American Journal of Physiology-Lung Cellular and Molecular Physiology

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.

ObjectiveReliable animal models of tracheal stenosis are necessary for the development and translational testing of anti-fibrotic and regenerative therapies, but existing rabbit models frequently demonstrate substantial variability in stenosis severity, which limits their translational utility. The objective of our study was to determine whether airway diameter-matched mechanical injury improves the severity and reproducibility of experimental tracheal stenosis in a rabbit model, and to evaluate whether rabbit body weight is a reliable surrogate for tracheal luminal diameter during model creation. MethodsFourteen male New Zealand White rabbits (weight range, 2.7-3.5 kg) underwent tracheal injury using steel-bristle brushes introduced through a tracheotomy. Animals were assigned to receive either airway diameter-matched injury, in which brush size was selected to closely approximate the directly measured tracheal lumen diameter, or non-matched injury, in which brush size was selected without regard to measured lumen diameter. At postoperative day 21 (POD21), the injured tracheal segment and a native uninjured segment from the same animal were harvested and compared. Stenosis degree was quantified grossly, and lamina propria-to-cartilage (LP:C) ratio was quantified histologically by three blinded reviewers. The relationship between rabbit weight and airway diameter was assessed, and inter-rater reliability was calculated using the intraclass correlation coefficient (ICC). ResultsTwelve of fourteen rabbits reached the POD21 endpoint; two were euthanized early for severe airway compromise meeting humane endpoint criteria, both with approximately 80% stenosis. Injured tracheas demonstrated significantly greater stenosis than native controls (66.0 {+/-} 13.0% vs 16.0 {+/-} 2.7%; p = 0.00012), with a corresponding increase in LP:C ratio (p = 0.031). Airway diameter-matched injury produced significantly greater stenosis than non-matched injury (74.6 {+/-} 6.1% vs 50.6 {+/-} 4.0%; p = 0.001), while LP:C ratio did not differ between injury techniques (p = 1.0). Rabbit weight did not correlate with airway diameter (r = 0.176, p = 0.515; R2 = 0.031). Inter-rater reliability was excellent for both stenosis degree (ICC = 0.989) and LP:C ratio (ICC = 0.992). ConclusionsDirect measurement and matching of injury instrument diameter to native airway diameter substantially improves both the severity and the reproducibility of stenosis in a rabbit tracheal injury model, whereas body weight is an unreliable surrogate for airway size. This optimized, standardized protocol offers a reproducible platform for future translational studies of airway fibrosis and anti-fibrotic or regenerative therapies.

The acute respiratory distress syndrome is a heterogenous syndrome characterized by the rapid development of respiratory failure. Nearly 40% of patients who develop ARDS will die, and there is growing interest in identification of biomarkers to identify patients at risk of death and/or inform treatment decisions. Most prior work on biomarkers in ARDS has focused on the plasma compartment, but there is concern that circulating biomarkers may not reflect alveolar pathobiology. The anti-inflammatory microRNA-146a has been shown to be upregulated in inflammatory cells in human bronchoalveolar lavage fluid, but it is not known if these levels correspond with outcomes. We measured miR-146a expression by digital droplet PCR in human biospecimens from four different cohorts of patients with respiratory failure requiring mechanical ventilation - two plasma cohorts, one bronchoalveolar lavage cohort, and one heat moisture exchange (HME) filter fluid cohort. We found that miR-146a was detectible in plasma, bronchoalveolar lavage fluid, and HME fluid. However, only when measured in the alveolar space, was miR-146a expression significantly lower in older adults and those who died. It did not correlate with outcomes when measured in plasma. To our knowledge, this is the first report that nucleotides can be measured in HME fluid and builds upon expanding literature that circulating biomarkers may not reflect complex biology of the alveolar microenvironment during ARDS.

Collagens are key components of the extracellular matrix (ECM) that play a crucial role in maintaining structure, strength, and function of the lungs. Fibrillar collagens are crosslinked by enzymes such as lysyl oxidases and transglutaminases and organized into networks by proteoglycans and glycoproteins. Collagens are the main load-bearing components and along with elastin may impart a non-linear strain hardening behavior to the lung. In disease, collagen crosslinking and organization can be disrupted, possibly due to abnormal levels of enzymes or ECM components. Few studies have examined collagen crosslinking and organization in healthy and diseased human lungs. In this study, alterations in collagen crosslinking and organization were investigated in human lung control, fibrotic and chronic obstructive pulmonary disease (COPD) tissue sections. Ultra-performance liquid chromatography and second harmonic generation microscopy measured pyridinoline crosslinks and the distribution of mature and immature collagens within the decellularized scaffolds, respectively. Fibrotic scaffolds had higher total collagen but less crosslinking per mole of collagen compared with COPD donors. Image analysis by second harmonic generation microscopy showed mature collagens populated airway or blood vessel walls in all three groups and in the parenchyma of fibrotic scaffolds. Immature collagens, on the other hand, were mainly localized to parenchymal regions in control and COPD scaffolds, with fewer immature collagens in fibrotic parenchyma. Additionally, quantification of the mature to immature collagen ratio in defined regions of control and diseased scaffolds showed increased organized collagen in fibrotic tissue. Our study shows that collagen crosslinking and organization are disrupted in fibrotic and COPD lungs and these changes may be compartment specific and can contribute to aberrant mechanical properties of diseased lungs. Our findings highlight that along with total collagen content, collagen crosslinking and organization are equally important while investigating collagen-mediated pathological changes in lung tissue. These changes may have implications for developing ECM-based therapeutics for patients with lung diseases.

Tissue hypoperfusion is common in clinical settings, but how tissues respond to hypoperfusion on a microphysiological scale is not clear. We used real-time confocal microscopy of live, perfused lungs to gain insights into the effects of hypoperfusion on the microcirculation and microphysiology of lung alveoli, where gas exchange occurs. We focused on effects of hypoperfusion on alveolar liquid secretion, since alveolar liquid secretion is important for alveolar homeostatic functions. Our findings show lung hypoperfusion stimulated a reversal of alveolar liquid transport, from homeostatic liquid secretion to absorption. Specifically, lung perfusion at or near physiological perfusion pressure led to alveolar liquid secretion that depended on the alveolar epithelial cystic fibrosis transmembrane conductance regulator (CFTR), Na+-K+-Cl- cotransporters, and the Na+/K+-ATPase. Within minutes of halting lung perfusion or majorly reducing it, alveoli stopped secreting liquid and instead absorbed it via the epithelial Na+ channel, CFTR, and K+-Cl- cotransporters. We provide evidence that hypoperfusion caused alveolar microvessel lumens to shrink and airspaces to expand, leading to epithelial stretch that stimulated liquid absorption. These findings show lung hypoperfusion initiates mechanical signals that stimulate the alveolar epithelium to absorb liquid, and they may inform the pathogenesis of lung diseases characterized by acute microvascular hypoperfusion.

Wildland firefighters experience repeated exposure to wildfire smoke, yet the pathophysiological mechanisms underlying chronic inhalation injury remain poorly understood. Although prior studies report parenchymal destruction following prolonged woodsmoke exposure, the temporal relationship between molecular, structural, and functional decline following inhalation of smoke from needles/leaves remains unclear. To address this gap, we characterized coordinated changes in lung structure, function, and underlying molecular disruptions using a dosimetry-based murine model approximating 7-14 years of firefighter service. Male apolipoprotein E-deficient mice were exposed to smoldering Douglas fir needle smoke (40 mg/m3, 2 h/day, 5 days/week) for 8 or 16 weeks. Immunofluorescence analyses revealed an early elastolytic response at 8 weeks, with increased neutrophil elastases and matrix metalloproteinases-9 and -12, accompanied by elevated surfactant protein-D, compared to air controls. These changes were resolved by 16 weeks despite progressive tissue injury. Airspace enlargement was evident at 8 weeks, progressed by 16 weeks, and included increased alveolar blunting and septal wall thickening at the later time point. Cleaved caspase-3 was elevated at 16 weeks, indicative of advanced parenchymal damage and apoptosis. Epithelial tight-junction protein ZO-1 intensity was reduced at both evaluation points, whereas the epithelial-to-mesenchymal marker N-cadherin remained undetectable in the alveolar epithelium. Functional impairment as evident by increased static compliance and upward shifts in pressure-volume curves was only significant after 16 weeks of exposure. Findings indicate that molecular and structural injury of tissue destruction preceded measurable functional decline, underscoring the need for early biomarkers to identify smoke-induced lung injury in wildland firefighters before function loss occurs.

BackgroundCalcitonin gene related peptide (CGRP) hyperpolarizes pulmonary arterial smooth muscle cells (SMCs) and endothelial cells (ECs) through PKA-dependent activation of KATP channels. CGRP can diminish the severity of pulmonary fibrosis (PF), however, the effects on vascular signaling were poorly defined. We hypothesized that hyperpolarization to CGRP would be augmented in a mouse model of PF. MethodsPF was induced in male and female C57BL/6 mice by intratracheal delivery of bleomycin (3 wk), with saline used as control (sham). Pulmonary arteries (PAs; 100-150 {micro}m diameter) were cannulated and pressurized to 16 cmH2O, and endothelial tubes were studied in complementary experiments to eliminate the influence of SMCs. Membrane potential (Vm) was recorded continuously using intracellular microelectrodes. Responses were also evaluated in isolated lungs preconstricted with U46619 ([~]10 mmHg). ResultsPF led to greater indices of PH in males vs. females. Isolated lungs and PAs from male PF mice had enhanced vasodilation and hyperpolarization of Vm to CGRP, although no effect was observed in females. The greater vasodilation and hyperpolarization of SMCs to CGRP in males persisted in endothelium-disrupted PAs and during treatment with L-NAME indicating that ECs are not required for greater responsiveness to CGRP. With no effect on resting Vm, inhibition of KATP channels or PKA significantly attenuated hyperpolarization of SMCs and ECs, attenuated vasodilation to CGRP in PAs, and eliminated differences between groups in males. Direct activation of PKA, but not KATP, evoked greater Vm hyperpolarization and vasodilation in PF vs. sham PAs and lungs. Although no difference in sensory nerves was observed in fibrotic mice, perivascular nerve stimulation evoked greater vasodilation in PAs. ConclusionsIn a mouse model of PF, CGRP-dependent hyperpolarization of pulmonary arterial SMCs and ECs is augmented through increased PKA-dependent activation of KATP channels leading to increased vasodilator sensitivity.

I.BackgroundPulmonary arterial hypertension (PAH) is a debilitating cardiopulmonary disease characterized by progressive remodeling of the pulmonary vasculature. Pathologic transforming growth factor-{beta} (TGF-{beta}) signaling is an essential driver of vascular remodeling in PAH. While global inhibitors of TGF-{beta} exist, their clinical application is limited by systemic adverse effects. Therefore, a critically unmet need in PAH is to identify pulmonary vascular-specific regulators of the TGF-{beta} axis, which would selectively enhance clinical efficacy while minimizing adverse effects. As the clinical care of PAH largely promotes vasodilation, and only one FDA-approved agent targets vascular remodeling, this study aimed to identify selective, therapeutically targetable regulators of the TGF-{beta} axis in the PAH pulmonary vasculature. MethodsCD248 was identified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics in human lungs. CD248 levels were assessed across human, rat, and mouse lung tissues using western blotting, RTqPCR, and/or immunofluorescence techniques. CD248-null (CD248-/-) mice were used to study the contribution of CD248 to hypoxia-sugen (H/S)-induced PAH. The mechanistic role of CD248 in PAH vascular remodeling and TGF-{beta} signaling was assessed by genetic (siRNA knockdown; overexpression) and pharmacologic (Ontuxizumab) manipulation of primary human pulmonary vascular cells. ResultsLC-MS/MS proteomics coupled with pathway enrichment analysis of human lung tissue identified CD248 as a putative mediator of vascular remodeling that is elevated in PAH lungs. CD248 was elevated in PAH pulmonary artery smooth muscle cells (PASMCs) across human, rat, and mouse lung tissue. CD248-/- mice were protected from H/S-induced elevations in right ventricular (RV) systolic pressure (RVSP), RV hypertrophy, and pulmonary artery muscularization. CD248 knock-down reduced cell proliferation and migration of primary PAH PASMCs. CD248 was essential for phospho-activation of TGF-{beta} receptor I (T{beta}RI) at S165 and canonical phosphorylation of SMAD3 at S423/425. CD248 loss blunted TGF-{beta}-induced gene expression (FN1, Col11, -SMA) and activated expression of the vasoprotective matrix metalloprotease, MMP-8. Mechanistically, CD248 interacted with and enhanced de novo phosphorylation and stability of T{beta}RI, blocking its ubiquitin-mediated proteasomal degradation. Ontuxizumab promoted T{beta}RI instability and attenuated the production of FN1, Col11, and -SMA in primary PAH PASMCs. ConclusionsThis work identifies CD248 as a previously unrecognized co-activator of T{beta}RI in PAH. As CD248 is largely quiescent in most adult tissues yet pathologically upregulated in the PAH pulmonary vasculature, this study supports the potential of anti-CD248 therapy as a novel pulmonary vascular-specific alternative to systemic TGF-{beta} inhibition.

Mammalian lungs operate within a thoracic cage composed of parietal pleura, rib cage, skeletal muscle, and diaphragm, yet clinical ventilator metrics largely reflect the combined mechanics of lung and surrounding structures and the thoracic cage. We hypothesized that thoracic boundary conditions selectively alter measured lung biomechanics. We performed paired pulmonary function testing (FlexiVent) in C57BL6 mice of both sexes spanning development through adulthood, measuring quasi-static pressure-volume behavior and dynamic forced-oscillation parameters in vivo (supine, mechanically ventilated) and again ex vivo in the same lungs. In a subset, we additionally compared in vivo and ex vivo microCT-derived lung volumes, including a pressure-fixed ex vivo protocol using snap freezing at controlled inflation pressure. Quasi-static pressure-volume curves were similar between conditions, with near-identity at higher pressures and only modest divergence at low pressures, consistent with thoracic structures primarily modulating recruitment/de-recruitment rather than intrinsic elastic recoil. Maximal volume at 30 cmH2O showed strong in vivo-ex vivo correlation and minimal bias, and static compliance and PV-loop hysteresis exhibited small biases relative to reported disease-model effect sizes. In contrast, dynamic mechanics demonstrated a clear in vivo elevation of tissue damping (G) with only modest change in tissue elastance (H) and little change in Newtonian resistance (Rn), producing a meaningful increase in hysteresivity (G/H). This dissociation implicates frequency-dependent mechanical heterogeneity (time-constant mismatch/pendelluft) imposed or amplified by nonuniform thoracic loading. Ex vivo microCT enabled reliable whole-lung segmentation and correlated with ex vivo PFT volumes at matched pressures, whereas in vivo volumetry showed weaker agreement. These results indicate that thoracic structures contribute modest restriction but disproportionately increase dynamic dissipation and heterogeneity, suggesting that ex vivo functional testing and oscillometry-like metrics may better detect biomechanical changes inherent to lung parenchyma.

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease (ILD) characterized by progressive fibrosis, irreversible loss of lung elasticity, and chronic respiratory failure, with a mean survival of 3-5 years. The disease is believed to result from repeated alveolar epithelial injury that sustains transforming growth factor-beta (TGF-{beta}) signaling, driving fibroblast-to-myofibroblast differentiation and excessive collagen deposition. Although current IPF models--including animal studies, 2D cultures, and basic 3D systems--have enhanced understanding of disease mechanisms, they inadequately replicate epithelial-fibroblast interactions, extracellular matrix (ECM) remodeling, and epithelial barrier dysfunction. To address this limitation, we engineered a 3D lung co-culture model that simulates the physiological epithelial-fibroblast crosstalk and ECM remodeling characteristic of IPF. Our model embeds fibroblasts within a collagen-hyaluronic acid matrix overlaid with an epithelial monolayer cultured at an air-liquid interface. Basolateral TGF-{beta} exposure generated a profibrotic microenvironment that weakened epithelial barrier integrity and drove myofibroblast differentiation marked by elevated -SMA and vimentin. Elevated pro-inflammatory cytokine secretion and increased collagen disorganization further demonstrated active fibrogenesis. Together, these features show that our model captures key early events in IPF pathogenesis and offers a versatile platform for next-generation lung-on-a-chip studies in fibrotic disease.

RationaleHeterogeneous alveolar collapse is prevalent in inflammatory lung conditions such as chronic obstructive pulmonary disease, acute respiratory distress syndrome, and pneumonia. Although neutrophil-released proteases contribute to the tissue remodeling that leads to alveolar collapse, how this altered mechanical environment in turn affects neutrophil migration remains largely unexplored. ObjectivesIn this study, we investigate how alveolar collapse and stretch influence neutrophil migration and identify the mechanical and biochemical factors that govern regional migration differences. MethodsWe developed a novel precision-cut lung slice platform that generates collapsed vs non-collapsed regions within the same slice. Neutrophils in both regions were longitudinally imaged for up to 5 hours to quantify motility behavior. Migration mechanisms were probed using migration-related inhibitors, collagenase, and cigarette smoke extract. A crystal ribcage system, which preserves intact alveolar shape and the air-liquid interface, was also used to assess the effects of ventilation on neutrophil migration. ResultsNeutrophil migration was faster in the collapsed region compared to not-collapsed regions. This regional difference was eliminated by Rho-associated protein kinase (ROCK) inhibition, which selectively increased migration speed in the non-collapsed region. The regional difference persisted with the addition of collagenase and cigarette smoke extract, both of which significantly increased the migration speed in both regions. In the crystal ribcage, the preserved air-liquid interface and ventilation together enhanced neutrophil migration compared with a collapsed lung. ConclusionsAlveolar collapse and stretch facilitate neutrophil migration, indicating the role of localized tissue remodeling in driving neutrophil activity and further disease progression.

BACKGROUND Bronchopulmonary dysplasia-associated pulmonary hypertension (BPD-PH) complicates prematurity and carries substantial morbidity in extremely preterm infants. Pulmonary microvascular endothelial cell (PMVEC) dysfunction promotes capillary rarefaction and vascular remodeling, but epigenetic mechanisms after neonatal hyperoxia are poorly defined. Baf60c (SMARCD3), a SWI/SNF subunit supporting vascular homeostasis, and Smarcc2 (BAF170), a PBAF scaffold subunit linked to proliferative signaling, have not been studied together in BPD-PH. METHODS Neonatal C57BL/6 mice were exposed to 85% oxygen for 14 days. Right ventricular systolic pressure (RVSP), right ventricular hypertrophy, lung weight index, and pulmonary histopathology were assessed; PMVEC proliferation, migration, and invasion were measured. Transcriptome sequencing with GO/KEGG analyses, siRNA knockdown, LY294002 inhibition, coimmunoprecipitation, and Western blotting mapped the Baf60c-Smarcc2-PI3K-Akt-mTOR axis. A Tie1-driven, lung-tropic adeno-associated virus delivered by superficial facial vein injection at postnatal day 1 enabled PMVEC-specific Baf60c overexpression. RESULTS Hyperoxia increased RVSP, right ventricular hypertrophy, and lung weight index, impaired alveolarization, reduced capillary density, and promoted arteriolar remodeling. PMVEC function was impaired, with PI3K-Akt pathway enrichment and suppressed signaling. Hyperoxia decreased Baf60c and increased Smarcc2. Baf60c knockdown upregulated Smarcc2, suppressed PI3K-Akt-mTOR, and phenocopied hyperoxia; Smarcc2 knockdown had opposite effects. Baf60c bound Smarcc2 but not PI3K. PMVEC-specific Baf60c overexpression attenuated pulmonary hypertension and right ventricular hypertrophy and partially improved alveolar and microvascular injury. CONCLUSIONS Hyperoxia-induced BPD-PH is associated with reduced Baf60c, increased Smarcc2, and suppressed PI3K-Akt-mTOR signaling in PMVECs. Baf60c may indirectly regulate this pathway through Smarcc2. Endothelial Baf60c is a potential therapeutic target in BPD-PH.

Rationale: Preclinical familial pulmonary fibrosis (FPF) represents an early stage of fibrotic lung disease, yet the compartment- and cell-specific molecular programs preceding fibrosis remain poorly understood. Objective: To define spatially organized molecular signatures associated with preclinical FPF and identify tissue-informed circulating biomarkers linked to early fibrotic remodeling. Methods: We performed integrated multi-omic profiling of histologically preserved and remodeled lung regions from subjects with preclinical FPF, Idiopathic Pulmonary Fibrosis (IPF), and controls using spatial transcriptomics, single-nucleus RNA sequencing (snRNAseq), and blood proteomics. Differential expression and pathway enrichment analyses were performed across spatial compartments and epithelial cell states. Results: Histologically preserved lung regions in preclinical FPF demonstrated transcriptional abnormalities including stress-response, ciliary, and extracellular matrix-associated programs despite minimal architectural distortion. Spatial analyses identified alterations in alveolar niche molecular programs accompanied by increasing profibrotic signaling across preserved and tissue remodeled lung compartments. Compared with advanced IPF, preclinical FPF retained epithelial repair and surfactant-associated signatures. Integration with snRNAseq demonstrated enrichment of alveolar and airway epithelial cell dysregulated states associated with transitional phenotypes previously implicated in IPF. Compartment- and epithelial-associated transcriptional signatures identified in lung tissue were partially represented in the peripheral blood. Conclusion: Preclinical FPF is characterized by compartment- and cell-specific molecular programs that precede established fibrosis. We identified distinct alveolar, airway, and vascular molecular signatures and epithelial remodeling states represented in the peripheral blood. These findings provide an initial framework for molecular classification of early stages of pulmonary fibrosis and support future studies evaluating minimally invasive approaches for disease stratification and precision therapeutics.

Alveolar capillary endothelial cells are positioned adjacent to the alveolar epithelium and contribute to lung homeostasis and injury responses. Single-cell studies have identified aerocyte capillary endothelial cells (aCap), which are specialized for gas exchange, and general capillary endothelial cells (gCap), which contribute to endothelial maintenance and inflammatory signaling. Apelin and its receptor are differentially enriched across these endothelial compartments, but their roles in emphysema development remain incompletely understood. Using an elastase-induced emphysema model in male C57BL/6J mice, we combined bulk RNA sequencing, CIBERSORTx-based cell-type deconvolution, histology, inflammatory assays, pulmonary function testing, and pharmacologic activation of the apelin receptor with [Pyr1]-Apelin-13. At 24 hours after elastase exposure, the inferred fraction of gCap was reduced, and lung expression of apelin and the apelin receptor was decreased. Early [Pyr1]-Apelin-13 administration reduced lung inflammatory mediator expression, Ly6G-positive neutrophil accumulation, bronchoalveolar lavage neutrophil counts, and matrix metalloproteinase-9 activity. Early treatment also attenuated subsequent airspace enlargement, whereas treatment initiated after emphysema was established did not improve physiological or histological outcomes. In a chronic {beta}ENaC-transgenic mouse model, the inferred gCap fraction was maintained, the aCap fraction was reduced, and apelin receptor activation did not improve disease phenotypes. These findings suggest that early activation of the apelin receptor modifies acute inflammatory and endothelium-associated responses following elastase injury and limits emphysematous remodeling in mice. Together, these results support a time-sensitive role for apelin-APJ signaling during the early phase of emphysema development.

Biological sex has systemic effects on gene expression, cell behavior, and disease etiology. Despite these widespread effects, sex as a biological variable is understudied, particularly in chronic lung diseases. In idiopathic pulmonary fibrosis (IPF), 70% of patients are male, and male patients have overall worse survival post-diagnosis. While behavioral differences between sexes might account for some of the epidemiological differences, the contribution of underlying biology is not known. In this study, we performed regional proteomic analysis via laser-captured microdissection-coupled mass spectrometry and analyzed the data for sex-biased protein expression. We discovered that even in control lung, sex differences existed in both airway and alveolar regions. Sex differences became more pronounced in diseased regions, with sex-biased expression of diverse proteins including those involved in extracellular vesicle secretion, cellular metabolism, and extracellular matrix remodeling. These data suggest that baseline sex differences in lung proteome may contribute to sex-specific susceptibility, progression, and clinical outcomes in IPF, underscoring the need for future mechanistic and clinical studies to account for sex as a biological variable.

BackgroundHyaluronan (HA) is a major extracellular matrix glycosaminoglycan that regulates vascular integrity and immune signaling in the lung. Its biological effects are strongly size-dependent, with high-molecular-weight HA (HMW-HA) generally protective and low-molecular-weight HA (LMW-HA) pro-inflammatory. However, how different HA sizes and concentrations globally remodel endothelial cell signaling remains poorly understood. MethodsHuman lung microvascular endothelial cells (HULEC-5a) were treated with physiologic (200 ng/mL) or supraphysiologic (1 {micro}g/mL) concentrations of LMW-, medium-molecular-weight (MMW-), or HMW-HA. Cell viability was confirmed by LDH assay. Quantitative proteomics with downstream Ingenuity Pathway Analysis (IPA) was used to profile HA-induced signaling networks. ResultsProteomic analysis revealed a conserved HA-response signature across all conditions involving cell cycle regulation, senescence, and immune modulation, with distinct size-and dose-dependent differences. At supraphysiologic concentrations, HMW-HA suppressed proliferative and inflammatory pathways, consistent with a protective, quiescent phenotype. LMW-HA induced the broadest stress-associated proteomic changes, consistent with its role as a damage-associated molecular pattern. Unexpectedly, physiologic MMW-HA elicited the strongest responses, driving metabolic and cytoskeletal pathways including insulin signaling and Rho GTPase activity. Network analysis highlighted 176 overlapping pathways across HA treatments, with unique contributions of LMW- and HMW-HA to stress- versus barrier-stabilizing signaling, respectively. ConclusionHA is not a passive structural molecule but an active regulator of endothelial signaling, with effects shaped by both molecular weight and concentration. Our findings identify a distinct role for MMW-HA at physiologic levels and highlight how HA fragmentation and accumulation may contribute to endothelial dysfunction in lung injury, with implications for targeted HA-based therapies.

Background: Alveolar macrophage (AM) dysfunction contributes to Acute Respiratory Distress Syndrome (ARDS) pathogenesis. We investigated the role of extracellular vesicles (EVs) in mediating this dysfunction. Methods: Pulmonary EVs were isolated from broncho-alveolar lavage and non-directed bronchial lavage samples of ventilated sepsis patients with and without ARDS, and post-operative control patients via ultracentrifugation. AMs were isolated from lung tissue resections of lobectomy patients. AMs were treated with pooled EVs for 24 hours prior to functional, metabolic and autophagy profiling. EV cargo was profiled via small RNA transcriptomics and proteomics. Mechanistic role of EV microRNAs was assessed via mimic / antagomir transfection. Results: Pulmonary EVs from sepsis patients with ARDS impaired AM efferocytosis, and control EVs had no effect. ARDS EV treatment enhanced AM mitochondrial-linked respiration, but not glycolysis. ARDS EV treatment impaired LC3B-II and LAMP1 expression, indicating dysregulated AM autophagy-lysosomal machinery. Proteomics revealed downregulation of innate immune pathways in ARDS EVs. Transcriptomics revealed enrichment of 24 microRNAs in ARDS EVs; miR-652-3p was the most enriched, validated by RT-qPCR. EV miR-652-3p was associated with 90-day mortality (9.20 vs 0.59 RQ, p=0.0295) and inversely correlated with oxygenation (PaO2/FiO2). AM transfection with miR-652-3p mimic induced similar dysregulation of function and autophagy as ARDS EVs. Transfection of ARDS EVs with antagomirs to miR-652-3p prior to AM treatment partially rescued efferocytosis and autophagy. Conclusions: Targeting EV miR-652-3p may restore alveolar macrophage function and reduce excessive inflammation, thus offering a novel therapeutic strategy for patients with ARDS.

The increasing prevalence of antibiotic-resistant bacterial infections highlights the need for physiologically relevant in vitro models that recapitulate host-pathogen interactions. Pseudomonas aeruginosa is a clinically important opportunistic pathogen associated with hospital-acquired infections and chronic airway diseases, including cystic fibrosis, where dysregulated inflammatory responses contribute to disease progression. While air-liquid interface (ALI) models have advanced the study of airway epithelium, most of these modes lack immune components, limiting their ability to capture immune-epithelial interactions. Here, we expanded a previously established dual-cell ALI model incorporating human monocyte-derived macrophages to investigate how immune context, bacterial dose, and time influence early infection dynamics. Standard BCi-NS1.1 epithelial monocultures and macrophage co-cultures were infected with P. aeruginosa (PAO1) at low (100 colony-forming units (CFU) and high (1000 CFU) inoculum and analyzed over 10, 16, and 24 h post-infection (hpi). Macrophage presence did not significantly alter total bacterial burden but markedly influenced cytokine responses and bacterial spatial organization. Pro-inflammatory cytokines (interleukin (IL)-1, IL-1{beta}, Tumor Necrosis Factor (TNF)-) were enhanced in dual-cell culture models, while IL-6 exhibited a threshold-dependent response detectable only at higher bacterial loads. Confocal imaging revealed that macrophages altered bacterial distribution, promoting a more dispersed pattern compared to the clustered organization observed in epithelial monocultures. These effects were most pronounced at lower bacterial inocula. Together, our findings demonstrate that macrophages reshape early infection dynamics by modulating inflammatory signaling and bacterial spatial organization without affecting overall bacterial burden. This study highlights the importance of incorporating immune cells into in vitro airway models.

Genome-wide association studies have identified rare and common mutations associated with increased risk of pulmonary arterial hypertension (PAH), but the mechanism by which impaired SOX17 expression increases PAH risk is not known. Notably, SOX17 plays a critical role in endothelial identity during development by suppressing RUNX1 through binding to its promoter and directing stem and progenitor cells toward an endothelial rather than a hematopoietic cell fate. RUNX1 functions as a key regulator of myeloid differentiation, aberrant angiogenesis and adverse cardiac remodeling. Previously, we found that RUNX1 inhibition reverses pulmonary hypertension (PH) in multiple animal models. Here, we hypothesize that impaired expression of SOX17 in PAH leads to endothelial cell (EC) dysfunction by failing to suppress RUNX1. METHODSHuman pulmonary artery endothelial cells (HPAECs) with stable SOX17 CRISPR/Cas9 knockout or RUNX1 overexpression were generated and examined for endothelial and hematopoietic gene expression, proliferation, migration, apoptosis, and angiogenesis. Immortalized lymphoblastoid cell lines (LCLs) from PAH patients with SOX17 mutations and healthy controls were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into ECs. The effect of RUNX1 inhibition on Sugen/hypoxia-PH was examined in rats, SOX17 enhancer knockout (SOX17enhKO) mice, and Cdh5-CreERT2;Runx1(flox/flox);SOX17enhKO triple transgenic mice. SOX17 and RUNX1 expression were analyzed in peripheral blood samples from PAH patients (n=359). RESULTSHPAECs with SOX17 deletion or RUNX1 overexpression exhibited decreased expression of EC markers, enhanced proliferation and migration, defective angiogenesis, and decreased apoptosis. RUNX1 siRNA knockdown or RUNX1 inhibition by Ro5-3335 partially restored the endothelial properties in SOX17 KO HPAECs. ECs differentiated from SOX17 mutant PAH patient iPSCs exhibited upregulated RUNX1 expression and loss of endothelial identity, which was also partially restored by RUNX1 siRNA or Ro5-3335. In addition, SOX17enhKO mice had increased RUNX1 expression and susceptibility to Sugen/hypoxia-induced PH (SuHx-PH). Treatment with RUNX1 inhibitors or inducible endothelial-specific deletion of RUNX1 rescued SuHx-PH susceptibility in SOX17enhKO mice. RUNX1 inhibitors Ro5-3335 and Ro24-7429 also reversed SuHx-PH in wild-type rats. In addition, plasma RUNX1 expression was higher in PAH patients lacking detectable SOX17 expression than in patients with detectable SOX17 expression. CONCLUSIONSImpaired SOX17 expression increases the risk of PAH through insufficient suppression of RUNX1, leading to pulmonary endothelial dysfunction. RUNX1 inhibition mitigates PH associated with SOX17 deficiency and may represent a novel therapeutic strategy for PAH, especially those with rare or common SOX17 mutations.

Ozone (O3)-driven pulmonary inflammation is partly regulated by damage associated molecular patterns (DAMPs) binding to scavenging receptors (SRs). However, how SRs and DAMPs regulate O3-induced pulmonary inflammation remains incompletely understood. CD163 is a SR responsible for clearing cell free hemoglobin (CFH), a DAMP which accumulates during acute pulmonary injury and is associated with worsening respiratory outcomes. We hypothesized that increased CD163 is necessary for reducing CFH levels and resolving O3-induced pulmonary injury. To test this hypothesis, we defined CD163 and CFH responses to O3 exposure in C57BL/6N (WT) and CD163 deficient (Cd163-/-) mice, as well as in human bronchoalveolar lavage fluid (BALF). In WT mice, lung Cd163 expression was significantly increased by O3 during peak inflammation and declined 24 hours post exposure. Human exposure studies revealed a diversity of Cd163 expression and a reduction of CFH following O3 exposure, suggesting regulation of this pathway in humans. When compared to WT mice, Cd163-/- mice had augmented O3-induced pulmonary injury, inflammation, and oxidative stress. Further, the antioxidant EUK-134 did not reduce O3-induced pulmonary oxidative stress in Cd163-/- mice, suggesting a role for CD163 in the pulmonary response to oxidative insults. Furthermore, compared to WT controls, Cd163-/- mice receiving an oropharyngeal aspiration of CFH had a significant increase in airspace inflammation. Combined, these findings suggest that CD163 mediated clearance of CFH is involved in resolving O3-induced pulmonary injury, inflammation, and oxidative stress. New & NoteworthyOzone (O3) is known to induce damage associated molecular patterns (DAMPs) which drive lung inflammation. The scavenging receptor, CD163, binds and clears the DAMP cell free hemoglobin (CFH), which accumulates during sterile lung injury. Our findings indicate that O3 exposure alters CD163 expression in the lung and that mice lacking Cd163 expression have more lung inflammation. Our data indicate that CD163 serves a protective role in response to acute O3 exposure perhaps through CFH clearance.