Human Lung Tissue Research Articles

Ischemia/Reperfusion Upregulates Genes Related to PANoptosis in Human Lung Transplants.

Activation of multiple programmed cell death (PCD) pathways has been reported in cellular and animal studies of ischemia/reperfusion injury in lung transplantation. However, the status of these pathways in human lung transplants remains unknown. This study investigates the involvement of PCD pathways and their relationship with inflammation and signaling pathways in human lung transplants. Transcriptomic analysis was conducted on 54 paired human lung tissue samples at the end of cold preservation time and 2 h after reperfusion, collected between 2008 and 2011. Gene Set Enrichment Analysis (GSEA) and single-sample GSEA were used to examine the activation of genes in 6 PCD pathways. The relationships between PCD pathways and inflammation, as well as signaling pathways, were assessed via single-gene GSEA. GSEA results indicated that apoptosis and necroptosis were significantly upregulated after reperfusion in human lung transplants, whereas the gene sets related to pyroptosis, ferroptosis, autophagy, and cuproptosis were not significantly upregulated. Notably, single-sample GSEA demonstrated an intricate interplay among pyroptosis, apoptosis, and necroptosis, collectively referred to as PANoptosis, which is further supported by enrichment of genes related to PANoptosome, inflammatory response, and nuclear factor-κB and interferon signaling pathways, via single-gene GSEA assays. This study demonstrated the genes of PANoptosis are upregulated in human lung grafts during reperfusion. The discovery of PANoptosis as an underlying mechanism of cell death in human lung grafts implies that effective therapeutics to prevent or reduce PANoptosis may alleviate ischemia/reperfusion injury and improve clinical lung transplant outcomes.

Computer vision applications for the detection or analysis of tuberculosis using digitised human lung tissue images - a systematic review

ObjectiveTo conduct a systematic review of the computer vision applications that detect, diagnose, or analyse tuberculosis (TB) pathology or bacilli using digitised human lung tissue images either through automatic or semi-automatic methods. We categorised the computer vision platform into four technologies: image processing, object/pattern recognition, computer graphics, and deep learning. In this paper, the focus is on image processing and deep learning (DL) applications for either 2D or 3D digitised human lung tissue images. This review is useful for establishing a common practice in TB analysis using human lung tissue as well as identifying opportunities for further research in this space. The review brings attention to the state-of-art techniques for detecting TB, with emphasis on the challenges and limitations of the current techniques. The ultimate goal is to promote the development of more efficient and accurate algorithms for the detection or analysis of TB, and raise awareness about the importance of early detection.DesignWe searched five databases and Google Scholar for articles published between January 2017 and December 2022 that focus on Mycobacterium tuberculosis detection, or tuberculosis pathology using digitised human lung tissue images. Details regarding design, image processing and computer-aided techniques, deep learning models, and datasets were collected and summarised. Discussions, analysis, and comparisons of state-of-the-art methods are provided to help guide future research. Further, a brief update on the relevant techniques and their performance is provided.ResultsSeveral studies have been conducted to develop automated and AI-assisted methods for diagnosing Mtb and TB pathology from digitised human lung tissue images. Some studies presented a completely automated method of diagnosis, while other studies developed AI-assisted diagnostic methods. Low-level focus areas included the development of a novel μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}CT scanner for soft tissue image contract, and use of multiresolution computed tomography to analyse the 3D structure of the human lung. High-level focus areas included the investigation the effects of aging on the number and size of small airways in the lungs using CT and whole lung high-resolution μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}CT, and the 3D microanatomy characterisation of human tuberculosis lung using μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}CT in conjunction with histology and immunohistochemistry. Additionally, a novel method for acquiring high-resolution 3D images of human lung structure and topology is also presented.ConclusionThe literature indicates that post 1950s, TB was predominantly studied using animal models even though no animal model reflects the full spectrum of human pulmonary TB disease and does not reproducibly transmit Mtb infection to other animals (Hunter, 2011). This explains why there are very few studies that used human lung tissue for detection or analysis of Mtb. Nonetheless, we found 10 studies that used human tissues (predominately lung) of which five studies proposed machine learning (ML) models for the detection of bacilli and the other five used CT on human lung tissue scanned ex-vivo.

Utilizing collagen-coated hydrogels with defined stiffness as calibration standards for AFM experiments on soft biological materials: the case of lung cells and tissue

Abstract Atomic Force Microscopy (AFM) is crucial in mechanobiology for high-resolution imaging and nanomechanical measurements of biological samples, providing insights into their mechanical properties. However, AFM faces challenges such as tip damage and cantilever selection errors, impacting measurement accuracy. This study proposes a methodology using collagen-coated hydrogels with predefined stiffness for calibrating AFM measurements on soft biological materials. By facilitating appropriate cantilever selection, assessing systematic errors, and evaluating tip damage, this approach ensures reliable Young’s modulus measurements. The proof of concept with human lung cells and tissue specimens demonstrates improved accuracy and reliability of AFM-based nanomechanical characterizations, essential for understanding cellular mechanics and disease progression.

Differential transcriptomic host responses in the early phase of viral and bacterial infections in human lung tissue explants ex vivo

BackgroundThe first 24 h of infection represent a critical time window in interactions between pathogens and host tissue. However, it is not possible to study such early events in human lung during natural infection due to lack of clinical access to tissue this early in infection. We, therefore, applied RNA sequencing to ex vivo cultured human lung tissue explants (HLTE) from patients with emphysema to study global changes in small noncoding RNA, mRNA, and long noncoding RNA (lncRNA, lincRNA) populations during the first 24 h of infection with influenza A virus (IAV), Mycobacterium bovis Bacille Calmette-Guerin (BCG), and Pseudomonas aeruginosa.ResultsPseudomonas aeruginosa caused the strongest expression changes and was the only pathogen that notably affected expression of microRNA and PIWI-associated RNA. The major classes of long RNAs (> 100 nt) were represented similarly among the RNAs that were differentially expressed upon infection with the three pathogens (mRNA 77–82%; lncRNA 15–17%; pseudogenes 4–5%), but lnc-DDX60-1, RP11-202G18.1, and lnc-THOC3-2 were part of an RNA signature (additionally containing SNX10 and SLC8A1) specifically associated with IAV infection. IAV infection induced brisk interferon responses, CCL8 being the most strongly upregulated mRNA. Single-cell RNA sequencing identified airway epithelial cells and macrophages as the predominant IAV host cells, but inflammatory responses were also detected in cell types expressing few or no IAV transcripts. Combined analysis of bulk and single-cell RNAseq data identified a set of 6 mRNAs (IFI6, IFI44L, IRF7, ISG15, MX1, MX2) as the core transcriptomic response to IAV infection. The two bacterial pathogens induced qualitatively very similar changes in mRNA expression and predicted signaling pathways, but the magnitude of change was greater in P. aeruginosa infection. Upregulation of GJB2, VNN1, DUSP4, SerpinB7, and IL10, and downregulation of PKMYT1, S100A4, GGTA1P, and SLC22A31 were most strongly associated with bacterial infection.ConclusionsHuman lung tissue mounted substantially different transcriptomic responses to infection by IAV than by BCG and P. aeruginosa, whereas responses to these two divergent bacterial pathogens were surprisingly similar. This HLTE model should prove useful for RNA-directed pathogenesis research and tissue biomarker discovery during the early phase of infections, both at the tissue and single-cell level.

SPP1 induces idiopathic pulmonary fibrosis and NSCLC progression via the PI3K/Akt/mTOR pathway

BackgroundThe prevalence of non-small cell lung cancer (NSCLC) is notably elevated in individuals diagnosed with idiopathic pulmonary fibrosis (IPF). Secreted phosphoprotein 1 (SPP1), known for its involvement in diverse physiological processes, including oncogenesis and organ fibrosis, has an ambiguous role at the intersection of IPF and NSCLC. Our study sought to elucidate the function of SPP1 within the pathogenesis of IPF and its subsequent impact on NSCLC progression.MethodsFour GEO datasets was analyzed for common differential genes and TCGA database was used to analyze the prognosis. The immune infiltration was analyzed by TIMER database. SPP1 expression was examined in human lung tissues, the IPF fibroblasts and the BLM-induced mouse lung fibrosis model. Combined with SPP1 gene gain- and loss-of-function, qRT-PCR, Western blot, EdU and CCK-8 experiments were performed to evaluate the effects and mechanisms of SPP1 in IPF progression. Effect of SPP1 on NSCLC was detected by co-cultured IPF fibroblasts and NSCLC cells.ResultsThrough bioinformatics analysis, we observed a significant overexpression of SPP1 in both IPF and NSCLC patient datasets, correlating with enhanced immune infiltration of cancer-associated fibroblasts in NSCLC. Elevated levels of SPP1 were detected in lung tissue samples from IPF patients and bleomycin-induced mouse models, with partial colocalization observed with α-smooth muscle actin. Knockdown of SPP1 inhibits TGF-β1-induced differentiation of fibroblasts to myofibroblasts and the proliferation of IPF fibroblasts. Conversely, SPP1 overexpression promoted IPF fibroblast proliferation via PI3K/Akt/mTOR pathway. Furthermore, IPF fibroblasts promoted NSCLC cell proliferation and activated the PI3K/Akt/mTOR pathway; these effects were attenuated by SPP1 knockdown in IPF fibroblasts.ConclusionsOur findings suggest that SPP1 functions as a molecule promoting both fibrosis and tumorigenesis, positioning it as a prospective therapeutic target for managing the co-occurrence of IPF and NSCLC.

Experimental evolution under different nutritional conditions changes the genomic architecture and virulence of Acinetobacter baumannii

This study uncovers the molecular processes governing the adaptive evolution of multidrug-resistant (MDR) pathogens without antibiotic pressure. Genomic analysis of MDR Acinetobacter baumannii cells cultured for 8000 generations under starvation conditions (EAB1) or nutrient-rich conditions (EAB2) revealed significant genomic changes, primarily by insertion sequence (IS)-mediated insertions and deletions. Only two Acinetobacter-specific prophage-related deletions and translocations were observed in the EAB1 strain. Both evolved strains exhibited higher virulence in mouse infection studies, each with different modes of action. The EAB1 strain displayed a heightened ability to cross the epithelial barrier of human lung tissue, evade the immune system, and spread to lung tissues, ultimately resulting in cellular mortality. In contrast, the EAB2 strain strongly attached to epithelial cells, leading to increased synthesis of proinflammatory cytokines and chemokines. The genomic alterations and increased virulence observed in evolved strains during short-term evolution underscore the need for caution when handling these pathogens, as these risks persist even without antibiotic exposure.

Graph-Based Spatial Proximity of Super-Resolved Protein-Protein Interactions Predicts Cancer Drug Responses in Single Cells.

Current bulk molecular assays fail to capture spatial signaling activities in cancers, limiting our understanding of drug resistance mechanisms. We developed a graph-based super-resolution protein-protein interaction (GSR-PPI) technique to spatially resolve single-cell signaling networks and evaluate whether higher resolution microscopy enhances the biological study of PPIs using deep learning classification models. Single-cell spatial proximity ligation assays (PLA, ≤ 9 PPI pairs) were conducted on EGFR mutant (EGFRm) PC9 and HCC827 cells (>10,000 cells) treated with 100nM Osimertinib. Multiplexed PPI images were obtained using wide-field and super-resolution microscopy (Zeiss Airyscan, SRRF). Graph-based deep learning models analyzed subcellular protein interactions to classify drug treatment states and test GSR-PPI on clinical tissue samples. GSR-PPI triangulated PPI nodes into 3D relationships, predicting drug treatment labels. Biological discriminative ability (BDA) was evaluated using accuracy, AUC, and F1 scores. The method was also applied to 3D spatial proteomic molecular pixelation (PixelGen) data from T cells. GSR-PPI outperformed baseline models in predicting drug responses from multiplexed PPI imaging in EGFRm cells. Super-resolution data significantly improved accuracy over localized wide-field imaging. GSR-PPI classified drug treatment states in cancer cells and human lung tissues, with performance improving as imaging resolution increased. It differentiated single and combination drug therapies in HCC827 cells and human tissues. Additionally, GSR-PPI accurately distinguished T-cell stimulation states, identifying key nodes such as CD44, CD45, and CD54. The GSR-PPI framework provides valuable insights into spatial protein interactions and drug responses, enhancing the study of signaling biology and drug resistance. The online version contains supplementary material available at 10.1007/s12195-024-00822-1.

Identification of SNHG11 as a Therapeutic Target in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a life-threatening condition characterized by pulmonary vascular remodeling and endothelial dysfunction. Current therapies primarily target vasoactive imbalances but often fail to address adverse vascular remodeling. Long non-coding RNA (lncRNA), which are key regulators of various cellular processes, remain underexplored in the context of PH. To investigate the role of lncRNA in PH, we performed a comprehensive analysis using Weighted Gene Co-expression Network Analysis (WGCNA) on the GSE113439 dataset, comprising human lung tissue samples from different PH subtypes. Our analysis identified the lncRNA SNHG11 as consistently downregulated in PH. Functional assays in human pulmonary artery endothelial cells (HPAECs) demonstrated that SNHG11 plays a critical role in modulating inflammation, cell proliferation, apoptosis, and the JAK/STAT and MAPK signaling pathways. Mechanistically, SNHG11 influences the stability of PRPF8, a crucial mRNA spliceosome component, thereby affecting multiple cellular functions beyond splicing. In vivo experiments using a hypoxic rat model showed that knockdown of SNHG11 alleviates PH development and improves right ventricular function. These findings highlight SNHG11 as a key regulator in PH pathogenesis and suggest it as a potential therapeutic target.

PAI-1 Derived from Alveolar Type 2 Cells Drives Aging-Associated Pulmonary Fibrosis

Pulmonary fibrosis (PF) is a lethal lung disease that predominantly affects older adults; however, whether and how aging triggers fibrosis remains unclear. To pinpoint the predominant initiating factors of PF, we first analyzed single-cell RNA sequencing (scRNA-seq) data from the lung tissues of 45 normal donors and 51 PF patients and found that aging might serve as the primary catalyst for PF development. To further investigate the influence of aging on PF formation, we conducted a comprehensive and thorough study employing a natural aging mouse model. We found that dynamic alterations in the quantity and types of collagen fibers during aging-induced PF progression, especially in collagenous (Col) I, emerged as the predominant driver of PF. We then investigated the regulation of Col I synthesis during aging using primary alveolar type 2 (AT2) cells and A549 cells line through conditioned media and Transwell coculture, and found that secretions—particularly plasminogen activator inhibitor (PAI)-1—from aged AT2 cells promoted fibrosis and enhanced collagen type I alpha 1 (Col1al) production via the transforming growth factor (TGF)-β/small mother against decapentaplegic (Smad)2/3 pathway. Furthermore, scRNA-seq and a histological analysis of human lung tissue demonstrated a significant upregulation of SERPINE1 (the gene encoding PAI-1) and PAI-1 expression in both aging lung tissue and AT2 cells, which was consistent with our findings from animal experiments, providing additional evidence for the pivotal role of PAI-1 during aging and the development of PF. Our research demonstrates that PAI-1, a crucial factor secreted by aging AT2 cells, exerts a pivotal role in promoting the synthesis of Col1a1 in fibroblasts, subsequently leading to Col I deposition, and in driving the progression of PF by mediating the TGF-β/Smad2/3 pathway. Our findings offer critical evidence for the involvement of epithelial dysfunction in age-related PF and provides potential novel therapeutic targets for clinical intervention.

The Potential of Ambroxol as a Panacea for Neonatal Diseases: A Scoping Review.

Ambroxol, a commonly used mucolytic agent, has been extensively studied for its clinical effectiveness in managing respiratory conditions in pediatric and adult patients. The existing body of research on ambroxol demonstrates its safety and efficacy. However, its potential role in preventing and treating neonatal diseases still needs to be explored. This scoping review aims to shed light on the unexplored potential of ambroxol, particularly its applications in perinatal and neonatal care. We aim to offer valuable insights for healthcare professionals, researchers, and academics, thus presenting a positive perspective. Key scientific databases such as Google Scholar, PubMed, Cochrane Library, and Europe PMC were meticulously searched for relevant literature on ambroxol in perinatal and neonatal medicine. Gray literature was also surveyed, and the search encompassed all study designs and languages up to June 2024. Furthermore, citations and reference lists of relevant articles were scrutinized to identify additional pertinent literature. Ambroxol has demonstrated promising effects in preventing and managing respiratory distress syndrome (RDS). It can enter the placental circulation and rapidly build up in human lung tissue to a much greater extent than in plasma. It promotes fetal lung maturation, surfactant production, and alveolar expansion. Numerous studies have demonstrated the efficacy of antenatal and postnatal ambroxol in the prevention and treatment of RDS. Ambroxol has the potential to be administered intravenously or through nebulization, offering the hopeful possibility of reducing the high failure rate typically associated with non-invasive ventilation in extremely preterm infants, instilling a sense of hope and optimism about the potential of ambroxol. It also shows potential in treating bronchopulmonary dysplasia, meconium aspiration syndrome, and neonatal infections. Ambroxol has been observed to assist in the closure of patent ductus arteriosus in preterm infants by inhibiting vasodilator agents such as nitric oxide and exerting vasoconstrictive properties. However, these biological actions may raise concerns regarding the potential induction of pulmonary hypertension and an increased risk of necrotizing enterocolitis. The present scoping review also examines the clinical evidence and the potential of ambroxol in reducing the incidence of intraventricular hemorrhage in preterm infants. Ambroxol may have potential analgesic properties in managing neonatal pain, and as it can penetrate the blood-brain barrier, it suggests potential neuroprotective properties. These properties may encompass the modulation of microglial activation and the antagonistic impact on glutamate receptors. Ambroxol's attributes could contribute to a decreased susceptibility to neurological complications and have demonstrated anticonvulsant effects in preclinical studies. While low-to-moderate-quality evidence indicates potential applications of ambroxol in neonatal care, further research is needed to determine the drug's optimal dosing, timing, and safety profiles in this patient population. We need to investigate ambroxol's potential synergistic effects with antenatal steroids. Exploration is required to assess ambroxol's potential in reducing the high failure rate associated with non-invasive respiratory support for RDS. Lastly, comprehensive studies on the long-term neurodevelopmental outcomes of neonates exposed to ambroxol are essential.

3D Bioprinitng technologies for the enhancement and application of functional lung organoid models

Respiratory diseases, ranging from minor infections to serious chronic diseases and malignancies, negatively affect the respiratory system and are influenced by various environmental factors such as air pollution, occupational hazards, and tobacco smoke, as well as lifestyle, genetic causes, and infectious agents. The prevalence and severity of respiratory diseases requires the development of advanced models to better understand their pathophysiology and develop effective treatments. In this context, 3D bioprinting technology is emerging as an innovative tool to create functional lung organoid models. The use of induced pluripotent stem cells (iPSCs) and extracellular matrix (ECM) in bioprinting enables the development of organoids that closely mimic human lung tissue. Bioprinting-based organoids can better replicate the dynamic environment of the human lung, facilitating more accurate disease modeling and drug testing. In this review, we highlight the potential of bioprinted lung organoids in understanding the mechanisms of chronic respiratory diseases, testing the efficacy and safety of new drugs, and exploring regenerative medicine approaches. The integration of advanced bioprinting and organoid technologies is a promising field in respiratory disease research and treatment, offering new hope for patients suffering from lung diseases.

A Novel Human Extravascular Monocyte Subset with Antiviral Functions Is Crucial for Resolving Lung Tissue Infection.

We identified a novel human subset of lung extravascular monocytes with antiviral functions that play a critical role in resolving SARS-CoV-2 infection from human lung tissues in an immunologically naïve context.

Microwave dielectric properties of normal, fibroelastotic, and malignant human lung tissue

Technological development of microwave treatment and detection techniques for lung cancer requires accurate and comprehensive knowledge of the microwave dielectric properties of human lung tissue. We characterize the dielectric properties of room temperature human lung tissue from 0.5 to 10 GHz for three lung tissue groups: normal, fibroelastotic, and malignant. We fit a two-pole Debye model to the measured frequency-dependent complex permittivity and calculate the median Debye parameters for the three groups. We find that malignant lung tissue is approximately 10% higher in relative permittivity and conductivity compared to normal lung tissue; this trend matches previously reported normal versus malignant data for other biological tissues. There is little contrast between benign lung tissue with fibroelastosis and malignant lung tissue. We extrapolate our data from room temperature to 37 °C using a temperature-dependence model for animal lung tissue and use the Maxwell-Garnett dielectric mixing model to predict the dielectric properties of inflation-dynamic human lung tissue; both approximations correspond with previously reported dielectric data of bovine and porcine lung tissue.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a critical regulator of inflammatory signalling through toll-like receptors 4 and 7/8 in murine and human lungs.

Toll-like receptors 4 (TLR4) and TLR7/TLR8 play an important role in mediating the inflammatory effects of bacterial and viral pathogens. Interleukin-1 receptor-associated kinase 4 (IRAK4) is an important regulator of signalling by toll-like receptor (TLR) and hence is a potential therapeutic target in diseases characterized by increased lung inflammatory signalling. We used an established murine model of acute lung inflammation, and studied human lung tissue ex vivo, to investigate the effects of inhibiting IRAK4 on lung inflammatory pathways. We show that TLR4 stimulation produces an inflammatory response characterized by neutrophil influx and tumour necrosis factor-α (TNF-α) production in murine lungs and that these responses are markedly reduced in IRAK4 kinase-dead mice. In addition, we characterize a novel selective IRAK4 inhibitor, BI1543673, and show that this compound can reduce lipopolysaccharide (LPS)-induced airway inflammation in wild-type mice. Additionally, BI1543673 reduced inflammatory responses to both TLR4 and TLR7/8 stimulation in human lung tissue studied ex vivo. These data demonstrate a key role for IRAK4 signalling in lung inflammation and suggest that IRAK4 inhibition has potential utility to treat lung diseases characterized by inflammatory responses driven through TLR4 and TLR7/8.

Heavy Metal Exposure-Mediated Dysregulation of Sphingolipid Metabolism.

Exposure to heavy metals (HMs) is often associated with inflammation and cell death, exacerbating respiratory diseases including asthma. Most inhaled particulate HM exposures result in the deposition of HM-bound fine particulate matter, PM2.5, in pulmonary cell populations. While localized high concentrations of HMs may be a causative factor, existing studies have mostly evaluated the effects of systemic or low-dose chronic HM exposures. This report investigates the impact of local high concentrations of specific HMs (NaAsO2, MnCl2, and CdCl2) on sphingolipid homeostasis and oxidative stress, as both play a role in mediating responses to HM exposure and have been implicated in asthma. Utilizing an in vitro model system and three-dimensional ex vivo human tissue models, we evaluated the expression of enzymatic regulators of the salvage, recycling, and de novo synthesis pathways of sphingolipid metabolism, and observed differential modulation in these enzymes between HM exposures. Sphingolipidomic analyses of specific HM-exposed cells showed increased levels of anti-apoptotic sphingolipids and reduced pro-apoptotic sphingolipids, suggesting activation of the salvage and de novo synthesis pathways. Differential sphingolipid regulation was observed within HM-exposed lung tissues, with CdCl2 exposure and NaAsO2 exposure activating the salvage and de novo synthesis pathway, respectively. Additionally, using spatial transcriptomics and quantitative real-time PCR, we identified HM exposure-induced transcriptomic signatures of oxidative stress in epithelial cells and human lung tissues.

PAI-1 Interaction with Sortilin Related Receptor-1 is Required for Lung Fibrosis.

Plasminogen activator inhibitor-1 (PAI-1) has been previously shown to promote lung fibrosis via a mechanism that requires an intact vitronectin (VTN) binding site. In the present study, employing two distinct murine fibrosis models, we find that VTN is not required for PAI-1 to drive lung scarring. This result suggested the existence of a previously unrecognized profibrotic PAI-1-protein interaction involving the VTN-binding site for PAI-1. Using an unbiased proteomic approach, we identified sortilin related receptor 1 (SorlA) as the most highly enriched PAI-1 interactor in the fibrosing lung. We next investigated the role of SorlA in pulmonary fibrosis and found that SorlA deficiency protected against lung scarring in a murine model. We further show that, while VTN deficiency does not influence fibrogenesis in the presence or absence of PAI-1, SorlA is required for PAI-1 to promote scarring. These results, together with data showing increased SorlA levels in human IPF lung tissue, support a novel mechanism through which the potent profibrotic mediator PAI-1 drives lung fibrosis and implicate SorlA as a new therapeutic target in IPF treatment.

Unbound Fractions of PFAS in Human and Rodent Tissues: Rat Liver a Suitable Proxy for Evaluating Emerging PFAS?

Adverse health effects associated with exposures to perfluoroalkyl and polyfluoroalkyl substances (PFAS) are a concern for public health and are driven by their elimination half-lives and accumulation in specific tissues. However, data on PFAS binding in human tissues are limited. Accumulation of PFAS in human tissues has been linked to interactions with specific proteins and lipids in target organs. Additional data on PFAS binding and unbound fractions (funbound) in whole human tissues are urgently needed. Here, we address this gap by using rapid equilibrium dialysis to measure the binding and funbound of 16 PFAS with 3 to 13 perfluorinated carbon atoms (ηpfc = 3-13) and several functional headgroups in human liver, lung, kidney, heart, and brain tissue. We compare results to mouse (C57BL/6 and CD-1) and rat tissues. Results show that funbound decreases with increasing fluorinated carbon chain length and hydrophobicity. Among human tissues, PFAS binding was generally greatest in brain > liver ≈ kidneys ≈ heart > lungs. A correlation analysis among human and rodent tissues identified rat liver as a suitable surrogate for predicting funbound for PFAS in human tissues (R2 ≥ 0.98). The funbound data resulting from this work and the rat liver prediction method offer input parameters and tools for toxicokinetic models for legacy and emerging PFAS.

Molecular Changes Implicate Angiogenesis and Arterial Remodeling in Systemic Sclerosis-Associated and Idiopathic Pulmonary Hypertension.

Pulmonary hypertension (PH) is a common complication of systemic sclerosis (SSc) and a leading cause of mortality among patients with this disease. PH can also occur as an idiopathic condition (idiopathic pulmonary arterial hypertension). Investigation of transcriptomic alterations in vascular populations is critical to elucidating cellular mechanisms underlying pathobiology of SSc-associated and idiopathic PH. We analyzed single-cell RNA sequencing profiles of endothelial and perivascular mesenchymal populations from explanted lung tissue of patients with SSc-associated PH (n=16), idiopathic pulmonary arterial hypertension (n=3), and healthy controls (n=15). Findings were validated by immunofluorescence staining of explanted human lung tissue. Three disease-associated endothelial populations emerged. Two angiogenic endothelial cell (EC) subtypes markedly expanded in SSc-associated PH lungs: tip ECs expressing canonical tip markers PGF and APLN and phalanx ECs expressing genes associated with vascular development, endothelial barrier integrity, and Notch signaling. Gene regulatory network analysis suggested enrichment of Smad1 (SMAD family member 1) and PPAR-γ (peroxisome proliferator-activated receptor-γ) regulon activities in these 2 populations, respectively. Mapping of potential ligand-receptor interactions highlighted Notch, apelin-APJ (apelin receptor), and angiopoietin-Tie (tyrosine kinase with immunoglobulin-like and EGF-like domains 1) signaling pathways between angiogenic ECs and perivascular cells. Transitional cells, expressing both endothelial and pericyte/smooth muscle cell markers, provided evidence for the presence of endothelial-to-mesenchymal transition. Transcriptional programs associated with arterial endothelial dysfunction implicated VEGF-A (vascular endothelial growth factor-A), TGF-β1 (transforming growth factor beta-1), angiotensin, and TNFSF12 (tumor necrosis factor ligand superfamily member 12)/TWEAK (TNF-related weak inducer of apoptosis) in the injury/remodeling phenotype of PH arterial ECs. These data provide high-resolution insights into the complexity and plasticity of the pulmonary endothelium in SSc-associated PH and idiopathic pulmonary arterial hypertension and provide direct molecular insights into soluble mediators and transcription factors driving PH vasculopathy.

3D Culture Analysis of Cancer Cell Adherence to Ex Vivo Lung Microexplants.

Ex vivo 3D culture of human tissue explants addresses many limitations of traditional monolayer cell culture techniques, namely the lack of cellular heterogeneity and absence of 3D intercellular spatial relationships, but presents challenges with regard to repeatability owing to the difficulty of acquiring multiple tissue samples from the same donor. In this study, we used a cryopreserved bank of human lung microexplants, ∼1 mm3 fragments of peripheral lung from donors undergoing lung resection surgery, and a liquid-like solid 3D culture matrix to describe a method for the analysis of non-small-cell lung cancer adhesion to human lung tissue. H226 (squamous cell carcinoma), H441 (lung adenocarcinoma), and H460 (large cell carcinoma) cell lines were cocultured with lung microexplants. Confocal fluorescence microscopy was used to visualize the adherence of each cell line to lung microexplants. Adherent cancer cells were quantified following filtration of nonadherent cells, digestion of cultured microexplants, and flow cytometry. This method was used to evaluate the role of integrins in cancer cell adherence. A statistically significant decrease in the adherence of H460 cells to lung microexplants was observed when anti-integrins were administered to H460 cells before coculture with lung microexplants.

Quantification of circadian rhythms in mammalian lung tissue snapshot data

Healthy mammalian cells have a circadian clock, a gene regulatory network that allows them to schedule their physiological processes to optimal times of the day. When healthy cells turn into cancer cells, the circadian clock often becomes cancer specifically disturbed, so there is an interest in the extraction of circadian features from gene expression data of cancer. This is challenging, as clinical gene expression samples of cancer are snapshot-like and the circadian clock is best examined using gene expression time series. In this study, we obtained lists of intersecting circadian genes in public gene expression time series data of lung tissue of mouse and baboon. We base our circadian gene lists on correlations of gene expression levels of circadian genes, which are closely associated to the phase differences between them. Combining circadian gene expression patterns of diurnal and nocturnal species of different ages provides circadian genes that are also important in healthy and cancerous human lung tissue. We tested the quality of the representation of the circadian clock in our gene lists by PCA-based reconstructions of the circadian times of the mouse and baboon samples. Then we assigned potential circadian times to the human lung tissue samples and find an intact circadian clock in the healthy human lung tissue, but an altered, weak clock in the adjacent cancerous lung tissue.