Sulfation Patterns Research Articles

Investigation of the pharmacokinetic properties of synthetic heparan sulfate oligosaccharides.

Heparan sulfate (HS) is a sulfated polysaccharide with a wide range of biological activities. There is an increasing interest in the development of structurally homogeneous HS oligosaccharides as therapeutics. However, the factors influencing the pharmacokinetic properties of HS-based therapeutics remain unknown. Here, we report the pharmacokinetic properties of a panel of dodecasaccharides (12-mers) with varying sulfation patterns in healthy mice and uncover the pharmacokinetic properties of an octadecasaccharide (18-mer) in acutely injured mice. In the 12-mer panel, 1 12-mer, known as dekaparin, is anticoagulant, and 3 12-mers are nonanticoagulant. The concentrations of 12-mers in plasma and urine were determined by the disaccharide analysis using liquid chromatography coupled with tandem mass spectrometry. We observed a striking difference between anticoagulant and nonanticoagulant oligosaccharides in the 12-mer panel, showing that anticoagulant dekaparin had a 4.6-fold to 8.6-fold slower clearance and 4.4-fold to 8-fold higher plasma exposure compared to nonanticoagulant 12-mers. We also observed that the clearance of HS oligosaccharides is impacted by disease. Using an antiinflammatory 18-mer, we discovered that the clearance of 18-mer is reduced 2.8-fold in a liver failure mouse model compared to healthy mice. Our results suggest that oligosaccharides are rapidly cleared renally if they have low interaction with circulating proteins. We observed that the clearance rate of oligosaccharides is inversely associated with the degree of binding to target proteins, which can vary in response to pathophysiological conditions. Our findings uncover a contributing factor for the plasma and renal clearance of oligosaccharides which will aid the development of HS-based therapeutics.

Interactions of marine sulfated glycans with antithrombin and platelet factor 4.

The molecular interactions of sulfated glycans, such as heparin, with antithrombin (AT) and platelet factor 4 (PF4) are essential for certain biological events such as anticoagulation and heparin induced thrombocytopenia (HIT). In this study, a library including 84 sulfated glycans (polymers and oligomers) extracted from marine algae along with several animal-originated polysaccharides were subjected to a structure-activity relationship (SAR) study regarding their specific molecular interactions with AT and PF4 using surface plasmon resonance. In this SAR study, multiple characteristics were considered including different algal species, different methods of extraction, molecular weight, monosaccharide composition, sulfate content and pattern and branching vs. linear chains. These factors were found to influence the binding affinity of the studied glycans with AT. Many polysaccharides showed stronger binding than the low molecular weight heparin (e.g., enoxaparin). Fourteen polysaccharides with strong AT-binding affinities were selected to further investigate their binding affinity with PF4. Eleven of these polysaccharides showed strong binding to PF4. It was observed that the types of monosaccharides, molecular weight and branching are not very essential particularly when these polysaccharides are oversulfated. The sulfation levels and sulfation patterns are, on the other hand, the primary contribution to strong AT and PF4 interaction.

Overexpression of NDST1 Attenuates Fibrotic Response in Murine Adipose-Derived Stem Cells.

Adipose-derived stem cells (ADSCs) hold tremendous potential for treating diseases and repairing damaged tissues. Heparan sulfate (HS) plays various roles in cellular signaling mechanisms. The importance of HS in stem cell function has been reported and well documented. However, there has been little progress in using HS for therapeutic purposes. We focused on one of the sulfotransferases, NDST1, which influences overall HS chain extent and sulfation pattern, with the expectation to enhance stem cell function by increasing the N-sulfation level. We herein performed transfections of a green fluorescent protein-vector control and NDST1-vector into mouse ADSCs to evaluate stem cell functions. Overexpression of NDST1 suppressed the osteogenic differentiation of ADSCs. There was no pronounced effect observed on the stemness, inflammatory gene expression, nor any noticeable effect in adipogenic and chondrogenic differentiation. Under the tumor necrosis factor-alpha stimulation, NDST1 overexpression induced several chemokine productions that attract neutrophils and macrophages. Finally, we identified an antifibrotic response in ADSCs overexpressing NDST1. This study provides a foundation for the evaluation of HS-related effects in ADSCs undergoing ex vivo gene manipulation.

Comprehensive structural assignment of glycosaminoglycan oligo- and polysaccharides by protein nanopore

Glycosaminoglycans are highly anionic functional polysaccharides with information content in their structure that plays a major role in the communication between the cell and the extracellular environment. The study presented here reports the label-free detection and analysis of glycosaminoglycan molecules at the single molecule level using sensing by biological nanopore, thus addressing the need to decipher structural information in oligo- and polysaccharide sequences, which remains a major challenge for glycoscience. We demonstrate that a wild-type aerolysin nanopore can detect and characterize glycosaminoglycan oligosaccharides with various sulfate patterns, osidic bonds and epimers of uronic acid residues. Size discrimination of tetra- to icosasaccharides from heparin, chondroitin sulfate and dermatan sulfate was investigated and we show that different contents and distributions of sulfate groups can be detected. Remarkably, differences in α/β anomerization and 1,4/1,3 osidic linkages can also be detected in heparosan and hyaluronic acid, as well as the subtle difference between the glucuronic/iduronic epimers in chondroitin and dermatan sulfate. Although, at this stage, discrimination of each of the constituent units of GAGs is not yet achieved at the single-molecule level, the resolution reached in this study is an essential step toward this ultimate goal.

Profiling Heparan Sulfate-Heavy Metal Ions Interaction Using Electrochemical Techniques.

Heparan sulfate glycosaminoglycans provides extracellular matrix defense against heavy metals cytotoxicity. Identifying the precise glycan sequences that bind a particular heavy metal ion is a key for understanding those interactions. Here, electrochemical and surface characterization techniques were used to elucidate the relation between the glycans structural motifs, uronic acid stereochemistry, and sulfation regiochemistry to heavy metal ions binding. A divergent strategy was employed to access a small library of structurally well-defined tetrasaccharides analogs with different sulfation patterns and uronic acid compositions. These tetrasaccharides were electrochemically grafted onto glassy carbon electrodes and their response to heavy metal ions was monitored by electrochemical impedance spectroscopy. Key differences in the binding of Hg(II), Cd(II), and Pb(II) were associated with a combination of the uronic acid type and the sulfation pattern.

Glycosaminoglycan Mimetic Precision Glycomacromolecules with Sequence-Defined Sulfation and Rigidity Patterns.

Sulfated glycosaminoglycans (sGAGs) such as heparan sulfate (HS) are structurally diverse linear polysaccharides that are involved in many biological processes and have gained interest as antiviral compounds. Their recognition is driven by a complex orchestra of structural parameters that are still under intense investigation. One distinct characteristic is the incorporation of sulfation patterns including highly sulfated and non-sulfated sequences that provide variations in flexibility and conformation, which in turn impact the biological function of sGAGs. However, these distinct features have not yet been fully realized in the synthetic preparation of sGAG mimetics. Here, we present the synthesis of three groups of sulfated glycomacromolecules as sGAG mimetics: (i) globally sulfated glycooligomers, (ii) glycooligomers with sequence-defined sulfation patterns, and (iii) a globally sulfated glycooligomer-oligo-L-proline hybrid structure. The complete synthesis, including chemical sulfation, was conducted on solid support, enabled by the introduction of a commercially available photocleavable linker allowing for the preservation of sensitive sulfates during cleavage of the products. Structures were obtained in good purity and with high degrees of sulfation demonstrating the wide applicability of this methodology to prepare tailor-made sulfated glycomacromolecules and similar sGAG mimetics. Structures were tested for their anticoagulant properties showing activity similar to their natural HS counterpart and significantly lower than HP.

Harnessing Human Papillomavirus' Natural Tropism to Target Tumors.

Human papillomaviruses (HPV) are small non-enveloped DNA tumor viruses established as the primary etiological agent for the development of cervical cancer. Decades of research have elucidated HPV’s primary attachment factor to be heparan sulfate proteoglycans (HSPG). Importantly, wounding and exposure of the epithelial basement membrane was found to be pivotal for efficient attachment and infection of HPV in vivo. Sulfation patterns on HSPG’s become modified at the site of wounds as they serve an important role promoting tissue healing, cell proliferation and neovascularization and it is these modifications recognized by HPV. Analogous HSPG modification patterns can be found on tumor cells as they too require the aforementioned processes to grow and metastasize. Although targeting tumor associated HSPG is not a novel concept, the use of HPV to target and treat tumors has only been realized in recent years. The work herein describes how decades of basic HPV research has culminated in the rational design of an HPV-based virus-like infrared light activated dye conjugate for the treatment of choroidal melanoma.

Using NMR to Dissect the Chemical Space and O-Sulfation Effects within the O- and S-Glycoside Analogues of Heparan Sulfate.

Heparan sulfate (HS), a sulfated linear carbohydrate that decorates the cell surface and extracellular matrix, is ubiquitously distributed throughout the animal kingdom and represents a key regulator of biological processes and a largely untapped reservoir of potential therapeutic targets. The temporal and spatial variations in the HS structure underpin the concept of “heparanome” and a complex network of HS binding proteins. However, despite its widespread biological roles, the determination of direct structure-to-function correlations is impaired by HS chemical heterogeneity. Attempts to correlate substitution patterns (mostly at the level of sulfation) with a given biological activity have been made. Nonetheless, these do not generally consider higher-level conformational effects at the carbohydrate level. Here, the use of NMR chemical shift analysis, NOEs, and spin–spin coupling constants sheds new light on how different sulfation patterns affect the polysaccharide backbone geometry. Furthermore, the substitution of native O-glycosidic linkages to hydrolytically more stable S-glycosidic forms leads to observable conformational changes in model saccharides, suggesting that alternative chemical spaces can be accessed and explored using such mimetics. Employing a series of systematically modified heparin oligosaccharides (as a proxy for HS) and chemically synthesized O- and S-glycoside analogues, the chemical space occupied by such compounds is explored and described.

Re-understanding of structure and anticoagulation: Fucosylated chondroitin sulfate from sea cucumber Ludwigothurea grisea

Fucosylated chondroitin sulfate (FCS) from sea cucumber Ludwigothurea grisea (FCSLg) is the first one that reported to bear the di-fucosyl branches. Here we deciphered it by analyzing the physicochemical properties and its derivatives. Oligosaccharides prepared by selective cleavage of glycosidic linkages presented the mono-fucose and heterodisaccharide branches in FCSLg. The disaccharide branch was determined as d-GalNAcR1-(α1,2)-l-FucR2 rather than the di-fucosyl branch, where R1 was 4-mono-O- or 4,6-di-O-sulfation, and R2 was 3-mono-O- or 3,4-di-O-sulfation, respectively. The diversity of sulfation patterns in branches complicated the structure. These results give us a new understanding of FCSLg and provided a reliable method to decipher the FCS with complex branches. Bioanalysis of chemically modified derivatives showed that modulating the molecular mass could enhance the Xase target selectivity. Side chains conferred the Xase complex inhibition by binding to FIXa with a high affinity. Whether monosaccharide and disaccharide branches have differential effects needs to be further explored.

Mild acid hydrolysis on Fucan sulfate from Stichopus herrmanni: Structures, depolymerization mechanism and anticoagulant activity

Fucan sulfate (FS) from sea cucumbers possesses linear sequences with repeating units that differ principally in the pattern of sulfation and position of glycosidic linkage. FS from Stichopus herrmanni (ShFS) was preliminarily identified as the relatively simple structure {-3)-L-Fuc2S-(α1-}n. Herein, mild acid hydrolysis was employed on ShFS to yield 21 oligosaccharides. Analyses on their structures complemented the features of ShFS to refine its spectral signal assignments. Combining with the methylation analysis, unit L-Fuc2S4S was determined as the micro-component in its intact structure. The irregularity came from minor sulfation on O-4. Temperature and acid concentration were the critical parameters to the depolymerization and formation of oligosaccharides. Meanwhile, a three-step cleavage of the hydrolysis mechanism involving the partial O-2 de-sulfation and preferential cleavage between Fuc0S and Fuc2S4S was proposed. Bioactivity assays revealed that the Mw 15–16 kDa conferred the potent anticoagulation via inhibiting the thrombin activity mediated by heparin cofactor II.

Abstract 259: Study of a low anticoagulant bovine heparin as a drug capable of disrupting tumor cell-platelet interaction and preventing hematogenous metastasis

Abstract Metastasis is the leading cause of death in cancer patients. During metastatic dissemination, circulating tumor cells need to survive in the bloodstream and their interaction with platelets through P-selectin-ligand seems to be crucial. It has been demonstrated that heparin (from porcine mucosa, porcine UFH), a glycosaminoglycan composed by repetitive disaccharides of uronic acid and glucosamine, may interfere with P-selectin interaction. Recently our research group purified by ion exchange chromatography a fraction of bovine heparin displaying very low anticoagulant potential, named LABH (~20 IU/mg LABH x ~200 IU/mg porcine UFH x ~100 IU/mg bovine UFH). The advantage of bovine UFH and, especially of LABH, is their reduced risk of bleeding side effect. Our aim in this work was to investigate the potential use of LABH as an antimetastatic agent in vitro and in vivo, evaluating its ability to interfere with tumor cell-platelet interaction, tumor cell binding to P-selectin and preventing induced metastasis in mice. For this, we challenged 8-12 weeks C57Bl/6 mice with 4 or 8 mg/kg of porcine UFH, bovine UFH or LABH followed by intravenous injection of B16F10 cells (murine melanoma cell line). After 21 days, lungs were analyzed and metastatic foci were counted. The number of metastatic foci was significantly reduced when animals were pretreated with heparins (50-70 foci in control mice and 10-15 in treated mice). In other attempt, isolated platelets from healthy volunteers were incubated for 1h with MV3 cells (human melanoma cell line) in vitro in the presence or absence of heparin. All three heparins tested were efficient in blocking MV3-platelet interaction in a dose-response manner, but LABH required higher doses than porcine and bovine UFH. When analyzing the direct binding of U937 cell (human lymphoma cell line) to immobilized P-selectin, we could observe that porcine and bovine UFH inhibited at the same level (~ 60% of inhibition with 100 μg/mL), while LABH was less effective (~ 45% of inhibition with 100 μg/mL). Collectively our results demonstrate that despite presenting distinct patterns of sulfation and anticoagulant potential, all heparins significantly reduced lung metastasis, platelet-tumor cell adhesion and P-selectin binding. On the other hand, we hypothesize that LABH may act suppressing metastasis also by other mechanisms since its effect on in vitro experiments was less pronounced in comparison to porcine and bovine UFH. Citation Format: Juliana Maria Motta, Carlos Roberto-Fernandes, Kayene Vitória Micheli, Ana Maria Tovar, Paulo Antônio Mourão, Mariana Sá Pereira. Study of a low anticoagulant bovine heparin as a drug capable of disrupting tumor cell-platelet interaction and preventing hematogenous metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 259.

Fucose-Rich Sulfated Polysaccharides from Two Vietnamese Sea Cucumbers Bohadschia argus and Holothuria (Theelothuria) spinifera: Structures and Anticoagulant Activity.

Fucosylated chondroitin sulfates (FCSs) FCS-BA and FCS-HS, as well as fucan sulfates (FSs) FS-BA-AT and FS-HS-AT were isolated from the sea cucumbers Bohadschia argus and Holothuria (Theelothuria) spinifera, respectively. Purification of the polysaccharides was carried out by anion-exchange chromatography on DEAE-Sephacel column. Structural characterization of polysaccharides was performed in terms of monosaccharide and sulfate content, as well as using a series of non-destructive NMR spectroscopic methods. Both FCSs were shown to contain a chondroitin core [→3)-β-d-GalNAc-(1→4)-β-d-GlcA-(1→]n bearing sulfated fucosyl branches at O-3 of every GlcA residue in the chain. These fucosyl residues were different in pattern of sulfation: FCS-BA contained Fuc2S4S, Fuc3S4S and Fuc4S at a ratio of 1:8:2, while FCS-HS contained these residues at a ratio of 2:2:1. Polysaccharides differed also in content of GalNAc4S6S and GalNAc4S units, the ratios being 14:1 for FCS-BA and 4:1 for FCS-HS. Both FCSs demonstrated significant anticoagulant activity in clotting time assay and potentiated inhibition of thrombin, but not of factor Xa. FS-BA-AT was shown to be a regular linear polymer of 4-linked α-L-fucopyranose 3-sulfate, the structure being confirmed by NMR spectra of desulfated polysaccharide. In spite of considerable sulfate content, FS-BA-AT was practically devoid of anticoagulant activity. FS-HS-AT cannot be purified completely from contamination of some FCS. Its structure was tentatively represented as a mixture of chains identical with FS-BA-AT and other chains built up of randomly sulfated alternating 4- and 3-linked α-L-fucopyranose residues.

Alpine ecosystem response to climate warming: Long-term monitoring data of stream chemistries revisited

Alpine ecosystems are considered to be more sensitive to climate change than are downstream habitats because they exhibit less resilience to climate warming. Long-term monitoring of stream chemistries serves as a powerful tool to discern environmentally driven changes, in the context of cryospheric processes. According to the research hypothesis, the chemistry trends in the Green Lakes Valley, Colorado Front Range resulted from bedrock weathering in response to permafrost thawing coupled with exogenous sources. The median pH values during the 36 years of monitoring (1984–2020) have increased significantly in all five sampling sites from the headwater (5.7 at ~3800 m a.s.l) to the outlet (6.9 at ~3345 m a.s.l). The ANC increased significantly from a median of 10.6 μeq L−1 at the headwater to a median of 129 μeq L−1 at the outlet. These trends have occurred in spite of high sulfate concentrations in meltwater generated by oxidative weathering of pyrite (OWP). Analysis of the major cations and anions in the downstream sites revealed fairly consistent multi-segment trends. The first segment from 1984 to the mid 1990s exhibited a slight decrease in the electrolytes concentrations. Remarkably rapid upswings in electrolyte concentrations were observed in the mid 1990s to the mid 2000s. However, these trends significantly decreased from the mid 2000s onward. If climate warming is the mechanism that controls the rate of mineral weathering, then the electrolytes release rates from meltwater and permafrost thawing should increase over time, rather than flatten off or even decrease, as observed here. These spatiotemporal patterns of calcium and sulfate can be explained by a combination of mineral weathering and exogenous additions. Permafrost thawing is an important mechanism that adversely affects the OWP; consequently, it releases sulfuric acid and increases mineral weathering. However, the influence of eolian dust on alpine stream chemistry should not be overlooked.

Effect of Sulfation on the Conformational Dynamics of Dermatan Sulfate Glycosaminoglycan: A Gaussian Accelerated Molecular Dynamics Study.

Glycosaminoglycans (GAGs) are anionic biopolymers present on cell surfaces as a part of proteoglycans. The biological activities of GAGs depend on the sulfation pattern. In our study, we have considered three octadecasaccharide dermatan sulfate (DS) chains with increasing order of sulfation (dp6s, dp7s, and dp12s) to illuminate the role of sulfation on the GAG units and its chain conformation through 10 μs-long Gaussian accelerated molecular dynamics simulations. DS is composed of repeating disaccharide units of iduronic acid (IdoA) and N-acetylgalactosamine (N-GalNAc). Here, N-GalNAc is linked to IdoA via β(1-4), while IdoA is linked to N-GalNAc through α(1-3). With the increase in sulfation, the DS structure becomes more rigid and linear, as is evident from the distribution of root-mean-square deviations (RMSDs) and end-to-end distances. The tetrasaccharide linker region of the main chain shows a rigid conformation in terms of the glycosidic linkage. We have observed that upon sulfation (i.e., dp12s), the ring flip between two chair forms vanished for IdoA. The dynamic cross-correlation analysis reveals that the anticorrelation motions in dp12s are reduced significantly compared to dp6s or dp7s. An increase in sulfation generates relatively more stable hydrogen-bond networks, including water bridging with the neighboring monosaccharides. Despite the favorable linear structures of the GAG chains, our study also predicts few significant bendings related to the different puckering states, which may play a notable role in the function of the DS. The relation between the global conformation with the micro-level parameters such as puckering and water-mediated hydrogen bonds shapes the overall conformational space of GAGs. Overall, atomistic details of the DS chain provided in this study will help understand their functional and mechanical roles, besides developing new biomaterials.

Functions of chondroitin/dermatan sulfate containing GalNAc4,6-disulfate.

Chondroitin sulfate (CS) and dermatan sulfate (DS) containing GalNAc4,6-disulfate (GalNAc4S6S) were initially discovered in marine animals. Following the discovery, these glycosaminoglycans have been found in various animals including human. In the biosynthesis of CS/DS containing GalNAc4S6S, 3 groups of sulfotransferases are involved; chondroitin 4-sulfotransferases (C4STs), dermatan 4-sulfotransferase-1 (D4ST-1), and GalNAc 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST). GalNAc4S-6ST and its products have been shown to play important roles in the abnormal pathological conditions such as central nervous system injury, cancer development, abnormal tissue fibrosis, development of osteoporosis, and infection with viruses or nematodes. CS/DS containing GalNAc4S6S has been shown to increase with the functional differentiation of mast cells, macrophages, and neutrophils. Genetic approaches using knockout or knockdown of GalNAc4S-6ST, blocking of the epitopes containing GalNAc4S6S by specific antibodies and chemical technology that enabled the synthesis of oligosaccharides with defined sulfation patterns, have been applied successfully to these investigations. These studies contributed significantly to the basic understanding of the functional roles of CS/DS containing GalNAc4S6S in various abnormal conditions and appear to provide promising clues to the development of possible measures to treat them.

Maternal Zika Virus Infection Alters Offspring Hippocampal Glycan Sulfation Patterns in Nonhuman Primates

Zika virus (ZIKV) infection during pregnancy can impair offspring hippocampal neurocircuit formation and cognitive function, but underlying mechanisms driving this neuronal miswiring remain unknown. During fetal development, axonal migration, pathfinding, and synaptogenesis are strongly influenced by extracellular chondroitin and dermatan sulfate‐glycosaminoglycans (CS/DS‐GAGs), which consist of six differentially sulfated isomers (CS‐A, B (DS), C, D, E, O). The relative abundance of these CS/DS isomers can influence functions ranging from circuit plasticity (CS‐C) and maturation (CS‐A, B) to axonal growth attraction (CS‐D) and repulsion (CS‐E). Using LC‐MS/MS to analyze the relative abundance of each CS/DS isomer in cortical and hippocampal tissue obtained from normal 145d fetal Macaca nemestrina nonhuman primates (NHP), a key difference between cortex and hippocampus included the presence of a less ‘mature’ glycan matrix environment in the latter region. This finding is supported by a 1.6‐fold increase in the neuroplastic CS‐C (p=0.002) and 1.7‐fold increase in the neuronal attractant CS‐D (p=0.0001) isomers, along with a 39%‐decrease in axonal repulsive CS‐E (p<0.0001), a 22%‐decrease in synapse stabilizing CS‐B/DS (p=0.06), and a 6%‐decrease in the matrix maturing CS‐A (p=0.02) isomers. The hippocampus also exhibited a 51%‐decrease in the nonsulfated CS‐O (p=0.0001) isomer compared to the adjacent cortex, with the resultant hypersulfation of hippocampal CS/DS‐GAGs (p=0.0001) predicted to increase binding of positively charged extracellular growth factors. These differences in CS/DS‐GAGs between brain regions suggest that whereas the cortex provides a matured matrix environment capable of supporting newly formed circuitry, hippocampal CS/DS‐GAGs retain a developmental sulfation patterning that allows for additional neuronal migration, axon growth, and synapse formation. With this background, we then tested whether 3rd trimester congenital ZIKV infection (fetal d120‐d124) in M. nemestrina influences offspring CS/DS‐GAG sulfation patterns 21d later. We found that whereas no change was observed in the cortex, offspring exposed to congenital ZIKV exhibited a 33%‐decrease in CS‐D (p=0.01) and a 1.5x‐increase in CS‐O (p=0.03) in the hippocampus. This ZIKV‐driven reduction of the axonal attractant CS‐D can be predicted to limit hippocampal neurocircuit formation, potentially exacerbated by reduced retention of growth factors resulting from the increase in nonsulfated CS‐O, which is consistent with previous evidence of reduced hippocampal neurocircuit formation after maternal ZIKV infection. Overall, these results imply that maternal ZIKV infection during late‐stage fetal neurodevelopment 1) targets the immature hippocampal CS/DS‐GAGs over the matured cortex, 2) identifies recoding of CS/DS‐GAGs as a novel target for ZIKV infection, and 3) offers a novel and plausible mechanism to explain the deleterious impact of maternal ZIKV on offspring development

6‐O‐sulfation of endothelial glycocalyx heparan sulfates regulates systemic coagulation

ObjectiveThe endothelial glycocalyx is a glycosaminoglycan‐rich layer that lines the surface of all vessels and is critical for vascular homeostasis, including the maintenance of an anticoagulant vascular surface. Heparan sulfate (HS), a linear polysaccharide composed of repeating disaccharide units of glucosamine and iduronic acid, is the major glycosaminoglycan comprising the endothelial glycocalyx. The mechanism by which glycocalyx HS regulates coagulation is unknown. As the biological function of HS derives from its unique pattern of sulfation (2‐O‐, 6‐O‐, and/or N‐sulfation) within constituent disaccharides, we hypothesized that the degree of heparan sulfate sulfation critically regulates systemic hemostasis.MethodsWe performed thromboelastography on whole blood collected from tamoxifen‐treated VE‐Cadherin‐Cre‐ERT2‐Sulf1/2 knockout mice, which have loss of endothelial sulf‐1 (a 6‐O specific HS sulfatase) and thus increased heparan sulfate 6‐O‐sulfation at the endothelial surface. Tamoxifen‐treated floxed mice (no Cre recombinase) served as controls. To determine the effects of HS in clot initiation and propagation, separate from platelet contributions, we treated platelet‐poor plasma from healthy donors (pooled and deidentified) with highly‐sulfated HS (HS09) and lower‐sulfated HS (HS01) and measured endogenous thrombin potential with calibrated automated thrombography. To explore the potential mechanism by which HS functions as anticoagulant, we repeated calibrated automated thrombography with antithrombin‐III‐, heparin cofactor‐II depleted plasma.ResultsMice lacking endothelial sulf‐1 demonstrated increased R‐time, decreased angle, and increased K‐time compared to tamoxifen‐treated controls, suggesting that increased endothelial‐surface 6‐O HS sulfation decreases coagulation. In platelet poor plasma, highly‐sulfated HS09 attenuated thrombin generation in dose‐dependent manner while such effect was not observed with the same dose of less‐sulfated HS01. Repeated analysis using platelet poor plasma depleted of antithrombin‐III and of heparin cofactor‐II showed dose‐dependent decrease of thrombin generation with HS09, suggesting that HS exerts this anticoagulant effect independently of heparin‐like activation of antithrombin‐III.ConclusionThe anticoagulant effect of endothelial‐surface HS is dependent on 6‐O sulfation of its constituent disaccharides, and this effect is mediated by the mechanisms distinct from well‐known heparin anticoagulation mechanisms.

Compositional analysis of chondroitin/dermatan sulfate in rhesus monkeys (Macaca mulatta).

Glycosaminoglycans (GAGs) are found in various tissues and are involved in many physiological functions. Since the rhesus monkey (Macaca mulatta) is the most widely used nonhuman primate in biomedical research, an understanding of the compositions of GAGs in their tissues is important. The aim of this study was to determine the content and sulfation pattern of disaccharides contained in several tissues of the rhesus monkey. The chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chain was extracted from several tissues of female and male rhesus monkeys. Compositional analysis was performed after digestion with chondroitinases ABC and ACI to reveal the sulfation pattern of the CS/DS hybrid chain. This study revealed that the major CS/DS disaccharide units present in the tissues were A and C types. The E and iE types were specifically distributed not only in the tracheal tissue but also in gastrointestinal tissues.

Glycosaminoglycans in cancer therapy.

Glycosaminoglycans (GAGs) are an important component of the tumor microenvironment (TME). GAGs can interact with a variety of binding partners and thereby influence cancer progression on multiple levels. GAGs can modulate growth factors and chemokine signaling, invasion, and metastasis formation. Moreover, GAGs are able to change the physical property of the extracellular matrix (ECM). Abnormalities in GAG abundance and structure (e.g., sulfation patterns and molecular weight) are found across various cancer types and show biomarker potential. Targeting GAGs, as well as the usage of GAGs and their mimetics, are promising approaches to interfere with cancer progression. In addition, GAGs can be used as drug and cytokine carriers to induce an antitumor response. In this review, we summarize the role of GAGs in cancer and the potential use of GAGs and GAG derivatives to target cancer.

Context Dependent Sulf1/Sulf2 Functional Divergence in Endothelial Cell Activity.

Signalling activities are tightly regulated to control cellular responses. Heparan sulfate proteoglycans (HSPGs) at the cell membrane and extracellular matrix regulate ligand availability and interaction with a range of key receptors. SULF1 and SULF2 enzymes modify HSPG sulfation by removing 6-O sulfates to regulate cell signalling but are considered functionally identical. Our in vitro mRNA and protein analyses of two diverse human endothelial cell lines, however, highlight their markedly distinct regulatory roles of maintaining specific HSPG sulfation patterns through feedback regulation of HS 6-O transferase (HS6ST) activities and highly divergent roles in vascular endothelial growth factor (VEGF) and Transforming growth factor β (TGFβ) cell signalling activities. Unlike Sulf2, Sulf1 over-expression in dermal microvascular HMec1 cells promotes TGFβ and VEGF cell signalling by simultaneously upregulating HS6ST1 activity. In contrast, Sulf1 over-expression in venous ea926 cells has the opposite effect as it attenuates both TGFβ and VEGF signalling while Sulf2 over-expression maintains the control phenotype. Exposure of these cells to VEGF-A, TGFβ1, and their inhibitors further highlights their endothelial cell type-specific responses and integral growth factor interactions to regulate cell signalling and selective feedback regulation of HSPG sulfation that additionally exploits alternative Sulf2 RNA-splicing to regulate net VEGF-A and TGFβ cell signalling activities.