A.-M. Robert

Evidence by in vivo and in vitro studies that binding of pycnogenols to elastin affects its rate of degradation by elastases

Procyanidol oligomers and (+) catechin bound to insoluble elastin markedly affect its rate of degradation by elastases. Insoluble elastin pretreated with procyanidol oligomers (PCO) was resistant to the hydrolysis induced by both porcine pancreatic and human leukocyte elastases. The quantitative adsorption of pancreatic elastase was similar on either untreated or PCO-treated elastin suggesting that the binding of this compound to elastin increases the non-productive catalytic sites of elastase molecules. (+) Catechin-insoluble elastin complexes were partially resistant to the degradation induced by human leukocyte elastase but were hydrolysed at the same rate as untreated samples by a constant amount of pancreatic elastase. In addition, the coacervation profile of kappa-elastin peptides as a function of temperature is greatly modified in presence of these flavonoids. We conclusively evidenced that PCOs bind to skin elastic fibres when injected intradermally into young rabbits. As a result, these elastic fibres were found more resistant to the hydrolytic action of porcine pancreatic elastase when injected to the same site. These in vivo studies further emphasized the potential effect of these compounds in preventing elastin degradation by elastase(s) as occurred in inflammatory processes.

Biological effects of hyaluronan in connective tissues, eye, skin, venous wall. Role in aging

Hyaluronan, as most macromolecules of the extracellular matrix, are produced by the differentiated mesenchymal cells. These cells produce also enzymes degrading hyaluronan. This results in the presence of several hyaluronan pools of different molecular weights, all capable of interacting with surrounding cells, mediated by hyaluronan binding proteins and receptors. These interactions modulate cell phenotype and produce a variety of effects conditioning the specific functions of tissues. We shall discuss here several examples studied in our laboratory, concerning skin, cornea and the venous wall. Some of these actions might even be harmful, and could play an important role in aging of connective tissues with loss of function. Some of these age-dependent modifications mediated by hyaluronan will be reviewed and commented, especially the upregulation of matrix degrading enzymes as MMP-2 and MMP-9. We shall also mention some of our experiments for finding molecules capable of counteracting the harmful effects mediated by hyaluronan.

Expression of senescence-associated β-galactosidase (SA-β-Gal) by human skin fibroblasts, effect of advanced glycation end-products and fucose or rhamnose-rich polysaccharides

Expression by cells of the SA-beta-Gal was shown to be a reliable indicator of the switch mechanism used by cells to enter the senescent phenotype. We used this method in order to explore the variation of SA-beta-Gal-positive cells with passage number and time spent in culture. Both parameters produced an increase of SA-beta-Gal-positive cells. The addition of a Maillard-product (advanced glycation end-product=AGE) to the fibroblast cultures also increased SA-beta-Gal expression. Fucose- and rhamnose-rich oligo- and polysaccharides (FROPs and RROPs, respectively) provided a significant protection against this AGE-induced increase of SA-beta-Gal-positive cells. It is speculated that these processes might well play an important role in skin aging.

The Maillard reaction. From nutritional problems to preventive medicine.

The questions we were asked by Dr Edeas, president of the French Society of Antioxidants to discuss in this introductory lecture are the following: (a) the metabolism of glycation; (b) what are its consequences at the cellular level, and (c) their effect on health. As a recent and vast literature is available on these subjects, in the following we present a short survey of some basic data on the proposed subjects, insisting on our own experiments on the cytotoxicity of Maillard products and on a new approach to prevent the aggravation and acceleration of age-related diseases, essentially diabetes type II and its consequences on the cardiovascular system.

Collagen biosynthesis in cell culture: Comparison of corneal keratocytes and skin fibroblasts: Effect of rhamnose-rich oligo- and polysaccharides

Corneal keratocytes are often confounded with fibroblasts, although their matrix-synthetic phenotype is quite different as shown by the nature and relative amount of the different collagens and proteoglycans-glycosaminoglycans synthesized. In these experiments, we compared the concentration of collagens excreted in the culture medium by human corneal keratocytes and skin fibroblasts at three consecutives passages. Although the keratocytes excreted less collagen at earlier passages, they approached and reached fibroblasts at later passages. This can be taken as an indication of the progressive loss of a specific keratocyte phenotype with increasing passages (in vitro aging). The effect of rhamnose-rich oligo- and polysaccharides on collagen secretion also confirmed the subtle differences between these two cell-types, as well as the difference between saturation density of both cell types at confluence and the proportion of dead cells floating on top of the culture medium. These differences were also attenuated with passage number without disappearing completely. The significance of these findings will be discussed in the light of previous results in our and other laboratories on matrix-secreting phenotypes and aging.

The effect of procyanidolic oligomers on the composition of normal and hypercholesterolemic rabbit aortas

Rabbits were fed with normal (group 1 and 2) and cholesterol rich diets (group 3 and 4) concomitantly to a daily peroral administration of 50 mg/kg procyanidolic oligomers (PCO) to groups 2 and 4. After 10 weeks, the cholesterol content of the blood serum and the excised aortic intima-media were significantly higher in groups 3 and 4 than in groups 1 and 2. The DNA, hydroxyproline, uronic acid contents were similar in aortic dry weight basis in all four groups. The intima-media samples were extracted successively with 0.15 M NaCl, 0.02 M sodium phosphate pH 7.4 (NaCl extract) and with 4 M guanidinium chloride, 0.05 M sodium acetate pH 5.8 prior (G1 extract) and following (G2 extract) hydrolysis of the collagen with collagenase. The cholesterol contents of G1 extracts were higher in groups 2 and 4 than in groups 1 and 3. The cholesterol content of aortic elastin increased with cholesterol feeding (group 3). With simultaneous administration of cholesterol and PCO the cholesterol content of aortic elastin in group 4 was significantly lower than in group 3. The uronic acid contents increased in G1 extracts and in the collagenase digest with PCO treatment of both normal and hypercholesterolemic rabbits. The ratio of dermatan-sulphate to chondroitin-sulphate decreased with hypercholesterolemia (group 3) and with PCO (group 2 and 4). The parallelism between increased cholesterol and uronic acid contents and modified glycosaminoglycan composition in G1 extract, indicate that the interaction of cholesterol with macromolecules of the aorta can be modulated by PCO. This drug modifies the extractibility of aortic cholesterol and glycosaminoglycans and reduces the association of cholesterol to elastin.

Age- and passage-dependent upregulation of fibroblast elastase-type endopeptidase activity. Role of advanced glycation endproducts, inhibition by fucose- and rhamnose-rich oligosaccharides

It could be shown using the in vitro cell culture aging model, that elastase-type endopeptidase activity is progressively upregulated with successive passages (in vitro aging). Similar results were obtained previously by determining elastase-type activity as a function of age in aorta extracts (human) and skin extracts (mouse). Among the possible mechanisms involved we tested the role of advanced glycation endproducts (AGEs) on this process. AGE-production was shown to increase with age, exemplified by the exponential age-dependent crosslinking of collagen, demonstrated by Fritz Verzár, already in 1963. Several AGEs significantly upregulated elastase-type activity when added to the culture medium of fibroblasts. This effect appears to be mediated by some AGE-receptors as shown previously, and could be inhibited by a 5 kDa rhamnose-rich oligosaccharide (RROP-3) as well as by a fucose-rich oligosaccharide (FROP-3). When present in the culture media, RROP-3 and FROP-3 efficiently inhibited the passage-dependent upregulation of elastase-type activity expressed by human skin fibroblasts. The use of specific inhibitors and zymography suggested that matrix metalloproteinases (MMP)-9 activation and expression are mainly involved. A detailed discussion is proposed for the interpretation of age-dependent modifications of tissues as vascular wall and skin in the light of these and related experiments, highlighting the role of several specific receptors in the mediation of the observed reactions.

Receptors and aging: Structural selectivity of the rhamnose-receptor on fibroblasts as shown by Ca2+-mobilization and gene-expression profiles

Qualitative and quantitative modifications of receptors were shown to play a key role in cell and tissue aging. We recently described the properties of a rhamnose-recognizing receptor on fibroblasts involved in the mediation of age-dependent functions of these cells. Using Ca(2+)-mobilization and DNA-microarrays we could show in the presence of rhamnose-rich oligo- and polysaccharides (RROPs) Ca(2+)-mobilization and changes in gene regulation. Here, we compared the effects of several RROPs, differing in their carbohydrate sequence and molecular weights, in normal human dermal fibroblasts (NHDFs). It appeared that different structural features were required for maximal effects on Ca(2+)-mobilization and gene-expression profiles. Maximal effect on Ca(2+) influx and intracellular free calcium regulation was exhibited by RROP-1, a 50 kDa average molecular weight polysaccharide, and RROP-3, a 5 kDa average molecular weight oligosaccharide with a different carbohydrate sequence. Maximal effect on gene-expression profiles was obtained with RROP-3. These results suggest the possibility of several different transmission pathways from the rhamnose-receptor to intracellular targets, differentially affecting these two intracellular functions, with potential consequences on aging. Although of only relative specificity, this receptor site exhibits a high affinity for rhamnose, absent from vertebrate glycoconjugates. The rhamnose-receptor might well represent an evolutionary conserved conformation of a prokaryote lectin.

Original articleCollagen biosynthesis in cell culture: Comparison of corneal keratocytes and skin fibroblasts: Effect of rhamnose-rich oligo- and polysaccharidesBiosynthèse du collagène en culture cellulaire : comparaison entre kératocytes cornéens et fibroblastes cutanés: Effet des oligo- et polysaccharides riches en rhamnose

Corneal keratocytes are often confounded with fibroblasts, although their matrix-synthetic phenotype is quite different as shown by the nature and relative amount of the different collagens and proteoglycans-glycosaminoglycans synthesized. In these experiments, we compared the concentration of collagens excreted in the culture medium by human corneal keratocytes and skin fibroblasts at three consecutives passages. Although the keratocytes excreted less collagen at earlier passages, they approached and reached fibroblasts at later passages. This can be taken as an indication of the progressive loss of a specific keratocyte phenotype with increasing passages (in vitro aging). The effect of rhamnose-rich oligo- and polysaccharides on collagen secretion also confirmed the subtle differences between these two cell-types, as well as the difference between saturation density of both cell types at confluence and the proportion of dead cells floating on top of the culture medium. These differences were also attenuated with passage number without disappearing completely. The significance of these findings will be discussed in the light of previous results in our and other laboratories on matrix-secreting phenotypes and aging.

The survival–reproduction association becomes stronger when conditions are good

Positive covariations between survival and reproductive performance (S–R covariation) are generally interpreted in the context of fixed or dynamic demographic heterogeneity (i.e. persistent differences between individuals, or dynamic variation in resource acquisition), but the processes underlying covariations are still unknown. We used multi-event modelling to investigate how environmental and individual features influence S–R covariation patterns in a long-lived seabird, the Monteiros storm petrel (Oceanodroma monteiroi). Our analysis reveals that a strong positive association between individual breeding success and subsequent survival occurs only when conditions are favourable to reproduction (in favourable years, in high-quality nests and in nest-faithful breeders). This finding reflects differences in the main causes of breeding failure and mortality under favourable and unfavourable conditions, which in turn lead to distinct patterns of S–R covariation. We suggest, in particular, that resource-related sources of demographic heterogeneity do not generate a strong S–R covariation, in contrast with hidden and unpredictable sources of variation.