Patrick Kern

Imbalance in the Synthesis of Collagen Type I and Collagen Type III in Smooth Muscle Cells Derived from Human Varicose Veins

Varicose veins have a thickening wall. Their smooth muscle cells are disorganized as regards proliferation and production of extracellular matrix protein. An imbalance between the synthesis of collagen type I protein (collagen I) and collagen type III protein (collagen III) could explain the lack of elasticity of varicose veins. Therefore, collagen synthesis was compared in the media and in cultured smooth muscle cells derived from human control and varicose saphenous veins. An increase in total collagen synthesis was observed in the media and in smooth muscle cells derived from varicose veins. This augmentation was due to an overproduction of collagen I in cultured cells from varicose veins consistent with an increase in the release of collagen I metabolites in the media. A concomitant decrease in collagen III was observed in cultures of smooth muscle cells from varicose veins. The increase in the synthesis of collagen I in cells from varicose veins was correlated with an overexpression of the gene since mRNAs for collagen I were augmented without change in mRNA-half-life. This augmentation in the synthesis of collagen I was reduced by the addition of exogenous collagen III in cultures from varicose veins. These findings suggest a dysregulation of the synthesis of collagen I and III in smooth muscle cells derived from varicose veins.

Age-dependent variations of the biosyntheses of fibronectin and fibrous collagens in mouse skin

Skin explant cultures from hairless mice of increasing age were incubated with radioactive precursors in order to determine the age-dependent variations of the biosyntheses of fibronectin and fibrous collagens (types I and III). Total collagen synthesis expressed as a percentage of total protein synthesis did not vary with age but, if expressed as micrograms hydroxyproline per mg wet weight of skin, decreased by about 30% between 2 and 22 months of age. Hydroxylation of collagen, expressed as the ratio of 3H-hypro over 3H (pro + hypro) incorporated in freshly synthesized collagen, decreased with age by about 40% between 2 and 22 months of age. The proportion of type III collagen expressed as % of type I + type III collagens increased progressively with age by about 25% at 12 months to 60% at 22 months of age. Fibronectin biosynthesis, determined by immunoprecipitation of 35S-methionine labeled peptides in SDS-extracts of skin increased progressively with age from about 2% of total incorporated radioactivity in fibronectin at 2 months to 4% at 22 months. Plasma fibronectin, of hepatic origin, was shown already to increase with age in humans. It appears thus that the expression of genes coding for extracellular matrix macromolecules is under age-dependent regulation. This regulation appears to be different for the investigated macromolecules.

Effect of high-glucose concentrations on the expression of collagens and fibronectin by fibroblasts in culture

Extracellular matrix macromolecules such as collagen and fibronectin are progressively altered during aging and age-related diseases like diabetes. We investigated the effect of high-glucose concentration (mimicking diabetic conditions) and the influence of in vitro cell aging [comparing 4th-passage fibroblasts (P4) to 15th-passage fibroblasts (P15)] on collagen and fibronectin synthesis. Fibroblasts were incubated at postconfluency with radiolabeled precursors, [3H] proline for collagen, [35S] methionine for fibronectin. We report that in control conditions (5 mM glucose) collagen III production increased with in vitro cell aging. High glucose concentrations (10 and 15 mM) increased specifically collagen III synthesis both at the mRNA and protein levels, without alteration of collagen I production in P4 and P15 cells. Fibronectin synthesis was also increased both during in vitro cell aging and in high glucose-treated P4 fibroblasts. Taken together, these data suggest similarities between changes of phenotypic expression of collagen and fibronectin induced by in vitro cell aging and conditions imitating diabetes.

Structurally different RGTAs modulate collagen-type expression by cultured aortic smooth muscle cells via different pathways involving fibroblast growth factor-2 or transforming growth factor-β1

We have engineered polymers called ReGeneraTing Agents (RGTAs), which mimic the protecting and potentiating properties of heparan sulfates toward heparin‐binding growth factors (HBGF). RGTAs have been shown to optimize cell growth and regulate collagen production in vitro. Here, we studied relationships between RGTA structure and collagen‐type expression in aortic smooth muscle cells by using two RGTAs, the carboxylmethylsulfate dextran RG‐1503 and the carboxylmethylsulfate dextran with added benzylamide RG‐1192. RG‐1192 specifically induced a fivefold decrease in collagen III synthesis. This effect was abolished by FGF‐2 neutralizing antibody. RG‐1192 and FGF‐2 acted synergistically to decrease collagen III. RG‐ 1192 was more effective than heparin in this process. RG‐1192 increased the pericellular localization of FGF‐2 and protected FGF‐2 from proteolysis. Surface plasmon resonance analysis indicated a Kd of 15.7 nM for the RG‐1192/FGF‐2 interaction (10.6 nM for the heparin/FGF‐2 interaction). The structurally different RG‐1503 (without benzylamide) did not interact with FGF‐2 and worked synergistically with TGF‐β1 to specifically induce a twofold increase in collagen V. RGTAs with different structures exert different modulating effects on the collagen phenotype. Selection of appropriate RGTAs, which had been shown to enhance in vivo tissue repair, may provide a mean of correcting collagen abnormalities in vascular disorders and more generally in fibrotic diseases.

In vivo EVALUATION OF PHOTOPROTECTION AGAINST CHRONIC ULTRAVIOLET-A IRRADIATION BY A NEW SUNSCREEN MEXORYL® SX*

Abstract— In a previous study on the hairless mouse it was shown that sub‐erythemal doses of pure UV‐A enhanced the numerous changes normally observed during chronological aging. A new sunscreen (a bis‐benzylidene campho sulfonic acid derivative) has been synthesized in our research laboratory (Λmax: 345 nm, ε: 47000). Its photoprotective properties against UV‐A induced damages were assessed in our mouse model. Three month old albino hairless mice were exposed for 1 y to suberythemal doses (35 J/cm2) of UV‐A obtained from a xenon source filtered through a WG 345 filter. One group of animals was exposed untreated, the other received a formulation containing 5% of the sunscreen prior to irradiation. At the end of the study the cutaneous properties of protected mice were compared to those of unprotected animals and to 3 and 15 month old unirradiated controls. We found that the visible changes induced by UV‐A irradiation were mainly sagging and wrinkling. Histological and electron microscopic alterations consisted of hyperkeratosis, increased density of elastic fibers with alteration of fiber orientation and increased glycosaminoglycan deposits. Biochemical changes consisted of decreases in total collagen and collagen hydroxylation and increases in both collagen III/I + III ratio and fibronectin biosynthesis. All these changes were reduced or abolished by the sunscreen.

UVA- and UVB-induced changes in collagen and fibronectin biosynthesis in the skin of hairless mice

The modifications induced in hairless mouse skin by chronic UV irradiation were investigated. Skin explant cultures were used to study UVA- and UVB-induced changes occurring in interstitial collagen (type I and type III) and fibronectin biosynthesis. To study the long-term effects, albino hairless mice were irradiated with UVA radiation alone from two sources with different spectral qualities or with UVB. UVA and UVB radiation produced a significant increase in the ratio of type III to type I collagen (more than 100% for UVA-irradiated skin and about 60% for UVB-irradiated skin) accompanied by a significantly increased fibronectin biosynthesis (50% or more in all irradiated groups). Irradiation with either UVA or UVB alone had no significant effect on the total collagen synthesis and resulted in only a slight decrease in the total collagen content of the skin determined as hydroxyproline. This decrease was significant only in the case of the group irradiated with UVA (xenon) (decrease of 25%, expressed as micrograms of hydroxyproline per milligram wet weight). A significant decrease in collagen hydroxylation (expressed as radioactive hydroxyproline/radioactive hydroxyproline plus proline in neosynthesized collagen) was observed of about 50% in skin irradiated with UVA (xenon) but not in UVB-treated skin. Several of the above modifications (increased fibronectin biosynthesis, increased collagen type III to type I ratio) correspond to the modifications observed during the aging of non-irradiated hairless mice. Therefore it appears that UV irradiation accelerates the modifications of extracellular matrix biosynthesis observed during aging.

Synthesis and biological activities of a library of glycosaminoglycans mimetic oligosaccharides

Biologically active oligosaccharides related to glycosaminoglycans are accumulating increased attention because of their therapeutic potential and for their value in mechanistic studies. Heparan mimetics (HMs) are a family of dextran based polymer known to mimic the properties of glycosaminoglycans, and particularly those of heparan sulfates, as to interact with heparin binding proteins. HMs have shown to stimulate tissue repair in various animal models. Here, we use different methods to depolymerize HMs in order to produce a library of related oligosaccharides and study their biological activities. Since HMs were resistant to endoglycanases activities, depolymerization was achieved by chemical approaches. In vitro biological studies showed that HM oligosaccharides can differentially potentiate FGF-2 mitogenic and antithrombotic activities. In vivo, a selected oligosaccharide (H-dp12) showed to be able to regenerate tissue almost as well as the related polymeric product. The very low anticoagulant activity and high biological activity of low mass oligosaccharides give to these products a new therapeutic potential.

Heparan mimetic regulates collagen expression and TGF-β1 distribution in gamma-irradiated human intestinal smooth muscle cells

Radiation‐induced intestinal fibrosis is characterized by collagen accumulation, a process in which TGF‐β1 plays a key role. We analyzed the effects of gamma radiation on collagen expression and TGF‐βl distribution in human intestinal smooth muscle cells (HISM). We investigated the activity of a car‐boxymethylated and sulfated dextran (RG‐1503), exhibiting antifibrotic properties and promoting in vivo intestinal tissue repair, on irradiated HISM. After 60Co irradiation (10 Gy), HISM were labeled with [3H] proline (±RG‐1503). Radiolabeled collagen I, III, and V were quantified by SDS‐PAGE. TGF‐β1 was quantified by ELISA in culture medium, pericellular and intracellular compartments. Irradiation induced a specific 2.85‐fold increase in collagen III production by HISM. Collagen V decreased by 80% 72 h after irradiation. Pericellular TGF‐β1 was increased (up to twofold) in irradiated HISM. RG‐1503 added before or after irradiation reversed both mRNA and protein levels of collagen III and V to control values. RG‐1503 decreased the amount of TGF‐β1 in the cell layer below the control values. Irradiation of HISM induced the development of a fibrotic phenotype in terms of collagen production and TGF‐β1 distribution. The antifibrotic RG‐1503 restored HISM physiological characteristics and may represent a promising therapeutic approach for radiation‐induced intestinal fibrosis.—Alexakis, C., Guettoufi, A., Mestries, P., Strup, C., Mathé, D., Barbaud, C., Barritault, D., Caruelle, J.‐ P., Kern, P. Heparan mimetic regulates collagen expression and TGF‐β1 distribution in gamma‐irradiated human intestinal smooth muscle cells. FASEB J. 15, 1546–1554 (2001)

Collagen synthesis by vascular smooth muscle cells in the presence of antiproliferative polysaccharides.

Production of various components of the extracellular matrix (ECM) modulates biological functions of the vascular tissue. This process is generally amplified in pathologic states as atherosclerosis. Atheroma originates from smooth muscle cells (SMC) which have migrated and proliferated in the vascular intima. In this study we investigated protein synthesis, collagen synthesis, and types I, III, and V collagen distribution by SMC in the presence of three families of watersoluble polysaccharides, heparin, fucans, and derivatized dextrans. We observed that fucan and derivatized dextran were able, as was heparin, to inhibit rat aortic SMC growth in culture. We then analyzed collagen modulation by measuring the incorporation of the radiolabeled precursor (3H)-proline into vascular SMC. Our results showed uncoupling of the antiproliferative capacity with collagen biosynthesis. However, fucan, the most antiproliferative polysaccharide, was also the most active in inhibiting protein and collagen synthesis. In addition, compounds that decreased total collagen synthesis preferentially increased the proportion of cell-associated collagen. Interestingly, only the antiproliferative polysaccharides inhibited significantly type V collagen biosynthesis. These new biomaterials appear to be valuable tools to study and control extracellular-matrix interactions with cells from the vascular walls.

Chemically modified dextrans modulate expression of collagen phenotype by cultured smooth muscle cells in relation to the degree of carboxymethyl, benzylamide, and sulfation substitutions.

We developed regenerating agents (RGTAs) corresponding to polysaccharides derived from dextran and containing defined amounts of carboxymethyl (CM), carboxymethyl sulfate (CMS), carboxymethyl benzylamide (CMB), or carboxymethyl benzylamide sulfate (CMBS) groups with varying degrees of substitution. These compounds mimicked some effects of heparin on smooth muscle cell (SMC) proliferation and promoted in vivo tissue remodeling. We demonstrated that only RGTAs containing both CM and sulfate groups decreased SMC proliferation, in correlation with increased sulfation level. This effect was amplified by the presence of benzylamide. Independent of this activity on cell proliferation (i.e., with postconfluent cells), RGTAs modulated collagen biosynthesis by SMCs. On the one hand, CMBS more than CMS RGTAs induced a decrease of collagen III synthesis at the level of mRNA steady state and protein production. On the other hand, CMS to a greater extent than CMBS RGTAs increased both collagen V mRNA and protein production. In addition, only benzylamide-containing RGTAs increased accumulation of collagen I and III in the cell layer. In conclusion, RGTA bioactivities required the presence of CM functions, increased with the sulfation level, and varied with benzylamide substitution. RGTAs that modulate cell proliferation and collagen biosynthesis by differential mechanisms may represent potential antifibrotic agents.