NADPH Oxidase 4 Mediates TGFß1-induced CCN2 in Gingival Fibroblasts.

Transforming growth factor ß (TGFß) plays a central role in the pathogenesis of gingival overgrowth (GO). Connective tissue growth factor (CTGF; or CCN2) is induced by TGFß in human gingival fibroblasts (HGFs) and is overexpressed in GO tissues. CCN2 creates an environment favorable for fibrogenesis and is required for the maximal profibrotic effects of TGFß. We previously showed that Src, JNK, and Smad3 mediate TGFß1-induced CCN2 protein expression in HGFs. Moreover, Src is an upstream signaling transducer of JNK and Smad3. Recent studies suggested that NADPH oxidase (NOX)-dependent redox mechanisms are involved in mediating the profibrotic effects of TGFß. In this study, we demonstrated that TGFß1 upregulated NOX4 protein expression and increased reactive oxygen species (ROS) production in HGFs. Genetic or pharmacologic targeting of NOX4 abrogated TGFß1-induced ROS production; Src, JNK, and Smad3 activation; and CCN2 and type I collagen protein expression in HGFs. Our results indicated that NOX4-derived ROS play pivotal roles in activating Src kinase activity leading to the activation of canonical (Smad3) and noncanonical (JNK) cascades that cooperate to attain maximum CCN2 expression. Furthermore, we demonstrated that curcumin significantly inhibited the TGFß1-induced NOX4 protein expression in HGFs. Curcumin potentially qualifies as an agent to control GO by suppressing TGFß1-induced NOX4 expression in HGFs.

Local Inflammation Alters MMP-2 and MMP-9 Gelatinase Expression Associated with the Severity of Nifedipine-Induced Gingival Overgrowth: a Rat Model Study.

Nifedipine-induced gingival overgrowth (NIGO) is characterized by cell proliferation and extracellular matrix (ECM) component accumulation in gingival connective tissues, with varying degrees of inflammation and fibrosis. Impaired collagen and ECM homeostasis may be among the underlying molecular mechanisms that lead to the fibrotic changes that occur in drug-induced gingival overgrowth (DIGO). Because matrix metalloproteinases (MMPs) play vital roles in regulating collagen and ECM metabolism, many studies have been performed to reveal the relationship between MMPs and DIGO. It is thought that the gelatinases MMP-2 and MMP-9, both type IV collagenases, are involved in the development of tissue inflammation and organ fibrosis. However, the few studies regarding gelatinase expression in DIGO are controversial. Recent studies have demonstrated the inhibitory effect of cyclosporine A (CsA) on gelatinase expression and/or activity; however, similar changes have yet to be detected in Nif-treated gingival tissues. In this study, we verified that Nif treatment could lead to gingival overgrowth in rats and that gingival inflammation played a pro-proliferative role in NIGO development. Additionally, we examined the temporal expression of gelatinases on days 0, 7, 14, 21, 30, and 40 during NIGO development. The aim was to investigate whether MMP-2 and MMP-9 played significant roles in regulating NIGO development and progression. MMP-2 gene expression was not altered by Nif treatment alone but was significantly inhibited by Nif treatment for 30 days in the presence of local inflammation. However, no significant alterations in MMP-2 protein expression were detected in the Nif-treated gingival tissue, regardless of the presence or absence of local inflammation. Moreover, Nif treatment could lead to transient and significant increases in MMP-9 gene and protein expression levels in the presence of local inflammation. In particular, active MMP-9 expression increased significantly in the gingival tissue that received the combined effect of Nif and ligation treatment; besides, a temporal, but not significant, change was also observed in the gingival tissue that received Nif treatment alone. Taken together, these results provided evidence that temporal changes in MMP-2 and MMP-9 expression occurred during NIGO development. Nif treatment accompanied by local inflammation regulated MMP-2 and MMP-9 expression, primarily MMP-9, which was most likely associated with NIGO severity. Thus, MMP-9 is a potential contributing factor in the process of NIGO development.

Elevated levels of cyclooxygenase 1 and 2 in human cyclosporine induced gingival overgrowth.

OBJECTIVE: This study was carried out to immuno-localize and estimate the levels of cyclooxygenase 1 and 2 in human gingival tissue samples from healthy individuals, chronic periodontitis patients and patients with cyclosporine induced gingival overgrowth. METHODS: Group I consisted of individuals with healthy gingiva (n=6), Group II - cyclosporine induced gingival overgrowth (n=9) and Group III - chronic periodontitis patients (n=6). Gingival tissue samples were collected from subjects of all the three groups. COX-1, COX-2 levels were estimated in tissue homogenates by enzyme activity assay. Immuno-localization for COX-1 and COX-2 was also done in sections of gingival tissue. RESULTS: The study results demonstrated a significantly higher mean levels of COX-1 and 2 in drug induced gingival overgrowth samples (p<0.05). COX-1 and COX-2 was localized to epithelium and connective tissue in human gingival tissue sections from cyclosporine induced gingival overgrowth. CONCLUSION: Cyclooxygenase enzymes appear to be potential mediators involved in the pathogenesis of cyclosporine induced gingival overgrowth.

Matrix metalloproteinases, tissue inhibitors of matrix metalloproteinases, and inflammation in cyclosporine A-induced gingival enlargement: a pilot in vitro study using a three-dimensional model of the human oral mucosa.

BACKGROUND: It has been suggested that cyclosporine A (CsA) induces gingival enlargement by promoting an increase in the gingival extracellular matrix (ECM). Nonetheless, the variable occurrence of CsA-induced gingival enlargement in patients receiving this medication indicates a multifactorial pathogenesis. Clinical observations suggest that local inflammation is associated with the development and severity of CsA-induced gingival enlargement. Therefore, the purpose of this study is to investigate the effects of CsA and inflammation on the production of ECM homeostatic mediators. METHODS: The effects of CsA and inflammation (as assessed using interleukin [IL]-1ß) on the secretion of mediators involved in ECM homeostasis were determined using fibroblast monolayers and three-dimensional (3D) models of the human oral mucosa. Fibroblast monolayers and 3D cultures were treated with CsA alone or in combination with IL-1ß for up to 72 hours, and the secretion of matrix metalloproteinases (MMPs) 1, 2, 3, 8, 9, 10, and 13 and tissue inhibitors of MMPs (TIMPs) 1, 2, and 4 into the culture medium was assessed using enzyme-linked immunoassay-based antibody arrays. RESULTS: Fibroblast monolayers responded to CsA with no changes in the secretion of ECM mediators. Conversely, 3D cultures responded to CsA treatment with a reduction in MMP-10 secretion. IL-1ß alone triggered higher secretory levels of MMPs in both fibroblast monolayers (MMP-3 and MMP-10) and 3D cultures (MMP-9 and MMP-10). Importantly, fibroblast monolayers and 3D cultures treated with a combination of IL-1ß and CsA showed a decrease in the MMP-1/TIMP-1 ratio. CONCLUSIONS: These data support the hypothesis that inflammation may alter the pathogenesis of CsA-induced gingival enlargement by promoting a synergistic decrease in the MMP-1/TIMP-1 ratio.

Correlations between the gingival crevicular fluid MMP8 levels and gingival overgrowth in patients with fixed orthodontic devices.

UNLABELLED: Levels of metalloproteinase 8 (MMP8) in gingival crevicular fluid were studied in case of treatment with fixed orthodontic appliances. It were found a relationship between its levels and stages of the treatment under a good control of the bacterial plaque. Gingival overgrowth (GO) during the orthodontic treatment was traditionally considered as an inflammatory reaction consecutive of bacterial plaque accumulation because of difficult hygiene in those patients. Our study starts from the hypothesis that the gingival volume growth during the fixed orthodontic treatment appears at the beginning, without any inflammatory signs, as a result of the mechanical stress and periodontal remodeling during the orthodontic dental movement, the MMP8 acting as an indicator of this situation. MATERIAL AND METHODS: Twenty-two patients received a fixed orthodontic treatment. Periodontal examination took place one hour before and one hour, four and eight hours and weekly after until eight weeks. At each session gingival crevicular fluid (GCF) was sampled and the level of MMP8 was determined. At the appearance of gingival overgrowth (GO) gingivectomy was performed. RESULTS: In the 13 patients that did not develop gingival overgrowth, the levels of MMP8 increased in the first 4-8 hours after orthodontic appliance and then fall to the initial level. In the nine patients with gingival overgrowth, the MMP8 levels in GCF continued to rise until the appearance of GO. In cases of GO with inflammation the levels of MMP8 were significantly higher than in cases of GO without inflammation. The expression of MMP8 in hypertrophied gingiva was more intensive in cases of GO with inflammation. CONCLUSIONS: It is possible that the MMP8 values in GCF to be a marker of the GO onset. MMP8 determination and monitoring at shorter time intervals may lead to a better control of the bacterial plaque and avoidance of gingival inflammation.

GCF and serum myeloperoxidase and matrix metalloproteinase-13 levels in renal transplant patients.

AIM: The rationale of this study was to address whether local or systemic changes reflect proteolytic (matrix metalloproteinase-13) or oxidative (myeloperoxidase) stress in renal transplant patients receiving cyclosporine-A (CsA) and having gingival overgrowth (GO), in patients receiving CsA therapy and having no GO and patients receiving tacrolimus therapy. MATERIAL AND METHODS: Gingival crevicular fluid (GCF) samples were collected from sites with (GO+) and without GO (GO-) in CsA patients having GO; GO- sites in CsA patients having no GO; sites from tacrolimus, gingivitis and healthy subjects. GCF and serum myeloperoxidase (MPO) and matrix metalloproteinase-13 (MMP-13) levels were determined by ELISA. RESULTS: GO+ sites in CsA patients having GO had elevated GCF MPO levels than those of CsA patients having no GO, tacrolimus and healthy subjects (p<0.005), but comparable to those of gingivitis. GCF MPO levels were higher in GO+ compared to GO- sites in CsA patients having GO (p<0.05). Patient groups had similar, but higher GCF MMP-13 levels than healthy group. CONCLUSIONS: These results show that CsA and tacrolimus therapy have not a significant effect on GCF MPO and MMP-13 levels, and gingival inflammation seems to be the main reason for their elevations.

Matrix metalloproteinase-3 gene polymorphism in renal transplant patients with gingival overgrowth.

BACKGROUND AND OBJECTIVE: Gingival enlargement frequently occurs in transplant patients receiving immunosuppressive drugs. It was hypothesized that gingival enlargement associated with cyclosporine use results from reduced degradation of extracellular matrix in the gingiva. Matrix metalloproteinase-3 (MMP-3) is involved in biodegradation of the extracellular matrix, and its inhibition may contribute to an abnormal accumulation of fibronectin and proteoglycans, which are MMP-3 substrates. The aim of this study was to investigate whether an association exists between MMP-3 genotypes and gingival enlargement in kidney transplant patients medicated with cyclosporine A. MATERIAL AND METHODS: Sixty-four unrelated kidney transplant patients suffering from gingival overgrowth, as well as 111 control transplant patients without gingival overgrowth, were enrolled in the study. Gingival overgrowth was assessed 6 mo after transplantation. During the post-transplant period all patients were given cyclosporine A as a principal immunosuppressive agent. MMP-3 polymorphism was determined using a PCR restriction fragment length polymorphism assay. RESULTS: In kidney transplant patients suffering from gingival overgrowth the mean gingival overgrowth score was 1.35 +/- 0.57, whereas in control subjects the mean gingival overgrowth score was 0.0. The distribution of MMP-3-1178A/dupA alleles among all kidney transplant patients, as well as in the two study subgroups, did not differ significantly from Hardy-Weinberg equilibrium. The frequency of the MMP-3-1171A/A genotype (28.1% for gingival overgrowth vs. 26.1% for controls) and of the MMP-3-1171dupA/dupA genotype (32.8% for gingival overgrowth vs. 22.5% for controls) was similar for both study groups. The risk of gingival overgrowth was lowest among patients carrying the MMP-3-1171A/dupA genotype (odds ratio 0.52), but this did not differ markedly from the other genotypes. CONCLUSION: No association between MMP-3 gene polymorphism and gingival overgrowth was revealed in kidney transplant patients administered cyclosporine A.

Immunohistochemical analysis of inducible and endothelial forms of nitric oxide synthase in cyclosporin A-induced gingival overgrowth.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: CsA differentially upregulated iNOS, but not eNOS, in overgrown gingiva, which may play a pivotal role in the pathogenesis of CsA-induced GO.

Differential gene and protein expression of tissue inhibitors of metalloproteinases (TIMP)-3 and TIMP-4 in gingival tissues from drug induced gingival overgrowth.

OBJECTIVES: The purpose of this study was to analyse mRNA expression and protein localization of tissue inhibitors of metalloproteinases (TIMP)-3 and TIMP-4 in gingival tissues removed from drug (calcium-channel blocker) induced gingival overgrowth and periodontitis patients. DESIGN: Employing RT-PCR, we evaluated TIMP-3 and TIMP-4 mRNA levels of 20 human gingival tissue samples taken from patients suffering gingival overgrowth (GO) and periodontitis (P). Then, using immunohistochemistry we investigated the TIMP-3 and TIMP-4 protein localization of five sample tissues from each group. RESULTS: TIMP-4 mRNA levels in GO-gingiva tended to be lower than in P-gingiva but the results differed little (p = 0.22). Varying degrees of inflammation in the protein localization of TIMP-3 and TIMP-4 were found. TIMP-4 immunoreactivity (IR) was weak in the endothelial cells, fibroblasts, epithelial basal and parabasal cells while the degree of inflammation differed as well. TIMP-3 and TIMP-4 IR in inflammatory cells, including lymphocytes, plasma cells, and macrophages, were faint and intense respectively. For P-gingiva, both TIMP-3 and TIMP-4 IR expression was weak in the endothelial cells, fibroblasts, basal and parabasal epithelial layers. Expression of TIMP-3 was faint in the inflammatory cells, whereas TIMP-4 IR was strong. CONCLUSION: Our findings suggest that TIMP-3 and TIMP-4 expression differs in GO and P-gingival tissues, both of which are potentially involved in pathogenesis.

Cyclosporine A inhibits the expression of membrane type-I matrix metalloproteinase in gingiva.

BACKGROUND AND OBJECTIVE: Membrane type-I matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) regulate the activation of MMP-2; however, their roles in the activation of MMP-2 in gingiva during treatment with cyclosporine A are still unknown. Therefore, the expressions of membrane type-I MMP and TIMP-2, as well as MMP-2, in gingivae upon treatment with cyclosporine A were examined in vivo and in vitro. MATERIAL AND METHODS: Thirty-four rats were divided into two groups after edentulous ridges were established. The experimental group received 30 mg/kg/d of cyclosporine A and the control group received vehicle. At the end of the experimental period, the rats were killed, the gingivae were obtained and the expression of mRNA and protein of membrane type-I MMP, TIMP-2 and MMP-2 in gingiva were examined using real-time polymerase chain reaction and immunohistochemistry. In human gingival fibroblasts, the activity of MMP-2 and the expression of MMP-2, membrane type-I MMP and TIMP-2 mRNAs were examined (using zymography and reverse transcription-polymerase chain reaction, respectively) after treatment with cyclosporine A. RESULTS: In gingivae of rats, cyclosporine A significantly decreased the expression of mRNA and protein of membrane type-I MMP, but not of TIMP-2. The expression of MMP-2 mRNA was unaffected but the expression of MMP-2 protein showed a significant decrease upon treatment with cyclosporine A. In fibroblast culture medium, the presence of cyclosporine A induced a decrease in MMP-2 activity in a dose-dependent manner. The expression of MMP-2, membrane type-I MMP and TIMP-2 mRNAs in fibroblasts was not significantly affected by cyclosporine A; however, in fibroblasts the ratio of mRNA expression of membrane type-I MMP to that of TIMP-2 decreased as the cyclosporine A dose was increased. CONCLUSION: Cyclosporine A inhibits the expression of membrane type-I MMP in gingiva and it may further reduce the activation of MMP-2.

Mechanism of azithromycin treatment on gingival overgrowth.

Azithromycin is effective for the remission of cyclosporine A-induced gingival overgrowth (CIGO) in persons who have undergone renal transplant. To explain its mechanism in alleviating the clinical symptoms of these individuals, we examined the effect of azithromycin on cell proliferation and collagen turnover modified by cyclosporin A in human gingival fibroblasts from healthy persons and from persons who had undergone renal transplant. Cyclosporin A-induced proliferation of renal transplant fibroblasts and normal fibroblasts was inhibited by azithromycin. Azithromycin elevated the reduced metalloproteinase (MMP)-1 and MMP-2 activities in cyclosporine A-treated renal transplant fibroblasts and normal fibroblasts. In cyclosporine A-treated renal transplant fibroblasts, azithromycin blocked the accumulation of total collagen in culture media and the increase in type I collagen mRNA level, but recovered the reduced MMP-2 mRNA level to the control. These results suggest that azithromycin may improve CIGO by blocking cyclosporine A-induced cell proliferation and collagen synthesis, and by activating MMP-2 in gingival fibroblasts of persons with cyclosporine A-induced gingival overgrowth.

Effects of cyclosporin, phenytoin, and nifedipine on the synthesis and degradation of gingival collagen in tufted capuchin monkeys (Cebus apella): histochemical and MMP-1 and -2 and collagen I gene expression analyses.

BACKGROUND: The purpose of this experimental study was to evaluate the collagen fiber distribution histologically after phenytoin, cyclosporin, or nifedipine therapy and to correlate it with collagen I and matrix metalloproteinase (MMP)-1 and -2 gene expression levels. METHODS: Gingival samples from the canine area were obtained from 12 male monkeys (Cebus apella). The mesial part of each sample was assessed by reverse transcription-polymerase chain reaction, whereas the distal part was processed histologically for picrosirius red and hematoxylin and eosin stainings, as well as for collagen IV immunostaining. One week after the first biopsy, the animals were assigned to three groups that received daily oral dosages of cyclosporin, phenytoin, or nifedipine for 120 days. Additional gingival samples were obtained on days 52 and 120 of treatment from two animals from each group on the opposite sides from the first biopsies. RESULTS: Picrosirius red staining showed a predominance of mature collagen fibers in the control group. Conversely, there was an enlargement of areas occupied by immature collagen fibers in all groups at days 52 and 120, which was not uniform over each section. There was a general trend to lower levels of MMP-1 gene expression on day 52 and increased levels on day 120. Phenytoin led to increased levels of MMP-2 and collagen I gene expression on day 120, whereas the opposite was observed in the nifedipine group. CONCLUSION: Cyclosporin, phenytoin, and nifedipine led to phased and drug-related gene expression patterns, resulting in impaired collagen metabolism, despite the lack of prominent clinical signs.

Apoptosis and expression of caspase-3 in cyclosporin-induced gingival overgrowth.

BACKGROUND: The pathogenesis of epithelial thickening in gingival overgrowth remains obscure. Apoptosis plays an important role in maintaining tissue hemostasis. The aim of the present study was to investigate apoptosis via immunohistochemical analyses in cyclosporin A-induced gingival overgrowth tissue samples to determine whether these processes play a role in the pathogenesis of gingival overgrowth. METHODS: Gingival biopsies (one per person) were harvested from 22 renal transplant recipients (eight men and 14 women; mean age, 36.4 +/- 13.3 years) who had been diagnosed with cyclosporin A-induced gingival enlargement and from 12 systemically healthy persons (seven men and five women; mean age, 27.0 +/- 16.0 years) with plaque-induced gingivitis. Distributions of caspase-3 and apoptosis were determined immunologically. RESULTS: Significant differences were found with regard to caspase-3 levels and the extent of apoptosis between the cyclosporin A group and the control group. Plaque index, gingival index, and probing depths were significantly lower in the control group. CONCLUSION: The extent of keratinocyte apoptosis and decreased levels of caspase-3 may be an important factor affecting the gingiva of kidney transplant recipients with cyclosporin A-induced gingival overgrowth.

Matrix metalloproteinase-1 gene polymorphism in renal transplant patients with and without gingival enlargement.

BACKGROUND: Gingival enlargement frequently occurs in transplant patients receiving immunosuppressive drugs. It was hypothesized that gingival enlargement associated with cyclosporin use results in a disturbance of the homeostatic balance, which is characterized by an increase in the number of fibroblasts and volume of the extracellular matrix. Matrix metalloproteinase (MMP) serves as an initiator of extracellular matrix destruction. The aim of this study was to determine whether there is an association between genotypes of the MMP-1 gene and gingival enlargement in kidney transplant patients. METHODS: Sixty-one unrelated kidney transplant patients with gingival enlargement and 121 control transplant patients without enlargement were enrolled in the study. Six months after transplantation, all patients were given medication, which included cyclosporin A, and gingival enlargement was assessed. MMP-1 polymorphism was determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. RESULTS: In kidney transplant patients with gingival enlargement, the mean score of gingival enlargement was 1.42+/-0.63, whereas in control subjects, it was 0.0. There were no significant differences in the frequency of -1607 1G>2G alleles and genotypes between patients with and without gingival enlargement. In all subjects (N=182) and in patients without gingival enlargement, the genotype distribution met Hardy-Weinberg equilibrium criteria, whereas in patients with gingival enlargement, it was markedly different (P<0.06). There was a trend for carriers of at least the 1G allele to have an increased risk of gingival enlargement, but the trend was not statistically significant (odds ratio, 2.32; P<0.073). CONCLUSION: No association between the MMP-1 gene polymorphism and gingival enlargement was revealed in kidney transplant patients who were administered cyclosporin A as a principal immunosuppressive agent.

Elevated gene expression of MMP-1, MMP-10, and TIMP-1 reveal changes of molecules involved in turn-over of extracellular matrix in cyclosporine-induced gingival overgrowth.

In humans, pathogenesis in cyclosporine A (CsA)-induced gingival overgrowth (GO) includes the accumulation of extracellular matrix (ECM) constituents, viz., collagen type-1 and type-3 and proteoglycans, in subgingival connective tissue. However, whether this increase is associated with alterations of molecules pivotal for the turn-over of collagens and proteoglycans remains unclear. The present study explores the status of matrix metalloproteinase MMP-1 and MMP-10, which are important for fibrillar collagen and proteoglycan turn-over, and their tissue inhibitor TIMP-1, on their gene expression and protein levels in frozen sections derived from GO and matched normal tissue. In situ hybridization (ISH) revealed elevated levels of MMP-1 gene expression in the connective tissue of GO compared with normal tissue. This elevation also applied to MMP-10 and TIMP-1, the latter exhibiting the strongest gene transcription in the deep connective tissue. These differences detected by ISH were corroborated by quantitative reverse transcription/polymerase chain reaction; relative gene expression analysis indicated a 1.9-fold increase for MMP-1, a 2.3-fold increase for MMP-10, and a 4.8-fold increase for TIMP-1. Detection of the protein by indirect immunofluorescence showed that normal gingival tissue was devoid of all three proteins, although they were detectable in GO tissue, with emphasis on TIMP-1. Analysis of our data indicates elevated levels of MMP-1 and-10, and particularly TIMP-1. With respect to TIMP-1, this elevation may in turn lead to alterations in ECM turn-over by abrogating MMP-1 and MMP-10, thereby contributing to ECM accumulation associated with GO.

Effect of cyclosporin A on the expression of inducible nitric oxide synthase in the gingiva of rats.

BACKGROUND: The role of nitric oxide (NO) in the pathogenesis of cyclosporin A (CsA)-induced gingival overgrowth is still unknown. The purpose of this study was to evaluate the effect of CsA on the expression of nitric oxide synthases (NOS) in the gingival tissue of rats. METHODS: Thirty male Sprague-Dawley rats were randomly assigned to a control and two test groups. Rats in each group received CsA (0, 10, or 30 mg/kg) daily by gastric feeding for 4 weeks. The plasma NO and the NOS enzyme activities were assayed at week 4 in the blood samples and in the gingiva and lung tissue specimens, respectively. The distribution of inducible nitric oxide synthase (iNOS) was further evaluated in tissues obtained from the gingiva and lung at the end of weeks 1 and 4 by immunohistochemistry. RESULTS: In the CsA-treated animals, increased levels of plasma nitrites/nitrates were measured in comparison to those in control rats. Significantly greater iNOS enzyme activities were detected in lung and gingival tissues obtained from CsA-treated animals than from control animals. In addition, cells positively staining for iNOS were clearly observed in both gingival and lung tissues obtained from the CsA-treated animals by immunohistochemistry, whereas a few stained cells were found in those from the control group. The quantity of cells positively stained for iNOS was greater in tissue from week 4 than week 1. CONCLUSIONS: The effect of CsA on gingival iNOS expression was evaluated in rats for 4 weeks. A greater iNOS expression in the gingiva was observed after CsA therapy by both enzyme activities and immunohistochemica staining. Therefore, we suggest that CsA can increase gingival iNOS expression, which may play an important role in cyclosporin-induced gingival overgrowth.

Cyclosporin A specifically affects nuclear PLCbeta1 in immunodepressed heart transplant patients with gingival overgrowth.

One of the most commonly observed adverse effects of cyclosporin A (CsA) is the development of gingival overgrowth (GO). Fibroblasts are involved in GO, but the question why only a percentage of patients undergoing CsA treatment shows this side-effect remains unanswered. In a previous study, CsA has been demonstrated to induce over-expression of phospholipase C (PLC) beta(1) in fibroblasts of patients with clinical GO, in cells from both enlarged and clinically healthy gingival sites. In this work, we assessed the expression of PLCbeta isoforms to investigate whether the exaggerated fibroblast response to CsA related to increased PLCbeta(1) expression could also be detected in CsA-treated patients without clinical signs of GO. Our results support the hypothesis of a multi-factorial origin of gingival overgrowth, including specific changes within the gingival tissues orchestrating fibroblastic hyper-responsiveness as a consequence of a long-term in vivo exposure to cyclosporin A.

Evaluation of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 levels in gingival fibroblasts of cyclosporin A-treated patients.

BACKGROUND: Cyclosporin A (CsA) is a potent immunosuppressant used to prevent organ transplant rejection and to treat various autoimmune diseases. CsA-induced gingival overgrowth (CsA GO) is the most widely seen side effect of this drug; its pathogenesis is not completely understood. The aim of this study was to identify and compare matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels in gingival fibroblast cultures of tissues derived from renal transplant patients receiving CsA and exhibiting gingival overgrowth and from periodontally healthy control subjects. METHODS: Gingival overgrowth samples were obtained from patients undergoing therapy with CsA, and control tissues were obtained from systemically healthy donors. Gingival fibroblasts were grown using explant cultures. Three different study groups were identified: 1) CsA GO fibroblast culture; 2) CsA-treated healthy gingival fibroblast culture (H+CsA); and 3) healthy gingival fibroblast culture (H). The levels of MMP-1 and TIMP-1 in these groups of gingival fibroblasts were analyzed by enzyme-linked immunoabsorbent assay (ELISA). RESULTS: The levels of TIMP-1 were significantly lower in CsA GO than H (P < 0.05). There was no statistically significant difference in the levels of MMP-1 between H and CsA GO (P = 0.505). The ratio of MMP-1 to TIMP-1 was significantly higher in CsA GO than H (P < 0.05). CONCLUSIONS: The results of this study indicate that CsA therapy does not have a significant effect on MMP-1 levels. However, low TIMP-1 levels can be an important factor in the pathogenesis of CsA GO, since the balance between MMP-1 and TIMP-1 levels was changed by CsA.

Expression of matrix metalloproteinases in cyclosporin-treated gingival fibroblasts is regulated by transforming growth factor (TGF)-beta1 autocrine stimulation.

BACKGROUND: Gingival overgrowth is a common side effect following the administration of cyclosporin A (CsA). The pathogenesis of this condition is not fully understood; however, recent studies show that CsA regulates the transcription of several cytokines including transforming growth factor-beta1 (TGF-beta1). The aim of this study was to investigate the potential role of TGF-beta1 in the pathogenesis of CsA-induced gingival overgrowth, exploring a possible autocrine stimulation of TGF-beta1 as a cellular regulator of synthesis of matrix metalloproteinases (MMPs) and its tissue inhibitors (TIMPs). METHODS: Gingival fibroblasts from human normal gingiva were incubated with increasing concentrations of CsA, cultured for 24 hours, and the expression and production of TGF-beta1 determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. MMP and TIMP mRNA expression levels were also analyzed by RT-PCR. To determine the effect of TGF-beta1 on the expression of MMP and TIMP by human gingival fibroblasts under CsA treatment, human gingival fibroblast cultures were treated with sense oligonucleotides (SON) or antisense oligonucleotides (AON). RESULTS: CsA simultaneously stimulated TGF-beta1 expression and production and inhibited expression of MMP-1 and MMP-2 by human gingival fibroblasts, whereas CsA has a slight effect on TIMP-1 and TIMP-2 expression. AON reduced TGF-beta1 production as demonstrated by ELISA, whereas TGF-beta1 mRNA expression levels were not significantly modified. The inhibition of TGF-beta1 production by AON modulated MMP expression, demonstrating the autocrine inhibitory effect of TGF-beta1 in CsA-treated human gingival fibroblasts. CONCLUSIONS: The data presented here suggest that TGF-beta1 in an autocrine fashion may contribute to a reduction of proteolytic activity of human gingival fibroblasts in CsA-induced gingival overgrowth, which favors the accumulation of extracellular matrix.

Phenytoin-mediated androgen metabolism in gingival fibroblasts. Effects of the antiandrogen finasteride and the alkaline phosphatase inhibitor levamisole.

OBJECTIVES: This investigation attempts to identify the role of the alkaline phosphatase inhibitor levamisole (L) and the antiandrogen finasteride (F) on 5alpha-reductase activity in gingival fibroblasts, to elucidate mechanisms for phenytoin-induced gingival overgrowth. MATERIAL AND METHODS: Human gingival fibroblasts were incubated with Eagle's MEM and 14C-testosterone/14C-4-androstenedione as substrates; effective concentrations of phenytoin (Ph), levamisole (L) and finasteride (F), alone and in combinations of (Ph + F) (Ph + L) were added to the incubate. After 24 h, the medium was analysed for steroid metabolites and quantified using a radioisotope scanner. RESULTS: The metabolites isolated were 5alpha-dihydrotestosterone (DHT), 4-androstenedione (4-A) or testosterone (T) from each substrate. With 14C-T as substrate, Ph stimulated DHT synthesis by 1.7-fold, while F and L inhibited this activity by 1.8-fold and 34%, respectively (n = 6; P < 0.001). The combination of Ph + F reduced yields by 2.7-fold compared with Ph alone and Ph + L reduced DHT synthesis by 2.4-fold compared with Ph alone (n = 6; P < 0.001). When 14C-4-androstenedione was used as substrate, similar trends were identified. CONCLUSION: These results suggest that the alkaline phosphatase inhibitor levamisole and the 5alpha-reductase inhibitor finasteride can substantially decrease the yields of DHT in fibroblasts, stimulated by phenytoin. This could be a potential target for reducing the gingival overgrowth caused by phenytoin.