Maria Ringvall

Abnormal mast cells in mice deficient in a heparin-synthesizing enzyme

Heparin is a sulphated polysaccharide, synthesized exclusively by connective-tissue-type mast cells and stored in the secretory granules in complex with histamine and various mast-cell proteases. Although heparin has long been used as an antithrombotic drug, endogenous heparin is not present in the blood, so it cannot have a physiological role in regulating blood coagulation. The biosynthesis of heparin involves a series of enzymatic reactions, including sulphation at various positions,. The initial modification step, catalysed by the enzyme glucosaminyl N -deacetylase/N -sulphotransferase-2, NDST-2 (refs 4–7), is essential for the subsequent reactions. Here we report that mice carrying a targeted disruption of the gene encoding NDST-2 are unable to synthesize sulphated heparin. These NDST-2-deficient mice are viable and fertile but have fewer connective-tissue-type mast cells; these cells have an altered morphology and contain severely reduced amounts of histamine and mast-cell proteases. Our results indicate that one site of physiological action for heparin could be inside connective-tissue-type mast cells, where its absence results in severe defects in the secretory granules.

Heparan sulfate and development: differential roles of the N-acetylglucosamine N-deacetylase/N-sulfotransferase isozymes.

Heparan sulfates (HSs) are N- and O-sulfated polysaccharide components of proteoglycans, which are important constituents of the cell surface as well as the extracellular matrix. Heparin, with extensive clinical application as an anticoagulant, is a highly sulfated form of HS present within the granules of connective tissue type mast cells. The diverse functions of HS, which include the modulation of growth factor/cytokine activity, interaction with matrix proteins and binding of enzymes to cell surfaces, depend greatly on the presence of specific, high affinity regions on the chains. N-acetylglucosamine N-deacetylase/N-sulfotransferases, NDSTs, are an important group of enzymes in HS biosynthesis, initiating the sulfation of the polysaccharide chains and thus determining the generation of the high affinity sites. Here, we review the role of the four vertebrate NDSTs in HS biosynthesis as well as their regulated expression. The main emphasis is the phenotypes of mice lacking one or more of the NDSTs.

Zebrafish genes for neuropeptide Y and peptide YY reveal origin by chromosome duplication from an ancestral gene linked to the homeobox cluster

Abstract: Neuropeptide Y (NPY) and peptide YY (PYY) are related 36‐amino acid peptides. NPY is widely distributed in the nervous system and has several physiological roles. PYY serves as an intestinal hormone as well as a neuropeptide. We report here cloning of the npy and pyy genes in zebrafish (Danio rerio). NPY differs at only one to four amino acid positions from NPY in other jawed vertebrates. Zebrafish PYY differs at three positions from PYY from other fishes and at 10 positions from mammals. In situ hybridization showed that neurons containing NPY mRNA have a widespread distribution in the brain, particularly in the telencephalon, optic tectum, and rhombencephalon. PYY mRNA was found mainly in brainstem neurons, as reported previously for vertebrates as divergent as the rat and the lamprey, suggesting an essential role for PYY in these neurons. PYY mRNA was observed also in the telencephalon. These results were confirmed by immunocytochemistry. As in the human, the npy gene is located adjacent to homeobox (hox) gene cluster A (copy a in zebrafish), whereas the pyy gene is located close to hoxBa. This suggests that npy and pyy arose from a common ancestral gene in a chromosomal duplication event that also involved the hox gene clusters. As zebrafish has seven hox clusters, it is possible that additional NPY family genes exist or have existed. Also, the NPY receptor system seems to be more complex in zebrafish than in mammals, with at least two receptor genes without known mammalian orthologues.

Enzymatically Active N-Deacetylase/N-Sulfotransferase-2 Is Present in Liver but Does Not Contribute to Heparan Sulfate N-Sulfation

Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with growth factors and morphogens. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. NDST (glucosaminyl N-deacetylase/N-sulfotransferase), responsible for HS N-sulfation, is a key enzyme directing further modifications including O-sulfation. To elucidate the roles of the different NDST isoforms in HS biosynthesis, we took advantage of mice with targeted mutations in NDST1 and NDST2 and used liver as our model organ. Of the four NDST isoforms, only NDST1 and NDST2 transcripts were shown to be expressed in control liver. The absence of NDST1 or NDST2 in the knock-out mice did not affect transcript levels of other NDST isoforms or other HS modification enzymes. Although the sulfation level of HS synthesized in NDST1–/– mice was drastically lowered, liver HS from wild-type mice, from NDST1+/–, NDST2–/–, and NDST1+/–/NDST2–/– mice all had the same structure despite greatly reduced NDST enzyme activity (30% of control levels in NDST1+/–/NDST2–/– embryonic day 18.5 embryos). Enzymatically active NDST2 was shown to be present in similar amounts in wild-type, NDST1–/–, and NDST1+/– embryonic day 18.5 liver. Despite the substantial contribution of NDST2 to total NDST enzyme activity in embryonic day 18.5 liver (≈40%), its presence did not appear to affect HS structure as long as NDST1 was also present. In NDST1–/– embryonic day 18.5 liver, in contrast, NDST2 was responsible for N-sulfation of the low sulfated HS. A tentative model to explain these results is presented.

Activated Platelets Provide a Functional Microenvironment for the Antiangiogenic Fragment of Histidine-Rich Glycoprotein

The angiogenesis inhibitor histidine-rich glycoprotein (HRG) constitutes one of several examples of molecules regulating both angiogenesis and hemostasis. The antiangiogenic properties of HRG are mediated via its proteolytically released histidine- and proline-rich (His/Pro-rich) domain. Using a combination of immunohistochemistry and mass spectrometry, we here provide biochemical evidence for the presence of a proteolytic peptide, corresponding to the antiangiogenic domain of HRG, in vivo in human tissue. This finding supports a role for HRG as an endogenous regulator of angiogenesis. Interestingly, the His/Pro-rich peptide bound to the vessel wall in tissue from cancer patients but not to the vasculature in tissue from healthy persons. Moreover, the His/Pro-rich peptide was found in close association with platelets. Relesate from in vitro–activated platelets promoted binding of the His/Pro-rich domain of HRG to endothelial cells, an effect mediated by Zn2+. Previous studies have shown that zinc-dependent binding of the His/Pro-rich domain of HRG to heparan sulfate on endothelial cells is required for inhibition of angiogenesis. We describe a novel mechanism to increase the local concentration and activity of an angiogenesis inhibitor, which may reflect a host response to counteract angiogenesis during pathologic conditions. Our finding that tumor angiogenesis is elevated in HRG-deficient mice supports this conclusion. (Mol Cancer Res 2009;7(11):1792–802)

Mast Cell Differentiation and Activation Is Closely Linked to Expression of Genes Coding for the Serglycin Proteoglycan Core Protein and a Distinct Set of Chondroitin Sulfate and Heparin Sulfotransferases

Serglycin (SG) proteoglycan consists of a small core protein to which glycosaminoglycans of chondroitin sulfate or heparin type are attached. SG is crucial for maintaining mast cell (MC) granule homeostasis through promoting the storage of various basic granule constituents, where the degree of chondroitin sulfate/heparin sulfation is essential for optimal SG functionality. However, the regulation of the SG core protein expression and of the various chondroitin sulfate/heparin sulfotransferases during MC differentiation and activation are poorly understood. Here we addressed these issues and show that expression of the SG core protein, chondroitin 4-sulfotransferase (C4ST)-1, and GalNAc(4S)-6-O-sulfotransferase (GalNAc4S6ST) are closely linked to MC maturation. In contrast, the expression of chondroitin 6-sulfotransferase correlated negatively with MC maturation. The expression of N-deacetylase/N-sulfotransferase (NDST)-2, a key enzyme in heparin synthesis, also correlated strongly with MC maturation, whereas the expression of the NDST-1 isoform was approximately equal at all stages of maturation. MC activation by either calcium ionophore or IgE ligation caused an up-regulated expression of the SG core protein, C4ST-1, and GalNAc4S6ST, accompanied by increased secretion of chondroitin sulfate as shown by biosynthetic labeling experiments. In contrast, NDST-2 was down-regulated after MC activation, suggesting that MC activation modulates the nature of the glycosaminoglycan chains attached to the SG core protein. Taken together, these data show that MC maturation is associated with the expression of a distinct signature of genes involved in SG proteoglycan synthesis, and that MC activation modulates their expression.

Vaccination against the extra domain-B of fibronectin as a novel tumor therapy

Monoclonal antibody‐based therapies have made an important contribution to current treatment strategies for cancer and autoimmune disease. However, the cost for these new drugs puts a significant strain on the healthcare economy, resulting in limited availability for patients. Therapeutic vaccination, defined as induction of immunity against a disease‐related self‐molecule, is therefore an attractive alternative. To analyze the potential of such an approach, we have developed a vaccine against the extra domain‐B (ED‐B) of fibronectin. This 91‐aa domain, inserted by alternative splicing, is expressed during vasculogenesis in the embryo, but essentially undetectable under normal conditions in the adult. However, ED‐B is highly expressed around angiogenic vasculature, such as in tumorigenesis. Here, we demonstrate that it is possible to break self‐tolerance and induce a strong antibody response against ED‐B by vaccination. Nineteen of 20 vaccinated mice responded with production of anti‐ED‐B antibodies and displayed a 70% reduction in tumor size compared to those lacking anti‐ED‐B antibodies. Analysis of the tumor tissue revealed that immunization against ED‐B induced several changes, consistent with an attack by the immune system. These data show that tumor vascular antigens are highly interesting candidates for development of therapeutic vaccines targeting solid tumors.—Huijbers, E. J. M., Ringvall, M., Femel, J., Kalamajski, S., Lukinius, A., Abrink, M., Hellman, L., Olsson, A.‐K. Vaccination against the extra domain‐B of fibronectin as a novel tumor therapy. FASEB J. 24, 4535–4544 (2010). www.fasebj.org

Identification of potent biodegradable adjuvants that efficiently break self-tolerance - a key issue in the development of therapeutic vaccines.

Monoclonal antibodies are used successfully in the treatment of many human disorders. However, these antibodies are expensive and have in many countries put a major strain on the health care economy. Therapeutic vaccines, directed against the same target molecules, may offer a solution to this problem. Vaccines usually involve lower amount of recombinant protein, approximately 10,000-20,000 times less, which is significantly more cost effective. Attempts to develop such therapeutic vaccines have also been made. However, their efficacy has been limited by the lack of potent immunostimulatory compounds, adjuvants, for human use. To address this problem we have conducted a broad screening for adjuvants that can enhance the efficacy of therapeutic vaccines, whilst at the same time being non-toxic and biodegradable. We have now identified adjuvants that show these desired characteristics. A combination of Montanide ISA720 and phosphorothioate stabilized CpG stimulatory DNA, induced similar or even higher anti-self-antibody titers compared to Freunds adjuvant, currently the most potent, but also toxic, adjuvant available. This finding removes one of the major limiting factors in the field and facilitates the development of a broad range of novel therapeutic vaccines.

Mice Deficient in Heparan Sulfate N-Deacetylase/N-Sulfotransferase 1

Ndsts (N-deacetylase/N-sulfotransferases) are enzymes responsible for N-sulfation during heparan sulfate (HS) and heparin biosynthesis. In this review, basic features of the Ndst1 enzyme are covered and a brief description of HS biosynthesis and its regulation is presented. Effects of Ndst1 deficiency on embryonic development are described. These include immature lungs, craniofacial dysplasia and eye developmental defects, branching defect during lacrimal gland development, delayed mineralization of the skeleton, and reduced pericyte recruitment during vascular development. A brief account of the effects of Ndst1 deficiency in selective cell types in adult mice is also given.

Enhanced Platelet Activation Mediates the Accelerated Angiogenic Switch in Mice Lacking Histidine-Rich Glycoprotein

Background The heparin-binding plasma protein histidine-rich glycoprotein (HRG; alternatively, HRGP/HPRG) can suppress tumor angiogenesis and growth in vitro and in vivo. Mice lacking the HRG gene are viable and fertile, but have an enhanced coagulation resulting in decreased bleeding times. In addition, the angiogenic switch is significantly enhanced in HRG-deficient mice. Methodology/Principal Findings To address whether HRG deficiency affects tumor development, we have crossed HRG knockout mice with the RIP1-Tag2 mouse, a well established orthotopic model of multistage carcinogenesis. RIP1-Tag2 HRG−/− mice display significantly larger tumor volume compared to their RIP1-Tag2 HRG+/+ littermates, supporting a role for HRG as an endogenous regulator of tumor growth. In the present study we also demonstrate that platelet activation is increased in mice lacking HRG. To address whether this elevated platelet activation contributes to the increased pathological angiogenesis in HRG-deficient mice, they were rendered thrombocytopenic before the onset of the angiogenic switch by injection of the anti-platelet antibody GP1bα. Interestingly, this treatment suppressed the increase in angiogenic neoplasias seen in HRG knockout mice. However, if GP1bα treatment was initiated at a later stage, after the onset of the angiogenic switch, no suppression of tumor growth was detected in HRG-deficient mice. Conclusions Our data show that increased platelet activation mediates the accelerated angiogenic switch in HRG-deficient mice. Moreover, we conclude that platelets play a crucial role in the early stages of tumor development but are of less significance for tumor growth once angiogenesis has been initiated.