As a 5-methylcytosine (m5C) methyltransferase, increased NOP2/Sun RNA methyltransferase 2 (NSUN2) has been revealed to promote the progression of non-small cell lung cancer (NSCLC) through m5C modification. Herein, this study aimed to investigate the potential molecular mechanisms underlying the high NSUN2 expression in NSCLC, and the potential downstream m5C mRNAs of NSUN2 in promoting NSCLC progression. Functional analyses were conducted using in vitro MTT, EdU, transwell, wound healing, sphere and tube formation assays, and in vivo murine model. m5C modification was determined by MeRIP assay. RIP assay determined the binding between NSUN2 and SLC7A5 mRNA. The upstream molecular mechanism of the upregulation of NSUN2 expression was explored using ChIP, Co-immunoprecipitation (Co-IP), and luciferase reporter assays. NSUN2 was highly expressed in NSCLC and predicted poor outcomes in NSCLC patients. Functionally, NSUN2 silencing suppressed cancer cell proliferation, migration, stemness properties, angiogenic ability and glutamine metabolism. Mechanistically, NSUN2 induced m5C methylation modification of SLC7A5, and stabilized SLC7A5 mRNA via a YBX1-dependent mechanism. Accordingly, SLC7A5 overexpression reversed the anticancer effects of NSUN2 on above oncogenic phenotypes. Further upstream molecular mechanism analysis showed that P300 could bind to and cooperate with transcription factor SP1 to increase NSUN2 expression by Histone H3 Lysine 27 acetylation (H3K27ac). Further in vivo analyses suggested that NSUN2 silencing suppressed ESCC growth and metastasis in vivo by regulating SLC7A5 expression. In conclusion, increased NSUN2 derived by P300/SP1 complex-mediated histone acetylation promoted the growth, metastasis and glutamine metabolism of NSCLC by stabilizing SLC7A5 mRNA via m5C modification.

We used spatial transcriptomics from idiopathic pulmonary fibrosis (IPF) and unaffected control lung tissue to further understand the pathogenesis of MUC5B-driven lung fibrosis. We captured 43 fields of view in 15 IPF and 13 controls with and without the promoter variant using the CosMx platform and identified 19 cell types via semi-supervised clustering. was ectopically expressed in AT2 cells in controls with the risk variant. We observed a decreased proportion of AT2 cells in controls and an increased proportion of aberrant basaloid cells in IPF associated with the risk variant. We identified co-localized expression of in respiratory bronchioles with 13 genes including the endoplasmic reticulum (ER) stress marker and distal secretory markers and . Experimentally, we demonstrated a direct relationship between expression and ER stress in bronchiolar epithelia and validated the co-expression of and in the IPF lung. Based on our results, we conclude that injures alveolar and bronchiolar epithelia that results in loss of AT2 cells and an increase in aberrant basaloid cells which initiates ER stress and a secretory phenotype in the terminal respiratory bronchiole, establishing a persistently injured distal airspace.

Pulmonary fibrosis is increasingly understood to involve dysfunction within and across multiple cellular compartments, with recent attention highlighting the involvement of pulmonary vascular dysfunction in failed repair and progression of fibrosis. Formulation and delivery of lung-targeting lipid nanoparticles may provide a means to selectively target the lung but not systemic vasculature. However, the feasibility and efficacy of such approaches in the fibrotic lung are unknown. We sought to test whether intravenously administered lung-targeting lipid nanoparticles can safely deliver mRNA to the healthy and fibrotic lung vasculature in young and aged mice and whether delivery of mRNA encoding a matricellular protein could promote fibrosis resolution. We used a Selective Organ Targeting (SORT) LNP formulation and characterized cell-specificity of delivery after bleomycin-induced lung fibrosis. We then delivered Ccn3 mRNA (encoding cellular communication network factor 3) to aged mice in the setting of established lung fibrosis and evaluated fibrotic regression and vascular repair. The matricellular protein encoded by was previously identified by our group as an important regulator of lung endothelial function. We found that LNP delivery was lung specific and predominantly endothelial targeting in the setting of lung fibrosis. Delivery of mRNA to aged mice via LNPs modestly reduced fibrosis and improved microvascular density in the lungs. Our results support the concept that cell-specific and repair-promoting cargos delivered via lung targeting LNPs may have utility for treatment of established fibrosis.

Extracellular vesicles (EVs) have emerged as versatile carriers of therapeutic cargo, including nucleic acids, proteins, and small molecules. However, their potential to deliver bioactive lipid mediators remains largely unexplored. Here, we present a novel synthetic biology-based strategy to selectively load EVs with pro-resolving lipid mediators of the Resolvin D and E series by co-expressing the resolvin biosynthetic enzymes cyclooxygenase 2 (COX-2), 5-lipoxygenase (5-LOX), and 15-lipoxygenase (15-LOX) using a custom-designed multigene expression vector. Human embryonic kidney 293T (HEK293T) cells transfected with the multigene expression vector and cultured in the presence of fatty acid free bovine serum albumen (BSA)-complexed docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and aspirin produced multiple members of the Resolvin D, aspirin-triggered Resolvin D series, and Resolvin E1 and E2, along with their biosynthetic precursors, which were subsequently packaged into EVs (referred to as Resolvin-EVs). Resolvin-EVs attenuated neutrophil adhesion to endothelial cells both under static and flow conditions and preserved endothelial barrier integrity by upregulating VE-cadherin. In macrophages, Resolvin-EVs suppressed nuclear factor kappa B (NF-κB) reporter activity, release of IL6 and TNFα. Effects of Resolvin-EVs on endothelial permeability and macrophage activation were abrogated by pharmacological inhibition of EV uptake using nystatin and cytochalasin D. Furthermore, Resolvin-EVs enhanced efferocytosis in THP-1 derived macrophages compared to Control-EVs. Notably, post-injury administration of Resolvin-EVs attenuated pulmonary inflammation in LPS-treated mice without inducing systemic or pulmonary toxicity. Together, these findings establish a novel, scalable platform for generating Resolvin-loaded EVs and highlight their therapeutic potential for acute lung injury and other chronic inflammatory disorders.

Cystic fibrosis (CF) is an autosomal recessive disease caused by variants in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. Delivery of a functional CFTR transgene to airway epithelial cells (AEC) offers the potential to provide durable restoration of normal CFTR function. Adeno-associated virus (AAV) vectors are the leading platform for the delivery of gene therapy; however, wild-type AAV vectors exhibit a limited capacity to transduce airway cells and evade pre-existing human neutralizing antibodies (NAb). We therefore employed a directed evolution platform to invent a novel AAV capsid (A101) with the capacity to efficiently transduce AECs, including in the presence of NAbs, following aerosolized administration to nonhuman primates (NHP). We then engineered 4D-710, a gene therapy comprising the A101 vector and a transgene with a partial deletion in the regulatory domain () to facilitate vector packaging. 4D-710 exhibited efficient transduction of human bronchial epithelial (HBE) cell air-liquid interface (ALI) cultures in vitro and robust functional activity in CF HBE ALI cultures. Aerosolized administration of 4D-710 to NHPs was well tolerated and resulted in dose-dependent transgene expression and increased CFTR protein in diverse AEC types compared to vehicle controls. No significant differences in mRNA levels were observed in lung samples from NHPs with pre-existing serum anti-capsid NAbs compared to NAb-negative NHPs. These findings demonstrate the tolerability and feasibility of A101-mediated transgene delivery and expression in primate airways. A clinical trial evaluating aerosol delivery of 4D-710 in adults with CF (NCT05248230) is underway.

Pulmonary blood volumes (PBV), currently not assessed by computed tomography pulmonary angiography (CTPA), could provide additional information to routine investigations performed for chronic thromboembolic pulmonary hypertension (CTEPH). We investigated CTPA-based PBV in evaluating hemodynamic outcome from pulmonary endarterectomy (PEA) surgery. A deep learning-based CTPA vascular segmentation model, differentiating arteries and veins, was applied for automated PBV measurements in CTEPH patients who underwent PEA at UK's national CTEPH service. Pulmonary arteries were compartmentalised into "central" (main pulmonary and proximal lobar) and "intrapulmonary". Mean pulmonary arterial pressure >30 mmHg post-PEA defined "clinically relevant" residual PH. Logistic regression models applying CTPA-based PBV to identify residual PH were trained and tested on the discovery and validation cohorts respectively. Paired pre- and postoperative CTPA, in the discovery (n=71) and validation (n=102) cohorts showed that central pulmonary artery volume and total artery to vein volume ratio (A-VR) decreased and pulmonary vein volume increased with hemodynamic improvement post-PEA. Preoperative central pulmonary artery volume and A-VR helped identify patients at risk for clinically relevant residual PH post-PEA (AUROC 0.88 and 0.82 in the discovery and validation cohorts). Postoperative central pulmonary artery volume, A-VR and pulmonary vein volume helped to non-invasively identify patients without clinically relevant residual PH (AUROC 0.91 and 0.88 in the discovery and validation cohorts). Automated quantification of CTPA-based PBV at diagnosis can help stratify risk for residual PH in patients managed with PEA. Utilizing CTPA-derived PBV post-PEA to identify patients without residual PH can potentially reduce the need for routine postoperative right heart catheterization.

Endurance exercise is broadly beneficial to cardiopulmonary function, with these benefits thought to be driven by extrapulmonary factors rather than direct structural changes in the lungs. Thus, to address how endurance exercise training and sex impact molecular responses in the lungs, we used a multi-omics approach to study 6-month-old Fischer 344 rats that undertook a progressive endurance treadmill training protocol for 1 to 8 weeks. Specifically, we reannotated publicly accessible transcriptomics, metabolomics, proteomics and phosphoproteomics data from the Molecular Transducers of Physical Activity Consortium (MoTrPAC) and integrated newly analyzed acetylproteomics data to assess multi-omic sex differences in sedentary and treadmill trained rats. Female rats displayed enrichment in immune-related features and pathways at the transcriptome and proteome level that were largely maintained with training. However, both sexes exhibited decreases in immune pathway activity following 8 weeks of training, although the effect was more pronounced in males. Shared responses to training included increased enrichment in transcriptomic pathways related to type I alveoli, proteomic pathways related to cilia, and decreased acetylation of pathways linked to mitochondrial function. Furthermore, features known to be enriched in lung diseases were attenuated with training in both sexes. Together, our findings provide novel insight into responses to endurance exercise training in the healthy rat lung and may offer translational insight into sex-specific differences in lung disease pathogenesis and treatment.

Allergic asthma is a widespread disease of the airway stemming from the actions of multiple cell types, including eosinophils and epithelial cells. The urokinase plasminogen activator receptor (uPAR) is a membrane bound protein that can contribute to the activation and mobilization of leukocytes and is present at increased levels in asthmatics. However, its role in allergic asthma remains poorly understood. Here, we used multiple mouse strains and different models of allergic airway disease to study the function of uPAR in the pathogenesis of this disease. , the gene encoding uPAR, was rapidly induced following allergic sensitization through the airway, and again following subsequent allergen challenge. -deficient mice displayed both increased numbers of eosinophils and heightened airway hyperresponsiveness (AHR) in multiple models of allergic asthma. Mice selectively lacking in eosinophils also had more robust eosinophilia than did WT mice, and eosinophils lacking displayed increased activity in an ex vivo assay of chemokine-dependent migration. However, those mice did not have increased AHR compared with WT mice. Conversely, although mice selectively lacking Plaur in lung epithelial cells did not have increased inflammation compared with wild type (WT) mice, they displayed heightened AHR. These findings suggest that uPAR controls both airway inflammation and AHR, but through distinct mechanisms. Targeting uPAR might have therapeutic potential for treating inflammation and AHR in asthma.

Lipid metabolism disorder is increasingly recognized as hallmarks of Pulmonary hypertension (PH). Fatty acid-binding proteins (FABPs), particularly FABP4 and FABP5, which regulate lipid transport and metabolism of fatty acid, are thought to contribute to the development of PH. However, it remains unclear whether FABP4 and FABP5 serve as therapeutic targets for the treatment of PH. In the present study, the levels of FABP4/5 were elevated in the plasma and lung tissues of IPAH patients, as well as in the lung tissues of the PH rat model compared with control. The circulating levels of FABP4 of IPAH patients were correlated with mean pulmonary arterial pressure (mPAP). To determine the preventive or therapeutic effect of FABP4 and FABP5 inhibition, FABP4 and FABP5 inhibitors alone or combination were administered at early (days 2 following monocrotaline (MCT) injection) and late (day 12 following MCT injection) stage of PH rat model, respectively. Combined treatment with FABP4/5 inhibitors in the early stage of the MCT-PH rat model effectively reduced right ventricular systolic pressure (RVSP) and improved right ventricular function, accompanied by reductions in pulmonary vascular and right ventricular fibrosis, as well as blood lipid levels, lipid peroxidation, and inflammation. Combined treatment with FABP4/5 inhibitors at the late stage of MCT-PH improved right ventricular function, suppressed pulmonary vascular and right ventricular fibrosis, and lowered blood lipid levels, but did not affect RVSP. In conclusion, our study indicates that combined inhibition of FABP4 and FABP5 can prevent the pathogenesis of PH, representing a potential therapeutic strategy for PH.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are heterogeneous and potentially fatal consequences of serious conditions such as trauma and sepsis, an exacerbated inflammatory response to infection. There are no effective treatments for ALI/ARDS, partly due to an incomplete understanding of its pathogenesis across different patient sub-populations, contributing to mortality rates of 25-40%. ALI/ARDS is characterized by lung hypoxia, inflammatory cell infiltration, edema, and endothelial cell injury and death. Lung endothelial viability is essential for gas exchange, nutrient delivery, and immune cell migration, as well as the prevention of proteinaceous fluid accumulation. Given that lung endothelial death is a predominant feature of ALI/ARDS, its inhibition could represent a novel therapeutic strategy. In this article, we review studies examining pulmonary endothelium death during sepsis-induced ALI/ARDS, including studies of lung endothelium apoptosis, pyroptosis, necroptosis, and ferroptosis. We also highlight gaps in current knowledge that, if addressed, could facilitate the development of effective treatments for sepsis-induced ALI/ARDS. Future studies of the mechanisms regulating lung endothelial death may uncover novel therapeutic targets for ALI/ARDS. These targets could be leveraged in precision medicine approaches to treat patient sub-populations most likely to benefit from inhibiting specific forms of lung endothelial death.

Osteopontin, also known as secreted phosphoprotein 1 (Spp1), is a key molecule involved in lung fibrosis; however, the mechanism underlying the exacerbation caused by Spp1-producing cells remains unclear. In the present study, we investigated the detailed functions of Spp1-producing macrophages in lung fibrosis. Analysis of published single-cell RNA sequencing (scRNA-seq) datasets revealed the fibrogenic role of the interaction between -expressing macrophages and fibroblasts in patients with idiopathic pulmonary fibrosis. In addition, interstitial macrophages (IMs) were identified as the primary Spp1 source in the bleomycin-treated lungs of -enhanced green fluorescent protein (EGFP) knock-in reporter mice; their IMs promote lung fibrosis by enhancing fibroblast activation. -EGFP IMs expanded, peaking 7 days post-bleomycin administration and engrafting as inflammatory resident macrophages. Multi-omics analysis revealed that -EGFP IMs produced glycoprotein non-metastatic melanoma protein b (Gpnmb)-a fibrogenic, pro-inflammatory protein. Furthermore, Spp1-producing macrophages expressed the interleukin (IL)7 receptor on their surface in the fibrotic lungs of humans and mice. In the bleomycin-induced lung fibrosis model of Il7r Csf1r-iCre mice, macrophage expression of and was reduced, and lung fibrosis was attenuated, compared with those of Il7r mice. These profibrotic Spp1-producing macrophages and the IL-7/macrophage/Spp1 axis may represent therapeutic targets for lung fibrosis.

Checkpoint inhibitor pneumonitis (CIP) is a highly morbid complication of immune checkpoint immunotherapy, characterized by acute lung injury leading, in severe cases, to hypoxic respiratory failure and death. CIP incidence in lung cancer is high (10-15%). Yet, the pathophysiology of CIP is poorly understood. To investigate the mechanisms underlying alveolar inflammation in patients with CIP, human bronchoalveolar fluid (BALF) samples from control and CIP patients were analyzed using flow cytometry, single cell RNA sequencing (scRNA seq), and ELISA. Findings were validated using multiple external cohorts. In vitro experiments and in vivo rodent models were employed to investigate the mechanisms driving alveolar inflammation in CIP. Analysis of scRNA seq and flow cytometry data demonstrated increased macrophages in patients with CIP compared to controls. Several distinct pro-inflammatory alveolar macrophage subsets were increased in CIP. CIP macrophages expressed increased chemokine ligand-18 (CCL18) at the transcript (scRNAseq), cellular (flow cytometry) and secreted protein (BALF ELISA) level. BALF CCL18 levels were associated with clinical CIP severity. CCL18 over-expression in mice promoted lung inflammation that phenocopied human CIP, including upregulation of pro-inflammatory macrophage subsets. These findings suggest that BALF macrophages and CCL18 protein levels are increased in patients with CIP and associate with greater CIP severity. Additionally, CCL18 promotes lung inflammation in mice that mimics human CIP, suggesting a causal role for CCL18 in CIP.