搜索结果：American journal of respiratory cell and molecular biology

Over the past two decades there have been remarkable advances in stem cell biology, bioengineering, and lung regenerative research, transforming our understanding of pulmonary biology from development to repair, and disease. Strategies using endogenous lung progenitor cells, pluripotent stem cell technologies, and engineered tissue platforms have become central tools for interrogating lung biology. Major breakthroughs have included the identification of diverse cell populations that coordinate lung homeostasis and repair, facilitated by the extensive adoption of single cell, multiomic and spatialomics approaches. Simultaneous progress in biomaterials, organoid systems, decellularized lung scaffolds, and lung-on-chip platforms has uncovered how extracellular matrix composition, mechanical forces, and tissue architecture contribute to the regulation of cell fate and function. These advances have enabled increasingly physiologically relevant in vitro, and ex vivo models while informing tissue engineering strategies aimed ultimately at functional lung replacement. Translation toward the clinic has advanced through both cell-based and cell-free therapeutic strategies. Early efforts focused largely on mesenchymal stromal cell-based approaches and extracellular vesicles, which have demonstrated safety and context-dependent efficacy in inflammatory lung diseases, alongside emerging preclinical evidence of functional engraftment of induced pluripotent stem cell-derived lung lineages. The past twenty years of progress, captured at the 20th Anniversary Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, highlights the power of interdisciplinary collaboration in advancing lung regeneration from foundational discovery toward therapeutic reality.

WNT ligands have been critically implicated in many aspects of lung cancer progression. This study explores the impacts of Wnt family member 10A (WNT10A) in progression and immune response in lung adenocarcinoma (LUAD) and investigates the underpinning mechanisms. Expression, prognosis, and immune correlations of WNT10A in LUAD were explored using TCGA-LUAD, GEO datasets, and tissue microarrays. In vitro, WNT10A was knocked down in LUAD cell lines (H441 and H1299), followed by RNA sequencing and functional assays. Tumor-bearing mouse models with WNT10A knockdown were used to assess tumor growth and immune response. The functions of WNT10A on CD8 + T cell cytotoxicity were further explored through co-culture experiments and flow cytometry. FRAT1 overexpression, Wnt activators (BML-284), and CXCL12 manipulations were employed to verify mechanistic links. High WNT10A expression showed a trend toward poor overall survival, reduced CD8 + T cell infiltration, and unfavorable prognosis in LUAD patients. WNT10A loss in LUAD cells reduced growth, mobility, and inhibited Wnt signaling. In vivo, WNT10A loss prolonged animal survival in immunocompetent mice, reduced tumorigenic activity of mouse 3LL cells, and enhanced CD8 + T cell activity and tumor cell apoptosis. Mechanistically, WNT10A promoted FRAT1-mediated suppression of GSK3β, facilitating CTNNB1 activation and CXCL12 expression, which restricted CD8 + T cell function. WNT10A silencing synergized with anti-PD-1, reducing tumor burden and boosting CD8 + T cell infiltration/function. Generally, WNT10A promotes LUAD progression and CD8 + T cell dysfunction through FRAT1-mediated Wnt signaling and CXCL12 activation. Focusing on WNT10A could offer a compelling approach to boosting T cell-driven anti-tumor responses for the treatment of LUAD.

Interstitial lung disease (ILD) disproportionately affects older adults, yet the contribution of immunosenescence to disease pathogenesis remains poorly understood. In fibrotic ILDs (fILDs), CD8 + T cells accumulate in fibrotic regions, where they may drive disease by promoting cytotoxic inflammation, impairing epithelial repair, and sustaining senescence. CD8 + T cell exhaustion (CD8 + Tex) has also emerged as a hallmark of chronic lung disease, although its relationship to immunosenescence in ILD remains unclear. Here, we highlight the heterogeneity among CD8 + T cells in fILD, including effector- and senescent-like subsets, and identify programmed death (PD)-1 as a protective "brake" limiting tissue-damaging immunopathology. Functional profiling indicates that CD8 + T cells in fILD exhibit features consistent with ex-tissue-resident and effector memory CD8 + T cell subsets. Recent evidence from severe and post-acute viral injury demonstrates that PD-1hiCD8 + T cells balance protective immunity with restraint of fibrotic sequelae while also driving maladaptive epithelial remodeling through expansion of dysplastic basal-like cells and impaired alveolar regeneration. These observations suggest that CD8 + T cells in fILD may directly regulate the balance between tissue repair and fibrosis. Our recent studies have shown that the antifibrotic effects of pirfenidone and nintedanib may arise from selective modulation of profibrotic programs in CD8+/CD4 + T cells, lymphoid endothelial cells and dendritic cells. Collectively, these findings support a paradigm shift in which fILD reflects a dysregulation of local immune networks rather an inevitable consequence of aging. Most importantly, these networks are modifiable, offering opportunities for early detection, patient stratification, and stage-specific immunomodulatory interventions, with maladaptive memory CD8 + T cell functional states serving as potential biomarkers of disease susceptibility.

Recent evidence suggests that bronchial epithelial cells from individuals with asthma exhibit altered metabolic signatures. This metabolic shift of energetically demanding cells leads to increased inflammation, excessive reactive oxygen species production (ROS), and oxidative stress-all hallmarks of mitochondrial dysfunction. While mitochondrial dysfunction has been implicated in disruption in epithelial cell function in asthma, the mechanistic link between bronchoconstriction observed in asthma and these metabolic alterations remains poorly defined. Club cell secretory protein (CC16) is the most abundant protein found in the lung and exerts key anti-inflammatory and antioxidant functions contributing to protection against airway remodeling. Decreased levels of CC16 in both serum and bronchial alveolar lavage fluid (BALF) are characteristic of asthma and worsening respiratory disease. Using a well-established transmembrane compression system to model bronchoconstriction coupled with mass spectrometry and quantitative proteomics, we investigated how modeling bronchoconstriction in airway cells impacts CC16 expression and cell metabolic pathway changes over time. Using naive mouse tracheal epithelial cells (MTECs) and normal human bronchial epithelial cells (HBECs), we observed that recombinant (r)CC16 induces the expression of proteins related to various metabolic pathways, such as glycolysis, gluconeogenesis, and the pentose phosphate pathway and that compression of airway cells results in acute decreases in CC16 expression, as well as decreases in metabolic processes. MTECs deficient in CC16 (CC16-/-) had lower mitochondrial oxygen consumption rate (OCR) compared to WT cells. Exogenous addition of rCC16 significantly increased OCR of both WT and CC16 deficient MTECs. Our findings suggest a novel role for CC16 in mediating airway epithelial cell metabolic processes, which could be decreased by bronchoconstrictive events in human asthma. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD067703.

Benzodiazepines are widely used for sedation and anxiolysis but may also exert unrecognized beneficial effects on airway smooth muscle (ASM) tone in clinical settings where they are routinely administered. While their primary mechanism involves GABAA receptor (GABAAR) modulation in the central nervous system, their direct effects on peripheral airways remain poorly understood. Using mouse precision-cut lung slices (PCLS) and video phase-contrast microscopy, we show that diazepam, lorazepam, and midazolam induce robust, reversible, and dose-dependent relaxation of methacholine (MCh)-constricted peripheral airways with IC50 of 12, 22, and 23 µM, respectively. We selected diazepam to further investigate the cellular and molecular mechanisms underlying airway relaxation. GABAAR antagonists picrotoxin and flumazenil failed to block relaxation, and diazepam inhibited MCh-induced airway constriction even in the absence of extracellular Ca2+, consistent with a GABAAR-independent mechanism. Diazepam-induced relaxation correlated with strong inhibition of concurrent intracellular Ca2+ oscillations in ASM cells. Diazepam inhibited airway constriction and Ca2+ transients elicited by intracellular IP3 uncaging but not by caffeine, indicating specific modulation of IP3-receptor mediated Ca2+ signaling. Furthermore, low concentrations of diazepam (1 µM) significantly prolonged terbutaline-induced airway relaxation, mirroring the effects of selective phosphodiesterase 4 (PDE4) inhibitor rolipram. Diazepam inhibited purified PDE4D2 activity and potentiated forskolin-induced cAMP accumulation in human ASM cells. These findings indicate that diazepam produces bronchodilation at least in part through direct PDE4 inhibition and modulation of IP3 receptor-mediated Ca2+ oscillations in ASM. The synergistic interaction between benzodiazepines and β2-adrenoreceptor agonists at therapeutic concentrations has important clinical implications for bronchodilation in high-risk patients.

The pulmonary vasculature develops in close association with the airways and this network expands through the interactions between endothelial cells and the surrounding mesenchymal cells, the pericytes. Emerging evidence suggests that pericytes play a significant role in various lung diseases, such as congenital diaphragmatic hernia and chronic obstructive pulmonary disease. However, characterizing pericytes remains challenging, impeding our understanding of their exact role in lung development and disease. Therefore, we used a novel cell tracing technology based on a bacterial DNA cytosine methyltransferase (Dcm) fused to RNA polymerase II (DCM-TM) to methylate active genes. The doxycycline inducible Dcm-PolII fusion protein was activated at specific time points during gestation, while the epigenetically labeled genes were analyzed at later time points. This retrospective cell tracing was coupled to single-cell RNA sequencing to track the development of mouse pulmonary pericytes at the single cell level. This revealed the paths to differentiation of perivascular progenitors into pericytes and vascular smooth muscle cells. Temporal analysis uncovered dynamic gene expression profiles during pericyte differentiation, highlighting pathways crucial for pulmonary vascular development. Further analysis showed intricate signaling interactions between pericyte progenitors and mature pericytes, and we validated MCAM as a bona fide pulmonary pericyte marker. Our findings challenge conventional views on pericyte origin and underscore the importance of accurate pericyte identification in developmental and disease contexts. Overall, this study enhances our understanding of pulmonary pericyte ontogeny and differentiation, offering insights into their potential as therapeutic targets in pericyte-associated lung diseases.

Lung injury after hematopoietic stem cell transplantation (HCT) occurs due to infection, chemotherapy toxicity, and alloreactive inflammation. Analyses of bronchoalveolar lavage (BAL) fluid have revealed dominant pathobiologic signatures, but minimally-invasive diagnostics are needed. To determine whether microbiome and gene expression perturbations are shared along the respiratory tract or isolated to the alveoli in pediatric HCT patients with lung injury. We performed bulk RNA sequencing on 206 paired nasal and BAL samples from 160 HCT patients and 17 healthy controls enrolled at 28 children's hospitals (2016-2025). Microbial and human transcripts were compared using multivariable models accounting for age, sex, and paired sampling. HCT BAL and nasal transcriptomes differed across 13,698 genes, 48 cellular components, and network interactions linking inflammation, reactive oxygen species, and immunometabolism. Minimal BAL-nasal correlation was observed in gene expression levels (median ρ = 0.03, IQR -0.03 to 0.08) or fractional abundance of key cells such as neutrophils and CD8 + T-cells. BAL microbiomes harbored fewer commensal bacteria and more fungi and DNA viruses. BAL bacterial RNA was associated with diminished immune signaling whereas nasal bacterial RNA aligned with inflammatory gene expression. Further, only BAL microbial RNA was linked to transcriptional shifts in epithelial injury response, keratinization, and collagen deposition. Finally, BAL commensal microbiome depletion, epithelial injury, and immune dysregulation signatures were associated with death or prolonged mechanical ventilation, whereas nasal samples provided minimal prognostic information. These data support alveolar compartmentalization in pediatric HCT and emphasize the ongoing need for minimally-invasive but informative diagnostics.

Idiopathic pulmonary fibrosis (IPF) is a progressive and often fatal lung disease characterized by the scarring and stiffening of lung tissue. While its exact cause remains elusive, emerging evidence underscores the significant role of mechanical forces and aberrant epithelial responses in its pathogenesis. The airway epithelium serves as a critical barrier and regulator of lung function, exhibiting abnormal responses to mechanical stress. Recent studies suggest that the abnormal airway epithelial response to mechanical stressors contributes to the progression of IPF and serves as an early indicator of disease onset. Understanding the complex interplay between mechanical forces and these cells in IPF not only sheds light on disease mechanisms but also opens avenues for novel therapeutic interventions targeting these pathways. The goal of this review is to highlight the most recent advances in understanding mechanobiology and the interaction between biomechanical properties and the airway epithelium in the pathogenesis of IPF. We seek to describe a positive feedback loop underlying pulmonary fibrosis, in which pro-fibrotic mechanobiological responses of airway epithelial cells drive biomechanical alterations in the lung airways. In parallel, these biomechanical changes further stimulate pro-fibrotic responses in the epithelial cells, perpetuating the cycle. Additionally, we aim to provide a comprehensive perspective on the role of lung biomechanics in the progression of fibrosis, emphasizing the critical need to disrupt this insidious feedback loop to halt or prevent the advancement of lung fibrosis.

Primary Ciliary Dyskinesia (PCD) is a genetically heterogeneous disorder leading to destructive airway disease with severe bronchiectasis and chronic lung failure in adulthood. Pathogenic variants in CCDC40 are associated with more severe reduction of lung function compared to most other PCD types. Currently, no therapies correcting the underlying disease mechanism are available. Here we investigate the efficacy of lipidoid nanoparticle-formulated mRNA encoding human CCDC40 (LNP-CCDC40-mRNA) as a corrective measure for structural and functional defects in vitro (human cells) and in vivo (zebrafish). Human nasal respiratory epithelial cells cultured at air-liquid-interface from five CCDC40-deficient individuals and a newly generated vertebrate animal model (ccdc40-/- zebrafish) were treated with LNP-CCDC40-mRNA. CCDC40-deficient cells were analyzed by high-speed video microscopy and immunofluorescence microscopy. ccdc40-/- zebrafish olfactory pit cilia were analyzed by high-speed video microscopy and fluid flow assays. Topical application of exogenous LNP-CCDC40-mRNA to CCDC40-deficient cells results in endogenous CCDC40 expression (10-74% of ciliated cells), enabling axonemal integration of CCDC40-associated proteins (CCDC39, GAS8/DRC4, DNALI1). Consistently, ciliary beat frequencies were significantly increased in treated CCDC40-deficient cells and comparable to healthy control cells. Further, we showed improved ciliary transport of fluorescent particles. Injection or topical application of human LNP-CCDC40-mRNA to ccdc40-/- zebrafish significantly increased ciliary motility and established directional flow in olfactory pits. We provide structural and functional evidence in vitro and in vivo for the biological efficacy of LNP-CCDC40-mRNA in CCDC40-deficient respiratory cells and zebrafish. Based on our results, an in vivo human study (Phase 1 trial) is planned in individuals with pathogenic variants in CCDC40.

Acute respiratory distress syndrome (ARDS) is a life-threatening inflammatory lung injury. Regulatory T-cells (Tregs) and their extracellular vesicles (Treg-EVs) possess immunomodulatory properties that may be therapeutic in ARDS. However, evidence is scattered across individual studies, hindering the assessment of their safety and efficacy. To address this gap, we applied a systematic review (SR) methodology, a common approach in clinical research that has increasingly been recognized for its value in preclinical evidence synthesis. Specifically, we synthesized studies of Tregs/Treg-EVs in animal models of acute lung injury (ALI) or patients with ARDS. Importantly, we provide explanatory text throughout for readers less familiar with SRs. The primary preclinical and clinical outcomes were histological lung injury and mortality, respectively. Study selection and data extraction were performed in duplicate. Twenty-two preclinical and two clinical studies met the inclusion criteria, all using whole-cell Tregs. Meta-analysis of animal studies demonstrated that, compared to controls, Treg treatment significantly reduced histological lung injury (≤7 days: standardized mean difference (SMD) = -2.06 [95% confidence interval (CI): -2.96, -1.15]; >7 days: SMD = -2.18 [95% CI: -3.28, -1.08]). Tregs also reduced bronchoalveolar lavage fluid pro-inflammatory cytokines, total protein, total cells, and neutrophil counts. Clinically, early-phase study designs precluded meta-analysis; however, safety and tolerability of Tregs for ARDS were supported. Identified mechanisms underlying Treg effects included immunomodulation, cytokine regulation, and epigenetic pathways. Our review demonstrates the utility of formal preclinical evidence synthesis and supports the therapeutic potential of Tregs. Further investigations are justified to refine Tregs as a cellular therapy for ARDS.

Pathologically altered physical properties of the extracellular matrix are increasingly recognized as an active player in fibrosis inception and progression. Fibroblasts produce an increasingly stiff matrix, which in turn perpetuates fibroblast activation and transdifferentiation into myofibroblasts. Yet, there is still an unmet need for accessible technologies allowing detailed characterization of tissue stiffness to study this relationship. In our current study, we demonstrate the feasibility of a Brillouin microscopy-based quantitative spatial stiffness measurement for the characterization of lung tissue samples with variable degrees of fibrosis. First, we validated our Brillouin microscopy setup using hydrogels with defined levels of stiffness. We then devised a workflow to measure native murine lung tissue cryosections and successfully characterized stiffness levels of lung tissue sections with fibrosis and in non-fibrotic controls. Finally, we successfully applied our setup to fibrotic human lung tissue sections. In conclusion, our proof-of-concept study shows the feasibility of quantitative spatial lung tissue stiffness assessment by Brillouin microscopy in a setup that can easily be integrated into common research workflows. Stiffness measurements through label- and contact-free Brillouin microscopy in combination with techniques such as immunofluorescence staining, RNA-scope, spatial transcriptomics, spatial proteomics, and others have great potential to generate new insights into the mechanobiology of pulmonary fibrosis and a multitude of other diseases in the future.

Neutrophil extracellular trap (NET) formation has been implicated in the pathogenesis of rheumatoid arthritis-associated interstitial lung disease (RA-ILD), yet the specific neutrophil subsets involved and the mechanisms remain incompletely understood. Here, we identified a significantly expanded population of microsomal glutathione S-transferase 2-high NET-releasing polymorphonuclear neutrophils (Mgst2hi PMNs) in the lungs, bronchoalveolar lavage fluid (BALF), and peripheral blood (PB) of zymosan A-induced interstitial pneumonia-arthritis mouse models. Pharmacologic inhibition of Mgst2 with coniferyl ferulate (CF) and genetic knockdown using an adeno-associated virus (AAV9)-delivered shRNA under the Cd11b promoter (AAV9-shRNA-Mgst2) markedly attenuated pulmonary progression by suppressing NET formation. Mechanistically, Mgst2 promoted NET release in neutrophil-differentiated HL-60 cells and primary human peripheral blood neutrophils from healthy donors through a NADPH oxidases 2 (NOX2)-dependent pathway. NETs derived from these cells induced transdifferentiation of primary human pulmonary microvascular pericytes into myofibroblasts by activating transforming growth factor-beta (TGF-β) pathway. Clinically, circulating Mgst2hi NET-releasing PMNs were significantly elevated in the PB of patients with RA-ILD. Of note, more abundant Mgst2hi PMNs were found in RA patients with nonspecific interstitial pneumonia (NSIP) than those with usual interstitial pneumonia (UIP), organizing pneumonia (OP) and other ILD patterns. Moreover, the proportion of circulating Mgst2hi PMNs positively correlated with RA-NSIP severity, as assessed by and mean lung vessel diameter (6 mm), fibrosis score, and vessel score. Collectively, these findings demonstrate a critical pathogenic role for Mgst2hi PMNs in RA-ILD and suggest their utility as a potential therapeutic target through modulation of NET formation.

Bronchopulmonary Dysplasia (BPD) arises from disrupted lung development after preterm birth and produces structural deficits at every level of the respiratory tree. Lower airway disease is emerging as a clinically significant BPD phenotype with increased mortality, yet the molecular mechanisms whereby preterm birth disrupts airway development remain poorly defined. To develop a human model of lower airway disease following preterm birth and to define a molecular endotype of evolving BPD (eBPD) at baseline and in response to injury. An ex vivo organotypic Airway Epithelial Cell (AEC) model was combined with well-characterized pathologic and transcriptomic patient samples for quantitative immunohistochemistry and RNA sequencing analyses. Compared to AECs from healthy controls, eBPD-derived AECs exhibited reduced proliferation, impaired differentiation to ciliated epithelium, and expansion of a vimentin-positive population with a transcriptional profile associated with impaired AEC differentiation. Following hyperoxia exposure, eBPD-derived AECs mounted a robust vimentin response ex vivo, paralleling increased vimentin expression observed in airway cells from lung tissue of human infants with BPD. Using an organotypic model of neonatal airway differentiation, we demonstrate eBPD is associated with impaired AEC differentiation, increased vimentin-expression and concomitant loss of ciliated cells, and an exaggerated vimentin response to hyperoxic injury. These findings mimic the effects of prematurity in airway cells in human patients. These data support a mechanism whereby hyperoxia leads to impaired epithelial differentiation and associated lower airway dysfunction in BPD and inform future mechanistic studies interrogating the role of intermediate filaments in maladaptive epithelial repair.

A pseudostratified epithelium lines the upper airways and is maintained by stem cells that express a basal cell phenotype. These airway stem cells share molecular and functional similarities with other tissue stem cells, particularly those that are involved in development or postnatal maintenance of the esophagus and epidermis. Thus, analysis of gene function in the airway is complicated by the potential for lethal non-respiratory phenotypes. Conditional genetic approaches employing cell type-specific and/or temporally controlled expression systems provide some options, but limitations remain. To overcome these concerns, we tested a novel microsurgical approach in which a chimeric airway is created by orthotopic transplantation of transgenic mouse tissue into the trachea. We hypothesized that precise temporal and spatial control of gene expression in the pseudostratified tracheal epithelium would prevent life-threatening collateral tissue injury and allow analysis of essential genes. First, we demonstrated that the graft was revascularized allowing parenteral administration of tamoxifen. Second, we transplanted tracheal tissue from Krt5CreERT2; Rosa26mTmG or Col1a2CreERT2; Rosa26mTmG donors into wild type mice and established that tamoxifen-dependent recombination in basal cells or fibroblasts was highly efficient and restricted to the graft. Finally, we assessed the feasibility of knocking out an essential gene, Itgb1, by transplanting tracheal segments from Krt5CreERT2; Rosa26mTmG; Itgb1flox/flox transgenic mice into wild type mice. We demonstrated highly selective recombination in the graft and long-term survival. We conclude that the chimeric airway model allows analysis of essential gene function in the airway and has the potential to be a versatile tool for preclinical testing of targeted gene therapies.

This study aimed to investigate the role of the FBXW7/SPI1/MFAP4 axis in pulmonary arterial hypertension (PAH). An in vivo PAH model was constructed using a hypoxia chamber. The expression levels of FBXW7, SPI1 and MFAP4 were detected by immunofluorescence and Western blotting. Cardiopulmonary function was assessed; pathological changes in lung tissue were examined by HE staining, and inflammatory factors levels were measured using ELISA. For in vitro studies, a PAH model was simulated using hypoxia-induced pulmonary arterial smooth muscle cells (PASMCs). MFAP4-stable knockdown cells were constructed. Cell proliferation was evaluated with CCK8 and EdU assays, and cell migration was assessed using scratch wound and transwell assays. The targeting relationship between SPI1 and MFAP4 was verified by dual-luciferase reporter and ChIP-qPCR assays. Further, co-immunoprecipitation (Co-IP) and protein stability experiments were performed to confirm FBXW7-mediated ubiquitination of SPI1. In vivo, MFAP4 knockdown led to decreases in right ventricular systolic pressure (RVSP), mean pulmonary arterial pressure (mPAP), right ventricular (RV) contraction index, and pulmonary arterial (PA) wall thickness. In vitro, MFAP4 knockdown suppressed PASMC proliferation and migration. SPI1 was identified as an upstream transcription factor of MFAP4. FBXW7 was shown to promote SPI1 ubiquitination and its degradation in vitro. Overexpression of FBXW7 suppressed PAH progression both in vitro and in vivo, while simultaneous overexpression of either SPI1 or MFAP4 counteracted the protective effects of FBXW7 overexpression. Decreased FBXW7 expression in PAH inhibits SPI1 ubiquitination and protein degradation, leading to increased MFAP4 levels, which ultimately promotes PAH progression.

Lymphangioleiomyomatosis (LAM) is a rare, progressive lung disease that predominantly affects women, particularly during their reproductive years. It is characterised by the proliferation of abnormal smooth muscle-like cells that infiltrate the lungs, leading to cystic destruction of lung tissue and a decline in respiratory function. The pathogenic defect in LAM occurs due to mutations in the tuberous sclerosis complex (TSC) genes, resulting in failure to suppress the mechanistic target of rapamycin (mTOR) pathway activation, which drives abnormal cell proliferation and lymphangiogenesis. Although mTOR inhibitors, such as sirolimus, have improved clinical management by slowing disease progression, they are not curative and do not reverse existing lung damage. Recent research has expanded our understanding of LAM pathogenesis by revealing substantial genetic and cellular heterogeneity within LAM lesions. Beyond mTOR dysregulation, pathways involving estrogen signalling, metabolic adaptation, and immune evasion contribute to disease development. These insights open new avenues for treatment. A better understanding of these pathways will pave the way for more durable and individualised treatments for LAM.

Endothelial-to-mesenchymal transition (EndMT) is a biological process through which lung vascular endothelial cells (ECs) transdifferentiate into mesenchymal-like cells. EndMT has recently been implicated in the development and progression of pulmonary vascular remodeling in pulmonary hypertension (PH); however, its underlying regulatory mechanisms remain incompletely understood. MicroRNAs (miRNAs) are key post-transcriptional regulators of EC gene expression and cellular responses to various stimuli. Notably, microRNA-153 (miR-153) has been shown to directly target SNAI1 to modulate epithelial-to-mesenchymal transition (EMT), a process closely related to EndMT and extensively studied in cancer. Whether miR-153 also participates in EndMT regulation, however, remains unknown. In this study, we demonstrate that 72-hour hypoxic exposure induces SNAI1-mediated EndMT in human lung vascular ECs. Hypoxia also increased cell proliferation and disrupted intercellular junctions, leading to enhanced endothelial permeability. Reduced miR-153 expression was observed in both hypoxia- and TGF-β1-induced EndMT, as well as in ECs isolated from PH patients exhibiting an EndMT phenotype. Similar to hypoxia, TGF-β1 promoted EC permeability. Loss of miR-153 enhanced SNAI1-mediated EndMT, endothelial survival, and permeability under normoxic conditions, whereas miR-153 overexpression attenuated EndMT induced by hypoxia or TGF-β1. However, miR-153 restoration did not completely restore endothelial barrier integrity disrupted by these stimuli. In vitro findings were validated in experimental PH model. In conclusion, miR-153 serves as a critical regulator of EndMT, maintaining endothelial identity and barrier function. Therapeutic delivery of miR-153 may therefore represent a novel strategy to inhibit EndMT and attenuate pulmonary vascular remodeling in PH.

Recent investigations show that mitochondrial impairment significantly contributes to endothelial damage in septic acute respiratory distress syndrome (ARDS). Humanin (HN) and its derivative Humanin-G (HNG) are mitochondrial polypeptides which have been identified as inhibitors of cellular apoptosis and neuroprotective agents against oxidative stress. This study aims to elucidate the effects of HNG on pulmonary vascular endothelial damage. In septic ARDS patients, serum concentrations of HN increased markedly on Day 1, followed by a progressive decrease from Day 3 to Day 7. A murine model of septic ARDS was established through intraperitoneal injection of lipopolysaccharide. The results showed that HNG pretreatment significantly reduced inflammatory factor expression in both in vivo and in vitro settings, as confirmed by qPCR and Western blot. Furthermore, HNG treatment conferred protection against lung injury, restored mitochondrial morphology, improved mitochondrial respiratory function, and corrected impaired membrane potential, as assessed by H&E staining, transmission electron microscopy, Seahorse analysis, and JC-1 staining, respectively. Additionally, protein-peptide interaction analysis suggested that HNG binds to the interleukin-6 receptor alpha, and immunoprecipitation confirmed that HNG competitively interacts with the IL-6 receptor family in comparison to IL-6. Furthermore, WB analysis indicated that the protective effects of HNG on mitochondria may be largely due to the suppression of STAT3 expression in septic lung endothelial cells. In summary, this study suggests that the administration of the mitochondrial peptide HNG confers protective effects and mitigates mitochondrial damage by inhibiting the downstream pro-inflammatory pathways of IL-6/STAT3 in the pulmonary vascular endothelial cells of septic ARDS.

The identification of clinically predictive serum biomarkers for pulmonary fibrosis is a significant challenge and important goal. Multiple recent proteomic biomarker studies have identified latent transforming growth factor binding protein-2 (LTBP2) as a circulating factor associated with disease progression in fibrotic lung diseases in humans (including IPF), but its role in the development of fibrosis is incompletely defined. LTBP2 competes with the large latent transforming growth factor-beta (TGFβ) complex (LLC) for binding to the N-terminus of fibrillin and is thought to promote the release of active TGFβ. We hypothesized that LTBP2 deficiency would promote LLC sequestration in matrix and reduce TGFβ signaling. We recently reported an LTBP2 knockout (Ltbp2-/-) mouse with no baseline lung abnormalities. Here we show that Ltbp2-/- mice exposed to either bleomycin or silica have a significant reduction in fibrosis compared to wild type controls. Consistent with reduced fibrosis, after bleomycin Ltbp2-/- mouse lungs have reduced TGFβ signaling and isolated fibroblasts from Ltbp2-/- mice exhibit impaired migration in an in vitro wound closure assay. Transcriptomic analysis of bleomycin-treated control and Ltbp2-/-mouse lung tissue identified multiple LTBP2-regulated genes, including the lncRNA antisense of IGFR2 non-coding RNA (Airn) which has reported antifibrotic effects. Interestingly, we also observed that Ltbp2-/-mice had impaired epithelial repair after bleomycin treatment, a phenotype that also occurred in a naphthalene model of club cell injury. These findings provide evidence that LTBP2 is profibrotic and facilitates TGFβ signaling but is also required for normal airway epithelial repair.