Search

Pseudorabies (also called Aujeszky's disease) is a highly infectious viral disease caused by the pseudorabies virus (PRV, or Suid herpesvirus 1). Although the disease has been controlled by immunization with the PRV-attenuated vaccine, the emerging PRV variants can escape the immune surveillance in the vaccinated pig, resulting in recent outbreaks. Furthermore, the virus has been detected in other animals and humans, indicating cross-transmission of PRV. However, the mechanism of PRV cross-species transmission needs further study. In this study, we compared the amino acid sequences of glycoproteins (gD), gL, and thymidine kinase (TK) of PRV strains, human PRV hSD-1 2019 strain, and the attenuated strain Bartha-K61, followed by predication of their spatial conformation. In addition, the interactions between the viral gD protein and host nectin-1, nectin-2, and HS were also evaluated via molecular docking. The results showed that the amino acid sequence homology of the gD, gL, and TK proteins of hSD-1 2019 and JL-CC was 97.5%, 94.4%, and 99.1%, respectively. Moreover, there were mutations in the amino acid sequences of gD, gL, and TK proteins of hSD-1 2019 and JL-CC compared with the corresponding reference sequences of the Bartha strain. The mutations of gD, gL, and TK might not affect the spatial conformation of the protein domain but may affect the recognition of antibodies and antigen epitopes. Moreover, the gD protein of JL-CC, isolated previously, can bind to human nectin-1, nectin-2, and HS, suggesting the virus may be highly infectious and pathogenic to human beings. [ABSTRACT FROM AUTHOR]

Despite recent advances in radiotherapeutic strategies, acquired resistance remains a major obstacle, leading to tumor recurrence for many patients. Once thought to be a strictly cancer cell intrinsic property, it is becoming increasingly clear that treatment-resistance is driven in part by complex interactions between cancer cells and non-transformed cells of the tumor microenvironment. Herein, we report that radiotherapy induces the production of extracellular vesicles by breast cancer cells capable of stimulating tumor-supporting fibroblast activity, facilitating tumor survival and promoting cancer stem-like cell expansion. This pro-tumor activity was associated with fibroblast production of the paracrine signaling factor IL-6 and was dependent on the expression of the heparan sulfate proteoglycan CD44v3 on the vesicle surface. Enzymatic removal or pharmaceutical inhibition of its heparan sulfate side chains disrupted this tumor-fibroblast crosstalk. Additionally, we show that the radiation-induced production of CD44v3+ vesicles is effectively silenced by blocking the ESCRT pathway using a soluble pharmacological inhibitor of MDA-9/Syntenin/SDCBP PDZ1 domain activity, PDZ1i. This population of vesicles was also detected in the sera of human patients undergoing radiotherapy, therefore representing a potential biomarker for radiation therapy and providing an opportunity for clinical intervention to improve treatment outcomes. [ABSTRACT FROM AUTHOR]

Synthesis of glycosaminoglycans, such as heparan sulfate (HS) and chondroitin sulfate (CS), occurs in the lumen of the Golgi, but the relationship between Golgi structural integrity and glycosaminoglycan synthesis is not clear. In this study, we disrupted the Golgi structure by knocking out GRASP55 and GRASP65 and determined its effect on the synthesis, sulfation, and secretion of HS and CS. We found that GRASP depletion increased HS synthesis while decreasing CS synthesis in cells, altered HS and CS sulfation, and reduced both HS and CS secretion. Using proteomics, RNA-seq and biochemical approaches, we identified EXTL3, a key enzyme in the HS synthesis pathway, whose level is upregulated in GRASP knockout cells; while GalNAcT1, an essential CS synthesis enzyme, is robustly reduced. In addition, we found that GRASP depletion decreased HS sulfation via the reduction of PAPSS2, a bifunctional enzyme in HS sulfation. Our study provides the first evidence that Golgi structural defect may significantly alter the synthesis and secretion of glycosaminoglycans. [ABSTRACT FROM AUTHOR]

Abstract Exostosin-like 3 (EXTL3) encodes the glycosyltransferases responsible for the biosynthesis of the backbone structure of heparan sulfate (HS), a sulfated polysaccharide that is ubiquitously distributed on the animal cell surface and in the extracellular matrix. A lack of EXTL3 reduces HS levels and causes embryonic lethality, indicating its indispensable role in the biosynthesis of HS. EXTL3 has also been identified as a receptor molecule for regenerating islet-derived (REG) protein ligands, which have been shown to stimulate islet β-cell growth. REG proteins also play roles in keratinocyte proliferation and/or differentiation, tissue regeneration and immune defenses in the gut as well as neurite outgrowth in the central nervous system. Compared with the established function of EXTL3 as a glycosyltransferase in HS biosynthesis, the REG-receptor function of EXTL3 is not conclusive. Genetic diseases caused by biallelic mutations in the EXTL3 gene were recently reported to result in a neuro-immuno-skeletal dysplasia syndrome. EXTL3 is a key molecule for the biosynthesis of HS and may be involved in the signal transduction of REG proteins.

Glycosaminoglycans (GAGs) including chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate, except for hyaluronan that is a free polysaccharide, are covalently attached to core proteins to form proteoglycans. More than 50 gene products are involved in the biosynthesis of GAGs. We recently developed a comprehensive glycosylation mapping tool, GlycoMaple, for visualization and estimation of glycan structures based on gene expression profiles. Using this tool, the expression levels of GAG biosynthetic genes were analyzed in various human tissues as well as tumor tissues. In brain and pancreatic tumors, the pathways for biosynthesis of chondroitin and dermatan sulfate were predicted to be upregulated. In breast cancerous tissues, the pathways for biosynthesis of chondroitin and dermatan sulfate were predicted to be up- and down-regulated, respectively, which are consistent with biochemical findings published in the literature. In addition, the expression levels of the chondroitin sulfate-proteoglycan versican and the dermatan sulfate-proteoglycan decorin were up- and down-regulated, respectively. These findings may provide new insight into GAG profiles in various human diseases including cancerous tumors as well as neurodegenerative disease using GlycoMaple analysis.

Complement dysregulation is characteristic of the renal diseases atypical hemolytic uremic syndrome (aHUS) and complement component 3 glomerulopathy (C3G). Complement regulatory protein Factor H (FH) inhibits complement activity, whereas FH-related proteins (FHRs) lack a complement regulatory domain. FH and FHRs compete for binding to host cell glycans, in particular heparan sulfates (HS). HS is a glycosaminoglycan with an immense structural variability, where distinct sulfation patterns mediate specific binding of proteins. Mutations in FH, FHRs, or an altered glomerular HS structure may disturb the FH : FHRs balance on glomerular endothelial cells, thereby leading to complement activation and the subsequent development of aHUS/C3G. In this study, we aimed to identify specific HS structures that could specifically compete off FHRs from HS glycocalyx (HSGlx), without interfering with FH binding. FH/FHR binding to human conditionally immortalized glomerular endothelial cells (ciGEnCs) and HSGlx purified from ciGEnC glycocalyx was assessed. HS modifications important for FH/FHR binding to HSGlx were analyzed using selectively desulfated heparins in competition with purified HSGlx. We further assessed effects of heparinoids on FHR1- and FHR5-mediated C3b deposition on ciGEnCs. In the presence of C3b, binding of FH, FHR1 and FHR5 to ciGEnCs was significantly increased, whereas binding of FHR2 was minimal. FHR1 and 5 competitively inhibited FH binding to HSGlx, leading to alternative pathway dysregulation. FHR1 and FHR5 binding was primarily mediated by N-sulfation while FH binding depended on N-, 2-O- and 6-O-sulfation. Addition of 2-O-desulfated heparin significantly reduced FHR1- and FHR5-mediated C3b deposition on ciGEnCs. We identify 2-O-desulfated heparin derivatives as potential therapeutics for C3G and other diseases with dysregulated complement.

Complement dysregulation is characteristic of the renal diseases atypical hemolytic uremic syndrome (aHUS) and complement component 3 glomerulopathy (C3G). Complement regulatory protein Factor H (FH) inhibits complement activity, whereas FH-related proteins (FHRs) lack a complement regulatory domain. FH and FHRs compete for binding to host cell glycans, in particular heparan sulfates (HS). HS is a glycosaminoglycan with an immense structural variability, where distinct sulfation patterns mediate specific binding of proteins. Mutations in FH, FHRs, or an altered glomerular HS structure may disturb the FH : FHRs balance on glomerular endothelial cells, thereby leading to complement activation and the subsequent development of aHUS/C3G. In this study, we aimed to identify specific HS structures that could specifically compete off FHRs from HS glycocalyx (HSGlx), without interfering with FH binding. FH/FHR binding to human conditionally immortalized glomerular endothelial cells (ciGEnCs) and HSGlx purified from ciGEnC glycocalyx was assessed. HS modifications important for FH/FHR binding to HSGlx were analyzed using selectively desulfated heparins in competition with purified HSGlx. We further assessed effects of heparinoids on FHR1- and FHR5-mediated C3b deposition on ciGEnCs. In the presence of C3b, binding of FH, FHR1 and FHR5 to ciGEnCs was significantly increased, whereas binding of FHR2 was minimal. FHR1 and 5 competitively inhibited FH binding to HSGlx, leading to alternative pathway dysregulation. FHR1 and FHR5 binding was primarily mediated by N-sulfation while FH binding depended on N-, 2-O- and 6-O-sulfation. Addition of 2-O-desulfated heparin significantly reduced FHR1- and FHR5-mediated C3b deposition on ciGEnCs. We identify 2-O-desulfated heparin derivatives as potential therapeutics for C3G and other diseases with dysregulated complement. [ABSTRACT FROM AUTHOR]

Heparan sulfate (HS) is a complex polysaccharide abundantly found in extracellular matrices and cell surfaces. HS participates in major cellular processes, through its ability to bind and modulate a wide array of signaling proteins. HS/ligand interactions involve saccharide domains of specific sulfation pattern. Assembly of such domains is orchestrated by a complex biosynthesis machinery and their structure is further regulated at the cell surface by post-synthetic modifying enzymes. Amongst them, extracellular sulfatases of the Sulf family catalyze the selective removal of 6-O-sulfate groups, which participate in the binding of many proteins. As such, increasing interest arose on the regulation of HS biological properties by the Sulfs. However, studies of the Sulfs have so far been essentially restricted to the fields of development and tumor progression. The aim of this review is to survey recent data of the literature on the still poorly documented role of the Sulfs during inflammation, and to widen the perspectives for the study of this intriguing regulatory mechanism toward new physiopathological processes.

Astrocytes are major producers of the extracellular matrix (ECM), which is involved in the plasticity of the developing brain. In utero alcohol exposure alters neuronal plasticity. Glycosaminoglycans (GAGs) are a family of polysaccharides present in the extracellular space; chondroitin sulfate (CS)- and heparan sulfate (HS)-GAGs are covalently bound to core proteins to form proteoglycans (PGs). Hyaluronic acid (HA)-GAGs are not bound to core proteins. In this study we investigated the contribution of astrocytes to CS-, HS-, and HA-GAG production by comparing the makeup of these GAGs in cortical astrocyte cultures and the neonatal rat cortex. We also explored alterations induced by ethanol in GAG and core protein levels in astrocytes. Finally, we investigated the relative expression in astrocytes of CS-PGs of the lectican family of proteins, major components of the brain ECM, in vivo using translating ribosome affinity purification (TRAP) (in Aldh1l1-EGFP-Rpl10a mice. Cortical astrocytes produce low levels of HA and show low expression of genes involved in HA biosynthesis compared to the whole developing cortex. Astrocytes have high levels of chondroitin-0-sulfate (C0S)-GAGs (possibly because of a higher sulfatase enzyme expression) and HS-GAGs. Ethanol upregulates C4S-GAGs as well as brain-specific lecticans neurocan and brevican, which are highly enriched in astrocytes of the developing cortex in vivo. These results begin to elucidate the role of astrocytes in the biosynthesis of CS- HS- and HA-GAGs, and suggest that ethanol-induced alterations of neuronal development may be in part mediated by increased astrocyte GAG levels and neurocan and brevican expression. [ABSTRACT FROM AUTHOR]

Heparan sulfate (HS) is a complex polysaccharide abundantly found in extracellular matrices and cell surfaces. HS participates in major cellular processes, through its ability to bind and modulate a wide array of signaling proteins. HS/ligand interactions involve saccharide domains of specific sulfation pattern. Assembly of such domains is orchestrated by a complex biosynthesis machinery and their structure is further regulated at the cell surface by post-synthetic modifying enzymes. Amongst them, extracellular sulfatases of the Sulf family catalyze the selective removal of 6- O -sulfate groups, which participate in the binding of many proteins. As such, increasing interest arose on the regulation of HS biological properties by the Sulfs. However, studies of the Sulfs have so far been essentially restricted to the fields of development and tumor progression. The aim of this review is to survey recent data of the literature on the still poorly documented role of the Sulfs during inflammation, and to widen the perspectives for the study of this intriguing regulatory mechanism toward new physiopathological processes. [ABSTRACT FROM AUTHOR]

Many mammalian herpes viruses utilize heparan sulfate (HS) moieties present on cell surface proteoglycans as receptors for cell entry, and this process also requires viral glycoprotein C (gC) homologues. However, our understanding of the role of gC in facilitating attachment of other alpha-herpes viruses such as the duck plague virus (DPV) remains preliminary. To study the role of gC during DPV infection, we used a gC-deleted mutant virus (DPV-ΔgC-EGFP). Examination of the viral copy number by real-time PCR, as well as time course studies of viral adsorption and proliferation revealed that gC was involved in the viral binding to the cell surface. The affinity of viral glycoproteins (gB-DPV, gC-DPV, and gE-DPV) to HS was assessed using a prokaryotic expression system and HiTrap™ Heparin HP column chromatography. In addition, to confirm that gC played a role in the interaction between DPV and HS, viruses were treated with the HS analogue heparin and host cells were treated with its inhibitors heparinase prior to exposure to DPV-ΔgC-EGFP or wild-type strain Chinese virulent duck plague virus (DPV-CHv). The effects of heparin and heparinase on virus infectivity demonstrated that function of gC on viral adsorption is independent of interactions between gC and heparin sulfate on cell surface. All in all, this study demonstrated that the gC of DPV can mediate viral adsorption in an HS-independent manner, which distinguish it from the gC of some other alpha-herpes viruses. Future studies will be required to identify the receptors involved in gC protein binding to cells. This work provides us a foundation for further studies of examining the roles of gC in the adsorption during duck plague virus infection.

The polysaccharide heparan sulfate is ubiquitously expressed as a proteoglycan in extracellular matrices and on cell surfaces. In the glomerular filtration barrier, the action of the heparan sulfate is directly related to the function of glomerular filtration, mostly attributed to the sulfated domains that occur along the polysaccharide chain, as evidenced by fact that release of fragments of heparan sulfate by heparanase significantly increases the permeability of albumin passage through the glomerular endothelium, event that originates proteinuria. This review aims to show the importance of the structural domains of heparan sulfate in the process of selective permeability and to demonstrate how these domains may be altered during the glomerular inflammation processes that occur in preeclampsia.

Glycosaminoglycan (GAG), proteoglycans (PG), immune response, inflammation, heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), hyaluronan. [Extracted from the article]

The polysaccharide heparan sulfate is ubiquitously expressed as a proteoglycan in extracellular matrices and on cell surfaces. In the glomerular filtration barrier, the action of the heparan sulfate is directly related to the function of glomerular filtration, mostly attributed to the sulfated domains that occur along the polysaccharide chain, as evidenced by fact that release of fragments of heparan sulfate by heparanase significantly increases the permeability of albumin passage through the glomerular endothelium, event that originates proteinuria. This review aims to show the importance of the structural domains of heparan sulfate in the process of selective permeability and to demonstrate how these domains may be altered during the glomerular inflammation processes that occur in preeclampsia. [ABSTRACT FROM AUTHOR]

Heparan sulfate proteoglycans (HSPGs) are implicated as inflammatory mediators in a variety of settings, including chemokine activation, which is required to recruit circulating leukocytes to infection sites. Heparan sulfate (HS) polysaccharide chains are highly interactive and serve co-receptor roles in multiple ligand:receptor interactions. HS may also serve as a storage depot, sequestering ligands such as cytokines and restricting their access to binding partners. Heparanase, through its ability to fragment HS chains, is a key regulator of HS function and has featured prominently in studies of HS's involvement in inflammatory processes. This review focuses on recent discoveries regarding the role of HSPGs, HS, and heparanase during inflammation, with particular focus on the brain. HS chains emerge as critical go-betweens in multiple aspects of the inflammatory response—relaying signals between receptors and cells. The molecular interactions proposed to occur between HSPGs and the pathogen receptor toll-like receptor 4 (TLR4) are discussed, and we summarize some of the contrasting roles that HS and heparanase have been assigned in diseases associated with chronic inflammatory states, including Alzheimer's disease (AD). We conclude by briefly discussing how current knowledge could potentially be applied to augment HS-mediated events during sustained neuroinflammation, which contributes to neurodegeneration in AD. [ABSTRACT FROM AUTHOR]

Abstract: Background: MicroRNAs are small noncoding RNAs which regulate gene expression at the posttranscriptional level by inducing mRNA degradation or translational repression. MicroRNA-dependent modulation of the extracellular matrix and its cellular receptors has emerged as a novel mechanism of regulating numerous matrix-dependent processes, including cell proliferation and apoptosis, cell adhesion and migration, cell differentiation and stem cell properties. Scope of review: In this review, we will present different mechanisms by which microRNAs and extracellular matrix constituents mutually regulate their expression, and we will demonstrate how these expression changes affect cell behavior. We will also highlight the importance of dysregulated matrix-related microRNA expression for the pathogenesis of inflammatory and malignant disease, and discuss the potential for diagnostic and therapeutic applications. Major conclusions: MicroRNAs and matrix-dependent signal transduction processes form novel regulatory circuits, which profoundly affect cell behavior. As misexpression of microRNAs targeting extracellular matrix constituents is observed in a variety of diseases, a pharmacological intervention with these processes has therapeutic potential, as successfully demonstrated in vitro and in advanced animal models. However, a deeper mechanistic understanding is required to address potential side effects prior to clinical applications in humans. General significance: A full understanding of the role and function of microRNA-dependent regulation of the extracellular matrix may lead to new targeted therapies and new diagnostics for malignant and inflammatory diseases in humans. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. [Copyright &y& Elsevier]

Abstract: Background: Breast cancer–endothelium interactions provide regulatory signals facilitating tumor progression. The endothelial cells have so far been mainly viewed in the context of tumor perfusion and relatively little is known regarding the effects of such paracrine interactions on the expression of extracellular matrix (ECM), proteasome activity and properties of endothelial cells. Methods: To address the effects of breast cancer cell (BCC) lines MDA-MB-231 and MCF-7 on the endothelial cells, two cell culture models were utilized; one involves endothelial cell culture in the presence of BCCs-derived conditioned media (CM) and the other co-culture of both cell populations in a Transwell system. Real-time PCR was utilized to evaluate gene expression, an immunofluorescence assay for proteasome activity, and functional assays (migration, adhesion and invasion) and immunofluorescence microscopy for cell integrity and properties. Results: BCC-CM decreases the cell migration of HUVEC. Adhesion and invasion of BCCs are favored by HUVEC and HUVEC-CM. HA levels and the expression of CD44 and HA synthase-2 by HUVEC are substantially upregulated in both cell culture approaches. Adhesion molecules, ICAM-1 and VCAM-1, are also highly upregulated, whereas MT1-MMP and MMP-2 expressions are significantly downregulated in both culture systems. Notably, the expression and activity of the proteasome β5 subunit are increased, especially by the action of MDA-MB-231-CM on HUVEC. Conclusions and general significance: BCCs significantly alter the expression of matrix macromolecules, proteasome activity and functional properties of endothelial cells. Deep understanding of such paracrine interactions will help to design novel drugs targeting breast cancer at the ECM level. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. [Copyright &y& Elsevier]

Abstract: Background: The solid melanoma tumor consists of transformed melanoma cells, and the associated stromal cells including fibroblasts, endothelial cells, immune cells, as well as, soluble macro- and micro-molecules of the extracellular matrix (ECM) forming the complex network of the tumor microenvironment. Heparan sulfate proteoglycans (HSPGs) are an important component of the melanoma tumor ECM. Importantly, there appears to be both a quantitative and a qualitative shift in the content of HSPGs, in parallel to the nevi–radial growth phase–vertical growth phase melanoma progression. Moreover, these changes in HSPG expression are correlated to modulations of key melanoma cell functions. Scope of review: This review will critically discuss the roles of HSPGs/heparin in melanoma development and progression. Major conclusions: We have correlated HSPGs' expression and distribution with melanoma cell signaling and functions as well as angiogenesis. General significance: The current knowledge of HSPGs/heparin biology in melanoma provides a foundation we can utilize in the ongoing search for new approaches in designing anti-tumor therapy. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. [Copyright &y& Elsevier]

Binding to negatively charged heparan sulfates (HS) at the cell surface is considered the first step in the internalization of cationic cell-penetrating peptides (CPPs). However, little is known about the relation of the characteristics of the HS-CPP interaction such as affinity, stoichiometry, and clustering with uptake. In this study, we investigated a collection of mutants of a cyclic CPP derived from human lactoferrin with respect to HS binding and uptake. The thermodynamic parameters of HS binding were determined by isothermal titration calorimetry, clustering of HS was investigated by dynamic light scattering, and cellular uptake by flow cytometry and confocal microscopy. Whereas mutations of non-arginine amino acids that are conserved across lactoferrins of different mammalia only had a minor effect on uptake efficiency, changes in the number of arginine residues influenced the uptake significantly. In general, introduction of arginine residues and cyclization improved the HS affinity and the ability to cluster HS. In particular, there was a strong negative correlation between stoichiometry and uptake, indicating that crosslinking of HS is the driving force for the uptake of arginine-rich CPPs. Using glycan microarrays presenting a collection of synthetic HS, we show that a minimal chain length of HS is required for peptide binding. [ABSTRACT FROM AUTHOR]

Highlights: [•] A simple fluorimetric heparanase activity assay using the synthetic sulfated pentasaccharide fondaparinux as substrate. [•] The assay was used for testing potential heparanase inhibitors. [•] The inhibitory activity of sulfated glycans showed to be strongly dependent on their degree of sulfation as well as their basic glycan structure, but independent of their molecular mass. [•] The semisynthetic β-1,3-glucan sulfate PS3 showed the strongest inhibitory effect with an IC50 being 160 times lower than that of suramin. [ABSTRACT FROM AUTHOR]

Highlights: [•] Glycosaminoglycans (GAGs) were isolated from the lumpsucker fish, Cyclopterus lumpus. [•] Two key GAG components were isolated by ion-exchange and preparative chromatography. [•] The structures were characterised by analytical techniques and confirmed by NMR as HA and CD/DS. [•] Biological activity of the polymers was determined and is in keeping with the low sulfate content. [•] The potential commercial use of lumpsucker by-catch, as a new source of HA and CS, will be explored. [ABSTRACT FROM AUTHOR]

Background: Mucopolysaccharidoses (MPSs) are a group of lysosomal storage diseases caused by the deficiency/absence of enzymes which catalyze the degradation of glycosaminoglycans (GAGs). The use of biological samples dried on filter paper has been increasing because it makes it easy to ship them to reference laboratories. Urinary GAGs are the main biomarkers of MPS and, thus, we studied the correlations of determinations to GAGs and creatinine, as well as compared the GAGs' profile on electrophoresis, between urine and dried urine in filter paper (DUFP) samples. We also assessed the GAG stability over time under different storage temperatures. Methods: We quantified the GAG concentration in both sample types and compared the results by Pearson correlation. Results: The results were very similar, with r=0.97 for creatinine and with r=0.94 and r=0.98 for GAGs for controls and patients, respectively, with similar electrophoretic profiles. The GAG stability in DUFP was up to 30days at −20, 4, and 25°C and up to 21days at 37°C. Conclusion: Our proposal assessed urinary GAGs in DUFP and concluded that these samples can be used in the investigation of MPS, replacing urine samples in neonatal screening and monitoring of therapies, due to ease of transportation and storage. [ABSTRACT FROM AUTHOR]

Highlights: [•] SS is an autoimmune disease with unknown etiology. [•] There is evidence for innate immune activation in early SS disease. [•] Defects in salivary gland development are implicated in disease development. [•] TLR activation in salivary and lacrimal tissue may contribute to SS pathogenesis. [•] Understanding early events in SS will improve diagnosis and therapy for SS patients. [ABSTRACT FROM AUTHOR]

Most research strategies for cartilage tissue engineering use extended culture with complex media loaded with costly GFs (growth factors) to drive tissue assembly and yet they result in the production of cartilage with inferior mechanical and structural properties compared with the natural tissue. Recent evidence suggests that GAGs (glycosaminoglycans) incorporated into tissue engineering scaffolds can sequester and/or activate GFs and thereby more effectively mimic the natural ECM (extracellular matrix). Such approaches may have potential for the improvement of cartilage engineering. However, natural GAGs are structurally complex and heterogeneous, making structure-function relationships hard to determine and clinical translation difficult. Importantly, subfractions of GAGs with specific chain lengths and sulfation patterns have been shown to activate key signalling processes during stem cell differentiation. In addition, recently, GAGs have been bound to synthetic biomaterials, such as electrospun scaffolds and hydrogels, in biologically active conformations, and methods to purify and select affinity-matched GAGs for specific GFs have also been developed. The identification and use of specific GAG moieties to promote chondrogenesis is therefore an exciting new avenue of research. Combining these with synthetic biomaterials may allow a more effective mimicry of the natural ECM, reduction in the need for expensive GFs, and perhaps the deposition of an articular cartilage-like matrix in a clinically relevant manner. [ABSTRACT FROM AUTHOR]

Abstract: Human circulating endothelial progenitor cells isolated from peripheral blood generate in culture cells with features of endothelial cells named late-outgrowth endothelial colony-forming cells (ECFC). In adult blood, ECFC display a constant quantitative and qualitative decline during life span. Even after expansion, it is difficult to reach the cell dose required for cell therapy of vascular diseases, thus limiting the clinical use of these cells. Glycosaminoglycans (GAG) are components from the extracellular matrix (ECM) that are able to interact and potentiate heparin binding growth factor (HBGF) activities. According to these relevant biological properties of GAG, we designed a GAG mimetic having the capacity to increase the yield of ECFC production from blood and to improve functionality of their endothelial outgrowth. We demonstrate that the addition of [OTR4131] mimetic during the isolation process of ECFC from Cord Blood induces a 3 fold increase in the number of colonies. Moreover, addition of [OTR4131] to cell culture media improves adhesion, proliferation, migration and self-renewal of ECFC. We provide evidence showing that GAG mimetics may have great interest for cell therapy applied to vascular regeneration therapy and represent an alternative to exogenous growth factor treatments to optimize potential therapeutic properties of ECFC. [Copyright &y& Elsevier]

Abstract: Hepatocellular carcinoma (HCC) is resistant to conventional chemotherapy, and is rarely amenable to radiotherapy. Heparanase, enzyme attacks heparan sulfate proteoglycans (HSPGs), is preferentially expressed in human tumors and its overexpression in low-metastatic tumor confers a highly invasive phenotype in experimental animals. Meanwhile, high doses of suramin dramatically increase tissue glycosaminoglycans due, in part, to inhibition of heparanase enzymes. Therefore, the following study was conducted to evaluate the chemopreventive and hepatoprotective effects of suramin in in-vivo model of HCC. Therefore, HCC was induced in SD rats by thioacetamide (200mg/kg) in presence/absence of suramin (20mg/kg). Liver impairment was assessed by measuring serum ?-fetoprotein and investigating liver sections stained with Hematoxylin/Eosin. Hepatic HSPGs and heparanse were measured by ELISA. Glucosamine and glucuronic acid were measured by chemical methods. Gene expression of fibroblast growth factor (FGF)-2 and caspase-3 was measured. Apoptotic pathway was evaluated by measuring the activity of caspase-3/8/9. Suramin increased the animal survival and decreased serum ?-fetoprotein. In addition, suramin ameliorated fibrosis and massive hepatic tissue breakdown. Suramin restored hepatic HSPGs and reduced the activity of hepatic heparanase leading to decreased hepatic levels of glucosamine and glucuronic acid. Moreover, suramin reduced the gene expression of FGF-2 and caspase-3. Finally, suramin blocked the elevated activity of caspase-3/8/9. In conclusion, surmain showed antitumor activity as well as hepatoprotective effects. Besides its antioxidant activity, other mechanisms are involved including restoration of HSPGs and inhibition of heparanase and FGF-2. Suramin inhibits intrinsic and extrinsic apoptotic pathway. Targeting HSPGs expression is potential therapeutic target for HCC. [Copyright &y& Elsevier]

Abstract: Impaired heparan sulfate (HS) synthesis in vertebrate development causes complex malformations due to the functional disruption of multiple HS-binding growth factors and morphogens. Here, we report developmental heart defects in mice bearing a targeted disruption of the HS-generating enzyme GlcNAc N-deacetylase/GlcN N-sulfotransferase 1 (NDST1), including ventricular septal defects (VSD), persistent truncus arteriosus (PTA), double outlet right ventricle (DORV), and retroesophageal right subclavian artery (RERSC). These defects closely resemble cardiac anomalies observed in mice made deficient in the cardiogenic regulator fibroblast growth factor 8 (FGF8). Consistent with this, we show that HS-dependent FGF8/FGF-receptor2C assembly and FGF8-dependent ERK-phosphorylation are strongly reduced in NDST1−/− embryonic cells and tissues. Moreover, WNT1-Cre/LoxP-mediated conditional targeting of NDST function in neural crest cells (NCCs) revealed that their impaired HS-dependent development contributes strongly to the observed cardiac defects. These findings raise the possibility that defects in HS biosynthesis may contribute to congenital heart defects in humans that represent the most common type of birth defect. [Copyright &y& Elsevier]

Abstract: In hormone-dependent breast cancer, estrogen receptors are the principal signaling molecules that regulate several cell functions either by the genomic pathway acting directly as transcription factors in the nucleus or by the non-genomic pathway interacting with other receptors and their adjacent pathways like EGFR/IGFR. It is well established in literature that EGFR and IGFR signaling pathways promote cell proliferation and differentiation. Moreover, recent data indicate the cross-talk between ERs and EGFR/IGFR signaling pathways causing a transformation of cell functions as well as deregulation on normal expression pattern of matrix molecules. Specifically, proteoglycans, a major category of extracellular matrix (ECM) and cell surface macromolecules, are modified during malignancy and cause alterations in cancer cell signaling, affecting eventually functional cell properties such as proliferation, adhesion and migration. The on-going strategies to block only one of the above signaling effectors result cancer cells to overcome such inactivation using alternative signaling pathways. In this article, we therefore review the underlying mechanisms in respect to the role of ERs and the involvement of cross-talk between ERs, IGFR and EGFR in breast cancer cell properties and expression of extracellular secreted and cell bound proteoglycans involved in cancer progression. Understanding such signaling pathways may help to establish new potential pharmacological targets in terms of using ECM molecules to design novel anticancer therapies. [Copyright &y& Elsevier]

Abstract: Heparan sulfate proteoglycans (HSPGs), expressed on the cell surface and in the extracellular matrix of most animal tissues, have essential functions in development and homeostasis, and have been implicated in several pathological conditions. The functions of HSPGs are mainly mediated through interactions of the heparan sulfate (HS) polysaccharide side chains with different protein ligands. The molecular structure of HS is highly diverse, expressed in a cell-type specific manner. The flexible yet controlled structure of HS is primarily generated through a strictly regulated biosynthesis process and is further modified post-synthetically, such as desulfation by endosulfatases and fragmentation by heparanase. Heparanase is an endo-glucuronidase expressed in all tissues. The enzyme has been found up-regulated in a number of pathological conditions, implying a role in diseases mainly through degradation of HS. Emerging evidence demonstrates important roles of HS and heparanase in inflammatory reactions, particularly in the regulation of leukocyte activation and extravasation. Neuroinflammation is a common feature of various central nervous system disorders, thus it is a great interest to understand the implications of HS and heparanase in neuroinflammation. [Copyright &y& Elsevier]

Abstract: Smooth muscle cell proliferation can be inhibited by heparan sulfate proteoglycans whereas the removal or digestion of heparan sulfate from perlecan promotes their proliferation. In this study we characterized the glycosaminoglycan side chains of perlecan isolated from either primary human coronary artery smooth muscle or endothelial cells and determined their roles in mediating cell adhesion and proliferation, and in fibroblast growth factor (FGF) binding and signaling. Smooth muscle cell perlecan was decorated with both heparan sulfate and chondroitin sulfate, whereas endothelial perlecan contained exclusively heparan sulfate chains. Smooth muscle cells bound to the protein core of perlecan only when the glycosaminoglycans were removed, and this binding involved a novel site in domain III as well as domain V/endorepellin and the α2β1 integrin. In contrast, endothelial cells adhered to the protein core of perlecan in the presence of glycosaminoglycans. Smooth muscle cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains and promoted the signaling of FGF2 but not FGF1. Also endothelial cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains, but in contrast, promoted the signaling of both growth factors. Based on this differential bioactivity, we propose that perlecan synthesized by smooth muscle cells differs from that synthesized by endothelial cells by possessing different signaling capabilities, primarily, but not exclusively, due to a differential glycanation. The end result is a differential modulation of cell adhesion, proliferation and growth factor signaling in these two key cellular constituents of blood vessels. [Copyright &y& Elsevier]

Abstract: Numerous extracellular proteins, growth factors, chemokines, cytokines, enzymes, lipoproteins, involved in a variety of biological processes, interact with heparin and/or heparan sulfate at the cell surface and in the extracellular matrix (ECM). The goal of this study is to investigate the relationship(s) between affinity and kinetics of heparin–protein interactions and the localization of the proteins, their intrinsic disorder and their biological roles. Most proteins bind to heparin with a higher affinity than their fragments and form more stable complexes with heparin than with heparan sulfate. Lipoproteins and matrisome-associated proteins (e.g. growth factors and cytokines) bind to heparin with very high affinity. Matrisome-associated proteins form transient complexes with heparin. However they bind to this glycosaminoglycan with a higher affinity than the proteins of the core matrisome, which contribute to ECM assembly and organization, and than the secreted proteins which are not associated with the ECM. The association rate of proteins with heparin is related to the intrinsic disorder of heparin-binding sites. Enzyme inhibitor activity, protein dimerization, skeletal system development and pathways in cancer are functionally associated with proteins displaying a high or very high affinity for heparin (K D <100nM). Besides their use in investigating molecular recognition and functions, kinetics and affinity are essential to prioritize interactions in networks and to build network models as discussed for the interaction network established at the surface of endothelial cells by endostatin, a heparin-binding protein regulating angiogenesis. [Copyright &y& Elsevier]

Abstract: The gene products of two members of the EXT gene family, EXT1 and EXT2, function together as a polymerase in the biosynthesis of heparan sulfate. EXTL2, one of the three EXT-like genes in the human genome that are homologous to EXT1 and EXT2, encodes an N-acetylhexosaminyltransferase. However, both the role of EXTL2 in glycosaminoglycan (GAG) biosynthesis and the biological significance of EXTL2 remain unclear. Interestingly, EXTL2 can transfer a GlcNAc residue to the tetrasaccharide linkage region when this region is phosphorylated by a xylose kinase 1 (FAM20B) and thereby terminate chain elongation. Production of GAGs was significantly higher in EXTL2-knockout mice than in wild-type mice. EXTL2-knockout mice are viable and apparently healthy during development and after birth. Therefore, EXTL2-knockout mice were analyzed following the experimental induction of two separate pathological conditions. Carbon tetrachloride (CCl4) was used to induce liver failure, and 5/6th nephrectomy in combination with a high-phosphate diet was used to induce chronic kidney disease (CKD). Under conditions of CCl4-induced liver failure, hepatocyte proliferation following CCl4 treatment was lower in EXTL2-knockout mice than in wild-type mice; consequently, liver regeneration was impaired in EXTL2-knockout mice. This reduction in hepatocyte proliferation resulted partially because EXTL2-knockout mice experienced less hepatocyte-growth-factor-mediated signaling than did wild-type mice. Under conditions of induced CKD, matrix mineralization in vascular smooth muscle cells (VSMCs) in aortic rings of EXTL2-knockout mice was enhanced relative to that in wild-type mice. Altered biosynthesis of GAGs in EXTL2-knockout mice affected bone-morphogenetic-protein signaling, and consequently enhanced the differentiation of VSMCs into osteoblasts. Taken together, these results indicated that the EXTL2-dependent mechanism that regulates GAG biosynthesis is important for the maintenance of tissue homeostasis under pathological conditions, that is, lack of EXTL2 causes GAG overproduction and structural changes of GAGs associated with pathological processes. [Copyright &y& Elsevier]

Abstract: In the review article presented here, we demonstrate that the connective tissue is more than just a matrix for cells and a passive scaffold to provide physical support. The extracellular matrix can be subdivided into proteins (collagen, elastin), glycoconjugates (structural glycoproteins, proteoglycans) and glycosaminoglycans (hyaluronan). Our main focus rests on the anionic biopolyelectrolytes of the perlecan/syndecan superfamily which belongs to extracellular matrix and cell membrane integral proteoglycans. Though the extracellular domain of the syndecans may well be performing a structural role within the extracellular matrix, a key function of this class of membrane intercalated proteoglycans may be to act as signal transducers across the plasma membrane and thus be more appropriately included in the group of cell surface receptors. Nevertheless, there is a continuum in functions of syndecans and perlecans, especially with respect to their structural role and biomedical significance. HS/CS proteoglycans are receptor sites for lipoprotein binding thus intervening directly in lipid metabolism. We could show that among all lipoproteins, HDL has the highest affinity to these proteoglycans and thus instals a feedforward forechecking loop against atherogenic apoB100 lipoprotein deposition on surface membranes and in subendothelial spaces. Therefore, HDL is not only responsible for VLDL/IDL/LDL cholesterol exit but also controls thoroughly the entry. This way, it inhibits arteriosclerotic nanoplaque formation. The ternary complex ‘lipoprotein receptor (HS/CS-PG) – lipoprotein (LDL, oxLDL, Lp(a)) – calcium’ may be interpreted as arteriosclerotic nanoplaque build-up on the molecular level before any cellular reactivity, possibly representing the arteriosclerotic primary lesion combined with endothelial dysfunction. With laser-based ellipsometry we could demonstrate that nanoplaque formation is a Ca2+-driven process. In an in vitro biosensor application of HS-PG coated silica surfaces we tested nanoplaque formation and size in clinical trials with cardiovascular high-risk patients who underwent treatment with ginkgo or fluvastatin. While ginkgo reduced nanoplaque formation (size) by 14.3% (23.4%) in the isolated apoB100 lipid fraction at a normal blood Ca2+ concentration, the effect of the statin with a reduction of 44.1% (25.4%) was more pronounced. In addition, ginkgo showed beneficial effects on several biomarkers of oxidative stress and inflammation. Besides acting as peripheral lipoprotein binding receptor, HS/CS-PG is crucially implicated in blood flow sensing. A sensor molecule has to fulfil certain mechanochemical and mechanoelectrical requirements. It should possess viscoelastic and cation binding properties capable of undergoing conformational changes caused both mechanically and electrostatically. Moreover, the latter should be ion-specific. Under no-flow conditions, the viscoelastic polyelectrolyte at the endothelium — blood interface assumes a random coil form. Blood flow causes a conformational change from the random coil state to the directed filament structure state. This conformational transition effects a protein unfurling and molecular elongation of the GAG side chains like in a ‘stretched’ spring. This configuration is therefore combined with an increase in binding sites for Na+ ions. Counterion migration of Na+ along the polysaccharide chain is followed by transmembrane Na+ influx into the endothelial cell and by endothelial cell membrane depolarization. The simultaneous Ca2+ influx releases NO and PGI2, vasodilatation is the consequence. Decrease in flow reverses the process. Binding of Ca2+ and/or apoB100 lipoproteins (nanoplaque formation) impairs the flow sensor function. The physicochemical and functional properties of proteoglycans are due to their amphiphilicity and anionic polyelectrolyte character. Thus, they potently interact with cations, albeit in a rather complex manner. Utilizing 23Na+ and 39 K+ NMR techniques, we could show that, both in HS-PG solutions and in native vascular connective tissue, the mode of interaction for monovalent cations is competition. Mg2+ and Ca2+ ions, however, induced a conformational change leading to an increased allosteric, cooperative K+ and Na+ binding, respectively. Since extracellular matrices and basement membranes form a tight-fitting sheath around the cell membrane of muscle and Schwann cells, in particular around sinus node cells of the heart, and underlie all epithelial and endothelial cell sheets and tubes, a release of cations from or an adsorption to these polyanionic macromolecules can transiently lead to fast and drastic activity changes in these tiny extracellular tissue compartments. The ionic currents underlying pacemaker and action potential of sinus node cells are fundamentally modulated. Therefore, these polyelectrolytic ion binding characteristics directly contribute to and intervene into heart rhythm. [Copyright &y& Elsevier]

Abstract: Adipogenesis depends on growth factors controlling proliferation/differentiation of mesenchymal stem cells (MSCs). Membrane binding and endocytosis of growth factors are often coupled to receptor activation and downstream signaling leading to specific cellular responses. The novel adipokine tartrate-resistant acid phosphatase (TRAP) 5a exhibits a growth factor-like effect on MSCs and pre-adipocytes and induces hyperplastic obesity in vivo. However its molecular interaction with pre-adipocytes remains unknown. Therefore, this study aimed to investigate membrane interaction of TRAP and its endocytosis routes in pre-adipocytes. Confocal and/or electron microscopy were used to detect TRAP in untreated or TRAP 5a/b treated pre-adipocytes under conditions that allow or inhibit endocytosis in combination with co-staining of endocytotic vesicles. TRAP interaction with heparin/heparan sulfate was verified by gel filtration. It could be shown that TRAP 5a, but not 5b, binds to the membrane of pre-adipocytes where it co-localizes with heparin-sulfate proteoglycan glypican-4. Also in vitro, TRAP 5a exhibited affinity for both heparin and heparan sulfate with heparin inhibiting its enzyme activity. Upon caveolae-mediated endocytosis of saturating levels of TRAP 5a, TRAP 5a co-localized intracellularly with glypican-4 and late endosomal marker Rab-7 positive vesicles. The protein was also located in multivesicular bodies (MVBs) but did not co-localize with lysosomal marker LAMP-1. TRAP 5a endocytosis was also detectable in pre-osteoblasts, but not fibroblasts, embryonic MSCs or mature adipocytes. These results indicate that TRAP 5a exhibits binding to cell surface, endocytosis and affinity to glucosaminoglycans (GAGs) in pre-adipocyte and pre-osteoblast lineage cells in a manner similar to other heparin-binding growth factors. [Copyright &y& Elsevier]

Highlights: [•] In a clinical trial, metabolic syndrome (MS) patients were treated with Ginkgo biloba. [•] Ellipsometry, photometric methods and ELISAs were applied for biosensor profiling of MS risk, status and treatment outcome. [•] After medication, biomarkers of oxidative stress, inflammation and arteriosclerosis were significantly diminished. [•] Multiple correlations unraveled the network of biomarker interactions and demonstrated its usefulness in personalized medicine. [Copyright &y& Elsevier]

Abstract: Roundabout 1 (Robo1) is the cognate receptor for secreted axon guidance molecule, Slits, which function to direct cellular migration during neuronal development and angiogenesis. The Slit2–Robo1 signaling is modulated by heparan sulfate, a sulfated linear polysaccharide that is abundantly expressed on the cell surface and in the extracellular matrix. Biochemical studies have further shown that heparan sulfate binds to both Slit2 and Robo1 facilitating the ligand–receptor interaction. The structural requirements for heparan sulfate interaction with Robo1 remain unknown. In this report, surface plasmon resonance (SPR) spectroscopy was used to examine the interaction between Robo1 and heparin and other GAGs and determined that heparin binds to Robo1 with an affinity of ∼650 nM. SPR solution competition studies with chemically modified heparins further determined that although all sulfo groups on heparin are important for the Robo1–heparin interaction, the N-sulfo and 6-O-sulfo groups are essential for the Robo1–heparin binding. Examination of differently sized heparin oligosaccharides and different GAGs also demonstrated that Robo1 prefers to bind full-length heparin chains and that GAGs with higher sulfation levels show increased Robo1 binding affinities. [Copyright &y& Elsevier]

Abstract: FGFs, in a complex with their receptors (FGFRs) and heparan sulfate (HS), are responsible for a range of cellular functions, from embryogenesis to metabolism. Both germ line and somatic FGFR mutations are known to play a role in a range of diseases, most notably craniosynestosis dysplasias, dwarfism and cancer. Because of the ability of FGFR signalling to induce cell proliferation, migration and survival, FGFRs are readily co-opted by cancer cells. Mutations in, and amplifications of, these receptors are found in a range of cancers with some of the most striking clinical findings relating to their contribution to pathogenesis and progression of female cancers. Here, we outline the molecular mechanisms of FGFR signalling and discuss the role of this pathway in women's cancers, focusing on breast, endometrial, ovarian and cervical carcinomas, and their associated preclinical and clinical data. We also address the rationale for therapeutic intervention and the need for FGFR-targeted therapy to selectively target cancer cells in view of the fundamental roles of FGF signalling in normal physiology. [Copyright &y& Elsevier]

Abstract: The tetrasaccharide heparan sulfate (HS) mimetic PG545, a clinical anti-cancer candidate, is an inhibitor of the HS-degrading enzyme heparanase. The kinetics of heparanase inhibition by PG545 and three structural analogues were investigated to understand their modes of inhibition. The cholestanol aglycon of PG545 significantly increased affinity for heparanase and also modified the inhibition mode. For the tetrasaccharides, competitive inhibition was modified to parabolic competition by the addition of the cholestanol aglycon. For the trisaccharides, partial competitive inhibition was modified to parabolic competition. A schematic model to explain these findings is presented. [Copyright &y& Elsevier]

Abstract: Recently, we have disclosed that human KIAA1199 (hKIAA1199) is a hyaluronan (HA) binding protein implicated in HA depolymerization. Although a murine homologue (mKiaa1199) was previously cloned, no information about the function of the molecule was available. Here, we show that cells transfected with mKiaa1199 cDNA selectively catabolized HA via the clathrin-coated pit pathway. A glycosaminoglycan-binding assay demonstrated the specific binding of mKiaa1199 to HA. These results were similar to our observations with hKIAA1199, although slight differences were found in the peak sizes of the minimum degradates of HA. We conclude that like hKIAA1199, mKiaa1199 is a hyaladherin, leading to HA depolymerization. [Copyright &y& Elsevier]

Highlights: [•] Four synthetic mimics of bovine bactenecin were analyzed for anti-HSV-2 properties. [•] All four peptides blocked viral attachment and entry in vitro. [•] Two peptides, 1018 and HH-2, blocked genital HSV-2 infection in mice when admixed with virus. [Copyright &y& Elsevier]

Highlights: [•] Plasmatic galactosaminoglycans were determined by a highly sensitive HPLC. [•] Plasmatic galactosaminoglycans decrease significantly with age in healthy. [•] Patients all presented higher significantly galactosaminoglycans than healthy. [•] Plasmatic galactosaminoglycans may be useful markers for mucopolysaccharidoses. [ABSTRACT FROM AUTHOR]

Highlights: [•] ROS and oxidative stress affect cancer progression. [•] ROS participate in the remodeling of tumor/stroma ECM. [•] ECM-tumor cell interface is moulded by ROS. [•] ROS activate stroma cells. [•] ECM remodeling and tumor escape. [ABSTRACT FROM AUTHOR]