| 1. |
- Jakobsson, Lars, et al.
(författare)
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Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis
- 2006
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Ingår i: Developmental Cell. - 1534-5807 .- 1878-1551. ; 10:5, s. 625-634
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Tidskriftsartikel (refereegranskat)abstract
- Several receptor tyrosine kinases require heparan sulfate proteoglycans (HSPGs) as coreceptors for efficient signal transduction. We have studied the role of HSPGs in the development of blood capillary structures from embryonic stem cells, a process strictly dependent on signaling via vascular endothelial growth factor receptor-2 (VEGFR-2). We show, by using chimeric cultures of embryonic stem cells defective in either HS production or VEGFR-2 synthesis, that VEGF signaling in endothelial cells is fully supported by HS expressed in trans by adjacent perivascular smooth muscle cells. Transactivation of VEGFR-2 leads to prolonged and enhanced signal transduction due to HS-dependent trapping of the active VEGFR-2 signaling complex. Our data imply that direct signaling via HSPG core proteins is dispensable for a functional VEGF response in endothelial cells. We propose that transactivation of tyrosine kinase receptors by HSPGs constitutes a mechanism for crosstalk between adjacent cells.
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| 2. |
- Johansson, Karl-Axel, et al.
(författare)
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The quality assurance process for the ARTSCAN head and neck study - a practical interactive approach for QA in 3DCRT and IMRT.
- 2008
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Ingår i: Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology. - : Elsevier BV. - 0167-8140 .- 1879-0887. ; 87:2, s. 290-9
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Tidskriftsartikel (refereegranskat)abstract
- AIM: This paper describes the quality assurance (QA) work performed in the Swedish multicenter ARTSCAN (Accelerated RadioTherapy of Squamous cell CArcinomas in the head and Neck) trial to guarantee high quality in a multicenter study which involved modern radiotherapy such as 3DCRT or IMRT. MATERIALS AND METHODS: The study was closed in June 2006 with 750 randomised patients. Radiation therapy-related data for every patient were sent by each participating centre to the QA office where all trial data were reviewed, analysed and stored. In case of any deviation from the protocol, an interactive process was started between the QA office and the local responsible clinician and/or physicist to increase the compliance to the protocol for future randomised patients. Meetings and workshops were held on a regular basis for discussions on various trial-related issues and for the QA office to report on updated results. RESULTS AND DISCUSSION: This review covers the 734 patients out of a total of 750 who had entered the study. Deviations early in the study were corrected so that the overall compliance to the protocol was very high. There were only negligible variations in doses and dose distributions to target volumes for each specific site and stage. The quality of the treatments was high. Furthermore, an extensive database of treatment parameters was accumulated for future dose-volume vs. endpoint evaluations. CONCLUSIONS: This comprehensive QA programme increased the probability to draw firm conclusions from our study and may serve as a concept for QA work in future radiotherapy trials where comparatively small effects are searched for in a heterogeneous tumour population.
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| 3. |
- Kawamura, Harukiyo, et al.
(författare)
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Neuropilin-1 in regulation of VEGF-induced activation of p38MAPK and endothelial cell organization
- 2008
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 112:9, s. 3638-49
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Tidskriftsartikel (refereegranskat)abstract
- Vascular endothelial growth factor (VEGF)-A regulates vascular development and angiogenesis. VEGF isoforms differ in ability to bind coreceptors heparan sulfate (HS) and neuropilin-1 (NRP1). We used VEGF-A165 (which binds HS and NRP1), VEGF-A121 (binds neither HS nor NRP1), and parapoxvirus VEGF-E-NZ2 (binds NRP1 but not HS) to investigate the role of NRP1 in organization of endothelial cells into vascular structures. All 3 ligands induced similar level of VEGFR-2 tyrosine phosphorylation in the presence of NRP1. In contrast, sprouting angiogenesis in differentiating embryonic stem cells (embryoid bodies), formation of branching pericyte-embedded vessels in subcutaneous matrigel plugs, and sprouting of intersegmental vessels in developing zebrafish were induced by VEGF-A165 and VEGF-E-NZ2 but not by VEGF-A121. Analyses of recombinant factors with NRP1-binding gain- and loss-of-function properties supported the conclusion that NRP1 is critical for VEGF-induced sprouting and branching of endothelial cells. Signal transduction antibody arrays implicated NRP1 in VEGF-induced activation of p38MAPK. Inclusion of the p38MAPK inhibitor SB203580 in VEGF-A165-containing matrigel plugs led to attenuated angiogenesis and poor association with pericytes. Our data strongly indicate that the ability of VEGF ligands to bind NRP1 influences p38MAPK activation, and formation of functional, pericyte-associated vessels.
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| 4. |
- Le Jan, Sébastien, et al.
(författare)
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Functional Overlap Between Chondroitin and Heparan Sulfate Proteoglycans During VEGF-Induced Sprouting Angiogenesis
- 2012
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Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - 1079-5642 .- 1524-4636. ; 32:5, s. 1255-1263
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Heparan sulfate proteoglycans regulate key steps of blood vessel formation. The present study was undertaken to investigate if there is a functional overlap between heparan sulfate proteoglycans and chondroitin sulfate proteoglycans during sprouting angiogenesis.METHODS AND RESULTS: Using cultures of genetically engineered mouse embryonic stem cells, we show that angiogenic sprouting occurs also in the absence of heparan sulfate biosynthesis. Cells unable to produce heparan sulfate instead increase their production of chondroitin sulfate that binds key angiogenic growth factors such as vascular endothelial growth factor A, TGFβ, and platelet-derived growth factor B. Lack of heparan sulfate proteoglycan production however leads to increased pericyte numbers and reduced adhesion of pericytes to nascent sprouts, likely due to dysregulation of TGFβ and platelet-derived growth factor B signal transduction.CONCLUSIONS: The present study provides direct evidence for a previously undefined functional overlap between chondroitin sulfate proteoglycans and heparan sulfate proteoglycans during sprouting angiogenesis. Our findings provide information relevant for potential future drug design efforts that involve targeting of proteoglycans in the vasculature.
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| 5. |
- Abramsson, Alexandra, 1973, et al.
(författare)
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Defective N-sulfation of heparan sulfate proteoglycans limits PDGF-BB binding and pericyte recruitment in vascular development
- 2007
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Ingår i: GENES & DEVELOPMENT. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 21:3, s. 316-331
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Tidskriftsartikel (refereegranskat)abstract
- During vascular development, endothelial platelet-derived growth factor B (PDGF-B) is critical for pericyte recruitment. Deletion of the conserved C-terminal heparin-binding motif impairs PDGF-BB retention and pericyte recruitment in vivo, suggesting a potential role for heparan sulfate (HS) in PDGF-BB function during vascular development. We studied the participation of HS chains in pericyte recruitment using two mouse models with altered HS biosynthesis. Reduction of N-sulfation due to deficiency in N-deacetylase/N-sulfotransferase-1 attenuated PDGF-BB binding in vitro, and led to pericyte detachment and delayed pericyte migration in vivo. Reduced N-sulfation also impaired PDGF-BB signaling and directed cell migration, but not proliferation. In contrast, HS from glucuronyl C5-epimerase mutants, which is extensively N- and 6-O-sulfated, but lacks 2-O-sulfated L-iduronic acid residues, retained PDGF-BB in vitro, and pericyte recruitment in vivo was only transiently delayed. These observations were supported by in vitro characterization of the structural features in HS important for PDGF-BB binding. We conclude that pericyte recruitment requires HS with sufficiently extended and appropriately spaced N-sulfated domains to retain PDGF-BB and activate PDGF receptor β (PDGFRβ) signaling, whereas the detailed sequence of monosaccharide and sulfate residues does not appear to be important for this interaction.
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| 6. |
- Angerth, Tomas, et al.
(författare)
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Cloning and structural analysis of a gene encoding a mouse mastocytoma proteoglycan core protein : Analysis of its evolutionary relation to three cross hybridizing regions in the mouse genome
- 1990
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Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 93:2, s. 235-240
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Tidskriftsartikel (refereegranskat)abstract
- Serglycin (SGC) is a Ser-Gly-repeat-containing protein, used as proteoglycan core protein in the parietal yolk sac and in mast cell, where glycosaminoglycan side chains are attached to the serine residues of the repeat region. In this article, the structure of the gene SGC encoding mouse SGC is reported. The gene is divivided into three exons, which are all contained within a region of approximately 13 kb. Nucleotide (nt) sequence analysis was carried out on a region of 1.2 kb upstream from the first exon. The region containing the two promoters (active in parietal yolk sac and in mast cells, respectively) was analyzed for the presence of recognition sites for known DNA-binding proteins. A number of sequence closely related to known recognition sites were found in both promoters, and one consensus octamer-binding site could be identified in the putative yolk-sac promoter. Multiple regions in the mouse genome hybridizing with DNA fragments covering the Ser-Gly repeat region have previously been described, and it has been sugggested that these loci may represent other proteoglycan core proteins. Analysis of nt sequence was carried out on three out of the more than 15 of three regions present in the mouse genome. However, none of the clones analyzed was found to have any open reading frame in the region of cross-hybridization which possibly could code for a SGC protein. Instead, one of the clones was found to contain an exon encoding a highly basic protein, unrelated to SGC protein. Instead, one of the clones was found to contain an exon a highly basic protein, unrelated to SGC. Hence, no evidence ws found for a multigene family of Ser-Gly-repeat-containing proteoglycan-encoding genes.
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| 7. |
- Bachvarova, Velina, et al.
(författare)
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Chondrocytes respond to an altered heparan sulfate composition with distinct changes of heparan sulfate structure and increased levels of chondroitin sulfate
- 2020
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Ingår i: Matrix Biology. - : ELSEVIER. - 0945-053X .- 1569-1802. ; 93, s. 43-59
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate (HS) regulates the activity of many signaling molecules critical for the development of endochondral bones. Even so, mice with a genetically altered HS metabolism display a relatively mild skeletal phenotype compared to the defects observed in other tissues and organs pointing to a reduced HS dependency of growth-factor signaling in chondrocytes. To understand this difference, we have investigated the glycosaminoglycan (GAG) composition in two mouse lines that produce either reduced levels of HS (Ext1(gt/gt) mice) or HS lacking 2-O-sulfation (Hs2st1(-/-) mice). Analysis by RPIP-HPLC revealed an increased level of sulfated disaccarides not affected by the mutation in both mouse lines indicating that chondrocytes attempt to restore a critical level of sulfation. In addition, in both mutant lines we also detected significantly elevated levels of CS. Size exclusion chromatography further demonstrated that Ext1(gt/gt) mutants produce more but shorter CS chains, while the CS chains produced by (Hs2st1(-/-) mice) mutants are of similar length to that of wild type littermates indicating that chondrocytes produce more rather than longer CS chains. Expression analysis revealed an upregulation of aggrecan, which likely carries most of the additionally produced CS. Together the results of this study demonstrate for the first time that not only a reduced HS synthesis but also an altered HS structure leads to increased levels of CS in mammalian tissues. Furthermore, as chondrocytes produce 100-fold more CS than HS the increased CS levels point to an active, precursor-independent mechanism that senses the quality of HS in a vast excess of CS. Interestingly, reducing the level of cell surface CS by chondroitinase treatment leads to reduced Bmp2 induced Smad1/5/9 phosphorylation. In addition, Erk phosphorylation is increased independent of Fgf18 treatment indicating that both, HS and CS, affect growth factor signaling in chondrocytes in distinct manners.
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| 8. |
- Barkefors, Irmeli, 1981-
(författare)
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Directing Angiogenesis : Cellular Responses to Gradients in vitro
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Blood vessels are essential for the delivery of nutrients and oxygen to tissues, as well as for the removal of waste products. Patients with tumors, wounds or diabetes all have active angiogenesis, formation and remodeling of blood vessels, a process that is initiated and manipulated by gradients of secreted signaling proteins. This thesis describes the development of new microfluidic in vitro assays where directed migration of single endothelial cells and three dimensional vascular structures can be monitored in real time. Combining these assays with live imaging microscopy we have studied the behavior of endothelial cells in gradients of proangiogenic factors as well as directed sprouting in embryonic kidneys and stem cell cultures. With the 2D assay we have quantified endothelial cell chemotaxis towards FGF2, VEGFA165 and VEGFA121 and we also demonstrate that constant levels of VEGFA165, but not of FGF2, are able to reduce chemokinesis of endothelial cells. In the 3D migration chamber we have studied directed endothelial cell sprouting in mouse embryonic kidneys and embryoid bodies in response to VEGFA gradients. In both models directed angiogenesis is detected towards increasing levels of growth factor. Using the microarray technique on differentiating embryonic stem cells we have been able to identify the gene exoc3l2 as potentially involved in angiogenesis and endothelial cell migration and we present evidence that ExoC3l2 is associated with the exocyst complex; an important regulator of cell polarity. We have also shown that siRNA mediated gene silencing of exoc3l2 results in impaired VEGFR2 phosphorylation as well as loss of directionality in response to a VEGFA gradient.
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| 9. |
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| 10. |
- Berthelsen, Anne Kiil, et al.
(författare)
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What's new in target volume definition for radiologists in ICRU Report 71? How can the ICRU volume definitions be integrated in clinical practice?
- 2007
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Ingår i: Cancer Imaging. - : E-MED LTD. - 1470-7330. ; 7:1, s. 104-104
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Tidskriftsartikel (refereegranskat)abstract
- The optimal definition of the size, shape and location of gross tumour volume is one of the most important steps in the planning of radiation therapy, and necessitates a proper understanding of the procedure from both the oncologic radiologist and the radiation oncologist. This overview reports on the different terms and concepts that have been recommended in the ICRU Reports for this purpose; the latest Report 71 focuses on both previously given recommendations, and especially on electron beam therapy. This paper also highlights some of the problems that are encountered in the use of the International Commission on Radiation Units and Measurements (ICRU) recommendations in clinical practice, and at the interface between the radiation oncologist and the diagnostic oncologist.
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| 11. |
- Busse, Marta, et al.
(författare)
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Contribution of EXT1, EXT2, and EXTL3 to heparan sulfate chain elongation
- 2007
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 282:45, s. 32802-32810
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Tidskriftsartikel (refereegranskat)abstract
- The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate biosynthesis. Five human members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are believed to form a Golgi-located hetero-oligomeric complex that catalyzes the chain elongation step in heparan sulfate biosynthesis, whereas the EXTL proteins exhibit overlapping glycosyl-transferase activities in vitro, so that it is not apparent what reactions they catalyze in vivo. We used gene-silencing strategies to investigate the roles of EXT1, EXT2, and EXTL3 in heparan sulfate chain elongation. Small interfering RNAs (siRNAs) directed against the human EXT1, EXT2, or EXTL3 mRNAs were introduced into human embryonic kidney 293 cells. Compared with cells transfected with control siRNA, those transfected with EXT1 or EXT2 siRNA synthesized shorter heparan sulfate chains, and those transfected with EXTL3 siRNA synthesized longer chains. We also generated human cell lines overexpressing the EXT proteins. Overexpression of EXT1 resulted in increased HS chain length, which was even more pronounced in cells coexpressing EXT2, whereas overexpression of EXT2 alone had no detectable effect on heparan sulfate chain elongation. Mutations in either EXT1 or EXT2 are associated with hereditary multiple exostoses, a human disorder characterized by the formation of cartilage-capped bony outgrowths at the epiphyseal growth plates. To further investigate the role of EXT2, we generated human cell lines overexpressing mutant EXT2. One of the mutations, EXT2-Y419X, resulted in a truncated protein. Interestingly, the capacity of wild type EXT2 to enhance HS chain length together with EXT1 was not shared by the EXT2-Y419X mutant.
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| 12. |
- Carlsson, Pernilla, 1976-
(författare)
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Cellular design of heparan sulfate : The NDST enzymes and their regulation
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.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.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: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.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.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.
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| 13. |
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| 14. |
- Carlsson, Pernilla, et al.
(författare)
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Heparan sulfate biosynthesis: Characterization of an NDST1 splice variant
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Annan publikation (populärvet., debatt m.m.)abstract
- N-Deacetylase/N-sulfotransferases (NDSTs) are Golgi-located enzymes involved in the biosynthesis of heparan sulfate. They are bifunctional enzymes responsible for N-deacetylation of N-acetylglucosamine residues followed by N-sulfation of the generated free amino groups. In this paper we have identified and characterized a splice variant of NDST1 mRNA. The alternatively spliced mRNA transcript was shown to be present in varying amounts in different adult and embryonic mouse tissues. The protein resulting from translation of the spliced transcript (NDST1S) lacks the C-terminal half of fullength NDST and appears to be devoid of enzyme activity. As shown in HEK 293 cells overexpressing NDST1, a high expression of the splice variant resulted in reduced levels of NDST1. Unexpectedly, the level of N-sulfation was largely unaltered in heparan sulfate produced in NDST1S overexpressing cells while 6-O-sulfation was elevated and 2-O-sulfation was reduced. NDST1S shares the ability of NDST1 to interact with EXT2, one of the components of the heparan sulfate copolymerase. We speculate that NDST1S may alter the composition of the tentaive enzyme complex, the GAGosome, resulting in changes in the structure of heparan sulfate synthesized.
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| 15. |
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| 16. |
- Carlsson, Pernilla, et al.
(författare)
-
Heparin biosynthesis
- 2012
-
Ingår i: Handbook of experimental pharmacology. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 0171-2004. ; 207, s. 23-41
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Tidskriftsartikel (refereegranskat)abstract
- Heparin and heparan sulfate share the same polysaccharide backbone structure but differ in sulfation degree and expression pattern. Whereas heparan sulfate is found in virtually all cells of the human body, heparin expression is restricted to mast cells, where it has a function in storage of granular components such as histamine and mast cell specific proteases. Although differing in charge and sulfation pattern, current knowledge indicates that the same pathway is used for synthesis of heparin and heparan sulfate, with a large number of different enzymes taking part in the process. At present, little is known about how the individual enzymes are coordinated and how biosynthesis is regulated. These questions are addressed in this chapter together with a review of the basic enzymatic steps involved in initiation, elongation, and modification of the polysaccharides.
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| 17. |
- Carlsson, Pernilla, et al.
(författare)
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Heparin/heparan sulfate biosynthesis : Processive formation of N-sulfated domains
- 2008
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 283:29, s. 20008-20014
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate (HS) proteoglycans influence embryonic development as well as adult physiology through interactions with various proteins, including growth factors/morphogens and their receptors. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. A key step is the initial generation of N-sulfated domains, primary sites for further polymer modification and ultimately for functional interactions with protein ligands. Such domains, generated through action of a bifunctional GlcNAc N-deacetylase/N-sulfotransferase (NDST) on a [GlcUA-GlcNAc](n) substrate, are of variable size due to regulatory mechanisms that remain poorly understood. We have studied the action of recombinant NDSTs on the [GlcUA-GlcNAc](n) precursor in the presence and absence of the sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). In the absence of PAPS, NDST catalyzes limited and seemingly random N-deacetylation of GlcNAc residues. By contrast, access to PAPS shifts the NDST toward generation of extended N-sulfated domains that are formed through coupled N-deacetylation/N-sulfation in an apparent processive mode. Variations in N-substitution pattern could be obtained by varying PAPS concentration or by experimentally segregating the N-deacetylation and N-sulfation steps. We speculate that similar mechanisms may apply also to the regulation of HS biosynthesis in the living cell.
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| 18. |
- Chang, Ya-Ting, et al.
(författare)
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Versican accumulates in vascular lesions in pulmonary arterial hypertension
- 2016
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Ingår i: PULMONARY CIRCULATION. - : Wiley. - 2045-8932 .- 2045-8940. ; 6:3, s. 347-359
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Tidskriftsartikel (refereegranskat)abstract
- Pulmonary arterial hypertension (PAH) is a lethal condition for which there is no effective curative pharmacotherapy. PAH is characterized by vasoconstriction, wall thickening of pulmonary arteries, and increased vascular resistance. Versican is a chondroitin sulfate proteoglycan in the vascular extracellular matrix that accumulates following vascular injury and promotes smooth-muscle cell proliferation in systemic arteries. Here, we investigated whether versican may play a similar role in PAH. Paraffin-embedded lung sections from patients who underwent lung transplantation to treat PAH were used for immunohistochemistry. The etiologies of PAH in the subjects involved in this study were idiopathic PAH, scleroderma, and congenital heart disease (atrial septal defect) with left-to-right shunt. Independent of the underlying etiology, increased versican immunostaining was observed in areas of medial thickening, in neointima, and in plexiform lesions. Western blot of lung tissue lysates confirmed accumulation of versican in patients with PAH. Double staining for versican and CD45 showed only occasional colocalization in neointima of high-grade lesions and plexiform lesions. In vitro, metabolic labeling with [S-35] sulfate showed that human pulmonary artery smooth-muscle cells (hPASMCs) produce mainly chondroitin sulfate glycosaminoglycans. In addition, hypoxia, but not cyclic stretch, was demonstrated to increase both versican messenger RNA expression and protein synthesis by hPASMCs. Versican accumulates in vascular lesions of PAH, and the amount of versican correlates more with lesion severity than with underlying etiology or inflammation. Hypoxia is a possible regulator of versican accumulation, which may promote proliferation of pulmonary smooth-muscle cells and vascular remodeling in PAH.
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| 19. |
- Dagälv, Anders, et al.
(författare)
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Cell surface mast cell proteoglycans identified as heparin-substituted syndecan-2
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Annan publikation (populärvet., debatt m.m.)abstract
- Connective tissue type mast cells isolated from the peritoneal cavity of mice and then cultured in vitro have been used to answer the question if one cell at a given time point can synthesize heparan sulfate chains with different structure. Characterization of cell surface proteoglycans made by the cells demonstrated that they were identical to syndecan-2, substituted with heparin chains. Ion exchange chromatography showed that the syndecan heparin chains behaved identically as heparin chains recovered from serglycin, inside the cells. This was also the case when mast cells from NDST2 deficient mice were studied. This time, syndecan-2 as well as serglycin derived polysaccharide chains had a lower but identical charge density. We conclude that mast cells only synthesize one kind of heparan sulfate/heparin chain at a time and that polysaccharide chains of identical structure will be found at the cell surface and inside the cell.
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| 20. |
- Dagälv, Anders, et al.
(författare)
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Lack of both lethality and defective in vitro differentiation of embryonic stem cells N-deacetylase/N-sulfotransferase 1 and 2 causes early embryonic
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Annan publikation (populärvet., debatt m.m.)abstract
- NDSTs (N-deacetylase/N-sulfotransferases) are enzymes responsible for N-sulfation during heparan sulfate and heparin biosynthesis. While lack of NDST2 results in defective mast cells and NDST1 deficiency causes neonatal death and lung, skeletal and brain defects, lack of both isoforms is not compatible with embryonic development. We here show that NDST1/2-/- embryos die before E6.5 and that embryos dissected out at E5.5 lack parts of the embryo/extraembryonic tissue. Consistent with their in vivo behavior, in vitro cultured NDST1/2 deficient embryos displayed impaired ability of inner cell mass proliferation. In addition, markers for all the three germ layers had a disturbed expression pattern in isolated NDST1/2 deficient embryonic stem (ES) cells. Characterization of heparan sulfate (HS) structure in control ES cells and in ES cells lacking NDST1, NDST2 or both NDST1 and NDST2 revealed big differences. As expected, control cells synthesized HS with the highest degree of sulfation closely followed by HS from NDST2-/- cells, which in turn was more sulfated than HS produced by NDST1-/- cells. HS from NDST1/2-/- cells was almost devoid of sulfate groups. Notably, lack of one NDST isoform did not result in increased expression of any of the others. While all cell types except the NDST1/2-/- cells produced HS with a higher degree of sulfation when allowed to differentiate for 8 days, HS from control cells was still more heavily sulfated than that produced by NDST2-/- cells followed by the HS of NDST1-/- cells. The increase in sulfation was paralleled by increased expression of NDST transcripts and could also be recorded as increased N-sulfotransferase activity of cell lysates. While NDST1/2 deficient ES cells were unable to differentiate into beating cardiomyocytes all NDST1-/- and control embryoid bodies had started to beat after 4 days of culture. Surprisingly, NDST2 deficiency resulted in delayed cardiomyocyte differentiation.
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| 21. |
- Dagälv, Anders, et al.
(författare)
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Lowered Expression of Heparan Sulfate/Heparin Biosynthesis Enzyme N-Deacetylase/N-Sulfotransferase 1 Results in Increased Sulfation of Mast Cell Heparin
- 2011
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 286:52, s. 44433-44440
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Tidskriftsartikel (refereegranskat)abstract
- Deficiency of the heparan sulfate biosynthesis enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) in mice causes severely disturbed heparan sulfate biosynthesis in all organs, whereas lack of NDST2 only affects heparin biosynthesis in mast cells (MCs). To investigate the individual and combined roles of NDST1 and NDST2 during MC development, in vitro differentiated MCs derived from mouse embryos and embryonic stem cells, respectively, have been studied. Whereas MC development will not occur in the absence of both NDST1 and NDST2, lack of NDST2 alone results in the generation of defective MCs. Surprisingly, the relative amount of heparin produced in NDST1(+/-) and NDST1(-/-) MCs is higher (approximate to 30%) than in control MCs where approximate to 95% of the (35)S-labeled glycosaminoglycans produced is chondroitin sulfate. Lowered expression of NDST1 also results in a higher sulfate content of the heparin synthesized and is accompanied by increased levels of stored MC proteases. A model of the GAGosome, a hypothetical Golgi enzyme complex, is used to explain the results.
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| 22. |
- Dagälv, Anders
(författare)
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Role of Heparan Sulfate N-sulfation in Mouse Embryonic Development
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Heparan sulfate (HS) is a sulfated glycosaminoglycan expressed by all cells in the body. It is found at the cell surface and in the extracellular matrix where it binds a large amount of various ligands including growth factors and morphogens. HS is important for building up morphogen gradients during embryonic development and to act as coreceptors for signaling molecules. Many different Golgi enzymes are involved in the biosynthesis of HS. It is known that some of these enzymes interact with each other but not how the whole biosynthesis machinery works or how the cell regulates the structure of the HS that it produces. In this thesis, cells and mice deficient in two of these biosynthetic enzymes, glucosaminyl N-deacetylase/N-sulfotransferase-1 (NDST1) and the isoform NDST2 have been studied. NDSTs perform the first modifications during biosynthesis where they replace N-acetyl groups on N-acetyl-glucosamine units with sulfate groups. It is known that deficiency of NDST1 is lethal, while lack of NDST2 only results in abnormal connective tissue type mast cells. Here it is shown that deficiency of both NDST1 and NDST2 is embryonically lethal. The embryonic stem (ES) cells extracted from the inner cell mass of double knockout blastocysts show in addition an impaired differentiation capacity compared to wild-type ES cells and fail completely to differentiate into cardiac muscle cells which NDST1-/-, NDST2-/- and wild-type ES cells all do. Cultured mast cells that lack NDST2 produce heparin that is low-sulfated compared to wild-type HS. To our surprise, we could show that mast cells deficient in NDST1 instead produce a more highly sulfated heparin than wild-type cells. We use a model that predicts that the biosynthesis enzymes work together in a multienzyme complex, the GAGosome, to explain our results. We hypothesize that NDST1 has a higher affinity for the GAGosome than NDST2 which only in the absence of NDST1 gets incorporated into the enzyme complex. When all GAGosomes contain NDST2, a more highly sulfated glycosaminoglycan chain will be synthesized. A splice variant of NDST1, NDST1S, has also been studied. We could show that NDST1S lacks enzyme activity but that it probably has the capacity to incorporate into GAGosomes. Overexpression of NDST1S results in altered structure of the HS produced by the cells. We speculate that expression of the splice variant during development may be one way to regulate HS structure.
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| 23. |
- Deligny, Audrey, et al.
(författare)
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NDST2 (N-Deacetylase/N-Sulfotransferase-2) Enzyme Regulates Heparan Sulfate Chain Length
- 2016
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 291:36, s. 18600-18607
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Tidskriftsartikel (refereegranskat)abstract
- Analysis of heparan sulfate synthesized by HEK 293 cells overexpressing murine NDST1 and/or NDST2 demonstrated that the amount of heparan sulfate was increased in NDST2-but not in NDST1-overexpressing cells. Altered transcript expression of genes encoding other biosynthetic enzymes or proteoglycan core proteins could not account for the observed changes. However, the role of NDST2 in regulating the amount of heparan sulfate synthesized was confirmed by analyzing heparan sulfate content in tissues isolated from Ndst2(-/-) mice, which contained reduced levels of the polysaccharide. Detailed disaccharide composition analysis showed no major structural difference between heparan sulfate from control and Ndst2(-/-) tissues, with the exception of heparan sulfate from spleen where the relative amount of trisulfated disaccharides was lowered in the absence of NDST2. In vivo transcript expression levels of the heparan sulfate-polymerizing enzymes Ext1 and Ext2 were also largely unaffected by NDST2 levels, pointing to a mode of regulation other than increased gene transcription. Size estimation of heparan sulfate polysaccharide chains indicated that increased chain lengths in NDST2-overexpressing cells alone could explain the increased heparan sulfate content. A model is discussed where NDST2-specific substrate modification stimulates elongation resulting in increased heparan sulfate chain length.
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| 24. |
- Dierker, Tabea, et al.
(författare)
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Altered heparan sulfate structure in Glce(-/-) mice leads to increased Hedgehog signaling in endochondral bones
- 2016
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Ingår i: Matrix Biology. - : Elsevier BV. - 0945-053X .- 1569-1802. ; 49, s. 82-92
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Tidskriftsartikel (refereegranskat)abstract
- One of the key regulators of endochondral ossification is Indian hedgehog (Ihh), which acts as a long-range morphogen in the developing skeletal elements. Previous studies have shown that the distribution and signaling activity of Ihh is regulated by the concentration of the extracellular glycosaminoglycan heparan sulfate (HS). An essential step during biosynthesis of HS is the epimerization of D-glucuronic to L-iduronic acid by the enzyme glucuronyl C5-epimerase (Hsepi or Glce). Here we have investigated chondrocyte differentiation in Glce deficient mice and found increased regions of proliferating chondrocytes accompanied by a delayed onset of hypertrophic differentiation. In addition, we observed increased expression levels of the Ihh target genes Patched1 (Ptch1) and Parathyroid hormone related peptide (Pthrp; Parathyroid hormone like hormone (Pthlh)) indicating elevated Ihh signaling. We further show that Ihh binds with reduced affinity to HS isolated from Glce(-/-) mice. Together our results strongly indicate that not only the level, but also the structure of HS is critical in regulating the distribution and signaling activity of Ihh in chondrocytes.
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| 25. |
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