Alveolar Epithelial Cell Proliferation Research Articles

Transcriptome Analysis of Rat Lungs Exposed to Moxa Smoke after Acute Toxicity Testing.

The increasing use of moxibustion has led to a debate concerning the safety of this treatment in human patients. Inhalation of cigarette smoke induces lung inflammation and granulomas, the proliferation of alveolar epithelial cells, and other toxic effects; therefore, it is important to assess the influence of inhaled moxa smoke on the lungs. In the present study, a novel poisoning cabinet was designed and used to assess the acute toxicity of moxa smoke in rats. We evaluated pathological changes in rat lung tissue and analyzed differentially expressed genes (DEGs) using RNA-seq and transcriptomic analyses. Our results show that the maximum tolerable dose of moxa smoke was 290.036 g/m³ and LC50 was 537.65 g/m³. Compared with that of the control group, the degree of inflammatory cell infiltration in the lung tissues of group A rats (all dead group) was increased, while that in group E rats (all live group) remained unchanged. GO and KEGG enrichment analyses showed that the DEGs implicated in cell components, binding, and cancer were significantly enriched in the experimental groups compared with the profile of the control group. The expressions of MAFF, HSPA1B, HSPA1A, AOC1, and MX2 determined using quantitative real-time PCR were similar to those determined using RNA-seq, confirming the reliability of RNA-seq data. Overall, our results provide a basis for future evaluations of moxibustion safety and the development of moxibustion-based technology.

Insight Into the Roles of Non-coding RNA in Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease most commonly occurring in premature infants, and its pathological manifestations are alveolar hypoplasia and dysregulation of pulmonary vasculature development. The effective treatment for BPD has not yet been established. Non-coding RNAs, including microRNAs and long non-coding RNAs do not encode proteins, but can perform its biological functions at the RNA level. Non-coding RNAs play an important role in the incidence and development of BPD by regulating the expression of genes related to proliferation, apoptosis, angiogenesis, inflammation and other cell activities of alveolar epithelial cells and vascular endothelial cells. Here we summarize the role of non-coding RNAs in BPD, which provides possible molecular marker and therapeutic target for the diagnosis and treatment of BPD.

STAT3 Promotes Apoptosis of Alveolar Epithelial Cells by Inhibiting AKT Signaling

Type II alveolar epithelial cells are a crucial component of alveolar epithelium, and transcriptional activator 3 (STAT3) have functions in regulating alveolar epithelial cell proliferation. Therefore, based on the modular approach, we analyzed the effects of silencing STAT3 on type II alveolar epithelial cells and studied its mechanism of action. Initially, in the GEO database, we downloaded data on type II alveolar epithelial cells. For transcript to me data in alveolar epithelial cell samples, we performed a differential analysis. Secondly, protein interaction network analysis (PPIs) were performed on the differential genes, and the PPIs were analyzed modularly. The module gene was subjected to enrichment analysis of GO function and KEGG pathway. Non-coding RNAs and transcription factors that regulate the module are predicted based on hyper geometric testing. Thus, we have a total of 13 dysfunction modules. These modular genes are significantly involved in biological processes such as nuclear membranes, embryonic organ development, and regulate the insulin signaling pathway and the PI3K-Akt signaling pathway substantially. We identified vital ncRNA pivots (miR-205-5p) and TF pivot (Eomes, Etsl, Nfkbl, Spi1, Statl, Usfl) to regulate dysfunction modules significantly. Our work deciphered a co-expression network that involved essential gene regulation of type II alveolar epithelial cell apoptosis. It helps to reveal the regulation of silencing STAT3 gene on alveolar epithelial cell apoptosis and deepen our understanding of the mechanism. More importantly, we explained that the silencing gene STAT3 inhibits the apoptosis of alveolar epithelial cells by activating the AKT signaling pathway, providing a new theoretical reference for the study of alveolar epithelial cells.

Extracellular vesicles derived from umbilical cord mesenchymal stromal cells alleviate pulmonary fibrosis by means of transforming growth factor-\u03b2 signaling inhibition

BackgroundPulmonary fibrosis (PF), the end point of interstitial lung diseases, is characterized by myofibroblast over differentiation and excessive extracellular matrix accumulation, leading to progressive organ dysfunction and usually a terminal outcome. Studies have shown that umbilical cord-derived mesenchymal stromal cells (uMSCs) could alleviate PF; however, the underlying mechanism remains to be elucidated.MethodsThe therapeutic effects of uMSC-derived extracellular vesicles (uMSC-EVs) on PF were evaluated using bleomycin (BLM)-induced mouse models. Then, the role and mechanism of uMSC-EVs in inhibiting myofibroblast differentiation were investigated in vivo and in vitro.ResultsTreatment with uMSC-EVs alleviated the PF and enhanced the proliferation of alveolar epithelial cells in BLM-induced mice, thus improved the life quality, including the survival rate, body weight, fibrosis degree, and myofibroblast over differentiation of lung tissue. Moreover, these effects of uMSC-EVs on PF are likely achieved by inhibiting the transforming growth factor-β (TGF-β) signaling pathway, evidenced by decreased expression levels of TGF-β2 and TGF-βR2. Using mimics of uMSC-EV-specific miRNAs, we found that miR-21 and miR-23, which are highly enriched in uMSC-EVs, played a critical role in inhibiting TGF-β2 and TGF-βR2, respectively.ConclusionThe effects of uMSCs on PF alleviation are likely achieved via EVs, which reveals a new role of uMSC-EV-derived miRNAs, opening a novel strategy for PF treatment in the clinical setting.

Hyperoxia-induced miR-342-5p down-regulation exacerbates neonatal bronchopulmonary dysplasia via the Raf1 regulator Spred3.

Bronchopulmonary dysplasia (BPD) is the most prevalent chronic paediatric lung disease and is linked to the development of chronic obstructive pulmonary disease. MicroRNA-based regulation of type II alveolar epithelial cell (T2AEC) proliferation and apoptosis is an important factor in the pathogenesis of BPD and warrants further investigation. Two murine models of hyperoxic lung injury (with or without miR-342-5p or Sprouty-related, EVH1 domain-containing protein 3 [Spred3] modulation) were employed: a hyperoxia-induced acute lung injury model (100% O2 on postnatal days 1-7) and the BPD model (100% O2 on postnatal days 1-4, followed by room air for 10 days). Tracheal aspirate pellets from healthy control and moderate/severe BPD neonates were randomly selected for clinical miR-342-5p analysis. Hyperoxia decreased miR-342-5p levels in primary T2AECs, MLE12 cells and neonatal mouse lungs. Transgenic miR-342 overexpression in neonatal mice ameliorated survival rates and improved the BPD phenotype and BPD-associated pulmonary arterial hypertension (PAH). T2AEC-specific miR-342 transgenic overexpression, as well as miR-342-5p mimic therapy, also ameliorated the BPD phenotype and associated PAH. miR-342-5p targets the 3'UTR of the Raf1 regulator Spred3, inhibiting Spred3 expression. Treatment with recombinant Spred3 exacerbated the BPD phenotype and associated PAH. Notably, miR-342-5p inhibition under room air conditions did not mimic the BPD phenotype. Moderate/severe BPD tracheal aspirate pellets exhibited decreased miR-342-5p levels relative to healthy control pellets. These findings suggest that miR-342-5p mimic therapy may show promise in the treatment or prevention of BPD.

Roles of lung-recruited monocytes and pulmonary Vascular Endothelial Growth Factor (VEGF) in resolving Ventilator-Induced Lung Injury (VILI).

Monocytes and vascular endothelial growth factor (VEGF) have profound effects on tissue injury and repair. In ventilator-induced lung injury (VILI), monocytes, the majority of which are Ly6C+high, and VEGF are known to initiate lung injury. However, their roles in post-VILI lung repair remain unclear. In this study, we used a two-hit mouse model of VILI to identify the phenotypes of monocytes recruited to the lungs during the resolution of VILI and investigated the contributions of monocytes and VEGF to lung repair. We found that the lung-recruited monocytes were predominantly Ly6C+low from day 1 after the insult. Meanwhile, contrary to inflammatory cytokines, pulmonary VEGF decreased upon VILI but subsequently increased significantly on days 7 and 14 after the injury. There was a strong positive correlation between VEGF expression and proliferation of alveolar epithelial cells in lung sections. The expression pattern of VEGF mRNA in lung-recruited monocytes was similar to that of pulmonary VEGF proteins, and the depletion of monocytes significantly suppressed the increase of pulmonary VEGF proteins on days 7 and 14 after VILI. In conclusion, during recovery from VILI, the temporal expression patterns of pulmonary growth factors are different from those of inflammatory cytokines, and the restoration of pulmonary VEGF by monocytes, which are mostly Ly6C+low, is associated with pulmonary epithelial proliferation. Lung-recruited monocytes and pulmonary VEGF may play crucial roles in post-VILI lung repair.

A novel coronavirus—from basic research to clinic

<p indent="0mm">The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is now sweeping the globe like the toppling Dominoes, and has become an alarming public health issue worldwide. To date, there have been more than 76 million confirmed cases and more than 1,690,000 deaths worldwide. SARS-CoV-2 might originate from bats and is usually transmitted through respiratory droplets and contact. The SARS-CoV-2 genome is about 30 kilobases in length, encoding 16 non-structural proteins and 4 structural proteins. The receptor-binding domain (RBD) in the S1 subunit of the spike (S) protein of SARS-CoV-2 interacts with the entry receptor angiotensin-converting enzyme 2 (ACE2) on host alveolar type II epithelial cells. The interaction causes structural changes in the S2 subunit leading to virus-host cell fusion. RNA-dependent RNA polymerase (RdRp) is vital to replication of the viral genome. ACE2 may be downregulated after infection, leading to lung edema and injury. Common symptoms of SARS-CoV-2 infection include fever, dry cough, dyspnea, fatigue, and myalgia, that is, a pneumonia that has been termed coronavirus disease-2019 (COVID-19) by the World Health Organization. COVID-19 can be fatal. The imaging findings of the lung are mostly multiple ground glass shadows and subsegmental consolidation near the pleura. SARS-CoV-2 can be detected by nucleic acid and antibody tests, virus isolation, and electron microscopy. The pathological features of COVID-19 are diffuse alveolar injury, fibrous myxoid exudate, interstitial inflammation, alveolar epithelial cell proliferation and shedding, and hyaline membrane formation. At present, antivirals are non-specific and mainly include lopinavir/ritonavir, remdesivir, and umifenovir. EK1 peptide and SARS-CoV-2-HR2P may be novel antiviral agents that can prevent infection. Convalescent plasma is generally a valid option. Although no vaccine is officially available, there are inactivated vaccines, vectored vaccines and nucleic acid-based vaccines in clinical trials. Prompt and strong measures have been taken to prevent the transmission of SARS-CoV-2 in China.

Astragalus Polysaccharide Regulates Proliferation, Migration and Invasion of Lung Cancer Cells via Janus Kinase/Signal Transducer and Activator of Transcription Signaling Pathway

To evaluate the regulatory effects of Astragalus polysaccharide on the proliferation, migration and invasion of lung cancer cells via the Janus kinase/signal transducer and activator of transcription signaling pathway. The inhibitory effects of treatment with Astragalus polysaccharide at different concentrations (0, 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 mg/ml) for different time (24, 48 and 72 h) on the proliferation of adenocarcinomic human alveolar basal epithelial cells in the logarithmic growth phase were detected by methyl thiazolyl tetrazolium assay and its inhibitory effects on the migration and invasion of adenocarcinomic human alveolar basal epithelial cells were determined by wound healing assay and Transwell migration and invasion assays respectively. In contrast with control group, the proliferation of adenocarcinomic human alveolar basal epithelial cells treated with different concentrations of Astragalus polysaccharide for 24, 48 and 72 h was remarkably inhibited in a dose and time dependent manner under the same drug concentration, longer the action time, higher the proliferation inhibition rate of adenocarcinomic human alveolar basal epithelial cells; under the same action time, higher the drug concentration, higher the proliferation inhibition rate of adenocarcinomic human alveolar basal epithelial cells. The number of migrating cells dropped significantly in Astragalus polysaccharide groups (p<0.05), which was more obvious with the increase of Astragalus polysaccharide concentration (p<0.05). Astragalus polysaccharide can dramatically inhibit the proliferation of lung cancer adenocarcinomic human alveolar basal epithelial cells in a concentration and time-dependent manner and also effectively suppress their migration and invasion. The mechanism may be related to its ability to up-regulate the protein expressions of Src homology region 2 containing protein tyrosine phosphatase 2 and suppressor of cytokine signaling 3 and inhibit the over-activation of the Janus kinase/signal transducer and activator of transcription signaling pathway in lung cancer adenocarcinomic human alveolar basal epithelial cells.

MicroRNA 198 Inhibits Invasion and Metastasis in Non-Small Cell Lung Cancer by Targeting Toll-Like Receptor 4

The expression of microRNA 198 which is localized in human chromosome 13q13.3 is downregulated in many malignant tumors. The purpose of this study is to investigate the regulatory effects of microRNA 198 on non-small cell lung cancer and the associated regulatory mechanism. The expression levels of microRNA 198 in non-small cell lung cancer cell lines and tumor tissues were determined using the reverse transcription polymerase chain reaction method. Overexpression microRNA in adenocarcinomic human alveolar basal epithelial cells, non-small cell lung cancer cells and cell proliferation in each group were measured with the cell counting kit-8 method. Cell migration was determined with a cell scratch test, while transwell assay was used for the determination of cell migration and invasion capacities. Bioinformatics website was used to predict microRNA 198 target genes and verified with double luciferase assay. Western blot method was used to assay the protein expression level of toll-like receptor 4 in the cells and the effects of overexpression of microRNA 198 on the regulation of cell proliferation and migration by microRNA 198. The expressions of microRNA 198 in non-small cell lung cancer cell lines and tissues were significantly downregulated. However, overexpression of microRNA 198 through transfection significantly inhibited the proliferation, migration and invasion of lung cancer cell lines adenocarcinomic human alveolar basal epithelial cells and non-small cell lung cancer cells and directly targeted and inhibited the expression of toll-like receptor 4. Overexpression of toll-like receptor 4 reversed the regulatory effect of microRNA 198 to some extent. MicroRNA 198 inhibits the proliferation, migration and invasion of non-small cell lung cancer cells by targeting toll-like receptor 4, thereby playing the role of tumor suppressor gene in the development of non-small cell lung cancer.

Dual roles of TGF-β signaling in the regulation of dental epithelial cell proliferation.

The purpose of this study is to investigate the molecular mechanisms and biological function of TGF-β-activated Smad1/5 in dental epithelium. Immunohistochemistry was used to detect the expressions of TGF-β signaling-related gene in mice molar germ. Primary dental epithelial cells were cultured and treated with TGF-β1 at a concentration of 0.5 or 5ng/mL. Small molecular inhibitors, SB431542 and ML347, was used to inhibite ALK5 and ALK1/2, respectively. Small interfering RNA was used to knock down Smad1/5 or Smad2/3. The proliferation rate of cells was evaluated by EdU assay. In the basal layer of dental epithelial bud TGF-β1 and p-Smad1/5 were highly expressed, and in the interior of the epithelial bud TGF-β1 was lowly expressed, whereas p-Smad2/3 was highly expressed. In primary cultured dental epithelial cells, low concentration of TGF-β1 activated Smad2/3 but not Smad1/5, while high concentration of TGF-β1 was able to activate both Smad2/3 and Smad1/5. SB431542 but not ML347 was able to block the phosphorylation of Smad2/3 by TGF-β1. Either SB431542 or ML347 was able to block the phosphorylation of Smad1/5 by TGF-β1. EdU staining showed that high concentration of TGF-β1 promoted dental epithelial cell proliferation, which was reversed by silencing Smad1/5, whereas low concentration of TGF-β1 inhibited cell proliferation, which was reversed by silencing Smad2/3. In conclusions, TGF-β exhibits dual roles in the regulation of dental epithelial cell proliferation through two pathways. On the one hand, TGF-β activates canonical Smad2/3 signaling through ALK5, inhibiting the proliferation of internal dental epithelial cells. On the other hand, TGF-β activates noncanonical Smad1/5 signaling through ALK1/2-ALK5, promoting the proliferation of basal cells in the dental epithelial bud.

MicroRNA-31 regulates dental epithelial cell proliferation by targeting Satb2

MicroRNAs (miRNAs) exhibit strong potential clinical application owing to their extensive regulation and flexible delivery properties. MicroRNA-31 (miR-31) is an evolutionarily conserved miRNA expressed during tooth development, and it is highly expressed in mouse incisor epithelium. The specific role of miR-31 in odontogenesis has not been elucidated comprehensively, and the aim of the present study was to investigate its activity. Our results showed that miR-31 suppressed LS8 cell proliferation by inhibiting the cell cycle at the G1/S transition. Mutation of Special AT-rich sequence-binding protein 2 (SATB2) gene is responsible for human SATB2-associated syndrome (SAS), which is often accompanied by dental abnormities. Here, it was identified as a direct target of miR-31 in LS8 cells and a promoter of cell proliferation. The expression and distribution of SATB2 in mouse molars and incisors were explored using immunofluorescence, which showed strong signals in the nuclei of incisor epithelial cells and weak signals in the cytoplasm of molar epithelial cells. Moreover, rescue experiments demonstrated that Satb2 could mitigate the inhibitory effect of miR-31 on cell proliferation by promoting the expression of CDK4. Collectively, our results suggested that miR-31 regulates dental epithelial cell proliferation by targeting Satb2, highlighting the biological importance of miR-31 in odontogenesis.

Wnt/β-catenin signaling is critical for regenerative potential of distal lung epithelial progenitor cells in homeostasis and emphysema

Wnt/β-catenin signaling regulates progenitor cell fate decisions during lung development and in various adult tissues. Ectopic activation of Wnt/β-catenin signaling promotes tissue repair in emphysema, a devastating lung disease with progressive loss of parenchymal lung tissue. The identity of Wnt/β-catenin responsive progenitor cells and the potential impact of Wnt/β-catenin signaling on adult distal lung epithelial progenitor cell function in emphysema are poorly understood. Here, we used TCF/ Lef:H2B/GFP reporter mice to investigate the role of Wnt/β-catenin signaling in lung organoid formation. We identified an organoid-forming adult distal lung epithelial progenitor cell population characterized by a low Wnt/β-catenin activity, which was enriched in club and alveolar epithelial type (AT)II cells. Endogenous Wnt/β-catenin activity was required for the initiation of multiple subtypes of distal lung organoids derived from the Wntlow epithelial progenitors. Further ectopic Wnt/β-catenin activation specifically led to an increase in alveolar organoid number; however, the subsequent proliferation of alveolar epithelial cells in the organoids did not require constitutive Wnt/β-catenin signaling. Distal lung epithelial progenitor cells derived from the mouse model of elastase-induced emphysema exhibited reduced organoid forming capacity. This was rescued by Wnt/β-catenin signal activation, which largely increased the number of alveolar organoids. Together, our study reveals a novel mechanism of lung epithelial progenitor cell activation in homeostasis and emphysema.

Yes-associated protein upregulates filopodia formation to promote alveolar epithelial-cell phagocytosis

Cells engulf particles larger than 0.5 μm in diameter by phagocytosis, which is driven by cytoskeletal rearrangements. Phagocytosis by alveolar epithelial cells (AECs) helps to maintain the alveolar homeostasis. Yes-associated protein (YAP), a transcriptional coactivator of the Hippo pathway, affects proliferation, differentiation, and cytoskeletal rearrangement of AECs, but it is not clear whether YAP regulates phagocytosis. In this study, interference with YAP expression inhibited phagocytosis in MLE-12 cells and in primary cultures of AEC. Filopodia formation promoted phagocytosis in AECs, and YAP enhanced filopodia formation in AECs. Blocking PI3K signaling resulted in reduced YAP protein expression and inhibition of phagocytosis. The results indicate that YAP expression was regulated by PI3K signaling and promoted phagocytosis in AECs by upregulating filopodia formation.

Blockade of Pan-Fibroblast Growth Factor Receptors Mediates Bidirectional Effects in Lung Fibrosis.

FGFs (fibroblast growth factors) are major factors associated with the pathogenesis of pulmonary fibrosis. On the one hand, nintedanib, a tyrosine kinase inhibitor targeting several growth factor receptors, including the FGF receptor (FGFR), has been approved for the treatment of idiopathic pulmonary fibrosis. On the other hand, recent reports suggest that FGFs are required for epithelial recovery. In this study, we focused on FGF signaling to both fibroblasts and alveolar epithelial cells (AECs), and we examined the effect of a pan-FGFR blocker on experimental pulmonary fibrosis in mice. The effects of BGJ398, a pan-FGFR inhibitor, on the migration and proliferation of fibroblasts and AECs were assessed using Transwell migration or [3H]thymidine incorporation assays. The expression of FGFR was analyzed using IB or flow cytometry. We also investigated the effect of BGJ398 on pulmonary fibrosis induced by bleomycin in mice. Both lung fibroblasts and AECs expressed FGFRs. BGJ398 significantly inhibited the proliferation and migration of lung fibroblasts stimulated with FGF2. BGJ398 also reduced the proliferation of AECs in response to FGF2. Although the administration of BGJ398 ameliorated pulmonary fibrosis in bleomycin-treated mice, it increased mortality resulting from alveolar injury and inhibition of AEC regeneration. These data suggest that the total inhibition of FGFR signaling can suppress lung fibrosis by inhibiting fibroblast activities, although alveolar injury is simultaneously caused.

Senescence of IPF Lung Fibroblasts Disrupt Alveolar Epithelial Cell Proliferation and Promote Migration in Wound Healing.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease marked by excessive accumulation of lung fibroblasts (LFs) and collagen in the lung parenchyma. The mechanisms that underlie IPF pathophysiology are thought to reflect repeated alveolar epithelial injury leading to an aberrant wound repair response. Recent work has shown that IPF-LFs display increased characteristics of senescence including growth arrest and a senescence-associated secretory phenotype (SASP) suggesting that senescent LFs contribute to dysfunctional wound repair process. Here, we investigated the influence of senescent LFs on alveolar epithelial cell repair responses in a co-culture system. Alveolar epithelial cell proliferation was attenuated when in co-culture with cells or conditioned media from, senescence-induced control LFs or IPF-LFs. Cell-cycle analyses showed that a larger number of epithelial cells were arrested in G2/M phase when co-cultured with IPF-LFs, than in monoculture. Paradoxically, the presence of LFs resulted in increased A549 migration after mechanical injury. Our data suggest that senescent LFs may contribute to aberrant re-epithelialization by inhibiting proliferation in IPF.

Regeneration of Pulmonary Tissue in a Calf Model of Fibrinonecrotic Bronchopneumonia Induced by Experimental Infection with Chlamydia Psittaci.

Pneumonia is a cause of high morbidity and mortality in humans. Animal models are indispensable to investigate the complex cellular interactions during lung injury and repair in vivo. The time sequence of lesion development and regeneration is described after endobronchial inoculation of calves with Chlamydia psittaci. Calves were necropsied 2–37 days after inoculation (dpi). Lesions and presence of Chlamydia psittaci were investigated using histology and immunohistochemistry. Calves developed bronchopneumonia at the sites of inoculation. Initially, Chlamydia psittaci replicated in type 1 alveolar epithelial cells followed by an influx of neutrophils, vascular leakage, fibrinous exudation, thrombosis and lobular pulmonary necrosis. Lesions were most extensive at 4 dpi. Beginning at 7 dpi, the number of chlamydial inclusions declined and proliferation of cuboidal alveolar epithelial cells and sprouting of capillaries were seen at the periphery of necrotic tissue. At 14 dpi, most of the necrosis had been replaced with alveoli lined with cuboidal epithelial cells resembling type 2 alveolar epithelial cells and mild fibrosis, and hyperplasia of organized lymphoid tissue were observed. At 37 dpi, regeneration of pulmonary tissue was nearly complete and only small foci of remodeling remained. The well-defined time course of development and regeneration of necrotizing pneumonia allows correlation of morphological findings with clinical data or treatment regimen.

Enhancement of FAK alleviates ventilator-induced alveolar epithelial cell injury

Mechanical ventilation induces lung injury by damaging alveolar epithelial cells (AECs), but the pathogenesis remains unknown. Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase that is involved in cell growth and intracellular signal transduction pathways. This study explored the potential role of FAK in AECs during lung injury induced by mechanical ventilation. High-volume mechanical ventilation (HMV) was used to create a mouse lung injury model, which was validated by analysis of lung weight, bronchoalveolar lavage fluid and histological investigation. The expression of FAK and Akt in AECs were evaluated. In addition, recombinant FAK was administered to mice via the tail vein, and then the extent of lung injury was assessed. Mouse AECs were cultured in vitro, and FAK expression in cells under stretch was investigated. The effects of FAK on cell proliferation, migration and apoptosis were investigated. The results showed that HMV decreased FAK expression in AECs of mice, while FAK supplementation attenuated lung injury, reduced protein levels/cell counts in the bronchoalveolar lavage fluid and decreased histological lung injury and oedema. The protective effect of FAK promoted AEC proliferation and migration and prevented cells from undergoing apoptosis, which restored the integrity of the alveoli through Akt pathway. Therefore, the decrease in FAK expression by HMV is essential for injury to epithelial cells and the disruption of alveolar integrity. FAK supplementation can reduce AEC injury associated with HMV.

Glycogen Synthase Kinase-3\u03b2 Inhibition with 9-ING-41 Attenuates the Progression of Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with a median survival of 3 years after diagnosis. Although the etiology of IPF is unknown, it is characterized by extensive alveolar epithelial cell apoptosis and proliferation of myofibroblasts in the lungs. While the origins of these myofibroblast appear to be diverse, fibroblast differentiation contributes to expansion of myofibroblasts and to disease progression. We found that agents that contribute to neomatrix formation and remodeling in pulmonary fibrosis (PF); TGF-β, Factor Xa, thrombin, plasmin and uPA all induced fibroblast/myofibroblast differentiation. These same mediators enhanced GSK-3β activation via phosphorylation of tyrosine-216 (p-Y216). Inhibition of GSK-3β signaling with the novel inhibitor 9-ING-41 blocked the induction of myofibroblast markers; α-SMA and Col-1 and reduced morphological changes of myofibroblast differentiation. In in vivo studies, the progression of TGF-β and bleomycin mediated PF was significantly attenuated by 9-ING-41 administered at 7 and 14 days respectively after the establishment of injury. Specifically, 9-ING-41 treatment significantly improved lung function (compliance and lung volumes; p < 0.05) of TGF-β adenovirus treated mice compared to controls. Similar results were found in mice with bleomycin-induced PF. These studies clearly show that activation of the GSK-3β signaling pathway is critical for the induction of myofibroblast differentiation in lung fibroblasts ex vivo and pulmonary fibrosis in vivo. The results offer a strong premise supporting the continued investigation of the GSK-3β signaling pathway in the control of fibroblast-myofibroblast differentiation and fibrosing lung injury. These data provide a strong rationale for extension of clinical trials of 9-ING-41 to patients with IPF.

Study on the expression level of zonula occludens 1 and zonula occludens 1 related nucleic acid binding protein in alveolar epithelial cells in hyperoxia induced bronchopulmonary dysplasia newborn rats

Objective Previous studies have found abnormal proliferation and transdifferentiation of alveolar epithelial cells(AECs)in hyperoxic lung injury of neonatal rats.The purpose of this study was to clarify the expression of zonula occludens 1(ZO-1) and ZO-1 related nucleic acid binding protein(ZONAB)in AECs in hyperoxic lung injury model, in order to investigate its effect on the proliferation and transdifferentiation of AECs in the injured lung tissue. Methods Full-term neonatal Wistar rats were randomly divided into two groups within 12 h after birth, model group(inhaled oxygen concentration 85%)and control group(inhaled air). Lung specimens were collected at 7, 14 and 21 days after exposure.The expression of ZONAB in typeⅡalveolar epithelial cells(AECⅡ)was observed by double immunofluorescence staining.At the same time, AEC Ⅱ was isolated from lung tissues of animal models at these time points, and the expression levels of ZO-1, ZONAB protein and mRNA in lung tissues and AECⅡof the two groups were detected by Western blot and Real-Time PCR.In addition, AEC Ⅱ was isolated from lung tissue of normal newborn rats and then divided into model group(85% oxygen concentration)and control group(21% oxygen concentration). After 48 hours of culture in vitro, the expression levels of ZO-1, ZONAB protein and mRNA were detected, and the expression level and location of ZONAB were observed by immunofluorescence staining. Results Double immunofluorescence staining showed that the expression of ZONAB in AECⅡin model group was significantly lower than that in control group.The protein and mRNA expression levels of ZO-1 and ZONAB in AECⅡisolated from lung tissue of model group were both significantly lower than those from control group, starting from 7 d after hyperoxia exposure.AECⅡisolated from lung tissue of normal newborn rats, were then incubated for 48 hours under hyperoxia or normoxia in vitro, the protein and mRNA expression levels of ZO-1 and ZONAB significantly decreased in model group compared with those in control group.The results of immunofluorescence staining showed that the expression of ZONAB was higher in AECⅡof the control group, and ZONAB was mostly located in the junction and nucleus of cells, while the expression of ZONAB in the model group significantly decreased than that in the control group, and the expression sites were clustered in the cytoplasm, with little expression in the junction and nucleus. Conclusion ZO-1, as a tight junction-related protein, is down-regulated in hyperoxic lung injury model.In addition to destroying pulmonary epithelial barrier to mediate pulmonary edema, it also participated in the regulation of proliferation and differentiation of AECs by regulating transcription factor ZONAB, suggesting that this may be another pathway leading to hyperoxic lung injury. Key words: Hyperoxia; Alveolar epithelial cell; Transdifferentiation; Zonula occludens 1; Zonula occludens 1 related nucleic acid binding protein

Improving the viability of tissue-resident stem cells using an organ-preservation solution.

Human clinical specimens are a valuable source of tissue‐resident stem cells, but such cells need to be collected immediately after tissue collection. To extend the timescale for collection from fresh human samples, we developed a new extracellular fluid (ECF)‐type preservation solution based on a high‐sodium and low‐potassium solution containing low‐molecular‐weight dextran and glucose, which is used for preservation of organs for transplantation. In this study, we compared the preservation of tissue‐resident stem cells using our ECF solution with that using three other solutions: PBS, Dulbecco’s modified Eagle’s medium and Euro‐Collins solution. These solutions represent a common buffer, a common culture medium and a benchmark organ‐preservation solution, respectively. Lung tissues were removed from mice and preserved for 72 h under low‐temperature conditions. Of the solutions tested, only preservation in the ECF‐type solution could maintain the proliferation and differentiation capacity of mouse lung tissue‐resident stem cells. In addition, the ECF solution could preserve the viability and proliferation of human alveolar epithelial progenitor cells when stored for more than 7 days at 4 °C. The mean viability of human alveolar type II cells at 2, 5, 8 and 14 days of low‐temperature preservation was 90.9%, 84.8%, 85.7% and 66.3%, respectively, with no significant differences up to 8 days. Overall, our findings show that use of our ECF‐type preservation solution may maintain the viability and function of tissue‐resident stem cells. Use of this preservation solution may facilitate the investigation of currently unobtainable human tissue specimens for human stem cell biology.