American Journal of Respiratory and Critical Care Medicine

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic interstitial lung disease associated with failed alveolar epithelial repair with an expansion of aberrant airway-like epithelium in the alveolar space leading to lung function decline usually resulting in death within 3-5 years of diagnosis. While single-cell and spatial transcriptomic approaches have been used to characterize disease-emergent cell populations, less is known about the regulation of transcriptional programs that drive failed alveolar epithelial cell repair in IPF. DNA methylation is a fundamental layer of gene regulation that stabilizes differentiated cell identity; however, changes in methylation in the epithelial compartment in IPF have not been studied. To identify novel epigenetic mediators of epithelial cell dysfunction, we performed high-resolution DNA methylation profiling of purified distal lung epithelial cells from 10 age-matched control and 14 IPF lungs using Oxford Nanopore Technologies (ONT) whole-genome, long-read sequencing. We identified widespread methylome remodeling in the IPF lung epithelium, with 84% of differentially methylated regions (DMRs) hypomethylated. DMRs were largely found outside of promoters, with 88% outside of 3 kb from the transcription start site (TSS), consistent with altered distal regulatory element activity. DMRs were enriched for transcription factor (TF) binding site motifs and gene associations consistent with dysregulation of polycomb repressive complex 2 (PRC2) and increases in p63 activity. DNA methylation at DMRs associated with p63 target genes, including KRT5, and genes implicated in failed epithelial repair, including MUC5B, MMP7, and S100A2 inversely correlated with gene expression. Consistent with the observed dysregulation of the IPF epithelial methylome, an experimental co-culture model of alveolar type II epithelial cell (AT2s) to basal-like epithelial cell transdifferentiation revealed widespread hypomethylation. Further, sites associated with NKX2.1 and FOXA1/2 binding, TFs involved in alveolar fate maintenance, were hypermethylated, suggesting loss of epigenetic regulation of alveolar identity. Together, these data implicate DNA methylation in the failed alveolar epithelial repair processes in IPF, potentially providing future therapeutic strategies by identifying putative regulatory elements associated with aberrant transcriptional programs.

Rationale: Conventional CT-derived pulmonary vascular metrics quantify vascular pruning but do not assess the spatial heterogeneity of vascular architecture. In COPD, peripheral microvascular pruning coexists with compensatory central vessel dilation, paradoxically preserving total vessel volume while reducing effective vascular penetration into lung parenchyma. Objectives: To develop and evaluate Vascular Proximity Coverage (VPC), a distance-based CT biomarker quantifying the spatial efficiency of pulmonary vascular distribution. Methods: This retrospective analysis included 7,881 COPDGene participants [PHASE] with complete vascular segmentations. VPC was estimated by integrating the area under the cumulative lung tissue coverage curve at distances of 0-10 mm from segmented vessel voxels. Multivariable models assessed associations with lung function, six-minute walk distance (6MWD), St. George's Respiratory Questionnaire (SGRQ) score, mMRC dyspnea score, and all-cause mortality over a median follow-up of [10.8] years, adjusting for demographics, smoking, emphysema, scanner type, and BV5. The cohort was stratified at median BV5 and VPC into concordant (high/high, low/low) and discordant (high-BV5/low-VPC, low-BV5/high-VPC) vascular phenotypes. Results: VPC declined across disease severity (5.82 {+/-} 0.50 [UNITS] in non-smokers to 5.21 {+/-} 0.64 in GOLD 4; p < 0.001). Among spirometrically normal smokers, current smokers had lower VPC than former smokers (5.31 vs. 5.68; p < 0.001). Each standard deviation decrease in VPC was independently associated with reduced 6MWD ({beta} = -16.8 m, 95% CI: -19.5 to -14.0), worse SGRQ ({beta} = 1.9, 95% CI: 1.4 to 2.4), and greater dyspnea (RR = 1.05, 95% CI: 1.03 to 1.08; all p < 0.001). The lowest VPC quartile was associated with 38% higher mortality than the reference quartile (HR = 1.38, 95% CI: 1.18 to 1.62; p < 0.001). The high-BV5/low-VPC phenotype showed 24% higher mortality than high-BV5/high-VPC (HR = 1.24, 95% CI: 1.08 to 1.42; p = 0.002) despite preserved vessel volume. Conclusions: VPC captures spatial heterogeneity of vascular architecture independent of vessel volume, is reduced in smokers without airflow obstruction, and identifies excess mortality risk among individuals whose total vessel volume appears preserved.

Pulmonary hypertension (PH) is a progressive and fatal disease characterized by pulmonary vascular remodeling, inflammation, and immune dysregulation. Myeloid-derived suppressor cells (MDSCs) and macrophages contribute to PAH pathobiology; however, the molecular regulators of their pathological activation remain poorly defined. The triggering receptor expressed on myeloid cells 2 (TREM2) is an immunomodulatory receptor that shapes myeloid cell metabolism, survival, and immunosuppressive function, yet its role in PH has not been investigated. Methods: Single-cell RNA sequencing (scRNA-seq) data from human pulmonary artery tissue (GSE210248; n=3 PAH, n=3 donors) were analyzed to characterize TREM2 expression across cell populations. Wild-type (WT) and global TREM2 knockout (TREM2 KO) mice were exposed to chronic hypoxia (10% FiO2, 28 days). Hemodynamic, histological, flow cytometric, and ex vivo functional assessments were performed. TREM2 expression was measured by flow cytometry in circulating MDSCs from PAH patients (n=22) and healthy controls (n=13). Results: TREM2 was markedly enriched in monocyte/macrophage populations in PH pulmonary arteries and TREM2-high immune cells exhibited transcriptional downregulation of chemotaxis programs and upregulation of antigen processing and MHC II presentation pathways. TREM2 deficiency significantly attenuated hypoxia-induced RVSP increase, right ventricular dysfunction, pulmonary inflammation, and vascular remodeling. MDSCs from TREM2 KO mice exerted significantly less suppression of CD4+ and CD8+ T cell proliferation. TREM2 was significantly elevated in circulating MDSCs from PAH patients and showed a directional association with hemodynamic severity. Conclusions: TREM2 is a novel regulator of myeloid-driven immunosuppression and vascular remodeling in PAH and warrants investigation as a therapeutic target and biomarker.

Redox homeostasis requires both inducible antioxidant defense and constitutive machinery that continuously regenerates cellular reducing capacity. How these systems are altered across lung cell populations in chronic obstructive pulmonary disease (COPD) remains unclear. We integrated donor-level single-nucleus lung transcriptomes with smoke-exposure models, independent cohorts, and large-scale pharmacologic perturbation data. Both redox programs were reduced in established COPD, but constitutive redox support showed the greater loss, concentrated in fibroblasts and lymphatic endothelium. In fibroblasts, PGC-1 target-program output showed the most negative effect among 515 regulator programs despite preserved PPARGC1A expression. Smoke exposure activated inducible antioxidant defense across experimental models, whereas this adaptive program was reduced in established COPD. Pharmacologic perturbations further separated the two arms of redox homeostasis, with redox-directed interventions producing larger transcriptional responses in inducible defense and a distinct set of compounds increasing constitutive support together with PGC-1 target output and movement away from an independently defined COPD fibroblast state. These findings identify a cell-selective loss of constitutive redox support in COPD and implicate reduced PGC-1 target-program output in the fibroblast redox phenotype.

Pulmonary lymphatic vessels are essential for interstitial fluid balance and macromolecular clearance, yet the endothelial mechanisms regulating lymphatic vessel function during chronic fibroproliferative lung injury remain undefined. Here, we demonstrate that the lineage-defining transcription factor ERG in lymphatic endothelial cells (LECs) governs junctional plasticity and fluid drainage in pulmonary fibrosis. In human idiopathic pulmonary fibrosis (IPF) lungs, lymphatic ERG expression is markedly downregulated. Lineage-specific inducible deletion of Erg in murine LECs (Erg-CKO) unexpectedly conferred robust protection against bleomycin-induced pulmonary fibrosis, significantly dampening acute inflammation, reducing edema, and preserving pulmonary compliance. Mechanistically, loss of ERG enhanced lymphatic drainage capacity in vivo. Transcriptomic profiling of isolated LECs revealed that ERG deficiency activates actomyosin contractile pathways and selectively upregulates Endothelin Receptor Type B (Ednrb), driving junctional remodeling from continuous zippers into discontinuous button-like configurations that facilitate interstitial fluid entry. Pharmacological blockade of EDNRB with BQ-788 abolished junctional reorganization, eliminated the enhanced lymphatic drainage, and completely reversed the anti-fibrotic protection in Erg-CKO mice. Together, our findings identify an endothelial ERG-EDNRB regulatory axis that controls lymphatic junctional architecture and demonstrate that promoting lymphatic clearance via endothelial junctional remodeling represents a viable therapeutic strategy for fibrotic vascular remodeling.

Background: Chronic obstructive pulmonary disease (COPD) is characterized by airway remodeling, which may extend to the central airways and alter tracheal morphology. Whether tracheal shape relates to disease severity, quality of life, functional capacity, or survival is not established. Objective: To determine whether CT-derived tracheal lumen eccentricity is associated with cross-sectional and longitudinal clinical outcomes in COPD. Methods: We analyzed 8,383 participants from the multicenter COPDGene study. At each axial cross-section of the trachea, lumen eccentricity ({epsilon}) was computed from the major and minor axis lengths, and the participant-level value was taken as the arithmetic mean across all tracheal slices; {epsilon} = 0 denotes a circular lumen and values approaching 1 denote progressively elongated ellipses. Baseline outcomes were spirometry, 6-minute walk distance (6MWD), St. George's Respiratory Questionnaire (SGRQ) total score, and modified Medical Research Council (mMRC) dyspnea score. Longitudinal outcomes were annualized change in FEV1 and all-cause mortality. Multivariable linear and Cox proportional hazards models were adjusted for [age, sex, race, BMI, smoking status, pack-years, scanner model]. Results: Tracheal eccentricity rose monotonically with severity of airflow obstruction (0.60{+/-}0.07, 0.61{+/-}0.07, 0.62{+/-}0.07, 0.64{+/-}0.08, and 0.64{+/-}0.08 in GOLD stages 0 through 4, respectively). Higher eccentricity was independently associated with lower FEV1 ({beta} = -0.30 L, 95% CI -0.47 to -0.14; p < 0.001), lower FEV1/FVC ({beta} = -0.06, 95% CI -0.09 to -0.04; p < 0.001), shorter 6MWD ({beta} = -15.7 m, 95% CI -25.2 to -6.2; p = 0.001), higher SGRQ score ({beta} = 16.9, 95% CI 11.2 to 22.5; p < 0.001), higher mMRC dyspnea score ({beta} = 0.4, 95% CI 0.1 to 0.6; p = 0.007), and accelerated lung function decline ({beta} = -23.8 mL/year, 95% CI -45.13 to -2.48; p = 0.029). Eccentricity was also associated with all-cause mortality (adjusted HR 1.81, 95% CI 1.01 to 3.26; p = 0.047). Conclusions: Tracheal lumen eccentricity is associated with worse quality of life, reduced functional capacity, accelerated lung function decline, and mortality in COPD, supporting central airway morphology as a candidate imaging biomarker.

Background: Obesity is associated with airway disease, but the contribution of specific thoracic adipose depots remains unclear. We examined whether intrathoracic adipose tissue (ITAT), defined as adipose tissue within the thoracic cavity excluding pericardial adipose tissue (PAT), was associated with airway phenotypes. Methods: We included Framingham Heart Study participants with CT-derived thoracic adipose tissue measures. Cross-sectional associations of ITAT with CT-based airway wall and lumen measurements, spirometry, and respiratory symptoms were evaluated using multivariable regression. Primary analyses used sex-specific ITAT z-scores and adjusted for waist circumference to account for central obesity. Secondary analyses examined sex-specific ITAT quintiles and compared ITAT and PAT in individual-depot and mutually adjusted models. Results: Among 2,825 adult participants (52.0% female), after adjustment for waist circumference, each 1-SD higher ITAT, relative to the sex-specific distribution, was associated with greater segmental ({beta} [95% CI]: 0.010 mm [0.006, 0.014]) and subsegmental airway wall thickness (0.006 mm [0.002, 0.009]), narrower segmental airway lumen diameter (-0.049 [-0.088, -0.011]), lower FEV1 percent predicted (-1.92 percentage points [-2.72, -1.12]), and higher odds of wheezing (OR [95% CI]: 1.16 [1.02, 1.31]) and dyspnea (1.22 [1.05, 1.42]). Associations were similar after further adjustment for PAT and across ITAT quintiles. In contrast, PAT was not independently associated with airway structural measures after adjustment for ITAT. Conclusion: Higher ITAT volume was associated with greater airway wall thickness and narrower airway lumen, as well as lower FEV1 and greater respiratory symptoms, independent of central obesity and pericardial adiposity. These findings support a depot-specific association of ITAT with airway disease.

Goblet cell hyperplasia (GCH) is a hallmark of chronic obstructive pulmonary disease (COPD) and contributes to morbidity and mortality. We investigated the cellular and molecular origins of GCH in COPD using single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and in vitro models. We identified "basal-to-goblet transitional cells" (BGTC) which are transcriptionally and physically located between basal and goblet cells and are characterized by expression of clade B serpins. In vitro studies indicate that inflammatory cytokines, including IL-1{beta}, stimulate basal-to-goblet differentiation through a SERPINB3/4+ intermediate and SERPINB3 overexpression induces differentiation of airway basal cells into IL1B-producing inflammatory goblet cells. In addition, we identified a subset of SERPINB4+ "goblet-variant basal cells" from lungs of COPD patients that express goblet cell and inflammatory genes ex vivo. Together, these results indicate clade B serpins contribute to altered epithelial differentiation and inflammatory signaling in COPD and support therapeutic targeting of clade B serpins to reduce GCH.

The alveolar sac architecture is essential for efficient gas exchange and must be precisely maintained throughout life; however, how this delicate structure is preserved during adult regeneration remains poorly understood. Using a mouse pneumonectomy model, we found that Lgr6+ Hhip+ ductal myofibroblasts, a poorly characterized mesenchymal population, are indispensable for maintaining alveolar integrity during lung regrowth. Comprehensive characterization using single-cell transcriptomics, mouse genetics, and pharmacological assays demonstrated that these ductal myofibroblasts secrete myogenic factors, most notably CCN4, to reactivate a myogenic program that converts them into contractile PA-DMFs, thereby preserving alveolar architecture. Lineage-tracing further revealed that these ductal myofibroblasts originate from embryonic MCAM- SMA+ distal progenitors via subepithelial TGF-{beta} signaling, serving as a lifelong guardian of alveolar structural integrity. Notably, cross-species analysis identified an analogous population of LGR6+ fibromyocytes in human respiratory bronchioles. Together, these findings indicate ductal myofibroblasts as a developmentally programmed cell population that reactivate a contractile program to structurally support the regeneration of adult lungs.

Tissue homeostasis requires mechanisms that preserve quiescence while allowing rapid activation after injury, but how tissues actively restrain injury responses during homeostasis remains poorly understood. Here we identify alveolar type 1 (AT1) cells as organizers of the distal lung niche through tonic expression of PDGFA, which maintains alveolar fibroblast identity and restrains myofibroblast activation. Cell type-specific ablation revealed that loss of AT1s, but not other major alveolar cell populations, was sufficient to evoke spontaneous fibroblast activation and tissue-wide responses that recapitulate injury. In silico ligand-receptor analysis, ex vivo screening, and conditional genetic models identified AT1-derived PDGFA as a key homeostatic signal. Loss of AT1-derived PDGFA caused spontaneous myofibroblast induction and tissue remodeling, whereas increasing PDGFA promoted fibroblast proliferation while suppressing myofibroblast differentiation. Integrated phosphoproteomic, transcriptomic, and chromatin accessibility analyses showed that PDGFA engages SHP2-ERK signaling, suppresses MRTFA-associated SRF programs, and promotes E2F-associated cell-cycle programs, thereby opposing the pro-fibrotic effects of TGF{beta}-driven fibroblast activation. Restoring PDGFA signaling abrogates myofibroblast features from human idiopathic pulmonary fibrotic lungs and attenuated fibrotic remodeling in vivo. These findings define a tonic AT1-derived PDGFA signal that maintains alveolar homeostasis and offers a therapeutic target to prevent or revert lung fibrosis.

Pulmonary lymphocytosis is a common feature of chronic lung disease, but the mechanisms governing lymphocyte accumulation within the lung remain poorly understood. Here, we identify granulocyte-macrophage colony-stimulating factor (GM-CSF) as a central regulator of pulmonary lymphocyte homeostasis. Using genetic models, single-nucleus and spatial transcriptomics, cell trafficking analyses, and pulmonary macrophage replacement studies, we show that loss of GM-CSF signaling causes profound pulmonary lymphocytosis characterized by lymphoid follicle formation, local immunoglobulin production, and expansion of multiple lymphocyte lineages. Mechanistically, GM-CSF signaling deficiency blocks terminal alveolar macrophage differentiation and reprograms alveolar macrophages into inflammatory states marked by reduced PU.1 and PPAR{gamma} expression, enhanced NF-{kappa}B signaling, and expression of lymphocyte-recruiting chemokines. These changes promote lymphocyte recruitment, local proliferation, and lymphoid lineage commitment. Restoration of GM-CSF-responsive alveolar macrophages re-established macrophage identity, suppressed inflammatory chemokine programs, and reversed pulmonary lymphocytosis. Together, these findings identify alveolar macrophages as gatekeepers of adaptive immune homeostasis in the lung.

Background. Short telomeres are an established cause of idiopathic pulmonary fibrosis (IPF), but the genetic evidence rests on seven instruments, six of which lie at telomere-maintenance loci, and so cannot separate an effect of telomere length from an effect of the telomerase pathway itself - a distinction that determines whether telomere length is a tractable target or only a marker of the pathway. Whether other measures of biological ageing share the effect, and whether accelerated ageing causes immune-mediated disease more generally, has not been tested systematically. Methods. Two-sample Mendelian randomisation of six ageing measures - GrimAge, PhenoAge, Hannum-age and intrinsic epigenetic age acceleration, proteomic age acceleration, and leukocyte telomere length - against six immune-mediated diseases in FinnGen R12: giant cell arteritis (GCA), polymyalgia rheumatica, primary Sjoegren's disease, interstitial lung disease (ILD), Crohn's disease and ulcerative colitis. All 36 pairs were analysed in both directions using inverse-variance weighted (IVW), MR-Egger, weighted-median and weighted-mode estimators, with Cochran's Q, I2, leave-one-out and single-variant diagnostics, a pre-specified analysis excluding HLA-region instruments, and iterative Q-based outlier removal. Instruments were annotated against telomere-maintenance and pulmonary-fibrosis loci and that class excluded. Minimum detectable effects were computed for every pair before estimates were examined. Any pair surviving correction was re-estimated in two independent IPF case-control datasets. A two-step analysis tested mediation of ILD risk by seven gut microbial taxa acting through telomere length. Results. One of 36 forward pairs survived correction. Longer telomeres reduced ILD risk: outlier-robust IVW odds ratio (OR) 0.59 per standard deviation (SD), 95% CI 0.49-0.71, p = 2.7 x 10^-8, on 77 instruments, with no residual heterogeneity (I2 = 6%) and concordance across estimators. Four pleiotropic instruments - in TERT, TERC, APOE and ATM - inflated the unfiltered estimate to OR 0.46 (0.37-0.57) and accounted for all of its heterogeneity. The effect was reproduced in 5,159 clinician-diagnosed European IPF cases (OR 0.18, 0.12-0.25, p = 5.0 x 10^-23) and in 1,210 East Asian cases (OR 0.54, 0.35-0.83, p = 0.005). Excluding every telomere-maintenance and pulmonary-fibrosis locus left OR 0.26 (0.17-0.40), p = 1.6 x 10^-9, on 72 instruments, establishing that the effect does not depend on TERT or TERC. The effect was three times larger for clinically defined IPF than for the broad registry ILD endpoint (p = 1.8 x 10^-9), consistent with dilution of a fibrosis-specific mechanism by case mix. No methylation clock and no proteomic age measure affected any disease; smallest detectable ORs ranged from 1.29 to 5.24 per SD, so these nulls exclude only large effects. The reverse direction could not be tested at informative precision. One apparent clock-autoimmunity association was produced entirely by a single HLA-region instrument. Mediation was null at its first step. Conclusions. Telomere attrition causally increases fibrotic lung disease risk, with an effect that concentrates in clinically defined IPF and that survives removal of every telomere-maintenance locus - shown here for the first time - and is therefore attributable to telomere length rather than to the telomerase genes that dominate conventional instrument sets. Four methylation clocks and proteomic age acceleration show no such effect, so these measures cannot be treated as interchangeable proxies for biological ageing.

Background: Sex differences in asthma are well recognized, but sex-associated airway DNA methylation (DNAm) patterns and their relationships with gene expression remain incompletely characterized. We therefore conducted cis- and trans-expression quantitative trait methylation (eQTM) analyses using sex-stratified and CpG-by-sex interaction models. Methods: We analyzed nasal airway epithelial DNAm and transcriptomic data from 298 participants in the CAAPA discovery cohort. Asthma-associated differentially methylated CpGs (DMCs) were identified using sex-combined and sex-stratified models. Cis- and trans-eQTM analyses were then performed in participants with asthma using sex-combined, sex-stratified, and CpG-by-sex interaction models. To prioritize asthma-associated regulatory relationships, eQTMs that were also significant in non-asthmatic controls were excluded from downstream analyses. Replication in an independent bronchial epithelial cohort was defined by concordant effect direction and P < 0.05. Replicated targets underwent pathway and network analyses. Findings: Asthma-associated DMCs were predominantly hypomethylated in females and hypermethylated in males. Among trans-eQTMs, 379 associations were shared across all four models, whereas the majority were model-specific, indicating extensive sex-dependent regulatory architecture. We replicated 3, 1, 9, and 2 cis-eQTMs and 96, 84, 216, and 41 trans-eQTMs in the sex-combined, female-stratified, male-stratified, and interaction models, respectively. Sex-combined targets implicated innate immune and inflammatory signaling (TNF, IRF8); female-stratified targets implicated cytoskeletal remodeling, focal adhesion, and PI3K-Akt signaling (ITGA10); male-stratified targets implicated transcriptional regulation and post-translational modification (MOV10, HDAC7, CSF1, KAT2A); and interaction targets implicated lipid-mediator metabolism and inflammatory signaling and resolution (CYP2C9, LTB4R, CCL19). Our sex-dependent analyses showed that the majority of eQTMs resided within the trans-regulatory architecture, suggesting that sex-dependent gene expression differences are associated predominantly with trans rather than cis regulatory mechanisms. Pathway analyses revealed enrichment of innate immunity in the sex-combined model, structural remodeling in females, transcriptional control in males, and inflammatory resolution in the sex-interaction model. Interpretation: Sex-specific and sex-dependent airway methylation-expression associations were largely undetected by sex-combined analysis. Sex-aware analytical models may help resolve epigenetic mechanisms underlying sex differences in asthma biology.

BackgroundReactive oxygen species (ROS) overproduction, inflammatory cytokines, and plasma mediators disrupt alveolar tight junction integrity and perpetuate oxidative injury in acute respiratory distress syndrome (ARDS). Biological heterogeneity of ARDS, reflected by hyper- and hypoinflammatory subphenotypes with distinct plasma mediator profiles, may contribute to heterogeneity of treatment effect. Stem cell-derived EVs have been shown to exert regenerative and immunomodulatory effects by horizontal transfer of proteins, lipids and genetic material to injured target cells. This study investigated whether extracellular vesicles derived from bone marrow (MSC-EVs) and adipose tissue (ASC-EVs) mesenchymal stem cells restore barrier integrity and redox homeostasis, and whether efficacy differs across subphenotypes. MethodsBronchial epithelial cells (BEAS-2B), microvascular endothelial cells (HMEC-1), and small airway epithelial cells (SAEC) were challenged with plasma from patients with hyperinflammatory or hypoinflammatory ARDS subphenotypes, including those treated with venovenous ECMO. MSC-EVs or ASC-EVs were added concomitantly with plasma exposure or six hours later, at a dose of 5,000 particles/cell. After 24 h, the expression of actin and tight junction proteins, zonula occludens (ZO)-1, occludin, and vascular endothelial (VE)-cadherin, was evaluated by immunofluorescence. Oxidative stress parameters, antioxidant defenses, and antioxidant enzyme activities, were measured. ResultsPlasma from patients with the hyperinflammatory ARDS subphenotype induced marked disruption of ZO-1, occludin, and VE-cadherin expression, together with actin cytoskeletal rearrangement, across BEAS-2B, HMEC-1, and SAEC cells. Barrier disruption was accompanied by increased oxidative stress markers and altered antioxidant enzyme activity. Both MSC-EVs and ASC-EVs restored tight junction protein expression and cytoskeletal organization when administered either concurrently with ARDS plasma or 6 hours after exposure, and both effectively modulated redox parameters. Delayed administration 6 hours after injury produced greater effects for several oxidative stress endpoints, particularly in cells challenged with hypoinflammatory plasma. ConclusionARDS-associated plasma mediators induce subphenotype-dependent impairment of epithelial-endothelial barrier integrity through oxidative mechanisms. Both MSC-EVs and ASC-EVs mitigate this injury, with efficacy that varies according to ARDS subphenotype and timing of administration.

BackgroundMacitentan is a dual endothelin receptor antagonist used in pulmonary arterial hypertension (PAH) and has also been studied in other forms of pulmonary hypertension (PH). Evidence across PH phenotypes has been heterogeneous. This study aims to evaluate the efficacy and safety of macitentan compared with concurrent control therapies across PH phenotypes and to explore whether treatment effects differ according to PH classification. MethodsPubMed, Embase, and the Cochrane database were searched from inception through June 2026. Randomized and non-randomized comparative studies of patients with PH receiving macitentan were eligible. Prespecified efficacy outcomes were 6-minute walk distance (6MWD), pulmonary vascular resistance (PVR), mean pulmonary arterial pressure (mPAP), and cardiac index; prespecified safety outcomes included mortality and adverse events. Random-effects meta-analyses were performed using mean differences (MDs) for continuous outcomes and risk ratios (RRs) for dichotomous outcomes. ResultsFourteen studies met the eligibility criteria. Macitentan was associated with a greater reduction in PVR (MD, -134.50 dyn.s.cm; 95% CI, -226.10 to -42.86) and an increase in cardiac index (MD, 0.44 L/min/m; 95% CI, 0.22 to 0.67). No statistically significant difference was observed for 6MWD (MD, 5.04 m; 95% CI, -15.06 to 25.14) or mPAP (MD, -2.70 mmHg; 95% CI, -6.46 to 1.05). In pooled safety analyses, mortality was lower and risks of anemia, peripheral edema, and discontinuation due to adverse events were higher with macitentan. Heterogeneity and the small number of studies within several phenotype-specific analyses limited the precision of subgroup findings. ConclusionsMacitentan-containing therapy was associated with favorable changes in selected hemodynamic outcomes but not with a statistically significant improvement in 6MWD or mPAP. The safety findings were characterized by higher risks of anemia, peripheral edema, and treatment discontinuation. These results should be interpreted in the context of differences in PH phenotype, dose, comparator, study design, and outcome measurement.

Objective: The maturation of an autologous arteriovenous fistula (AVF) depends on the coordinated hemodynamic adaptation of the inflow artery and the outflow vein. While venous remodeling has been extensively studied, the mechanisms of arterial adaptation are poorly understood. We addressed this knowledge gap with the first temporal single-cell atlas of the porcine AVF model. Methods: Six Yorkshire pigs underwent bilateral femoro-femoral AVF creation for tissue harvest at 2 and 21 days postoperatively or sham operation to collect native vessels after 2 days. Arteries and veins were analyzed by droplet-based single-cell RNA sequencing, histology, and immunohistochemistry. Results: All fistulas were patent, with flows reaching 1185.0 +/- 170.6 mL/min despite mild to moderate neointima formation in the veins. Arteries and veins demonstrated >60% endothelial denudation and increased neovascularization. Arterial remodeling was characterized by a progressive increase in venous-like transcriptional features. Arteries presented a venous-like endothelial cell (EC) composition at 21 days postop and a temporal increase in venous-associated phenotypes of myofibroblasts and fibroblasts. In both vessels, the latter populations were the main bearers of mechanosensitive signatures at 2 days. Despite the temporal convergence in phenotypes, arteries and veins retained vessel-selective characteristics. Arterial ECs expressed high levels of nitric oxide synthase, whereas venous ECs were largely inflammatory. Arterial myofibroblasts and fibroblasts had higher expression of Notch signaling transducers (HEY2, HEYL) and cellular communication network factors (CCN3, CCN5), while Wnt signaling regulators (DKK2, SFRP4) and complement genes (C3, C7, CFD) were upregulated in veins. Anti-inflammatory macrophages increased in both vessels during remodeling, likely contributing to the maturation of the fistulas. Conclusion: These analyses challenge the traditional paradigm of exclusive vein arterialization and support a complementary process of arterial venification after anastomosis. Recognition of this underappreciated phenomenon may reshape our understanding of AVF biology and guide the discovery of both vascular-wide and vessel-selective anti-stenotic therapies.

Type 2 inflammation is central to chronic rhinosinusitis with nasal polyps (CRSwNP), but it does not fully explain persistent stromal and vascular remodeling. We asked whether complement, adhesion, stromal, and vascular signals converge across tissue, cellular, and spatial scales. We integrated four bulk-transcriptomic cohorts with a single-cell atlas and GeoMx spatial transcriptomics. Network analysis identified eight contrast-associated hub genes and an ABCC9-ACTA2-GPM6A score that separated study-defined groups across cohorts. Single-cell mapping localized these genes mainly to pericytes, fibroblasts, and endothelial cells, while CellChat linked structural and immune compartments through extracellular-matrix and inflammatory signals. In spatial data, C3-C3AR1 showed closer-than-random proximity after multiple-testing correction, whereas ICAM1-ITGAL/ITGB2 did not. Anatomical-site sensitivity analyses retained all eight hub-gene directions. These findings support CASVR as an association framework linking complement-related signals to structural-vascular compartments and identify C3-C3AR1 for independent mechanistic testing.

Study Question. COPD exacerbations with respiratory failure have an outsized impact on morbidity, mortality, and healthcare costs. Physicians treat these severe events with high doses of corticosteroids out of concern for intrinsic corticosteroid resistance. This study was designed to determine whether people who suffer from exacerbations with respiratory failure have intrinsic steroid resistance that could affect that decision. Patients and Methods. A matched, case-control study was performed at University of Colorado. Subjects were enrolled into the Exacerbation group if they were hospitalized for an exacerbation requiring ventilation, or into the Stable COPD group if they had no inpatient exacerbation within 12 months. Stable COPD subjects were frequency-matched to Exacerbation subjects for age, smoking status and FEV1%. Corticosteroid sensitivity was assessed ex vivo and in vivo >45 days following the last exacerbation during a period of relative quiescence. The primary outcome was the group difference in methylprednisolone IC50 for LPS-stimulated IL-8 release by mononuclear cells. Measurements and Main Results. The Exacerbation group (N=8) had more exacerbations in the prior year (6.0 vs. 0.5; p<0.01) and had higher baseline blood lymphocytes (p<0.01) versus Stable controls (N=8). Ex vivo, methylprednisolone equally suppressed IL-8, IL-6, TNFalpha and IL-1bets release by mononuclear cells and whole blood across groups. In vivo, methylprednisolone decreased plasma mediators and mononuclear cells in both groups but had a small differential effect on mononuclear cell gene expression. Answer to the Question. Exacerbations with respiratory failure have persistent lymphocytosis, an altered gene response to corticosteroids, but no corticosteroid resistance to suppress inflammation.

Background. Polygenic risk scores (PRS) capture inherited susceptibility to asthma, whereas methylation risk scores (MRS) may reflect disease-associated epigenetic variation. We assessed whether these scores differ in patients requiring biologic therapy and whether MRS change after mepolizumab initiation. Methods. Cross-sectional analyses in the GEMAS study included 699 participants with genotype data (428 with asthma, 271 controls) and 668 with blood DNA methylation data (408 with asthma, 260 controls). We evaluated 35 existing asthma PRS and derived six MRS from published epigenomic asthma studies. Longitudinal MRS changes after mepolizumab initiation were assessed in 16 GEMAS patients and were replicated in 20 patients from the MEGA study. Results. Twenty-eight PRS differentiated biologic-treated patients from controls (areas under the curve [AUC], 0.68-0.75), but none distinguished biologic-treated and non-treated patients after multiple-testing correction. MRS was higher in asthma without biologic than in controls (odds ratio [OR], 1.64; 95% CI, 1.34-1.99) and lower in biologic-treated than in asthma without biologic patients (OR, 0.54; 95% CI, 0.35-0.81). MRS did not differ significantly between biologic-treated patients and controls. Following mepolizumab initiation, MRS decreased at 6 and 12 months in GEMAS (p=4.81x10-5 and p=2.40x10-5, respectively). The 6-month decrease was also observed in MEGA (p=3.25x10-9). MRS epigenetic markers were predominantly hypermethylated, opposite to their described pattern in asthma risk. Conclusions. Asthma PRS did not differ significantly between biologic-treated and non-biologic-treated patients, but asthma MRS were lower in biologic-treated patients. The MRS decreased after mepolizumab initiation in two independent cohorts, consistent with a reversal of asthma-associated epigenetic patterns.

Pulmonary arterial hypertension (PAH) is characterized by the hyperproliferative phenotype of pulmonary arterial smooth muscle cells (PAVSMCs) and extensive extracellular matrix (ECM) remodeling. Vacuolar protein sorting 34 (VPS34), a Class III Phosphatidylinositol-3 Kinase (PI3K), regulates cell proliferation, but its specific function in pulmonary vascular remodeling remains poorly understood. We aim to determine the role of VPS34 activation and regulation in PAVSMC proliferation, matrix deposition, and the progression of PAH. First, we found a marked decrease in the inhibitory phosphorylation of VPS34 at Ser164 (P-S164-VPS34), indicative of constitutive VPS34 activation, in remodeled pulmonary arteries (PAs) from human PAH patients and male mice exposed to Sugen/Hypoxia (SuHx). In human PAH PAVSMCs, siRNA-mediated knockdown of VPS34 or pharmacological inhibition via the selective inhibitor SAR405 significantly blunted cell proliferation and reduced the production of fibronectin, a major component of the remodeled ECM. Mechanistically, we found that Akt activation in PAH PAVSMCs promotes VPS34 activation; Protein Phosphatase 1 Catalytic Subunit Alpha is a potential effector in VPS34 activation; and active VPS34 led to the activation of the mTOR/S6 signaling pathway with tuberous sclerosis complex 2 downregulation. Finally, in vivo treatment with SAR405 attenuated right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling in SuHx mice, while concurrently decreasing vascular fibronectin deposition. Our study demonstrates that VPS34 activation, driven by an Akt-mediated loss of the inhibitory P-S164, is crucial in PAVSMC proliferation and fibrotic vascular remodeling in PAH. Targeting the VPS34 axis represents a promising anti-remodeling therapeutic strategy for the treatment of PAH. Keywords: Pulmonary arterial hypertension, vacuolar protein sorting 34, pulmonary vascular remodeling, pulmonary arterial vascular smooth muscle