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Background/Purpose: Gingival overgrowth can occur as a result of poor oral hygiene or a side effect of taking certain medications, such as cyclosporine A (CsA). It has been shown that this immunosuppressant drug induces epithelial-to-mesenchymal transition (EMT) in the gingival epithelium but the associated molecular mechanism remains to be elucidated. Methods: We first assessed the relative expression of microRNA-200a (miR-200a) in response to the CsA treatment using qRT-PCR. Next, luciferase reporter assay was applied to examine whether miR-200a was able to regulate ZEB2 and Western blot was utilized to measure the expression of ZEB2 in normal human gingival fibroblasts (HGFs). To confirm the significance of miR-200a and ZEB2 in the CsA-induced gingival overgrowth, miR-200a inhibitor and shRNA mediated knockdown of ZEB2 were used and cell proliferation in HGFs was assessed by MTT assay. Results: The expression of miR-200a was dose-dependently downregulated following the CsA treatment. Luciferase reporter assay confirmed that ZEB2 was a direct downstream target regulated by miR-200a and ZEB2 was indeed increased after the administration of CsA. We demonstrated that knockdown of ZEB2 hampered the CsA-induced HGFs proliferation and the elevated cell proliferation due to inhibition of miR-200a was reversed by repression of ZEB2. Conclusion: Our results showed that insufficient miR-200a in HGFs caused by CsA administration may lead to gingival enlargement mediated by the upregulation of ZEB2. This finding supported that CsA-induced EMT contributed to the adverse effect of using CsA and miR-200a may serve as an upstream target to prevent the overgrowth of the gingiva.

Background/purpose: Gingival overgrowth can occur as a result of poor oral hygiene or a side effect of taking certain medications, such as cyclosporine A (CsA). It has been shown that this immunosuppressant drug induces epithelial-to-mesenchymal transition (EMT) in the gingival epithelium but the associated molecular mechanism remains to be elucidated.Methods: We first assessed the relative expression of microRNA-200a (miR-200a) in response to the CsA treatment using qRT-PCR. Next, luciferase reporter assay was applied to examine whether miR-200a was able to regulate ZEB2 and Western blot was utilized to measure the expression of ZEB2 in normal human gingival fibroblasts (HGFs). To confirm the significance of miR-200a and ZEB2 in the CsA-induced gingival overgrowth, miR-200a inhibitor and shRNA mediated knockdown of ZEB2 were used and cell proliferation in HGFs was assessed by MTT assay.Results: The expression of miR-200a was dose-dependently downregulated following the CsA treatment. Luciferase reporter assay confirmed that ZEB2 was a direct downstream target regulated by miR-200a and ZEB2 was indeed increased after the administration of CsA. We demonstrated that knockdown of ZEB2 hampered the CsA-induced HGFs proliferation and the elevated cell proliferation due to inhibition of miR-200a was reversed by repression of ZEB2.Conclusion: Our results showed that insufficient miR-200a in HGFs caused by CsA administration may lead to gingival enlargement mediated by the upregulation of ZEB2. This finding supported that CsA-induced EMT contributed to the adverse effect of using CsA and miR-200a may serve as an upstream target to prevent the overgrowth of the gingiva. [ABSTRACT FROM AUTHOR]

Background/purpose: Cyclosporine A (CsA) has been used as an immunosuppressive agent with a side effect of gingival overgrowth. It has been known that CsA-induced epithelial-mesenchymal transition (EMT) in gingiva, but the molecular mechanism has not been fully unveiled. The purpose of the study is to investigate functional roles of microRNAs in gingival overgrowth.Methods: The effect of CsA on the expression of microRNA-200b (miR-200b) in normal human gingival fibroblasts (HGFs) was determined using qRT-PCR. Luciferase reporter assay and Western blot were utilized to examine the relationship between miR-200b and EMT inducer Slug. Cell proliferation was assessed by MTT assay.Results: CsA was found to downregulate the miR-200b transcript in HGFs in a dose-dependent manner. Luciferase reporter assay confirmed that Slug was a direct target of miR-200b, and the CsA-induced cell proliferation and Slug upregulation were inhibited by overexpression of miR-200b. Additionally, the silence of Slug reversed the increased proliferation of HGFs by miR-200b inhibitor.Conclusion: Repression of miR-200b after CsA administration led to an increase in Slug expression. Our results suggested that miR-200b was an upstream effector of the CsA-induced EMT and may act as a therapeutic target for CsA-induced gingival overgrowth. [ABSTRACT FROM AUTHOR]

Background/purpose: Many fibrotic processes are associated with an increased level of transforming growth factor-β1 (TGF-β1). TGF-β1 can increase synthesis of matrix proteins and enhance secretion of protease inhibitors, resulting in matrix accumulation. Connective tissue growth factor (CTGF) is a downstream profibrotic effector of TGF-β1 and is associated with the fibrosis in several human organs. Curcumin has been applied to reduce matrix accumulation in fibrotic diseases. This study was aimed to evaluate whether curcumin could suppress TGF-β1-induced CTGF expression and its related signaling pathway involving in this inhibitory action in primary human gingival fibroblasts.Methods: The differences in CTGF expression among three types of gingival overgrowth and normal gingival tissues were assessed by immunohistochemistry. Gingival fibroblast viability in cultured media with different concentrations of curcumin was studied by MTT assay. The effect of curcumin on TGF-β1-induced CTGF expression in primary human gingival fibroblasts was examined by immunoblotting. Moreover, the proteins involved in TGF-β1 signaling pathways including TGF-β1 receptors and Smad2 were also analyzed by immunoblotting.Results: CTGF was highly expressed in fibroblasts, epithelial cells and some of endothelial cells, smooth muscle cells, and inflammatory cells in phenytoin-induced gingival overgrowth tissues rather than in those of hereditary and inflammatory gingival overgrowth tissues. Moreover, CTGF expression in the epithelial and connective tissue layers was higher in phenytoin-induced gingival overgrowth tissues than in normal gingival tissues. Curcumin was nontoxic and could reduce TGF-β1-induced CTGF expression by attenuating the phosphorylation and nuclear translocation of Smad2.Conclusion: Curcumin can suppress TGF-β1-induced CTGF expression through the interruption of Smad2 signaling. [ABSTRACT FROM AUTHOR]

Gingival overgrowth occurs as a side effect of systemic medication with immunosuppressant cyclosporine A (CsA). Slug, a master regulator of epithelial–mesenchymal transition, is dramatically upregulated in a variety of fibrotic diseases. The aim of this study is to investigate the role of epithelial–mesenchymal transition marker Slug in the pathogenesis of CsA-induced gingival overgrowth. Methods: Clinically healthy gingiva and CsA-induced gingival overgrowth specimens were analyzed by immunohistochemistry. The effect of CsA on normal human gingival fibroblasts (HGFs) was used to elucidate whether Slug expression could be affected by CsA by real-time reverse transcription-polymerase chain reaction and western blot. Cell proliferation in CsA-treated HGFs with Slug lentiviral-mediated shRNAi knockdown was evaluated by tetrazolium bromide reduction assay. Results: Slug expression was higher in CsA-induced gingival overgrowth specimens than in clinical healthy gingiva (p

Cyclosporine A (CsA) is used as an immunosuppressive agent, and its prominent side effect is the induction of gingival overgrowth. Snail is a master regulator of epithelial–mesenchymal transition (EMT). EMT under pathological processes could lead to fibrotic changes. The purpose of this study was to investigate the role of Snail in the pathogenesis of CsA-induced gingival overgrowth. Methods: The effect of CsA on normal human gingival fibroblasts (HGFs) was used to elucidate whether Snail expression could be induced by CsA by using quantitative real-time reverse transcription–polymerase chain reaction and western blot. The cell proliferation rate in CsA-treated HGFs with Snail lentiviral-mediated short hairpin RNA interference (shRNAi) knockdown was evaluated by tetrazolium bromide reduction assay. Results: CsA increased the Snail transcript and Snail protein expression in HGFs in a dose-dependent manner (p

The prominent side effect of the immunosuppressive drug cyclosporine A (CsA) is gingival overgrowth. Hypoxia-inducible factor (HIF)-1α regulates a wide variety of profibrogenic genes, which are closely associated with tissue fibrosis. The aim of this study was to compare HIF-1α expression in normal gingival tissues and CsA-induced gingival overgrowth specimens and further explore the potential mechanisms that may lead to induction of HIF-1α expression. Methods: Fifteen CsA-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. Western blot was used to investigate the effects of CsA on the expression of HIF-1α in cultured human gingival fibroblasts. The effects of CsA on plasminogen activator inhibitor (PAI)-1 expression were evaluated in environmental hypoxia. Results: HIF-1α staining in gingival tissue was stronger in CsA-induced gingival overgrowth group than normal gingival group (p

Transforming growth factor-β (TGF-β) plays an important role in the pathogenesis of cyclosporine A (CsA)-induced gingival overgrowth (GO). Connective tissue growth factor (CTGF/CCN2) acts as a cofactor with TGF-β to induce the maximal profibrotic effects of TGF-β. We investigated the effects of CsA on CCN2 expression in human gingival fibroblasts (HGFs) and the potential chemopreventive agent for CsA-induced GO. Methods: Western blot analyses were used to examine the signaling pathways of CsA-induced CCN2 expression in HGFs and whether epigallocatechin-3-gallate (EGCG), curcumin, or lovastatin can inhibit CsA-induced CCN2 expression. Results: CsA significantly stimulated CCN2 synthesis in HGFs. This effect can be inhibited by c-Jun NH2-terminal kinase (JNK) and Smad3 inhibitors but not by TGF-β neutralizing antibody and TGF-β type I receptor inhibitor. Furthermore, EGCG completely blocked CsA-induced CCN2 expression. Conclusion: CsA-induced CCN2 protein expression is mediated through JNK and Smad signaling. CsA may contribute to the pathogenesis of GO through upregulation of CCN2 expression in HGFs. EGCG could be an adjuvant for the prevention of CsA-induced GO.

Connective tissue growth factor (CTGF/CCN2) is involved in the development and progression of fibrotic diseases, including gingival overgrowth (GO). Recent studies indicate that lysophosphatidic acid (LPA) is also significantly involved in wound healing and the development of fibrosis. This study investigated whether epigallocatechin-3-gallate (EGCG) can inhibit LPA-induced CCN2 expression in human gingival fibroblast (GF) and its mechanism. Methods: Western blot analyses were used to study the signaling pathways of LPA-induced CCN2 expression in human GFs and the effects of EGCG on this pathway. Results: LPA stimulated CCN2 synthesis in human GFs. This effect can be significantly inhibited bytransforming growth factor-β type I receptor/ALK5, Smad3, and JNK inhibitors but not ERK, P38, and MAPK inhibitors. EGCG completely inhibited LPA-induced CCN2 expression through attenuating the LPA-induced JNK and Smad3 phosphorylation in human GFs. Conclusion: LPA produced at the surgical wound may contribute to the recurrence of GO by upregulating CCN2 expression in human GFs. This effect was mediated by Smad3 and JNK activation and ALK5 transactivation. EGCG could be a useful agent for reducing the recurrence of GO after surgery through suppression of JNK and Smad3 activations.

Gingival overgrowth can be induced in patients treated with cyclosporine-A (CsA), an immunosuppressant often used following organ transplantation. A pre-existing rat model designed to mimic CsA-induced gingival overgrowth in humans was used to test the effectiveness of frequent application of a chlorhexidine antiplaque solution in reducing the overgrowth. Methods: Four groups of rats were fed CsA. One group received chlorhexidine mouthwash twice a day, the second group received chlorhexidine mouthwash once a day, the third group received chlorhexidine mouthwash every other day, and the fourth group did not receive chlorhexidine mouthwash all. A fifth negative control group received only mineral oil. Overgrowth was determined by measuring the changes in the gingival probing depth and the keratinized gingival widthon molars. A gingival histological examination was performed. Results: Rats treated with mouthwash twice daily exhibited decreased probing depths and gingival widths without statistical significance. Histological examination revealed that CsA treatment caused gingival enlargement, whereas chlorhexidine treatment twice a day diminished the enlargement. Conclusion: These findings suggest that chlorhexidine mouthwash used twice daily may reduce the severity of CsA-induced gingival overgrowth. Further research is warranted to determine the optimal dose and treatment regimen.

Background/purpose: Gingival overgrowth occurs as a side effect of systemic medication with immunosuppressant cyclosporine A (CsA). Slug, a master regulator of epithelial-mesenchymal transition, is dramatically upregulated in a variety of fibrotic diseases. The aim of this study is to investigate the role of epithelial-mesenchymal transition marker Slug in the pathogenesis of CsA-induced gingival overgrowth.Methods: Clinically healthy gingiva and CsA-induced gingival overgrowth specimens were analyzed by immunohistochemistry. The effect of CsA on normal human gingival fibroblasts (HGFs) was used to elucidate whether Slug expression could be affected by CsA by real-time reverse transcription-polymerase chain reaction and western blot. Cell proliferation in CsA-treated HGFs with Slug lentiviral-mediated shRNAi knockdown was evaluated by tetrazolium bromide reduction assay.Results: Slug expression was higher in CsA-induced gingival overgrowth specimens than in clinical healthy gingiva (p < 0.05). Slug expression was significantly higher in CsA-induced gingival overgrowth specimens with higher levels of inflammatory infiltrates (p < 0.05). CsA was found to increase Slug transcript and protein expression in HGFs in a dose-dependent manner (p < 0.05). In addition, knockdown of Slug significantly suppressed CsA-induced cell proliferation in HGFs (p < 0.05).Conclusion: Taken together, upregulation of Slug in HGFs stimulated by CsA may play an important role in the pathogenesis of CsA-induced gingival overgrowth. [ABSTRACT FROM AUTHOR]

Background/purpose: Cyclosporine A (CsA) is used as an immunosuppressive agent, and its prominent side effect is the induction of gingival overgrowth. Snail is a master regulator of epithelial-mesenchymal transition (EMT). EMT under pathological processes could lead to fibrotic changes. The purpose of this study was to investigate the role of Snail in the pathogenesis of CsA-induced gingival overgrowth.Methods: The effect of CsA on normal human gingival fibroblasts (HGFs) was used to elucidate whether Snail expression could be induced by CsA by using quantitative real-time reverse transcription-polymerase chain reaction and western blot. The cell proliferation rate in CsA-treated HGFs with Snail lentiviral-mediated short hairpin RNA interference (shRNAi) knockdown was evaluated by tetrazolium bromide reduction assay.Results: CsA increased the Snail transcript and Snail protein expression in HGFs in a dose-dependent manner (p < 0.05). In addition, downregulation of Snail by lentiviral infection significantly reduced CsA-stimulated cell proliferation in HGFs (p < 0.05).Conclusion: CsA stimulated Snail expression and cell proliferation in HGFs, while silencing Snail could effectively reverse these phenomena. These results may provide new avenues for the design of novel antifibrotic therapies for CsA-induced gingival overgrowth through targeting Snail. [ABSTRACT FROM AUTHOR]

Background/Purpose The prominent side effect of the immunosuppressive drug cyclosporine A (CsA) is gingival overgrowth. Hypoxia-inducible factor (HIF)-1α regulates a wide variety of profibrogenic genes, which are closely associated with tissue fibrosis. The aim of this study was to compare HIF-1α expression in normal gingival tissues and CsA-induced gingival overgrowth specimens and further explore the potential mechanisms that may lead to induction of HIF-1α expression. Methods Fifteen CsA-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. Western blot was used to investigate the effects of CsA on the expression of HIF-1α in cultured human gingival fibroblasts. The effects of CsA on plasminogen activator inhibitor (PAI)-1 expression were evaluated in environmental hypoxia. Results HIF-1α staining in gingival tissue was stronger in CsA-induced gingival overgrowth group than normal gingival group ( p < 0.05). The expression of HIF-1α was significantly higher in CsA-induced gingival overgrowth specimens with higher levels of inflammatory infiltrates ( p = 0.041). CsA was found to upregulate HIF-1α protein in a dose-dependent manner ( p < 0.05). Hypoxia increased CsA-induced PAI-1 protein expression than normoxic conditions ( p < 0.05). Conclusion These results suggest that HIF-1α expression is significantly upregulated in CsA-induced gingival overgrowth specimens. The activation of HIF-1α may promote fibrogenesis by an increase of PAI-1 expression and a subsequent elevation of extracellular matrix production in gingival tissues. [ABSTRACT FROM AUTHOR]

Background/Purpose Transforming growth factor-β (TGF-β) plays an important role in the pathogenesis of cyclosporine A (CsA)-induced gingival overgrowth (GO). Connective tissue growth factor (CTGF/CCN2) acts as a cofactor with TGF-β to induce the maximal profibrotic effects of TGF-β. We investigated the effects of CsA on CCN2 expression in human gingival fibroblasts (HGFs) and the potential chemopreventive agent for CsA-induced GO. Methods Western blot analyses were used to examine the signaling pathways of CsA-induced CCN2 expression in HGFs and whether epigallocatechin-3-gallate (EGCG), curcumin, or lovastatin can inhibit CsA-induced CCN2 expression. Results CsA significantly stimulated CCN2 synthesis in HGFs. This effect can be inhibited by c-Jun NH 2 -terminal kinase (JNK) and Smad3 inhibitors but not by TGF-β neutralizing antibody and TGF-β type I receptor inhibitor. Furthermore, EGCG completely blocked CsA-induced CCN2 expression. Conclusion CsA-induced CCN2 protein expression is mediated through JNK and Smad signaling. CsA may contribute to the pathogenesis of GO through upregulation of CCN2 expression in HGFs. EGCG could be an adjuvant for the prevention of CsA-induced GO. [ABSTRACT FROM AUTHOR]

Background/Purpose: Connective tissue growth factor (CTGF/CCN2) is involved in the development and progression of fibrotic diseases, including gingival overgrowth (GO). Recent studies indicate that lysophosphatidic acid (LPA) is also significantly involved in wound healing and the development of fibrosis. This study investigated whether epigallocatechin-3-gallate (EGCG) can inhibit LPA-induced CCN2 expression in human gingival fibroblast (GF) and its mechanism. Methods: Western blot analyses were used to study the signaling pathways of LPA-induced CCN2 expression in human GFs and the effects of EGCG on this pathway. Results: LPA stimulated CCN2 synthesis in human GFs. This effect can be significantly inhibited bytransforming growth factor-β type I receptor/ALK5, Smad3, and JNK inhibitors but not ERK, P38, and MAPK inhibitors. EGCG completely inhibited LPA-induced CCN2 expression through attenuating the LPA-induced JNK and Smad3 phosphorylation in human GFs. Conclusion: LPA produced at the surgical wound may contribute to the recurrence of GO by upregulating CCN2 expression in human GFs. This effect was mediated by Smad3 and JNK activation and ALK5 transactivation. EGCG could be a useful agent for reducing the recurrence of GO after surgery through suppression of JNK and Smad3 activations. [ABSTRACT FROM AUTHOR]

Background/Purpose: Gingival overgrowth can be induced in patients treated with cyclosporine-A (CsA), an immunosuppressant often used following organ transplantation. A pre-existing rat model designed to mimic CsA-induced gingival overgrowth in humans was used to test the effectiveness of frequent application of a chlorhexidine antiplaque solution in reducing the overgrowth. Methods: Four groups of rats were fed CsA. One group received chlorhexidine mouthwash twice a day, the second group received chlorhexidine mouthwash once a day, the third group received chlorhexidine mouthwash every other day, and the fourth group did not receive chlorhexidine mouthwash all. A fifth negative control group received only mineral oil. Overgrowth was determined by measuring the changes in the gingival probing depth and the keratinized gingival widthon molars. A gingival histological examination was performed. Results: Rats treated with mouthwash twice daily exhibited decreased probing depths and gingival widths without statistical significance. Histological examination revealed that CsA treatment caused gingival enlargement, whereas chlorhexidine treatment twice a day diminished the enlargement. Conclusion: These findings suggest that chlorhexidine mouthwash used twice daily may reduce the severity of CsA-induced gingival overgrowth. Further research is warranted to determine the optimal dose and treatment regimen. [Copyright &y& Elsevier]