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Cyclosporin A (CyA) is an immunosuppressive agent used to prevent allograft rejection, but unfortunately it causes adverse effects such as bone diseases, osteoporosis and osteomalacia. These pathologies involve an imbalance between synthesis, degradation and mineralization of extracellular matrix. CyA can modify extracellular matrix components such as glycosaminoglycans (GAG) and collagen fibers. In addition, normal cell activity is dependent on cell morphology and substrate cell attachment. We treated normal human osteoblasts with CyA and analyzed: (i) gene expression by a microarray method; (ii) extracellular GAG and collagen after (3)H-glucosamine and Western blot analysis; and (iii) cytoskeletal changes, using actin and tubulin fluorescent antibodies. CyA increased intra- and extracellular GAG and extracellular GAG classes such as hyaluronic acid, chondroitin sulphate, and dermatan sulphate; there was no noteworthy effect on heparan sulphate and the ratio of non-sulphated to sulphated GAG. In osteoblast cultures the drug reduced cytoskeletal actin, while tubulin did not change. In vivo the osteoblasts showed morphological changes with different extracellular matrix synthesis. Microarray analysis indicated the inhibition of gene pathways related to Wnt signaling molecules, and the cytoskeletal and focal adhesion cascade. In in vitro human osteoblasts CyA modified gene expression related to cytoskeletal pattern organization and cell morphology. Since in bone pathologies osteoblasts show different morphology related to cell size, these data suggest that in vivo osteoblast different functions could be dependent on alteration of osteoblast differentiation.

Background: Cyclosporin A is a powerful immunosuppressive drug with considerable impact on transplants and is able to modify extracellular matrix (ECM) composition. It has recently been demonstrated that cyclosporin A stimulates the production of the cytokine family. Cytokines such as interleukin, transforming growth factor beta(1), and bone morphogenetic protein induce the deposition of glycosaminoglycans (GAGs), proteoglycans, and collagen fibers in the connective ECM. ECM composition is very important for normal tissue development and function. In this work, we examine the effects caused by cyclosporin A on cultures of normal human palate fibroblasts in order to evaluate interleukin, transforming growth factor beta II, and bone morphogenetic protein II membrane receptor induction and extracellular GAG changes such as hyaluronic acid, heparin sulfate, and chondroitin sulfate., Methods: Palate fibroblasts were maintained for 24 h in serum-free 199 medium containing 5 microg/mL (3)H glucosamine hydrochloride. After this time, TGF II and BMP II receptors were determined by microarray analysis and GAG classes by the biochemical method., Results: The results show that TGFbeta(1) II and BMP II membrane receptors are significantly inhibited in cyclosporin A-treated cultures as compared to controls, whereas IL-1R2 membrane receptors are stimulated. The behavior of total intra- and extracellular GAGs is significantly increased in cyclosporin A-treated cultures, whereas the ratio between non-sulfated/sulfated GAGs decreases (p

Background: Secreted protein acidic and rich in cysteine (SPARC) is a glycoprotein that mediates cell-matrix interactions. In adults, its expression is mostly limited to tissue undergoing remodeling. During the development of Cyclosporin A (CsA)-induced gingival overgrowth (GO) a remodeling of the connective compartment occurs. By contrast, clinical trials showed that FK506 is not related to GO. SPARC expression and its involvement in GO is unknown. Our aim was, therefore, to analyze the effect of CsA and FK506 on SPARC gene expression., Methods: Cultured human gingival fibroblasts were incubated with CsA, FK506 or with their vehicle (VH) for 24, 48 and 72 h. SPARC gene expression was determined by RT-PCR., Results: SPARC mRNA levels tended to increase 72 h after CsA treatment, whilst they are undetectable in FK506-treated fibroblasts, compared to VH., Conclusion: This gene expression profile is consistent with the involvement of SPARC in the mechanisms leading to the development of CsA-induced GO. By contrast, the undetectable SPARC mRNA levels in FK506-treated fibroblasts suggest that FK506 may be associated with a role of ECM stabilization, that does not induce GO.

Aim: Our research was focused on the ultrastructural features of gingival epithelium in kidney transplant patients and, in particular, on Langerhans cells, with the aim of verifying whether ultrastructural modifications might explain gingival overgrowth., Methods: Using electron microscopy and immunohistochemical S100 staining at optical microscopy, we examined gingival samples obtained from 18 kidney transplant patients who presented with gingival overgrowth following cyclosporin A treatment., Results: The hyperkeratosis shown by the epithelium, and especially the absence of Birbeck granules in the Langerhans cells observed in serial sections, lead us to correlate these data to an immunodeficiency which affects the epithelium in the complex mechanism determining overgrowth., Conclusions: In our previous studies we attributed the responsibility of overgrowth to the connective tissue alone. However, in the light of the present results, we cannot exclude a contribution of the epithelium to gingival overgrowth.

In a significant number of cases (25-81%) immunosuppressant treatment with cyclosporin A (CsA) is associated with gingival overgrowth, seriously interfering with the functions of mastication and speech. In CsA-induced gingival enlargement, quantitative modifications of the extracellular matrix components occur, and collagen (COL) metabolism and matrix metalloproteinases (MMPs) have been suggested as being the main targets. Since the mechanisms at the basis of CsA-induced gingival overgrowth are not yet completely understood, our aim was to analyze the effect of CsA on COL turnover in cultured human gingival fibroblasts. Cultured human gingival fibroblasts from four healthy volunteers were incubated with CsA (800 ng/ml) or with its vehicle (VH) for variable intervals of time (24, 48, 72 h). Fibroblast morphology was studied by light and electron microscope. Collagen type I (COL-I), MMP-1, MMP-2, TIMP-1 and TGF-beta1 mRNA were determined by RT-PCR; COL-I and MMP-1 by dot blot, and MMP-2 by zymography. Our results evidenced an up-regulation of COL-I and TGF-beta1 gene expression 72 h after CsA treatment. MMP-1, MMP-2 and TIMP-1 mRNA levels are affected but not significantly. Protein analysis revealed COL-I increase at all the considered times and, 72 h after CsA treatment, reduced collagenolytic levels. Our data suggest that COL accumulation during CsA-induced gingival overgrowth may be mainly sustained by an altered COL-I degradation due to decreased MMP-1 activity. However, interindividual differences of collagenase levels after CsA treatment suggest that a genetic predisposition to develop gingival overgrowth may be relevant.

Cyclosporin A is a powerful immunosuppressive drug that has had a major impact on transplant therapy. It apparently links to different enzymatic pathways, and affects multiple enzymatic systems. Transforming growth factor beta induces the deposition of glycosaminoglycans, proteoglycans, and collagen fibers in the extracellular matrix. The aim of this study of normal and hypertrophic human gingival fibroblast cultures was to evaluate the cytoskeletal and extracellular changes in glycosaminoglycan secretion due to the presence of cyclosporin A and transforming growth factor beta. The results showed that there is an increase in total and individual classes of extracellular glycosaminoglycans in the presence of cyclosporin A and transforming growth factor beta, but the action of the latter was significantly greater. Immunohistochemical analysis of the cytoskeleton did not reveal any morphological differences between treated and control cells. Our data suggest that the biochemical changes in the extracellular matrix are caused more by cytokine, and that cyclosporin A does not induce any morphological changes in fibroblast cultures derived from hypertrophic and normal gingiva.

Patients treated with immunosuppressive drugs can develop cancers. The authors present two cases of Kaposi's sarcoma in kidney transplant patients who had been treated with azathioprine, steroids and cyclosporin-A; during this treatment the Langerhans cells decreased and Kaposi's sarcoma appeared. Discontinuation or reduction of the dosage of cyclosporin-A led to complete regression of the illness, and the Langerhans cells reappeared. We suggest that cyclosporin-A damages the immunological function of the epidermal Langerhans cells, and that this is the primary factor in the development of Kaposi's sarcoma.

The overgrowth-affected gingiva of patients treated with cyclosporin A after kidney transplant was examined with ultrastructural and histochemical methods to evaluate the involvement of connective tissue. Gingival overgrowth has the same clinical signs as local edema. The ultrastructural study showed that the dimensional increase was largely due to increased production of amorphous ground substance by fibroblasts, possibly resulting from an increased release of histamine by mast cells. The histochemical data revealed that the affected tissues contained higher levels of glycosaminoglycans and that cyclosporin A induced comparably high levels of glycosaminoglycans in in vitro cultures of fibroblasts obtained from normal gingiva. The combination of ultrastructural and histochemical data, therefore, strongly suggests that the response of the connective tissue in gingival overgrowth cannot be ignored and may be the main cause of the observed pathological condition.

The effect of Cyclosporin A (CyA) in culture of human gingival fibroblasts was assessed. Extracellular and intracellular glycosaminoglycans decrease in treated cells. The ratio of non sulphated over sulphated GAGs (glycosaminoglycans) increased only in the medium. Cytoskeleton organization of treated fibroblasts was also studied by immunohistochemical means and by ultrastructural observations. Minor alterations were documented in the structure and distribution of cytoplasmic organelles. The organization of tubulin and vimentin filaments did not change in Cy A treated cells either. This study confirms the capability of Cyclosporin A to induce alterations of GAG production and synthesis with no interference in cytoplasm organization. Normal pathway GAG secretion is suggested.

J Oral Pathol Med (2004) 33: 346–53 It has been demonstrated that cyclosporin A (CyA) blocks the immune system, acts on cytoskeleton and stimulates the production of extracellular matrix (ECM) and transforming growth factor-β1 (TGF- β1). This cytokine, such as transforming growth factor-α (TGF- α), induces deposition of glycosaminoglycans (GAG), proteoglycans and collagen fibres in the ECM. In this work, we examined the effect induced by CyA, TGF- β1 and TGF- α on cultures of healthy and overgrown human gingival fibroblasts in order to evaluate the glycosaminoglycan, cytoskeletal changes and the behaviour of fibroblasts after concanavalin A (Con A) treatment. Moreover, we examined gingival biopsies by Alcian blue histochemical staining and electron transmission microscopy. Total and extracellular sulphated GAG in overgrown gingiva specimens and in derived fibroblast cultures treated with CyA and cytokines were significantly higher than controls. The action of cytokines was increased ( P ≤ 0.01) compared with CyA with a greater effect of TGF- α in comparison with TGF- β1; the electron microscopy showed ECM accumulation. The agglutinations showed the heterogeneity of fibroblast populations. Stimulation with Con A showed that the fibroblast population had cell surface heterogeneity, and could respond in a different way to both CyA and cytokine stimulus. Moreover, increased synthesis of GAG in overgrown gingiva compared with synthesis in normal fibroblasts before CyA treatment suggests a possible genetic origin of damage. As not all CyA-treated patients develop gingival overgrowth, a genetic predisposition may explain the different responses of gingival fibroblast populations. [ABSTRACT FROM AUTHOR]