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BackgroundPCSK9 (proprotein convertase subtilisin-kexin type 9) chaperones the hepatic LDLR (low-density lipoprotein receptor) for lysosomal degradation, elevating serum LDL (low-density lipoprotein) cholesterol and promoting atherosclerotic heart disease. Though the major effect on the hepatic LDLR comes from secreted PCSK9, the details of PCSK9 reuptake into the hepatocyte remain unclear. In both tissue culture and animal models, HSPGs (heparan sulfate proteoglycans) on hepatocytes act as co-receptors to promote PCSK9 reuptake. We hypothesized that if this PCSK9:HSPG interaction is important in humans, disrupting it with unfractionated heparin (UFH) would acutely displace PCSK9 from the liver and increase plasma PCSK9.MethodsWe obtained remnant plasma samples from 160 subjects undergoing cardiac catheterization before and after administration of intravenous UFH. PCSK9 levels were determined using a commercial enzyme-linked immunosorbent assay.ResultsMedian plasma PCSK9 was 113 ng/mL prior to UFH and 119 ng/mL afterward. This difference was not significant (P=0.83 [95% CI, -6.23 to 6.31 ng/mL]). Equivalence testing provided 95% confidence that UFH would not raise plasma PCSK9 by > 4.7%. Among all subgroups, only subjects with the lowest baseline PCSK9 concentrations exhibited a response to UFH (8.8% increase, adj. P=0.044). A modest correlation was observed between baseline plasma PCSK9 and the change in plasma PCSK9 due to UFH (RS=-0.3634; P

An oral antiviral against SARS-CoV-2 that also attenuates inflammatory instigators of severe COVID-19 is not available to date. Herein, we show that the apoA-I mimetic peptide 4 F inhibits Spike mediated viral entry and has antiviral activity against SARS-CoV-2 in human lung epithelial Calu3 and Vero-E6 cells. In SARS-CoV-2 infected Calu3 cells, 4 F upregulated inducers of the interferon pathway such as MX-1 and Heme oxygenase 1 (HO-1) and downregulated mitochondrial reactive oxygen species (mito-ROS) and CD147, a host protein that mediates viral entry. 4 F also reduced associated cellular apoptosis and secretion of IL-6 in both SARS-CoV-2 infected Vero-E6 and Calu3 cells. Thus, 4 F attenuates in vitro SARS-CoV-2 replication, associated apoptosis in epithelial cells and secretion of IL-6, a major cytokine related to COVID-19 morbidity. Given established safety of 4 F in humans, clinical studies are warranted to establish 4 F as therapy for COVID-19.

Neovascularization is a key feature of ischemic retinal diseases and the wet form of age-related macular degeneration (AMD), all leading causes of severe vision loss. Vascular endothelial growth factor (VEGF) inhibitors have transformed the treatment of these disorders. Millions of patients have been treated with these drugs worldwide. However, in real-life clinical settings, many patients do not experience the same degree of benefit observed in clinical trials, in part because they receive fewer anti-VEGF injections. Therefore, there is an urgent need to discover and identify novel long-acting VEGF inhibitors. We hypothesized that binding to heparan-sulfate proteoglycans (HSPG) in the vitreous, and possibly other ocular structures, may be a strategy to promote intraocular retention, ultimately leading to a reduced burden of intravitreal injections. We designed a series of VEGF receptor 1 variants and identified some with strong heparin-binding characteristics and ability to bind to vitreous matrix. Our data indicate that some of our variants have longer duration and greater efficacy in animal models of intraocular neovascularization than current standard of care. Our study represents a systematic attempt to exploit the functional diversity associated with heparin affinity of a VEGF receptor.

Heparan sulfate proteoglycans consist of a small family of proteins decorated with one or more covalently attached heparan sulfate glycosaminoglycan chains. These chains have intricate structural patterns based on the position of sulfate groups and uronic acid epimers, which dictate their ability to engage a large repertoire of heparan sulfate-binding proteins, including extracellular matrix proteins, growth factors and morphogens, cytokines and chemokines, apolipoproteins and lipases, adhesion and growth factor receptors, and components of the complement and coagulation system. This review highlights recent progress in the characterization of the so-called heparan sulfate interactome, with a major focus on systems-wide strategies as a tool for discovery and characterization of this subproteome. In addition, we compiled all heparan sulfate-binding proteins reported in the literature to date and grouped them into a few major functional classes by applying a networking approach.

Proteoglycans are a specific subset of glycoproteins found at the cell surface and in the extracellular matrix, where they interact with a plethora of proteins involved in metabolic homeostasis and meta-inflammation. Over the last decade, new insights have emerged on the mechanism and biological significance of these interactions in the context of diet-induced disorders such as obesity and type-2 diabetes. Complications of energy metabolism drive most diet-induced metabolic disorders, which results in low-grade chronic inflammation, thereby affecting proper function of many vital organs involved in energy homeostasis, such as the brain, liver, kidney, heart and adipose tissue. Here, we discuss how heparan, chondroitin and keratan sulfate proteoglycans modulate obesity-induced metabolic dysfunction and low-grade inflammation that impact the initiation and progression of obesity-associated morbidities.

Heparan sulfate proteoglycans are found at the cell surface and in the extracellular matrix, where they interact with a plethora of proteins involved in lipid homeostasis and inflammation. Over the last decade, new insights have emerged regarding the mechanism and biological significance of these interactions in the context of cardiovascular disease. The majority of cardiovascular disease-related deaths are caused by complications of atherosclerosis, a disease that results in narrowing of the arterial lumen, thereby reducing blood flow to critical levels in vital organs, such as the heart and brain. Here, we discuss novel insights into how heparan sulfate proteoglycans modulate risk factors such as hyperlipidemia and inflammation that drive the initiation and progression of atherosclerotic plaques to their clinical critical endpoint.

The present study was designed to characterize transduction of non-human primate brain and spinal cord with a modified adeno-associated virus serotype 2, incapable of binding to the heparan sulfate proteoglycan receptor, referred to as AAV2-HBKO. AAV2-HBKO was infused into the thalamus, intracerebroventricularly or via a combination of both intracerebroventricular and thalamic delivery. Thalamic injection of this modified vector encoding GFP resulted in widespread CNS transduction that included neurons in deep cortical layers, deep cerebellar nuclei, several subcortical regions, and motor neuron transduction in the spinal cord indicative of robust bidirectional axonal transport. Intracerebroventricular delivery similarly resulted in widespread cortical transduction, with one striking distinction that oligodendrocytes within superficial layers of the cortex were the primary cell type transduced. Robust motor neuron transduction was also observed in all levels of the spinal cord. The combination of thalamic and intracerebroventricular delivery resulted in transduction of oligodendrocytes in superficial cortical layers and neurons in deeper cortical layers. Several subcortical regions were also transduced. Our data demonstrate that AAV2-HBKO is a powerful vector for the potential treatment of a wide number of neurological disorders, and highlight that delivery route can significantly impact cellular tropism and pattern of CNS transduction.

The misfolding and accumulation of tau protein into intracellular aggregates known as neurofibrillary tangles is a pathological hallmark of neurodegenerative diseases such as Alzheimer's disease. However, while tau propagation is a known marker for disease progression, exactly how tau propagates from one cell to another and what mechanisms govern this spread are still unclear. Here, we report that cellular internalization of tau is regulated by quaternary structure and have developed a cellular assay to screen for genetic modulators of tau uptake. Using CRISPRi technology we have tested 3200 genes for their ability to regulate tau entry and identified enzymes in the heparan sulfate proteoglycan biosynthetic pathway as key regulators. We show that 6-O-sulfation is critical for tau-heparan sulfate interactions and that this modification regulates uptake in human central nervous system cell lines, iPS-derived neurons, and mouse brain slice culture. Together, these results suggest novel strategies to halt tau transmission.

BACKGROUND:HSPGs are glycoproteins containing covalently attached heparan sulfate (HS) chains which bind to growth factors, chemokines, etc., and regulate various aspects of inflammation including cell recruitment. We previously showed that deletion of endothelial N-acetylglucosamine N-deacetylase-N-sulfotransferase-1 (Ndst1), an enzyme responsible for N-sulfation during HS biosynthesis, reduces allergic airway inflammation (AAI). Here, we investigated the importance of O-sulfation mediated by uronyl 2-O-sulfotransferase (Hs2st) in development of AAI relative to N-sulfation. METHODS:Mice deficient in endothelial and leukocyte Hs2st (Hs2stf/fTie2Cre+) or Ndst1 (Ndst1f/fTie2Cre+) and WT mice were challenged with Alternaria alternata and evaluated for airway inflammation. Trafficking of murine eosinophils on lung endothelial cells was examined in vitro under conditions of flow. RESULTS:Exposure to Alternaria decreased expression level of Hs2st in WT mice while level of Ndst1 remained unchanged. Compared to WT mice, Alternaria-challenged Hs2stf/fTie2Cre+ mice exhibited significantly increased eosinophils in the bone marrow, bronchoalveolar lavage fluid [BALF] and lung tissue associated with persistent airway hyperresponsiveness, airway mucus hypersecretion and elevated Th2 cytokines. In contrast, Alternaria-challenged Ndst1f/fTie2Cre+ mice exhibited a marked reduction in airway eosinophilia, mucus secretion and smooth muscle mass compared to WT counterparts. While BALF eotaxins were lower in Alternaria-challenged Hs2stf/fTie2Cre+ relative to WT mice, they were not reduced to background levels as in allergen-challenged Ndst1f/fTie2Cre+ mice. Trafficking of murine eosinophils under conditions of flow in vitro was similar on Hs2st-deficient and WT endothelial cells. Expression of ZO-1 in Hs2st-deficient lung blood vessels in control and allergen-challenged mice was significantly lower than in WT counterparts. CONCLUSIONS:Our study demonstrates that allergen exposure reduces expression of Hs2st; loss of uronyl 2-O-sulfation in endothelial and leukocyte HSPG amplifies recruitment of eosinophils likely due to a compromised vascular endothelium resulting in persistent inflammation whereas loss of N-sulfation limits eosinophilia and attenuates inflammation underscoring the importance of site-specific sulfation in HSPG to their role in AAI.

Background: In our previous study, N end of the Circumsporozoite protein (CSP I-plus) modified recombinant human Endostatin (rEndostatin, endostar) (rES-CSP) was constructed, which had antiangiogenic capability and bound to hepatocellular carcinoma in vivo and in vitro. In this study, the inhibition of rES-CSP on hepatocellular carcinoma metastasis was verified in vivo and in vitro, and its possible mechanism was explored.Methods: Firstly, the impact of rES-CSP on the migration, adhesion of hepatoma cell HCCLM3 was identified by wound healing, transwell, and on metastasis of orthotopic xenograft model was identified in nude mouse. Then the expression of metastasis-associated molecules (MMP2, E-cadherin, integrinβ1) and angiogenesis-related factors (VEGFA) in vitro and in vivo were detected by real-time PCR, western blotting, immunohistochemistry.Results: Finally, we found that rES-CSP could inhibit the migration and invasion of HCCLM3, and decrease tumor metastasis and growth in nude mouse orthotopic xenograft models. The tumor inhibiting rates of rES-CSP and Endostar were 42.46 ± 5.39% and 11.1 ± 1.88%. The lung metastasis rates of the control, Endostar and rES-CSP were 71, 50, and 42.8%, respectively. Compared with Endostar, rES-CSP significantly down-regulated the expression of VEGFA and integrinβ1. Heparin, a competitive inhibitor of CSP I-plus, which can be bind to the highly-sulfated heparan sulfate proteoglycans (HSPGs) over-expressed in liver and hepatocellular carcinoma, alleviated the down-regulation of VEGFA and integrinβ1.Conclusions: These indicate that rES-CSP may play a role in inhibiting tumor growth and metastasis by down-regulating the angiogenic factor VEGF and the metastasis-related molecules or by interfering with HSPGs-mediated tumor metastasis. [ABSTRACT FROM AUTHOR]

In mice lacking glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1), the LPL secreted by adipocytes and myocytes remains bound to heparan sulfate proteoglycans (HSPGs) on all cells within tissues. That observation raises a perplexing issue: Why isn't the freshly secreted LPL in wild-type mice captured by the same HSPGs, thereby preventing LPL from reaching GPIHBP1 on capillaries? We hypothesized that LPL-HSPG interactions are transient, allowing the LPL to detach and move to GPIHBP1 on capillaries. Indeed, we found that LPL detaches from HSPGs on cultured cells and moves to: 1) soluble GPIHBP1 in the cell culture medium; 2) GPIHBP1-coated agarose beads; and 3) nearby GPIHBP1-expressing cells. Movement of HSPG-bound LPL to GPIHBP1 did not occur when GPIHBP1 contained a Ly6 domain missense mutation (W109S), but was almost normal when GPIHBP1's acidic domain was mutated. To test the mobility of HSPG-bound LPL in vivo, we injected GPIHBP1-coated agarose beads into the brown adipose tissue of GPIHBP1-deficient mice. LPL moved quickly from HSPGs on adipocytes to GPIHBP1-coated beads, thereby depleting LPL stores on the surface of adipocytes. We conclude that HSPG-bound LPL in the interstitial spaces of tissues is mobile, allowing the LPL to move to GPIHBP1 on endothelial cells.

The Eph receptors and their cognate ephrin ligands play key roles in many aspects of nervous system development. These interactions typically occur within an individual tissue type, serving either to guide axons to their terminal targets or to define boundaries between the rhombomeres of the hindbrain. We have identified a novel role for the Caenorhabditis elegans ephrin EFN-4 in promoting primary neurite outgrowth in AIY interneurons and D-class motor neurons. Rescue experiments reveal that EFN-4 functions non-cell autonomously in the epidermis to promote primary neurite outgrowth. We also find that EFN-4 plays a role in promoting ectopic axon branching in a C. elegans model of X-linked Kallmann syndrome. In this context, EFN-4 functions non-cell autonomously in the body-wall muscle and in parallel with HS modification genes and HSPG core proteins. This is the first report of an epidermal ephrin providing a developmental cue to the nervous system.

Recently, we showed that endothelial heparan sulfate facilitates entry of a bacterial pathogen into the central nervous system. Here, we show that normal bactericidal activity of neutrophils is influenced by the sulfation pattern of heparan sulfate. Inactivation of heparan sulfate uronyl 2-O-sulfotransferase (Hs2st) in neutrophils substantially reduced their bactericidal activity, and Hs2st deficiency rendered mice more susceptible to systemic infection with the pathogenic bacterium group B Streptococcus. Specifically, altered sulfation of heparan sulfate in mutant neutrophils affected formation of neutrophil extracellular traps while not influencing phagocytosis, production of reactive oxygen species, or secretion of granular proteases. Heparan sulfate proteoglycan(s) is present in neutrophil extracellular traps, modulates histone affinity, and modulates their microbial activity. Hs2st-deficient brain endothelial cells show enhanced binding to group B Streptococcus and are more susceptible to apoptosis, likely contributing to the observed increase in dissemination of group B Streptococcus into the brain of Hs2st-deficient mice following intravenous challenge. Taken together, our data provide strong evidence that heparan sulfate from both neutrophils and the endothelium plays important roles in modulating innate immunity.

Macrophages are important mediators of diseases associated with metabolic inflammation such as obesity and atherosclerosis. In this Stimulus we discuss recent findings showing that heparan sulfate proteoglycans on macrophages serve as an important inflammatory rheostat. This observation has significant implications as the degree of macrophage proteoglycan sulfation can determine and possibly predict disease outcomes of metabolic inflammatory disorders.

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(-/-)Ndst1(f/f)LysMCre(+) 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 upregulated genes suggested increased type I interferon signaling, which was confirmed by elevation of STAT1 phosphorylation induced by IFN-β. The proinflammatory macrophages derived from Ndst1(f/f)LysMCre(+) 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.

In our juvenile traumatic brain injury (jTBI) model, emergence of cognitive dysfunctions was observed up to 6 months after trauma. Here we hypothesize that early brain injury induces changes in the neurovascular unit (NVU) that would be associated with amyloid-beta (Aβ) accumulation. We investigated NVU changes for up to 6 months in a rat jTBI model, with a focus on the efflux protein P-glycoprotein (P-gp) and on the basement membrane proteins perlecan and fibronectin, all known to be involved in Aβ clearance. Rodent-Aβ staining is present and increased after jTBI around cerebral blood microvessels, and the diameter of those is decreased by 25% and 34% at 2 and 6 months, respectively, without significant angiogenesis. P-glycoprotein staining in endothelium is decreased by 22% and parallels an increase of perlecan and fibronectin staining around cerebral blood vessels. Altogether, these results strongly suggest that the emergence of long-term behavioral dysfunctions observed in rodent jTBI may be related to endothelial remodeling at the blood-brain barrier alongside vascular dysfunction and altered Aβ trafficking. This study shows that it is important to consider jTBI as a vascular disorder with long-term consequences on cognitive functions.

Hair follicle (HF) morphogenesis and cycling are a result of intricate autonomous epithelial-mesenchymal interactions. Once the first HF cycle is complete it repeatedly undergoes cyclic transformations. Heparan sulfate (HS) proteoglycans are found on the cell surface and in the extracellular matrix where they influence a variety of biological processes by interacting with physiologically important proteins, such as growth factors. Inhibition of heparanase (an HS endoglycosidase) in in vitro cultured HFs has been shown to induce a catagen-like process. Therefore, this study aimed to elucidate the precise role of HS in HF morphogenesis and cycling. An inducible tetratransgenic mouse model was generated to excise exostosin glycosyltransferase 1 (Ext1) in keratin 14-positive cells from P21. Interestingly, EXT1(StEpiΔ/StEpiΔ) mice presented solely anagen HFs. Moreover, waxing the fur to synchronize the HFs revealed accelerated hair regrowth in the EXT1(StEpiΔ/StEpiΔ) mice and hindered cycling into catagen. The ablation of HS in the interfollicular epidermal cells of mature skin led to the spontaneous formation of new HFs and an increase in Sonic Hedgehog expression resembling wild-type mice at P0, thereby indicating that the HS/Sonic Hedgehog signaling pathway regulates HF formation during embryogenesis and prevents HF formation in mature skin. Finally, the knock-out of HS also led to the morphogenesis and hyperplasia of sebaceous glands and sweat glands in mature mice, leading to exacerbated sebum production and accumulation on the skin surface. Therefore, our findings clearly show that an intricate control of HS levels is required for HF, sebaceous gland, and sweat gland morphogenesis and HF cycling.

Guanidinoglycosides, a family of cellular transporters capable of delivering high Mw biopolymers, have previously been shown to display high selectivity for cell-surface heparan sulfate proteoglycans and promote their clustering. Herein, the internalization mechanism of amphiphilic guanidinoglycoside derivatives was investigated by cell-surface FRET analysis. Unexpectedly, although the heparan sulfate selectivity is maintained, the cellular uptake of these derivatives does not appear to involve clustering of the proteoglycans on the cell surface. This suggests a distinct uptake mechanism when compared to the parent guanidinoglycoside-based carriers.

Heparan sulfate proteoglycans have been shown to modulate podocyte adhesion to--and pedicel organization on--the glomerular basement membrane. Recent studies showed that foot process effacement developed in a mutant mouse model whose podocytes were unable to assemble heparan sulfate glycosaminoglycan chains. This study, a further refinement, explored the role of heparan N-sulfation on podocyte behavior. A novel mutant mouse (Ndst1(-/-)) was developed, having podocyte-specific deletion of Ndst1, the enzyme responsible for N-sulfation of heparan sulfate chains. Podocytes having this mutation had foot process effacement and abnormal adhesion to Bowman's capsule. Although glomerular hypertrophy did develop in the kidneys of mutant animals, mesangial expansion was not seen. The lack of heparan N-sulfation did not affect the expression of agrin or perlecan proteoglycan core proteins. Loss of N-sulfation did not result in significant proteinuria, but the increase in the albumin/creatinine ratio was coincident with the development of the enlarged lysosomes in the proximal tubules. Thus, although the renal phenotype of the Ndst1(-/-) mouse is mild, the data show that heparan chain N-sulfation plays a key role in podocyte organization.

ObjectiveChylomicron 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 resultsTriglyceride-rich lipoprotein clearance was measured in AlbCre(+)Ndst1(f/f), Ldlr(-/-), and AlbCre(+)Lrp1(f/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 3 receptors (AlbCre(+)Ndst1(f/f) Lrp1(f/f) Ldlr(-/-)) displayed dramatic hyperlipidemia (870 ± 270 mg triglyceride/dL; 1300 ± 350 mg of total cholesterol/dL) and exhibited persistent elevated postprandial triglyceride levels because of 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), whereas LDLR and LRP1 clear larger particles (≈ 40-60 nm diameter). Finally, we show that HSPGs play a major role in clearance of triglyceride-rich lipoproteins in mice fed normal chow or under postprandial conditions but seem to play a less significant role on a high-fat diet.ConclusionsThese data show that HSPGs, LDLR, and LRP1 clear distinct subsets of particles, that HSPGs work independently of LDLR and LRP1, and that HSPGs, LDLR, and LRP1 are the 3 major hepatic triglyceride-rich lipoprotein clearance receptors in mice.

BackgroundLipoprotein lipase (Lpl) acts on triglyceride-rich lipoproteins in the peripheral circulation, liberating free fatty acids for energy metabolism or storage. This essential enzyme is synthesized in parenchymal cells of adipose tissue, heart, and skeletal muscle and migrates to the luminal side of the vascular endothelium where it acts upon circulating lipoproteins. Prior studies suggested that Lpl is immobilized by way of heparan sulfate proteoglycans on the endothelium, but genetically altering endothelial cell heparan sulfate had no effect on Lpl localization or lipolysis. The objective of this study was to determine if extracellular matrix proteoglycans affect Lpl distribution and triglyceride metabolism.Methods and findingsWe examined mutant mice defective in collagen XVIII (Col18), a heparan sulfate proteoglycan present in vascular basement membranes. Loss of Col18 reduces plasma levels of Lpl enzyme and activity, which results in mild fasting hypertriglyceridemia and diet-induced hyperchylomicronemia. Humans with Knobloch Syndrome caused by a null mutation in the vascular form of Col18 also present lower than normal plasma Lpl mass and activity and exhibit fasting hypertriglyceridemia.ConclusionsThis is the first report demonstrating that Lpl presentation on the lumenal side of the endothelium depends on a basement membrane proteoglycan and demonstrates a previously unrecognized phenotype in patients lacking Col18.

Heparan sulfate (HS) proteoglycans (HSPGs) bind to multiple growth factors/morphogens and regulate their signaling. 6-O-sulfation (6S) of glucosamine within HS chains is critical for many of these ligand interactions. Sulf-1 and Sulf-2, which are extracellular neutral-pH sulfatases, provide a novel post-synthetic mechanism for regulation of HSPG function by removing 6S from intact HS chains. The Sulfs can thereby modulate several signaling pathways, including the promotion of Wnt signaling. We found induction of SULF2 transcripts and Sulf-2 protein in human lung adenocarcinoma and squamous cell carcinoma, the two major classes of non-small-cell lung carcinomas (NSCLCs). We confirmed widespread Sulf-2 protein expression in tumor cells of 10/10 surgical specimens of human lung squamous carcinomas. We studied five Sulf-2(+) NSCLC cell lines, including two, which were derived by cigarette-smoke transformation of bronchial epithelial cells. shRNA-mediated Sulf-2 knockdown in these lines caused an increase in 6S on their cell surface and in parallel reversed their transformed phenotype in vitro, eliminated autocrine Wnt signaling and strongly blunted xenograft tumor formation in nude mice. Conversely, forced Sulf-2 expression in non-malignant bronchial epithelial cells produced a partially transformed phenotype. Our findings support an essential role for Sulf-2 in lung cancer, the leading cancer killer.

Endostatin, a collagen XVIII fragment, is a potent anti-angiogenic protein. We sought to identify its endothelial cell surface receptor(s). Alkaline phosphatase- tagged endostatin bound endothelial cells revealing two binding affinities. Expression cloning identified glypican, a cell surface proteoglycan as the lower-affinity receptor. Biochemical and genetic studies indicated that glypicans' heparan sulfate glycosaminoglycans were critical for endostatin binding. Furthermore, endostatin selected a specific octasulfated hexasaccharide from a sequence in heparin. We have also demonstrated a role for endostatin in renal tubular cell branching morphogenesis and show that glypicans serve as low-affinity receptors for endostatin in these cells, as in endothelial cells. Finally, antisense experiments suggest the critical importance of glypicans in mediating endostatin activities.

Glypicans are major cell surface heparan sulfate proteoglycans, the structures of which are characterized by the presence of a cysteine-rich globular domain, a short glycosaminoglycan (GAG) attachment region, and a glycosylphosphatidylinositol membrane anchor. Despite strong evolutionary conservation of the globular domains of glypicans, no function has yet been attributed to them. By using a novel quantitative approach for assessing proteoglycan glycosylation, we show here that removal of the globular domain from rat glypican-1 converts the proteoglycan from one that bears approximately 90% heparan sulfate (HS) to one that bears approximately 90% chondroitin sulfate. Mutational analysis shows that sequences at least 70 amino acids away from the glypican-1 GAG attachment site are required for preferential HS assembly, although more nearby sequences also play a role. The effects of the glypican-1 globular domain on HS assembly could also be demonstrated by fusing this domain to sequences representing the GAG attachment sites of other proteoglycans or, surprisingly, simply by expressing the isolated globular domain in cells and analyzing effects either on an exogenously expressed glypican-1 GAG attachment domain or on endogenous proteoglycans. Quantitative analysis of the effect of the globular domain on GAG addition to proteoglycan core proteins suggested that preferential HS assembly is achieved, at least in part, through the inhibition of chondroitin sulfate assembly. These data identify the glypican-1 globular domain as a structural motif that potently influences GAG class determination and suggest that an important role of glypican globular domains is to ensure a high level of HS substitution of these proteoglycans.

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8-fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400-fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.

ARH-77 cells do not adhere to type I collagen and readily invade into collagen gels, but following expression of the transmembrane heparan sulfate proteoglycan syndecan-1, they bind collagen and fail to invade. We now show that cells transfected with syndecan-2 or syndecan-4 also bind collagen and are non-invasive. In contrast, cells transfected with the glycosylphosphatidylinositol-anchored proteoglycan glypican-1 do not bind to collagen and remain invasive, even though glypican- and syndecan-expressing cells have similar surface levels of heparan sulfate, and their proteoglycans have similar affinities for collagen. Analysis of cells expressing syndecan-1-glypican-1 chimeric proteoglycans reveals that inhibition of invasion requires the extracellular domain of syndecan but not its transmembrane or cytoplasmic domain. Surprisingly, cells bearing a chimera composed of the glypican extracellular domain fused to the syndecan transmembrane and cytoplasmic domains bind to collagen but remain invasive, implying that adhesion to collagen is not by itself sufficient to inhibit invasion. Apparently, the extracellular domain of syndecan-1, presumably by interacting with cell-surface signal transducing molecules, directly regulates complex cell behaviors such as motility and invasiveness. These results also show for the first time that syndecans and glypicans can have distinct functions, even when expressed by the same cell type.

The glypicans are a family of glycosylphosphatidylinositol (GPI)-anchored proteoglycans that, by virtue of their cell-surface localization and possession of heparan sulfate chains, may regulate the responses of cells to numerous heparin-binding growth factors, cell adhesion molecules, and extracellular matrix components. Mutations in one glypican cause a syndrome of human birth defects, suggesting important roles for these proteoglycans in development. Glypican-1, the first-discovered member of this family, was originally found in cultured fibroblasts, and later shown to be a major proteoglycan of the mature and developing brain. Here we examine the pattern of glypican-1 mRNA and protein expression more widely in the developing rodent, concentrating on late embryonic and early postnatal stages. High levels of glypican-1 expression were found throughout the brain and skeletal system. In the brain, glypican-1 mRNA was widely, and sometimes only transiently, expressed by zones of neurons and neuroepithelia. Glypican-1 protein localized strongly to axons and, in the adult, to synaptic terminal fields as well. In the developing skeletal system, glypican-1 was found in the periosteum and bony trabeculae in a pattern consistent with expression by osteoblasts, as well as in the bone marrow. Glypican-1 was also observed in skeletal and smooth muscle, epidermis, and in the developing tubules and glomeruli of the kidney. Little or no expression was observed in the developing heart, lung, liver, dermis, or vascular endothelium at the stages examined. The tissue-, cell type-, and in some cases stage-specific expression of glypican-1 revealed in this study are likely to provide insight into the functions of this proteoglycan in development.

Heparan sulfate is ubiquitous at the cell surface, where it is expressed predominantly on proteoglycans of either the transmembrane syndecan family or the glycosylphosphatidylinositol (GPI)-anchored glypican family, and has been proposed to function as a "coreceptor" for a number of "heparin-binding" growth factors. Although little is known about functional differences between individual members of the glypican gene family, mutations in both the Drosophila gene dally and the human gene for glypican-3 strongly suggest that at least some glypicans do function in cellular growth control and morphogenesis. In particular, deletion of the human glypican-3 gene is responsible for Simpson-Golabi-Behmel syndrome, and its associated pre- and postnatal tissue overgrowth, increased risk of embryonal tumors during early childhood, and numerous visceral and skeletal anomalies. We have identified and characterized, by sequencing of EST clones and products of rapid amplification of cDNA ends (RACE), an mRNA that encodes a 572-amino-acid member of the glypican gene family (glypican-5) that is most related (50% amino acid similarity, 39% identity) to glypican-3. Glypican-5 mRNA is detected as a 3.9- and 4.4-kb transcript in adult and neonatal mouse brain total RNA, and in situ hybridization results localize transcript primarily to restricted regions of the developing central nervous system, limb, and kidney in patterns consistent with a role in the control of cell growth or differentiation. Interestingly, glypican-5 localizes to 13q31-32 of the human genome, deletions of which are associated with human 13q- syndrome, a developmental disorder with a pattern of defects that shows significant overlap with the pattern of glypican-5 expression.

Cerebroglycan is a glycosylphosphatidylinositol-linked integral membrane heparan sulfate proteoglycan found exclusively in the developing nervous system. In the rodent, cerebroglycan mRNA first appears in regions containing newly generated neurons and typically disappears 1 to several days later (Stipp et al., 1994, J. Cell Biol. 124:149-160). To gain insight into the roles that cerebroglycan plays in the developing nervous system, monospecific antibodies were prepared and used to localize cerebroglycan protein. In the rat, cerebroglycan was prominantly expressed on axon tracts throughout the developing brain and spinal cord, where it was found at times when axons are actively growing, but generally not after axons have reached their targets. Cerebroglycan was also found on neuronal growth cones both in vivo and in vitro. Interestingly, cerebroglycan immunoreactivity was rarely seen in or around neuronal cell bodies. Indeed, by examining the hippocampus at a late stage in development-when most neurons no longer express cerebroglycan but newly generated granule neurons do-evidence was obtained that cerebroglycan is strongly polarized to the axonal, and excluded from the somatodendritic, compartment of neurons. The timing and pattern of cerebroglycan expression are consistent with a role for this cell-surface heparan sulfate proteoglycan in regulating the growth or guidance of axons.

The amyloid precursor protein (APP) of Alzheimer's disease has been shown to stimulate neurite outgrowth in vitro. The effect of APP on neurite outgrowth can be enhanced if APP is presented to neurons in substrate-bound form, in the presence of heparan sulfate proteoglycans. To identify specific heparan sulfate proteoglycans that bind to APP, conditioned medium from neonatal mouse brain cells was subjected to affinity chromatography with recombinant APP695 as a ligand. Glypican bound strongly to the APP affinity column. Purified glypican bound to APP with an equilibrium dissociation constant of 2.8 nM and inhibited APP-induced neurite outgrowth from chick sympathetic neurons. The effect of glypican was specific for APP, as glypican did not inhibit laminin-induced neurite outgrowth. Furthermore, treatment of cultures with 4-methylumbelliferyl-beta-D-xyloside, a competitive inhibitor of proteoglycan glycanation, inhibited APP-induced neurite outgrowth but did not inhibit laminin-induced neurite outgrowth. This result suggests that endogenous proteoglycans are required for substrate-bound APP to stimulate neurite outgrowth. Secreted glypican may act to inhibit APP-induced neurite outgrowth in vivo by competing with endogenous proteoglycans for binding to APP.

Glycosaminoglycan (GAG)-collagen interactions play important roles in cell adhesion and extracellular matrix assembly; however, the chemical bases for these interactions are not fully understood. We have used affinity co-electrophoresis (ACE) (Lee, M.K. and Lander, A.D., Proc. Natl. Acad. Sci, USA, 88, 2768-2772, 1991) to study the binding of the heparan sulphate proteoglycan syndecan-1 and heparin to human collagens. [35S]Syndecan-1 [from normal murine mammary gland (NMuMG) epithelial cells] and low-M(r) (approximately 6 kDa) [125I]heparin were subjected to electrophoresis through agarose gel lanes containing human collagens at various concentrations, and binding affinities were measured from shifts in migration of the labelled materials. Results demonstrate that the affinities of each collagen for syndecan-1 and low-M(r) heparin were similar, and followed the order: type V >> type IV approximately type III approximately type I > type VI >> type II, and ranged in Kd from approximately 10(-8) to approximately 3 x 10(-6) M. These data suggest that syndecan-1 and heparin may contain similar collagen-binding determinants. It was also found that the same heparin subpopulation was selectively bound with high affinity by each of the collagens. The published amino acid sequences of the six collagens were examined for what are thought to be heparin-binding consensus sequences (Cardin, A.D. and Weintraub, H.J.R., Arteriosclerosis, 9, 21-32, 1989). The presence of such sequences did not correlate with affinity for heparin or syndecan-1, and collagens I, II and III lacked such sequences entirely. The data suggest that collagens may use novel types of binding sites to interact with GAGs.

Cell-surface proteoglycans have been implicated in cell responses to growth factors, extracellular matrix, and cell adhesion molecules. M12, one of the most abundant membrane-associated proteoglycans in the adult rat brain, is a approximately 65 kDa glycosylphosphatidylinositol-linked protein that bears heparan sulfate chains (Herndon and Lander, 1990). To assess its identity, M12 was purified and internal peptide sequences obtained. Comparison of the results with protein sequence predicted by a cDNA cloned from PC12 cells indicated that M12 is rat glypican, a proteoglycan first cloned from human fibroblasts. In addition, antibodies raised against a rat glypican fusion protein specifically detected the 65 kDa brain proteoglycan core protein, both by immunoprecipitation and by Western blotting. Northern blot analysis using a rat glypican probe also detected glypican message in the adult, as well as the developing rat brain. In situ hybridization with glypican RNA probes showed that glypican is expressed in a subset of structures in the adult rat nervous system. These include the hippocampus, dorsal thalamus, amygdala, cerebral cortex, piriform cortex, olfactory tubercle, several cranial nerve nuclei, the ventral horn of the spinal cord, and the dorsal root ganglia. Several other brain regions exhibited little or no hybridization over background. In most cases where glypican hybridization was observed, the signal could be localized specifically to the cell bodies of identifiable neurons, for example, spinal motoneurons, hippocampal pyramidal cells. In the cerebral cortex, glypican hybridization was found in layers 2/3, 5, and 6, but was missing from 1 and 4. The data suggest that glypican is expressed primarily by subpopulations of projection neurons in the adult rat nervous system.

Heparan sulfate proteoglycans (HSPGs) are found on the surface of all adherent cells and participate in the binding of growth factors, extracellular matrix glycoproteins, cell adhesion molecules, and proteases and antiproteases. We report here the cloning and pattern of expression of cerebroglycan, a glycosylphosphatidylinositol (GPI)-anchored HSPG that is found in the developing rat brain (previously referred to as HSPG M13; Herndon, M. E., and A. D. Lander. 1990. Neuron. 4:949-961). The cerebroglycan core protein has a predicted molecular mass of 58.6 kD and five potential heparan sulfate attachment sites. Together with glypican (David, G., V. Lories, B. Decock, P. Marynen, J.-J. Cassiman, and H. Van den Berghe. 1990. J. Cell Biol. 111:3165-3176), it defines a family of integral membrane HSPGs characterized by GPI linkage and conserved structural motifs, including a pattern of 14 cysteine residues that is absolutely conserved. Unlike other known integral membrane HSPGs, including glypican and members of the syndecan family of transmembrane proteoglycans, cerebroglycan is expressed in only one tissue: the nervous system. In situ hybridization experiments at several developmental stages strongly suggest that cerebroglycan message is widely and transiently expressed by immature neurons, appearing around the time of final mitosis and disappearing after cell migration and axon outgrowth have been completed. These results suggest that cerebroglycan may fulfill a function related to the motile behaviors of developing neurons.