Effect of cellular aging on collagen biosynthesis: I. Methodological considerations and pharmacological applications.

The study of the age and passage dependent modifications of collagen biosynthesis requires a simple, rapid and reproducible procedure adaptable to serial cell cultures. To make such a method comparable to other methods of collagen determination, we calibrated a colorimetric procedure both by hydroxyproline (HYP) determinations and in terms of collagen concentration. For collagen types I and IV, widely different slopes were obtained with the colorimetric procedure. To further refine the procedure, we tempted to completely inhibit collagen synthesis by beta-aminopropionitrile (beta APN) added to cultures in order to obtain a negative control. Using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl 2H-tetrazolium bromide (MTT-test), it could be shown that relatively high concentrations of beta APN are tolerated by the cells. It appeared, however, that even the highest concentration of beta APN (1mM) still tolerated by the fibroblasts did not completely inhibit collagen synthesis. At low concentrations, beta APN even stimulated cell-proliferation. The colorimetric procedure calibrated in terms of collagen type I concentration, was therefore retained for the serial determination of collagen synthesis and accumulation. We shall here describe the methodological details of its validation as well as its application for the pharmacological study of the effect of aging on collagen biosynthesis. Among the factors involved, the accumulation of advanced glycation end-products (AGEs) might well play an important role. Several of such AGE-products showed a significant inhibition of collagen deposition. On the contrary, retinol, ascorbic acid as well as the rhamnose-rich oligo- and polysaccharides (RROPs) did produce a significant upregulation collagen deposition. Polysaccharide preparations, rich in rhamnose and fucose (the EROB-mixture) could protect against the AGEs-induced inhibition of collagen accumulation.

[Demonstration of the cytotoxic effect of Advanced Glycation Endproducts (AGE-s)]. / Démonstration de l'effet cytotoxique des produits avancés de la glycation (AGE-s).

Advanced Glycation End-products (AGE-s) were shown to exhibit a number of potentially harmful properties in contact with cells and tissues. As their concentrations increases with age, faster even in hyperglycemic individuals, they are considered important for aging- and age-associated pathologies, especially for athero-arteriosclerosis and type II diabetes. We describe here the methods used for the demonstration of a direct cytotoxicity of several AGE-products when added to human skin fibroblast cultures. This cytotoxicity was still demonstrable when cells, previously cultured with AGE-s, were transferred to new medium without AGE-s. This effect, the remanence of cytotoxicity in absence of AGE-s, suggests a certain degree of inheritance, possibly by epigenetic mechanisms, of the cytotoxic effect of AGE-s, mediated by the AGE-receptors (RAGE-s) and inhibited by free radical-scavengers, such as L-Carnosine, Catalase and Rhamnose-rich oligo- and polysaccharides. Such cytotoxicity can occur not only on the skin but also in other tissues. It appears thus that besides the crosslinking of collagen and other macromolecules, the products of the Maillard reaction can exert their harmful cytotoxic effects directly on the cells.

[The importance of the Maillard reaction in ophtalmology]. / Importance de la réaction de Maillard en ophtalmologie.

Non enzymatic glycosylation( glycation) of proteins, described by L. C. Maillard in 1912, results in the formation of advanced glycation end products (AGE-s). These exhibit a number of harmful reactions, increasing with age and involved in several age-associated pathologies. In ocular pathology, their role was demonstrated at several levels of age-associated eye-diseases, such as the rigidification of cornea, in the separation of vitreous fibers from the hyaluronan jelly, which might result in retinal detachment. AGE-s are involved also in retinal microvascular alterations in diabetics as well as in age-related macular degeneration. We compared the cytotoxic effect of several AGE-s on human skin fibroblasts and corneal keratocytes. Keratocytes were shown to be much more resistant to the cytotoxic effect of several AGE-products than fibroblasts. This higher resistance of keratocytes to the free radical mediated cytotoxic effect of AGE-s might be the result of the constant exposure of cornea to UV-light possibly mediating the appearance of more efficient protective mechanisms during evolution.

Effect of advanced glycation end-products on cell proliferation and cell death.

The effect of advanced glycation end products (AGE-s) was studied on the proliferation and cell death of human skin fibroblasts in culture. Several AGE-products were prepared from proteins, a peptide and amino acids, using Glucose or Fructose, with or without Fe2+. The AGE preparations increased cell death at the 7th day, after only 72 hours of incubation. Some of these glycation products modified also proliferation. This effect of AGE-s was even maintained without these products in fresh medium for a second period of incubation up to 10 days from the start of the experiment. In order to explore the role of AGE-receptors, especially of AGE-receptor and of growth factor receptors (fibroblast and epidermal growth factors receptors), antibodies to these receptors were added to cell cultures and their effect on both cell death and proliferation were determined as for the AGE-s. These anti-receptor antibodies imitated to some extent the results obtained with AGE-s, producing increase of cell death and proliferation, followed above a certain concentration of antibodies by a decrease and a new increase or plateau. This might correspond to the internalization of the receptors followed by a re-expression on the cell membrane. The role of receptor-mediated Reactive Oxygen Species-production was also explored using scavengers: N-acetyl-cysteine (NAC), L-Carnosine, superoxide dismutase (SOD) and Catalase. Several of these scavengers decreased cell death, suggesting that Reactive Oxygen Species-production is partially involved in the observed phenomena.

Pharmacological properties of rhamnose-rich polysaccharides, potential interest in age-dependent alterations of connectives tissues.

Rhamnose-rich oligo- and polysaccharides (RROPs) were tested for their potential pharmacological properties using human skin fibroblasts in serial cultures. The substances tested were shown to stimulate cell proliferation, decrease elastase-type activity, stimulate collagen biosynthesis, and protect hyaluronan against free radical mediated degradation. These reactions appear to be triggered by the mediation of a specific alpha-L-rhamnose recognizing lectin-site acting as a receptor, transmitting signals to the cell-interior. The rapid increase of intracellular free calcium after addition of RROP-1 and preliminary data using micro arrays appear also to confirm this contention.

Studies on skin aging. Preparation and properties of fucose-rich oligo- and polysaccharides. Effect on fibroblast proliferation and survival.

Skin aging represents an important chapter of connective tissue aging and concerns an organ of vital importance. Here we describe the preparation as well as the biological properties of fucose-rich oligo- and polysaccharides (FROPs), composed of polymers of a trisaccharide containing galactose, acetyl galacturonic acid and fucose, from the original high molecular weight bacterial polysaccharide (Fucogel), Solabia, France). Using endoglycosidases, oligo- and polysaccharides were prepared and characterized by physical and chemical procedures. The non-reducing end-groups comprise equal amounts of galactose and fucose. The here-described biological properties are: stimulation of cell proliferation of cultured human skin fibroblasts, protection of cells against ascorbate-induced cytotoxicity due to the release of reactive oxygen species (ROS). Properties elsewhere described concern the inhibition of matrix metallo-proteinases 2 and 9 (MMP-2 and MMP-9), their expression and activation. Using fluorescein isothiocyanate (FITC)-labeled polysaccharides, their interaction with cell membranes and also their penetration and accumulation in cells, especially in the cell nucleus could be demonstrated, probably via cell-membrane receptor-mediated mechanisms. We describe some of the symptoms of skin aging and show, that the here-described polysaccharide preparations are susceptible to slow down some of the cellular and molecular mechanisms involved, partly by the mediation of the above-mentioned receptors, partly by acting directly on the regulation of gene expression.

Pharmacological properties of fucose. Applications in age-related modifications of connective tissues.

Fucose is the only component of glycoconjugates of vertebrates in the L-configuration. It exhibits a number of unique and interesting biological properties reviewed briefly in this article. Its constant end-standing position on glycan chains predisposes fucose to play a key role in cell-cell and cell-matrix interactions, mediated by several receptors such as those recognising the Lewis-type blood group substances, fucose-recognising lectines and the mannose-fucose receptors. Some of the as yet unstudied or less well understood properties of L-fucose were explored in the present study, as its non-enzymatic interaction with amine-groups on macromolecules, its cellular uptake attributed to specific transport mechanisms and its effect on fibroblast cell cultures. We could document the stimulation of cell-proliferation and the inhibition of MMP-expression and activation, both for MMP-2 and MMP-9. These and the other shortly reviewed properties of L-fucose may play an important role in its biological applications and actions.

Protection by L-fucose and fucose-rich polysaccharides against ROS-produced cell death in presence of ascorbate.

It was shown previously, that millimolar concentrations of ascorbate have cytotoxic and anti-proliferative effects (Eur. J. Clin. Invest. 32 (2002) 372). With increasing concentrations of ascorbate an increasing number of fibroblasts was detached from the culture dish and shown to be lysed by the effect of ascorbate-induced generation of reactive oxygen species (ROS-s). It also could be shown, that this cytotoxic effect is partly due to the dose-dependent inhibition by ascorbate of fibronectin biosynthesis. Superoxide dismutase (SOD) and catalase were shown to salvage cells from ROS-induced cell-death by preventing the inhibition of fibronectin biosynthesis. We used this model system to test the cyto-protective effect of L-fucose and fucose-rich oligo- and polysaccharides (FROP-s). It appeared that relatively low concentrations of L-fucose and FROP-3 (Biomed. Pharmacother. in press) could efficiently protect fibroblasts from the ascorbate-induced cell-death. These novel pharmacological properties of L-fucose and FROP-3 might well be related to their accelerating effect of wound healing.

Regulation of elastase-type endopeptidase activity, MMP-2 and MMP-9 expression and activation in human dermal fibroblasts by fucose and a fucose-rich polysaccharide.

Tissue loss during ageing and age-dependent pathologies are the result of a disturbed regulation of proteolytic activities. Elastase-type endopeptidases, especially MMP-2 and -9, play an important role in this respect. Dermal fibroblast cultures and skin explant cultures were used in order to measure the efficiency of fucose and fucose-rich polysaccharides to downregulate the elastase-type endopeptidase activity. Fucose and fucose-rich polysaccharides were shown to downregulate this elastase-type activity, the basic activity and also the hyaluronan or kappa-elastin-stimulated activity. In skin explant cultures, we could demonstrate that fucose and fucose-rich polysaccharides produced an inhibition of the activation of the pro-form to the active form of MMP-9. Here, we show that mono-, di-, oligo- and polysaccharides acting on the elastin-laminin receptor and/or on the fucose-mannose receptor are efficient inhibitors of such enzymes by downregulating elastase-type endopeptidase activity, both at the level of their biosynthesis and at the level of the activation of the pro-enzymes. Fucose and fucose-rich polysaccharide preparations were shown to be efficient modulators of MMP-2 and MMP-9, activity with potential therapeutic applications in age-related pathologies accompanied by tissue loss.

Inhibition of cell proliferation and fibronectin biosynthesis by Na ascorbate.

BACKGROUND: The importance of ascorbate on the production of extracellular matrix proteins (as elastin and collagens) is now well documented, but no studies have been published concerning its effects on fibronectin biosynthesis. Fibronectin is important for cell attachment and for proliferation. MATERIALS AND METHODS: The effects of Na ascorbate were investigated on cell attachment, proliferation, viability and fibronectin biosynthesis by human skin fibroblasts in vitro. Proliferation was followed by the monitoring of [(3)H]-thymidine incorporation; viability by the MTT-test, cell adherence by counting adherent and nonadherent cells and fibronectin biosynthesis by immunoprecipitation of biosynthetically labelled fibronectin. RESULTS: In the presence of ascorbate, the fibroblasts showed a biphasic growth pattern. At 500 microM ascorbate, [(3)H]-thymidine incorporation was stimulated by 15% as compared to the controls. Higher concentrations gradually decreased proliferation up to 36% of the control value at 5 mM. These effects of ascorbate on DNA synthesis were followed to > 1.25 mM by a strong inhibition, cytotoxic effect and cell death. The non-adherent cell count increased to 10% of the total population at 2.5 mM and to 31% at 5.0 mM ascorbate.Increasing concentrations of ascorbate resulted in a dose-dependent decrease of fibronectin biosynthesis, both in the culture supernates and cell extracts. This inhibition mainly concerned cell membrane-associated fibronectin.Superoxide-dismutase or catalase could inhibit Na ascorbate-induced cytotoxicity and partially re-establish fibronectin biosynthesis. Desferrioxamine, ergothionein and vitamin E were inefficient. CONCLUSIONS: Our results indicate that ascorbate decreases fibronectin biosynthesis of cultured human skin fibroblasts, thereby producing cell detachment and decreased proliferation. This effect is mainly mediated by the reactive oxygen species and can be inhibited by superoxide-dismutase and catalase.

Cell death by overload of the elastin-laminin receptor on human activated lymphocytes: protection by lactose and melibiose.

BACKGROUND: Activated human lymphocytes were shown to express the elastin-laminin receptor in vitro and also in vivo in atherosclerotic plaques. In the presence of the agonist, elastin peptides, this receptor was shown to mediate an increased cell proliferation and an increased synthesis and excretion of an elastase-type serine endopeptidase. In this study, we investigated the variation of the above reaction as a function of agonist concentration. MATERIALS AND METHODS: Human lymphocytes were obtained by tonsillectomy and cultured in the presence of phytohaemagglutinin and elastin peptides. Cell viability was evaluated by vital dye exclusion. Elastase and cathepsin G activities were determined in culture supernates and cell lysates using synthetic substrates. Apoptotic cells were identified by the TUNEL method and by electron microscopy. RESULTS: At increasing concentrations of elastin peptides, a dose-dependent increase in cell death was observed. Up to 100 micrograms mL-1 elastin peptides and an increasing fraction of lymphocytes were found permeable to trypan blue, and a large proportion was in apoptosis. Elastin peptide-induced cell death was inhibited by 1 microgram mL-1 lactose and melibiose. CONCLUSION: We describe here cell death of human activated lymphocytes expressing the elastin-laminin receptor in the presence of increasing concentrations of elastin peptides, agonists of the receptor. The mechanism of cell death appears to be related to the triggering of the release of elastase and free radicals mediated by the elastin-laminin receptor. Antagonists of this receptor, lactose and melibiose, protected the lymphocytes from the receptor-mediated cell death.

Cell death induced in lymphocytes expressing the elastin-laminin receptor by excess agonists: necrosis and apoptosis.

This manuscript summarizes our experiments carried out during the last years on the expression of the elastin-laminin receptor on human activated lymphocytes and cell death triggered by the activation of this receptor by its agonists, elastin peptides. We could distinguish two types of cell reactions, depending on the elastin peptide concentration added to the cell culture media of lymphocytes. At low concentrations (1-10 micrograms/mL, 1.3-13 x 10(-8) M) of kappa-elastin, there was a stimulation of cell proliferation, elastase biosynthesis and release. As the concentration of kappa-elastin was increased in the culture medium up to 100 micrograms/mL, lymphocyte proliferation and elastase production decreased and the proportion of dead cells increased. Cell death was shown to be due to both apoptotic and non-apoptotic mechanisms. Apoptotic cell death increased with agonist concentration and reached approximately 60% of the lymphocyte population at mg/mL elastin peptide concentrations. This observation was confirmed by the concomitant use of several different methodologies, such as flow cytometry and electron microscopy. The precise nature of the non-apoptotic cell death remains to be established.

Aging and matrix biology.

This introduction to a theme issue of Pathologie Biologie on the extracellular matrix starts with a brief overview of the advances made over the last few years and of the increasing specialization they have resulted in. A review is then presented of cell-matrix interactions, with emphasis on those mediated by the elastin-laminin receptor during physiologic processes and during aging and age-related diseases. The activated human lymphocyte expressing the elastin-laminin receptor is used as an example. When exposed to low levels of elastin peptides (kappa-elastin, 75 kappa D, 1-10 micrograms/ml, i.e., 1.4 to 14.10(-8)M), this receptor mediates increases in cell growth and in the production of serine-elastase. Levels of about 100 micrograms/ml are associated with cell death due to necrosis and to apoptosis. This example illustrates the key role played by epigenetic phenomena in aging of cells and tissues.

Lymphocytes in human atherosclerotic plaque exhibit the elastin-laminin receptor: potential role in atherogenesis.

The purpose of this immuno-histochemical study was to investigate if lymphocytes, present in the human atherosclerotic plaque, exhibit the elastin-laminin receptor. We showed recently that human activated lymphocytes in vitro express this receptor. Briefly, we demonstrated by immuno-localization experiments and by flow cytometry that this receptor is available on the cell surface of human activated lymphocytes, free to react with ligands and show capping. The activation of this receptor by elastin peptides triggers several cellular reactions of biological interest as shown previously such as chemotactic movement to an elastin peptide gradient, modulation of the biosynthesis of connective tissue macromolecules, increase of protease synthesis and release of free radicals (O2-., NO.) from mononuclear and endothelial cells, modifications of ion fluxes and also increase of cell proliferation. All these processes may contribute to the development of the atherosclerotic lesion. Two of the previously demonstrated cell reactions mediated by the receptor could be demonstrated also on PHA-stimulated human lymphocytes namely stimulation of cell proliferation and increase of elastase activity. We demonstrated in the present immuno-histological study that about 50-60% of lymphocytes of the human atherosclerotic plaque obtained by endarterectomy express the 67 kDa subunit of the elastin-laminin receptor confirming that the above described phenomena could contribute to the chronicity of the lesion.

Human helper and memory lymphocytes exhibit an inducible elastin-laminin receptor.

We showed recently that human activated lymphocytes express the elastin-laminin receptor. In this study, we were interested in the kinetics of the induction of this receptor on human activated lymphocytes in vitro and in the quantification of its expression on different human lymphocyte subsets. It appears that the expression of the elastin-laminin receptor is a general property of most activated human lymphocytes but strongly dependent on the lymphocyte subsets. It appeared that the helper (CD4+) and memory (CD45RO+) lymphocytes exhibited the strongest increase of elastin-laminin receptor expression when cultured for 72 h in the presence of 2 microg/ml elastin peptides (2.7x10[-8] M) as compared to control cells. Activation of this receptor by elastin peptides triggers the stimulation of biosynthesis and release of a PMN-like elastase. Activated T lymphocytes (mostly helper and memory T cells) are present from early stages of the atherosclerotic process and this release could contribute to the progression of the lesion by engaging a vicious circle with more elastin peptides released attracting more mononuclear cells and increasing their elastase production.

Increased elastase and cathepsin G activity in activated lymphocytes from aged patients: role of denutrition and dementia.

Elastase and cathepsin G activities in cell-lysates and in culture supernates of activated human lymphocytes (incubated for 96 h in the presence of 5 microg/ml of phytohemagglutinin with/without 2 microg/ml of elastin peptides) of 25 old, hospitalized patients (average age: 88.48+/-6.81 years), suffering from vascular-type dementia or denutrition were examined, in comparison with samples of 11 young, healthy donors. Elastase activity was found to be significantly elevated, the excreted fraction of elastase activity strongly decreased in lymphocytes of these patients as compared to young donors. The highest values of enzymatic activities in the culture supernates and also in the cell-lysates were obtained for samples of undernourished patients. Lymphocytes from young, healthy donors showed a significant increase both of excreted and intracellular elastase activity when cultured in presence of elastin peptides. The activation of elastolytic activity by elastin peptides decreased with the age of donors. The results for cathepsin G are essentially similar but the differences between age-categories are less pronounced than for elastase activity.

Presence of the elastin-laminin receptor on human activated lymphocytes.

A variety of cells - fibroblasts, vascular smooth muscle cells, endothelial cells, monocytes and polymorphonuclear leukocytes (PMNs) - carry the elastin-laminin receptor. The activation of this receptor by elastin peptides triggers a variety of reactions as chemotactic movements to an elastin peptide gradient, release of lytic enzymes and oxygen-free radicals, modifications of ion fluxes. We now show that human lymphocytes also express this receptor. Membrane labelling of the receptor by specific antibodies shows capping. In the presence of elastin peptides lymphocytes show increased proliferation and increased production of an elastase type serine protease apparently identical to PMN-elastase, inhibited by cycloheximide and by anti-PMN elastase antibodies. T-lymphocytes are present in atherosclerotic plaques where elastin degradation occurs and could contribute to the chronicity of the lesion by the above mechanism.

Alterations in nucleoside monophosphate concentrations in 3LL tumours after combined treatment with tiazofurin and 5-hexyl-2'-deoxyuridine.

The IMP and GMP concentrations were compared after treatment with tiazofurin alone and in combination with 5-hexyl-2'-deoxyuridine (HUdR) in 3LL-HH adenocarcinoma in vivo. The elevation in IMP/GMP ratio, indicating guanylate depletion and increase of inosine-5'-monophosphate concentration, showed a dose dependence and was the highest at the 7th hour after treatment with tiazofurin. HUdR application alone caused only a modest change in the nucleotide concentration of LL-HH tumour. However, the rise of IMP but not the reduction of guanylate concentration induced by tiazofurin was remarkably mitigated by HUdR treatment, without affecting the antitumour potency of tiazofurin. Thus HUdR showed modifying activity on some of the tiazofurin-induced changes in nucleotide metabolism which appeared not to be associated with the antiproliferative activity of tiazofurin. It follows that reduced GMP concentration and not the elevation of IMP/GMP ratio could predict therapeutic responses to tiazofurin.