heparitin-sulfate and aminoquinuride

The arrangement of lens cells is regulated by ocular growth factors. Although the effects of these inductive molecules on lens cell behavior (proliferation, survival, and fiber differentiation) are well-characterized, the precise mechanisms underlying the regulation of growth factor-mediated signaling in lens remains elusive. Increasing evidence highlights the importance of heparan sulfate proteoglycans (HSPGs) for the signaling regulation of growth factors; however, the identity of the different lens HSPGs and the specific roles they play in lens biology are still unknown.. Semiquantitative real-time (RT)-PCR and immunolabeling were used to characterize the spatial distribution of all known HSPG core proteins and their associated glycosaminoglycans (heparan and chondroitin sulfate) in the postnatal rat lens. Fibroblast growth factor (FGF)-2-treated lens epithelial explants, cultured in the presence of Surfen (an inhibitor of heparan sulfate [HS]-growth factor binding interactions) were used to investigate the requirement for HS in FGF-2-induced proliferation, fiber differentiation, and ERK1/2-signaling.. The lens expresses all HSPGs. These HSPGs are differentially localized to distinct functional regions of the lens. In vitro, inhibition of HS-sulfation with Surfen blocked FGF-2-mediated ERK1/2-signaling associated with lens epithelial cell proliferation and fiber differentiation, highlighting that these cellular processes are dependent on HS.. These findings support a requirement for HSPGs in FGF-2 driven lens cell proliferation and fiber differentiation. The identification of specific HSPG core proteins in key functional lens regions, and the divergent expression patterns of closely related HSPGs, suggests that different HSPGs may differentially regulate growth factor signaling networks leading to specific biological events involved in lens growth and maintenance.

Topics: Animals; Animals, Newborn; Blotting, Western; Cell Differentiation; Cell Proliferation; Chondroitin Sulfates; Epithelial Cells; Fibroblast Growth Factor 2; Gene Expression Regulation; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Lens, Crystalline; MAP Kinase Signaling System; Organ Culture Techniques; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Signal Transduction; Urea

Mucopolysaccharidosis (MPS) are rare inherited diseases characterized by accumulation of lysosomal glycosaminoglycans, including heparan sulfate (HS). Patients exhibit progressive multi-visceral dysfunction and shortened lifespan mainly due to a severe cardiac/respiratory decline. Cathepsin V (CatV) is a potent elastolytic protease implicated in extracellular matrix (ECM) remodeling. Whether CatV is inactivated by HS in lungs from MPS patients remained unknown. Herein, CatV colocalized with HS in MPS bronchial epithelial cells. HS level correlated positively with the severity of respiratory symptoms and negatively to the overall endopeptidase activity of cysteine cathepsins. HS bound tightly to CatV and impaired its activity. Withdrawal of HS by glycosidases preserved exogenous CatV activity, while addition of Surfen, a HS antagonist, restored elastolytic CatV-like activity in MPS samples. Our data suggest that the pathophysiological accumulation of HS may be deleterious for CatV-mediated ECM remodeling and for lung tissue homeostasis, thus contributing to respiratory disorders associated to MPS diseases.

Topics: Adolescent; Animals; Bronchi; Cathepsins; Child; Child, Preschool; CHO Cells; Cricetulus; Cysteine Endopeptidases; Epithelial Cells; Extracellular Matrix; Female; Heparitin Sulfate; Humans; Male; Mucopolysaccharidoses; Severity of Illness Index; Urea; Young Adult

Glycosylation is a ubiquitous post-translational modification that decorates proteins and lipids with glycans. These glycans can play critical roles in regulating biological events, and therefore, the discovery of strategies that target these molecules represent an important advancement toward understanding and controlling glycan-mediated cellular phenotypes. We describe the use of a small molecule, surfen, to temporarily silence the functions mediated by heparan sulfate glycosaminoglycans in mouse embryonic stem cells. Surfen binds heparan sulfate to antagonize growth factor interactions, thereby inhibiting signal transduction events that lead to differentiation. The strategies outlined in this chapter allow the characterization of resulting antagonistic effects caused by glycan-small molecule binding events toward maintaining embryonic stem cell pluripotency, curbing differentiation, and inhibiting signaling events.

Topics: Animals; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Glycosaminoglycans; Heparitin Sulfate; Intercellular Signaling Peptides and Proteins; Mice; Mouse Embryonic Stem Cells; Polymerase Chain Reaction; Signal Transduction; Urea

Hereditary multiple exostoses (HME) is a pediatric disorder caused by heparan sulfate (HS) deficiency and is characterized by growth plate-associated osteochondromas. Previously, we found that osteochondroma formation in mouse models is preceded by ectopic bone morphogenetic protein (BMP) signaling in the perichondrium, but the mechanistic relationships between BMP signaling and HS deficiency remain unclear. Therefore, we used an HS antagonist (surfen) to investigate the effects of this HS interference on BMP signaling, ligand availability, cell-surface BMP receptor (BMPR) dynamics, and BMPR interactions in Ad-293 and C3H/10T1/2 cells. As observed previously, the HS interference rapidly increased phosphorylated SMAD family member 1/5/8 levels. FACS analysis and immunoblots revealed that the cells possessed appreciable levels of endogenous cell-surface BMP2/4 that were unaffected by the HS antagonist, suggesting that BMP2/4 proteins remained surface-bound but became engaged in BMPR interactions and SMAD signaling. Indeed, surface mobility of SNAP-tagged BMPRII, measured by fluorescence recovery after photobleaching (FRAP), was modulated during the drug treatment. This suggested that the receptors had transitioned to lipid rafts acting as signaling centers, confirmed for BMPRII via ultracentrifugation to separate membrane subdomains.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein Receptors, Type II; Cells, Cultured; Chondrogenesis; Exostoses, Multiple Hereditary; Gene Expression Regulation; Heparitin Sulfate; Humans; Mice; Mice, Inbred C3H; Phosphorylation; Signal Transduction; Urea

During limb skeletogenesis the cartilaginous long bone anlagen and their growth plates become delimited by perichondrium with which they interact functionally. Yet, little is known about how, despite being so intimately associated with cartilage, perichondrium acquires and maintains its distinct phenotype and exerts its border function. Because perichondrium becomes deranged and interrupted by cartilaginous outgrowths in Hereditary Multiple Exostoses (HME), a pediatric disorder caused by EXT mutations and consequent heparan sulfate (HS) deficiency, we asked whether EXT genes and HS normally have roles in establishing its phenotype and function. Indeed, conditional Ext1 ablation in perichondrium and lateral chondrocytes flanking the epiphyseal region of mouse embryo long bone anlagen - a region encompassing the groove of Ranvier - caused ectopic cartilage formation. A similar response was observed when HS function was disrupted in long bone anlagen explants by genetic, pharmacological or enzymatic means, a response preceded by ectopic BMP signaling within perichondrium. These treatments also triggered excess chondrogenesis and cartilage nodule formation and overexpression of chondrogenic and matrix genes in limb bud mesenchymal cells in micromass culture. Interestingly, the treatments disrupted the peripheral definition and border of the cartilage nodules in such a way that many nodules overgrew and fused with each other into large amorphous cartilaginous masses. Interference with HS function reduced the physical association and interactions of BMP2 with HS and increased the cell responsiveness to endogenous and exogenous BMP proteins. In sum, Ext genes and HS are needed to establish and maintain perichondrium's phenotype and border function, restrain pro-chondrogenic signaling proteins including BMPs, and restrict chondrogenesis. Alterations in these mechanisms may contribute to exostosis formation in HME, particularly at the expense of regions rich in progenitor cells including the groove of Ranvier.

Topics: Animals; Bone and Bones; Bone Morphogenetic Protein 2; Cartilage; Chondrogenesis; Choristoma; Embryo, Mammalian; Exostoses, Multiple Hereditary; Gene Deletion; Gene Expression Regulation, Developmental; Heparitin Sulfate; Humans; Kinetics; Mice; Models, Biological; N-Acetylglucosaminyltransferases; Phenotype; Protein Binding; Signal Transduction; Urea

VEGF was first described as vascular permeability factor, a potent inducer of vascular leakage. Genetic evidence indicates that VEGF-stimulated endothelial proliferation in vitro and angiogenesis in vivo depend on heparan sulfate, but a requirement for heparan sulfate in vascular hyperpermeability has not been explored. Here we show that altering endothelial cell heparan sulfate biosynthesis in vivo decreases hyperpermeability induced by both VEGF(165) and VEGF(121). Because VEGF(121) does not bind heparan sulfate, the requirement for heparan sulfate suggested that it interacted with VEGF receptors rather than the ligand. By applying proximity ligation assays to primary brain endothelial cells, we show a direct interaction in situ between heparan sulfate and the VEGF receptor, VEGFR2. Furthermore, the number of heparan sulfate-VEGFR2 complexes increased in response to both VEGF(165) and VEGF(121). Genetic or heparin lyase-mediated alteration of endothelial heparan sulfate attenuated phosphorylation of VEGFR2 in response to VEGF(165) and VEGF(121), suggesting that the functional VEGF receptor complex contains heparan sulfate. Pharmacological blockade of heparan sulfate-protein interactions inhibited hyperpermeability in vivo, suggesting heparan sulfate as a potential target for treating hyperpermeability associated with ischemic disease.

Topics: Animals; Blood Vessels; Endothelial Cells; Heparitin Sulfate; Humans; Mice; Neoplasms; Permeability; Phosphorylation; Skin; Sulfotransferases; Urea; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

In a search for small molecule antagonists of heparan sulfate, we examined the activity of bis-2-methyl-4-amino-quinolyl-6-carbamide, also known as surfen. Fluorescence-based titrations indicated that surfen bound to glycosaminoglycans, and the extent of binding increased according to charge density in the order heparin > dermatan sulfate > heparan sulfate > chondroitin sulfate. All charged groups in heparin (N-sulfates, O-sulfates, and carboxyl groups) contributed to binding, consistent with the idea that surfen interacted electrostatically. Surfen neutralized the anticoagulant activity of both unfractionated and low molecular weight heparins and inhibited enzymatic sulfation and degradation reactions in vitro. Addition of surfen to cultured cells blocked FGF2-binding and signaling that depended on cell surface heparan sulfate and prevented both FGF2- and VEGF(165)-mediated sprouting of endothelial cells in Matrigel. Surfen also blocked heparan sulfate-mediated cell adhesion to the Hep-II domain of fibronectin and prevented infection by HSV-1 that depended on glycoprotein D interaction with heparan sulfate. These findings demonstrate the feasibility of identifying small molecule antagonists of heparan sulfate and raise the possibility of developing pharmacological agents to treat disorders that involve glycosaminoglycan-protein interactions.

Topics: Animals; Cell Adhesion; CHO Cells; Cricetinae; Cricetulus; Factor Xa; Fibroblast Growth Factor 2; Glycosaminoglycans; Heparin Lyase; Heparin, Low-Molecular-Weight; Heparitin Sulfate; Herpesvirus 1, Human; Humans; Mice; Neovascularization, Physiologic; Neutralization Tests; Signal Transduction; Solutions; Sulfotransferases; Sulfur; Swine; Urea