Modulation of heparan sulfate biosynthesis by sodium butyrate in recombinant CHO cells

Sodium butyrate, a histone deacetylase inhibitor, has been used to improve transgene expression in Chinese hamster ovary (CHO) cells. The current study explores the impact of butyrate treatment on heparan sulfate (HS) biosynthesis and structural composition in a recombinant CHO-S cell line expressing enzymes in the heparin (HP)/(HS) biosynthetic pathway (Dual-10 stably expressing NDST2 and HS3st1). Flow cytometric analysis showed that antithrombin binding was increased in Dual-10 cells and basic fibroblast growth factor binding was decreased in response to sodium butyrate treatment. The results were in agreement with the AMAC-LCMS (2-aminoacridine-tagged HS/HP analysis by liquid chromatography mass spectrometry) data that showed that there was an increase in heparan sulfate tri-sulfated disaccharides and a decrease in N-sulfated disaccharides in the butyrate-treated cells. However, we could not detect any changes in the chondroitin sulfate pathway in Dual-10 cells treated with butyrate. The current study is the first to report the effect of butyrate on glycosaminoglycan profiles.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-013-9677-9) contains supplementary material, which is available to authorized users.     

Antibody-based assay for N-deacetylase activity of heparan sulfate/heparin N-deacetylase/N-sulfotransferase (NDST): novel characteristics of NDST-1 and -2

A new assay was developed to measure the N-deacetylase activity of the glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs), which are key enzymes in sulfation of heparan sulfate (HS)/heparin. The assay is based on the recognition of NDST-generated N-unsubstituted glucosamine units in Escherichia coli K5 capsular polysaccharide or in HSs by monoclonal antibody JM-403. Substrate specificity and potential product inhibition of the NDST isoforms 1 and 2 were analyzed by comparing lysates of human 293 kidney cells stably transfected with mouse NDST-1 or -2. We found HSs to be excellent substrates for both NDST enzymes. Both NDST-1 and -2 N-deacetylate heparan sulfate from human aorta ( approximately 0.6 sulfate groups/disaccharide) with comparable high efficiency, apparent Km values of 0.35 and 0.76 microM (calculation based on [HexA]) being lower (representing a higher affinity) than those for K5 polysaccharide (13.3 and 4.7 microM, respectively). Comparison of various HS preparations and the unsulfated K5 polysaccharide as substrates indicate that both NDST-1 and -2 can differentially N-sulfate polysaccharides already modified to some extent by various other enzymes involved in HS/heparin synthesis. Both enzymes were equally inhibited by N-sulfated sequences (>or=6 sugar residues) present in N-sulfated K5, N-deacetylated N-resulfated HS, and heparin. Our primary findings were confirmed in the conventional N-deacetylase assay measuring the release of 3H-acetate of radiolabeled K5 or HS as substrates. We furthermore showed that NDST N-deacetylase activity in crude cell/tissue lysates can be partially blocked by endogenous HS/heparin. We speculate that in HS biosynthesis, some NDST variants initiate HS modification/sulfation reactions, whereas other (or the same) NDST isoforms later on fill in or extend already modified HS sequences.     

Bioengineered Chinese Hamster Ovary Cells with Golgi-targeted 3-O-Sulfotransferase-1 Biosynthesize Heparan Sulfate with an Antithrombin-binding Site

HS3st1 (heparan sulfate 3-O-sulfotransferase isoform-1) is a critical enzyme involved in the biosynthesis of the antithrombin III (AT)-binding site in the biopharmaceutical drug heparin. Heparin is a highly sulfated glycosaminoglycan that shares a common biosynthetic pathway with heparan sulfate (HS). Although only granulated cells, such as mast cells, biosynthesize heparin, all animal cells are capable of biosynthesizing HS. As part of an effort to bioengineer CHO cells to produce heparin, we previously showed that the introduction of both HS3st1 and NDST2 (N-deacetylase/N-sulfotransferase isoform-2) afforded HS with a very low level of anticoagulant activity. This study demonstrated that untargeted HS3st1 is broadly distributed throughout CHO cells and forms no detectable AT-binding sites, whereas Golgi-targeted HS3st1 localizes in the Golgi and results in the formation of a single type of AT-binding site and high anti-factor Xa activity (137 ± 36 units/mg). Moreover, stable overexpression of HS3st1 also results in up-regulation of 2-O-, 6-O-, and N-sulfo group-containing disaccharides, further emphasizing a previously unknown concerted interplay between the HS biosynthetic enzymes and suggesting the need to control the expression level of all of the biosynthetic enzymes to produce heparin in CHO cells.     

Lowered expression of heparan sulfate/heparin biosynthesis enzyme N-deacetylase/n-sulfotransferase 1 results in increased sulfation of mast cell heparin

Deficiency of the heparan sulfate biosynthesis enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) in mice causes severely disturbed heparan sulfate biosynthesis in all organs, whereas lack of NDST2 only affects heparin biosynthesis in mast cells (MCs). To investigate the individual and combined roles of NDST1 and NDST2 during MC development, in vitro differentiated MCs derived from mouse embryos and embryonic stem cells, respectively, have been studied. Whereas MC development will not occur in the absence of both NDST1 and NDST2, lack of NDST2 alone results in the generation of defective MCs. Surprisingly, the relative amount of heparin produced in NDST1(+/-) and NDST1(-/-) MCs is higher (≈30%) than in control MCs where ≈95% of the (35)S-labeled glycosaminoglycans produced is chondroitin sulfate. Lowered expression of NDST1 also results in a higher sulfate content of the heparin synthesized and is accompanied by increased levels of stored MC proteases. A model of the GAGosome, a hypothetical Golgi enzyme complex, is used to explain the results.     

Undersulfation of heparan sulfate restricts differentiation potential of mouse embryonic stem cells

Heparan sulfate proteoglycans, present on cell surfaces and in the extracellular matrix, interact with growth factors and morphogens to influence growth and differentiation of cells. The sulfation pattern of the heparan sulfate chains formed during biosynthesis in the Golgi compartment will determine the interaction potential of the proteoglycan. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes have a key role during biosynthesis, greatly influencing total sulfation of the heparan sulfate chains. The differentiation potential of mouse embryonic stem cells lacking both NDST1 and NDST2 was studied using in vitro differentiation protocols, expression of differentiation markers, and assessment of the ability of the cells to respond to growth factors. The results show that NDST1 and NDST2 are dispensable for mesodermal differentiation into osteoblasts but necessary for induction of adipocytes and neural cells. Gene expression analysis suggested a differentiation block at the primitive ectoderm stage. Also, GATA4, a primitive endoderm marker, was expressed by these cells. The addition of FGF4 or FGF2 together with heparin rescued the differentiation potential to neural progenitors and further to mature neurons and glia. Our results suggest that the embryonic stem cells lacking both NDST1 and NDST2, expressing a very low sulfated heparan sulfate, can take the initial step toward differentiation into all three germ layers. Except for their potential for mesodermal differentiation into osteoblasts, the cells are then arrested in a primitive ectoderm and/or endoderm stage.     

Synthesis of Heparan Sulfate with Cyclophilin B-binding Properties Is Determined by Cell Type-specific Expression of Sulfotransferases

Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4⁺ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells.     

Heparan sulfate glycosaminoglycans are receptors sufficient to mediate the initial binding of adenovirus types 2 and 5

Cell infection by adenovirus serotypes 2 and 5 (Ad2/5) initiates with the attachment of Ad fiber to the coxsackievirus and Ad receptor (CAR) followed by alpha(v) integrin-mediated entry. We recently demonstrated that heparan sulfate glycosaminoglycans (HS GAGs) expressed on cell surfaces are involved in the binding and infection of Ad2/5 (M. C. Dechecchi, A. Tamanini, A. Bonizzato, and G. Cabrini, Virology 268:382-390, 2000). The role of HS GAGs was investigated using extracellular soluble domain 1 of CAR (sCAR-D1) and heparin as soluble receptor analogues of CAR and HS GAGs in A549 and recombinant CHO cell lines with differential levels of expression of the two receptors and cultured to various densities. Complete inhibition of binding and infection was obtained by preincubating Ad2/5 with both heparin (10 microg/ml) and sCAR-D1 (200 microg/ml) in A549 cells. Partial inhibition was observed when heparin and sCAR-D1 were preincubated separately with Ad. The level of heparin-sensitive [(3)H]Ad2/5 binding doubled in sparse A549 cells (50 to 70,000 cells/cm(2)) with respect to that of cells grown to confluence (200 to 300,000 cells/cm(2)), in parallel with increased expression of HS GAGs. [(3)H]Ad2 bound to sparse CAR-negative CHO cells expressing HS GAGs (CHO K1). No [(3)H]Ad2 binding was observed in CHO K1 cells upon competitive inhibition with heparin and in HS GAG-defective CHO A745, D677, and E606 clones. HS-sensitive Ad2 infection was obtained in CAR-negative sparse CHO K1 cells but not in CHO A745 cells, which were permissive to infection only upon transfection with CAR. These results demonstrate that HS GAGs are sufficient to mediate the initial binding of Ad2/5.     

The heparan and heparin metabolism pathway is involved in regulation of fatty acid composition

Six genes involved in the heparan sulfate and heparin metabolism pathway, DSEL (dermatan sulfate epimerase-like), EXTL1 (exostoses (multiple)-like 1), HS6ST1 (heparan sulfate 6-O-sulfotransferase 1), HS6ST3 (heparan sulfate 6-O-sulfotransferase 3), NDST3 (N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 3), and SULT1A1 (sulfotransferase family, cytosolic, 1A, phenol-preferring, member 1), were investigated for their associations with muscle lipid composition using cattle as a model organism. Nineteen single nucleotide polymorphisms (SNPs)/multiple nucleotide length polymorphisms (MNLPs) were identified in five of these six genes. Six of these mutations were then genotyped on 246 Wagyu x Limousin F(2) animals, which were measured for 5 carcass, 6 eating quality and 8 fatty acid composition traits. Association analysis revealed that DSEL, EXTL1 and HS6ST1 significantly affected two stearoyl-CoA desaturase activity indices, the amount of conjugated linoleic acid (CLA), and the relative amount of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) in skeletal muscle (P<0.05). In particular, HS6ST1 joined our previously reported SCD1 and UQCRC1 genes to form a three gene network for one of the stearoyl-CoA desaturase activity indices. These results provide evidence that genes involved in heparan sulfate and heparin metabolism are also involved in regulation of lipid metabolism in bovine muscle. Whether the SNPs affected heparan sulfate proteoglycan structure is unknown and warrants further investigation.     

Sulf loss influences N-, 2-O-, and 6-O-sulfation of multiple heparan sulfate proteoglycans and modulates fibroblast growth factor signaling

Sulf1 and Sulf2 are two heparan sulfate 6-O-endosulfatases that regulate the activity of multiple growth factors, such as fibroblast growth factor and Wnt, and are essential for mammalian development and survival. In this study, the mammalian Sulfs were functionally characterized using overexpressing cell lines, in vitro enzyme assays, and in vivo Sulf knock-out cell models. Analysis of subcellular Sulf localization revealed significant differences in enzyme secretion and detergent solubility between the human isoforms and their previously characterized quail orthologs. Further, the activity of the Sulfs toward their native heparan sulfate substrates was determined in vitro, demonstrating restricted specificity for S-domain-associated 6S disaccharides and an inability to modify transition zone-associated UA-GlcNAc(6S). Analysis of heparan sulfate composition from different cell surface, shed, glycosylphosphatidylinositol-anchored and extracellular matrix proteoglycan fractions of Sulf knock-out cell lines established differential effects of Sulf1 and/or Sulf2 loss on nonsubstrate N-, 2-O-, and 6-O-sulfate groups. These findings indicate a dynamic influence of Sulf deficiency on the HS biosynthetic machinery. Real time PCR analysis substantiated differential expression of the Hs2st and Hs6st heparan sulfate sulfotransferase enzymes in the Sulf knock-out cell lines. Functionally, the changes in heparan sulfate sulfation resulting from Sulf loss were shown to elicit significant effects on fibroblast growth factor signaling. Taken together, this study implicates that the Sulfs are involved in a potential cellular feed-back mechanism, in which they edit the sulfation of multiple heparan sulfate proteoglycans, thereby regulating cellular signaling and modulating the expression of heparan sulfate biosynthetic enzymes.     

Altered heparan sulfate structure in mice with deleted NDST3 gene function

We report the generation and analysis of mutant mice bearing a targeted disruption of the heparan sulfate (HS)-modifying enzyme GlcNAc N-deacetylase/N-sulfotransferase 3 (NDST3). NDST3(-/-) mice develop normally, are fertile, and show only subtle hematological and behavioral abnormalities in agreement with only moderate HS undersulfation. Compound mutant mice made deficient in NDST2;NDST3 activities also develop normally, showing that both isoforms are not essential for development. In contrast, NDST1(-/-);NDST3(-/-) compound mutant embryos display developmental defects caused by severe HS undersulfation, demonstrating NDST3 contribution to HS synthesis in the absence of NDST1. Moreover, analysis of HS composition in dissected NDST3 mutant adult brain revealed regional changes in HS sulfation, indicating restricted NDST3 activity on nascent HS in defined wild-type tissues. Taken together, we show that NDST3 function is not essential for development or adult homeostasis despite contributing to HS synthesis in a region-specific manner and that the loss of NDST3 function is compensated for by the other NDST isoforms to a varying degree.     

Blockage of undesirable endocytosis of recombinant human growth/differentiation factor-5 in Chinese hamster ovary cell cultures requires heparin analogs with specific chain lengths

Cell surface heparan sulfate proteoglycan (HSPG)-mediated endocytosis lowers the yield of recombinant human bone morphogenetic proteins (rhBMPs), such as rhBMP-2 and rhBMP-4, from Chinese hamster ovary (CHO) cell cultures. Exogenous recombinant human growth/differentiation factor-5 (rhGDF-5), a member of the BMP family, bound to cell surface HSPGs and was actively internalized into CHO cells. Knockdown of heparan sulfate (HS) synthesis enzymes in CHO cells revealed that the chain length and N-sulfation of HS affected the binding of rhGDF-5 to HSPGs and subsequent rhGDF-5 internalization. To increase product yield by minimizing rhGDF-5 internalization in recombinant CHO (rCHO) cell cultures, heparin, and dextran sulfate (DS) of various polysaccharide chain lengths, which are structural analogs of HS, were examined for blockage of rhGDF-5 internalization. Heparin fragments of four monosaccharides (MW of 1.2 kDa) and DS (MW of 15 kDa) did not inhibit rhGDF-5 internalization whereas unfractionated heparin and DS of 200 kDa could significantly inhibit it. Compared to the control cultures, supplementation with unfractionated heparin or DS of 200 kDa at 1 g L^-1 resulted in more than a 10-fold increase in the maximum rhGDF-5 concentration. Taken together, the supplementation of structural HS analogs improved rhGDF-5 production in rCHO cell cultures by inhibiting rhGDF-5 internalization.     

The biosynthesis of anticoagulant heparan sulfate by the heparan sulfate 3-O-sulfotransferase isoform 5

The role of heparan sulfate (HS) in regulating blood coagulation has a wide range of clinical implications. In this study, we investigated the role of 3-O-sulfotransferase isoform 5 (3-OST-5) in generating anticoagulant HS in vivo. A Chinese hamster ovary cell line (3OST5/CHO) stably expressing 3-OST-5 was generated. The expression of 3-OST-5 in 3OST5/CHO cells was confirmed by Northern blot analysis, RT-PCR, and the disaccharide analyses of the HS from the cells. We also determined the effects of the HS from 3OST5/CHO on antithrombin-mediated inhibition of factor Xa. Fluorescently labeled antithrombin bound to the surface of 3OST5/CHO cells, suggesting that the antithrombin-binding HS is indeed present on the cell surface. Our results demonstrate that the 3-OST-5 gene is capable of synthesizing anticoagulant HS in CHO cells and has the potential to contribute to the biosynthesis of anticoagulant HS in humans.     

Specific and flexible roles of heparan sulfate modifications in Drosophila FGF signaling

Specific sulfation sequence of heparan sulfate (HS) contributes to the selective interaction between HS and various proteins in vitro. To clarify the in vivo importance of HS fine structures, we characterized the functions of the Drosophila HS 2-O and 6-O sulfotransferase (Hs2st and Hs6st) genes in FGF-mediated tracheal formation. We found that mutations in Hs2st or Hs6st had unexpectedly little effect on tracheal morphogenesis. Structural analysis of mutant HS revealed not only a loss of corresponding sulfation, but also a compensatory increase of sulfation at other positions, which maintains the level of HS total charge. The restricted phenotypes of Hsst mutants are ascribed to this compensation because FGF signaling is strongly disrupted by Hs2st; Hs6st double mutation, or by overexpression of 6-O sulfatase, an extracellular enzyme which removes 6-O sulfate groups without increasing 2-O sulfation. These findings suggest that the overall sulfation level is more important than strictly defined HS fine structures for FGF signaling in some developmental contexts.     

2-O Heparan Sulfate Sulfation by Hs2st Is Required for Erk/Mapk Signalling Activation at the Mid-Gestational Mouse Telencephalic Midline

Heparan sulfate (HS) is a linear carbohydrate composed of polymerized uronate-glucosamine disaccharide units that decorates cell surface and secreted glycoproteins in the extracellular matrix. In mammals HS is subjected to differential sulfation by fifteen different heparan sulfotransferase (HST) enzymes of which Hs2st uniquely catalyzes the sulfation of the 2-O position of the uronate in HS. HS sulfation is postulated to be important for regulation of signaling pathways by facilitating the interaction of HS with signaling proteins including those of the Fibroblast Growth Factor (Fgf) family which signal through phosphorylation of extracellular signal-regulated kinases Erk1/2. In the developing mouse telencephalon Fgf2 signaling regulates proliferation and neurogenesis. Loss of Hs2st function phenocopies the thinned cerebral cortex of mutant mice in which Fgf2 or Erk1/2 function are abrogated, suggesting the hypothesis that 2-O-sulfated HS structures play a specific role in Fgf2/Erk signaling pathway in this context in vivo. This study investigated the molecular role of 2-O sulfation in Fgf2/Erk signaling in the developing telencephalic midline midway through mouse embryogenesis at E12.5. We examined the expression of Hs2st, Fgf2, and Erk1/2 activity in wild-type and Hs2st^-/- mice. We found that Hs2st is expressed at high levels at the midline correlating with high levels of Erk1/2 activation and Erk1/2 activation was drastically reduced in the Hs2st^-/- mutant at the rostral telencephalic midline. We also found that 2-O sulfation is specifically required for the binding of Fgf2 protein to Fgfr1, its major cell-surface receptor at the rostral telencephalic midline. We conclude that 2-O sulfated HS structures generated by Hs2st are needed to form productive signaling complexes between HS, Fgf2 and Fgfr1 that activate Erk1/2 at the midline. Overall, our data suggest the interesting possibility that differential expression of Hs2st targets the rostral telencephalic midline for high levels of Erk signaling by increasing the sensitivity of cells to an Fgf2 signal that is rather more widespread.     

Systemic inactivation of Hs6st1 in mice is associated with late postnatal mortality without major defects in organogenesis

Heparan sulfate (HS) proteoglycans modulate the biological activity of a number of growth factors in development, homeostasis, and cancer. Specific modifications of HS chains by HS biosynthetic enzymes have been implicated in growth factor signaling in multiple aspects of organogenesis. Although the role of HS 6-O-sulfotransferases has been described in processes such as trachea formation in Drosophila and vasculogenesis in zebrafish, little is known about how HS 6-O-sulfotransferases (Hs6st1-3 in mice) influence mouse development. To address this issue, we generated a conditionally mutant Hs6st1 mouse line and then generated mice with systemic inactivation of Hs6st1. Hs6st1-null pups were viable and grossly normal at birth. The lack of obvious abnormalities in lung, liver, and kidney, which express high levels of Hs6st1 during development, suggests that at least during embryonic life, the loss of Hs6st1 function may be compensated for by mechanisms involving other HS modifying enzymes. During early adulthood, however, Hs6st1-null mice failed to thrive and exhibited growth retardation, body weight loss, enlargement of airspaces in the lung and, in some cases, lethality. Our results suggest a potentially critical role for HS 6-O sulfation by Hs6st1 in postnatal processes.     

The Role of Drosophila Heparan Sulfate 6-O-Endosulfatase in Sulfation Compensation

The biosynthesis of heparan sulfate proteoglycans is tightly regulated by multiple feedback mechanisms, which support robust developmental systems. One of the regulatory network systems controlling heparan sulfate (HS) biosynthesis is sulfation compensation. A previous study using Drosophila HS 2-O- and 6-O-sulfotransferase (Hs2st and Hs6st) mutants showed that loss of sulfation at one position is compensated by increased sulfation at other positions, supporting normal FGF signaling. Here, we show that HS sulfation compensation rescues both Decapentaplegic and Wingless signaling, suggesting a universal role of this regulatory system in multiple pathways in Drosophila. Furthermore, we identified Sulf1, extracellular HS 6-O-endosulfatase, as a novel component of HS sulfation compensation. Simultaneous loss of Hs2st and Sulf1 led to 6-O-oversulfation, leading to patterning defects, overgrowth, and lethality. These phenotypes are caused at least partly by abnormal up-regulation of Hedgehog signaling. Thus, sulfation compensation depends on the coordinated activities of Hs2st, Hs6st, and Sulf1.     

Drosophila heparan sulfate 3-0 sulfotransferase B Null Mutant Is Viable and Exhibits No Defects in Notch Signaling

Heparan sulfate proteoglycans （HSPGs） are critically involved in a variety of biological events. The functions of HSPGs are determined by the nature of the core proteins and modifications of heparan sulfate （HS） glycosaminoglycan （GAG） chains. The distinct O-sulfo- transferases are important for nonrandom modifications at specific positions. Two HS 3-0 sulfotransferase （Hs3st） genes, Hs3st-A and Hs3st-B, were identified in Drosophila. Previous experiments using RNA interference （RNAi） suggested that Hs3st-B was required for Notch signaling. Here, we generated a null mutant of Hs3st-B via ends-out gene targeting and examined its role（s） in development. We found that homozygous Hs3st-B mutants have no neurogenic defects or alterations in the expression of Notch signaling target gene. Thus, our results strongly argue against an essential role for Hs3st-B in Notch signaling. Moreover, we have generated two independent Hs3st-A RNAi lines which worked to deplete Hs3st-A. Importantly, Hs3st-A RNAi combined with Hs3st-B mutant flies did not alter the expression of Notch signaling components, arguing that both Hs3st-A and Hs3st-B were not essential for Notch signaling. The establishment of Hs3st-B mutant and effective Hs3st-A RNAi lines provides essential tools for further studies of the physiological roles of Hs3st-A and Hs3st-B in development and homeostasis.     

Glycanogenomics: a qPCR-approach to investigate biological glycan function

As an indirect approach towards glycan structures, qRT-PCR analyses using the ΔΔC_T method were performed to investigate changes in expression levels of heparan sulfate-synthesising enzymes of stimulated and unstimulated HMVECs. We chose NDSTs as early enzymes initiating sulfation and 3OSTs which act late generating specific binding sites. Major changes in expression patterns were found for the NDST3 and 3OST1 isoforms. Both enzymes were down-regulated 7- and 6-fold, respectively, following TNF-α stimulation, and 3.5- and 7.6-fold following LPS-stimulation suggesting a common restructuring process of HS in inflammation leading to a less diverse sulfation pattern. Immunostaining of TNF-α-stimulated cells using a phage display-derived antibody specific for 3-O-sulfation and unsulfated regions of HS resulted in significant fluorescence changes between unstimulated and stimulated.