Shinsaku Togo

Lung Fibroblast Repair Functions in Patients with Chronic Obstructive Pulmonary Disease Are Altered by Multiple Mechanisms

RATIONALE Fibroblasts are believed to be the major cells responsible for the production and maintenance of extracellular matrix. Alterations in fibroblast functional capacity, therefore, could play a role in the pathogenesis of pulmonary emphysema, which is characterized by inadequate maintenance of tissue structure. OBJECTIVES To evaluate the hypothesis that deficient fibroblast repair characterizes cells obtained from individuals with chronic obstructive pulmonary disease (COPD) compared with control subjects. METHODS Fibroblasts were cultured from lung tissue obtained from individuals undergoing thoracotomy and were characterized in vitro. MEASUREMENTS AND MAIN RESULTS Fibroblasts from individuals with COPD, defined by reduced FEV(1), manifested reduced chemotaxis toward fibronectin and reduced contraction of three-dimensional collagen gels, two bioassays associated with fibroblast repair function. At least two mechanisms appear to account for these differences. Prostaglandin E (PGE), a known inhibitor of fibroblast repair functions, was produced in increased amount by fibroblasts from subjects with COPD, which also expressed increased amounts of the receptors EP2 and EP4, both of which signal through cyclic AMP. Incubation of fibroblasts with indomethacin or with the PKA inhibitor KT-5720 partially restored COPD subject fibroblast function. In addition, fibroblasts from subjects with COPD produced more transforming growth factor (TGF)-beta1, but manifested reduced response to TGF-beta1. The functional alterations in fibroblasts correlated with both lung function assessed by FEV(1) and, for the data available, with severity of emphysema assessed by Dl(CO). CONCLUSIONS Fibroblasts from individuals with COPD have reduced capability to sustain tissue repair, which suggests that this may be one mechanism that contributes to the development of emphysema.

Carcinoma-Associated Fibroblasts Are a Promising Therapeutic Target

Human carcinomas frequently exhibit significant stromal reactions such as the so-called “desmoplastic stroma” or “reactive stroma”, which is characterised by the existence of large numbers of stromal cells and extracellular matrix proteins. Carcinoma-associated fibroblasts (CAFs), which are rich in activated fibroblast populations exemplified by myofibroblasts, are among the predominant cell types present within the tumour-associated stroma. Increased numbers of stromal myofibroblasts are often associated with high-grade malignancies with poor prognoses in humans. CAF myofibroblasts possess abilities to promote primary tumour development, growth and progression by stimulating the processes of neoangiogenesis as well as tumour cell proliferation, survival, migration and invasion. Moreover, it has been demonstrated that CAFs serve as a niche supporting the metastatic colonisation of disseminated carcinoma cells in distant organs. Their contribution to primary and secondary malignancies makes these fibroblasts a potential therapeutic target and they also appear to be relevant to the development of drug resistance and tumour recurrence. This review summarises our current knowledge of tumour-promoting CAFs and discusses the therapeutic feasibility of targeting these cells as well as disrupting heterotypic interactions with other cell types in tumours that may improve the efficacy of current anti-tumour therapies.

NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract

BackgroundWe have previously reported that low concentrations of cigarette smoke extract induce DNA damage without leading to apoptosis or necrosis in human bronchial epithelial cells (HBECs), and that IL-6/STAT3 signaling contributes to the cell survival. Since NF-κB is also involved in regulating apoptosis and cell survival, the current study was designed to investigate the role of NF-κB in mediating cell survival in response to cigarette smoke exposure in HBECs.MethodsBoth the pharmacologic inhibitor of NF-κB, curcumin, and RNA interference targeting p65 were used to block NF-κB signaling in HBECs. Apoptosis and cell survival were then assessed by various methods including COMET assay, LIVE/DEAD Cytotoxicity/Viability assay and colony formation assay.ResultsCigarette smoke extract (CSE) caused DNA damage and cell cycle arrest in S phase without leading to apoptosis in HBECs as evidenced by TUNEL assay, COMET assay and DNA content assay. CSE stimulated NF-κB -DNA binding activity and up-regulated Bcl-XL protein in HBECs. Inhibition of NF-κB by the pharmacologic inhibitor curcumin (20 μM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure. Furthermore, cells lacking p65 were incapable of forming cellular colonies when these cells were exposed to CSE, while they behaved normally in the regular culture medium.ConclusionThe current study demonstrates that CSE activates NF-κB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65. These results suggest that activation of NF-κB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.

PDE4 inhibitors roflumilast and rolipram augment PGE2 inhibition of TGF-β1-stimulated fibroblasts

Fibrotic diseases are characterized by the accumulation of extracellular matrix together with distortion and disruption of tissue architecture. Phosphodiesterase (PDE)4 inhibitors, by preventing the breakdown of cAMP, can inhibit fibroblast functions and may be able to mitigate tissue remodeling. Transforming growth factor (TGF)-beta1, a mediator of fibrosis, can potentially modulate cAMP by altering PGE(2) metabolism. The present study assessed whether PDE4 inhibitors functionally antagonize the profibrotic activity of fibroblasts stimulated by TGF-beta1. The PDE4 inhibitors roflumilast and rolipram both inhibited fibroblast-mediated contraction of three-dimensional collagen gels and fibroblast chemotaxis toward fibronectin in the widely studied human fetal lung fibroblast strain HFL-1 and several strains of fibroblasts from adult human lung. Roflumilast was approximately 10-fold more potent than rolipram. There was a trend for PDE4 inhibitors to inhibit more in the presence of TGF-beta1 (0.05 < P < 0.08). The effect of the PDE4 inhibitors was mediated through cAMP-stimulated protein kinase A (PKA), although a PKA-independent effect on gel contraction was also observed. The effect of PDE4 inhibitors depended on fibroblast production of PGE(2) and TGF-beta1-induced PGE(2) production. PDE4 inhibitors together with TGF-beta1 resulted in augmented PGE(2) production together with increased expression of COX mRNA and protein. The present study supports the concept that PDE4 inhibitors may attenuate fibroblast activities that can lead to fibrosis and that PDE4 inhibitors may be particularly effective in the presence of TGF-beta1-induced fibroblast stimulation.

Prostacyclin analogs stimulate VEGF production from human lung fibroblasts in culture

Prostacyclin is a short-lived metabolite of arachidonic acid that is produced by several cells in the lung and prominently by endothelial cells. It increases intracellular cAMP levels activating downstream signaling thus regulating vascular mesenchymal cell functions. The alveolar wall contains a rich capillary network as well as a population of mesenchymal cells, i.e., fibroblasts. The current study evaluated the hypothesis that prostacyclin may mediate signaling between endothelial and mesenchymal cells in the alveolar wall by assessing the ability of prostacyclin analogs to modulate fibroblast release of VEGF. To accomplish this study, human lung fibroblasts were cultured in routine culture on plastic support and in three-dimensional collagen gels with or without three prostacyclin analogs, carbaprostacyclin, iloprost, and beraprost, and the production of VEGF was evaluated by ELISA and quantitative real-time PCR. Iloprost and beraprost significantly stimulated VEGF mRNA levels and protein release in a concentration-dependent manner. These effects were blocked by the adenylate cyclase inhibitor SQ-22536 and by the protein kinase A (PKA) inhibitor KT-5720 and were reproduced by a direct PKA activator but not by an activator of exchange protein directly activated by cAMP (Epac), indicating that cAMP-activated PKA signaling mediated the effect. Since VEGF serves to maintain the pulmonary microvasculature, the current study suggests that prostacyclin is part of a bidirectional signaling network between the mesenchymal and vascular cells of the alveolar wall. Prostacyclin analogs, therefore, have the potential to modulate the maintenance of the pulmonary microcirculation by driving the production of VEGF from lung fibroblasts.

Statins inhibit matrix metalloproteinase release from human lung fibroblasts

Pleiotropic effects of statins have been reported to include inhibition of matrix metalloproteinase (MMP) release from macrophages and endothelial cells. We evaluated whether statins would inhibit MMP release from human lung fibroblasts, which play a major role in remodelling processes. Monolayer and three-dimensional (3D) collagen gel cultures of fibroblasts were used. Cytokines (tumour necrosis factor-α and interleukin-1α) were used to induce MMP release and mRNA expression. Collagen degradation induced by cytokines and neutrophil elastase (NE) was evaluated by quantifying hydroxyproline. Atorvastatin inhibited MMP-1 and -3 release and mRNA expression in both culture systems. Similar results were obtained with simvastatin and fluvastatin. In 3D cultures where cytokines also stimulated MMP-9 release, atorvastatin also inhibited MMP-9 release. In 3D cultures, cytokines together with NE induced collagen degradation, which was also inhibited by atorvastatin. The effect of atorvastatin was reversed by mevalonate and geranylgeranyl-pyrophosphate but not by farnesyl-pyrophosphate. The current data suggest that statins may modulate remodelling processes mediated by fibroblasts by inhibiting MMP release.

N-acetyl-l-cysteine inhibits TGF-β1-induced profibrotic responses in fibroblasts

BACKGROUND Excessive production of TGF-beta(1) plays a key role in the tissue remodeling or fibrotic process observed in bronchial asthma, chronic pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF). TGF-beta(1) has been reported to decrease the intracellular glutathione level and stimulate the production of reactive oxygen species. OBJECTIVES The aim of this study was to evaluate whether the antioxidant N-acetyl-l-cysteine (NAC) can affect TGF-beta(1)-mediated tissue remodeling in fibroblasts or modulate the production of fibronectin and vascular endothelial growth factor (VEGF) which are believed to be important mediators of tissue repair and remodeling. METHODS To accomplish this, human fetal lung fibroblasts (HFL-1) were used to assess the effect of NAC on the TGF-beta(1)-mediated contraction of floating gels and the TGF-beta(1)-induced mediator production. In addition, the effect of NAC on the TGF-beta(1)-induced differentiation to myofibroblasts was evaluated by assessing alpha-smooth muscle actin (alpha-SMA) expression. RESULTS NAC significantly abolished the TGF-beta(1)-augmented gel contraction (at 3mM, gel size 63.4+/-2.6% vs. 39.1+/-4.1%; p<0.01) compared with control in a concentration-dependent manner. NAC also significantly inhibited the TGF-beta(1)-augmented fibronectin (p<0.01) and VEGF (p<0.01) production in the media of both the three-dimensional gel and monolayer culture. Furthermore, NAC reversed the TGF-beta(1)-stimulated alpha-SMA expression (p<0.01). CONCLUSION These results suggest that NAC can affect the TGF-beta(1)-induced tissue remodeling or fibrotic process in vitro.

Prostaglandin E2 protects human lung fibroblasts from cigarette smoke extract-induced apoptosis via EP2 receptor activation

Prostaglandin E2 (PGE2) has been shown to have a strong cytoprotective effect, inhibiting apoptosis. In the present study, we evaluated whether PGE2 has a protective effect on cigarette smoke extract (CSE)‐induced apoptosis in human lung fibroblasts. Apoptosis was assessed by various methods, including DNA content analysis. CSE (15%–20%) led to apoptosis and induced imbalance in favor of pro‐ over anti‐apoptotic protein expression and activated caspases. PGE2 blocked CSE‐induced apoptosis and modulated the balance of pro‐ and anti‐apoptotic proteins and decreased the activation of caspases. This anti‐apoptotic effect was mediated via EP2 receptor activation as the EP2 agonist butaprost mimicked PGE2 activity and siRNA for the EP2 receptor blocked it. An adenylyl cyclase inhibitor was found to abolish the PGE2‐mediated cytoprotective effect. Correspondingly, c‐AMP analogs blocked CSE‐induced apoptosis. Consistently, the protein kinase A (PKA) inhibitor KT‐5720 abolished PGE2‐mediated protection. PGE2 and butaprost phosphorylated Bad and KT‐5720 blocked phosphorylation. These results suggest that PGE2 inhibits CSE‐induced apoptosis via EP2 receptor activation and activation of PKA, which leads to an alteration in the balance between pro‐ and anti‐apoptotic factors. Through such a mechanism, PGE2 may alter survival of cells in the smoke‐exposed lungs, thus affecting the pathogenesis of cigarette smoke‐induced disease. J. Cell. Physiol. 210: 99–110, 2007.

Prostaglandin E2 Stimulates the Production of Vascular Endothelial Growth Factor through the E-Prostanoid–2 Receptor in Cultured Human Lung Fibroblasts

Fibroblasts are the major mesenchymal cells present within the interstitium of the lung and are a major source of vascular endothelial growth factor (VEGF), which modulates the maintenance of pulmonary microvasculature. Prostaglandin E(2) (PGE(2)) acts on a set of E-prostanoid (EP) receptors that activate multiple signal transduction pathways leading to downstream responses. We investigated the modulation by PGE(2) of VEGF release by human lung fibroblasts. Human lung fibroblasts were cultured until reaching 90% confluence in tissue culture plates, after which the culture media were changed to serum-free Dulbeccos modified Eagles medium, with or without PGE(2), and with specific agonists or antagonists for each EP receptor. After 2 days, culture media were assayed for VEGF by ELISA. The results demonstrated that PGE(2) and the EP2 agonist ONO-AE1-259-01 significantly stimulated the release of VEGF in a concentration-dependent manner. Agonists for other EP receptors did not stimulate the release of VEGF. The stimulatory effect of PGE(2) was blocked by the EP2 antagonist AH6809, but was not blocked by antagonists for other EP receptors. The protein kinase-A (PKA) inhibitor KT-5720 also blocked the stimulatory effect of PGE(2). The increased release of VEGF induced by PGE(2) was accompanied by a transient increase in the concentration of VEGF mRNA. These findings demonstrate that PGE(2) can modulate the release of VEGF by human lung fibroblasts through its actions in the EP2 receptor/PKA pathway. This activity may contribute to the maintenance of pulmonary microvasculature in the alveolar wall.

Oncostatin M Modulates Fibroblast Function via Signal Transducers and Activators of Transcription Proteins–3

Oncostatin M (OSM), an inflammatory cytokine of the interleukin-6 (IL-6) superfamily, plays a key role in various biological processes such as modulation of extracellular matrix (ECM), cell proliferation, cell survival, and induction of inflammation. It has been reported that OSM was increased in asthma and pulmonary fibrosis, and thus OSM may play a role in airway remodeling and the development of lung parenchymal fibrosis. Recruitment of lung fibroblasts to the sites of airway injury and subsequent differentiation into myofibroblasts is believed to contribute to excess ECM deposition. In the current study, we assessed the ability of OSM to modulate fibroblast collagen gel contraction, migration toward fibronectin, and expression of α-smooth muscle actin (α-SMA). We demonstrated that OSM augments gel contraction, chemotaxis, and α-SMA expression. OSM-augmented fibroblast chemotaxis was mediated by the signal transducer and activator of transcription (STAT3) and p38 mitogen-activated protein kinase, while augmentation on gel contraction and α-SMA expression was mediated by STAT3. Neither transforming growth factor-β1 nor PGE2 was involved in mediating OSM effect on the cells. The Th2 cytokines IL-4 and IL-13, which also are believed to play an important role in promoting lung fibrosis and airway remodeling, act through STAT3, and we demonstrated the potential for additive effects of OSM with IL-4 and IL-13. The present study supports the concept that OSM may contribute to tissue remodeling, which may be additive with IL-4 or IL-13. Blockade of OSM or OSM-mediated STAT3 signaling could be a therapeutic target to regulate lung fibrotic mechanisms.