Jie-ming Qu

Pro-inflammatory Phenotype of COPD Fibroblasts not Compatible with Repair in COPD Lung

Chronic obstructive pulmonary disease (COPD) is characterized by loss of elastic fibres from small airways and alveolar walls, with the decrease in elastin increasing with disease severity. It is unclear why there is a lack of repair of elastic fibres. We have examined fibroblasts cultured from lung tissue from subjects with or without COPD to determine if the secretory profile explains lack of tissue repair. In this study, fibroblasts were cultured from lung parenchyma of patients with mild COPD [Global initiative for chronic Obstructive Lung Disease (GOLD) 1, n= 5], moderate to severe COPD (GOLD 2–3, n= 12) and controls (non‐COPD, n= 5). Measurements were made of proliferation, senescence‐associated β‐galactosidase‐1, mRNA expression of IL‐6, IL‐8, MMP‐1, tropoelastin and versican, and protein levels for IL‐6, IL‐8, PGE2, tropoelastin, insoluble elastin, and versican. GOLD 2–3 fibroblasts proliferated more slowly (P < 0.01), had higher levels of senescence‐associated β‐galactosidase‐1 (P < 0.001) than controls and showed significant increases in mRNA and/or protein for IL‐6 (P < 0.05), IL‐8 (P < 0.01), MMP‐1 (P < 0.05), PGE2 (P < 0.05), versican (P < 0.05) and tropoelastin (P < 0.05). mRNA expression and/or protein levels of tropoelastin (P < 0.01), versican (P < 0.05), IL‐6 (P < 0.05) and IL‐8 (P < 0.05) were negatively correlated with FEV1% of predicted. Insoluble elastin was not increased. In summary, fibroblasts from moderate to severe COPD subjects display a secretory phenotype with up‐regulation of inflammatory molecules including the matrix proteoglycan versican, and increased soluble, but not insoluble, elastin. Versican inhibits assembly of tropoelastin into insoluble elastin and we conclude that the pro‐inflammatory phenotype of COPD fibroblasts is not compatible with repair of elastic fibres.

Intrapleural delivery of MSCs attenuates acute lung injury by paracrine/endocrine mechanism

Two different repair mechanisms of mesenchymal stem cells (MSCs) are suggested to participate in the repair of acute lung injury (ALI): (i) Cell engraftment mechanism, (ii) Paracrine/endocrine mechanism. However, the exact roles they play in the repair remain unclear. The aim of the study was to evaluate the role of paracrine/endocrine mechanism using a novel intrapleural delivery method of MSCs. Either 1 × 106 MSCs in 300 μl of PBS or 300 μl PBS alone were intrapleurally injected into rats with endotoxin‐induced ALI. On days 1, 3 or 7 after injections, samples of lung tissues and bronchoalveolar lavage fluid (BALF) were collected from each rat for assessment of lung injury, biochemical analysis and histology. The distribution of MSCs was also traced by labelling the cells with 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI). MSCs intrapleural injection significantly improved LPS‐induced lung histopathology compared with PBS‐treated group at day 3. There was also a significant decrease in total cell counts and protein concentration in BALF at day 7 in the MSCs ‐treated rats compared to PBS control group. Tracking the DAPI‐marked MSCs showed that there were no exotic MSCs in the lung parenchyma. MSCs administration resulted in a down‐regulation of pro‐inflammatory response to endotoxin by reducing TNF‐α both in the BALF and in the lung, while up‐regulating the anti‐inflammatory cytokine IL‐10 in the lung. In conclusion, treatment with intrapleural MSCs administration markedly attenuates the severity of endotoxin‐induced ALI. This role is mediated by paracrine/endocrine repair mechanism of MSCs rather than by the cell engraftment mechanism.

Inhibited proliferation of human lung fibroblasts by LPS is through IL-6 and IL-8 release.

Through the consideration of decreased proliferation of lung fibroblasts from subjects with chronic obstructive pulmonary disease (COPD) and the proinflammatory role of lipopolysaccharide (LPS) in the COPD development, we hypothesized that LPS might inhibit proliferation in lung fibroblasts and the possible mechanism was investigated. Primary human lung fibroblasts were cultured from peripheral lung tissue and then treated with or without LPS. Proliferation was measured by AlamarBlue® assay. Levels of TNF-α, IL-6, IL-8, IL-12p70, IL-1β and IL-10 in the supernatants were measured by ELISA. The mRNA of histone deacetylases 2 (HDAC2) was analyzed using real-time PCR. LPS appeared to have a dose-dependent inhibitory effect on fibroblasts proliferation. The concentrations of IL-6 and IL-8 in the treatment culture media were significantly increased, accompanied by a reduced mRNA expression of HDAC2. IL-6 or IL-8 itself led to the reduction of fibroblasts proliferation. Treatment with 1 ng/ml TNF-α in fibroblasts also caused a significant decrease in proliferation and an increase in the production of IL-8 and IL-6. Our data suggest that LPS can inhibit the proliferation of in vitro human lung fibroblasts at least through a production of IL-6 and IL-8. The cytokine response is related to the decreased HDAC2 transcription.

A therapeutic role for mesenchymal stem cells in acute lung injury independent of hypoxia-induced mitogenic factor

Bone marrow mesenchymal stem cells (BM‐MSCs) have therapeutic potential in acute lung injury (ALI). Hypoxia‐induced mitogenic factor (HIMF) is a lung‐specific growth factor that participates in a variety of lung diseases. In this study, we evaluated the therapeutic role of BM‐MSC transplantation in lipopolysaccharide (LPS)‐ induced ALI and assessed the importance of HIMF in MSC transplantation. MSCs were isolated and identified, and untransduced MSCs, MSCs transduced with null vector or MSCs transduced with a vector encoding HIMF were transplanted into mice with LPS‐induced ALI. Histopathological changes, cytokine expression and indices of lung inflammation and lung injury were assessed in the various experimental groups. Lentiviral transduction did not influence the biological features of MSCs. In addition, transplantation of BM‐MSCs alone had significant therapeutic effects on LPS‐induced ALI, although BM‐MSCs expressing HIMF failed to improve the histopathological changes observed with lung injury. Unexpectedly, tumour necrosis factor α levels in lung tissues, lung oedema and leucocyte infiltration into lungs were even higher after the transplantation of MSCs expressing HIMF, followed by a significant increase in lung hydroxyproline content and α‐smooth muscle actin expression on day 14, as compared to treatment with untransduced MSCs. BM‐MSC transplantation improved LPS‐induced lung injury independent of HIMF.

High temperature requirement A3 (HTRA3) expression predicts postoperative recurrence and survival in patients with non- small-cell lung cancer

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, and its recurrence rate after complete resection is high, owing to local or distant metastases. Low expression of high temperature requirement A3 (HTRA3) has been reported to promote tumorigenesis, diminish the effects of anti-tumor treatments, and correlate with a malignant phenotype. To assess the involvement of HTRA3 in the prognosis of postoperative NSCLC, we obtained tumors from 78 patients who had undergone complete surgical resection, and immunohistochemically examined them for HTRA3 expression. HTRA3 was significantly down-regulated in lung cancer tissues compared with normal lung tissues, and only six tumor cases(7.7%) exhibited relatively high levels of HTRA3 (P < 0.001). Notably, high-HTRA3 patients were at significantly lower risk of postoperative recurrence than low-HTRA3 or HTRA3-negative patients (0% versus 31.2% and 35.0%; P = 0.044, 0.029, respectively). High expression of HTRA3 also independently indicated longer disease-free survival in Cox regression analysis (hazard ratio 0.39, 95%CI 0.16-0.95, P = 0.038). Ectopic expression of the long isoform of HTRA3 attenuated the invasion of an NSCLC cell line in a Transwell assay, while knockdown of HTRA3 had the converse effect. Thus, HTRA3 suppresses tumor cell invasiveness and may serve as a prognostic biomarker for postoperative recurrence or survival in NSCLC.

Knockdown of versican V1 induces a severe inflammatory response in LPS-induced acute lung injury via the TLR2-NF-κB signaling pathway in C57BL/6J mice

The versican family is important in the modulation of inflammation, however, the role of versican V1 (V1) in lipo-polysaccharide (LPS)-induced acute lung injury (ALI) and the underlying mechanisms remain to be elucidated. To investigate this, the present study performed experiments in male C57BL/6J mice, which were randomly divided into a normal control group (control; n=6), an LPS-stimulated ALI group (LPS; n=6), a scramble small interfering (si)RNA group (scramble; n=6), a V1-siRNA group (V1-siRNA; n=6), a scramble siRNA and LPS-stimulated group (scramble+LPS; n=6) and a V1-siRNA and LPS-stimulated group (V1-siRNA+LPS; n=6). On day 1, the mice were anesthetized, and 5 nmol scramble siRNA or V1-siRNA were administered intratracheally. On day 3, LPS (1 mg/kg) or phosphate-buffered saline (50 µl per mouse) were injected intratracheally. All the mice were anesthetized and sacrificed on day 4, and samples were collected and analyzed. The mRNA and protein expression levels were examined using reverse transcription-quantitative polymerase chain reaction analysis, immunohistochemical staining and western blot analysis. ALI was evaluated based on lung injury scores, cell counts and total protein concentrations in the bronchoalveolar lavage fluid (BALF). Inflammatory mediators were detected using an enzyme-linked immunosorbend assay. V1 was increased by LPS in the mouse ALI model, whereas specific V1 knockdown induced higher lung injury scores, and higher total cell counts and protein concentrations in the BALF. Tumor necrosis factor-α (TNF)-α was upregulated, and interleukin-6 exhibited an increasing trend. The expression of toll-like receptor 2 (TLR2), but not TLR4, increased, and the nuclear factor (NF)-κB pathway subunit, P65, was phosphorylated. Taken together, the expression of V1 was upregulated by LPS, and V1 inhibition resulted in the aggravation of LPS-induced ALI via the activation of TLR2-NF-κB and release of TNF-α.

Pulmonary fibroblasts from COPD patients show an impaired response of elastin synthesis to TGF-β1

Insufficiency of tissue repair by pulmonary fibroblasts may contribute to the decrease in elastic fibres in chronic obstructive pulmonary disease (COPD). In this study, the repair function of COPD fibroblasts was assessed by examining the response to transforming growth factor (TGF)-β1. Primary pulmonary fibroblasts were cultured from lung tissue of COPD patients and smoking control subjects. Cellular proliferation was measured with Alamar Blue reduction method. Levels of tropoelastin mRNA and soluble elastin was measured using real-time RT-PCR and Fastin elastin assay respectively. The percentage of increase in proliferation and elastin production after TGF-β1 (1 ng/ml) treatment was calculated for fibroblasts from each subject. COPD fibroblasts showed slower proliferation than control fibroblasts, and a reduced response to TGF-β1 stimulation. The promotive effect of TGF-β1 on elastin synthesis in control fibroblasts was significantly diminished in fibroblasts from COPD patients. Our findings indicate that COPD lung fibroblasts have a significantly decreased response to TGF-β1 in terms of proliferation and elastin production.

Knockdown of versican 1 blocks cigarette-induced loss of insoluble elastin in human lung fibroblasts

COPD lung is characterized by loss of alveolar elastic fibers and an increase in the chondroitin sulfate (CS) matrix proteoglycan versican V1 (V1). V1 is a known inhibitor of elastic fiber deposition and this study investigates the effects of knockdown of V1, and add-back of CS, on CCL-210 lung fibroblasts treated with cigarette smoke extract (CSE) as a model for COPD. CSE inhibited fibroblast proliferation, viability, tropoelastin synthesis, and elastin deposition, and increased V1 synthesis and secretion. V1 siRNA decreased V1 and constituent CS, did not affect tropoelastin production, but blocked the CSE-induced loss in insoluble elastin. Exogenous CS reduced insoluble elastin, even in the presence of V1 siRNA. These findings confirm that V1 and CS impair the assembly of tropoelastin monomers into insoluble fibers, and further demonstrate that specific knockdown of V1 alleviates the impaired assembly of elastin seen in cultures of pulmonary fibroblasts exposed to CSE, indicating a regulatory role for this protein in the pathophysiology of COPD.