The effect of synovial membrane products and mechanical pressure upon the metabolism of articular cartilage proteoglycans has been studied in vitro. The degradation of cartilage proteoglycans was studied in an organ culture system and measured as the release of [35S ] sulphate from prelabelled cartilage. The effect of synovial membrane products upon the synthesis of proteoglycans was studied in a chondrocyte monolayer system and the effect of mechanical pressure upon the synthesis of proteoglycans in an organ culture system. In both types of experiments [35S] sulphate was used as precursor. The findings may be summarized as follows 1 Conditioned synovial medium (control-SM) enhanced the degradation and reduced the synthesis of cartilage proteoglycans. In addition the degradation was further enhanced when the synovial tissue had been cultured in the presence of dextran sulphate. 2 Conditioned medium from synovial tissue cultured in the presence of indo-methacin (indo-SM), significantly reduced the synthesis of cartilage proteoglycans in chondrocyte cultures and reduced, although non-significantly, the degradation of proteoglycans in whole cartilage cultures. 3 Addition o f the prostaglandins E1 or E2 (PGE1 or PGE2 ) together with indo-SM to the cartilage cultures greatly enhanced cartilage degradation whereas the addition of PGE1 or PGE2 together with control-SM had no effect compared with that of control-SM alone. 4 Conditioned medium from synovial tissue cultured in the presence of low doses of glucocorticoids reduced cartilage degradation compared with control-SM. However, addition of control-SM together w ith low concentrations of glucocorticoids to the cartilage cultures significantly enhanced cartilage degradation. 5 Conditioned medium from synovial tissue cultured with actinomycin D or cycloheximide did not enhance cartilage degradation compared with cartilage cultured alone. 6 A continuous pressure of approximately 30 kgfcm-2 on cultures of cartilage reduced both the synthesis and the degradation o f cartilage proteoglycans. Although it is difficult to extrapolate from the in vitro to the in vivo situation, it is proposed that some factor(s) from the synovial membrane have the capacity to enhance the degradation and reduce the synthesis o f articular cartilage proteoglycans. From these experiments it cannot be completely excluded that treatm ent of arthritic joints with non-steroidal or streroidal anti-inflammatory drugs may result under certain conditions in enhanced joint damage. It is also suggested that under certain conditions the metabolism o f cartilage proteoglycans could be directly affected by mechanical stress. / <p>Diss. Umeå, Umeå universitet, 1982, härtill 6 uppsatser</p> / digitalisering@umu

<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>

En raison d’une abondante matrice extracellulaire chondrogénique, peu vascularisée et hypoxique, le chondrosarcome, cancer du cartilage, est une tumeur chimio et radiorésistante. Une stratégie bispécifique s’appuyant sur deux caractéristiques spécifiques du chondrosarcome, une forte densité de charges négatives, ainsi qu’une hypoxie chronique sévère de la matrice extracellulaire, est actuellement développée au laboratoire et consiste à fonctionnaliser une prodrogue activée en hypoxie avec un ammonium quaternaire (AQ). Sur la base de résultats précliniques, une prodrogue, ICF05016, a été identifiée et fait actuellement l’objet d’une étude d’optimisation.Ce projet vise à identifier des relations structure-activité dans une série de moutardes phosphorodiamidates vectorisées par une fonction AQ. Dans une première partie, nous nous sommes intéressés à faire varier la nature des substituants de la fonction ammonium quaternaire ainsi que la longueur du bras espaceur séparant cette dernière de la moutarde. Ainsi, 27 dérivés ont été synthétisés par une séquence commune multi-étapes impliquant la phosphorylation d’un dérivé 2-nitro-5-hydroxyméthylimidazole.Un screening par résonance plasmonique de surface a permis de mettre en évidence 12 composés plus affins envers l’aggrécane que le composé de référence ICF05016. Cette étude a révélé une dépendance importante entre l’affinité à l’aggrécane et la nature de l’ammonium quaternaire, les composés benzyléspossédant tous des affinités supérieures. In vitrosur cultures cellulaires HEMC-SS, les composés ont affichés des ratios de cytotoxicité normoxie vs hypoxie (HCR)supérieurs à celui du lead ICF05016, et notamment le composé benzylé31f.Avec une constante de dissociation à l’aggrécane de 2,10 µM et un différentiel de cytotoxicité normoxie/hypoxie équivalent à celui de l’évosfosfamide en étude clinique, la composé 31f a été évalué en termes de stabilité et de clivage en conditions réductrices, mimant l’hypoxie tumorale. Démontrant une excellente stabilité sur 24 heures dans un tampon phosphate, le composé 31f s’est révélé être réduit par réduction chimique ou enzymatique via une nitroréductase, et ce avec une cinétique rapide. Ce dernier a été ainsi identifié au terme de l’évaluation in vitro et de cet axe de recherche comme un nouveau « lead ».Dans une seconde partie, nous avons choisi de moduler l’halogène et la position des bras alkylants portés par la moutarde phosphorodiamidate. Compte tenu de l’instabilité des composés de la série bromée, par rapport à la série chlorée, les analogues bromés des composés ICF05016 et 31f n’ont pu faire l’objet d’une évaluation in vitro. Pour les isomères de type moutarde ifosfamide, l’instabilité des précurseurs phosphoramidiques et les difficultés de synthèse inhérentes à l’introduction d’une fonction amine secondaire sur un chlorure de phosphoryle préalablement fonctionnalisé, nous ont contraints à réorienter nos travaux vers des structures issues d’une étape de chimie de click, permettant d’introduire le bras vecteur portant la fonction amine tertiaire indépendamment de l’étape de phosphorylation. Cette séquence réactionnelle pourrait être étendue à la synthèse de nouveaux analogues du composé 31f, identifié lors de ces travaux. / Due to an abundant chondrogenic, poorly vascularized and particularly hypoxic extracellular matrix, chondrosarcoma, a malignant cartilaginous tumour, is chemo- and radio-resistant. A dual targeted therapy leveraging specific chondrosarcoma hallmarks, an extensive cartilaginous extracellular matrix, namely the high negative fixed charge density and severe chronic hypoxia,was investigatedby conjugation of a hypoxia-activated prodrug (HAP) to quaternary ammonium (QA) functions. Based on preclinical results, animidazole prodrug, ICF05016, was identified and provided the basis for a lead optimization study.This project was designed to identify structure-activity relationships in a series of QA-targeted phosphorodiamidate mustard conjugates. In a first part, a series of 27 conjugates, with different QA function and length of the alkyl linker, was synthesizedvia a common multi-step sequence involvingphosphorylation of a key 2-nitroimidazole alcohol intermediate. Then, biomolecular interactions between these QA derivatives and aggrecan were assessed using thesurface plasmon resonance technology. This screeningrevealed that the affinity depends more on the natureof the QA function, rather than on the linker length. The most promising results were obtainedwith QA bearing a benzyle group. Twelves compounds were then evaluated in terms of hypoxia selective cytotoxicityonthe HEMC-SS cell line. For all prodrugs, an overall improvement in hypoxic selectivity compared with the ICF05016 was obtained and the positive impact of thebenzyle QA function was again highlighted. With a dissociation constant of 2.10 µM in the SPR experiment and an attractive hypoxic selectivity, compound 31fwas further selected for a stability and reductive activation study. Activation of the prodrug and phosphoramide mustard release under reductive chemical conditions, and nitroreductase-based activation were demonstrated. From this study, compound 31f emerged as the most effective PG-targeted HAPs. In a second part, the nature of the halogen and the position of the alkylating arms carried by the phosphorodiamidic mustard were modified. Given the high instability of the final compounds of the brominated series, compared to the chlorinated series, brominated analogs of the compounds ICF05016 and 31fcould not be obtained with sufficient purity to be evaluated in vitro.For ifosfamide-like isomers, the design of the prodrugs was modified due to the instability of the phosphoramidic precursors or difficulties of introducing a secondary amine function on a phosphoryl chloride previously functionalized.A click chemistry approach was developed to tether the spacer arm carrying the tertiary amine function independently of the phosphorylation step and could be extended to the synthesis of triazoleanalogues of the new lead 31f.

Perlecan is an important basement membrane heparan sulfate (HS) proteoglycan that is essential for various cell signaling events involved in tissue development. Heparanase is a lysosomal enzyme involved in the turnover of HS. This project aimed to assist in researching the structure of HS on perlecan and how this structure changes with tissue development. This will be achieved by generating monoclonal antibodies that have an altered affinity for perlecan after heparanase treatment. Recombinant perlecan domain I was characterized by ELISA and western blotting and used as the antigen for two fusions. The first fusion was focused on the production of IgM the common subtype of anti-glycosaminoglycans antibodies. However, no clones were produced, which may have been due to the lack of feeder layers. In order to address this problem, the fibroblast cell line MRC-5 was used as a feeder layer in the second fusion. From this fusion, we obtained 216 positive cultures, which were screened against full length perlecan from endothelial cells. Of these, 26 cultures were tested against heparanase treated perlecan, and then 2 cultures were chosen for subcloning based on the different immunoreactivity between enzyme treated and nontreated perlecan. From the 2 chosen cultures, 13 sub clones were derived and 10 of them were adapted into a serum free culture environment. The 10 monoclonal antibodies displayed strong immunoreactivity with full length perlecan in ELISA and Western Blotting. When they were used as primary antibodies in Immunocytochemistry, they were able to recognize the native perlecan deposited by human chondrocytes. When the cells were incubated with heparanase, antibody 5D7-2E4 and 13E9-3G5 showed an increase in immunoreactivity while antibody 13E9-3B3 gave a decrease. These three antibodies will be the potential tools used in the future to study perlecan turnover in different cells and tissue. The remaining seven antibodies will also be very useful in the research of perlecan as they have been shown to bind to the protein core. In the future, it will be worth subcloning some of the frozen stored stocks of uncloned hybridomas, where there are potential opportunities to select antibodies, which will react with the carbohydrate chains on perlecan.

Trauma to the central nervous system (CNS) results in an irreversible disruption of axon tracts, often leading to lifelong functional deficits. Despite a large body of research into the mechanisms that underlie the lack of axonal regeneration after CNS injury, there are currently no effective treatments. One major obstacle involves the presence at injury sites of CNS growth-inhibitory molecules, such as myelin proteins and astrocyte-derived chondroitin sulfate proteoglycans (CSPGs), which act as environmental barriers to axonal regeneration. Our lab recently described the identification and characterization of a novel compound, F05, which promotes growth on inhibitory substrates in vitro. I show that F05 improves regeneration in vivo after acute sensory axon transection as well as after optic nerve crush injury. F05 does not target known signaling molecules involved in CSPG or myelin mediated inhibition but does affect growth cone microtubule dynamics, suggesting a potentially novel mechanism of growth promotion. Using a protein microarray, I show that apoptotic signaling pathways may underlie glial-derived inhibition and its relief by F05. In addition, I employed a comparative gene microarray to show that F05 induces similar changes in gene expression as antipsychotics of the piperazine phenothiazine structural class (PhAPs). Indeed, PhAPs share F05’s ability to overcome glial-derived inhibition of cultured CNS neurons and do so through a mechanism dependent on antagonism of calmodulin. These studies have led to the identification of potentially novel clinical treatments for CNS injury as well as a better understanding of environmentally derived growth-inhibitory signaling mechanisms.

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.

Flexor tendon injuries in zone II of the hand (i.e. between the distal volar crease and the distal interphalangeal joint) can be costly for both the afflicted individual and society because of the high cost of a long rehabilitation period, complicated by tendon ruptures or scarring with adhesion formation, causing impaired range of motion. The aim of the present thesis was to characterize more fully the deep flexor tendon, the tendon sheath and their response to injury in a rabbit model in order to find potential targets to improve the outcome of repair. The intrasynovial rabbit deep flexor tendon differed from the extrasynovial peroneus tendon in the expression of collagens and transforming growth factor-β1 gene expression. Differences were also found in collagen III and proteoglycans between regions of the flexor tendon subjected to either compressive or tensile load. After laceration and subsequent repair of the flexor tendon, a shift in collagen gene expression from type I to type III occurred. Proteoglycans were generally increased with the notable exception of decorin, a potential inhibitor of the profibrotic transforming growth factor-β1 which was markedly increased during the first two weeks after repair in tendon tissue but remained unaltered in the sheaths. Both vascular endothelial growth factor and basic fibroblast growth factor mRNA levels remained essentially unaltered, whereas insulin-like growth factor-1 increased later in the healing process, suggesting potential beneficial effects of exogenous addition, increasing tendon strength through stimulating tenocyte proliferation and collagen synthesis. Matrix metalloproteinase-13 mRNA levels increased and remained high in both tendon and sheath, whereas there was only a transient increase of matrix metalloproteinase-3 mRNA in tendon. We could also demonstrate a significant increase of the proportion of myofibroblasts, mast cells and neuropeptide containing nerve fibers in the healing tendon tissue, all components of the profibrotic myofibroblast-mast cell-neuropeptide pathway. / Biomolecular aspects of flexor tendon healing

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.

Heparan sulfate (HS) is a highly sulfated polysaccharide expressed on the cell surface and in the extracellular matrix, interacting with a large number of proteins. HS is implicated in human diseases, including different types of cancer and amyloid diseases such as Alzheimer's disease (AD). The aims of this thesis were to gain deeper insights into AD and cancer progression by elucidating the roles of HS in amyloid-β (Aβ) pathology and hypoxia. The toxic Aβ-peptide is a key molecule in AD due to its ability to aggregate and form amyloid plaques in the brains of diseased patients. It has been reported that HS accumulates with Aβ in these amyloid plaques. We have found that HS is differentially accumulated with Aβ species within the amyloid plaques in the brains of AD patients. We also identified that the HS in the plaques originated from glial cells. Further, we investigated the role of HS in Aβ toxicity using cell models that either lack HS or express abnormal HS. The results show that cell surface HS mediates Aβ internalization and cytotoxicity. Upregulation of heparanase, an endo-glucuronidase that specifically cleaves HS chains, in human cancers increases the potential of tumor cells to metastasize. Spalax, an animal model for hypoxic tolerance, expresses high levels of heparanase. Analysis of HS from different Spalax organs revealed a high sulfation degree and an atypical domain structure, likely modulated by high heparanase expression in the organs. Cells cultured under hypoxic conditions showed a similar HS domain structure and had an increase in heparanase mRNA. We propose that hypoxia-induced heparanase expression is relevant for tumor progression, a process often associated with oxygen deficiency. Altogether, the findings in this thesis are important for future development of therapeutics aiming at interfering with HS functions in AD and cancer.

Aggregating proteoglycans (PG) bearing chondroitin sulfate (CS) side chains are well-known inhibitors of neural plasticity and associate with hyaluronan and tenascin-R to form a complex of extracellular matrix (ECM) in the central nervous system (CNS). Little is known about whether proteolytic cleavage of the core protein affects neural plasticity. Several members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of metalloproteinases are glutamyl-endopeptidases that cleave aggregating PGs. Our initial studies determined that neural cultures secrete a brevican-containing matrix, and that these neural cultures also produced ADAMTS4, a protease that cleaves brevican. Furthermore, this brevican-containing matrix in astrocytes could be modulated by treatment with transforming growth factor beta (TGFbeta) through the inhibition of the activity of the ADAMTSs.Once it was established that neural cultures produce a brevican-rich matrix, we s ought to utilize this matrix to determine whether cleavage of aggregating PGs, especially brevican, by the ADAMTSs influences neurite outgrowth in cultured neurons. Transfection of rat neurons with ADAMTS4 cDNA induced longer neurites, and interestingly, this effect proved to be independent of the proteolytic action of the ADAMTSs. Addition of recombinant ADAMTS4 or ADAMTS5 protein to immature neuronal cultures similarly enhanced neurite extension, an action dependent on the activation of extracellular signal-related kinase (ERK)1/2 (MAP kinase 42/44), resulting in the first evidence that ADAMTSs may induce intracellular signaling events. Studies of dendritic spine morphology and levels of synaptic proteins in response to ADAMTS4 treatment were also undertaken. Neuronal cultures treated with ADAMTS4 showed increased length of dendritic spines and increased percent of immature spines detected. A concurrent decrease in post-synaptic protein staining was detected on the neurites of yo ung neurons overexpressing ADAMTS4 or expressing proteolytically-inactive mutant ADAMTS4 protein. Thus, ADAMTS4 may promote plasticity in neurons in vitro by preventing the formation, maturation, and/or stabilization of synapses. Overall, these experiments provide evidence that implicate the ADAMTSs as mediators of neural plasticity, and while primarily known only as proteases, these studies demonstrate that the ADAMTSs exert actions distinct from these proteolytic properties that require the induction of intracellular signaling events.