A common theme in amyloid disorders is the deposition of disease-specific protein aggregates in tissues. Amyloid proteins bind to heparan sulfate (HS), a sulfated glycosaminoglycan, and HS has been found to promote the aggregation process. The present work relates to HS mediated mechanisms of amyloidosis, particularly transthyretin (TTR) amyloidosis, AA-amyloidosis and Alzheimer’s disease (AD). TTR is a transport protein present in the blood and cerebrospinal fluid, which under unclear circumstances can deposit as amyloid in the myocardium of elderly individuals. Examination of cardiac tissue from a 70 year old patient with reported cardiomyopathy reveald co-deposition of TTR amyloid and HS. Studies revealed that HS promotes TTR fibrillization through interaction with a basic motif in the protein. Empolyment of a cell model demonstrated that cell surface HS mediates internalization of TTR, an effect likely facilitated by HS-binding to the basic motif on TTR. Collectively, HS-TTR interactions at the cell surface may have dual outcomes, resulting in either fibrillization or internalization, respectively. During inflammatory conditions, serum amyloid A (SAA), an acute-phase protein associated with the high-density lipoprotein (HDL), can assemble into insoluble amyloid fibrils, causing AA-amyloidosis. We found that HS structures exceeding 12-14 sugar units in length separates SAA from HDL and induces subsequent aggregation of the polypeptide. Our result proposes a novel role for HS in AA-amyloidosis in which a critical length of HS is required for separation of SAA from HDL. Late-onset AD patients show reduced ability to clear cerebral amyloid-β (Aβ) aggregates, a pathological hallmark of the disease. Althought the pathway of Aβ clearance is still unclear, several cell-surface receptors are implicated in Aβ internalization. We found that ApoE facilitated Aβ uptake through interactions with HS-proteoglycans and low-density lipoprotein receptor-related protein 1. The ApoE interaction with Aβ likely promotes Aβ clearance in the brain, but, if unbalanced, may contribute to the pathology of AD.     These findings are in accord with the concept of HS as a promoter of amyloid protein aggregation, but also point to more complex relationship.

Heparan sulfates (HSs) are a class of anionic carbohydrate chains found at cell surfaces and in the extracellular matrix where they interact with a number of proteins. HS is characterized by extreme structural heterogeneity, and has been implicated in a number of biological phenomenon like embryogenesis, morphogen gradient formation and signalling of growth factors such as FGF, PDGF etc. Despite the characteristic structural heterogeneity, evidence from compositional studies show that the HS structure is expressed in a tightly regulated manner, implying a functional significance, which is most likely in the modulation of cell behaviour through HS-protein interactions. The lack of molecular tools has, however, hampered the understanding of HS structures with functional significance. This work therefore aims at characterizing the structural requirements on HS involved in the interaction with the anti-HS phage display antibodies HS4C3, AO4B08 and HS4E4 and a selected growth factor PDGF-BB. The characterization was done with the help of tailored oligosaccharide libraries generated from sources bearing structural resemblance to HS. The work has thus made available tools that preferentially recognize certain structural features on the HS chain and will aid in the further study of HS structure and its regulation. Evidence is also provided to support the notion that HS protein interactions can occur in multiple manners, utilizing any of the structural features on the HS chain.

<p>Growth factors and their receptors coordinate neuronal differentiation during development, yet their roles in the embyronal tumor neuroblastoma, where differentiation is a validated treatment strategy, remain unclear. The neuroblastoma tumor stroma is thought to suppress neuroblast growth via release of soluble differentiating factors. Here we identify critical components of the differentiating stroma secretome and describe preclinical testing of a novel therapeutic strategy based on their mechanism of action.</p><p>Expression of heparan sulfate proteoglycans (HSPGs), including T&#946;RIII, GPC1, GPC3, SDC3, and SDC4, is decreased in neuroblastoma, high in the stroma, and suppresses tumor growth. High expression of T&#946;RIII, GPC1, and SDC3 is associated with improved patient prognosis. HSPGs signal via heparan sulfate binding to FGFR1 and FGF2, which leads to phosphorylation of FGFR1 and Erk MAPK, and upregulation of the transcription factor inhibitor of DNA binding 1 (Id1). Surface expression and treatment with soluble HSPGs promotes neuroblast differentiation via this signaling complex. Expression of individual HSPGs positively correlates with Id1 expression in neuroblastoma patient samples and multivariate regression demonstrates that expression of HSPGs as a group positively correlates with Id1 expression, underscoring the clinical relevance of this pathway. HSPGs also enhance differentiation from FGF2 released by the stroma and FGF2 is identified as a potential serum prognostic biomarker in neuroblastoma patients. </p><p>The anticoagulant heparin has similar differentiating effects to HSPGs, decreasing neuroblast proliferation and reducing tumor growth while extending survival in an orthotopic xenograft model of neuroblastoma. Dissection of individual sulfation sites identifies 2-O-, 3-O-de-sulfated heparin (ODSH) as a differentiating agent that suppresses orthotopic xenograft growth and metastasis in two models while avoiding anticoagulation. These studies form the preclinical rationale for a multicenter clinical trial currently being proposed.</p><p>In conclusion, these studies translate mechanistic insights in neuroblast HSPG function to identify heparins as differentiating agents for clinical development in neuroblastoma, while demonstrating that tumor stroma biology can inform design of targeted molecular therapeutics.</p> / Dissertation

Cartilage development relies on the coordinated presentation of biological signals to direct chondrocyte morphology and function. This is largely controlled by perlecan, a heparan sulfate proteoglycan (HSPG). Understanding the role of perlecan and its pendant glycosaminoglycan chains (GAG) in cartilage development is essential for advances in tissue engineered cartilage replacement strategies. Perlecan was immunolocalised to the pericellular matrix of prehypertrophic and hypertrophic chondrocytes in human fetal feet. Human fetal chondrocytes were isolated and cultured in 3-dimensional (3D) scaffolds for a period of 4 weeks. Their chondrogenic phenotype, based on extracellular matrix (ECM) components, was assessed and compared to 2D cultures. Chondrocyte perlecan was immunopurified from human fetal chondrocytes grown in vitro and fetal cartilage tissue and characterised using a combination of antibody-based techniques (ELISA, Western blotting) and gel electrophoresis. The biological function of chondrocyte perlecan was determined by its ability to form ternary complexes with fibroblast growth factors (FGF) and their receptors (FGFR) using an antibody-based technique as well as a cell proliferation assay using cells expressing FGFR isotypes. Perelcan was restricted to the prehypertrophic and hypertrophic zones of cartilage. This zonal organisation of chondrocytes and chondrogenic properties, determined by their morphology and PG deposition, was recapitulated in the 3D constructs while 2D cultures displayed dedifferentiated chondrocytes. Exogenous FGF2 promoted chondrocyte proliferation, while FGF18 stimulated the synthesis of perlecan, reflecting chondrocyte hypertrophy. Chondrocyte perlecan (630kDa) contained HS, chondroitin sulfate (CS) and keratan sulfate (KS) chains. Chondrocyte perlecan formed HS dependent ternary complexes with FGF2-FGFR1c and FGF18-FGFR3c, while FGF18-FGFR3c binding to perlecan protein core was also observed. Binding of FGF18-FGFR3c to chondrocyte perlecan HS was more promiscuous than FGF2-FGFR1c. Furthermore, chondrocyte perlecan HS mediated biological activity with FGF18 via FGFR3c, which was modulated by mammalian heparanase, while no biological activity was elicited by FGF2-FGFR1c. The findings underline how perlecan and its GAGs interact with FGF and FGFR in a spatio-temporal manner to promote signalling, effecting chondrocyte behaviour and morphology in cartilage development. This insight can be utilised in tissue engineering to improve the development of biologically functional cartilage replacements.

Cartilage development relies on the coordinated presentation of biological signals to direct chondrocyte morphology and function. This is largely controlled by perlecan, a heparan sulfate proteoglycan (HSPG). Understanding the role of perlecan and its pendant glycosaminoglycan chains (GAG) in cartilage development is essential for advances in tissue engineered cartilage replacement strategies. Perlecan was immunolocalised to the pericellular matrix of prehypertrophic and hypertrophic chondrocytes in human fetal feet. Human fetal chondrocytes were isolated and cultured in 3-dimensional (3D) scaffolds for a period of 4 weeks. Their chondrogenic phenotype, based on extracellular matrix (ECM) components, was assessed and compared to 2D cultures. Chondrocyte perlecan was immunopurified from human fetal chondrocytes grown in vitro and fetal cartilage tissue and characterised using a combination of antibody-based techniques (ELISA, Western blotting) and gel electrophoresis. The biological function of chondrocyte perlecan was determined by its ability to form ternary complexes with fibroblast growth factors (FGF) and their receptors (FGFR) using an antibody-based technique as well as a cell proliferation assay using cells expressing FGFR isotypes. Perelcan was restricted to the prehypertrophic and hypertrophic zones of cartilage. This zonal organisation of chondrocytes and chondrogenic properties, determined by their morphology and PG deposition, was recapitulated in the 3D constructs while 2D cultures displayed dedifferentiated chondrocytes. Exogenous FGF2 promoted chondrocyte proliferation, while FGF18 stimulated the synthesis of perlecan, reflecting chondrocyte hypertrophy. Chondrocyte perlecan (630kDa) contained HS, chondroitin sulfate (CS) and keratan sulfate (KS) chains. Chondrocyte perlecan formed HS dependent ternary complexes with FGF2-FGFR1c and FGF18-FGFR3c, while FGF18-FGFR3c binding to perlecan protein core was also observed. Binding of FGF18-FGFR3c to chondrocyte perlecan HS was more promiscuous than FGF2-FGFR1c. Furthermore, chondrocyte perlecan HS mediated biological activity with FGF18 via FGFR3c, which was modulated by mammalian heparanase, while no biological activity was elicited by FGF2-FGFR1c. The findings underline how perlecan and its GAGs interact with FGF and FGFR in a spatio-temporal manner to promote signalling, effecting chondrocyte behaviour and morphology in cartilage development. This insight can be utilised in tissue engineering to improve the development of biologically functional cartilage replacements.

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

Heparan sulfate (HS) is a class of linear, sulfated polysaccharides located on cell surface, secretory granules, and in extracellular matrices found in all animal organ systems. It consists of alternately repeating disaccharide units, expressed in animal species ranging from hydra to higher vertebrates including humans. HS binds and mediates the biological activities of over 300 proteins, including growth factors, enzymes, chemokines, cytokines, adhesion and structural proteins, lipoproteins and amyloid proteins. The binding events largely depend on the fine structure - the arrangement of sulfate groups and other variations - on HS chains. With the activated electron dissociation (ExD) high-resolution tandem mass spectrometry technique, researchers acquire rich structural information about the HS molecule. Using this technique, covalent bonds of the HS oligosaccharide ions are dissociated in the mass spectrometer. However, this information is complex, owing to the large number of product ions, and contains a degree of ambiguity due to the overlapping of product ion masses and lability of sulfate groups; as a result, there is a serious barrier to manual interpretation of the spectra. The interpretation of such data creates a serious bottleneck to the understanding of the biological roles of HS. In order to solve this problem, I designed HS-SEQ - the first HS sequencing algorithm using high-resolution tandem mass spectrometry. HS-SEQ allows rapid and confident sequencing of HS chains from millions of candidate structures and I validated its performance using multiple known pure standards. In many cases, HS oligosaccharides exist as mixtures of sulfation positional isomers. I therefore designed MULTI-HS-SEQ, an extended version of HS-SEQ targeting spectra coming from more than one HS sequence. I also developed several pre-processing and post-processing modules to support the automatic identification of HS structure. These methods and tools demonstrated the capacity for large-scale HS sequencing, which should contribute to clarifying the rich information encoded by HS chains as well as developing tailored HS drugs to target a wide spectrum of diseases.

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.

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.