Human cleft lip and palate fibroblasts and normal nicotine-treated fibroblasts show altered in vitro expressions of genes related to molecular signaling pathways and extracellular matrix metabolism.

Nonsyndromic cleft lip with or without cleft palate (CLP) is a frequent craniofacial malformation caused by both genetic and environmental factors. Maternal smoking during pregnancy is a known risk factor, due to the teratogenic role of nicotine. To assess and compare the impact of CLP and nicotine, we studied the quantitative expression of genes involved in signaling pathways and extracellular matrix (ECM) metabolism in human normal nicotine-treated (NicN) and CLP fibroblasts compared to normal control (CTRL) cells. Palatal fibroblast cultures from seven CLP children and seven age-matched CTRL subjects were established and subconfluent cells incubated for 24 h without (CTRL and CLP fibroblasts) or with (NicN fibroblasts) 0.6 mM nicotine. Gene expressions were analyzed by real-time quantitative PCR. For the first time, a regulated cholinergic signaling in our human fibroblasts in vitro was demonstrated. Members of TGF-beta, retinoic acid (RA), and GABA-ergic signaling systems were also differently regulated. Among the ECM genes, fibronectin, syndecan, integrin alpha2, and MMP13 genes were concordantly modulated, while integrin beta5, and decorin genes were discordantly modulated. Interestingly, nicotine treatment regulated gene expressions of CD44 and CLPTM1, two candidate genes for CLP. Our findings show a positive association between nicotine treatment and CLP phenotype. Results suggest that nicotine deranges normal palate development, which might contribute to the development of a CLP malformative phenotype, through the impairment of some important signaling systems and ECM composition.

New insights in collagen turnover in orofacial cleft patients.

OBJECTIVE: We aimed to characterize the fibroblast phenotype of patients by analyzing gene and protein expression of cleft lip and/or cleft palate fibroblasts in relation to collagen turnover and extracellular matrix remodeling. PATIENTS: Human palatal fibroblasts were obtained from three healthy subjects without cleft lip and/or cleft palate and from three subjects with nonsyndromic cleft lip and/or cleft palate. Collagen turnover-related gene and protein expression were analyzed by real-time polymerase chain reaction, Western and dot blots, and sodium dodecyl sulfate zymography. RESULTS: Cleft lip and/or cleft palate fibroblasts, compared with controls, displayed a down-regulation of collagens type I and III messenger RNA (p < .0001 and p < .001, respectively) but an opposite tendency to increase protein levels. Cleft lip and/or cleft palate cells had higher lysyl hydroxylase-2b messenger RNA levels expressed in relation to collagen type I messenger RNA, down-regulated matrix metalloproteinase-1, tissue inhibitor of matrix metalloproteinase-1, and Secreted Protein Acidic and Rich in Cysteine messenger RNA (p < .0001 and p < .01, respectively). Pro-matrix metalloproteinase-1 tended to decrease, and pro-matrix metalloproteinase-2 and -9 were down-regulated (p < .01, p < .05, respectively), as was Secreted Protein Acidic and Rich in Cysteine protein expression (p < .05). CONCLUSIONS: Our results suggest that the cleft lip and/or cleft palate fibroblast phenotype is characterized by a tendency toward interstitial collagen deposition due to posttranslational modifications, such as decreased collagen degradation by matrix metalloproteinases and increased collagen cross-links. These findings may contribute to the knowledge of the cleft lip and/or cleft palate fibroblast phenotype and may be useful to the surgeon when considering the potential wound contraction and subsequent undesired scarring in cleft lip and/or cleft palate ocurring after the surgical closure of a cleft palate.

Patterns of some extracellular matrix gene expression are similar in cells from cleft lip-palate patients and in human palatal fibroblasts exposed to diazepam in culture.

Prenatal exposure to diazepam, a prototype sedative drug that belongs to Benzodiazepines, can lead to orofacial clefting in human newborns. By using real-time PCR, in the present study we investigated whether diazepam elicits gene expression alterations in extracellular matrix (ECM) components, growth factors and gamma-aminobutyric acid receptor (GABRB3), implicated in the coordinate regulation of palate development. Palate fibroblasts were treated with diazepam (Dz-N fibroblasts) and compared to cleft lip-palate (CLP) fibroblasts obtained from patients with no known exposure to diazepam or other teratogens. Untreated fibroblasts from non-CLP patients were used as control. The results showed significant convergences in gene expression pattern of collagens, fibromodulin, vitronectin, tenascin C, integrins and metalloprotease MMP13 between Dz-N and CLP fibroblasts. Among the growth factors, constitutive Fibroblast Growth Factor 2 (FGF2) was greatly enhanced in Dz-N and CLP fibroblasts and associated with a higher reduction of FGF receptor. Transforming Growth Factor beta 3 (TGFbeta(3)) resulted up-regulated in CLP fibroblasts and decreased in Dz-N fibroblasts. We found phenotypic differences exhibited by Dz-N and CLP fibroblasts in GABRB3 gene regulation, so further studies are necessary to determine whether GABAergic system could be involved in the development of diazepam mediated CLP phenotype. Taken together the results elucidate the molecular mechanisms underlying possible toxicology effects induced by diazepam. Counselling of women on the safety of diazepam exposure is clinically important, also for the forensic consequences.

Toremifene decreases type I, type II and increases type III receptors in desmoid and fibroma and inhibits TGFbeta1 binding in desmoid fibroblasts.

Tissue infiltration is different in desmoid and fibroma tumours. Both produce high levels of transforming growth factor beta1 (TGFbeta1), which is related to extracellular matrix (ECM) accumulation which in turn regulates cell function and cell migration. Interactions between collagen, proteoglycans and cell surface fibronectin are involved in the assembly and functions of the ECM. As toremifene inhibits collagen and TGFbeta1 synthesis, we tested it in normal, desmoid and fibroma fibroblasts. We will report the changes in glycosaminoglycan (GAG) and collagen synthesis, TGFbeta1 activity, fibronectin mRNA expression and TGFbeta1 receptors after toremifene treatment in normal, fibroma and desmoid fibroblasts. We evaluated GAG and collagen synthesis with 3H-glucosamine and 3H-proline incorporation, TGFbeta1 activity with the ELISA method, TGFbeta1 receptor affinity with 125I-TGFbeta1 binding and total RNA with Northern blot analysis. GAG and collagen synthesis, TGFbeta1 activity and fibronectin levels were higher in fibroma and desmoid than normal fibroblasts. The increase was greater in desmoid than fibroma tumour cells. Toremifene treatment reduced GAG and collagen synthesis, TGFbeta1 activity and fibronectin levels in all cell cultures. The percentage reduction in GAG was similar in all cultures; the reduction in collagen synthesis and TGFbeta1 activity was the highest in desmoid fibroblasts. TGFbeta1 receptors were higher in fibroma and desmoid cells than controls. Toremifene reduced TGFbeta1 receptors only in desmoid fibroblasts, with no effect on the changes in type I, II, and III receptors. Our data show that toremifene modifies the ECM components that regulate cytokine activity and cell migration. The reduction in receptor number only in desmoid cells suggests that toremifene may reduce TGFbeta1's affinity for its receptors. Synthesis of a substance regulating protein kinase activity, which is directly involved in the link between TGFbeta1 and its receptors, cannot be excluded.

Downregulated gene expression in human palate fibroblasts after cyclosporin A treatment.

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

Glycosaminoglycan, collagen, and glycosidase changes in human osteoblasts treated with interleukin 1, and osteodystrophy.

Normal bone homeostasis involves a balance between osteoblast and osteoclast action, regulated by hormones and cytokine stimuli. Hemodialysis patients appear to have increased production of interleukin-1 (IL-1), interleukin-6 (IL-6) and glycosaminoglycans (GAG) in serum. IL-1 plays a role in the synthesis, degradation and degree of sulphatation of ECM components such as glycosaminoglycans. Also, continuous changes in the ECM involve enzymes such as beta-N-acetyl-d-glucosaminidase (beta-NAG) and beta-d-glucuronidase (beta-GLU) which act on different GAG classes and collagen fibers. We examined the effects of IL-1alpha on ECM synthesis and the related enzymes in human uremic osteoblast cultures. We also measured the levels of IL-1beta, and IL-6 and alkaline phosphatase activity. In biopsies of uremic bone there was less ECM deposition than resorption associated with changes in osteoblast morphology. In vitro osteoblast proliferation was higher (P< or =0.01), and extracellular GAG lower (P< or =0.01) than in controls. The enzyme beta-NAG was high (P< or =0.05) but there were no noteworthy changes in beta-GLU. ELISA of the medium indicated spontaneous production of IL-1beta and IL-6, which significantly increased after IL-1alpha treatment compared to controls. IL-1alpha reduced alkaline phosphatase activity (P< or =0.01) in uremic osteoblast cultures. IL-1 acts on osteoblasts with decreases in GAG synthesis and alkaline phosphatase activity, while beta-NAG increases. This lead to a reduction in the organic component in ECM and its mineralization, and to changes in the regulation of cytokine activity by GAG. The enzymatic breakdown might be facilitated by metabolic acidosis and failed osteoblast differentiation; these factors could be correlated with different degrees of osteodystrophy.

Desmoid and fibroma tumors differently respond to TGFbeta(1) stimulus and ECM macromolecule accumulation.

Desmoid and fibroma tumours are characterized by cell proliferation, glycosaminoglycan and collagen fibre accumulation, high levels of transforming growth factor beta(1) (TGFbeta(1)) and different patterns of tissue infiltration. TGFbeta(1) is related to extracellular matrix (ECM) composition which, in turn, regulates cell functions and cell migration. In this study we report changes in cell proliferation, glycosaminoglycan (GAG) and collagen synthesis, TGFbeta(1) mRNA expression and fibronectin levels in normal, desmoid and fibroma fibroblast cultures before and after TGFbeta(1) stimulation. Our data showed cell proliferation, GAG and collagen synthesis, transforming growth factor beta(1) mRNA expression and fibronectin levels were significantly higher in desmoid than in fibroma cultures. TGFbeta(1) treatment had no effect on cell proliferation, but increased TGFbeta(1) mRNA expression, GAG, fibronectin and collagen synthesis in desmoid and fibroma fibroblasts. Its effects were more marked in desmoid cells. Fibronectin favours cell migration, while changes in GAG composition alter cell behaviour and ECM organization. In conclusion our data suggest that the different patterns of infiltration in desmoid and fibroma tumours are due to changes in ECM components and cell-ECM interactions which can be ascribed to altered TGFbeta(1) mRNA expression and TGFbeta(1) activity.

Survivin as prognostic factor in squamous cell carcinoma of the oral cavity.

A series of 78 cases of oral squamous cell carcinoma was analysed by immunohistochemistry for expression of survivin, a recent apoptosis inhibitor. All cases were positive for survivin expression and were divided into two groups using a system of scores. Disease-specific survival curves were calculated according to Kaplan-Meier algorithm, and log rank test was used to compare survival curves. Then, Cox regression analysis was applied to determine the single contribution of covariates on survival rate. So, Cox analysis allowed us to detect the variables most associated to survival. Among the studied variables, such as grade of differentiation, tumor size, stage, recurrence of disease, lymph node presence, only stage and recurrence of disease were predictors of outcome; however, when we analyzed the survival without considering recurrence (that was the stronger predictor of death), a stepwise Cox analysis showed that Survivin, stage and grade of differentiation are significantly associated to survival, with a higher value for Survivin. These data suggest that survivin expression may identify cases of oral squamous cell carcinoma with more aggressive and invasive phenotype and, therefore, could influence the decision for the therapy at the time of diagnosis.

Human desmoid fibroblasts: matrix metalloproteinases, their inhibitors and modulation by Toremifene.

BACKGROUND: Desmoid tumour is a benign, non metastasising neoplasm characterised by an elevated deposition of organic macromolecules in the extracellular matrix (ECM). The matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases involved in the degradation of ECM macromolecules. The MMPs and their natural inhibitors (TIMPs) have been implicated in tumour growth, invasion and metastasis. In this study we provide evidence that the in vitro cultured cell line from desmoid tumour accumulates more collagen fibres in the ECM than healthy fibroblasts. METHODS: We investigated collagen accumulation by 3H-thymidine incorporation, MMP expression by substrate gel zymography and TIMP expression by Western blot analysis. RESULTS: Desmoid fibroblasts showed a reduction in MMP activity and an increase of type I and III collagen and TIMPs compared to normal fibroblasts. CONCLUSION: The increase in collagen in desmoid fibroblasts was due to inhibited collagen degradation (reduction of MMP activity) rather than to increased collagen synthesis. Adding toremifene, an anti-estrogen triphenylethylene derivate, to desmoid fibroblasts reduced collagen accumulation by decreasing mRNA expression and increasing collagen degradation.

Genetic portrait of mild and severe lingual dysplasia.

Squamous cell carcinoma is the most frequent malignant tumor of the oral cavity and often arises from premalignant lesions. Traditional methods used by the pathologist are subjective and lack the sensitivity to predict accurately which precancers may progress with time. Therefore, it is important to search for markers that may identify progression of premalignant lesions. Microarray technology can be use with this aim. Here, we define the genetic expression profile of lingual dysplasia (DS) progression. By using cDNA microarray containing 19.2K clones and a baseline of 11 normal tissues, we compared 5 mild and 4 severe DS. We identified 270 genes differentially expressed in normal tissue vs. mild DS (i.e. 161 up- and 109 down-regulated) and 181 genes differentially expressed in mild vs. severe DS (i.e. 63 up- and 118 down-regulated). The described genes cover a broad range of functional activities: (a) anti-oxidative, (b) DNA-repair, (c) inflammatory response, (d) cell-adhesion/mobility, (e) extracellular matrix depolymerization, and (f) cell-cycle regulation. The data reported better define DS progression and can help in classifying premalignant lesions.

Differential effect of Cyclosporin A and FK506 on SPARC mRNA expression by human gingival fibroblasts.

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.

Depletion of polyamines and increase of transforming growth factor-beta1, c-myc, collagen-type I, matrix metalloproteinase-1, and metalloproteinase-2 mRNA in primary human gingival fibroblasts.

BACKGROUND: The polyamines spermidine, spermine, and putrescine are known to be deeply linked with growth processes, gene expression, and extracellular matrix synthesis. Their cellular content depends primarily on the activity of the enzyme ornithine decarboxylase. High levels of ornithine decarboxylase and polyamines have been found in proliferative, inflammatory, and neoplastic pathologies of the oral cavity and in gingival fluid. Difluoromethylornithine (DFMO) selectively inhibits ornithine decarboxylase, thus depleting polyamine content and preventing cell proliferation and synthesis activity. The aim of this study was to investigate whether DFMO treatment could modify the genes involved in cell proliferation and extracellular matrix turnover. METHODS: Fibroblasts derived from non-inflamed gingiva were maintained in Dulbecco's modified Eagle's medium (DMEM) plus alpha-difluoromethylornithine for 4 days. At 0, 24, 48, 72, and 96 hours cell number was assessed, polyamine levels were quantified with high performance liquid chromatography (HPLC) method, and transforming growth factor-beta1 (TGF-beta1), c-myc, matrix metalloproteinases (MMP)-1 and 2, collagen type I (COL-I) and tissue inhibitor of matrix metalloproteinases (TIMP)-1 were evaluated by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Fibroblasts treated with DFMO significantly decreased cell proliferation, ornithine decarboxylase activity, and putrescine levels at all treatment times, spermidine after 72 and 96 hours, and spermine after 96 hours of culture. Total polyamines decreased (P < or =0.01) at 96 hours after DFMO treatment, while c-myc, TGF-beta1, MMP-1 and 2, COL-I mRNA significantly increased. Conversely, TIMP-1 did not show any significant change. The polyamines trend was not correlated to c-myc, TGF-beta1, MMP-1 and -2, and TIMP-1 mRNA levels. Transforming growth factor-beta1 and c-myc mRNA expression were related and correlated to MMP-1 and 2, COL-I and TIMP-1 mRNA trend after DFMO treatment. CONCLUSIONS: Our data show that as the polyamine content decreases, TGF-beta1, c-myc, MMP-1 and -2, and COL-I mRNA levels increase, therefore a negative regulatory role of the polyamines on the mRNA expression could be suggested.

Effect of Vitamin C on pre-osteoblast gene expression.

Ascorbic acid (AA), also known as Vitamin C, is a cofactor required for the function of several hydroxylases. It is not synthesised in humans and has to be provided by diet. Its absence is responsible for scurvy, a condition related to the defective synthesis of collagen by the reduced function of prolylhydroxylase. AA is also a risk factor for periodontal disease. Recently, it has been shown that AA induces embryonic stem cells to differentiate into osteoblasts. The mechanism by which AA sustains pre-osteoblast proliferation and commitment is mediated through the synthesis of collagen type I, interaction with alpha2- and beta1-integrin, activation of the mitogen-activated protein kinase pathway, and phosphorylation of osteoblast-specific transcription factors. However, the multifunctional role of AA is not fully elucidated. MC3T3-E1 mouse calvaria-derived cell line is a well-defined in vitro model of pre-osteoblast differentiation, and AA is essential for the proliferation and differentiation of MC3T3-E1. By using DNA micro-arrays containing 15,000 genes, we identified several genes in MC3T3-E1 cultured with AA for 24h whose expression was significantly up or downregulated. The differentially expressed genes covered a broad range of functional activities: (1) cell growth; (2) metabolism; (3) morphogenesis; (4) cell death; (5) cell communication. The data reported are, to our knowledge, the first genetic portrait of early stage stimulation of pre-osteoblasts by AA, and may be relevant to better understand the molecular mechanism of pre-osteoblast proliferation and commitment. Elucidation of the molecular mechanism has important clinical implications because it may facilitate the correct use of AA to accelerate bone regeneration.

Basic fibroblast growth factor: effects on matrix remodeling, receptor expression, and transduction pathway in human periosteal fibroblasts with FGFR2 gene mutation.

The Crouzon syndrome, which is associated with fibroblast growth factor receptor (FGFR2) mutations, is characterized by premature fusion of cranial sutures. We used an in vitro model of cultured periosteal fibroblasts from normal subjects and from Crouzon patients with FGFR2 mutation. We analyzed the matrix turnover rate and the effects of adding FGF2 by evaluating fibronectin synthesis and the activity of some proteolytic enzymes. To assess the role of some FGF signaling molecules involved in FGFR2 regulation, we studied Grb2 tyrosine phosphorylation and the phosphotyrosine proteins associated with Grb2. The iodinate FGF binding assay was performed to quantify FGFR expression. Compared with normal fibroblasts, fibronectin synthesis was decreased in Crouzon fibroblasts, and protease activities in cells and medium were enhanced, suggesting that excess fibronectin catabolism is present. Differences were more marked when FGF2 was added. Very few phosphoproteins were visible in anti-Grb2 immunoprecipitations from Crouzon fibroblasts, which showed a significant increase in the number of high-affinity and low-affinity FGF2 receptors. These results suggest that the abnormal genotype and the Crouzon cellular phenotype are related. To compensate the low levels of tyrosine phosphorylation, Crouzon cells might increase the numbers of FGFR2, thus increasing the cell surface binding sites for FGF2.

Cyclosporin A and transforming growth factor beta modify the pattern of extracellular glycosaminoglycans without causing cytoskeletal changes in human gingival fibroblasts.

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.

Biocompatibility of collagen membranes crosslinked with glutaraldehyde or diphenylphosphoryl azide: an in vitro study.

Crosslinking of collagen biomaterials increases their resistance to degradation in vivo. Glutaraldehyde (GA) is normally used to crosslink collagen biomaterial, but is often cytotoxic. Diphenylphosphoryl azide (DPPA) has recently been proposed as reagent, but little is known about its effects on cell behavior. In this study, we determined which collagen membrane was the most biocompatible: Paroguide which is crosslinked with DPPA and contains chondroitin sulfate; Opocrin which is crosslinked with DPPA; Biomed Extend which is crosslinked with GA; and Bio-Gide which is left untreated. Cell proliferation and extracellular matrix macromolecule deposition were evaluated in human fibroblasts cultured on the membranes. The GA-crosslinked Biomed Extend membrane and the not-crosslinked Bio-Gide membrane reduced cell growth and collagen secretion compared with DPPA-crosslinked biomembranes. When Paroguide and Opocrin were compared, better results were obtained with Paroguide. The greatest amount of transforming growth factor beta1, a growth factor involved in extracellular matrix macromolecule accumulation and in tissue regeneration, was produced by cells cultured on Paroguide, with Opocrin second. Our data suggest that the DPPA method is more biocompatible than the GA for crosslinking collagen biomaterials and that membranes made of collagen plus chondroitin sulfate are better than membranes made of pure collagen.

Ultrastructural changes in Langerhans cells in gingival overgrowth in cyclosporin A-treated renal transplant patients.

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.

Effect of cyclosporin A on human gingival fibroblast collagen turnover in relation to the development of gingival overgrowth: an in vitro study.

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.

Ornithine decarboxylase, polyamines and CD11b expression in HL-60 cells during differentiation induced by retinoic acid.

Polyamines (PA) and retinoic acid affect mammalian cell growth, differentiation and apoptosis. Retinoic acid induces granulocytic differentiation of mieloid cell lines and, during this process, is responsible for the expression of CD11b, a surface antigen. In this study we investigate the effects of retinoic acid on HL-60 cells, monitoring ornithine decarboxylase (ODC) activity (enzyme rate of PA), putrescine (PUT), spermidine (SPD), spermine (SPM) levels, CD11b myeloid surface marker differentiation, cell cycle, and apoptosis. ODC activity and PUT levels are correlated with mieloid cell differentiation induced by retinoic acid treatment. Only the ODC/PUT ratio is connected with retinoic acid treated HL-60 cells. Treated cultures show a decrease of proliferation and a cell block in the G0/G1 phase, with consequent diminished S phase. The G0/G1 and S phases are significantly related to ODC activity and to PUT and SPD behavior, whereas in differentiating condition only the decrease of PUT is related to the S phase. CD11b expression, stimulated by retinoic acid treatment, is associated with the SPM trend. Total PA behavior agrees with apoptotic cell increase after 96 h of stimulation. Our data show that retinoic acid treatment modifies ODC activity and the turnover of PA. PUT, SPD and SPM, therefore, have a different role, and may be involved in the differentiative/apoptotic program of retinoic acid treated HL-60 cells.

Effects of interleukin-1beta on chondroblast viability and extracellular matrix changes in bovine articular cartilage explants.

Osteoarthritis is a degenerative disease of joint cartilage, characterized by the progressive and permanent degeneration of cartilage due to an imbalance in normal extracellular matrix turnover. Interleukin-1 beta is a proinflammatory agent, which is present in an elevated amount in osteoarthritic cartilage, and is thought to play a decisive role in osteoarthritis. Interleukin-1 beta acts as an important mediator of extracellular matrix changes where its activity is regulated by glycosaminoglycan composition. The aim of this study was to investigate the extracellular matrix changes in bovine cartilage explants following interleukin-1 beta treatment by morphological, histochemical and biochemical methods. Interleukin-1 beta stimulated the release of matrix sulfated proteoglycans in the culture medium, and significantly inhibited sulfated proteoglycan synthesis. These events were associated to a strong stimulation of nitric oxide production. Interleukin-1 beta-treated cartilage showed evident collagen fibers around the chondrocytes, together with diminished glycosaminoglycan sulfate content in the extracellular matrix of the explants. Moreover, the ultrastructure and viability of cells did not change in treated cartilage. Our data show that interleukin-1 beta modifies the ECM turnover without toxic effect on chondrocytes.