Elevation of collagen type I in fibroblast-keratinocyte cocultures emphasizes the decisive role of fibroblasts in the manifestation of the phenotype of cyclosporin A-induced gingival overgrowth

Background and Objective: Collagen type I elevation in cyclosporin A‐induced gingival overgrowth supports evidence that gingival fibroblasts play a decisive role in the manifestation of the phenotype. To analyze the role of gingival fibroblasts under more in vivo‐like conditions, we evaluated the effect of cyclosporin A on collagen type I gene and protein expression in gingival overgrowth‐derived gingival fibroblasts established as cocultures with gingival keratinocytes as well as in matched gingival fibroblast monolayers. Material and methods: Monolayers and cocultures of primary gingival fibroblasts were treated with cyclosporin A for 6 and 72 h. The expression of collagen type I mRNA was analyzed by quantitative real time polymerase chain reaction, while expression and secretion of collagen type I protein was analyzed by indirect immunofluorescence and western blotting. Results: Compared with controls, significant elevation of collagen type I mRNA was restricted to cocultures after 6 and 72 h of treatment with cyclosporin A. In keratinocytes, collagen type I remained undetectable. In monolayers and cocultures, indirect immunofluorescence showed a slightly higher level of collagen type I protein in gingival fibroblasts in response to stimulation with cyclosporin A. Semiquantitative detection of collagen type I by western blotting demonstrated a nonsignificant increase for cell extracts in monolayers and cocultures. For secreted collagen type I, western blot analysis of the supernatants revealed elevated protein levels in cultures stimulated with cyclosporin A. Compared with the corresponding monolayers, the stimulatory effect of cyclosporin A on protein secretion was significant only in coculture. Conclusion: Our results indicate that collagen type I is a target of cyclosporin A and that gingival fibroblasts are decisive for the manifestation of the gingival overgrowth‐phenotype. Furthermore, the results suggest that cocultures of gingival overgrowth‐derived gingival fibroblasts and gingival keratinocytes permit analysis of cyclosporin A‐induced effects under more in vivo‐like conditions.     

Plasminogen activators and plasminogen activator inhibitors in gingival crevicular fluid of cyclosporin A-treated patients

BACKGROUND: The plasminogen activator (PA) system plays many roles in the inflammatory process and tissue remodelling and repair and is considered to play a significant role in periodontal tissue destruction and healing. The aim of this study was to evaluate the role of the PA system in cyclosporin A (CsA)-induced gingival overgrowth in renal transplant patients. METHODS: Eighteen renal transplant patients exhibiting moderate to severe CsA-induced gingival overgrowth, 10 other renal transplant patients receiving CsA therapy but showing no sign of CsA-induced gingival overgrowth (CsA-H), 16 chronic gingivitis patients (CG) and 16 systemically and periodontally healthy control subjects (H) were included in the study. Gingival crevicular fluid (GCF) samples were obtained from four randomly selected sites in each subject with the exception of the CsA-induced gingival overgrowth group, where four GCF samples were harvested from sites with severe overgrowth (CsA GO+) and from four sites without any gingival overgrowth (CsA GO-). The GCF levels of albumin, tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 2 (PAI-2) were analysed by enzyme-linked immunosorbent assay. The results were tested for statistical differences. RESULTS: In CsA GO+ sites t-PA levels were significantly elevated in comparison with gingivitis and healthy sites, while PAI-2 levels in these sites showed statistically significant differences only with CsA-H and gingivitis sites (p<0.05). The levels of t-PA and PAI-2 were significantly higher in CsA GO- sites compared with those of CsA-H, gingivitis and healthy sites (p<0.05). The levels of u-PA and PAI-1 failed to show significant differences between the study groups. CONCLUSIONS: The findings of the present study indicate alterations in GCF t-PA and PAI-2 levels in CsA-induced gingival overgrowth and might suggest involvement of the plasminogen activating system in the pathogenesis of this side-effect of CsA therapy. However, to what extent these molecules contribute to the pathogenesis of CsA-induced gingival overgrowth remains to be determined.     

Effect of lipopolysaccharide from periodontal pathogens on the production of tissue plasminogen activator and plasminogen activator inhibitor 2 by human gingival fibroblasts

Both tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) are important proteolysis factors present in inflamed human periodontal tissues. The aim of the present study was to investigate the effect of lipopolysaccharide (LPS) on the synthesis of t-PA and PAI-2 by human gingival fibroblasts (HGF). LPS from different periodontal pathogens including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum were extracted by the hot phenol water method. The levels of t-PA and PAI-2 secreted into the cell culture media were measured by enzyme-linked immunosorbent assays (ELISA). The mRNA for t-PA and PAI-2 were measured by RT-PCR. The results showed t-PA synthesis was increased in response to all types of LPS studied and PAI-2 level was increased by LPS from A. actinomycetemcomitans and F. nucleatum, but not P. gingivalis. When comparing the effects of LPS from non-periodontal bacteria (Escherichia coli and Salmonella enteritidis) with the LPS from periodontal pathogens, we found that the ratio of t-PA to PAI-2 was greater following exposure of the cells to LPS from periodontal pathogens. The highest ratio of t-PA to PAI-2 was found in those cells exposed to LPS from P. gingivalis. These results indicate that LPS derived from periodontal pathogens may cause unbalanced regulation of plasminogen activator and plasminogen activator inhibitor by HGF and such an effect may, in part, contribute to the destruction of periodontal connective tissue through dysregulated pericellular proteolysis.     

LPS调控人牙龈成纤维细胞表达uPA的研究

目的：观察LPS对牙龈成纤维细胞表达尿激酶型纤溶酶原激活剂（uPA)的影响。方法：采用Western blotting和Northern blotting分别观察LPS对牙龈成纤维细胞内uPA蛋白质表达水平和mRNA表达水平的影响。结果：经1.0μg/mL LPS处理8h后，牙龈成纤维细胞内uPA蛋白表达量明显增强，且这一增强作用可持续24h；经1.0μg/mL LPS处理4h后，牙龈成纤维细胞内uPA mRNA表达量明显增强。结论：LPS能够促进牙龈成纤维细胞表达uPA，参与牙周组织的破坏。     

Immunohistochemical localization of tissue-type plasminogen activator and type I plasminogen activator inhibitor in radicular cysts

BACKGROUND: The plasminogen/plasmin proteolytic system participates in a wide variety of extracellular matrix degradation. Detailed knowledge of plasminogen activators (PAs) and their inhibitors may be important for understanding the pathogenesis of radicular cysts. The purpose of this study was to investigate the in situ localization of tissue-type PA (t-PA) and type I PA inhibitor (PAI-1) in radicular cysts. METHODS: Thirty formalin-fixed, paraffin-embedded specimens of radicular cysts were examined using immunohistochemistry. In addition, another section from each radicular cyst specimen was stained with hematoxylin and eosin to assess the presence of inflammatory infiltrates. Differences in t-PA and PAI-1 expression between tissues with low and high levels of inflammation were subsequently analyzed using Fisher's exact test. RESULTS: Both t-PA- and PAI-1-positive cells were detected in the lining epithelium, connective tissue, inflammatory infiltrates, and endothelium. In addition, the t-PA signal was mainly expressed in epithelial cells. However, the PAI-1 signal was mainly expressed in fibroblasts. Moreover, significantly greater t-PA as well as PAI-1 expression was noted in radicular cysts with high levels of inflammation as compared to tissues with low levels of inflammatory cell infiltrates (P < 0.05). CONCLUSIONS: The present study confirms earlier indications of local production of PA and its inhibitor in radicular cysts. In addition, this study further shows the tissue localization of the antigens for t-PA as well as PAI-1, and demonstrates that the expression of both t-PA and PAI-1 increases with the grade of inflammation in radicular cysts.     

Tissue plasminogen activator (t-PA) and placental plasminogen activator inhibitor (PAI-2) in gingival fluid from 8–9-year-old children

High concentrations of tissue plasminogen activator (t-PA) and placental type plasminogen activator inhibitor (PAI-2) have previously been found in gingival crevicular fluid (GCF) of adults. In the present study, the levels were examined in 16 children aged 8–9 yr. Sampling of GCF was performed with small disks of Millipore-filter. t-PA and PAI-2 were analyzed with enzyme-linked immunosorbent assays with low method errors. The mean concentration of t-PA was slightly higher than in adults, while the mean PAI-2-concentration was slightly lower. An intraindividual study comparing healthy and inflamed sites in the children showed slightly higher concentrations in GCF from inflamed sites. No change was observed in the balance between t-PA and PAI-2.     

Immunohistochemical demonstration of the plasminogen activator system in human gingival tissues and gingival fibroblasts

The relative distribution of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) was studied in cultured human gingival fibroblasts, healthy gingival tissues and inflamed gingival tissues by immunohistochemistry. In cultured gingival fibroblasts t-PA, u-PA and PAI-1 were expressed in cytoplasm; u-PA and PAI-1 were more intensely stained than t-PA; PAI-2 was not detectable in gingival fibroblasts. Following interleukin 1β (IL-1β) stimulation, the intensity of intracellular staining for t-PA was increased and a number of cells staining strongly for PAI-2 were seen; no difference in the intensity of immunostaining level was noted for the expression of u-PA and PAI-1 between IL-1β stimulated cells and unstimulated cells. In healthy gingival tissues, u-PA and PAI-1 displayed a wide distribution throughout all the connective tissue and epithelium; t-PA localized mainly in the connective tissue while PAI-2 showed little association with the connective tissue but did faintly stain in the epithelial layer. In inflamed gingival tissues, staining for t-PA was significantly increased in the extracellular matrix of the connective tissue, whereas staining for u-PA, PAT-I and PAI-2 was found to be slightly increased, but no significant difference was noted for staining when compared with the healthy gingival tissues. A granular distribution of t-PA, u-PA, PAI-1 and PAI-2 was noted around areas of inflammatory cell infiltration. These immunohistochemical findings indicate that the plasminogen activator system produced by fibroblasts may be influenced by the presence of the inflammatory mediator IL-1β. In addition, the significant increase of t-PA in inflamed connective tissue and the wide expression of these components around inflamed cells may contribute to connective tissue degradation and may relate to the migration and localization of monocytes/macrophages in inflamed tissue.     

Enhancement of LPS-stimulated plasminogen activator production in aged gingival fibroblasts

Plasminogen activator (PA) converts plasminogen to plasmin, and plasmin activates the kinin cascade and latent methalloproteases. It is known that the alteration of the PA-plasmin system affects the progression of periodontal disease. We have reported previously that LPS from Campylobacter rectus, which is associated with adult periodontitis, increased PA production in human gingival fibroblasts (hGF). The effects of in vitro- and in vivo-cellular ageing on PA production from human and rat gingival fibroblasts (rGF) were studied. In vitro cellular aged hGF were prepared by subcultivations of hGF, and in vivo aged rGF was cultured primarily from the gingival tissue of aged rats. The cells were challenged with LPS and PA released into the cultured medium was measured as PA activity. Both in vitro and in vivo cellular aged GFs produced a significantly higher PA activity by LPS compared with young GFs cell. In RT-PCR experiments, tissue type PA (tPA) mRNA levels in both aged hGF and rGF were higher than in young cells, whereas plasminogen activator inhibitor 1 (PAI-1) mRNA remained unchanged and urotype PA (uPA) mRNA was not detected. Since LPS-stimulated PA activity from gingival fibroblasts was stimulated in aged cells using both in vitro- and in vivo-experimental models, the ageing of gingival fibroblasts may have an effect on the severity of inflammation and degradation of the extracellular matrix of gingival tissues by producing a large amount of PA in response to LPS.     

Reduction in gingival overgrowth associated with conversion from cyclosporin A to tacrolimus

BACKGROUND: Unsightly gingival overgrowth affects many individuals immunosuppressed with cyclosporin A (CsA). Current management involves repeated periodontal surgery and intensive hygienist support. Tacrolimus is an effective alternative immunosuppressive agent for renal transplantation which does not appear to produce gingival enlargement. AIMS: The purpose of the present study was to monitor the gingival response of 4 renal transplant patients (RTPs), with clinically significant CsA-induced gingival overgrowth, after their immunosuppressive therapy was switched to tacrolimus. METHODS: Intra-oral photographs and alginate impressions were taken both prior to the drug conversion and again, 6 to 9 months later. Gingival overgrowth scores were determined, from plaster models on both these occasions. RESULTS: All of the RTPs experienced significant resolution of their gingival enlargement within the time period studied; however, only one had complete regression. CONCLUSION: It is concluded that conversion of RTPs with gingival overgrowth from CsA to tacrolimus may provide an effective management strategy for this clinical problem.     

Aggravation of gingival inflammatory symptoms during pregnancy associated with the concentration of plasminogen activator inhibitor type 2 (PAI-2) in gingival fluid

Gingival inflammatory symptoms are aggravated during pregnancy. In vitro studies suggest a hormonal influence on the plasminogen activator inhibitor type 2 (PAI-2), and a disturbed balance of the fibrinolytic system could help to explain pregnancy gingivitis. Gingival crevicular fluid (GCF) was sampled in 14 women in pregnant and post-pregnant states. The gingival condition was assessed by the gingival index of Løe & Silness (GI) and the amount of bacterial plaque by the plaque index of Silness & Løe (PI). The ratio of sites with gingivitis to sites with bacterial plaque was calculated (G/P-ratio). Antigen levels of tissue plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitors type 1 (PAI-1) and PAI-2 in GCF were determined with ELISAs and 17 β-oestradiol and progesterone in serum with radioimmunoassays. For each individual the differences (Δ) in hormone levels and PAs and PAIs between pregnancy and post-pregnancy were calculated. Based on differences in G/P-ratio between pregnancy and post-pregnancy, subgrouping was done into a high-reacting and a low-reacting group. For the total group, the mean G/P-ratio was 2.0 during and 1.2 after pregnancy (p=0.064). A statistically significant correlation between Δprogesterone and ΔPAI-2 was noted: the higher Δprogesterone, the lower ΔPAI-2. No other significant correlations between hormone levels and components of the fibrinolytic system were found. For the total group of women, the concentrations of PAI-2, PAI-1 and t-PA were significantly higher during than after pregnancy. The individuals in the high-reacting group, however, showed a lower or unchanged production of PAI-2 during pregnancy, while those in the low-reacting group showed a greatly increased production. The lower inhibitory capacity in terms of a low production of PAI-2 during pregnancy in women with a higher inflammatory reaction indicates that the components of the fibrinolytic system may be involved in the development of pregnancy gingivitis and implies that PAI-2 serves as an inhibitor of importance for tissue proteolysis. The present finding contributes to the explanation of pregnancy gingivitis.     

慢性牙龈炎及慢性牙周炎治疗前后患牙龈沟液中t-PA及PAI活性的比较

目的 :比较慢性牙龈炎、慢性牙周炎患牙治疗前后龈沟液中t PA及PAI活性。方法 :选择慢性龈炎(n =2 8)及慢性牙周炎 (n =5 2 )患牙两组 ,在治疗前及治疗后收集龈沟液 (GCF)并记录临床指标 ,用发色底物法测定GCF中t PA及PAI的活性。结果 :t PA活性在牙龈炎组治疗前后差异无显著性 ,在牙周炎组治疗前后差异有显著性 ;PAI活性在牙龈炎组及牙周炎组治疗前后差异均有显著性 ;PAI/t PA比值在牙周炎组治疗前后差异有显著性 ,牙龈炎组治疗前后差异无显著性。结论 :PAI可以作为判断牙龈炎症程度及治疗效果较为客观的指标 ;牙周炎组中治疗后PAI/t PA显著降低的部位是否预示较差的预后 ,尚需进一步纵向研究。     

The calcium channel blocker used with cyclosporin has an effect on gingival overgrowth

BACKGROUND/AIMS: To investigate whether the choice of calcium channel blocker, used in conjunction with cyclosporin A, affected the prevalence of gingival overgrowth. METHOD: A cohort of 135 renal transplant recipients who had been medicated with cyclosporin A in combination with either nifedipine (89) or amlodipine (46) since transplant, took part in the study. The inclusion criteria were that eligible subjects had been in receipt of a kidney transplant for at least 12 months, had at least 10 teeth and had not received specialist periodontal treatment. The age, gender, current drug regimen and dosage were recorded for each participant and alginate impressions taken of both arches. The presence and severity of gingival overgrowth were scored from plaster models. RESULTS: A higher proportion (72%) of the amlodipine group were categorised as having gingival overgrowth compared with only 53% of the nifedipine group, chi square=4.5, p<0.05. Logistic regression analysis was used to explore the relationship between the presence or absence of gingival overgrowth (dependent variable) and age, gender, time since transplant, dose of cyclosporin A, centre in which the patient was treated, and the calcium channel blocker used (independent variables). Independent predictors of gingival overgrowth in this multivariate analysis were whether the individual was treated with amlodipine or nifedipine (p=0.01) and whether the individual was young or old (p=0.01). Within the multivariate analysis, the odds ratio for amlodipine to be associated with gingival overgrowth compared with nifedipine was 3.0 (confidence interval 1.3-6.9). CONCLUSIONS: The prevalence of gingival overgrowth in renal transplant recipients maintained on cyclosporin A and nifedipine is lower than those treated with cyclosporin A and amlodipine.     

环孢菌素A导致牙龈过度生长机理的研究近况

牙龈过度生长是环孢菌素A的重要的不良反应之一,其病理特征是牙龈组织上皮层的增厚和胞外基质的蓄积,但其确切的病理机制仍不清楚。该文就环孢菌素A导致的牙龈过度生长的病因及病理机制的研究现状做一综述。     

The up-regulation of heme oxygenase-1 expression in human gingival fibroblasts stimulated with nicotine

BACKGROUND: Cigarette smoking is a major risk factor in the development and further progression of periodontal diseases. Heme oxygenase-1 (HO-1) is known as a stress-inducible protein and functions as an antioxidant enzyme. There is limited information on the expression of HO-1 in smoking-associated periodontal disease. OBJECTIVES: The aim of the present study was to investigate the effects of nicotine on the expression of HO-1 protein in cultured human gingival fibroblasts in vitro and further to compare HO-1 expression in gingival tissues obtained from cigarette smokers and non-smokers in vivo. METHODS: Western blot assay was used to investigate the effects on human gingival fibroblasts exposed to nicotine. In addition, antioxidants catalase, superoxide dismutase (SOD), and N-acetyl-l-cysteine (NAC) were added to test how they modulated the effects on nicotine-induced HO-1 expression. Gingival biopsies taken from the flap surgery of 20 male patients with periodontal disease (10 cigarette smokers and 10 non-smokers) were examined by immunohistochemistry. RESULTS: The exposure of quiescent human gingival fibroblasts to 10 mm nicotine resulted in the induction of HO-1 protein expression in a time-dependent manner (p < 0.05). The addition of glutathione (GSH) precursor NAC inhibited the nicotine-induced HO-1 protein expression (p < 0.05). However, SOD and catalase did not decrease the nicotine-induced HO-1 protein expression (p > 0.05). The results from immunohistochemistry demonstrated that HO-1 expression was significantly higher in cigarette smokers (p < 0.05). HO-1 was noted in the basal layers of epithelium, inflammatory cells, and fibroblasts in specimens from cigarette smoking. CONCLUSIONS: Taken together, these results suggest that HO-1 expression is significantly up-regulated in gingival tissues from cigarette smokers, and nicotine may, among other constituents, be responsible for the enhanced HO-1 expression in vivo. The regulation of HO-1 expression induced by nicotine is critically dependent on the intracellular GSH concentration.     

Detection of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2(PAI-2) in gingival crevicular fluid from healthy, gingivitis and periodontitis patients

Background: The regulation of plasminogen activation is a key element in controlling proteolytic events in the extracellular matrix. Our previous studies had demonstrated that in inflamed gingival tissues, tissue‐type plasminogen activator (t‐PA) is significantly increased in the extracellular matrix of the connective tissue and that interleukin 1β (IL‐1β) can up regulate the level of t‐PA and plasminogen activator inhibitor‐2 (PAI‐2) synthesis by human gingival fibroblasts. Method: In the present study, the levels of t‐PA and PAI‐2 in gingival crevicular fluid (GCF) were measured from healthy, gingivitis and periodontitis sites and compared before and after periodontal treatment. Crevicular fluid from106 periodontal sites in 33 patients were collected. 24 sites from 11 periodontitis patients received periodontal treatment after the first sample collection and post‐treatment samples were collected 14 days after treatment. All samples were analyzed by enzyme‐linked immunosorbent assay (ELISA) for t‐PA and PAI‐2. Results: The results showed that significantly high levels of t‐PA and PAI‐2 in GCF were found in the gingivitis and periodontitis sites. Periodontal treatment led to significant decreases of PAI‐2, but not t‐PA, after 14 days. A significant positive linear correlation was found between t‐PA and PAI‐2 in GCF (r=0.80, p<0.01). In the healthy group, different sites from within the same subject showed little variation of t‐PA and PAI‐2 in GCF. However, the gingivitis and periodontitis sites showed large variation. These results suggest a good correlation between t‐PA and PAI‐2 with the severity of periodontal conditions. Conclusion: This study indicates that t‐PA and PAI‐2 may play a significant rôle in the periodontal tissue destruction and tissue remodeling and that t‐PA and PAI‐2 in GCF may be used as clinical markers to evaluate the periodontal diseases and assess treatment.     

Morphological evaluation of combined effects of cyclosporin and nifedipine on gingival overgrowth in rats

It has previously been shown that, while cyclosporin A (CsA) and nifedipine both cause gingival overgrowth in the rat, the combined use of these drugs increases the severity of overgrowth. The aim of this study was to describe the histometry and densities of fibroblasts, collagen fibers and vessels in the gingival tissue of rats that were treated with CsA and nifedipine, either alone or in combination. Rats were treated for 60 days with a daily subcutaneous injection of 10 mg/kg body weight of CsA and/or with 50 mg/kg body weight of nifedipine added to the chow. The results confirmed that CsA causes a more severe overgrowth than nifedipine, and that the combined use of these drugs increases the overgrowth severity. All the rat groups that were studied showed that, as the severity of overgrowth increased, there was a parallel increase in fibroblasts and collagen, and a decrease in vessel content. Therefore, independently of whether the gingival overgrowth was caused by CsA alone, nifedipine alone, or both treatments in combination, the fibroblast and collagen density increased in parallel with the severity of the overgrowth.     

Morphometric evaluation of gingival overgrowth and regression caused by cyclosporin in rats

Cyclosporin A is a selective immunosuppressant, used in organ transplants to prevent graft rejection. Cyclosporin A can cause various side effects including gingival overgrowth. The aim of this work was to evaluate gingival overgrowth of rats treated daily with 10 mg/kg bodyweight of cyclosporin A for 60 days, as well as the regression after the interruption of treatment. All rats treated with cyclosporin A developed gingival overgrowth, with increased thickness of the epithelium, height and width of the connective tissue. The density of fibroblasts and collagen fibers also increased. Five to 90 days after the interruption of treatment with cyclosporin A, there was a progressive reduction of the gingival volume and of collagen fibers and fibroblast densities. The reduction was more pronounced in the initial periods and after 90 days did not return to the normal values.