Drug-Induced Gingival Overgrowth: A Pilot Study on the Effect of Diphenylhydantoin and Gabapentin on Human Gingival Fibroblasts.

INTRODUCTION: The administration of several classes of drugs can lead to the onset of gingival overgrowth: anticonvulsants, immunosuppressants, and calcium channel blockers. Among the anticonvulsants, the main drug associated with gingival overgrowth is diphenylhydantoin. MATERIALS AND METHODS: In this study, we compared the effects of diphenylhydantoin and gabapentin on 57 genes belonging to the "Extracellular Matrix and Adhesion Molecule" pathway, present in human fibroblasts of healthy volunteers. RESULTS: Both molecules induce the same gene expression profile in fibroblasts as well as a significant upregulation of genes involved in extracellular matrix deposition like COL4A1, ITGA7, and LAMB3. The two treatments also induced a significant downregulation of genes involved in the expression of extracellular matrix metalloproteases like MMP11, MMP15, MMP16, MMP24, and transmembrane receptor ITGB4. CONCLUSIONS: Data recorded in our study confirmed the hypothesis of a direct action of these drugs at the periodontium level, inducing an increase in matrix production, a reduction in its degradation, and consequently resulting in gingival hyperplasia.

Phenytoin and gingival mucosa: A molecular investigation.

Several distinct classes of drugs, such as anticonvulsants, immunosuppressants, and calcium channel blockers, caused gingival overgrowth. One of the main drugs associated with the gingival overgrowth is the anti-epileptic such as phenytoin, which affects gingival tissues by altering extracellular matrix metabolism. In our study, we evaluate the effect of phenytoin, a drug whose active substance is phenytoin, on gingival fibroblasts of healthy volunteers. Gene expression of 29 genes was investigated in gingival fibroblasts' cell culture treated with phenytoin compared with untreated cells. Among the studied genes, only 13 genes (CXCL5, CXCL10, CCR1, CCR3, CCR5, CCR6, IL-1A, IL-1B, IL-5, IL-7, IL-6R, BMP-2, and TNFSF-10) were statistically significant. All but one gene resulted downregulated after 24 h of treatment with phenytoin. BPM2 was the only, although weakly, up-expressed gene. Probably, we have not highlighted overexpression of the other inflammatory molecules because the study was performed on healthy people. Many studies show that phenytoin induces the overexpression of these cytokines but, probably, in our study, the drug does not have the same effect because we used gingival fibroblasts of healthy people.

Evaluation of inductive effects of different concentrations of cyclosporine A on MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 in fetal and adult human gingival fibroblasts.

Background The etiology of gingival overgrowth due to cyclosporine A (CsA) is still unknown. The aim of this study was to determine the possible role of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) on extra-cellular matrix (ECM) homeostasis when treated with different levels of CsA and its difference between fetal and adult human gingival fibroblasts (HGFs). Methods Each group of cells (adult and fetal) was cultured in 40 wells that consisted of four different CsA treatment concentrations. Every 10 wells were treated with 0, 50, 100, and 150 ng/mL of CsA which makes a total of 80 wells. Supernatants of every well were used to determine the concentration of MMPs and TIMPs using the Elisa kits from Boster, CA, USA. Results MMP-1 level increased with the treatment of CsA when treated with 50 and 150 ng/mL of CsA (p = 0.02 and p = 0.04) as TIMP-1 decreased (p < 0.0001) in adult group; while in the fetal group, TIMP-1 level increased with treatment of 150 ng/mL (p < 0.0001). MMP-2 level increased in both adult and fetal groups (p < 0.0001). MMP-3 level decreased in adult group (p < 0.0001) but went up in fetal HGFs (p = 0.01) when treated with 150 ng/mL CsA. TIMP-2 level increased in all wells significantly when treated with CsA (p < 0.0001). The study showed that CsA affects secretion of MMPs and TIMPs. MMP-1 increment and TIMP-1 decrement were observed, which indicate more degradation of ECM. This may be due to single donor use in this study. TIMP-2 and MMP-2 were both more active when treated with CsA which may be due to the gelatinase activity of them and that in CsA gingival overgrowth. There was more inflammation rather than fibrosis.

Phenytoin activates Smad3 phosphorylation and periostin expression in drug-induced gingival enlargement.

Drug-induced gingival enlargement (DIGE) is a fibrotic condition associated with systemic administration of the anti-epileptic drug, phenytoin. We have previously demonstrated that periostin, which is transforming growth factor-beta (TGF-ß) inducible gene, is upregulated in various fibrotic conditions including gingival enlargement associated with nifedipine. The objective of this study was to assess periostin expression in phenytoin-induced gingival enlargement (PIGE) tissues and to investigate the mechanisms underlying periostin expression. Human PIGE tissues were assessed using Masson's trichrome, with cell infiltration and changes in extracellular matrix composition characterized through labeling with antibodies to periostin, phospho-SMAD 3, TGF-ß, as well as the macrophage markers CD68 and RM3/1. Using human gingival fibroblasts (HGFs) in vitro we examined the pathways through which phenytoin acts on fibroblasts. In PIGE tissues, which demonstrate altered collagen organization and increased inflammatory cell infiltration, periostin protein was increased compared with healthy tissues. p-SMAD2/3, the transcription factor associated with canonical TGF-ß signaling, is localized to the nuclei in both gingival fibroblasts and oral epithelial cells in PIGE tissues, but not in healthy tissue. In vitro culture of HGFs with 15 and 30 µg/ml of phenytoin increased periostin protein levels, which correlated with p-SMAD3 phosphorylation. Inhibition of canonical TGF-ß signaling with SB431542 significantly reduced phenytoin induction of SMAD3 phosphorylation and periostin expression in HGFs. Analysis of PIGE tissues showed a subset of CD68 stained macrophages were TGF-ß positive and that RM1/3 regenerative macrophages were present in the tissues. Our results demonstrate that phenytoin up-regulates periostin in HGFs in a TGF-ß-dependent manner.

4PBA strongly attenuates endoplasmic reticulum stress, fibrosis, and mitochondrial apoptosis markers in cyclosporine treated human gingival fibroblasts.

Cyclosporine induces overgrowth of human gingiva. Previously we have shown (i) cyclosporine-inducing ER stress in human gingival fibroblasts (HGF), (ii) increased matrix protein expression, and (iii) interference with mitochondrial pro- and anti-apoptotic factors. This study was undertaken to assess the effects of melatonin (an antioxidant), 4PBA (an ER stress inhibitor), and simvastatin on the expression of ER Stress markers as well as on matrix and mitochondrial markers. HGF incubated with cyclosporine, or without melatonin/4PBA/statin. After 24 hr of incubation, mRNA expression of ER stress markers (GRP78, CHOP, XBP1, and XBPs) and matrix protein markers (like α-SMA, VEGF, TGF-ß, CTGF), and mitochondrial apoptosis markers estimated and compared with housekeeping gene GAPDH. Compared to the control cyclosporine significantly augmented ER Stress and matrix proteins, which decreased significantly with the use of melatonin, 4PBA, and simvastatin. The mitochondrial proapoptotic molecule cyclophilin D, as well as Bcl2 expression also decreased after PBA treatment, paralleling an increase in cytochrome c expression. The effect of 4PBA was much more pronounced than the influence of other two. In conclusion, 4PBA could be a viable therapeutic option for drug-induced gingival overgrowth.

Biological roles of KGF, CTGF and TGF-ß in cyclosporine-A- and phenytoin- induced gingival overgrowth: A comparative experimental animal study.

OBJECTIVE: To identify the possible biological roles of keratinocyte growth factor (KGF), connective tissue growth factor (CTGF) and transforming growth factor-ß (TGF-ß) in cyclosporine-A (CsA) and phenytoin (PNT)-induced gingival overgrowth (GO) and to correlate them with each other. METHODS: Sixty adult male albino rats were selected and divided into 3 equal groups. Group I rats received no treatment. Group II rats were administrated CsA for 7 weeks. Group III were administrated PNT for the same period. Rats were euthanized at the end of the experiment and routine tissue processing was carried out. The obtained specimens were stained with H&E, KGF, CTGF and TGF-ß antibodies. RESULTS: One-way MANOVA test for KGF, CTGF and TGF-ß revealed an overall significant difference between the different groups (P<0.001). LSD post hoc test for multiple comparisons revealed a significant difference between each two groups. Two-tailed Pearson correlation for group II revealed non-significant weak positive correlations between KGF & CTGF and between CTGF & TGF-ß. Non-significant weak negative correlation was found between KGF & TGF-ß. Meanwhile, group III revealed non-significant weak positive correlation between KGF & TGF-ß and between CTGF & TGF-ß. Significant moderate positive correlation was found between KGF & CTGF. CONCLUSION: The findings of the present study indicated that KGF, CTGF and TGF-ß have biological roles in progression of CsA- and PNT- induced GO. KGF plays a greater role in CsA- induced GO than in PNT- induced GO. Meanwhile, CTGF and TGF-ß play a role in PNT- induced GO greater than in CsA- induced GO.

Transglutaminase 2 May Be Associated with Peri-implant Gingival Overgrowth: Preliminary Assessments.

UNLABELLED: Tissue transglutaminase 2 (TG2) is ubiquitously expressed in normal tissues and plays an important role in the pathophysiology of wound healing. An increase in periodontal tissues has been previously reported in cyclosporine-induced gingival overgrowth. PURPOSE: The aim of this study was to explore associations between TG2 expression and the vascularization and maturation processes of peri-implant soft tissues over time. MATERIALS AND METHODS: Edentulous patients proposed for mandibular implant-retained overdentures were included in the study. Biopsies of the peri-implant mucosa were performed at the first surgical stage and at 4, 8, and 12 months after prosthetic load. A follow-up program was directed to record plaque indexes, bleeding on probing data, and pocket probing depth around implants. An evaluation of the vessels' density was carried out by digital virtual microscopy and using an immunohistochemistry approach (antibodies anti-CD31, anti-TG2). A robust multivariable regression model was implemented. RESULTS: According to model results, blood vessel count and probing (as a marker of gingival overgrowth in absence of plaque) significantly decrease over time and are associated with TG2, particularly for values above the median. CONCLUSION: The association of an increased TG2 expression in the extracellular matrix might have a significant impact in the development of gingival overgrowth around a loaded implant.

Prevention of phenytoin-induced gingival overgrowth by lovastatin in mice.

Drug-induced gingival overgrowth is caused by the antiseizure medication phenytoin, calcium channel blockers, and ciclosporin. Characteristics of these drug-induced gingival overgrowth lesions differ. We evaluate the ability of a mouse model to mimic human phenytoin-induced gingival overgrowth and assess the ability of a drug to prevent its development. Lovastatin was chosen based on previous analyses of tissue-specific regulation of CCN2 production in human gingival fibroblasts and the known roles of CCN2 in promoting fibrosis and epithelial to mesenchymal transition. Data indicate that anterior gingival tissue overgrowth occurred in phenytoin-treated mice based on gross tissue observations and histomorphometry of tissue sections. Molecular markers of epithelial plasticity and fibrosis were regulated by phenytoin in gingival epithelial tissues and in connective tissues similar to that seen in humans. Lovastatin attenuated epithelial gingival tissue growth in phenytoin-treated mice and altered the expressions of markers for epithelial to mesenchymal transition. Data indicate that phenytoin-induced gingival overgrowth in mice mimics molecular aspects of human gingival overgrowth and that lovastatin normalizes the tissue morphology and the expression of the molecular markers studied. Data are consistent with characterization of phenytoin-induced human gingival overgrowth in vivo and in vitro characteristics of cultured human gingival epithelial and connective tissue cells. Findings suggest that statins may serve to prevent or attenuate phenytoin-induced human gingival overgrowth, although specific human studies are required.

Cellular crosstalk mechanism of Toll-like receptors in gingival overgrowth (review).

Gingival overgrowth is an undesirable outcome of systemic medication and is evidenced by the accretion of collagenous components in gingival connective tissues along with diverse degrees of inflammation. Phenytoin therapy has been found to induce the most fibrotic lesions in gingiva, cyclosporine caused the least fibrotic lesions, and nifedipine induced intermediate fibrosis in drug­induced gingival overgrowth. In drug­induced gingival overgrowth, efficient oral hygiene is compromised and has negative consequences for the systemic health of the patients. Toll­like receptors (TLRs) are involved in the effective recognition of microbial agents and play a vital role in innate immunity and inflammatory signaling responses. TLRs stimulate fibrosis and tissue repairs in several settings, although with evident differences between organs. In particular, TLRs exert a distinct effect on fibrosis in organs with greater exposure to TLR ligands, such as the gingiva. Cumulative evidence from diverse sources suggested that TLRs can affect gingival overgrowth in several ways. Numerous studies have demonstrated the expression of TLRs in gingival tissues and suggested its potential role in gingival inflammation, cell proliferation and synthesis of the extracellular matrix which is crucial to the development of gingival overgrowth. In the present review, we assessed the role of TLRs on individual cell populations in gingival tissues that contribute to the progression of gingival inflammation, and the involvement of TLRs in the development of gingival overgrowth. These observations suggest that TLRs provide new insight into the connection among infection, inflammation, drugs and gingival fibrosis, and are therefore efficient therapeutic target molecules. We hypothesize that TLRs are critical for the development and progression of gingival overgrowth, and thus blocking TLR expression may serve as a novel target for antifibrotic therapy.

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 ß1). 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 ß1 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.

Role of transforming growth factor ß-connective tissue growth factor pathway in dihydropyridine calcium channel blockers-induced gingival overgrowth.

BACKGROUND: Gingival overgrowth was reported as a side effect after chronic administration of several drugs, which, despite their different pharmacological effect, seem to have the gingival mucosa as a secondary target. The thickness of the gingival epithelium and fibrosis in the lamina propria are unspecific changes that together determine the enlargement of the gingival mucosa, but the molecular mechanisms responsible for the imbalance of collagen synthesis/breakdown are still uncertain. The aim of our study was to assess the role of TGF-ß1-CTGF pathway in the activation of cells with a fibrilogenetic phenotype responsible for the gingival fibrosis developed after chronic administration of dihydropyridine calcium channel blockers. MATERIALS AND METHODS: Fragments of gingival tissue collected from patients clinically diagnosed with gingival overgrowth after chronic administration of nifedipine and amlodipine were processed for paraffin embedding. Serial sections were used for routine staining Masson and Gömöri's silver impregnation in order to reveal collagen accumulation and for immunohistochemical reactions to label TGF-ß1, CTGF, Ki67 and α-SMA. RESULTS: Routine histological staining for collagen revealed the presence of gingival fibrosis and a change between type I collagen/type III collagen ratio. Regardless of the drug involved, many slides showed extended TGF-ß1 positive areas, mainly in the profound - spinous and basal - layers, but also in some cells from the subjacent connective tissue. CTGF exposed intense positive reaction in the basal and parabasal layers, but also in resident cells from the connective tissue. Ki67 immunolabeling did not reveal an increased fibroblast proliferation in the lamina propria. We noticed the presence of a small number of myofibroblasts in the lamina propria. CONCLUSIONS: These findings suggest that TGF-ß1-CTGF axis is activated in dihydropyridine calcium channel blockers-induced gingival overgrowth and exerts a different control on the activation of fibroblasts with a synthetic phenotype. These results also have implications for better understanding mechanisms of fibrosis and the future use of this pathogenic pathway as a therapeutic target in order to limit gingival fibrosis.

The role of phosphatase and tensin homolog in drug-induced gingival overgrowth.

BACKGROUND AND OBJECTIVE: We proposed that phosphatase and tensin homolog (PTEN) might be one of the signaling proteins that alter the balance between cell growth and cell death in drug-induced gingival overgrowth. The aim of this study was to investigate the expression of PTEN in subjects using cyclosporine A and to analyze the relationship between PTEN and cell proliferation marker, proliferating cell nuclear antigen (PCNA), in cyclosporine A-induced gingival overgrowth. MATERIAL AND METHODS: In total, samples from 36 subjects, i.e. 24 cyclosporine A-mediated renal transplant patients with gingival overgrowth (n = 12) or without gingival overgrowth (n = 12) and 12 matched periodontally healthy subjects, were included in the study. PTEN and PCNA expressions in gingival tissues were analyzed using immunohistochemistry, PTEN expression was also analyzed by western blot. PTEN immunoreactivity was calculated with a histologic score (HSCORE) value and PCNA immunoreactivity was calculated with the PCNA-proliferative index. RESULTS: Phosphatase and tensin homolog HSCORE for the group with gingival overgrowth was found to be significantly lowest compared to the group without gingival overgrowth and the control group (p < 0.001) while the highest PTEN HSCORE was found in the control group. In addition, the PTEN HSCORE for the group without gingival overgrowth was significantly lower compared to controls (p < 0.001). The highest PCNA-proliferative index score was observed in the group with gingival overgrowth while the lowest score was observed in the control group (p < 0.001). The immunoblot signal for PTEN was significantly decreased in the group with gingival overgrowth compared to the group without gingival overgrowth and the control group (p < 0.001). Western blot results were different from immunohistochemistry and revealed there was no significant difference between the without gingival overgrowth and the control group (p > 0.05). CONCLUSION: Our results showing decreased PTEN levels in patients with gingival overgrowth supported with increased PCNA expression suggested that PTEN might take part in the imbalance between cell proliferation and death in drug-induced gingival overgrowth.

Effects of cyclosporine and phenytoin on biomarker expressions in gingival fibroblasts of children and adults: an in vitro study.

BACKGROUND: Both phenytoin (PhT) and cyclosporine (CsA) have been related to gingival overgrowth, but the presence and incidence of cytokines in gingival tissues are associated with different mechanisms. On the basis of a few epidemiologic data, children are more prone to have gingival overgrowth than adults and there is no obvious plausibility to justify this difference. The aim of this study was to investigate the effect of PhT and CsA on the some biological marker expression and compare it among adults and children. METHODS: Gingival fibroblasts that had been harvested from adults and children with normal gingiva were incubated with CsA and PhT and then cultured for 48 h. Matrix metalloproteinases (MMP-1 and MMP-2), tissue inhibitor of metalloproteinases (TIMP), collagen (CoL), elastin (Eln), lysyl oxidase (Lysyl), cathepsin (Cat) L and B, and mRNA levels in culture were determined by reverse transcription polymerase chain reaction. The amounts of transforming growth factor-ß (TGF-ß) and epidermal growth factor (EGF) were assessed by enzyme-linked immunosorbent assay. RESULTS: CsA and PhT stimulated TGF-ß and Cat B production and inhibited expression of MMP-1 by fibroblasts. CsA suppressed TIMP in children, but PhT stimulated its expression. In adults, both CSA and PHT increased TGF-ß, Lysyl, and EGF levels. CsA reduced Eln level, whereas PhT increased it. CONCLUSIONS: The results suggest that CsA and PhT have different effects on biogene marker expression in adults and children, or drug-induced gingival overgrowth is affected by different cellular pathways in children in contrast to that in adults. It seems that in children the MMP-1/TIMP system, and in adults the Lysyl/Eln pathway, plays an important role in impaired CoL metabolism.

Nifedipine induces periostin expression in gingival fibroblasts through TGF-beta.

Gingival enlargement is a fibrotic condition that can arise from systemic administration of the dihydropyridine calcium channel blocker nifedipine. Periostin, a transforming growth factor-beta (TGF-ß)-inducible matricellular protein, has been associated with fibrosis in numerous tissues, but its expression has never been examined in nifedipine-influenced gingival enlargement (NIGE). The objective of this study was to assess if periostin up-regulation is associated with NIGE and whether nifedipine induces periostin expression in gingival fibroblasts. In NIGE tissue (n = 6), periostin is overexpressed in the gingival connective tissue compared with healthy control tissue (n = 6). The transcription factor p-SMAD2/3, which is associated with canonical TGF-ß signaling, localizes to the nuclei in both HGFs and oral epithelial cells in NIGE tissues, but not in control healthy tissue. In vitro culture of HGFs with 30 and 100 ng/mL of nifedipine significantly increased periostin mRNA and protein levels, which correlated with increased levels of active TGF-ß and increased phosphorylation and nuclear localization of SMAD3. Blocking of canonical TGF-ß signaling through inhibition of the TGF-ß receptor I with SB431542 significantly reduced nifedipine-induced SMAD3 phosphorylation and periostin expression. Our results demonstrate that nifedipine up-regulates periostin in HGFs in a TGF-ß-dependent manner.

Immunolocalization of Bcl-2 oncoprotein in amlodipine-induced gingival overgrowth.

BACKGROUND: Drug-induced gingival overgrowth (DIGO) is one of the unwanted side effects of amlodipine therapy, but the pathogenesis still remains unclear. Apoptosis, which plays a ubiquitous role in tissue homeostasis, including gingiva, may be involved in the development of gingival enlargement. AIMS AND OBJECTIVES: (i) To study the distribution of Bcl-2 in healthy and overgrown gingival tissues. (ii) To compare and correlate the Bcl-2 expression in gingival samples from subjects on amlodipine therapy to the findings in healthy controls. MATERIALS AND METHODS: A total of 25 subjects were recruited for the study - 15 hypertensive patients and 10 systemically healthy subjects. Both the groups were analyzed for Bcl-2 expression using immunohistochemistry. RESULTS: Few of the control specimens showed weak positivity to Bcl-2 antibody, with the distribution limited to the basal cell layers alone, whereas 10 hyperplastic specimens expressed Bcl-2 and, unlike the control group, the distribution pattern was seen in both basal and suprabasal layers. CONCLUSION: The results indicate that the pathogenesis of amlodipine-induced gingival overgrowth might involve inhibition of apoptosis, especially with morphogenesis of hyperplastic gingival epithelia.

The possible potential therapeutic targets for drug induced gingival overgrowth.

Gingival overgrowth is a side effect of certain medications. The most fibrotic drug-induced lesions develop in response to therapy with phenytoin, the least fibrotic lesions are caused by cyclosporin A, and the intermediate fibrosis occurs in nifedipine-induced gingival overgrowth. Fibrosis is one of the largest groups of diseases for which there is no therapy but is believed to occur because of a persistent tissue repair program. During connective tissue repair, activated gingival fibroblasts synthesize and remodel newly created extracellular matrix. Proteins such as transforming growth factor (TGF), endothelin-1 (ET-1), angiotensin II (Ang II), connective tissue growth factor (CCN2/CTGF), insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF) appear to act in a network that contributes to the development of gingival fibrosis. Since inflammation is the prerequisite for gingival overgrowth, mast cells and its protease enzymes also play a vital role in the pathogenesis of gingival fibrosis. Drugs targeting these proteins are currently under consideration as antifibrotic treatments. This review summarizes recent observations concerning the contribution of TGF-ß, CTGF, IGF, PDGF, ET-1, Ang II, and mast cell chymase and tryptase enzymes to fibroblast activation in gingival fibrosis and the potential utility of agents blocking these proteins in affecting the outcome of drug-induced gingival overgrowth.

Expression of angiotensin II and its receptors in cyclosporine-induced gingival overgrowth.

BACKGROUND AND OBJECTIVES: The renin-angiotensin system (RAS) is considered as a hormonal circulatory system involved in maintaining blood pressure, electrolyte and fluid homeostasis. RAS components can be synthesized in local tissues and are found to play a role in gingival overgrowth. The drug-induced gingival overgrowth (DIGO) is a fibrotic condition, which is associated with multiple factors, including inflammation and adverse drug effects such as cyclosporine A. This study was directed forward to the identification of the angiotensinogen, angiotensin II (Ang II) and its receptors AT1 /AT2 expression in DIGO tissues and cyclosporine-treated human gingival fibroblast cells. MATERIAL AND METHODS: Gingival samples were obtained from patients with cyclosporine-induced gingival overgrowth, chronic periodontitis and normal healthy subjects. The total RNA was isolated and reverse transcription-polymerase chain reaction was performed for angiotensinogen, Ang II and AT1 /AT2 receptor. Ang II protein was estimated from tissue by enzyme immunoassay. The expression of Ang II and its receptors were also examined in gingival fibroblast cells treated with cyclosporine. RESULTS: Ang II mRNA and protein expression was significantly higher in patients with DIGO than in patients with periodontitis and healthy subjects. The AT1 mRNA was expressed more than AT2 in all examined tissues. In gingival fibroblasts, Ang II and AT1 expressions were increased with cyclosporine incorporation compared to controls. CONCLUSION: These results suggest that cyclosporine can modulate local expression of RAS components such as angiotensinogen, Ang II and its receptors in gingival tissues and gingival fibroblast cells.

Nitric oxide inhibits androgen receptor-mediated collagen production in human gingival fibroblasts.

UNLABELLED: Lin S-J, Lu H-K, Lee H-W, Chen Y-C, Li C-L, Wang L-F. Nitric oxide inhibits androgen receptor-mediated collagen production in human gingival fibroblasts. J Periodont Res 2012; 47: 701-710. © 2012 John Wiley & Sons A/S Background and Objective: In our previous study, we found that flutamide [an androgen receptor (AR) antagonist] inhibited the up-regulation of collagen induced by interleukin (IL)-1ß and/or nifedipine in gingival fibroblasts. The present study attempted to verify the role of nitric oxide (NO) in the IL-1ß/nifedipine-AR pathway in gingival overgrowth. MATERIAL AND METHODS: Confluent gingival fibroblasts derived from healthy individuals (n = 4) and those with dihydropyridine-induced gingival overgrowth (DIGO) (n = 6) were stimulated for 48 h with IL-1ß (10 ng/mL), nifedipine (0.34 µm) or IL-1ß + nifedipine. Gene and protein expression were analyzed with real-time RT-PCR and western blot analyses, respectively. Meanwhile, Sircol dye-binding and the Griess reagent were, respectively, used to detect the concentrations of total soluble collagen and nitrite in the medium. RESULTS: IL-1ß and nifedipine simultaneously up-regulated the expression of the AR and type-I collagen α1 [Colα1(I)] genes and the total collagen concentration in DIGO cells (p < 0.05). IL-1ß strongly increased the expression of inducible nitric oxide synthase (iNOS) mRNA and the nitrite concentration in both healthy and DIGO cells (p < 0.05). However, co-administration of IL-1ß and nifedipine largely abrogated the expression of iNOS mRNA and the nitrite concentration with the same treatment. Spearman's correlation coefficients revealed a positive correlation between the AR and total collagen (p < 0.001), but they both showed a negative correlation with iNOS expression and the NO concentration (p < 0.001). The iNOS inhibitor, 1400W, enhanced IL-1ß-induced AR expression; furthermore, the NO donor, NONOate, diminished the expression of the AR to a similar extent in gingival fibroblasts derived from both healthy patients and DIGO patients (p < 0.05). CONCLUSION: IL-1ß-induced NO attenuated AR-mediated collagen production in human gingival fibroblasts. The iNOS/NO system down-regulated the axis of AR/Colα1(I) mRNA expression and the production of AR/total collagen proteins by DIGO cells.

The involvement of TGF-ß1 and CTGF in regional gingival overgrowth.

Gingival overgrowth is a multifactorial and invalidating condition. Our research is about gingival overgrowth caused by gingival plaque, its purpose being the evaluation of the presence of gingivitis and/or parodontitis in patients with gingival growth and the extent in which there is a connection between gingival overgrowth and the inflammatory process that can contribute to an exceedingly stimulation of the overgrowth. Immunohistological study was conducted on human material--gingival mucosa that came from patients with ages between 20-65 years, divided into three groups: group I--control group, group II--patients with gingivitis, group III--patients with local or general periodontitis. The intensity of immunohistochemical staining of TGF-ß1 and CTGF varies from one group to another, and also depends on the area of gingival mucosa that was observed. TGF-ß1 has a crucial role in periodontal disease fibrogenesis by intensifying the action of CTGF.

Curcumin inhibits thrombin-stimulated connective tissue growth factor (CTGF/CCN2) production through c-Jun NH2-terminal kinase suppression in human gingival fibroblasts.

BACKGROUND: Connective tissue growth factor (CTGF/CCN2), associated with multiple human fibrotic diseases, is overexpressed in the tissue of gingival overgrowth. Although surgical excision is the current treatment modality for gingival overgrowth, the recurrent rate is high despite proper recall programs. Thrombin plays a key role in wound repair, remodeling, and fibrosis after injury and exerts profibrotic effects by activating protease-activated receptors (PARs). Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] is a natural plant phenolic compound that possesses both anti-inflammatory and antioxidant properties. This study investigates the signaling pathway of thrombin-induced CCN2 expression and inhibition of CCN2 expression by curcumin. METHODS: The signaling pathway of thrombin-induced CCN2 expression in human gingival fibroblasts (HGFs) was studied using Western blot analysis. The CCN2 mRNA level was determined by quantitative reverse transcription-polymerase chain reaction. RESULTS: Thrombin induced CCN2 expression in HGFs by activating PAR1. Pretreatment with antioxidant N-acetyl-l-cysteine, apoptosis signal-regulating kinase 1 (ASK1) inhibitor thioredoxin, and c-Jun NH2-terminal kinase (JNK) inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one) significantly reduced thrombin-induced CCN2 expression in HGFs. Curcumin dose dependently inhibited thrombin-induced CCN2 expression through JNK suppression in HGFs. CONCLUSIONS: The results of this study suggest that thrombin-induced CCN2 expression may occur through PAR1, reactive oxygen species, ASK1, and JNK signaling in HGFs. Curcumin could effectively inhibit CCN2 expression through JNK suppression. These signaling events are important for wound healing and fibrosis. Additional research, including animal studies, is required to confirm the inhibiting role of curcumin in the development of gingival overgrowth.