MacArthur JM, Bishop JR, Stanford KI, Wang L, Bensadoun A, Witztum JL, et al. Liver heparan sulfate proteoglycans mediate clearance of triglyceride-rich lipoproteins independently of LDL receptor family members. J Clin Invest 2007;117:153-164. (Reprinted with permission.)
We examined the role of hepatic heparan sulfate in triglyceride-rich lipoprotein metabolism by inactivating the biosynthetic gene GlcNAc N-deacetylase/N-sulfotransferase 1 (Ndst1) in hepatocytes using the Cre-loxP system, which resulted in an approximately 50% reduction in sulfation of liver heparan sulfate. Mice were viable and healthy, but they accumulated triglyceride-rich lipoprotein particles containing apoB-100, apoB-48, apoE, and apoCI-IV. Compounding the mutation with LDL receptor deficiency caused enhanced accumulation of both cholesterol- and triglyceride-rich particles compared with mice lacking only LDL receptors, suggesting that heparan sulfate participates in the clearance of cholesterol-rich lipoproteins as well. Mutant mice synthesized VLDL normally but showed reduced plasma clearance of human VLDL and a corresponding reduction in hepatic VLDL uptake. Retinyl ester excursion studies revealed that clearance of intestinally derived lipoproteins also depended on hepatocyte heparan sulfate. These findings show that under normal physiological conditions, hepatic heparan sulfate proteoglycans play a crucial role in the clearance of both intestinally derived and hepatic lipoprotein particles.
Experimental and clinical evidence indicates that the proteoglycan protein superfamily plays a crucial role in homeostasis at the cellular and tissue level. Indeed, modulation of cell/extracellular matrix interactions by these glycoconjugates influences cell growth, embryonic development, and postnatal tissue remodeling and seems to be directly implicated in various disease conditions. A recent report highlighting the influence of heparan sulfate proteoglycans (HSPGs) on plasma lipoprotein metabolism1 brings these macromolecules back to the arena of liver biology. By reducing the liver content of mature heparan sulfate (HS) through liver-specific gene targeting, MacArthur et al. elegantly showed that hepatic HSPGs have a physiologically relevant role in the clearance of different classes of plasma lipoproteins.1 The contribution of these macromolecules to lipoprotein metabolism was previously suggested by in vitro and in vivo studies.2–4 However, confirmatory in vivo studies have been extremely elusive, mainly because of the intrinsic complexity of proteoglycan biology.
Proteoglycan structure consists of a core protein with covalent substitutions of one or several glycosaminoglycan (GAG) chains. Even though the protein component itself may display binding sites for specific ligands, the refined chemical structure of GAGs defines most of the PG binding properties, which are very diverse, leading to exquisitely specific ligand recognition. The structural features of GAGs are cell type–specific and tissue-specific. For instance, liver HSPGs hold almost 2-fold more sulfate content than those derived from other tissues. Furthermore, multiple GAGs within a single PG molecule determine a large number of low-affinity binding sites, turning PGs into virtual reservoirs of their ligands. In particular, HSPGs bind several growth factors, morphogens, and extracellular proteases as well as some key modulators of lipoprotein metabolism such as lipases and apolipoproteins. Interestingly, the particular distribution of sulfate groups along the liver HSPGs confers increased affinity for apolipoprotein E (apoE) compared to HSPGs expressed in the vascular bed.
Among the PG superfamily, HSPGs are the predominant proteoglycan class found in the liver, accounting for more than two-thirds of total tissue GAG content, followed by chondroitin/dermatan sulfate PGs; no hepatic keratan sulfate PGs have been reported thus far. The relative GAG content in the liver is altered (significantly increased in chondroitin sulfate PGs) under different physiological and pathological conditions, such as hepatic regeneration,5 cholestasis,6 liver cirrhosis,7 and HCC.8 These changes in hepatic PG composition may have substantial pathogenic implications for liver diseases, because it can affect the normal interaction between several growth factors (e.g., hepatocyte growth factor, transforming growth factor-β) with their respective liver cell surface receptors, thus modulating hepatocyte proliferation and fibrogenesis.
In addition, the potential role of hepatic HSPGs in lipoprotein metabolism in vivo was postulated, based on the pioneering work led by Mahley.2–4 After establishing that the interaction between apoE and HSPG was important for the uptake of triglyceride-rich lipoproteins by cultured cells,2, 3 a classic study showed that direct infusion of HS-degrading enzymes into the portal circulation of mice dramatically reduced plasma clearance and hepatic uptake apoE-enriched lipoproteins,4 suggesting a functional role for cell surface–associated HSPGs, either directly as lipoprotein receptors or indirectly by a bridging interaction with the low-density lipoprotein receptor (LDLR)-related protein. From a clinical perspective, supporting evidence was also provided by the analysis of HSPG binding activities of dominant variants of apoE that cause human type III hyperlipoproteinemia.9
By targeting genes encoding different HS-processing enzymes, it has been possible to gain significant insights about the biological functions of HSPGs in vivo.10 Germline disruption of the Ndst1 (GlcNAc N-deacetylase/N-sulfotransferase) gene, which encodes an enzyme isoform that plays a key role in the maturation of HS, leads to a dramatic decrease in N-sulfated HSPGs in all tissues, resulting in embryonic and perinatal death. To overcome lethality, liver-specific Ndst1 null mice generated by MacArthur et al.1 are an extraordinary tool to establish more definitively the importance of hepatic HSPGs in lipoprotein physiology. These mice exhibited a 2.5-fold increase in plasma total triglyceride levels accumulated in remnant lipoproteins under chow diet as well as significantly higher plasma cholesterol levels transported in LDL particles when crossbred to LDLR-deficient mice. This work indicates that hepatic HSPGs are indeed critical for LDLR-independent chylomicron and very low density lipoprotein (VLDL) remnant clearance in vivo. This study also reveals that HSPGs may also play a significant role in hepatic LDL uptake when the classic LDL receptor pathway is absent or down-regulated.
Additional studies have begun to address the molecular identity of specific hepatic HSPGs that may account for their effects on lipid metabolism. As a sinusoidal transmembrane HSPG facing the plasma compartment, syndecan-1 is a very attractive candidate to be involved in lipoprotein physiology. Indeed, syndecan-1 mediates clathrin-independent endocytic uptake of lipoprotein lipase-enriched LDL11 and apoE-rich and triglyceride-rich VLDL12 in cultured cells. In addition, inhibition of syndecan-1 expression decreases binding of remnant lipoproteins in HepG2 cells.13 To further assess the role of liver syndecan-1 in vivo, we overexpressed this HSPG by adenoviral gene transfer in mice,14 which exhibited a significant hyperlipidemic response due to accumulation of all plasma lipoprotein classes that were also enriched in apoE. We postulated that excessive hepatic overexpression of syndecan-1 may lead to abnormal sequestration of lipoproteins within the space of Disse, precluding their physical interaction and clearance through sinusoidal receptors of the LDLR family.14 Whether other HSPGs (e.g., perlecan) and additional PG classes (e.g., decorin) are also critical for hepatic lipoprotein uptake and plasma lipid level regulation in vivo remains an open question.
The study by MacArthur et al.1 may have important implications for pathophysiology and therapy of human primary and secondary dyslipidemias. Interestingly, animal models of insulin resistance and diabetes exhibit altered expression of hepatic PGs (lower HSPG/chondroitin sulfate PG ratio) as well as reduced HSPG levels associated with decreased sulfation of GAG chains.15, 16 Whether these findings are also present and pathogenically relevant in patients with diabetes and metabolic syndrome remains to be established. If HSPGs indeed are critical players in controlling lipoprotein metabolism, polymorphism analysis of genes encoding HSPG protein cores and/or GAG biosynthetic enzymes may define novel markers of predisposition to dyslipidemias or response to lipid-lowering therapy. On the other hand, nuclear farnesoid X receptor agonism transcriptionally stimulates the expression of syndecan-1 in cultured cells, including cultured hepatocytes.17 It would be interesting to evaluate if the hypotriglyceridemic effect induced by these farnesoid X receptor compounds in rodents is indeed dependent on increased expression of hepatic HSPGs. If so, these glycoconjugated protein macromolecules may represent novel targets for development of lipid-lowering drugs.