Apoptosis in gingival overgrowth tissues.

Variations in the balance between cell proliferation and apoptosis could contribute to the etiology of gingival overgrowth. The aim of this study was to test the hypothesis that, in fibrotic gingival lesions, fibroblast proliferation is stimulated and apoptosis is decreased. Apoptotic index, caspase 3 expression, the proliferative index, FOXO1 expression, and histological inflammation were measured in situ. Analysis of data showed that apoptosis decreased in all forms of gingival overgrowth examined (p < 0.05), and inflammation caused a small but significant increase compared with non-inflamed tissues (p < 0.05). The greatest decrease of apoptosis occurred in the most fibrotic tissues. Cell proliferation was elevated in all forms of gingival overgrowth tested, independent of inflammation (p < 0.05). To identify potential mechanisms of transcriptional regulation of apoptosis, we assessed FOXO1 and caspase 3 expression levels and found them to correlate well with diminished apoptosis. Analysis of data suggests that increased fibroblast proliferation and a simultaneous decrease in apoptosis contribute to gingival overgrowth.

Connective tissue growth factor in drug-induced gingival overgrowth.

BACKGROUND: Drug-induced gingival overgrowth is a known side effect of certain chemotherapeutic agents used for the treatment of systemic disorders. The pathogenesis and mechanisms responsible for this condition are not fully understood. This study assesses for the presence and localization of connective tissue growth factor (CTGF) in drug-induced gingival overgrowth tissues. CTGF immunostaining was compared with sections stained with transforming growth factor (TGF)-beta1 and CD31 antibodies in order to investigate possible pathogenic mechanisms. METHODS: Gingival overgrowth samples were obtained from patients undergoing therapy with phenytoin (n = 9), nifedipine (n = 4), cyclosporin A (n = 5), and control tissues from systemically healthy donors (n = 9). Tissue sections were subjected to peroxidase immunohistochemistry and were stained with CTGF and TGF-beta1 polyclonal primary antibodies. Possible relationships between CTGF staining and angiogenesis were also studied using an anti-CD31 antibody as a marker for endothelial cells. Staining was analyzed by computer-assisted quantitative and semiquantitative methodology at 5 defined sites in all samples based on the location of specific landmarks including epithelium and underlying connective tissues. RESULTS: Cellular and extracellular CTGF content in phenytoin gingival overgrowth tissues was significantly (P<0.05) higher compared to the other gingival overgrowth tissues and the controls. Higher CTGF staining in phenytoin gingival overgrowth tissues was accompanied by an increased abundance of fibroblasts and connective tissue fibers. No strong association of CTGF staining with TGF-beta1 or CD31 staining was found. CONCLUSIONS: The data from the present study show significantly higher CTGF staining in phenytoin-induced gingival overgrowth tissues compared to controls, cyclosporin A-, or nifedipine-induced gingival overgrowth. Moreover, semiquantitative analyses of histologic samples support the concept that the phenytoin overgrowth tissues are fibrotic. These associations suggest a possible role for CTGF in promoting development of fibrotic lesions in phenytoin-induced gingival overgrowth.

Regulation of lysyl oxidase, collagen, and connective tissue growth factor by TGF-beta1 and detection in human gingiva.

Gingival overgrowth is characterized by excess extracellular matrix accumulation and elevated levels of cytokines, including transforming growth factor-beta1 (TGF-beta1). The functional relationships between altered cytokine levels and extracellular matrix accumulation have not been extensively investigated in gingival cells and tissues. Lysyl oxidase catalyzes the final known enzymatic step required for cross-linking collagen and elastin in the synthesis of a functional extracellular matrix. This study investigated the regulation by TGF-beta1 of lysyl oxidase and its collagen and elastin substrates in early passage human gingival fibroblasts. In addition, TGF-beta1 regulation of connective tissue growth factor (CTGF) was assessed in human gingival cells and tissues. The results show that TGF-beta1 increases lysyl oxidase enzyme activity and mRNA levels for lysyl oxidase and alpha-1-type I collagen, but not elastin, in a dose- and time-dependent manner. Maximal stimulation of lysyl oxidase activity and mRNA levels for both lysyl oxidase and collagen occurs after 48 hours of treatment of gingival fibroblastic cells with 400 pM of TGF-beta1. This study shows for the first time that CTGF mRNA and protein are strongly and rapidly induced by TGF-beta1 in human gingival fibroblasts. Exogenous addition of 1 to 50 ng/ml CTGF to gingival fibroblasts stimulates production of lysyl oxidase enzyme activity up to 1.5-fold after 48 hours, and 50 ng/ml CTGF stimulated insoluble collagen accumulation 1.5- to 2.0-fold after 4, 11, and 18 days of treatment. It is interesting to note that the addition of CTGF-blocking antibodies in the presence of TGF-beta did not block TGF-beta stimulation of collagen mRNA levels. Thus, although CTGF itself contributes to increased insoluble collagenous extracellular matrix accumulation, CTGF does not mediate all of the effects of TGF-beta1 on stimulation of collagen mRNA levels in human gingival fibroblasts. Immunohistochemistry studies of gingival overgrowth tissue samples indicate for the first time detectable levels of CTGF protein in Dilantin-induced hyperplasia tissues also positive for TGF-beta1. CTGF was not found in TGF-beta1-negative samples. In addition, extracellular lysyl oxidase protein was detected in vivo. Taken together, these studies support mostly independent roles for TGF-beta1 and CTGF in stimulating collagenous extracellular matrix accumulation in human gingival fibroblasts and tissues.