Cyclosporin A regulates interleukin-1beta and interleukin-6 expression in gingiva: implications for gingival overgrowth

BACKGROUND: Gingival overgrowth is a common side effect following the administration of cyclosporin A (CsA); however, the cellular mechanisms remain poorly understood. CsA's immunosuppressant properties involve the regulation of synthesis and cellular response to cytokines. A CsA-induced alteration in the cytokine profile within gingival tissue could provide a mechanism for gingival hyperplasia. The aim of this study was to investigate the effects of CsA on the production of 2 cytokines - interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) - by both gingival fibroblasts and peripheral blood mononuclear cells (PBMC). METHODS: Cells were stimulated for 24 hours in the presence of CsA over a concentration range of 100 to 2,000 ng/ml and the resultant cytokine production determined by ELISA. In addition, levels of both cytokines within normal, inflamed, and overgrown gingival tissue were determined. RESULTS: CsA inhibited IL-6 production by gingival fibroblasts in a dose-dependent manner. In contrast, at a concentration of 2,000 ng/ml, CsA stimulated IL-6 production by PBMC (P <0.05). Fibroblasts derived from overgrown gingiva produced significantly higher levels of IL-6 than their normal counterparts (P <0.05). CsA inhibited IL-1beta production by PBMC over the whole concentration range (P <0.05). IL-1beta was not found in measurable quantities in any of the fibroblast cultures. Levels of IL-6 extracted from overgrown gingival tissue were significantly higher than in inflamed or normal tissue. In contrast IL-1beta levels in overgrown tissue were not statistically significantly greater than those in inflamed tissue. CONCLUSIONS: These results show that CsA does regulate cytokine expression in gingival tissue. This effect may play an important role in the pathogenesis of CsA-induced gingival overgrowth.     

Molecular events associated with ciclosporin A-induced gingival overgrowth are attenuated by Smad7 overexpression in fibroblasts

Sobral LM, Aseredo F, Agostini M, Bufalino A, Pereira MCC, Graner E, Coletta RD. Molecular events associated with ciclosporin A‐induced gingival overgrowth are attenuated by Smad7 overexpression in fibroblasts. J Periodont Res 2012; 47: 149–158. © 2011 John Wiley & Sons A/S Background and Objective: Ciclosporin A (CsA)‐induced gingival overgrowth is attributed to an exaggerated accumulation of extracellular matrix, which is mainly due to an increased expression of transforming growth factor‐β1 (TGF‐β1). Herein, the in vitro investigation of effects of overexpression of Smad7, a TGF‐β1 signaling inhibitor, in the events associated with CsA‐induced extracellular matrix accumulation was performed. Material and Methods: The effects of Smad7 were assessed by stable overexpression of Smad7 in fibroblasts from normal gingiva. Smad7‐overexpressing cells and control cells were incubated with CsA, and synthesis of type I collagen, production and activity of MMP‐2 and cellular proliferation were evaluated by ELISA, zymography, growth curve, bromodeoxyuridine incorporation assay and cell cycle analysis. The effects of CsA on cell viability and apoptosis of fibroblasts from normal gingiva were also evaluated. Western blot and immunofluorescence for phospho‐Smad2 were performed to measure the activation of TGF‐β1 signaling. Results: Although the treatment with CsA stimulated TGF‐β1 production in both control and Smad7‐overexpressing fibroblasts, its signaling was markedly inhibited in Smad7‐overexpressing cells, as revealed by low levels of phospho‐Smad2. In Smad7‐overexpressing cells, the effects of CsA on proliferation, synthesis of type I collagen and the production and activity of MMP‐2 were significantly blocked. Smad7 overexpression blocked CsA‐induced fibroblast proliferation via p27 regulation. Neither CsA nor Smad7 overexpression induced cell death. Conclusion: The data presented here confirm that TGF‐β1 expression is related to the molecular events associated with CsA‐induced gingival overgrowth and suggest that Smad7 overexpression is effective in blocking these events, including proliferation, type I collagen synthesis and MMP‐2 activity.     

环孢素A与牙龈卟啉菌内毒素对人牙龈成纤维细胞增殖及TGF-β1分泌影响的研究

目的 探讨环孢素A(CsA)与牙龈卟啉菌内毒素(Pg LPS)对人牙龈成纤维细胞增殖及分泌转化生长因子-β1(TGF-β1)的影响。方法 用组织块法原代培养健康人牙龈成纤维细胞,分别用800 ng/ml CsA、100 ng/ml LPS、800 ng/ml CsA+100 ng/ml LPS作用于生长良好的第4~8代细胞,用MTT法测定作用后第2、3、5天的吸光值。ELISA法检测第3天时细胞上清中的TGF-β1分泌的变化。结果 细胞经CsA或LPS作用后形态无明显变化,CsA能促进细胞增殖和分泌TGF-β1,LPS作用与其相反,二者联合作用能明显促进细胞的生长及TGF-β1分泌,比单纯使用CsA更明显。结论 CsA与LPS在人牙龈成纤维细胞增殖和TGF-β1分泌方面可能有协同作用。     

Cyclosporin A and hydroxycyclosporine (M-17) affect the secretory phenotype of human gingival fibroblasts

The responsiveness of human gingival fibroblast populations to cyclosporin A (CsA) and its principal metabolite, hydroxycyclosporine (M17), was evaluated in cell culture. Gingival fibroblasts exhibited a dose-dependent accumulation and bell-shaped distribution of dansylated CsA. A 100-fold excess of non-labeled CsA prevented the accumulation of the fluorescent probe in the fibroblasts. Both CsA (400 ng/ml) and M17 (100 ng/ml) stimulated mean gingival fibroblast cell number to 23.2% and 36.7% above controls, and reduced mean collagen production by 37.7% and 37.4% below controls, respectively; however, neither CsA nor M17 affected mean protean production in comparison to control cultures. Analyses of responses to CsA and M17 by ligand-accumulating and non-accumulating fibroblasts sorted out from the parent cultures did not provide consistent interstrain responses either by cells representing the upper quartile of fluorescence or cells representing the bottom quartiles of fluorescence. These data demonstrate that CsA is accumulated by gingival fibroblasts and that CsA and M17 are potent modulators of gingival fibroblast phenotype.     

Effect of tranilast on matrix metalloproteinase-1 secretion from human gingival fibroblasts in vitro

BACKGROUND: Some drugs such as phenytoin, calcium blockers, or cyclosporins are known to cause gingival fibrous hyperplasia, an unwanted side effect. Decreased collagen catabolism in overgrown gingival tissue has been proposed as one of the reasons causing the disease. The effect of tranilast, which suppresses collagen synthesis and cell proliferation, on matrix metalloproteinase (MMP-1) secretion from human gingival fibroblast, was studied in vitro. METHODS: Human gingival fibroblasts were cultured from specimens taken from healthy, periodontal, and overgrown gingival tissues. The effects of tranilast on cell proliferation and MMP-1 secretion from gingival fibroblast were assessed. Inhibitory effect of transforming growth factor (TGF)-beta secretion from gingival fibroblast by tranilast was also evaluated. RESULTS: Tranilast did not interfere with cell proliferation at the low concentrations. MMP-1 concentration significantly increased at the lower doses of tranilast up to about 2-fold compared to controls (P < 0.05). In contrast, higher doses of tranilast significantly decreased activity to 30% and 20%, respectively. MMP-1 secretion was inhibited significantly by phenytoin, nifedipine, and cyclosporin A and the depressed MMP-1 recovered to the control level with tranilast. The amount of secretion from normal and periodontitis gingival fibroblast specimens did not differ, but that from the overgrown gingiva was significantly less than the other types. Moreover, TGF-beta secretion was significantly inhibited by 300 microM of tranilast. CONCLUSIONS: Tranilast upregulates the expression of type 1 collagenase suppressed by gingival overgrowth-inducing drugs, and inhibits TGF-beta secretion from gingival fibroblasts. Therefore, tranilast could be considered as an agent for controlling gingival over-growth.     

Regulation of fibroblast-induced collagen gel contraction by interleukin-1β

Fibroblasts incorporated within collagen gels induce a cell-mediated contraction of the gel to form a three-dimensional, tissue-like structure by a mechanism thought to mimic wound contraction in vivo . In this study a gel contraction model was used to investigate the ability of fibroblasts derived from adult gingiva, adult skin and fetal skin to organise a collagen matrix. In addition the effects of interleukin-1β (IL-1β) on the contraction process was also investigated. Over the concentration range 5-50 U/ml, IL-1β induced a statistically significant inhibition of gel contraction in all fibroblast cell types ( P <0.05), although fetal fibroblasts appeared least responsive and gingival fibroblasts most responsive to the inhibitory effects of this cytokine. Comparison of gel contraction by the different fibroblast strains indicated that fetal and gingival fibroblasts shared similar contraction kinetics. For the adult skin fibroblasts, three of five strains studied showed significantly diminished levels of gel contraction compared to fetal and gingival cells. This apparent difference in fibroblast phenotype may, at least in part, explain the fetal-like wound healing pattern seen in the oral mucosa.     

姜黄素对环孢素A诱导激活的大鼠牙龈成纤维细胞TGF-β1/Smad3通路的抑制作用

目的：体外实验研究姜黄素（curcumin,Cur）对环孢素A（cyclosporine A,CsA）作用大鼠牙龈成纤维细胞TGF-β1/Smad3通路的影响,为进一步探讨Cur抑制CsA所致药物性牙龈增生的发生机制提供理论依据。方法：使用CCK-8法观察0、5、10、20、30 μmol/L Cur对大鼠牙龈成纤维细胞增殖的影响, 20 μmol/L Cur+200 ng/mL CsA共同作用对大鼠牙龈成纤维细胞增殖的影响。实时荧光定量PCR检测20 μmol/L Cur+200 ng/mL CsA共同作用下,牙龈成纤维细胞中转化生长因子TGF-β1、Smad3、平滑肌肌动蛋白α-SMA和I型胶原蛋白COL-I的mRNA表达变化;蛋白免疫印迹实验检测TGF-β1、Smad3、p-Smad3、α-SMA和COL-I的蛋白表达变化。细胞划痕实验观察20 μmol/L Cur+200 ng/mL CsA 对牙龈成纤维细胞迁移能力的影响。采用SPSS 23.0 软件包对数据进行统计学分析。结果：大鼠牙龈成纤维细胞在20 μmol/L Cur+200 ng/mL CsA共同作用下,细胞增殖和迁移能力明显降低;20 μmol/L Cur显著下调了牙龈成纤维细胞中TGF-β1、α-SMA 和COL-I的mRNA 表达,蛋白免疫印迹实验提示,TGF-β1、p-Smad3、α-SMA 和COL-I的表达同样显著下调。结论：Cur可能通过抑制CsA激活的牙龈成纤维细胞TGF-β1/Smad3 信号通路,从而降低牙龈成纤维细胞增殖、迁移、平滑肌肌动蛋白和胶原分泌,改善牙龈增生。     

Phenotypic differences in growth, matrix synthesis and response to nifedipine between fibroblasts derived from clinically healthy and overgrown gingival tissue

Gingival overgrowth is a disfiguring condition affecting 10–20% of patients on nifedipine therapy. The pathogenesis of this condition, although unclear, is thought to involve an interaction between the drug and resident gingival fibroblasts. The aim of the present study was to investigate the cellular mechanisms underlying this condition using cell culture techniques. Gingival fibroblast cell lines were derived by explant culture from two patients on long-term nifedipine therapy exhibiting gingival overgrowth ('responders') and from two patients on similar therapy with clinically healthy gingiva ('non responders'). Comparative studies showed phenotypic differences between the two cell types, 'responded cells having an increased growth potential and producing increased levels of protein and collagen compared to 'non responded lines. Addition of exogenous nifedipine (10–1000 ng/ml) to cultures had no effect on 'non-responder' cells but induced a significant inhibitory response in the 'responder' cells. Although adding support to the concept that nifedipine-sensitive fibroblasts reside within overgrown connective tissue, the inhibitory effect of the drug on cell growth and matrix synthesis was surprising in view of the clinical appearance of this condition.     

Enamel matrix derivative induces production of vascular endothelial cell growth factor in human gingival fibroblasts

Enamel matrix derivative (EMD) may enhance periodontal wound healing by inducing angiogenesis. We sought to investigate the effect and the mechanism of action of EMD on vascular endothelial growth factor (VEGF) production by human gingival fibroblasts. Cells were stimulated with EMD, transforming growth factor‐β1 (TGF‐β1), or fibroblast growth factor 2 (FGF‐2), with or without antibodies to TGF‐β1 or FGF‐2. The levels of VEGF in the culture media were measured using an ELISA. We examined the effects of SB203580 [a p38 mitogen‐activated protein kinase (MAPK) inhibitor], U0126 [an extracellular signal‐regulated kinase (ERK) inhibitor], SP600125 [a c‐Jun N‐terminal kinase (JNK) inhibitor], and LY294002 [a phosphatidylinositol 3‐kinase (PI3K)/Akt inhibitor] on EMD‐induced VEGF production. Enamel matrix derivative stimulated the production of VEGF in a dose‐ and time‐dependent manner. Treatment of human gingival fibroblasts with antibodies to TGF‐β1 or FGF‐2 significantly decreased EMD‐induced VEGF production, whereas the addition of exogenous TGF‐β1 and FGF‐2 stimulated VEGF production. Enamel matrix derivative‐induced VEGF production was significantly attenuated by SB203580, U0126, and LY294002. Our results suggest that EMD stimulates VEGF production partially via TGF‐β1 and FGF‐2 in human gingival fibroblasts and that EMD‐induced VEGF production is regulated by ERK, p38 MAPK, and PI3K/Akt pathways. Enamel matrix derivative‐induced production of VEGF by human gingival fibroblasts may be involved in the enhancement of periodontal wound healing by inducing angiogenesis.     

Immunohistochemical analysis of epidermal growth factor receptor in cyclosporin A-induced gingival overgrowth

Cyclosporin A (CsA)-induced gingival overgrowth represents a tissue of fibrosis and epidermal growth factor (EGF) has been shown to induce extracellular matrix synthesis by fibroblasts. The purpose of this study was to evaluate the expression of EGF-receptor (EGF-r) in frozen sections of CsA-induced overgrown gingival tissue using immunohistochemical and semiquantitative techniques. Gingival biopsies were obtained from 12 renal transplant patients receiving CsA as well as 9 systemically and periodontally healthy individuals. Immunohistochemical staining procedures were carried out in frozen sections of gingival tissue and the expression of EGF-r was compared between the two study groups. The expression of EGF-r was more pronounced in the oral gingival epithelium of CsA-induced overgrown gingiva as compared to those of the clinically healthy gingival specimens. The reactivity in the inflammatory infiltrate and connective tissue cells of both of the study groups was similar. In conclusion, the results of the present study may suggest that CsA affects EGF-r metabolism in gingival keratinocytes resulting in an increased number of cell surface receptors, which may eventually play a role in the pathogenesis of gingival tissue alterations.     

Immunohistochemical analysis of epidermal growth factor receptor in cyclosporin A-induced gingival overgrowth

Cyclosporin A (CsA)induced gingival overgrowth represents a tissue of fibrosis and epidermal growth factor (EGF) has been shown to induce extracellular matrix synthesis by fibroblasts. The purpose of this study was to evaluate the expression of EGF-receptor (EGF-r) in frozen sections of CsA-induced overgrown gingival tissue using immunohistochemical and semiquantitative techniques. Gingival biopsies were obtained from 12 renal transplant patients receiving CsA as well as 9 systemically and periodontally healthy individuals. Immunohistochemical staining procedures were carried out in frozen sections of gingival tissue and the expression of EGF-r was compared between the two study groups. The expression of EGF-r was more pronounced in the oral gingival epithelium of CsA-induced overgrown gingiva as compared to those of the clinically healthy gingival specimens. The reactivity in the inflammatory infiltrate and connective tissue cells of both of the study groups was similar. In conclusion, the results of the present study may suggest that CsA affects EGF-r metabolism in gingival keratinocytes resulting in an increased number of cell surface receptors, which may eventually play a role in the pathogenesis of gingival tissue alterations.     

Cyclosporin A upregulates prostaglandin E2 production in human gingival fibroblasts challenged with tumor necrosis factor alpha in vitro

The effect of Cyclosporin A (CsA) on prostaglandin E₂ (PGE₂) production in human gingival fibroblasts challenged with tumor necrosis factor alpha (TNF-α) was studied. TNF-α (1-100 ng/ml) dose-dependently stimulated PGE₂; formation in 24 h cultures. CsA (1-100 ng/ml) did not induce PGE₂; formation itself but potentiated TNF-α induced PGE; formation in gingival fibroblasts in a manner dependent on the concentrations of both CsA and TNF-α. TNF-α (10 ng/ml) stimulated the release of [³H]-arachidonic acid (A.A) from prelabelled fibroblasts that was potentiated by CsA (100 ng/ml). Addition of exogenous unlabelled AA (5-20 μM/ml) to the cells resulted in enhanced PGE₂: formation that was not potentiated by CsA (100 ng/mi). Furthermore. CsA (100 ng/ml) did not further increase the level of cyclooxygenase-2 mRNA induced by TNF-α (10 ng/ml). although PGE₂ formation was enhanced. The results indicate that CsA and TNF-α act in concert on PGE₂ formation in gingival fibroblasts. which may be of importance in the pathogenesis of gingival overgrowth induced by the drug.     

Extracellular glycosaminoglycan changes in healthy and overgrown gingiva fibroblasts after cyclosporin A and cytokine treatments

BACKGROUND: It has been demonstrated that cyclosporin A (CyA) blocks the immune system, acts on cytoskeleton and stimulates the production of extracellular matrix (ECM) and transforming growth factor-beta1 (TGF-beta1). This cytokine, such as transforming growth factor-alpha (TGF-alpha), induces deposition of glycosaminoglycans (GAG), proteoglycans and collagen fibres in the ECM. METHODS: In this work, we examined the effect induced by CyA, TGF-beta1 and TGF-alpha on cultures of healthy and overgrown human gingival fibroblasts in order to evaluate the glycosaminoglycan, cytoskeletal changes and the behaviour of fibroblasts after concanavalin A (Con A) treatment. Moreover, we examined gingival biopsies by Alcian blue histochemical staining and electron transmission microscopy. RESULTS: Total and extracellular sulphated GAG in overgrown gingiva specimens and in derived fibroblast cultures treated with CyA and cytokines were significantly higher than controls. The action of cytokines was increased (P < or = 0.01) compared with CyA with a greater effect of TGF-alpha in comparison with TGF-beta1; the electron microscopy showed ECM accumulation. The agglutinations showed the heterogeneity of fibroblast populations. CONCLUSIONS: Stimulation with Con A showed that the fibroblast population had cell surface heterogeneity, and could respond in a different way to both CyA and cytokine stimulus. Moreover, increased synthesis of GAG in overgrown gingiva compared with synthesis in normal fibroblasts before CyA treatment suggests a possible genetic origin of damage. As not all CyA-treated patients develop gingival overgrowth, a genetic predisposition may explain the different responses of gingival fibroblast populations.     

Effect of cell donor age on the synthetic properties of fibroblasts obtained from phenytoin-induced gingival hyperplasia

Diploid fibroblasts obtained from explants of human gingiva and maintained in vitro undergo a several-fold decrease in protein and collagen synthesis as a function of increasing donor age. Using drug-induced gingival hyperplasia as a model, we performed experiments to learn whether fibroblasts derived from hyperplastic tissue behave in a similar manner. Fibroblast strains were established from explants of hyperplastic gingiva obtained from 10 patients chronically ingesting phenytoin and ranging in age from 9 to 45 years. Protein production and degradation were compared to previously reported data similarly obtained from periodontally normal donors ranging in age from 12 to 68 yr. The total quantity of protein and collagen produced by the phenytoin cells was significantly greater than previously reported for cells from normal gingiva. No donor age-related decrease in protein and collagen production nor in the proportion of cell synthetic activity committed to collagen production was observed for cultures of phenytoin cells. The gross pattern of proteins produced, as assessed by 2-dimensional gel electrophoresis, was unrelated to donor age in both normal and phenytoin cells, but three polypeptides ranging in size from about 20 kD to 40 kD that were not found in the cultures of normal cells were produced by five of seven phenytoin cells strains. The observations demonstrate that the phenytoin cells do not undergo the donor age-dependent decrease in synthesis observed for normal cells. This abnormality may account in part for the phenytoin-induced hyperplasia. The phenytoin cells appear to be a unique phenotype.     

Role of Transforming Growth Factor‐beta1 in Cyclosporine‐Induced Epithelial‐to‐Mesenchymal Transition in Gingival Epithelium

Background: It has been proposed that cyclosporin A (CsA) may induce epithelial‐to‐mesenchymal transition (EMT) in gingiva. The aims of the present study are to confirm the notion that EMT occurs in human gingival epithelial (hGE) cells after CsA treatment and to investigate the role of transforming growth factor beta1 (TGF‐β1) on this CsA‐induced EMT. Methods: The effects of CsA, with and without TGF‐β1 inhibitor, on the morphologic changes of primary culture of hGE cells were examined in vitro. The changes of protein and messenger RNA (mRNA) expressions of two EMT markers (E‐cadherin and alpha‐smooth muscle actin) in the hGE cells after CsA treatment with and without TGF‐β1 inhibitor were evaluated with immunocytochemistry and real‐time polymerase chain reaction. Results: The epithelial cells became spindle‐like, elongated, and disassociated from neighboring cells and lost their original cobblestone monolayer pattern when CsA was added. However, the epithelial cells stayed in their original cobblestone morphology with treatment of TGF‐β1 inhibitor on top of the CsA treatment. When CsA was given, the protein and mRNA expressions of E‐cadherin and α‐SMA were significantly altered, and these alterations were significantly reversed with pretreatment of TGF‐β1 inhibitor. Conclusions: CsA could induce Type 2 EMT in gingiva by changing the morphology of epithelial cells and altering the EMT markers/effectors. The CsA‐induced gingival EMT is dependent or at least partially dependent on TGF‐β1.     

Abnormal cellular property of fibroblasts from congenital gingival fibromatosis

Two strains of cultured cells were isolated and characterized from the gingiva of two siblings with congenital gingival fibromatosis. The growth rate of both fibroblast strains was slower than that of comparable cells obtained from the normal gingiva of control individuals. The amounts of substances, including collagen and glycosaminoglycans, biosynthesized by the diseased cells were much greater than those by the control cells from normal gingivae. Namely, 11.7-13.7% of the protein synthesized by diseased cells was collagen, whereas collagen accounted for only 6.1-8.5% of the total protein produced by normal cells. Moreover, the production of a large amount of extracellular substances by the diseased cells was confirmed by electron microscopic examination. These observations suggest that the fibromatosis tissues contain affected fibroblasts which have low growth activity but are active in the production of much greater amounts of collagen and other extracellular substances compared to normal fibroblasts.