Ji Young Um

Metformin reduces TGF-β1-induced extracellular matrix production in nasal polyp-derived fibroblasts.

Background and Objects Metformin is widely used to treat type 2 diabetes mellitus, and adenosine monophosphate–activated protein kinase (AMPK) is thought to be the target that mediates its effects. Recently, it has been demonstrated that metformin has antifibrotic effects beyond its antihyperglycemic action. The purposes of this study were to investigate the effect of metformin on TGF-β1–induced myofibroblast differentiation (α-smooth muscle actin [α-SMA]) and extracellular matrix (ECM) production and to determine the underlying mechanism of the action of metformin in nasal polyp–derived fibroblasts (NPDFs). Study Design Basic research. Setting The rhinology laboratory of Korea University Guro Hospital, Seoul, Korea. Methods NPDFs from 7 patients were incubated with TGF-β1 and treated with metformin or compound C, an inhibitor of AMPK. To determine the proliferation rate of nasal fibroblasts, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed. The expression levels of α-SMA and fibronectin were determined by reverse transcription–polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescent staining. Phosphorylation of AMPK and phosphorylation of Smad2/3 were evaluated by Western blot analysis. Results In TGF-β1–induced NPDFs, metformin inhibited the expression of α-SMA and fibronectin, as confirmed by both RT-PCR and Western blot analysis. Metformin increased the phosphorylation of AMPK and the expression levels of α-SMA and fibronectin. However, compound C reversed these effects. Metformin inhibited TGF-β1–induced phosphorylation of Smad2/3. Conclusions This study showed that metformin inhibits TGF-β1–induced myofibroblast differentiation and ECM production in NPDFs via the Smad2/3 pathway. AMPK can be a therapeutic target for the prevention of ECM remodeling in nasal polyps.

Effects of histone deacetylase inhibitor on extracellular matrix production in human nasal polyp organ cultures.

Background Nasal polyposis is associated with a chronic inflammatory condition of the sinonasal mucosa and involves myofibroblast differentiation and extracellular matrix (ECM) accumulation. Epigenetic modulation by histone deacetylase (HDAC) inhibitors including trichostatin A (TSA) has been reported to have inhibitory effects on myofibroblast differentiation in lung and renal fibroblasts. The purpose of this study was to investigate the inhibitory effect of TSA on myofibroblast differentiation and ECM production in nasal polyp organ cultures. Methods Nasal polyp tissues from 18 patients were acquired during endoscopic sinus surgery. After organ culture, nasal polyps were stimulated with TGF-beta1 and then treated with TSA. Alpha-smooth muscle actin (α-SMA), fibronectin, and collagen type I expression levels were examined by reverse transcription–polymerase chain reaction (PCR), real-time PCR, Western blot, and immunofluorescent staining. HDAC2, HDAC4, and acetylated H4 expression levels were assayed by Western blot. Cytotoxicity was analyzed by the terminal deoxynucleotidyl transferase biotin–dUTP nick end labeling assay. Results The expression levels of α-SMA, fibronectin, and collagen type 1 were increased in nasal polyp after transforming growth factor (TGF) beta1 treatment. TSA-inhibited TGF-beta1 induced these gene and protein expression levels. Furthermore, TSA suppressed protein expression levels of HDAC2 and HDAC4. However, TSA induced hyperacetylation of histones H4. Treatment with TGF-beta1 with or without TSA did not have cytotoxic effect. Conclusion These findings provide novel insights into the epigenetic regulation in myofibroblast differentiation and ECM production of nasal polyp. TSA could be a candidate of a therapeutic agent for reversing the TGF-beta1–induced ECM synthesis that leads to nasal polyp development.

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.

Inhibitory effect of ginsenoside Rg1 on extracellular matrix production via extracellular signal-regulated protein kinase/activator protein 1 pathway in nasal polyp-derived fibroblasts

Nasal polyps are associated with chronic inflammation of the sinonasal mucosa and are involved in myofibroblast differentiation and extracellular matrix (ECM) accumulation. Ginsenoside Rg1, a compound derived from Panax ginseng, shows antifibrotic and anticancer effects. However, the molecular effects of Rg1 on myofibroblast differentiation and ECM production remain unknown. The aims of this study were to investigate the effect of Rg1 on transforming growth factor (TGF)-β1-induced myofibroblast differentiation and ECM production and to determine the molecular mechanism of Rg1 in nasal polyp-derived fibroblasts (NPDFs). NPDFs were isolated from nasal polyps of seven patients who had chronic rhinosinusitis with nasal polyp. NPDFs were exposed to TGF-β1 with or without Rg1. Expression levels of α-smooth muscle actin (SMA), fibronectin and collagen type Iα1 were determined by reverse transcription polymerase chain reaction, Western blot and immunofluorescent staining. TGF-β1 signaling molecules, including Smad2/3, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were analyzed by Western blotting. Transcription factors involved with TGF-β1 signaling, nuclear factor (NF)-κB and activator protein 1 (AP-1) were also assessed by Western blot. The cytotoxic effect of Rg1 was measured by an established viability assay. The mRNA and protein expression levels of α-SMA, fibronectin and collagen type Iα1 were increased in TGF-β1-induced NPDFs. Rg1 inhibited these effects. The inhibitory molecular mechanism of Rg1 was involved in the ERK pathway. Rg1 inhibited the transcription factor activation of AP-1. Rg1 itself was not cytotoxic. The ginsenoside Rg1 has inhibitory effects on myofibroblast differentiation and ECM production. The inhibitory mechanism of Rg1 is involved with the ERK and AP-1 signaling pathways. Rg1 may be useful as an inhibitor of ECM deposition, and has potential to be used as a novel treatment option for nasal polyps.

Histamine Promotes the Release of Interleukin-6 via the H1R/p38 and NF-κB Pathways in Nasal Fibroblasts.

Purpose Based on the close relationship between histamine and interleukin 6 (IL-6), we hypothesized that histamine may regulate the production of cytokines, such as IL-6, during allergic inflammation. Here, we examined the role of histamine in IL-6 production and histamine receptor activity in nasal fibroblasts, along with the mechanisms underlying these effects. Methods Experiments were performed using nasal fibroblasts from 8 normal patients. RT-PCR was used to identify the major histamine receptors expressed in nasal fibroblasts. Fibroblasts were then treated with histamine with or without histamine-receptor antagonists, and monitored for IL-6 production using an ELISA. Four potential downstream signaling molecules, p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and NF-κB, were evaluated by Western blot, and a luciferase reporter assay. Results Elevated expression was seen for all histamine receptors, with IL-6 protein levels increasing significantly following histamine stimulation. Among the histamine-receptor specific antagonists, only the H1R antagonist significantly decreased IL-6 production in histamine-stimulated nasal fibroblasts. Histamine increased the expression level of phosphorylated p38 (pp38), pERK, and pJNK, as well as NF-κB induction. The H1R antagonist actively suppressed pp38 and NF-κB expression in histamine-induced nasal fibroblasts, but not pERK and pJNK. The p38 inhibitor strongly attenuated IL-6 production in histamine-stimulated nasal fibroblasts. Conclusions The data presented here suggest that antihistamines may be involved in the regulation of cytokines, such as IL-6, due to the role of histamine as an inflammatory mediator in nasal fibroblasts.

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.

Berberine Inhibits Myofibroblast Differentiation in Nasal Polyp-Derived Fibroblasts via the p38 Pathway

The purposes of this study were to determine whether berberine has any effect on phenotype changes and extracellular matrix (ECM) production in nasal polyp‐derived fibroblasts (NPDFs) and to investigate the underlying molecular mechanism. NPDFs were pre‐treated with berberine prior to induction by transforming growth factor (TGF)‐β1. The expression of α‐smooth muscle actin (SMA) and collagen type I mRNA was determined by a reverse transcription‐polymerase chain reaction, and the expression of α‐SMA protein and collagen type I was determined by western blotting and/or immunofluorescent staining. The total soluble collagen production was analysed by the SirCol collagen assay. The expression of several signaling molecules of the TGF‐β1 pathway was evaluated by western blot analysis. In TGF‐β1‐induced NPDFs, berberine significantly inhibited the expression of α‐SMA and collagen type I mRNA and reduced α‐SMA and collagen protein levels. Berberine only suppressed the expression of pp38 among the evaluated signaling molecules. SB203580 (a specific inhibitor of p38 kinase) markedly suppressed the increased expression of collagen type I and α‐SMA in TGF‐β1‐induced NPDFs. Berberine exerts suppressive effects on phenotype changes and ECM production in NPDFs via p38 signaling pathway interference. The findings provide new therapeutic options for ECM production in nasal polyps. Copyright

Rapamycin inhibits transforming growth factor beta 1 induced myofibroblast differentiation via the phosphorylated'phosphatidylinositol 3-kinase mammalian target of rapamycin signal pathways in nasal polyp'derived fibroblasts

Purpose Rapamycin has antiproliferative and antifibrogenic effects in vitro and in vivo. The purpose of this study was to evaluate the effects of rapamycin on transforming growth factor (TGF) beta 1 induced myofibroblast differentiation (alpha smooth-muscle actin [SMA]), extracellular matrix production, and collagen contraction in nasal polyp–derived fibroblasts (NPDF). The underlying molecular mechanisms of rapamycin were also determined in NPDFs. Methods NPDFs were grown in culture and transformed into myofibroblasts by using TGF beta 1 (5 ng/mL). For cytotoxicity evaluation, a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay was used. Expression levels of alpha SMA, phosphorylated phosphatidylinositol 3-kinase (PI3K), and phosphorylated mammalian target of rapamycin (mTOR) were determined by using Western blot, reverse transcription–polymerase chain reaction, and immunofluorescence staining. The total amount of collagen was analyzed by using the Sircol collagen assay, and contractile activity was measured with a collagen gel contraction assay. Silencing mTOR with mTOR-specific small interference RNA was determined by using reverse transcription–polymerase chain reaction. Results Whereas rapamycin (range, 0–400 nM) had no significant cytotoxic effects on TGF beta 1 induced NPDFs, it significantly reduced the expression levels of alpha–SMA in TGF beta 1 induced NPDFs in a dose-dependent manner. TGF beta 1 induced collagen production and collagen contraction were significantly inhibited by rapamycin treatment. Rapamycin also attenuated the TGF beta 1 induced activation of PI3K and mTOR, and its inhibitory effects were similar to those of mTOR silencing and a specific PI3K inhibitor. Conclusions Rapamycin inhibited TGF beta 1 induced myofibroblast differentiation, extracellular matrix production, and collagen contraction through the PI3K/mTOR signal pathways in NPDFs.

Stimulatory effects of histamine on migration of nasal fibroblasts.

Fibroblast migration is crucial for normal wound repair after sinonasal surgery. Histamine is known to be involved in wound healing by its effects on cell proliferation and migration. This study aimed to determine whether histamine affects the migration of nasal fibroblasts and to investigate the mechanism of action of histamine on nasal fibroblasts.

Inhibitory effect of prostaglandin E(2) on the migration of nasal fibroblasts.

Background Fibroblast migration is crucial for normal wound repair after sinonasal surgery. Prostaglandin E2 (PGE2) is a potent inhibitor of fibroblast functions including chemotaxis, proliferation, and matrix production. The purpose of this study was to determine whether PGE2 affects the migration of nasal fibroblasts and to investigate the mechanism of action of PGE2 on nasal fibroblasts. Methods Primary cultures of nasal fibroblasts were established from inferior turbinate samples. Fibroblast migration was evaluated with scratch assays. Reverse-transcription polymerase chain reaction was performed for E prostanoid (EP) 1, EP2, EP3, and EP4 receptors. EP receptor–selective agonists and antagonists were used to evaluate receptor functions. Stimulatory G (Gs) proteins were activated to evaluate mechanisms. Intracellular cyclic adenosine monophosphate (cAMP) levels were measured by ELISA, and fibroblast cytoskeletal structures were visualized with immunocytochemistry. Results PGE2 significantly reduced the migration of nasal fibroblasts. Agonists selective for the EP2 and EP4 receptors significantly reduced the nasal fibroblast migration. Antagonists of the EP2 and EP4 receptors inhibited the effect of PGE2 on nasal fibroblast migration. Activation of Gs protein and adenyl cyclase reduced nasal fibroblast migration. Conclusion PGE2 inhibited the migration of nasal fibroblasts via the EP2 and EP4 receptors, and this inhibition was mediated by cAMP elevation. Targeting specific EP receptors could offer therapeutic opportunities for conditions such as delayed wound healing after nasal surgery.