Proliferation Of Bronchial Epithelial Cells Research Articles

Investigate the potential impact of Hemagglutinin from the H1N1 strain on severe pneumonia

The most prevalent glycoprotein on the influenza virus envelope is called hemagglutinin (HA), yet little is known about its involvement in the pathophysiology and etiology of severe influenza pneumonia. Here, after stimulating human bronchial epithelial cells (16-HBE) and mice with HA of H1N1 for 12 h, we investigated the proliferation, migration, inflammatory cytokines expression, and apoptosis in 16-HBE and the pathological damage in mouse lung tissue. The expression of inflammatory cytokines plasminogen activator inhibitor 1(PAI-1), urokinase‐type (uPA) and tissue‐type (tPA) plasminogen activators, and apoptosis were all enhanced by HA, which also prevented the proliferation and migration of bronchial epithelial cells. HA enhanced up-regulated PAI-1, uPA, and tPA protein expression within mouse lung tissue and caused lung injury. In conclusion, HA alone, but not the whole H1N1 virus, induces lung tissue injury by inhibiting cell proliferation and migration, while promoting the expression of inflammatory cytokines and apoptosis.

ALKBH5 Modulates Asthma Progression by Downregulating N6-methyladenosine Methylation.

Asthma is a chronic respiratory disease that is characterized by airway inflammation, excessive mucus production, and airway remodeling. Prevention and treatment for asthma is an urgent issue in clinical studies. In recent years, N6-methyladenosine methylation (m6A) has emerged as a promising regulatory approach involved in multiple diseases. ALKBH5 (alkB homolog 5) is a demethylase widely studied in disease pathologies. This work aimed to explore the regulatory mechanisms underlying the ALKBH5-regulated asthma. We established an interleukin-13 (IL-13)-stimulated cell model to mimic the in vitro inflammatory environment of asthma. ALKBH5 knockdown in bronchial epithelial cells was performed using siRNAs, and the knockdown efficacy was analyzed by quantitative PCR (qPCR). Cell viability and proliferation were measured by cell counting kit 8 (CCK-8) and colony formation assay. The ferroptosis was assessed by measuring the total iron, Fe2+, lipid reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) levels. The enrichment of N6-methyladenosine methylation (m6A) modification was detected by the MeRIP assay. Knockdown of ALKBH5 significantly elevated the survival and colony formation ability of bronchial epithelial cells in the IL-13 induction model. The levels of total iron, Fe2+, lipid ROS, and MDA were remarkedly elevated, and the SOD level was reduced in IL-13-induced bronchial epithelial cells, and depletion of ALKBH5 reversed these effects. Knockdown of ALKBH5 elevated the enrichment of m6A modification and expression of glutathione peroxidase 4 (GPX4). Knockdown of GPX4 abolished the pro-proliferation and anti-ferroptosis effects of siALKBH5. Knockdown of ALKBH5 improved the proliferation of bronchial epithelial cells and alleviated cell ferroptosis.

Effect of photodynamic therapy mediated by hematoporphyrin derivatives on small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells

To investigate the effects of photodynamic therapy (PDT) mediated by hematoporphyrin derivatives (HPD) on the proliferation of small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells. H446 cells and BEAS-2B cells were cultured in vitro with different concentrations of HPD(0, 5, 10, 12, 15, 20 μg/mL) for 4 h, and then irradiated with 630 nm laser with different energy densities (0, 25, 50, 75, 100 mW/cm2). Cell viability of H446 cells and BEAS-2B cells were detected by CCK8 assay. The cell apoptosis was observed with Annexin V-FTTC/PI double staining and Hoechst 33258. The RT-PCR examination was applied to detect the transcriptional changes of the mRNA of Bax、Bcl-2, and Caspase-9. The results of CCK8 showed that when the HPD was 15 μg/mL and the laser power density reached 50 mW/cm2, the cell viability was significantly decreased compared with the black control group. Hoechst 33258 staining showed that with the increase of HPD concentration, the cell density was reduced, and apoptotic cells increased. Flow cytometry assay revealed that the apoptotic rates of the HPD-PDT group of H446 cells and BEAS-2B cells were significantly different from those of the blank control group. The RT-PCR examination showed that the expression levels of Bax and Caspase-9 mRNA in the HPD-PDT group were up-regulated, while the expression levels of Bcl-2 mRNA were down-regulated significantly. HPD-PDT can inhibit H446 cells and BEAS-2B cells growth. The mechanism may be related to up-regulating the expression levels of Bax and Caspase-9 mRNA and down-regulating the expression levels of Bcl-2 mRNA.

Methyltransferase-like 3 (METTL3) Epigenetically Modulates Glutathione Peroxidase 4 (GPX4) Expression to Affect Asthma.

Asthma, a prevalent chronic airway inflammatory condition, poses a significant health challenge. In this study, we delved into the regulatory mechanisms governing asthma, focusing on Methyltransferase-like 3 (METTL3). Through an ovalbumin (OVA)-induced mouse model and interleukin-13 (IL-13)-induced cell model, we mimicked the in vivo and in vitro functions of METTL3 in asthma. Our research revealed that METTL3 expression significantly decreased in asthma-induced mice and IL-13-stimulated cells compared to the control group. Moreover, METTL3 overexpression enhanced bronchial epithelial cell viability and proliferation. Mechanistically, we observed elevated levels of total iron, Fe2+, malondialdehyde (MDA), lipid reactive oxygen species (ROS), alongside reduced glutathione (GSH) levels in IL-13-stimulated cells. Remarkably, METTL3 overexpression counteracted these effects, suggesting a pivotal role in mitigating asthma-related oxidative stress. Furthermore, our study highlighted the involvement of N6-methyladenosine methylation (m6A) modification, where METTL3 regulated the m6A modification of glutathione peroxidase 4 (GPX4) RNA, impacting RNA stability. Knockdown of METTL3 suppressed m6A modification on GPX4 RNA, impairing its stability and contributing to IL-13-induced ferroptosis. Interestingly, METTL3 overexpression not only inhibited cell ferroptosis but also alleviated asthma symptoms. Our findings shed light on the epigenetic regulation of asthma through METTL3-mediated m6A modification, offering potential therapeutic avenues for this prevalent inflammatory disease.

Panax notoginseng saponin R1 improves glucocorticoid-inhibited airway epithelium repair via glucocorticoid receptor β

BackgroundPanax notoginseng saponin R1(PNS-R1), derived from Panax notoginseng roots, promotes wound repair, whereas glucocorticoids can inhibit the repair of airway epithelial damage in asthma. ObjectiveThis study investigated whether PNS-R1 counteracts the inhibitory effects of glucocorticoids on the repair of airway epithelial damage in asthma. MethodsIn vivo, female C57BL/6 mice were sensitized, challenged with house dust mites (HDM), and treated with dexamethasone, PNS-R1, and/or adenovirus GRβ-shRNA. Airway epithelium damage was examined using pathological sections of the trachea and bronchi, markers of airway inflammation, epithelial cells in bronchoalveolar lavage fluid, and expression of the E-cadherin protein. In vitro, we treated 16HBE cells with dexamethasone, PNS-R1, and/or GRβ-siRNA and detected cell proliferation and migration. The expression of GRβ and key components of MKP-1 and Erk1/2 were detected by western blotting. ResultsIn vivo, PNS-R1 reduced airway inflammation, hyperresponsiveness, and mucus hypersecretion; the combination of PNS-R1 and dexamethasone promoted airway epithelial integrity and reduced cell detachment. In vitro, PNS-R1 alleviated the inhibition of bronchial epithelial cell growth, migration, and proliferation by dexamethasone; PNS-R1 promoted GRβ expression, inhibited MKP-1 protein expression, and activated MAPK signaling, thereby promoting airway epithelial cell proliferation and repair. ConclusionsPanax notoginseng saponin R1 alleviated the inhibitory effect of dexamethasone on the repair of airway epithelial damage in asthmatic mice, likely by promoting the proliferation of airway epithelial cells by stimulating GRβ expression and activating the MAPK pathway.

The effect of multiple outgrowths from bronchial tissue explants on progenitor/stem cell number in primary bronchial epithelial cell cultures from smokers and patients with COPD.

Although studies suggest a deficiency in stem cell numbers in chronic airway diseases such as chronic obstructive pulmonary disease (COPD), the role of bronchial epithelial progenitor/stem (P/S) cells is not clear. The objectives of this study were to investigate expression of progenitor/stem (P/S) cell markers, cytokeratin (CK) 5, CK14 and p63 in bronchial epithelial explants and cell cultures obtained from smokers with and without COPD following multiple outgrowths, and to study this effect on bronchial epithelial cell (BEC) proliferation. Bronchial epithelial explants were dissected from lung explants and cultured on coverslips. Confluent cultures were obtained after 3-4 weeks' (transfer, Tr1), explants were then transferred and cultured for a second (Tr2) and third (Tr3) time, respectively. At each stage, expression of CK5, CK14 and p63 in explants and BEC were determined by immunostaining. In parallel experiments, outgrowing cells from explants were counted after 4wks, and explants subsequently transferred to obtain new cultures for a further 3 times. As the transfer number advanced, CK5, CK14 and p63 expression was decreased in both explants and BEC from both smokers without COPD and patients with COPD, with a more pronounced decrease in BEC numbers in the COPD group. Total cell numbers cultured from explants were decreased with advancing outgrowth number in both groups. Smoking status and lung function parameters were correlated with reduced P/S marker expression and cell numbers. Our findings suggest that the number of P/S cells in airway epithelium may play a role in the pathogenesis of COPD, as well as a role in the proliferation of airway epithelial cells, in vitro.

Bone Morphogenic Proteins and Their Antagonists in the Lower Airways of Stable COPD Patients.

Bone morphogenic proteins (BMPs) and their antagonists are involved in the tissue development and homeostasis of various organs. To determine transcriptomic and protein expression of BMPs and their antagonists in stable COPD. We measured the expression and localization of BMPs and some relevant antagonists in bronchial biopsies of stable mild/moderate COPD (MCOPD) (n = 18), severe/very severe COPD (SCOPD) (n = 16), control smokers (CS) (n = 13), and control non-smokers (CNS) (n = 11), and in lung parenchyma of MCOPD (n = 9), CS (n = 11), and CNS (n = 9) using immunohistochemistry and transcriptome analysis, in vitro after the stimulation of the 16HBE cells. In bronchial biopsies, BMP4 antagonists CRIM1 and chordin were increased in the bronchial epithelium and lamina propria of COPD patients. BMP4 expression was decreased in the bronchial epithelium of SCOPD and MCOPD compared to CNS. Lung transcriptomic data showed non-significant changes between groups. CRIM1 and chordin were significantly decreased in the alveolar macrophages and alveolar septa in COPD patients. External 16HBE treatment with BMP4 protein reduced the bronchial epithelial cell proliferation. These data show an imbalance between BMP proteins and their antagonists in the lungs of stable COPD. This imbalance may play a role in the remodeling of the airways, altering the regenerative-reparative responses of the diseased bronchioles and lung parenchyma.

Host–device interactions: exposure of lung epithelial cells and fibroblasts to nickel, titanium, or nitinol affect proliferation, reactive oxygen species production, and cellular signaling

Endoscopic implantation of medical devices for the treatment of lung diseases, including airway stents, unidirectional valves and coils, is readily used to treat central airway disease and emphysema. However, granulation and fibrotic tissue formation impairs treatment effectiveness. To date little is known about the interaction between implanted devices, often made from metals, such as nickel, titanium or nitinol, and cells in the airways. Here, we study the response of lung epithelial cells and fibroblasts to implant device materials. The adhesion and proliferation of bronchial epithelial cells and lung fibroblasts upon exposure to 10 × 3 × 1 mm pieces of nickel, titanium or nitinol is examined using light and scanning electron microscopy. Pro-inflammatory cytokine mRNA expression and release, signaling kinase activity and intracellular free radical production are assessed. Nitinol, and to a lesser extent nickel and titanium, surfaces support the attachment and growth of lung epithelial cells. Nitinol induces a rapid and significant alteration of kinase activity. Cells directly exposed to nickel or titanium produce free radicals, but those exposed to nitinol do not. The response of lung epithelial cells and fibroblasts depends on the metal type to which they are exposed. Nitinol induces cellular surface growth and the induction of kinase activity, while exposure of lung epithelial cells to nickel and titanium induces free radical production, but nitinol does not.Graphical

Mechanical bacterial lysate enhances antimicrobial barrier mechanisms in human airway epithelial cells.

Polyvalent mechanical bacterial lysate is effective in the prevention of respiratory tract infections, although its mechanism of action is not entirely elucidated. Because epithelial cells constitute the frontline defense against infections, we investigated the molecular mechanisms of innate response exerted by bronchial epithelial cells in the presence of polyvalent mechanical bacterial lysate. By using primary human bronchial epithelial cells, we observed that polyvalent mechanical bacterial lysate was able to increase the expression of cellular adhesion molecules such as ICAM-1 and E-cadherin, as well as the expression of amphiregulin, a growth factor able to support human bronchial epithelial cell proliferation. Remarkably, polyvalent mechanical bacterial lysate promoted in human bronchial epithelial cells the de novo expression of human β-defensin-2, a major antimicrobial peptide, conferring them a direct antimicrobial activity. Moreover, polyvalent mechanical bacterial lysate-stimulated human bronchial epithelial cells provided signals for increased IL-22 production by innate lymphoid cells via IL-23, which could further contribute to the release of antimicrobial peptides by epithelial cells. In agreement with these in vitro data, the concentration of both IL-23 and antimicrobial peptides (human β-defensin-2 and LL-37) increased in the saliva of healthy volunteers after sublingual administration of polyvalent mechanical bacterial lysate. Altogether, these results indicate that polyvalent mechanical bacterial lysate administration might support mucosal barrier integrity and promote mechanisms of antimicrobial activity in airway epithelial cells.

Coal dust exposure induces proliferation and migration of human bronchial epithelial cells

Coal dust exposure induces proliferation and migration of human bronchial epithelial cells

MiRNA-34c-5p targets Fra-1 to inhibit pulmonary fibrosis induced by silica through p53 and PTEN/PI3K/Akt signaling pathway.

Silica dust particles are representative of air pollution and long-term inhalation of silicon-containing dust through the respiratory tract can cause pulmonary fibrosis. Epithelial-mesenchymal transformation (EMT) plays an important role in the development of fibrosis. This process can relax cell-cell adhesion complexes and enhance cell migration and invasion properties of these cells. Dysregulation of microRNA-34c (miR-34c) is highly correlated with organ fibrosis including pulmonary fibrosis. In this study, we found that miR-34c-5p could alleviate the occurrence and development of silica-mediated EMT. Fos-related antigen 1 was identified as a functional target of miR-34c-5p by bioinformatics analysis and the dual luciferase gene reporting assay. Importantly, chemically induced up-regulation of hsa-miR-34c-5p correlated inversely with the expression of Fra-1 and further exploration found that the miR-34c-5p/Fra-1 axis inhibits the activation of the phosphatase and tensin homolog deleted on chromosome 10/phosphatidylinositol-4,5-bisphosphate3-kinase/protein kinase B (PTEN/PI3K/AKT) signaling pathway. In addition, through interaction with PTEN/p53 it inhibits the proliferation and migration of human bronchial epithelial cells stimulated by silica, and promotes cell apoptosis, thereby preventing EMT. This finding provides a promising biomarker for the diagnosis and prognosis of pulmonary fibrosis. Furthermore, overexpression of miR-34c-5p represents a potential therapeutic approach.

Toxic effect of silica nanoparticles on bronchial epithelial cells

Nanometer silica dioxide (nano-SiO2) is a widely utilized material in everyday applications; however, it exhibits toxic side effects on the human body. This study aims to provide reliable research basis for theoretical guidance in mitigating the toxic effects of nano-SiO2 in future applications by analyzing the mechanism of its effect on bronchial epithelial cells. In this experiment, under nano-SiO2 intervention, the proliferation and migration capacities of bronchial epithelial cells decreased (P < 0.05); apoptosis capacity increased (P < 0.05); the P13K/AKT/mTOR signaling pathway was inhibited (P < 0.05), and the oxidative stress reaction and inflammatory response was intensified (P < 0.05). Thus, we can tentatively infer that nano-SiO2 causes apoptosis of the bronchial epithelial cells by inhibiting the P13K/AKT/mTOR signaling pathway and accelerates the activation of oxidative stress, thus leading to toxic effects on bronchial epithelial cells.

Effect of Peroxiredoxin 1 on the biological function of airway epithelial cells and epithelial-mesenchymal transition

Peroxiredoxin 1 (PRDX1) participates in tumor cell proliferation, apoptosis, migration, invasion, and the epithelial-to-mesenchymal transition (EMT). This study aimed to investigate the effect of PRDX1 on the EMT of airway epithelial cells stimulated with lipopolysaccharide (LPS) and transforming growth factor-beta 1 (TGF-β1). PRDX1 overexpression significantly increased the proliferation and migration of human bronchial epithelial (BEAS-2B) cells, reduced cell apoptosis (p < 0.01), and induced EMT and collagen deposition by upregulating the expression of the matrix metallopeptidase (MMP)2, MMP9, α-smooth muscle actin (α-SMA), N-cadherin, vimentin and twist proteins and inhibiting E-cadherin expression (p < 0.05). PRDX1 overexpression promoted TGF-β1-mediated inhibition of cell proliferation and migration and significantly enhanced the TGF-β1-induced EMT and collagen synthesis (p < 0.05). Knockdown of PRDX1 inhibited cell proliferation, migration, EMT, and collagen synthesis (p < 0.01), reversed LPS-mediated inhibition of cell proliferation and migration, and significantly suppressed LPS-induced EMT and collagen synthesis (p < 0.01). The result indicating that PRDX1 may be involved in LPS/TGF-1-induced EMT and collagen synthesis in human bronchial epithelial cells.

C-EBP\u03b2 mediates in cigarette/IL-17A-induced bronchial epithelial\u2013mesenchymal transition in COPD mice

BackgroundCigarettes smoking and IL-17A contribute to chronic obstructive pulmonary disease (COPD), and have synergistical effect on bronchial epithelial cell proliferation. CCAAT/enhancer-binding protein β (C-EBPβ) could be induced by IL-17A and is up-regulated in COPD. We explored the effect of cigarettes and IL-17 on bronchial epithelial–mesenchymal transition (EMT) in COPD mice and potential mechanism involved with C-EBPβ in this study.MethodsCOPD model was established with mice by exposing to cigarettes. E-Cadherin, Vimentin, IL-17A and C-EBPβ distributions were detected in lung tissues. Primary bronchial epithelial cells were separated from health mice and cocultured with cigarette smoke extract (CSE) or/and IL-17A. E-Cadherin, Vimentin and IL-17 receptor (IL-17R) expressions in vitro were assessed. When C-EBPβ were silenced by siRNA in cells, E-Cadherin, Vimentin and C-EBPβ expressions were detected.ResultsE-Cadherin distribution was less and Vimentin distribution was more in bronchus of COPD mice than controls. IL-17A and C-EBPβ expressions were higher in lung tissues of COPD mice than controls. In vitro, C-EBPβ protein expression was highest in CSE + IL-17A group, followed by CSE and IL-17A groups. E-cadherin expression in vitro was lowest and Vimentin expression was highest in CSE + IL-17A group, followed by CSE or IL-17A group. Those could be inhibited by C-EBPβ silenced.ConclusionsC-EBPβ mediates in cigarette/IL-17A-induced bronchial EMT in COPD mice. Our findings contribute to a better understanding on the progress from COPD to lung cancers, which will provide novel avenues in preventing tumorigenesis of airway in the context of cigarette smoking.

O3‐3: miR‐21 regulates the proliferation and apoptosis of human bronchial epithelial cells by targeting PARP‐1

O3‐3: miR‐21 regulates the proliferation and apoptosis of human bronchial epithelial cells by targeting PARP‐1

Long noncoding RNA PTTG3P/miR-192-3p/CCNB1 axis is a potential biomarker of childhood asthma

BackgroundIncreasing evidence suggests that long non-coding RNAs (lncRNAs) affect the regulation of immune responses, airway inflammation, and other pathological processes involved in asthma. LncRNA PTTG3P is associated with the development of various tumors, but its role in childhood asthma remains unknown. In this study, we investigated the functions of the lncRNA PTTG3P in the progression of childhood asthma. MethodsTwenty-six healthy children and 26 asthmatic children were monitored for disease progression for 2 years. We obtained blood samples during the chronic phase of disease for lncRNA/mRNA expression microarray analysis. A competitive endogenous RNA network (PTTG3P/miR-192-3p/CCNB1) was identified using bioinformatics analyses. Real-time qPCR and western blot were used to quantify gene and protein expression levels, respectively. Cell counting kit‑8 and transwell assays were used to evaluate the proliferation and migration of bronchial epithelial (16HBE) cells. Double luciferase reporter gene assay was used to validate the predictive targets in PTTG3P, miR-192-3p, and CCNB1. ResultsPTTG3P was highly expressed in the peripheral blood of asthmatic children. Knocking down PTTG3P inhibited epithelial-mesenchymal transition, proliferation, and migration of 16HBE cells. PTTG3P promoted progression of childhood asthma by targeting the miR-192-3p/CCNB1 axis. ConclusionsChildhood asthma was associated with the PTTG3P/miR-192-3p/CCNB1 axis. This study provides potential diagnostic and treatment biomarkers for childhood asthma.

MiRNA-21 Regulates Bronchial Epithelial Cell Proliferation by Activating Tgfβ1/Smad Signaling Pathway and Its Correlation with Asthma Severity in Children

Background:This research was designed to probe into the role of miRNA-21 in the pathogenesis of childhood asthma and its correlation with the severity.Methods:Fifty-four children with bronchial asthma admitted to the Third Affiliated Hospital of Qiqihar Medical University from Jun 2018 to Dec 2019 were included. Forty nine healthy children underwent physical examination at this time period were also enrolled. The miR-21 expression in peripheral blood serum was analyzed by qRT-PCR. The relationship between the expression and severity of asthma in children was explored by Spearman correlation analysis and ROC curve. Bronchial epithelial cell lines were cultured in vitro and divided into blank control group, negative control group and miR-21 inhibition and activation group. The changes of cell proliferation after treatment were detected by CCK-8 test in different groups. The expression of TGF-β1/Smad signaling pathway protein in cells was assessed by Western blot (WB).Results:Compared with that of healthy children, the miR-21 expression in peripheral blood serum of asthmatic children was higher (P<0.001). MiR-21 expression was positively correlated with the severity of illness (r=0.853, P<0.001). The results of cell experiments in vitro signified that miR-21 can promote the proliferation of bronchial epithelial cells, and may be involved in regulating the expression of TGF-β1/Smad3 signaling pathway, thus affecting cell proliferation.Conclusion:miRNA-21 regulates the proliferation of bronchial epithelial cells by activating TGFβ1/Smad signaling pathway. And it is positively correlated with the severity of asthma in children.

Evaluation of Phage Therapy for Pulmonary Infection of Mouse by Liquid Aerosol-Exposure Pseudomonas aeruginosa.

BackgroundPseudomonas aeruginosa is an important nosocomial infectious bacterium, more and more multidrug resistant P. aeruginosa have been isolated and posed severe challenges to clinical antibiotic treatment, bringing additional morbidity, mortality, and economic burden. Bacteriophages can lyse bacteria specificity and are feasible alternatives to antibiotics.MethodsA Pseudomonas aeruginosa-infecting phage vB_PaeP_PA01EW was isolated. Phage plaque assays, transmission electron microscopy, host-range determination, infection assay analyses, whole-genome sequencing and annotation were performed for the phage. Mice pneumonia model using liquid aerosol-exposure Pseudomonas aeruginosa was established, and phage therapy was evaluated.ResultsvB_PaeP_PA01EW belongs to the family Podoviridae according to transmission electron microscopy and was identified as a Luz24likevirus according to the genome analysis. For the phage therapy, compared with the bacteria-infected group, the phage-rescue group has some characteristics. First, adventitial edema and diffuse infiltration of inflammatory cells in tissues were alleviated, Second, bronchial epithelial cell proliferation was reduced. Third, the bacterial burden was significantly decreased.ConclusionThis study provided data support and theoretical basis for the clinical application of bacteriophages. It has important guiding significance and reference value for the application of bacteriophage therapy of other pathogenic bacteria.

Nitric Oxide System and Bronchial Epithelium: More Than a Barrier.

Airway epithelium forms a physical barrier that protects the lung from the entrance of inhaled allergens, irritants, or microorganisms. This epithelial structure is maintained by tight junctions, adherens junctions and desmosomes that prevent the diffusion of soluble mediators or proteins between apical and basolateral cell surfaces. This apical junctional complex also participates in several signaling pathways involved in gene expression, cell proliferation and cell differentiation. In addition, the airway epithelium can produce chemokines and cytokines that trigger the activation of the immune response. Disruption of this complex by some inflammatory, profibrotic, and carcinogens agents can provoke epithelial barrier dysfunction that not only contributes to an increase of viral and bacterial infection, but also alters the normal function of epithelial cells provoking several lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) or lung cancer, among others. While nitric oxide (NO) molecular pathway has been linked with endothelial function, less is known about the role of the NO system on the bronchial epithelium and airway epithelial cells function in physiological and different pathologic scenarios. Several data indicate that the fraction of exhaled nitric oxide (FENO) is altered in lung diseases such as asthma, COPD, lung fibrosis, and cancer among others, and that reactive oxygen species mediate uncoupling NO to promote the increase of peroxynitrite levels, thus inducing bronchial epithelial barrier dysfunction. Furthermore, iNOS and the intracellular pathway sGC-cGMP-PKG are dysregulated in bronchial epithelial cells from patients with lung inflammation, fibrosis, and malignancies which represents an attractive drug molecular target. In this review we describe in detail current knowledge of the effect of NOS-NO-GC-cGMP-PKG pathway activation and disruption in bronchial epithelial cells barrier integrity and its contribution in different lung diseases, focusing on bronchial epithelial cell permeability, inflammation, transformation, migration, apoptosis/necrosis, and proliferation, as well as the specific NO molecular pathways involved.

A Robust Protocol for Decellularized Human Lung Bioink Generation Amenable to 2D and 3D Lung Cell Culture

Decellularization efforts must balance the preservation of the extracellular matrix (ECM) components while eliminating the nucleic acid and cellular components. Following effective removal of nucleic acid and cell components, decellularized ECM (dECM) can be solubilized in an acidic environment with the assistance of various enzymes to develop biological scaffolds in different forms, such as sheets, tubular constructs, or three-dimensional (3D) hydrogels. Each organ or tissue that undergoes decellularization requires a distinct and optimized protocol to ensure that nucleic acids are removed, and the ECM components are preserved. The objective of this study was to optimize the decellularization process for dECM isolation from human lung tissues for downstream 2D and 3D cell culture systems. Following protocol optimization and dECM isolation, we performed experiments with a wide range of dECM concentrations to form human lung dECM hydrogels that were physically stable and biologically responsive. The dECM based-hydrogels supported the growth and proliferation of primary human lung fibroblast cells in 3D cultures. The dECM is also amenable to the coating of polyester membranes in Transwell™ Inserts to improve the cell adhesion, proliferation, and barrier function of primary human bronchial epithelial cells in 2D. In conclusion, we present a robust protocol for human lung decellularization, generation of dECM substrate material, and creation of hydrogels that support primary lung cell viability in 2D and 3D culture systems