Jacques-Paul Borel

Stimulation of collagen synthesis in fibroblast cultures by the tripeptide‐copper complex glycyl‐L‐histidyl‐L‐lysine‐Cu2+

Glycyl‐L‐histidyl‐L‐lysine (GHK) is a tripeptide with affinity for copper(II) ions and was isolated from human plasma. This peptide appears to play a physiological role in wound healing. We report the stimulating effect of GHK‐Cu on collagen synthesis by fibroblasts. The stimulation began between 10−12 and 10−11 M, maximized at 10−9 M, and was independent of any change in cell number. The presence of a GHK triplet in the α2(I) chain of type I collagen suggests that the tripeptide might be liberated by proteases at the site of a wound and exert in situ healing effects.

Fibronectin dependence of the contraction of collagen lattices by human skin fibroblasts

The role of fibronectin in the contraction of collagen lattices by human skin fibroblasts has been investigated. Incubation of lattice cultures in Dulbeccos modified Eagles medium supplemented with increasing concentrations of non-dialysed or dialysed fetal calf serum demonstrated that the rate of contraction was dependent on non-dialysable serum components. The suppression of contraction observed when fibronectin was eliminated from serum, either by affinity chromatography on gelatin-agarose columns or by precipitation with anti-fibronectin antibodies, showed that fibronectin is critical for the contraction. When collagen lattices were incubated in a serum-free culture medium totally devoid of fibronectin, no contraction occurred. When fibronectin was added to this medium, their contraction was correlated with the concentration of fibronectin added. The contraction was inhibited by cycloheximide, tunicamycin, and monensin. These results demonstrate that the contraction of collagen lattices by human skin fibroblasts is dependent on fibronectin, and that other protein factors synthesized by the cells or contained in serum are also necessary.

Does oxygen free radical increased formation explain long term complications of diabetes mellitus

Oxygen free radicals (OFR) can form by reaction of glycated proteins with molecular oxygen. We hypothesize that this mechanism operates in tissues of diabetic patients when their content of glycated proteins is significantly increased. OFR are harmful to polyunsaturated fatty acids of lipid membranes, proteins, sugars and DNA. The most significant complications of diabetes, for example polyneuritis, retinopathy, microangiopathy, perforating ulcers, impaired healing, may depend on the excessive production of OFR by glycated proteins. Clues to these effects may be deduced from the decrease of glutathione stores in red blood cells, and the increases of lipid peroxidation and malondialdehyde formation, all of which have been documented to occur in the course of diabetes mellitus.

Collagen degradation by superoxide anion in pulse and gamma radiolysis

Delipidated collagen fibrils reconstituted from acid-soluble calf skin collagen, suspended in 50 mM phosphate buffer, pH 7.4, containing 100 mM sodium formate, were submitted to pulse radiolysis in Febetron devices or to gamma radiolysis in a 60Co irradiator. A collagen degradation process was found. The kinetics of this degradation was followed by evaluation of the amount of 4-hydroxyproline present in the small peptides liberated during the irradiation period. The yield of 4-hydroxyproline small peptides was low (0.1 mol/100 eV for an initial collagen concentration 3.2 microM). It increased linearly with the dose of irradiation and the concentration of collagen in suspension. The kinetic competition between O2-. dismutation and O2-. reaction with collagen was studied by pulse radiolysis at several concentrations of collagen. A value of the kinetic constant of k(O2-. + collagen) = 4.8 . 10(6) mol-1.l.s-1 was determined.

Chronic UVB- and all-trans retinoic-acid-induced qualitative and quantitative changes in hairless mouse skin

Histochemical and ultrastructural studies have already demonstrated that chronic exposure to UV radiation induces profound alterations in all structural elements of the skin and that topical all-trans retinoic acid (tRA) can substantially correct much of the tissue damage. However, previous biochemical studies on dermal components of the extracellular matrix have led to contradictory results, particularly with regard to the effect of chronic UV exposure. The aim of our study was to investigate changes in collagen content and other dermal modifications induced by tRA in irradiated and non-irradiated hairless mouse skin. Hairless mice were exposed to increasing doses of UVB for 10 weeks (the cumulative total dose was 4.6 J cm-2). After the UV irradiation period the animals were treated with 0.05% tRA or with ethanol-polyethylene glycol vehicle alone three times a week for up to 10 weeks. Non-irradiated animals underwent the same treatments. The main clinical and histological changes induced by UVB exposure were erythema, wrinkling, keratosis and epidermal thickening. Following UVB exposure, tRA treatment did not improve the clinical aspect but increased the width of the dermal repair zone. Fibronectin, laminin and type I and VI collagens were detected by indirect immunofluorescence techniques in this zone. Type I and III collagens were quantitated in skin fragments after cyanogen bromide digestion and polyacrylamide gel electrophoresis. Under our experimental conditions, UVB irradiation alone induced neither changes in total collagen nor in type I and III collagen levels. tRA treatment of irradiated skin significantly increased both type I and III collagen levels by factors of 1.33 and 1.88 respectively. The ratio of type III to types I + III increased significantly. Topical tRA also increased collagen type levels in non-irradiated hairless mouse skin. Type I collagen increased proportionally to type III. This study leads to the conclusion that topical tRA exerts direct or indirect effects on collagen metabolism in irradiated as well as non-irradiated hairless mouse skin.

Effects of preformed proline and proline amino acid precursors (including glutamine) on collagen synthesis in human fibroblast cultures

A technique of derivatizing proline and 4-hydroxyproline with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was used to measure the radioactivities, concentrations and specific activities of proline and hydroxyproline. The technique was used to study the conditions of procollagen synthesis in cultured human foreskin fibroblasts. Procollagen synthesis appeared to be independent of the proline concentration in the medium, in the presence of glutamine, when monitored by the assay of non-dialyzable hydroxyproline, but not when monitored by [14C]proline incorporation. In the absence of unlabelled proline added to labelled proline in the medium, the specific activity of the secreted procollagen did not reach a plateau over a 24-h period. When the medium was supplemented with glutamine, glutamic acid, or aspartic acid, both the radioactivity and concentration of intracellular free proline decreased. Pyrrolidone-2-carboxylic acid and ornithine both induced a slight increase in concentration of the intracellular free proline. Glutamine competed with [14C]proline for incorporation into prolyl-tRNA and procollagen, independently of free intracellular proline, and it stimulated the biosynthesis of procollagen (expressed as non-dialyzable hydroxyproline) by a factor of 2.3.

Variability in the retraction of collagen lattices by scleroderma fibroblasts—relationship to protein synthesis and clinical data

Skin fibroblasts from 18 scleroderma patients were seeded into collagen lattices and their ability to retract their substratum was compared with that of control fibroblasts from healthy donors. When considered as a whole, scleroderma fibroblasts retracted lattices earlier and more intensely than controls. Analysis of individual results demonstrated that morphea and diffuse systemic sclerosis (dSSc) fibroblasts had different kinetics of lattice retraction. Fibroblasts which contracted lattices more intensely than controls were found to produce increased levels of fibronectin. A comparison of the retraction of collagen lattices by fibroblasts from involved (IS) and uninvolved skin (US) of the same patients (n= 4) showed that those from IS retracted the lattices more than fibroblasts from normal donors, whereas a high variability was found with fibroblasts from US. The increased retraction of collagen lattices seems to be a feature of the more severe forms of scleroderma.

Inhibition of collagen synthesis by interleukin-1 in three-dimensional collagen lattice cultures of fibroblasts

Interleukin-1 (II-1) was added to collagen lattice cultures of human skin fibroblasts. No cell division was induced, the ability of fibroblasts to contract the lattices was decreased and a dose-related inhibition of collagen synthesis without effect on non-collagen proteins was found. Indomethacin had no influence on these effects.

Effect of transforming growth factor β on fibroblasts in three‐dimensional lattice cultures

Human skin fibroblasts were cultivated in three‐dimensional fibrin or collagen lattices, under retracting or non‐retracting conditions, and the influence of transforming growth factor β (TGFβ) was tested. TGFβ stimulated the synthesis of non‐collagen protein and of collagen in all the systems. However, only in non‐retracting fibrin lattices did it restore a level of protein synthesis comparable to that found in monolayers. The effects of TGFβ greatly depended on the type of substratum and on the presence or absence of retraction.

Nonisotopic Evaluation of Collagen in Fibroblasts Cultures

A method for the evaluation of collagen concentrations in the medium of fibroblasts in culture was developed. Collagen was precipitated with other proteins by addition of ethanol and hydrolyzed by 6 M HCl. The primary amino acids of the hydrolyzate were reacted with o-phthalaldehyde (OPA) and secondary amino acids (Pro, Hyp) were derivatized with 9-fluorenylmethyl-chloroformate (FMOC-Cl). The mixture was separated by isocratic HPLC on a reverse-phase column. FMOC-derivatives were detected by fluorometry, whereas OPA-derivatives were not. This method is suitable for the monitoring of collagen metabolism in fibroblast cultures exposed to various effectors.