Different localization of P2X4 and P2X7 receptors in native mouse lung - lack of evidence for a direct P2X4-P2X7 receptor interaction.

Introduction: P2X receptors are a family of homo- and heterotrimeric cation channels gated by extracellular ATP. The P2X4 and P2X7 subunits show overlapping expression patterns and have been involved in similar physiological processes, such as pain and inflammation as well as various immune cell functions. While formation of P2X2/P2X3 heterotrimers produces a distinct pharmacological phenotype and has been well established, functional identification of a P2X4/P2X7 heteromer has been difficult and evidence for and against a physical association has been found. Most of this evidence stems, however, from in vitro model systems. Methods: Here, we used a P2X7-EGFP BAC transgenic mouse model as well as P2X4 and P2X7 knock-out mice to re-investigate a P2X4-P2X7 interaction in mouse lung by biochemical and immunohistochemical experiments as well as quantitative expression analysis. Results: No detectable amounts of P2X4 could be co-purified from mouse lung via P2X7-EGFP. In agreement with these findings, immuno-histochemical analysis using a P2X7-specific nanobody revealed only limited overlap in the cellular and subcellular localizations of P2X4 and P2X7 in both the native lung tissue and primary cells. Comparison of P2X4 and P2X7 transcript and protein levels in the respective gene-deficient and wild type mice showed no mutual interrelation between their expression levels in whole lungs. However, a significantly reduced P2rx7 expression was found in alveolar macrophages of P2rx4 -/- mice. Discussion: In summary, our detailed analysis of the cellular and subcellular P2X4 and P2X7 localization and expression does not support a physiologically relevant direct association of P2X4 and P2X7 subunits or receptors in vivo.

Downregulation of HDAC6 mitigates lung ischemia/reperfusion injury depending on activation of Nrf2/HO-1 signaling pathway and inactivation of ERK/NF-κB signaling pathway.

INTRODUCTION: Lung ischemia/reperfusion injury (LIRI) is a pathological process caused by the deficiency and subsequent reperfusion of oxygen and blood to the lung. Literature reports that the catalytic activity and expression of HDAC6 can be induced in response to IRI. HDAC6 inhibition confers protective effects against a series of IRI and also exerts pulmonary protection against various lung damage. The present study was formulated to investigate the functional role of HDAC6 inhibitor in LIRI and to probe into the intrinsic mechanisms underlying the protective role of HDAC6 inhibitor against LIRI. METHODS: Lung epithelial cell line MLE-12 cells were subjected to H/R injury to construct in vitro cell culture model of LIRI. For functional experiments, MLE-12 cells were pre-treated with various concentrations of selective HDAC6 inhibitor ACY-1215 (1, 5, 10 µM) to evaluate the biological role of HDAC6 in LIRI. For rescue experiments, MLE-12 cells were pre-treated with Nrf2 inhibitor ML385 (10 µM) or ERK activator LM22B-10 (50 µM) to discuss the molecular mechanisms. RESULTS: It was verified that HDAC6 inhibition repressed H/R-induced apoptosis, oxidative stress, inflammation and mitochondrial dysfunction of MLE-12 cells. HDAC6 inhibition activated Nrf2/HO-1 signaling pathway and inactivated ERK/NF-κB signaling pathway in MLE-12 cells. The repressing effects of HDAC6 inhibition on H/R-induced apoptosis, oxidative stress, inflammation and mitochondrial dysfunction of MLE-12 cells were partially abolished upon pre-treatment with Nrf2 inhibitor ML385 or ERK activator LM22B-10. CONCLUSION: HDAC6 inhibition may mitigate H/R-induced lung epithelial cell injury depending on activation of Nrf2/HO-1 signaling pathway and inactivation of ERK/NF-κB signaling pathway.

The Roles and Regulatory Mechanisms of Tight Junction Protein Cingulin and Transcription Factor Forkhead Box Protein O1 in Human Lung Adenocarcinoma A549 Cells and Normal Lung Epithelial Cells.

Tight junction (TJ) protein cingulin (CGN) and transcription factor forkhead box protein O1 (FOXO1) contribute to the development of various cancers. Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for some cancers. HDAC inhibitors affect the expression of both CGN and FOXO1. However, the roles and regulatory mechanisms of CGN and FOXO1 are unknown in non-small cell lung cancer (NSCLC) and normal human lung epithelial (HLE) cells. In the present study, to investigate the effects of CGN and FOXO1 on the malignancy of NSCLC, we used A549 cells as human lung adenocarcinoma and primary human lung epithelial (HLE) cells as normal lung tissues and performed the knockdown of CGN and FOXO1 by siRNAs. Furthermore, to investigate the detailed mechanisms in the antitumor effects of HDAC inhibitors for NSCLC via CGN and FOXO1, A549 cells and HLE cells were treated with the HDAC inhibitors trichostatin A (TSA) and Quisinostat (JNJ-2648158). In A549 cells, the knockdown of CGN increased bicellular TJ protein claudin-2 (CLDN-2) via mitogen-activated protein kinase/adenosine monophosphate-activated protein kinase (MAPK/AMPK) pathways and induced cell migration, while the knockdown of FOXO1 increased claudin-4 (CLDN-4), decreased CGN, and induced cell proliferation. The knockdown of CGN and FOXO1 induced cell metabolism in A549 cells. TSA and Quisinostat increased CGN and tricellular TJ protein angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) in A549. In normal HLE cells, the knockdown of CGN and FOXO1 increased CLDN-4, while HDAC inhibitors increased CGN and CLDN-4. In conclusion, the knockdown of CGN via FOXO1 contributes to the malignancy of NSCLC. Both HDAC inhibitors, TSA and Quisinostat, may have potential for use in therapy for lung adenocarcinoma via changes in the expression of CGN and FOXO1.

In vitro lung epithelial cell model reveals novel roles for Pseudomonas aeruginosa siderophores.

The multidrug-resistant pathogen Pseudomonas aeruginosa is a common nosocomial respiratory pathogen that continues to threaten the lives of patients with mechanical ventilation in intensive care units and those with underlying comorbidities such as cystic fibrosis or chronic obstructive pulmonary disease. For over 20 years, studies have repeatedly demonstrated that the major siderophore pyoverdine is an important virulence factor for P. aeruginosa in invertebrate and mammalian hosts in vivo. Despite its physiological significance, an in vitro, mammalian cell culture model that can be used to characterize the impact and molecular mechanisms of pyoverdine-mediated virulence has only been developed very recently. In this study, we adapt a previously-established, murine macrophage-based model to use human bronchial epithelial (16HBE) cells. We demonstrate that conditioned medium from P. aeruginosa induced rapid 16HBE cell death through the pyoverdine-dependent secretion of cytotoxic rhamnolipids. Genetic or chemical disruption of pyoverdine biosynthesis decreased rhamnolipid production and mitigated cell death. Consistent with these observations, chemical depletion of lipids or genetic disruption of rhamnolipid biosynthesis abrogated the toxicity of the conditioned medium. Furthermore, we also examine the effects of exposure to purified pyoverdine on 16HBE cells. While pyoverdine accumulated within cells, it was largely sequestered within early endosomes, resulting in minimal cytotoxicity. More membrane-permeable iron chelators, such as the siderophore pyochelin, decreased epithelial cell viability and upregulated several pro-inflammatory genes. However, pyoverdine potentiated these iron chelators in activating pro-inflammatory pathways. Altogether, these findings suggest that the siderophores pyoverdine and pyochelin play distinct roles in virulence during acute P. aeruginosa lung infection. IMPORTANCE: Multidrug-resistant Pseudomonas aeruginosa is a versatile bacterium that frequently causes lung infections. This pathogen is life-threatening to mechanically-ventilated patients in intensive care units and is a debilitating burden for individuals with cystic fibrosis. However, the role of P. aeruginosa virulence factors and their regulation during infection are not fully understood. Previous murine lung infection studies have demonstrated that the production of siderophores (e.g., pyoverdine and pyochelin) is necessary for full P. aeruginosa virulence. In this report, we provide further mechanistic insight into this phenomenon. We characterize distinct and novel ways these siderophores contribute to virulence using an in vitro human lung epithelial cell culture model.

circGRHPR inhibits aberrant epithelial-mesenchymal transformation progression of lung epithelial cells associated with idiopathic pulmonary fibrosis.

Air pollution has greatly increased the risk of idiopathic pulmonary fibrosis (IPF). Circular RNAs (circRNAs) have been found to play a significant role in the advancement of IPF, but there is limited evidence of correlation between circRNAs and lung epithelial cells (LECs) in IPF. This research aimed to explore the influence of circRNAs on the regulation of EMT progression in LECs, with the objective of elucidating its mechanism and establishing its association with IPF. Our results suggested that the downregulation of circGRHPR in peripheral blood of clinical cases was associated with the diagnosis of IPF. Meanwhile, we found that circGRHPR was downregulated in transforming growth factor-beta1 (TGF-ß1)-induced A549 and Beas-2b cells. It is a valid model to study the abnormal EMT progression of IPF-associated LECs in vitro. The overexpression of circGRHPR inhibited the abnormal EMT progression of TGF-ß1-induced LECs. Furthermore, as the sponge of miR-665, circGRHPR released the expression of E3 ubiquitin-protein ligase NEDD4-like (NEDD4L), thus promoting its downstream transforming growth factor beta receptor 2 (TGFBR2) ubiquitination. It is helpful to reduce the response of LECs to TGF-ß1 signaling. In summary, circGRHPR/miR-665/NEDD4L axis inhibited the abnormal EMT progression of TGF-ß1-induced LECs by promoting TGFBR2 ubiquitination, which provides new ideas and potential targets for the treatment of IPF.

Small extracellular vesicles from irradiated lung epithelial cells promote the activation of fibroblasts in pulmonary fibrosis.

BACKGROUND: Alveolar epithelial injury and dysfunction are the risk factors for radiation-induced pulmonary fibrosis (RIPF). However, it is not clear about the relationship between RIPF and the small extracellular vesicles (sEV) secreted by irradiated alveolar epithelial cells. Based on the activation of fibroblasts, this study explored the role of sEV derived from alveolar epithelial cells in RIPF and the potential mechanisms. METHODS: Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting were used to characterize sEV. Western blotting was used to detect fibrosis-associated proteins. Cell counts and transwell assays were used to evaluate the proliferation and migration ability of fibroblasts. RT-PCR was used to observe the extracellular matrix (ECM) synthesized by fibroblasts, miRNA changes in the sEV were determined by second-generation sequencing. RESULTS: TEM, NTA, and western blotting showed the extracellular vesicles with a double-layer membrane structure of approximately 100 nm in diameter. The sEV derived from irradiated A549, HBEC3-KT, and MLE12 cells upregulated FN1 and alpha-SMA proteins expression in fibroblasts and drove the fibroblast to myofibroblast transition, and the sEV from irradiated mouse bronchoalveolar lavage fluid (BALF) affirmed the same results. In addition, the sEV derived from irradiated alveolar epithelial cells significantly increased the migration ability of fibroblasts and the expression of extracellular matrix proteins such as FN1. The results of miRNA sequencing of sEV in BALF of rats with RIPF showed that the metabolic pathway may be important for miRNA to regulate the activation of fibroblasts. CONCLUSION: The sEV derived from radiated pulmonary epithelial cells promote the activation, migration and extracellular matrix proteins expression of lung fibroblasts; miRNA in sEV may be an important molecular that affects the activation of lung fibroblasts.

TRAF3 gene regulates macrophage migration and activation by lung epithelial cells infected with Aspergillus fumigatus.

IMPORTANCE: Aspergillus fumigatus can infect immunocompromised individuals and cause chronic and fatal invasive fungal infections. A better understanding of the molecular mechanisms of A. fumigatus-host interactions may provide new references for disease treatment. In this study, we demonstrated that the TRAF3 gene plays an important role in the early infection of A. fumigatus by regulating the resistance of lung epithelial cells to A. fumigatus. Macrophages are the most abundant innate immune cells in the alveoli; however, few studies have reported on the interactions between lung epithelial cells and macrophages in response to A. fumigatus invasion. In our study, it was demonstrated that the TRAF3 gene reduces migration to macrophages and cytokine production by negatively regulating lung epithelial cell adhesion and internalization of A. fumigatus spores. Together, our results provide new insights into lung epithelial cell-macrophage interactions during A. fumigatus infection.

miR-616-3p alleviates inflammatory response by targeting C-X-C motif chemokine ligand 5.

C/EBP homologous protein (CHOP) is a key regulator in ER stress-mediated signaling pathway via PERK-dependent unfolded protein response. It has been known that microRNA-616 (miR-616) is produced from the intron of the human DDIT3 gene encoding CHOP and increased by ER stress. However, the role of miR-616 and its targets are not fully addressed yet. Here we try to identify a novel target of miR-616 in human lung epithelial cells. Microarray analysis showed that CXCL5 is the most downregulated gene by miR-616 overexpression in A549 cells. We also found that CXCL5 mRNA and protein levels were significantly reduced by miR-616 mimic in the presence or absence of TNFα, while anti-miR-616 enhanced CXCL5 expression. In addition, miR-616-3p targeting sequence in 3'UTR of CXCL5 was confirmed by luciferase reporter assay suggesting that miR-616-3p directly binds to 3'UTR of CXCL5 and inhibits CXCL5 expression. Finally, we confirmed that conditioned medium from A549 cells treated with TNFα or Streptococcus pneumoniae lysates increased intra-alveolar neutrophil infiltration in a mouse model of pulmonary inflammation, while this induction was significantly reduced in a conditioned medium from cells transfected with miR-616-3p. These results suggest that miR-616-3p can alleviate CXCL5-induced pulmonary inflammatory response via targeting 3'UTR of CXCL5 gene.

Acute and repeated exposures of normal human bronchial epithelial (NHBE) cells culture to particles from a coloured pyrotechnic smoke.

Coloured pyrotechnic smokes are frequently used in the military field and occasionally by civilians, but their health hazards have been little studied. The main concern could rise from inhalation of smoke particles. Our previous study showed that acute exposure to particles from a red signalling smoke (RSS) induced an antioxidant and inflammatory responses in small airway epithelial cells. The aim of this study was to further explore the toxicity of RSS particles at a more proximal level of the respiratory tract, using normal human bronchial epithelial cells grown at the Air-Liquid Interface. Acute exposure (24 h) induced an oxidative stress that persisted 24 h post-exposure, associated with particle internalization and epithelium morphological changes (cuboidal appearance and loss of cilia). Repeated exposures (4×16h) to RSS particles did not trigger oxidative stress but cell morphological changes occurred. Overall, this study provides a better overview of the toxic effects of coloured smoke particles.

Endocytosis inhibitors block SARS-CoV-2 pseudoparticle infection of mink lung epithelium.

Introduction: Both spill over and spill back of SARS-CoV-2 virus have been reported on mink farms in Europe and the United States. Zoonosis is a public health concern as dangerous mutated forms of the virus could be introduced into the human population through spillback. Methods: The purpose of our study was to determine the SARS-CoV-2 entry mechanism using the mink lung epithelial cell line (Mv1Lu) and to block entry with drug inhibitors. Results: Mv1Lu cells were susceptible to SARS-CoV-2 viral pseudoparticle infection, validating them as a suitable disease model for COVID-19. Inhibitors of TMPRSS2 and of endocytosis, two pathways of viral entry, were tested to identify those that blocked infection. TMPRSS2 inhibitors had minimal impact, which can be explained by the apparent lack of activity of this enzyme in the mink and its localization within the cell, not on the cell surface. Discussion: Dyngo4a, a small molecule endocytosis inhibitor, significantly reduced infection, supporting the conclusion that the entry of the SARS-CoV-2 virus into Mv1Lu cells occurs primarily through endocytosis. The small molecule inhibitors that were effective in this study could potentially be used therapeutically to prevent SARS-CoV-2 infection in mink populations. This study will facilitate the development of therapeutics to prevent zoonotic transmission of SARS-CoV-2 variants to other animals, including humans.

JTE-013 Alleviates Pulmonary Fibrosis by Affecting the RhoA/YAP Pathway and Mitochondrial Fusion/Fission.

Pulmonary fibrosis may be due to the proliferation of fibroblasts and the aggregation of extracellular matrix, resulting in the stimulation of inflammation damage, destroying lung tissue structure, seriously affecting the patient's respiratory function, and even leading to death. We investigated the role and mechanism of JTE-013 in attenuating bleomycin (BLM)-induced pulmonary fibrosis. BLM-induced pulmonary fibrosis was established in mice. Type 2 alveolar epithelial cells (MLE-12) were stimulated with sphingosine monophosphate (S1P) in vitro. JTE-013, an S1PR2 (sphingosine 1-phosphate receptor 2) antagonist, and Verteporfin were administered in vivo and in vitro. IL-4, IL-5, TNF-α, and IFN-γ were measured by ELISA. IL-4 and IFN-γ positive cells were detected by flow cytometry. Inhibition of S1PR2 with JTE-013 significantly ameliorated BLM-induced pathological changes and inflammatory cytokine levels. JTE-013 also significantly reduced the expression of RHOA/YAP pathway proteins and mitochondrial fission protein Drp1, apoptosis, and the colocalization of α-SMA with YAP, Drp1, and Tom20, as detected by immunohistochemistry, immunofluorescence staining, TUNEL, and Western blot. In vitro, S1PR2 and YAP knockdown downregulated RHOA/YAP pathway protein expression, Drp1 phosphorylation, and Drp1 translocation, promoted YAP phosphorylation and phenotypic transformation of MFN2, and inhibited the up-regulation of mitochondrial membrane potential, reactive oxygen species production, and cell apoptosis (7.13% vs. 18.14%), protecting the integrity of the mitochondrial dynamics. JTE-013 also inhibited the expression of fibrosis markers α-SMA, MMP-9, and COL1A1, and alleviated the symptoms of pulmonary fibrosis. Conclusively, JTE-013 has great anti-pulmonary fibrosis potential by regulating RHOA/YAP and mitochondrial fusion/fission.

Particulate matter promotes the epithelial to mesenchymal transition in human lung epithelial cells via the ROS pathway.

OBJECTS: Epidemiologic studies have linked exposure to airborne pollutant particulate matter (PM) with increased rates of chronic cardiopulmonary diseases, including asthma and idiopathic pulmonary fibrosis (IPF). Several investigations have suggested that the epithelial-to-mesenchymal transition (EMT) may contribute to the complex pathobiology of environmental exposure-mediated pulmonary fibrosis. The present study was designed to characterize the mechanisms of PM-mediated EMT in human lung epithelial cells (HBECs). METHODS AND RESULTS: PM induced significant dose (0-100 µg/ml) and time (0-72 h)-dependent increases in transforming growth factor ß (TGFß) and fibronectin (FN) protein levels in HBECs lysates. PM-activated TGFß and FN protein production in HBECs was prevented by the antioxidant N-acetyl-cysteine (NAC, 5 mM). Furthermore, the NF-κB inhibitor BAY11-7082 (5 µM) abolished PM-induced FN production in HBECs. Biomarkers of EMT (ACTA2, SNAIL1 and SNAIL2) in PM-treated HBECs were significantly increased at the mRNA level compared to control cells. CONCLUSIONS: These results demonstrate that PM increases protein levels of TGFß and FN via reactive oxygen species (ROS)-dependent pathways. In addition, PM exposure induces EMT in human lung epithelial cells, supporting a novel mechanism for PM-induced pulmonary fibrosis.

Novel Long Non-Coding RNA (lncRNA) Transcript AL137782.1 Promotes the Migration of Normal Lung Epithelial Cells through Positively Regulating LMO7.

The role of long non-coding RNA (lncRNAs) in biological processes remains poorly understood, despite their significant impact. Our previous research discovered that the expression of AL137782.1, a long transcript of the novel lncRNA ENSG00000261553, is upregulated in lung epithelial cells upon exposure to microbes. Furthermore, the expression of AL137782.1 exhibits variability between para-cancerous and lung adenocarcinoma samples. These findings imply that this lncRNA may play a role in both normal lung epithelial cellular processes and pathophysiology. To elucidate the function of AL137782.1 in lung epithelial cells, we utilized bioinformatics retrieval and analysis to examine its expression. We then analyzed its subcellular localization using fluorescence in situ hybridization (FISH) and subcellular fractionation. Through rapid amplification of cDNA ends (RACE), we confirmed the presence of a 4401 nt lncRNA AL137782.1 in lung epithelial cells. Moreover, we discovered that this lncRNA positively regulates both mRNA and the protein expression of LMO7, a protein that may regulate the cell migration of normal lung epithelial cells. Although the overexpression of AL137782.1 has been shown to enhance the migration of both normal lung epithelial cells and lung adenocarcinoma cells in vitro, our study revealed that the expression of this lncRNA was significantly decreased in lung cancers compared to adjacent tissues. This suggests that the cell migration pattern regulated by the AL137782.1-LMO7 axis is more likely to occur in normal lung epithelial cells, rather than being a pathway that promotes lung cancer cell migration. Therefore, our study provides new insights into the mechanism underlying cell migration in human lung epithelial cells. This finding may offer a potential strategy to enhance normal lung epithelial cell migration after lung injury.

Targeting Spike Glycoprotein S1 Mediated by NLRP3 Inflammasome Machinery and the Cytokine Releases in A549 Lung Epithelial Cells by Nanocurcumin.

Chronic inflammation and tissue damage can result from uncontrolled inflammation during SARS-CoV-2 or COVID-19 infections, leading to post-acute COVID conditions or long COVID. Curcumin, found in turmeric, has potent anti-inflammatory properties but limited effectiveness. This study developed nanocurcumin, a curcumin nanoparticle, to enhance its physical and chemical stability and investigate its in vitro anti-inflammatory properties upon CoV2-SP induction in lung epithelial cells. Nanocurcumin was prepared by encapsulating curcumin extract in phospholipids. The particle size, polydispersity index, and zeta potential of nanocurcumin were measured using dynamic light scattering. The encapsulated curcumin content was determined using HPLC analysis. The encapsulation efficiency of curcumin was 90.74 ± 5.35% as determined by HPLC. Regarding the in vitro release of curcumin, nanocurcumin displayed a higher release content than non-nanoparticle curcumin. Nanocurcumin was further investigated for its anti-inflammatory properties using A549 lung epithelial cell line. As determined by ELISA, nanocurcumin showed inhibitory effects on inflammatory cytokine releases in CoV2-SP-stimulated conditions, as evidenced by a significant decrease in IL-6, IL-1ß and IL-18 cytokine secretions compared with the spike-stimulated control group (p < 0.05). Additionally, as determined by RT-PCR, nanocurcumin significantly inhibited the CoV2-SP-stimulated expression of inflammatory genes (IL-6, IL-1ß, IL-18, and NLRP3) compared with the spike-stimulated control group (p < 0.05). Regarding the inhibition of NLRP3 inflammasome machinery proteins by Western blot, nanocurcumin decreased the expressions of inflammasome machinery proteins including NLRP3, ASC, pro-caspase-1, and the active form of caspase-1 in CoV2-SP-stimulated A549 cells compared with the spike-stimulated control group (p < 0.05). Overall, the nanoparticle formulation of curcumin improved its solubility and bioavailability, demonstrating anti-inflammatory effects in a CoV2-SP-induced scenario by inhibiting inflammatory mediators and the NLRP3 inflammasome machinery. Nanocurcumin shows promise as an anti-inflammatory product for preventing COVID-19-related airway inflammation.

Cytokine induction of HIF-1α during normoxia in A549 human lung carcinoma cells is regulated by STAT1 and JNK signalling pathways.

Hypoxia inducible factor-1ɑ (HIF-1ɑ) is the regulatory subunit of the HIF-1 transcription factor that is a regulator of cell physiological responses to hypoxia. However, the biological function and regulatory mechanisms controlling HIF-1α in normoxia are poorly understood. Here, we first examined the role of HIF-1α in the inflammatory activation of A549 human lung carcinoma cells in normoxia. Inactivation of the HIF-1α gene by CRISPR/Cas9 reduced the secretion of CXCL8 induced by stimulation with a cytokine mixture (CM) consisting of IL-1, TNFα and IFNγ. We next determined that cytokines act co-operatively to induce expression and nuclear accumulation of HIF-1α. To investigate the signalling mechanisms by which cytokines induce HIF-1α in normoxia, pharmacological inhibitors against the Jak/STAT, PI3K, NFκB, MEK/ERK, and JNK pathways were used. Inhibition of the Jak/STAT and JNK pathways inhibited the induction and nuclear accumulation of HIF-1ɑ by cytokines. Furthermore, siRNA knockdown of STAT1 and JNK also reduced the induction of HIF-1α by cytokines. Finally, pharmacological inhibition of these two pathways also blocked the trans-activation of HIF-1. These findings have implications for understanding the role and regulatory mechanisms of HIF-1ɑ in inflammation and cell biology.

Effect of FTY-720 on Pulmonary Fibrosis in Mice via the TGF-ß1 Signaling Pathway and Autophagy.

We investigated whether FTY-720 might have an effect on bleomycin-induced pulmonary fibrosis through inhibiting TGF-ß1 pathway, and up-regulating autophagy. The pulmonary fibrosis was induced by bleomycin. FTY-720 (1 mg/kg) drug was intraperitoneally injected into mice. Histological changes and inflammatory factors were observed, and EMT and autophagy protein markers were studied by immunohistochemistry and immunofluorescence. The effects of bleomycin on MLE-12 cells were detected by MTT assay and flow cytometry, and the related molecular mechanisms were studied by Western Blot. FTY-720 considerably attenuated bleomycin-induced disorganization of alveolar tissue, extracellular collagen deposition, and α-SMA and E-cadherin levels in mice. The levels of IL-1ß, TNF-α, and IL-6 cytokines were attenuated in bronchoalveolar lavage fluid, as well as protein content and leukocyte count. COL1A1 and MMP9 protein expressions in lung tissue were significantly reduced. Additionally, FTY-720 treatment effectively inhibited the expressions of key proteins in TGF-ß1/TAK1/P38MAPK pathway and regulated autophagy proteins. Similar results were additionally found in cellular assays with mouse alveolar epithelial cells. Our study provides proof for a new mechanism for FTY-720 to suppress pulmonary fibrosis. FTY-720 is also a target for treating pulmonary fibrosis.

Downregulation of Lysosome-Associated Membrane Protein-2A Contributes to the Pathogenesis of COPD.

Background: Macroautophagy plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD), but the role of chaperone-mediated autophagy (CMA) has not been investigated. We investigated if and how CMA is involved in the pathogenesis of COPD. Methods: We measured the level of lysosome-associated membrane protein-2A (LAMP-2A), which is a critical component of CMA that functions as a receptor for cytosolic substrate proteins, in total lung tissues and primary human bronchial epithelial cells (HBECs) from healthy never smokers, smokers, and COPD patients. We assessed the effects of LAMP-2A knock-down on cigarette smoke extract (CSE)-induced aging, cell cycle arrest, and apoptosis in BEAS-2B cells and the expression levels of apoptosis hallmarks in primary HBECs and lung tissue sections. Results: We found that the protein levels of LAMP-2A in lung homogenates and primary HBECs from smokers and COPD patients were lower than those from never smokers. In addition, its level in primary HBECs was negatively correlated with years of smoking. CSE caused degradation of LAMP-2A protein via the lysosomal pathway by activating macroautophagy. Knock-down of LAMP-2A markedly enhanced CSE-induced expression of senescence markers such as p16, p21, p27, and p53. G2/M cell cycle arrest, up-regulation of cyclin B1, and apoptosis in BEAS-2B cells. Apoptosis was increased in CSE-treated primary HBECs and in lung tissues from smokers and COPD patients. Conclusion: Cigarette smoke-induced down-regulation of LAMP-2A is involved in acceleration of aging and apoptosis of lung epithelial cells, which might at least partially contribute to COPD pathogenesis.

The effect of airborne Palladium nanoparticles on human lung cells, endothelium and blood - A combinatory approach using three in vitro models.

A better understanding of the mechanisms behind adverse health effects caused by airborne fine particles and nanoparticles (NP) is essential to improve risk assessment and identification the most critical particle exposures. While the use of automobile catalytic converters is decreasing the exhausts of harmful gases, concentrations of fine airborne particles and nanoparticles (NPs) from catalytic metals such as Palladium (Pd) are reaching their upper safe level. Here we used a combinatory approach with three in vitro model systems to study the toxicity of Pd particles, to infer their potential effects on human health upon inhalation. The three model systems are 1) a lung system with human lung cells (ALI), 2) an endothelial cell system and 3) a human whole blood loop system. All three model systems were exposed to the exact same type of Pd NPs. The ALI lung cell exposure system showed a clear reduction in cell growth from 24 h onwards and the effect persisted over a longer period of time. In the endothelial cell model, Pd NPs induced apoptosis, but not to the same extent as the most aggressive types of NPs such as TiO2. Similarly, Pd triggered clear coagulation and contact system activation but not as forcefully as the highly thrombogenic TiO2 NPs. In summary, we show that our 3-step in vitro model of the human lung and surrounding vessels can be a useful tool for studying pathological events triggered by airborne fine particles and NPs.

The role of potassium current in the pulmonary response to environmental oxidative stress.

Exposure of human lung epithelial cells (A549 cell line) to the oxidant pollutant ozone (O3) alters cell membrane currents inducing its decrease, when the cell undergoes to a voltage-clamp protocol ranging from -90 to +70mV. The membrane potential of these cells is mainly maintained by the interplay of potassium and chloride currents. Our previous studies indicated the ability of O3 to activate ORCC (Outward Rectifier Chloride Channel) and consequently increases the chloride current. In this paper our aim was to understand the response of potassium current to oxidative stress challenge and to identify the kind potassium channel involved in O3 induced current changes. After measuring the total membrane current using an intracellular solution with or without potassium ions, we obtained the contribution of potassium to the overall membrane current in control condition by a mathematical approach. Repeating these experiments after O3 treatment we observed a significant decrease of Ipotassium. Treatment of the cells with Iberiotoxin (IbTx), a specific inhibitor of BK channel, we were able to verify the presence and the functionality of BK channels. In addition, the administration of 4-Aminopyridine (an inhibitor of voltage dependent K channels but not BK channels) and Tetraethylammonium (TEA) before and after O3 treatment we observed the formation of BK oxidative post-translation modifications. Our data suggest that O3 is able to inhibit potassium current by targeting BK channel. Further studies are needed to better clarify the role of this BK channel and its interplay with the other membrane channels under oxidative stress conditions. These findings can contribute to identify the biomolecular pathway induced by O3 allowing a possible pharmacological intervention against oxidative stress damage in lung tissue.

Genistein and Procyanidin B2 Reduce Carcinogen-Induced Reactive Oxygen Species and DNA Damage through the Activation of Nrf2/ARE Cell Signaling in Bronchial Epithelial Cells In Vitro.

Cancer is one of the leading causes of death worldwide. Chemotherapy and radiation therapy are currently providing the basis for cancer therapies, although both are associated with significant side effects. Thus, cancer prevention through dietary modifications has been receiving growing interest. The potential of selected flavonoids in reducing carcinogen-induced reactive oxygen species (ROS) and DNA damage through the activation of nuclear factor erythroid 2 p45 (NF-E2)-related factor (Nrf2)/antioxidant response element (ARE) pathway was studied in vitro. Dose-dependent effects of pre-incubated flavonoids on pro-carcinogen 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKAc)-induced ROS and DNA damage in human bronchial epithelial cells were studied in comparison to non-flavonoids. The most effective flavonoids were assessed for the activation of Nrf2/ARE pathway. Genistein, procyanidin B2 (PCB2), and quercetin significantly suppressed the NNKAc-induced ROS and DNA damage. Quercetin significantly upregulated the phosphorylated protein kinase B/Akt. PCB2 significantly upregulated the activation of Nrf2 and Akt through phosphorylation. Genistein and PCB2 significantly upregulated the phospho-Nrf2 nuclear translocation and catalase activity. In summary, genistein and PCB2 reduced the NNKAc-induced ROS and DNA damage through the activation of Nrf2. Further studies are required to understand the role of dietary flavonoids on the regulation of the Nrf2/ARE pathway in relation to carcinogenesis.