Jae Min Shin

Trichostatin A inhibits epithelial mesenchymal transition induced by TGF-β1 in airway epithelium

Background and Objectives Tissue remodeling is believed to cause recalcitrant chronic rhinosinusitis (CRS). Epithelial-mesenchymal transition (EMT) is a novel clinical therapeutic target in many chronic airway diseases related with tissue remodeling. The aim of this study was to investigate the effect of trichostatin A (TSA) on transforming growth factor (TGF)-β1-induced EMT in airway epithelium and nasal tissue. Materials and Methods A549 cells, primary nasal epithelial cells (PNECs), or inferior nasal turbinate organ culture were exposed to TSA prior to stimulation with TGF-β1. Expression levels of E-cadherin, vimentin, fibronectin, α-smooth muscle actin (SMA), histone deacetylase 2 (HDAC2), and HDAC4 were determined by western blotting and/or immunofluorescent staining. Hyperacetylation of histone H2 and H4 by TSA was measured by western blotting. After siHDAC transfection, the effects of HDAC2 and HDAC4 silencing on expression of E-cadherin, vimentin, fibronectin, α-SMA, HDAC2, and HDAC4 in TGF-β1-induced A549 were determined by RT-PCR and/or western blotting. We assessed the change in migration capacity of A549 cells by using cell migration assay and transwell invasion assay. Results TGF-β1 altered mRNA and protein expression levels of EMT markers including E-cadherin, vimentin, fibronectin, α-SMA, slug, and snail in A549 cells. Inhibition and silencing of HDAC2 and HDAC4 by TSA and siRNA enhanced TGF-β1-induced EMT in A549 cells. TSA blocked the effect of TGF-β1 on the migratory ability of A549 cells. In experiments using PNECs and inferior turbinate organ cultures, TSA suppressed expression of EMT markers induced by TGF-β1. Conclusions We showed that EMT is induced by TGF-β1 in airway epithelial cells and nasal tissue via activation of HDAC2 and HDAC4, and that inhibition of HDAC2 and HDAC4 by TSA reduces TGF-β1-induced EMT. This observation indicates that histone deacetylase inhibitors such as TSA could be potential candidates for treatment of recalcitrant CRS related with tissue remodeling.

Pirfenidone Inhibits Transforming Growth Factor β1-induced Extracellular Matrix Production in Nasal Polyp-derived Fibroblasts:

Purpose Pirfenidone has been shown to have antifibrotic and anti-inflammatory effects in the lungs. The purpose of this study was to evaluate the inhibitory effects of pirfenidone on transforming growth factor (TGF)-β1-induced myofibroblast differentiation and extracellular matrix accumulation. We also determined the molecular mechanisms of pirfenidone in nasal polyp-derived fibroblasts (NPDF). Methods NPDFs were isolated from nasal polyps from eight patients who had chronic rhinosinusitis with nasal polyp. Pirfenidone was used to treat TGF-β1-induced NPDFs. Cytotoxicity was evaluated by using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Fibroblast migration was evaluated with scratch assays. Expression levels of α-smooth muscle actin (SMA), fibronectin, and phosphorylated Smad2/3 were determined by Western blot and/or reverse transcription-polymerase chain reaction and immunofluorescent staining. Total collagen production was analyzed with the Sircol collagen assay and contractile activity was measured by a collagen gel contraction assay. Results Pirfenidone (0-2 mg/mL) has no significant cytotoxic effects in TGF-β1-induced NPDFs. Migration of NPDFs was significantly inhibited by pirfenidone treatment. The expression levels of α-SMA and fibronectin were significantly reduced in pirfenidone-treated NPDFs. Collagen contraction and production were also significantly decreased by pirfenidone treatment. Finally, pirfenidone significantly inhibited phosphorylation of the Smad2/3 pathway in TGF-β1-induced NPDFs. Conclusions Pirfenidone has an inhibitory effect on TGF-β1-induced migration, myofibroblast differentiation (α-SMA), extracellular matrix accumulation, and collagen contraction by blocking the phosphorylation of Smad2/3 pathways in NPDFs. Thus, pirfenidone may inhibit TGF-β1-induced extracellular matrix by regulating Smad2/3.

Increased expression of high-mobility group protein B1 in chronic rhinosinusitis.

Background Chronic rhinosinusitis (CRS) is an inflammation of the sinonasal mucosa and many inflammatory cells and cytokines are involved in its pathogenesis. High-mobility group protein B1 (HMGB1) is a DNA-binding protein that has a proinflammatory function when secreted into extracellular space. The purpose of this study was to evaluate the expression of HMGB1 in paranasal sinus mucosa and to determine the difference of HMGB1 expression between CRS patients and normal controls. Methods Paranasal sinus mucosa was obtained from 10 patients with CRS and 10 patients without CRS. Semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR), real-time PCR and Western blot analysis were performed to detect mRNA and protein. Sections of the mucosa were immunostained for localization of HMGB1 and image analysis was performed. Results RT-PCR and real-time PCR showed that the expression level of HMGB1 mRNA was significantly increased in the tissues of patients with CRS compared with controls. Western blot analysis showed that the expression level of HMGB1 protein was significantly increased in the tissues of CRS. In immunohistochemical staining, the HMGB1 protein was expressed in epithelial cells and inflammatory cells and the expression intensity of HMGB1 protein was stronger in CRS. Conclusion HMGB1 is increased in the paranasal sinus mucosa of patients with CRS. These results suggest a possible contribution of HMGB1 in the pathophysiology of CRS.

Baicalin Down-Regulates IL-1β-Stimulated Extracellular Matrix Production in Nasal Fibroblasts

Purpose Baicalin, a Chinese herbal medicine, has anti-fibrotic and anti-inflammatory effects. The aims of present study were to investigate the effects of baicalin on the myofibroblast differentiation, extracellular matrix production, migration, and collagen contraction of interleukin (IL)-1β-stimulated nasal fibroblasts and to determine the molecular mechanism of baicalin in nasal fibroblasts. Methods Nasal fibroblasts were isolated from the inferior turbinate of patients. Baicalin was used to treat IL-1β-stimulated nasal fibroblasts. To evaluate cytotoxicity, a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay was used. The expression levels of α-smooth muscle actin (SMA), fibronectin, phospho-mitogen-activated protein kinase (p-MAPK), p-Akt, p-p50, p-p65, and p-IκBα were measured by western blotting, reverse transcription-polymerase chain reaction (RT—PCR),or immunofluorescence staining. Fibroblast migration was analyzed with scratch assays and transwell migration assays. Total collagen was evaluated with the Sircol collagen assay. Contractile activity was measured with a collagen gel contraction assay. Results Baicalin (0–50 μM) had no significant cytotoxic effects in nasal fibroblasts. The expression of α–SMA and fibronectin were significantly down-regulated in baicalin-treated nasal fibroblasts. Migration, collagen production, and contraction of IL-1β-stimulated nasal fibroblasts were significantly inhibited by baicalin treatment. Baicalin also significantly down-regulated p-MAPK, p-Akt, p-p50, p-p65, and p-IκBα in IL-1β-stimulated nasal fibroblasts. Conclusions We showed that baicalin down-regulated myofibroblast differentiation, extracellular matrix production, migration, and collagen contraction via the MAPK and Akt/ NF-κB pathways in IL-1β-stimulated nasal fibroblasts.

Inhibitory Effect of Delphinidin on Extracellular Matrix Production via the MAPK/NF-κB Pathway in Nasal Polyp-Derived Fibroblasts.

Purpose Nasal polyps are associated with chronic inflammation of the mucous membranes in the nose and paranasal sinuses and involved in extracellular matrix (ECM) accumulation. Delphinidin promotes ECM degradation in hepatitis and cardiac fibrosis. The aims of this study were to examine the inhibitory effect of delphinidin on TGF-β1-induced myofibroblast differentiation and ECM accumulation, and to determine the underlying mechanisms in nasal polyp-derived fibroblasts (NPDFs). Methods NPDFs were stimulated with TGF-β1, with or without delphinidin, and the expression levels of α-SMA, fibronectin, and collagen type I were determined by RT-PCR, Western blot analysis, and collagen assay. The expression of α-SMA protein was measured by immunocytochemical staining. Mitogen-activated protein kinase and NF-κB activation induced by TGF-β1 were determined by Western blot analysis. The transcriptional activity of NF-κB was measured by luciferase assay. Results The expression levels of α-SMA, fibronectin, and collagen type I increased in TGF-β1-stimulated NPDFs. In TGF-β1-induced NPDFs, delphinidin inhibited the expression of α-SMA, fibronectin, and collagen. Inhibitors of MAPK and NF-κB blocked the expression of α-SMA, fibronectin, and collagen type I. Delphinidin suppressed the activation of MAPK and NF-κB induced by TGF-β1 stimulation. Conclusions These results suggest that delphinidin may inhibit TGF-β1-induced myofibroblast differentiation and ECM production through the MAPK/NF-κB signaling pathway in NPDFs.

Diesel Exhaust Particles Upregulate Interleukins IL-6 and IL-8 in Nasal Fibroblasts.

Background Diesel exhaust particles (DEP) are a major source of air pollution. Nasal fibroblasts are known to produce various cytokines and chemokines. The aim of this study was to evaluate DEP-induced cytokines and chemokines in nasal fibroblasts and to identify the signaling pathway involved. Methods A cytokine and chemokine array performed after stimulation of nasal fibroblasts with DEP revealed that levels of IL-6 and IL-8 were increased most significantly among various cytokines and chemokines. RT—PCR and ELISA were used to determine the mRNA and protein expression levels of IL-6 and IL-8. Signaling pathways of p-38, Akt, and NF-κB were analyzed by western blotting, luciferase assay, and ELISA. Organ cultures of nasal interior turbinate were also developed to demonstrate the ex vivo effect of DEP on the expression of IL-6 and IL-8 and the associated signaling pathway. Results DEP increased the expressions of IL-6 and IL-8 in nasal fibroblasts at mRNA and protein levels. DEP induced phosphorylation of p38, Akt, and NF-κB, whereas inhibitors of p38, Akt, and NF-κB blocked these phophorylations and the expressions of IL-6 and IL-8. These findings were also observed in ex vivo organ culture of nasal inferior turbinate. Conclusions DEP induces expression of IL-6 and IL-8 via p38, Akt, and NF-κB signaling pathways in nasal fibroblasts. This finding suggests that air pollution might induce or aggravate allergic rhinitis or chronic rhinosinusitis.

Wogonin inhibits transforming growth factor β1-induced extracellular matrix production via the p38/activator protein 1 signaling pathway in nasal polyp-derived fibroblasts.

Background Wogonin has been shown to have antifibrotic and anti-inflammatory effects in the lower airway. The purpose of this study was to evaluate the effects of wogonin on transforming growth factor (TGF) β1–induced myofibroblast differentiation, extracellular matrix production, migration, and collagen contraction, and to determine the molecular mechanisms of wogonin in nasal polyp–derived fibroblasts (NPDF). Methods NPDFs were isolated from nasal polyps from eight patients. TGF-β1–induced NPDFs were treated with wogonin. Cytotoxicity was evaluated by using a 3-(4,5- dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Fibroblast migration was evaluated with transwell and scratch migration assays. The expression levels of α-smooth muscle actin, fibronectin, phosphorylated-p38, and c-Fos were determined by Western blot and/or reverse transcription–polymerase chain reaction. The total collagen amount was analyzed with the Sircol collagen assay, and contractile activity was measured by a collagen gel contraction assay. Results Wogonin (0–60 μM) had no significant cytotoxic effects on TGF-β1–induced NPDFs. Migration of NPDFs was significantly inhibited by wogonin treatment. The expression levels of α-smooth muscle actin and fibronectin were significantly reduced in wogonin-treated NPDFs. Collagen production and contraction were also significantly decreased by wogonin treatment. Wogonin markedly inhibited activation of the p38/activator protein 1 pathway in TGF-β1–induced NPDFs. Conclusion These results indicated that wogonin may inhibit TGF-β–induced myofibroblast differentiation, extracellular matrix production, migration, and collagen contraction through the p38/activator protein-1 pathway in NPDFs.

Glucocorticoids ameliorate TGF-β1-mediated epithelial-To-mesenchymal transition of airway epithelium through MAPK and Snail/Slug signaling pathways

Chronic rhinosinusitis with nasal polyps (CRSwNP) is closely associated with tissue remodeling. Epithelial-to-mesenchymal transition (EMT), a process of tissue remodeling, can be a therapeutic target of CRSwNP. Glucocorticoids are a type of steroid hormone that is used primarily in medical therapy for patients with CRSwNP; however, their effects on EMT in the airway epithelium remain unknown. To investigate the effects of dexamethasone and fluticasone propionate, a class of glucocorticoids, on transforming growth factor-β1 (TGF-β1) -induced EMT, we used A549 cells, human primary nasal epithelial cells (hPNECs) and ex vivo organ culture of the inferior turbinate. TGF-β1 induced changes in cell morphology, suppressed the expression of E-cadherin and enhanced the expression of a-smooth muscle actin, vimentin and fibronectin in A549 cells. However, glucocorticoids inhibited EMT, migration and invasion enhancement by TGF-β1. We found that the induction of phosphorylated ERK, p38 and the activity of Snail and Slug transcription factors by TGF-β1 were suppressed by glucocorticoids. Glucocorticoids also had a similar effect in hPNECs and ex vivo organ cultures of the inferior turbinate. These findings suggest that glucocorticoids might be a useful therapy for preventing tissue remodeling by blocking the EMT initiated by TGF-β1-induced MAPK and Snail/Slug signaling pathways in CRSwNP.

Cigarette smoke extract increases vascular endothelial growth factor production via TLR4/ROS/MAPKs/NF-kappaB pathway in nasal fibroblast

Purpose Cigarette smoke is a complex mixture of various chemical compounds, including free radicals and highly toxic compounds. Cigarette smoke exposure has been shown to be associated with chronic rhinosinusitis and tissue remodeling in upper airway. Vascular endothelial growth factor (VEGF) is one of the cytokines with a crucial role in tissue remodeling of airway. The aims of this study were to determine the effects of cigarette smoke extract (CSE) on VEGF expression and to investigate the underlying molecular mechanisms of CSE in nasal fibroblasts. Methods Nasal fibroblasts were stimulated with CSE. Cytotoxicity was evaluated by 3-(4,5- dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. The expression level of VEGF was measured using reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay. Messenger RNA (mRNA) expression level of TLR4 were determined by RT-PCR. Small interfering RNA (siRNA) for TLR4 was transfected to suppress TLR4 expression. Activation of reactive oxygen species (ROS) was analyzed by using dichlorodihydro-fluorescein diacetate assay. Mitogen-activated protein kinase (MAPK) and NF-kappaB activations were determined by using western blot and/or luciferase assay. Results CSE had no significant cytotoxic effect in nasal fibroblast up to 5%. CSE significantly increased both VEGF mRNA and protein expression dose-dependently. The down-regulation of TLR4 transcription by siRNA treatment suppressed CSE-induced expressions of both TLR4 and VEGF. Pretreatment with ROS scavengers, specific inhibitors of each MAPK, and NF-kappaB inhibitor significantly decreased CSE-induced VEGF expression. Conclusions CSE has a stimulatory effect on VEGF expression through the TLR4, ROS, MAPK, and NF-kappaB signaling pathway in nasal fibroblasts.

Tumor necrosis factor‐α regulates interleukin‐33 expression through extracellular signal‐regulated kinase, p38, and nuclear factor–κB pathways in airway epithelial cells

Interleukin (IL)‐33 plays an important role in controlling immune responses in barrier tissues, and is a potent mediator of inflammatory diseases such as asthma, rheumatoid disease, and chronic rhinosinusitis. The aims of the present study were 2‐fold: (1) to determine the stimulatory effect of tumor necrosis factor‐α (TNF‐α) on IL‐33 production in nasal epithelial and A549 cells; and (2) to identify downstream pathways that activate IL‐33 production.