We present a short review of human skin aging with a complete list of our previous publications as well as on fibroblasts and their aging process.Age-dependent skin loss was measured on biopsy samples from a relatively large number of Caucasian Europeans, males and females, showing a loss with age of about 7% of the "original" (0 age) skin thickness every 10 years.The age-dependent loss of two major constituents of the skin extracellular matrix, collagen and elastin and their age-dependent modifications are described in some detail.We insisted on the age-dependent loss of hyaluronan, the most important reason of loss of hydration and wrinkling.

Oriented collagen biosynthesis is one of the major mechanisms involved in tissue and organ formation during development. Corneal biogenesis is one example. Defects in this process lead to anomalies in tissue structure and function. The transparency of cornea and its achievement are a good example as well as its pathological modifications. Keratoconus is one example of this type of pathologies, involving also inappropriate cross-linking of collagen fibers. Among the tentatives to correct this anomaly, the riboflavin-potentiated UV-cross-linking (CXL) of keratoconus corneas appears clinically satisfactory, although none of the experiments and clinical results published prove effective cross-linking. The published results are reviewed in this article.

The three major symptoms of the irido-corneo-endothelial syndrome are the alterations of the corneal endothelium and of the iris with a loss of the regulation of the cell cycle, and the progressive obstruction of the irido-corneal angle. This rare pathology attacks mainly young adult women. Most of the symptoms and complications originate from the excessive proliferation of the corneal endothelial cells accompanied by the evolution of their phenotype towards that of the epithelial cells. In normal conditions the corneal endothelial cells do not divide, they are blocked in the G1 stage of the cell cycle, mainly because of the action of the inhibitors of cyclin-dependent kinases. Still these cells retain a good capacity for proliferation, which can be induced by the down-regulation of the expression of the inhibitors of the cyclin-dependent kinases. This proliferative capacity declines with age and is also different according to the localization of the cells: it is more intense with those originating from the central area then in those from the peripheral area of the cornea. The age-related decline of the proliferative capacity is not due to the shortening of the telomers, but to the stress-induced accelerated senescence of the cells. (C) 2012 Elsevier Masson SAS. All rights reserved.

The science of connective tissues has (at least) a double origin. Collagen, their major constituent was first studied in conjunction with the leather industry. Acid mucopolysaccharides (now glycosaminoglycans) were characterised by (bio)-chemists interested in glycoconjugates. They joined mainly hospital-based rheumatology departments. Later started the study of elastin with the discovery of elastases and of connective tissue-born (structural) glycoproteins. Besides rhumatologists and leather-chemists mainly pathologists became involved in this type of research, followed closely by ophthalmology research. The first important meetings of these diverse specialists were organised under the auspices of NATO, first in Saint-Andrew's in GB in 1964 [1] and a few years later (1969) in Santa Margareta, Italy [2]. With the discovery of fibronectin, a "structural glycoprotein", started the study of cell-matrix interactions, reinforced by the identification of cell-receptors mediating them and the "cross-talk" between cells and matrix constituents. The first initiative to organise societies for this rapidly growing discipline was that of Ward Pigman in New York in 1961, restricted however to glycol-conjugates. Next year, in 1962 was founded the first European Connective Tissue Society in Paris: the "Club francais du tissu conjonctif', which played a crucial role in the establishment of schools, laboratories, national and international meetings in the major cities of France: Paris, Lyon, Reims, Caen,Toulouse. A second European society was born in Great Britain, and at a joint meeting with the French society at the Paris Pasteur Institute, was founded in 1967 by these societies the Federation of European Connective Tissue Societies (FECTS). Their meetings, organised every second year, drained a wide attendance from all over the world. An increasing number of young scientists joined since then this branch of biomedical discipline with several international journals devoted to connective tissue research, to matrix biology. The increasing number and quality of the young generation of scientists engaged in research related to the extracellular matrix or better Biomatrix and cell-matrix interactions is a further guarantee for the continued interest in this crucial field of science at the interface of basic and medically oriented research. (C) 2011 Elsevier Masson SAS. All rights reserved.

Aging of the extracellular matrix (ECM) became an important topic over the last decades as a consequence of the rapid increase of human life expectancy. Another motivation is the fact that most age-related diseases concern directly or indirectly ECM-rich tissues (connective tissues) such as bone, cartilage, vessel walls and skin. A further complicating factor is the demonstration over the last decades of the 20th century of large number of macromolecular constituents of ECM. The age-dependent modifications of some of them were not yet studied systematically. The aging of ECM can be divided in two major chapters: the age-dependent modifications of their biosynthesis, and the age-dependent post-synthetic modifications of ECM-components. We shall present in this review both these aspects of the age-dependent modifications of ECM. And finally, we shall discuss the relevance of the age-dependent modifications of ECM in respect with the development of age-associated pathologies.

Connective tissues play an important role in the physiological functions of the organism. The integrity of the macromolecular components of these tissues, also called extracellular matrix, is necessary for their functional efficiency. A number of proteinases present in the organism, and the activity of which increases with age and with several pathologies, specifically degrade the components of the extracellular matrix. For a long time, tentatives for the protection of the matrix-components against degradation were made with low molecular weight inhibitors, not very efficient in vivo and not devoid of inconveniencies. We initiated a different approach for the preservation of the macromolecules of the extracellular matrix against proteolytic degradation with substances which exert an intense antiproteolytic activity not only in vitro, but also in vivo. The particularity of these substances is the fact that they do not act on the enzymes, but combine with the macromolecules. This is the type of combination of substances with the macromolecules of the matrix that prevents their degradation by the proteinases. Because of this affinity of such antiproteolytic agents not for the enzymes but for the substrates, we called them "substrate protectors" (Robert et al., 1979) [1]. The aim of the present review is to summarise the essential of our experiments which led to the description of substrate protectors. (C) 2011 Elsevier Masson SAS. All rights reserved.

We present a review of our early work on the Maillard reaction, at the interface of food chemistry and tissue biochemistry, as well as the reinterpretation of our early findings in the light of recent advances in the chemistry of the involved reactions. These concern specifically the role of lower aldehydes, produced during the glycolytic pathways and especially acetaldehyde. We also review some of our recent findings on the cytotoxic and genotoxic aspect of these "illicit" organic reactions, taking place in tissues (and also in food products) besides the genetically "programmed" metabolic pathways. Some recent results in organic-pharmaceutical chemistry confirm the potential importance of the reviewed reactions both in food chemistry and in tissues as well as the pathological importance of reactions taking place in tissues.

The initiation and evolution of the receptor concept and its application in pharmacology can be traced back to Paul Ehrlich's original experiments. Since several decades the receptor concept is in the foreground of cell biology and pharmacology. We present here a short reminder of Ehrlich's concepts on receptor action, its evolution and modifications as a result of increasing life expectancy of human societies. Results obtained by several teams on the age-dependent modifications of receptor function are reviewed with special emphasis on the age-dependent loss of receptors and of uncoupling of receptors from their intracellular transmission pathway. As a special example we summarize our results on the elastin receptor and its age-dependent modifications. These modifications result in the loss of the physiologically helpful functions mediated by this receptor, such as vasodilation by coupling with the inducible nitric oxide synthase (iNOS)-inhibition of cellular cholesterol synthesis and modulation of free radical production by inhibition of the guanine nucleotide binding protein (Gi protein)-mediated transmission pathway. Only the harmful effects such as free radical release and up-regulation of elastase production remain in "old" cells. The age-dependent modifications of receptor function play an important role in the increasing frequency and severity of age-related diseases such as athero-arteriosclerosis and emphysema as well as the loss of hormone- and drug actions. These processes and their inhibition or correction represent a new challenge for cellular pharmacology.

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.

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.

Gerontological experimentation is and was always strongly influenced by "theories". The early decades of molecular genetics inspired deterministic thinking, based on the "Central Dogma" (DNA -> RNA -> Proteins). With the progress of detailed knowledge of gene-function a much more complicated picture emerged. Regulation of gene-expression turned out to be a highly complicated process. Experimental gerontology produced over the last decades several "paradigms" incompatible with simple genetic determinism. The increasing number of such detailed experimental "facts" revealed the importance of epigenetic factors and of posttranslational modifications in the age-dependent decline of physiological functions. We shall present in this review a short but critical analysis of genetic and epigenetic processes applied to the interpretation of the more and more precisely elucidated experimental paradigms of aging followed by some of the most relevant aging-mechanisms at the post-translational level, the posttranslational modifications of proteins such as the Maillard reaction, the proteolytic production of harmful peptides and the molecular mechanisms of the aging of elastin with the role of the age-dependent uncoupling of the elastin receptor, as well as the loss of several other receptors. We insist also on the well documented influence of posttranslational modifications on gene expression and on the role of non-coding RNA-s. Altogether, these data replace the previous simplistic concepts on gene action as related to aging by a much more complicated picture, where epigenetic and posttranslational processes together with environmentally influenced genetic pathways play key-roles in aging and strongly influence gene expression.

La découverte du vieillissement cellulaire par L. Hayflick et son analyse au niveau cytogénétique par Macieira Coelho, auteurs des deux premières revues de ce numéro spécial consacré à ce sujet, a ouvert une nouvelle voie dans l’étude du vieillissement et ont créé un modèle in vitro qui a permis d’aborder au laboratoire des problèmes que pose ce phénomène complexe qu’est la sénescence cellulaire et son extrapolation de la cellule à l’organisme pour élucider son rôle dans le vieillissement de l’individu et dans l’émergence des maladies qui l’accompagnent. Cette complexité fait l’objet de la revue de B. Carnes et de ses collègues (1992) [34] et constitue aussi le fond de nos réflexions qui suivent les trois précédentes. Nous aborderons en particulier les mécanismes et les conséquences fonctionnelles du vieillissement cellulaire et de son rôle dans le vieillissement des tissus et des organes. Cela nous amène à analyser au niveau cellulaire et moléculaire les maladies qui accompagnent le vieillissement, surtout les maladies cardiovasculaires et les tumeurs malignes. Ces deux classes de pathologies sont en effet essentiellement responsables du décès des personnes âgées. Dans notre discussion et conclusions, nous confronterons l’essentiel de nos connaissances acquises au niveau fondamental avec les données classiques pour préciser le rôle du vieillissement cellulaire dans les pathologies liées à l’âge.

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.

Expression by cells of the SA-β-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-β-Gal-positive cells with passage number and time spent in culture. Both parameters produced an increase of SA-β-Gal-positive cells. The addition of a Maillard-product (advanced glycation end-product = AGE) to the fibroblast cultures also increased SA-β-Gal expression. Fucose- and rhamnose-rich oligo- and polysaccharides (FROPs and RROPs, respectively) provided a significant protection against this AGE-induced increase of SA-β-Gal-positive cells. It is speculated that these processes might well play an important role in skin 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.

La peau, à part ses fonctions physiologiques importantes, joue aussi un rôle social de premier plan d’où l’importance accordée à l’étude de son vieillissement. C’est aussi le tissu le plus volumineux de l’organisme, son épaisseur varie cependant selon sa localisation sur le corps. Nous avons mesuré la perte de tissu cutané avec l’âge sur des biopsies au niveau de la face interne du bras de volontaires et trouvé une perte moyenne de 7 % de l’épaisseur cutanée par décade, avec cependant des variations individuelles importantes. Cette perte concerne l’épiderme et le derme et résulte à la fois de la perte de cellules et de la matrice extracellulaire. La perte de la matrice cutanée est due à la perte de cellules qui la fabriquent, à la baisse de la capacité synthétique des cellules avec l’âge et surtout à l’augmentation de l’activité élastasique cutanée. Dans la peau jeune, les fibres élastiques du derme papillaire forment des faisceaux en forme de candélabre qui s’ancrent dans la lame basale dermoépidermique. Avec l’âge, ce réseau vertical est lysé et progressivement remplacé par un réseau horizontal de fibres plus épaisses et dépourvues d’élasticité. Nous avons mis au point une méthode morphométrique pour quantifier les fibres élastiques cutanées avec une coloration spécifique qui laisse le fond incolore. Grâce à cette méthode, nous avons pu montrer que la densité du réseau élastique cutané augmente avec l’âge. En revanche, l’élasticité cutanée mesurée par indentométrie baisse linéairement ave l’âge. Ces deux paramètres montrent cependant une forte variabilité individuelle justifiant l’examen des personnes à la recherche de mécanismes sous-jacents pouvant accélérer la perte d’élasticité cutanée avec l’âge.

Skin is the most voluminous organ of the body. It assumes several important physiological functions and represents also a "social interface" between an individual and other members of society. This is the main reason its age-dependent modifications are in the forefront of dermatological research and of the "anti-aging" cosmetic industry. Here we concentrate on some aspects only of skin aging, as far as the cellular and extracellular matrix components of skin are concerned. Most well studied mechanisms of skin aging can be situated at the postgenetic level, both epigenetic and post-translational mechanisms being involved. Some of these mechanisms will be reviewed as well as the capacity of fucose- and rhamnose-rich oligo- and polysaccharides (FROP and RROP) to counteract several of the mechanisms involved in skin aging.

The postponement of the most frequent age-related diseases stimulated speculations of the possibility of "dying of old age". The selective decline of individual physiological functions-aging in spare-parts-indicates however the potential limitation of the life-span by the rapid decline of some of the vital parameters. We explored a possibility of such a limitation of maximal life-span by the age-related alteration of elastin, consisting in Ca-accumulation, lipid deposition and elastolytic degradation. The quantitative evaluation of these processes suggests an approximative upper limit for the elastic properties of the cardio-respiratory system of about 100-120 years, at least, as far as elastin is involved. This process, age-related alterations of elastic fibers, is however not the only one limiting the functional value of the cardiovascular system. Crosslinking of collagen fibers by advanced glycation end-products certainly contributes also to the age-dependent rigidification of the cardiovascular system. Therefore the answer to the initial question, can age-dependent alterations of a single matrix macromolecule be limiting such vital functions as the cardio-respiratory system-is a cautious yes, with however the caveat that other, independent mechanisms, such as the Maillard reaction, can also interfere with and limit further the functional value of such vital physiological functions.

Collagen synthesis and accumulation were studied by serial cultures of human dermal fibroblasts. A freshly seeded strain of cells was compared to cryopreserved cells from the same donor. Up to 25 passages there was no clear sign of reaching phase III, the decline of the culture. This was ascertained by the count of the dead cells floating on top of the culture fluid, the time to reach saturation density and the number of cells at confluence, counted after trypsinization. Although collagen synthesis oscillated to some extent, the average value of collagen deposited by the cells did not show any clear sign of decrease of collagen synthesis, on the contrary, there was an increase between the 15th and 25th passages. A review of the literature revealed that recent experiments on the age-dependent variation of maximal passage number did not confirm previous results showing a progressive decline, which was much delayed in recent studies as compared to previous records. The same appears to happen with collagen synthesis, found by earlier investigators to decline with increasing passage numbers. This is not confirmed, at least up to passage 25 in the present experiments. As skin tissue is progressively lost with age, our results are more in favor of increasing matrix degradation with age as an important factor of the age-dependent loss of skin tissue, more than decrease of matrix synthesis.