Stabilisation of collagen by betel nut polyphenols as a mechanism in oral submucous fibrosis

Summary Treatment of reconstituted collagen fibrils and pieces of rat dermis with the crude extract, purified tannins or (+)-catechin from betel nut (Areca catechu) increases their resistance to both human and bacterial collagenases in a concentration-dependent manner. These tanning agents may stabilise collagen in vivo following damage to the oral epithelium and promote the sub-epithelial fibrosis which occurs in betel nut chewers.     

Carcinogenicity examination of betel nuts and piper betel leaves.

A dry powder of betel nuts, piper betel leaves and lime was administered to rats. Epidermal thickening was frequently observed in the upper digestive tracts of rats in groups fed the betel nut diet mixed with lime and the betel leaves diet, and a forestomach papilloma was seen in 1 rat given betel leaves diet. These epidermal changes were scarcely seen in rats given either betel nut or normal diet alone.     

Carcinogenicity examination of betel nuts and piper betel leaves

Summary A dry powder of betel nuts, piper betel leaves and lime was administered to rats. Epidermal thickening was frequently observed in the upper digestive tracts of rats in groups fed the betel nut diet mixed with lime and the betel leaves diet, and a forestomach papilloma was seen in 1 rat given betel leaves diet. These epidermal changes were scarcely seen in rats given either betel nut or normal diet alone.This work was supported by a grant from the U.S.-Japan Cooperative Medical Science Program     

Betel nut residues in archaeological samples of human teeth from the Mariana Islands

Two tetrahydropyridine alkaloids, arecaidine and guvacine that are characteristic of betel nut (Areca catechu L.) have been detected in the deliberately stained labial surface of female teeth excavation on Rota, Mariana Islands. This was accomplished using selected ion monitoring techniques in conjunction with gas chromatography/electron impact-mass spectrometry. These alkaloids were not present on the buccal surface of the teeth and indicate the use of betel nut to effect the staining.     

Collagen in aging muscles.

The salt, acid and insoluble collagen fractions were estimated in red, white and cardiac muscles of 10-, 15- and 20-month-old albino rats. The total collagen level with reference to total proteins is more in red than in white and cardiac muscle. Accumulation of more of insoluble collagen and decrease in salt extractable collagen is seen in all three muscles with aging.     

The growth of human fibroblasts and A431 epidermoid carcinoma cells on gamma-irradiated human amnion collagen substrata

Summary Human fibroblasts and A431 human epidermoid carcinoma cells were cultured on gamma-irradiated human amnion collagen as well as on plastic dishes and non-irradiated collagen coated dishes. The morphology, attachment, growth and short-term cytotoxicity of these culture conditions have been determined. Both irradiated and non-irradiated amnion collagen enhanced the attachment and proliferation of fibroblasts as compared to the plastic dishes. No differences in these properties were observed for A431 cells cultured on irradiated collagen when compared with culture on non-irradiated collagen substrates. Cytotoxicity assays showed that irradiated and non-irradiated collagens were not cytotoxic for either fibroblasts or A431 cells. The results demonstrated that amnion collagen irradiated at doses of 0.25–2.0 Mrads is optimal for cell growth.     

Effects of gamma irradiation on dermal equivalents in vitro

Summary Dermal equivalents (DE), collagen lattices, were produced in vitro and used as a model for studying the possible role of a pure population of fibroblasts in post-radiotherapeutic dermal fibrosis. Single doses of gamma irradiation induced a partial inhibition of the collagen lattice retraction and of protein synthesis. The collagen production was less inhibited than was synthesis of non-collagen protein, which resulted in an increase of the relative amount of collagen synthesized by irradiated fibroblasts. These data suggest that gamma irradiation might be able to select some fibroblast clones able to produce increasing amounts of collagen. This selection process could be involved in the development of tissue fibrosis after therapeutic radiation.     

The growth of human fibroblasts and A431 epidermoid carcinoma cells on gamma-irradiated human amnion collagen substrata

Human fibroblasts and A431 human epidermoid carcinoma cells were cultured on gamma-irradiated human amnion collagen as well as on plastic dishes and non-irradiated collagen coated dishes. The morphology, attachment, growth and short-term cytotoxicity of these culture conditions have been determined. Both irradiated and non-irradiated amnion collagen enhanced the attachment and proliferation of fibroblasts as compared to the plastic dishes. No differences in these properties were observed for A431 cells cultured on irradiated collagen when compared with culture on non-irradiated collagen substrates. Cytotoxicity assays showed that irradiated and non-irradiated collagens were not cytotoxic for either fibroblasts or A431 cells. The results demonstrated that amnion collagen irradiated at doses of 0.25-2.0 Mrads is optimal for cell growth.     

Effects of gamma irradiation on dermal equivalents in vitro

Dermal equivalents (DE), collagen lattices, were produced in vitro and used as a model for studying the possible role of a pure population of fibroblasts in post-radiotherapeutic dermal fibrosis. Single doses of gamma irradiation induced a partial inhibition of the collagen lattice retraction and of protein synthesis. The collagen production was less inhibited than was synthesis of non-collagen protein, which resulted in an increase of the relative amount of collagen synthesized by irradiated fibroblasts. These data suggest that gamma irradiation might be able to select some fibroblast clones able to produce increasing amounts of collagen. This selection process could be involved in the development of tissue fibrosis after therapeutic radiation.     

Differential regulation of collagen types I and III expression in cardiac fibroblasts by AGEs through TRB3/MAPK signaling pathway

Advanced glycation end products (AGEs) play an important role in collagen deposition in diabetic cardiomyopathy. TRB3, a mammalian homolog of Drosophila tribbles, functions to increase glucose intolerance and regulates cell proliferation. We demonstrated that AGEs induce collagen type I expression but inhibit collagen type III expression, accompanied by increased TRB3 expression. Furthermore, the collagen type I induced byAGEs was down-regulated after inhibition of ERK and p38-MAPK, the collagen type III reduced by AGEs was up-regulated after inhibition of ERK. The expression of collagen types I and III regulated by AGEs through MAPK was partly reversed after treatment with TRB3 siRNA. It suggests that the TRB3/MAPK signaling pathway participates in the regulation of collagen types I and III by AGEs and may provide new therapeutic strategies for diabetic cardiomyopathy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 08 May 2008; received after revision 25 June 2008; accepted 22 July 2008 M. Tang, M. Zhong: These two authors contributed equally to this work.     

Collagen treated with (+)-catechin becomes resistant to the action of mammalian collagenase

Treatment of radioactively labeled guinea-pig skin soluble collagen or calf skin collagen with the flavonoid (+)-catechin makes the collagen resistant to the action of mammalian collagenase but not to the action of bacterial collagenase. Complete resistance to the action of the mammalian enzyme may be achieved by incubating 0.6 mg of collagen (dry weight) with 0.1 mM (+)-catechin, followed by dialysis to remove the unbound flavonoid. Since incubation of the mammalian enzyme with (+)-catechin does not inhibit its activity, it is postulated that (+)-catechin binds tightly to collagen and modifies its structure sufficiently to make it resistant to enzyme degradation.     

Independent modulation of collagen fibrillogenesis by decorin and lumican

The leucine-rich proteoglycans (also known as "small, leucine-rich proteoglycans," or SLRPs) lumican and decorin are thought to be involved in the regulation of collagen fibril assembly. Preparation of these proteoglycans in chemical amounts without exposure to denaturants has recently been achieved by infecting HT-1080 cells with vaccinia virus that contains an expression cassette for these molecules. Addition of lumican and decorin to a collagen fibrillogenesis assay based on turbidity demonstrated that lumican accelerated initial fibril formation while decorin retarded initial fibril formation. At the end of fibrillogenesis, both proteoglycans resulted in an overall reduced turbidity, suggesting that fibril diameter was lower. The presence of both proteoglycans had a synergistic effect, retarding fibril formation to a greater degree than either proteoglycan individually. Competitive binding studies showed that lumican did not compete for decorin-binding sites on collagen fibrils. Both proteoglycans increased the stability of fibrils to thermal denaturation to approximately the same degree. These studies show that lumican does not compete for decorin-binding sites on collagen, that decorin and lumican modulate collagen fibrillogenesis, and that, in the process, they also enhance collagen fibril stability.     

Renewal of pulmonary insoluble collagen in the adult rat]

Study of polymeric pulmonary collagen in adult Rats showed that about 70% of collagen was renewed with a half-time equal to 525 days. This value is to be compared with the median life-span of this rat strain, 890 days. The remaining 30% of polymeric collagen is renewed with a shorter half time, about 30 days.     

Annexin V interactions with collagen

Annexin V was originally identified as a collagen-binding protein called anchorin CII and was isolated from chondrocyte membranes by affinity chromatography on native type II collagen. The binding of annexin V to native collagen type II is stable at physiological ionic strength when annexin V is reconstituted in liposomes. The binding to native collagen types II and X, and to some extent to type I as well, was confirmed using recombinant annexin V. A physiological role for annexin V interactions with extracellular collagen is consistent with the localization of annexin V on the outer cell surface of chondrocytes, microvilli of hypertrophic chondrocytes, fibroblasts and osteoblasts. A breakthrough in our understanding of the function of annexin V was made with the discovery of its calcium channel activity. At least one of several putative functions of annexin V became obvious from studies on matrix vesicles derived from calcifying cartilage. It was found that calcium uptake by matrix vesicles depend on collagen type II and type X binding to annexin V in the vesicles and was lost when collagens were digested with collagenase; calcium influx was reconstituted after adding back native collagen II or V. These findings indicate that annexin V plays a major role in matrix vesicle-initiated cartilage calcification as a collagen-regulated calcium channel.     

Degradation of collagen by the cariogenic bacteria, Streptococcus mutans

The behaviour of cariogenic Bacteria (Streptococcus mutans) is studied with regard to collagen, which represents 90% of the dentine organic matrix. Collagenase activity of cariogenic Bacteria is measured with radioactive precursors and gel electrophoresis and compared to reference bacterial collagenase (Clostridium histolyticum). Labelled collagen substrate has been prepared with two different methods: extraction by 0,5 M acetic acid from young Rat skin, previously labelled with L-proline 14C, or reduction by Na B3H4. Both collagen sutstrates have been incubated for 2 h in Terleckyj medium in which the Streptococcus mutans have been inoculated. The experiments show a proteolytic activity of Streptococcus Mutans on the collagen substrate.     

Degradation of type I collagen fibrils synthesized by human dental pulp cells in explant culture exposed toActinomyces viscosus. Electron microscope immunotyping

Summary Actinomyces viscosus Be 66, added to pulpal cells in culture, does not cause apparent cellular damage. The extracellular matrix consists of altered collagen fibrils and thin filaments, immunochemically identified as type I collagen. They probably represent the first steps of collagen degradation.This work was supported by INSERM (ATP: 77-85) and CNRS (RCP: 533).     

Do Schwann cells produce collagen type III?

Summary The fact that collagen from both normal nerve endoneurium and Schwann cell tumours present characteristics of collagen type III, suggests that Schwann cells produce this type of collagen.     

Collagen treated with (+)-catechin becomes resistant to the action of mammalian collagenase

Summary Treatment of radioactively labeled guinea-pig skin soluble collagen or calf skin collagen with the flavonoid (+)-catechin makes the collagen resistant to the action of mammalian collagenase but not to the action of bacterial collagenase. Complete resistance to the action of the mammalian enzyme may be achieved by incubating 0.6 mg of collagen (dry weight) with 0.1 mM (+)-catechin, followed by dialysis to remove the unbound flavonoid. Since incubation of the mammalian enzyme with (+)-catechin does not inhibit its activity, it is postulated that (+)-catechin binds tightly to collagen and modifies its structure sufficiently to make it resistant to enzyme degradation.Acknowledgments. This work was supported by grants from the Easter Seal Research Foundation of the National Easter Seal Society for Crippled Children and Adults (R-7821), and the National Institutes of Health (HL-20447). (+)-Catechin was a generous gift from Zyma SA, CH-1260 Nyon, Switzerland.     

Effects of isaxonine phosphate and analogs on fibroblast metabolism in culture

Human skin fibroblasts in confluent cultures were incubated for 24 h in the presence of isaxonine phosphate (Nerfactor) and several related factors. The incorporation of 14C-proline into secreted proteins and the release of collagen into the medium were inhibited. When the cells were incubated for an additional period of 24 h after thorough washing, protein and collagen syntheses were found to be identical to those of controls, demonstrating that the inhibition of protein synthesis was independent of any toxic effect. When cells were incubated in the presence of both isaxonine and colchicine, the secretion of collagen was more inhibited than by colchicine alone, and proteins accumulated in the cells.     

X-ray diffraction of the collagen contained in blood vessels]

An X-ray diffraction study has been undertaken on Human and Rabbit blood-vessels. Vessels are stretched 500% and the diagrams present a great analogy with those obtained from collagen. Thus, it appears, that it is possible to determine by X-Rays some pathological anomalies of the collagen in vessels.