Explore the vast range of titles available.

Heparanase, a mammalian endo-beta-D-glucuronidase, specifically degrades heparan sulfate proteoglycans ubiquitously associated with the cell surface and extracellular matrix. This single gene encoded enzyme is over-expressed in most human cancers, promoting tumor metastasis and angiogenesis. We report that targeted disruption of the murine heparanase gene eliminated heparanase enzymatic activity, resulting in accumulation of long heparan sulfate chains. Unexpectedly, the heparanase knockout (Hpse-KO) mice were fertile, exhibited a normal life span and did not show prominent pathological alterations. The lack of major abnormalities is attributed to a marked elevation in the expression of matrix metalloproteinases, for example, MMP2 and MMP14 in the Hpse-KO liver and kidney. Co-regulation of heparanase and MMPs was also noted by a marked decrease in MMP (primarily MMP-2,-9 and 14) expression following transfection and over-expression of the heparanase gene in cultured human mammary carcinoma (MDA-MB-231) cells. Immunostaining (kidney tissue) and chromatin immunoprecipitation (ChIP) analysis (Hpse-KO mouse embryonic fibroblasts) suggest that the newly discovered co-regulation of heparanase and MMPs is mediated by stabilization and transcriptional activity of beta-catenin. The lack of heparanase expression and activity was accompanied by alterations in the expression level of MMP family members, primarily MMP-2 and MMP-14. It is conceivable that MMP-2 and MMP-14, which exert some of the effects elicited by heparanase (i.e., over branching of mammary glands, enhanced angiogenic response) can compensate for its absence, in spite of their different enzymatic substrate. Generation of viable Hpse-KO mice lacking significant abnormalities may provide a promising indication for the use of heparanase as a target for drug development.

Defective amyloid-β (Aβ) clearance from the brain is a major contributing factor to the pathophysiology of Alzheimer’s disease (AD). Aβ clearance is mediated by macrophages, enzymatic degradation, perivascular drainage along the vascular basement membrane (VBM) and transcytosis across the blood–brain barrier (BBB). AD pathology is typically associated with cerebral amyloid angiopathy due to perivascular accumulation of Aβ. Heparan sulfate (HS) is an important component of the VBM, thought to fulfill multiple roles in AD pathology. We previously showed that macrophage-mediated clearance of intracortically injected Aβ was impaired in the brains of transgenic mice overexpressing heparanase (Hpa-tg). This study revealed that perivascular drainage was impeded in the Hpa-tg brain, evidenced by perivascular accumulation of the injected Aβ in the thalamus of Hpa-tg mice. Furthermore, endogenous Aβ accumulated at the perivasculature of Hpa-tg thalamus, but not in control thalamus. This perivascular clearance defect was confirmed following intracortical injection of dextran that was largely retained in the perivasculature of Hpa-tg brains, compared to control brains. Hpa-tg brains presented with thicker VBMs and swollen perivascular astrocyte endfeet, as well as elevated expression of the BBB-associated water-pump protein aquaporin 4 (AQP4). Elevated levels of both heparanase and AQP4 were also detected in human AD brain. These findings indicate that elevated heparanase levels alter the organization and composition of the BBB, likely through increased fragmentation of BBB-associated HS, resulting in defective perivascular drainage. This defect contributes to perivascular accumulation of Aβ in the Hpa-tg brain, highlighting a potential role for heparanase in the pathogenesis of AD.

Amyloid diseases encompass >20 medical disorders that include amyloid protein A (AA) amyloidosis, Alzheimer's disease, and type 2 diabetes. A common feature of these conditions is the selective organ deposition of disease-specific fibrillar proteins, along with the sulfated glycosaminoglycan, heparan sulfate. We have generated transgenic mice that overexpress human heparanase and have tested their susceptibility to amyloid induction. Drastic shortening of heparan sulfate chains was observed in heparanase-overproducing organs, such as liver and kidney. These sites selectively escaped amyloid deposition on experimental induction of inflammation-associated AA amyloidosis, as verified by lack of material staining with Congo Red, as well as lack of associated polysaccharide, whereas the same tissues from control animals were heavily infiltrated with amyloid. By contrast, the spleens of transgenic mice that failed to significantly overexpress heparanase contained heparan sulfate chains similar in size to those of control spleen and remained susceptible to amyloid deposition. Our findings provide direct in vivo evidence that heparan sulfate is essential for the development of amyloid disease.

Heparan sulfate proteoglycans (HSPGs) interact with numerous proteins of importance in animal development and homeostasis 1 , 2 , 3 . Heparanase, which is expressed in normal tissues and upregulated in angiogenesis, cancer and inflammation, selectively cleaves β-glucuronidic linkages in HS chains. In a previous study, we transgenically overexpressed heparanase in mice to assess the overall effects of heparanase on HS metabolism. Metabolic labeling confirmed extensive fragmentation of HS in vivo 4 , 5 . In the current study we found that in liver showing excessive heparanase overexpression, HSPG turnover is accelerated along with upregulation of HS N- and O-sulfation, thus yielding heparin-like chains without the domain structure typical of HS. Heparanase overexpression in other mouse organs and in human tumors correlated with increased 6-O-sulfation of HS, whereas the domain structure was conserved. The heavily sulfated HS fragments strongly promoted formation of ternary complexes with fibroblast growth factor 1 (FGF1) or FGF2 and FGF receptor 1. Heparanase thus contributes to regulation of HS biosynthesis in a way that may promote growth factor action in tumor angiogenesis and metastasis.

Heparin, discovered as an anticoagulant more than 100 years ago, is composed of the same structural units as heparan sulfate, and their biosynthesis in the Golgi compartment is carried out by the same families of enzymes. However, while heparin is restricted to mast cells, heparan sulfate is widely distributed and is found at the cell surface of most, if not all, cells in the body and also in basement membranes and the extracellular matrix surrounding cells. Compared to the highly sulfated heparin, heparan sulfate has a complex and variable structure that yet appears regulated in tissue‐specific fashion. Interactions of proteins with heparan sulfate may be controlled through subtle structural changes of binding saccharide domains, resulting in modulated functional response. Unpredicted effects of enzyme overexpression on heparan sulfate structure point to key roles for interacting enzyme networks—the \"GAGosome\"—in regulation of heparan sulfate biosynthesis.

Mutation of the melanocortin-receptor 4 (MC4R) is the most frequent cause of severe obesity in humans. Binding of agouti-related peptide (AgRP) to MC4R involves the co-receptor syndecan-3, a heparan sulfate proteoglycan. The proteoglycan can be structurally modified by the enzyme heparanase. Here we tested the hypothesis that heparanase plays a role in food intake behaviour and energy balance regulation by analysing body weight, body composition and food intake in genetically modified mice that either lack or overexpress heparanase. We also assessed food intake and body weight following acute central intracerebroventricular administration of heparanase; such treatment reduced food intake in wildtype mice, an effect that was abolished in mice lacking MC4R. By contrast, heparanase knockout mice on a high-fat diet showed increased food intake and maturity-onset obesity, with up to a 40% increase in body fat. Mice overexpressing heparanase displayed essentially the opposite phenotypes, with a reduced fat mass. These results implicate heparanase in energy balance control via the central melanocortin system. Our data indicate that heparanase acts as a negative modulator of AgRP signaling at MC4R, through cleavage of heparan sulfate chains presumably linked to syndecan-3.

The tumor suppressors EXT1 and EXT2 are associated with hereditary multiple exostoses and encode bifunctional glycosyltransferases essential for chain polymerization of heparan sulfate (HS) and its analog, heparin (Hep). Three highly homologous EXT-like genes, EXTL1-EXTL3, have been cloned, and EXTL2 is an α1,4-GlcNAc transferase I, the key enzyme that initiates the HS/Hep synthesis. In the present study, truncated forms of EXTL1 and EXTL3, lacking the putative NH2-terminal transmembrane and cytoplasmic domains, were transiently expressed in COS-1 cells and found to harbor α-GlcNAc transferase activity. EXTL3 used not only N-acetylheparosan oligosaccharides that represent growing HS chains but also GlcAβ1-3Galβ1-O-C2H 4NH-benzyloxycarbonyl (Cbz), a synthetic substrate for α-GlcNAc transferase I that determines and initiates HS/Hep synthesis. In contrast, EXTL1 used only the former acceptor. Neither EXTL1 nor EXTL3 showed any glucuronyltransferase activity as examined with N-acetylheparosan oligosaccharides. Heparitinase I digestion of each transferase-reaction product showed that GlcNAc had been transferred exclusively through an α1,4-configuration. Hence, EXTL3 most likely is involved in both chain initiation and elongation, whereas EXTL1 possibly is involved only in the chain elongation of HS and, maybe, Hep as well. Thus, their acceptor specificities of the five family members are overlapping but distinct from each other, except for EXT1 and EXT2 with the same specificity. It now has been clarified that all of the five cloned human EXT gene family proteins harbor glycosyltransferase activities, which probably contribute to the synthesis of HS and Hep.

The D ‐glucuronyltransferase and N ‐acetyl‐ D ‐glucosaminyltransferase reactions in heparan sulfate biosynthesis have been associated with two genes, EXT1 and EXT2 , which are also implicated in the inherited bone disorder, multiple exostoses. Since the cell systems used to express recombinant EXT proteins synthesize endogenous heparan sulfate, and the EXT proteins tend to associate, it has not been possible to define the functional roles of the individual protein species. We therefore expressed EXT1 and EXT2 in yeast, which does not synthesize heparan sulfate. The recombinant EXT1 and EXT2 were both found to catalyze both glycosyltransferase reactions in vitro . Coexpression of the two proteins, but not mixing of separately expressed recombinant EXT1 and EXT2, yields hetero‐oligomeric complexes in yeast and mammalian cells, with augmented glycosyltransferase activities. This stimulation does not depend on the membrane‐bound state of the proteins.

Biosynthesis of heparin, a mast cell–derived glycosaminoglycan with widespread importance in medicine, has not been fully elucidated. In biosynthesis of heparan sulfate (HS), a structurally related polysaccharide, HS glucuronyl C5-epimerase (Hsepi) converts D -glucuronic acid (GlcA) to L -iduronic acid (IdoA) residues. We have generated Hsepi -null mouse mutant mast cells, and we show that the same enzyme catalyzes the generation of IdoA in heparin and that 'heparin' lacking IdoA shows a distorted O -sulfation pattern.

PurposeThis study aimed to explore physicians’ use of drug information in professional work, with special focus on those working in primary care, and also in relation to personal characteristics of physicians.MethodsA web-based questionnaire was distributed by e-mail to physicians in five regions in Sweden. The questions concerned drug-related queries at issue when searching for information, sources used, and factors of importance for the choice of source, as well as responder characteristics.ResultsA total of 3254 (85%) out of 3814 responding physicians stated that they searched for drug information every week. For physicians working in primary health care, the corresponding number was 585 (96%). The most common drug-related issues searched for by 76% of physicians every week concerned pharmacotherapeutic aspects (e.g., dosing), followed by adverse drug reactions (63%). For 3349 (88%) physicians, credibility was the most important factor for the choice of sources of drug information, followed by easy access online (n = 3127, 82%). Further analyses among physicians in primary care showed that some personal characteristics, like seniority, sex, and country of education, as well as research experience, were associated with usage and preferences of drug information sources.ConclusionsThis study confirms that physicians often use drug information sources in professional work, in particular those who work in primary health care. Credibility and easy access are key factors for usage. Among physicians in primary care, personal factors influenced the choice of drug information sources.