A competitive alphascreen assay for detection of hyaluronan.

A method for specific quantification of hyaluronan (HA) concentration using AlphaScreen® (Amplified Luminescent Proximity Homogeneous Assay) technology is described. Two types of hydrogel-coated and chromophore-loaded latex nanobeads are employed. The proximity of the beads in solution is detected by excitation of the donor bead leading to the production of singlet oxygen, and chemiluminescence from the acceptor bead upon exposure to singlet oxygen. In the HA assay, the donor bead is modified with streptavidin, and binds biotin-labeled HA. The acceptor bead is modified with Ni(II), and is used to bind a specific recombinant HA-binding protein (such as HABP; aggrecan G1-IGD-G2) with a His-tag. Competitive inhibition of the HA-HABP interaction by free unlabeled HA in solution is used for quantification. The assay is specific for HA, and not dependent on HA molecular mass above the decasaccharide. HA can be quantified over a concentration range of approximately 30-1600 ng/mL using 2.5 µL of sample, for a detectable mass range of approximately 0.08-4 ng HA. This sensitivity of the AlphaScreen assay is greater than existing ELISA-like methods, due to the small volume requirements. HA can be detected in biological fluids using the AlphaScreen assay, after removal of bound proteins from HA and dilution or removal of other interfering proteins and lipids.

Suppression of Ischemia-Induced Hippocampal Pyramidal Neuron Death by Hyaluronan Tetrasaccharide through Inhibition of Toll-Like Receptor 2 Signaling Pathway.

Toll-like receptors (TLRs) are one of the main contributors that induce inflammation under tissue injury and infection. Because excessive inflammation can aggravate disease states, it is important to control inflammation at a moderate level. Herein, we show that hyaluronan (HA) oligomer, HA tetrasaccharide (HA4), could suppress the expression of proinflammatory cytokine IL-1ß when stimulated with both TLR2- and TLR4-specific agonists in primary hippocampal neurons. To understand the effect of HA4 against ischemic insult, we performed hypoxic-ischemic (H/I) brain injury against neonatal mice. HA4 treatment significantly prevented hippocampal pyramidal cell death even 7 days after H/I injury, compared with the control mice. Although TLR2 and TLR4 are known as receptors for HA and also act as a receptor for inducing inflammation, only TLR2-deficient mice showed tolerance against H/I injury. Moreover, HA4 administration suppressed gliosis by inhibiting the activation of NF-κB, the downstream target of TLR2, which led to the suppression of IL-1ß expression. Taken together, our data suggest that the neuroprotective effect of HA4 relies on antagonizing the TLR2/NF-κB pathway to reduce inflammation through suppressing the expression of proinflammatory cytokines after neonatal H/I brain injury.

No influence of exogenous hyaluronan on the behavior of human cancer cells or endothelial cell capillary formation.

UNLABELLED: Hyaluronan (HA), a type of glycosaminoglycan used to construct the extracellular matrix, is involved in the proliferation and motility of cells, including cancer cells. The aim of this study was to determine whether exogenous HA has an influence on cancer in vitro and in vivo. High-molecular-weight HA (900 kDa) and low-molecular-weight HA (10 kDa) were added to several types of cancer cell lines in vitro, and proliferation and invasion were assessed. The effect of HA on capillary formation by human umbilical vein endothelial cells was also analyzed. The results showed that both types of HA had no apparent effect on cellular proliferation, invasion, or capillary formation. In an animal study, the 2 types of HA were orally administered to tumor-bearing mice at a dosage of 200 mg/kg/d for 4 wk. Analysis using an in vivo imaging system revealed that tumor proliferation and metastasis were not greatly altered by HA administration. Furthermore, CD31 immunohistochemical staining revealed no obvious change in tumor microvessels. Taken together, these results demonstrate that exogenously administered HA has little effect on cancer. This study may support the safety of various forms of HA administration, including oral intake. PRACTICAL APPLICATION: Orally administered hyaluronan was recently found to have beneficial effects. However, the effect of exogenous hyaluronan on cancer remains unclear. Our findings support the safety of orally administered hyaluronan and its use as a functional food ingredient.

Hyaluronan oligosaccharides perturb lymphocyte slow rolling on brain vascular endothelial cells: implications for inflammatory demyelinating disease.

Inflammatory demyelinating diseases like multiple sclerosis are characterized by mononuclear cell infiltration into the central nervous system. The glycosaminoglycan hyaluronan and its receptor, CD44, are implicated in the initiation and progression of a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Digestion of hyaluronan tethered to brain vascular endothelial cells by a hyaluronidase blocks the slow rolling of lymphocytes along activated brain vascular endothelial cells and delays the onset of EAE. These effects could be due to the elimination of hyaluronan or the generation of hyaluronan digestion products that influence lymphocytes or endothelial cells. Here, we found that hyaluronan dodecasaccharides impaired activated lymphocyte slow rolling on brain vascular endothelial cells when applied to lymphocytes but not to the endothelial cells. The effects of hyaluronan dodecasaccharides on lymphocyte rolling were independent of CD44 and a receptor for degraded hyaluronan, Toll-like receptor-4. Subcutaneous injection of hyaluronan dodecasaccharides or tetrasaccharides delayed the onset of EAE in a manner similar to subcutaneous injection of hyaluronidase. Hyaluronan oligosaccharides can therefore act directly on lymphocytes to modulate the onset of inflammatory demyelinating disease.

Oral administration of high molecular weight hyaluronan (900 kDa) controls immune system via Toll-like receptor 4 in the intestinal epithelium.

Low molecular weight hyaluronan enhances or induces inflammation through toll-like receptor 4 (TLR-4). However, the effects of high molecular weight hyaluronan (HA900) on TLR-4 are unknown. In this study, HA900 (900 kDa) was administered orally to MRL-lpr/lpr mice, a Th-1-type autoimmune disease model. Lymphoaccumulation of double-negative T cells, which is enhanced by proinflammatory cytokines, was suppressed by HA900 treatment. Cytokine array analysis showed that HA900 treatment enhanced production of interleukin-10, an anti-inflammatory cytokine, and down-regulated chemokine production. HA900 colocalized with TLR-4 on the luminal surface of epithelial cells in the large intestine. These cells are parts of the immune system and express cytokines. DNA array analysis of the tissue from the large intestine showed that HA900 treatment up-regulated suppressor of cytokine signaling 3 (SOCS3) expression and down-regulated pleiotrophin expression. Treatment of cultured double-negative T cells from MRL-lpr/lpr mice with pleiotrophin rescued these cells. SOCS3, which is known to suppress inflammation, was enhanced by HA900 treatment. In TLR-4 knockdown HT29 cells (a cell line derived from large intestinal cells), HA900 did not bind to HT29 cells and did not up-regulate SOCS3 expression. Our results suggest that oral administration of HA900 modulates Th-1-type autoimmune disease and inflammation by up-regulating SOCS3 expression and down-regulating pleiotrophin expression via TLR-4 in intestinal epithelial cells.

Hyaluronan fragments: an information-rich system.

Hyaluronan is a straight chain, glycosaminoglycan polymer of the extracellular matrix composed of repeating units of the disaccharide [-D-glucuronic acid-beta1,3-N-acetyl-D-glucosamine-beta1,4-]n. Hyaluronan is synthesized in mammals by at least three synthases with products of varying chain lengths. It has an extraordinary high rate of turnover with polymers being funneled through three catabolic pathways. At the cellular level, it is degraded progressively by a series of enzymatic reactions that generate polymers of decreasing sizes. Despite their exceedingly simple primary structure, hyaluronan fragments have extraordinarily wide-ranging and often opposing biological functions. There are large hyaluronan polymers that are space-filling, anti-angiogenic, immunosuppressive, and that impede differentiation, possibly by suppressing cell-cell interactions, or ligand access to cell surface receptors. Hyaluronan chains, which can reach 2 x 10(4) kDa in size, are involved in ovulation, embryogenesis, protection of epithelial layer integrity, wound repair, and regeneration. Smaller polysaccharide fragments are inflammatory, immuno-stimulatory and angiogenic. They can also compete with larger hyaluronan polymers for receptors. Low-molecular-size polymers appear to function as endogenous "danger signals", while even smaller fragments can ameliorate these effects. Tetrasaccharides, for example, are anti-apoptotic and inducers of heat shock proteins. Various fragments trigger different signal transduction pathways. Particular hyaluronan polysaccharides are also generated by malignant cells in order to co-opt normal cellular functions. How the small hyaluronan fragments are generated is unknown, nor is it established whether the enzymes of hyaluronan synthesis and degradation are involved in maintaining proper polymer sizes and concentration. The vast range of activities of hyaluronan polymers is reviewed here, in order to determine if patterns can be detected that would provide insight into their production and regulation.

Application of fluorophore-assisted carbohydrate electrophoresis to analysis of disaccharides and oligosaccharides derived from glycosaminoglycans.

Various combinations of fluorescent dyes, polyacrylamide gels, and electrophoresis buffers were tested by fluorophore-assisted carbohydrate electrophoresis (FACE) for the purpose of analyzing sulfated and nonsulfated glycosaminoglycan (GAG) oligosaccharides in which disaccharides and low-molecular weight oligosaccharides were included. A nonionic fluorescent dye was found to be suitable for analyzing sulfated disaccharides derived from sulfated GAGs (e.g., chondroitin sulfate, dermatan sulfate) because sulfated disaccharides themselves had enough anionic potential for electrophoresis. The migration rates of chondroitin sulfate (CS) disaccharides in polyacrylamide gels were affected by the number of sulfate residues and the conformation of each disaccharide. When an anionic fluorescent dye, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS), was coupled with sulfated GAG oligosaccharides, nearly all of the conjugates migrated at the electrophoretic front due to the added anionic potential. Nonsulfated hyaluronan (HA) oligosaccharides (2-16 saccharides) were subjected to electrophoresis by coupling with a nonionic fluorescent dye, 2-aminoacridone (AMAC), but did not migrate in the order of their molecular size. Especially di-, tetra-, hexa-, and octasaccharides of HA migrated in the reverse order of their molecular size. HA/CS oligosaccharides were able to migrate in the order of their chain lengths by coupling with an anionic fluorescent dye in a nonborate condition.

The keratan sulfate disaccharide Gal(6S03) beta1,4-GlcNAc(6S03) modulates interleukin 12 production by macrophages in murine Thy-1 type autoimmune disease.

It has been reported that disaccharides of the glycosaminoglycans (GAGs), heparin, or heparan sulfate suppress the production of cytokines. Therefore, we examined the effects of GAGs (keratan sulfate, hyaluronan, chondroitin, chondroitin sulfate, and heparin sulfate) disaccharides on production of interleukin (IL)-12, a pivotal cytokine in the Th-1 type immune system. Among the GAG disaccharides, only a keratan sulfate disaccharide, Gal(6-SO(3))-GlcNAc(6-SO(3)) (L4), suppressed IL-12 production in macrophages stimulated with lipopolysaccharides and interferon-gamma. Neither keratan sulfate chains nor keratan sulfate tetrasaccharides elicited any change in the IL-12 production. N-Acetyl-lactosamine, Gal-GlcNAc (LacNAc), also did not change IL-12 production. These results indicated that a certain size, i.e. disaccharide and sulfate, are essential to suppress IL-12 production. L4 was then applied to MRL-lpr/lpr mice, a Th-1 type autoimmune disease model. The treatment of MRL-lpr/lpr mice with L4 1) decreased in serum IL-12, 2) induced apoptosis in T cells in lymph nodes thereby suppressing lymphoaccumulation, and 3) suppressed hypergammaglobulinemia and glomerulonephritis. We showed previously that IL-12 suppresses cell death of T cells, thereby enhancing the lymphoaccumulation in MRL-lpr/lpr mice. Moreover, it has been reported that IL-12 deficiency in MRL-lpr/lpr mice diminishes lymphoaccumulation and delays glomerulonephritis. The treatment with L4 suppressed phosphoprotein kinase C and phosphoinositide 3-kinase expression in macrophages, suggesting that L4 suppresses IL-12 production by inhibiting phosphoprotein kinase C and phosphoinositide 3-kinase pathways.

Direct hepatic fate specification from mouse embryonic stem cells.

The molecules responsible for hepatic differentiation from embryonic stem (ES) cells have yet to be elucidated. Here we have identified growth factors that allow direct hepatic fate-specification from ES cells by using simple adherent monolayer culture conditions. ES cell-derived hepatocytes showed liver-specific characteristics, including several metabolic activities, suggesting that ES cells can differentiate into functional hepatocytes without the requirement for embryoid body (EB) formation, in vivo transplantation, or a coculture system. Most importantly, transplantation of ES cell-derived hepatocytes in mice with cirrhosis showed significant therapeutic effects. In conclusion, this novel system for hepatic fate specification will help elucidate the precise molecular mechanisms of hepatic differentiation in vitro and could represent an attractive approach for developing stem cell therapies for treatment of hepatic disease in humans. Supplementary material for this article can be found on the HEPATOLOGY website ( http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Long-term maintenance of liver-specific functions in cultured ES cell-derived hepatocytes with hyaluronan sponge.

This study investigated the three-dimensional culture of hepatocytes differentiated from mouse embryonic stem (ES) cells with a porous hyaluronan (HA) sponge support. Hepatocytes were immobilized within the pores of the support. Spheroids could be observed within the support, each containing between 20 and 50 hepatocytes. To examine the liver-specific functions of the hepatocytes in the culture, the levels of albumin secreted into the medium were analyzed. The secretion of albumin was stable over the course of 32 days, longer than that in both conventional monolayer and collagen sponge cultures. To elucidate further the liver-specific functions of hepatocytes embedded in the HA sponge, metabolic activities of the hepatocytes were examined for their ability to eliminate ammonia from culture media and the synthesis of urea nitrogen. While rates of ammonia removal and urea nitrogen synthesis were similar to those in both conventional monolayer and in collagen sponge cultures, these functions were maintained for longer duration in cells embedded in the HA sponge. These results demonstrate that the porous HA sponge is an effective support for the in vitro culture of ES-derived hepatocytes used for both basic and applied studies for cell transplantation.

Isolation and partial characterization of fucan sulfates from the body wall of sea cucumber Stichopus japonicus and their ability to inhibit osteoclastogenesis.

Two types of fucan sulfate were isolated from chloroform/methanol extract of the body wall of the sea cucumber Stichopus japonicus. One type (type A) contained 3.41 mmol fucose/g and 2.35 mmol sulfate/g, and the molecular mass was determined to be 9 kDa by gel permeation chromatography (GPC). Structural analysis suggested that type A consists of a backbone of (1-->3)-linked fucosyl residues that are substituted at C-4 with fucosyl residues, and that fucosyl residues are sulfated at C-2 and/or C-4. Another type (type B) contained 3.90 mmol fucose/g and 3.07 mmol sulfate/g, and the molecular mass was determined to be 32kDa by GPC. Structural analysis showed that type B is largely composed of unbranched (1-->3)-linked fucosyl residues, and that sulfate substitution(s) occur at C-2 and/or C-4. The potential of both types to inhibit osteoclastogenesis was examined by an in vitro assay system, showing that both types of fucan sulfate inhibit osteoclastogenesis more than 95% at 50 microg/mL concentration. These results suggest that types A and B fucan sulfate from sea cucumber are potent inhibitors of osteoclastogenesis.

Specificity of the tumor necrosis factor-induced protein 6-mediated heavy chain transfer from inter-alpha-trypsin inhibitor to hyaluronan: implications for the assembly of the cumulus extracellular matrix.

The formation of the hyaluronan-rich cumulus extracellular matrix is crucial for female fertility and accompanied by a transesterification reaction in which the heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI)-related proteins are covalently transferred to hyaluronan. Tumor necrosis factor-induced protein-6 (TNFIP6) is essential for this transfer reaction. Female mice deficient in TNFIP6 are infertile due to the lack of a correctly formed cumulus matrix. In this report, we characterize the specificity of TNFIP6-mediated HC transfer from IalphaI to hyaluronan. Hyaluronan oligosaccharides with eight or more monosaccharide units are potent acceptors in the HC transfer, with longer oligosaccharides being somewhat more efficient. Epimerization of the N-acetyl-glucosamine residues to N-acetyl-galactosamines (i.e. in chondroitin) still allows the HC transfer although at a significantly lower efficiency. Sulfation of the N-acetyl-galactosamines in dermatan-4-sulfate or chondroitin-6-sulfate prevents the HC transfer. Hyaluronan oligosaccharides disperse cumulus cells from expanding cumulus cell-oocyte complexes with the same size specificity as their HC acceptor specificity. This process is accompanied by the loss of hyaluronan-linked HCs from the cumulus matrix and the appearance of oligosaccharide-linked HCs in the culture medium. Chondroitin interferes with the expansion of cumulus cell-oocyte complexes only when added with exogenous TNFIP6 before endogenous hyaluronan synthesis starts, supporting that chondroitin is a weaker HC acceptor than hyaluronan. Our data indicate that TNFIP6-mediated HC transfer to hyaluronan is a prerequisite for the correct cumulus matrix assembly and hyaluronan oligosaccharides and chondroitin interfere with this assembly by capturing the HCs of the IalphaI-related proteins.

Differentiation of embryonic stem cells into hepatocytes: biological functions and therapeutic application.

Embryonic stem (ES) cells provide a unique source for tissue regeneration. We examined whether mouse ES cells can efficiently differentiate into transplantable hepatocytes. ES cells were implanted into mouse livers 24 hours after carbon tetrachloride intoxication; ES-derived cells with several hepatocyte-cell-markers were generated. They were able to grow in vitro and showed morphology consistent with typical mature hepatocytes and expressed hepatocyte-specific genes. After transplantation into the carbon tetrachloride-injured mouse liver, ES-derived green fluorescent protein-positive cells were incorporated into liver tissue and rescued mice from hepatic injury. No teratoma formation was observed in the transplant recipients. In conclusion, ES cells can provide a valuable tool for studying the molecular basis for differentiation of hepatocytes and form the basis for cell therapies.

Large-scale preparation, purification, and characterization of hyaluronan oligosaccharides from 4-mers to 52-mers.

Hyaluronan (HA) was depolymerized by partial digestion with testicular hyaluronidase and separated into size-uniform HA oligosaccharides from 4-mers to 52-mers by anion exchange chromatography after removal of the hyaluronidase. The purity and size of each HA oligosaccharide was confirmed by using HPLC analyses, FACE, and ESI-MS. (1)H and (13)C NMR assignments and elemental analyses were obtained for each HA oligosaccharide. Endotoxins, proteins, and DNA were absent or in trace amounts in these HA oligosaccharides. Gram/mg-scale hyaluronan oligosaccharides were obtained from 200 g of HA starting material. These pure, size-uniform, and large range of HA oligosaccharides will be available for investigating important biological functions of HA, such as for the determination of the size(s) of HA oligosaccharides that induce angiogenesis or mediate inflammatory responses, and to interact with HA-binding proteins and receptors both in in vitro and in vivo studies.

Effect of hyaluronan oligosaccharides on the expression of heat shock protein 72.

We have previously shown that intraarticular treatment with a hyaluronan (HA) preparation (840 kDa), HA84, up-regulates heat shock protein 72 (Hsp72) expression and suppresses degeneration of synovial cells in an arthritis model. In that study, the HA84 administered was degraded into HA oligosaccharides in the synovial tissue, suggesting that HA84 or degradation products of HA may up-regulate Hsp72 expression. Thus, in the present study, we examined the effects of HA of various molecular sizes on Hsp72 expression and cell death in stressed cells. Western blotting analysis showed that treatment of K562 cells with HA tetrasaccharides up-regulated Hsp72 expression after exposure to hyperthermia. On the other hand, treatment of the cells with HA of other sizes (di-, hexa-, deca-, dodecasaccharides), HA84, or tetrasaccharides of keratan sulfate did not elicit any change in expression of the Hsp72 protein. Treatment of the cells with tetrasaccharides of HA up-regulated not only expression of the Hsp72 protein but also Hsp72 mRNA expression and enhanced activation of HSF1, a transcription factor controlling Hsp72 expression, after exposure to hyperthermia. Because the level of Hsp72 protein was not affected by tetrasaccharides of HA when the K562 cells were kept at 37 degrees C without any stress, it is evident that tetrasaccharides of HA did not act as a stress factor. In addition, tetrasaccharides of HA suppressed cell death in the case of K562 cells exposed to hyperthermia and of PC12 cells under serum deprivation. These results suggest that a certain size of oligosaccharides, i.e. the tetrasaccharides of HA, up-regulates Hsp72 expression by enhancing the activation of HSF1 under stress conditions and suppresses cell death.