CsA-induced Gingival Overgrowth Research Articles

Cyclosporine A-induced gingival overgrowth in renal transplant patients accompanied by epithelial-to-mesenchymal transition.

To investigate the association between the prevalence of cyclosporin A-induced gingival overgrowth and the expression of the epithelial-to-mesenchymal transition factors in the gingival tissues of renal transplant patients. Gingival overgrowth (GO) is a frequent complication in organ transplant patients treated with the immunosuppressant cyclosporin A (CsA). The epithelial-to-mesenchymal transition (EMT) is considered a factor contributing to CsA-induced GO. However, current knowledge on this topic is sparse. Sixty-three renal transplant patients were divided into two groups according to the occurrence of GO: those with gingival overgrowth (GO+ group) and those without gingival overgrowth (GO- group). Data on age, sex, and use of immunosuppressant and calcium channel blocker medications, serum creatinine values, peak concentrations of blood CsA, and gingival hyperplasia scores were recorded to identify clinically pathogenic factors. Gingival tissues from five patients with CsA-induced GO and five healthy subjects were selected for histomorphological observation with hematoxylin-eosin staining, Masson staining, and immunohistochemical staining. The mRNA expression of EMT factors was detected with reverse transcription-quantitative PCR. The use of CsA significantly increased the prevalence of GO in renal transplant patients. The expression of α-SMA, SMAD4, and TGM2 was upregulated and that of E-cadherin was downregulated in the gingival tissues of patients with CsA-induced GO compared with those of the corresponding controls. Treatment with CsA is closely related to the occurrence of GO in renal transplant patients and EMT plays an important role in CsA-induced gingival tissue hyperplasia.

Antioxidants protect against gingival overgrowth induced by cyclosporine A.

We investigated the importance of reactive oxygen species (ROS) on developing gingival overgrowth (GO) and then introduced the antioxidant strategy to prevent, or even reduce GO. Gingival overgrowth is a common side effect of the patients receiving cyclosporine A (CsA), an immune suppressant. Although it has been broadly investigated, the exact pathogenesis of the induced GO is still uncertain. We cultured human primary gingival fibroblasts and used animal model of GO to investigate the ameliorative effects of antioxidants on CsA-induced GO. To examine the CsA-induced oxidative stress, associated genes and protein expression, and the overgrown gingiva of rats by using immunocytochemistry, confocal laser scanning microscopy, real-time PCR, ELISA, gelatin zymography, gingival morphological, and immunohistochemical analysis. We found for the first time that ROS was responsible for the CsA-induced oxidative stress and TGF-β1 expression in human primary gingival fibroblasts, as well as the GO of rats. The antioxidants (oxidative scavenger of vitamin E and an antioxidative enzyme inducer of hemin) ameliorated CsA-induced pathological and morphological alterations of GO without affected the CsA-suppressed il-2 expression in rats. CsA-induced oxidative stress, HO-1, TGF-β1, and type II EMT were also rescued by antioxidants treatment. We concluded that CsA repetitively stimulating the production of ROS is the cause of CsA-GO which is ameliorated by treating antioxidants, including vitamin E and sulforaphane. Furthermore, the immunosuppressive effect of CsA is not interfered by antioxidant treatments in rats. This finding may thus help the clinician devise better prevention strategies in patients susceptible to GO.

MiR-200a inhibits proliferation rate in drug-induced gingival overgrowth through targeting ZEB2

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. 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. 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. 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.

Down-regulation of miR-200b-targeting Slug axis by cyclosporine A in human gingival fibroblasts

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. 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. 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. 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.

Cyclosporine A – Induced gingival overgrowth and proliferating cell nuclear antigen expression in experimental periodontitis

The most important microscopic characteristic of Cyclosporine A-induced gingival overgrowth is fibroepithelial hyperplasia. Objective: The objective was to investigate the influence of previous exposure to Cyclosporine A over gingival epithelium in experimental periodontitis in rats. Methods: Twenty Wistar rats with 12 weeks-old were divided into four groups with 5 animals each: Control Group (CG); Cyclosporine Group (CsAG); Ligature group (LG) and Cyclosporine and Ligature Group (CsALG). Daily doses of CsA (10 mg/kg) were applied to CsAG and CsALG during 60 days since the beginning of the experiment and, a ligature was placed in LG and CsALG 30 days after the beginning of the experiment. After 60 days, animals were euthanized and gingival tissue was processed to histomorphometric analysis of epithelial thickness (mm2), immunohistochemical expression of PCNA (%) and inflammatory response. Data were analyzed by Kruskal-Wallis and Mann Whitney at 0.05 significance level. Results: Considering epithelial thickness, CG was thinner than all groups, CsALG was the largest and CsAG and LG were similar between each other. Regarding the PCNA expression CG (16.46 ± 9.26) was similar to CsAG (34.47 ± 19.75) and, LG (59.02 ± 10.33) was similar to CsALG (40.59 ± 18.25). Significant difference (p < 0.05) occurred only in inflammation presence comparing CG/LG and CsAG/CsALG. A weak positive correlation between the number of PCNA+ and inflammatory cells (p = 0.001; r = 0.611) was observed. Conclusion: Based on these results it was concluded that the enlargement of gingival epithelium observed in experimental periodontitis can be increased by previous exposition to CsA and inflammatory conditions enhanced proliferative activity of the keratinocytes.

A novel gingival overgrowth mouse model induced by the combination of CsA and ligature-induced inflammation

Drug-induced gingival overgrowth (DIGO) is a side effect of the enlargement of gingival tissue by phenytoin, nifedipine, and cyclosporine A (CsA). Gingival inflammation has been identified as a key factor that initiates DIGO. However, a sufficient animal model for clarifying the role of inflammation in DIGO has not yet been generated. We herein describe a novel CsA-induced gingival overgrowth mouse model to evaluate the role of inflammation. A ligature was placed around the second molar in maxillae for 7days to induce gingival inflammation, and CsA (50mg/kg/day) was administered to mice during each experimental period. The severity of gingival overgrowth and mRNA expression of inflammatory cytokines in gingiva were assessed by the gingival overgrowth degree, histological analyses, and RT-PCR. The administration of CsA for 28days in combination with ligation significantly increased the gingival overgrowth degree and expanded the connective tissue area. Increases in the gingival overgrowth degree continued in a time-dependent manner until 21days. Furthermore, the cessation of CsA reduced gingival overgrowth. Thin ligatures (7–0 size) induced weaker tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 mRNA expression and less gingival overgrowth than thick ligatures (5–0 ligature). Moreover, the administration of an antibiotic cocktail, which suppressed the expression of these inflammatory cytokines in gingiva, attenuated gingival overgrowth induced by ligatures and CsA. These results suggest that inflammation in gingival tissue plays a role in initiating CsA-induced gingival overgrowth. This gingival overgrowth mouse model has potential for elucidating the etiology of DIGO from the view point of gingival inflammation.

Increased Advanced Oxidation Protein Products Generation by Cyclosporine-A and Angiotensin II in Human Gingival Fibroblasts - Ex-vivo Study.

Cyclosporin-A (CsA), an immunosuppressant, induces renal fibrosis and Renin Angiotensin System (RAS) is known to play a major role. CsA has the potential to increase the oxidative stress; specifically through the Advanced Oxidation Protein Products (AOPP) which could possibly stimulate fibrosis. A similar type of pathology occurs even in the gingiva known as CsA Induced Gingival Overgrowth (CIGO). This study was undertaken to estimate the AOPP generation by Human Gingival Fibroblasts (HGF) under the influence of CsA and Angiotensin II (Ang II). Six healthy gingival tissue samples were obtained during crown lengthening procedure and primary HGF were cultured using enzymatic digestion method. The ideal non-cytotoxic concentrations of CsA and Ang II were identified using cytotoxicity assay. Later, HGF were incubated with CsA and Ang II for 12 hours and AOPP assay was performed at zero and one hour interval. There was a statistically significant increase in AOPP production in both the CsA and Ang II when compared to the control group with a p value<0.05. CsA can induce oxidative stress and preventing/controlling it may be necessary to prevent untoward effect of the drug.

Systematic review of cyclosporin A-induced gingival overgrowth and genetic predisposition.

This systematic review aimed to investigate the influence of gene polymorphisms on the development of gingival overgrowth in renal transplant patients treated with cyclosporin A. Electronic and hand literature searches were conducted by two independent reviewers in MEDLINE-Pubmed, Cochrane Library, ISI Web of Science, and SCOPUS Elsevier for prospective (case-control studies, cohort studies), cross-sectional, and retrospective studies published up to June 2016 (first week) in any language. Data were reviewed and extracted in duplicate independently. Methodologic quality assessment of the included studies was performed during the data extraction process. Due to the estimated high risk of bias and the heterogeneity of the included studies in regards to the variety of medications administered to study patients, a systematic review of the literature and not a meta-analysis of the data was performed. Fourteen articles meeting study inclusion criteria were selected for data extraction that examined the association between various genetic polymorphisms and gingival overgrowth in kidney transplant patients receiving cyclosporin A. Interleukin-1A, interleukin-10, transforming growth factor-β1 and androgen receptor gene polymorphisms may have a significant effect on an individual susceptibility to cyclosporin A-induced gingival overgrowth in renal transplant patients. Genetic polymorphisms seem to affect the development of cyclosporin A-induced gingival overgrowth in renal transplant patients. Pharmacogenetics and pharmacogenomics have the potential to determine the clinical outcome of a medication, the drug efficacy, and adverse drug reactions such as gingival overgrowth.

Upregulation of embryonic stem cell marker Nanog in human gingival fibroblasts stimulated with cyclosporine A: An in vitro study

Background/purposeGingival overgrowth is a common side effect of medication with the immunosuppressant cyclosporine A (CsA). This study proposed to verify whether Nanog, an embryonic stem cell marker, contributes to gingival overgrowth stimulated with CsA in human gingival fibroblasts (HGFs). Materials and methodsThe effect of CsA on HGFs was used to elucidate whether Nanog expression could be induced by CsA using quantitative real-time reverse transcription-polymerase chain reaction and Western blotting. Cell growth in CsA-treated HGFs with Nanog lentivirus-mediated short hairpin RNA interference knockdown was evaluated by tetrazolium bromide reduction assay. ResultsCsA upregulated Nanog transcript in HGFs in a dose-dependent manner (P < 0.05). CsA was also shown to increase Nanog protein expression in HGFs in a dose-dependent manner (P < 0.05). In addition, downregulation of Nanog by lentiviral infection significantly inhibited CsA-stimulated cell growth in HGFs (P < 0.05). ConclusionCsA upregulated Nanog expression and cell growth in HGFs, while silencing Nanog effectively reversed these phenomena. Nanog may act as a major switch in the pathogenesis of CsA-induced gingival overgrowth.

Gingival Crevicular Fluid and Plasma Levels of Transglutaminase-2 and Oxidative Stress Markers in Cyclosporin A-Induced Gingival Overgrowth.

Transglutaminase (TGM)-2 has been shown to contribute to fibrosis by extracellular matrix accumulation in some organs and is activated by intracellular reactive oxygen species. The aim of this study is to investigate levels of gingival crevicular fluid (GCF) and plasma TGM-2 and oxidative stress markers (OSMs) in cyclosporin A (CsA)-induced gingival overgrowth (GO). The study enrolled 20 healthy (H) individuals; 20 patients with gingivitis (G); 20 CsA-medicated patients with GO (CsA GO+); and 20 CsA-medicated patients without GO (CsA GO-). GCF and plasma levels of TGM-2 were analyzed by enzyme-linked immunosorbent assay. Spectrofluorometry was used to analyze thiobarbituric acid reactive substance (TBARS); ferric-reducing antioxidant power (FRAP); total oxidant status (TOS); and total antioxidant capacity (TAC). GCF TGM-2 level was elevated in CsA GO+ compared with G (P = 0.048) and H (P = 0.001) groups. GCF TBARS level was elevated in CsA GO+ compared with other groups (CsA GO- group: P = 0.003; G group: P <0.001; and H group: P <0.001) and was higher in CsA GO- than in H (P = 0.048). GCF FRAP level was lower in CsA GO- than in H (P = 0.04). Both CsA GO+ and CsA GO- groups had lower GCF TOS levels than H (P <0.001 and P = 0.002) and G (P = 0.003 and P = 0.04). GCF TAC was higher in CsA GO+ than in H (P = 0.02). Plasma TGM-2 level was elevated in CsA GO+ compared with G (P = 0.048) and H (P = 0.002). Plasma FRAP level was higher in H and CsA GO- than in CsA GO+ (P = 0.008 and P = 0.02). CsA use significantly alters GCF and plasma levels of TGM-2 and OSMs. TGM-2 may contribute to CsA-induced GO in CsA-treated patients by changing GCF and plasma levels of OSMs. Further studies are needed to prove causality and its direction.

Upregulation of Slug expression by cyclosporine A contributes to the pathogenesis of gingival overgrowth.

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. 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. 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). Taken together, upregulation of Slug in HGFs stimulated by CsA may play an important role in the pathogenesis of CsA-induced gingival overgrowth.

Efficacy of azithromycin and metronidazole combined therapy on rats’ gingival overgrowth induced by cyclosporine-A: An experimental animal study

AimsTo evaluate the therapeutic efficacy of azithromycin (azm) and/or metronidazole (mtz) on the histopathological features of rats’ gingival overgrowth (GO) induced by cyclosporine-A (CsA) in an animal model. MethodsNinety male albino rats were divided randomly into six equal groups. The rats of group I received corn oil via gastric feeding for 7 weeks. Group II rats were administered CsA for the same period. Groups III, IV, and V rats received CsA for 6 weeks and simultaneously in the 7th week received a monotherapy of placebo gel, azm suspension, mtz gel, respectively. Group VI rats were handled as groups III, IV, and V and instead received a combined therapy of azm suspension, and mtz gel. Rats were euthanized at the end of the experiment and routine tissue processing was carried out. The obtained specimens were stained with H&E, TGF-β, MMP-1, and IL-6 antibodies. ResultsOne-way MANOVA test for TGF-β, MMP-1, and IL-6 revealed an overall significant difference between the different groups (P=0.000). LSD post hoc test for multiple comparisons of TGF-β revealed nonsignificant difference between groups I and VI and between groups IV and V. Nonsignificant difference was found between groups II and III considering the amount of MMP-1 immune expression. In addition, nonsignificant difference was found between groups V and VI regarding the amount of immune expression for IL-6. ConclusionCombined therapy of azm suspension and mtz gel significantly improved the histopathological features of CsA-induced GO better than a monotherapy of azm suspension or mtz gel.

Cyclosporine A promotes cell proliferation, collagen and α-smooth muscle actin expressions in rat gingival fibroblasts by Smad3 activation and miR-29b suppression.

Transforming growth factor (TGF)-β/Smad3 signaling promotes tissue fibrosis, and miR-29b is a downstream inhibitor of TGF-β/Smad3-mediated fibrosis, both of which may be involved in the pathogenesis of cyclosporine A-induced gingival overgrowth. The aim of this study is to investigate the effect of miR-29b and TGF-β/Smad3 signaling in cyclosporine A-stimulated rat gingival fibroblasts. We explored the effect of cyclosporine A on cell proliferation, type Ι collagen (COL1) and alpha-smooth muscle actin (α-SMA) expressions in rat gingival fibroblasts with the Cell Counting Kit-8 assay, real-time polymerase chain reaction, western blot and immunofluorescence. The expressions of TGF-β1, Smad3 and miR-29b were also evaluated after cyclosporine A treatment. The effects of Smad3 and miR-29b on cyclosporine A-induced alterations were further determined by Smad3 knockdown and miR-29b overexpression using transient transfection experiments. We found that cyclosporine A increased cell proliferation, COL1 and α-SMA expressions in rat gingival fibroblasts. TGF-β1 mRNA and protein expressions were upregulated and phosphorylated Smad3 was activated by cyclosporine A treatment, while miR-29b expression was significantly suppressed. Moreover, Smad3 knockdown and miR-29b overexpression reversed cyclosporine A-enhanced cell proliferation, COL1 and α-SMA expressions in gingival fibroblasts. In addition, the suppression of miR-29b upon cyclosporine A exposure was significantly elevated by Smad3 knockdown, whereas cyclosporine A-induced Smad3 activation was not altered by miR-29b overexpression. These results suggest that cyclosporine A promotes cell proliferation, collagen and α-SMA expressions in rat gingival fibroblasts by Smad3 activation and miR-29b suppression.

Elevated Snail expression in human gingival fibroblasts by cyclosporine A as the possible pathogenesis for gingival overgrowth.

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. 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. 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). 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.

Tanshinone IIA treatment alleviated the rat gingival connective tissue overgrowth induced by cyclosporine A.

The use of cyclosporine A induces fibrous enlargement of the gingival connective tissue. Existing treatment modalities, although effective, do not necessarily prevent the recurrence of the lesion. Emerging evidence indicates that tanshinone IIA (Tan IIA) could effectively attenuate a variety of fibrotic diseases. The present research aims to assess whether Tan IIA can effectively alleviate the gingival fibrous overgrowth induced by cyclosporine A. Forty-five Wistar rats were divided into the no-treatment control group, cyclosporine A-treated group and the group treated with a combination of cyclosporine A and Tan IIA. Paraffin-embedded sections of mandibular first molar regions were selected for hematoxylin and eosin staining, Masson's trichrome staining, picro-sirius red staining and immunohistochemistry analyses of transforming growth factor-β1 (TGF-β1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinase-1 (MMP-1). The gingival connective tissue area was measured and numbers of the TGF-β1-, TIMP-1- and MMP-1-positive cells were counted. The analysis of variance for factorial designs for testing the overall differences and Fisher least significant difference test for post hoc analysis were used to determine the significance levels. Cyclosporine A treatment led to overgrowth of gingival connective tissue in rats. In the cyclosporine A-treated rats, the expression of TGF-β1 and TIMP-1 was significantly upregulated, whereas expression of the MMP-1 was downregulated, along with thicker and denser collagen fibers. In rats treated with a combination of cyclosporine A and Tan IIA, the cyclosporine A-induced changes were alleviated. Cyclosporine A enhanced gingival fibrous overgrowth via upregulation of the TGF-β1 and TIMP-1 expression, and downregulation of MMP-1 expression. Tan IIA can effectively prevent cyclosporine A-induced gingival fibrous overgrowth in rats by downregulating TGF-β1 and TIMP-1 expression, and upregulating MMP-1 expression.

Altered oxidative status and integrin expression in cyclosporine A-treated oral epithelial cells

Background and objective: Cyclosporine A (CsA) is an immunosuppressive agent administered to transplant patients. A well-known reported oral side effect of CsA consumption is gingival overgrowth (GO). Changes in the expression of integrins occurring in the gingiva following CsA treatment have been reported but these reports are mainly concerned with the connective tissue of the gingiva. In this study we targeted the alterations in the oral epithelium using KB cells, an oral epithelial cell line.Methods: Cultured oral epithelial cells were treated with increasing concentrations of CsA (0.1, 1 and 10 µg/mL) and the molecular changes involving antioxidant enzymes [glutathione peroxidase (GPx) and glutathione reductase (GR)] and the level of reactive oxygen species (ROS) were measured. Quantitative real-time PCR was used to assess the expression of selected integrins (α2, α5 and β1).Results: At CsA concentration above 0.1 µg/mL GPx demonstrated an increase in activity while GR activity and the level of reduced glutathione were diminished (p < 0.05). α5 and β1 integrin were downregulated at all treatment concentrations of CsA while α2 integrin presented this effect at concentrations above 1 µg/mL (p < 0.05).Conclusion: The results suggest a possible role for oxidative stress and the altered expression of integrins in the pathology of CsA-induced gingival overgrowth.

Are antimicrobial peptides related to cyclosporine A-induced gingival overgrowth?

ObjectiveThe aim of the present study was to investigate the effect of cyclosporine-A (CsA) medication on gingival crevicular fluid (GCF) LL-37, human neutrophil peptide (HNP)1-3 and adrenomedullin (ADM) levels. DesignCsA-treated renal transplant recipients with GO (CsA GO+) and without GO (CsA GO−), tacrolimus-medicated renal transplant recipients (n=20/group), systemically healthy subjects with gingivitis (n=21) and individuals free of periodontal and systemic diseases (n=20) were included in the present study. Periodontal parameters were recorded and GCF samples were obtained from the study participants. GCF LL-37, HNP1-3 and ADM levels were analyzed by enzyme-linked immunosorbent assay. ResultsGCF LL-37 total amount was higher at GO+ sites than the other study sites (p<0.05). Total amount of GCF HNP1-3 was higher in immunosuppressive treatment groups than healthy and gingivitis groups, regardless of GO presence (p<0.05). GCF ADM total amount was similar in all study groups. GCF volume, papillary bleeding index and hyperplastic index (p<0.05) were significantly correlated with GCF LL-37 total amounts (p<0.05), but not with GCF HNP1-3 and ADM total amount at GO+ sites (p>0.05). ConclusionNeutrophil infiltration due to extended inflammation might have increased GCF LL-37 levels at GO+ sites and contributed to the pathogenesis of CsA-induced GO.

Cyclosporine A induces connective tissue growth factor expression in human gingival fibroblasts: Suppression by epigallocatechin-3-gallate

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. 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. 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. 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.

Gingival Tissue and Apoptosis

Background: Several factors can influence gingival tissues. Among them: disrupting tissue homeostasis and occurrence of pathological conditions. The aim of our study was to investigate and compare the presence of apoptosis in patients treated with immunosuppressive therapy, patients with periodontitis and on the other hand, healthy patients, as well as to better understand the role of apoptosis in the these processes. Methods: The first objective group consisted of 21 patients (10 males and 11 females; mean age 37.4 ± 10.2 years old) with neither kidney diseases nor treated with cyclosporine A (CsA) therapy, who had a verified periodontal disease. The second group consisted of 21 kidney-transplant patients (9 males and 12 females; mean age 36.2 ± 9.5 years old), with diagnosed gingival overgrowth (GO) subjected to continuous immunosuppressive therapy. The control group consisted of the same number of patients, clinically healthy subjects (15 males and 6 females; mean age 29 ± 14.0 years old) with plaque-induced gingivitis. The following indexes were analyzed: plaque index (PI) Sillnes-Loe, index of gingival inflammation (GI) according to Loe-Sillnes, and gingival overgrowth index (GOI) according to MacGaw et al. The determination of CsA in blood was performed by a fluorescence polarised immunoassay (FPIA).The tissue samples were estimated by semiquantitative analysis in order to determine the presence of apoptotic cells and immunohistochemical expression of the bcl-2 and p53 proteins. Results: We found statistical differences in bcl-2 and apoptotic index, among the groups: greatest expression of bcl-2 and apoptotic index was registered in the group treated with CsA, and the lowest expression was noted in the gingivitis group(p 0,05 ; r = 0.187). Conclusion: Our findings suggest that increased apoptosis may have a role in the pathogenesis of CsA-induced gingival overgrowth in the cases of patients on high dose of CsA.

Clinical factors of cyclosporine A-induced gingival overgrowth and serum cyclophilin A in renal transplant recipients

Clinical factors of cyclosporine A-induced gingival overgrowth and serum cyclophilin A in renal transplant recipients