Cyclosporine A enhances gingival ß-catenin stability via Wnt signaling.

BACKGROUND: Cyclosporine A (CsA) increases ß-catenin messenger RNA (mRNA) and protein expression. The present study demonstrates that Wnt/ß-catenin signaling inhibits ß-catenin degradation in the gingiva. METHODS: Forty 5-week-old male Sprague-Dawley rats were assigned to two study groups after healing from right maxillary molar extractions. The rats in the experimental group were fed 30 mg/kg CsA daily for 4 weeks, whereas the control rats were fed mineral oil. At the end of the study, all rats were sacrificed, and the gingivae were obtained. The gingival morphology after CsA treatment was evaluated by histology, and the genes related to Wnt/ß-catenin signaling were initially screened by microarray. Polymerase chain reaction, Western blotting, and immunohistochemistry were used to examine the mRNA and protein expression of proliferating cell nuclear antigen, cyclin D1, E-cadherin, ß-catenin, Dvl-1, glycogen synthase kinase-3ß, axin-1, and adenomatous polyposis coli (APC). Phosphoserine and ubiquitinylated ß-catenin were detected after immunoprecipitation. RESULTS: In rats treated with CsA, overgrowth of gingivae was observed, and altered expression of genes related to Wnt/ß-catenin signaling was detected by the microarray. The gingival mRNA and protein expression profiles for genes associated with Wnt/ß-catenin signaling further confirmed the effect of CsA: ß-catenin and Dvl-1 expression increased, but APC and axin-1 expression decreased. Western blotting and immunohistochemistry showed decreases in ß-catenin serine phosphorylation (33/37) and ubiquitinylation in the gingivae of CsA-treated rats. CONCLUSION: CsA-enhanced gingival ß-catenin stability may be involved in gene upregulation or ß-catenin degradation via the Wnt/ß-catenin pathway.

Releasing growth factors from activated human platelets after chitosan stimulation: a possible bio-material for platelet-rich plasma preparation.

INTRODUCTION: Thrombin is commonly used for activating the platelets and releasing the growth factors on the application of platelet-rich plasma (PRP). We have previously reported that chitosan can enhance rabbit platelet aggregation. In this study, the effects of chitosan on the subsequent growth factors release after human platelets activation were examined to evaluate the possibility of chitosan being used as a substitute for thrombin during PRP preparation. MATERIAL AND METHODS: Human platelet activation was determined by aggregation, adhesion and alpha-granule membrane glycoprotein expression. Platelet aggregation was measured by the turbidimetric method, the adhesion was directly examined on chitosan-coated glass plates under light microscope and scanning electron microscope (SEM), and the alpha-granule membrane glycoprotein was detected by fluorescent isothiocyanate (FITC)-conjugated anti-CD61 antibody through flow cytometry. The subsequent epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets were assayed by ELISA after mixing with chitosan. RESULTS: The enhancing effects on the platelet adhesion and the aggregation from chitosan were observed. Under both microscopes, the adhesive platelets on the chitosan-coated plates were not only greater in number but also earlier in activation than those on the control plates. With flow cytometry, increased glycoprotein IIIa expression in platelets was detected after chitosan treatment. Greater concentrations of growth factors were measured from PRP after chitosan treatment than after the solvent treatment. CONCLUSION: Because of the observations of growth factors releasing from activated human platelets after chitosan stimulation, we suggest that chitosan may be an appropriate substitute for thrombin in PRP preparation.

Cyclosporin-induced downregulation of the expression of E-cadherin during proliferation of edentulous gingival epithelium in rats.

BACKGROUND: To examine the role of E-cadherin in epithelial hyperplasia of cyclosporin A (CsA)-induced gingival enlargement, mRNA and protein levels of E-cadherin, beta-catenin, proliferating cell nuclear antigen (PCNA), and Cyclin D1 were examined in the edentulous gingiva of rats following CsA treatment. METHODS: Three weeks after the extraction of all maxillary molars, 20 male Sprague-Dawley rats were assigned to a CsA-fed group (30 mg/kg daily) or a control group. Five rats per group were sacrificed at weeks 1 and 4. Edentulous ridge specimens were taken, and the expression levels of E-cadherin, beta-catenin, Cyclin D1, and PCNA mRNAs were estimated by reverse transcription-polymerase chain reaction (RT-PCR). Tissue specimens of the week 4 groups were examined using immunohistochemical (IHC) staining for proteins. RESULTS: The mRNA expression of E-cadherin was significantly weaker in the CsA-treated group than the control group at both times. Using IHC staining, a weaker level of membrane-bonded E-cadherin was also observed in the gingival epithelial cells in the CsA group than in controls. By contrast, significantly stronger beta-catenin and Cyclin D1 mRNA expressions and protein levels were found in CsA-treated rats than controls by RT-PCR and immunohistochemistry at week 4, whereas PCNA production was stronger at both times. CONCLUSIONS: CsA treatment reduced the production of E-cadherin but increased the production of beta-catenin, Cyclin D1, and PCNA. Thus, CsA may downregulate E-cadherin gene expression, leading to the epithelial cell proliferation of gingival overgrowth.

Upregulation of the expression of epidermal growth factor and its receptor in gingiva upon cyclosporin A treatment.

BACKGROUND: To understand the roles of epidermal growth factor (EGF) and EGF receptor (EGF-R) in cyclosporin A (CsA)-induced gingival overgrowth, expression of EGF and EGF-R upon CsA treatment was examined in an oral epidermoid carcinoma cell line of humans (OECM-1) and in edentulous gingiva of rats. METHODS: In vitro study: after CsA treatment, OECM-1 cells were harvested to evaluate their mRNA and protein expression of EGF and EGF-R with reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry (ICC). In vivo study: 3 weeks after extraction of all maxillary molars, 20 male Sprague-Dawley rats were assigned to a CsA group (30 mg/kg, fed daily) and a control group. Five rats per group were sacrificed at weeks 1 and 4. Edentulous ridge specimens were obtained for evaluating their mRNAs and protein expression with RT-PCR, real-time RT-PCR, and immunohistochemistry (IHC). In both in vitro and in vivo experiments, the proliferating potential of epithelial cells was examined by the presence of proliferating cell nuclear antigen (PCNA). RESULTS: In vitro: dose-dependently increased mRNA expression of EGF and EGF-R in OECM-1 cells was noted after CsA treatment. Protein expressions of EGF and EGF-R were higher in OECM-1 with CsA treatment than without CsA. In vivo: higher mRNA and protein expressions of EGF and EGF-R were also observed in the gingival tissues of CsA-treated rats. In both in vitro and in vivo experiments, greater PCNA expression after CsA treatment was demonstrated. CONCLUSIONS: Higher expression of EGF and EGF-R upon CsA therapy was observed in OECM-1 epithelial cells of humans and in edentulous gingiva of rats. We suggest that CsA could upregulate gene and protein expression of EGF and EGF-R, and the upregulation may play a role in gingival overgrowth.

Upregulation of transforming growth factor-beta1 and vascular endothelial growth factor gene and protein expression in cyclosporin-induced overgrown edentulous gingiva in rats.

BACKGROUND: To examine the effects of cyclosporin A (CsA) on the expression of growth factors in induced gingival overgrowth with limited contributing factors arising from local inflammation caused by bacterial plaque, this study of gingival overgrowth was designed on the edentulous ridge of rats. METHODS: After a 3-week healing period following maxillary molar extractions, 16 five-week-old male Sprague-Dawley rats were assigned to CsA and control groups. Animals in the CsA group were fed 30 mg/kg CsA daily, whereas the control rats received a mineral oil vehicle instead. After 4 weeks, all animals were sacrificed, and the morphology of edentulous ridges was recorded by dental impression. The gingivae on the left-hand side were dissected and stored for mRNA analysis, whereas the gingivae on the right-hand side were fixed in 4% paraformaldehyde for immunohistochemistry (IHC) analysis of transforming growth factor-beta1 (TGF-beta1), platelet-derived growth factor beta (PDGF-beta), insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor (VEGF). RESULTS: The edentulous gingivae were enlarged and the body weights were reduced in the CsA-treated animals compared to controls. The mRNA expressions of TGF-beta1, IGF-1, and VEGF were higher in the gingivae of the CsA group than in the control group. In addition, a greater mRNA expression (7.21-fold) of VEGF was demonstrated in the CsA group than in the control group by real-time polymerase chain reaction (PCR). The percentages of cells staining positive for TGF-beta1 and VEGF were significantly greater in the CsA rats than in the control rats. CONCLUSIONS: Greater mRNA expression and positive staining for TGF-beta1 and VEGF were observed in the edentulous gingivae of rats that received CsA. Therefore, CsA may upregulate TGF-beta1 and VEGF gene expression and protein secretion in CsA-induced gingival overgrowth.

Dual biological functions of an interleukin-1 receptor antagonist-interleukin-10 fusion protein and its suppressive effects on joint inflammation.

The aim of this study was to construct and purify a novel interleukin-1 receptor antagonist (IL-1ra)-interleukin-10 (IL-10) fusion protein and determine its biological function and anti-inflammatory effects. The isolated cDNAs of two inhibitory cytokines (IL-1ra, IL-10) were used to construct a cDNA for the IL-1ra-IL-10 fusion protein. The expressed recombinant cytokines and fusion product were purified and their biological properties analysed. The anti-IL-1 effect was evaluated by using a thymocyte-proliferation assay, and the IL-10 effect was investigated by the inhibition of interferon-gamma (IFN-gamma) production from splenocytes. The clinical response and histological analyses were studied in an adjuvant arthritic rat model. The fusion protein was 38 000 molecular weight in size. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting demonstrated that the purified protein was recognized by both IL-1ra and IL-10 antibodies. The fusion protein significantly inhibited IL-1-mediated thymocyte proliferation and concanavalin A (ConA)-primed IFN-gamma production from splenocytes. The fusion protein also suppressed joint swelling (paw circumference reduced from 5.0 +/- 0.2 to 4.1 +/- 0.1 cm; paw thickness approximately 2 mm in difference) and synovial inflammation in adjuvant arthritis of rats. Our investigations indicate that this fusion protein effectively suppresses inflammatory arthritis and may initiate a trend for future clinical application to target multiple molecules at the same time.