NAMPT regulation and extracellular NAMPT's (eNAMPT's) role in PAH vascular remodeling. NAMPT transcription and protein expression in human lung endothelial cells were assessed in response to PAH-relevant stimuli (PDGF [platelet-derived growth factor], VEGF [vascular endothelial growth factor], TGF-ß1 [transforming growth factor-ß1], and hypoxia). Endothelial-to-mesenchymal transition was detected by SNAI1 (snail family transcriptional repressor 1) and PECAM1 (platelet endothelial cell adhesion molecule 1) immunofluorescence. An eNAMPT-neutralizing polyclonal antibody was tested in a PAH model of monocrotaline challenge in rats. Plasma eNAMPT concentrations, significantly increased in patients with idiopathic pulmonary arterial hypertension, were highly correlated with indices of PAH severity. eNAMPT increased endothelial-to-mesenchymal transition, and each PAH stimulus significantly increased endothelial cell NAMPT promoter activity involving transcription factors STAT5 (signal transducer and activator of transcription 5), SOX18 (SRY-box transcription factor 18), and SOX17 (SRY-box transcription factor 17), a PAH candidate gene newly defined by genome-wide association study. The hypoxia-induced transcription factor HIF-2α (hypoxia-inducible factor-2α) also potently regulated NAMPT promoter activity, and HIF-2α binding sites were identified between -628 bp and -328 bp. The PHD2 (prolyl hydroxylase domain-containing protein 2) inhibitor FG-4592 significantly increased NAMPT promoter activity and protein expression in an HIF-2α-dependent manner. Finally, the eNAMPT-neutralizing polyclonal antibody significantly reduced monocrotaline-induced vascular remodeling, PAH hemodynamic alterations, and NF-κB activation. eNAMPT is a novel and attractive therapeutic target essential to PAH vascular remodeling.

Development of a biomarker mortality risk model in acute respiratory distress syndrome.

BACKGROUND: There is a compelling unmet medical need for biomarker-based models to risk-stratify patients with acute respiratory distress syndrome. Effective stratification would optimize participant selection for clinical trial enrollment by focusing on those most likely to benefit from new interventions. Our objective was to develop a prognostic, biomarker-based model for predicting mortality in adult patients with acute respiratory distress syndrome. METHODS: This is a secondary analysis using a cohort of 252 mechanically ventilated subjects with the diagnosis of acute respiratory distress syndrome. Survival to day 7 with both day 0 (first day of presentation) and day 7 sample availability was required. Blood was collected for biomarker measurements at first presentation to the intensive care unit and on the seventh day. Biomarkers included cytokine-chemokines, dual-functioning cytozymes, and vascular injury markers. Logistic regression, latent class analysis, and classification and regression tree analysis were used to identify the plasma biomarkers most predictive of 28-day ARDS mortality. RESULTS: From eight biologically relevant biomarker candidates, six demonstrated an enhanced capacity to predict mortality at day 0. Latent-class analysis identified two biomarker-based phenotypes. Phenotype A exhibited significantly higher plasma levels of angiopoietin-2, macrophage migration inhibitory factor, interleukin-8, interleukin-1 receptor antagonist, interleukin-6, and extracellular nicotinamide phosphoribosyltransferase (eNAMPT) compared to phenotype B. Mortality at 28 days was significantly higher for phenotype A compared to phenotype B (32% vs 19%, p = 0.04). CONCLUSIONS: An adult biomarker-based risk model reliably identifies ARDS subjects at risk of death within 28 days of hospitalization.

Genomic and Genetic Approaches to Deciphering Acute Respiratory Distress Syndrome Risk and Mortality.

Significance: Acute respiratory distress syndrome (ARDS) is a severe, highly heterogeneous critical illness with staggering mortality that is influenced by environmental factors, such as mechanical ventilation, and genetic factors. Significant unmet needs in ARDS are addressing the paucity of validated predictive biomarkers for ARDS risk and susceptibility that hamper the conduct of successful clinical trials in ARDS and the complete absence of novel disease-modifying therapeutic strategies. Recent Advances: The current ARDS definition relies on clinical characteristics that fail to capture the diversity of disease pathology, severity, and mortality risk. We undertook a comprehensive survey of the available ARDS literature to identify genes and genetic variants (candidate gene and limited genome-wide association study approaches) implicated in susceptibility to developing ARDS in hopes of uncovering novel biomarkers for ARDS risk and mortality and potentially novel therapeutic targets in ARDS. We further attempted to address the well-known health disparities that exist in susceptibility to and mortality from ARDS. Critical Issues: Bioinformatic analyses identified 201 ARDS candidate genes with pathway analysis indicating a strong predominance in key evolutionarily conserved inflammatory pathways, including reactive oxygen species, innate immunity-related inflammation, and endothelial vascular signaling pathways. Future Directions: Future studies employing a system biology approach that combines clinical characteristics, genomics, transcriptomics, and proteomics may allow for a better definition of biologically relevant pathways and genotype-phenotype connections and result in improved strategies for the sub-phenotyping of diverse ARDS patients via molecular signatures. These efforts should facilitate the potential for successful clinical trials in ARDS and yield a better fundamental understanding of ARDS pathobiology.

The enactment stage of end-of-life decision-making for children.

OBJECTIVES: Typically pediatric end-of-life decision-making studies have examined the decision-making process, factors, and doctors' and parents' roles. Less attention has focussed on what happens after an end-of-life decision is made; that is, decision enactment and its outcome. This study explored the views and experiences of bereaved parents in end-of-life decision-making for their child. Findings reported relate to parents' experiences of acting on their decision. It is argued that this is one significant stage of the decision-making process. METHODS: A qualitative methodology was used. Semi-structured interviews were conducted with bereaved parents, who had discussed end-of-life decisions for their child who had a life-limiting condition and who had died. Data were thematically analysed. RESULTS: Twenty-five bereaved parents participated. Findings indicate that, despite differences in context, including the child's condition and age, end-of-life decision-making did not end when an end-of-life decision was made. Enacting the decision was the next stage in a process. Time intervals between stages and enactment pathways varied, but the enactment was always distinguishable as a separate stage. Decision enactment involved making further decisions - parents needed to discern the appropriate time to implement their decision to withdraw or withhold life-sustaining medical treatment. Unexpected events, including other people's actions, impacted on parents enacting their decision in the way they had planned. Several parents had to re-implement decisions when their child recovered from serious health issues without medical intervention. SIGNIFICANCE OF RESULTS: A novel, critical finding was that parents experienced end-of-life decision-making as a sequence of interconnected stages, the final stage being enactment. The enactment stage involved further decision-making. End-of-life decision-making is better understood as a process rather than a discrete once-off event. The enactment stage has particular emotional and practical implications for parents. Greater understanding of this stage can improve clinician's support for parents as they care for their child.

BAL Cell Gene Expression Is Indicative of Outcome and Airway Basal Cell Involvement in Idiopathic Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a fatal disease with a variable and unpredictable course. OBJECTIVES: To determine whether BAL cell gene expression is predictive of survival in IPF. METHODS: This retrospective study analyzed the BAL transcriptome of three independent IPF cohorts: Freiburg (Germany), Siena (Italy), and Leuven (Belgium) including 212 patients. BAL cells from 20 healthy volunteers, 26 patients with sarcoidosis stage III and IV, and 29 patients with chronic obstructive pulmonary disease were used as control subjects. Survival analysis was performed by Cox models and component-wise boosting. Presence of airway basal cells was tested by immunohistochemistry and flow cytometry. MEASUREMENTS AND MAIN RESULTS: A total of 1,582 genes were predictive of mortality in the IPF derivation cohort in univariate analyses adjusted for age and sex at false discovery rate less than 0.05. A nine-gene signature, derived from the discovery cohort (Freiburg), performed well in both replication cohorts, Siena (P < 0.0032) and Leuven (P = 0.0033). nCounter expression analysis confirmed the array results (P < 0.0001). The genes associated with mortality in BAL cells were significantly enriched for genes expressed in airway basal cells. Further analyses by gene expression, flow cytometry, and immunohistochemistry showed an increase in airway basal cells in BAL and tissues of IPF compared with control subjects, but not in chronic obstructive pulmonary disease or sarcoidosis. CONCLUSIONS: Our results identify and validate a BAL signature that predicts mortality in IPF and improves the accuracy of outcome prediction based on clinical parameters. The BAL signature associated with mortality unmasks a potential role for airway basal cells in IPF.

Single nucleotide polymorphisms in the MYLKP1 pseudogene are associated with increased colon cancer risk in African Americans.

INTRODUCTION: Pseudogenes are paralogues of functional genes historically viewed as defunct due to either the lack of regulatory elements or the presence of frameshift mutations. Recent evidence, however, suggests that pseudogenes may regulate gene expression, although the functional role of pseudogenes remains largely unknown. We previously reported that MYLKP1, the pseudogene of MYLK that encodes myosin light chain kinase (MLCK), is highly expressed in lung and colon cancer cell lines and tissues but not in normal lung or colon. The MYLKP1 promoter is minimally active in normal bronchial epithelial cells but highly active in lung adenocarcinoma cells. In this study, we further validate MYLKP1 as an oncogene via elucidation of the functional role of MYLKP1 genetic variants in colon cancer risk. METHODS: Proliferation and migration assays were performed in MYLKP1-transfected colon and lung cancer cell lines (H441, A549) and commercially-available normal lung and colon cells. Fourteen MYLKP1 SNPs (MAFs >0.01) residing within the 4 kb MYLKP1 promoter region, the core 1.4 kb of MYLKP1 gene, and a 4 kb enhancer region were selected and genotyped in a colorectal cancer cohort. MYLKP1 SNP influences on activity of MYLKP1 promoter (2kb) was assessed by dual luciferase reporter assay. RESULTS: Cancer cell lines, H441 and A549, exhibited increased MYLKP1 expression, increased MYLKP1 luciferase promoter activity, increased proliferation and migration. Genotyping studies identified two MYLKP1 SNPs (rs12490683; rs12497343) that significantly increase risk of colon cancer in African Americans compared to African American controls. Rs12490683 and rs12497343 further increase MYLKP1 promoter activity compared to the wild type MYLKP1 promoter. CONCLUSION: MYLKP1 is a cancer-promoting pseudogene whose genetic variants differentially enhance cancer risk in African American populations.

Validation of a 52-gene risk profile for outcome prediction in patients with idiopathic pulmonary fibrosis: an international, multicentre, cohort study.

BACKGROUND: The clinical course of idiopathic pulmonary fibrosis (IPF) is unpredictable. Clinical prediction tools are not accurate enough to predict disease outcomes. METHODS: We enrolled patients with IPF diagnosis in a six-cohort study at Yale University (New Haven, CT, USA), Imperial College London (London, UK), University of Chicago (Chicago, IL, USA), University of Pittsburgh (Pittsburgh, PA, USA), University of Freiburg (Freiburg im Breisgau, Germany), and Brigham and Women's Hospital-Harvard Medical School (Boston, MA, USA). Peripheral blood mononuclear cells or whole blood were collected at baseline from 425 participants and from 98 patients (23%) during 4-6 years' follow-up. A 52-gene signature was measured by the nCounter analysis system in four cohorts and extracted from microarray data (GeneChip) in the other two. We used the Scoring Algorithm for Molecular Subphenotypes (SAMS) to classify patients into low-risk or high-risk groups based on the 52-gene signature. We studied mortality with a competing risk model and transplant-free survival with a Cox proportional hazards model. We analysed timecourse data and response to antifibrotic drugs with linear mixed effect models. FINDINGS: The application of SAMS to the 52-gene signature identified two groups of patients with IPF (low-risk and high-risk), with significant differences in mortality or transplant-free survival in each of the six cohorts (hazard ratio [HR] range 2·03-4·37). Pooled data showed similar results for mortality (HR 2·18, 95% CI 1·53-3·09; p<0·0001) or transplant-free survival (2·04, 1·52-2·74; p<0·0001). Adding 52-gene risk profiles to the Gender, Age, and Physiology index significantly improved its mortality predictive accuracy. Temporal changes in SAMS scores were associated with changes in forced vital capacity (FVC) in two cohorts. Untreated patients did not shift their risk profile over time. A simultaneous increase in up score and decrease in down score was predictive of decreased transplant-free survival (3·18, 1·16-8·76; p=0·025) in the Pittsburgh cohort. A simultaneous decrease in up score and increase in down score after initiation of antifibrotic drugs was associated with a significant (p=0·0050) improvement in FVC in the Yale cohort. INTERPRETATION: The peripheral blood 52-gene expression signature is predictive of outcome in patients with IPF. The potential value of the 52-gene signature in predicting response to therapy should be determined in prospective studies. FUNDING: The Pulmonary Fibrosis Foundation, the Harold Amos Medical Faculty Development Program of the Robert Wood Johnson Foundation, and the National Heart, Lung, and Blood Institute of the US National Institutes of Health.