Elevated plasma triglyceride levels represent a risk factor for premature atherosclerosis. In mice, accumulation of triglyceride-rich lipoproteins can occur if sulfation of heparan sulfate in hepatocytes is diminished, as this alters hepatic lipoprotein clearance via heparan sulfate proteoglycans (HSPGs). However, the relevant HSPG has not been determined. In this study, we found by RT-PCR analysis that mouse hepatocytes expressed the membrane proteoglycans syndecan-1, -2, and -4 and glypican-1 and -4. Analysis of available proteoglycandeficient mice showed that only syndecan-1 mutants (Sdc1 -/-mice) accumulated plasma triglycerides. Sdc1 -/-mice also exhibited prolonged circulation of injected human VLDL and intestinally derived chylomicrons. We found that mice lacking both syndecan-1 and hepatocyte heparan sulfate did not display accentuated triglyceride accumulation compared with single mutants, suggesting that syndecan-1 is the primary HSPG mediating hepatic triglyceride clearance. Immunoelectron microscopy showed that syndecan-1 was expressed specifically on the microvilli of hepatocyte basal membranes, facing the space of Disse, where lipoprotein uptake occurs. Abundant syndecan-1 on wild-type murine hepatocytes exhibited saturable binding of VLDL and inhibition by heparin and facilitated degradation of VLDL. Furthermore, adenovirus-encoded syndecan-1 restored binding, uptake, and degradation of VLDL in isolated Sdc1 -/-hepatocytes and the lipoprotein clearance defect in Sdc1 -/-mice. These findings provide the first in vivo genetic evidence that syndecan-1 is the primary hepatocyte HSPG receptor mediating the clearance of both hepatic and intestinally derived triglyceride-rich lipoproteins.

Summary Heparan sulfate proteoglycans (HSPGs) are an important constituent of the macrophage glycocalyx and extracellular microenvironment. To examine their role in atherogenesis, we inactivated the biosynthetic gene N-acetylglucosamine N-deacetylase-N-sulfotransferase 1 (Ndst1) in macrophages and crossbred the strain to Ldlr−/− mice. When placed on an atherogenic diet, Ldlr−/−Ndst1f/fLysMCre+ mice had increased atherosclerotic plaque area and volume compared to Ldlr−/− mice. Diminished sulfation of heparan sulfate resulted in enhanced chemokine expression, increased macrophages in plaques, increased expression of ACAT2, a key enzyme in cholesterol ester storage, and increased foam cell conversion. Motif analysis of promoters of up-regulated genes suggested increased Type I Interferon signaling, which was confirmed by elevation of STAT1 phosphorylation induced by IFN-β. The pro-inflammatory macrophages derived from Ndst1f/fLysMCre+ mice also sensitized the animals to diet-induced obesity. We propose that macrophage HSPGs control basal activation of macrophages by maintaining Type I interferon reception in a quiescent state through sequestration of IFN-β.

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Objective Chylomicron and very low-density lipoprotein remnants are cleared from the circulation in the liver by heparan sulfate proteoglycan (HSPG) receptors (syndecan-1), the low-density lipoprotein receptor (LDLR), and LDLR-related protein-1 (LRP1), but the relative contribution of each class of receptors under different dietary conditions remains unclear. Approach and Results Triglyceride-rich lipoprotein clearance was measured in AlbCre+Ndst1f/f, Ldlr−/−, and AlbCre+Lrp1f/f mice and mice containing combinations of these mutations. Triglyceride measurements in single and double mutant mice showed that HSPGs and LDLR dominate clearance under fasting conditions and postprandial conditions, but LRP1 contributes significantly when LDLR is absent. Mice lacking hepatic expression of all three receptors (AlbCre+Ndst1f/f Lrp1f/f Ldlr−/−) displayed dramatic hyperlipidemia (870 ± 270 mg triglyceride/dL; 1300 ± 350 mg of total cholesterol/dL) and exhibited persistent elevated postprandial triglyceride levels due to reduced hepatic clearance. Analysis of the particles accumulating in mutants showed that HSPGs preferentially clear a subset of small triglyceride-rich lipoproteins (~20-40 nm diameter), while LDLR and LRP1 clear larger particles (~40-60 nm diameter). Finally, we show that HSPGs play a major role in clearance of TRLs in mice fed normal chow or under postprandial conditions but appear to play a less significant role on a high fat diet. Conclusion These data show that HSPGs, LDLR, and LRP1 clear distinct subsets of particles, that HSPGs work independently from LDLR and LRP1, and that HSPGs, LDLR, and LRP1 are the three major hepatic TRL clearance receptors in mice.

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We recently showed that the heparan sulfate proteoglycan syndecan-1 mediates hepatic clearance of triglyceride-rich lipoproteins in mice based on systemic deletion of syndecan-1 and hepatocyte-specific inactivation of sulfotransferases involved in heparan sulfate biosynthesis (MacArthur et al. (2007) J. Clin. Invest. 117:153–164; Stanford et al. (2009) J. Clin. Invest. 119:3236–3245; Stanford et al. (2010) J. Biol. Chem. 285:286–294). In this report we show that syndecan-1 expressed on primary human hepatocytes and Hep3B human hepatoma cells can mediate binding and uptake of VLDL. Syndecan-1 also undergoes spontaneous shedding from primary human and murine hepatocytes and Hep3B cells. In human cells, phorbol myristic acid (PMA) induces syndecan-1 shedding, resulting in accumulation of syndecan-1 ectodomains in the medium. Shedding occurs through a protein kinase C-dependent activation of A Disintegrin and Metalloproteinase-17. PMA-stimulation significantly decreases DiD-VLDL binding to cells, and shed syndecan-1 ectodomains bind to VLDL. Although mouse hepatocytes appear resistant to induced-shedding in vitro, injection of lipopolysaccharide into mice results in loss of hepatic syndecan-1, accumulation of ectodomains in the plasma, impaired VLDL catabolism, and hypertriglyceridemia. Conclusion These findings suggest that syndecan-1 mediates hepatic VLDL turnover in humans as well as in mice and that shedding might contribute to hypertriglyceridemia in patients with sepsis.

Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lack Ext1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes. A better understanding of how viral vectors enter cells in vivo is critical to improve their therapeutic use. Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) vectors have shown promise in clinical trials for treatment of a wide variety of diseases (1, 2). Both vectors, when injected intravenously into mice, exhibit transgene expression in liver (3-5). Heparan sulfate proteoglycans (HSPGs) are the primary receptors currently thought to facilitate AAV2 and Ad5 entry into hepatocytes (6-8).HSPGs are present both on the cell surface and in the extracellular matrix (9, 10). They consist of a protein core posttranslationally modified to contain heparan sulfate (HS) chains (11). HS biosynthesis occurs by polymerization of alternating glucuronic acid and N-acetylglucosamine residues (12-14), catalyzed by an enzyme complex composed of EXT1 and EXT2 (15). EXT1 and EXT2 are essential molecules required for HS synthesis; cells lacking either molecule do not synthesize HS (16,49).AAV2 binds directly to cell surface HSPGs via an HS-binding motif on the virus capsid (3,17,18). AAV capsid modifications that alter the cluster of positive amino acids that constitute the HS binding motif abrogate liver transduction (3,19,20), suggesting that the ability of the capsid to bind to HS is critical for AAV2 liver transduction in vivo. In contrast, Ad5 binding to HSPGs requires the presence of blood coagulation factor X (FX), which binds to the Ad5 hexon when the virus comes in contact with blood (7,(21)(22)(23). The interaction of Ad.FX and HS is mediated by electrostatic interactions between the heparin binding exosite of the FX serine protease domain and the sulfate groups of HS (6,(23)(24)(25). FX is required for Ad5 transduction in vivo in wild-type mice. In the absence of FX, or when viruses with mutant hexon proteins unable to bind FX are used, Ad5 liver transduction is essentially completely abrogated (7, 21-23, 26, 27).

Diabetes -associated hyperlipidemia is generally attributed to reduced clearance of plasma lipoproteins, especially remnant lipoproteins enriched in cholesterol and triglycerides. Hepatic clearance of remnants occurs via low density lipoprotein receptors and the heparan sulfate proteoglycan, syndecan-1. Previous studies have suggested alterations in heparan sulfate proteoglycan metabolism in rat and mouse diabetic models, consistent with the idea that diabetic dyslipidemia might be caused by alterations in proteoglycan expression in the liver. In this study we analyzed the content and composition of liver heparan sulfate in streptozotocin-induced insulin-deficient diabetic mice that displayed fasting hypertriglyceridemia and delayed clearance of dietary triglyceride-rich lipoproteins. No differences between normal and diabetic littermates in liver heparan sulfate content, sulfation, syndecan-1 protein levels, or affinity for heparin-binding ligands, such as apolipoprotein E or fibroblast growth factor-2, were noted. Decreased incorporation of [35 S]sulfate in insulin-deficient mice in vivo was observed, but the decrease was due to increased plasma inorganic sulfate, which reduced the efficiency of labeling of liver heparan sulfate. These results show that hyperlipidemia in insulin-deficient mice is not due to changes in hepatic heparan sulfate composition.Hypertriglyceridemia is a significant complication of insulindependent diabetes mellitus (IDDM) 2 that likely contributes to cardiovascular disease in affected individuals, but its cause remains unknown (1-3). Insulin deficiency suppresses hepatic triglyceride production (4, 5), suggesting that increased plasma triglyceride levels might result from decreased catabolism of triglyceride-rich lipoproteins (TRL). In various diabetic models delayed TRL remnant clearance has been attributed to altered expression of heparan sulfate proteoglycans (HSPGs) in the liver (6 -14).We recently showed that mutant mice lacking the plasma membrane HSPG, syndecan-1, exhibit hypertriglyceridemia due to delayed clearance of TRL remnants from the circulation associated with reduced VLDL binding, uptake, and degradation in isolated hepatocytes (15). Furthermore, mice with undersulfated liver heparan sulfate have the same phenotype (16,17). One of mutants lacked the enzyme N-acetylglucosamine N-deacetylase/N-sulfotransferase 1 (Ndst1), a biosynthetic enzyme that regulates the overall level of sulfation of heparan sulfate glycosaminoglycans. In vivo studies have suggested that IDDM causes reduced expression of Ndst1 (6,10,11,13,14,18), leading to the hypothesis that hypertriglyceridemia was caused by undersulfated heparan sulfate in the liver.In this work, we show that mice with IDDM exhibit reduced [ 35 S]sulfate incorporation into hepatic heparan sulfate. However, the reduction was caused by changes in plasma sulfate concentration after the onset of diabetes rather than any change in heparan sulfate biosynthesis. The application of mass spectrometry showed that hepatic heparan sulf...