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Abstract: Fibroblasts play an important role in the repair and remodeling processes following injury. Leukotriene D4 (LTD4) is a potent mediator in inflammatory processes, but the direct effect of cysteinyl leukotrienes on fibroblast migration remains unelucidated. In this study, the effect of the LTD4 on normal human lung fibroblasts (NHLF) chemotaxis induced by human plasma fibronectin (HFn) was investigated using the modified Boyden''s chamber technique. LTD4 potentiated NHLF chemotaxis to HFn in concentration-dependent manner. A specific cysteinyl leukotriene receptor type 1 antagonist, pranlukast inhibited this effect, indicating that LTD4 affected cell migration via its specific receptor. The potentiating effect of LTD4 on fibroblast chemotaxis was completely abolished by pertussis toxin (PTX), suggesting that LTD4-induced effect was dependent on PTX-sensitive Gi/o signaling. These findings suggest that LTD4 has a potential to augment fibroblast chemotaxis, and to contribute to regulation of the wound healing and following remodeling in fibrotic processes of the lung. [Copyright &y& Elsevier]

Fibroblasts, as a major source of extracellular interstitial connective tissue matrix, play an important role in wound healing and the development of fibrosis. The phosphodiesterase (PDE) 4 inhibitor cilomilast inhibits fibroblast chemotaxis and fibroblast-mediated gel contraction. Using the Boyden blindwell chamber chemotaxis assay and the type I collagen gel contraction model, this study investigated whether specific cytokines modulate cilomilast''s inhibitory effect through regulation of endogenous PGE2 production. Human recombinant IL-1β stimulated PGE2 production and shifted the cilomilast concentration–dependence curve to the left in both assay systems, indicating increased sensitivity to cilomilast. In contrast, human recombinant IL-4 inhibited PGE2 production and shifted the cilomilast concentration–dependence curve to the right in both systems. In summary, the inhibitory effect of cilomilast on fibroblast migration and collagen gel contraction is modulated by IL-1β and IL-4 through regulation of PGE2 production. [Copyright &y& Elsevier]

Abstract: Fibrosis is an abnormal response to organ injury, characterized by accumulation of activated fibroblasts at the sites of injury. Fibroblasts arise from several sources, including resident fibroblasts and circulating fibrocytes that infiltrate organ tissue. Recently, epithelial–mesenchymal transition (EMT) has been recognized as a source of mesenchymal cells. EMT is induced by various growth factors, such as transforming growth factor (TGF)-β1, and enhanced by inflammatory cytokines. Recently the tumor necrosis factor superfamily member LIGHT has been implicated in the pathogenesis of inflammatory disease and airway remodeling in severe asthma. We hypothesized that LIGHT might contribute to the pathogenesis of airway fibrosis via enhancement of EMT. Therefore, we investigated LIGHT’s ability to induce EMT. A549 cells were stimulated with LIGHT, TGF-β1 or both for 48h. To estimate EMT, we evaluated the expression of epithelial and mesenchymal markers using immunocytochemistry, Western blotting and quantitative RT-PCR. Signaling pathways for EMT were characterized by Western analysis to detect phosphorylation of Erk1/2 and smad2. LIGHT enhanced TGF-β1-induced EMT both morphologically, by suppressing E-cadherin and enhancing vimentin, and functionally, by enhancing cell contractility. Additionally, LIGHT induced EMT without TGF-β1. Evaluation of the mechanism showed that LIGHT did not induce TGF-β1 production or affect the smad-snai1 pathway. Inhibition of Erk1/2 phosphorylation reduced LIGHT-induced EMT, indicating the Erk1/2 pathway to be a key pathway in LIGHT-induced EMT. In summary, LIGHT enhanced TGF-β1-induced EMT but also induced EMT via the Erk1/2 pathway by itself, without TGF-β1 signaling. LIGHT may contribute to the pathogenesis of airway fibrosis through enhancement of EMT. [Copyright &y& Elsevier]

Abstract: Lung cancer is the most common cause of cancer-related death worldwide. Stromal cancer-associated fibroblasts (CAFs) play crucial roles in carcinogenesis, proliferation, invasion, and metastasis of non-small cell lung carcinoma, and targeting of CAFs could be a novel strategy for cancer treatment. However, the characteristics of human CAFs still remain to be better defined. In this study, we established patient-matched CAFs and normal fibroblasts (NFs), from tumoral and non-tumoral portions of resected lung tissue from lung cancer patients. CAFs showed higher α-smooth muscle actin (α-SMA) expression than NFs, and CAFs clearly enhanced collagen gel contraction. Furthermore, we employed three-dimensional co-culture assay with A549 lung cancer cells, where CAFs were more potent in inducing collagen gel contraction. Hematoxylin and eosin staining of co-cultured collagen gel revealed that CAFs had the potential to increase invasion of A549 cells compared to NFs. These observations provide evidence that lung CAFs have the tumor-promoting capacity distinct from NFs. [Copyright &y& Elsevier]

Abstract: We have recently reported that disruption of nuclear erythroid 2 P45-related factor 2 (Nrf2) enhances susceptibility to airway inflammatory responses induced by low-dose diesel exhaust particles (DEP) in mice. C57BL/6 Nrf2 knockout (Nrf2−/−) mice and wild-type (Nrf2+/+) mice were further exposed to low-dose DEP for 7h/day, 5days/week, for a maximum of 8weeks. After exposure to DEP for 5weeks, allergic airway inflammation was generated in the mice by intraperitoneal sensitization with OVA followed by intranasal challenge. Nrf2−/− mice exposed to relatively low-dose DEP showed significantly increased percentage changes relative to the OVA alone group in terms of airway hyperresponsiveness (AHR) and inflammatory cells, levels of IL-5 and thymus and activation regulated chemokine (TARC) in bronchoalveolar lavage (BAL) fluid than did Nrf2+/+ mice. Lung tissues of Nrf2−/− mice after DEP exposure showed inflammatory cell infiltrates, and increased PAS staining-positive mucus cell hyperplasia. In contrast, the percentage changes relative to the OVA group in the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in whole blood was higher in Nrf2+/+ mice than in Nrf2−/− mice. By using Nrf2−/− mice, it was shown for the first time that relatively low-dose DEP exposure induces oxidant stress, and that host anti-oxidant responses play a key role in the development of DEP-induced exacerbation of allergic airway inflammation. [ABSTRACT FROM AUTHOR]

Abstract: To test our hypothesis that diesel exhaust particle (DEP)-induced oxidative stress and host antioxidant responses play a key role in the development of DEP-induced airway inflammatory diseases, C57BL/6 nuclear erythroid 2 P45-related factor 2 (Nrf2) knockout (Nrf2−/−) and wild-type mice were exposed to low-dose DEP for 7 h/day, 5 days/week, for 8 weeks. Nrf2−/− mice exposed to low-dose DEP showed significantly increased airway hyperresponsiveness and counts of lymphocytes and eosinophils, together with increased concentrations of IL-12 and IL-13, and thymus and activation-regulated chemokine (TARC), in BAL fluid than wild-type mice. In contrast, expression of antioxidant enzyme genes was significantly higher in wild-type mice than in Nrf2−/− mice. We have first demonstrated that disruption of Nrf2 enhances susceptibility to airway inflammatory responses induced by inhalation of low-dose DEP in mice. These results strongly suggest that DEP-induced oxidative stress and host antioxidant responses play some role in the development of DEP-induced airway inflammation. [Copyright &y& Elsevier]

Abstract: Transforming growth factor-β1 (TGF-β1) is a key mediator in tissue repair and fibrosis. Using small interference RNA (siRNA), the role of Smad2 and Smad3 in TGF-β stimulation of human lung fibroblast contraction of collagenous matrix and induction of α-SMA and the role of α-SMA in contraction were assessed. HFL-1 cells were transfected with Smad2, Smad3 or control-siRNA, and cultured in floating Type I collagen gels ±−TGF-β1. TGF-β1 augmented gel contraction in Smad2-siRNA- and control-siRNA-treated cells, but had no effect in Smad3-siRNA-treated cells. Similarly, TGF-β1 upregulated α-SMA in Smad2-siRNA- and control-siRNA-treated cells, but had no effect on Smad3-siRNA-treated cells. α-SMA-siRNA-treated cells did not contact the collagen gels with or without TGF-β1, suggesting α-SMA is required for gel contraction. Thus, Smad3 mediates TGF-β1-induced contraction and α-SMA induction in human lung fibroblasts. Smad3, therefore, could be a target for blocking contraction of human fibrotic tissue induced by TGF-β1. [Copyright &y& Elsevier]

Abstract: Transforming growth factor-β1 (TGF-β1) is a key factor in a variety of physiological and pathological processes. Vascular endothelial growth factor (VEGF) is a key angiogenic factor, and vascular change is one of the features of airway remodeling. We examined the effect of TGF-β1 on VEGF production by fibroblasts from mice lacking expression of Smad2 or Smad3 as well as human lung fibroblasts treated with or without Smad2 or Smad3 siRNA. TGF-β1 stimulated VEGF production by fibroblasts from Smad2 deficient animals and wildtype animals. In contrast, TGF-β1 did not affect VEGF production by fibroblasts from Samd3 deficient mice. Similarly, TGF-β1 failed to stimulate VEGF production by HFL-1 cells treated with Samd3 siRNA but significantly increased VEGF production by the cells treated with Smad2 siRNA. These result suggest that TGF-β1 stimulation of VEGF production by fibroblasts is regulated by Smad3 but not by Smad2 signaling. [Copyright &y& Elsevier]