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Clinical manipulation of angiogenesis (the formation of new blood vessels from pre-existing vasculature) is of interest to treat diseases such as cancer and ischemic tissue where it is not properly regulated. Several treatments targeting vascular endothelial growth factor (VEGF) and its receptors - which are abundant at sites of angiogenesis - are currently in use to treat various types of cancer, however they have severe vascular side effects. Conversely, VEGF has been used clinically to promote angiogenesis to treat ischemic tissue. However, despite encouraging data from pre-clinical models, trials in humans have been disappointing. For further therapies to be developed, more information on how VEGF interacts with its receptors is required. Heparan sulfate (HS) is a ubiquitous glycosaminoglycan involved in a number of physiological processes including angiogenesis. HS facilitates the interaction of VEGF with its receptors, which is crucial for angiogenesis. Modification of this interaction via synthetic mimetics of HS may allow clinical intervention of angiogenesis. The current investigation aims first, to clarify the requirement for the interaction between VEGF and HS in angiogenesis; second to characterise the structure of HS that binds to VEGF so that mimetics can be developed; and third, to determine the effect of HS mimetics on angiogenesis in vivo. To determine the requirement for VEGF/HS interaction in angiogenesis, several mutants of VEGF165 that had lower affinities for HS were assayed for their ability to induce ectopic angiogenesis in the subintestinal baskets of zebrafish embryos. Wild type VEGF165 induced a 200-250% increase in ectopic vessels, which was matched only by a control mutant. Other mutants did not induce ectopic vessels, suggesting that this interaction is required for angiogenesis. To characterise the structure of HS that binds to VEGF, various HS mimetics were assayed against heparin in a VEGF competition assay using Biacore. Of these, the strongest inhibition (IC¬50 =~16nM) was with 2O10, an oligosaccharide that consisted of two highly sulfated octasaccharide domains (NS domains) that flanked an unsulfated dodecasaccharide region. To determine the type of sulfation required for this interaction, HS fragments were assayed for interaction with VEGF165 using the filter binding assay, and analysed by HPLC which indicated 6-O sulfation may be preferential for VEGF binding to HS.To investigate the ability of HS to affect angiogenesis, the effects of HS mimetics on zebrafish embryo subintestinal baskets were measured. The most interesting of these was with 2O10, which had a biphasic response whereby low doses (3ng) increased basket vasculature by 30% and high doses (30ng) decreased the endogenous vessels by 20%. As 2O10 had a high affinity for VEGF, its effects on the vasculature may be due to interaction with endogenous VEGF, which would indicate that HS mimetics can be used to control angiogenesis by modification of growth factor signalling. The investigation concludes that the interaction between VEGF and HS is critical for angiogenesis, and that this can be modulated by the application of HS mimetics that bind strongly to VEGF.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The Y402H polymorphism in complement factor H (CFH) is a common and important risk factor, where CFH is an inhibitor of the alternative complement pathway. The disease-associated protein variant (CFH402H) binds poorly to aged human macular Bruch’s membrane (BM), a site of AMD formation. Heparan sulphate (HS) is the major binding site for CFH in this extracellular matrix. Unlike CFH402Y, CFH402H binds poorly to lowly sulphated HS. The aim of this research was to investigate the presence and distribution of proteoglycan (PG) core proteins and glycosaminoglycans (GAGs) in the normal adult human macula, and to analyse potential changes with age in the quantity and composition of HS and other potential molecular determinants of disease in BM. Post mortem human eye tissue was obtained from consenting donors (age range 18-93 years), and either dissected into tissue layers or used to produce frozen macular tissue sections. Proteomic analysis of different retinal tissue layers was performed by tandem mass spectrometry. Immunofluorescence microscopy was undertaken on the macular tissue sections. Compositional analysis of HS in BM was performed by 2-aminoacridone labelling of HS disaccharides and reverse phase high performance liquid chromatography against reference HS disaccharide standards. PG core proteins were identified in BM and other macular tissue layers, including members of the basement membrane, hyalectan and short leucine-rich repeat PG families. HS, chondroitin sulphate, dermatan sulphate and hyaluronan were present throughout the retina and choroid, but keratan sulphate only in the sclera. The mean quantity of HS in BM was 47% lower (p=0.006) in old donors (n=13, 64-92 years), compared to young donors (n=6; 26-39 years). The mean level of HS sulphation was also lower in old donors, e.g. 34% vs. 39% (p=0.02) N-sulphated HS. The mean level of HS in macular BM by immunohistochemistry was approximately 50% lower (p=0.02) in old donors (n=10, 18-93 years), and the mean level of the HS PG core protein perlecan was reduced by 85% (p=0.01; n=18, 27-90 years). High levels of complement activation (C3b and membrane attack complex) were observed in some young donors. Reduced HS was associated with increased complement activation in some donors (r2 0.30). A combination of proteomics and immunohistochemistry approaches has provided the first comprehensive analysis of the presence and distribution of PG core proteins and their associated GAG chains throughout the macular layers of the normal adult human retina. These demonstrate a differential distribution according to PG core protein, GAG class and GAG sulphation state. The quantity of HS decreases substantially with age in human BM, and its sulphation level also decreases. The presence of less HS in old BM would make fewer binding sites available for CFH, and could contribute to AMD pathogenesis through increased complement activation. This idea is supported by the observation that reduced HS is associated in some individuals with increased C3b in BM. These findings have important implications for unravelling mechanisms of ocular disease and planning novel therapeutic strategies, particularly in the case of AMD.

Ageing poses one of the largest risk factors for the development of cardiovascular disease (CVD). The increased propensity towards vascular pathology with advancing age maybe explained, in part, by a reduction in the ability of circulating endothelial progenitor cells (EPCs) to contribute to vascular repair and regeneration. Among all current putative EPC populations, outgrowth endothelial cells (OECs) display the most features consistent with a human postnatal vasculogenic cell. Cell-surface heparan sulfate (HS) proteoglycans, by virtue of specific sulfated domains within the glycosaminoglycan chain, are able to bind and modulate the activities of a variety of proteins important for EPC mobilisation, homing and function at sites requiring neovascularization. This study aimed to determine if human OEC function is impaired with age, and to ascertain whether this is accompanied by changes in the fine structure of OEC HS.Using in vitro cell culture methods, OECs were isolated from healthy subjects across an age range and cell phenotype was verified by the demonstration of numerous endothelial, but not hematopoietic, cell characteristics. The functional capacity of peripheral blood derived OECs from young and old subjects, and comparative cord blood derived OECs, was assessed in terms of their susceptibility to apoptosis, proliferative, migratory and tube-forming capabilities. In vitro scratch and transwell migration assays revealed that the migratory capacity of peripheral blood derived OECs isolated from old subjects was impaired in comparison to those from young subjects and cord blood derived OECs. Structural analysis of HS by high performance liquid chromatography (HPLC) demonstrated a significant reduction in the relative percentage of the trisulfated disaccharide, 2-O-sulfated-uronic acid, N, 6-O-sulfated-glucosamine (UA[2S]-GlcNS[6S]), within OEC HS with age (r = -0.847, p=<0.01). Moreover, a decline in the migratory response of OECs towards a gradient of VEGF significantly correlated with the percentage expression of this disaccharide (r = 0.840, p<0.01). Disruption of cell surface HS by pre-treatment with heparinase I and III was found to significantly reduce the VEGF-induced migratory response of peripheral blood derived OECs isolated from young subjects to levels similar to that observed for OECs from older individuals. Understanding the role of HS in regulating the directional migration of EPCs to sites requiring neovascularization and developing approaches to facilitate EPC migration may aid in the design of more successful strategies to optimise the regenerative capacity of these cells in the ageing vasculature.

Heparan sulphate (HS) is critical for embryonic development with involvement in a myriad of biological processes, centrally mediating morphogenic movements and facilitating the specification and differentiation of tissues. Complicated by its structural micro-heterogeneity along with expression on numerous different proteoglycan cores, the plethora of roles for HS in biology and their underlying mechanisms have not yet been fully defined. The discovery and characterisation of new reagents and methods for modification of HS expression and/or structure will aid efforts in elucidating the structure and activity of this glycosaminoglycan. Until now, azido sugars have been utilised as labelling reagents for various types of glycosylation, including N-glycans, O-linked mucin-type glycosylation and O-GlcNAcetylation of proteins. Incorporation of the unnatural azido sugar into the glycan of interest inserts a chemically reactive abiotic azide for subsequent detection via Staudinger ligation or click chemistries. However, to our knowledge, application of these azido sugars has not been explored for glycosaminoglycans. A metabolic labelling approach using Ac4GalNAz yields UDP-GalNAz and UDP-GlcNAz (Boyce et al., 2011), ready to target CS/DS and HS, respectively. We hypothesised that HS synthesis might be altered in the presence of UDP-GlcNAz due to the location of the azide on the acetyl group and the potential for interference with endogenous N-deacetylase-N-sulphotransferase biosynthetic enzyme activity. In mammalian cell culture (Chinese hamster ovary cells), treatment with Ac4GalNAz led to a decrease in total HS abundance accompanied by significant increases in 6-O-sulphation within the chains. Incorporation of a radiolabelled metabolic precursor revealed that average HS chain length was decreased in azido sugar-treated CHO cells. The modifications to HS were dose-dependent and HS inhibition was transient. Following removal of Ac4GalNAz from cell culture, HS expression returned to baseline levels within 24 hours. Previous work from the Bertozzi group has demonstrated the utility of Ac4GalNAz for visualising GalNAc- and O-GlcNAc-modified proteins in vivo. Using Xenopus, we were able to show that treatment of fertilised eggs with Ac4GalNAz decreased the abundance of HS in a similar way to that seen in vitro, with an associated impact on embryonic development. Embryonic axial elongation was impaired, with defective myotomal development and aberrant axonal patterning along the trunk and tail. Posterior somite cell nuclei were disorganised, with loss of distinct chevron patterning and skeletal muscle development was impaired with muscle fibres spanning some of the somite boundaries. Removal of the inhibitor partially rescued tail extension defects, as well as muscle development, but not axonal patterning. Therefore, these experiments illustrate a novel application for Ac4GalNAz as a soluble and reversible inhibitor of HS synthesis for in vitro and in vivo studies. The observed potential for control of inhibition via time- and dose-dependent effects enables targeted and selective inhibition of HS and potentially provides a powerful new inhibitor for the study of HS-mediated events.

A síntese noturna de melatonina pela glândula pineal é inibida por padrões moleculares associados à patógenos ou à danos, como por exemplo lipopolissacarídeo (LPS) ou peptídeo &#946;-amilóide. A interação destas moléculas com receptores toll 4 (TLR4) ativa o eixo imune-pineal, favorecendo a migração de leucócitos para o local da injúria. Heparan sulfato (HS) é um glicosaminoglicano da matrix extracelular que por dano tecidual, inflamação generalizada ou migração de células tumorais, liberam dissacarídeos que podem ligar a TLR4, levando a formação de uma resposta inflamatória. Avaliamos se HS poderia prejudicar a atividade da melatonina. HS é capaz de inibir a síntese noturna de melatonina, através da supressão da expressão gênica e do conteúdo enzimático de acetilserotonina O-metiltransferase (ASMT). Este efeito é modulado pela interação de HS com TLR4, mas não envolve a via de translocação nuclear de NF-&#954;B. Estes dados sugerem que um aumento de moléculas de HS na matriz da glândula pineal é traduzido a todo o organismo por uma redução no pico noturno de melatonina. / The nocturnal synthesis of melatonin by the pineal gland is inhibited by pathogen or damage-associated molecular patterns, such as lipopolysaccharide (LPS) and &#946;-amyloid peptide. The interaction of these molecules with toll like receptors 4 (TLR4) activates the immune-pineal axis, favoring the migration of leukocytes for the site of lesion. Heparan sulfate (HS), a glycosaminoglycan of the extracellular matrix, that in case of tissue injury, generalized inflammation or migration of tumor cells, releases disaccharide, which can bind to TLR4 triggering an inflammatory response. Here we evaluated if HS could impair nocturnal melatonin activity. HS is capable of inhibit the melatonin synthesis by the suppression of the gene expression and enzymatic content of acetylserotonin O-methyltransferase (ASMT). This effect is modulated by the interaction of HS with TLR4, but does not involve the NF-&#954;B nuclear translocation pathway. This data suggest that the increase in HS in pineal gland matrix is translated to the whole organism by a reduction in the nocturnal melatonin peak.

Nervous system function is closely tied to its structure, which ensures proper connectivity and neural activity. Neuronal architecture is assembled by a series of morphogenetic events, including the coordinated migrations of neurons and axons during development. Subsequently, the neuronal architecture established earlier must persist in the face of further growth, maturation of the nervous system, and the mechanical stress of body movements. In this work, we have shed light on the molecular mechanisms governing both the initial assembly of the nervous system and the long-term maintenance of neural circuits. In particular, we identified heparan sulfate proteoglycans (HSPGs) as regulators of neuronal migrations. Our discovery and analysis of viable mutations in the two subunits of the heparan sulfate co-polymerase reveals the importance of the coordinated and dynamic action of HSPGs in neuronal and axon guidance during development. Furthermore, we uncovered that the HSPG LON-2/glypican functions as a modulator of UNC-6/netrin signaling through interactions with the UNC-40/DCC receptor. During larval and adult life, molecules such as the protein SAX-7, homologous to mammalian L1CAM, function to protect the integrity of nervous system architecture. Indeed, loss of sax-7 leads to progressive disorganization of neuronal architecture. Through a forward genetic screen, we identified LON-1 as a novel maintenance molecule that functions post-embryonically with SAX-7 to maintain the architecture of the nervous system. Together, our work highlights the importance of extracellular interactions to modulate signaling events during the initial development of the nervous system, and to subsequently maintain neuronal architecture for the long-term.

<p>Heparan sulfate (HS) belongs to the glycosaminoglycan family of polysaccharides and is found attached to protein cores on cell surfaces and in the extracellular matrix. The HS backbone consists of alternating hexuronic acid and glucosamine units and undergoes a number of modification reactions creating HS chains with alternating highly and low modified domains, where high degree of modification correlates with high negative charge. Fibroblast growth factors (FGFs) and their receptors (FRs) both bind to HS, which affect formation of the FGF–FR complexes on the cell surfaces. Activated FRs can trigger several intracellular signaling pathways leading thereby to diverse cellular responses. </p><p>Work presented in this thesis focuses on the effect of HS and its structures on FGF–FR complex formation and FGF-induced signaling. Studies with short, highly modified oligosaccharides and FGF1 and 2 combined with FR1c, 2c, 3c or 4 showed a correlation between the overall degree of modification and amount/stability of FGF–FR complexes. Our findings imply that several HS structures, differently modified but with the same negative charge density are equal in their ability to support complex formation. Co-application of oligosaccharides with FGF2 to HS-deficient cells and investigation of the thereby induced cell signaling confirmed our findings with a cell-free system. The oligosaccharide with the highest modification degree displayed the biggest impact on cell signaling, which was FGF2 concentration dependent. Studies with long HS polysaccharides with preserved high and low modified domains suggest that the proportion between these two types of domains and also the structure of the low modified domains are of importance for the FGF–HS–FR complex formation and cell activation capacity. </p><p>This work illuminates several aspects in how HS structure influences the interplay between FGFs and FRs and contributes to the understanding of what factors affect a cell’s response following FGF stimulation.</p>

<p>Heparan sulfate proteoglycans are proteins with long, unbranched heparan sulfate (HS) polysaccharide chains attached to them. They are found on cell surfaces and in basement membranes where they exert their action by interacting with a wide range of enzymes and signaling molecules and are thereby involved in a range of various processes both during embryonic development and in adult physiology.</p><p>A great part of the biological functionality of proteoglycans can be directly related to the polysaccharide part. HS chains display very variable sulfation patterns where highly sulfated regions are responsible for a large part of the biological activity. The biosynthesis of HS is a complex process in which a number of enzymes are involved. Better comprehension of how this process is regulated could reveal clues to how formation of HS sulfation patterns occurs, and thereby how HS functionality is controlled.</p><p>This thesis is focusing on regulation of one of the enzymes responsible for HS sulfation, glucosaminyl N-deacetylase/N-sulfotransferase (NDST), in an attempt to understand these mechanisms better. Different aspects of NDST regulation were studied in three projects:</p><p>I) “Heparin/heparan sulfate biosynthesis: Processive formation of N-sulfated domains”, where the sulfate donor PAPS is shown to influence the manner in which NDST modifies the substrate, affecting the domain structure of the polysaccharide.</p><p>II) “Heparan sulfate biosynthesis: Characterization of an NDST1 splice variant”, where a splice variant of NDST1 which appears to influence NDST1 protein levels and affect HS structure is described.</p><p>III) “Heparan sulfate biosynthesis in zebrafish: Five NDST genes with distinct expression patterns during embryonic development”, in which five zebrafish NDSTs were cloned and shown to be expressed in a temporally and spatially regulated manner.</p>

<p>Neuroblastoma is a pediatric cancer arising from undifferentiated neural crest-derived precursor cells. Treatment strategies for neuroblastoma aim to promote neuroblast differentiation, however current therapies available are only modestly effective. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that act to promote neuroblast differentiation, though the precise factors released and their mechanism of action in neuroblastoma remains unclear. Here, we identify a novel component of the differentiating stroma secretome and harness stroma biology to inform the use of a combination therapy for neuroblastoma treatment.</p><p>HBEGF expression is decreased in neuroblastoma compared to benign disease, correlating to an increase in mortality. HBEGF protein is expressed only in stromal compartments of tumor specimens, with tissue from late-stage disease containing very little stroma or HBEGF. Addition of soluble HBEGF to neuroblastoma cell lines leads to increased neuroblast differentiation and decreased proliferation. Heparan sulfate proteoglycans (HSPGs) and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor (EGFR), leading to activation of the ERK1/2 and STAT3 pathways and upregulation of the inhibitor of DNA binding 1 transcription factor. </p><p>Expression of the type III TGF-β receptor (TβRIII), an HSPG, is epigenetically regulated in neuroblastoma cells via direct binding of the N-Myc transcription factor to Sp-1 sites on the TβRIII promoter. Analysis of patient microarray data demonstrate that other members of the differentiating stroma secretome, including HBEGF and EGFR, are positively correlated with TβRIII expression, suggesting that these proteins may be co-regulated. Treatment with inhibitors aimed at blocking N-Myc function, including inhibitors of histone deacetylases, DNA methyltransferases (DNMTs), and aurora kinase A (AurkA) can promote neuroblast differentiation and decrease proliferation. The combination of the DNMT inhibitor decitabine with the AurkA inhibitor MLN8237 enhances differentiation and reduces proliferation compared to either agent alone. Importantly, the combination of clinically achievable doses of these targeted agents dramatically reduces tumor growth in orthotopic xenograft models of neuroblastoma, identifying a novel combination therapy that may benefit children with this disease.</p><p>In conclusion, these studies dissect the tumor microenvironment to identify an important member of the differentiating stroma secretome, while also revealing a combination therapy for clinical development that has the potential to decrease adverse side effects and increase effectiveness of neuroblastoma treatment.</p> / Dissertation