You Mi Moon

Epigenetic regulation of myofibroblast differentiation and extracellular matrix production in nasal polyp-derived fibroblasts.

Nasal polyposis is a multi‐factorial disease associated with chronic inflammatory condition of the paranasal sinuses. Myofibroblast differentiation and extracellular matrix (ECM) accumulation are involved in the pathogenesis of nasal polyposis.

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.

Effect of simvastatin on transforming growth factor beta-1-induced myofibroblast differentiation and collagen production in nasal polyp-derived fibroblasts.

Background Statins are the most commonly prescribed drugs for the treatment of hypercholesterolemia. Statins exert not only lipid-lowering but also other cellular effects, including antifibrotic properties. The purpose of this study was to determine the effect of simvastatin on transforming growth factor (TGF)-beta-1–induced myofibroblast differentiation and collagen production in nasal polyp-derived fibroblasts (NPDFs) and to verify the mechanism of the effect of simvastatin in TGF-beta-1–induced myofibroblast differentiation in NPDFs. Methods NPDFs were pretreated with simvastatin with or without mevalonate or Y-27643 for 2 hours before induction by TGF-beta-1. The expression of alpha-smooth muscle actin (SMA) and collagen type IV mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of alpha-SMA protein was determined by immunofluorescent cytochemical staining. Total soluble collagen production was analyzed by the SirCol collagen dye-binding assay (Biocolor, Belfast, U.K.). Phosphorylation of Smad 2/3 was evaluated by Western blot analysis. Results In TGF-beta-1–induced NPDFs, simvastatin significantly inhibited the expressions of α-SMA and collagen type IV mRNA and reduced alpha-SMA and collagen protein levels. Pretreatment with mevalonate reversed the effect of simvastatin. The expression of alpha-SMA mRNA and protein was significantly decreased by pretreatment with Y-27632. The TGF-beta-1–induced expression of pSmad 2/3 protein was notably decreased by pretreatment with simvastatin. Conclusion We showed that simvastatin inhibits TGF-beta-1–induced myofibroblast differentiation (expression of alpha-SMA) and collagen production in NPDFs and Rho/Rock and TGF-β/Smad signaling is involved as an underlying mechanism. The results of our study suggest that simvastatin is a possible candidate for the suppression of nasal polyp formation.

Role of Reactive Oxygen Species in Transforming Growth Factor Beta1-Induced Alpha Smooth-Muscle Actin and Collagen Production in Nasal Polyp-Derived Fibroblasts

Background: Myofibroblasts are detected in nasal polyps and are involved in nasal polyp formation by inducing extracellular matrix accumulation. Reactive oxygen species (ROS) are released during the differentiation of fibroblasts to myofibroblasts. The purpose of this study was to investigate ROS production and nicotinamide adenine dinucleotide phosphate oxidase (NOX) expression in nasal polyp-derived fibroblasts (NPDFs) and to evaluate whether ROS from NOX mediates transforming growth factor (TGF)-β1-induced production of alpha smooth-muscle actin (α-SMA) and collagen production. Methods: NPDFs were incubated and treated with TGF-β1. The mRNA expression of NOXs, α-SMA, and collagen type I and IV was determined by reverse transcription-polymerase chain reaction, and the expression of α-SMA protein was determined by immunofluorescence microscopy. The amount of total soluble collagen production was analyzed by the SirCol assay. The ROS generation of cells was investigated using the 2′,7′-dichlorfluorescein-diacetate. The fluorescence was captured by fluorescent microscope and measured using a fluorometer. Results: Stimulation with TGF-β1 increased ROS production by NPDFs compared with NPDFs not treated with TGF-β1. Stimulation with TGF-β1 increased the expression of NOX4 mRNA most potently among various Nox enzymes. siNOX4 was able to decrease the level of ROS production. Myofibroblast differentiation and the production of collagen in NPDFs were prevented by inhibition of ROS generation with diphenyliodonium, N-acetylcysteine, ebselen, and siNox4. Conclusions: This study showed that NOX4 and ROS have a role in myofibroblast differentiation and collagen production of TGF-β1-induced NPDFs and that these processes are inhibited by the elimination of ROS.

Nox4 Mediates Hypoxia-Stimulated Myofibroblast Differentiation in Nasal Polyp-Derived Fibroblasts

Background: Chronic hypoxia is associated with remodeling in various organs. Reactive oxygen species (ROS) derived from NADPH oxidases (Nox), and transforming growth factor-β1 (TGF-β1) have been implicated in the pathogenesis of hypoxia-induced remodeling. The aims of this study were to determine in hypoxia-stimulated nasal polyp-derived fibroblasts (NPDF) the effect of hypoxia on the differentiation of myofibroblasts, the role of ROS, the major Nox homolog mediating myofibroblast differentiation, and the role of TGF-β1. Methods: Eight primary cultures of NPDF were established from nasal polyps, which were incubated under hypoxic conditions. Reverse transcription polymerase chain reaction for αSMA, Nox1, Nox3, Nox4, Nox5, and fibronectin mRNA was performed. Western blotting for α-SMA and fibronectin was done. ROS production was detected using a fluorometer. NPDF were pretreated with ROS scavengers and transfected with siNox4. The TGF-β1 protein level was measured by ELISA. The effect of treatment with TGF-β1 type I tyrosine kinase inhibitor SB431542 on myofibroblast differentiation was observed. Results: Hypoxic stimulation of NPDF significantly increased α-SMA and fibronectin mRNA and protein expression. ROS production was increased by hypoxia, and ROS scavengers inhibited myofibroblast differentiation. Nox4 mRNA was the only Nox homolog increased by hypoxia. Transfection with siNox4 inhibited myofibroblast differentiation. TGF-β1 was secreted endogenously by hypoxic NPDF. SB431542 significantly inhibited myofibroblast differentiation. Conclusions: Hypoxia induces myofibroblast differentiation of NPDF through a signaling pathway involving Nox4-dependent ROS generation and TGF-β1. Therapies targeting Nox4 may be effective against remodeling of nasal polyps.

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.

Effect of [6]-gingerol on myofibroblast differentiation in transforming growth factor beta 1-induced nasal polyp-derived fibroblasts

Background [6]-Gingerol is one of the major pungent principles of ginger and has diverse effects, including anti-inflammatory, and antioxidative effects. Reactive oxygen species (ROS) are released during the phenotypic transformation of fibroblasts to myofibroblasts, a process that is involved in the growth of nasal polyps by inducing extracellular matrix (ECM) accumulation. The purpose of this study was to determine the effect of [6]-gingerol on myofibroblast differentiation and collagen production of nasal polyp–derived fibroblasts (NPDFs) and to determine if the effect of [6]-gingerol is linked to an antioxidant effect. Methods NPDFs were incubated and treated with transforming growth factor (TGF) beta 1. The ROS generated by NPDFs were determined using 2″,7″-dichlorfluorescein-diacetate. The fluorescence was captured by a fluorescent microscope and measured using a fluorometer. The expression of alpha-smooth muscle actin (SMA) and collagen type IV mRNA was determined by a reverse transcription-polymerase chain reaction, and the expression of α-SMA protein and pSmad2/3 was determined by immunofluorescence microscopy and or Western blotting. The amount of total soluble collagen production was analyzed by the SirCol collagen dye-binding assay. Results TGF-beta 1 stimulation increased ROS production by NPDFs. [6]-Gingerol decreased the production of ROS in TGF-beta 1–induced NPDFs. Myofibroblast differentiation, collagen production, and phosphorylation of Smad2/3 were prevented by [6]-gingerol and inhibition of ROS generation with antioxidant such as diphenyliodonium, N-acetylcysteine, and ebselen. Conclusion These results suggest the possibility that [6]-gingerol may play an important role in inhibiting the production of the ECM in the development of nasal polyps through an antioxidant effect.

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

Effect of Prostaglandin E2 on Vascular Endothelial Growth Factor Production in Nasal Polyp Fibroblasts

Purpose Angiogenesis is involved in the pathogenesis of chronic rhinosinusitis with nasal polyps. We aimed to investigate the effects of prostaglandin E2 (PGE2) on vascular endothelial growth factor (VEGF) production, the role of E-prostanoid (EP) 4 receptors, and the signal transduction pathway mediating VEGF production in nasal polyp-derived fibroblasts (NPDFs). Methods Eight primary NPDF cultures were established from nasal polyps, which were incubated with or without PGE2. Reverse transcription-polymerase chain reaction amplification of EP receptors (EP1, EP2, EP3, and EP4) and immunofluorescence staining for VEGF production were performed. VEGF production via the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K) pathways was evaluated by enzyme-linked immunosorbent assay. Results All EP receptors were expressed in NPDFs. PGE2 significantly increased VEGF production concentration- and time dependently, and VEGF production was regulated by an EP4 receptor. Activation of intracellular cAMP regulated VEGF production. VEGF production was decreased by PKA and PI3K inhibitors via intracellular cAMP. Conclusions PGE2 stimulates VEGF production via the EP4 receptor in NPDFs. These results indicate that PGE2-induced VEGF production is mediated, at least partially, through cAMP-dependent signaling pathways. Therapies targeting the EP4 receptor may be effective in inhibiting the development of nasal polyps.

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.