CsA-induced Gingival Overgrowth Research Articles

The role of androgen receptor gene in cyclosporine induced gingival overgrowth

Gingival overgrowth is a prominent side effect of cyclosporine (CsA) therapy in renal transplant patients. Although the exact mechanism by which this drug induces gingival overgrowth is uncertain, marked variations in individual susceptibility to this drug suggest a genetic predisposition. Studies have shown that genetic variation (polymorphism) in the trinucleotide cytosine-adenine- guanine (CAG) sequence in exon 1 of the androgen receptor (AR) gene is related to altered activity of the AR as a transcription factor. However, the relationship between the length of the CAG repeat and gingival overgrowth has not yet been studied. The present study was carried out to determine whether there is an association between CsA-induced gingival overgrowth and the length of the CAG repeats in the AR gene. Genomic DNA samples were prepared from the blood of 50 renal transplant patients with CsA-induced gingival overgrowth and from the blood of 100 renal transplant patients on CsA with no gingival overgrowth. The difference in allele distribution among the subjects with gingival overgrowth and control samples was statistically significant (p = 0.001). The findings suggest a link between CsA7induced gingival overgrowth and a smaller size of CAG repeat in the AR gene.

The Upregulation of Transglutaminase‐2 by Cyclosporin A in Human Gingival Fibroblasts Is Augmented by Oxidative Stress

Transglutaminase-2 (TGM-2) has been implicated in several fibrotic disorders and can be induced by reactive oxygen species (ROS). Hence, the authors hypothesize that cyclosporin A (CsA) may regulate TGM-2 via ROS, and this regulation may have a role in the pathogenesis of CsA-induced gingival overgrowth. Cytotoxicity, 2',7'-dichlorodihydrofluorescein diacetate assay, and Western blot were used to investigate the effects of CsA in human gingival fibroblasts (HGFs). In addition, extracellular signal-regulated kinase (ERK) inhibitor PD98059, phosphatidylinositol 3-kinase inhibitor LY294002, glutathione precursor N-acetyl-L-cysteine (NAC), curcumin, epigallocatechin-3 gallate (EGCG), and p38 inhibitor SB203580 were added to find the possible regulatory mechanisms. Concentrations of CsA >500 ng/mL demonstrated cytotoxicity to HGFs (P < 0.05). CsA enhanced the generation of intracellular ROS at concentrations >200 ng/mL (P <0.05). TGM-2 protein induced by CsA was found in HGFs in a dose- and time-dependent manner (P <0.05). The addition of PD98059, LY294002, NAC, curcumin, EGCG, and SB203580 markedly inhibited TGM-2 expression induced by CsA (P <0.05). These results demonstrate that CsA significantly upregulates intracellular ROS generation and elevates TGM-2 expression in HGFs. In addition, TGM-2 induced by CsA is downregulated by PD98059, LY294002, NAC, curcumin, EGCG, and SB203580.

Effect of adjunctive roxithromycin therapy on interleukin‐1β, transforming growth factor‐β1 and vascular endothelial growth factor in gingival crevicular fluid of cyclosporine A‐treated patients with gingival overgrowth

Systemic macrolide antibiotic administration has been shown to result in the elimination or reduction cyclosporine A-induced gingival overgrowth. Roxithromycin (ROX) is known to have anti-inflammatory, immunomodulatory and tissue reparative effects. This study was to evaluate the effect of adjunctive ROX therapy on cyclosporine A-induced gingival overgrowth and interleukin (IL)-1β, transforming growth factor (TGF)-β1 and vascular endothelial growth factor (VEGF) levels in gingival crevicular fluid of renal transplant patients. Thirty-one patients with clinically significant overgrowth and 16 periodontally healthy subjects were included in this randomized, double-blind, placebo-controlled, parallel-arm study. Patients received scaling and root planing (SRP) at baseline and randomized to take either ROX or placebo for 5 d. The clinical parameters, including plaque index, papillary bleeding index, probing depth and gingival overgrowth scores, were recorded. The amounts of IL-1β, TGF-β1 and VEGF in gingival crevicular fluid were detected by ELISA. Periodontal parameters as well as gingival crevicular fluid biomarker levels were evaluated at baseline and at 1 and 4 wk post-therapy. Following SRP plus ROX and SRP plus placebo therapy, significant improvements in clinical periodontal parameters of both study groups were observed (p < 0.025). In the ROX group, adjunctive ROX therapy resulted in a greater gingival overgrowth scores reduction compared with those in the placebo group at 4 wk (p < 0.017). Initial amounts of IL-1β, TGF-β1 and VEGF for both the ROX and placebo groups were significantly higher than those for healthy subjects (p < 0.017), with no statistical difference between the two study groups. At 1 and 4 wk post-therapy, significant decreases in the amounts of IL-1β, TGF-β1 and VEGF were observed in both study groups when compared with baseline (p < 0.025), but there was no difference in the levels of IL-1β and VEGF between the two study groups. The amount of decrease in TGF-β1 levels for the ROX group was statistically significant compared to that for the placebo group at 4 wk after treatment (p < 0.017). Our study indicated that combination of ROX with non-surgical therapy improves gingival overgrowth status and decreases gingival crevicular fluid TGF-β1 levels in patients with severe gingival overgrowth. The reduction of gingival crevicular fluid TGF-β1 following ROX therapy suggests an anti-inflammatory/immunomodulatory effect of ROX on the treatment of cyclosporine A-induced gingival overgrowth.

Interleukin-10 gene promoter polymorphisms are associated with cyclosporin A-induced gingival overgrowth in renal transplant patients

ObjectiveInterleukin-10 (IL-10) is an anti-inflammatory cytokine whose genetic polymorphisms are associated with the production of IL-10 and the susceptibility to periodontal diseases. The aim of this study was to investigate the possible association of IL-10 single nucleotide polymorphisms (SNPs) and cyclosporin A (CsA)-induced gingival overgrowth (GO) in renal transplant patients in a Chinese population, taking into account subgingival microbiota as additional variables. Material and methodsA total of 202 patients were dichotomized into two groups: 122 with GO and 80 without GO. The IL-10-1082 SNP, -819 SNP and -592 SNP were measured using an allele-specific PCR method. The levels of subgingival bacteria were measured by real-time PCR. Genotype and allele frequencies were analyzed using the Chi-square test and logistic regression analysis. ResultsThe frequency of IL-10-819TT (-592AA) genotype was statistically higher in patients with GO than that in patients without GO (P<0.05). Multiple logistic regression analysis demonstrated that the prevalence of GO is not dependent on age, gender, and pharmacological variables, being significantly associated with the carriers of ATA haplotype (OR=2.425, 95%CI=1.214–4.845, P=0.012). Moreover, ATA positive carriers in the GO group presented significantly higher levels of Porphyromonas gingivalis and Treponema denticola than those negative carriers. ConclusionsOur results show that IL-10-819TT (-592AA) genotype and ATA halpotype are associated with susceptibility to CsA-induced GO. Meanwhile, ATA haplotype is associated with a higher detection of P. gingivalis and T. denticola in GO patients, and may increase the risk of developing GO.

Effect of periodontal therapy on the course of cyclosporin-induced gingival overgrowth: role of ABCB1 and PAI-1 gene polymorphisms.

Etiological periodontal therapy is effective in reducing cyclosporin A-induced gingival overgrowth, but a high variability among subjects has been observed. This study aimed to evaluate the role of polymorphisms in PAI-1 and A BCB1 genes on the course of this side effect following periodontal therapy. Forty-five transplant patients were subjected to nonsurgical periodontal therapy and evaluated for hypertrophy index, probing depths, bleeding, and plaque scores at baseline, and after 3 and 6 months. A BCB1 (C3435T and G2677T) and PAI-1 (4G/5G) polymorphisms were studied with polymerase chain reaction-restriction fragment length polymorphism and allele-specific polymerase chain reaction respectively. All the monitored periodontal indexes decreased significantly during the six months. Modeling of hypertrophy index by linearmixed- effect models (allowing non-normal distribution of the outcome variable hypertrophy index) resulted in the selection as the most significant model, of the one comprising the independent variables: time, C 3435T genotype, and their interaction term. This model indicated that C 3435T-mutated patients had significantly higher baseline hypertrophy index values (90% Markov chain Monte C arlo empirical confidence intervals: 5.08, 30.00). The decrease in hypertrophy index values over time showed a trend toward being faster in mutated than nonmutated patients (interaction time: C 3435T nonmutated, 90% Markov chain Monte C arlo empirical confidence interval: -11.08, -0.40). When hypertrophy index values were normalized, the significance and trend were lost. No effect of the A BCB1 G2677T and PAI-1 4G/5G polymorphisms was observed. These preliminary results suggest that C 3435T polymorphism is a genetic factor that could influence the course of cyclosporin A-induced gingival overgrowth in transplant patients subjected to periodontal therapy.

The role of inflammation and apoptosis in cyclosporine A-induced gingival overgrowth.

Cyclosporin A(CsA) - induced gingival overgrowth(GO) is a current problem of tissue-specific mechanism which is still incompletely explained. The apoptotic process has been of particular interest like a new concept in the etiology of this unwanted effect. The aim of our study was to detect the level of apoptosis, expression bcl-2 and p53, associated with the different doses of CsA. in gingival stroma. A cohort of 84 kidney transplant recipients was divided into four subgroups based on average daily dose of therapeutically applied CsA (Neoral®), (100 mg, 125 mg, 150 mg and 175 mg). The control group consisted of 21 patients, clinically diagnosed with periodontitis, who were not subjected to any medicamentous treatment causing gingival overgrowth. The following indexes were analyzed: plaque index (PI), index of gingival inflammation (GI) according to Loe-Silnes, and gingival overgrowth index (GOI) according to MacGaw et al. The tissue samples were subjected to a semiquantitative analysis to detect apoptotical cells and immunohistochemically stained to detect the expression of the bcl-2 and p53 proteins. The difference in percentage of apoptotic cells between the group taking 175 mg and other subgroups, as well as the control group was statistically significant (p<0.05). There was a significant difference in percentage of expression bcl-2 between the 175 mg group compared to the other three subgroups and the control (p=0.001). However, a statistically significant positive correlation between the medicament dose, p53, apoptosis, and bcl-2 was registered (p<0.05). Inflammation plays the most important role in the induction of apoptosis and proliferation in gingival tissues.

Proliferative and inductive effects of Cyclosporine a on gingival fibroblast of child and adult.

Background:Gingival overgrowth is a serious side-effect that accompanies the use of Cyclosporin A (CsA). Up to 97% of the transplant recipient children, who were submitted to CsA therapy, have been reported to suffer from this side-effect. Several conflicting theories have been proposed to explain the fibroblast's function in CsA-induced gingival overgrowth. The aim of this study is to assess the proliferation of gingival fibroblasts and levels of released cytokines after being exposed to CsA, in both adults and pediatric groups, and to make a comparison between the results of the two groups.Materials and Methods:The adult fibroblast samples were derived from four healthy adults, aged 35 to 42 years and pediatric samples were obtained from four healthy children, age between four and eleven years. Tissue samples were plated in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), Streptomycin and Penicillin. The samples were cultured in 25 cm2 plates containing 5% CO2, and incubated at 37°C. The cells used for all the experiments were at the fourth passage. The concentration of PGE2, IL-1β, IL-6, IL-8, TNF-α, and TGF-β1 was determined by the enzyme-linked immunosorbent assay (ELISA) and the proliferation rate was assessed by the MTT assay. Alpha error levels were set as 0.05.Results:CsA stimulated significantly higher levels of IL-6, IL-8 and TGF-β1 in adult gingival fibroblasts than it did in the control group; whereas, the expression of IL-1β and PGE2 in the fibroblasts exposed to CsA was significantly weaker (P < 0.05). The fibroblasts in the two groups did not reveal any noticeable difference in the production of TNF-α. Furthermore, cell proliferation in the CsA group was not significantly higher than that in the control group. No significant differences in cytokines TNF-α and IL-1β were noted between the two groups. The results indicated that CsA stimulated cell proliferation in the pediatric fibroblast cell line. Comparison between the results in the adult and pediatric groups demonstrated that the levels of IL-1β, IL-6, IL-8, and PGE2 were significantly higher in the pediatric group than in the adult group; however, statistics showed no significant difference in the levels of TNF-α and TGF-β1 and CsA-induced proliferation between these two groups.Conclusions:The mechanism of a CsA-induced fibroblast overgrowth may converge on the steps involving fibroblast proliferation and cytokine network including IL-6, IL-8, IL-1β, TGF-β1, and PGE2, in both adults and pediatrics. As the prevalence and intensity of drug-induced gingival overgrowth is more serious in the pediatrics. As group than in adults, we suggest that more studies be conducted on the pediatric group.

The modulation of hypoxia-inducible factor-1α/plasminogen activator inhibitor-1 axis in human gingival fibroblasts stimulated with cyclosporine A.

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. 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. 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). 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.

Association of CD14‐260 polymorphisms, red‐complex periodontopathogens and gingival crevicular fluid cytokine levels with cyclosporine A‐induced gingival overgrowth in renal transplant patients

BACKGROUD AND OBJECTIVE: Genetic factors may influence the colonization of pathogenic bacteria, therefore increasing the risk for the initiation and development of periodontal disease. The present study was carried out to investigate the association of CD14-260 polymorphisms, subgingival microbiota, and gingival crevicular fluid (GCF) cytokine levels with cyclosporine A (CsA)-induced gingival overgrowth (GO) in renal transplant patients. A total of 204 patients were dichotomized into two groups: 124 with GO and 80 without GO. The CD14-260 polymorphisms were measured using an allele-specific PCR method. The levels of periodontal pathogens were determined by real-time PCR of subgingival samples. GCF levels of IL-1β and sCD14 were detected by ELISA. The frequency of CD14-260 genotype CT + TT was found to be similar in both groups. Patients with GO presented increased prevalence of Pg, Td, and Tf (red complex) and significantly higher levels of interleukin -1β than those without GO. Patients with GO carrying CT + TT genotypes were found to have higher frequencies of Pg, Td, and Tf than those carrying the CC genotype. Furthermore, in the presence of red complex, CT + TT genotypes were associated with higher interleukin -1β levels and severe GO. Multiple logistic regression analysis demonstrated that the severity of GO is not dependent on age, gender and pharmacological variables, being only associated with CD14-260 genotype and red complex periodontopathogens. No association between CD14-260 polymorphisms and the prevalence of GO was revealed in renal transplant patients administered CsA. However, CD14-260 CT + TT genotypes are associated with the prevalence of red complex periodontopathogens in patients with GO, and may thus play some role in the development of severe CsA-induced GO.

Can chlorhexidine mouthwash twice daily ameliorate cyclosporine-induced gingival overgrowth?

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. 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 width on molars. A gingival histological examination was performed. 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. 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.

Immunohistochemical Analysis of CD45RO+ T Cells and Vascular Endothelial Growth Factor Expression in Cyclosporin A–Induced Rat Gingival Tissue

The aim of this study is to evaluate CD4(+), CD8(+), and CD45RO(+) T cells, and vascular endothelial growth factor (VEGF) expression in cyclosporin A (CsA)-induced rat overgrown gingival tissue during an 8-week period. Sixty male Sprague-Dawley rats weighing 200 to 250 g were used in this study. Mandibular first molars were ligated with 3-0 silk suture. The rats received daily doses of 0.09% NaCl (control group) or 10 mg/kg body weight of CsA (test group) by intraperitoneal injections. Five rats from the control group and 10 rats from the test group were sacrificed at each experimental period (2, 4, 6, and 8 weeks after the beginning of CsA treatment). The specimens were examined immunohistochemically. CD4(+), CD8(+), and CD45RO(+) T cells, and VEGF expression were more prevalent in the CsA-treated group than in the control group (P <0.05). VEGF was significantly correlated with CD4(+) T cells, CD4(+)/CD8(+) ratio, and CD45RO(+) cells (P <0.05). Based on our findings, we conclude that VEGF, a major regulator of angiogenesis, and CD4(+), CD8(+), and CD45RO(+) memory T cells play a key role in CsA-induced gingival overgrowth.

Transglutaminase 2 expression is significantly increased in cyclosporine-induced gingival overgrowth.

Cyclosporine A is a potent immunosuppressant used to prevent organ transplant rejection and treat various autoimmune diseases. However, cyclosporine A can also induce gingival overgrowth, which is characterized by increased extracellular matrix due to an altered balance between collagen synthesis and degradation. This study proposed to verify whether trans-glutaminase 2, an enzyme thought to be responsible for the assembly and remodelling of extracellular matrix, plays any role in the pathogenesis of cyclosporine A-induced gingival overgrowth. Cyclosporine A-induced gingival overgrowths were collected from 21 liver transplant patients and case-controlled with 20 non-hyperplastic gingival biopsies from healthy patients who had previous periodontal treatment. In both groups, the presence and tissue distribution of transglutaminase 2 were determined by immunohistochemistry and analyzed in comparison with the tissue morphology and expression of lymphocyte-related antigens (CD3 and CD20) and a vessel-related marker (CD34). Transglutaminase 2 expression showed a significant increase (2.6-fold) in the stromal component of cyclosporine A-treated patients compared with controls (p<0.001), which suggested that transglutaminase 2 had a role in the pathogenesis of the disease. Further studies should investigate the therapeutic effect of anti-transglutaminase 2 drugs (putrescine or 1,4-diamino-butane) in these patients.

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

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. 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. 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. 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.

Toll like receptor 4 and membrane-bound CD14 expressions in gingivitis, periodontitis and CsA-induced gingival overgrowth

ObjectiveImmune cell recognition of lipopolysaccharides via CD14 and Toll-like receptor 4 (TLR4) complexes plays a crucial role in linking innate and adaptive immune responses. This study was aimed to investigate the expression of TLR4 and membrane-bound CD14 (mCD14) in the gingival tissues of patients with gingivitis, periodontitis and CsA-induced gingival overgrowth. DesignGingival tissues were obtained from 10 renal transplant patients receiving cyclosporine-A (CsA) and having gingival overgrowth (GO), 10 patients with chronic periodontitis, 10 generalized aggressive periodontitis, 10 gingivitis and 10 healthy subjects. Immunohistochemistry was performed in order to determine the localization of TLR4 and mCD14 in tissue specimens. ResultsTLR4 and mCD14 expressions were detected in all tissues including healthy gingival biopsies. TLR4 and mCD14 positive cells were predominantly confined to the epithelium–connective tissue interface area, and were highly expressed in the basal cell layer of patients with CsA GO and chronic periodontitis, compared to healthy group (P<0.05). ConclusionThe present study suggests that TLR4 and mCD14 protein expressions may be interrelated and appear to be associated with periodontal disease. CsA usage seemed not to affect TLR4 and mCD14 expressions in CsA induced GO tissues.

Epithelial to Mesenchymal Transition in Gingival Overgrowth

Epithelial to mesenchymal transition (EMT) occurs normally in development. In pathology, EMT drives cancer and fibrosis. Medication with phenytoin, nifedipine, and cyclosporine-A often causes gingival overgrowth. Based partly on the histopathology of gingival overgrowth, the present study investigates the hypothesis that EMT could contribute to its development. We found that phenytoin-induced human gingival overgrowth tissues, the most fibrotic drug-induced variety, contain diminished epithelial E-cadherin expression, whereas fibroblast-specific protein-1 (FSP-1) and alphavbeta6 integrin levels are up-regulated. In connective tissue stroma, fibronectin and alternatively spliced fibronectin extra type III domain A (FN-ED-A) levels are increased in overgrowth lesions. Transforming growth factor (TGF)-beta1 treatment of primary human gingival epithelial cells cultured in transwell plates resulted in inhibited barrier function as determined by reduced electrical resistance, paracellular permeability assays, and cell surface E-cadherin expression. Moreover, TGF-beta1 altered the expression of other markers of EMT determined at the mRNA and protein levels: E-cadherin decreased, whereas SLUG, fibronectin, matrix metalloproteinase (MMP)2, MMP9, and MMP13 increased. Nifedipine- and cyclosporine A-induced gingival overgrowth tissues similarly contain diminished E-cadherin and elevated levels of FSP-1 and fibronectin, but normal levels of alphavbeta6 integrin. In summary, data in vitro support that human gingival epithelial cells undergo functional and gene expression changes consistent with EMT in response to TGF-beta1, and in vivo studies show that important EMT markers occur in clinical gingival overgrowth tissues. These findings support the hypothesis that EMT likely occurs in drug-induced gingival overgrowth.

Cyclosporin A-induced gingival overgrowth is not associated with myofibroblast transdifferentiation

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.

Regulation of type I plasminogen activator inhibitor in human gingival fibroblasts with cyclosporine A

Cyclosporine A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of gingival overgrowth. Type I plasminogen activator inhibitor (PAI-1) has shown to play an important role in CsA-induced gingival overgrowth. However, little is known about whether factors can modulate CsA-induced PAI-1 expression. Cytotoxicity, reverse transcriptase-polymerase chain reaction, and enzyme-linked immunosorbent assay were used to investigate the effects of Human gingival fibroblasts (HGFs) exposed to CsA. In addition, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, interlukin-1alpha, tumor necrosis factor-alpha, mitogen-activated protein kinase kinase (MEK) inhibitor U0126, signal-regulated protein kinase (ERK) inhibitor PD98059 and cell-permeable glutathione precursor N-acetyl-L-cysteine (NAC) were added to test how they modulated the effects of CsA-induced PAI-1 expression. The concentration of CsA higher than 500 ng ml(-1) demonstrated cytotoxicity to HGFs (P < 0.05). Periodontal pathogens as well as proinflammatory cytokines were found to increase the CsA-induced PAI-1 mRNA and protein expression (P < 0.05). Pharmacological agents NAC, U0126, and PD98059 were found to decrease the CsA-induced PAI-1 mRNA and protein expression (P < 0.05). Cyclosporine A (CsA) may predispose to gingival overgrowth under inflammatory environments. The regulation of PAI-1 expression induced by CsA might be critically related with the intracellular glutathione and the ERK-MAPK pathway.

Expression of TNF-α and RANTES in drug-induced human gingival overgrowth

Objectives:Regulated on activation, normal T cell expressed and secreted (RANTES) is a chemokine that is produced by fibroblasts, lymphoid and epithelial cells of the mucosa in response to various external stimuli. RANTES expression has been demonstrated in a variety of diseases characterized by inflammation, including asthma, transplantationassociated accelerated atherosclerosis, endometriosis and fibrosis. RANTES mRNA is quickly up-regulated by tumor necrosis factor (TNF)-α stimulation. Cyclosporine A (CsA) is widely used in organ transplant patients, often causing various side-effects including gingival overgrowth, which is fibrotic in nature. This study was carried out to assess the mRNA expression of TNF-α and RANTES in healthy individual, chronic periodontitis and CsAinduced gingival overgrowth tissues.Materials and Methods:Gingival tissue samples were collected from chronic periodontitis, CsAinduced gingival overgrowth patients and healthy individuals. Total RNA was isolated and reverse transcription polymerase chain reaction (RT-PCR) was performed for TNF-α and RANTES expression.Results:The results suggest that CsAinduced gingival overgrowth tissues expressed significantly increased TNF-α and RANTES compared to control and chronic periodontitis.Conclusion:The findings of the present study suggest that CsA can modify the expression of TNF-α and RANTES in drug-induced human gingival overgrowth.

The upregulation of heat shock protein 47 in human gingival fibroblasts stimulated with cyclosporine A

Heat shock protein 47 (Hsp47), a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen. Heat shock protein 47 is consistently and dramatically upregulated in a variety of fibrotic diseases. The aim of this study was to compare Hsp47 expression in normal gingival tissues and cyclosporine A-induced gingival overgrowth specimens and further explore the potential mechanisms that may lead to induction of Hsp47 expression. Fifteen cyclosporine A-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. Western blot was used to investigate the effects of cyclosporine A on the expression of Hsp47 in human gingival fibroblasts. In addition, Aggregatibacter actinomycetemcomitans, interleukin-1 alpha (IL-1 alpha) and mitogen-activated protein kinase kinase (MEK) inhibitor U0126 were added to seek the possible regulatory mechanisms of Hsp47 expression. A significantly higher percentage of cells positively stained for Hsp47 was noted in the cyclosporine A-induced gingival overgrowth group than in the normal gingival group (p < 0.05). Expression of Hsp47 was observed mainly in the cytoplasm of fibroblasts, endothelial cells, epithelial cells and inflammatory cells. Expression of Hsp47 was significantly higher in cyclosporine A-induced gingival overgrowth specimens with higher levels of inflammatory infiltrates (p < 0.05). Cyclosporine A upregulated Hsp47 expression in human gingival fibroblasts in a dose-dependent manner (p < 0.05). The addition of A. actinomycetemcomitans or interleukin-1 alpha significantly increased Hsp47 expression compared with cyclosporine A alone (p < 0.05). The MEK inhibitor U0126 was found to inhibit cyclosporine A-induced Hsp47 expression (p < 0.05). Expression of Hsp47 is significantly upregulated in cyclosporine A-induced gingival overgrowth specimens, and Hsp47 expression induced by cyclosporine A in fibroblasts may be mediated by the MEK signal transduction pathway. The expression of Hsp47 could be significantly enhanced by A. actinomycetemcomitans and interleukin-1 alpha.

Immunohistochemical Analysis of Inducible and Endothelial Forms of Nitric Oxide Synthase in Cyclosporin A‐Induced Gingival Overgrowth

The contribution of nitric oxide (NO) to immune response and matrix degradation in the periodontal environment suggests a role for NO and NO-synthase (NOS) activity in the pathogenesis of cyclosporin A (CsA)-induced gingival overgrowth (GO). However, current knowledge on this topic is limited to experimental animal studies. The present study was undertaken on the basis of a hypothesis whether altered nitrite/nitrate levels in gingival crevicular fluid (GCF) and endothelial NOS (eNOS) and inducible NOS (iNOS) immunoreactivity in gingiva of CsA-treated patients contribute to the pathogenesis of CsA-induced GO. Twenty-four CsA-medicated renal transplant patients with GO (GO+; n = 12) or without GO (GO-; n = 12), 10 gingivitis, and 10 healthy subjects were included in the study. GCF samples from two proximal sites facing interdental papilla were collected, and papilla was excised. iNOS and eNOS were determined by immunohistochemistry. GCF nitrite/nitrate levels were analyzed based on the Griess reaction. Weak iNOS immunostaining was observed in the healthy and GO- groups. In the gingivitis and GO+ groups, iNOS immunostaining significantly increased in connective tissue. Epithelial immunostaining of iNOS was localized to basal keratinocytes and the lower layer of stratum (str.) spinosum in the gingivitis group. In the GO+ group, iNOS immunostaining was differentially localized to keratinocytes of str. superficiale but considerably decreased in the str. basale. Weak eNOS immunostaining was found in the healthy and GO- groups, whereas higher immunostaining was observed in the gingivitis and GO+ groups. No intergroup differences were observed regarding nitrite/nitrate levels in GCF. CsA differentially upregulated iNOS, but not eNOS, in overgrown gingiva, which may play a pivotal role in the pathogenesis of CsA-induced GO.