Wei-Guo Xu

Mesenchymal stem cells protect cigarette smoke-damaged lung and pulmonary function partly via VEGF–VEGF receptors†‡

Progressive pulmonary inflammation and emphysema have been implicated in the progression of chronic obstructive pulmonary disease (COPD), while current pharmacological treatments are not effective. Transplantation of bone marrow mesenchymal stem cells (MSCs) has been identified as one such possible strategy for treatment of lung diseases including acute lung injury (ALI) and pulmonary fibrosis. However, their role in COPD still requires further investigation. The aim of this study is to test the effect of administration of rat MSCs (rMSCs) on emphysema and pulmonary function. To accomplish this study, the rats were exposed to cigarette smoke (CS) for 11 weeks, followed by administration of rMSCs into the lungs. Here we show that rMSCs infusion mediates a down‐regulation of pro‐inflammatory mediators (TNF‐α, IL‐1β, MCP‐1, and IL‐6) and proteases (MMP9 and MMP12) in lung, an up‐regulation of vascular endothelial growth factor (VEGF), VEGF receptor 2, and transforming growth factor (TGFβ‐1), while reducing pulmonary cell apoptosis. More importantly, rMSCs administration improves emphysema and destructive pulmonary function induced by CS exposure. In vitro co‐culture system study of human umbilical endothelial vein cells (EA.hy926) and human MSCs (hMSCs) provides the evidence that hMSCs mediates an anti‐apoptosis effect, which partly depends on an up‐regulation of VEGF. These findings suggest that MSCs have a therapeutic potential in emphysematous rats by suppressing the inflammatory response, excessive protease expression, and cell apoptosis, as well as up‐regulating VEGF, VEGF receptor 2, and TGFβ‐1. J. Cell. Biochem. 114: 323–335, 2013.

Mesenchymal stem cells alleviate airway inflammation and emphysema in COPD through down-regulation of cyclooxygenase-2 via p38 and ERK MAPK pathways

Bone marrow-derived mesenchymal stem cells (MSCs) have been identified as one possible strategy for the treatment of chronic obstructive pulmonary disease (COPD). Our previous studies have demonstrated that MSC administration has therapeutic potential in airway inflammation and emphysema via a paracrine mechanism. We proposed that MSCs reverse the inflammatory process and restore impaired lung function through their interaction with macrophages. In our study, the rats were exposed to cigarette smoke (CS), followed by the administration of MSCs into the lungs for 5 weeks. Here we show that MSC administration alleviated airway inflammation and emphysema through the down-regulation of cyclooxygenase-2 (COX-2) and COX-2-mediated prostaglandin E2 (PGE2) production, possibly through the effect on alveolar macrophages. In vitro co-culture experiments provided evidence that MSCs down-regulated COX-2/PGE2 in macrophages through inhibition of the activation-associated phosphorylation of p38 MAPK and ERK. Our data suggest that MSCs may relieve airway inflammation and emphysema in CS-exposed rat models, through the inhibition of COX-2/PGE2 in alveolar macrophages, mediated in part by the p38 MAPK and ERK pathways. This study provides a compelling mechanism for MSC treatment in COPD, in addition to its paracrine mechanism.

Cigarette smoke-induced skeletal muscle atrophy is associated with up-regulation of USP-19 via p38 and ERK MAPKs†

Ubiquitin‐specific proteases (USPs) deubiquitinate ubiquitin–protein conjugates in the ubiquitin–proteasome system. Previous research shows that ubiquitin‐specific protease‐19 (USP‐19) is up‐regulated in mammalian skeletal muscle in some degradative conditions, such as including fasting, diabetes, dexamethasone treatment, and cancer, and its function is associated with muscle atrophy. However, it is still unclear whether USP‐19 is involved in muscle atrophy induced by chronic obstructive pulmonary disease. Rats exposed to chronic cigarette smoke and L6 myotubes incubated with cigarette smoke extract (CSE) were studied here. Using western blot analysis and quantitative real‐time polymerase chain reaction (qPCR), we observed over‐expression of USP‐19 and down‐regulation of myosin heavy chain (MHC) in both models. Moreover, CSE exposure inhibited myogenic differentiation and myotube formation in L6 myotubes. To explore the mechanism underlying these effects, we investigated the levels of phosphorylated mitogen‐activated protein kinases (MAPKs) and total MAPKs. Exposing myotubes to CSE resulted in the general activation of MAPKs such as p38, JNK, and ERK1/2. The ERK inhibitor PD98059 and the p38 inhibitor SB203580 significantly blocked the increase in USP‐19 gene expression induced by CSE. Our findings suggest that USP‐19 is associated with muscle atrophy in response to cigarette smoke and is a potential therapeutic target. CSE promotes myotube wasting in culture partly by inhibiting myogenic differentiation and acts via p38 and ERK MAPK to stimulate expression of USP‐19 in vitro. J. Cell. Biochem. 112: 2307–2316, 2011.

Yes-Associated Protein 1 Promotes Adenocarcinoma Growth and Metastasis through Activation of the Receptor Tyrosine Kinase Axl:

Yes-associated protein 1 (YAP1), a nuclear effector of the Hippo pathway, plays an important role in tumorigenesis and progression of multiple cancers. The present study aimed to investigate the clinical significance of YAP1 and receptor tyrosine kinase Axl expression in human lung adenocarcinomas (LAC). We further explored possible molecular mechanisms mediated by YAP1 in LAC and gastric adenocarcinoma (GAC) cells. Forty-nine cases of human LAC and normal lung tissue (NLT) were collected. The expression of YAP1 and Axl was assessed by immunohistochemical assay through tissue microarray procedure and the clinicopathologic characteristics of all patients were analyzed. Using a loss of function approach, we investigated the effects of small hairpin RNA (shRNA)-mediated knockdown of YAP1 on the expression of Axl, proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase-9 (MMP-9), and the proliferative activities and invasive potential in LAC A549 and GAC SGC-7901 cell lines. As a result, the expression of YAP1 and Axl was found in LAC tissues with higher strong reactivity rate compared to the NLT (87.8% vs.60.8%, p=0.000;77.6% vs 0.0%, P=0.000), but they did not associate with the age, gender, tumor size, TNM staging or lymph node metastases of LAC patients (each p>0.05). Spearman rank correlation analysis showed a positive correlation between YAP1 and Axl expression. Furthermore, knockdown of YAP in vitro markedly down-regulated the expression of Axl, PCNA and MMP-9, and inhibited the proliferation and invasion of LAC and GAC cells. Taken together, YAP1 and Axl are highly expressed in LAC compared to the NLT, and knockdown of YAP1 may inhibit the proliferation and invasion of adenocarcinoma cells through downregulation of the Axl pathway, representing a potential therapeutic target for the treatment of cancer.

Histone modifications of Notch1 promoter affect lung CD4+ T cell differentiation in asthmatic rats.

Asthma is an inflammatory disease of the airways, and the current treatment in managing asthma is the control of inflammation. Notch signaling pathway has been linked to T-cell imbalance. The present study aimed to explore the histone modifications of Notch1 promoter in normal and asthmatic lung CD4+ T cells. Chromatin immunoprecipitation analysis showed that the acetylation levels of total H3, H4, site-specific H3K9, H3K14, H3K27, H3K18, H4K16, and the trimethylation levels of H3K4, H3K79 of Notchl gene promoter were increased significantly in asthmatic lung CD4+ T cells compared to the control group, which correlated with increased P300, PCAF activity and decreased HDAC1, HDAC2 activity. After intervention of garcinol, a potent inhibitor of histone acetyltransferases, in asthmatic lung CD4+ T cells, HAT activity decreased significantly and the increased Notch1 and hes-1 expression was reversed. The total H3ac, H4ac, site-specific H3K9ac, H3K14ac, H3K27ac, H3K18ac, H4K16ac and H3K79me3 levels of Notch1 gene promoter decreased significantly, and the H3K4me3, H3K9me3, H4K20me3 levels had no significant difference. We further investigated the suppressive effects of GAR on asthmatic parameters. Results showed that the levels of IL-4, 1L-5 and IL-13 were significantly reduced and a small reverse trend was found in the level of IFN-g after GAR treatment. Furthermore, the expression of NF-κB and AP-1 reduced significantly. These results suggest that asthma is associated with changes in the epigenetic status of Notchl promoter, including abnormal histone acetylation and methylation, and GAR may have applications in the treatment of asthma.

Blockade of Notch Signalling by γ-Secretase Inhibitor in Lung T Cells of Asthmatic Mice Affects T Cell Differentiation and Pulmonary Inflammation

Notch is a single-pass transmembrane receptor protein expressed by T cells, which contributes to the pathogenesis of asthma through regulation of the development and differentiation of T cells. γ-Secretase inhibitor (GSI) acts as an effective blocker of Notch signalling. The present study aimed to investigate the role of GSI MW167 in T cell differentiation and antigen-induced airway inflammation. An OVA-induced airway inflammation mouse model was established. Blockade of Notch signalling was achieved using MW167. The expression of IL-4, IL-5, IFN-γ, Notch1 signalling and pro-inflammatory transcription factors in activated lung T cells was evaluated. Finally, the therapeutic effect of MW167 was investigated by haematoxylin and eosin staining, real-time PCR and ELISA. The expression of IL-4 and IL-5 decreased and that of IFN-γ increased significantly, and the protein expression levels of pro-inflammatory transcription factors reduced in active lung T cells after administration of MW167, compared to the control group. MW167 treatment prevented OVA-induced airway inflammation and histological changes. The serum and bronchoalveolar lavage fluid (BALF) levels of IL-4 and IL-5 in MW167-treated mice decreased significantly, whereas those of IFN-γ increased, relative to the levels in OVA-challenged animals treated with PBS. Our findings indicate that Notch signalling plays an important role in the pathogenesis of asthma and that MW167 may be a potential therapeutic target for allergen-induced airway inflammation.

Simvastatin alleviates airway inflammation and remodelling through up‐regulation of autophagy in mouse models of asthma

Statins have been widely used in inflammatory diseases including asthma, because of their anti‐inflammatory and immunomodulatory properties. It has been shown that simvastatin induces autophagy and cell death in some circumstances. However, the possible cross‐talk between simvastatin and autophagic processes in lung disease is largely unknown. Thus, we investigated the impact of simvastatin on airway inflammation and airway remodelling and the possible relationship of these processes to a simvastatin‐induced autophagic pathway in mouse models of asthma.

Troglitazone upregulates PTEN expression and induces the apoptosis of pulmonary artery smooth muscle cells under hypoxic conditions

The increased proliferation and decreased apoptosis of pulmonary artery smooth muscle cells (PASMCs) are the main causes of hypoxic pulmonary hypertension. In this study, we investigated the role of troglitazone [peroxisome proliferator-activated receptor γ (PPARγ) agonist] in the regulation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression and the apoptosis of PASMCs under hypoxic conditions. Normal human PASMCs were cultured in growth medium (GM) and treated with troglitazone (0.5-80 µM) under hypoxic conditions (5% CO2+94% N2+1% O2) for 72 h. The gene expression of PTEN, AKT-1 and AKT-2 was determined by quantitative reverse transcription PCR (qRT-PCR). The protein expression level of PTEN, AKT and phosphorylated AKT (p-AKT) was determined by western blot analysis. The apoptosis of PASMCs was determined by measuring the activities of caspase-3, -8 and -9 and by TUNEL assay. The proliferation rate of the PASMCs was altered in a concentration-dependent manner by troglitazone. A significantly reduced proliferation rate was observed at troglitazone concentrations starting from 20 µM under hypoxic conditions (72 ± 5.8%). Although the gene expression levels of PTEN were increased, the gene expression levels of AKT-1 and AKT-2 remained unaltered. Consistent with this, PTEN protein expression was also altered in a concentration-dependent manner by troglitazone. Although AKT expression was unaltered in all the cell samples, reduced p-AKT expression was observed in the troglitazone-treated PASMCs. Troglitazone increased the activities of caspase-3, -8 and -9 in the PASMCs. bpV(HOpic) (PTEN inhibitor) and GW9662 (PPARγ inhibitor) inhibited PTEN protein expression and recovered the proliferation rate of the PASMCs. TUNEL assay demonstrated that troglitazone significantly increased the apoptosis of PASMCs under hypoxic conditions. In conclusion, troglitazone increases PTEN expression under hypoxic conditions in a concentration-dependent manner. Troglitazone increases the apoptosis of PASMCs under hypoxic conditions. The increase in PTEN expression is mediated through the PPARγ signaling pathway.

Ginsenoside Rg1 Ameliorates Cigarette Smoke-Induced Airway Fibrosis by Suppressing the TGF-β1/Smad Pathway In Vivo and In Vitro

Small airway fibrosis is a key pathological process accompanying chronic obstructive pulmonary disease (COPD) and includes fibroblast/myofibroblast transdifferentiation and excessive extracellular matrix deposition. Ginsenoside Rg1, one of the main active ingredients of Panax ginseng, has been shown to exert an antifibrotic effect in many tissues. However, little is known about the underlying mechanism and whether ginsenoside Rg1 can exert an effect on small airway fibrosis. We investigated the anti-small airway fibrosis effects of ginsenoside Rg1 in human embryonic lung fibroblasts and in COPD rats. We found that ginsenoside Rg1 effectively reduced the degree of pulmonary fibrosis, decreased the expression of α-smooth muscle actin, collagen I, and matrix metalloproteinase 9, and maintained the ratio of matrix metalloproteinase 9 to tissue inhibitor of metalloproteinase 1. Importantly, ginsenoside Rg1 significantly attenuated cigarette smoke extract-induced upregulation of transforming growth factor β1, TGF-β receptor I, phospho-Smad2, and phospho-Smad3. In addition, ginsenoside Rg1 mimicked the effect of SB525334, a TGF-β receptor I-Smad2/3 inhibitor. Collectively, these results suggest that ginsenoside Rg1 may suppress cigarette smoke-induced airway fibrosis in pulmonary fibroblasts and COPD rats by inhibiting the TGF-β1/Smad signaling pathway.

Differences in airway remodeling and airway inflammation among moderate-severe asthma clinical phenotypes

Background To identify asthma clinical phenotypes using cluster analysis and improve our understanding of heterogeneity in asthma. Methods Clustering approaches were applied to 203 patients who were diagnosed with asthma in XinHua Hospital (January 2012 to December 2015). One hundred and twenty patients underwent multi-slice spiral computed tomography (MSCT) examination and 30 underwent bronchial mucosal biopsy for evaluation of airway remodeling and airway inflammation among the phenotypes. Results Four groups were identified. Patients in cluster 1 (n=52) had early onset atopic asthma and patients in cluster 2 (n=65) had small airway obstruction and atopic asthma. Cluster 3 (n=52) was a unique group of patients with late-onset and non-atopic asthma. Patients in cluster 4 (n=34) had severe airflow obstruction and obvious airway remodeling as observed on MSCT (P<0.05). According to the immunohistochemistry of IL-5 and IL-17 (P<0.05), the results of clusters 1 and 2 may be attributable to the Th2 immune response, whereas those of clusters 3 and 4 to the Th17 immune response. Conclusions Four distinct clinical phenotypes of asthma were identified by cluster analysis. The results of the MSCT and pathological examinations may suggest specific pathogeneses among the phenotypes.