Background Primary graft dysfunction (PGD) usually occurs within 72 hours after lung transplantation and is primarily caused by ischemia-reperfusion injury (IRI). Patients who develop PGD after lung transplantation tend to have a poor prognosis. However, effective clinical strategies to reduce the incidence of primary graft dysfunction remain limited. Therefore, a comprehensive understanding of the mechanisms underlying lung ischemia-reperfusion injury is essential for improving outcomes in lung transplant recipients. Methods In this study, we explored the differential expression of metabolism-related genes in lung transplantation induced IRI and identify its potential molecular mechanisms by bioinformatics analysis. Next, we used two machine learning algorithms and further screened for key genes in them. The outside dataset GSE8021 was used to validated the accuracy of the model established by metabolism-related genes machine learning genes. In addition, we observed the distribution and localization of metabolism-related machine learning genes in the single-cell dataset GSE220797 and analyzed the correlation between metabolism-related machine learning genes and immune cells by the CIBERSORT immune infiltration algorithm. Finally, we validated the nine metabolism-related machine learning genes by rat orthotopic left lung transplantation model and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR), we found that seven of these metabolism-related machine learning genes were consistent with the results of the bioinformatics analysis. Results We identified multiple metabolism-related genes machine learning genes (PDE4B, CDA, HMOX1, EHHADH, AMD1, GUCY1A1, GUCY1B1, UGCG, and FPGT). Significant changes were observed in some of these genes following ischemia-reperfusion. They represent important biomarkers in ischemia-reperfusion injury induced by lung transplantation and hold promise as therapeutic targets for mitigating lung ischemia-reperfusion injury and reducing the incidence of primary graft dysfunction.

Tissue-resident immune cells play a crucial role in chronic lung diseases, yet a comprehensive profile of these cells in human lungs is lacking. Here, we explore alterations of resident immune cells in idiopathic pulmonary fibrosis (IPF), a fatal lung disease characterized by tissue inflammation and progressive scarring. Utilizing ex-vivo human lung perfusion with single-cell RNA-sequencing, we successfully segregate the resident immune cells. By analyzing approximately 100,000 resident immune cells from seven patients with IPF and five healthy control lungs, we identify 13 distinct cell types. Previously unrecognized aberrant lymphocyte phenotypes are uncovered. Specifically, among T lymphocytes, we observe an enrichment of GZMK+ CD8+ T cells in IPF lungs, possessing a potential pro-fibrotic function. The fraction of pro-inflammatory HSPhi CD4+ conventional T cells increases in IPF lungs, while the quiescent subset decreases. Despite an increased presence of Tregs in IPF lungs, these cells show reduced expression of genes associated with immune suppression. Moreover, significant B cell expansion and activation occur, with continuous differentiation into IgG-producing plasma cells. Stromal niche interaction analysis shows that IPF fibroblasts, especially the CTHRC1hi subset, exert stronger effects on lymphocytes. These findings provide evidence of dysregulated immune cell populations in IPF, advancing our understanding of its immunopathology.

Tissue-resident immune cells are crucial in chronic lung diseases, yet a comprehensive profile in human lungs is lacking. Here, we defined alterations of resident immune cells in idiopathic pulmonary fibrosis (IPF), a fatal interstitial lung disease characterized by tissue inflammation and progressive scarring. Utilizing ex-vivo human lung perfusion coupled with single-cell RNA-sequencing, we successfully segregated the resident immune cells. Analyzing approximately 100,000 resident immune cells from 7 IPF and 5 control lungs, we identified 13 distinct cell types. Previously unrecognized aberrant lymphocyte phenotypes were uncovered. Specifically, among T lymphocytes, we observed an enrichment of GZMK + CD8 + T cells in IPF lungs, possessing a potential pro-fibrotic function. The fraction of pro-inflammatory HSP hi CD4 + T cells was increased in IPF lungs, while the quiescent subset decreased. Despite an increased presence of Tregs in IPF lungs, these cells showed reduced expression of genes associated with immune suppression. Moreover, significant B cell expansion and activation occurred, with continuous differentiation into IgG-producing plasma cells. Stromal niche interaction analysis showed that IPF fibroblasts, especially the CTHRC1 hi subset, exerted stronger effects on lymphocytes. These findings offer novel insights into dysregulated immune populations in IPF, advancing understanding of its immunopathology.

BACKGROUND:Rapid On-Site Evaluation (ROSE) has been widely used in clinical applications. However, in the field of lung transplantation, there have been no comparative experiments to confirm and quantify its effectiveness. To this end, ROSE was used to detect donor lung infection or colonization and analyze its influence on the prognosis of lung transplantation. METHODS:This study retrospectively analyzed 15 patients who received our center from March 2023 to May 2023. Fibrobronchoscopy and ROSE of bronchoalveolar lavage fluid(BALF) were performed. Postoperative survival rate index was collected for prognostic analysis. The coincidence rate of ROSE and traditional test culture was compared. RESULTS:① The 15 BALF samples were divided into infection group and colonization group according to the presence of infection and phagocytosis.② The average time of ROSE report was 10.40 min, and the average time of Sputum culture test report was 4663 min, and the difference between the two groups was statistically significant (p < 0.01). ③ The results of ROSE's evaluation of donor lung infection were in good agreement with those of traditional Sputum culture, and the difference was statistically significant (p < 0.01). (4) There were no significant differences in postoperative survival rate index between the infection group and the colonization group after targeted antibiotics were applied in advance. CONCLUSION:ROSE has a high heterogeneity in the evaluation of donor lung transplantation infection. It can be used as one of the important auxiliary examination techniques before and after lung transplantation.

Idiopathic pulmonary fibrosis (IPF) is a chronic, irreversible, and fatal disease characterized by progressive interstitial lung fibrosis. Given its insidious onset and poor outcome, there is an urgent need to elucidate the molecular mechanisms underlying IPF and identify effective therapeutic targets and diagnosis and prognosis biomarkers. Ferroptosis is an iron-dependent form of programmed cell death that occurs as lipid peroxides accumulate. Growing evidence suggests that ferroptosis is important in IPF. Human ferroptosis PCR array was performed on IPF and control lung tissue. The differentially expressed ferroptosis-related genes (DE-FRGs) were identified, underwent functional enrichment analyses, protein–protein interaction network construction, and potential drug target prediction. The DE-FRGs were validated and their value as diagnostic and prognostic blood biomarkers were evaluated using the Gene Expression Omnibus dataset GSE28042. The array identified 13 DE-FRGs. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that the DE-FRGs were mainly related to iron ion transport, blood microparticles, and oxidoreductase activity, and were involved in porphyrin metabolism, necroptosis, and the p53 signaling pathway in addition to ferroptosis. The 13 DE-FRGs were analyzed using the Drug–Gene Interaction Database to explore novel IPF therapeutic agents, yielding 42 potential drugs. Four DE-FRGs (BBC3, STEAP3, EPRS, SLC39A8) in the peripheral blood of IPF patients from the GSE28042 dataset demonstrated the same expression pattern as that observed in the lung tissue array. The receiver operating characteristic analysis demonstrated that the area under the curve of STEAP3 and EPRS were > 0.75. The survival analysis demonstrated that STEAP3 and EPRS were significantly different between the IPF and control groups. The FRG expression profiles in IPF and control lung tissue were characterized. The findings provided valuable ideas to elucidate the role of ferroptosis in IPF and aided the identification of novel IPF therapeutic targets and biomarkers.

BACKGROUND:Pulmonary fibrosis (PF) is the leading cause of death in many lung diseases due to inflammation, tissue damage, infection, or other contributing factors. Iron metabolism and ferroptosis have been reported to participate in some PF diseases, but the universality remains elusive. METHODS:Herein, comparative studies were conducted among idiopathic pulmonary fibrosis (IPF), immune-associated systemic sclerosis (SSc), and infectious COVID-19. The iron level was evaluated by Perls' staining and ferritin level. Ferroptosis was detected by immunohistochemistry (malondialdehyde, oxidizing lipids, GPX4, and FSP1) and transmission electron microscopy. The results were also validated by public datasets analysis. Furthermore, the iron homeostasis and ferroptosis signatures were studied in the SARS-Cov-2 spike protein-induced PF cell model. The iron-mediated inflammation and fibrosis in PF were evaluated both in vitro and in vivo. RESULTS:We found that COVID-19 patients showed the most severe pulmonary damage and fibrosis signature. COVID-19 and SSc-PF patients have more obvious immune cell infiltration with CD11c+ monocytes and CD68 + macrophages. Iron overload and ferroptosis were common in PF, while COVID-19 patients showed distinct iron metabolism signatures with higher expression of HO-1. Among all ferroptosis markers, IPF patients showed the highest E06 level, COVID-19 and SSc-PF had both higher levels of MDA and 4HNE. Further studies showed iron overload and ferroptosis occurred mainly in alveolar type II cells and macrophages. Deferoxamine (DFO) and Ferrostatin-1 (Fer1) effectively prevented malondialdehyde production and IL-6 upregulation. DFO and Fer1 alleviated fibrosis in mice. CONCLUSIONS:Our study demonstrates that iron overload and ferroptosis are common signatures in PF and represent potential therapeutic targets.

Introduction:Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disease with a poor prognosis and no effective pharmacological treatments. Cytokines are a class of small-molecule proteins with diverse biological activities. Many cytokines-most notably transforming growth factor β-have been demonstrated to play an important role in IPF. However, a few studies have systematically described the relationship between cytokines and IPF. Methods:Lung tissues from controls and patients with IPF were collected during lung transplantation. The expression profiles of 440 cytokines in lung tissues were obtained using protein microarrays. Proteomic analysis was performed, and differentially expressed proteins (DEPs) were identified. Furthermore, an integrative bioinformatics analysis was performed and included functional enrichment analysis, protein-protein interaction (PPI) network construction, hub protein determination, immune cell infiltration analysis, potential drug prediction, and single-cell analysis. The hub protein expression was validated through Gene Expression Omnibus (GEO) database evaluation and immunochemical analysis. Results:32 DEPs were identified from the two groups. They were mainly enriched in cell chemotaxis, basal part of cell, and growth factor binding and were involved in PI3K-Akt signaling. The PPI network was constructed for the DEPs, and five hub proteins (FGF2, HGF, HBEGF, ERBB3, and ANGPT2) were identified. The immune infiltration analysis demonstrated a significantly higher percentage of resting NK cells in IPF lung tissue. The drug prediction analyses identified 13 potential candidates targeting the five hub proteins. The single-cell analysis predicted the cellular localization of each key cytokine. Conclusions:Using protein microarrays, we obtained comprehensive cytokine expression profiles in control and IPF lung tissues and conducted an integrated bioinformatics analysis of the proteomic data. Our findings may improve the comprehension of the role of cytokines in IPF and the underlying mechanisms. Moreover, they provide novel targets for developing safe and efficacious drugs for treating IPF.

Mesenchymal stem cell therapy involves the secretion of various factors to regulate the local microenvironment in various of diseases. This therapy offers hope for treating acute myocardial infarction (MI), which poses a serious threat to human health. However, challenges such as low paracrine efficiency and poor cell survival persist due to the harsh post-infarction conditions, such as hypoxia. Recently, enhanced cell therapy, in which vascular endothelial growth factor A (VEGFA) and basic fibroblast growth factor (bFGF) are used as therapeutic agents to limit myocardial injury and simultaneously induce neovascularisation, has been recognised as a promising new strategy to improve the efficacy of cell therapy. Chemically synthetic modified messenger RNA (modRNA), a novel protein expression technology, enables safe, rapid, efficient and pulsatile expression of target proteins in vivo and in vitro settings. It has been widely applied in the fields of vaccine research and tissue regeneration. In this study, human adipose-derived stem cells (hADSCs) were transfected with VEGFA and bFGF modRNA to transiently overexpress these proteins before transplantation. This modification enhanced the paracrine effect of transplanted hADSCs and promoted stability in the vascular network at the transplantation site. Overexpression of VEGFA and bFGF in hADSCs not only inhibited apoptosis but also reduced ventricular remodelling and improved cardiac function and left ventricular conduction. Overall, the additive effects of VEGFA modRNA, bFGF modRNA and hADSCs hold promise for comprehensive cardiac repair post-MI and show substantial potential for treating ischemic heart diseases. KEY POINTS: ModRNAs-transfected hADSCs exhibit pulsed and transient expression, enabling efficient production of functional VEGFA and bFGF proteins. Intracardiac injection of these engineered hADSCs leads to the enhancement of cardiac function and the improvement of electrical conduction. The hADSCsdual mainly exerts its effect on myocardial infarction by promoting stable vascular regeneration and suppressing cell apoptosis.

BACKGROUND: The infiltration of macrophages into the lungs is a common characteristic of perivascular inflammation, contributing to vascular remodeling in pulmonary hypertension (PH). Peli1 (pellino E3 ubiquitin-protein ligase 1) plays a critical role in regulating the production of proinflammatory cytokines and the polarization of macrophages in various diseases. However, the role of Peli1 in PH remains to be investigated. METHODS: The expression and biological function of Peli1 were investigated in both human and experimental models of PH. Peli1-deficient mice and bone marrow transplant mice were utilized to explore the roles of Peli1 in macrophages in vivo. Proteomic analysis and molecular biology techniques were used to uncover the underlying mechanisms. RESULTS: The upregulation of Peli1 in the lungs and alveolar macrophages was observed in hypoxia-treated mice. Peli1 knockout mice and myeloid Peli1-deficient mice significantly ameliorated hypoxia-induced right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling. Mechanistically, Peli1 facilitated the ubiquitination and subsequent proteasomal degradation of Foxp1 (forkhead box p1), thereby alleviating its suppression of IL ( interleukin ) -6 transcription and contributing to macrophage activation. Furthermore, myeloid Foxp1 deficiency partially eliminates the protective effect of myeloid Peli1 deficiency in hypoxia-induced PH mice. CONCLUSIONS: Our findings demonstrate that the Peli1-Foxp1-IL-6 pathway plays a crucial role in macrophage activation and recruitment during the development of PH, underscoring the potential of Peli1 as a therapeutic target for PH.

Objective: To investigate the high-risk factors for early pulmonary bacterial infection following lung transplantation and their association with long-term mortality. Methods: A retrospective analysis was conducted on 142 lung transplant recipients treated at Wuxi People's Hospital between January 2018 and July 2022. After applying predefined inclusion and exclusion criteria, 111 cases were analyzed. Univariate and multivariate logistic regression analyses were performed to identify independent risk factors for early pulmonary infection post-transplantation. Additionally, univariate and multivariate Cox regression analyses were used to identify independent prognostic facin donor lungs, and operation duration as risk factors for early pulmonary infection (all P < 0.05). Multivariate analysis was confirmatory for these as independent risk factors (all P < 0.05). Univariate analysis also showed that intraoperative blood loss and oxygenation index impacted one-year survival (P < 0.05). Multivariate analysis was confirmatory for these as independent risk factors (P < 0.05). Conclusion: Early pulmonary bacterial infection was not found to be an independent factor affecting 1-year survival. However, substantial intraoperative blood loss and a reduced oxygenation index were identified as independent risk factors associated with increased mortality within 1 year post-transplantation.

Macrophages act as the first immune defense line of the host against Mycobacterium tuberculosis (Mtb). A previous study showed that circRNA_SLC8A1 was significantly upregulated in Mtb-infected macrophages, but its regulatory mechanism in anti-tuberculosis infection is unclear. Therefore, this study aimed to investigate the role of circRNA_SLC8A1 in the anti-tuberculosis activity of macrophages. We showed that circRNA_SLC8A1 was upregulated in tuberculosis patients. Moreover, the binding sites of miR-20b-5p on circRNA_SLC8A1 and Sequestosome 1 (SQSTM1/p62) mRNA were predicted by StarBase and verified by the double luciferase reporter gene assay. Next, we found that miR-20b-5p expression was decreased, while SQSTM1 protein expression was increased in a time- and dose-dependent manner in the human macrophage U937 in response to Mtb infection. Furthermore, circRNA_SLC8A1 overexpression vector (circRNA_SLC8A1) or shRNA (sh-circRNA_SLC8A1) and/or miR-20b-5p mimic or inhibitor and/or SQSTM1 overexpression vector (SQSTM1) or small interfering RNA (si-SQSTM1) or its corresponding control were transfected into Mtb-infected macrophages. Results showed that overexpression of circRNA_SLC8A1 or miR-20b-5p inhibitor promoted the secretion of pro-inflammatory factors IL-1β, IL-6, and TNF-α, increased Nitric Oxide (NO) content and inducible nitric oxide synthase (iNOS) expression, inhibited Reactive oxygen species (ROS) production. Cleaved-caspase-3 protein expression, and cell apoptosis, and promoted Mtb survival. Silencing SQSTM1 inhibited secretion of pro-inflammatory factors and activation of the NF-κB pathway. Overexpression of miR‐20b‐5p blocked the promoting of circ‐SLC8A1 on SQSTM1 protein expression. In summary, circRNA_SLC8A1 sponged miR‐20b‐5p to upregulate SQSTM1/p62 expression and promoted Mtb survival in macrophages through the NF-κB signaling pathway.

BACKGROUND:The N6-methyladenosine (m6A) modification of RNA and its regulators have important roles in the pathogenesis of pulmonary hypertension (PH). Ythdf2 (YTH N6-methyladenosine RNA binding protein 2) is best known for its role in degrading m6A-modified mRNAs such as Hmox1 mRNA, which leads to alternative activation of macrophages in PH. Recent studies have also linked Ythdf2 to the proliferation of pulmonary artery smooth muscle cells (PASMCs). However, its specific roles in PASMCs and downstream targets during the development of PH remain unclear. METHODS:The expression and biological function of Ythdf2 in PASMCs were investigated in human and experimental models of PH. Smooth muscle cell-specific Ythdf2-deficient mice were used to assess the roles of Ythdf2 in PASMCs in vivo. Proteomic analysis, m6A sequencing, and RNA immunoprecipitation analysis were used to screen for potential downstream targets. RESULTS:Ythdf2 was significantly upregulated in human and rodent PH-PASMCs, and smooth muscle cell-specific Ythdf2 deficiency ameliorated PASMC proliferation, right ventricular hypertrophy, pulmonary vascular remodeling, and PH development. Higher expression of Ythdf2 promoted PASMC proliferation and PH by paradoxically stabilizing Myadm mRNA in an m6A-dependent manner. Loss of Ythdf2 decreased the expression of Myadm in PASMCs and pulmonary arteries, both in vitro and in vivo. Additionally, silencing Myadm inhibited the Ythdf2-dependent hyperproliferation of PASMCs by upregulating the cell cycle kinase inhibitor p21. CONCLUSIONS:We have identified a novel mechanism where the increased expression of Ythdf2 stimulates PH-PASMC proliferation through an m6A/Myadm/p21 pathway. Strategies targeting Ythdf2 in PASMCs might be useful additions to the therapeutic approach to PH.

Aim of the studyExploring the protective effect of ARC@DPBNP on lipopolysaccharides (LPS)-induced ALI and its underlying mechanism.Materials and MethodsALI model was established by intransally administrating LPS (4 mg/kg) into C57BL/6 mice. The suppression effects of ALI was first compared between ARC (intragastric administrated, with doses ranging from 10 to 80 mg/kg) and ARC@BPBNPs (intratracheally administrated, with doses ranging from 1 to 4 mg/kg). Changes in lung histology post intratracheal intervention of 3 mg/kg ARC@DPBNPs were detected. The expression of pyrotosis pathway-related proteins in lungs as well as in RAW264.7 cells was detected by western blotting. The ASC expression in lung macrophages was examined using immune-fluorescent staining. The polarization of RAW264.7 cells and lung macrophages were detected by flow cytometry. The network pharmacology was constructed by Cytoscape, and the molecular docking was perfomed by AutoDock Vina.ResultsDocking predicted the high affinity of ARC to MAPK1 (ERK2). HE staining showed that ARC@DPBNPs attenuated LPS-induced ALI at a remarkably lower dose than ARC. The improved histopathological changes, lung W/D weight ratio, and decreased of inflammatory factor levels in lung collectively demonstrated the alleviation effects of ARC@DPBNPs. Compared with the LPS group, ARC@DPBNPs down-regulated the ERK pathway, resulted in a suppression of the macrophage pyroptosis and M1 polarization. This suppression effects could be removed by the ERK activator Ro 67-7476.ConclusionARC@DPBNPs attenuated ALI by suppressing LPS-induced macrophage pyroptosis and polarization, probably through down-regulation of the ERK pathway.

The 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. Four 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. Through 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. Our 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.

Pulmonary fibrosis (PF) is a severe pulmonary disease with limited available therapeutic choices. Recent evidence increasingly points to abnormal lipid metabolism as a critical factor in PF pathogenesis. Our latest research identifies the dysregulation of low-density lipoprotein (LDL) is a new risk factor for PF, contributing to alveolar epithelial and endothelial cell damage, and fibroblast activation. In this study, we first integrative summarize the published literature about lipid metabolite changes found in PF, including phospholipids, glycolipids, steroids, fatty acids, triglycerides, and lipoproteins. We then reanalyze two single-cell RNA-sequencing (scRNA-seq) datasets of PF, and the corresponding lipid metabolomic genes responsible for these lipids’ biosynthesis, catabolism, transport, and modification processes are uncovered. Intriguingly, we found that macrophage is the most active cell type in lipid metabolism, with almost all lipid metabolic genes being altered in macrophages of PF. In type 2 alveolar epithelial cells, lipid metabolic differentially expressed genes (DEGs) are primarily associated with the cytidine diphosphate diacylglycerol pathway, cholesterol metabolism, and triglyceride synthesis. Endothelial cells are partly responsible for sphingomyelin, phosphatidylcholine, and phosphatidylethanolamines reprogramming as their metabolic genes are dysregulated in PF. Fibroblasts may contribute to abnormal cholesterol, phosphatidylcholine, and phosphatidylethanolamine metabolism in PF. Therefore, the reprogrammed lipid profiles in PF may be attributed to the aberrant expression of lipid metabolic genes in different cell types. Taken together, these insights underscore the potential of targeting lipid metabolism in developing innovative therapeutic strategies, potentially leading to extended overall survival in individuals affected by PF.

ObjectivesExtracorporeal membrane oxygenation (ECMO) has become an important life support technique during lung transplantation. We aimed to develop a rat model for lung transplantation using venoarterial (VA) ECMO support.MethodsAdult male Sprague-Dawley rats weighing 400 to 450 g were used in this study. ECMO circuits were created by obtaining venous access from the femoral vein with subsequent extracorporeal oxygen exchange, which was then returned to the circulatory system through the left carotid artery (ie, VA-ECMO). Simultaneously, the donor lungs were retrieved and immersed in cold, low-potassium dextran lung preservation solution. Orthotopic left lung transplantation supported by VA-ECMO was performed. Thereafter, a respiratory failure rat model was constructed using ventilation with a hypoxic and hypercapnic gas mixture, consisting of 6% oxygen, 8% carbon dioxide, and 86% nitrogen, before lung transplantation. Similarly, left lung transplantation supported by VA-ECMO was performed in rats with respiratory failure. Arterial blood gas levels were measured at designated time points throughout the experiment.ResultsWe found that VA-ECMO provided sufficient oxygenation and carbon dioxide removal to allow for smooth left lung transplantation in healthy rats and those with respiratory failure.ConclusionsWe established a rat model for lung transplantation using VA-ECMO. Left lung transplantation using VA-ECMO support is also feasible and safe in rat models of respiratory failure. These models provide efficient and economical models for translational medicine for lung transplantation using ECMO. Moreover, it will be invaluable to evaluate the physiological and pathophysiological roles of ECMO during lung transplantation.

OBJECTIVE:Acute lung injury (ALI) caused by sepsis is a life-threatening condition characterized by uncontrollable lung inflammation. The current study sought to investigate the mechanism of adipose-derived mesenchymal stem cell-derived exosomes (ADMSC-Exos) in attenuating sepsis-induced ALI through TGF-β secretion in macrophages. METHODS:Adipose-derived mesenchymal stem cell-derived exosomes (ADMSC-Exos) were extracted from ADMSCs and identified. Septic ALI mouse models were established via cecal ligation and puncture (CLP), followed by administration of ADMSC-Exos or sh-TGF-β lentiviral vector. Mouse macrophages (cell line RAW 264.7) were treated with lipopolysaccharide (LPS), co-cultured with Exos and splenic T cells, and transfected with TGF-β siRNA. The lung injury of CLP mice was evaluated, and levels of inflammatory indicators and macrophage markers were measured. The localization of macrophage markers and TGF-β was determined, and the level of TGF-β in lung tissues was measured. The effect of TGF-β knockdown on sepsis-induced ALI in CLP mice was evaluated, and the percentages of CD4+CD25+Foxp3+ Tregs in mononuclear cells/macrophages and Foxp3 levels in lung tissues/co-cultured splenic T cells were examined. RESULTS:ADMSC-Exos were found to alleviate sepsis-induced ALI, inhibit inflammatory responses, and induce macrophages to secrete TGF-β in CLP mice. TGF-β silencing reversed the alleviating effect of ADMSC-Exos on sepsis-induced ALI. ADMSC-Exos also increased the number of Tregs in the spleen of CLP mice and promoted M2 polarization and TGF-β secretion in LPS-induced macrophages. After knockdown of TGF-β in macrophages in the co-culture system, the number of Tregs decreased, suggesting that ADMSC-Exos increased the Treg number by promoting macrophages to secrete TGF-β. CONCLUSION:Our findings suggest ADMSC-Exos can effectively alleviate sepsis-induced ALI in CLP mice by promoting TGF-β secretion in macrophages.

Background. Primary graft dysfunction, which is directly related to cold ischemia–reperfusion (CI/R) injury, is a major obstacle in lung transplantation (LTx). Ferroptosis, a novel mode of cell death elicited by iron-dependent lipid peroxidation, has been implicated in ischemic events. This study aimed to investigate the role of ferroptosis in LTx-CI/R injury and the effectiveness of liproxstatin-1 (Lip-1), a ferroptosis inhibitor, in alleviating LTx-CI/R injury. Methods. LTx-CI/R-induced signal pathway alterations, tissue injury, cell death, inflammatory responses, and ferroptotic features were examined in human lung biopsies, the human bronchial epithelial (BEAS-2B) cells, and the mouse LTx-CI/R model (24-h CI/4-h R). The therapeutic efficacy of Lip-1 was explored and validated both in vitro and in vivo. Results. In human lung tissues, LTx-CI/R activated ferroptosis-related signaling pathway, increased the tissue iron content and lipid peroxidation accumulation, and altered key protein (GPX4, COX2, Nrf2, and SLC7A11) expression and mitochondrial morphology. In BEAS-2B cells, the hallmarks of ferroptosis were significantly evidenced at the setting of both CI and CI/R compared with the control, and the effect of adding Lip-1 only during CI was much better than that of only during reperfusion by Cell Counting Kit-8. Furthermore, Lip-1 administration during CI markedly relieved LTx-CI/R injury in mice, as indicated by significant improvement in lung pathological changes, pulmonary function, inflammation, and ferroptosis. Conclusions. This study revealed the existence of ferroptosis in the pathophysiology of LTx-CI/R injury. Using Lip-1 to inhibit ferroptosis during CI could ameliorate LTx-CI/R injury, suggesting that Lip-1 administration might be proposed as a new strategy for organ preservation.

Pulmonary hypertension (PH) is a devastating disease characterized by irreversible pulmonary vascular remodeling (PVR) that causes right ventricular failure and death. The early alternative activation of macrophages is a critical event in the development of PVR and PH, but the underlying mechanisms remain elusive. Previously we have shown that N6-methyladenosine (m6A) modifications of RNA contribute to phenotypic switching of pulmonary artery smooth muscle cells and PH. In the current study, we identify Ythdf2, an m6A reader, as an important regulator of pulmonary inflammation and redox regulation in PH. In a mouse model of PH, the protein expression of Ythdf2 was increased in alveolar macrophages (AMs) during the early stages of hypoxia. Mice with a myeloid specific knockout of Ythdf2 (Ythdf2Lyz2 Cre) were protected from PH with attenuated right ventricular hypertrophy and PVR compared to control mice and this was accompanied by decreased macrophage polarization and oxidative stress. In the absence of Ythdf2, heme oxygenase 1 (Hmox1) mRNA and protein expression were significantly elevated in hypoxic AMs. Mechanistically, Ythdf2 promoted the degradation of Hmox1 mRNA in a m6A dependent manner. Furthermore, an inhibitor of Hmox1 promoted macrophage alternative activation, and reversed the protection from PH seen in Ythdf2Lyz2 Cre mice under hypoxic exposure. Together, our data reveal a novel mechanism linking m6A RNA modification with changes in macrophage phenotype, inflammation and oxidative stress in PH, and identify Hmox1 as a downstream target of Ythdf2, suggesting that Ythdf2 may be a therapeutic target in PH.