American Journal of Physiology-Lung Cellular and Molecular Physiology

BackgroundChronic obstructive pulmonary disease (COPD) is a chronic lung condition characterised by accelerated lung aging. Extracellular vesicles (EVs), which can be categorised into large EVs (LEVs) and small EVs (SEVs), may play a critical role in intercellular communication. They contribute to the pathogenesis of COPD by transporting and transferring microRNAs (miRNAs). This study profiles cells and EV-associated miRNAs from both healthy and COPD small airway (SA)-epithelial cells and SA-fibroblasts and identifies the biological pathways associated with these miRNAs. MethodsEVs were isolated from conditioned media of healthy and COPD SA-epithelial cells and SA-fibroblasts, both at baseline and following H2O2 exposure. MiRNAs were extracted from cells and EVs and analysed by small RNA (smRNA) sequencing. ResultsSmRNA sequencing of COPD SA-epithelial cells and EVs revealed that four miRNAs were upregulated and fourteen were downregulated in the cells compared to healthy controls. COPD LEVs displayed nine upregulated and ten downregulated miRNAs, while SEVs showed ten upregulated and eleven downregulated miRNAs. Only one miRNA consistently upregulated in COPD SA-epithelial cells, LEVs, and SEVs. The various differentially expressed miRNAs were primarily associated with cellular senescence pathways. In SA-fibroblasts 39 miRNAs were upregulated in COPD compared to healthy cells. 14 miRNAs were upregulated in COPD LEVs and 11 downregulated, whereas SEVs exhibited twenty upregulated and eleven downregulated miRNAs. Overlap was limited, with only three miRNAs consistently upregulated in SA-fibroblasts and EVs. These miRNAs were linked to pathways related to fibrosis and cellular senescence. Furthermore, oxidative stress alters the miRNA profiles detected in cells and EVs differently between cells from healthy individuals and COPD patients. ConclusionsCOPD alters miRNA signatures in cells and their EVs, with limited overlap between compartments. These COPD-associated miRNAs are enriched in pathways driving cellular senescence and fibrosis, suggesting a potential role in disease progression.

Breastfeeding provides health benefits in childhood, reducing the frequency of gastrointestinal and respiratory infections. Breastmilk (BM) is a rich source of bioactive molecules including extracellular vesicles (EVs), which exert immunomodulatory signalling in recipient cells, with cargo that is affected by maternal characteristics. Here we investigated the biophysical characteristics of BM-EVs from mothers with (asthmatic BM-EVs) or without asthma (control BM-EVs) and their effect on the release of cytokines from primary human hTERT-immortalized airway smooth muscle cells (hASMs) from asthmatic or non-asthmatic (control) donors. BM-EVs were isolated using size exclusion chromatography (N=5/group), characterized biophysically and by EV-specific protein markers. In addition, BM-EV were co-cultured (48h) with primary hASM cells from both non-asthmatic (control) and asthmatic donors to determine the effect on cytokine release. All participants were Caucasian and the BM was collected 12-15 weeks postpartum. BM-EVs showed the presence of intact and small-EVs ([~]100 nm). Asthmatic BM-EVs appeared to have a smaller average EV size (135.6 nm) vs. controls (148.3 nm, p=0.0613), but [~]5-fold higher concentration of both total (p=0.0014) and small EVs (p=0.0016). The expression of EV subtype protein expression was reduced in asthmatic BM-EVs vs. control BM-EVs: CD63 by 86% (p=0.0224), flotillin-1 by 40% (p=0.0196), CD9 by 24% (p=0.0646) and HSP70 by 69% (p=0.0873). Asthmatic BM-EVs co-cultured with hASMs from control donors decreased pro-inflammatory cytokine release: MCP-1 by 55% (p=0.0286), IL-6 by 45% (p=0.0801) and IL-2 by 32% (p=0.0970) vs. control-BM-EVs. Conversely, asthmatic BM-EVs co-cultured with hASMs from asthmatic donors increased secretion of anti-inflammatory cytokine IL-10 by 32% (p=0.0660), and IL-1Ra by 75% (p=0.0875), and pro-inflammatory IL-2 by 57% (p=0.0688) vs. control-BM-EVs. Internalization of control and asthmatic BM-EVs was confirmed by labelled EV uptake experiments. No detrimental effects on cell viability with BM-EV treatment were observed. In summary, asthmatic BM-EVs are smaller and enriched in BM, and exert differential effects on cytokine release in a BM-donor and recipient-cell specific manner. Given that BM can enter infant airways, the immunomodulatory effects of BM-EVs on hASMs warrants further investigation to delineate the under underlying mechanisms.

AimThe mammalian tracheal epithelium is composed by different cell types unevenly distributed along the proximal-distal axis. Nevertheless, variations in expression and function of ion channels and transporters participating in fluid absorption and secretion had never been studied separately in proximal and distal sections of the mouse trachea. In this work, we aim to characterize basal and stimulated absorption and secretion of fluid obtained from proximal and distal trachea from the same animal. MethodsUssing chamber experiments were performed using a custom-made tissue slider that allowed the mounting small tracheal sections, where response to agonists and blockers was recorded. The role of the NKCC1 co-transporter was studied using the Slc12a2-/- mouse. A genetically tomato-induced mouse model was used to assess co-expression of NKCC1 and ASCL3 by immunofluorescence. Animals were instilled with different interleukins (ILs) to determine changes in absorption, secretion and mucus properties. ResultsProximal trachea didnt participate in sodium absorption but exhibited higher cAMP- and succinate-induced anion secretion than the distal section. NBCe1-dependent bicarbonate and TMEM16A-driven chloride secretion was significantly higher in the distal section. NKCC1+ cells were found in the submucosal glands (SMGs) and abundant patches of NKCC1+ cells in the distal region. Isolated NKCC1+ cells co-expressing ASCL3 were also detected. ILs treatment changed the electrophysiological properties of the distal but not the proximal trachea. ConclusionsOur experiments determined that the mouse trachea organizes its functions differentially in the proximal and distal sections, based in the functional distribution of channels, transporters and receptors. While the distal trachea drastically changed its responses to agonists inducing anion secretion the proximal trachea was unperturbed by the action of ILs.

IntroductionIn acute lung injury (ALI), clinical data show that while mortality rates are similar between sexes, women require shorter ventilation times and intensive care unit stays than men, yet preclinical studies show conflicting sex-specific vulnerabilities. We reasoned that a hidden dosing bias may explain the inconsistency, as intratracheal bleomycin is scaled to body weight, even though lung mass grows more slowly than total body mass, so age-matched males, whose body mass outpaces lung growth, inevitably receive more drug per gram of lung than females. MethodsWe compared age-matched (12-week) and body-weight-matched ([~]300g) Sprague-Dawley rats receiving intratracheal bleomycin (2.5mg/kg) or saline. Both cohorts underwent functional assessments (plethysmography, lung mechanics, arterial gases, histology) at day 7; weight-matched animals exclusively underwent mechanistic profiling (BALF analysis, cytokine multiplex, paired mRNA/miRNA-sequencing, immunoblotting). ResultsMales developed worse hypoxemia (PaO2: age-matched p = 0.045; weight-matched p = 0.027) with higher respiratory rates (both cohorts p < 0.05). Weight-matched males showed greater compliance loss (p = 0.029), increased BALF protein (p = 0.008), and elevated IL-1{beta} (p =0.005) and TNF- (p = 0.017). RNA-sequencing identified 2,393 male versus 1,533 female differentially-expressed genes, with males activating complement-coagulation cascades while females enriched ECM-remodeling/BMP-signaling pathways. Males exhibited significant miR-672-3p suppression (p < 0.0001), inversely correlating with inflammatory targets. SERPINA3 and its upstream regulator STAT3 showed significantly higher induction in males (both p < 0.0001), whereas females exhibited higher BMPR2 protein levels (p = 0.009) and preserved IL-10 (p = 0.023). ConclusionsBody-weight matching corrects unrecognized allometric bias affecting preclinical ALI sex-difference studies. Both cohorts demonstrated male vulnerability with worse hypoxemia and increased respiratory rates. Weight-matched molecular analyses revealed distinct responses: males showed significant miR-672-3p suppression with concurrent inflammatory mediator upregulation, including higher SERPINA3, IL-1{beta}, and TNF-. In contrast, females maintained higher miR-672-3p levels alongside elevated BMPR2/IL-10, suggesting that divergent post-transcriptional regulation contributes to functional differences and may inform sex-specific therapeutic strategies.

Previous studies indicate that pigs with CFTR-null and CFTR-{Delta}F508 mutations develop multiorgan disease similar to that in people with cystic fibrosis (CF). At birth, their airways exhibit host defense defects that predispose to airway infection, inflammation, and mucus accumulation. The CFTR-G551D mutation causes CF by producing CFTR channels that localize correctly but have reduced channel activity. Ivacaftor (VX-770) is a small molecule drug developed to potentiate CFTR activity. To test the phenotype of the CFTR-G551D mutation in pigs and determine whether ivacaftor can rescue CF abnormalities, we developed CFTRG551D/G551D (CF-G551D) pigs through homologous recombination in fetal fibroblasts and somatic cell nuclear transfer. Newborn CF-G551D piglets exhibited phenotypes similar to CF-null piglets, including meconium ileus, exocrine pancreatic destruction, micro-gallbladder, vas deferens destruction, and airway structural abnormalities. Compared to wild-type pigs, CF-G551D pigs had reduced forskolin-stimulated short-circuit current in airway and intestinal tissues. Ivacaftor increased the single-channel open state probability of CFTR-G551D and increased short-circuit current to near wild-type levels. Similar to our other CF pig models, we found that 100% of CF-G551D pigs were born with meconium ileus. To test whether in utero ivacaftor treatment could prevent or alleviate meconium ileus, pregnant sows were treated with ivacaftor beginning at day 35 of gestation and continuing until delivery. This treatment rescued the pancreas, gallbladder, and vas deferens phenotype in the majority of CF-G551D pigs. Animals that were spared from meconium ileus were able to survive without ivacaftor treatment. Airway disease developed similar to other CF pig models. These findings indicate that this model may be useful for studies in which CFTR function can be reversed, for investigating in utero CFTR correction strategies, and for longitudinal studies in CF pigs.

The Acute Respiratory Distress Syndrome (ARDS) is a life-threatening cause of respiratory failure, and patients who develop ARDS frequently require mechanical ventilation, which puts them at risk of developing ventilator induced lung injury (VILI). Both VILI and ARDS can induce pulmonary surfactant dysfunction, but the mechanisms are not known. Here we report a novel role for a phospholipid binding protein, Annexin A2 (AnxA2), in the regulation of surfactant composition and function following injurious ventilation. Wild type and AnxA2-/-mice were subjected to injurious ventilation and we found that AnxA2-/- mice developed stiffer lungs following VILI that was not due to differences in barrier permeability or inflammation. Furthermore, we found that pulmonary surfactant from AnxA2-/- mice had reduced surface tension lowering properties and that this was due to a reduction in 1-palmitoyl-2-oleoylphosphatidylglycerol, or POPG. Quantitative analysis of surface tension-surface area hysteresis loops obtained from surfactant isolated from AnxA2-/- mice showed a defect in phase transitions during compression. In summary, Annexin A2 regulates surfactant function during injurious ventilation and may serve as a novel therapeutic target to prevent surfactant dysfunction in patients with ARDS who require mechanical ventilation.

BackgroundLung ischemia-reperfusion (IR) injury drives early morbidity after lung transplantation and cardiothoracic surgery, yet targeted preventive therapies are lacking. The gut-lung axis and microbiota-derived tryptophan metabolites, including indole-3-propionate (IPA), may regulate pulmonary immunity and inflammation. We investigated whether a tryptophan-rich (Trp-Rich) diet attenuates sterile lung IR injury by increasing microbiota-derived indole metabolites and reprogramming alveolar macrophage (AM) inflammatory responses. MethodsC57BL/6 mice received isocaloric tryptophan-standard (Trp-Std; 0.18%) or Trp-Rich (0.60%) diets for 14 days, then underwent unilateral left lung IR (60 min ischemia followed by 60 min reperfusion). Oxygen saturation, lung cytokines, and aryl hydrocarbon receptor (AhR) signaling readouts (Cyp1a1/Cyp1b1) were evaluated. Gut microbiota was profiled by 16S rRNA sequencing, and targeted metabolomics quantified tryptophan metabolites in feces, portal vein (PV) plasma, and lung tissue. To further assess inflammatory priming in vivo, mice were additionally challenged with intratracheal lipopolysaccharide (LPS). Mechanistic studies compared IPA with related indole metabolites in MH-S cells and primary human AMs, including ex vivo nutritional IR, LPS stimulation, and AhR stimulation and blockade using synthetic agonists and antagonists. ResultsTrp-Rich feeding improved post-IR oxygenation, reduced lung IL-1{beta}, and increased pulmonary Cyp1a1/Cyp1b1 gene expression. Trp-Rich diet remodeled the gut microbiota, including enrichment of Bifidobacterium and Lactobacillus, and increased IPA levels across feces, PV plasma, and lung tissue, with lower kynurenine/IPA ratios across matrices. In the LPS intratracheal challenge, Trp-Rich feeding reduced IL-6 levels in lung tissue and systemic plasma. Primary murine AMs isolated from Trp-Rich mice also showed reduced IL-1{beta} and IL-6 release in an ex vivo nutritional IR model. Among tested indole metabolites, IPA showed the strongest dose-dependent suppression of LPS-induced cytokines and chemokines in MH-S cells and primary human AMs, remained active in the ex vivo nutritional IR model, and its anti-inflammatory effect was abrogated by AhR blockade and enhanced by co-treatment with other indole metabolites. ConclusionsA Trp-Rich diet attenuated sterile lung IR injury, coinciding with gut microbiota remodeling, increased systemic and pulmonary IPA, reduced inflammatory priming, and reprogrammed AM responses. These data support diet- or microbiome-directed strategies targeting IPA-AhR signaling to mitigate perioperative lung IR injury. Caption for graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/714281v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@1b06a9corg.highwire.dtl.DTLVardef@1273f33org.highwire.dtl.DTLVardef@1a63a2borg.highwire.dtl.DTLVardef@350e1c_HPS_FORMAT_FIGEXP M_FIG A tryptophan-rich diet remodels the gut microbiota and indole metabolite profiles, including IPA, enhances alveolar macrophage AhR signaling, and attenuates sterile lung ischemia-reperfusion injury. C_FIG

BackgroundThe extracellular matrix (ECM) is a dynamic network that provides structural support and maintains tissue homeostasis. Collagens are the main structural components of the ECM, occupying distinct tissue compartments and serving specialized roles. Dysregulated ECM remodeling involves an imbalance between collagen production and degradation, generating neoepitope-specific fragments that can be released into circulation. Serological measurements of these fragments can be used as biomarkers of disease and have been associated with progression and mortality in different fibrotic diseases, including pulmonary fibrosis (PF). This study aimed to investigate whether these systemic biomarkers originate from human lung tissue in patients with PF and non-fibrotic controls. MethodsLung tissue was collected from patients with PF (n = 21) and non-fibrotic controls (n = 21) and processed in parallel as formalin-fixed paraffin-embedded or snap-frozen samples. Serum samples were collected from patients with PF and healthy controls (n = 21). Neoepitope-specific biomarkers reflecting type III, IV, and VI collagen production (PRO-C3, PRO-C4, and PRO-C6) and degradation (C3M, C4M, C4Ma3, and C6M) were quantified in serum and proteolytically degraded lung tissue, and their spatial distribution was assessed by immunohistochemistry in lung tissue sections. ResultsAll collagen remodeling biomarkers were significantly increased in serum of patients with PF compared with healthy controls (PRO-C3: p = 0.0006, all others: p < 0.0001). Collagen degradation fragments (C3M, C4M, and C6M) could be generated and released from both non-fibrotic and fibrotic human lung tissue following proteolytic cleavage with pepsin, collagenase, and/or MMP-9. All biomarkers were detected in lung tissue by immunohistochemical staining, with widespread distribution of type III and IV collagen fragments, whereas type VI collagen (PRO-C6) production showed a more compartment-specific pattern. ConclusionsThese findings demonstrated that neoepitope-specific collagen remodeling biomarkers, usually detected in circulation, are present and can be released from human lung tissue. Their spatial distribution suggests that ECM remodeling is heterogeneous and differs according to collagen type and distinct tissue compartments. Collectively, our findings support the use of collagen remodeling biomarkers as tools to assess ECM remodeling in pulmonary disease.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by aberrantly activated, apoptosis-resistant profibrotic lung (myo)fibroblasts. Prior research has demonstrated that lung fibroblasts from patients with IPF exhibit resistance to DNA damage, suggesting that this behavior contributes to their persistent survival and continuous proliferation. We propose that elevated levels of the DNA damage repair protein RAD51 regulate myofibroblast activation and apoptosis and provide a potential therapeutic target to impede fibrosis progression. MethodsHuman lung fibroblasts were transfected with siRNA against RAD51 or treated with RAD51-specific inhibitor B02 and markers of fibrosis, DNA damage, apoptosis, metabolic reprogramming, and mitochondrial dynamics were assessed. The preclinical efficacy of B02 was evaluated in human precision cut lung slices (PCLS) and in a mouse model of pulmonary fibrosis. FindingsRAD51 expression was significantly upregulated in the lungs and lung fibroblasts of IPF patients. Knockdown or inhibition of RAD51 in fibroblasts reduced profibrotic marker expression, suppressed mTORC1 signaling and mitochondrial function, and increased apoptosis susceptibility. Pharmacological inhibition of RAD51 shifted the profibrotic phenotype towards a fibrosis-resolving state in human and mouse PCLS, and in a bleomycin-induced mouse model of lung fibrosis. InterpretationThe inhibition of RAD51 exerts therapeutic benefits in lung fibrosis by promoting apoptosis. Our findings identify that inhibiting RAD51 with B02 in fibroblasts impairs DNA repair and induces metabolic reprogramming, making it a potential therapeutic target. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSPulmonary fibrosis (PF) is characterized by excessive fibroblast activation and subsequent deposition of extracellular matrix (ECM) proteins, which ultimately disrupt normal lung architecture. A significant contributing factor to the pathogenesis of pulmonary fibrosis is the presence of fibroblasts that are resistant to apoptosis, preventing normal wound healing. Recent studies highlight the DNA repair protein RAD51 as effective in protecting fibroblasts from death induced by chemotherapy and ionizing radiation. These finding suggested that RAD51 could have a role in fibroblast activation and apoptosis resistance in pulmonary fibrosis. Added value of this studyWe demonstrated that RAD51 is important for maintaining apoptosis-resistant fibrotic fibroblasts and their metabolic abnormalities. Our findings indicated that TGF{beta}-mediated upregulation of RAD51 reduces DNA damage, activates multiple pathways related to fibroblast activation and proliferation, and induces metabolic reprogramming, ultimately regulating apoptosis. Mechanistically, RAD51 inhibition enhanced p53 acetylation at lysine 120 and upregulated the expression proapoptotic proteins PUMA/BAK in mitochondria, promoting apoptosis. Pharmacological inhibition of RAD51 using the specific inhibitor B02 during the fibrotic phase of experimental lung disease effectively ameliorated pulmonary fibrosis. Implications of all the available evidenceOur findings establish that RAD51 plays an important role in the survival of apoptosis-resistant fibrotic fibroblasts. We propose that reducing RAD51 expression leads to the metabolic reprogramming of activated fibroblasts, resulting in decreased mitochondrial respiration, reduced ATP levels, and diminished glycolysis or glutaminolysis. These observations suggest that targeting energy metabolism through RAD51 inhibition could be a viable strategy to enhance apoptosis, thereby creating a therapeutically targetable pathway in fibrotic cells. These findings highlight the potential of RAD51 as a therapeutic target for the treatment of IPF.

Chronic obstructive pulmonary disease (COPD) is characterized by neutrophilic inflammation, emphysema, and mild pulmonary hypertension (PH). Oxidative/nitrosative stress are key drivers, but specific mitochondrial mechanisms remain unclear. We show increased expression of the regulatory mitochondrial cytochrome c oxidase subunit 4 isoform 2 (COX4I2) in an early murine model and human COPD. After 8 months of cigarette smoke exposure, Cox4i2-/- mice were completely protected from emphysema but not from PH, associated with reduced nitrosative stress, inflammation, and apoptosis. Using a novel Cox4i2 reporter mouse and in situ hybridization of human lungs, COX4I2 was detected in precapillary ACTA2+ cells and capillary pericytes. COX4I2 promotes mitochondrial reactive oxygen species (mtROS) production in these cells, thereby enhancing neutrophil migration and alveolar type II cell apoptosis, and modulates angiogenesis. In contrast to Cox4i2-/-, mitochondria-targeted antioxidant MitoQ reversed emphysema and PH, suggesting pericyte-specific regulation of COPD pathologies and mtROS inhibition as a therapeutic approach in COPD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/703513v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@83bca4org.highwire.dtl.DTLVardef@d5ebaborg.highwire.dtl.DTLVardef@632d1borg.highwire.dtl.DTLVardef@1267a13_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease characterized by epithelial cell senescence. Pirfenidone and nintedanib are approved drugs for the treatment of IPF. They significantly slow disease progression, but their mechanisms of action, especially on alveolar type 2 (AT2) cells, are poorly understood. We addressed this question by evaluating colony formation and growth of human AT2 cells co-cultured with fibroblasts in organoid culture in the presence of pirfenidone and nintedanib. We further evaluated molecular changes induced by these drugs via single cell RNA-seq of treated organoids. MethodsAT2 cells isolated from normal donor lungs or IPF patients were mixed with human fibroblasts in 3D culture and grown in the absence or presence of pirfenidone or nintedanib. After 14 days in culture, the organoids were quantified and cells extracted from Matrigel for single cell RNA-seq. ResultsAT2 cell organoids cultured in the presence of pirfenidone or nintedanib resulted in increased colony formation and, in the case of nintedanib, in larger colonies. We observed that untreated or pirfenidone treated AT2 cells lost surfactant protein C (SFTPC) expression and acquired an expression profile consistent with keratin (KRT)17high/KRT5- basaloid cells, whereas a larger proportion of nintedanib treated cells retained SFTPC expression. In contrast, AT2 cells treated with TGF{beta} inhibitor exhibited intermediate (SFTPC-/KRT17low) gene expression profile. ConclusionThese results suggest that nintedanib maintains an AT2-like expression state in culture and acts proximal to TGF{beta}. Conflict of Interest StatementPJW was supported by grants from Boehringer Ingelheim, Roche, Sanofi, Pliant and Arda Therapeutics and received personal fees from Boehringer Ingelheim and Sanofi. None of these companies had a role in the design or analysis of the study or in the writing of the manuscript. ASM, GP, JRR and DG are employees of Genetech. The other authors have no conflicts of interest to declare.

RationaleIdiopathic pulmonary fibrosis (IPF) is a progressive, incurable scarring disease of the lung. A common genetic variant near AKAP13, a multifunctional scaffold protein that integrates intracellular signalling through its interactions with RhoA and protein kinase A (PKA), has been associated with IPF susceptibility and elevated AKAP13 mRNA expression in lung tissue from patients. However, its contribution to the pathogenesis of IPF remains unclear. ObjectiveThis study investigates how an AKAP13 variant alters epithelial signalling and evaluates the therapeutic potential of targeting AKAP13. Findingsrs62025270-bearing iHBECs exhibited selective upregulation of AKAP13 isoforms, accompanied by increased cell adhesion and reduced proliferation. Transcriptomic profiling revealed upregulated fibrosis-related genes in rs62025270-bearing iHBECs, including SAA1, FGF2, MMP1, CTSB, COL4A1, and CDKN1A. rs62025270-bearing iHBECs also displayed increased RhoA activation and SMAD2 phosphorylation following LPA stimulation. Furthermore, cells harbouring the AKAP13 variant showed reduced intracellular cAMP levels. Pharmacological inhibition of AKAP13 with A13 reversed the pro-adhesive phenotype and reduced RhoA activation in iHBECs. Moreover, in IPF-derived PCLS, A13 suppressed SERPINE1, CCN2, and MMP7 expression, reduced SMAD2 nuclear translocation, and decreased hydroxyproline levels. ConclusionsPresence of an AKAP13 variant disrupts epithelial homeostasis and promotes pro-fibrotic signalling. Inhibition of AKAP13s RhoGEF domain with A13 restores epithelial function and attenuates fibrotic activation, supporting AKAP13 as a therapeutic target in IPF.

Testosterone seems protective against asthma, but the underlying mechanisms are uncertain. Herein, the effect of testosterone was investigated on several features of experimental asthma. Systemic testosterone was first altered to subphysiological, physiological, or supraphysiological levels in male BALB/c mice through orchiectomy and testosterone supplementation. Testosterone (0.25 mg/day/30 g of body weight) was delivered continuously during 20 days using an implanted pump. At day 10, each group was exposed intranasally to either saline or house dust mite (HDM) once daily for 10 consecutive days to induce allergic lung inflammation. The day after the last exposure, respiratory mechanics was measured at baseline and in response to nebulized methacholine. Bronchoalveolar lavages (BAL) and lung tissues were also collected to quantify inflammation. Baseline respiratory mechanics were altered in mice with subphysiological levels of testosterone, with signs of small airway narrowing heterogeneity and closure. Testosterone drastically inhibited the HDM-induced inflammation. Yet, testosterone also increased the response to methacholine, as well as hysteresis, which are both indicators of enhanced airway smooth muscle activity. While it suggests that testosterone increases the contractility of the smooth muscle, it simultaneously and markedly inhibits inflammation. Explanations as to how these outcomes may lead to protection in asthma are discussed.

BackgroundPulmonary arterial hypertension is a progressive and fatal cardiopulmonary disease marked by excessive proliferation of pulmonary artery smooth muscle cells (PASMCs), pathological vascular remodeling, and ultimately right heart failure. Dysregulated BMPR2 signaling is a central molecular hallmark of PAH and is often associated with epigenetic suppression of BMPR2 expression. Switch-independent 3a (SIN3a), a transcriptional co-regulator and chromatin-modifying scaffold protein, has emerged as a key regulator of BMPR2 expression, yet its role in PAH pathogenesis remains poorly defined. MethodsWe generated smooth muscle cell-specific SIN3a knockout mice (SIN3aSMC-/-) and subjected them to the Sugen/hypoxia protocol to induce PAH. A cohort received Sotatercept treatment. In parallel, human PASMCs engineered to overexpress SIN3a were exposed to TGF{beta}1 or hypoxia (1% O2) in vitro. Comprehensive transcriptomic profiling and pathway analyses identified molecular networks regulated by SIN3a and Sotatercept. Hemodynamic measurements and detailed morphometric analyses were used to assess disease severity and treatment response. ResultsSIN3a overexpression in PASMCs suppressed hypoxia-inducible factor-1 and TGF-{beta}/SMAD2/3 signaling, restored BMPR2 expression, and activated canonical BMP signaling through SMAD1/5/9 phosphorylation, while reducing pro-inflammatory, oxidative, and fibrotic gene programs. Transcriptomic analyses revealed that SIN3a and Sotatercept converge on gene networks that regulate BMPR2 signaling, ID isoforms, extracellular matrix remodeling, oxidative stress, and inflammation. In vivo, smooth muscle-specific SIN3a deletion exacerbated Sugen/hypoxia-induced PAH, increasing right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling, and fibrosis. Sotatercept treatment reversed these pathological features, restored SIN3a and BMPR2 expression, reactivated BMP signaling, and attenuated HIF-1 and TGF-{beta} signaling in SIN3a-deficient mice. ConclusionsSIN3a is a central epigenetic regulator of PASMC homeostasis that integrates oxidative stress, inflammation, and fibrotic signaling. Loss of SIN3a accelerates PAH progression, whereas Sotatercept restores SIN3a expression, rebalances BMPR2 and TGF-{beta} signaling, and attenuates pulmonary vascular remodeling and right ventricular dysfunction. Together, these findings identify SIN3a as a disease-relevant therapeutic target and support the use of Sotatercept as a disease-modifying approach for pulmonary vascular disease.

Mechanical ventilation (MV) can induce or exacerbate ventilator-induced lung injury (VILI), particularly in mechanically heterogeneous lungs with pre-existing injury. We investigated VILI in a rat model of bleomycin-induced lung injury and compared it with healthy controls using a combined in-vivo and ex-vivo imaging approach. Previously acquired in-vivo data from four-dimensional (4D) phase-contrast synchrotron micro-computed tomography (microCT) and forced oscillation measurements showed increased lung elastance and reduced local acinar strain in bleomycin-induced injured lungs at baseline and after injurious MV. To identify structural and mechanical correlates, we performed automated three-dimensional (3D) pore analysis and atomic force microscopy (AFM) on formalin-fixed, paraffin-embedded lung tissue, complemented by histology and spatial co-registration. Ex-vivo analysis revealed pronounced airspace enlargement after injurious MV of healthy lungs, whereas this effect was attenuated in fibrotic lungs. AFM demonstrated region-specific mechanical responses, and correlation analyses linked pore geometry and nanoscale stiffness to in-vivo lung mechanics. Spatial analysis further showed colocalization of VILI-associated airspace damage with injured regions. Overall, extracellular matrix remodelling modifies the lungs mechanical response to injurious MV. This multiscale correlative approach provides mechanistic insight into the interplay between lung injury and VILI and informs ventilation strategies in structurally altered lungs.

BackgroundIdiopathic pulmonary fibrosis (IPF) remains a fatal interstitial lung disease with limited diagnostic specificity and therapeutic options. This study integrates bulk and single-cell RNA sequencing (RNA-seq) to identify novel biomarkers and elucidate molecular mechanisms underlying IPF pathogenesis. MethodsWe prospectively enrolled 14 treatment-naive IPF patients and 6 controls. Bulk RNA-seq was performed on bronchoalveolar lavage fluid (BALF), while single-cell RNA-seq analyzed lung tissues from 4 IPF patients and 3 controls. Differentially expressed genes (DEGs) were identified (|log2FC| >1, FDR <0.05), followed by functional enrichment, protein-protein interaction (PPI) network analysis, and cell-type-specific expression profiling. Results1. DEG Identification: Bulk RNA-seq revealed 108 DEGs (24 upregulated, 84 downregulated). KEGG enrichment analysis of DEGs revealed that upregulated genes were mainly enriched in inflammation and immune pathways (such as NF-{kappa}B signaling pathway, Fc epsilon RI signaling pathway, B cell receptor signaling pathway, phagosome, Fc gamma R-mediated phagocytosis), pyrimidine metabolism, cell cycle, and PI3K-Akt signaling pathway. 2. PPI Network: Module analysis identified a proliferative gene module 1 (NUF2, CEP55, ANLN, TTK, TK1, MYBL2, CCNA2, RRM2, CDT1) linked to cell division and cycle regulation. 3. Single-Cell Insights: scRNA-seq of 30,477 cells delineated 11 populations. Module 1 genes exhibited predominant expression in proliferating cells, Module 1 signature score of proliferating cells was significantly higher in IPF than in control group. 4. Pathogenic Links: Key genes (e.g., CEP55, TTK) were associated with PI3K/AKT signaling, epithelial-mesenchymal transition (EMT), and anti-apoptotic pathways, mirroring oncogenic mechanisms. ConclusionThis multi-omics approach uncovers a proliferation-centric gene module in IPF, revealing shared molecular pathways with tumorigenesis. Our findings highlight novel diagnostic biomarkers and suggest repurposing cell cycle inhibitors as potential therapies. Future studies should validate these targets in preclinical models to advance precision medicine for IPF.

Asthma is the most common chronic respiratory disease of childhood and is strongly associated with genetic variants at the 17q21 locus that increase expression of ORMDL3, a negative regulator of serine palmitoyl-CoA transferase (SPT), the rate-limiting enzyme in de novo sphingolipid synthesis. Reduced sphingolipid production has been linked to airway hyperreactivity, a key physiological feature of asthma, but the mechanisms connecting altered sphingolipid metabolism to airway dysfunction remain unclear. We examined whether sphingolipid metabolites regulate airway smooth muscle reactivity. Circulating sphingolipids were quantified in children with asthma carrying 17q21 risk alleles and in mice with reduced SPT activity. Functional airway responses were assessed in precision-cut lung slices exposed to sphingosine-1-phosphate (S1P), sphinganine-1-phosphate (Sa1P), and S1P receptor antagonists. Homozygous carriers of the rs7216389 risk allele and SPT-deficient mice displayed an increased S1P-to-Sa1P ratio. In functional assays, Sa1P opposed S1P-induced airway contraction, and increasing Sa1P availability reduced airway hyperresponsiveness. These findings identify the S1P/Sa1P axis as a metabolic rheostat regulating airway smooth muscle tone and suggest that targeting sphingolipid metabolism may offer a therapeutic strategy to mitigate intrinsic airway hyperreactivity in asthma. One sentence summaryAn imbalance between sphingosine-1-phosphate and sphinganine-1-phosphate links the asthma risk locus 17q21 to airway hyperreactivity and reveals sphingolipid metabolism as a potential therapeutic target.

Idiopathic pulmonary fibrosis (IPF) is characterized by impaired alveolar type 2 cell regeneration. However, robust in vitro models of human distal lung epithelium are limited. In this study, we generated immortalized AT2 cell lines from healthy and IPF lungs using HTII-280 sorting and SV40 large T antigen transduction. These lines retain key features of alveolar epithelial biology in both 2D and 3D cultures, including self-renewal, differentiation, and transitional cell states. They form 3D organoids efficiently under optimized feeder-free, serum-free medium conditions, with higher colony-forming capacity in healthy AT2 cell lines comparing with IPF AT2 cell lines. These accessible models recapitulate alveolar epithelial biology, offering a platform for cell-biology research and therapeutic development in lung diseases.

To explore the mechanism of Hsa_circ_0000629 adsorbing miR-212-5p/ nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) through sponge in bronchial asthma. Twenty BALB/C mice were randomly divided into a normal control group and an asthma group. Pathological changes in lung tissue were observed via HE staining. Human bronchial epithelial cells (16HBE) were transfected with Hsa_circ_0000629 overexpression group (Hsa_circ_0000629-over), Hsa_circ_0000629 siRNA (Hsa_circ_0000629-si), mimic NC, miR-212-5p mimic, inhibitor NC, miR-212-5p inhibitor, and LPS+Hsa_circ_0000629 si. LPS-induced asthmatic cell models (LPS group) and untransfected 16HBE cells (NC group) served as controls. qRT-PCR was used to measure Hsa_circ_0000629, miR-212-5p and NLRP3 expression. ELISA assessed interleukin 18 (IL-18), interleukin 1{beta} (IL-1{beta}), interleukin 6 (IL-6) and tumor necrosis factor - (TNF-) levels. Cell proliferation and the apoptosis were evaluated by EDU assay and flow cytometry, respectively. Western blot analyzed Cleaved-caspase 1, 3 and 9 proteins expression. Dual-luciferase assay verified the binding sites of Hsa_circ_0000629 to miR-212-5p and NLRP3 to miR-212-5p. HE staining revealed inflammatory cell infiltration, bronchial wall thickening, smooth muscle hyperplasia, and alveolar destruction in asthmatic mice. Compared with the controls, Hsa_circ_0000629 and NLRP3 expression were significantly increased, while miR-212-5p expression was decreased in asthmatic lung tissues. In 16HBE cells, Hsa_circ_0000629-over and LPS groups showed elevated Hsa_circ_0000629 and NLRP3 expression but reduced miR-212-5p levels. Silencing Hsa_circ_0000629 in LPS-treated cells (LPS+Hsa_circ_0000629-si) reversed these effects. Overexpression of miR-212-5p counteracted Hsa_circ_0000629-induced NLRP3 upregulation, while miR-212-5p inhibition enhanced NLRP3 expression. LPS exposure increased TNF-, IL-18, IL-6, and IL-1{beta} levels, reduced cell proliferation, and promoted apoptosis. These changes were attenuated by Hsa_circ_0000629 silencing or miR-212-5p overexpression. Western blot confirmed that Hsa_circ_0000629 overexpression upregulated Cleaved-Caspase 1, 3, and 9, whereas miR-212-5p mimic or Hsa_circ_0000629-si reversed this trend. Dual-luciferase assays demonstrated targeted interactions among Hsa_circ_0000629, miR-212-5p, and NLRP3. Interference with Hsa_circ_0000629 expression can alleviate LPS induced apoptosis in 16HBE cells and inhibit the expression of inflammatory factors by targeting the miR-212-5p/NLRP pathway, which may be a new target for the treatment of asthma.

Right ventricular failure (RVF) is a serious disease with a high mortality but no effective pharmacologic treatments. We reported RVF was reversed by chronic treatment with an 1A-adrenergic receptor (1A-AR) agonist. Recent studies suggest mitochondrial dysfunction contributes to RVF. Therefore, we investigated if reversal of RVF by chronic 1A-AR agonist treatment involved improved mitochondrial function. A mouse model of RVF caused by pulmonary artery constriction (PAC) for 2 wk was chronically treated for a further 2 wk. with a low dose of the 1A-AR agonist A61603 (10 ng/kg/day) or vehicle (no drug control). RV dysfunction was assessed from the fractional shortening of the RV outflow tract (RVOT FS). RVOT FS for sham controls (46.5 {+/-} 1.3 %, n = 9) was reduced 4 wk after PAC (27.6 {+/-} 1.5 %, n = 13, P < 0.0001), but was higher after PAC plus 2 wk A61603 treatment (34.5 {+/-} 0.6 %, n = 14, P < 0.001). RV myocardial respiration rate (O2 consumption) for sham controls (776 {+/-} 51 pM/s/mg, n = 9) was reduced 4 wk after PAC (493 {+/-} 28 pM/s/mg, n = 15, P <0.0001), but was higher after PAC plus 2 wk A61603 treatment (634 {+/-} 30 pM/s/mg, n = 11, P <0.05). RV myocardial ATP level for sham controls (3.3 {+/-} 0.1 mM, n = 10) was reduced 4 wk after PAC (1.9 {+/-} 0.1 mM, n = 6, P < 0.0001), but was higher after PAC plus 2 wk A61603 treatment (2.6 {+/-} 0.13 mM, n = 7, P < 0.01). In conclusion, reversal of RVF after chronic A61603 treatment involved reversal of mitochondrial dysfunction. Consistent with our previous studies, this study suggests that the 1A-AR is a therapeutic target to treat RVF. HighlightsRV failure is reported to involve mitochondrial dysfunction which might impair RV contraction by decreasing cardiomyocyte ATP level. Using the pulmonary artery constriction model of RV failure, we found that chronic treatment with an 1A-adrenergic receptor agonist increased RV myocardial respiration rate, increased RV myocardial ATP level, and increased RV function. These findings suggest that the 1A-adrenergic receptor is a therapeutic target for treating RV failure, and that the mechanism involves improved RV cardiomyocyte bioenergetic status.