Studies on corneal wound healing. Effect of fucose on iodine vapor-burnt rabbit corneas.

Corneal wound healing often leads to the development of scar tissue with loss of transparency. Reconstitution of transparent corneal stroma depends on the regulation of the biosynthetic activities of postlesional keratocytes and also to a large extent on the limitation of matrix degradation, attributed essentially to the upregulation of matrix metalloproteases and especially MMP-9. Using a standardized method for the production of reproducible corneal lesions by burning with iodine vapors, we could show that the local application of 0.5 mg/ml L-fucose reduced significantly MMP-9 upregulation and accelerated the recovery of the epithelial layer of the cornea. The iodine vapor used in the experiments produces a rapid loss of epithelium with no or slight effect below the basement membrane. A relatively rapid regrowth of epithelium was observed. The speed of this reepithelialization was stimulated by the local application of fucose. At 48 h after burn, there was a difference between fucose-treated and control corneas (epithelial thickness was about 50 mum for fucose-treated corneas and 37 microm for control corneas). Culture media of in vivo fucose-treated corneas showed an important decrease of MMP-9 activity (-51%, n = 6, p < 0.01). It appears that the in vivo fucose treatment reduced the MMP-9 activity released in the media. This effect is significant 24 h after iodine vapor burn. In order to study the effect of fucose on normal corneas, it was added to rabbit as well as human cornea explant cultures, and the production and release of MMP-9 was determined by zymography. Fucose at a concentration of 0.5 mg/ml produced a 70% decrease of MMP-9 activity released in the medium by corneal explant cultures. Other mono- and oligosaccharides were also tested. Besides lactose, fucose-rich oligosaccharides also produced significant inhibition. Galactose, melibiose, mannose and glucose were inactive. These results justify the use of fucose for the local treatment of corneal wounds.

MMP-type endopeptidase activity in the cornea. Its evolution during organ culture storage at the Eye Bank. Effect of hyaluronan.

About 46% of total corneas obtained from donors in the French Eye Bank cannot be grafted for several reasons as loss of endothelium or other. Corneal cells express proteolytic enzymes, essentially matrix metallo-proteinase MMP-2 and MMP-9. In presence of hyaluronan and some other GAG-s their activity increases as could be shown on keratocyte cultures. Hyaluronan concentration increases during in vitro preservation and can represent a serious hazard for corneal conservation. The control of MMP-release and activation might well be one of the factors involved in graft deterioration. We could show however that only a slight fraction (< or =12%) of total, relatively high endopeptidase activity of the cornea is released in the media during storing. It appears therefore that most of the proteolytic activity determined in corneal extracts remains confined to the stroma and might not represent an important risk for preservation, at least for the endothelium.

Pharmacology of skin aging. Stimulation of glycosaminoglycan biosynthesis by L-fucose and fucose-rich polysaccharides, effect of in vitro aging of fibroblasts.

The effect of L-fucose and fucose-rich polysaccharides (FROP-s [Biomed. Pharmacother., 2003; 57: 187-94]) was investigated, using human skin fibroblast cultures at several passages. The cells were incubated with radioactive glucosamine for 24 h, followed by the determination of individual glycosaminoglycans (GAG-s) by selective hydrolysis using specific enzymes. The effect of L-fucose and of FROP-3 [Biomed. Pharmacother., in 2003; 57: 187-94], both at 1 and 10 microg/ml, added to the culture medium, was investigated. L-Fucose stimulated the incorporation of the tracer in heparan sulfates by fibroblasts at the ninth passage by 20%. FROP-3 stimulated incorporation in keratan sulfates by 45% by fibroblasts of the fifth passage. This effect was identical at both concentrations tested. For fibroblasts at the ninth passage FROP-3 stimulated incorporation in dermatan sulfate. This effect was dose dependent of the order of +67% at 1 microg/ml and +128% at 10 microg/ml. Incorporation in hyaluronan was also stimulated by about +27%. These stimulations of GAG-biosynthesis might play a role in the increase of total skin thickness of hairless rats treated with L-fucose, as well as in several other favorable results recorded for FROP-3 such as the increased hydration (resistance to pressure) and elasticity of human skin (Robert C, Robert AM, Robert L).

Tropoelastin biosynthesis by corneal cells. Epithelial inhibition of keratocyte tropoelastin biosynthesis.

The vertebrate cornea is an avascular tissue and does not contain elastic fibers. We tested the capacity of corneal epithelial cells and stromal keratocytes to synthesize tropoelastin. Explant cultures and cell cultures were obtained from these two cell types in standard culture conditions. Their elastin-synthetic activity was compared to skin explant cultures and to dermal fibroblast cell cultures. Both corneal cell types synthesized tropoelastin as shown by the incorporation of a radioactive precursor followed by immunoprecipitation of tropoelastin. When serial cultures of keratocytes were tested, tropoelastin biosynthesis strongly increased after the 3rd passage and was at the 9th passage more than the double of that of the first passage. When cocultures were studied with or without cell contact, epithelial cells partially inhibited tropoelastin biosynthesis by keratocytes. This inhibition was somewhat stronger (-36%, p < 0.005) with cell-to-cell contact than keeping separate epithelial cells and keratocytes bathing in the same medium (-18%, p < 0.005). When human skin fibroblasts were substituted for keratocytes with cell-to-cell contact, their tropoelastin biosynthesis was also inhibited by corneal epithelial cells (-42%, p < 0.005), to the same extent as for keratocytes. In Transwell culture, this inhibition was again somewhat lower (-36%, p < 0.005). Some diffusible factor produced by epithelial cells is apparently involved. The epithelial inhibition of tropoelastin biosynthesis by stromal keratocytes might represent one of the mechanisms keeping corneal stroma exempt of elastin fibers.

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.

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.

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.

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.

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.