Biomedical subjects

L Robert

Publications and source records attributed to L Robert.

At least 37 records · Page 2Linked to original sources

PubMed2003

Aging, from basic research to pathological applications.

This introductory chapter tentatively draws a parallel between some of the highlights of basic aging research and their incidence for the understanding of age-associated pathologies. Some of the most important mechanisms described for cell- and tissue-aging, such as limited cell-proliferation, "parasitic" reactions, such as non-enzymatic glycation, ROS-production, uncontrolled proteolysis involving matricryptic sites, receptor loss and uncoupling appear to be the most promising links between those two approaches to fundamental and clinical gerontology. Some of the "parasitic", epigenetic, post-translational reactions lead to vicious circles which over the years might well produce cell- and tissue-damage as seen in most age-associated diseases. There is little doubt that the better understanding of these relationships will also prove helpful for pharmacological research as related to the age-associated pathologies.

Aged↗

PubMed2003

Effect of a fucose-rich polysaccharide preparation on the age-dependent evolution of the skin surface micro-relief.

It was demonstrated previously that the evaluation of the microdepressionary skin surface relief by semi-automated computerised morphometry enables the determination of several of its geometrical parameters, such as, the average number of polygons in a given microscopic field, and/or the average surface of the polygons. Using this procedure, it could be shown in a study on about 100 persons, males and females, aged from 6 months to 89 years that these parameters undergo age-related alterations. These alterations are faster at younger ages, slow down at middle ages and accelerate after 50-60 years again. Taken at a sun-protected site, the interior face of the forearm, close to the elbow, this test reflects the effect of chronological ageing of the skin surface micro-relief. In the present work we tested the effect of a preparation containing a fucose-rich polysaccharide as active principle on the microdepressionary skin surface micro-relief evaluated by a morphometric method, comparing results before treatment and after 4 weeks of treatment. We could demonstrate on 20 female volunteers, aged from 39 to 71 years, that after 4 weeks of treatment with the abovementioned preparation there was a significant improvement of the skin surface relief, as shown by the displacement of geometrical characteristics of 17 treated skins out of 20, towards a "younger" pattern, as for instance the increase of the average number of polygons in a given microscopic field. This improvement was less than 15% of the untreated value for two persons, and more than 15% for 15 women out of 20. The average improvement was 37.16%, and for the 15 persons showing more than 15% improvement, it was nearly 50%, corresponding to a decrease of apparent age by 10-15 years. These results suggest that a treatment with the fucose-rich polysaccharide preparation can slow down human skin ageing and even can reverse age-dependent skin alterations.

Adult↗

PubMed2003

Effect of sulfated GAGs on the expression and activation of MMP-2 and MMP-9 in corneal and dermal explant cultures.

It has been shown previously that hyaluronan (HA) added to fibroblast and keratocyte cell cultures or corneal explant cultures produces an up-regulation of MMP-2 and MMP-9 expression and activation. Here, we examine the effect of sulfated GAG-s, chondroitin 4 and 6 sulfate (CS4, CS6), dermatan sulfate (DS), keratan sulfate (KS) and heparan sulfate (HS) on MMP-2 and 9 expression and activation under the same culture conditions. It appears that CS4 has only minor effects, KS inhibits MMP-2 activation and CS6, DS and HS increase MMP-2 activation in corneal explant cultures. For skin explant cultures, DS, KS and HS strongly increase MMP-9 activation, whereas KS inhibits and DS increases MMP-2 activation. All these effects can be strongly inhibited by the addition of an antibody to CD44, except CS6 and DS. Activation by these two GAGs was only slightly affected, supporting the contention that the effects of HA, CS4, KS and HS are mediated by one of the isoforms of this CD44 receptor. The physio-pathological significance of these results is discussed for cornea and skin ageing, because of the divergent evolution with in vitro ageing of the relative proportions of GAGs synthesised by these two cell types.

Cells, Cultured↗

PubMed2003

Hohenheimer Consensus Talk. Oxidative and premature skin ageing.

To elucidate the scientific state of the art with respect to the role of nutrition in skin ageing, nine experts from different disciplines discussed the role of micronutrients on 'oxidative and premature skin ageing'. In this 25th Hohenheim Consensus Meeting, 13 questions were discussed and, based on published valid data, answered by mutual agreement. The consensus answers achieved during the meeting are justified by a scientific background text. The importance of in vitro and in vivo models regarding oxidative and premature skin ageing was critically evaluated. There was a special focus on prevention and intervention of skin ageing with nutrition. Finally, the paper summarizes the scientific background from different areas related to oxidative and premature skin ageing.

Animals↗

PubMed2002

Modulation of cell-phenotype during in vitro aging. Glycosaminoglycan biosynthesis by skin fibroblasts and corneal keratocytes.

The aim of this study was to compare keratocyte and fibroblast phenotypes during in vitro aging by comparing their biosynthesis of glycosaminoglycans using explant and cell cultures. Human skin and corneal explant cultures were realised with Dulbecco Modified Eagle's medium containing 3H glucosamine. Sequential cell cultures were studied at different passages for GAGs biosynthesis by 3H glucosamine incorporation followed by selective degradation with specific hydrolases. Radioactivity was determined and each GAG fraction evaluated. KS and DS are the major components synthesised by corneal explant culture. During in vitro aging, keratocytes synthesised 41% less KS between passages 4-9 with a decrease by 26% of the proportion of DS observed in the same conditions. In skin explant cultures, as expected the major components are CS and hyaluronan (HA). In the first cell passage studied compared with skin organ cultures we could notice a strong decrease of the proportions of DS and KS compensated by an increase of the proportion of HA. During the successive passages of fibroblasts, the proportions of DS and HS decreased (-30 and -62%, respectively) and those of KS increased (+90%). These results indicate that there remain measurable differences between keratocyte and fibroblast phenotypes as far as GAG-synthesis is concerned all though the successive passages, starting from explant cultures and up to the limits of in vitro cell passages.

Cells, Cultured↗

PubMed2002

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.

Child, Preschool↗

PubMed2002

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.

Antioxidants↗

PubMed2002

[Protection of cornea against proteolytic damage. Experimental study of procyanidolic oligomers (PCO) on bovine cornea].

AIM OF THE STUDY: The purpose of this work was the development of an antiprotease treatment for the cornea and later for other ocular tissues. Indeed, many proteases participate in various ocular pathologies and no really effective treatments are currently available. METHODS: We worked on bovine corneas in organ cultures, incubated in the presence and absence of bacterial collagenase. We used procyanidolic oligomers from grape seeds (PCO) as the antiproteolytic agent, added to the cultures in two different concentrations. RESULTS: The corneas incubated in the presence of collagenase were quickly attacked and their degradation was practically complete after 24 hours. With the low concentration used (0.066 mg/ml), proteolysis was only slightly inhibited. With the higher concentration of PCO (1 mg/ml), the collagenolysis was completely prevented and the corneas were completely preserved. CONCLUSION: These results show that the protective effect of the PCO against proteolytic attack, demonstrated previously on other tissues, proved to be effective also for the cornea in vitro. An adequate method of administration in clinical practice must now be developed in order to obtain an effective clinical antiproteolytic treatment.

Animals↗

PubMed2001

Chemotactic penetration of keratocytes in ePTFE polymer in vitro.

Expanded polytetrafluoroethylene (ePTFE) is used as a support for artificial corneas. Implanted in corneas, most of the time this polymer is colonized by corneal host cells. The absence of colonization often coincides with extrusion of the polymer. Therefore, we decided to introduce keratocytes into ePTFE in vitro before implantation. Because keratocytes do not spontaneously enter ePTFE, we used several chemoattractants, separately and in a mixture, to stimulate the penetration of cultured keratocytes into the polymer. The influence of the passage number on cell penetration was also studied. No significant differences were observed up to the seventh passage, although seventh-passage cells penetrated somewhat more slowly than younger cells. Satisfactory results were obtained with four of the tested chemotactic factors: IL-6, type alpha transforming growth factor (TGF-alpha), platelet derived growth factor isoform BB (PDGF-BB), and fibroblast growth factor-2 (FGF-2). Under our experimental conditions, two to more than six million keratocytes entered the polymer discs with a volume of 706.5 mm(3) in the presence of these four chemoattractants. TGF-alpha was the most efficient and was selected for further in vitro and in vivo studies.

Biocompatible Materials↗

PubMed2001

Effect of hyaluronan on MMP expression and activation.

Matrix metalloproteases (MMPs) play a crucial role in tissue remodelling in a variety of physiological and pathological processes. Hyaluronan is also involved in the same processes. Several cytokines and growth factors are involved in the regulation of the biosynthesis of hyaluronan and also of MMPs. The activity of MMPs has been shown to be regulated at the level of transcription and activation of the zymogen form. In order to explore the possible relationship between matrix components and especially hyaluronan, we studied the effect of hyaluronan on MMP expression (biosynthesis and activation) in the culture of human skin fibroblasts and corneal keratocytes (explant cultures and cell cultures). These cells were shown to exhibit distinct phenotypes as far as matrix biosynthesis is concerned. Using a synthetic substrate N-Suc(ala)3pNA, we measured elastase-type endopeptidase activity produced by fibroblasts and keratocytes and characterized the MMPs by zymography. Hyaluronan added to fibroblast cultures stimulated the membrane-bound elastase-type endopeptidase activity in a dose dependent fashion. Similar results were obtained with keratocyte cultures. In the presence of 1 mg/ml hyaluronan there was an increase in MMP expression and also an activation of latent MMPs both by fibroblasts and keratocytes.

Cells, Cultured↗

PubMed2001

Matrix biology: past, present and future.

Matrix biology (the biology of extracellular matrix) is a relatively recent branch of biomedical sciences and comprises a number of subspecialties. From molecular-cell biology, biochemistry, genetics and clinical science of diseases localised at or affecting the matrix rich tissues (connective tissues) as bone, cartilage, vessel wall, skin, eye and some others. The rapid expansion of all these branches of matrix biology is the combined result of the availability of advanced methods of cell and molecular biology and the increasing awareness of the importance of this field for a number of basic and applied sciences. This introduction is a review for the special issue of Pathologie Biologie devoted to 'Matrix Biology' and brushes an impressionistic landscape of the major advances accomplished over the finishing century and tries to predict some of the most important advances to be expected during the coming century.

Animals↗

PubMed2001

Age-related alterations in the signal transduction pathways of the elastin-laminin receptor.

With aging we assist to alterations in the vascular structure and function. One important factor in these vascular wall changes is the degradation of the elastin fibre major protein: elastin. Elastin peptides derived from the degradation are present in human sera. Elastin peptides induce on fibroblasts, phagocytic cells, lymphocytes, smooth muscle cells and endothelial cells, a variety of biological effects mediated by the elastin-laminin receptor which has been demonstrated to be present on the membrane of these cells. The transduction pathway of the ELR receptor involves the activation of phospholipase C (PLC) by a pertussis toxin sensitive G-protein. PLC induces the production of inositol trisphosphate (IP3) leading to the increase of the intracellular free calcium on one hand, and of diacylglycerol (DAG) which stimulates the translocation to the membrane of PKC leading to the phosphorylation of members of the MAPK family, such as p42/p44 MAPK. A progressive age dependent uncoupling of the elastin-laminin receptor occurs impairing its transduction pathway and which results in alteration of the calcium signaling and loss in calcium homeostasis of the cells. These alterations in the signal transduction of the elastin-laminin receptor result in modified activities of parenchymal and phagocytic cells with aging, such as free radical production and elastase release. Thus, these age-related alterations in the elastin-laminin receptor signal transduction may be involved in the atherogenesis.

Aging↗

PubMed2001

Corneal collagens.

Cornea is a highly differentiated tissue rich in extracellular matrix (ECM) specifically distributed in space in order to insure its dual role--transparency and protection of inner eye-tissues. Corneal ECM is especially rich in collagens. Since the characterisation of a number of distinct collagen types it appeared that most of them are present in the cornea. Their synthesis follows a specific program of sequential expression of the different collagen types to be synthesised during the development and maturation of the cornea. The precise regulation of the diameter and orientation of fibers, and of the interfibrillar spaces is partially at least attributed to interactions between glycosaminoglycans and collagens. The 'program' of vectorial collagen synthesis and GAG-collagen interactions changes also with age and in several pathological conditions as corneal dystrophies and wound healing. The Maillard reaction, especially in diabetes, is one of these important factors involved in age-dependent modifications of corneal structure and function. Far from being inert, corneal collagens were shown to have relatively short half-lives. The biosynthesis of corneal collagens was studied also during wound healing. The refibrillation of wounded corneas does not follow the original 'program' of ECM-synthesis as shown by the comparative study of wound healing using biochemical and morphometric methods. This review recapitulates briefly previous and recent studies on corneal collagens in order to present to clinicians and scientists an overview of the state of the art of this important field at the intersection of eye research and matrix biology.

Aging↗

PubMed2001

Effect of an alpha-blocker (Nicergoline) and of a beta-blocker (Acebutolol) on the in vitro biosynthesis of vascular extracellular matrix.

The effect of an alpha-blocking agent and of a beta-blocking agent on the biosynthesis of extracellular matrix macromolecules of the arterial wall was investigated. Rabbit aorta explants were cultured up to 48 hours with radioactive proline, lysine or glucosamine. In presence of these drugs, at concentration shown to be effective for the inhibition of platelet-endothelial cell interactions (10(-7) M), the incorporation of 14C proline in total macromolecular proline was higher than in macromolecular hydroxyproline suggesting a relatively higher rate of biosynthesis of non-collagenous proteins as compared to collagens. The alpha-blocking increased the incorporation of 14C proline in collagenous and non-collagenous proteins after 18 hours of incubation. beta-blocking also increased the incorporation of proline in macromolecular proline and hydroxyproline as compared to control cultures. Both increased the incorporation of 3H glucosamine in newly synthesised glycosaminoglycans. beta-blocking increased mainly the neosynthesis of heparan sulphate, alpha-blocking that of hyaluronan. The incorporation of 14C-lysine in crosslinked, insoluble elastin was not modified. These experiments confirm that alpha and beta-blocking agents can influence not only the tonus of aortic smooth muscle cells but also the relative rates of biosynthesis of extracellular matrix macromolecules. This effect should be taken in consideration for the evaluation of the long range effect of alpha and beta-blocking drugs on the vascular wall.

Acebutolol↗

PubMed2001

Effect of procyanidolic oligomers on the permeability of the blood-brain barrier.

Blood-brain barrier (BBB) is the site of regulatory mechanisms which control the exchange of substances between the brain and the blood through the wall of 'true' brain capillaries with tight junctions between endothelial cells. In some pathological situations the permeability of the BBB is increased because of a partial proteolytic degradation of some constituents of the capillary basement lamina. In such cases it is important to restore normal permeability. The effect of procyanidolic oligomers (PCO) on the BBB was investigated in vivo with quantitative morphologic procedures. We also investigated the action of this drug on collagen and basement lamina constituents (Matrigel) in vitro. Collagenase injected in lateral brain ventricles was shown to increase BBB permeability. Per os administration of PCO to rats greatly increased the resistance of brain capillaries to bacterial collagenase, as shown by the inhibition of the diffusion of fluorescein-isothiocyanate-marked dextran particles from the blood-stream into the brain tissues. Calf skin collagen pretreated in vitro with PCO became more resistant to the hydrolytic action of collagenase. Similar, even more intense protective effect was seen when basal lamina constituents containing type IV collagen was incubated with PCO before exposure to pronase. These in vitro effects may partly explain the in vivo protective effect of PCO against the alteration of brain capillaries by i.v. injected bacterial collagenase.

Animals↗

PubMed2001

[Elastin and elastases. Past, present, and future].

Extracellular matrix (ECM) is composed of a large number of macromolecules belonging to one of the four classes comprising ECM, collagens and elastin, the fibrous elements, proteoglycans and glycosaminoglycans and structural glycoproteins. Three of these 4 classes emerged during the Cambrian explosion, at the level of the first invertebrates, the sponges. Elastin appeared only in vertebrates. This protein is also the first to undergo post-synthetic modifications responsible for the progressive loss of its essential rheological properties involved in its physiological functions in the circulatory and respiratory system of vertebrates. The essential mechanisms of its emergence and decay are sufficiently understood in order to integrate them in the processes of development and age-dependent decay of organisms.

Aging↗

PubMed2001

[The elastin-laminin receptor].

Elastin is a major component of the extracellular matrix. Elastin peptides derived from its degradation are present in human sera. Elastin peptides induce on fibroblasts, phagocytic cells, lymphocytes, smooth muscle cells and endothelial cells, a variety of biological effects mediated by the elastin-laminin receptor which has been demonstrated to be present on the membrane of these cells. The transduction pathway of the ELR receptor involves the activation of phospholipase C (PLC) by a pertussis toxin sensitive G-protein. PLC induces the production of inositol trisphosphate (IP3) leading to the increase of the intracellular free calcium on one hand, and of diacylglycerol (DAG) which stimulates the translocation to the membrane of PKC leading to the phosphorylation of members of the MAPK family, such as p42/p44 MAPK. Considering the multiple biological effects of ELR the elucidation of the complexity of the signaling pathways will help to better modulate it, mainly in pathological situations such as atherosclerosis.

Animals↗

PubMed2000

Plasma fibronectin in French centenarians.

Plasma fibronectin was shown to increase with age, the difference between individuals (the SD of the mean) also increases with age. Fibronectin is highly sensitive to proteolytic degradation and several of the degradation products were shown to have noxious effects as proper proteolytic activity, activation of IL-1 and collagenase expression and also activation of fibronectin biosynthesis. It was therefore interesting to compare the plasma fibronectin values of centenarians in relatively good health with an elderly population in a geriatric hospital, somewhat younger (70-96 years) but with a variety of pathologies. A third population of men and women between 59 and 70 in good health (the EVA-epidemiological study) was also used for comparison. Plasma fibronectin was determined by a specific and highly sensitive Elisa assay. Fibronectin fragments were characterized by immunoblot. It could be shown that plasma fibronectin in centenarians had a lower distribution with lower average values than the geriatric population. Fibronectin fragments could be demonstrated in the plasma of a selection of geriatric patents but not in the plasma of centenarians. These results suggest a more moderate increase of plasma fibronectin in the relatively healthy centenarians as compared to a younger but pathological population. They also show that the plasma fibronectin of the investigated centenarians was better protected from proteolytic degradation than in the geriatric population. The above results also confirm the contention that epigenetic mechanisms such as an age-dependent increase of fibronectin synthesis and degradation and the potential noxious effects of degradation products may well play an important role in the age-dependent decline of physiological functions.

Journal Article↗