Dulce Papy-Garcia

The 37-kDa/67-kDa Laminin Receptor Acts as a Receptor for Infectious Prions and Is Inhibited by Polysulfated Glycanes

BACKGROUND Recently, we showed that the 37-kDa/67-kDa laminin receptor (LRP/LR) acts as the receptor of the cellular prion protein. METHODS For the present study, we investigated the binding of the murine scrapie prion protein (moPrP27-30) to baby hamster kidney (BHK) cells, using the Semliki Forest virus system. RESULTS The enhanced binding of moPrP27-30 to BHK cells expressing moLRP::FLAG was inhibited by the LRP/LR-specific antibody W3, which suggests that LRP/LR acts as a receptor for the scrapie form of the prion protein, PrP(Sc). This finding was confirmed by a parallel study that showed that bovine prions are internalized by human enterocytes via LRP/LR. The heparan sulfate mimetics HM5004 and HM2602 reduced PrP27-30 binding to moLRP-expressing cells to approximately 30% and approximately 20%, respectively, at a concentration of 10 microg/mL, whereas pentosan polysulfate (SP54) and phycarin sulfate (PS3) both reduced the binding to approximately 40% at a concentration of 100 microg/mL. CONCLUSIONS We suggest that the inhibition reported elsewhere of PrP(Sc) synthesis and the incubation times prolonged in rodent models by these sulfated glycans are due to the inhibition of the LRP/LR-dependent binding of prions to the target cells.

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.

Live imaging of prions reveals nascent PrPSc in cell-surface, raft-associated amyloid strings and webs

PrPSc forms micrometer long amyloidic strings that can congregate in clusters and webs at the surface of living cells.

New agents for the treatment of infarcted myocardium

Local delivery of angiogenic growth factors for the treatment of myocardial ischemia has been well documented in various animal models, and clinical trials are now in progress. Our strategy was radically different, based on selective protection of some of the growth factors naturally present within the injured tissue. This protection was obtained by applying a chemically defined substitute for Dextran called RGTA11 (for ReGeneraTing Agent). RGTA is a family of agents, which has properties mimicking those of heparan sulfates toward heparin‐binding growth factors (HBGF) and which stimulate tissue repair and protection. Indeed, we have previously shown that RGTA prevents most of the damage resulting from acute skeletal muscle ischemia [FASEB J. (1999) 13, 761–766]. We now show that the same agent can be used for the treatment of myocardial infarction. Acute myocardial infarction was induced in pigs by ligation of the left circumflex artery. One hour later, a single injection of 10 μg of RGTA11 was made in the center of the infarcted area. Three weeks later we observed 1) recovery of 84% of the initial left ventricular ejection fraction (only 55% in saline‐treated controls), 2) an almost 50% reduction in the infarct size, 3) a reduction in fibrotic tissue formation, 4) significant preservation of myocytes, and 5) an increase in the number of blood vessels. The treatment of ischemic heart disease with RGTA would have clear advantages over other therapies such as growth factor, gene, or cell transplants, based on a stable, simple, and easy‐to‐develop chemical product.

Glycosaminoglycans and their synthetic mimetics inhibit RANTES-induced migration and invasion of human hepatoma cells

The CC-chemokine regulated on activation, normal T-cell expressed, and presumably secreted (RANTES)/CCL5 mediates its biological activities through activation of G protein–coupled receptors, CCR1, CCR3, or CCR5, and binds to glycosaminoglycans. This study was undertaken to investigate whether this chemokine is involved in hepatoma cell migration or invasion and to modulate these effects in vitro by the use of glycosaminoglycan mimetics. We show that the human hepatoma Huh7 and Hep3B cells express RANTES/CCL5 G protein–coupled receptor CCR1 but not CCR3 nor CCR5. RANTES/CCL5 binding to these cells depends on CCR1 and glycosaminoglycans. Moreover, RANTES/CCL5 strongly stimulates the migration and the invasion of Huh7 cells and to a lesser extent that of Hep3B cells. RANTES/CCL5 also stimulates the tyrosine phosphorylation of focal adhesion kinase and activates matrix metalloproteinase-9 in Huh7 hepatoma cells, resulting in increased invasion of these cells. The fact that RANTES/CCL5-induced migration and invasion of Huh7 cells are both strongly inhibited by anti-CCR1 antibodies and heparin, as well as by β-d-xyloside treatment of the cells, suggests that CCR1 and glycosaminoglycans are involved in these events. We then show by surface plasmon resonance that synthetic glycosaminoglycan mimetics, OTR4120 or OTR4131, directly bind to RANTES/CCL5. The preincubation of the chemokine with each of these mimetics strongly inhibited RANTES-induced migration and invasion of Huh7 cells. Therefore, targeting the RANTES-glycosaminoglycan interaction could be a new therapeutic approach for human hepatocellular carcinoma. [Mol Cancer Ther 2007;6(11):2948–58]

Glycosaminoglycans mimetics potentiate the clonogenicity, proliferation, migration and differentiation properties of rat mesenchymal stem cells

Successful use of stem cell-based therapeutic products is conditioned by transplantation of optimized cells in permissive microenvironment. Mesenchymal stem cell (MSC) fates are tightly regulated by humoral factors, cellular interactions and extracellular matrix (ECM) components, such as glycosaminoglycans (GAG), which are complex polysaccharides with structural heterogeneity. During osteogenesis, a temporally controlled expression of particular GAG species is required to interact with specific growth promoting and differentiating factors to regulate their biological activities. As a comparative tool to study natural GAG, we used structurally and functionally related synthetic GAG mimetics. One of these compounds [OTR(4120)] was previously shown to stimulate bone repair in rat models. Here, we demonstrate that structurally distinct GAG mimetics stimulate differentially clonogenicity, proliferation, migration and osteogenic phenotype of MSC in vitro, according to their specific chemical signature, underlying the role of sulfate and acetyl groups in specific interactions with heparin binding factors (HBF). These effects are dependent on FGF-2 interactions since they are inhibited by a FGF receptor 1 signaling pathway blocker. These data suggest that the in vivo [OTR(4120)] bone regenerative effect could be due to its ability to induce MSC migration and osteogenic differentiation. To conclude, we provide evidences showing that GAG mimetics may have great interest for bone regeneration therapy and represent an alternative to exogenous growth factor treatments to optimize potential therapeutic properties of MSC.

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.

Age-related Changes in Rat Myocardium Involve Altered Capacities of Glycosaminoglycans to Potentiate Growth Factor Functions and Heparan Sulfate-altered Sulfation

Background: Heparan sulfates (HS) are important cell behavior regulators. Results: With age, HS structural changes affect myocardial growth factor functionalities. Conclusion: This reveals the importance of HS on the control of essential tissue repair effectors during aging. Significance: Changes in cardiac HS may alter tissue homeostasis and impair heart function. This might also limit the success of protein therapies and implantation of therapeutic cells. Glycosaminoglycans (GAGs) are essential components of the extracellular matrix, the natural environment from which cell behavior is regulated by a number or tissue homeostasis guarantors including growth factors. Because most heparin-binding growth factor activities are regulated by GAGs, structural and functional alterations of these polysaccharides may consequently affect the integrity of tissues during critical physiological and pathological processes. Here, we investigated whether the aging process can induce changes in the myocardial GAG composition in rats and whether these changes can affect the activities of particular heparin-binding growth factors known to sustain cardiac tissue integrity. Our results showed an age-dependent increase of GAG levels in the left ventricle. Biochemical and immunohistological studies pointed out heparan sulfates (HS) as the GAG species that increased with age. ELISA-based competition assays showed altered capacities of the aged myocardial GAGs to bind FGF-1, FGF-2, and VEGF but not HB EGF. Mitogenic assays in cultured cells showed an age-dependent decrease of the elderly GAG capacities to potentiate FGF-2 whereas the potentiating effect on VEGF165 was increased, as confirmed by augmented angiogenic cell proliferation in Matrigel plugs. Moreover, HS disaccharide analysis showed considerably altered 6-O-sulfation with modest changes in N- and 2-O-sulfations. Together, these findings suggest a physiological significance of HS structural and functional alterations during aging. This can be associated with an age-dependent decline of the extracellular matrix capacity to efficiently modulate not only the activity of resident or therapeutic growth factors but also the homing of resident or therapeutic cells.

Glycosaminoglycan mimetics-induced mobilization of hematopoietic progenitors and stem cells into mouse peripheral blood: structure/function insights.

OBJECTIVE Glycosaminoglycans (GAG) are major components of bone marrow extracellular matrix because they have the property to interact with cells and growth factors in hematopoietic niches. In this study, we investigated the effect of two different chemically defined GAG mimetics on mobilization of hematopoietic stem and progenitor cells (HSPCs) in mice peripheral blood. MATERIALS AND METHODS Mobilization was achieved by intraperitoneal injection of GAG mimetics. Mobilized cells were characterized phenotypically by reverse transcription polymerase chain reaction and fluorescence-activated cell sorting analysis and functionally by colony-forming cell, cobblestone area-forming cell and long-term culture-initiating cell assays in vitro. Radioprotection assays were performed to confirm the functionality of primitive hematopoietic cells in vivo. Involvement of stromal-derived factor-1 (SDF-1) and matrix metalloproteinase-9 (MMP-9) were investigated. RESULTS GAG mimetics treatment induces hyperleukocytosis and mobilization of HSPC. They synergize with the effects of granulocyte colony-stimulating factor or AMD3100 on hematopoietic progenitors mobilization. Reconstitution of lethally irradiated recipient mice with peripheral blood mononuclear cells from GAG mimetic-treated donor mice improves engraftment and survival. BiAcore studies indicate that the mimetics interact directly with SDF-1. In addition, GAG mimetics-induced mobilization is associated with increased levels of pro- and active MMP-9 from bone marrow cells and increased level of SDF-1 in peripheral blood. Finally, mobilization is partially inhibited by co-injection with anti-SDF-1 antibody. CONCLUSION This study demonstrates that GAG mimetics induce efficient mobilization of HSPCs, associated with an activation of pro-MMP-9 and a modification in the SDF-1 concentration gradient between bone marrow and peripheral blood. We suggest that structural features of GAGs can modify the nature of mobilized cells.

A synthetic glycosaminoglycan mimetic (RGTA) modifies natural glycosaminoglycan species during myogenesis.

Crucial events in myogenesis rely on the highly regulated spatiotemporal distribution of cell surface heparan sulfate proteoglycans to which are associated growth factors, thus creating a specific microenvironment around muscle cells. Most growth factors involved in control of myoblast growth and differentiation are stored in the extracellular matrix through interaction with specific sequences of glycosaminoglycan oligosaccharides, mainly heparan sulfate (HS). Different HS subspecies revealed by specific antibodies, have been shown to provide spatiotemporal regulation during muscle development. We have previously shown that glycosaminoglycan (GAG) mimetics called RGTA (ReGeneraTing Agent), stimulate muscle precursor cell growth and differentiation. These data suggest an important role of GAGs during myogenesis; however, little is yet known about the different species of GAGs synthesized during myogenesis and their metabolic regulation. We therefore quantified GAGs during myogenesis of C2.7 cells and show that the composition of GAG species was modified during myogenic differentiation. In particular, HS levels were increased during this process. In addition, the GAG mimetic RGTA, which stimulated both growth and differentiation of C2.7 cells, increased the total amount of GAG produced by these cells without significantly altering their rate of sulfation. RGTA treatment further enhanced HS levels and changed its sub-species composition. Although mRNA levels of the enzymes involved in HS biosynthesis were almost unchanged during myogenic differentiation, heparanase mRNA levels decreased. RGTA did not markedly alter these levels. Here we show that the effects of RGTA on myoblast growth and differentiation are in part mediated through an alteration of GAG species and provide an important insight into the role of these molecules in normal or pathologic myogenic processes.