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.

Epithelial and connective tissue cell CTGF/CCN2 expression in gingival fibrosis.

Gingival overgrowth is a side effect of certain medications and occurs in non-drug-induced forms either as inherited (human gingival fibromatosis) or idiopathic gingival overgrowth. The most fibrotic drug-induced lesions develop in response to therapy with phenytoin; the least fibrotic lesions are caused by cyclosporin A; and intermediate fibrosis occurs in nifedipine-induced gingival overgrowth. Connective tissue growth factor (CTGF/CCN2) expression is positively related to the degree of fibrosis in these tissues. The present study has investigated the hypothesis that CTGF/CCN2 is expressed in human gingival fibromatosis tissues and contributes to this form of non-drug-induced gingival overgrowth. Histopathology/immunohistochemistry studies showed that human gingival fibromatosis lesions are highly fibrotic, similar to phenytoin-induced lesions. Connective tissue CTGF/CCN2 levels were equivalent to the expression in phenytoin-induced gingival overgrowth. The additional novel observation was made that CTGF/CCN2 is highly expressed in the epithelium of fibrotic gingival tissues. This finding was confirmed by in situ hybridization. Real-time polymerase chain reaction (PCR) analyses of RNA extracted from drug-induced gingival overgrowth tissues for CTGF/CCN2 were fully consistent with these findings. Finally, normal primary gingival epithelial cell cultures were analysed for basal and transforming growth factor beta1 (TGF-beta1) or lysophosphatidic acid-stimulated CTGF/CCN2 expression at protein and RNA levels. These data indicate that fibrotic human gingival tissues express CTGF/CCN2 in both the epithelium and connective tissues; that cultured gingival epithelial cells express CTGF/CCN2; and that lysophosphatidic acid further stimulates CTGF/CCN2 expression. These findings suggest that interactions between epithelial and connective tissues could contribute to gingival fibrosis.

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.

Future caries susceptibility in children with early childhood caries following treatment under general anesthesia.

PURPOSE: The purpose of this study was to assess the susceptibility of children to the future development of caries following comprehensive treatment for early childhood caries (ECC) under general anesthesia. METHODS: The patients selected for this retrospective study were identified by analyzing dental records of children receiving treatment at the Franciscan Children's Hospital & Rehabilitation Center, Boston, MA (FCH & RC). In total, 4,143 records were reviewed. Of these, ECC was diagnosed in 42 patients before their admission to the operating room. Thirty-one control children were selected randomly from the dental records reviewed at FCH & RC. The control group was initially caries-free. The caries status of the children diagnosed with ECC was evaluated and compared with the control group. Children in both groups were seen for recall at intervals of six to nine months over a two-year period. The carious lesions were recorded in two categories; new smooth surface caries (NSSC) and new pit and fissure caries (NPFC). RESULTS: Thirty-three of 42 (79%) ECC children compared to nine of 31 (29%) control children had detectable carious lesions at subsequent recall visits. Children with ECC demonstrated a mean number of 3.2 +/- 3.3 new carious lesions compared to a mean of only 0.8 +/- 1.6 carious lesions in the control group. These differences were statistically significant (t71 = 3.8; P < 0.001). In addition, of the 42 patients treated for ECC under general anesthesia, seven (17%) required retreatment under general anesthesia within two years following their initial full-mouth rehabilitation. The prevalence of NSSC in the ECC group was significantly higher than the control group (t71 = 3.5; P < 0.001). CONCLUSIONS: Despite increased preventive measures implemented for children who experienced ECC, this study concluded that this group of children is still highly predisposed to greater caries incidence in later years. These findings strongly suggest that more aggressive preventive therapies may be required to prevent the future development of carious lesions in children who experienced ECC.

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.

Selective inhibition of sheep kidney 11 beta-hydroxysteroid dehydrogenase isoform 2 activity by 5 alpha-reduced (but not 5 beta) derivatives of adrenocorticosteroids.

We have previously reported that 5 alpha and 5 beta pathways of steroid metabolism are controlled in vivo by dietary Na+ and glycyrrhetinic acid, see Gorsline et al. 1988; Latif et al. 1990. The present investigations provide evidence supporting the suggestion that endogenous substances may regulate the glucocorticoid inactivating isoenzymes, 11 beta-HSD (hydroxysteroid dehydrogenase) 1 (liver) and 11 beta-HSD2 (kidney). The activity of 11 beta-HSD is impaired in essential hypertension, following licorice ingestion, and in patients with apparent mineralocorticoid excess where 11 beta-HSD2 is particularly affected. In all three conditions, excretion of the less common 5 alpha metabolites is elevated in urine. We now report on the differential abilities of a series of Ring A reduced (5 alpha and 5 beta) adrenocorticosteroid and progesterone metabolites to inhibit these isoenzymes. Using liver microsomes with NADP+ as co-factor (11 beta-HSD1), and sheep kidney microsomes with NAD+ as co-factor (11 beta-HSD2), we have systematically investigated the abilities of a number of adrenocorticosteroids and their derivatives to inhibit the individual isoforms of 11 beta-HSD. A striking feature is the differential sensitivity of the two isoenzymes to inhibition by 5 alpha and 5 beta derivatives. 11 beta-HSD1 is inhibited by both 5 alpha and certain 5 beta derivatives. 11 beta-HSD-2 was selectively inhibited only by 5 alpha derivatives: 5 beta derivatives were without inhibitory activity toward this isoform of 11 beta-HSD. These results indicate the importance of the structural conformation of the A and B Rings in conferring specific inhibitory properties on these compounds. In addition, we discuss the effects of additions or substitutions of other functional groups on the inhibitory potency of these steroid molecules against 11 beta-HSD1 and 11 beta-HSD2.

Kidney 11 beta-HSD2 is inhibited by glycyrrhetinic acid-like factors in human urine.

We have previously shown that human urine contains substances that, like glycyrrhetinic acid, inhibit 11 beta-HSD1. We have named these substances "glycyrrhetinic acid-like factors" or GALFs. We now have found that human urine contains measurable quantities of both 11 beta(HSD1)- and 11 beta(HSD2)-GALF inhibitory substances. Both are markedly elevated in pregnancy. Their chemical and high-performance liquid chromatography (HPLC) characteristics suggest that several of the GALFs are steroidal. Large quantities of neutral 11 beta(HSD1)- and 11 beta(HSD2)-GALFs can be extracted directly from urine into ethyl acetate, yielding fraction EA1. Hydrolysis of the GALFs remaining in the aqueous phase by beta-glucuronidase markedly increases the total amounts of GALFs, with the majority now being ethyl acetate extractable (fraction EA2). These EA2 post-hydrolysis GALFs can be separated by HPLC resulting in at least six components with inhibitory activity against each isoenzyme. Only two GALF peaks are active against both 11 beta-HSD1 and 11 beta-HSD2. The others are peaks with specific 11 beta(HSD1)- and 11 beta(HSD2)-GALF inhibitory activity. The GALFs in the same posthydrolysis EA2 extract are also inhibitory toward the 11 beta-HSD1 that is present in vascular smooth muscle where they may play a role in the mechanisms controlling blood pressure. We have also found that 11 beta-HSD2 is selectively inhibited by 5 alpha- (but not by 5 beta-) reduced steroids. GC-MS analysis of the 11 beta(HSD2)-GALFs in EA2 is now being performed to determine whether this group includes 3 alpha,5 alpha-ring A-tetrahydro-reduced derivatives of steroids.

Inhibitors of 11 beta-hydroxysteroid dehydrogenase and 5 beta-steroid reductase in urine from patients with congestive heart failure.

Recently, the current authors reported the presence in normotensive male and female urines of reproducibly measurable levels of naturally occurring substances in partially purified extracts of urine with inhibitory activity like glycyrrhetic acid (GA) towards both 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) and steroid 5 beta-reductase (5 beta-SR) in vitro. Since these substances mimic two known inhibitory activities of GA, they have been named 'Glycyrrhetic Acid-Like Factors', abbreviated as 'GALFs' or, more specifically 11 beta-GALF for substance(s) active against 11 beta-OHSD, and 5 beta-GALF for those inhibitory to 5 beta-SR. Administration of glycyrrhetic acid in man leads to cortisol-dependent mineralocorticoid hypertension, owing to impaired inactivation of cortisol by 11 beta-OHSD, and may be associated with increased sensitivity to mineralocorticoids owing to impaired 5 beta-SR. In this preliminary report, the results are described of a study on the presence of GALF factors in urines collected from patients with congestive heart failure (CHF) and mild essential hypertension. The results show that in such patients there are increased amounts of both 11 beta- and 5 beta- GALFs compared to normotensive. The possible physiological significance of these results is discussed.

Detection of glycyrrhetinic acid-like factors (GALFs) in human urine.

Patients with the syndrome of apparent mineralocorticoid excess and those who ingest licorice show markedly decreased 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) and 5 beta-reductase activity; both are important for the deactivation of glucocorticoids and other steroid hormones. Glycyrrhetinic acid (GA), present as its glycoside in licorice, is a potent inhibitor of both 11 beta-OHSD and 5 beta-reductase and, as we have also shown, confers Na(+)-retaining properties on glucocorticoids and amplifies those of aldosterone and deoxycorticosterone. We report the results of our initial studies demonstrating the presence of naturally occurring substances, which inhibit both 5 beta-reductase and 11 beta-OHSD as does GA, in partially purified extracts of urine from normotensive men and nonpregnant and pregnant women. Since these substances exhibit GA-like activity, we have termed them GA-like factors (GALFs). This "inhibitory" material is heat stable and does not react with ninhydrin; the majority is not extractable with ethyl acetate and thus is not a "free" steroid. When further purified by high-performance liquid chromatography with a methanol/water gradient, the majority of these GALFs appeared in two regions of inhibitory activity. The chemical nature of this material is currently being investigated. These experiments indicate that normal human urine contains GALFs that may play a role in Na+ homeostasis and regulation of blood pressure.

The proteoglycanase from human cartilage and cultured rabbit chondrocytes and its relation to osteoarthritis.

The neutral metal dependent proteoglycanase predominates as the chief neutral protease present in human patellar cartilage or released by cultured rabbit chondrocytes into the culture medium. The cultured chondrocytes released the proteoglycanase mostly in latent form. Its activation resulted in splitting off a 10,000 dalton fragment. The chondrocytes also released inhibitory activity against the proteoglycanase. Most of it was released in the first 24 hours of culture, while most of the enzyme was released in the following 48 hours. Both the human cartilage and the rabbit chondrocyte enzyme occur in two molecular weight sizes, in equilibrium with each other.

Soluble age-related factors from skeletal muscle which influence muscle development.

Successful regeneration of damaged striated muscle in adult mice is dependent on the regeneration of newly differentiated myofibers from proliferating satellite cells and inhibition of scar tissue formation by fibroblasts. As with most tissues, the ability of skeletal muscle to regenerate decreases in older animals. In this study, we have analysed soluble extracts from intact and regenerating skeletal muscle from mice of different ages for their ability to affect avian myogenesis in tissue culture. We were interested in determining whether an age-dependent difference could be detected with this tissue culture bioassay system. Total cell proliferation in the cultures, measured by [3H]thymidine incorporation was increased equally by muscle extracts from both young and older mice but the resulting cell populations differed in proportion of cell types. The ratio of myoblasts to fibroblasts was significantly greater in cultures exposed to extracts from younger mouse muscle as compared with cultures exposed to extracts from older animals. This age-related activity was found to reside in a low molecular weight (MW) (greater than 12 kD) component of the extract. This fraction had dissimilar effects on myoblasts and fibroblasts. Relative to saline controls, myoblast proliferation was increased and fibroblast proliferation decreased. The low MW fraction from younger mouse muscle extracts stimulated myogenic cell proliferation and myotube formation to a greater extent than the similar fraction prepared from older mouse muscle. Conversely, younger mouse muscle fractions had significantly greater inhibitory activity against fibroblast proliferation than did older mouse muscle fractions.

'Gelatinase-like' activity from articular chondrocytes in monolayer culture.

In addition to releasing collagenase and proteoglycanase activity, rabbit articular chondrocytes in monolayer culture released into the culture medium, latent, neutral enzyme activity which when activated by p-aminophenylmercuric acetate degraded fluorescein-labeled polymeric rat tail tendon Type I collagen and the tropocollagen TCA and TCB fragments of human Type II collagen into smaller peptides at 37 degrees C. Enzyme activity was abolished if p-aminophenylmercuric acetate-activated culture medium was preincubated with 1.10-phenanthroline, a metal chelator. Thus, articular chondrocytes in monolayer culture are capable of producing neutral proteinases which acting together can result in complete degradation of tendon and cartilage collagen to small peptides.

Red blood cell glucose-6-phosphate dehydrogenase activity in aged humans.

The enzyme glucose-6-phosphate dehydrogenase (G6PD) was assayed in the circulating erythrocytes of 38 20-to 30-year-old subjects and 29 80- to 90-year-old subjects. The red blood cell G6PD activity of an aged-unbiased erythrocyte population in the young group was found to be significantly higher than in the older group (5.46 +/- 1.06) vs (4.31 +/- 0.99) (P less than 0.001), respectively (mean +/- SD). No significant difference was observed between the mean enzyme values of the male and female subjects in the two age groups. Since the mature red blood cell is devoid of regenerative capabilities, one may propose an explanation for this observation that implicates either an alteration in G6PD synthesis in the erythrocyte precursor cells in aged hosts, and/or the presence of an inhibitory factor that has the effect of decreasing the assayed activity for this enzyme.

Interstitial space of mouse skeletal muscle.

1. A new preparation of mouse skeletal muscle, prepared from pectoral muscles, is described.2. The sorbitol space of this muscle, both in vivo and in vitro, has been measured with dynamic loading of the muscle in vitro as an experimental variable.3. The Na(+) and K(+) contents of the muscle have been determined and the apparent intracellular concentration for these ions calculated both in vivo and after incubation in vitro.4. Histological studies on the incubated muscle have been made so as to permit comparison of the changes in the chemical measurements with changes in the ultrastructure of the muscle.5. The results of these experiments show that there is an increase in the apparent extracellular space of the muscle following incubation. This increase is constant, and independent of the load, with the important exception that unloaded muscles do not reach an equilibrium during the period of incubation and have a much greater apparent extracellular space.6. Intracellular Na(+) and K(+) concentrations are consistent with the sorbitol being restricted to an extracellular phase in the loaded muscle; but the evidence implies that sorbitol in the unloaded muscle penetrates into a space from which Na(+) is excluded.7. The total water content of the muscle per unit weight is unchanged by incubation, indicating that the apparent change in sorbitol space is in the ratio of intracellular space to extracellular space rather than by addition of water to the extracellular space. The significance of these results is discussed with reference to the use of such preparations for in vitro studies.

Periosteal and metaplastic bone formation in mouse minced muscle regeneration.

Structures resembling exostoses derived from periosteum and metaplastic nodules of cartilage and bone were induced in mouse legs by isotopic implants of minced skeletal muscle or heterotopic implants of nonlimb skeletal and cardiac muscle that does not regenerate. Liver mince implants produced no periosteal response. Control experiments demonstrated that, after excision of the muscle without the placing of any implant, no periosteal response or metaplastic bone occurred unless the periosteum was deliberately injured. Metaplastic nodules of cartilage and bone arose from the fibrous stroma that constituted the greater part of the implant when muscle mince regeneration was unsuccessful. The development of both the periosteal response and the metaplastic bony nodules is inversely related to the efficiency of phagocytosis by macrophages that remove the implanted debris and the concomitant degree of regeneration of new muscle. These phenomena, and, therefore, bone and cartilage formation, are strongly age related. The rapidity and extent of development of both abnormal bone and cartilage--less than 7 days--and its reproducibility may make this phenomenon a useful model for studies on the pathophysiology of bone and cartilage formation, particularly with regard to age.

Age-related impeded regeneration of mouse minced anterior tibial muscle.

The effect of increasing age on the completeness of anterior tibial muscle regeneration from autotransplants of minced muscle has been studied in Swiss Webster and C57/B6J mice aged 18 to 120 days. A progressively declining capability to regenerate new myofibers was associated with a decreasing phagocytic clearance of implanted myofiber debris. Concurrently, there was decreased presumptive myoblast proliferation and new myofiber formation. The importance of age-related host factors, including nonspecific macrophage activity, in muscle mince regeneration was demonstrated by: (1) the successful regeneration of muscle in heterotransplanted muscle minces from older mice implanted in younger animals and (2) the failure of muscle regeneration when the reverse experiment was performed in syngeneic animals. Heterologous striated muscle from the diaphragm regenerated in the bed of the excised anterior tibial muscle, whereas heterologous cardiac muscle failed to regenerate as expected because of the absence of satellite cells. The failure of phagocytic clearance of implanted myofiber mince and concurrent retardation of regeneration suggests a major age-related nonimmune role of phagocytic macrophages in the early stages of regeneration of anterior tibial muscle from isotopic minced muscle implants.