Gaston Godeau

Marine Polysaccharides: A Source of Bioactive Molecules for Cell Therapy and Tissue Engineering

The therapeutic potential of natural bioactive compounds such as polysaccharides, especially glycosaminoglycans, is now well documented, and this activity combined with natural biodiversity will allow the development of a new generation of therapeutics. Advances in our understanding of the biosynthesis, structure and function of complex glycans from mammalian origin have shown the crucial role of this class of molecules to modulate disease processes and the importance of a deeper knowledge of structure-activity relationships. Marine environment offers a tremendous biodiversity and original polysaccharides have been discovered presenting a great chemical diversity that is largely species specific. The study of the biological properties of the polysaccharides from marine eukaryotes and marine prokaryotes revealed that the polysaccharides from the marine environment could provide a valid alternative to traditional polysaccharides such as glycosaminoglycans. Marine polysaccharides present a real potential for natural product drug discovery and for the delivery of new marine derived products for therapeutic applications.

Picrosirius Red Staining A Useful Tool to Appraise Collagen Networks in Normal and Pathological Tissues

Specific staining of the extracellular matrix components is especially helpful in studying tissue remodeling, particularly in the case of connective tissue pathologies. As developed by Junqueira and colleagues in 1979, specific staining by Picrosirius red is one of the most important stains to study collagen networks in different tissues. Under polarized light, collagen bundles appear green, red or yellow, and are easily differentiated from the black background, thus allowing for quantitative morphometric analysis. As Junqueira and colleagues point out, many studies use color staining to differentiate collagen bundles and to specify collagen types, yet other studies report that polarized colors only reflect fiber thickness and packing. Using a simple histological example, our study illustrates the inability of Picrosirius red staining to differentiate collagen types, since the absorbed amount of polarized light by this dye strictly depends on the orientation of the collagen bundles.

Micrometer scale Ex Vivo multiphoton imaging of unstained arterial wall structure

We characterize the application of multiphoton microscopy to the observation of the extracellular matrix of fresh unstained vessels.

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.

Analysis of the ex vivo specificity of human gelatinases A and B towards skin collagen and elastic fibers by computerized morphometry.

Cutaneous aging and chronic exposure to UV irradiation leads to alterations in the appearance and biochemical composition of the skin. Members of the MMP family have been involved in the destruction of the extracellular matrix. Among them, gelatinases A and B were found to display elastolytic activity, in vitro. In this study, we first determined the ex vivo elastolytic potential of both endopeptidases, using human skin tissue sections and computerized morphometric analyses, and compared it with those of neutrophil elastase. In such conditions, gelatinase B (50 nM) induced 50% elastolysis. The percentage of elastic fibers degraded by gelatinase A (10-100 nM) never exceeded 10%. Elastolysis by gelatinase B and leukocyte elastase was characterized by a decrease in fiber length and an increase in the average diameter of the fibers. In addition, gelatinase B exhibited fibrillin-degrading activities. On the contrary, gelatinase A (50 nM) elicited up to 50% hydrolysis of collagen fibers, preferentially degrading type III collagen fibers. Gelatinase B did not promote any collagen degrading activity. Our data suggested that in vivo gelatinases could disrupt most extracellular matrix structures of human skin. Gelatinase B and to a much lesser extent, gelatinase A would degrade components of the elastic fibers network while gelatinase A, but not gelatinase B, would alter mostly collagen fibers and also degrade constituents of the dermo-epidermal junction.

Immunohistological and morphometric analysis of intra-epithelial lymphocytes and Langerhans cells in healthy and diseased human gingival tissues.

Periodontal diseases are histologically characterized by an infiltration of several inflammatory cell populations into the gingival epithelium and connective tissue, associated with degradation of extracellular matrix components. The purpose of this in situ study was to evaluate the inflammatory state of gingival tissues by the number of intra-epithelial lymphocyte (IEL) subsets and the area fraction (AA%) occupied by collagen fibres in the upper gingival connective tissue, and also to evaluate the number of CD1a+ Langerhans cells (LC) in order to show correlation(s), if any, between these histological findings. The gingival samples were from 10 clinically healthy controls (group C), 8 patients with gingivitis (group G) and 9 with chronic adult periodontitis (group P). A quantitative evaluation of the number of cell populations (CD1a+, CD45RB+, CD3+, CD8+, CD20+, TIA-1+ and GrB+ cells) and the area fraction (AA%) occupied by collagen fibres in the upper gingival connective tissue was made by morphometric and automated image analysis. The results showed that, compared with group C, all IEL subset numbers were significantly increased (p<0.05) in G and P groups, CD20+ excepted. In addition, there was a significant increase in the cytotoxic TIA-1+ IEL number (p<0.05) in group P when compared with group G. The study also showed a significant decrease in the number of CD1a+ LC in groups G and P (p<0.02 and p<0.001, respectively) when compared with group C. No significant difference was found in CD1a+ LC number between groups G and P. The determination of coefficients of correlation (r) with data obtained for each patient showed that in group G, CD1a+ LC number was significantly correlated with CD45RB+ (p<0.05) and CD3+ (p<0.01) IEL numbers whereas during periodontitis, CD1a+ LC number was significantly and inversely correlated with CD20+ (p<0.01), cytotoxic TIA-1+ (p<0.01) and with activated cytotoxic GrB+ (p<0.01) IEL numbers. Moreover, in group P a significant (p<0.05) positive correlation was shown between CD1a+ LC number and the AA% occupied by collagen fibres. This work demonstrates a decrease in CD1a+ LC number according to the severity of the periodontal disease estimated by the number of IEL and by the area fraction occupied by collagen fibres in human gingiva. The decrease of such cells could represent a way to avoid immune overstimulation.

An Engineered Biopolymer Prevents Mucositis Induced by 5-Fluorouracil in Hamsters

Oral mucositis is a common, treatment-limiting, and costly side effect of cancer treatments whose biological underpinnings remain poorly understood. In this study, mucositis induced in hamsters by 5-fluorouracil (5-FU) was observed after cheek-pouch scarifications, with and without administration of RGTA (RG1503), a polymer engineered to mimic the protective effects of heparan sulfate. RG1503 had no effects on 5-FU-induced decreases in body weight, blood cell counts, or cheek-pouch and jejunum epithelium proliferation rates, suggesting absence of interference with the cytotoxic effects of 5-FU. Extensive mucositis occurred in all of the untreated animals, and consisted of severe damage to cheek pouch tissues (epithelium, underlying connective tissue, and muscle bundles). Only half of the RG1503-treated animals had mucositis, over a mean area 70% smaller than in the untreated animals. Basement membranes were almost completely destroyed in the untreated group but was preserved in the RG1503 group. RG1503 blunted or abolished the following 5-FU-induced effects: increases in matrix metalloproteinase (MMP)-2, MMP-9, and plasmin, and decreases in tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. These data indicate that mucositis lesions are related to massive release of proteolytic enzymes and are improved by RG1503 treatment, this effect being ascribable in part to restoration of the MMP-TIMP balance. RG1503 given with cancer treatment might protect patients from mucositis.

Effect of adenovirus-mediated overexpression of decorin on metalloproteinases, tissue inhibitors of metalloproteinases and cytokines secretion by human gingival fibroblasts

Decorin is a small leucine-rich proteoglycan that plays a role in control of cell proliferation, cell migration, collagen fibrillogenesis and modulation of the activity of TGF-beta. In the present study, we investigated the effects of decorin on the production of metalloproteinases (MMP-1, -2, -3, -9 and -13), tissue inhibitors of metalloproteinases (TIMP-1, -2) and cytokines (TGF-beta, IL-1beta, IL-4 and TNF-alpha). Decorin was overexpressed in cultured human gingival fibroblasts using adenovirus-mediated gene transfer. Decorin infection resulted in decreased protein levels of MMP-1 and MMP-3 whereas MMP-2 and TIMP-2 secretion was increased. MMP-9, MMP-13 and TIMP-1 were not affected by decorin infection. Cytokine measurements by ELISA showed that decorin overexpression reduced TGF-beta and IL-1beta. In contrast, IL-4 and TNF-alpha levels were markedly increased in decorin-infected cells. These results suggest that decorin could modulate the expression of certain metalloproteinases and their inhibitors, as well as the production of cytokines. Altogether, our data suggest that decorin might play a pivotal role in tissue remodeling by acting on the balance between extracellular matrix synthesis and degradation.

Anetoderma: An altered balance between metalloproteinases and tissue inhibitors of metalloproteinases

The amount of elastic fibers from lesional and healthy skin areas of five patients with anetoderma was determined by automated image analysis. Dermal elastic fibers were almost completely absent in anetodermic skin and preelastic fibers were undetectable or extremely rare. Organ cultures were performed using explants from affected and unaffected skin areas of the same patient. We identified and quantified proteases in the culture media of explants: MMP-1 (collagenase 1), MMP-2 and MMP-9 (gelatinases A and B), MMP-3 (stromelysin 1), MMP-7 (matrilysin 1), and tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2. The data were compared with those of two healthy donors. For the five samples of anetodermic skin, MMP-1 levels were significantly higher compared with the uninvolved cultures and the two healthy samples. A significant increase of TIMP-1 expression was also observed in the affected cultures. We demonstrated a significant increase in the production of gelatinase A in lesional skin when compared with nonlesional skin and healthy donor samples. We found no significant production of TIMP-2 in the five samples of anetodermic skin compared with the samples from the two healthy donors. There was a significant decrease in TIMP-2 expression in the five nonlesional samples compared with the control samples. These data are in favor of an altered balance in anetodermic patients between MMP-2 and TIMP-2. Levels of MMP-9, MMP-3, and MMP-7 were significantly higher in the culture-conditioned media of the anetodermic skin samples than the nonlesional skin cultures. Because MMP-3, MMP-7, MMP-9 are known to degrade elastin, and MMP-3 can activate the latent forms of MMP-7 and MMP-9, we propose that these metalloproteinases also participate in the degradation of elastic fibers in anetodermic skin.

Quantitative morphological analysis of Langerhans cells in healthy and diseased human gingiva

Langerhans cells (LC) are implicated in the initiation and maintenance of inflammatory periodontal diseases. The purpose of this immunohistological study using morphometric and automated image analysis was to determine the morphological features of CD1a+ LC in healthy and inflammatory gingiva according to their localisation in the upper epithelium or the basal layer. The study was on gingival samples from 11 healthy controls (C), eight patients with gingivitis (G) and 12 patients with severe chronic adult periodontitis (P). The results show that in the basal layer of all experimental groups, the perimeter, surface and equivalent diameter of CD1a+ LC were significantly decreased (P<0.005) when compared with those in the upper epithelium of the same group. Furthermore, CD1a+ LC had become more rounded, reflected by a significant increase in form factor (P<0.005), when located close to the epithelial basal membrane. In the upper epithelium of group P, the perimeter, surface and equivalent diameter of CD1a+ LC were significantly decreased (P<0. 05) and the form factor significantly increased (P<0.05) when compared with the upper epithelium of group C. This work provides evidence for important morphological variations in CD1a+ LC according to their location within the epithelium and the severity of the periodontal disease. The observed morphological changes may reflect a cellular adaptation during the epithelial transmigration and could eventually be involved in immune stimulation during periodontitis.