RESUMO
Cyclosporin A (CyA) induces gingival overgrowth via its stimulatory effects on expression of transforming growth factor-beta1 (TGF-beta1) and collagen. It is not known whether CyA has a direct effect on gingival fibroblasts or induces its effect indirectly via stimulation of myofibroblast transdifferentiation. The present study was undertaken to examine the in vivo and in vitro effect of CyA on myofibroblast transdifferentiation. Rats were treated for 60 days with a daily subcutaneous injection of CyA, and the gingival overgrowth tissue was analyzed by immunohistochemistry. In vitro, fibroblasts from normal gingiva (NG) were cultured in the presence of different concentrations of CyA, and subjected to semi-quantitative reverse transcriptase-polymerase chain reaction and western blot. Although CyA treatment stimulated TGF-beta1 expression by NG fibroblasts, it lacked to induce expression and production of isoform alpha of smooth muscle actin (alpha-SMA), the specific myofibroblast marker. The expression levels of connective tissue growth factor (CTGF), which has been considered a key molecule to promote the transdifferentiation of myofibroblasts via TGF-beta1 activation, were unaffected by CyA. Our results demonstrate that CyA-induced gingival overgrowth is not associated with activation of myofibroblast transdifferentiation, since CyA is not capable to increase CTGF expression.
Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Ciclosporina/farmacologia , Fibroblastos/efeitos dos fármacos , Crescimento Excessivo da Gengiva/induzido quimicamente , Imunossupressores/farmacologia , Actinas/metabolismo , Adulto , Animais , Western Blotting , Técnicas de Cultura de Células , Colágeno/metabolismo , Fator de Crescimento do Tecido Conjuntivo/análise , Meios de Cultura , Fibroblastos/citologia , Fibroblastos/metabolismo , Crescimento Excessivo da Gengiva/metabolismo , Humanos , Masculino , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/análise , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Cyclosporin A (CyA) induces gingival overgrowth via its stimulatory effects on expression of transforming growth factor-beta1 (TGF-â1) and collagen. It is not known whether CyA has a direct effect on gingival fibroblasts or induces its effect indirectly via stimulation of myofibroblast transdifferentiation. The present study was undertaken to examine the in vivo and in vitro effect of CyA on myofibroblast transdifferentiation. Rats were treated for 60 days with a daily subcutaneous injection of CyA, and the gingival overgrowth tissue was analyzed by immunohistochemistry. In vitro, fibroblasts from normal gingiva (NG) were cultured in the presence of different concentrations of CyA, and subjected to semi-quantitative reverse transcriptase-polymerase chain reaction and western blot. Although CyA treatment stimulated TGF-â1 expression by NG fibroblasts, it lacked to induce expression and production of isoform á of smooth muscle actin (á-SMA), the specific myofibroblast marker. The expression levels of connective tissue growth factor (CTGF), which has been considered a key molecule to promote the transdifferentiation of myofibroblasts via TGF-â1 activation, were unaffected by CyA. Our results demonstrate that CyA-induced gingival overgrowth is not associated with activation of myofibroblast transdifferentiation, since CyA is not capable to increase CTGF expression.
Assuntos
Adulto , Animais , Humanos , Masculino , Ratos , Transdiferenciação Celular/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Ciclosporina/farmacologia , Fibroblastos/efeitos dos fármacos , Crescimento Excessivo da Gengiva/induzido quimicamente , Imunossupressores/farmacologia , Actinas/metabolismo , Western Blotting , Técnicas de Cultura de Células , Meios de Cultura , Colágeno/metabolismo , Fator de Crescimento do Tecido Conjuntivo/análise , Fibroblastos/citologia , Fibroblastos/metabolismo , Crescimento Excessivo da Gengiva/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/análise , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Glycosaminoglycans (GAGs) play important roles in cell behavior and have the ability to bind and modulate cytokines. Using primary cultured fibroblasts from hereditary gingival fibromatosis (HGF), normal gingiva (NG), and NG treated with cyclosporin-A (NGc) we show changes in the expression and structural characteristics of GAGs as well as in the expression of enzymes involved in their biosynthesis and degradation. In addition, we show the over-expression of TGF-beta1 and TGF-beta type II receptor in HGF and NGc. There is an increase in the GAGs retained in the cellular fraction, and the fine structure of galactosaminoglycans show a decrease in alpha-l-iduronic acid content in HGF and NGc. Elevated extracellular levels of low molecular weight hyaluronan (HA) are found in HGF due to increase in the expression of HA synthase 3 and hyaluronidases 1 and 2. The results bring new insights to the accumulation of extracellular matrix related to TGF-beta over-expression.
Assuntos
Fibroblastos/metabolismo , Crescimento Excessivo da Gengiva/metabolismo , Glicosaminoglicanos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima , Células Cultivadas , Ciclosporina/farmacologia , Fibromatose Gengival/metabolismo , Gengiva/efeitos dos fármacos , Gengiva/metabolismo , Humanos , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
BACKGROUND: Gingival overgrowth (GO) is a common side effect of the chronic use of cyclosporine (CsA), an immunosuppressant widely used to prevent rejection in transplant patients. Recent studies have reported elevated levels of specific cytokines in gingival overgrowth tissue, particularly TGF-beta, suggesting that this growth factor plays a role in the accumulation of extracellular matrix materials. The effectiveness of azithromycin, a macrolide antibiotic, in the regression of this undesirable side effect has also been demonstrated. METHODS: In this study, we created an experimental model for assessing the therapeutic effect of roxithromycin in GO and the expression of transforming growth factor beta (TGF-beta2) through immunohistochemistry. We used four groups of rats totaling 32 individuals. GO was induced during five weeks and drug treatment was given on the 6th week as follows: group 1 received saline; group 2 received CsA and was treated with saline on the 6th week; group 3 received CsA and, on the 6th week, ampicilin; and group 4 received CsA during 5 weeks and, on the 6th week, was treated with roxithromycin. RESULTS: The results demonstrated that roxithromycin treatment was effective in reducing cyclosporine-induced GO in rats. Both epithelial and connective tissue showed a decrease in thickness and a significant reduction in TGF-beta2 expression, with a lower number of fibroblasts, reduction in fibrotic areas and decrease in inflammatory infiltrate. CONCLUSION: The present data suggest that the down-regulation of TGF-beta2 expression may be an important mechanism of action by which roxithromycin inhibits GO.
Assuntos
Antibacterianos/uso terapêutico , Crescimento Excessivo da Gengiva/tratamento farmacológico , Crescimento Excessivo da Gengiva/metabolismo , Roxitromicina/uso terapêutico , Fator de Crescimento Transformador beta2/biossíntese , Animais , Ciclosporina/efeitos adversos , Regulação para Baixo , Crescimento Excessivo da Gengiva/induzido quimicamente , Imuno-Histoquímica , Imunossupressores/efeitos adversos , Masculino , Distribuição Aleatória , Ratos , Ratos Wistar , Fator de Crescimento Transformador beta2/análiseRESUMO
BACKGROUND AND OBJECTIVE: Cyclosporin A-induced gingival overgrowth comprises a variety of signaling pathways (including growth factors and proteoglycans) that are still not completely understood. In the present study, gingival overgrowth was investigated in transplant patients receiving cyclosporin A (cyclosporin A group) and compared with gingival tissues never exposed to the drug (control group) by analyzing the gene expression of the cell-surface heparan sulfate proteoglycans syndecan-2, syndecan-4 and betaglycan. MATERIAL AND METHODS: mRNA analysis was carried out by reverse transcription-polymerase chain reaction amplification of pooled samples from nine patients of the cyclosporin A group and six control subjects. The groups were compared by the Student's t-test. RESULTS: The expression of heparan sulfate proteoglycans was increased in the cyclosporin A group (165% for syndecan-2, 308% for syndecan-4, and 42% for betaglycan) compared with the control group. CONCLUSION: Our findings agree with the current concept of cyclosporin A-induced gingival overgrowth and provide new evidence that its noncollagenous extracellular matrix is overexpressed.
Assuntos
Crescimento Excessivo da Gengiva/metabolismo , Proteoglicanas de Heparan Sulfato/biossíntese , Adulto , Estudos de Casos e Controles , Ciclosporina/efeitos adversos , Feminino , Expressão Gênica , Crescimento Excessivo da Gengiva/induzido quimicamente , Humanos , Imunossupressores/efeitos adversos , Transplante de Rim , Transplante de Fígado , Masculino , Pessoa de Meia-Idade , Proteoglicanas/biossíntese , RNA Mensageiro/análise , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas , Sindecana-2/biossíntese , Sindecana-4/biossínteseRESUMO
BACKGROUND: Cyclosporin (CsA) and tacrolimus (FK-506) are immunosuppressive drugs that specifically inhibit T-cell activation via calcineurin inhibition. Gingival overgrowth is a common side effect following the administration of CsA. The severity of gingival overgrowth seen in patients taking FK-506 is less than that observed with CsA. Little is known about the involvement of saliva in drug-induced gingival overgrowth. The purpose of this study was to investigate the salivary contents of tumor growth factor beta1 (TGF-beta1), epidermal growth factor (EGF), and interleukin-6 (IL-6) as well as the hystometry of gingival tissue obtained from rats treated with either FK-506 or CsA. METHODS: For 30 or 60 days rats received daily subcutaneous injection doses of either CsA or FK-506 (10 mg/kg). The concentrations of TGF-beta1, EGF, and IL-6 in saliva were determined by enzyme-linked immunosorbent assay, and after histological processing, the oral epithelium and connective tissue were assessed at the region of the lower first molars. RESULTS: The levels of TGF-beta1, EGF, and IL-6 in saliva were not significantly altered by any of the treatments after 30 days. After 60 days of treatment with CsA, gingival overgrowth and significant increase in salivary TGF-beta1, EGF, and IL-6 concentrations were observed; no statistically significant changes were induced by FK-506. CONCLUSION: Within the limits of this experimental study, it can be concluded that CsA, but not FK-506, induced gingival overgrowth associated with an increase of the salivary levels of the cytokines TGF-beta1, EGF, and IL-6.
Assuntos
Ciclosporina/efeitos adversos , Citocinas/metabolismo , Imunossupressores/farmacologia , Saliva/metabolismo , Tacrolimo/efeitos adversos , Animais , Fator de Crescimento Epidérmico/análise , Crescimento Excessivo da Gengiva/induzido quimicamente , Crescimento Excessivo da Gengiva/metabolismo , Imunossupressores/efeitos adversos , Interleucina-6/análise , Masculino , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta1RESUMO
Glycosaminoglycans are thought to accumulate in formative lesions like drug-induced gingival overgrowth. Recent evidences, however, suggest that the amounts of glycosaminoglycans are comparable in overgrown and healthy gingiva. Besides, alterations in the size distribution of glycosaminoglycan molecules isolated from phenytoin-induced overgrown samples have also been suggested. Therefore, we sought to determine possible differences in molecular size distribution of gingival glycosaminoglycans in other types of drug-induced overgrowths. Purified gingival glycosaminoglycans from healthy and cyclosporin- and nifedipine-induced overgrown gingival tissues were analyzed by agarose gel electrophoresis and their molecular-size distribution was evaluated by both gel filtration chromatography and polyacrylamide gel electrophoresis. Our results on the gingival glycosaminoglycan composition showed presence of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronic acid in all types of gingival tissues examined. In addition, hyaluronic acid was predominantly of a large size eluting near to the void volume of a Superose-6 column, while the sulfated glycosaminoglycans were mainly composed of low molecular size glycosaminoglycans. Our results show no differences in the molecular-size distribution of hyaluronic acid and sulfated glycosaminoglycans among healthy and drug-induced overgrown gingival tissues.
Assuntos
Bloqueadores dos Canais de Cálcio/efeitos adversos , Ciclosporina/efeitos adversos , Gengiva/química , Crescimento Excessivo da Gengiva/metabolismo , Glicosaminoglicanos/química , Imunossupressores/efeitos adversos , Nifedipino/efeitos adversos , Adolescente , Adulto , Sulfatos de Condroitina/isolamento & purificação , Cromatografia em Gel , Dermatan Sulfato/isolamento & purificação , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Glicosaminoglicanos/isolamento & purificação , Heparitina Sulfato/isolamento & purificação , Humanos , Ácido Hialurônico/isolamento & purificação , Pessoa de Meia-Idade , Estrutura Molecular , Peso Molecular , SefaroseRESUMO
BACKGROUND: Gingival overgrowth is one of the side effects associated with the systemic use of cyclosporin A (CsA). In vitro studies on the extracellular matrix of gingival tissues have demonstrated an altered composition, particularly an accumulation of proteoglycans and collagen. We investigated the gene expression of extracellular matrix proteoglycans in CsA-induced gingival tissue alterations. METHODS: mRNA expression of the proteoglycans perlecan, decorin, biglycan, and versican was analyzed by reverse transcription polymerase chain reaction (RT-PCR) in gingival samples obtained from 12 individuals, six with CsA-induced gingival overgrowth (CsA group) and six with a normal gingiva (control group). The RT-PCR products were subjected to 1% agarose gel electrophoresis containing ethidium bromide and analyzed qualitatively and semiquantitatively by densitometry. Density values were normalized by determining the expression of the housekeeping gene beta-actin in the same sample. Groups were compared by the Student's t test. RESULTS: Perlecan expression showed a marked increase (54%) in the CsA group compared to the control group (P < 0.01), while no significant differences were observed for the other proteoglycans. CONCLUSION: CsA-induced gingival overgrowth seems to be associated with increased expression of perlecan, a typical basement membrane proteoglycan, but not decorin, biglycan, or versican.
Assuntos
Ciclosporina/efeitos adversos , Proteínas da Matriz Extracelular/biossíntese , Crescimento Excessivo da Gengiva/induzido quimicamente , Crescimento Excessivo da Gengiva/metabolismo , Imunossupressores/efeitos adversos , Proteoglicanas/biossíntese , Adulto , Biglicano , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Decorina , Feminino , Expressão Gênica , Proteoglicanas de Heparan Sulfato/biossíntese , Humanos , Transplante de Rim/efeitos adversos , Lectinas Tipo C , Transplante de Fígado/efeitos adversos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , VersicanasRESUMO
Glycosaminoglycans in normal and cyclosporin-induced gingival overgrowth were extracted by papain digestion and purified by Mono Q-FPLC chromatography. The purified glycosaminoglycans were analyzed by agarose gel electrophoresis and by the pattern of degradation products formed by chondroitin lyases on HPLC chromatography. Our results on the glycosaminoglycan composition showed presence of chondroitin 4- and 6-sulfate, dermatan sulfate, heparan sulfate and hyaluronic acid in both normal gingiva and cyclosporin-induced gingival overgrowth. The total and relative amounts of glycosaminoglycans were similar between normal and overgrown gingiva. This suggests that the glycosaminoglycan composition is not changed in cyclosporin-induced gingival overgrowth. Our present biochemical results conflict with histochemical and biosynthetic data previously reported by other groups. Those studies suggested that the affected tissues contained higher levels of glycosaminoglycans and that cyclosporin induced comparably high levels of these compounds in in vitro cultures of gingival fibroblasts. Therefore, these discrepant results suggest that a cyclosporin-induced increase on gingival glycosaminoglycans still remains an open question. The implications of these conflicting results are discussed.