American Journal of Respiratory Cell and Molecular Biology

BackgroundExtensive comorbidity between cardiovascular (CVD) and respiratory (RT) diseases is well-documented, yet the shared genetic mechanisms remain elusive. Genetic pleiotropy may play a pivotal role in understanding the intricate comorbidity patterns associated with cardiovascular and respiratory conditions. MethodsOur comprehensive analysis encompasses the largest available GWAS dataset of European ancestry covering six major CVDs (atrial fibrillation, coronary artery disease, venous thromboembolism, heart failure, peripheral arterial disease, and stroke) and four prevalent RTs (asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and sleep apnea). Initially, we aimed to unveil the common genetic basis of major CVDs, through genome-wide and local genetic correlations and polygenic overlap. Subsequently, the shared genetic mechanisms between RTs and CVDs was investigated in terms of both horizontal and vertical pleiotropy. From a horizontal pleiotropy perspective, cross-trait analysis was utilized to identify pleiotropic genetic determinants including genomic loci, single nucleotide polymorphisms (SNPs), genes, biological pathways, and protein targets. From a vertical pleiotropic perspective, Mendelian randomization was employed to evaluate potential causal relationships between CVDs and RTs. ResultsOur study confirmed the significant existence of genetic correlations and overlaps between CVDs and RTs. Pleiotropy analysis under the composite null hypothesis identified 17,964 significant potential pleiotropic SNPs in 24 trait pairs, with 73 pleiotropic loci and 69 colocalized loci detected. Gene-based analysis revealed 59 candidate pleiotropic genes, highly enriched in unsaturated fatty acid biosynthetic processes and MHC class I-mediated antigen processing and presentation. Mendelian randomization analysis demonstrated a positive causal relationship only between chronic obstructive pulmonary disease and heart failure. Overall, the genetic basis between CVDs and RTs was inconsistent with vertical pleiotropy, suggesting the dramatic impact of horizontal pleiotropy. ConclusionsOur findings indicate widely distributed pleiotropic genetic determinants between RTs and CVDs across the genome. These results support a common genetic basis for RTs and CVDs and are important for intervention and therapeutic targets in comorbidities. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIA common genetic underpinning for CVDs and RTs has been identified using a variety of approaches and further explained as a shared genetic mechanism mediated by pleiotropy. C_LIO_LIThe systematic atlas of horizontal pleiotropy addressed key questions about pleiotropic SNPs, genomic loci, genes, functional features, and protein targets contributing to comorbidity between CVDs and RTs. C_LIO_LIThe systematic atlas of vertical pleiotropy highlighted causal associations between CVDs and RTs beyond the observed correlations. C_LI What Are the Clinical Implications?This study may help to elucidate the shared genetic mechanism between respiratory and cardiovascular diseases and further prioritize shared drug targets between RTs and CVDs.

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.

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.

Idiopathic Pulmonary Fibrosis (IPF) is characterized by scarring and remodeling of lung tissue, leading to progressive pulmonary dysfunction. Currently, very little is known about the steps involved in disease initiation and progression because models of IPF poorly replicate these processes. However, understanding the pathogenesis of IPF is essential to developing effective therapies. To address this, we developed a scaffold-based human co-culture alveolar organoid model that utilizes healthy and IPF human primary lung fibroblasts and induced pluripotent stem cell-derived alveolar type 2 (iAT2) cells carrying the surfactant protein C (SFTPC) 173T mutation of familial IPF, or their syngeneic corrected controls, to recapitulate epithelial-mesenchymal cellular communication during fibrosis initiation and progression. We found that the interaction between epithelial cells and fibroblasts plays a key role in inducing fibrotic responses in this model, with the secretion of chemokines, cytokines, TGF{beta}, and matrix metalloproteinases that mirror those observed in the serum of patients with pulmonary fibrosis. Single-cell RNA sequencing revealed the emergence of many cell subtypes observed in progressive lung fibrosis, along with key cellular interactions that correlated with the initial upregulation of fibrosis pathways, extracellular matrix (ECM) remodeling, inflammation, and changes in lipid metabolism. The anti-fibrotic compounds, Nintedanib and the TGF{beta} inhibitor, SB431542, demonstrated dose-dependent efficacy in the model, with IC50 values comparable to those observed in the clinic, and significantly reduced secretion of fibrosis-related factors. Overall, this study shows that the three-dimensional cell co-culture organoid effectively models progressive lung fibrosis, facilitating the investigation of epithelial-mesenchymal interactions and serving as a patient-relevant model to better predict the efficacy of therapeutics in the clinic.

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.

CFTR modulator therapies have transformed CF care, yet chronic airway inflammation persists in many people with cystic fibrosis (pwCF) even after long-term highly effective modulator therapy (HEMT). Because of the adverse side effects or the incompatibility with CFTR modulators, the use of traditional anti-inflammatory therapies is very limited in CF. Hence, new therapeutic strategies that rebalance inflammation without worsening infection with immunosuppression are needed. We evaluated the selective phosphodiesterase 4 (PDE4) inhibitor apremilast (Apr) for its ability to modulate dysregulated inflammation in humanized CF (G551D) rats acutely challenged with Pseudomonas aeruginosa. Apr is an approved anti-inflammatory therapeutic strategy for several chronic inflammatory conditions, but it has not been well studied in CF. In the humanized CF (G551D) rats, a short prophylactic Apr regimen significantly preserved lung function and reduced lung injury, accompanied by broad modulation of inflammatory responses, notably within Th1 and Th17 axes. Importantly, Apr did not cause a significant increase in bacterial burden. Just as importantly, Apr did not reduce CFTR mRNA or protein in vivo, and it increased G551D-CFTR phosphorylation critical for channel gating in vitro, supporting mechanistic compatibility with HEMT. These findings suggest Apr as a potential adjunct to CFTR modulators to rebalance airway inflammation while preserving host defense.

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.

Pulmonary hypertension (PH) is a progressive condition characterized by increased pulmonary arterial pressure. Endothelial cell dysfunction is one important characteristic of PH. Recently, capillary endothelial cells, including aerocytes (aCaps) and general capillary cell (gCaps), have been detected in developing lungs but their role and the regulatory mechanisms underlying PH remain poorly understood. The goal of this study was to identify changes in Caps and their effects on hypertensive pulmonary circulation. We set up a Capillary Alveoli Micro-physiological System (CAMS) incorporated with hPSCs(human pluripotent stem cells)-aCaps to show loss of Cap connection under dynamically cultured hypoxic condition. We employed single-cell RNA sequencing (scRNA-seq) and immunofluorescence to demonstrate impaired gCaps differentiation with increased expression of cell membrane receptor CD93 in PH patients and a Sugen 5416/hypoxia (SuHx) rat model. Conditional Knockdown or Lentiviral overexpression of CD93 alleviated the pathology observed in SuHx mice. We also revealed that CD93 overexpression upregulated SMAD2/3 to repress Apelin (APLN) expression by CHIP assay. Finally, supplementation with an APLNR agonist in the PH rat model promoted gCaps-to-aCaps differentiation and improved haemodynamic indices. Overall, our results highlight the potential for promoting capillary cell differentiation with G protein biased APLNR agonist as a therapeutic strategy for pulmonary vascular disease.

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.

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.

Pulmonary fibrosis (PF) involves excessive collagen accumulation, yet mechanisms shifting the balance of synthesis and degradation toward net deposition remain unclear. Myeloperoxidase (MPO) inversely correlates with survival in PF. Using the bleomycin model, we found MPO knockout (MPOko) mice were protected from fibrosis, and pharmacological MPO inhibition after peak inflammation (day 7) recapitulated this protection. MPO persisted in lung tissue 21 days post-injury despite neutrophil efflux, linking acute inflammation to sustained remodeling. Mechanistically, we identified that MPO inhibits Cathepsin K (CatK), a potent collagenolytic enzyme involved in fibrosis resolution. Notably, CatK gene expression (CTSK) is elevated in PF, suggesting post-translational inhibition of CatK. MPOko and inhibitor-treated mice exhibited elevated CatK activity after bleomycin; exogenous addition of pathophysiologic concentrations of MPO reduced CatK activity in mouse precision-cut lung slices and human fibroblasts. Biochemically, MPO reduced CatK activity to 33% of control. In two distinct cohorts of PF patients, we observed significantly increased MPO protein levels in platelet poor plasma and in lung tissue. In PF patients, 62% had MPO levels in platelet poor plasma exceeding healthy controls, while lung tissue from other PF patients showed significantly elevated MPO staining. Plasma levels were inversely correlated with decreased survival, FVC, and DLCO. These findings establish MPO as a post-translational inhibitor of CatK-mediated collagenolysis, revealing a mechanism linking acute inflammation to sustained fibrosis and suggest a patient subpopulation that may benefit from MPO-targeted therapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=54 SRC="FIGDIR/small/713467v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@d8fc5eorg.highwire.dtl.DTLVardef@1a088fcorg.highwire.dtl.DTLVardef@818b7dorg.highwire.dtl.DTLVardef@ecdca0_HPS_FORMAT_FIGEXP M_FIG C_FIG Myeloperoxidase persists in lung tissue after injury and inhibits cathepsin K activity, impairing collagen degradation and promoting extracellular matrix accumulation during pulmonary fibrosis.

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.

Chronic obstructive pulmonary disease (COPD) is a debilitating and progressive lung disease that affects millions of people worldwide. There is a continuing clinical need to characterize COPD at the molecular level to be able to identify the multi-omic biomarkers of its pathogenesis and to enable more accurate diagnoses and more effective treatment. We used Multi-Omics Factor Analysis (MOFA) to jointly analyze genomic, blood transcriptomic, and plasma proteomic data collected from 1,872 participants in the Genetic Epidemiology of COPD study who had moderate to very severe COPD. Five latent factors identified by MOFA were associated with COPD-related lung function, chest computed tomography (CT) imaging, and blood count phenotypes, as well as all-cause mortality. The top genetic, transcriptomic and proteomic contributors to these latent factors were also individually associated with COPD-related outcomes. Moreover, factor loadings and expression levels of top omic drivers helped distinguish between patient subgroups. Quantitative trait loci analysis of a latent factor that was jointly driven by transcriptomics and proteomics revealed potential common genetic control of gene expression and protein abundance. Polygenic risk scores derived from a genomics-driven latent factor were associated with chest CT imaging and lung function phenotypes, and these associations were replicated in an independent COPD cohort. Together, our results suggest the potential of integrative omic approaches to identify the major axes of heterogeneity in COPD and uncover the multi-omic interplay between the contributors to each axis.

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.

Precision-cut lung slices (PCLS) retain the native cells and extracellular matrix that contribute to the structural and functional integrity of lung tissue. This technique enables the study of cell-matrix interactions and is particularly useful for pre-clinical pharmacological studies. More specifically, PCLS are widely used to model the complex pathophysiology of pulmonary fibrosis, an uncurable and progressive interstitial lung disease. Current ex vivo pulmonary fibrosis models expose PCLS to pro-fibrotic biochemical cues over a short timeframe (hours to days) and quickly collect samples for analysis due to viability concerns. This condensed timeline is a limitation to understanding chronic disease mechanisms. To extend the utility of ex vivo pulmonary fibrosis models, PCLS were embedded in engineered hydrogels and exposed to pro-fibrotic biochemical and biophysical cues. Hydrogel-embedded PCLS maintained greater than 80% total cell viability over 3 weeks in culture. Gene expression patterns in samples exposed to pro-fibrotic cues matched trends measured in human fibrotic lung tissue. Finally, treatment with Nintedanib, a Food and Drug Administration approved pulmonary fibrosis drug, moderately reduced fibroblast activation and influenced epithelial cell differentiation. Collectively, these results show that hydrogel-embedded PCLS models of pulmonary fibrosis extend our ability to study fibrotic processes ex vivo and, when applied to human tissues, present a new approach methodology for studying lung disease and treatment.

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) 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.

Pulmonary fibrosis (PF) can arise from mutations in alveolar epithelial type 2 (AT2) cell-specific genes, but manifests in fibrotic activation of mesenchymal cells, thus involving fibrogenic epithelial-mesenchymal crosstalk. The ligand-receptor interactions underlying the onset and early progression of PF remain poorly understood. Induced pluripotent stem cell (iPSC)-derived models are powerful tools to study respiratory diseases, yet are currently limited to reductionist single lineage epithelial models or multi-lineage systems that lack purity and lung-specificity of the mesenchyme. Here we generate a human iPSC line carrying both a lung mesenchyme-specific reporter (TBX4-LERtdTomato) and a reporter for mesenchymal activation/differentiation (ACTA2GFP). Applying this line, we develop a directed differentiation protocol capable of generating cells that express key molecular and functional features of primary human developing lung mesenchyme across multiple iPSC genetic backgrounds. We then establish co-cultures of these iPSC-derived lung mesenchymal cells (iLM) with patient-specific iPSC-derived alveolar epithelial type 2 cells (iAT2s) carrying an SFTPCI73T mutation as a model for PF. We find increased expression of fibrotic markers in co-cultures with mutant iAT2s as compared to co-cultures with gene-corrected iAT2s. Moreover, mutant iAT2s express markers of alveolar-basal intermediate (ABI) cells only in the presence of iLM, suggesting that bidirectional crosstalk promotes this aberrant cell state. We identify ligand-receptor pairs enriched in co-cultures with mutant iAT2s, including TGF{beta}, multiple integrins, and additional genes that have not been previously linked to PF. Finally, we show that small molecule-mediated inhibition of TGF{beta} or integrins V{beta}1/V{beta}6 attenuates both fibrotic mesenchymal activation and the presence of ABI cells in iLM/iAT2 co-cultures. Thus, we have established a human iPSC-derived co-culture system that recapitulates key molecular hallmarks of bidirectional fibrogenic epithelial-mesenchymal crosstalk in pulmonary fibrosis, and enables the identification and study of potentially druggable pathways involved in disease initiation and progression.

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.

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