Role of Heparanase on Hepatic Uptake of Intestinal Derived Lipoprotein and Fatty Streak Formation in Mice

BACKGROUND: Heparanase modulates the level of heparan sulfate proteoglycans (HSPGs) which have an important role in multiple cellular processes. Recent studies indicate that HSPGs have an important function in hepatic lipoprotein handling and processes involving removal of lipoprotein particles. PRINCIPAL FINDINGS: To determine the effects of decreased HSPGs chain length on lipoprotein metabolism and atherosclerosis, transgenic mice over-expressing the human heparanase gene were studied. Hepatic lipid uptake in hpa-Tg mice were evaluated by giving transgenic mice oral fat loads and labeled retinol. Sections of aorta from mice over-expressing heparanase (hpa-Tg) and controls (C57/BL6) fed an atherogenic diet were examined for evidence of atherosclerosis. Heparanase over-expression results in reduced hepatic clearance of postprandial lipoproteins and higher levels of fasting and postprandial serum triglycerides. Heparanase over-expression also induces formation of fatty streaks in the aorta. The mean lesion cross-sectional area in heparanase over-expressing mice was almost 6 times higher when compared to control mice (23,984 µm(2)±5,922 vs. 4,189 µm(2)±1,130, p<0.001). CONCLUSIONS: Over-expression of heparanase demonstrates the importance of HSPGs for the uptake of intestinal derived lipoproteins and its role in the formation of fatty streaks

Syndecans Reside in Sphingomyelin-Enriched Low-Density Fractions of the Plasma Membrane Isolated from a Parathyroid Cell Line

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) are one of the basic constituents of plasma membranes. Specific molecular interactions between HSPGs and a number of extracellular ligands have been reported. Mechanisms involved in controlling the localization and abundance of HSPG on specific domains on the cell surface, such as membrane rafts, could play important regulatory roles in signal transduction. METHODOLOGY/PRINCIPAL FINDINGS: Using metabolic radiolabeling and sucrose-density gradient ultracentrifugation techniques, we identified [(35)S]sulfate-labeled macromolecules associated with detergent-resistant membranes (DRMs) isolated from a rat parathyroid cell line. DRM fractions showed high specific radioactivity ([(35)S]sulfate/mg protein), implying the specific recruitment of HSPGs to the membrane rafts. Identity of DRM-associated [(35)S]sulfate-labeled molecules as HSPGs was confirmed by Western blotting with antibodies that recognize heparan sulfate (HS)-derived epitope. Analyses of core proteins by SDS-PAGE revealed bands with an apparent MW of syndecan-4 (30-33 kDa) and syndecan-1 (70 kDa) suggesting the presence of rafts with various HSPG species. DRM fractions enriched with HSPGs were characterized by high sphingomyelin content and found to only partially overlap with the fractions enriched in ganglioside GM1. HSPGs could be also detected in DRMs even after prior treatment of cells with heparitinase. CONCLUSIONS/SIGNIFICANCE: Both syndecan-1 and syndecan-4 have been found to specifically associate with membrane rafts and their association seemed independent of intact HS chains. Membrane rafts in which HSPGs reside were also enriched with sphingomyelin, suggesting their possible involvement in FGF signaling. Further studies, involving proteomic characterization of membrane domains containing HSPGs might improve our knowledge on the nature of HSPG-ligand interactions and their role in different signaling platforms

Heparan Sulfate Regrowth Profiles Under Laminar Shear Flow Following Enzymatic Degradation

The local hemodynamic shear stress waveforms present in an artery dictate the endothelial cell phenotype. The observed decrease of the apical glycocalyx layer on the endothelium in atheroprone regions of the circulation suggests that the glycocalyx may have a central role in determining atherosclerotic plaque formation. However, the kinetics for the cells’ ability to adapt its glycocalyx to the environment have not been quantitatively resolved. Here we report that the heparan sulfate component of the glycocalyx of HUVECs increases by 1.4-fold following the onset of high shear stress, compared to static cultured cells, with a time constant of 19 h. Cell morphology experiments show that 12 h are required for the cells to elongate, but only after 36 h have the cells reached maximal alignment to the flow vector. Our findings demonstrate that following enzymatic degradation, heparan sulfate is restored to the cell surface within 12 h under flow whereas the time required is 20 h under static conditions. We also propose a model describing the contribution of endocytosis and exocytosis to apical heparan sulfate expression. The change in HS regrowth kinetics from static to high-shear EC phenotype implies a differential in the rate of endocytic and exocytic membrane turnover.National Heart, Lung, and Blood Institute (Grant HL090856-01)Singapore-MIT Allianc

Cell surface proteoglycan expression during maturation of human monocytes-derived dendritic cells and macrophages

Cell surface proteoglycans play an important part in the functional and metabolic behaviour of leucocytes. We studied the expression of cell surface proteoglycans in human monocytes, in monocyte-derived immature and mature dendritic cells and in macrophages by metabolic labelling with [35S]-sulphate, reverse transcriptase–polymerase chain reaction (RT–PCR) and Western blotting. Immature dendritic cells had the highest metabolic activity for the synthesis of cell surface proteoglycans. The major part of these proteoglycans was in phosphatidylinositol-anchored form and was released after treatment with phospholipase C. A minor part was released by trypsin. Digestion with chondroitinase ABC and mild HNO2 treatment showed that cell surface proteoglycans had a higher proportion of chondroitin sulphate, both in the phospholipase C and trypsin fractions, suggesting that at least some glypicans contained chondroitin sulphate chains. RT–PCR detected the transcripts of glypicans 1, 3, 4 and 5 and all syndecans. Immature dendritic cells expressed a most complex spectrum of glypicans and syndecans, glypican-1 and syndecan-1 being expressed preferentially by this type of cells. Mature dendritic cells expressed glypican-3, which was not present in other lineages. These results suggest that different mononuclear cells synthesize cell surface proteoglycans actively with characteristic expression of different syndecans and glypicans genes, depending on the degree of cell differentiation and/or maturation

6-O sulfated and N-sulfated Syndecan-1 promotes baculovirus binding and entry into mammalian cells.

Baculoviruses are insect-specific viruses commonly found in nature. They are not able to replicate in mammalian cells but can transduce them when equipped with an appropriate mammalian cell active expression cassette. Although the viruses have been studied in several types of mammalian cells from different origins, the receptor that baculovirus uses to enter or interact with mammalian cells has not yet been identified. Due to the wide tropism of the virus, the receptor has been suggested to be a generally found cell surface molecule. In this article, we investigated the interaction of baculovirus and mammalian cell surface heparan sulfate proteoglycans (HSPG) in more detail. Our data show that baculovirus requires HSPG sulfation, particularly N- and 6-O-sulfation, to bind to and transduce mammalian cells. According to our results, baculovirus binds specifically to syndecan-1 (SDC-1) but does not interact with SDC-2 to SDC-4 or with glypicans. Competition experiments performed with SDC-1 antibody or recombinant SDC-1 protein inhibited baculovirus binding, and SDC-1 overexpression enhanced baculovirus-mediated transduction. In conclusion, we show that SDC-1, a commonly found cell surface HSPG molecule, has a role in the binding and entry of baculovirus in vertebrate cells. The results presented here reveal important aspects of baculovirus entry and can serve as a basis for next-generation baculovirus vector development for gene delivery.peerReviewe

Inhibition of glycosaminoglycan synthesis using rhodamine B in a mouse model of mucopolysaccharidosis type IIIA

Reduction of an enzyme activity required for the lysosomal degradation of glycosaminoglycan (gag) chains will result in a mucopolysaccharidosis (MPS) disorder. Substrate deprivation therapy (SDT), a potential therapy option for MPS with residual enzyme activity, aims to reduce the synthesis of gag chains, the natural substrate for the deficient enzyme. Reduced substrate levels would balance the reduced level of enzyme in patient cells, resulting in normalized gag turnover. Rhodamine B, a nonspecific inhibitor, reduced gag synthesis in a range of normal and MPS cells and also decreased lysosomal storage of gag in MPS VI (72%) and MPS IIIA (60%) cells. Body weight gain of male MPS IIIA mice treated with 1 mg/kg rhodamine B was reduced compared with untreated MPS IIIA mice and was indistinguishable from that of normal mice. Liver size, total gag content, and lysosomal gag was reduced in treated MPS IIIA animals as was urinary gag excretion. Lysosomal gag content in the brain was also reduced by treatment. The alteration in MPS IIIA clinical pathology by rhodamine B, combined with the observation that treatment had no effect on the health of normal animals, demonstrates the potential for SDT in general as a therapy for MPS disorders