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Sample records for acid ha hyaluronan

  1. Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts.

    PubMed

    Nagaoka, Aya; Yoshida, Hiroyuki; Nakamura, Sachiko; Morikawa, Tomohiko; Kawabata, Keigo; Kobayashi, Masaki; Sakai, Shingo; Takahashi, Yoshito; Okada, Yasunori; Inoue, Shintaro

    2015-12-25

    Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We recently reported that HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-β1, basic FGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly produced HA is dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-β1 were abrogated by blockade of the MAPK and/or Smad signaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 up-regulated HAS1/2 expression but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower molecular weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-β1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-β1 most efficiently contributes to production of high molecular weight HA by HAS up-regulation and HYBID down-regulation and suggests that inefficient down-regulation of HYBID by TGF-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients. PMID:26518873

  2. Hyaluronan (HA) Interacting Proteins RHAMM and Hyaluronidase Impact Prostate Cancer Cell Behavior and Invadopodia Formation in 3D HA-Based Hydrogels

    PubMed Central

    Gurski, Lisa A.; Nguyen, Ngoc T.; Xiao, Longxi; van Golen, Kenneth L.; Jia, Xinqiao; Farach-Carson, Mary C.

    2012-01-01

    To study the individual functions of hyaluronan interacting proteins in prostate cancer (PCa) motility through connective tissues, we developed a novel three-dimensional (3D) hyaluronic acid (HA) hydrogel assay that provides a flexible, quantifiable, and physiologically relevant alternative to current methods. Invasion in this system reflects the prevalence of HA in connective tissues and its role in the promotion of cancer cell motility and tissue invasion, making the system ideal to study invasion through bone marrow or other HA-rich connective tissues. The bio-compatible cross-linking process we used allows for direct encapsulation of cancer cells within the gel where they adopt a distinct, cluster-like morphology. Metastatic PCa cells in these hydrogels develop fingerlike structures, “invadopodia”, consistent with their invasive properties. The number of invadopodia, as well as cluster size, shape, and convergence, can provide a quantifiable measure of invasive potential. Among candidate hyaluronan interacting proteins that could be responsible for the behavior we observed, we found that culture in the HA hydrogel triggers invasive PCa cells to differentially express and localize receptor for hyaluronan mediated motility (RHAMM)/CD168 which, in the absence of CD44, appears to contribute to PCa motility and invasion by interacting with the HA hydrogel components. PCa cell invasion through the HA hydrogel also was found to depend on the activity of hyaluronidases. Studies shown here reveal that while hyaluronidase activity is necessary for invadopodia and inter-connecting cluster formation, activity alone is not sufficient for acquisition of invasiveness to occur. We therefore suggest that development of invasive behavior in 3D HA-based systems requires development of additional cellular features, such as activation of motility associated pathways that regulate formation of invadopodia. Thus, we report development of a 3D system amenable to dissection of

  3. Synthesis of novel amphiphilic hyaluronan containing-aromatic fatty acids for fabrication of polymeric micelles.

    PubMed

    Matelová, Alena; Huerta-Angeles, Gloria; Šmejkalová, Daniela; Brůnová, Zdislava; Dušek, Jan; Vícha, Robert; Velebný, Vladimír

    2016-10-20

    Novel hydrophobized hyaluronan (HA) derivatives, containing ω-phenylalkanoic acids (ω-PAA, 4-phenylbutyric acid, 6-phenylhexanoic, 8-phenyloctanoic or 11-tolylundecanoic acids) were prepared by esterification. Mixed anhydrides obtained after reaction of the carboxyl acid moiety and benzoyl chloride were found to be active acylating agents, affording hydrophobized HA in good yield and under mild conditions. The reactivity of the aromatic fatty acids towards esterification has decreased with the increasing length of the aliphatic spacer between the aromatic substituent and carboxylic acid moiety. The novel HA derivatives self-assembled from very low concentrations and were found to be non-cytotoxic. The potential use of ω-phenylalkanoic acids grafted-HA towards drug delivery applications was demonstrated by hydrophobic drugs (resveratrol and retinyl palmitate) encapsulation. The drug loading capacity of the novel HA derivatives was significantly improved most likely because of π⋯π interactions between the micelle core and loaded hydrophobic aromatic compound.

  4. Synthesis of novel amphiphilic hyaluronan containing-aromatic fatty acids for fabrication of polymeric micelles.

    PubMed

    Matelová, Alena; Huerta-Angeles, Gloria; Šmejkalová, Daniela; Brůnová, Zdislava; Dušek, Jan; Vícha, Robert; Velebný, Vladimír

    2016-10-20

    Novel hydrophobized hyaluronan (HA) derivatives, containing ω-phenylalkanoic acids (ω-PAA, 4-phenylbutyric acid, 6-phenylhexanoic, 8-phenyloctanoic or 11-tolylundecanoic acids) were prepared by esterification. Mixed anhydrides obtained after reaction of the carboxyl acid moiety and benzoyl chloride were found to be active acylating agents, affording hydrophobized HA in good yield and under mild conditions. The reactivity of the aromatic fatty acids towards esterification has decreased with the increasing length of the aliphatic spacer between the aromatic substituent and carboxylic acid moiety. The novel HA derivatives self-assembled from very low concentrations and were found to be non-cytotoxic. The potential use of ω-phenylalkanoic acids grafted-HA towards drug delivery applications was demonstrated by hydrophobic drugs (resveratrol and retinyl palmitate) encapsulation. The drug loading capacity of the novel HA derivatives was significantly improved most likely because of π⋯π interactions between the micelle core and loaded hydrophobic aromatic compound. PMID:27474668

  5. 4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3

    SciTech Connect

    Kultti, Anne; Pasonen-Seppaenen, Sanna; Jauhiainen, Marjo; Rilla, Kirsi J.; Kaernae, Riikka; Pyoeriae, Emma; Tammi, Raija H.; Tammi, Markku I.

    2009-07-01

    Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.

  6. New amphiphilic lactic acid oligomer-hyaluronan conjugates: synthesis and physicochemical characterization.

    PubMed

    Pravata, Laurent; Braud, Christian; Boustta, Mahfoud; El Ghzaoui, Abdelsalam; Tømmeraas, Kristoffer; Guillaumie, Fanny; Schwach-Abdellaoui, Khadija; Vert, Michel

    2008-01-01

    The "grafting onto" strategy was used to conjugate DL-lactic acid oligomers (OLA) to hyaluronan (HA) for the sake of developing novel degradable HA-based self-assembling polymeric systems. Grafting was achieved by reacting COCl-terminated OLA with cetyltrimethylammonium hyaluronate (CTA-HA) in dimethyl sulfoxide (DMSO). The resulting CTA-HAOLA conjugates were purified and turned to sodium form (Na-HAOLA) by dissolution in a phosphate buffer-DMSO mixture and successive dialyses against DMSO, ethanol, and water. In contrast, when the same protocol was applied to CTA-HAOLA, phase separation with gel formation was observed. The solution phase was composed of Na-HAOLA whereas the gel phase was made of mixed CTA-Na-HAOLA salt with ca. 25% of the carboxyl groups neutralized by CTA. Gelation was assigned to intramolecular hydrophobic associations between OLA and cetyl alkyl chains that complemented electrostatic interactions between CTA and HA COO- groups synergistically. Therefore, the corresponding stabilized CTA ions required more drastic conditions to be released. Under the selected dialysis conditions, the CTA-Na-HAOLA gels formed tiny tubes. Na-HAOLA and CTA-Na-HAOLA were characterized by FTIR, one-dimensional 1H and two-dimensional 1H NMR. The extent of grafting was ca. 5% per disaccharidic repeating unit, regardless of the molecular weight, as determined by NMR and capillary zone electrophoresis. Amphiphilic Na-HAOLA molecules were aggregated and formed spherical species in water according to size exclusion chromatography combined with multiangle laser light scattering detection. The critical aggregation concentration ranged between 0.2 and 0.35% (w/v), depending of the molecular weight of the parent hyaluronan. PMID:18047288

  7. Linolenic acid grafted hyaluronan: Process development, structural characterization, biological assessing, and stability studies.

    PubMed

    Huerta-Angeles, Gloria; Brandejsová, Martina; Kulhánek, Jaromír; Pavlík, Vojtěch; Šmejkalová, Daniela; Vágnerová, Hana; Velebný, Vladimír

    2016-11-01

    In this study, hyaluronan (HA) was grafted with alpha-linolenic acid (αLNA) by benzoyl mixed anhydrides methodology, which allowed the derivatization of HA under mild reaction conditions. The reaction was optimized and transferred from laboratory to semi-scale production. The derivative revealed an unexpected cytotoxicity after oven drying and storage at 40°C. For this reason, the storage conditions of sodium linolenyl hyaluronate (αLNA-HA) were optimized in order to preserve the beneficial effect of the derivative. Oven, spray dried and lyophilized samples were prepared and stored at -20°C, 4°C and 25°C up to 6 months. A comprehensive material characterization including stability study of the derivative, as well as evaluation of possible changes on chemical structure and presence of peroxidation products were studied by Nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GC-MS), thermogravimetric analysis (TGA) and complemented with assessment of in vitro viability on mouse fibroblasts NIH-3T3. The most stable αLNA-HA derivative was obtained after spray drying and storage at ambient temperature under inert atmosphere. The choice of inert atmosphere is recommended to suppress oxidation of αLNA supporting the positive influence of the derivative on cell viability. The encapsulation of hydrophobic drugs of αLNA-HA were also demonstrated. PMID:27516333

  8. Rheological study of in-situ crosslinkable hydrogels based on hyaluronanic acid, collagen and sericin.

    PubMed

    Vulpe, Raluca; Le Cerf, Didier; Dulong, Virginie; Popa, Marcel; Peptu, Catalina; Verestiuc, Liliana; Picton, Luc

    2016-12-01

    The elaboration of chemically crosslinked hydrogels based on collagen (C), hyaluronanic acid (HA) and sericin (S) with different polymer ratios was investigated by in-situ rheology. This reaction was performed via amide or ester bond reaction activated by carbodiimide, in pure water. Prior to molecule crosslinking, the rheological behaviour of the biopolymers (alone or in mixture) was characterized in a semi-dilute concentration regime. Both flow and dynamic measurements showed that uncrosslinked collagen alone appears to be rather elastic with yield stress properties, whereas uncrosslinked HA alone appears to be rather shear thinning and viscoelastic in agreement with entangled polymer behaviour. Sericin exhibited Newtonian low viscosity behaviour according to its very low molar mass. Before crosslinking, HA exhibited viscoelastic behaviour at concentrations above the critical entangled concentration (C*) in the mixtures, thus HA shows promise as a matrix for future crosslinked networks, whereas sericin did not significantly modify the rheology. During the reaction, followed by rheology, the kinetics were slower for pure HA systems compared with the mixtures (i.e., with added collagen and/or to a lesser extent sericin). At the same time, the final network of hydrogels (i.e., the elastic modulus) was more structured in the mixture based systems. This result is explained by ester bonds (the only possibility for pure HA systems), which are less favourable and reactive than amide bonds (possible with sericin and collagen). The presence of collagen in the HA matrix reinforced the hydrogel network. SEM studies confirmed the structure of the hydrogels, and in vitro degradability was globally consistent with the effect of the selected enzyme according to the hydrogel composition. All the elaborated hydrogels were non-cytotoxic in vitro. PMID:27612727

  9. Hypotheses on the evolution of hyaluronan: A highly ironic acid

    PubMed Central

    Csoka, Antonei B; Stern, Robert

    2013-01-01

    Hyaluronan is a high-molecular-weight glycosaminoglycan (GAG) prominent in the extracellular matrix. Emerging relatively late in evolution, it may have evolved to evade immune recognition. Chondroitin is a more ancient GAG and a possible hyaluronan precursor. Epimerization of a 4-hydroxyl in N-acetylgalactosamine in chondroitin to N-acetylglucosamine of hyaluronan is the only structural difference other than chain length between these two polymers. The axial 4-hydroxyl group extends out perpendicular from the equatorial plane of N-acetylgalactosamine in chondroitin. We suspect that this hydroxyl is a prime target for immune recognition. Conversion of a thumbs-up hydroxyl group into a thumbs-down position in the plane of the sugar endows hyaluronan with the ability to avoid immune recognition. Chitin is another potential precursor to hyaluronan. But regardless whether of chondroitin or of chitin origin, an ancient chondroitinase enzyme sequence seems to have been commandeered to catalyze the cleavage of the new hyaluronan substrate. The evolution of six hyaluronidase-like sequences in the human genome from a single chondroitinase as found in Caenorhabditis elegans can now be traced. Confirming our previous predictions, two duplication events occurred, with three hyaluronidase-like sequences occurring in the genome of Ciona intestinalis (sea squirt), the earliest known chordate. This was probably followed by en masse duplication, with six such genes present in the genome of zebra fish onwards. These events occurred, however, much earlier than predicted. It is also apparent on an evolutionary time scale that in several species, this gene family is continuing to evolve. PMID:23315448

  10. Hyaluronan: from biomimetic to industrial business strategy.

    PubMed

    Murano, Erminio; Perin, Danilo; Khan, Riaz; Bergamin, Massimo

    2011-04-01

    Hyaluronan (hyaluronic acid) is a naturally occurring polysaccharide of a linear repeating disaccharide unit consisting of beta-(1-->4)-linked D-glucopyranuronic acid and beta-(1-->3)-linked 2-acetamido-2-deoxy-D-glucopyranose, which is present in extracellular matrices, the synovial fluid of joints, and scaffolding that comprises cartilage. In its mechanism of synthesis, its size, and its physico-chemical properties, hyaluronan is unique amongst other glycosaminoglycans. The network-forming, viscoelastic and its charge characteristics are important to many biochemical properties of living tissues. It is an important pericellular and cell surface constituent; its interaction with other macromolecules such as proteins, participates in regulating cell behavior during numerous morphogenic, restorative, and pathological processes in the body. The knowledge of HA in diseases such as various forms of cancers, arthritis and osteoporosis has led to new impetus in research and development in the preparation of biomaterials for surgical implants and drug conjugates for targeted delivery. A concise and focused review on hyaluronan is timely. This review will cover the following important aspects of hyaluronan: (i) biological functions and synthesis in nature; (ii) current industrial production and potential biosynthetic processes of hyaluronan; (iii) chemical modifications of hyaluronan leading to products of commercial significance; and (iv) and the global market position and manufacturers of hyaluronan.

  11. Toll-like receptor 2 (TLR2), transforming growth factor-β, hyaluronan (HA), and receptor for HA-mediated motility (RHAMM) are required for surfactant protein A-stimulated macrophage chemotaxis.

    PubMed

    Foley, Joseph P; Lam, David; Jiang, Hongmei; Liao, Jie; Cheong, Naeun; McDevitt, Theresa M; Zaman, Aisha; Wright, Jo Rae; Savani, Rashmin C

    2012-10-26

    The innate immune system protects the host from bacterial and viral invasion. Surfactant protein A (SPA), a lung-specific collectin, stimulates macrophage chemotaxis. However, the mechanisms regulating this function are unknown. Hyaluronan (HA) and its receptors RHAMM (receptor for HA-mediated motility, CD168) and CD44 also regulate cell migration and inflammation. We therefore examined the role of HA, RHAMM, and CD44 in SPA-stimulated macrophage chemotaxis. Using antibody blockade and murine macrophages, SPA-stimulated macrophage chemotaxis was dependent on TLR2 but not the other SPA receptors examined. Anti-TLR2 blocked SPA-induced production of TGFβ. In turn, TGFβ1-stimulated chemotaxis was inhibited by HA-binding peptide and anti-RHAMM antibody but not anti-TLR2 antibody. Macrophages from TLR2(-/-) mice failed to migrate in response to SPA but responded normally to TGFβ1 and HA, effects that were blocked by anti-RHAMM antibody. Macrophages from WT and CD44(-/-) mice had similar responses to SPA, whereas those from RHAMM(-/-) mice had decreased chemotaxis to SPA, TGFβ1, and HA. In primary macrophages, SPA-stimulated TGFβ production was dependent on TLR2, JNK, and ERK but not p38. Pam3Cys, a specific TLR2 agonist, stimulated phosphorylation of JNK, ERK, and p38, but only JNK and ERK inhibition blocked Pam3Cys-stimulated chemotaxis. We have uncovered a novel pathway for SPA-stimulated macrophage chemotaxis where SPA stimulation via TLR2 drives JNK- and ERK-dependent TGFβ production. TGFβ1, in turn, stimulates macrophage chemotaxis in a RHAMM and HA-dependent manner. These findings are highly relevant to the regulation of innate immune responses by SPA with key roles for specific components of the extracellular matrix.

  12. Hyaluronan viscosupplementation: state of the art and insight into the novel cooperative hybrid complexes based on high and low molecular weight HA of potential interest in osteoarthritis treatment

    PubMed Central

    Schiraldi, Chiara; Stellavato, Antonietta; de Novellis, Francesca; La Gatta, Annalisa; De Rosa, Mario

    2016-01-01

    Summary Osteoarthritis (OA) represents a group of chronic, painful, disabling conditions affecting synovial joints. It is characterized by degeneration of articular cartilage, alterations of peri-articular and subchondral bone, low-grade synovial inflammation (synovitis). Despite OA is commonly described as a non-inflammatory disease, it is known that its progression and the subsequent increment of symptoms correlate to the production of inflammatory factors that induce the secretion of enzymes responsible for cartilage degradation. In clinical practice, to alleviate pain and stiffness, not only during acute phases but also as maintenance therapy, intra-articular injections of corticosteroids or similar drugs are used, besides it is well diffused the viscosupplementation procedure based on hyaluronan gel. There are many different products containing high molecular weight linear HA or cross-linked derivatives, however the novelty in the field consist in the hybrid cooperative complexes derived from high and low molecular weight HA through a patented processing. This technique permit to double the amount of HA delivered to the injured site without increasing the injected volume, beside in vitro assay on human chondrocytes suggested hybrid complexes as effective in the modulation of several inflammatory cytokines in joints. PMID:27252742

  13. Cytokine regulation of human lung fibroblast hyaluronan (hyaluronic acid) production. Evidence for cytokine-regulated hyaluronan (hyaluronic acid) degradation and human lung fibroblast-derived hyaluronidase.

    PubMed Central

    Sampson, P M; Rochester, C L; Freundlich, B; Elias, J A

    1992-01-01

    We characterized the mechanisms by which recombinant (r) tumor necrosis factor (TNF), IFN-gamma, and IL-1, alone and in combination, regulate human lung fibroblast hyaluronic acid (HA) production. Each cytokine stimulated fibroblast HA production. The combination of rTNF and rIFN-gamma resulted in a synergistic increase in the production of high molecular weight HA. This was due to a synergistic increase in hyaluronate synthetase activity and a simultaneous decrease in HA degradation. In contrast, when rTNF and rIL-1 were combined, an additive increase in low molecular weight HA was noted. This was due to a synergistic increase in hyaluronate synthetase activity and a simultaneous increase in HA degradation. Human lung fibroblasts contained a hyaluronidase that, at pH 3.7, depolymerized high molecular weight HA to 10-40 kD end products of digestion. However, hyaluronidase activity did not correlate with fibroblast HA degradation. Instead, HA degradation correlated with fibroblast-HA binding, which was increased by rIL-1 plus rTNF and decreased by rIFN-gamma plus rTNF. Recombinant IL-1 and rTNF weakly stimulated and rIL-1 and rTNF in combination further augmented the levels of CD44 mRNA in lung fibroblasts. In contrast, rIFN-gamma did not significantly alter the levels of CD44 mRNA in unstimulated or rTNF stimulated cells. These studies demonstrate that rIL-1, rTNF, and rIFN-gamma have complex effects on biosynthesis and degradation which alter the quantity and molecular weight of the HA produced by lung fibroblasts. They also show that fibroblast HA degradation is mediated by a previously unrecognized lysosomal-type hyaluronidase whose function may be regulated by altering fibroblast-HA binding. Lastly, they suggest that the CD44 HA receptor may be involved in this process. Images PMID:1401082

  14. Increased mucociliary differentiation and aquaporins formation of respiratory epithelial cells on retinoic acid-loaded hyaluronan-derivative membranes.

    PubMed

    Huang, Tsung-Wei; Chan, Yen-Hui; Su, Huang-Wei; Chou, Ya-Shuan; Young, Tai-Horng

    2013-06-01

    While playing a major role in maintaining the mucociliary phenotype of respiratory epithelial cells (RECs), retinoids are critical determinants of their normal function. However, despite being a powerful biological agent, retinoic acid (RA) is generally not used in regenerative medicine due to its scarce bioavailability via conventional administration. Therefore, the ability to incorporate RA into biomaterials allows for a combination of the biological effects of RA and biomaterials in influencing cellular behavior. This study attempts to develop RA-loaded hyaluronan-derivative membrane (RA-HAm) and investigates how this membrane affects the mucociliary differentiation and aquaporins (AQP) formation of RECs. In a simulated in vitro culture condition, the RA release from membranes is maintained for 7days. On the seventh day, the cumulative release rate of RA from supportive biomaterials is ~87% under detect limitation. RECs cultured on RA-HAm reveal numerous mature ciliated cells and microvilli compared to aggregated cilia-like structures on hyaluronan-derivative membrane (HAm). Moreover, the expression levels of MUC5AC and AQP on RA-HAm are higher than those on HAm. The proposed model elucidates the release of hydrophobic RA from hyaluronan-derivative biomaterials. We believe that RA-loaded hyaluronan biomaterials are highly promising biomaterials for use in sinonasal surgery and tissue engineering of the respiratory system.

  15. Hyaluronan in Tubular and Interstitial Nephrocalcinosis

    NASA Astrophysics Data System (ADS)

    Verkoelen, Carl F.

    2007-04-01

    Hyaluronan (HA) is the major glycosaminoglycan (GAG) component of the renal medullary interstitium. HA is extremely large (up to 104 kDa) and composed of thousands repeating disaccharides of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc). HA is synthesized by hyaluronan synthases (HASs) and degraded by hyaluronidases (Hyals). The production of HA by renomedullary interstitial cells is mediated by local osmolality. When excess water needs to be excreted, increased interstitial HA seems to antagonize water reabsorption, while the opposite occurs during water conservation. Hence, papillary interstitial HA is low and Hyal high during anti-diuresis, whereas during diuresis HA is high and Hyal low. The polyanion HA plays a role in the reabsorption of hypotonic fluid by immobilizing cations (Na+) via the carboxylate (COO-) groups of GlcUA. The binding of Ca2+ to anionic HA is probably also responsible for the fact that the papilla does not become a stone despite the extremely high interstitial phosphate and oxalate. HA is also an excellent crystal binding molecule. The expression of HA at the luminal surface of renal tubular cells leads to tubular nephrocalcinosis (tubular NC). Calcium staining methods (Von Kossa, Yasue) demonstrated that crystallization inhibitors cannot avoid the occasional precipitation of calcium phosphate in the papillary interstitium (interstitial NC). These crystals are probably immediately immobilized by the gel-like HA matrix. After ulcerating through the pelvic wall the calcified matrix becomes a Randall's plaque. The attachment of calcium oxalate crystals from the primary urine to plaque may ultimately lead to the development of clinical stones in the renal calyces (nephrolithiasis).

  16. Interaction of Hyaluronan Binding Peptides with Glycosaminoglycans in Poly(ethylene glycol) Hydrogels

    PubMed Central

    2015-01-01

    This study investigates the incorporation of hyaluronan (HA) binding peptides into poly(ethylene glycol) (PEG) hydrogels as a mechanism to bind and retain hyaluronan for applications in tissue engineering. The specificity of the peptide sequence (native RYPISRPRKRC vs non-native RPSRPRIRYKC), the role of basic amino acids, and specificity to hyaluronan over other GAGs in contributing to the peptide–hyaluronan interaction were probed through experiments and simulations. Hydrogels containing the native or non-native peptide retained hyaluronan in a dose-dependent manner. Ionic interactions were the dominating mechanism. In diH2O the peptides interacted strongly with HA and chondroitin sulfate, but in phosphate buffered saline the peptides interacted more strongly with HA. For cartilage tissue engineering, chondrocyte-laden PEG hydrogels containing increasing amounts of HA binding peptide and exogenous HA had increased retention and decreased loss of cell-secreted proteoglycans in and from the hydrogel at 28 days. This new matrix-interactive hydrogel platform holds promise for tissue regeneration. PMID:24597474

  17. Interaction of hyaluronan binding peptides with glycosaminoglycans in poly(ethylene glycol) hydrogels.

    PubMed

    Roberts, Justine J; Elder, Robert M; Neumann, Alexander J; Jayaraman, Arthi; Bryant, Stephanie J

    2014-04-14

    This study investigates the incorporation of hyaluronan (HA) binding peptides into poly(ethylene glycol) (PEG) hydrogels as a mechanism to bind and retain hyaluronan for applications in tissue engineering. The specificity of the peptide sequence (native RYPISRPRKRC vs non-native RPSRPRIRYKC), the role of basic amino acids, and specificity to hyaluronan over other GAGs in contributing to the peptide-hyaluronan interaction were probed through experiments and simulations. Hydrogels containing the native or non-native peptide retained hyaluronan in a dose-dependent manner. Ionic interactions were the dominating mechanism. In diH2O the peptides interacted strongly with HA and chondroitin sulfate, but in phosphate buffered saline the peptides interacted more strongly with HA. For cartilage tissue engineering, chondrocyte-laden PEG hydrogels containing increasing amounts of HA binding peptide and exogenous HA had increased retention and decreased loss of cell-secreted proteoglycans in and from the hydrogel at 28 days. This new matrix-interactive hydrogel platform holds promise for tissue regeneration. PMID:24597474

  18. Hyaluronan in human malignancies

    SciTech Connect

    Sironen, R.K.; Tammi, M.; Tammi, R.; Auvinen, P.K.; Anttila, M.; Kosma, V-M.

    2011-02-15

    Hyaluronan, a major macropolysaccharide in the extracellular matrix of connective tissues, is intimately involved in the biology of cancer. Hyaluronan accumulates into the stroma of various human tumors and modulates intracellular signaling pathways, cell proliferation, motility and invasive properties of malignant cells. Experimental and clinicopathological evidence highlights the importance of hyaluronan in tumor growth and metastasis. A high stromal hyaluronan content is associated with poorly differentiated tumors and aggressive clinical behavior in human adenocarcinomas. Instead, the squamous cell carcinomas and malignant melanomas tend to have a reduced hyaluronan content. In addition to the stroma-cancer cell interaction, hyaluronan can influence stromal cell recruitment, tumor angiogenesis and epithelial-mesenchymal transition. Hyaluronan receptors, hyaluronan synthases and hyaluronan degrading enzymes, hyaluronidases, are involved in the modulation of cancer progression, depending on the tumor type. Furthermore, intracellular signaling and angiogenesis are affected by the degradation products of hyaluronan. Hyaluronan has also therapeutic implications since it is involved in multidrug resistance.

  19. Novel bioadhesive hyaluronan-itaconic acid crosslinked films for ocular therapy.

    PubMed

    Calles, J A; Tártara, L I; Lopez-García, A; Diebold, Y; Palma, S D; Vallés, E M

    2013-10-15

    New hyaluronic acid (HA)-itaconic acid (IT) films have been previously synthesized and used as potential topical drug delivery systems (DDS) for ocular administration. In this study we explored homogeneous and heterogeneous crosslinking reactions of HA using glutaraldehyde (GTA) and polyethylene glycol diglycidyl ether (PEGDE) in the presence of IT, a naturally occurring compound that is non-toxic and readily biodegradable. We have studied the morphology, mechanical properties and in vitro biocompatibility between these new materials and ocular surface cells (human corneal epithelial cell line) and evaluated the biopharmaceutical performance of the designed formulations. Although all the synthesized materials exhibited good mechanical properties, the PEGDE modified films exhibited the best biocompatibility, with in vivo assays showing good adhesive performance and minimal irritation. PEGDE films were also tested for their effects in the treatment of intraocular pressure (IOP) in rabbits using timolol maleate (TM) as the model drug. These results may be useful for further design of novel bioadhesive matrix containing drugs by topical application in ophthalmology. PMID:23911915

  20. Regulation of Synthesis and Roles of Hyaluronan in Peritoneal Dialysis

    PubMed Central

    Bowen, Timothy; Meran, Soma; Williams, Aled P.; Newbury, Lucy J.; Sauter, Matthias; Sitter, Thomas

    2015-01-01

    Hyaluronan (HA) is a ubiquitous extracellular matrix glycosaminoglycan composed of repeated disaccharide units of alternating D-glucuronic acid and D-N-acetylglucosamine residues linked via alternating β-1,4 and β-1,3 glycosidic bonds. HA is synthesized in humans by HA synthase (HAS) enzymes 1, 2, and 3, which are encoded by the corresponding HAS genes. Previous in vitro studies have shown characteristic changes in HAS expression and increased HA synthesis in response to wounding and proinflammatory cytokines in human peritoneal mesothelial cells. In addition, in vivo models and human peritoneal biopsy samples have provided evidence of changes in HA metabolism in the fibrosis that at present accompanies peritoneal dialysis treatment. This review discusses these published observations and how they might contribute to improvement in peritoneal dialysis. PMID:26550568

  1. Preparation of hyaluronan-DNA matrices and films.

    PubMed

    Chen, Weiliam

    2012-10-01

    Natural carbohydrate is a class of underexplored polymers for gene delivery. The noninflammatory and nonimmunogenic properties of hyaluronan (hyaluronic acid, HA) are important in clinical situations. It has a role in wound repair and has great lubricating ability. Moreover, the presence of hyaluronidase in vivo enables any vehicle fabricated from HA to be degraded by enzyme-mediated erosion. When DNA is entrapped in a cross-linked HA vehicle, HA-DNA fragments are released on digestion by hyaluronidase. These fragments could serve both as microcarriers of DNA and its protective mechanism. This protocol describes preparation of water-insoluble HA-DNA matrices and films designed for clinical applications, and assays for verification of their bioactivities. Plasmid DNA (pDNA) encoding platelet-derived growth factor (PDGF) is coupled to the matrices that could be implanted into chronic wounds to accelerate their healing. pDNA encoding hyaluronan synthase 2 (HAS2) is coupled to the film that could initially serve as a physical barrier and subsequently a pDNA reservoir for sustaining HAS2 transfection. This would lead to continual HA production for preventing postsurgical adhesion.

  2. Rheological properties of aqueous solutions of biopolymeric hyaluronan

    NASA Astrophysics Data System (ADS)

    Szwajczak, Elzbieta

    2004-09-01

    Aqueous solutions of hyaluronic acid (hyaluronan, HA) were studied. The HA compound is a natural polysaccharide, bipolymer. It plays an important role in numerous biological processes as a component of the extracellular matrix, connective tissues and, especially, human and animal synovial joints. Natural and artificial solutions of the HA have demonstrated the viscoelastic nature. These properties are shown to be related to the microstructure parameters (bulk concentration, molecular weight) and external parameters (temperature, stress, shear rate). We emphasize the role of the flow properties of polymeric systems. It is found a liquid crystalline "order" can be "induced" during the material flow. The dynamic properties, such as the elastic shear modulus and viscous shear modulus, are given. These results are discussed in relation to the postulated function of hyaluronic acid in synovial joint and with respect to possibilities o their application in medicine and pharmacology.

  3. Biomimetic hemocompatible coatings through immobilization of hyaluronan derivatives on metal surfaces.

    PubMed

    Thierry, Benjamin; Winnik, Françoise M; Merhi, Yahye; Griesser, Hans J; Tabrizian, Maryam

    2008-10-21

    Biomimetic coatings offer exciting options to modulate the biocompatibility of biomaterials. The challenge is to create surfaces that undergo specific interactions with cells without promoting nonspecific fouling. This work reports an innovative approach toward biomimetic surfaces based on the covalent immobilization of a carboxylate terminated PEGylated hyaluronan (HA-PEG) onto plasma functionalized NiTi alloy surfaces. The metal substrates were aminated via two different plasma functionalization processes. Hyaluronan, a natural glycosaminoglycan and the major constituent of the extracellular matrix, was grafted to the substrates by reaction of the surface amines with the carboxylic acid terminated PEG spacer using carbodiimide chemistry. The surface modification was monitored at each step by X-ray photoelectron spectroscopy (XPS). HA-immobilized surfaces displayed increased hydrophilicity and reduced fouling, compared to bare surfaces, when exposed to human platelets (PLT) in an in vitro assay with radiolabeled platelets (204.1 +/- 123.8 x 10 (3) PLT/cm (2) vs 538.5 +/- 100.5 x 10 (3) PLT/cm (2) for bare metal, p < 0.05). Using a robust plasma patterning technique, microstructured hyaluronan surfaces were successfully created as demonstrated by XPS chemical imaging. The bioactive surfaces described present unique features, which result from the synergy between the intrinsic biological properties of hyaluronan and the chemical composition and morphology of the polymer layer immobilized on a metal surface.

  4. HA Antibody-Mediated FcγRIIIa Activity Is Both Dependent on FcR Engagement and Interactions between HA and Sialic Acids

    PubMed Central

    Cox, Freek; Kwaks, Ted; Brandenburg, Boerries; Koldijk, Martin H.; Klaren, Vincent; Smal, Bastiaan; Korse, Hans J. W. M.; Geelen, Eric; Tettero, Lisanne; Zuijdgeest, David; Stoop, Esther J. M.; Saeland, Eirikur; Vogels, Ronald; Friesen, Robert H. E.; Koudstaal, Wouter; Goudsmit, Jaap

    2016-01-01

    Interactions with receptors for the Fc region of IgG (FcγRs) have been shown to contribute to the in vivo protection against influenza A viruses provided by broadly neutralizing antibodies (bnAbs) that bind to the viral hemagglutinin (HA) stem. In particular, Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) has been shown to contribute to protection by stem-binding bnAbs. Fc-mediated effector functions appear not to contribute to protection provided by strain-specific HA head-binding antibodies. We used a panel of anti-stem and anti-head influenza A and B monoclonal antibodies with identical human IgG1 Fc domains and investigated their ability to mediate ADCC-associated FcγRIIIa activation. Antibodies which do not interfere with sialic acid binding of HA can mediate FcγRIIIa activation. However, the FcγRIIIa activation was inhibited when a mutant HA, unable to bind sialic acids, was used. Antibodies which block sialic acid receptor interactions of HA interfered with FcγRIIIa activation. The inhibition of FcγRIIIa activation by HA head-binding and sialic acid receptor-blocking antibodies was confirmed in plasma samples of H5N1 vaccinated human subjects. Together, these results suggest that in addition to Fc–FcγR binding, interactions between HA and sialic acids on immune cells are required for optimal Fc-mediated effector functions by anti-HA antibodies. PMID:27746785

  5. CD44S-hyaluronan interactions protect cells resulting from EMT against anoikis

    PubMed Central

    Cieply, Benjamin; Koontz, Colton; Frisch, Steven M.

    2016-01-01

    The detachment of normal epithelial cells from matrix triggers an apoptotic response known as anoikis, during homeostatic turnover. Metastatic tumor cells evade anoikis, by mechanisms that are only partly characterized. In particular, the epithelial–mesenchymal transition (EMT) in a subset of invasive tumor cells confers anoikis-resistance. In some cases, EMT up-regulates the cancer stem cell marker CD44S and the enzyme hyaluronic acid synthase-2 (HAS2). CD44S is the major receptor for hyaluronan in the extracellular matrix. Herein, we demonstrate that CD44S, unlike the CD44E isoform expressed in normal epithelial cells, contributes to the protection against anoikis. This protection requires the interaction of CD44S with hyaluronan (HA). CD44S–HA interaction is proposed to play an important role in tumor metastasis through enhanced cell survival under detached conditions. PMID:25937513

  6. Hyaluronan and synovial joint: function, distribution and healing

    PubMed Central

    2013-01-01

    Synovial fluid is a viscous solution found in the cavities of synovial joints. The principal role of synovial fluid is to reduce friction between the articular cartilages of synovial joints during movement. The presence of high molar mass hyaluronan (HA) in this fluid gives it the required viscosity for its function as lubricant solution. Inflammation oxidation stress enhances normal degradation of hyaluronan causing several diseases related to joints. This review describes hyaluronan properties and distribution, applications and its function in synovial joints, with short review for using thiol compounds as antioxidants preventing HA degradations under inflammation conditions. PMID:24678248

  7. Eco-friendly microwave-assisted protocol to prepare hyaluronan-fatty acid conjugates and to induce their self-assembly process.

    PubMed

    Calce, Enrica; Mercurio, Flavia Anna; Leone, Marilisa; Saviano, Michele; De Luca, Stefania

    2016-06-01

    An environmentally sustainable and energy-efficient synthetic process has been developed to prepare hyaluronan-based nano-sized material. It consists in a microwave-promoted acylation of the hydroxyl function of the polysaccharide with natural fatty acids, performed under solvent-free conditions. The efficient interaction of the solid reagents with the MW radiation accounts for the obtained high yielded products. The self-assembly process of the obtained compounds very fast occurred in an aqueous medium under MW-radiation, thus allowing the development of a green protocol for the nano-particles preparation. PMID:27083346

  8. Hyaluronan: A Simple Polysaccharide with Diverse Biological Functions

    PubMed Central

    Dicker, Kevin T.; Gurski, Lisa A.; Pradhan-Bhatt, Swati; Witt, Robert L.; Farach-Carson, Mary C.; Jia, Xinqiao

    2014-01-01

    Hyaluronan (HA) is a linear polysaccharide with disaccharide repeats of D-glucuronic acid and N-acetyl-D-glucosamine. It is evolutionary conserved and abundantly expressed in the extracellular matrix (ECM), on the cell surface and even inside cells. Being a simple polysaccharide, HA exhibits an astonishing array of biological functions. HA interacts with various proteins or proteoglycans to organize the ECM and to maintain tissue homeostasis. The unique physical and mechanical properties of HA contribute to the maintenance of tissue hydration, the mediation of solute diffusion through the extracellular space and the lubrication of certain tissues. The diverse biological functions of HA are manifested through its complex interactions with matrix components and resident cells. Binding of HA with cell surface receptors activates various signaling pathways that regulate cell function, tissue development, inflammation, wound healing and tumor progression and metastasis. Taking advantage of the inherent biocompatibility and biodegradability of HA, as well as its susceptibility to chemical modification, researchers have developed various HA-based biomaterials and tissue constructs with promising and broad clinical potential. In this article, we illustrate the properties of HA from a matrix biology perspective by first introducing principles underlying the biosynthesis and biodegradation of HA, as well as the interactions of HA with various proteins and proteoglycans. We next highlight the roles of HA in physiological and pathological states, including morphogenesis, wound healing and tumor metastasis. A deeper understanding of the mechanisms underlying the roles of HA in various physiological processes can provide new insights and tools for the engineering of complex tissues and tissue models. PMID:24361428

  9. Identification, design and synthesis of tubulin-derived peptides as novel hyaluronan mimetic ligands for the receptor for hyaluronan-mediated motility (RHAMM/HMMR).

    PubMed

    Esguerra, Kenneth Virgel N; Tolg, Cornelia; Akentieva, Natalia; Price, Matthew; Cho, Choi-Fong; Lewis, John D; McCarthy, James B; Turley, Eva A; Luyt, Leonard G

    2015-12-01

    Fragments of the extracellular matrix component hyaluronan (HA) promote tissue inflammation, fibrosis and tumor progression. HA fragments act through HA receptors including CD44, LYVE1, TLR2, 4 and the receptor for hyaluronan mediated motility (RHAMM/HMMR). RHAMM is a multifunctional protein with both intracellular and extracellular roles in cell motility and proliferation. Extracellular RHAMM binds directly to HA fragments while intracellular RHAMM binds directly to ERK1 and tubulin. Both HA and regions of tubulin (s-tubulin) are anionic and bind to basic amino acid-rich regions in partner proteins, such as in HA and tubulin binding regions of RHAMM. We used this as a rationale for developing bioinformatics and SPR (surface plasmon resonance) based screening to identify high affinity anionic RHAMM peptide ligands. A library of 12-mer peptides was prepared based on the carboxyl terminal tail sequence of s-tubulin isoforms and assayed for their ability to bind to the HA/tubulin binding region of recombinant RHAMM using SPR. This approach resulted in the isolation of three 12-mer peptides with nanomolar affinity for RHAMM. These peptides bound selectively to RHAMM but not to CD44 or TLR2,4 and blocked RHAMM:HA interactions. Furthermore, fluorescein-peptide uptake by PC3MLN4 prostate cancer cells was blocked by RHAMM mAb but not by CD44 mAb. These peptides also reduced the ability of prostate cancer cells to degrade collagen type I. The selectivity of these novel HA peptide mimics for RHAMM suggest their potential for development as HA mimetic imaging and therapeutic agents for HA-promoted disease.

  10. Ingested hyaluronan moisturizes dry skin

    PubMed Central

    2014-01-01

    Hyaluronan (HA) is present in many tissues of the body and is essential to maintain moistness in the skin tissues, which contain approximately half the body’s HA mass. Due to its viscosity and moisturizing effect, HA is widely distributed as a medicine, cosmetic, food, and, recently marketed in Japan as a popular dietary supplement to promote skin moisture. In a randomized, double-blind, placebo-controlled clinical study it was found that ingested HA increased skin moisture and improved treatment outcomes for patients with dry skin. HA is also reported to be absorbed by the body distributed, in part, to the skin. Ingested HA contributes to the increased synthesis of HA and promotes cell proliferation in fibroblasts. These effects show that ingestion of HA moisturizes the skin and is expected to improve the quality of life for people who suffer from dry skin. This review examines the moisturizing effects of dry skin by ingested HA and summarizes the series of mechanisms from absorption to pharmacological action. PMID:25014997

  11. Hyaluronan Synthesis and Myogenesis

    PubMed Central

    Hunt, Liam C.; Gorman, Chris; Kintakas, Christopher; McCulloch, Daniel R.; Mackie, Eleanor J.; White, Jason D.

    2013-01-01

    Exogenous hyaluronan is known to alter muscle precursor cell proliferation, migration, and differentiation, ultimately inhibiting myogenesis in vitro. The aim of the current study was to investigate the role of endogenous hyaluronan synthesis during myogenesis. In quantitative PCR studies, the genes responsible for synthesizing hyaluronan were found to be differentially regulated during muscle growth, repair, and pathology. Although all Has genes (Has1, Has2, and Has3) were differentially regulated in these models, only Has2 gene expression consistently associated with myogenic differentiation. During myogenic differentiation in vitro, Has2 was the most highly expressed of the synthases and increased after induction of differentiation. To test whether this association between Has2 expression and myogenesis relates to a role for Has2 in myoblast differentiation and fusion, C2C12 myoblasts were depleted of Has2 by siRNA and induced to differentiate. Depletion of Has2 inhibited differentiation and caused a loss of cell-associated hyaluronan and the hyaluronan-dependent pericellular matrix. The inhibition of differentiation caused by loss of hyaluronan was confirmed with the hyaluronan synthesis inhibitor 4-methylumbelliferone. In hyaluronan synthesis-blocked cultures, restoration of the pericellular matrix could be achieved through the addition of exogenous hyaluronan and the proteoglycan versican, but this was not sufficient to restore differentiation to control levels. These data indicate that intrinsic hyaluronan synthesis is necessary for myoblasts to differentiate and form syncytial muscle cells, but the hyaluronan-dependent pericellular matrix is not sufficient to support differentiation alone; additional hyaluronan-dependent cell functions that are yet unknown may be required for myogenic differentiation. PMID:23493399

  12. Hyaluronan stimulates pancreatic cancer cell motility

    PubMed Central

    Cheng, Xiao-Bo; Kohi, Shiro; Koga, Atsuhiro; Hirata, Keiji; Sato, Norihiro

    2016-01-01

    Hyaluronan (HA) accumulates in pancreatic ductal adenocarcinoma (PDAC), but functional significance of HA in the aggressive phenotype remains unknown. We used different models to investigate the effect of HA on PDAC cell motility by wound healing and transwell migration assay. Changes in cell motility were examined in 8 PDAC cell lines in response to inhibition of HA production by treatment with 4-methylumbelliferone (4-MU) and to promotion by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or by co-culture with tumor-derived stromal fibroblasts. We also investigated changes in cell motility by adding exogenous HA. Additionally, mRNA expressions of hyaluronan synthases and hyaluronidases were examined using real time RT-PCR. Inhibition of HA by 4-MU significantly decreased the migration, whereas promotion of HA by TPA or co-culture with tumor-derived fibroblasts significantly increased the migration of PDAC cells. The changes in HA production by these treatments tended to be associated with changes in HAS3 mRNA expression. Furthermore, addition of exogenous HA, especially low-molecular-weight HA, significantly increased the migration of PDAC cells. These findings suggest that HA stimulates PDAC cell migration and thus represents an ideal therapeutic target to prevent invasion and metastasis. PMID:26684359

  13. Controlled Immobilization Strategies to Probe Short Hyaluronan-Protein Interactions

    PubMed Central

    Minsky, Burcu Baykal; Antoni, Christiane H.; Boehm, Heike

    2016-01-01

    Well-controlled grafting of small hyaluronan oligosaccharides (sHA) enables novel approaches to investigate biological processes such as angiogenesis, immune reactions and cancer metastasis. We develop two strategies for covalent attachment of sHA, a fast high-density adsorption and a two-layer system that allows tuning the density and mode of immobilization. We monitored the sHA adlayer formation and subsequent macromolecular interactions by label-free quartz crystal microbalance with dissipation (QCM-D). The modified surfaces are inert to unspecific protein adsorption, and yet retain the specific binding capacity of sHA. Thus they are an ideal tool to study the interactions of hyaluronan-binding proteins and short hyaluronan molecules as demonstrated by the specific recognition of LYVE-1 and aggrecan. Both hyaladherins recognize sHA and the binding is independent to the presence of the reducing end. PMID:26883791

  14. Controlled Immobilization Strategies to Probe Short Hyaluronan-Protein Interactions

    NASA Astrophysics Data System (ADS)

    Minsky, Burcu Baykal; Antoni, Christiane H.; Boehm, Heike

    2016-02-01

    Well-controlled grafting of small hyaluronan oligosaccharides (sHA) enables novel approaches to investigate biological processes such as angiogenesis, immune reactions and cancer metastasis. We develop two strategies for covalent attachment of sHA, a fast high-density adsorption and a two-layer system that allows tuning the density and mode of immobilization. We monitored the sHA adlayer formation and subsequent macromolecular interactions by label-free quartz crystal microbalance with dissipation (QCM-D). The modified surfaces are inert to unspecific protein adsorption, and yet retain the specific binding capacity of sHA. Thus they are an ideal tool to study the interactions of hyaluronan-binding proteins and short hyaluronan molecules as demonstrated by the specific recognition of LYVE-1 and aggrecan. Both hyaladherins recognize sHA and the binding is independent to the presence of the reducing end.

  15. Tracheal reconstruction using chondrocytes seeded on a poly(L-lactic-co-glycolic acid)-fibrin/hyaluronan.

    PubMed

    Hong, Hyun Jun; Chang, Jae Won; Park, Ju-Kyeong; Choi, Jae Won; Kim, Yoo Suk; Shin, Yoo Seob; Kim, Chul-Ho; Choi, Eun Chang

    2014-11-01

    Reconstruction of trachea is still a clinical dilemma. Tissue engineering is a recent and promising concept to resolve this problem. This study evaluated the feasibility of allogeneic chondrocytes cultured with fibrin/hyaluronic acid (HA) hydrogel and degradable porous poly(L-lactic-co-glycolic acid) (PLGA) scaffold for partial tracheal reconstruction. Chondrocytes from rabbit articular cartilage were expanded and cultured with fibrin/HA hydrogel and injected into a 5 × 10 mm-sized, curved patch-shape PLGA scaffold. After 4 weeks in vitro culture, the scaffold was implanted on a tracheal defect in eight rabbits. Six and 10 weeks postoperatively, the implanted sites were evaluated by bronchoscope and radiologic and histologic analyses. Ciliary beat frequency (CBF) of regenerated epithelium was also evaluated. None of the eight rabbits showed any sign of respiratory distress. Bronchoscopic examination did not reveal stenosis of the reconstructed trachea and the defects were completely recovered with respiratory epithelium. Computed tomography scan showed good luminal contour of trachea. Histologic data showed that the implanted chondrocytes successfully formed neocartilage with minimal granulation tissue. CBF of regenerated epithelium was similar to that of normal epithelium. Partial tracheal defect was successfully reconstructed anatomically and functionally using allogeneic chondrocytes cultured with PLGA-fibrin/HA composite scaffold.

  16. Cellular Uptake and Internalization of Hyaluronan-based Doxorubicin and Cisplatin Conjugates

    PubMed Central

    Cai, Shuang; Alhowyan, Adel Ali B; Yang, Qiuhong; Forrest, W.C. Melanie; Shnayder, Yelizaveta; Forrest, M. Laird

    2015-01-01

    Background Hyaluronan (HA) is a ligand for the CD44 receptor which is crucial to cancer cell proliferation and metastasis. High levels of CD44 expression in many cancers have encouraged the development of HA-based carriers for anti-cancer therapeutics. Purpose The objective of this study was to determine whether HA conjugation of anticancer drugs impacts CD44-specific HA-drug uptake and disposition by human head and neck cancer cells. Methods The internalization and cellular disposition of hyaluronan-doxorubicin (HA-DOX), hyaluronan-cisplatin (HA-Pt), and hyaluronan-cyanine7 (HA-Cy7) conjugates were investigated by inhibiting endocytosis pathways, and by inhibiting the CD44–mediated internalization pathways that are known to mediate hyaluronan uptake in vitro. Results Cellular internalization of HA was regulated by CD44 receptors. In mouse xenografts, HA conjugation significantly enhanced tumor cell uptake compared to unconjugated drug. Discussion The results suggested that the main mechanism of HA-based conjugate uptake may be active transport via CD44 in conjunction with a clathrin–dependent endocytic pathway. Other HA receptors, hyaluronan–mediated motility receptor (RHAMM) and lymphatic vessel endothelial hyaluronan receptor (LYVE-1), did not play a significant role in conjugate uptake. Conclusions HA conjugation significantly increased CD44 mediated drug uptake and extended the residence time of drugs in tumor cells. PMID:24892741

  17. Biology and biotechnology of hyaluronan.

    PubMed

    Viola, Manuela; Vigetti, Davide; Karousou, Evgenia; D'Angelo, Maria Luisa; Caon, Ilaria; Moretto, Paola; De Luca, Giancarlo; Passi, Alberto

    2015-05-01

    The hyaluronan (HA) polymer is a critical component of extracellular matrix with a remarkable structure: is a linear and unbranched polymer without sulphate or phosphate groups. It is ubiquitous in mammals showing several biological functions, ranging from cell proliferation and migration to angiogenesis and inflammation. For its critical biological functions the amount of HA in tissues is carefully controlled by different mechanisms including covalent modification of the synthetic enzymes and epigenetic control of their gene expression. The concentration of HA is also critical in several pathologies including cancer, diabetes and inflammation. Beside these biological roles, the structural properties of HA allow it to take advantage of its capacity to form gels even at concentration of 1 % producing scaffolds with very promising applications in regenerative medicine as biocompatible material for advanced therapeutic uses. In this review we highlight the biological aspects of HA addressing the mechanisms controlling the HA content in tissues as well as its role in important human pathologies. In the second part of the review we highlight the different use of HA polymers in the modern biotechnology.

  18. Revealing the Mechanisms of Protein Disorder and N-Glycosylation in CD44-Hyaluronan Binding Using Molecular Simulation

    PubMed Central

    Guvench, Olgun

    2015-01-01

    The extracellular N-terminal hyaluronan binding domain (HABD) of CD44 is a small globular domain that confers hyaluronan (HA) binding functionality to this large transmembrane glycoprotein. When recombinantly expressed by itself, HABD exists as a globular water-soluble protein that retains the capacity to bind HA. This has enabled atomic-resolution structural biology experiments that have revealed the structure of HABD and its binding mode with oligomeric HA. Such experiments have also pointed to an order-to-disorder transition in HABD that is associated with HA binding. However, it had remained unclear how this structural transition was involved in binding since it occurs in a region of HABD distant from the HA-binding site. Furthermore, HABD is known to be N-glycosylated, and such glycosylation can diminish HA binding when the associated N-glycans are capped with sialic acid residues. The intrinsic flexibility of disordered proteins and of N-glycans makes it difficult to apply experimental structural biology approaches to probe the molecular mechanisms of how the order-to-disorder transition and N-glycosylation can modulate HA binding by HABD. We review recent results from molecular dynamics simulations that provide atomic-resolution mechanistic understanding of such modulation to help bridge gaps between existing experimental binding and structural biology data. Findings from these simulations include: Tyr42 may function as a molecular switch that converts the HA-binding site from a low affinity to a high affinity state; in the partially disordered form of HABD, basic amino acids in the C-terminal region can gain sufficient mobility to form direct contacts with bound HA to further stabilize binding; and terminal sialic acids on covalently attached N-glycans can form charge-paired hydrogen bonding interactions with basic amino acids that could otherwise bind to HA, thereby blocking HA binding to glycosylated CD44 HABD. PMID:26136744

  19. Hyaluronan cable formation by ocular trabecular meshwork cells.

    PubMed

    Sun, Ying Ying; Keller, Kate E

    2015-10-01

    Hyaluronan (HA) in the ocular trabecular meshwork (TM) is a critical modulator of aqueous humor outflow. Individual HA strands in the pericellular matrix can coalesce to form cable-like structures, which have different functional properties. Here, we investigated HA structural configuration by TM cells in response to various stimuli known to stimulate extracellular matrix (ECM) remodeling. In addition, the effects of HA cable induction on aqueous outflow resistance was determined. Primary TM cell cultures grown on tissue culture-treated plastic were treated for 12-48 h with TNFα, IL-1α, or TGFβ2. TM cells grown on silicone membranes were subject to mechanical stretch, which induces synthesis and activation of ECM proteolytic enzymes. HA structural configuration was investigated by HA binding protein (HAbp) staining and confocal microscopy. HAbp-labeled cables were induced by TNFα, TGFβ2 and mechanical stretch, but not by IL-1α. HA synthase (HAS) gene expression was quantitated by quantitative RT-PCR and HA concentration was measured by ELISA assay. By quantitative RT-PCR, HAS-1, -2, and -3 genes were differentially up-regulated and showed temporal differences in response to each treatment. HA concentration was increased in the media by TNFα, TGFβ2 and IL-1α, but mechanical stretch decreased pericellular HA concentrations. Immunofluorescence and Western immunoblotting were used to investigate the distribution and protein levels of the HA-binding proteins, tumor necrosis factor-stimulated gene-6 (TSG-6) and inter-α-inhibitor (IαI). Western immunoblotting showed that TSG-6 and IαI were increased by TNFα, TGFβ2 and IL-1α, but mechanical stretch reduced their levels. The underlying substrate appears to affect the identity of IαI·TSG-6·HA complexes since different complexes were detected when TM cells were grown on a silicone substrate compared to a rigid plastic surface. Porcine anterior segments were perfused with 10 μg/ml polyinosinic

  20. Hyaluronan cable formation by ocular trabecular meshwork cells.

    PubMed

    Sun, Ying Ying; Keller, Kate E

    2015-10-01

    Hyaluronan (HA) in the ocular trabecular meshwork (TM) is a critical modulator of aqueous humor outflow. Individual HA strands in the pericellular matrix can coalesce to form cable-like structures, which have different functional properties. Here, we investigated HA structural configuration by TM cells in response to various stimuli known to stimulate extracellular matrix (ECM) remodeling. In addition, the effects of HA cable induction on aqueous outflow resistance was determined. Primary TM cell cultures grown on tissue culture-treated plastic were treated for 12-48 h with TNFα, IL-1α, or TGFβ2. TM cells grown on silicone membranes were subject to mechanical stretch, which induces synthesis and activation of ECM proteolytic enzymes. HA structural configuration was investigated by HA binding protein (HAbp) staining and confocal microscopy. HAbp-labeled cables were induced by TNFα, TGFβ2 and mechanical stretch, but not by IL-1α. HA synthase (HAS) gene expression was quantitated by quantitative RT-PCR and HA concentration was measured by ELISA assay. By quantitative RT-PCR, HAS-1, -2, and -3 genes were differentially up-regulated and showed temporal differences in response to each treatment. HA concentration was increased in the media by TNFα, TGFβ2 and IL-1α, but mechanical stretch decreased pericellular HA concentrations. Immunofluorescence and Western immunoblotting were used to investigate the distribution and protein levels of the HA-binding proteins, tumor necrosis factor-stimulated gene-6 (TSG-6) and inter-α-inhibitor (IαI). Western immunoblotting showed that TSG-6 and IαI were increased by TNFα, TGFβ2 and IL-1α, but mechanical stretch reduced their levels. The underlying substrate appears to affect the identity of IαI·TSG-6·HA complexes since different complexes were detected when TM cells were grown on a silicone substrate compared to a rigid plastic surface. Porcine anterior segments were perfused with 10 μg/ml polyinosinic

  1. Improve the Strength of PLA/HA Composite Through the Use of Surface Initiated Polymerization and Phosphonic Acid Coupling Agent

    PubMed Central

    Wang, Tongxin; Chow, Laurence C.; Frukhtbeyn, Stanislav A.; Ting, Andy Hai; Dong, Quanxiao; Yang, Mingshu; Mitchell, James W.

    2011-01-01

    Bioresorbable composite made from degradable polymers, e.g., polylactide (PLA), and bioactive calcium phosphates, e.g., hydroxyapatite (HA), are clinically desirable for bone fixation, repair and tissue engineering because they do not need to be removed by surgery after the bone heals. However, preparation of PLA/HA composite from non-modified HA usually results in mechanical strength reductions due to a weak interface between PLA and HA. In this study, a calcium-phosphate/phosphonate hybrid shell was developed to introduce a greater amount of reactive hydroxyl groups onto the HA particles. Then, PLA was successfully grafted on HA by surface-initiated polymerization through the non-ionic surface hydroxyl groups. Thermogravimetric analysis indiated that the amount of grafted PLA on HA can be up to 7 %, which is about 50 % greater than that from the literature. PLA grafted HA shows significantly different pH dependent ζ-potential and particle size profiles from those of uncoated HA. By combining the phosphonic acid coupling agent and surface initiated polymerization, PLA could directly link to HA through covalent bond so that the interfacial interaction in the PLA/HA composite can be significantly improved. The diametral tensile strength of PLA/HA composite prepared from PLA-grafted HA was found to be over twice that of the composite prepared from the non-modified HA. Moreover, the tensile strength of the improved composite was 23 % higher than that of PLA alone. By varying additional variables, this approach has the potential to produce bioresorbable composites with improved mechanical properties that are in the range of natural bones, and can have wide applications for bone fixation and repair in load-bearing areas. PMID:22399838

  2. Biotinylated hyaluronan: a versatile and highly sensitive probe capable of detecting nanogram levels of hyaluronan binding proteins (hyaladherins) on electroblots by a novel affinity detection procedure.

    PubMed

    Melrose, J; Numata, Y; Ghosh, P

    1996-01-01

    Hyaluronan influences cellular proliferation and migration in developing, regenerating and remodelling tissues and in tissues undergoing malignant tumour-cell invasion. The widespread occurrence of hyaluronan-binding proteins indicates that the recognition of hyaluronan is important to tissue organisation and the control of cellular behaviour. A number of extracellular matrix and cellular proteins, which have been termed the hyaladherins, have specific affinities for hyaluronan. These include cartilage link-protein, hyaluronectin, neurocan, versican and aggrecan, which all bind to HA within the extracellular matrix. Cellular receptors for hyaluronan such as CD44 and RHAMM (receptor for hyaluronate-mediated motility) have also been identified. In the present study biotinylated hyaluronan (bHA) was prepared by reacting adipic dihydrazide with a 170 kDa hyaluronan sample using the bifunctional reagent 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide. The resultant free amine moeity of the hydrazido-hyaluronan was then reacted with biotin succinimidyl ester (sulfo-NHS-biotin) to prepare the bHA. After 4-20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotting to nitrocellulose membranes, bHA and avidin alkaline phosphatase conjugate could be used in conjunction with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrates to specifically visualise with high sensitivity (> or = 2 ng), bovine nasal cartilage link-protein, aggrecan hyaluronan binding region, and human fibroblast hyaluronan receptors such as CD-44. Conventional Western blotting using specific monoclonal antibodies to these proteins was also used to confirm the identities of these proteins. PMID:8907541

  3. A Lanthanum-Tagged Chemotherapeutic Agent HA-Pt to Track the In Vivo Distribution of Hyaluronic Acid Complexes

    PubMed Central

    Forrest, W.C.; Cai, Shuang; Aires, Daniel; Forrest, M. Laird

    2015-01-01

    Hyaluronic acid drug conjugates can target anti-cancer drugs directly to tumor tissue for loco-regional treatment with enhanced bioavailability, local efficacy and reduced toxicity. In this study, the distribution and pharmacokinetics of hyaluronic acid carrier and a conjugated cisplatin anti-cancer drug were tracked by lanthanum (III) [La(III)] affinity tagging of the nanocarrier. The strong binding affinity of La(III) to HA enabled the simple preparation of a physiologically stable complex HA-Pt-La and straightforward simultaneous detection of HA-La and Pt in biological matrices using inductively coupled plasma-mass spectrometry (ICP-MS). Consequently, after subcutaneous injection of HA-Pt-La nanoparticles in human head and neck squamous cell carcinoma (HNSCC) tumor-bearing mice, the HA and Pt content were detected and quantified simultaneously in the plasma, primary tumor, liver and spleen. PMID:26756040

  4. Attachment of hyaluronan to metallic surfaces.

    PubMed

    Pitt, William G; Morris, Robert N; Mason, Mitchell L; Hall, Matthew W; Luo, Yi; Prestwich, Glenn D

    2004-01-01

    Metal implants are in general not compatible with the tissues of the human body, and in particular, blood exhibits a severe hemostatic response. Herein we present results of a technique to mask the surface of metals with a natural biopolymer, hyaluronan (HA). HA has minimal adverse interactions with blood and other tissues, but attachment of bioactive peptides can promote specific biological interactions. In this study, stainless steel was cleaned and then surface-modified by covalent attachment of an epoxy silane. The epoxy was subsequently converted to an aldehyde functional group and reacted with hyaluronan through an adipic dihydrazide linkage, thus covalently immobilizing the HA onto the steel surface. Fluorescent labeling of the HA showed that the surface had a fairly uniform covering of HA. When human platelet rich plasma was placed on the HA-coated surface, there was no observable adhesion of platelets. HA derivatized with a peptide containing the RGD peptide sequence was also bound to the stainless steel. The RGD-containing peptide was bioactive as exemplified by the attachment and spreading of platelets on this surface. Furthermore, when the RGD peptide was replaced with the nonsense RDG sequence, minimal adhesion of platelets was observed. This type of controlled biological activity on a metal surface has potential for modulating cell growth and cellular interactions with metallic implants, such as vascular stents, orthopedic implants, heart valve cages, and more. PMID:14661254

  5. The high and low molecular weight forms of hyaluronan have distinct effects on CD44 clustering.

    PubMed

    Yang, Cuixia; Cao, Manlin; Liu, Hua; He, Yiqing; Xu, Jing; Du, Yan; Liu, Yiwen; Wang, Wenjuan; Cui, Lian; Hu, Jiajie; Gao, Feng

    2012-12-14

    CD44 is a major cell surface receptor for the glycosaminoglycan hyaluronan (HA). Native high molecular weight hyaluronan (nHA) and oligosaccharides of hyaluronan (oHA) provoke distinct biological effects upon binding to CD44. Despite the importance of such interactions, however, the feature of binding with CD44 at the cell surface and the molecular basis for functional distinction between different sizes of HA is still unclear. In this study we investigated the effects of high and low molecular weight hyaluronan on CD44 clustering. For the first time, we provided direct evidence for a strong relationship between HA size and CD44 clustering in vivo. In CD44-transfected COS-7 cells, we showed that exogenous nHA stimulated CD44 clustering, which was disrupted by oHA. Moreover, naturally expressed CD44 was distributed into clusters due to abundantly expressed nHA in HK-2 cells (human renal proximal tubule cells) and BT549 cells (human breast cancer cell line) without exogenous stimulation. Our results suggest that native HA binding to CD44 selectively induces CD44 clustering, which could be inhibited by oHA. Finally, we demonstrated that HA regulates cell adhesion in a manner specifically dependent on its size. oHA promoted cell adhesion while nHA showed no effects. Our results might elucidate a molecular- and/or cellular-based mechanism for the diverse biological activities of nHA and oHA.

  6. Hyaluronan Participates in the Epidermal Response to Disruption of the Permeability Barrier in Vivo

    PubMed Central

    Maytin, Edward V.; Chung, Helen H.; Seetharaman, V. Mani

    2004-01-01

    Hyaluronan (hyaluronic acid, HA) is a glycosaminoglycan in the extracellular matrix of tissues that plays a role in cellular migration, proliferation and differentiation. Injury to the stratum corneum elicits an epidermal hyperproliferative response, a pathogenic feature in many cutaneous diseases including eczema and psoriasis. Because HA is abundant in the matrix between keratinocytes, we asked whether the presence of HA is required for epidermal hyperplasia to occur in response to barrier injury. Disruption of the stratum corneum, by acetone application on the skin of hairless mice, led to a marked accumulation of HA in the matrix between epidermal basal and spinous keratinocytes, and also within keratinocytes of the upper epidermis. To test whether HA may have a functional role in epidermal hyperplasia, we used Streptomyces hyaluronidase (StrepH), delivered topically, to degrade epidermal HA and blunt the accumulation of epidermal HA after acetone. StrepH signficantly reduced epidermal HA levels, and also significantly inhibited the development of epidermal hyperplasia. This reduction in epidermal thickness was not attributable to any decrease in keratinocyte proliferation, but rather to an apparent acceleration in terminal differentiation (ie, increased keratin 10 and filaggrin expression). Overall, the data show that HA is a significant participant in the epidermal response to barrier injury. PMID:15466397

  7. Regulated Hyaluronan Synthesis by Vascular Cells

    PubMed Central

    Viola, Manuela; Karousou, Evgenia; D'Angelo, Maria Luisa; Caon, Ilaria; De Luca, Giancarlo; Passi, Alberto; Vigetti, Davide

    2015-01-01

    Cellular microenvironment plays a critical role in several pathologies including atherosclerosis. Hyaluronan (HA) content often reflects the progression of this disease in promoting vessel thickening and cell migration. HA synthesis is regulated by several factors, including the phosphorylation of HA synthase 2 (HAS2) and other covalent modifications including ubiquitination and O-GlcNAcylation. Substrate availability is important in HA synthesis control. Specific drugs reducing the UDP precursors are able to reduce HA synthesis whereas the hexosamine biosynthetic pathway (HBP) increases the concentration of HA precursor UDP-N-acetylglucosamine (UDP-GlcNAc) leading to an increase of HA synthesis. The flux through the HBP in the regulation of HA biosynthesis in human aortic vascular smooth muscle cells (VSMCs) was reported as a critical aspect. In fact, inhibiting O-GlcNAcylation reduced HA production whereas increased O-GlcNAcylation augmented HA secretion. Additionally, O-GlcNAcylation regulates HAS2 gene expression resulting in accumulation of its mRNA after induction of O-GlcNAcylation with glucosamine treatments. The oxidized LDLs, the most common molecules related to atherosclerosis outcome and progression, are also able to induce a strong HA synthesis when they are in contact with vascular cells. In this review, we present recent described mechanisms involved in HA synthesis regulation and their role in atherosclerosis outcome and development. PMID:26448750

  8. Molecular evolution of the hyaluronan synthase 2 gene in mammals: implications for adaptations to the subterranean niche and cancer resistance.

    PubMed

    Faulkes, Christopher G; Davies, Kalina T J; Rossiter, Stephen J; Bennett, Nigel C

    2015-05-01

    The naked mole-rat (NMR) Heterocephalus glaber is a unique and fascinating mammal exhibiting many unusual adaptations to a subterranean lifestyle. The recent discovery of their resistance to cancer and exceptional longevity has opened up new and important avenues of research. Part of this resistance to cancer has been attributed to the fact that NMRs produce a modified form of hyaluronan--a key constituent of the extracellular matrix--that is thought to confer increased elasticity of the skin as an adaptation for living in narrow tunnels. This so-called high molecular mass hyaluronan (HMM-HA) stems from two apparently unique substitutions in the hyaluronan synthase 2 enzyme (HAS2). To test whether other subterranean mammals with similar selection pressures also show molecular adaptation in their HAS2 gene, we sequenced the HAS2 gene for 11 subterranean mammals and closely related species, and combined these with data from 57 other mammals. Comparative screening revealed that one of the two putatively important HAS2 substitutions in the NMR predicted to have a significant effect on hyaluronan synthase function was uniquely shared by all African mole-rats. Interestingly, we also identified multiple other amino acid substitutions in key domains of the HAS2 molecule, although the biological consequences of these for hyaluronan synthesis remain to be determined. Despite these results, we found evidence of strong purifying selection acting on the HAS2 gene across all mammals, and the NMR remains unique in its particular HAS2 sequence. Our results indicate that more work is needed to determine whether the apparent cancer resistance seen in NMR is shared by other members of the African mole-rat clade.

  9. Design and syntheses of hyaluronan oligosaccharide conjugates as inhibitors of CD44-Hyaluronan binding

    PubMed Central

    Huang, Xuefei

    2016-01-01

    Hyaluronan (HA) is an integral component of the extracellular matrix. Its interactions with a cell surface receptor CD44 has been shown to play important roles in a variety of biological events including cell proliferation and metastasis. As multivalent CD44-HA binding is critical for downstream signaling, compounds that can selectively disrupt the complex formation of HA polysaccharide with CD44 can serve as useful probes of CD44 mediated cellular events as well as potential leads for novel therapeutics. Herein, we report the synthesis of several series of HA conjugates to target the HA binding pocket of CD44. As a small library of HA disaccharide derivatives failed to exhibit any inhibitory activities, we focused on HA tetrasaccharide based analogs. Traditional synthetic strategies towards HA oligosaccharides involve the construction of backbone from the corresponding monosaccharide building blocks, which can be quite tedious. In order to expedite the synthesis, we designed a new synthetic route taking advantage of the ability of hyaluronidase to generate large quantities of HA tetrasaccharide through digestion of HA polysaccharides. The HA tetrasaccharide obtained was utilized to prepare multiple S-linked HA analogs bearing aromatic groups at the reducing end glycan. One such compound containing an m-benzyl phenyl moiety exhibited significant inhibition of CD44-HA binding. Our approach provides a new direction towards the design of HA based CD44 antagonists. PMID:25997408

  10. Hyaluronan and Stone Disease

    NASA Astrophysics Data System (ADS)

    Asselman, Marino

    2008-09-01

    Kidney stones cannot be formed as long as crystals are passed in the urine. However, when crystals are retained it becomes possible for them to aggregate and form a stone. Crystals are expected to be formed not earlier than the distal tubules and collecting ducts. Studies both in vitro and in vivo demonstrate that calcium oxalate monohydrate crystals do not adhere to intact distal epithelium, but only when the epithelium is proliferating or regenerating, so that it possesses dedifferentiated cells expressing hyaluronan, osteopontin (OPN) and their mutual receptor CD44 at the apical cell membrane. The polysaccharide hyaluronan is an excellent crystal binding molecule because of its negative ionic charge. We hypothesized that the risk for crystal retention in the human kidney would be increased when tubular cells express hyaluronan at their apical cell membrane. Two different patient categories in which nephrocalcinosis frequently occurs were studied to test this hypothesis (preterm neonates and kidney transplant patients). Hyaluronan (and OPN) expression at the luminal membrane of tubular cells indeed was observed, which preceded subsequent retention of crystals in the distal tubules. Tubular nephrocalcinosis has been reported to be associated with decline of renal function and thus further studies to extend our knowledge of the mechanisms of retention and accumulation of crystals in the kidney are warranted. Ultimately, this may allow the design of new strategies for the prevention and treatment of both nephrocalcinosis and nephrolithiasis in patients.

  11. Glycosylation of CD44 negatively regulates its recognition of hyaluronan

    PubMed Central

    1995-01-01

    Although CD44 is expressed on a wide variety of cell types, few of them use it to recognize the ligand hyaluronan (HA). A glycosylation- defective clone of Chinese hamster ovary cells (Lec 8) bound HA, demonstrating that complete processing of glycoproteins with addition of a full complement of sialic acid is not required. On the contrary, subsequent findings revealed that complex sugars on CD44 can actually inhibit ligand recognition. Two subclones of wild-type Chinese hamster ovary cells with similar amounts of surface CD44 were isolated on the basis of HA binding and found to differ with respect to CD44 size as well as staining with fluorescent lectins. Treatment of the nonbinding clone with tunicamycin reduced the size of the protein and allowed the cells to recognize HA via CD44. This function was also induced by treatment with deglycosylating enzymes (either a mixture of endoglycosidase F and N-glycosidase F or neuraminidase alone). A possible role for glycosylation in regulation of adhesion was then sought with a series of normal and transformed murine cells. Disruption of glycosylation or treatment with deglycosylating enzymes did not induce ligand binding in an interleukin 7-dependent pre-B cell line, and splenic B cells also appeared to be in an inactive state. Some normal B cells acquired the ability to recognize HA after stimulation with lipopolysaccharide or interleukin 5 and had distinctive surface characteristics (loss of immunoglobulin D and acquisition of CD43). An additional subset of activated cells might have been in a transitional state, because the cells bound ligand after neuraminidase treatment. The ligand-binding ability of a purified CD44-immunoglobulin fusion protein dramatically increased after neuraminidase treatment. Thus, differential glycosylation of this molecule is sufficient to influence its recognition function. Cell adhesion involving HA can be regulated by multiple mechanisms, one of which involves variable glycosylation of CD

  12. Chromosomal localization of the human and mouse hyaluronan synthase genes

    SciTech Connect

    Spicer, A.P.; McDonald, J.A.; Seldin, M.F.

    1997-05-01

    We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

  13. N-Terminal signal sequence is required for cellular trafficking and hyaluronan-depolymerization of KIAA1199.

    PubMed

    Yoshida, Hiroyuki; Nagaoka, Aya; Nakamura, Sachiko; Tobiishi, Megumi; Sugiyama, Yoshinori; Inoue, Shintaro

    2014-01-01

    Recently, we disclosed that KIAA1199-mediated hyaluronan (HA) depolymerization requires an acidic cellular microenvironment (e.g. clathrin-coated vesicles or early endosomes), but no information about the structural basis underlying the cellular targeting and functional modification of KIAA1199 was available. Here, we show that the cleavage of N-terminal 30 amino acids occurs in functionally matured KIAA1199, and the deletion of the N-terminal portion results in altered intracellular trafficking of the molecule and loss of cellular HA depolymerization. These results suggest that the N-terminal portion of KIAA1199 functions as a cleavable signal sequence required for proper KIAA1199 translocation and KIAA1199-mediated HA depolymerization. PMID:24269685

  14. Effects Of Polylactic Acid Coating and Compression Load on the Delivery of Protein and Steroid from HA Ceramic Devices.

    PubMed

    Benghuzzi, Hamed; Tucci, Michelle A; Ibrahim, Jamil

    2015-01-01

    Density variations, due to particle size and time and temperature of sintering, affect the delivery profile of substances from ceramic delivery devices. This investigation was conducted to study the effect of polylactic acid (PLA) impregnation on the porosity of hydroxyapatite (HA) capsules by studying the delivery rate of bovine serum albumin (BSA) and testosterone (TE). HA capsules were fabricated by cold compressing calcined particles at 1000, 3000, and 5000 lbs. Each group was subdivided into PLA-impregnated and non-PLA impregnated capsules. Each capsule was loaded with either 40 mg of TE (impregnated or nonimpregnated with PLA polymer) or BSA. Data obtained in this study suggest that: (1) PLA impregnation of HA ceramic capsules decreases the rate of release of drugs from the ceramic reservoir, (2) Physiochemical characteristics of the drugs to be delivered is an instrumental key in the sustained release profiles, (3) Compression load of HA reservoirs is a key factor in predicting the duration and level of sustained delivery, and (4) Polymer coating of HA ceramic capsules reduces the pore size, as well as, blocking some of the pores on the surface.

  15. Involvement of lipoxygenase in lysophosphatidic acid-stimulated hydrogen peroxide release in human HaCaT keratinocytes.

    PubMed Central

    Sekharam, M; Cunnick, J M; Wu, J

    2000-01-01

    Although it is now recognized that low levels of reactive oxygen species (ROS) are required for the mitogenic response, mitogen-induced signalling pathways that regulate ROS generation in non-phagocytic cells remain largely uncharacterized. Using a real-time assay for measuring hydrogen peroxide (H(2)O(2)) formation, we analysed H(2)O(2) release in human HaCaT keratinocytes in response to lysophosphatidic acid (LPA), a mitogen for keratinocytes. LPA rapidly increased H(2)O(2) release in HaCaT cells. Unlike LPA-induced mitogen-activated protein (MAP) kinase activation, LPA-stimulated H(2)O(2) release was independent of the tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Calcium chelators, phospholipase A(2) inhibitors, and lipoxygenase inhibitors effectively blocked LPA-stimulated H(2)O(2) release, whereas cyclooxygenase inhibitors were without effect. Addition of 5-lipoxygenase products 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene B(4), but not 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene C(4), restored LPA-stimulated H(2)O(2) release in cells treated with the lipoxygenase inhibitors nordihydroguaiaretic acid and Zileuton. These results suggest that the lipoxygenase products 5-HPETE and leukotriene B(4) are required for LPA-stimulated H(2)O(2) release in HaCaT cells. PMID:10698703

  16. Hyaluronan synthases and hyaluronidases in nasal polyps.

    PubMed

    Panogeorgou, T; Tserbini, E; Filou, S; Vynios, D H; Naxakis, S S; Papadas, T A; Goumas, P D; Mastronikolis, N S

    2016-07-01

    Nasal polyps (NPs) are benign lesions of nasal and paranasal sinuses mucosa affecting 1-4 % of all adults. Nasal polyposis affects the quality of patient's life as it causes nasal obstruction, postnasal drainage, purulent nasal discharge, hyposmia or anosmia, chronic sinusitis, facial pain and snoring. Without treatment, the disease can alter the craniofacial skeleton in cases of extended growth of polyps. The development of NPs is caused by the hyperplasia of nasal or paranasal sinuses mucosa, and edema of extracellular matrix. This is usually the result of high concentration of high molecular mass hyaluronan (HA) which is either overproduced or accumulated from blood supply. The size of HA presents high diversity and, especially in pathologic conditions, chains of low molecular mass can be observed. In NPs, chains of about 200 kDa have been identified and considered to be responsible for the inflammation. The purpose of the present study was the investigation, in NPs and normal nasal mucosa (NM), of the expression of the wild-type and alternatively spliced forms of hyaluronidases, their immunolocalization, and the expression of HA synthases to examine the isoform(s) responsible for the increased amounts of HA in NPs. Hyaluronidases' presence was examined on mRNA (RT-PCR analysis) and protein (immunohistochemistry) levels. Hyaluronan synthases' presence was examined on mRNA levels. Hyaluronidases were localized in the cytoplasm of epithelial and inflammatory cells, as well as in the matrix. On mRNA level, it was found that hyal-1-wt was decreased in NPs compared to NM and hyal-1-v3, -v4 and -v5 were substantially increased. Moreover, HAS2 and HAS3 were the only hyaluronan synthases detected, the expression of which was almost similar in NPs and NM. Overall, the results of the present study support that hyaluronidases are the main enzymes responsible for the decreased size of hyaluronan observed in NPs; thus they behave as inflammatory agents. Therefore, they

  17. The Roles of Hyaluronan/RHAMM/CD44 and Their Respective Interactions along the Insidious Pathways of Fibrosarcoma Progression

    PubMed Central

    Nikitovic, Dragana; Kouvidi, Katerina; Karamanos, Nikos K.; Tzanakakis, George N.

    2013-01-01

    Fibrosarcomas are rare malignant mesenchymal tumors originating from fibroblasts. Importantly, fibrosarcoma cells were shown to have a high content and turnover of extracellular matrix (ECM) components including hyaluronan (HA), proteoglycans, collagens, fibronectin, and laminin. ECMs are complicated structures that surround and support cells within tissues. During cancer progression, significant changes can be observed in the structural and mechanical properties of the ECM components. Importantly, hyaluronan deposition is usually higher in malignant tumors as compared to benign tissues, predicting tumor progression in some tumor types. Furthermore, activated stromal cells are able to produce tissue structure rich in hyaluronan in order to promote tumor growth. Key biological roles of HA result from its interactions with its specific CD44 and RHAMM (receptor for HA-mediated motility) cell-surface receptors. HA-receptor downstream signaling pathways regulate in turn cellular processes implicated in tumorigenesis. Growth factors, including PDGF-BB, TGFβ2, and FGF-2, enhanced hyaluronan deposition to ECM and modulated HA-receptor expression in fibrosarcoma cells. Indeed, FGF-2 through upregulation of specific HAS isoforms and hyaluronan synthesis regulated secretion and net hyaluronan deposition to the fibrosarcoma pericellular matrix modulating these cells' migration capability. In this paper we discuss the involvement of hyaluronan/RHAMM/CD44 mediated signaling in the insidious pathways of fibrosarcoma progression. PMID:24083250

  18. Interaction of Hyaluronan with Cationic Nanoparticles.

    PubMed

    Bano, Fouzia; Carril, Mónica; Di Gianvincenzo, Paolo; Richter, Ralf P

    2015-08-01

    The polysaccharide hyaluronan (HA) is a main component of peri- and extracellular matrix, and an attractive molecule for materials design in tissue engineering and nanomedicine. Here, we study the morphology of complexes that form upon interaction of nanometer-sized amine-coated gold particles with this anionic, linear, and regular biopolymer in solution and grafted to a surface. We find that cationic nanoparticles (NPs) have profound effects on HA morphology on the molecular and supramolecular scale. Quartz crystal microbalance (QCM-D) shows that depending on their relative abundance, cationic NPs promote either strong compaction or swelling of films of surface-grafted HA polymers (HA brushes). Transmission electron and atomic force microscopy reveal that the NPs do also give rise to complexes of distinct morphologies-compact nanoscopic spheres and extended microscopic fibers-upon interaction with HA polymers in solution. In particular, stable and hydrated spherical complexes of single HA polymers with NPs can be prepared when balancing the ionizable groups on HA and NPs. The observed self-assembly phenomena could be useful for the design of drug delivery vehicles and a better understanding of the reorganization of HA-rich synthetic or biological matrices. PMID:26146006

  19. Interaction of Hyaluronan with Cationic Nanoparticles.

    PubMed

    Bano, Fouzia; Carril, Mónica; Di Gianvincenzo, Paolo; Richter, Ralf P

    2015-08-01

    The polysaccharide hyaluronan (HA) is a main component of peri- and extracellular matrix, and an attractive molecule for materials design in tissue engineering and nanomedicine. Here, we study the morphology of complexes that form upon interaction of nanometer-sized amine-coated gold particles with this anionic, linear, and regular biopolymer in solution and grafted to a surface. We find that cationic nanoparticles (NPs) have profound effects on HA morphology on the molecular and supramolecular scale. Quartz crystal microbalance (QCM-D) shows that depending on their relative abundance, cationic NPs promote either strong compaction or swelling of films of surface-grafted HA polymers (HA brushes). Transmission electron and atomic force microscopy reveal that the NPs do also give rise to complexes of distinct morphologies-compact nanoscopic spheres and extended microscopic fibers-upon interaction with HA polymers in solution. In particular, stable and hydrated spherical complexes of single HA polymers with NPs can be prepared when balancing the ionizable groups on HA and NPs. The observed self-assembly phenomena could be useful for the design of drug delivery vehicles and a better understanding of the reorganization of HA-rich synthetic or biological matrices.

  20. Hyaluronan content in experimental carcinoma is not correlated to interstitial fluid pressure.

    PubMed

    Jacobson, Annica; Salnikov, Alexei; Lammerts, Ellen; Roswall, Pernilla; Sundberg, Christian; Heldin, Paraskevi; Rubin, Kristofer; Heldin, Nils-Erik

    2003-06-13

    Mechanism(s) for generation of the high tumor interstitial fluid pressure (TIFP) that is characteristic of carcinoma is not known. We investigated the role of hyaluronan, the major water-binding polysaccharide of the extracellular matrix, for the generation of a high TIFP. A human anaplastic thyroid carcinoma (KAT-4) xenografted to athymic mice and a syngeneic rat colon carcinoma (PROb) were used. Neither KAT-4 nor PROb cells produced hyaluronan (HA) in culture, however, both cell lines produced factors that stimulated HA-synthesis by cultured fibroblasts. Modulating hyaluronan levels by transfection of PROb carcinoma cells with hyaluronan synthase-2 revealed no correlation between hyaluronan content and TIFP. Furthermore, lowering of TIFP by treating KAT-4 tumors with a specific inhibitor of TGF-beta 1 and -beta 3 did not change the concentration of hyaluronan in the tumors. In summary, our results suggest that a modulation of hyaluronan content is not a major pathogenetic mechanism for the generation of the characteristically high TIFP in malignant carcinomas.

  1. Hyaluronan-coated extracellular vesicles--a novel link between hyaluronan and cancer.

    PubMed

    Rilla, Kirsi; Siiskonen, Hanna; Tammi, Markku; Tammi, Raija

    2014-01-01

    The synthesis of hyaluronan (HA) on the plasma membrane is a unique and still partly mysterious way of macromolecular biosynthesis. HA forms pericellular coats around many cell types and accumulates in the extracellular matrix (ECM) of growing and renewing tissues. It is secreted to high concentrations in body fluids with antifriction properties like pleural, peritoneal, and synovial fluids, but is also detectable in plasma, saliva, and urine. In pathological states, like cancer and inflammation, the amount of HA is increased around cells, in the ECM, and in the body fluids. HA is an indicator of poor prognosis for cancer patients and creates a favorable environment for cellular growth and motility. The recent finding that HA-coated extracellular vesicles act both as a product of HA synthase activity and as special vehicles for HA, and perhaps carry signals important for malignant growth, provides a novel link between HA and cancer. HA could be carried on the surface of these vesicles in tissues and body fluids, creating beneficial environments by itself, or by associated molecules, for the invasion and metastasis of cancer cells. The HA-coated plasma membrane protrusions and vesicles shed from them are potential biomarkers in cancer and other HA-associated disease states. PMID:25081528

  2. Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi

    PubMed Central

    Peng, Huan; Luo, Shujie; Huang, Wenkun; Cui, Jiangkuan; Li, Xin; Kong, Lingan; Jiang, Daohong; Chitwood, David J.; Peng, Deliang

    2016-01-01

    Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 (KU877266), Ha-far-2 (KU877267), Hf-far-1 (KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in

  3. Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi.

    PubMed

    Qiao, Fen; Luo, Lilian; Peng, Huan; Luo, Shujie; Huang, Wenkun; Cui, Jiangkuan; Li, Xin; Kong, Lingan; Jiang, Daohong; Chitwood, David J; Peng, Deliang

    2016-01-01

    Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 (KU877266), Ha-far-2 (KU877267), Hf-far-1 (KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in

  4. Molecular evolution of the hyaluronan synthase 2 gene in mammals: implications for adaptations to the subterranean niche and cancer resistance

    PubMed Central

    Faulkes, Christopher G.; Davies, Kalina T. J.; Rossiter, Stephen J.; Bennett, Nigel C.

    2015-01-01

    The naked mole-rat (NMR) Heterocephalus glaber is a unique and fascinating mammal exhibiting many unusual adaptations to a subterranean lifestyle. The recent discovery of their resistance to cancer and exceptional longevity has opened up new and important avenues of research. Part of this resistance to cancer has been attributed to the fact that NMRs produce a modified form of hyaluronan—a key constituent of the extracellular matrix—that is thought to confer increased elasticity of the skin as an adaptation for living in narrow tunnels. This so-called high molecular mass hyaluronan (HMM-HA) stems from two apparently unique substitutions in the hyaluronan synthase 2 enzyme (HAS2). To test whether other subterranean mammals with similar selection pressures also show molecular adaptation in their HAS2 gene, we sequenced the HAS2 gene for 11 subterranean mammals and closely related species, and combined these with data from 57 other mammals. Comparative screening revealed that one of the two putatively important HAS2 substitutions in the NMR predicted to have a significant effect on hyaluronan synthase function was uniquely shared by all African mole-rats. Interestingly, we also identified multiple other amino acid substitutions in key domains of the HAS2 molecule, although the biological consequences of these for hyaluronan synthesis remain to be determined. Despite these results, we found evidence of strong purifying selection acting on the HAS2 gene across all mammals, and the NMR remains unique in its particular HAS2 sequence. Our results indicate that more work is needed to determine whether the apparent cancer resistance seen in NMR is shared by other members of the African mole-rat clade. PMID:25948568

  5. Novel synthetic method for the preparation of amphiphilic hyaluronan by means of aliphatic aromatic anhydrides.

    PubMed

    Huerta-Angeles, Gloria; Bobek, Martin; Příkopová, Eva; Šmejkalová, Daniela; Velebný, Vladimír

    2014-10-13

    The present work describes a novel and efficient method of synthesis of amphiphilic hyaluronan (HA) by esterification with alkyl fatty acids. These derivatives were synthesized under mild aqueous and well controlled conditions using mixed aliphatic aromatic anhydrides. These anhydrides characterized by the general formula RCOOCOC6H2Cl3 can be easily prepared by the reaction of the corresponding fatty acid (R) with 2,4,6-trichlorobenzoyl chloride (TCBC) in the presence of triethylamine. The aliphatic aromatic anhydrides RCOOCOC6H2Cl3 then react with the polysaccharide and enable the synthesis of aliphatic acid esters of HA in good yields. No hydrolytic degradation of hyaluronic acid could be observed. Parameters controlling the degree of esterification were systematically studied. Fatty acids with different chain lengths can be introduced applying this methodology. The degree of substitution was decreasing with increasing length of hydrophobic chain. The reaction products were fully characterized by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), SEC-MALLS and chromatographic analyses. Although the esterified HA products exhibited aggregation in solution as demonstrated by NMR, microscopy and rheology, they were still water-soluble.

  6. Hyaluronan Synthase 1: A Mysterious Enzyme with Unexpected Functions

    PubMed Central

    Siiskonen, Hanna; Oikari, Sanna; Pasonen-Seppänen, Sanna; Rilla, Kirsi

    2015-01-01

    Hyaluronan synthase 1 (HAS1) is one of three isoenzymes responsible for cellular hyaluronan synthesis. Interest in HAS1 has been limited because its role in hyaluronan production seems to be insignificant compared to the two other isoenzymes, HAS2 and HAS3, which have higher enzymatic activity. Furthermore, in most cell types studied so far, the expression of its gene is low and the enzyme requires high concentrations of sugar precursors for hyaluronan synthesis, even when overexpressed in cell cultures. Both expression and activity of HAS1 are induced by pro-inflammatory factors like interleukins and cytokines, suggesting its involvement in inflammatory conditions. Has1 is upregulated in states associated with inflammation, like atherosclerosis, osteoarthritis, and infectious lung disease. In addition, both full length and splice variants of HAS1 are expressed in malignancies like bladder and prostate cancers, multiple myeloma, and malignant mesothelioma. Interestingly, immunostainings of tissue sections have demonstrated the role of HAS1 as a poor predictor in breast cancer, and is correlated with high relapse rate and short overall survival. Utilization of fluorescently tagged proteins has revealed the intracellular distribution pattern of HAS1, distinct from other isoenzymes. In all cell types studied so far, a high proportion of HAS1 is accumulated intracellularly, with a faint signal detected on the plasma membrane and its protrusions. Furthermore, the pericellular hyaluronan coat produced by HAS1 is usually thin without induction by inflammatory agents or glycemic stress and depends on CD44–HA interactions. These specific interactions regulate the organization of hyaluronan into a leukocyte recruiting matrix during inflammatory responses. Despite the apparently minor enzymatic activity of HAS1 under normal conditions, it may be an important factor under conditions associated with glycemic stress like metabolic syndrome, inflammation, and cancer. PMID

  7. Reaction of peroxynitrite with hyaluronan and related saccharides.

    PubMed

    Corsaro, Maria Michela; Pietraforte, Donatella; Di Lorenzo, Angela Serena; Minetti, Maurizio; Marino, Gennaro

    2004-04-01

    The effects of peroxynitrite on hyaluronan has been studied by using an integrated spectroscopical approach, namely electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and mass spectrometry (MS). The reaction has been performed with the polymer, the tetrasaccharide oligomer as well as with the monosaccharides N-acetylglucosamine and glucuronic acid. The outcome of the presence of molecular oxygen and carbon dioxide has been also evaluated. Although 1H-NMR and ESI-MS experiments did not revealed peroxynitrite-mediated modification of hyaluronan as well as of related saccharides, from spin-trapping EPR experiments it was concluded that peroxynitrite induce the formation of C-centered carbon radicals, most probably by the way of its hydroxyl radical-like reactivity. These EPR data support the oxidative pathway involved in the degradation of hyaluronan, a probable event in the development and progression of rheumatoid arthritis.

  8. Hyaluronan mediates airway hyperresponsiveness in oxidative lung injury

    PubMed Central

    Lazrak, Ahmed; Creighton, Judy; Yu, Zhihong; Komarova, Svetlana; Doran, Stephen F.; Aggarwal, Saurabh; Emala, Charles W.; Stober, Vandy P.; Trempus, Carol S.; Garantziotis, Stavros

    2015-01-01

    Chlorine (Cl2) inhalation induces severe oxidative lung injury and airway hyperresponsiveness (AHR) that lead to asthmalike symptoms. When inhaled, Cl2 reacts with epithelial lining fluid, forming by-products that damage hyaluronan, a constituent of the extracellular matrix, causing the release of low-molecular-weight fragments (L-HA, <300 kDa), which initiate a series of proinflammatory events. Cl2 (400 ppm, 30 min) exposure to mice caused an increase of L-HA and its binding partner, inter-α-trypsin-inhibitor (IαI), in the bronchoalveolar lavage fluid. Airway resistance following methacholine challenge was increased 24 h post-Cl2 exposure. Intratracheal administration of high-molecular-weight hyaluronan (H-HA) or an antibody against IαI post-Cl2 exposure decreased AHR. Exposure of human airway smooth muscle (HASM) cells to Cl2 (100 ppm, 10 min) or incubation with Cl2-exposed H-HA (which fragments it to L-HA) increased membrane potential depolarization, intracellular Ca2+, and RhoA activation. Inhibition of RhoA, chelation of intracellular Ca2+, blockade of cation channels, as well as postexposure addition of H-HA, reversed membrane depolarization in HASM cells. We propose a paradigm in which oxidative lung injury generates reactive species and L-HA that activates RhoA and Ca2+ channels of airway smooth muscle cells, increasing their contractility and thus causing AHR. PMID:25747964

  9. A single amino acid in the stalk region of the H1N1pdm influenza virus HA protein affects viral fusion, stability and infectivity.

    PubMed

    Cotter, Christopher R; Jin, Hong; Chen, Zhongying

    2014-01-01

    The 2009 H1N1 pandemic (H1N1pdm) viruses have evolved to contain an E47K substitution in the HA2 subunit of the stalk region of the hemagglutinin (HA) protein. The biological significance of this single amino acid change was investigated by comparing A/California/7/2009 (HA2-E47) with a later strain, A/Brisbane/10/2010 (HA2-K47). The E47K change was found to reduce the threshold pH for membrane fusion from 5.4 to 5.0. An inter-monomer salt bridge between K47 in HA2 and E21 in HA1, a neighboring highly conserved residue, which stabilized the trimer structure, was found to be responsible for the reduced threshold pH for fusion. The higher structural and acid stability of the HA trimer caused by the E47K change also conferred higher viral thermal stability and infectivity in ferrets, suggesting a fitness advantage for the E47K evolutionary change in humans. Our study indicated that the pH of HA fusion activation is an important factor for influenza virus replication and host adaptation. The identification of this genetic signature in the HA stalk region that influences vaccine virus thermal stability also has significant implications for influenza vaccine production.

  10. Carboxymethyl Hyaluronan-Stabilized Nanoparticles for Anticancer Drug Delivery

    PubMed Central

    Woodman, Jessica L.; Suh, Min Sung; Zhang, Jianxing; Kondaveeti, Yuvabharath; Burgess, Diane J.; White, Bruce A.; Prestwich, Glenn D.; Kuhn, Liisa T.

    2015-01-01

    Carboxymethyl hyaluronic acid (CMHA) is a semisynthetic derivative of HA that is recognized by HA binding proteins but contains an additional carboxylic acid on some of the 6-hydroxyl groups of the N-acetyl glucosamine sugar units. These studies tested the ability of CMHA to stabilize the formation of calcium phosphate nanoparticles and evaluated their potential to target therapy resistant, CD44+/CD24−/low human breast cancer cells (BT-474EMT). CMHA stabilized particles (nCaPCMHA) were loaded with the chemotherapy drug cis-diamminedichloroplatinum(II) (CDDP) to form nCaPCMHACDDP. nCaPCMHACDDP was determined to be poorly crystalline hydroxyapatite, 200 nm in diameter with a −43 mV zeta potential. nCaPCMHACDDP exhibited a two-day burst release of CDDP that tapered resulting in 86% release by 7 days. Surface plasmon resonance showed that nCaPCMHACDDP binds to CD44, but less effectively than CMHA or hyaluronan. nCaPCMHA-AF488 was taken up by CD44+/CD24− BT-474EMT breast cancer cells within 18 hours. nCaPCMHACDDP was as cytotoxic as free CDDP against the BT-474EMT cells. Subcutaneous BT-474EMT tumors were more reproducibly inhibited by a near tumor dose of 2.8 mg/kg CDDP than a 7 mg/kg dose nCaPCMHACDDP. This was likely due to a lack of distribution of nCaPCMHACDDP throughout the dense tumor tissue that limited drug diffusion. PMID:26448751

  11. Determination of Hyaluronan Molecular Mass Distribution in Human Breast Milk

    PubMed Central

    Yuan, Han; Amin, Ripal; Ye, Xin; De La Motte, Carol A.; Cowman, Mary K.

    2015-01-01

    Hyaluronan (HA) in human milk mediates host responses to microbial infection, via TLR4- and CD44-dependent signaling. Signaling by HA is generally size-specific. Because pure HA with average molecular mass (M) of 35 kDa can elicit a protective response in intestinal epithelial cells, it has been proposed that human milk HA may have a bioactive low M component. Here we report the size distribution of HA in human milk samples from twenty unique donors. A new method for HA analysis, employingion exchange (IEX) chromatography to fractionate HA by size, and specific quantification of each size fraction by competitive Enzyme Linked Sorbent Assay (ELSA), was developed. When separated into four fractions, milk HA with M ≤ 20 kDa, M ≈20-60 kDa, and M ≈ 60-110 kDa comprised an average of 1.5%, 1.4% and 2% of the total HA, respectively. The remaining 95% was HA with M≥110 kDa. Electrophoretic analysis of the higher M HA from thirteen samples showed nearly identical M distributions, with an average M of ∼440 kDa. This higher M HA component in human milk is proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low M HA components. PMID:25579786

  12. Glycolic Acid Silences Inflammasome Complex Genes, NLRC4 and ASC, by Inducing DNA Methylation in HaCaT Cells.

    PubMed

    Tang, Sheau-Chung; Yeh, Jih-I; Hung, Sung-Jen; Hsiao, Yu-Ping; Liu, Fu-Tong; Yang, Jen-Hung

    2016-03-01

    AHAs (α-hydroxy acids), including glycolic acid (GA), have been widely used in cosmetic products and superficial chemical peels. Inflammasome complex has been shown to play critical roles in inflammatory pathways in human keratinocytes. However, the anti-inflammatory mechanism of GA is still unknown. The aim of this study is to investigate the relationship between the expression of the inflammasome complex and epigenetic modification to elucidate the molecular mechanism of the anti-inflammatory effect of GA in HaCaT cells. We evaluated NLRP3, NLRC4, AIM2, and ASC inflammasome complex gene expression on real-time polymerase chain reaction (PCR). Methylation changes were detected in these genes following treatment with DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza) with or without the addition of GA using methylation-specific PCR (MSP). GA inhibited the expressions of these inflammasome complex genes, and the decreases in the expressions of mRNA were reversed by 5-Aza treatment. Methylation was detected in NLRC4 and ASC on MSP, but not in NLRP3 or AIM2. GA decreased NLRC4 and ASC gene expression by increasing not only DNA methyltransferase 3B (DNMT-3B) protein level, but also total DNMT activity. Furthermore, silencing of DNMT-3B (shDNMT-3B) increased the expressions of NLRC4 and ASC. Our data demonstrated that GA treatment induces hypermethylation of promoters of NLRC4 and ASC genes, which may subsequently lead to the hindering of the assembly of the inflammasome complex in HaCaT cells. These results highlight the anti-inflammatory potential of GA-containing cosmetic agents in human skin cells and demonstrate for the first time the role of aberrant hypermethylation in this process.

  13. Hyaluronan and the hyaluronan receptor RHAMM promote focal adhesion turnover and transient tyrosine kinase activity

    PubMed Central

    1994-01-01

    The molecular mechanisms whereby hyaluronan (HA) stimulates cell motility was investigated in a C-H-ras transformed 10T 1/2 fibroblast cell line (C3). A significant (p < 0.001) stimulation of C3 cell motility with HA (10 ng/ml) was accompanied by an increase in protein tyrosine phosphorylation as detected by anti-phosphotyrosine antibodies using immunoblot analysis and immunofluorescence staining of cells. Tyrosine phosphorylation of several proteins was found to be both rapid and transient with phosphorylation occurring within 1 min of HA addition and dissipating below control levels 10-15 min later. These responses were also elicited by an antibody generated against a peptide sequence within the HA receptor RHAMM. Treatment of cells with tyrosine kinase inhibitors (genistein, 10 micrograms/ml or herbimycin A, 0.5 micrograms/ml) or microinjection of anti-phosphotyrosine antibodies inhibited the transient protein tyrosine phosphorylation in response to HA as well as prevented HA stimulation of cell motility. To determine a link between HA-stimulated tyrosine phosphorylation and the resulting cell locomotion, cytoskeletal reorganization was examined in C3 cells plated on fibronectin and treated with HA or anti-RHAMM antibody. These agents caused a rapid assembly and disassembly of focal adhesions as revealed by immunofluorescent localization of vinculin. The time course with which HA and antibody induced focal adhesion turnover exactly paralleled the induction of transient protein tyrosine phosphorylation. In addition, phosphotyrosine staining colocalized with vinculin within structures in the lamellapodia of these cells. Notably, the focal adhesion kinase, pp125FAK, was rapidly phosphorylated and dephosphorylated after HA stimulation. These results suggest that HA stimulates locomotion via a rapid and transient protein tyrosine kinase signaling event mediated by RHAMM. They also provide a possible molecular basis for focal adhesion turnover, a process that is

  14. Hyaluronan decreases surfactant inactivation in vitro.

    PubMed

    Lu, Karen W; Goerke, Jon; Clements, John A; Taeusch, H William

    2005-02-01

    Hyaluronan (HA) is an anionic polymer and a constituent of alveolar fluid that can bind proteins, phospholipids, and water. Previous studies have established that nonionic polymers improve the surface activity of pulmonary surfactants by decreasing inactivation of surfactant. In this work, we investigate whether HA can also have beneficial effects when added to surfactants. We used a modified pulsating bubble surfactometer to measure mixtures of several commercially available pulmonary surfactants or native calf surfactant with and without serum inactivation. Surface properties such as equilibrium surface tension, minimum and maximum surface tensions on compression and expansion of a surface film, and degree of surface area reduction required to reach a surface tension of 10 mN/m were measured. In the presence of serum, addition of HA dramatically improved the surface activities of all four surfactants and in some cases in the absence of serum as well. These results indicate that HA reduces inactivation of surfactants caused by serum and add evidence that endogenous HAs may interact with alveolar surfactant under normal and abnormal conditions.

  15. Human milk hyaluronan enhances innate defense of the intestinal epithelium.

    PubMed

    Hill, David R; Rho, Hyunjin K; Kessler, Sean P; Amin, Ripal; Homer, Craig R; McDonald, Christine; Cowman, Mary K; de la Motte, Carol A

    2013-10-01

    Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human β-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human β-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hβ D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn.

  16. More than just a filler - the role of hyaluronan for skin homeostasis.

    PubMed

    Anderegg, Ulf; Simon, Jan C; Averbeck, Marco

    2014-05-01

    In recent years, hyaluronan (HA) has become an increasingly attractive substance as a non-immunogenic filler and scaffolding material in cosmetic dermatology. Despite its wide use for skin augmentation and rejuvenation, relatively little is known about the molecular structures and interacting proteins of HA in normal and diseased skin. However, a comprehensive understanding of cutaneous HA homeostasis is required for future the development of HA-based applications for skin regeneration. This review provides an update on HA-based structures, expression, metabolism and its regulation, function and pharmacological targeting of HA in skin.

  17. Hyaluronan Synthesis, Catabolism, and Signaling in Neurodegenerative Diseases.

    PubMed

    Sherman, Larry S; Matsumoto, Steven; Su, Weiping; Srivastava, Taasin; Back, Stephen A

    2015-01-01

    The glycosaminoglycan hyaluronan (HA), a component of the extracellular matrix, has been implicated in regulating neural differentiation, survival, proliferation, migration, and cell signaling in the mammalian central nervous system (CNS). HA is found throughout the CNS as a constituent of proteoglycans, especially within perineuronal nets that have been implicated in regulating neuronal activity. HA is also found in the white matter where it is diffusely distributed around astrocytes and oligodendrocytes. Insults to the CNS lead to long-term elevation of HA within damaged tissues, which is linked at least in part to increased transcription of HA synthases. HA accumulation is often accompanied by elevated expression of at least some transmembrane HA receptors including CD44. Hyaluronidases that digest high molecular weight HA into smaller fragments are also elevated following CNS insults and can generate HA digestion products that have unique biological activities. A number of studies, for example, suggest that both the removal of high molecular weight HA and the accumulation of hyaluronidase-generated HA digestion products can impact CNS injuries through mechanisms that include the regulation of progenitor cell differentiation and proliferation. These studies, reviewed here, suggest that targeting HA synthesis, catabolism, and signaling are all potential strategies to promote CNS repair.

  18. Eicosapentaenoic acid inhibits TNF-{alpha}-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

    SciTech Connect

    Kim, Hyeon Ho; Lee, Youngae; Eun, Hee Chul Chung, Jin Ho

    2008-04-04

    Eicosapentaenoic acid (EPA) is an omega-3 ({omega}-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-{kappa}B activation induced by tumor necrosis factor (TNF)-{alpha} or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-{alpha}-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-{alpha} induced MMP-9 expression by NF-{kappa}B-dependent pathway. Pretreatment of EPA inhibited TNF-{alpha}-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect I{kappa}B-{alpha} phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-{kappa}B. EPA inhibited TNF-{alpha}-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKK{alpha}-dependent event. Taken together, we demonstrate that EPA inhibits TNF-{alpha}-induced MMP-9 expression through inhibition of p38 and Akt activation.

  19. Evaluation of emulsion electrospun polycaprolactone/hyaluronan/epidermal growth factor nanofibrous scaffolds for wound healing.

    PubMed

    Wang, Zhenbei; Qian, Yuna; Li, Linhao; Pan, Lianhong; Njunge, Lucy W; Dong, Lili; Yang, Li

    2016-01-01

    Wound healing scaffolds provide cells with structural integrity and can also deliver biological agents to establish a skin tissue-specific microenvironment to regulate cell functions and to accelerate the healing process. In this study, we fabricated biodegradable nanofibrous scaffolds with an emulsion electrospinning technique. The scaffolds were composed of polycaprolactone, hyaluronan and encapsulating epidermal growth factor. The morphology and core-sheath structure of the nanofibers were characterized by scanning electron microscopy and transmission electron microscopy. The scaffolds were also characterized for chemical composition and hydrophilicity with a Fourier-transform infrared analysis, energy dispersive spectroscopy and the water contact angle. An in vitro model protein bovine serum albumin and epidermal growth factor release study was conducted to evaluate the sustained release potential of the core-sheath structured nanofibers with and without the hyaluronan component. Additionally, an in vitro cultivation of human skin keratinocytes (HaCaT) and fibroblasts on polycaprolactone/hyaluronan and polycaprolactone/hyaluronan-epidermal growth factor scaffolds showed a significant synergistic effect of hyaluronan and epidermal growth factor on cell proliferation and infiltration. Furthermore, there was an up-regulation of the wound-healing-related genes collagen I, collagen III and TGF-β in polycaprolactone/hyaluronan/epidermal growth factor scaffolds compared with control groups. In the full-thickness wound model, the enhanced regeneration of fully functional skin was facilitated by epidermal regeneration in the polycaprolactone/hyaluronan/epidermal growth factor treatment group. Our findings suggest that bioactivity and hemostasis of the hyaluronan-based nanofibrous scaffolds have the capability to encapsulate and control the release of growth factors that can serve as skin tissue engineering scaffolds for wound healing.

  20. Anti-obesity potential of enzymatic fragments of hyaluronan on high-fat diet-induced obesity in C57BL/6 mice.

    PubMed

    Park, Byong-Gon; Park, Yoon-Sun; Park, Joo Woong; Shin, Eunji; Shin, Woon-Seob

    2016-04-22

    Hyaluronan has diverse biological activities depending on its molecular size. The hyaluronan fragments (50 kDa) can decrease adipogenic differentiation in vitro. However, in vivo anti-obesitic effects of hyaluronan fragments have not been elucidated. Therefore, we examined the anti-obesity effects of hyaluronan fragments on high-fat diet induced obesity in C57BL/6 mice. Oral administration of hyaluronan fragments (200 mg/kg for 8 weeks) decreased body weight, adipose tissues, serum lipid (low-density lipoprotein cholesterol, triglyceride), and leptin level. Hyaluronan fragments decreased the hypertrophy of adipose tissue and ameliorated liver steatosis. The mRNA expression of leptin was reduced in adipocyte by treatment with hyaluronan fragments. Additionally, hyaluronan fragments enhanced the mRNA expression of PPAR-α and its target genes UCP-2 and decreased mRNA expression of PPAR- γ and fatty acid synthase in liver. In conclusions, hyaluronan fragments had marked effects on inhibiting the development of obesity in obese mice fed the high-fat diet. It suggested that enhancing PPAR-α and suppressing PPAR-γ expression are two possible mechanisms for the anti-obesitic effect of hyaluronan fragments.

  1. Efficient CD44-targeted magnetic resonance imaging (MRI) of breast cancer cells using hyaluronic acid (HA)-modified MnFe2O4 nanocrystals

    NASA Astrophysics Data System (ADS)

    Lee, Taeksu; Lim, Eun-Kyung; Lee, Jaemin; Kang, Byunghoon; Choi, Jihye; Park, Hyo Seon; Suh, Jin-Suck; Huh, Yong-Min; Haam, Seungjoo

    2013-04-01

    Targeted molecular imaging with hyaluronic acid (HA) has been highlighted in the diagnosis and treatment of CD44-overexpressing cancer. CD44, a receptor for HA, is closely related to the growth of cancer including proliferation, metastasis, invasion, and angiogenesis. For the efficient detection of CD44, we fabricated a few kinds of HA-modified MnFe2O4 nanocrystals (MNCs) to serve as specific magnetic resonance (MR) contrast agents (HA-MRCAs) and compared physicochemical properties, biocompatibility, and the CD44 targeting efficiency. Hydrophobic MNCs were efficiently phase-transferred using aminated polysorbate 80 (P80) synthesized by introducing spermine molecules on the hydroxyl groups of P80. Subsequently, a few kinds of HA-MRCAs were fabricated, conjugating different ratios of HA on the equal amount of phase-transferred MNCs. The optimized conjugation ratio of HA against magnetic content was identified to exhibit not only effective CD44 finding ability but also high cell viability through in vitro experiments. The results of this study demonstrate that the suggested HA-MRCA shows strong potential to be used for accurate tumor diagnosis.

  2. The Polyphenol Chlorogenic Acid Attenuates UVB-mediated Oxidative Stress in Human HaCaT Keratinocytes

    PubMed Central

    Cha, Ji Won; Piao, Mei Jing; Kim, Ki Cheon; Yao, Cheng Wen; Zheng, Jian; Kim, Seong Min; Hyun, Chang Lim; Ahn, Yong Seok; Hyun, Jin Won

    2014-01-01

    We investigated the protective effects of chlorogenic acid (CGA), a polyphenol compound, on oxidative damage induced by UVB exposure on human HaCaT cells. In a cell-free system, CGA scavenged 1,1-diphenyl-2-picrylhydrazyl radicals, superoxide anions, hydroxyl radicals, and intracellular reactive oxygen species (ROS) generated by hydrogen peroxide and ultraviolet B (UVB). Furthermore, CGA absorbed electromagnetic radiation in the UVB range (280–320 nm). UVB exposure resulted in damage to cellular DNA, as demonstrated in a comet assay; pre-treatment of cells with CGA prior to UVB irradiation prevented DNA damage and increased cell viability. Furthermore, CGA pre-treatment prevented or ameliorated apoptosis-related changes in UVB-exposed cells, including the formation of apoptotic bodies, disruption of mitochondrial membrane potential, and alterations in the levels of the apoptosis-related proteins Bcl-2, Bax, and caspase-3. Our findings suggest that CGA protects cells from oxidative stress induced by UVB radiation. PMID:24753819

  3. [Larval stages of Ascaris lumbricoides: hyaluronan-binding capacity].

    PubMed

    Ponce-León, Patricia; Foresto, Patricia; Valverde, Juana

    2009-03-01

    Hyaluronic acid has important functions in inflammatory and tissue reparation processes. Owing to the varied strategies of the parasites to evade the host's immune response, as well as the multiple functions and physiological importance of hyaluronic acid, the aim was to study the hyaluronan binding capacity by Ascaris lumbricoides larval stages. Larval concentrates were prepared by hatching A. lumbricoides eggs. The larvae were collected by the Baermann method. The test of serum soluble CD44 detection by Agregation Inhibition was modified. All the larval concentrates presented hyaluronan binding capacity. The obtained results allow to suppose the existence of an hyaluronic acid specific receptor in A. lumbricoides. This receptor eventually might compete with the usual receptors of the host. The parasite might use this mechanism to evade the immune response.

  4. The Rise and Fall of Hyaluronan in Respiratory Diseases

    PubMed Central

    Lauer, Mark E.; Dweik, Raed A.; Garantziotis, Stavros; Aronica, Mark A.

    2015-01-01

    In normal airways, hyaluronan (HA) matrices are primarily located within the airway submucosa, pulmonary vasculature walls, and, to a lesser extent, the alveoli. Following pulmonary injury, elevated levels of HA matrices accumulate in these regions, and in respiratory secretions, correlating with the extent of injury. Animal models have provided important insight into the role of HA in the onset of pulmonary injury and repair, generally indicating that the induction of HA synthesis is an early event typically preceding fibrosis. The HA that accumulates in inflamed airways is of a high molecular weight (>1600 kDa) but can be broken down into smaller fragments (<150 kDa) by inflammatory and disease-related mechanisms that have profound effects on HA pathobiology. During inflammation in the airways, HA is often covalently modified with heavy chains from inter-alpha-inhibitor via the enzyme tumor-necrosis-factor-stimulated-gene-6 (TSG-6) and this modification promotes the interaction of leukocytes with HA matrices at sites of inflammation. The clearance of HA and its return to normal levels is essential for the proper resolution of inflammation. These data portray HA matrices as an important component of normal airway physiology and illustrate its integral roles during tissue injury and repair among a variety of respiratory diseases. PMID:26448757

  5. Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

    PubMed

    Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

    2013-10-01

    Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

  6. A novel eliminase from a marine bacterium that degrades hyaluronan and chondroitin sulfate.

    PubMed

    Han, Wenjun; Wang, Wenshuang; Zhao, Mei; Sugahara, Kazuyuki; Li, Fuchuan

    2014-10-01

    Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ(4,5)HexUAα1-3GalNAc(6-O-sulfate)β1-4GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications.

  7. A Novel Eliminase from a Marine Bacterium That Degrades Hyaluronan and Chondroitin Sulfate*

    PubMed Central

    Han, Wenjun; Wang, Wenshuang; Zhao, Mei; Sugahara, Kazuyuki; Li, Fuchuan

    2014-01-01

    Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ4,5HexUAα1-3GalNAc(6-O-sulfate)β1-4GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications. PMID:25122756

  8. Structure of DPPC-hyaluronan interfacial layers - effects of molecular weight and ion composition.

    PubMed

    Wieland, D C Florian; Degen, Patrick; Zander, Thomas; Gayer, Sören; Raj, Akanksha; An, Junxue; Dėdinaitė, Andra; Claesson, Per; Willumeit-Römer, Regine

    2016-01-21

    Hyaluronan and phospholipids play an important role in lubrication in articular joints and provide in combination with glycoproteins exceptionally low friction coefficients. We have investigated the structural organization of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) Langmuir layers at the solution-air interface at different length scales with respect to the adsorption of hyaluronan (HA). This allows us to assemble a comprehensive picture of the adsorption and the resulting structures, and how they are affected by the molecular weight of HA and the presence of calcium ions. Brewster angle microscopy and grazing incident diffraction were used to determine the lateral structure at the micro- and macro scale. The data reveals an influence of HA on both the macro and micro structure of the DPPC Langmuir layer, and that the strength of this effect increases with decreasing molecular weight of HA and in presence of calcium ions. Furthermore, from X-ray reflectivity measurements we conclude that HA adsorbs to the hydrophilic part of DPPC, but data also suggest that two types of interfacial structures are formed at the interface. We argue that hydrophobic forces and electrostatic interactions play important rules for the association between DPPC and HA. Surface pressure area isotherms were used to determine the influence of HA on the phase behavior of DPPC while electrophoretic mobility measurements were used to gain insight into the binding of calcium ions to DPPC vesicles and hyaluronan.

  9. An alternative standard for Trolox-equivalent antioxidant-capacity estimation based on thiol antioxidants. Comparative 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] decolorization and rotational viscometry study regarding hyaluronan degradation.

    PubMed

    Hrabárová, Eva; Valachová, Katarína; Rapta, Peter; Soltés, Ladislav

    2010-09-01

    Comparison of the effectiveness of antioxidant activity of three thiol compounds, D-penicillamine, reduced L-glutathione, and 1,4-dithioerythritol, expressed as a radical-scavenging capacity based on the two independent methods, namely a decolorization 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] assay and a rotational viscometry, is reported. Particular concern was focused on the testing of potential free-radical scavenging effects of thiols against hyaluronan degradation, induced by hydroxyl radicals. A promising, solvent-independent, antioxidative function of 1,4-dithioerythritol, comparable to that of a standard compound, Trolox(®), was confirmed by the 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] assay. The new potential antioxidant 1,4-dithioerythritol exhibited very good solubility in a variety of solvents (e.g., H(2)O, EtOH, and DMSO) and could be widely accepted and used as an effective antioxidant standard instead of a routinely used Trolox(®) on 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] assay.

  10. Effect of a high molecular weight hyaluronic acid (HA) preparation on the stimulation of polymorphonulcear leukocytes (PMNL)

    SciTech Connect

    McNeil, J.; Chow, D.C.; Skosey, J.L.

    1986-03-01

    During the process of joint inflammation PMNL are attracted into the joint space by chemotactic agents and are stimulated by immune complexes, particular matter (eg, crystals, cartilage debris) and other phlogistic agents. This process occurs in an environment rich in HA. The authors have examined the effect of high molecular weight HA. They have examined the effect of high molecular weight HA upon PMNL stimulation. PMNL were isolated from human blood and stimulated with either opsonized zymosan or formyl-methionyl-leucyl-phenylalanine (fmlp). The authors assessed stimulation by measuring the ability of cell supernatants to promote the release of /sup 35/S from chips of rabbit articular cartilage labeled in vivo, and the enhancement of oxidation of (1-/sup 14/C)glucose to /sup 14/CO/sub 2/. Stimulation of cells with zym in the presence of HA, 0.125-2.5 mg/ml, resulted in enhanced /sup 35/S release (33-59% over zym alone) and /sup 14/CO/sub 2/ production (0.5-64%). However, HA failed to enhance responses when fmlp (+cytochalasin B) was used as the stimulus. It has been demonstrated that high molecular weight HA inhibits phagocytosis of both latex and aggregated IgG. In our studies, it is likely that HA interference with ingestion of zym leads to frustrated phagocytosis and enhancement of PMNL responses. Similar modification of responses of inflammatory mediator cells could occur in inflamed joints.

  11. Evaluation of bacterial cellulose/hyaluronan nanocomposite biomaterials.

    PubMed

    Li, Ying; Qing, Shuang; Zhou, Jianhai; Yang, Guang

    2014-03-15

    Bacterial cellulose (BC) is useful in the biomedical field because of its unique structure and properties. The high nano-porosity of BC allows other materials to be incorporated and form reinforced composites. Here we describe the preparation and characterization of novel BC/hyaluronan (HA) nanocomposites with a 3-D network structure. BC/HA was obtained using a solution impregnation method. Elemental and ATR-FTIR analyses showed that this method is highly effective to form composites with BC. Weight loss analysis showed that BC/HA have a lower water loss than BC at 37 °C. The total surface area and pore volume of BC/HA films gradually decreased with the HA content, as followed by FE-SEM analysis. The elongation at break of BC/HA films gradually increased as the HA content increased. Thermogravimetric analysis showed that the weight loss for the BC/HA composites were lower than for pure BC between 250 and 350 °C. The results of weight loss, elongation at break and thermal stability suggested that these novel BC/HA films could be applied potentially as wound dressing materials.

  12. A simple, nondestructive assay for bound hyaluronan.

    PubMed

    Johnston, J B

    2000-01-01

    A simple, convenient, nondestructive method is described for the quantitative determination of bound hyaluronan. The method is based on the binding of the cationic dye Toluidine Blue O to the D-glucuronate component of the hyaluronan repeat disaccharide. Quantification is accomplished without interference by the dye's metachromatic properties. The method is easily adapted to hyaluronan coated medical devices and should be useful to developers and manufacturers of such devices and coatings.

  13. The effects of hyaluronan and its fragments on lipid models exposed to UV irradiation.

    PubMed

    Trommer, Hagen; Wartewig, Siegfried; Böttcher, Rolf; Pöppl, Andreas; Hoentsch, Joachim; Ozegowski, Jörg H; Neubert, Reinhard H H

    2003-03-26

    The effects of hyaluronan and its degradation products on irradiation-induced lipid peroxidation were investigated. Liposomal skin lipid models with increasing complexity were used. Hyaluronan and its fragments were able to reduce the amount of lipid peroxidation secondary products quantified by the thiobarbituric acid (TBA) assay. The qualitative changes were studied by mass spectrometry. To elucidate the nature of free radical involvement electron paramagnetic resonance (EPR) studies were carried out. The influence of hyaluronan and its fragments on the concentration of hydroxyl radicals generated by the Fenton system was examined using the spin trapping technique. Moreover, the mucopolysaccharide's ability to react with stable radicals was checked. The quantification assay of 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) showed no concentration changes of the stable radical caused by hyaluronan. Hyaluronan was found to exhibit prooxidative effects in the Fenton assay in a concentration dependent manner. A transition metal chelation was proposed as a mechanism of this behavior. Considering human skin and its constant exposure to UV light and oxygen and an increased pool of iron in irradiated skin the administration of hyaluronan or its fragments in cosmetic formulations or sunscreens could be helpful for the protection of the human skin. PMID:12623198

  14. Effect of molecular weight and concentration of hyaluronan on cell proliferation and osteogenic differentiation in vitro

    SciTech Connect

    Zhao, Ningbo Wang, Xin Qin, Lei Guo, Zhengze Li, Dehua

    2015-09-25

    Hyaluronan (HA), the simplest glycosaminoglycan and a major component of the extracellular matrix, exists in various tissues. It is involved in some critical biological procedures, including cellular signaling, cell adhesion and proliferation, and cell differentiation. The effect of molecular weight (MW) and concentration of HA on cell proliferation and differentiation was controversial. In this study, we investigated the effect of MW and concentration of HA on the proliferation and osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro. Results showed that high MW HA decreased the cell adhesion rate in a concentration-dependant manner. The cell adhesion rate was decreased by increasing MW of HA. Cell proliferation was significantly enhanced by low MW HA (P < 0.05). The factorial analysis indicated that MW and concentration had an interactive effect on the cell adhesion rate and cell proliferation (P < 0.05). High MW HA increased the mRNA expressions of ALP, RUNX-2 and OCN. The higher the MW was, the higher the mRNA expressions were. The factorial analysis indicated that MW and concentration had an interactive effect on ALP mRNA expression (P < 0.05). HA of higher MW and higher concentration promoted bone formation. These findings provide some useful information in understanding the mechanism underlying the effect of MW and concentration of HA on cell proliferation and differentiation. - Highlights: • Effect of hyaluronan on cell proliferation and differentiation is evaluated in vitro. • Hyaluronan of low molecular weight increases cell proliferation. • Hyaluronan of high molecular weight promotes cell osteogenic differentiation. • Molecular weight and concentration of hyaluronan show interactive effect.

  15. Effectiveness of Losartan-Loaded Hyaluronic Acid (HA) Micelles for the Reduction of Advanced Hepatic Fibrosis in C3H/HeN Mice Model

    PubMed Central

    Thomas, Reju George; Moon, Myeong Ju; Kim, Jo Heon; Lee, Jae Hyuk; Jeong, Yong Yeon

    2015-01-01

    Advanced hepatic fibrosis therapy using drug-delivering nanoparticles is a relatively unexplored area. Angiotensin type 1 (AT1) receptor blockers such as losartan can be delivered to hepatic stellate cells (HSC), blocking their activation and thereby reducing fibrosis progression in the liver. In our study, we analyzed the possibility of utilizing drug-loaded vehicles such as hyaluronic acid (HA) micelles carrying losartan to attenuate HSC activation. Losartan, which exhibits inherent lipophilicity, was loaded into the hydrophobic core of HA micelles with a 19.5% drug loading efficiency. An advanced liver fibrosis model was developed using C3H/HeN mice subjected to 20 weeks of prolonged TAA/ethanol weight-adapted treatment. The cytocompatibility and cell uptake profile of losartan-HA micelles were studied in murine fibroblast cells (NIH3T3), human hepatic stellate cells (hHSC) and FL83B cells (hepatocyte cell line). The ability of these nanoparticles to attenuate HSC activation was studied in activated HSC cells based on alpha smooth muscle actin (α-sma) expression. Mice treated with oral losartan or losartan-HA micelles were analyzed for serum enzyme levels (ALT/AST, CK and LDH) and collagen deposition (hydroxyproline levels) in the liver. The accumulation of HA micelles was observed in fibrotic livers, which suggests increased delivery of losartan compared to normal livers and specific uptake by HSC. Active reduction of α-sma was observed in hHSC and the liver sections of losartan-HA micelle-treated mice. The serum enzyme levels and collagen deposition of losartan-HA micelle-treated mice was reduced significantly compared to the oral losartan group. Losartan-HA micelles demonstrated significant attenuation of hepatic fibrosis via an HSC-targeting mechanism in our in vitro and in vivo studies. These nanoparticles can be considered as an alternative therapy for liver fibrosis. PMID:26714035

  16. Effectiveness of Losartan-Loaded Hyaluronic Acid (HA) Micelles for the Reduction of Advanced Hepatic Fibrosis in C3H/HeN Mice Model.

    PubMed

    Thomas, Reju George; Moon, Myeong Ju; Kim, Jo Heon; Lee, Jae Hyuk; Jeong, Yong Yeon

    2015-01-01

    Advanced hepatic fibrosis therapy using drug-delivering nanoparticles is a relatively unexplored area. Angiotensin type 1 (AT1) receptor blockers such as losartan can be delivered to hepatic stellate cells (HSC), blocking their activation and thereby reducing fibrosis progression in the liver. In our study, we analyzed the possibility of utilizing drug-loaded vehicles such as hyaluronic acid (HA) micelles carrying losartan to attenuate HSC activation. Losartan, which exhibits inherent lipophilicity, was loaded into the hydrophobic core of HA micelles with a 19.5% drug loading efficiency. An advanced liver fibrosis model was developed using C3H/HeN mice subjected to 20 weeks of prolonged TAA/ethanol weight-adapted treatment. The cytocompatibility and cell uptake profile of losartan-HA micelles were studied in murine fibroblast cells (NIH3T3), human hepatic stellate cells (hHSC) and FL83B cells (hepatocyte cell line). The ability of these nanoparticles to attenuate HSC activation was studied in activated HSC cells based on alpha smooth muscle actin (α-sma) expression. Mice treated with oral losartan or losartan-HA micelles were analyzed for serum enzyme levels (ALT/AST, CK and LDH) and collagen deposition (hydroxyproline levels) in the liver. The accumulation of HA micelles was observed in fibrotic livers, which suggests increased delivery of losartan compared to normal livers and specific uptake by HSC. Active reduction of α-sma was observed in hHSC and the liver sections of losartan-HA micelle-treated mice. The serum enzyme levels and collagen deposition of losartan-HA micelle-treated mice was reduced significantly compared to the oral losartan group. Losartan-HA micelles demonstrated significant attenuation of hepatic fibrosis via an HSC-targeting mechanism in our in vitro and in vivo studies. These nanoparticles can be considered as an alternative therapy for liver fibrosis.

  17. Hyaluronan degrading silica nanoparticles for skin cancer therapy

    NASA Astrophysics Data System (ADS)

    Scodeller, P.; Catalano, P. N.; Salguero, N.; Duran, H.; Wolosiuk, A.; Soler-Illia, G. J. A. A.

    2013-09-01

    We report the first nanoformulation of Hyaluronidase (Hyal) and its enhanced adjuvant effect over the free enzyme. Hyaluronic acid (HA) degrading enzyme Hyal was immobilized on 250 nm silica nanoparticles (SiNP) maintaining specific activity of the enzyme via the layer-by-layer self-assembly technique. This process was characterized by dynamic light scattering (DLS), zeta potential, infrared and UV-Vis spectroscopy, transmission electron microscopy (TEM) and enzymatic activity measurements. The nanoparticles were tested in vivo as adjuvants of carboplatin (CP), peritumorally injected in A375 human melanoma bearing mice and compared with the non-immobilized enzyme, on the basis of equal enzymatic activity. Alcian Blue staining of A375 tumors indicated large overexpression of hyaluronan. At the end of the experiment, tumor volume reduction with SiNP-immobilized Hyal was significantly enhanced compared to non-immobilized Hyal. Field emission scanning electron microscopy (FE-SEM) images together with energy dispersive X-ray spectroscopy (EDS) spectra confirmed the presence of SiNP on the tumor. We mean a proof of concept: this extracellular matrix (ECM) degrading enzyme, immobilized on SiNP, is a more effective local adjuvant of cancer drugs than the non-immobilized enzyme. This could prove useful in future therapies using other or a combination of ECM degrading enzymes.We report the first nanoformulation of Hyaluronidase (Hyal) and its enhanced adjuvant effect over the free enzyme. Hyaluronic acid (HA) degrading enzyme Hyal was immobilized on 250 nm silica nanoparticles (SiNP) maintaining specific activity of the enzyme via the layer-by-layer self-assembly technique. This process was characterized by dynamic light scattering (DLS), zeta potential, infrared and UV-Vis spectroscopy, transmission electron microscopy (TEM) and enzymatic activity measurements. The nanoparticles were tested in vivo as adjuvants of carboplatin (CP), peritumorally injected in A375 human

  18. Effect of the administration of an oral hyaluronan formulation on clinical and biochemical parameters in young horses with osteochondrosis.

    PubMed

    Carmona, J U; Argüelles, D; Deulofeu, R; Martínez-Puig, D; Prades, M

    2009-01-01

    The aim of this study was to evaluate the clinical and biochemical effects of the administration of oral hyaluronan (Hyal-Joint [HJ]) on young horses with osteochondrosis (OC). Our hypotheses were that HJ administration is safe, would decrease the degree of synovial effusion and the concentration of nitric oxide (NO) and prostaglandin E2 (PGE2) in synovial fluid, and would increase the concentration of hyaluronic acid (HA) in plasma and synovial fluid. Eleven young horses with tarsocrural OC were included in a randomised, double-blinded, placebo-controlled pilot clinical trial. Six horses received 250 mg/day HJ for 60 days (T60) and five horses received a placebo. The initial values of the degree of synovial effusion, NO, PGE2 and HA concentrations in synovial fluid and HA concentration in plasma were obtained. The horses were evaluated in terms of the same parameters at the end of treatment (T60) and 30 days thereafter (T90). The differences between the groups for each of the parameters evaluated at T0, T60 and T90 were not significant. Nevertheless, the horses treated with HJ tended to show a lower score for synovial effusion as well as higher HA, NO and PGE2 concentrations in synovial fluid, but these differences were non-significant. At a dose of 250 mg/day, HJ did not produce any adverse clinical effects and was well tolerated by the horses.

  19. Sulfated hyaluronan improves bone regeneration of diabetic rats by binding sclerostin and enhancing osteoblast function.

    PubMed

    Picke, Ann-Kristin; Salbach-Hirsch, Juliane; Hintze, Vera; Rother, Sandra; Rauner, Martina; Kascholke, Christian; Möller, Stephanie; Bernhardt, Ricardo; Rammelt, Stefan; Pisabarro, M Teresa; Ruiz-Gómez, Gloria; Schnabelrauch, Matthias; Schulz-Siegmund, Michaela; Hacker, Michael C; Scharnweber, Dieter; Hofbauer, Christine; Hofbauer, Lorenz C

    2016-07-01

    Bone fractures in patients with diabetes mellitus heal poorly and require innovative therapies to support bone regeneration. Here, we assessed whether sulfated hyaluronan included in collagen-based scaffold coatings can improve fracture healing in diabetic rats. Macroporous thermopolymerized lactide-based scaffolds were coated with collagen including non-sulfated or sulfated hyaluronan (HA/sHA3) and inserted into 3 mm femoral defects of non-diabetic and diabetic ZDF rats. After 12 weeks, scaffolds coated with collagen/HA or collagen/sHA3 accelerated bone defect regeneration in diabetic, but not in non-diabetic rats as compared to their non-coated controls. At the tissue level, collagen/sHA3 promoted bone mineralization and decreased the amount of non-mineralized bone matrix. Moreover, collagen/sHA3-coated scaffolds from diabetic rats bound more sclerostin in vivo than the respective controls. Binding assays confirmed a high binding affinity of sHA3 to sclerostin. In vitro, sHA3 induced BMP-2 and lowered the RANKL/OPG expression ratio, regardless of the glucose concentration in osteoblastic cells. Both sHA3 and high glucose concentrations decreased the differentiation of osteoclastic cells. In summary, scaffolds coated with collagen/sHA3 represent a potentially suitable biomaterial to improve bone defect regeneration in diabetic conditions. The underlying mechanism involves improved osteoblast function and binding sclerostin, a potent inhibitor of Wnt signaling and osteoblast function. PMID:27131598

  20. Sulfated hyaluronan improves bone regeneration of diabetic rats by binding sclerostin and enhancing osteoblast function.

    PubMed

    Picke, Ann-Kristin; Salbach-Hirsch, Juliane; Hintze, Vera; Rother, Sandra; Rauner, Martina; Kascholke, Christian; Möller, Stephanie; Bernhardt, Ricardo; Rammelt, Stefan; Pisabarro, M Teresa; Ruiz-Gómez, Gloria; Schnabelrauch, Matthias; Schulz-Siegmund, Michaela; Hacker, Michael C; Scharnweber, Dieter; Hofbauer, Christine; Hofbauer, Lorenz C

    2016-07-01

    Bone fractures in patients with diabetes mellitus heal poorly and require innovative therapies to support bone regeneration. Here, we assessed whether sulfated hyaluronan included in collagen-based scaffold coatings can improve fracture healing in diabetic rats. Macroporous thermopolymerized lactide-based scaffolds were coated with collagen including non-sulfated or sulfated hyaluronan (HA/sHA3) and inserted into 3 mm femoral defects of non-diabetic and diabetic ZDF rats. After 12 weeks, scaffolds coated with collagen/HA or collagen/sHA3 accelerated bone defect regeneration in diabetic, but not in non-diabetic rats as compared to their non-coated controls. At the tissue level, collagen/sHA3 promoted bone mineralization and decreased the amount of non-mineralized bone matrix. Moreover, collagen/sHA3-coated scaffolds from diabetic rats bound more sclerostin in vivo than the respective controls. Binding assays confirmed a high binding affinity of sHA3 to sclerostin. In vitro, sHA3 induced BMP-2 and lowered the RANKL/OPG expression ratio, regardless of the glucose concentration in osteoblastic cells. Both sHA3 and high glucose concentrations decreased the differentiation of osteoclastic cells. In summary, scaffolds coated with collagen/sHA3 represent a potentially suitable biomaterial to improve bone defect regeneration in diabetic conditions. The underlying mechanism involves improved osteoblast function and binding sclerostin, a potent inhibitor of Wnt signaling and osteoblast function.

  1. Monoclonal antibodies to CD44 and their influence on hyaluronan recognition

    PubMed Central

    1995-01-01

    Antibodies to CD44 have been used to inhibit a variety of processes which include lymphohemopoiesis, lymphocyte migration, and tumor metastasis. Some, but not all, CD44-mediated functions derive from its ability to serve as a receptor for hyaluronan (HA). However, sites on CD44 that interact with either ligands or antibodies are poorly understood. Interspecies rat/mouse CD44 chimeras were used to analyze the specificity of 25 mAbs and to determine that they recognize at least seven epitopes. Amino acid substitutions that resulted in loss of antibody recognition were all located in the region of homology to other cartilage link family proteins. While at least five epitopes were eliminated by single amino acid replacements, multiple residues had to be changed to destroy binding by other antibodies. One antibody was sensitive to changes in any of three separate parts of the molecule and some antibodies to distinct epitopes cross-blocked each other. Certain antibodies had the ability to increase HA binding by lymphocytes but this did not correlate absolutely with antibody specificity and was only partially attributable to CD44 cross-linking. Antibodies that consistently blocked HA recognition were all sensitive to amino acid changes within a short stretch of CD44. Such blocking antibodies interacted with CD44 more strongly than ligand in competition experiments. One large group of antibodies blocked ligand binding, but only with a particular cell line. This detailed analysis adds to our understanding of functional domains within CD44 and requirements for antibodies to influence recognition of one ligand. PMID:7542251

  2. Extracellular Vesicles from Caveolin-Enriched Microdomains Regulate Hyaluronan-Mediated Sustained Vascular Integrity

    PubMed Central

    Mirzapoiazova, Tamara; Lennon, Frances E.; Mambetsariev, Bolot; Allen, Michael; Riehm, Jacob; Poroyko, Valeriy A.; Singleton, Patrick A.

    2015-01-01

    Defects in vascular integrity are an initiating factor in several disease processes. We have previously reported that high molecular weight hyaluronan (HMW-HA), a major glycosaminoglycan in the body, promotes rapid signal transduction in human pulmonary microvascular endothelial cells (HPMVEC) leading to barrier enhancement. In contrast, low molecular weight hyaluronan (LMW-HA), produced in disease states by hyaluronidases and reactive oxygen species (ROS), induces HPMVEC barrier disruption. However, the mechanism(s) of sustained barrier regulation by HA are poorly defined. Our results indicate that long-term (6–24 hours) exposure of HMW-HA induced release of a novel type of extracellular vesicle from HLMVEC called enlargeosomes (characterized by AHNAK expression) while LMW-HA long-term exposure promoted release of exosomes (characterized by CD9, CD63, and CD81 expression). These effects were blocked by inhibiting caveolin-enriched microdomain (CEM) formation. Further, inhibiting enlargeosome release by annexin II siRNA attenuated the sustained barrier enhancing effects of HMW-HA. Finally, exposure of isolated enlargeosomes to HPMVEC monolayers generated barrier enhancement while exosomes led to barrier disruption. Taken together, these results suggest that differential release of extracellular vesicles from CEM modulate the sustained HPMVEC barrier regulation by HMW-HA and LMW-HA. HMW-HA-induced specialized enlargeosomes can be a potential therapeutic strategy for diseases involving impaired vascular integrity. PMID:26447809

  3. Intravesical administration of combined hyaluronic acid (HA) and chondroitin sulfate (CS) for the treatment of female recurrent urinary tract infections: a European multicentre nested case–control study

    PubMed Central

    Ciani, Oriana; Arendsen, Erik; Romancik, Martin; Lunik, Richard; Costantini, Elisabetta; Di Biase, Manuel; Morgia, Giuseppe; Fragalà, Eugenia; Roman, Tomaskin; Bernat, Marian; Guazzoni, Giorgio; Tarricone, Rosanna; Lazzeri, Massimo

    2016-01-01

    Objectives To compare the clinical effectiveness of the intravesical administration of combined hyaluronic acid and chondroitin sulfate (HA+CS) versus current standard management in adult women with recurrent urinary tract infections (RUTIs). Setting A European Union-based multicentre, retrospective nested case–control study. Participants 276 adult women treated for RUTIs starting from 2009 to 2013. Interventions Patients treated with either intravesical administration of HA+CS or standard of care (antimicrobial/immunoactive prophylaxis/probiotics/cranberry). Primary and secondary outcome measures The primary outcome was occurrence of bacteriologically confirmed recurrence within 12 months. Secondary outcomes were time to recurrence, total number of recurrences, health-related quality of life and healthcare resource consumption. Crude and adjusted results for unbalanced characteristics are presented. Results 181 patients treated with HA+CS and 95 patients treated with standard of care from 7 centres were included. The crude and adjusted ORs (95% CI) for the primary end point were 0.77 (0.46 to 1.28) and 0.51 (0.27 to 0.96), respectively. However, no evidence of improvement in terms of total number of recurrences (incidence rate ratio (95% CI), 0.99 (0.69 to 1.43)) or time to first recurrence was seen (HR (95% CI), 0.99 (0.61 to 1.61)). The benefit of intravesical HA+CS therapy improves when the number of instillations is ≥5. Conclusions Our results show that bladder instillations of combined HA+CS reduce the risk of bacteriologically confirmed recurrences compared with the current standard management of RUTIs. Total incidence rates and hazard rates were instead non-significantly different between the 2 groups after adjusting for unbalanced factors. In contrast to what happens with antibiotic prophylaxis, the effectiveness of the HA+CS reinstatement therapy improves over time. Trial registration number NCT02016118. PMID:27033958

  4. Wound healing of different molecular weight of hyaluronan; in-vivo study.

    PubMed

    Fouda, Moustafa M G; Abdel-Mohsen, A M; Ebaid, Hossam; Hassan, Iftekhar; Al-Tamimi, Jameel; Abdel-Rahman, Rasha M; Metwalli, Ali; Alhazza, Ibrahim; Rady, Ahmed; El-Faham, Ayman; Jancar, J

    2016-08-01

    Recruitment of cells and mediators is altered during impaired wound healing, thereby delaying this process. To overcome this problem, the correlation of wound healing in older rats, and the impact of different molecular weight of hyaluronan without silver nanoparticles; (low-HA1), (High-HA2), (Medium- HA3) and with silver nanoparticles (High-HA4) is investigated. The superior HA were selected to be further investigated onto diabetic wounds. Our results pointed to a marked deficiency in wounds granulation in older rats, which was accompanied with impairment of healing process. In older rats group treated with HA2 or HA4, granulation and dermal construction were improved. Furthermore, the number of pathogenic bacteria on wounds was declined throughout the first 24h by HA2 and HA4. The wound size in HA4-treated older rats was significantly smaller than that in other HA1, HA2 or HA3-treated older ones. Also, diabetes impaired the level of inflammatory cytokine, in diabetic model. On contrary, HA4 was found to normalize the level of inflammatory cytokine, in the diabetic model. Furthermore, HA4 was found to recover all oxidative and toxicity markers in diabetic models. This data confirms the critical role of HA4 to improve granulation and inflammatory mediators in impaired older and diabetic rat wound healing. PMID:27174907

  5. Alginate-hyaluronan composite hydrogels accelerate wound healing process.

    PubMed

    Catanzano, O; D'Esposito, V; Acierno, S; Ambrosio, M R; De Caro, C; Avagliano, C; Russo, P; Russo, R; Miro, A; Ungaro, F; Calignano, A; Formisano, P; Quaglia, F

    2015-10-20

    In this paper we propose polysaccharide hydrogels combining alginate (ALG) and hyaluronan (HA) as biofunctional platform for dermal wound repair. Hydrogels produced by internal gelation were homogeneous and easy to handle. Rheological evaluation of gelation kinetics of ALG/HA mixtures at different ratios allowed understanding the HA effect on ALG cross-linking process. Disk-shaped hydrogels, at different ALG/HA ratio, were characterized for morphology, homogeneity and mechanical properties. Results suggest that, although the presence of HA does significantly slow down gelation kinetics, the concentration of cross-links reached at the end of gelation is scarcely affected. The in vitro activity of ALG/HA dressings was tested on adipose derived multipotent adult stem cells (Ad-MSC) and an immortalized keratinocyte cell line (HaCaT). Hydrogels did not interfere with cell viability in both cells lines, but significantly promoted gap closure in a scratch assay at early (1 day) and late (5 days) stages as compared to hydrogels made of ALG alone (p<0.01 and 0.001 for Ad-MSC and HaCaT, respectively). In vivo wound healing studies, conducted on a rat model of excised wound indicated that after 5 days ALG/HA hydrogels significantly promoted wound closure as compared to ALG ones (p<0.001). Overall results demonstrate that the integration of HA in a physically cross-linked ALG hydrogel can be a versatile strategy to promote wound healing that can be easily translated in a clinical setting.

  6. Hyaluronan modulates TRPV1 channel opening, reducing peripheral nociceptor activity and pain.

    PubMed

    Caires, Rebeca; Luis, Enoch; Taberner, Francisco J; Fernandez-Ballester, Gregorio; Ferrer-Montiel, Antonio; Balazs, Endre A; Gomis, Ana; Belmonte, Carlos; de la Peña, Elvira

    2015-01-01

    Hyaluronan (HA) is present in the extracellular matrix of all body tissues, including synovial fluid in joints, in which it behaves as a filter that buffers transmission of mechanical forces to nociceptor nerve endings thereby reducing pain. Using recombinant systems, mouse-cultured dorsal root ganglia (DRG) neurons and in vivo experiments, we found that HA also modulates polymodal transient receptor potential vanilloid subtype 1 (TRPV1) channels. HA diminishes heat, pH and capsaicin (CAP) responses, thus reducing the opening probability of the channel by stabilizing its closed state. Accordingly, in DRG neurons, HA decreases TRPV1-mediated impulse firing and channel sensitization by bradykinin. Moreover, subcutaneous HA injection in mice reduces heat and capsaicin nocifensive responses, whereas the intra-articular injection of HA in rats decreases capsaicin joint nociceptor fibres discharge. Collectively, these results indicate that extracellular HA reduces the excitability of the ubiquitous TRPV1 channel, thereby lowering impulse activity in the peripheral nociceptor endings underlying pain.

  7. Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

    SciTech Connect

    Nishida, Yoshihiro . E-mail: ynishida@med.nagoya-u.ac.jp; Knudson, Warren; Knudson, Cheryl B.; Ishiguro, Naoki

    2005-07-01

    Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.

  8. Hyaluronidase and Hyaluronan Oligosaccharides Promote Neurological Recovery after Intraventricular Hemorrhage

    PubMed Central

    Vinukonda, Govindaiah; Dohare, Preeti; Arshad, Arslan; Zia, Muhammad T.; Panda, Sanjeet; Korumilli, Ritesh; Kayton, Robert; Hascall, Vincent C.; Lauer, Mark E.

    2016-01-01

    Intraventricular hemorrhage (IVH) in premature infants results in inflammation, arrested oligodendrocyte progenitor cell (OPC) maturation, and reduced myelination of the white matter. Hyaluronan (HA) inhibits OPC maturation and complexes with the heavy chain (HC) of glycoprotein inter-α-inhibitor to form pathological HA (HC–HA complex), which exacerbates inflammation. Therefore, we hypothesized that IVH would result in accumulation of HA, and that either degradation of HA by hyaluronidase treatment or elimination of HCs from pathological HA by HA oligosaccharide administration would restore OPC maturation, myelination, and neurological function in survivors with IVH. To test these hypotheses, we used the preterm rabbit model of glycerol-induced IVH and analyzed autopsy samples from premature infants. We found that total HA levels were comparable in both preterm rabbit pups and human infants with and without IVH, but HA receptors—CD44, TLR2, TLR4—were elevated in the forebrain of both humans and rabbits with IVH. Hyaluronidase treatment of rabbits with IVH reduced CD44 and TLR4 expression, proinflammatory cytokine levels, and microglia infiltration. It also promoted OPC maturation, myelination, and neurological recovery. HC–HA and tumor necrosis factor-stimulated gene-6 were elevated in newborns with IVH; and depletion of HC–HA levels by HA oligosaccharide treatment reduced inflammation and enhanced myelination and neurological recovery in rabbits with IVH. Hence, hyaluronidase or HA oligosaccharide treatment represses inflammation, promotes OPC maturation, and restores myelination and neurological function in rabbits with IVH. These therapeutic strategies might improve the neurological outcome of premature infants with IVH. SIGNIFICANCE STATEMENT Approximately 12,000 premature infants develop IVH every year in the United States, and a large number of survivors with IVH develop cerebral palsy and cognitive deficits. The onset of IVH induces inflammation

  9. Effect of hyaluronan to inhibit caspase activation in porcine granulosa cells.

    PubMed

    Tunjung, Woro Anindito Sri; Yokoo, Masaki; Hoshino, Yumi; Miyake, Yuko; Kadowaki, Akane; Sato, Eimei

    2009-04-24

    We studied the ability of hyaluronan (HA) to inhibit apoptosis in porcine granulosa cells. The granulosa layer with cumulus-oocyte complex is cultured in media supplemented with follicle stimulating hormone (FSH) and 4-MU an inhibitor of hyaluronan synthases. The concentration of HA significantly increased after supplemented with FSH, but significantly decreased with 4-MU. CD44, receptor of HA, expressed after cultured with FSH, decreased in addition low concentration of 4-MU, whereas not detected in high concentration of 4-MU, indicating parallel relation between the amount of HA and CD44 expression. The 4-MU treatment also decreased the expression of procaspase-3, -8, -9 suggesting that inhibition of HA synthesis leads to activation of these caspases. Moreover, addition of anti-CD44 antibody decreased the expression of procaspases suggesting that perturbation of HA-CD44 binding leads activation of caspases. Hence, HA has ability to inhibit apoptosis and HA-CD44 binding is important on apoptosis inhibitory mechanism in porcine granulosa cells.

  10. A Clinical Evaluation of Efficacy and Safety of Hyaluronan Sponge with Vitamin C Versus Placebo for Scar Reduction

    PubMed Central

    Mahedia, Monali; Shah, Nilay

    2016-01-01

    Background: Scar formation after injury or surgery is a major clinical problem. Individually, hyaluronan, or hyaluronic acid (HA), and vitamin C have been shown to reduce scarring by means of different mechanisms. The authors evaluated the efficacy and safety of an HA sponge system containing an active derivative of vitamin C to determine whether the use of this product promotes healing and reduces inflammation and scarring after surgery. Methods: This double-blind, randomized, prospective study was approved by the local institutional review board. Participants who had unilateral or bilateral surgical scars more than 1 month but less than 18 months old were enrolled. Surgical scars were randomly assigned to receive placebo or HA sponge with vitamin C. Three blinded evaluators reviewed photographs of the incision lines and assessed the scars using a visual analog scale. A patient satisfaction survey was also administered. Participants were followed up at 4 weeks, 12 weeks, and 1 year. Results: Twenty-three patients were enrolled in the study. Six patients dropped out of the study, for a total of 17 patients included in final analysis. Mean (range) age of patient was 43.5 (25–67) years. Mean (range) body mass index was 27.4 (18–36.9) kg/m2. The mean visual analog scale score for scars receiving HA sponge with vitamin C was slightly lower than the scars receiving placebo, but the difference was not statistically significant (t test; P = 0.9). The HA sponge with vitamin C was found to have significant positive findings on a patient satisfaction survey. Conclusions: The HA sponge system with vitamin C is safe to use in any scars older than 4 weeks. It has high patient satisfaction in achieving a better scar after surgery. The micro-roller used to apply the product was easy to use to potentially increase the spread of the medication in older scars. PMID:27536471

  11. Differential Proteomic Analysis of Human Placenta-Derived Mesenchymal Stem Cells Cultured on Normal Tissue Culture Surface and Hyaluronan-Coated Surface

    PubMed Central

    Wong, Tzyy Yue; Chen, Ying-Hui; Liu, Szu-Heng; Solis, Mairim Alexandra; Yu, Chen-Hsiang; Chang, Chiung-Hsin; Huang, Lynn L. H.

    2016-01-01

    Our previous results showed that hyaluronan (HA) preserved human placenta-derived mesenchymal stem cells (PDMSC) in a slow cell cycling mode similar to quiescence, the pristine state of stem cells in vivo, and HA was found to prevent murine adipose-derived mesenchymal stem cells from senescence. Here, stable isotope labeling by amino acid in cell culture (SILAC) proteomic profiling was used to evaluate the effects of HA on aging phenomenon in stem cells, comparing (1) old and young passage PDMSC cultured on normal tissue culture surface (TCS); (2) old passage on HA-coated surface (CHA) compared to TCS; (3) old and young passage on CHA. The results indicated that senescence-associated protein transgelin (TAGLN) was upregulated in old TCS. Protein CYR61, reportedly senescence-related, was downregulated in old CHA compared to old TCS. The SIRT1-interacting Nicotinamide phosphoribosyltransferase (NAMPT) increased by 2.23-fold in old CHA compared to old TCS, and is 0.48-fold lower in old TCS compared to young TCS. Results also indicated that components of endoplasmic reticulum associated degradation (ERAD) pathway were upregulated in old CHA compared to old TCS cells, potentially for overcoming stress to maintain cell function and suppress senescence. Our data points to pathways that may be targeted by HA to maintain stem cells youth. PMID:27057169

  12. Differential Proteomic Analysis of Human Placenta-Derived Mesenchymal Stem Cells Cultured on Normal Tissue Culture Surface and Hyaluronan-Coated Surface.

    PubMed

    Wong, Tzyy Yue; Chen, Ying-Hui; Liu, Szu-Heng; Solis, Mairim Alexandra; Yu, Chen-Hsiang; Chang, Chiung-Hsin; Huang, Lynn L H

    2016-01-01

    Our previous results showed that hyaluronan (HA) preserved human placenta-derived mesenchymal stem cells (PDMSC) in a slow cell cycling mode similar to quiescence, the pristine state of stem cells in vivo, and HA was found to prevent murine adipose-derived mesenchymal stem cells from senescence. Here, stable isotope labeling by amino acid in cell culture (SILAC) proteomic profiling was used to evaluate the effects of HA on aging phenomenon in stem cells, comparing (1) old and young passage PDMSC cultured on normal tissue culture surface (TCS); (2) old passage on HA-coated surface (CHA) compared to TCS; (3) old and young passage on CHA. The results indicated that senescence-associated protein transgelin (TAGLN) was upregulated in old TCS. Protein CYR61, reportedly senescence-related, was downregulated in old CHA compared to old TCS. The SIRT1-interacting Nicotinamide phosphoribosyltransferase (NAMPT) increased by 2.23-fold in old CHA compared to old TCS, and is 0.48-fold lower in old TCS compared to young TCS. Results also indicated that components of endoplasmic reticulum associated degradation (ERAD) pathway were upregulated in old CHA compared to old TCS cells, potentially for overcoming stress to maintain cell function and suppress senescence. Our data points to pathways that may be targeted by HA to maintain stem cells youth. PMID:27057169

  13. Investigating triazine-based modification of hyaluronan using statistical designs.

    PubMed

    Liang, Jue; Cheng, Lulu; Struckhoff, Jessica J; Ravi, Nathan

    2015-11-01

    Hyaluronan (HA) and its derivatives have been extensively researched for many biomedical applications. To precisely tailor the property of HA by derivatizing it to a pre-determined extent is challenging, yet critical. In this paper, we used 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and N-methylmorpholine (NMM) to derivatize HA via a triazine-based coupling reaction. Using a fractional factorial (FF) design, we observed that water content in the solvent, and molar ratios of CDMT and NaHCO3 to the carboxylate were the significant factors controlling the derivatization. We investigated how the effect of each factor changes as reaction conditions change. Moreover, by altering the amount of CDMT and NaHCO3, we developed a cubic regression model for precise control of the extent of derivatization using a response surface methodology (RSM) with a D-optimal design. No spurious peaks were detected by (1)H NMR spectrum and only 10% decrease of molecular weight of the derivatized HA was determined by GPC. The HA with 6% modification was relatively biocompatible up to 15 mg/mL. PMID:26256372

  14. Lubrication and wear properties of grafted polyelectrolytes, hyaluronan and hylan, measured in the surface forces apparatus.

    PubMed

    Benz, Marcel; Chen, Nianhuan; Israelachvili, Jacob

    2004-10-01

    Hyaluronan is believed to have an important function in the boundary biolubrication of articular cartilage. Using a Surface Forces Apparatus, we tested the tribological properties of surface bound, rather than "free" hyaluronan. The grafting process of the polyelectrolyte included either a biological route via an HA-binding protein or a chemical reaction to covalently bind the polymer to a lipid bilayer coated surface. In another reaction, we constructed a surface with covalently grafted hylan (crosslinked hyaluronan). We studied the normal and shear forces between these surfaces. None of the systems demonstrated comparable lubrication to that found between cartilage surfaces except at very low loads. Both grafted hyaluronan and hylan generated coefficients of friction between 0.15 and 0.3. Thus, the polysaccharide, which is a constituent of the lamina splendens (outermost cartilage layer), is not expected to be the responsible molecule for the great lubricity of cartilage; however, it may contribute to the load bearing and wear protection of these surfaces. This was concluded from the results with hylan, where a thin gel layer was sufficient to shield the underlying surfaces from damage even at applied pressures of over 200 atmospheres during shear. Our study shows that a low coefficient of friction is not a requirement for, or necessarily a measure of, wear protection. PMID:15368250

  15. Characteristic Formation of Hyaluronan-Cartilage Link Protein-Proteoglycan Complex in Salivary Gland Tumors.

    PubMed

    Kuwabara, Hiroko; Nishikado, Akira; Hayasaki, Hana; Isogai, Zenzo; Yoneda, Masahiko; Kawata, Ryo; Hirose, Yoshinobu

    2016-01-01

    Hyaluronan (HA) and its binding molecules, cartilage link protein (LP) and proteoglycan (PG), are structural components of the hydrated extracellular matrix. Because these molecules play important roles in the tumor microenvironment, we examined the distribution of HA, LP, versican, and aggrecan in salivary gland tumors using histochemical and immunohistochemical methods, including double staining. LP was present in pleomorphic adenoma (PA) and adenoid cystic carcinoma (ACC) tissues, and aggrecan was absent in the malignant tumors that we investigated. LP colocalized with both HA and aggrecan in the chondromyxoid matrix of PA, suggesting the presence of a HA-LP-aggrecan complex. Furthermore, the HA-LP-versican complex could be observed in the pseudocystic space of the cribriform structures in ACC. The characteristic HA-LP-PG complex in PA and ACC might play a role in the behavior of tumors, and immunohistochemical analysis of these molecules could represent a diagnostic adjunct for salivary gland tumors.

  16. Hyaluronan-phosphatidylethanolamine polymers form pericellular coats on keratinocytes and promote basal keratinocyte proliferation.

    PubMed

    Symonette, Caitlin J; Kaur Mann, Aman; Tan, Xiao Cherie; Tolg, Cornelia; Ma, Jenny; Perera, Francisco; Yazdani, Arjang; Turley, Eva A

    2014-01-01

    Aged keratinocytes have diminished proliferative capacity and hyaluronan (HA) cell coats, which are losses that contribute to atrophic skin characterized by reduced barrier and repair functions. We formulated HA-phospholipid (phosphatidylethanolamine, HA-PE) polymers that form pericellular coats around cultured dermal fibroblasts independently of CD44 or RHAMM display. We investigated the ability of these HA-PE polymers to penetrate into aged mouse skin and restore epidermal function in vivo. Topically applied Alexa(647)-HA-PE penetrated into the epidermis and dermis, where it associated with both keratinocytes and fibroblasts. In contrast, Alexa(647)-HA was largely retained in the outer cornified layer of the epidermis and quantification of fluorescence confirmed that significantly more Alexa(647)-HA-PE penetrated into and was retained within the epidermis than Alexa(647)-HA. Multiple topical applications of HA-PE to shaved mouse skin significantly stimulated basal keratinocyte proliferation and epidermal thickness compared to HA or vehicle cream alone. HA-PE had no detectable effect on keratinocyte differentiation and did not promote local or systemic inflammation. These effects of HA-PE polymers are similar to those reported for endogenous epidermal HA in youthful skin and show that topical application of HA-PE polymers can restore some of the impaired functions of aged epidermis. PMID:25276814

  17. Hyaluronan Polymer Length, Grafting Density, and Surface Poly(ethylene glycol) Coating Influence in Vivo Circulation and Tumor Targeting of Hyaluronan-Grafted Liposomes

    PubMed Central

    2015-01-01

    Hyaluronan-grafted liposomes (HA-liposomes) preferentially target CD44-overexpressing tumor cells in vitro via receptor-mediated endocytosis. We investigated the pharmacokinetics and biodistribution of HA-liposomes with various sizes of HA (MW 5–8, 50–60, and 175–350 kDa) in mice. Incorporation of negatively charged HA on the liposome surface compromised its blood circulation time, which led to decreased tumor accumulation in CD44+ human breast cancer MDA-MB-231 xenografts compared to PEGylated liposomes (PEG-5000). Clearance of HA-liposomes was HA polymer length-dependent; high MW (175–350 kDa, highest ligand binding affinity) HA-liposomes displayed faster clearance compared to low MW (5–8, 50–60 kDa) HA-liposomes or PEGylated liposomes. Surface HA ligand density can also affect clearance of HA-liposomes. Thus, HA is not an effective stealth coating material. When dual coating of PEG and HA was used, the PEG-HA-liposomes displayed similar blood circulation time and tumor accumulation to that of the PEGylated liposomes; however, the PEG-HA-liposomes displayed better cellular internalization capability in vivo. Tumor histology showed that PEG-HA-liposomes had a more direct association with CD44+ cancer cells, while PEGylated liposomes located predominantly in the tumor periphery, with less association with CD44+ cells. Flow cytometry analysis of ex vivo tumor cells showed that PEG-HA-liposomes had significantly higher tumor cell internalization compared to PEGylated liposomes. This study demonstrates that a long blood circulation time is critical for active tumor targeting. Furthermore, the use of the tumor-targeting ligand HA does not increase total tumor accumulation of actively targeted liposomes in solid tumors; however, it can enhance intracellular delivery. PMID:24806526

  18. Hyaluronan polymer length, grafting density, and surface poly(ethylene glycol) coating influence in vivo circulation and tumor targeting of hyaluronan-grafted liposomes.

    PubMed

    Qhattal, Hussaini Syed Sha; Hye, Tanvirul; Alali, Amer; Liu, Xinli

    2014-06-24

    Hyaluronan-grafted liposomes (HA-liposomes) preferentially target CD44-overexpressing tumor cells in vitro via receptor-mediated endocytosis. We investigated the pharmacokinetics and biodistribution of HA-liposomes with various sizes of HA (MW 5-8, 50-60, and 175-350 kDa) in mice. Incorporation of negatively charged HA on the liposome surface compromised its blood circulation time, which led to decreased tumor accumulation in CD44+ human breast cancer MDA-MB-231 xenografts compared to PEGylated liposomes (PEG-5000). Clearance of HA-liposomes was HA polymer length-dependent; high MW (175-350 kDa, highest ligand binding affinity) HA-liposomes displayed faster clearance compared to low MW (5-8, 50-60 kDa) HA-liposomes or PEGylated liposomes. Surface HA ligand density can also affect clearance of HA-liposomes. Thus, HA is not an effective stealth coating material. When dual coating of PEG and HA was used, the PEG-HA-liposomes displayed similar blood circulation time and tumor accumulation to that of the PEGylated liposomes; however, the PEG-HA-liposomes displayed better cellular internalization capability in vivo. Tumor histology showed that PEG-HA-liposomes had a more direct association with CD44+ cancer cells, while PEGylated liposomes located predominantly in the tumor periphery, with less association with CD44+ cells. Flow cytometry analysis of ex vivo tumor cells showed that PEG-HA-liposomes had significantly higher tumor cell internalization compared to PEGylated liposomes. This study demonstrates that a long blood circulation time is critical for active tumor targeting. Furthermore, the use of the tumor-targeting ligand HA does not increase total tumor accumulation of actively targeted liposomes in solid tumors; however, it can enhance intracellular delivery. PMID:24806526

  19. Hyaluronan fragments as mediators of inflammation in allergic pulmonary disease

    PubMed Central

    Ghosh, Sumit; Hoselton, Scott A.; Dorsam, Glenn P.; Schuh, Jane M.

    2015-01-01

    Asthma is frequently caused and/or exacerbated by sensitization to allergens, which are ubiquitous in many indoor and outdoor environments. Severe asthma is characterized by airway hyperresponsiveness and bronchial constriction in response to an inhaled allergen, leading to a disease course that is often very difficult to treat with standard asthma therapies. As a result of interactions among inflammatory cells, structural cells, and the intercellular matrix of the allergic lung, patients with sensitization to allergens may experience a greater degree of tissue injury followed by airway wall remodeling and progressive, accumulated pulmonary dysfunction as part of the disease sequela. In addition, turnover of extracellular matrix (ECM) components is a hallmark of tissue injury and repair. This review focuses on the role of the glycosaminoglycan hyaluronan (HA), a component of the ECM, in pulmonary injury and repair with an emphasis on allergic asthma. Both the synthesis and degradation of the ECM are critical contributors to tissue repair and remodeling. Fragmented HA accumulates during tissue injury and functions in ways distinct from the larger native polymer. There is gathering evidence that HA degradation products are active participants in stimulating the expression of inflammatory genes in a variety of immune cells at the injury site. In this review, we will consider recent advances in the understanding of the mechanisms that are associated with HA accumulation and inflammatory cell recruitment in the asthmatic lung. PMID:25582403

  20. Optimization of hyaluronan-based eye drop formulations.

    PubMed

    Salzillo, Rosanna; Schiraldi, Chiara; Corsuto, Luisana; D'Agostino, Antonella; Filosa, Rosanna; De Rosa, Mario; La Gatta, Annalisa

    2016-11-20

    Hyaluronan (HA) is frequently incorporated in eye drops to extend the pre-corneal residence time, due to its viscosifying and mucoadhesive properties. Hydrodynamic and rheological evaluations of commercial products are first accomplished revealing molecular weights varying from about 360 to about 1200kDa and viscosity values in the range 3.7-24.2mPa s. The latter suggest that most products could be optimized towards resistance to drainage from the ocular surface. Then, a study aiming to maximize the viscosity and mucoadhesiveness of HA-based preparations is performed. The effect of polymer chain length and concentration is investigated. For the whole range of molecular weights encountered in commercial products, the concentration maximizing performance is identified. Such concentration varies from 0.3 (wt%) for a 1100kDa HA up to 1.0 (wt%) for a 250kDa HA, which is 3-fold higher than the highest concentration on the market. The viscosity and mucoadhesion profiles of optimized formulations are superior than commercial products, especially under conditions simulating in vivo blinking. Thus longer retention on the corneal epithelium can be predicted. An enhanced capacity to protect corneal porcine epithelial cells from dehydration is also demonstrated in vitro. Overall, the results predict formulations with improved efficacy.

  1. Optimization of hyaluronan-based eye drop formulations.

    PubMed

    Salzillo, Rosanna; Schiraldi, Chiara; Corsuto, Luisana; D'Agostino, Antonella; Filosa, Rosanna; De Rosa, Mario; La Gatta, Annalisa

    2016-11-20

    Hyaluronan (HA) is frequently incorporated in eye drops to extend the pre-corneal residence time, due to its viscosifying and mucoadhesive properties. Hydrodynamic and rheological evaluations of commercial products are first accomplished revealing molecular weights varying from about 360 to about 1200kDa and viscosity values in the range 3.7-24.2mPa s. The latter suggest that most products could be optimized towards resistance to drainage from the ocular surface. Then, a study aiming to maximize the viscosity and mucoadhesiveness of HA-based preparations is performed. The effect of polymer chain length and concentration is investigated. For the whole range of molecular weights encountered in commercial products, the concentration maximizing performance is identified. Such concentration varies from 0.3 (wt%) for a 1100kDa HA up to 1.0 (wt%) for a 250kDa HA, which is 3-fold higher than the highest concentration on the market. The viscosity and mucoadhesion profiles of optimized formulations are superior than commercial products, especially under conditions simulating in vivo blinking. Thus longer retention on the corneal epithelium can be predicted. An enhanced capacity to protect corneal porcine epithelial cells from dehydration is also demonstrated in vitro. Overall, the results predict formulations with improved efficacy. PMID:27561497

  2. Fourier transform infrared study on microemulsion system of potassium salt of bis(2-ethylhexyl) phosphinic acid (HA)

    NASA Astrophysics Data System (ADS)

    Zhou, Weijin; Shi, Nai; Wang, Yi; Chang, Zhiyuan; Wu, JinGuang

    1994-01-01

    To study microemulsion formation in a solvent extraction system is to probe into some basic principles of extraction chemistry in the light of combining extraction chemistry with surface chemistry. In our previous investigations, the microemulsions of the salts of HDEHP and PC88A have been studied systematically by FT-IR. In the experiment, we observed the change of peak positions and intensities of P equals O, P-O-C and P-O-H groups during saponification and hydration, and discovered that the peak of P-O-C splits apart into 1045 and 1075 cm-1. The vibration frequency of the P-O-C group in HDEHP and PC88A is quite close to the symmetric stretching frequency of the POO- group, and thus causes difficulties in the study of their peak position and absorbance variation. For this reason we synthesized bis(2-ethylhexyl) phosphinic acid without the P-O-C group. Infrared spectra in the range of 800 - 4000 cm-1 of this microemulsion system was studied.

  3. Dietary flavonoid fisetin increases abundance of high-molecular-mass hyaluronan conferring resistance to prostate oncogenesis.

    PubMed

    Lall, Rahul K; Syed, Deeba N; Khan, Mohammad Imran; Adhami, Vaqar M; Gong, Yuansheng; Lucey, John A; Mukhtar, Hasan

    2016-09-01

    We and others have shown previously that fisetin, a plant flavonoid, has therapeutic potential against many cancer types. Here, we examined the probable mechanism of its action in prostate cancer (PCa) using a global metabolomics approach. HPLC-ESI-MS analysis of tumor xenografts from fisetin-treated animals identified several metabolic targets with hyaluronan (HA) as the most affected. Efficacy of fisetin on HA was then evaluated in vitro and also in vivo in the transgenic TRAMP mouse model of PCa. Size exclusion chromatography-multiangle laser light scattering (SEC-MALS) was performed to analyze the molar mass (Mw) distribution of HA. Fisetin treatment downregulated intracellular and secreted HA levels both in vitro and in vivo Fisetin inhibited HA synthesis and degradation enzymes, which led to cessation of HA synthesis and also repressed the degradation of the available high-molecular-mass (HMM)-HA. SEC-MALS analysis of intact HA fragment size revealed that cells and animals have more abundance of HMM-HA and less of low-molecular-mass (LMM)-HA upon fisetin treatment. Elevated HA levels have been shown to be associated with disease progression in certain cancer types. Biological responses triggered by HA mainly depend on the HA polymer length where HMM-HA represses mitogenic signaling and has anti-inflammatory properties whereas LMM-HA promotes proliferation and inflammation. Similarly, Mw analysis of secreted HA fragment size revealed less HMM-HA is secreted that allowed more HMM-HA to be retained within the cells and tissues. Our findings establish that fisetin is an effective, non-toxic, potent HA synthesis inhibitor, which increases abundance of antiangiogenic HMM-HA and could be used for the management of PCa.

  4. Dietary flavonoid fisetin increases abundance of high-molecular-mass hyaluronan conferring resistance to prostate oncogenesis.

    PubMed

    Lall, Rahul K; Syed, Deeba N; Khan, Mohammad Imran; Adhami, Vaqar M; Gong, Yuansheng; Lucey, John A; Mukhtar, Hasan

    2016-09-01

    We and others have shown previously that fisetin, a plant flavonoid, has therapeutic potential against many cancer types. Here, we examined the probable mechanism of its action in prostate cancer (PCa) using a global metabolomics approach. HPLC-ESI-MS analysis of tumor xenografts from fisetin-treated animals identified several metabolic targets with hyaluronan (HA) as the most affected. Efficacy of fisetin on HA was then evaluated in vitro and also in vivo in the transgenic TRAMP mouse model of PCa. Size exclusion chromatography-multiangle laser light scattering (SEC-MALS) was performed to analyze the molar mass (Mw) distribution of HA. Fisetin treatment downregulated intracellular and secreted HA levels both in vitro and in vivo Fisetin inhibited HA synthesis and degradation enzymes, which led to cessation of HA synthesis and also repressed the degradation of the available high-molecular-mass (HMM)-HA. SEC-MALS analysis of intact HA fragment size revealed that cells and animals have more abundance of HMM-HA and less of low-molecular-mass (LMM)-HA upon fisetin treatment. Elevated HA levels have been shown to be associated with disease progression in certain cancer types. Biological responses triggered by HA mainly depend on the HA polymer length where HMM-HA represses mitogenic signaling and has anti-inflammatory properties whereas LMM-HA promotes proliferation and inflammation. Similarly, Mw analysis of secreted HA fragment size revealed less HMM-HA is secreted that allowed more HMM-HA to be retained within the cells and tissues. Our findings establish that fisetin is an effective, non-toxic, potent HA synthesis inhibitor, which increases abundance of antiangiogenic HMM-HA and could be used for the management of PCa. PMID:27335141

  5. Effect of hyaluronan supplementation on boar sperm motility and membrane lipid architecture status after cryopreservation.

    PubMed

    Peña, F J; Johannisson, A; Wallgren, M; Rodriguez-Martinez, H

    2004-01-01

    We investigated the effect of supplementing extended boar semen with different amounts of hyaluronan (HA) prior to freezing on post-thaw sperm characteristics. Using a split sample design, the effect of HA at a final concentration of 500 or 1000 microg/ml semen on post-thaw motility parameters, and membrane lipid architecture status assessed by merocyanine-540/YOPRO-1 and flow cytometry were evaluated. HA-supplementation improved motility parameters (P < 0.05 to P < 0.001) and decreased the percentage of hyperactivated spermatozoa (P < 0.05). HA-supplemented samples had more spermatozoa showing high lipid membrane stability as assessed with merocyanine-540. In conclusion, HA appeared to preserve post-thaw spermatozoa viability in vitro and maintained membrane stability after cryopreservation. PMID:14643862

  6. Isolation and characterisation of a hyaluronan binding protein, hyaluronectin, from human placenta and its colocalisation with hyaluronan.

    PubMed Central

    Ponting, J M; Kumar, S

    1995-01-01

    Hyaluronan (HA) is a major component of the extracellular matrix and is known to influence cell behaviour and to play a role in angiogenesis, morphogenesis and tissue remodelling, although little is known concerning the regulation of these effects. Until now its detection in the placenta has been by indirect methods, which has led to conflicting conclusions as to its distribution and hence its role. Hyaluronectin (HN) is one of a group of proteins with HA binding ability which may regulate the effects of HA. Although nervous tissue HN has been partly characterised with regard to its distribution, structure and biochemistry, little is known about the mesenchymal isoform and its distribution in placenta has not previously been reported. Using specific probes we have characterised the distribution of HA and HN in human placental tissue. At all stages of development studied (8, 10, 12, 30 and 38 wk gestation) HA and HN were unequivocally colocalised, being distributed in the extracellular matrix of stromal tissue of placental villi, chorioallantoic membranes and umbilical cord. Particularly strong immunoreactivity was observed in the villous stroma immediately adjacent to fibrinoid depositions at sites of denudation of the trophoblast layer. Extraction and characterisation of the HN from placental villi have revealed 4 major glycoproteins of 47, 52, 57 and 67 kDa, this being a different pattern and smaller molecular range than observed for the nervous tissue form. This is the first direct demonstration of the presence of HA and HN in the placenta and identifies an abundant new source of mesenchymal HN. The functions of mesenchymal HN are unknown but may include ion exchange, immunosuppression and regulation of the effects of HA in such roles as maintenance of tissue architecture, cell migration and angiogenesis. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7544332

  7. Multivalent dendrimers presenting spatially controlled clusters of binding epitopes in thermoresponsive hyaluronan hydrogels.

    PubMed

    Seelbach, Ryan J; Fransen, Peter; Peroglio, Marianna; Pulido, Daniel; Lopez-Chicon, Patricia; Duttenhoefer, Fabian; Sauerbier, Sebastian; Freiman, Thomas; Niemeyer, Philipp; Semino, Carlos; Albericio, Fernando; Alini, Mauro; Royo, Miriam; Mata, Alvaro; Eglin, David

    2014-10-01

    The controlled presentation of biofunctionality is of key importance for hydrogel applications in cell-based regenerative medicine. Here, a versatile approach was demonstrated to present clustered binding epitopes in an injectable, thermoresponsive hydrogel. Well-defined multivalent dendrimers bearing four integrin binding sequences and an azido moiety were covalently grafted to propargylamine-derived hyaluronic acid (Hyal-pa) using copper-catalyzed alkyne-azide cycloaddition (CuAAC), and then combined with pN-modified hyaluronan (Hyal-pN). The dendrimers were prepared by synthesizing a bifunctional diethylenetriamine pentaacetic acid core with azido and NHBoc oligo(ethylene glycol) aminoethyl branches, then further conjugated with solid-phase synthesized RGDS and DGRS peptides. Azido terminated pN was synthesized by reversible addition-fragmentation chain transfer polymerization and reacted to Hyal-pa via CuAAC. Nuclear magnetic resonance (NMR), high performance liquid chromatography, size exclusion chromatography and mass spectroscopy proved that the dendrimers had well-defined size and were disubstituted. NMR and atomic absorption analysis confirmed the hyaluronan was affixed with dendrimers or pN. Rheological measurements demonstrated that dendrimers do not influence the elastic or viscous moduli of thermoresponsive hyaluronan compositions at a relevant biological concentration. Finally, human mesenchymal stromal cells were encapsulated in the biomaterial and cultured for 21days, demonstrating the faculty of this dendrimer-modified hydrogel as a molecular toolbox for tailoring the biofunctionality of thermoresponsive hyaluronan carriers for biomedical applications.

  8. Characterization of glycidyl methacrylate – Crosslinked hyaluronan hydrogel scaffolds incorporating elastogenic hyaluronan oligomers

    PubMed Central

    Ibrahim, S.; Kothapalli, C.R.; Kang, Q.K.; Ramamurthi, A.

    2013-01-01

    Prior studies on two-dimensional cell cultures suggest that hyaluronic acid (HA) stimulates cell-mediated regeneration of extracellular matrix structures, specifically those containing elastin, though such biologic effects are dependent on HA fragment size. Towards being able to regenerate three-dimensional (3-D) elastic tissue constructs, the present paper studies photo-crosslinked hydrogels containing glycidyl methacrylate (GM)-derivatized bio-inert high molecular weight(HMW)HA (1 × 106 Da) and a bioactive HA oligomer mixture (HA-o: MW ~0.75 kDa). The mechanical (rheology, degradation) and physical (apparent crosslinking density, swelling ratio) properties of the gels varied as a function of incorporated HA oligomer content; however, overall, the mechanics of these hydrogels were too weak for vascular applications as stand-alone materials. Upon in vivo subcutaneous implantation, only a few inflammatory cells were evident around GM–HA gels, however their number increased as HA-o content within the gels increased, and the collagen I distribution was uniform. Smooth muscle cells (SMC) were encapsulated into GM hydrogels, and calcein acetoxymethyl detection revealed that the cells were able to endure twofold the level of UV exposure used to crosslink the gels. After 21 days of culture, SMC elastin production, measured by immunofluorescence quantification, showed HA-o to increase cellular deposition of elastic matrix twofold relative to HA-o-free GM–HAgels. These results demonstrate that cell response to HA/HA-o is not altered by their methacrylation and photo-crosslinking into a hydrogel, and that HA-o incorporation into cell-encapsulating hydrogel scaffolds can be useful for enhancing their production of elastic matrix structures in a 3-D space, important for regenerating elastic tissues. PMID:20709199

  9. Identification of daidzein as a ligand of retinoic acid receptor that suppresses expression of matrix metalloproteinase-9 in HaCaT cells.

    PubMed

    Oh, Hyeon-Jeong; Kang, Young-Gyu; Na, Tae-Young; Kim, Hyeon-Ji; Park, Jun Seong; Cho, Won-Jea; Lee, Mi-Ock

    2013-08-25

    Retinoids have been used as therapeutics for diverse skin diseases, but their side effects limit clinical usage. Here, we report that extracts of two soybeans, Glycine max and Rhynchosia nulubilis, and their ethyl acetate fractions increased the transcriptional activity of retinoic acid receptors (RARs), and that daidzin and genistin were the major constituents of the active fractions. Daidzin and its aglycone, daidzein, induced transcriptional activity of RAR and RARγ. FRET analysis demonstrated that daidzein, but not daidzin, bound both RAR and RARγ with EC50 values of 28μM and 40μM, respectively. Daidzein increased expression of mRNA of RARγ through direct binding of RAR and recruitment of p300 to the RARγ2 promoter. Further, mRNA and gelatinolytic activity of matrix metalloproteinase-9 were decreased by daidzein in HaCaT cells. Together, these results indicate that daidzein functions as a ligand of RAR that could be a candidate therapeutic for skin diseases.

  10. Hyaluronan as an Immune Regulator in Human Diseases

    PubMed Central

    NOBLE, PAUL W.; LIANG, JIURONG; JIANG, DIANHUA

    2010-01-01

    Accumulation and turnover of extracellular matrix components are the hallmarks of tissue injury. Fragmented hyaluronan stimulates the expression of inflammatory genes by a variety of immune cells at the injury site. Hyaluronan binds to a number of cell surface proteins on a variety of cell types. Hyaluronan fragments signal through both Toll-like receptor (TLR) 4 and TLR2 as well as CD44 to stimulate inflammatory genes in inflammatory cells. Hyaluronan is also present on the cell surface of epithelial cells and provides protection against tissue damage by interacting with TLR2 and TLR4 on these parenchymal cells. Hyaluronan and hyaluronan-binding proteins regulate inflammation, tissue injury and repair through regulating inflammatory cell recruitment, release of inflammatory cytokines, and stem cell migration. This review focuses on the role of hyaluronan as an immune regulator in human diseases. PMID:21248167

  11. Hyaluronan Expressed by the Hematopoietic Microenvironment Is Required for Bone Marrow Hematopoiesis*

    PubMed Central

    Goncharova, Valentina; Serobyan, Naira; Iizuka, Shinji; Schraufstatter, Ingrid; de Ridder, Audrey; Povaliy, Tatiana; Wacker, Valentina; Itano, Naoki; Kimata, Koji; Orlovskaja, Irina A.; Yamaguchi, Yu; Khaldoyanidi, Sophia

    2012-01-01

    The contribution of hyaluronan (HA) to the regulatory network of the hematopoietic microenvironment was studied using knock-out mice of three hyaluronan synthase genes (Has1, Has2, and Has3). The number of hematopoietic progenitors was decreased in bone marrow and increased in extramedullary sites of Prx1-Cre;Has2flox/flox;Has1−/−;Has3−/− triple knock-out (tKO) mice as compared with wild type (WT) and Has1−/−;Has3−/− double knock-out (dKO) mice. In line with this observation, decreased hematopoietic activity was observed in long term bone marrow cultures (LTBMC) from tKO mice, whereas the formation of the adherent layer and generation of hematopoietic cells in WT and dKO cultures was not different. 4-Methylumbelliferone (4MU) was used to pharmacologically inhibit the production of HA in LTBMC. Treatment with 4MU inhibited HA synthesis, decreased expression of HAS2 and HAS3, and eliminated hematopoiesis in LTBMC, and this effect was alleviated by the addition of exogenous HA. Exogenous HA also augmented the cell motility in LTBMC, which correlated with the HA-stimulated production of chemokines and growth factors. Conditioned media from HA-induced LTBMC enhanced the chemotaxis of hematopoietic stem/progenitor cells (HSPC) in response to SDF-1. Exposure of endothelial cells to 4MU decreased their ability to support HSPC rolling and adhesion. In addition, migration of transplanted HSPC into the marrow of 4MU-pretreated mice was lower than in untreated mice. Collectively, the results suggest that HA depletion reduces the ability of the microenvironment to support HSPC, and confirm a role for HA as a necessary regulatory element in the structure of the hematopoietic microenvironment. PMID:22654110

  12. Characterization of CD44-Mediated Cancer Cell Uptake and Intracellular Distribution of Hyaluronan-Grafted Liposomes

    PubMed Central

    Qhattal, Hussaini Syed Sha; Liu, Xinli

    2011-01-01

    Hyaluronan (HA) is a biocompatible and biodegradable linear polysaccharide which is of interest for tumor targeting through cell surface CD44 receptors. HA binds with high affinity to CD44 receptors, which are overexpressed in many tumors and involved in cancer metastasis. In the present study, we investigated the impact of HA molecular weight (MW), grafting density, and CD44 receptor density on endocytosis of HA-grafted liposomes (HA-liposomes) by cancer cells. Additionally, the intracellular localization of the HA-liposomes was determined. HAs of different MWs (5-8, 10-12, 175-350, and 1600 kDa) were conjugated to liposomes with varying degrees of grafting density. HA surface density was quantified using the hexadecyltrimethylammonium bromide turbidimetric method. Cellular uptake and subcellular localization of HA-liposomes were evaluated by flow cytometry and fluorescence microscopy. Mean particle sizes of HA-liposomes ranged from 120 to 180 nm and increased with the bigger size of HA. HA-liposome uptake correlated with HA MW (5-8 < 10-12 < 175-350 kDa), grafting density, and CD44 receptor density and exceeded that obtained with unconjugated plain liposomes. HA-liposomes were taken up into cells via lipid raft-mediated endocytosis, which is both energy- and cholesterol-dependent. Once within cells, HA-liposomes localized primarily to endosomes and lysosomes. The results demonstrate that cellular targeting efficiency of HA-liposomes depends strongly upon HA MW, grafting density, and cell surface receptor CD44 density. The results support a role of HA-liposomes for targeted drug delivery. PMID:21696190

  13. Hyaluronan and Hyaluronan-Binding Proteins Accumulate in Both Human Type 1 Diabetic Islets and Lymphoid Tissues and Associate With Inflammatory Cells in Insulitis

    PubMed Central

    Bogdani, Marika; Johnson, Pamela Y.; Potter-Perigo, Susan; Nagy, Nadine; Day, Anthony J.; Bollyky, Paul L.

    2014-01-01

    Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that is present in pancreatic islets, but little is known about its involvement in the development of human type 1 diabetes (T1D). We have evaluated whether pancreatic islets and lymphoid tissues of T1D and nondiabetic organ donors differ in the amount and distribution of HA and HA-binding proteins (hyaladherins), such as inter-α-inhibitor (IαI), versican, and tumor necrosis factor–stimulated gene-6 (TSG-6). HA was dramatically increased both within the islet and outside the islet endocrine cells, juxtaposed to islet microvessels in T1D. In addition, HA was prominent surrounding immune cells in areas of insulitis. IαI and versican were present in HA-rich areas of islets, and both molecules accumulated in diabetic islets and regions exhibiting insulitis. TSG-6 was observed within the islet endocrine cells and in inflammatory infiltrates. These patterns were only observed in tissues from younger donors with disease duration of <10 years. Furthermore, HA and IαI amassed in follicular germinal centers and in T-cell areas in lymph nodes and spleens in T1D patients compared with control subjects. Our observations highlight potential roles for HA and hyaladherins in the pathogenesis of diabetes. PMID:24677718

  14. 4-Methylumbelliferone Treatment and Hyaluronan Inhibition as a Therapeutic Strategy in Inflammation, Autoimmunity, and Cancer

    PubMed Central

    Nagy, Nadine; Kuipers, Hedwich F.; Frymoyer, Adam R.; Ishak, Heather D.; Bollyky, Jennifer B.; Wight, Thomas N.; Bollyky, Paul L.

    2015-01-01

    Hyaluronan (HA) is a prominent component of the extracellular matrix at many sites of chronic inflammation, including type 1 diabetes (T1D), multiple sclerosis, and numerous malignancies. Recent publications have demonstrated that when HA synthesis is inhibited using 4-methylumbelliferone (4-MU), beneficial effects are observed in several animal models of these diseases. Notably, 4-MU is an already approved drug in Europe and Asia called “hymecromone” where it is used to treat biliary spasm. However, there is uncertainty regarding how 4-MU treatment provides benefit in these animal models and the potential long-term consequences of HA inhibition. Here, we review what is known about how HA contributes to immune dysregulation and tumor progression. Then, we review what is known about 4-MU and hymecromone in terms of mechanism of action, pharmacokinetics, and safety. Finally, we review recent studies detailing the use of 4-MU to treat animal models of cancer and autoimmunity. PMID:25852691

  15. Effects of leflunomide on hyaluronan synthases (HAS): NF-kappa B-independent suppression of IL-1-induced HAS1 transcription by leflunomide.

    PubMed

    Stuhlmeier, Karl M

    2005-06-01

    Despite evidence that points to unfettered hyaluronic acid (HA) production as a culprit in the progression of rheumatic disorders, little is known about differences in regulation and biological functions of the three hyaluronan synthase (HAS) genes. Testing the effects of drugs with proven anti-inflammatory effects could help to clarify biological functions of these genes. In this study, we demonstrate that leflunomide suppresses HA release in fibroblast-like synoviocytes (FLS) in a dose-dependent manner. We further demonstrate that leflunomide suppresses HA synthase activity, as determined by (14)C-glucuronic acid incorporation assays. Additional experiments revealed that in FLS, leflunomide specifically blocked the induction of HAS1. HAS2 and HAS3, genes that are, in contrast to HAS1, constitutively expressed in FLS, are not significantly affected. Leflunomide can function as a NF-kappaB inhibitor. However, EMSA experiments demonstrate that at the concentrations used, leflunomide neither interferes with IL-1beta- nor with PMA-induced NF-kappaB translocation. Furthermore, reconstituting the pyrimidine synthase pathway did not lead to the restoration of IL-1beta-induced HAS1 activation. More importantly, two tyrosine kinase inhibitors mimicked the effect of leflunomide in that both blocked IL-1beta-induced HAS1 activation without affecting HAS2 or HAS3. These data point at HAS1 activation as the possible cause for unfettered HA production in rheumatoid arthritis and might explain, at least in part, the beneficial effects of leflunomide treatment. These findings also support the concept that IL-1beta-induced HAS1 activation depends on the activation of tyrosine kinases, and indicate that leflunomide blocks HA release by suppressing tyrosine kinases rather than through inhibition of NF-kappaB translocation.

  16. Single-Step Grafting of Aminooxy-Peptides to Hyaluronan: A Simple Approach to Multifunctional Therapeutics for Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Sestak, Joshua.; Mullins, Meagan; Northrup, Laura; Thati, Shara; Siahaan, Teruna; Berkland, Cory

    2013-01-01

    The immune response to antigens is directed in part by the presence or absence of costimulatory signals. The ability to coincidently present both antigen and, for example, a peptide that inhibits or activates the costimulatory pathway, would be a valuable tool for tolerization or immunization, respectively. A simple reaction scheme utilizing oxime chemistry was identified as a means to efficiently conjugate different peptide species to hyaluronan. Peptides synthesized with an aminooxy N-terminus reacted directly to hyaluronan under slightly acidic aqueous conditions without the need for a catalyst. The resulting oxime bond was found to rapidly hydrolyze at pH 2 releasing peptide, but was stable at higher pH values (5.5 and 7). Two different peptide species, a multiple sclerosis antigen (PLP) and an ICAM-1 ligand (LABL) known to block immune cell stimulation, were functionalized with the aminooxy end group. These peptides showed similar reactivity to hyaluronan and were conjugated in an equimolar ratio. The resulting hyaluronan with grafted PLP and LABL significantly inhibited disease in mice with experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Aminooxy-peptides facilitate simple synthesis of multifunctional hyaluronan graft polymers, thus enabling novel approaches to antigen-specific immune modulation. PMID:23541930

  17. Sargahydroquinoic acid inhibits TNFα-induced AP-1 and NF-κB signaling in HaCaT cells through PPARα activation.

    PubMed

    Jeon, Youngsic; Jung, Yujung; Kim, Min Cheol; Kwon, Hak Cheol; Kang, Ki Sung; Kim, Yong Kee; Kim, Su-Nam

    2014-08-01

    Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors and expressed in various cell types in the skin, including keratinocytes, fibroblasts and infiltrating immune cells. Thus, their ligands are targets for the treatment of various skin disorders, such as photo-aging and chronological aging of skin. Intensive studies have revealed that PPARα/γ functions in photo-aging and age-related inflammation by regulating matrix metalloproteinases (MMPs) via activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB). However, the detailed mechanism of PPARα/γ's role in skin aging has not yet been elucidated. In this study, we confirmed that sargahydroquinoic acid (SHQA) as a PPARα/γ ligand significantly decreased Tumor Necrosis Factor-alpha (TNFα)-induced MMP-2/-9 expression by downregulating TNFα-induced transcription factors, subsequently reducing IκBα degradation and blocking NF-κB p65 nuclear translocation in HaCaT human epidermal keratinocyte cells. Treatment of cells with SHQA and GW6471 (PPARα antagonist) not bisphenol A diglycidyl ether (PPARγ antagonists), reversed the effect on TNFα-induced inflammatory signaling pathway activation. Taken together, our data suggest that SHQA inhibit TNFα-induced MMP-2/-9 expression and age-related inflammation by suppressing AP-1 and NF-κB pathway via PPARα. PMID:25019995

  18. Immune escape mutants of Highly Pathogenic Avian Influenza H5N1 selected using polyclonal sera: identification of key amino acids in the HA protein.

    PubMed

    Sitaras, Ioannis; Kalthoff, Donata; Beer, Martin; Peeters, Ben; de Jong, Mart C M

    2014-01-01

    Evolution of Avian Influenza (AI) viruses--especially of the Highly Pathogenic Avian Influenza (HPAI) H5N1 subtype--is a major issue for the poultry industry. HPAI H5N1 epidemics are associated with huge economic losses and are sometimes connected to human morbidity and mortality. Vaccination (either as a preventive measure or as a means to control outbreaks) is an approach that splits the scientific community, due to the risk of it being a potential driving force in HPAI evolution through the selection of mutants able to escape vaccination-induced immunity. It is therefore essential to study how mutations are selected due to immune pressure. To this effect, we performed an in vitro selection of mutants from HPAI A/turkey/Turkey/1/05 (H5N1), using immune pressure from homologous polyclonal sera. After 42 rounds of selection, we identified 5 amino acid substitutions in the Haemagglutinin (HA) protein, most of which were located in areas of antigenic importance and suspected to be prone to selection pressure. We report that most of the mutations took place early in the selection process. Finally, our antigenic cartography studies showed that the antigenic distance between the selected isolates and their parent strain increased with passage number.

  19. Effects of Lipophilic Extract of Viscum album L. and Oleanolic Acid on Migratory Activity of NIH/3T3 Fibroblasts and on HaCat Keratinocytes

    PubMed Central

    Kuonen, R.; Weissenstein, U.; Urech, K.; Kunz, M.; Hostanska, K.; Estko, M.; Heusser, P.; Baumgartner, S.

    2013-01-01

    Viscum album L. lipophilic extract (VALE) contains pharmacologically active pentacyclic triterpenes that are known to exhibit immunomodulatory, antitumor, and wound healing activity. Preliminary clinical observations indicate that VALE was able to influence cutaneous wound healing in vivo. The objective of this study was to investigate wound closure related properties of VALE in vitro. As measured in a wound healing assay, VALE and its predominant triterpene oleanolic acid (OA) significantly and dose dependently promoted the migration of NIH/3T3 fibroblasts in vitro, thereby leading to an enhanced wound closure. Compared to the negative control, maximal stimulation by 26.1% and 26.2%, respectively, was attained with 10 μg/mL VALE and 1 μg/mL OA. Stimulation of proliferation in NIH/3T3 fibroblasts by VALE and OA could be excluded. At higher concentrations both substances affected proliferation and viability of NIH/3T3 fibroblasts and HaCat keratinocytes. In the toxic range of concentrations of VALE and OA, migration of NIH/3T3 fibroblasts was suppressed. The extent of the stimulatory effect on cell migration of VALE quite closely corresponded to the effect expected by the concentrations of OA contained in the crude extract VALE. These data support the casual observation that Viscum album L. lipophilic extract might modulate wound healing related processes in vivo. PMID:24379890

  20. Interactions between Hyaluronan and Its Receptors (CD44, RHAMM) Regulate the Activities of Inflammation and Cancer

    PubMed Central

    Misra, Suniti; Hascall, Vincent C.; Markwald, Roger R.; Ghatak, Shibnath

    2015-01-01

    The glycosaminoglycan hyaluronan (HA), a major component of extracellular matrices, and cell surface receptors of HA have been proposed to have pivotal roles in cell proliferation, migration, and invasion, which are necessary for inflammation and cancer progression. CD44 and receptor for HA-mediated motility (RHAMM) are the two main HA-receptors whose biological functions in human and murine inflammations and tumor cells have been investigated comprehensively. HA was initially considered to be only an inert component of connective tissues, but is now known as a “dynamic” molecule with a constant turnover in many tissues through rapid metabolism that involves HA molecules of various sizes: high molecular weight HA (HMW HA), low molecular weight HA, and oligosaccharides. The intracellular signaling pathways initiated by HA interactions with CD44 and RHAMM that lead to inflammatory and tumorigenic responses are complex. Interestingly, these molecules have dual functions in inflammations and tumorigenesis. For example, the presence of CD44 is involved in initiation of arthritis, while the absence of CD44 by genetic deletion in an arthritis mouse model increases rather than decreases disease severity. Similar dual functions of CD44 exist in initiation and progression of cancer. RHAMM overexpression is most commonly linked to cancer progression, whereas loss of RHAMM is associated with malignant peripheral nerve sheath tumor growth. HA may similarly perform dual functions. An abundance of HMW HA can promote malignant cell proliferation and development of cancer, whereas antagonists to HA-CD44 signaling inhibit tumor cell growth in vitro and in vivo by interfering with HMW HA-CD44 interaction. This review describes the roles of HA interactions with CD44 and RHAMM in inflammatory responses and tumor development/progression, and how therapeutic strategies that block these key inflammatory/tumorigenic processes may be developed in rodent and human diseases. PMID:25999946

  1. Characterization of three novel fatty acid- and retinoid-binding protein genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the cereal cyst nematodes Heterodera avenae and H. filipjevi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinoid-binding (FAR) proteins are nematode-spe...

  2. Antitumor effects of the hyaluronan inhibitor 4-methylumbelliferone on pancreatic cancer

    PubMed Central

    Yoshida, Eri; Kudo, Daisuke; Nagase, Hayato; Shimoda, Hiroshi; Suto, Shinichiro; Negishi, Mika; Kakizaki, Ikuko; Endo, Masahiko; Hakamada, Kenichi

    2016-01-01

    Hyaluronan (HA) is a major component of the extracellular matrix (ECM), and influences tumor invasion and metastasis. In a previous study, the present authors reported for the first time that 4-methylumbelliferone (MU) inhibited HA synthesis and suppressed tumor growth. However, the localization of HA and the changes in ECM morphology caused by MU in pancreatic cancer remain to be examined in detail. In the present study, the cytotoxicity of MU and its effect on cellular proliferation was evaluated in the human pancreatic cancer cell line MIA PaCa-2. The amount of HA synthesized and the retention of HA around the cells were quantitatively and immunohistochemically analyzed in vitro and in vivo. Structural changes in the ECM in the tumor tissue were investigated using an electron microscope. MU treatment led to a decrease in extracellular HA retention, as evidenced by a particle exclusion assay and immunohistochemical staining. Cell proliferation was suppressed by MU in a dose-dependent manner. The release of lactate dehydrogenase into the culture medium due to damage to the cellular membrane did not increase following MU administration. In tumor-inoculated mice, MU suppressed any increase in tumor volume and decreased the quantity of HA. Electron microscopy revealed that MU attenuated the intercellular space and caused it to be less cohesive. These data indicate that MU inhibits HA synthesis and reduces the amount of HA in the ECM while exhibiting no obvious cytotoxic effect. These findings suggest that MU has potential as a novel therapeutic agent for pancreatic cancer.

  3. Formation and properties of hyaluronan/nano Ag and hyaluronan-lecithin/nano Ag films.

    PubMed

    Khachatryan, Gohar; Khachatryan, Karen; Grzyb, Jacek; Fiedorowicz, Maciej

    2016-10-20

    A facile and environmentally friendly method of the preparation of silver nanoparticles embedded in hyaluronan (Hyal/Ag) and hyaluronan-lecithin (Hyal-L/Ag) matrix was developed. Thin, elastic foils were prepared from gels by an in situ synthesis of Ag in an aqueous solution of sodium hyaluronate (Hyal), using aq. d-(+)-xylose solution as a reducing agent. The gels were applied to a clean, smooth, defatted Teflon surface and left for drying in the air. The dry foils were stored in a closed container. UV-vis spectroscopy, transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) spectra confirmed formation of about 10nm ball-shaped Ag nanoparticles situated within the polysaccharide template. Thermal properties of the composites were characterized involving differential scanning calorimetry (DSC) and thermogravimetric (TGA) analyses, whereas molecular weights of polysaccharide chains of the matrix were estimated with the size exclusion chromatography coupled with multiangle laser light scattering and refractometric detectors (HPSEC-MALLS-RI). An increase in the molecular weight of the hyaluronate after generation of Ag nanoparticles was observed. The foils showed specific properties. The study confirmed that silver nanoparticles can be successfully prepared with environmentally friendly method, using hyaluronan as a stabilizing template. Hyaluronan and hyaluronan-lecithin matrices provide nanocrystals uniform in size and shape. The composites demonstrated a bacteriostatic activity. PMID:27474588

  4. A hexadecylamide derivative of hyaluronan (HYMOVIS®) has superior beneficial effects on human osteoarthritic chondrocytes and synoviocytes than unmodified hyaluronan

    PubMed Central

    2013-01-01

    Background Intra-articular hyaluronan (HA) injection provides symptomatic benefit in the treatment of osteoarthritis (OA). Previously we found superior beneficial effects in a large animal OA model of a hexadecylamide derivative compared with unmodified HA of the same initial molecular weight. The current study sought to define possible molecular mechanisms whereby this enhanced relief of symptoms was occurring. Methods Chondrocytes and synovial fibroblasts were isolated from tissues of patients undergoing arthroplasty for knee OA. Monolayer cultures of cells were treated with 0, 0.5, 1.0 or 1.5 mg/mL of unmodified HA (500–730 kDa) or a hexadecylamide derivative of HA of the same initial molecular weight (HYADD4®-G; HYMOVIS®) simultaneously or 1 hour before incubation with interleukin (IL)-1beta (2 ng/mL). Cultures were terminated 15 or 30 minutes later (chondrocytes and synovial fibroblasts, respectively) for quantitation of phosphorylated-(p)-JNK, p-NFkappaB, p-p38, or at 24 hours for quantitation of gene expression (MMP1 &13, ADAMTS4 &5, TIMP1 &3, CD44, COL1A1 &2A1, ACAN, PTGS2, IL6, TNF) and matrix metalloproteinase (MMP)-13 activity. Results The hexadecylamide derivative of HA had significantly better amelioration of IL-1beta-induced gene expression of key matrix degrading enzymes (MMP1, MMP13, ADAMTS5), and inflammatory mediators (IL6, PTGS2) by human OA chondrocytes and synovial fibroblasts. Pre-incubation of cells with the derivatized HA for 1 hour prior to IL-1beta exposure significantly augmented the inhibition of MMP1, MMP13, ADAMTS4 and IL6 expression by chondrocytes. The reduction in MMP13 mRNA by the amide derivative of HA was mirrored in reduced MMP-13 protein and enzyme activity in IL-1beta-stimulated chondrocytes. This was associated in part with a greater inhibition of phosphorylation of the cell signalling molecules JNK, p38 and NF-kappaB. Conclusions The present studies have demonstrated several potential key mechanisms whereby the

  5. Inter-α-inhibitor Impairs TSG-6-induced Hyaluronan Cross-linking*

    PubMed Central

    Baranova, Natalia S.; Foulcer, Simon J.; Briggs, David C.; Tilakaratna, Viranga; Enghild, Jan J.; Milner, Caroline M.; Day, Anthony J.; Richter, Ralf P.

    2013-01-01

    Under inflammatory conditions and in the matrix of the cumulus-oocyte complex, the polysaccharide hyaluronan (HA) becomes decorated covalently with heavy chains (HCs) of the serum glycoprotein inter-α-inhibitor (IαI). This alters the functional properties of the HA as well as its structural role within extracellular matrices. The covalent transfer of HCs from IαI to HA is catalyzed by TSG-6 (tumor necrosis factor-stimulated gene-6), but TSG-6 is also known as a HA cross-linker that induces condensation of the HA matrix. Here, we investigate the interplay of these two distinct functions of TSG-6 by studying the ternary interactions of IαI and TSG-6 with well defined films of end-grafted HA chains. We demonstrate that TSG-6-mediated cross-linking of HA films is impaired in the presence of IαI and that this effect suppresses the TSG-6-mediated enhancement of HA binding to CD44-positive cells. Furthermore, we find that the interaction of TSG-6 and IαI in the presence of HA gives rise to two types of complexes that independently promote the covalent transfer of heavy chains to HA. One type of complex interacts very weakly with HA and is likely to correspond to the previously reported covalent HC·TSG-6 complexes. The other type of complex is novel and binds stably but noncovalently to HA. Prolonged incubation with TSG-6 and IαI leads to HA films that contain, in addition to covalently HA-bound HCs, several tightly but noncovalently bound molecular species. These findings have important implications for understanding how the biological activities of TSG-6 are regulated, such that the presence or absence of IαI will dictate its function. PMID:24005673

  6. Hyaluronan and chondroitin sulfate proteoglycans in the supramolecular organization of the mammalian vitreous body.

    PubMed

    Theocharis, Dimitrios A; Skandalis, Spyros S; Noulas, Argiris V; Papageorgakopoulou, Nickoletta; Theocharis, Achilleas D; Karamanos, Nikos K

    2008-01-01

    The mammalian vitreous gel is a specialized type of highly hydrated extracellular matrix, which is composed of interwoven networks of uronic acid-containing polyanionic macromolecules, (i.e., hyaluronan, versican, and IX collagen) and collagen fibrils. Hyaluronan comprises the vast majority of the uronic acid-containing molecules, which contributes to structure and function of vitreous in at least two ways: its unique biophysical and hydrodynamic properties influence the vitreous homeostasis and biomechanics; it is also a template for assembly of other extracellular macromolecules, for example, versican. The other uronic acid-containing molecules namely versican and IX collagen--two chondroitin sulfate (CS) proteoglycans--occur in the vitreous without significant quantitative variations among different mammalians but with some marked variations on the molecular size and sulfation pattern of their chondroitin sulfate side chains. The contribution of versican and IX collagen (through their protein and their CS side chains) to the supramolecular organization of the vitreous gel is poorly understood. However, versican having the ability to bind hyaluronan via its N-terminal and other binding partners via its C-terminal region can play a crucial role on the structural stability and functionality of the vitreous.

  7. Sargahydroquinoic acid inhibits TNFα-induced AP-1 and NF-κB signaling in HaCaT cells through PPARα activation

    SciTech Connect

    Jeon, Youngsic; Jung, Yujung; Kim, Min Cheol; Kwon, Hak Cheol; Kang, Ki Sung; Kim, Yong Kee; Kim, Su-Nam

    2014-08-08

    Highlights: • SHQA increases PPARα/γ transactivation and inhibits MMP-2/-9 expression. • SHQA inhibits TNFα-induced AP-1 and MAPK signaling. • SHQA inhibits TNFα-induced p65 translocation and IκBα phosphorylation. • SHQA inhibits TNFα-induced AP-1 and NF-κB signaling via PPARα. - Abstract: Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors and expressed in various cell types in the skin, including keratinocytes, fibroblasts and infiltrating immune cells. Thus, their ligands are targets for the treatment of various skin disorders, such as photo-aging and chronological aging of skin. Intensive studies have revealed that PPARα/γ functions in photo-aging and age-related inflammation by regulating matrix metalloproteinases (MMPs) via activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB). However, the detailed mechanism of PPARα/γ’s role in skin aging has not yet been elucidated. In this study, we confirmed that sargahydroquinoic acid (SHQA) as a PPARα/γ ligand significantly decreased Tumor Necrosis Factor-alpha (TNFα)-induced MMP-2/-9 expression by downregulating TNFα-induced transcription factors, subsequently reducing IκBα degradation and blocking NF-κB p65 nuclear translocation in HaCaT human epidermal keratinocyte cells. Treatment of cells with SHQA and GW6471 (PPARα antagonist) not bisphenol A diglycidyl ether (PPARγ antagonists), reversed the effect on TNFα-induced inflammatory signaling pathway activation. Taken together, our data suggest that SHQA inhibit TNFα-induced MMP-2/-9 expression and age-related inflammation by suppressing AP-1 and NF-κB pathway via PPARα.

  8. Nanofilms of hyaluronan/chitosan assembled layer-by-layer: An antibacterial surface for Xylella fastidiosa.

    PubMed

    Hernández-Montelongo, Jacobo; Nascimento, Vicente F; Murillo, Duber; Taketa, Thiago B; Sahoo, Prasana; de Souza, Alessandra A; Beppu, Marisa M; Cotta, Monica A

    2016-01-20

    In this work, nanofilms of hyaluronan/chitosan (HA/CHI) assembled layer by layer were synthesized; their application as a potential antimicrobial material was demonstrated for the phytopathogen Xylella fastidiosa, a gram-negative bacterium, here used as a model. For the synthesis, the influence of pH and ionic strength of these natural polymer stem-solutions on final characteristics of the HA/CHI nanofilms was studied in detail. The antibacterial effect was evaluated using widefield fluorescence microscopy. These results were correlated with the chemical properties of the nanofilms, studied by FTIR and Raman spectroscopy, as well as with their morphology and surface properties characterized using SEM and AFM. The present findings can be extended to design and optimize HA/CHI nanofilms with enhanced antimicrobial behavior for other type of phytopathogenic gram-negative bacteria species, such as Xanthomonas citri, Xanthomas campestri and Ralstonia solanacearum. PMID:26572322

  9. Peptide-decorated polyvinyl alcohol/hyaluronan nanofibers for human induced pluripotent stem cell culture.

    PubMed

    Deng, Yi; Zhang, Xiaohong; Zhao, Yinghui; Liang, Shangshang; Xu, Anxiu; Gao, Xiang; Deng, Feng; Fang, Jing; Wei, Shicheng

    2014-01-30

    Realization of the full potential of human induced pluripotent stem cells (hiPSCs) in clinical applications requires development of well-defined conditions for their growth and differentiation. A novel fully defined polyvinyl alcohol/hyaluronan (PVA/HA) polysaccharide nanofiber was developed for hiPSCs culture in commercially available xeno-free, chemically defined medium. Vitronectin peptide (VP) was immobilized to PVA/HA nanofibers through NHS/EDC chemistry. The hiPSCs successfully grew and proliferated on the VP-decorated PVA/HA nanofibers, similar to those on Matrigel™. Such well-defined, xeno-free and safe nanofiber substrate that supports culture of hiPSCs will not only help to accelerate the translational perspectives of hiPSCs, but also provide a platform to investigate the cell-nanofiber interaction mechanisms that regulate stem cell proliferation and differentiation.

  10. Hyaluronan Inhibits Tlr-4-Dependent RANKL Expression in Human Rheumatoid Arthritis Synovial Fibroblasts

    PubMed Central

    Hirabara, Shinya; Ishiguro, Naoki; Kojima, Toshihisa

    2016-01-01

    The Toll-like receptor (TLR) signaling pathway is activated in synovial fibroblast cells in patients with rheumatoid arthritis (RA). The receptor activator of nuclear factor-κB (RANK) and its ligand, RANKL, are key molecules involved in the differentiation of osteoclasts and joint destruction in RA. Hyaluronan (HA) is a major extracellular component and an important immune regulator. In this study, we show that lipopolysaccharide (LPS) stimulation significantly increases RANKL expression via a TLR-4 signaling pathway. We also demonstrate that HA suppresses LPS-induced RANKL expression, which is dependent on CD44, but not intercellular adhesion molecule-1 (ICAM-1). Our study provides evidence for HA-mediated suppression of TLR-4-dependent RANKL expression. This could present an alternative target for the treatment of destructed joint bones and cartilages in RA. PMID:27054952

  11. Neocartilage formation from mesenchymal stem cells grown in type II collagen-hyaluronan composite scaffolds.

    PubMed

    Yeh, Hsi-Yi; Lin, Ting-Yu; Lin, Chen-Huan; Yen, B Linju; Tsai, Ching-Lin; Hsu, Shan-Hui

    2013-01-01

    Three-dimensional (3D) collagen type II-hyaluronan (HA) composite scaffolds (CII-HA) which mimics the extracellular environment of natural cartilage were fabricated in this study. Rheological measurements demonstrated that the incorporation of HA increased the compression modulus of the scaffolds. An initial in vitro evaluation showed that scaffolds seeded with porcine chondrocytes formed cartilaginous-like tissue after 8 weeks, and HA functioned to promote the growth of chondrocytes into scaffolds. Placenta-derived multipotent cells (PDMC) and gingival fibroblasts (GF) were seeded on tissue culture polystyrene (TCPS), CII-HA films, and small intestinal submucosa (SIS) sheets for comparing their chondrogenesis differentiation potentials with those of adipose-derived adult stem cells (ADAS) and bone marrow-derived mesenchymal stem cells (BMSC). Among different cells, PDMC showed the greatest chondrogenic differentiation potential on both CII-HA films and SIS sheets upon TGF-β3 induction, followed by GF. This was evidenced by the up-regulation of chondrogenic genes (Sox9, aggrecan, and collagen type II), which was not observed for cells grown on TCPS. This finding suggested the essential role of substrate materials in the chondrogenic differentiation of PDMC and GF. Neocartilage formation was more obvious in both PDMC and GF cells plated on CII-HA composite scaffolds vs. 8-layer SIS at 28 days in vitro. Finally, implantation of PDMC/CII-HA constructs into NOD-SCID mice confirmed the formation of tissue-engineered cartilage in vivo.

  12. Enhanced apoptotic effects of dihydroartemisinin-aggregated gelatin and hyaluronan nanoparticles on human lung cancer cells.

    PubMed

    Sun, Qian; Teong, Benjamin; Chen, I-Fen; Chang, Shwu Jen; Gao, Jimin; Kuo, Shyh-Ming

    2014-04-01

    Recent studies suggest that dihydroartemisinin (DHA), a derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua L., has anticancer properties. Due to poor water solubility, poor oral activity, and a short plasma half-life, large doses of DHA have to be injected to achieve the necessary bioavailability. This study examined increasing DHA bioavailability by encapsulating DHA within gelatin (GEL) or hyaluronan (HA) nanoparticles via an electrostatic field system. Observations from transmission electron microscopy show that DHA in GEL and HA nanoparticles formed GEL/DHA and HA/DHA aggregates that were approximately 30-40 nm in diameter. The entrapment efficiencies for DHA were approximately 13 and 35% for the GEL/DHA and HA/DHA aggregates, respectively. The proliferation of A549 cells was inhibited by the GEL/DHA and HA/DHA aggregates. Fluorescent annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining displayed low background staining with annexin V-FITC or PI on DHA-untreated cells. In contrast, annexin V-FITC and PI stains dramatically increased when the cells were incubated with GEL/DHA and HA/DHA aggregates. These results suggest that DHA-aggregated GEL and HA nanoparticles exhibit higher anticancer proliferation activities than DHA alone in A549 cells most likely due to the greater aqueous dispersion after hydrophilic GEL or HA nanoparticles aggregation. These results demonstrate that DHA can aggregate with nanoparticles in an electrostatic field environment to form DHA nanosized aggregates. PMID:24039154

  13. CD44 and hyaluronan expression in human cutaneous scar fibroblasts.

    PubMed Central

    Messadi, D. V.; Bertolami, C. N.

    1993-01-01

    Fibrotic disorders of skin and other organs are typically associated with an abnormal accumulation of extracellular matrix. This study focuses on a matrix constituent, hyaluronan-which is known to be altered in fibrotic disorders of skin- and on CD44, a cell adhesion molecule and putative receptor for hyaluronan. Tissue samples were obtained from biopsies of human normal skin, normal cutaneous scar; and hypertrophic cutaneous scar. After culturing, cells were studied by single- and double-labeling immunohistochemistry using the two anti-CD44 monoclonal antibodies, BU-52 and J173, and a biotinylated hyaluronan binding complex probe, b-HABR. Certain cultures were pretreated with Streptomyces hyaluronidase to assess the dependency of CD44 expression on the presence of endogenous hyaluronan. CD44 expression, both in the presence and the absence of exogenous hyaluronan, was quantitated by radioimmunobinding assay. Overall glycosaminoglycan synthesis and identification of hyaluronan were accomplished by precursor incorporation assays and by quantitative cellulose acetate electrophoresis. CD44 was found to be a normal human adult fibroblastic antigen whose expression is markedly increased for hypertrophic scar fibroblasts compared with normal skin fibroblasts. Although hyaluronan was found to be the predominant glycosaminoglycan constituent of the pericellular matrix for these fibroblasts, CD44 attachment to the cell surface is neither mediated by hyaluronan nor is the presence of hyaluronan a prerequisite for CD44 expression. Exogenous hyaluronan induced a decline in measurable CD44 expression for normal skin fibroblasts but not for hypertrophic scar fibroblasts. These observations are compatible with current understanding of the way cells manage the hyaluronan economy of the extracellular matrix and emphasize phenotypic heterogeneities between fibroblasts derived from normal versus scar tissues. Images Figure 1 Figure 4 PMID:8475990

  14. Hyaluronan-CD44 Interaction Promotes Growth of Decidual Stromal Cells in Human First-Trimester Pregnancy

    PubMed Central

    Zhu, Rui; Wang, Song-Cun; Sun, Chan; Tao, Yu; Piao, Hai-Lan; Wang, Xiao-Qiu; Du, Mei-Rong; Da-Jin Li

    2013-01-01

    Hyaluronan (HA) and its receptor CD44 are expressed at the maternal-fetal interface, but its role in early pregnancy remains unclear. Here, we found that primary decidual stromal cells (DSCs) continuously secreted HA and expressed its receptor CD44. Pregnancy-associated hormones up-regulated HA synthetase (HAS) 2 transcription and HA release from DSCs. High molecular weight-HA (HMW-HA), but not medium molecular weight (MMW-HA) or low molecular weight (LMW-HA), promoted proliferation and inhibited apoptosis of DSCs in a CD44-dependent manner. The in-cell Western analysis revealed HMW-HA activated PI3K/AKT and mitogen-activated protein kinase (MAPK)/ERK1/2 signaling pathways time-dependently. Blocking these pathways by specific inhibitor LY294002 or U0126 abrogated HMW-HA-regulated DSc proliferation and apoptosis. Finally, we have found that HA content, HA molecular weight, HAS2 mRNA level, and CD44 expression were significantly decreased in DSCs from unexplained miscarriage compared with the normal pregnancy. Collectively, our results indicate that higher level and greater molecular mass of HA at maternal-fetal interface contributes to DSc growth and maintenance of DSCs in human early pregnancy. PMID:24069351

  15. Tissue hyaluronan expression, as reflected in the sputum of lung cancer patients, is an indicator of malignancy.

    PubMed

    Rangel, M P; de Sá, V K; Martins, V; Martins, J R M; Parra, E R; Mendes, A; Andrade, P C; Reis, R M; Longatto-Filho, A; Oliveira, C Z; Takagaki, T; Carraro, D M; Nader, H B; Capelozzi, V L

    2015-06-01

    Hyaluronan (HA) shows promise for detecting cancerous change in pleural effusion and urine. However, there is uncertainty about the localization of HA in tumor tissue and its relationship with different histological types and other components of the extracellular matrix, such as angiogenesis. We evaluated the association between HA and degree of malignancy through expression in lung tumor tissue and sputum. Tumoral tissue had significantly increased HA compared to normal tissue. Strong HA staining intensity associated with cancer cells was significant in squamous cell carcinoma compared to adenocarcinoma and large cell carcinoma. A significant direct association was found between tumors with a high percentage of HA and MVD (microvessel density) in tumoral stroma. Similarly significant was the direct association between N1 tumors and high levels of HA in cancer cells. Cox multivariate analysis showed significant association between better survival and low HA. HA increased in sputum from lung cancer patients compared to cancer-free and healthy volunteers and a significant correlation was found between HA in sputum and HA in cancer tissue. Localization of HA in tumor tissue was related to malignancy and reflected in sputum, making this an emerging factor for an important diagnostic procedure in patients suspected to have lung cancer. Further study in additional patients in a randomized prospective trial is required to finalize these results and to validate our quantitative assessment of HA, as well as to couple it to gold standard sputum cytology.

  16. Tissue hyaluronan expression, as reflected in the sputum of lung cancer patients, is an indicator of malignancy

    PubMed Central

    Rangel, M.P.; de Sá, V.K.; Martins, V.; Martins, J.R.M.; Parra, E.R.; Mendes, A.; Andrade, P.C.; Reis, R.M.; Longatto-Filho, A.; Oliveira, C.Z.; Takagaki, T.; Carraro, D.M.; Nader, H.B.; Capelozzi, V.L.

    2015-01-01

    Hyaluronan (HA) shows promise for detecting cancerous change in pleural effusion and urine. However, there is uncertainty about the localization of HA in tumor tissue and its relationship with different histological types and other components of the extracellular matrix, such as angiogenesis. We evaluated the association between HA and degree of malignancy through expression in lung tumor tissue and sputum. Tumoral tissue had significantly increased HA compared to normal tissue. Strong HA staining intensity associated with cancer cells was significant in squamous cell carcinoma compared to adenocarcinoma and large cell carcinoma. A significant direct association was found between tumors with a high percentage of HA and MVD (microvessel density) in tumoral stroma. Similarly significant was the direct association between N1 tumors and high levels of HA in cancer cells. Cox multivariate analysis showed significant association between better survival and low HA. HA increased in sputum from lung cancer patients compared to cancer-free and healthy volunteers and a significant correlation was found between HA in sputum and HA in cancer tissue. Localization of HA in tumor tissue was related to malignancy and reflected in sputum, making this an emerging factor for an important diagnostic procedure in patients suspected to have lung cancer. Further study in additional patients in a randomized prospective trial is required to finalize these results and to validate our quantitative assessment of HA, as well as to couple it to gold standard sputum cytology. PMID:25992645

  17. Binding of Hyaluronan to the Native Lymphatic Vessel Endothelial Receptor LYVE-1 Is Critically Dependent on Receptor Clustering and Hyaluronan Organization*

    PubMed Central

    Lawrance, William; Banerji, Suneale; Day, Anthony J.; Bhattacharjee, Shaumick; Jackson, David G.

    2016-01-01

    The lymphatic endothelial receptor LYVE-1 has been implicated in both uptake of hyaluronan (HA) from tissue matrix and in facilitating transit of leukocytes and tumor cells through lymphatic vessels based largely on in vitro studies with recombinant receptor in transfected fibroblasts. Curiously, however, LYVE-1 in lymphatic endothelium displays little if any binding to HA in vitro, and this has led to the conclusion that the native receptor is functionally silenced, a feature that is difficult to reconcile with its proposed in vivo functions. Nonetheless, as we reported recently, LYVE-1 can function as a receptor for HA-encapsulated Group A streptococci and mediate lymphatic dissemination in mice. Here we resolve these paradoxical findings and show that the capacity of LYVE-1 to bind HA is strictly dependent on avidity, demanding appropriate receptor self-association and/or HA multimerization. In particular, we demonstrate the prerequisite of a critical LYVE-1 threshold density and show that HA binding may be elicited in lymphatic endothelium by surface clustering with divalent LYVE-1 mAbs. In addition, we show that cross-linking of biotinylated HA in streptavidin multimers or supramolecular complexes with the inflammation-induced protein TSG-6 enables binding even in the absence of LYVE-1 cross-linking. Finally, we show that endogenous HA on the surface of macrophages can engage LYVE-1, facilitating their adhesion and transit across lymphatic endothelium. These results reveal LYVE-1 as a low affinity receptor tuned to discriminate between different HA configurations through avidity and establish a new mechanistic basis for the functions ascribed to LYVE-1 in matrix HA binding and leukocyte trafficking in vivo. PMID:26823460

  18. Targeting Hyaluronic Acid Family for Cancer Chemoprevention and Therapy

    PubMed Central

    Lokeshwar, Vinata B.; Mirza, Summan; Jordan, Andre

    2016-01-01

    Hyaluronic acid or hyaluronan (HA) is perhaps one of the most uncomplicated large polymers that regulates several normal physiological processes and, at the same time, contributes to the manifestation of a variety of chronic and acute diseases, including cancer. Members of the HA signaling pathway (HA synthases, HA receptors, and HYAL-1 hyaluronidase) have been experimentally shown to promote tumor growth, metastasis, and angiogenesis, and hence each of them is a potential target for cancer therapy. Furthermore, as these members are also overexpressed in a variety of carcinomas, targeting of the HA family is clinically relevant. A variety of targeted approaches have been developed to target various HA family members, including small-molecule inhibitors and antibody and vaccine therapies. These treatment approaches inhibit HA-mediated intracellular signaling that promotes tumor cell proliferation, motility, and invasion, as well as induction of endothelial cell functions. Being nontoxic, nonimmunogenic, and versatile for modifications, HA has been used in nanoparticle preparations for the targeted delivery of chemotherapy drugs and other anticancer compounds to tumor cells through interaction with cell-surface HA receptors. This review discusses basic and clinical translational aspects of targeting each HA family member and respective treatment approaches that have been described in the literature. PMID:25081525

  19. Size Matters: Molecular Weight Specificity of Hyaluronan Effects in Cell Biology

    PubMed Central

    Cyphert, Jaime M.; Trempus, Carol S.; Garantziotis, Stavros

    2015-01-01

    Hyaluronan signaling properties are unique among other biologically active molecules, that they are apparently not influenced by postsynthetic molecular modification, but by hyaluronan fragment size. This review summarizes the current knowledge about the generation of hyaluronan fragments of different size and size-dependent differences in hyaluronan signaling as well as their downstream biological effects. PMID:26448754

  20. Purification, crystallization and preliminary X-ray analysis of an HA17–HA70 (HA2–HA3) complex from Clostridium botulinum type C progenitor toxin

    PubMed Central

    Iwasa, Chikako; Tonozuka, Takashi; Shinoda, Masaya; Sagane, Yoshimasa; Niwa, Koichi; Watanabe, Toshihiro; Yoshida, Hiromi; Kamitori, Shigehiro; Takao, Toshifumi; Oguma, Keiji; Nishikawa, Atsushi

    2014-01-01

    The haemagglutinin (HA) complex of Clostridium botulinum type C toxin is composed of three types of subcomponents: HA33, HA17 and HA70 (also known as HA1, HA2 and HA3, respectively). Here, a 260 kDa HA17–HA70 complex was crystallized. His-tagged HA17 and maltose-binding-protein-tagged HA70 were expressed in Escherichia coli and their complex was affinity-purified using a combination of amylose resin chromatography and nickel–nitrilotri­acetic acid agarose chromatography. Diffraction data were collected to 8.0 Å resolution and the crystal belonged to the tetragonal space group P41212. The molecular-replacement solution indicated that one molecule of HA17 was bound to each HA70 monomer. PMID:24419620

  1. Incorporation of Pentraxin 3 into Hyaluronan Matrices Is Tightly Regulated and Promotes Matrix Cross-linking

    PubMed Central

    Baranova, Natalia S.; Inforzato, Antonio; Briggs, David C.; Tilakaratna, Viranga; Enghild, Jan J.; Thakar, Dhruv; Milner, Caroline M.; Day, Anthony J.; Richter, Ralf P.

    2014-01-01

    Mammalian oocytes are surrounded by a highly hydrated hyaluronan (HA)-rich extracellular matrix with embedded cumulus cells, forming the cumulus cell·oocyte complex (COC) matrix. The correct assembly, stability, and mechanical properties of this matrix, which are crucial for successful ovulation, transport of the COC to the oviduct, and its fertilization, depend on the interaction between HA and specific HA-organizing proteins. Although the proteins inter-α-inhibitor (IαI), pentraxin 3 (PTX3), and TNF-stimulated gene-6 (TSG-6) have been identified as being critical for COC matrix formation, its supramolecular organization and the molecular mechanism of COC matrix stabilization remain unknown. Here we used films of end-grafted HA as a model system to investigate the molecular interactions involved in the formation and stabilization of HA matrices containing TSG-6, IαI, and PTX3. We found that PTX3 binds neither to HA alone nor to HA films containing TSG-6. This long pentraxin also failed to bind to products of the interaction between IαI, TSG-6, and HA, among which are the covalent heavy chain (HC)·HA and HC·TSG-6 complexes, despite the fact that both IαI and TSG-6 are ligands of PTX3. Interestingly, prior encounter with IαI was required for effective incorporation of PTX3 into TSG-6-loaded HA films. Moreover, we demonstrated that this ternary protein mixture made of IαI, PTX3, and TSG-6 is sufficient to promote formation of a stable (i.e. cross-linked) yet highly hydrated HA matrix. We propose that this mechanism is essential for correct assembly of the COC matrix and may also have general implications in other inflammatory processes that are associated with HA cross-linking. PMID:25190808

  2. Hyaluronan Is Crucial for Stem Cell Differentiation into Smooth Muscle Lineage.

    PubMed

    Simpson, Russell M L; Hong, Xuechong; Wong, Mei Mei; Karamariti, Eirini; Bhaloo, Shirin Issa; Warren, Derek; Kong, Wei; Hu, Yanhua; Xu, Qingbo

    2016-05-01

    Deciphering the extracellular signals that regulate SMC differentiation from stem cells is vital to further our understanding of the pathogenesis of vascular disease and for development of cell-based therapies and tissue engineering. Hyaluronan (HA) has emerged as an important component of the stem cell niche, however its role during stem cell differentiation is a complicated and inadequately defined process. This study aimed to investigate the role of HA in embryonic stem cell (ESC) differentiation toward a SMC lineage. ESCs were seeded on collagen-IV in differentiation medium to generate ESC-derived SMCs (esSMCs). Differentiation coincided with increased HA synthase (HAS) 2 expression, accumulation of extracellular HA and its assembly into pericellular matrices. Inhibition of HA synthesis by 4-methylumbelliferone (4MU), removal of the HA coat by hyaluronidase (HYAL) or HAS2 knockdown led to abrogation of SMC gene expression. HA activates ERK1/2 and suppresses EGFR signaling pathways via its principle receptor, CD44. EGFR inactivation coincided with increased binding to CD44, which was further augmented by addition of high molecular weight (HMW)-HA either exogenously or via HAS2 overexpression through adenoviral gene transfer. HMW-HA-stimulated esSMCs displayed a functional role in vascular tissue engineering ex vivo, vasculogenesis in a matrigel plug model and SMC accumulation in neointimal lesions of vein grafts in mice. These findings demonstrate that HAS2-induced HA synthesis and organization drives ESC-SMC differentiation. Thus, remodeling of the HA microenvironment is a critical step in directing stem cell differentiation toward a vascular lineage, highlighting HA as a potential target for treatment of vascular diseases. Stem Cells 2016;34:1225-1238. PMID:26867148

  3. Conserved miR-26b enhances ovarian granulosa cell apoptosis through HAS2-HA-CD44-Caspase-3 pathway by targeting HAS2

    PubMed Central

    Liu, Jiying; Tu, Fei; Yao, Wang; Li, Xinyu; Xie, Zhuang; Liu, Honglin; Li, Qifa; Pan, Zengxiang

    2016-01-01

    The hyaluronan synthase 2 (HAS2)-hyaluronic acid (HA)-CD44-Caspase-3 pathway is involved in ovarian granulosa cell (GC) functions in mammals. HAS2 is a key enzyme required for HA synthesis and is the key factor in this pathway. However, the regulation of HAS2 and the HAS2-mediated pathway by microRNAs in GCs is poorly understood. Here, we report that miR-26b regulates porcine GC (pGC) apoptosis through the HAS2-HA-CD44-Caspase-3 pathway by binding directly to the 3′- untranslated region of HAS2 mRNA. Knockdown of miR-26b reduced pGC apoptosis. Luciferase reporter assays demonstrated that HAS2 is a direct target of miR-26b in pGCs. Knockdown and overexpression of miR-26b increased and decreased, respectively, HA content, and HAS2 and CD44 expression in pGCs. At the same time, inhibition and overexpression of miR-26b decreased and increased the expression of Caspase-3, a downstream factor in the HAS2-HA-CD44 pathway. Moreover, knockdown of HAS2 enhanced pGC apoptosis, reduced the inhibitory effects of a miR-26b inhibitor on pGC apoptosis, repressed HA content and CD44 expression, and promoted Caspase-3 expression. In addition, overexpression of HAS2 has a opposite effect. Collectively, miR-26b positively regulates pGC apoptosis via a novel HAS2-HA-CD44-Caspase-3 pathway by targeting the HAS2 gene. PMID:26887530

  4. Hyaluronan modulates TRPV1 channel opening, reducing peripheral nociceptor activity and pain

    PubMed Central

    Caires, Rebeca; Luis, Enoch; Taberner, Francisco J.; Fernandez-Ballester, Gregorio; Ferrer-Montiel, Antonio; Balazs, Endre A.; Gomis, Ana; Belmonte, Carlos; de la Peña, Elvira

    2015-01-01

    Hyaluronan (HA) is present in the extracellular matrix of all body tissues, including synovial fluid in joints, in which it behaves as a filter that buffers transmission of mechanical forces to nociceptor nerve endings thereby reducing pain. Using recombinant systems, mouse-cultured dorsal root ganglia (DRG) neurons and in vivo experiments, we found that HA also modulates polymodal transient receptor potential vanilloid subtype 1 (TRPV1) channels. HA diminishes heat, pH and capsaicin (CAP) responses, thus reducing the opening probability of the channel by stabilizing its closed state. Accordingly, in DRG neurons, HA decreases TRPV1-mediated impulse firing and channel sensitization by bradykinin. Moreover, subcutaneous HA injection in mice reduces heat and capsaicin nocifensive responses, whereas the intra-articular injection of HA in rats decreases capsaicin joint nociceptor fibres discharge. Collectively, these results indicate that extracellular HA reduces the excitability of the ubiquitous TRPV1 channel, thereby lowering impulse activity in the peripheral nociceptor endings underlying pain. PMID:26311398

  5. The porcine sperm reservoir in relation to the function of hyaluronan

    PubMed Central

    TIENTHAI, Paisan

    2015-01-01

    The oviduct plays a role in successful animal reproduction not only in spermatozoa and ova transport to the fertilization site but also by affording a microenvironment for fertilization and early embryonic development. The sperm reservoir (SR) is restricted in the uterotubal junction (UTJ) and caudal isthmus. Billions of porcine spermatozoa are distributed to the female reproductive tract during/after insemination, and small amounts of them are stored for about 36–40 hours in the SR, which maintains sperm viability in the pre-ovulation period through its surface epithelium and production of fluid. The SR regulates the release of spermatozoa so that only a small population moves towards the fertilization site (ampulla) to decrease polyspermy. This review attempts to provide information about the structure and function of the porcine SR, its intraluminal content (hyaluronan, HA), and the influences of HA on porcine spermatozoa in vivo. In pigs, the spermatozoa are stored in a mucous-like fluid within the UTJ and caudal isthmus in the pre-ovulation period. The oviduct fluid contains sulfated glycosaminoglycans (GAGs) and non-sulfated GAGs, i.e., HA. It is interesting to note that HA is synthesized by hyaluronan synthase-3 (HAS-3), and its receptor, CD44, is found in the epithelium of the porcine SR site. Additionally, sperm capacitation does not occur in vivo in the SR during the pre- and peri-ovulation periods, but spermatozoa in the SR will attempt to capacitate if exposed to bicarbonate. However, capacitation in the SR will rise in the post-ovulation period, indicating the role of HA in modulating sperm capacitation after ovulation. All data support the understanding that the porcine SR ensures the viability of fertile spermatozoa and maintains the non-capacitated status during the pre-ovulation period. This basic knowledge about the SR is believed to be useful to advance sperm preparation procedures for in vitro fertilization (IVF) and improve the preservation

  6. Tumor targeting profiling of hyaluronan-coated lipid based-nanoparticles

    NASA Astrophysics Data System (ADS)

    Mizrahy, Shoshy; Goldsmith, Meir; Leviatan-Ben-Arye, Shani; Kisin-Finfer, Einat; Redy, Orit; Srinivasan, Srimeenakshi; Shabat, Doron; Godin, Biana; Peer, Dan

    2014-03-01

    Hyaluronan (HA), a naturally occurring high Mw (HMw) glycosaminoglycan, has been shown to play crucial roles in cell growth, embryonic development, healing processes, inflammation, and tumor development and progression. Low Mw (LMw, <10 kDa) HA has been reported to provoke inflammatory responses, such as induction of cytokines, chemokines, reactive nitrogen species and growth factors. Herein, we prepared and characterized two types of HA coated (LMw and HMw) lipid-based targeted and stabilized nanoparticles (tsNPs) and tested their binding to tumor cells expressing the HA receptor (CD44), systemic immunotoxicity, and biodistribution in tumor bearing mice. In vitro, the Mw of the surface anchored HA had a significant influence on the affinity towards CD44 on B16F10 murine melanoma cells. LMw HA-tsNPs exhibited weak binding, while binding of tsNPs coated with HMw HA was characterized by high binding. Both types of tsNPs had no measured effect on cytokine induction in vivo following intravenous administration to healthy C57BL/6 mice suggesting no immune activation. HMw HA-tsNPs showed enhanced circulation time and tumor targeting specificity, mainly by accumulating in the tumor and its vicinity compared with LMw HA-tsNPs. Finally, we show that methotrexate (MTX), a drug commonly used in cancer chemotherapy, entrapped in HMw HA-tsNPs slowly diffused from the particles with a half-life of 13.75 days, and improved the therapeutic outcome in a murine B16F10 melanoma model compared with NPs suggesting an active cellular targeting beyond the Enhanced Permeability and Retention (EPR) effect. Taken together, these findings have major implications for the use of high molecular weight HA in nanomedicine as a selective and safe active cellular targeting moiety.Hyaluronan (HA), a naturally occurring high Mw (HMw) glycosaminoglycan, has been shown to play crucial roles in cell growth, embryonic development, healing processes, inflammation, and tumor development and progression

  7. Correlation of hyaluronan deposition with infiltration of eosinophils and lymphocytes in a cockroach-induced murine model of asthma

    PubMed Central

    Cheng, Georgiana; Swaidani, Shadi; Sharma, Manisha; Lauer, Mark E; Hascall, Vincent C; Aronica, Mark A

    2013-01-01

    Asthma is a chronic inflammatory disease that exhibits airway remodeling with changes in the extracellular matrix (ECM). The role of the ECM in mediating these changes is poorly understood. Hyaluronan (HA), a major component of the ECM, has been implicated in many biological processes in diseases. This study investigates the processes involved in HA synthesis, deposition and localization during the propagation of cockroach-induced asthma. Mice were sensitized and challenged with cockroach antigen, and sacrificed at various time points during an 8-week challenge protocol. Analysis of bronchoalveolar lavage (BAL) fluid revealed an increase in total nucleated cells as early as 6 h, which peaked at 6 days. Histopathologic analysis of the lung tissue revealed an influx of inflammatory cells at the peribronchial and perivascular regions starting at 12 h, which peaked at 6 days and persisted to 8 weeks. Eosinophils predominated in the early time points while lymphocytes predominated during the late time points. Quantitative polymerase chain reaction (PCR) data showed that hyaluronan synthase 1 (HAS1) mRNA peaked within 6 h and then declined. HAS2 mRNA also peaked within 6 h but remained elevated throughout the 8-week exposure course. HA levels in lung tissue and BAL increased at 12 h and peaked by 6 and 8 days, respectively. Inflammatory cells and new collagen formation localized in areas of HA deposition. Taken together, these data support a role for HA in the pathogenesis in asthma. PMID:22917573

  8. Increased concentration of hyaluronan in tears after soaking contact lenses in Biotrue multipurpose solution

    PubMed Central

    Scheuer, Catherine A; Rah, Marjorie J; Reindel, William T

    2016-01-01

    Purpose This study was conducted to determine 1) the concentration of hyaluronan (HA) in the tear films of contact lens (CL) wearers versus non-CL wearers and 2) whether HA sorbed from Biotrue, an HA-containing multipurpose solution (MPS), onto senofilcon A lenses affects the concentration of HA in tears after 2 hours of wear. Patients and methods Tears of habitual CL wearers and non-CL wearers were collected on Schirmer strips at baseline and after 2 hours of wear of senofilcon A CLs that had first been either rinsed with Sensitive Eyes Saline or soaked in Biotrue MPS for 14 hours. HA concentrations were measured by enzyme-linked immunosorbent assay (ELISA) and adjusted for sample volumes. Results No difference in baseline concentrations of HA in tears was found between CL wearers and non-CL wearers (P=0.07), nor between males and females (P=0.06). However, age was significantly negatively associated with HA concentration (P<0.01), and mostly, CL wear contributed to a significant association (P<0.01). Among saline-rinsed CL wearers, no change in HA concentration in tears was observed after 2 hours of wear (P=0.38). By contrast, a significant increase in HA concentration was observed in the tears from eyes that had worn CLs soaked in Biotrue MPS when compared to baseline (P=0.01) or to saline-rinsed control (P=0.03). Conclusion 1) In this study population, no difference in baseline concentration of HA was observed between CL wearers and non-CL wearers, and 2) after 2 hours of wear of senofilcon A lenses that were soaked in Biotrue MPS, HA concentrations in the tear films of CL wearers increased. PMID:27784983

  9. Coating with artificial matrices from collagen and sulfated hyaluronan influences the osseointegration of dental implants.

    PubMed

    Schulz, Matthias C; Korn, Paula; Stadlinger, Bernd; Range, Ursula; Möller, Stephanie; Becher, Jana; Schnabelrauch, Matthias; Mai, Ronald; Scharnweber, Dieter; Eckelt, Uwe; Hintze, Vera

    2014-01-01

    Dental implants are an established therapy for oral rehabilitation. High success rates are achieved in healthy bone, however, these rates decrease in compromised host bone. Coating of dental implants with components of the extracellular matrix is a promising approach to enhance osseointegration in compromised peri-implant bone. Dental titanium implants were coated with an artificial extracellular matrix (aECM) consisting of collagen type I and either one of two regioselectively low sulfated hyaluronan (sHA) derivatives (coll/sHA1Δ6s and coll/sHA1) and compared to commercial pure titanium implants (control). After extraction of the premolar teeth, 36 implants were inserted into the maxilla of 6 miniature pigs (6 implants per maxilla). The healing periods were 4 and 8 weeks, respectively. After animal sacrifice, the samples were evaluated histomorphologically and histomorphometrically. All surface states led to a sufficient implant osseointegration after 4 and 8 weeks. Inflammatory or foreign body reactions could not be observed. After 4 weeks of healing, implants coated with coll/sHA1Δ6s showed the highest bone implant contact (BIC; coll/sHA1Δ6s: 45.4%; coll/sHA1: 42.2%; control: 42.3%). After 8 weeks, a decrease of BIC could be observed for coll/sHA1Δ6s and controls (coll/sHA1Δ6s: 37.3%; control: 31.7 %). For implants coated with coll/sHA1, the bone implant contact increased (coll/sHA1: 50.8%). Statistically significant differences could not be observed. Within the limits of the current study, aECM coatings containing low sHA increase peri-implant bone formation around dental implants in maxillary bone compared to controls in the early healing period.

  10. Hyaluronan Is Crucial for Stem Cell Differentiation into Smooth Muscle Lineage

    PubMed Central

    Simpson, Russell M.L.; Hong, Xuechong; Wong, Mei Mei; Karamariti, Eirini; Bhaloo, Shirin Issa; Warren, Derek; Kong, Wei; Hu, Yanhua

    2016-01-01

    Abstract Deciphering the extracellular signals that regulate SMC differentiation from stem cells is vital to further our understanding of the pathogenesis of vascular disease and for development of cell‐based therapies and tissue engineering. Hyaluronan (HA) has emerged as an important component of the stem cell niche, however its role during stem cell differentiation is a complicated and inadequately defined process. This study aimed to investigate the role of HA in embryonic stem cell (ESC) differentiation toward a SMC lineage. ESCs were seeded on collagen‐IV in differentiation medium to generate ESC‐derived SMCs (esSMCs). Differentiation coincided with increased HA synthase (HAS) 2 expression, accumulation of extracellular HA and its assembly into pericellular matrices. Inhibition of HA synthesis by 4‐methylumbelliferone (4MU), removal of the HA coat by hyaluronidase (HYAL) or HAS2 knockdown led to abrogation of SMC gene expression. HA activates ERK1/2 and suppresses EGFR signaling pathways via its principle receptor, CD44. EGFR inactivation coincided with increased binding to CD44, which was further augmented by addition of high molecular weight (HMW)‐HA either exogenously or via HAS2 overexpression through adenoviral gene transfer. HMW‐HA‐stimulated esSMCs displayed a functional role in vascular tissue engineering ex vivo, vasculogenesis in a matrigel plug model and SMC accumulation in neointimal lesions of vein grafts in mice. These findings demonstrate that HAS2‐induced HA synthesis and organization drives ESC‐SMC differentiation. Thus, remodeling of the HA microenvironment is a critical step in directing stem cell differentiation toward a vascular lineage, highlighting HA as a potential target for treatment of vascular diseases. Stem Cells 2016;34:1225–1238 PMID:26867148

  11. Coating with artificial matrices from collagen and sulfated hyaluronan influences the osseointegration of dental implants.

    PubMed

    Schulz, Matthias C; Korn, Paula; Stadlinger, Bernd; Range, Ursula; Möller, Stephanie; Becher, Jana; Schnabelrauch, Matthias; Mai, Ronald; Scharnweber, Dieter; Eckelt, Uwe; Hintze, Vera

    2014-01-01

    Dental implants are an established therapy for oral rehabilitation. High success rates are achieved in healthy bone, however, these rates decrease in compromised host bone. Coating of dental implants with components of the extracellular matrix is a promising approach to enhance osseointegration in compromised peri-implant bone. Dental titanium implants were coated with an artificial extracellular matrix (aECM) consisting of collagen type I and either one of two regioselectively low sulfated hyaluronan (sHA) derivatives (coll/sHA1Δ6s and coll/sHA1) and compared to commercial pure titanium implants (control). After extraction of the premolar teeth, 36 implants were inserted into the maxilla of 6 miniature pigs (6 implants per maxilla). The healing periods were 4 and 8 weeks, respectively. After animal sacrifice, the samples were evaluated histomorphologically and histomorphometrically. All surface states led to a sufficient implant osseointegration after 4 and 8 weeks. Inflammatory or foreign body reactions could not be observed. After 4 weeks of healing, implants coated with coll/sHA1Δ6s showed the highest bone implant contact (BIC; coll/sHA1Δ6s: 45.4%; coll/sHA1: 42.2%; control: 42.3%). After 8 weeks, a decrease of BIC could be observed for coll/sHA1Δ6s and controls (coll/sHA1Δ6s: 37.3%; control: 31.7 %). For implants coated with coll/sHA1, the bone implant contact increased (coll/sHA1: 50.8%). Statistically significant differences could not be observed. Within the limits of the current study, aECM coatings containing low sHA increase peri-implant bone formation around dental implants in maxillary bone compared to controls in the early healing period. PMID:24113890

  12. Two Cytoplasmic Acylation Sites and an Adjacent Hydrophobic Residue, but No Other Conserved Amino Acids in the Cytoplasmic Tail of HA from Influenza A Virus Are Crucial for Virus Replication

    PubMed Central

    Siche, Stefanie; Brett, Katharina; Möller, Lars; Kordyukova, Larisa V.; Mintaev, Ramil R.; Alexeevski, Andrei V.; Veit, Michael

    2015-01-01

    Recruitment of the matrix protein M1 to the assembly site of the influenza virus is thought to be mediated by interactions with the cytoplasmic tail of hemagglutinin (HA). Based on a comprehensive sequence comparison of all sequences present in the database, we analyzed the effect of mutating conserved residues in the cytosol-facing part of the transmembrane region and cytoplasmic tail of HA (A/WSN/33 (H1N1) strain) on virus replication and morphology of virions. Removal of the two cytoplasmic acylation sites and substitution of a neighboring isoleucine by glutamine prevented rescue of infectious virions. In contrast, a conservative exchange of the same isoleucine, non-conservative exchanges of glycine and glutamine, deletion of the acylation site at the end of the transmembrane region and shifting it into the tail did not affect virus morphology and had only subtle effects on virus growth and on the incorporation of M1 and Ribo-Nucleoprotein Particles (RNPs). Thus, assuming that essential amino acids are conserved between HA subtypes we suggest that, besides the two cytoplasmic acylation sites (including adjacent hydrophobic residues), no other amino acids in the cytoplasmic tail of HA are indispensable for virus assembly and budding. PMID:26670246

  13. Two Cytoplasmic Acylation Sites and an Adjacent Hydrophobic Residue, but No Other Conserved Amino Acids in the Cytoplasmic Tail of HA from Influenza A Virus Are Crucial for Virus Replication.

    PubMed

    Siche, Stefanie; Brett, Katharina; Möller, Lars; Kordyukova, Larisa V; Mintaev, Ramil R; Alexeevski, Andrei V; Veit, Michael

    2015-12-01

    Recruitment of the matrix protein M1 to the assembly site of the influenza virus is thought to be mediated by interactions with the cytoplasmic tail of hemagglutinin (HA). Based on a comprehensive sequence comparison of all sequences present in the database, we analyzed the effect of mutating conserved residues in the cytosol-facing part of the transmembrane region and cytoplasmic tail of HA (A/WSN/33 (H1N1) strain) on virus replication and morphology of virions. Removal of the two cytoplasmic acylation sites and substitution of a neighboring isoleucine by glutamine prevented rescue of infectious virions. In contrast, a conservative exchange of the same isoleucine, non-conservative exchanges of glycine and glutamine, deletion of the acylation site at the end of the transmembrane region and shifting it into the tail did not affect virus morphology and had only subtle effects on virus growth and on the incorporation of M1 and Ribo-Nucleoprotein Particles (RNPs). Thus, assuming that essential amino acids are conserved between HA subtypes we suggest that, besides the two cytoplasmic acylation sites (including adjacent hydrophobic residues), no other amino acids in the cytoplasmic tail of HA are indispensable for virus assembly and budding. PMID:26670246

  14. Enzymatic synthesis of hyaluronan hybrid urinary trypsin inhibitor.

    PubMed

    Kakizaki, Ikuko; Takahashi, Ryoki; Yanagisawa, Miho; Yoshida, Futaba; Takagaki, Keiichi

    2015-09-01

    Human urinary trypsin inhibitor is a proteoglycan that has a single low-sulfated chondroitin 4-sulfate chain at the seryl residue in position 10 of the core protein as a glycosaminoglycan moiety, and is used as an anti-inflammatory medicine based on the protease inhibitory activity of the core protein. However, the functions of the glycosaminoglycan moiety have not yet been elucidated in detail. In the present study, the glycosaminoglycan chains of a native urinary trypsin inhibitor were remodeled to hyaluronan chains, with no changes to the core protein, using transglycosylation as a reverse reaction of the hydrolysis of bovine testicular hyaluronidase, and the properties of the hybrid urinary trypsin inhibitor were then analyzed. The trypsin inhibitory activitiy of the hyaluronan hybrid urinary trypsin inhibitor was similar to that of the native type; however, its inhibitory effect on the hydrolysis of hyaluronidase were not as strong as that of the native type. This result demonstrated that the native urinary trypsin inhibitor possessed hyaluronidase inhibitory activity on its chondroitin sulfate chain. The hyaluronan hybrid urinary trypsin inhibitors obtained affinity to a hyaluronan-binding protein not exhibited by the native type. The interactions between the hyaluronan hybrid urinary trypsin inhibitors and phosphatidylcholine (abundant in the outer layer of plasma membrane) were stronger than that of the native type. Hyaluronan hybrid urinary trypsin inhibitors may be useful for investigating the functions of the glycosaminoglycan chains of urinary trypsin inhibitors and hyaluronan, and our hybrid synthesizing method may be used widely in research for future medical applications.

  15. Hyaluronan – A Functional and Structural Sweet Spot in the Tissue Microenvironment

    PubMed Central

    Monslow, James; Govindaraju, Priya; Puré, Ellen

    2015-01-01

    Transition from homeostatic to reactive matrix remodeling is a fundamental adaptive tissue response to injury, inflammatory disease, fibrosis, and cancer. Alterations in architecture, physical properties, and matrix composition result in changes in biomechanical and biochemical cellular signaling. The dynamics of pericellular and extracellular matrices, including matrix protein, proteoglycan, and glycosaminoglycan modification are continually emerging as essential regulatory mechanisms underlying cellular and tissue function. Nevertheless, the impact of matrix organization on inflammation and immunity in particular and the consequent effects on tissue healing and disease outcome are arguably under-studied aspects of adaptive stress responses. Herein, we review how the predominant glycosaminoglycan hyaluronan (HA) contributes to the structure and function of the tissue microenvironment. Specifically, we examine the evidence of HA degradation and the generation of biologically active smaller HA fragments in pathological settings in vivo. We discuss how HA fragments versus nascent HA via alternate receptor-mediated signaling influence inflammatory cell recruitment and differentiation, resident cell activation, as well as tumor growth, survival, and metastasis. Finally, we discuss how HA fragmentation impacts restoration of normal tissue function and pathological outcomes in disease. PMID:26029216

  16. Computational Study of Nanosized Drug Delivery from Cyclodextrins, Crown Ethers and Hyaluronan in Pharmaceutical Formulations.

    PubMed

    Torrens, Francisco; Castellano, Gloria

    2015-01-01

    The problem in this work is the computational characterization of cyclodextrins, crown ethers and hyaluronan (HA) as hosts of inclusion complexes for nanosized drug delivery vehicles in pharmaceutical formulations. The difficulty is addressed through a computational study of some thermodynamic, geometric and topological properties of the hosts. The calculated properties of oligosaccharides of D-glucopyranoses allow these to act as co-solvents of polyanions in water. In crown ethers, the central channel is computed. Mucoadhesive polymer HA in formulations releases drugs in mucosas. Geometric, topological and fractal analyses are carried out with code TOPO. Reference calculations are performed with code GEPOL. From HA to HA·3Ca and hydrate, the hydrophilic solvent-accessible surface varies with the count of H-bonds. The fractal dimension rises. The dimension of external atoms rises resulting 1.725 for HA. It rises going to HA·3Ca and hydrate. Nonburied minus molecular dimension rises and decays. Hydrate globularity is lower than O(water), Ca(2+) and O(HA). Ca(2+) rugosity is smaller than for hydrate, O(HA) and O(water). Ca(2+) and O(water) accessibilities are greater than hydrate. Conclusions are drawn on: (1) the relative stability of linear/cyclic and shorter/larger polymers; (2) the atomic analysis of properties allows determining the atoms with maximum reactivity.

  17. Mechanically strong triple network hydrogels based on hyaluronan and poly(N,N-dimethylacrylamide).

    PubMed

    Tavsanli, Burak; Can, Volkan; Okay, Oguz

    2015-11-21

    Hyaluronan (HA) is a natural polyelectrolyte with distinctive biological functions. Cross-linking of HA to generate less degradable hydrogels for use in biomedical applications has attracted interest over many years. One limitation of HA hydrogels is that they are very brittle and/or easily dissolve in physiological environments, which limit their use in load-bearing applications. Herein, we describe the preparation of triple-network (TN) hydrogels based on HA and poly(N,N-dimethylacrylamide) (PDMA) of high mechanical strength by sequential gelation reactions. TN hydrogels containing 81-91% water sustain compressive stresses above 20 MPa and exhibit Young's moduli of up to 1 MPa. HA of various degrees of methacrylation was used as a multifunctional macromer for the synthesis of the brittle first-network component, while loosely cross-linked PDMA was used as the ductile, second and third network components of TN hydrogels. By tuning the methacrylation degree of HA, double-network hydrogels with a fracture stress above 10 MPa and a fracture strain of 96% were obtained. Increasing the ratio of ductile-to-brittle components via the TN approach further increases the fracture stress above 20 MPa. Cyclic mechanical tests show that, although TN hydrogels internally fracture even under small strain, the ductile components hinder macroscopic crack propagation by keeping the macroscopic gel samples together.

  18. Visible Light Crosslinking of Methacrylated Hyaluronan Hydrogels for Injectable Tissue Repair

    PubMed Central

    Fenn, Spencer L.; Oldinski, Rachael A.

    2015-01-01

    Tissue engineering hydrogels are primarily cured in situ using ultraviolet (UV) radiation which limits the use of hydrogels as drug or cell carriers. Visible green light activated crosslinking systems are presented as a safe alternative to UV photocrosslinked hydrogels, without compromising material properties such as viscosity and stiffness. The objective of this study was to fabricate and characterize photocrosslinked hydrogels with well-regulated gelation kinetics and mechanical properties for the repair or replacement of soft tissue. An anhydrous methacrylation of hyaluronan (HA) was performed to control the degree of modification (DOM) of HA, verified by 1H-NMR spectroscopy. UV activated crosslinking was compared to visible green light activated crosslinking. While the different photocrosslinking techniques resulted in varied crosslinking times, comparable mechanical properties of UV and green light activated crosslinked hydrogels were achieved using each photocrosslinking method by adjusting time of light exposure. Methacrylated HA (HA-MA) hydrogels of varying molecular weight, DOM and concentration exhibited compressive moduli ranging from 1 kPa to 116 kPa, for UV crosslinking, and 3 kPa to 146 kPa, for green light crosslinking. HA-MA molecular weight and concentration were found to significantly influence moduli values. HA-MA hydrogels did not exhibit any significant cytotoxic affects towards human mesenchymal stem cells. Green light activated crosslinking systems are presented as a viable method to form natural-based hydrogels in situ. PMID:26097172

  19. Nanoparticles for localized delivery of hyaluronan oligomers towards regenerative repair of elastic matrix.

    PubMed

    Sylvester, Andrew; Sivaraman, Balakrishnan; Deb, Partha; Ramamurthi, Anand

    2013-12-01

    Abdominal aortic aneurysms (AAAs) are rupture-prone progressive dilations of the infrarenal aorta due to a loss of elastic matrix that lead to weakening of the aortic wall. Therapies to coax biomimetic regenerative repair of the elastic matrix by resident, diseased vascular cells may thus be useful to slow, arrest or regress AAA growth. Hyaluronan oligomers (HA-o) have been shown to induce elastic matrix synthesis by healthy and aneurysmal rat aortic smooth muscle cells (SMCs) in vitro but only via exogenous dosing, which potentially has side-effects and limitations to in vivo delivery towards therapy. In this paper, we describe the development of HA-o loaded poly(lactide-co-glycolide) nanoparticles (NPs) for targeted, controlled and sustained delivery of HA-o towards the elastogenic induction of aneurysmal rat aortic SMCs. These NPs were able to deliver HA-o over an extended period (>30 days) at previously determined elastogenic doses (0.2-20 μg ml(-1)). HA-o released from the NPs led to dose-dependent increases in elastic matrix synthesis, and the recruitment and activity of lysyl oxidase, the enzyme which cross-links elastin precursor molecules into mature fibers/matrix. Therefore, we were able to successfully develop a nanoparticle-based system for controlled and sustained HA-o delivery for the in vitro elastogenic induction of aneurysmal rat aortic smooth muscle cells. PMID:23917150

  20. Water-insoluble thin films from palmitoyl hyaluronan with tunable properties.

    PubMed

    Foglarová, Marcela; Chmelař, Josef; Huerta-Angeles, Gloria; Vágnerová, Hana; Kulhánek, Jaromír; Bartoň Tománková, Kateřina; Minařík, Antonín; Velebný, Vladimír

    2016-06-25

    Hyaluronan (HA) films exhibit properties suitable for various biomedical applications, but the solubility of HA limits their use in aqueous environments. Therefore, we developed water insoluble films based on palmitoyl esters of HA (pHA). Films were prepared from pHA samples with various degrees of substitution (DS) and molecular weights and their mechanical properties and swelling were characterized. Additionally, scanning electron microscopy and atomic force microscopy were used for visualization. Despite being prepared by solution casting, the films had a very smooth surface and were homogeneous in thickness. The film properties were in accordance with the polymer DS and molecular weight, enabling to tailor them for future applications by choosing a suitable pHA material. The behavior of the films toward cells was assessed in vitro. All films were non-cytotoxic and showed no adhesion of cells. These results show that the developed films are suitable candidates for various biomedical applications such as tissue engineering or wound healing. PMID:27083794

  1. Crosslinked hyaluronan with a protein-like polymer: novel bioresorbable films for biomedical applications.

    PubMed

    Pitarresi, G; Palumbo, F S; Calabrese, R; Craparo, E F; Giammona, G

    2008-02-01

    In this work, novel hydrogel films based on hyaluronan (HA) chemically crosslinked with the alpha,beta-poly(N-2-hydroxyethyl) (2-aminoethylcarbamate)-D,L-aspartamide (PHEA-EDA) were produced by solution casting method. The goal was to exploit both the biological key role of HA in tissue repair and regeneration, and the versatility of a synthetic protein-like polymer as the PHEA-EDA, in order to obtain biomaterials with physicochemical and biological properties suitable for a clinical use. By varying the molar ratio between the PHEA-EDA amino groups and HA carboxyl groups, three different films were obtained and characterized. Particularly FTIR, swelling, hydrolysis, and enzymatic degradation studies were performed. In addition, the cytocompatibility of HA/PHEA-EDA hydrogel films was evaluated using human derm fibroblasts, by means of MTT and trypan blue exclusion assays. The high swelling capability, the long-term hydrolysis resistance, and the resistance to hyaluronidase greater than that of only HA, together with the cell compatibility, have suggested the potential application of these novel HA-based hydrogel films in the biomedical field of tissue engineering.

  2. Oxidized low density lipoprotein (LDL) affects hyaluronan synthesis in human aortic smooth muscle cells.

    PubMed

    Viola, Manuela; Bartolini, Barbara; Vigetti, Davide; Karousou, Evgenia; Moretto, Paola; Deleonibus, Sara; Sawamura, Tatsuya; Wight, Thomas N; Hascall, Vincent C; De Luca, Giancarlo; Passi, Alberto

    2013-10-11

    Thickening of the vessel in response to high low density lipoprotein(s) (LDL) levels is a hallmark of atherosclerosis, characterized by increased hyaluronan (HA) deposition in the neointima. Human native LDL trapped within the arterial wall undergoes modifications such as oxidation (oxLDL). The aim of our study is to elucidate the link between internalization of oxLDL and HA production in vitro, using human aortic smooth muscle cells. LDL were used at an effective protein concentration of 20-50 μg/ml, which allowed 80% cell viability. HA content in the medium of untreated cells was 28.9 ± 3.7 nmol HA-disaccharide/cell and increased after oxLDL treatment to 53.9 ± 5.6. OxLDL treatments doubled the transcripts of HA synthase HAS2 and HAS3. Accumulated HA stimulated migration of aortic smooth muscle cells and monocyte adhesiveness to extracellular matrix. The effects induced by oxLDL were inhibited by blocking LOX-1 scavenger receptor with a specific antibody (10 μg/ml). The cholesterol moiety of LDL has an important role in HA accumulation because cholesterol-free oxLDL failed to induce HA synthesis. Nevertheless, cholesterol-free oxLDL and unmodified cholesterol (20 μg/ml) induce only HAS3 transcription, whereas 22,oxysterol affects both HAS2 and HAS3. Moreover, HA deposition was associated with higher expression of endoplasmic reticulum stress markers (CHOP and GRP78). Our data suggest that HA synthesis can be induced in response to specific oxidized sterol-related species delivered through oxLDL.

  3. Oxidized Low Density Lipoprotein (LDL) Affects Hyaluronan Synthesis in Human Aortic Smooth Muscle Cells*

    PubMed Central

    Viola, Manuela; Bartolini, Barbara; Vigetti, Davide; Karousou, Evgenia; Moretto, Paola; Deleonibus, Sara; Sawamura, Tatsuya; Wight, Thomas N.; Hascall, Vincent C.; De Luca, Giancarlo; Passi, Alberto

    2013-01-01

    Thickening of the vessel in response to high low density lipoprotein(s) (LDL) levels is a hallmark of atherosclerosis, characterized by increased hyaluronan (HA) deposition in the neointima. Human native LDL trapped within the arterial wall undergoes modifications such as oxidation (oxLDL). The aim of our study is to elucidate the link between internalization of oxLDL and HA production in vitro, using human aortic smooth muscle cells. LDL were used at an effective protein concentration of 20–50 μg/ml, which allowed 80% cell viability. HA content in the medium of untreated cells was 28.9 ± 3.7 nmol HA-disaccharide/cell and increased after oxLDL treatment to 53.9 ± 5.6. OxLDL treatments doubled the transcripts of HA synthase HAS2 and HAS3. Accumulated HA stimulated migration of aortic smooth muscle cells and monocyte adhesiveness to extracellular matrix. The effects induced by oxLDL were inhibited by blocking LOX-1 scavenger receptor with a specific antibody (10 μg/ml). The cholesterol moiety of LDL has an important role in HA accumulation because cholesterol-free oxLDL failed to induce HA synthesis. Nevertheless, cholesterol-free oxLDL and unmodified cholesterol (20 μg/ml) induce only HAS3 transcription, whereas 22,oxysterol affects both HAS2 and HAS3. Moreover, HA deposition was associated with higher expression of endoplasmic reticulum stress markers (CHOP and GRP78). Our data suggest that HA synthesis can be induced in response to specific oxidized sterol-related species delivered through oxLDL. PMID:23979132

  4. Hyaluronan production in human rheumatoid fibroblastic synovial lining cells is increased by interleukin 1β but inhibited by transforming growth factor β1

    PubMed Central

    Kawakami, M.; Suzuki, K.; Matsuki, Y.; Ishizuka, T.; Hidaka, T.; Konishi, T.; Matsumoto, M.; Kataharada, K.; Nakamura, H.

    1998-01-01

    OBJECTIVES—To investigate the regulatory roles of interleukin 1β (IL1β), tumour necrosis factor α (TNFα), interferon γ (IFNγ) or transforming growth factor β1 (TGFβ1) on hyaluronan (HA) synthesis by human fibroblastic synovial lining cells.
METHODS—Concentrations of HA in culture supernatants of fibroblastic synovial lining cell line (RAMAK-1 cell line) with or without stimulation by IL1β, TNFα, IFNγ or TGFβ1 were measured by sandwich binding protein assay. Levels of HA synthase mRNA of the cells with or without stimulation were detected by reverse transcribed polymerase chain reaction. Molecular weights of HA in the culture supernatants of the cells with or without stimulation were measured using high performance gel permeation liquid chromatography.
RESULTS—HA synthesis by the cells was not significantly augmented by TNFα or by IFNγ. It was significantly stimulated by IL1β but inhibited by TGFβ1. Molecular weights of HA in the culture supernatants of the cells were unchanged by stimulation with TNFα. They were remarkably increased by stimulation with IL1β and IFNγ, but reduced with TGFβ1.
CONCLUSION—IL1β is an up regulator of HA synthesis, while TGFβ1 is a down regulator. HA production in the synovial lining cells of inflamed joints (for example, rheumatoid arthritis) might be regulated by the balance of these cytokines.

 Keywords: synovial lining cells; hyaluronan, interleukin 1β; transforming growth factor β1 PMID:9893571

  5. Mannose reduces hyaluronan and leukocytes in wound granulation tissue and inhibits migration and hyaluronan-dependent monocyte binding.

    PubMed

    Jokela, Tiina A; Kuokkanen, Jukka; Kärnä, Riikka; Pasonen-Seppänen, Sanna; Rilla, Kirsi; Kössi, Jyrki; Laato, Matti; Tammi, Raija H; Tammi, Markku I

    2013-01-01

    Wound healing is a highly regulated process starting from coagulation and ending in tissue remodeling. The end result varies from perfectly restored tissue, such as in early fetal skin, to scars in adults. The balanced repair process is frequently disturbed by local or systemic factors, like infections and diabetes. A rapid increase of hyaluronan is an inherent feature of wounds and is associated with tissue swelling, epithelial and mesenchymal cell migration and proliferation, and induction of cytokine signaling. Hyaluronan extending from cell surface into structures called cables can trap leukocytes and platelets and change their functions. All these features of hyaluronan modulate inflammation. The present data show that mannose, a recently described inhibitor of hyaluronan synthesis, inhibits dermal fibroblast invasion and prevents the enhanced leukocyte binding to hyaluronan that takes place in cells treated with an inflammatory mediator interleukin-1β. Mannose also reduced hyaluronan in subcutaneous sponge granulation tissue, a model of skin wound, and suppressed its leukocyte recruitment and tissue growth. Mannose thus seems to suppress wounding-induced inflammation in skin by attenuating hyaluronan synthesis. PMID:23464634

  6. Over expression of hyaluronan promotes progression of HCC via CD44-mediated pyruvate kinase M2 nuclear translocation

    PubMed Central

    Li, Jing-Huan; Wang, Ying-Cong; Qin, Cheng-Dong; Yao, Rong-Rong; Zhang, Rui; Wang, Yan; Xie, Xiao-Ying; Zhang, Lan; Wang, Yan-Hong; Ren, Zheng-Gang

    2016-01-01

    Hyaluronan is expressed in hepatocellular carcinoma (HCC) as HCC generally arises from a cirrhotic liver in which excessive production and accumulation of HA leads to developing cirrhosis. Though it has been suggested HA is involved in progression of HCC, the mechanisms underlying the connection between HA and HCC progression are unclear. Since increased aerobic glycolysis is a metabolic trait of malignant cells and HA-CD44 can modulate glucose metabolism, we aim to investigate the roles of PKM2, a key enzyme in glucose metabolism, in the HA-CD44 axis facilitated the progress of HCC. We shown PKM2 was required for HA-promoted HCC progression, which was not modulated by PKM2 kinase activity but by nuclear translocation of PKM2. PKM2 translocation was Erk (Thr202/Tyr204) phosphorylation dependent, which functioned at the downstream of HA-CD44 binding. Furthermore, elevated HA expression significantly correlated with PKM2 nuclear location and was an independent factors predicting poor HCC prognosis. In conclusions PKM2 nuclear translocation is required for mediating the described HA biological effects on HCC progression and our results imply that inhibition of HA may have therapeutic value in treating HCC. PMID:27186420

  7. Hyaluronan Regulates Bone Morphogenetic Protein-7-dependent Prevention and Reversal of Myofibroblast Phenotype*

    PubMed Central

    Midgley, Adam C.; Duggal, Lucy; Jenkins, Robert; Hascall, Vincent; Steadman, Robert; Phillips, Aled O.; Meran, Soma

    2015-01-01

    Hyaluronan (HA) promotes transforming growth factor (TGF)-β1-driven myofibroblast phenotype. However, HA can also have disease-limiting activity. Bone morphogenetic protein-7 (BMP7) is an antifibrotic cytokine that antagonizes TGF-β1, and isolated studies have demonstrated that HA can both mediate and modulate BMP7 responses. In this study, we investigated whether BMP7 can modulate HA in a manner that leads to prevention/reversal of TGF-β1-driven myofibroblast differentiation in human lung fibroblasts. Results demonstrated that BMP7 prevented and reversed TGF-β1-driven myofibroblast differentiation through a novel mechanism. BMP7 promoted the dissolution and internalization of cell-surface HA into cytoplasmic endosomes. Endosomal HA co-localized with the HA-degrading enzymes, hyaluronidase-1 and hyaluronidase-2 (Hyal2). Moreover, BMP7 showed differential regulation of CD44 standard and variant isoform expression, when compared with TGF-β1. In particular, BMP7 increased membrane expression of CD44v7/8. Inhibiting CD44v7/8 as well as blocking Hyal2 and the Na+/H+ exchanger-1 at the cell-surface prevented BMP7-driven HA internalization and BMP7-mediated prevention/reversal of myofibroblast phenotype. In summary, a novel mechanism of TGF-β1 antagonism by BMP7 is shown and identifies alteration in HA as critical in mediating BMP7 responses. In addition, we identify Hyal2 and CD44v7/8 as new potential targets for manipulation in prevention and reversal of fibrotic pathology. PMID:25716319

  8. Antitumor effects of the hyaluronan inhibitor 4-methylumbelliferone on pancreatic cancer

    PubMed Central

    Yoshida, Eri; Kudo, Daisuke; Nagase, Hayato; Shimoda, Hiroshi; Suto, Shinichiro; Negishi, Mika; Kakizaki, Ikuko; Endo, Masahiko; Hakamada, Kenichi

    2016-01-01

    Hyaluronan (HA) is a major component of the extracellular matrix (ECM), and influences tumor invasion and metastasis. In a previous study, the present authors reported for the first time that 4-methylumbelliferone (MU) inhibited HA synthesis and suppressed tumor growth. However, the localization of HA and the changes in ECM morphology caused by MU in pancreatic cancer remain to be examined in detail. In the present study, the cytotoxicity of MU and its effect on cellular proliferation was evaluated in the human pancreatic cancer cell line MIA PaCa-2. The amount of HA synthesized and the retention of HA around the cells were quantitatively and immunohistochemically analyzed in vitro and in vivo. Structural changes in the ECM in the tumor tissue were investigated using an electron microscope. MU treatment led to a decrease in extracellular HA retention, as evidenced by a particle exclusion assay and immunohistochemical staining. Cell proliferation was suppressed by MU in a dose-dependent manner. The release of lactate dehydrogenase into the culture medium due to damage to the cellular membrane did not increase following MU administration. In tumor-inoculated mice, MU suppressed any increase in tumor volume and decreased the quantity of HA. Electron microscopy revealed that MU attenuated the intercellular space and caused it to be less cohesive. These data indicate that MU inhibits HA synthesis and reduces the amount of HA in the ECM while exhibiting no obvious cytotoxic effect. These findings suggest that MU has potential as a novel therapeutic agent for pancreatic cancer. PMID:27698797

  9. Hemocompatibility and Hemodynamics of Novel Hyaluronan-Polyethylene Materials for Flexible Heart Valve Leaflets.

    PubMed

    Prawel, David A; Dean, Harold; Forleo, Marcio; Lewis, Nicole; Gangwish, Justin; Popat, Ketul C; Dasi, Lakshmi Prasad; James, Susan P

    2014-03-01

    Polymeric heart valves (PHVs) hold the promise to be more durable than bioprosthetic heart valves and less thrombogenic than mechanical heart valves. We introduce a new framework to manufacture hemocompatible polymeric leaflets for HV (PHV) applications using a novel material comprised of interpenetrating networks (IPNs) of hyaluronan (HA) and linear low density polyethylene (LLDPE). We establish and characterize the feasibility of the material as a substitute leaflet material through basic hemodynamic measurements in a trileaflet configuration, in addition to demonstrating superior platelet response and clotting characteristics. Plain LLDPE sheets were swollen in a solution of silylated-HA, the silylated-HA was then crosslinked to itself before it was reverted back to native HA via hydrolysis. Leaflets were characterized with respect to (1) bending stiffness, (2) hydrophilicity, (3) whole blood clotting, and (4) cell (platelet and leukocyte) adhesion under static conditions using fresh human blood. In vitro hemodynamic testing of prototype HA/LLDPE IPN PHVs was used to assess feasibility as functional HVs. Bending stiffness was not significantly different from natural fresh leaflets. HA/LLDPE IPNs were more hydrophilic than LLDPE controls. HA/LLDPE IPNs caused less whole blood clotting and reduced cell adhesion compared to the plain LLDPE control. Prototype PHVs made with HA/LLDPE IPNs demonstrated an acceptable regurgitation fraction of 4.77 ± 0.42%, and effective orifice area in the range 2.34 ± 0.5 cm(2). These results demonstrate strong potential for IPNs between HA and polymers as future hemocompatible HV leaflets. Further studies are necessary to assess durability and calcification resistance. PMID:24729797

  10. Intermonomer Interactions in Hemagglutinin Subunits HA1 and HA2 Affecting Hemagglutinin Stability and Influenza Virus Infectivity

    PubMed Central

    DeFeo, Christopher J.; Alvarado-Facundo, Esmeralda; Vassell, Russell

    2015-01-01

    ABSTRACT Influenza virus hemagglutinin (HA) mediates virus entry by binding to cell surface receptors and fusing the viral and endosomal membranes following uptake by endocytosis. The acidic environment of endosomes triggers a large-scale conformational change in the transmembrane subunit of HA (HA2) involving a loop (B loop)-to-helix transition, which releases the fusion peptide at the HA2 N terminus from an interior pocket within the HA trimer. Subsequent insertion of the fusion peptide into the endosomal membrane initiates fusion. The acid stability of HA is influenced by residues in the fusion peptide, fusion peptide pocket, coiled-coil regions of HA2, and interactions between the surface (HA1) and HA2 subunits, but details are not fully understood and vary among strains. Current evidence suggests that the HA from the circulating pandemic 2009 H1N1 influenza A virus [A(H1N1)pdm09] is less stable than the HAs from other seasonal influenza virus strains. Here we show that residue 205 in HA1 and residue 399 in the B loop of HA2 (residue 72, HA2 numbering) in different monomers of the trimeric A(H1N1)pdm09 HA are involved in functionally important intermolecular interactions and that a conserved histidine in this pair helps regulate HA stability. An arginine-lysine pair at this location destabilizes HA at acidic pH and mediates fusion at a higher pH, while a glutamate-lysine pair enhances HA stability and requires a lower pH to induce fusion. Our findings identify key residues in HA1 and HA2 that interact to help regulate H1N1 HA stability and virus infectivity. IMPORTANCE Influenza virus hemagglutinin (HA) is the principal antigen in inactivated influenza vaccines and the target of protective antibodies. However, the influenza A virus HA is highly variable, necessitating frequent vaccine changes to match circulating strains. Sequence changes in HA affect not only antigenicity but also HA stability, which has important implications for vaccine production, as well

  11. Abnormally high body mass index and tobacco use are associated with poor sperm quality as revealed by reduced sperm binding to hyaluronan-coated slides.

    PubMed

    Wegner, Carole C; Clifford, Alicia L; Jilbert, Patricia M; Henry, Michael A; Gentry, William L

    2010-01-01

    Responses on a lifestyle questionnaire were correlated with results from traditional semen analysis and a newer functional sperm assay, namely, the ability of sperm to bind to a hyaluronan-coated slide. Increased percent normal morphology and motile sperm concentration were positively correlated with increased HA-binding score, whereas tobacco use (either current or previous history) and abnormally high body mass index were associated with lower binding scores.

  12. Hyaluronan's Role in Fibrosis: A Pathogenic Factor or a Passive Player?

    PubMed

    Albeiroti, Sami; Soroosh, Artin; de la Motte, Carol A

    2015-01-01

    Fibrosis is a debilitating condition that can lead to impairment of the affected organ's function. Excessive deposition of extracellular matrix (ECM) molecules is characteristic of most fibrotic tissues. Fibroblasts activated by cytokines or growth factors differentiate into myofibroblasts that drive fibrosis by depositing ECM molecules, such as collagen, fibronectin, and connective tissue growth factor. Transforming growth factor-β (TGF-β) is one of the major profibrotic cytokines which promotes fibrosis by signaling abnormal ECM regulation. Hyaluronan (HA) is a major ECM glycosaminoglycan that is regulated by TGF-β and whose role in fibrosis is emerging. Aside from its role as a hydrating, space filling polymer, HA regulates different cellular functions and is known to have a role in wound healing and inflammation. Importantly, HA deposition is increased in multiple fibrotic diseases. In this review we highlight studies that link HA to fibrosis and discuss what is known about the role of HA, its receptors, and its anabolic and catabolic enzymes in different fibrotic diseases.

  13. Distribution of versican and hyaluronan in the mouse uterus during decidualization.

    PubMed

    San Martin, S; Soto-Suazo, M; Zorn, T M T

    2003-08-01

    Preparation for embryo implantation requires extensive adaptation of the uterine microenvironment. This process consists of cell proliferation and cell differentiation resulting in the transformation of endometrial fibroblasts into a new type of cell called decidual cell. In the present study, we followed the space-time distribution of versican and hyaluronan (HA) in different tissues of the uterus before and after embryo implantation. Fragments of mouse uteri obtained on the fourth, fifth, sixth and seventh days of pregnancy were fixed in Methacarn, embedded in Paraplast and cut into 5-microm thick sections. HA was detected using a biotinylated fragment of the proteoglycan aggrecan, which binds to this glycosaminoglycan with high affinity and specificity. Versican was detected by a polyclonal antibody. Both reactions were developed by peroxidase methods. Before embryo implantation, both HA and versican were present in the endometrial stroma. However, after embryo implantation, HA disappeared from the decidual region immediately surrounding the implantation chamber, whereas versican accumulated in the same region. The differences observed in the expression of HA and versican suggest that both molecules may participate in the process of endometrial decidualization and/or embryo implantation.

  14. Matrix Hyaluronan Promotes Specific MicroRNA Upregulation Leading to Drug Resistance and Tumor Progression

    PubMed Central

    Bourguignon, Lilly Y. W.

    2016-01-01

    Solid tumor invasion, metastasis and therapeutic drug resistance are the common causes for serious morbidity and cancer recurrence in patients. A number of research studies have searched for malignancy-related biomarkers and drug targets that are closely linked to tumor cell properties. One of the candidates is matrix hyaluronan (HA), which is known as one of the major extracellular matrix (ECM) components. HA serves as a physiological ligand for surface CD44 molecule and also functions as a bio-regulator. The binding of HA to CD44 has been shown to stimulate concomitant activation of a number of oncogenic pathways and abnormal cellular processes in cancer cells and cancer stem cells (CSCs). MicroRNAs (miRNAs) belong to a class of small RNAs containing ~20–25 nucleotides and are known to promote aberrant cellular functions in cancer cells. In this article, I have focused on the role of HA interaction with CD44 and several important signaling molecules in the regulation of unique miRNAs (e.g., miR-21, miR-302 and miR-10b) and their downstream targets leading to multiple tumor cell-specific functions (e.g., tumor cell growth, drug resistance and metastasis) and cancer progression. This new knowledge could provide the groundwork necessary for establishing new tumor markers and developing important, novel drugs targeted against HA/CD44-associated tumor progression, which can be utilized in the therapeutic treatment of metastatic cancer patients. PMID:27070574

  15. Adiponectin resides in mouse skin and upregulates hyaluronan synthesis in dermal fibroblasts.

    PubMed

    Akazawa, Yumiko; Sayo, Tetsuya; Sugiyama, Yoshinori; Sato, Takashi; Akimoto, Noriko; Ito, Akira; Inoue, Shintaro

    2011-01-01

    Adipose tissue is a hormonally active tissue that produces adipokines that influence the activity of other tissues. Adiponectin is an adipocyte-specific adipokine involved in systemic metabolism. We detected the expression of adiponectin receptors (AdipoR1 and AdipoR2) mRNA in cultured dermal fibroblasts. The full-length adiponectin (fAd), but not the globular adiponectin (gAd), increased hyaluronan (HA) production and upregulated HA synthase (HAS) 2 mRNA expression. AdipoR1 and AdipoR2 mRNAs were also expressed in keratinocytes, though neither fAd nor gAd had any effect on HA synthesis. In mouse skin, we found that adiponectin was present and decreased markedly with aging. The age-dependent pattern of adiponectin decrease in skin, correlated well with that of HA in skin. Our experiments were also the first to identify adiponectin production in cultured mouse sebocytes, a finding that suggests that skin adiponectin may derive not only from plasma and/or subcutaneous adipose tissue, but also from the sebaceous gland. These results indicated that adiponectin plays an important role in the HA metabolism of skin. PMID:21117904

  16. Novel enzymatically cross-linked hyaluronan hydrogels support the formation of 3D neuronal networks.

    PubMed

    Broguiere, Nicolas; Isenmann, Luca; Zenobi-Wong, Marcy

    2016-08-01

    Hyaluronan (HA) is an essential component of the central nervous system's extracellular matrix and its high molecular weight (MW) form has anti-inflammatory and anti-fibrotic properties relevant for regenerative medicine. Here, we introduce a new hydrogel based on high MW HA which is cross-linked using the transglutaminase (TG) activity of the activated blood coagulation factor XIII (FXIIIa). These HA-TG gels have significant advantages for neural tissue engineering compared to previous HA gels. Due to their chemical inertness in the absence of FXIIIa, the material can be stored long-term, is stable in solution, and shows no cytotoxicity. The gelation is completely cell-friendly due to the specificity of the enzyme and the gelation rate can be tuned from seconds to hours at physiological pH and independently of stiffness. The gels are injectable, and attach covalently to fibrinogen and fibrin, two common bioactive components in in vitro tissue engineering, as well as proteins present in vivo, allowing the gels to covalently bind to brain or spinal cord defects. These optimal chemical and bioactive properties of HA-TG gels enabled the formation of 3D neuronal cultures of unprecedented performance, showing fast neurite outgrowth, axonal and dendritic speciation, strong synaptic connectivity in 3D networks, and rapidly-occurring and long-lasting coordinated electrical activity.

  17. Biophysical and biological characterization of a new line of hyaluronan-based dermal fillers: A scientific rationale to specific clinical indications.

    PubMed

    La Gatta, Annalisa; De Rosa, Mario; Frezza, Maria Assunta; Catalano, Claudia; Meloni, Marisa; Schiraldi, Chiara

    2016-11-01

    Chemico-physical and biological characterization of hyaluronan-based dermal fillers is of key importance to differentiate between numerous available products and to optimize their use. These studies on fillers are nowadays perceived as a reliable approach to predict their performance in vivo. The object of this paper is a recent line of hyaluronic acid (HA)-based dermal fillers, Aliaxin®, available in different formulations that claim a complete facial restoration. The aim of the study is to provide biophysical and biological data that may support the clinical indications and allow to predict performance possibly with respect to similar available products. Aliaxin® formulations were tested for their content in soluble HA, water uptake capacity, rheological behavior, stability to enzymatic degradation, and for in vitro capacity to stimulate extracellular matrix components production. The formulations were found to contain a low amount of soluble HA and were equivalent to each other regarding insoluble hydrogel concentration. The different crosslinking degree declared by the producer was consistent with the trend in water uptake capacity, rigidity, viscosity. No significant differences in stability to enzymatic hydrolysis were found. In vitro experiments, using a full thickness skin model, showed an increase in collagen production in the dermoepidermal junction. Results support the claims of different clinical indications, the classification of products regarding hydro-, lift-action and the specifically suggested needle gauge for the delivery. The biological outcomes also support products effectiveness in skin structure restoration. These data predicted a better performance regarding hydro-action, tissue integration, clinical management during delivery, and a high durability of the aesthetic effect when compared to data on marketed similar products. PMID:27524055

  18. Biophysical and biological characterization of a new line of hyaluronan-based dermal fillers: A scientific rationale to specific clinical indications.

    PubMed

    La Gatta, Annalisa; De Rosa, Mario; Frezza, Maria Assunta; Catalano, Claudia; Meloni, Marisa; Schiraldi, Chiara

    2016-11-01

    Chemico-physical and biological characterization of hyaluronan-based dermal fillers is of key importance to differentiate between numerous available products and to optimize their use. These studies on fillers are nowadays perceived as a reliable approach to predict their performance in vivo. The object of this paper is a recent line of hyaluronic acid (HA)-based dermal fillers, Aliaxin®, available in different formulations that claim a complete facial restoration. The aim of the study is to provide biophysical and biological data that may support the clinical indications and allow to predict performance possibly with respect to similar available products. Aliaxin® formulations were tested for their content in soluble HA, water uptake capacity, rheological behavior, stability to enzymatic degradation, and for in vitro capacity to stimulate extracellular matrix components production. The formulations were found to contain a low amount of soluble HA and were equivalent to each other regarding insoluble hydrogel concentration. The different crosslinking degree declared by the producer was consistent with the trend in water uptake capacity, rigidity, viscosity. No significant differences in stability to enzymatic hydrolysis were found. In vitro experiments, using a full thickness skin model, showed an increase in collagen production in the dermoepidermal junction. Results support the claims of different clinical indications, the classification of products regarding hydro-, lift-action and the specifically suggested needle gauge for the delivery. The biological outcomes also support products effectiveness in skin structure restoration. These data predicted a better performance regarding hydro-action, tissue integration, clinical management during delivery, and a high durability of the aesthetic effect when compared to data on marketed similar products.

  19. Effects of selected endocrine disruptors on meiotic maturation, cumulus expansion, synthesis of hyaluronan and progesterone by porcine oocyte-cumulus complexes.

    PubMed

    Mlynarcíková, Alzbeta; Nagyová, Eva; Ficková, Mária; Scsuková, Sona

    2009-04-01

    In most mammals, before ovulation, cumulus cells synthesize a large amount of hyaluronan (HA) that is organized into an extracellular matrix (ECM), which provides an essential microenvironment for in vivo oocyte fertilization. This process is called cumulus expansion. The present study assessed effects of selected endocrine disruptors (bisphenol A, BPA; 4-chloro-3-methyl phenol, CMP; di(2-ethylhexyl) phthalate, DEHP; and benzyl butyl phthalate, BBP) in a range of 100pM-100microM, on follicle-stimulating hormone (FSH)-induced meiotic maturation and cumulus expansion of porcine oocyte-cumulus complexes (OCC) cultured in vitro. Moreover, FSH-stimulated production of hyaluronic acid (HA) and progesterone by cumulus cells was measured. Both phenols, BPA and CMP (100microM), significantly affected meiotic maturation of oocytes. The number of oocytes that underwent germinal vesicle breakdown (GVBD) (78.7% and 72.4%, respectively) as well as the rate of oocytes that reached metaphase II stage (MII) (50% and 53.6%, respectively) after 44h culture were decreased compared to control (89.6% for GVBD and 81.5% for MII). FSH-stimulated expansion of cumulus was altered by the highest concentration of BPA and CMP (70% and 64%, respectively vs. 80.3% in control). Although BPA did not alter FSH-stimulated HA synthesis by cumulus cells, its incorporation within the complex was reduced to a half of control value. Progesterone production by OCC was significantly changed in the presence of BPA or DEHP. Finally, our results provide valuable information that oocyte meiotic progression was adversely affected during in vitro culture with endocrine disruptors. PMID:19162163

  20. Enhancing hyaluronan pseudoplasticity via 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride-mediated conjugation with short alkyl moieties.

    PubMed

    Petta, Dalila; Eglin, David; Grijpma, Dirk W; D'Este, Matteo

    2016-10-20

    Hyaluronan (HA) is widely used in the clinical practice and in biomedical research. Through chemical modification, HA shear-thinning properties, essential for injectability and additive manufacturing, can be optimized. In this study, we employed 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) for grafting propylamine and butylamine to HA. A parametric study was performed to identify the optimal reaction conditions. Results showed that DMTMM amidation gives reproducible and accurate control over a range of degrees of substitution (DS) from 1% to 50% and proved reliable to tune viscoelasticity. At DS=3.0% for HA-propylamine and 3.7% for HA-butylamine a maximum for storage modulus and pseudoplasticity was found, whereas above or below this DS, rheological features go back to baseline values of pristine HA. Due to their singular rheological profiles, these derivatives are valuable biomaterials candidates for preparing bioinks and hydrogels for drug delivery and regenerative medicine.

  1. Localized RNAi therapeutics of chemoresistant grade IV glioma using hyaluronan-grafted lipid-based nanoparticles.

    PubMed

    Cohen, Zvi R; Ramishetti, Srinivas; Peshes-Yaloz, Naama; Goldsmith, Meir; Wohl, Anton; Zibly, Zion; Peer, Dan

    2015-02-24

    Glioblastoma multiforme (GBM) is one of the most infiltrating, aggressive, and poorly treated brain tumors. Progress in genomics and proteomics has paved the way for identifying potential therapeutic targets for treating GBM, yet the vast majority of these leading drug candidates for the treatment of GBM are ineffective, mainly due to restricted passages across the blood-brain barrier. Nanoparticles have been emerged as a promising platform to treat different types of tumors due to their ability to transport drugs to target sites while minimizing adverse effects. Herein, we devised a localized strategy to deliver RNA interference (RNAi) directly to the GBM site using hyaluronan (HA)-grafted lipid-based nanoparticles (LNPs). These LNPs having an ionized lipid were previously shown to be highly effective in delivering small interfering RNAs (siRNAs) into various cell types. LNP's surface was functionalized with hyaluronan (HA), a naturally occurring glycosaminoglycan that specifically binds the CD44 receptor expressed on GBM cells. We found that HA-LNPs can successfully bind to GBM cell lines and primary neurosphers of GBM patients. HA-LNPs loaded with Polo-Like Kinase 1 (PLK1) siRNAs (siPLK1) dramatically reduced the expression of PLK1 mRNA and cumulated in cell death even under shear flow that simulate the flow of the cerebrospinal fluid compared with control groups. Next, a human GBM U87MG orthotopic xenograft model was established by intracranial injection of U87MG cells into nude mice. Convection of Cy3-siRNA entrapped in HA-LNPs was performed, and specific Cy3 uptake was observed in U87MG cells. Moreover, convection of siPLK1 entrapped in HA-LNPs reduced mRNA levels by more than 80% and significantly prolonged survival of treated mice in the orthotopic model. Taken together, our results suggest that RNAi therapeutics could effectively be delivered in a localized manner with HA-coated LNPs and ultimately may become a therapeutic modality for GBM.

  2. Inhibition of hyaluronan synthesis alters sulfated glycosaminoglycans deposition during chondrogenic differentiation in ATDC5 cells.

    PubMed

    Yoshioka, Yutaka; Kozawa, Eiji; Urakawa, Hiroshi; Arai, Eisuke; Futamura, Naohisa; Zhuo, Lisheng; Kimata, Koji; Ishiguro, Naoki; Nishida, Yoshihiro

    2015-08-01

    In chondrogenic differentiation, expression and collaboration of specific molecules, such as aggrecan and type II collagen, in extracellular matrix (ECM) are crucial. However, few studies have clarified the roles of hyaluronan (HA) in proteoglycan aggregation during chondrogenic differentiation. We assessed the roles of HA in sulfated glycosaminoglycans deposition during chondrogenic differentiation by means of 4-methylumbelliferone (4-MU), an HA synthase inhibitor, using ATDC5 cells. ATDC5 cells were treated with 0.5 mM 4-MU for 7 or 21 days after induction of chondrogenic differentiation with insulin. Depositions of sulfated glycosaminoglycans were evaluated with Alcian blue staining. mRNA expression of ECM molecules was determined using real-time RT-PCR. The deposition of aggrecan and versican was investigated with immunohistochemical staining using specific antibodies. Effects of 4-MU on HA concentrations were analyzed by HA binding assay. 4-MU suppressed the positivity of Alcian blue staining, although this delay was reversible. Interestingly, stronger positivity of Alcian blue staining was observed at day 21 in cultures with 4-MU discontinuation than in the control. 4-MU significantly increased the mRNA expression of aggrecan, versican, and type II collagen, which was consistent with increased deposition of aggrecan and versican. The HA concentration in ECM and cell-associated region was significantly suppressed with 4-MU treatment. We conclude that the inhibition of HA synthesis slows sulfated glycosaminoglycans deposition during chondrogenic differentiation despite the increased deposition of other ECM molecules. Transient starvation of HA with 4-MU accelerates chondrogenic ECM formation, suggesting its potential to stimulate chondrogenic differentiation with adequate use.

  3. The inhibition of the GTPase activating protein-Ha-ras interaction by acidic lipids is due to physical association of the C-terminal domain of the GTPase activating protein with micellar structures.

    PubMed Central

    Serth, J; Lautwein, A; Frech, M; Wittinghofer, A; Pingoud, A

    1991-01-01

    The effects of fatty acids and phospholipids on the interaction of the full-length GTPase activating protein (GAP) as well as its isolated C-terminal domain and the Ha-ras proto-oncogene product p21 were studied by various methods, viz. GTPase activity measurements, fluorescence titrations and gel permeation chromatography. It is shown that all fatty acids and acidic phospholipids tested, provided the critical micellar concentration and the critical micellar temperature are reached, inhibit the GAP stimulated p21 GTPase activity. This is interpreted to mean that it is not the molecular structure of acidic lipid molecules per se but rather their physical state of aggregation which is responsible for the inhibitory effect of lipids on the GTPase activity. The relative inhibitory potency of various lipids was measured under defined conditions with mixed Triton X-100 micelles to follow the order: unsaturated fatty acids greater than saturated acids approximately phosphatidic acids greater than or equal to phosphatidylinositol phosphates much greater than phosphatidylinositol and phosphatidylserine. GTPase experiments with varying concentrations of p21 and constant concentrations of GAP and lipids indicate that the binding of GAP by the lipid micelles is responsible for the inhibition, a finding which was confirmed by fluorescence titrations and gel filtrations which show that the C-terminal domain of GAP is bound by lipid micelles. PMID:2026138

  4. A ratiometric fluorescent probe for hyaluronidase detection via hyaluronan-induced formation of red-light emitting excimers.

    PubMed

    Hu, Qinghua; Zeng, Fang; Wu, Shuizhu

    2016-05-15

    Hyaluronidase (HAase), which is involved in various physiological and pathological processes, can selectively degrade hyaluronan (HA) into small fragments, and it has been reported as a diagnostic and prognostic biomarker for bladder cancer. Herein, a facile ratiometric fluorescent sensing system for HAase has been developed, which is based on hyaluronan-induced formation of red-light emitting excimers and can realize sensitive detection of HAase with a detection limit of 0.007 U/mL. A positively-charged pyrene analog (N-Py) has been synthesized and then mixed with the negatively-charged HA, due to electrostatic interaction between the two components, aggregation along with the N-Py excimers readily form which emits red light. While in the presence of HAase, the enzyme catalyzes the hydrolysis of HA into small fragments, which in turn triggers disassembly of excimers; consequently the N-Py excimer emission turns into monomer emission. The emission ratio resulted from the excimer-monomer transition can be used as the sensing signal for detecting HAase. The probe features visible-light excitation and red light emission (excimer), which is conducive to reducing possible interference from autofluorescence of biological samples. Furthermore, the assay system can be successfully used to determine HAase in human urine samples with satisfactory accuracy. This strategy may provide a suitable sensitive and accurate assay for HAase as well as an effective approach for developing fluorescent ratiometric assays for other enzymes. PMID:26774093

  5. The where, when, how, and why of hyaluronan binding by immune cells.

    PubMed

    Lee-Sayer, Sally S M; Dong, Yifei; Arif, Arif A; Olsson, Mia; Brown, Kelly L; Johnson, Pauline

    2015-01-01

    Hyaluronan is made and extruded from cells to form a pericellular or extracellular matrix (ECM) and is present in virtually all tissues in the body. The size and form of hyaluronan present in tissues are indicative of a healthy or inflamed tissue, and the interactions of hyaluronan with immune cells can influence their response. Thus, in order to understand how inflammation is regulated, it is necessary to understand these interactions and their consequences. Although there is a large turnover of hyaluronan in our bodies, the large molecular mass form of hyaluronan predominates in healthy tissues. Upon tissue damage and/or infection, the ECM and hyaluronan are broken down and an inflammatory response ensues. As inflammation is resolved, the ECM is restored, and high molecular mass hyaluronan predominates again. Immune cells encounter hyaluronan in the tissues and lymphoid organs and respond differently to high and low molecular mass forms. Immune cells differ in their ability to bind hyaluronan and this can vary with the cell type and their activation state. For example, peritoneal macrophages do not bind soluble hyaluronan but can be induced to bind after exposure to inflammatory stimuli. Likewise, naïve T cells, which typically express low levels of the hyaluronan receptor, CD44, do not bind hyaluronan until they undergo antigen-stimulated T cell proliferation and upregulate CD44. Despite substantial knowledge of where and when immune cells bind hyaluronan, why immune cells bind hyaluronan remains a major outstanding question. Here, we review what is currently known about the interactions of hyaluronan with immune cells in both healthy and inflamed tissues and discuss how hyaluronan binding by immune cells influences the inflammatory response. PMID:25926830

  6. Self-patterning of adipose-derived mesenchymal stem cells and chondrocytes cocultured on hyaluronan-grafted chitosan surface.

    PubMed

    Yeh, Hsi-Yi; Hsieh, Fu-Yu; Hsu, Shan-hui

    2016-03-01

    The articular cartilage, once injured, has a limited capacity for intrinsic repair. Preparation of functionally biocartilage substitutes in vitro for cartilage repair is an attractive concept with the recent advances in tissue engineering. In this study, adipose-derived adult stem cells (ADAS) and chondrocytes (Ch) were cocultured in different population ratios on the surface of hyaluronan-grafted chitosan (CS-HA) membranes. The two types of cells could self-assemble into cospheroids with different morphologies. In particular, when ADAS and Ch were cocultured at an initial ratio of 7:3 on CS-HA surface, the expression of chondrogenic markers was upregulated, leading to preferred chondrogenesis of the cospheroids. Therefore, using the ADAS/Ch 7:3 cospheroids derived on CS-HA surface instead of using only a single type of cells may be favorable for future therapeutic applications. PMID:26916660

  7. Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids

    PubMed Central

    McDonald, Braedon; McAvoy, Erin F.; Lam, Florence; Gill, Varinder; de la Motte, Carol; Savani, Rashmin C.; Kubes, Paul

    2008-01-01

    Adhesion molecules known to be important for neutrophil recruitment in many other organs are not involved in recruitment of neutrophils into the sinusoids of the liver. The prevailing view is that neutrophils become physically trapped in inflamed liver sinusoids. In this study, we used a biopanning approach to identify hyaluronan (HA) as disproportionately expressed in the liver versus other organs under both basal and inflammatory conditions. Spinning disk intravital microscopy revealed that constitutive HA expression was restricted to liver sinusoids. Blocking CD44–HA interactions reduced neutrophil adhesion in the sinusoids of endotoxemic mice, with no effect on rolling or adhesion in postsinusoidal venules. Neutrophil but not endothelial CD44 was required for adhesion in sinusoids, yet neutrophil CD44 avidity for HA did not increase significantly in endotoxemia. Instead, activation of CD44–HA engagement via qualitative modification of HA was demonstrated by a dramatic induction of serum-derived HA-associated protein in sinusoids in response to lipopolysaccharide (LPS). LPS-induced hepatic injury was significantly reduced by blocking CD44–HA interactions. Administration of anti-CD44 antibody 4 hours after LPS rapidly detached adherent neutrophils in sinusoids and improved sinusoidal perfusion in endotoxemic mice, revealing CD44 as a potential therapeutic target in systemic inflammatory responses involving the liver. PMID:18362172

  8. Formation of hyaluronan- and versican-rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells.

    PubMed

    Evanko, S P; Angello, J C; Wight, T N

    1999-04-01

    The accumulation of hyaluronan (HA) and the HA-binding proteoglycan versican around smooth muscle cells in lesions of atherosclerosis suggests that together these molecules play an important role in the events of atherogenesis. In this study we have examined the formation of HA- and versican-rich pericellular matrices by human aortic smooth muscle cells in vitro, using a particle-exclusion assay, and the role of the pericellular matrix in cell proliferation and migration. The structural dependence of the pericellular matrix on HA can be demonstrated by the complete removal of the matrix with Streptomyces hyaluronidase. The presence of versican in the pericellular matrix was confirmed immunocytochemically. By electron microscopy, the cell coat was seen as a tangled network of hyaluronidase-sensitive filaments decorated with ruthenium red-positive proteoglycan granules. Ninety percent of migrating cells in wounded cultures, and virtually all mitotic cells, displayed abundant HA- and versican-rich coats. Time-lapse video imaging revealed that HA- and versican-rich pericellular matrix formation is dynamic and rapid, and coordinated specifically with cell detachment and mitotic cell rounding. HA oligosaccharides, which inhibit the binding of HA to the cell surface and prevent pericellular matrix formation, significantly reduced proliferation and migration in response to platelet-derived growth factor, whereas larger HA fragments and high molecular weight HA had no effect. Treatment with HA oligosaccharides also led to changes in cell shape from a typical fusiform morphology to a more spread and flattened appearance. These data suggest that organization of HA- and versican-rich pericellular matrices may facilitate migration and mitosis by diminishing cell surface adhesivity and affecting cell shape through steric exclusion and the viscous properties of HA proteoglycan gels.

  9. Activation of the FGFR-STAT3 pathway in breast cancer cells induces a hyaluronan-rich microenvironment that licenses tumor formation

    PubMed Central

    Bohrer, Laura R.; Chuntova, Pavlina; Bade, Lindsey K.; Beadnell, Thomas C.; Leon, Ronald P.; Brady, Nicholas J.; Ryu, Yungil; Goldberg, Jodi E.; Schmechel, Stephen C.; Koopmeiners, Joseph S.; McCarthy, James B.; Schwertfeger, Kathryn L.

    2014-01-01

    Aberrant activation of fibroblast growth factor receptors (FGFRs) contributes to breast cancer growth, progression and therapeutic resistance. Due to the complex nature of the FGF/FGFR axis, and the numerous effects of FGFR activation on tumor cells and the surrounding microenvironment, the specific mechanisms through which aberrant FGFR activity contributes to breast cancer are not completely understood. We show here that FGFR activation induces accumulation of hyaluronan (HA) within the extracellular matrix (ECM) and that blocking HA synthesis decreases proliferation, migration and therapeutic resistance. Furthermore, FGFR-mediated HA accumulation requires activation of the signal transducer and activator of transcription 3 (STAT3) pathway, which regulates expression of hyaluronan synthase 2 (HAS2) and subsequent HA synthesis. Using a novel in vivo model of FGFR-dependent tumor growth, we demonstrate that STAT3 inhibition decreases both FGFR-driven tumor growth and HA levels within the tumor. Finally, our results suggest that combinatorial therapies inhibiting both FGFR activity and HA synthesis is more effective than targeting either pathway alone and may be a relevant therapeutic approach for breast cancers associated with high levels of FGFR activity. In conclusion, these studies indicate a novel targetable mechanism through which FGFR activation in breast cancer cells induces a pro-tumorigenic microenvironment. PMID:24197137

  10. Green synthesis, characterization, and anticancer activity of hyaluronan/zinc oxide nanocomposite.

    PubMed

    Namvar, Farideh; Azizi, Susan; Rahman, Heshu Sulaiman; Mohamad, Rosfarizan; Rasedee, Abdullah; Soltani, Mozhgan; Rahim, Raha Abdul

    2016-01-01

    The study describes an in situ green biosynthesis of zinc oxide nanocomposite using the seaweed Sargassum muticum water extract and hyaluronan biopolymer. The morphology and optical properties of the hyaluronan/zinc oxide (HA/ZnO) nanocomposite were determined by Fourier transform infrared spectroscopy, X-ray diffraction, field emission scanning electron microscopy, transmission electron microscopy, and ultraviolet-vis analysis. Electron microscopy and X-ray diffraction analysis showed that the zinc oxide nanoparticles were polydispersed with a mean size of 10.2±1.5 nm. The nanoparticles were mostly hexagonal in crystalline form. The HA/ZnO nanocomposite showed the absorption properties in the ultraviolet zone that is ascribed to the band gap of zinc oxide nanocomposite. In the cytotoxicity study, cancer cells, pancreatic adenocarcinoma (PANC-1), ovarian adenocarcinoma (CaOV-3), colonic adenocarcinoma (COLO205), and acute promyelocytic leukemia (HL-60) cells were treated with HA/ZnO nanocomposite. At 72 hours of treatment, the half maximal inhibitory concentration (IC50) value via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was 10.8±0.3 μg/mL, 15.4±1.2 μg/mL, 12.1±0.9 μg/mL, and 6.25±0.5 μg/mL for the PANC-1, CaOV-3, COLO-205, and HL-60 cells, respectively, showing that the composite is most toxic to the HL-60 cells. On the other hand, HA/ZnO nanocomposite treatment for 72 hours did not cause toxicity to the normal human lung fibroblast (MRC-5) cell line. Using fluorescent dyes and flow cytometry analysis, HA/ZnO nanocomposite caused G2/M cell cycle arrest and stimulated apoptosis-related increase in caspase-3 and -7 activities of the HL-60 cells. Thus, the study shows that the HA/ZnO nanocomposite produced through green synthesis has great potential to be developed into an efficacious therapeutic agent for cancers. PMID:27555781

  11. Green synthesis, characterization, and anticancer activity of hyaluronan/zinc oxide nanocomposite

    PubMed Central

    Namvar, Farideh; Azizi, Susan; Rahman, Heshu Sulaiman; Mohamad, Rosfarizan; Rasedee, Abdullah; Soltani, Mozhgan; Rahim, Raha Abdul

    2016-01-01

    The study describes an in situ green biosynthesis of zinc oxide nanocomposite using the seaweed Sargassum muticum water extract and hyaluronan biopolymer. The morphology and optical properties of the hyaluronan/zinc oxide (HA/ZnO) nanocomposite were determined by Fourier transform infrared spectroscopy, X-ray diffraction, field emission scanning electron microscopy, transmission electron microscopy, and ultraviolet–vis analysis. Electron microscopy and X-ray diffraction analysis showed that the zinc oxide nanoparticles were polydispersed with a mean size of 10.2±1.5 nm. The nanoparticles were mostly hexagonal in crystalline form. The HA/ZnO nanocomposite showed the absorption properties in the ultraviolet zone that is ascribed to the band gap of zinc oxide nanocomposite. In the cytotoxicity study, cancer cells, pancreatic adenocarcinoma (PANC-1), ovarian adenocarcinoma (CaOV-3), colonic adenocarcinoma (COLO205), and acute promyelocytic leukemia (HL-60) cells were treated with HA/ZnO nanocomposite. At 72 hours of treatment, the half maximal inhibitory concentration (IC50) value via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was 10.8±0.3 μg/mL, 15.4±1.2 μg/mL, 12.1±0.9 μg/mL, and 6.25±0.5 μg/mL for the PANC-1, CaOV-3, COLO-205, and HL-60 cells, respectively, showing that the composite is most toxic to the HL-60 cells. On the other hand, HA/ZnO nanocomposite treatment for 72 hours did not cause toxicity to the normal human lung fibroblast (MRC-5) cell line. Using fluorescent dyes and flow cytometry analysis, HA/ZnO nanocomposite caused G2/M cell cycle arrest and stimulated apoptosis-related increase in caspase-3 and -7 activities of the HL-60 cells. Thus, the study shows that the HA/ZnO nanocomposite produced through green synthesis has great potential to be developed into an efficacious therapeutic agent for cancers. PMID:27555781

  12. Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168.

    PubMed

    Jin, Peng; Kang, Zhen; Yuan, Panhong; Du, Guocheng; Chen, Jian

    2016-05-01

    Low-molecular-weight hyaluronan (LMW-HA) has attracted much attention because of its many potential applications. Here, we efficiently produced specific LMW-HAs from sucrose in Bacillus subtilis. By coexpressing the identified committed genes (tuaD, gtaB, glmU, glmM, and glmS) and downregulating the glycolytic pathway, HA production was significantly increased from 1.01gL(-1) to 3.16gL(-1), with a molecular weight range of 1.40×10(6)-1.83×10(6)Da. When leech hyaluronidase was actively expressed after N-terminal engineering (1.62×10(6)UmL(-1)), the production of HA was substantially increased from 5.96gL(-1) to 19.38gL(-1). The level of hyaluronidase was rationally regulated with a ribosome-binding site engineering strategy, allowing the production of LMW-HAs with a molecular weight range of 2.20×10(3)-1.42×10(6)Da. Our results confirm that this strategy for the controllable expression of hyaluronidase, together with the optimization of the HA synthetic pathway, effectively produces specific LMW-HAs, and could also be used to produce other LMW polysaccharides. PMID:26851304

  13. Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168.

    PubMed

    Jin, Peng; Kang, Zhen; Yuan, Panhong; Du, Guocheng; Chen, Jian

    2016-05-01

    Low-molecular-weight hyaluronan (LMW-HA) has attracted much attention because of its many potential applications. Here, we efficiently produced specific LMW-HAs from sucrose in Bacillus subtilis. By coexpressing the identified committed genes (tuaD, gtaB, glmU, glmM, and glmS) and downregulating the glycolytic pathway, HA production was significantly increased from 1.01gL(-1) to 3.16gL(-1), with a molecular weight range of 1.40×10(6)-1.83×10(6)Da. When leech hyaluronidase was actively expressed after N-terminal engineering (1.62×10(6)UmL(-1)), the production of HA was substantially increased from 5.96gL(-1) to 19.38gL(-1). The level of hyaluronidase was rationally regulated with a ribosome-binding site engineering strategy, allowing the production of LMW-HAs with a molecular weight range of 2.20×10(3)-1.42×10(6)Da. Our results confirm that this strategy for the controllable expression of hyaluronidase, together with the optimization of the HA synthetic pathway, effectively produces specific LMW-HAs, and could also be used to produce other LMW polysaccharides.

  14. Influenza Hemagglutinin (HA) Stem Region Mutations That Stabilize or Destabilize the Structure of Multiple HA Subtypes

    PubMed Central

    Byrd-Leotis, Lauren; Galloway, Summer E.; Agbogu, Evangeline

    2015-01-01

    ABSTRACT Influenza A viruses enter host cells through endosomes, where acidification induces irreversible conformational changes of the viral hemagglutinin (HA) that drive the membrane fusion process. The prefusion conformation of the HA is metastable, and the pH of fusion can vary significantly among HA strains and subtypes. Furthermore, an accumulating body of evidence implicates HA stability properties as partial determinants of influenza host range, transmission phenotype, and pathogenic potential. Although previous studies have identified HA mutations that can affect HA stability, these have been limited to a small selection of HA strains and subtypes. Here we report a mutational analysis of HA stability utilizing a panel of expressed HAs representing a broad range of HA subtypes and strains, including avian representatives across the phylogenetic spectrum and several human strains. We focused on two highly conserved residues in the HA stem region: HA2 position 58, located at the membrane distal tip of the short helix of the hairpin loop structure, and HA2 position 112, located in the long helix in proximity to the fusion peptide. We demonstrate that a K58I mutation confers an acid-stable phenotype for nearly all HAs examined, whereas a D112G mutation consistently leads to elevated fusion pH. The results enhance our understanding of HA stability across multiple subtypes and provide an additional tool for risk assessment for circulating strains that may have other hallmarks of human adaptation. Furthermore, the K58I mutants, in particular, may be of interest for potential use in the development of vaccines with improved stability profiles. IMPORTANCE The influenza A hemagglutinin glycoprotein (HA) mediates the receptor binding and membrane fusion functions that are essential for virus entry into host cells. While receptor binding has long been recognized for its role in host species specificity and transmission, membrane fusion and associated properties of HA

  15. The Adhesion and Neurite Outgrowth of Neurons on Poly(D-lysine)/Hyaluronan Multilayer Films.

    PubMed

    Shi, Haifei; Sheng, Guoping

    2016-06-01

    Poly(D-lysine)/hyaluronan (PDL/HA) films were prepared using layer-by-layer assembly technique and chemically cross-linked with a water soluble carbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS) through formation of amide bonds. Quartz crystal microbalance with dissipation (QCM-D) was used to follow the cross-linking reaction. Atomic force measurement, ellipsometry, and Fourier transform infrared (FTIR) spectroscopy were performed to study the chemical structure, topography, thickness and mechanical properties of the cross-linked films. QCM-D and Frictional force study were used to reveal the viscoelasticity of the films after cross-linking treatment. The stability of the films was studied via incubating the films in physiological environment. Finally, the neurons were used to evaluate the interaction between films and cells. The results indicated that the neurons were preferably proliferating and outgrowth neurite on cross-linked films while uncross-linked films are highly cell resistant. PMID:27427590

  16. Pulmonary surfactant adsorption is increased by hyaluronan or polyethylene glycol.

    PubMed

    Taeusch, H William; Dybbro, Eric; Lu, Karen W

    2008-04-01

    In acute lung injuries, inactivating agents may interfere with transfer (adsorption) of pulmonary surfactants to the interface between air and the aqueous layer that coats the interior of alveoli. Some ionic and nonionic polymers reduce surfactant inactivation in vitro and in vivo. In this study, we tested directly whether an ionic polymer, hyaluronan, or a nonionic polymer, polyethylene glycol, enhanced adsorption of a surfactant used clinically. We used three different methods of measuring adsorption in vitro: a modified pulsating bubble surfactometer; a King/Clements device; and a spreading trough. In addition we measured the effects of both polymers on surfactant turbidity, using this assay as a nonspecific index of aggregation. We found that both hyaluronan and polyethylene glycol significantly increased the rate and degree of surfactant material adsorbed to the surface in all three assays. Hyaluronan was effective in lower concentrations (20-fold) than polyethylene glycol and, unlike polyethylene glycol, hyaluronan did not increase apparent aggregation of surfactant. Surfactant adsorption in the presence of serum was also enhanced by both polymers regardless of whether hyaluronan or polyethylene glycol was included with serum in the subphase or added to the surfactant applied to the surface. Therefore, endogenous polymers in the alveolar subphase, or exogenous polymers added to surfactant used as therapy, may both be important for reducing inactivation of surfactant that occurs with various lung injuries.

  17. Interactions of hyaluronan grafted on protein surfaces studied using a quartz crystal microbalance and a surface force balance.

    PubMed

    Jiang, Lei; Han, Juan; Yang, Limin; Ma, Hongchao; Huang, Bo

    2015-10-01

    Vocal folds are complex and multilayer-structured where the main layer is widely composed of hyaluronan (HA). The viscoelasticity of HA is key to voice production in the vocal fold as it affects the initiation and maintenance of phonation. In this study a simple layer-structured surface model was set up to mimic the structure of the vocal folds. The interactions between two opposing surfaces bearing HA were measured and characterised to analyse HA's response to the normal and shear compression at a stress level similar to that in the vocal fold. From the measurements of the quartz crystal microbalance, atomic force microscopy and the surface force balance, the osmotic pressure, normal interactions, elasticity change, volume fraction, refractive index and friction of both HA and the supporting protein layer were obtained. These findings may shed light on the physical mechanism of HA function in the vocal fold and the specific role of HA as an important component in the effective treatment of the vocal fold disease. PMID:26274046

  18. Interactions of hyaluronan grafted on protein surfaces studied using a quartz crystal microbalance and a surface force balance.

    PubMed

    Jiang, Lei; Han, Juan; Yang, Limin; Ma, Hongchao; Huang, Bo

    2015-10-01

    Vocal folds are complex and multilayer-structured where the main layer is widely composed of hyaluronan (HA). The viscoelasticity of HA is key to voice production in the vocal fold as it affects the initiation and maintenance of phonation. In this study a simple layer-structured surface model was set up to mimic the structure of the vocal folds. The interactions between two opposing surfaces bearing HA were measured and characterised to analyse HA's response to the normal and shear compression at a stress level similar to that in the vocal fold. From the measurements of the quartz crystal microbalance, atomic force microscopy and the surface force balance, the osmotic pressure, normal interactions, elasticity change, volume fraction, refractive index and friction of both HA and the supporting protein layer were obtained. These findings may shed light on the physical mechanism of HA function in the vocal fold and the specific role of HA as an important component in the effective treatment of the vocal fold disease.

  19. Survival of cord blood haematopoietic stem cells in a hyaluronan hydrogel for ex vivo biomimicry.

    PubMed

    Demange, Elise; Kassim, Yusra; Petit, Cyrille; Buquet, Catherine; Dulong, Virginie; Cerf, Didier Le; Buchonnet, Gérard; Vannier, Jean-Pierre

    2013-11-01

    Haematopoietic stem cells (HSCs) and haematopoietic progenitor cells (HPCs) grow in a specified niche in close association with the microenvironment, the so-called 'haematopoietic niche'. Scaffolds have been introduced to overcome the liquid culture limitations, mimicking the presence of the extracellular matrix (ECM). In the present study the hyaluronic acid scaffold, already developed in the laboratory, has been used for the first time to maintain long-term cultures of CD34⁺ haematopoietic cells obtained from human cord blood. One parameter investigated was the impact on ex vivo survival of CD34⁺ cord blood cells (CBCs) on the hyaluronic acid surface, immobilized with peptides containing the RGD motif. This peptide was conjugated by coating the hyaluronan hydrogel and cultured in serum-free liquid phase complemented with stem cell factor (SCF), a commonly indispensable cytokine for haematopoiesis. Our work demonstrated that these hyaluronan hydrogels were superior to traditional liquid cultures by maintaining and expanding the HPCs without the need for additional cytokines, and a colonization of 280-fold increment in the hydrogel compared with liquid culture after 28 days of ex vivo expansion.

  20. XHAPLN3 plays a key role in cardiogenesis by maintaining the hyaluronan matrix around heart anlage.

    PubMed

    Ito, Yuzuru; Seno, Satsuki; Nakamura, Hiroaki; Fukui, Akimasa; Asashima, Makoto

    2008-07-01

    Hyaluronan matrix plays an important role during vertebrate cardiogenesis. Transcripts for the hyaluronan synthase Has2 gene are expressed in heart anlage, and disruption of either Has2 or versican, a hyaluronan matrix component, abrogates normal cardiac morphogenesis. However, the mechanisms by which hyaluronan matrix contributes to early heart development are largely unknown. Here we show that Xenopus hyaluronan and proteoglycan-binding link protein 3 (XHAPLN3) helps to maintain hyaluronan matrix around the cardiac anlage, and thereby contribute to cardiogenesis. XHAPLN3 mRNA transcript localization overlapped with the mRNA expression of both Xhas2 and Xversican at the heart anlage of early tailbud (stage 23) embryos. Furthermore, knockdown of XHAPLN3 or Xhas2 with morpholino antisense oligos caused a heart deficiency in developing tadpoles. Our results show when and how components of the hyaluronan matrix function in cardiogenesis, improving our understanding of how extracellular matrix participates in embryogenesis.

  1. Hyaluronan-conjugated liposomes encapsulating gemcitabine for breast cancer stem cells.

    PubMed

    Han, Na-Kyung; Shin, Dae Hwan; Kim, Jung Seok; Weon, Kwon Yeon; Jang, Chang-Young; Kim, Jin-Seok

    2016-01-01

    Investigation of potential therapeutics for targeting breast cancer stem cells (BCSCs) is important because these cells are regarded as culprit of breast cancer relapse. Accomplishing this kind of strategy requires a specific drug-delivery system using the distinct features of liposomes. Studies on targeted liposomal delivery systems have indicated the conjugation of hyaluronan (HA), a primary ligand for CD44 surface markers, as an appropriate method for targeting BCSCs. For this study, enriched BCSCs were obtained by culturing MCF-7 breast cancer cells in nonadherent conditions. The enriched BCSCs were challenged with HA-conjugated liposomes encapsulating gemcitabine (2, 2-difluoro-2-deoxycytidine, GEM). In vitro study showed that the HA-conjugated liposomes significantly enhanced the cytotoxicity, anti-migration, and anti-colony formation abilities of GEM through targeting of CD44 expressed on BCSCs. In pharmacokinetic study, area under the drug concentration vs time curve (AUC) of the immunoliposomal GEM was 3.5 times higher than that of free GEM, indicating that the HA-conjugated liposomes enhanced the stability of GEM in the bloodstream and therefore prolonged its half-life time. The antitumor effect of the immunoliposomal GEM was 3.3 times higher than that of free GEM in a xenograft mouse model, probably reflecting the unique targeting of the CD44 receptor by HA and the increased cytotoxicity and stability through the liposomal formulation. Furthermore, marginal change in body weight demonstrated that the use of liposomes considerably reduced the systemic toxicity of GEM on normal healthy cells. Taken together, this study demonstrates that HA-conjugated liposomes encapsulating GEM show promise for the therapy of breast cancer in vitro and in a xenograft model by targeting the BCSCs. PMID:27103799

  2. Hyaluronan-conjugated liposomes encapsulating gemcitabine for breast cancer stem cells

    PubMed Central

    Han, Na-Kyung; Shin, Dae Hwan; Kim, Jung Seok; Weon, Kwon Yeon; Jang, Chang-Young; Kim, Jin-Seok

    2016-01-01

    Investigation of potential therapeutics for targeting breast cancer stem cells (BCSCs) is important because these cells are regarded as culprit of breast cancer relapse. Accomplishing this kind of strategy requires a specific drug-delivery system using the distinct features of liposomes. Studies on targeted liposomal delivery systems have indicated the conjugation of hyaluronan (HA), a primary ligand for CD44 surface markers, as an appropriate method for targeting BCSCs. For this study, enriched BCSCs were obtained by culturing MCF-7 breast cancer cells in nonadherent conditions. The enriched BCSCs were challenged with HA-conjugated liposomes encapsulating gemcitabine (2, 2-difluoro-2-deoxycytidine, GEM). In vitro study showed that the HA-conjugated liposomes significantly enhanced the cytotoxicity, anti-migration, and anti-colony formation abilities of GEM through targeting of CD44 expressed on BCSCs. In pharmacokinetic study, area under the drug concentration vs time curve (AUC) of the immunoliposomal GEM was 3.5 times higher than that of free GEM, indicating that the HA-conjugated liposomes enhanced the stability of GEM in the bloodstream and therefore prolonged its half-life time. The antitumor effect of the immunoliposomal GEM was 3.3 times higher than that of free GEM in a xenograft mouse model, probably reflecting the unique targeting of the CD44 receptor by HA and the increased cytotoxicity and stability through the liposomal formulation. Furthermore, marginal change in body weight demonstrated that the use of liposomes considerably reduced the systemic toxicity of GEM on normal healthy cells. Taken together, this study demonstrates that HA-conjugated liposomes encapsulating GEM show promise for the therapy of breast cancer in vitro and in a xenograft model by targeting the BCSCs. PMID:27103799

  3. Targeted Delivery of Hyaluronan-Immobilized Magnetic Ceramic Nanocrystals.

    PubMed

    Wu, Hsi-Chin; Wang, Tzu-Wei; Hsieh, Shun-Yu; Sun, Jui-Sheng; Kang, Pei-Leun

    2016-01-01

    Effective cancer therapy relies on delivering the therapeutic agent precisely to the target site to improve the treatment outcome and to minimize side effects. Although surgery, chemotherapy, and radiotherapy are the standard methods commonly used in clinics, hyperthermia has been developed as a new and promising strategy for cancer therapy. In this study, magnetic bioceramic hydroxyapatite (mHAP) nanocrystals have been developed as heat mediator for intracellular hyperthermia. Hyaluronic acid (HA) modified mHAP nanocrystals are synthesized by a wet chemical precipitation process to achieve active targeting. The results demonstrate that the HA targeting moiety conjugated by a poly(ethylene glycol) (PEG) spacer arm is successfully immobilized on the surface of mHAP. The HA-modified mHAP possesses relatively good biocompatibility, an adequate biodegradation rate and superparamagnetic properties. The HA-modified mHAP could be localized and internalized into HA receptor-overexpressed malignant cells (e.g., MDA-MB-231 cell) and used as the heat generating agent for intracellular hyperthermia. The results from this study indicate that biocompatible HA-modified mHAP shows promise as a novel heat mediator and a specific targeting nanoagent for intracellular hyperthermia cancer therapy. PMID:27301176

  4. Hyaluronan synthesis is necessary for autoreactive T-cell trafficking, activation, and Th1 polarization

    PubMed Central

    Kuipers, Hedwich F.; Rieck, Mary; Gurevich, Irina; Nagy, Nadine; Negrin, Robert S.; Wight, Thomas N.; Steinman, Lawrence; Bollyky, Paul L.

    2016-01-01

    The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3+ regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS. PMID:26787861

  5. Conformational Analysis of the Streptococcus pneumoniae Hyaluronate Lyase and Characterization of Its Hyaluronan-specific Carbohydrate-binding Module*

    PubMed Central

    Suits, Michael D. L.; Pluvinage, Benjamin; Law, Adrienne; Liu, Yan; Palma, Angelina S.; Chai, Wengang; Feizi, Ten; Boraston, Alisdair B.

    2014-01-01

    For a subset of pathogenic microorganisms, including Streptococcus pneumoniae, the recognition and degradation of host hyaluronan contributes to bacterial spreading through the extracellular matrix and enhancing access to host cell surfaces. The hyaluronate lyase (Hyl) presented on the surface of S. pneumoniae performs this role. Using glycan microarray screening, affinity electrophoresis, and isothermal titration calorimetry we show that the N-terminal module of Hyl is a hyaluronan-specific carbohydrate-binding module (CBM) and the founding member of CBM family 70. The 1.2 Å resolution x-ray crystal structure of CBM70 revealed it to have a β-sandwich fold, similar to other CBMs. The electrostatic properties of the binding site, which was identified by site-directed mutagenesis, are distinct from other CBMs and complementary to its acidic ligand, hyaluronan. Dynamic light scattering and solution small angle x-ray scattering revealed the full-length Hyl protein to exist as a monomer/dimer mixture in solution. Through a detailed analysis of the small angle x-ray scattering data, we report the pseudoatomic solution structures of the monomer and dimer forms of the full-length multimodular Hyl. PMID:25100731

  6. Improvement and enhancement of antibladder carcinoma cell effects of heteronemin by the nanosized hyaluronan aggregation

    PubMed Central

    Huang, Han Hsiang; Kuo, Shyh Ming; Wu, Yi-Jhen; Su, Jui-Hsin

    2016-01-01

    The effects against tumors exerted by marine active compounds have been highlighted and investigated. Polymeric nanoparticles made from biodegradable and biocompatible molecules such as hyaluronan (HA) and chitosan (CHI) are able to aggregate the compounds to enhance their activities against tumor cells and reduce the toxicity on normal cells. Here, we extensively examined the antitumor activities and the mechanisms of HA/CHI nanoparticles-aggregated heteronemin (HET) extracted from the sponge Hippospongia sp. The half-maximal inhibitory concentration (IC50) of pure HET toward T24 bladder carcinoma cells is ~0.28 µg/mL. Pure HET from 0.2 to 0.8 µg/mL and HA nanoparticles-aggregated HET at 0.1 and 0.2 µg/mL significantly reduced T24 cell viability. Compared to pure HET, HA nanoparticles/HET aggregates showed much weaker viability-inhibitory effects on L929 normal fibroblasts. HET dose-dependently suppressed cancer cell migration as HA/CHI nanoparticles-aggregated HET displayed stronger migration-inhibitory effects than pure HET. Flow cytometric analysis showed that pure HET increased early/total apoptosis and JC-1 monomer fluorescence, while HA/CHI nanoparticles-aggregated HET induced higher apoptosis and JC-1 monomer rates than pure HET, suggesting that aggregation of HA nanoparticles offers HET stronger apoptosis-inducing capacity through mitochondrial depolarization. Western blot analysis showed that HA nanoparticles-aggregated HET further increased mitochondrial-associated, caspase-dependent and caspase-independent, as well as endoplasmic reticulum stress-related factors in comparison with pure HET. These data indicated that pure HET possesses cytotoxic, antimigratory, and apoptosis-inducing effects on bladder cancer cells in vitro, and its induction of apoptosis in bladder carcinoma cells is mainly caspase dependent. Moreover, HA nanoparticle aggregation reinforced the cytotoxic, antimigratory, and apoptosis-inducing activities against bladder carcinoma

  7. Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo- and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2, and HAS3*

    PubMed Central

    Bart, Geneviève; Vico, Nuria Ortega; Hassinen, Antti; Pujol, Francois M.; Deen, Ashik Jawahar; Ruusala, Aino; Tammi, Raija H.; Squire, Anthony; Heldin, Paraskevi; Kellokumpu, Sakari; Tammi, Markku I.

    2015-01-01

    In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1–3 (HAS1–3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647–23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis. PMID:25795779

  8. Properties of newly-synthesized cationic semi-interpenetrating hydrogels containing either hyaluronan or chondroitin sulfate in a methacrylic matrix.

    PubMed

    Gatta, Annalisa La; Schiraldi, Chiara; D'Agostino, Antonella; Papa, Agata; Rosa, Mario De

    2012-01-01

    Extracellular matrix components such as hyaluronan (HA) and chondroitin sulfate (CS) were combined with a synthetic matrix of p(HEMA-co-METAC) (poly(2-hydroxyethylmethacrylate-co-2-methacryloxyethyltrimethylammonium)) at 1% and 2% w/w concentration following a previously developed procedure. The resulting semi-interpenetrating hydrogels were able to extensively swell in water incrementing their dry weight up to 13 fold depending on the glycosamminoglycan content and nature. When swollen in physiological solution, materials water uptake significantly decreased, and the differences in swelling capability became negligible. In physiological conditions, HA was released from the materials up to 38%w/w while CS was found almost fully retained. Materials were not cytotoxic and a biological evaluation, performed using 3T3 fibroblasts and an original time lapse videomicroscopy station, revealed their appropriateness for cell adhesion and proliferation. Slight differences observed in the morphology of adherent cells suggested a better performance of CS containing hydrogels.

  9. Properties of newly-synthesized cationic semi-interpenetrating hydrogels containing either hyaluronan or chondroitin sulfate in a methacrylic matrix.

    PubMed

    Gatta, Annalisa La; Schiraldi, Chiara; D'Agostino, Antonella; Papa, Agata; Rosa, Mario De

    2012-01-01

    Extracellular matrix components such as hyaluronan (HA) and chondroitin sulfate (CS) were combined with a synthetic matrix of p(HEMA-co-METAC) (poly(2-hydroxyethylmethacrylate-co-2-methacryloxyethyltrimethylammonium)) at 1% and 2% w/w concentration following a previously developed procedure. The resulting semi-interpenetrating hydrogels were able to extensively swell in water incrementing their dry weight up to 13 fold depending on the glycosamminoglycan content and nature. When swollen in physiological solution, materials water uptake significantly decreased, and the differences in swelling capability became negligible. In physiological conditions, HA was released from the materials up to 38%w/w while CS was found almost fully retained. Materials were not cytotoxic and a biological evaluation, performed using 3T3 fibroblasts and an original time lapse videomicroscopy station, revealed their appropriateness for cell adhesion and proliferation. Slight differences observed in the morphology of adherent cells suggested a better performance of CS containing hydrogels. PMID:24955528

  10. Low Molecular Weight Hyaluronan-Pulsed Human Dendritic Cells Showed Increased Migration Capacity and Induced Resistance to Tumor Chemoattraction

    PubMed Central

    Rizzo, Manglio; Bayo, Juan; Piccioni, Flavia; Malvicini, Mariana; Fiore, Esteban; Peixoto, Estanislao; García, Mariana G.; Aquino, Jorge B.; Gonzalez Campaña, Ariel; Podestá, Gustavo; Terres, Marcelo; Andriani, Oscar; Alaniz, Laura; Mazzolini, Guillermo

    2014-01-01

    We have shown that ex vivo pre-conditioning of bone marrow-derived dendritic cells (DC) with low molecular weight hyaluronan (LMW HA) induces antitumor immunity against colorectal carcinoma (CRC) in mice. In the present study we investigated the effects of LMW HA priming on human-tumor-pulsed monocytes-derived dendritic cells (DC/TL) obtained from healthy donors and patients with CRC. LMW HA treatment resulted in an improved maturation state of DC/TL and an enhanced mixed leucocyte reaction activity in vivo. Importantly, pre-conditioning of DC/TL with LMW HA increased their ability to migrate and reduced their attraction to human tumor derived supernatants. These effects were associated with increased CCR7 expression levels in DC. Indeed, a significant increase in migratory response toward CCL21 was observed in LMW HA primed tumor-pulsed monocyte-derived dendritic cells (DC/TL/LMW HA) when compared to LWM HA untreated cells (DC/TL). Moreover, LMW HA priming modulated other mechanisms implicated in DC migration toward lymph nodes such as the metalloproteinase activity. Furthermore, it also resulted in a significant reduction in DC migratory capacity toward tumor supernatant and IL8 in vitro. Consistently, LMW HA dramatically enhanced in vivo DC recruitment to tumor-regional lymph nodes and reduced DC migration toward tumor tissue. This study shows that LMW HA –a poorly immunogenic molecule- represents a promising candidate to improve human DC maturation protocols in the context of DC-based vaccines development, due to its ability to enhance their immunogenic properties as well as their migratory capacity toward lymph nodes instead of tumors. PMID:25238610

  11. Relaxin Regulates Hyaluronan Synthesis and Aquaporins in the Cervix of Late Pregnant Mice

    PubMed Central

    Soh, Yu May; Tiwari, Anjana; Mahendroo, Mala; Conrad, Kirk P.

    2012-01-01

    Cervical ripening is associated with loss of structural integrity and tensile strength, thus enabling the cervix to dilate at term. It is characterized by changes in glycosaminoglycan composition, increased water content, and a progressive reorganization of the collagen network. The peptide hormone relaxin via interaction with its receptor, relaxin family peptide receptor 1 (RXFP1), promotes tissue hydration and increases cervical hyaluronan (HA) concentrations, but the mechanisms that regulate these effects are not known. This study in relaxin mutant (Rln−/−) mice tested the hypothesis that relaxin regulates HA synthase and aquaporin (AQP) expression in the cervix. We also assessed expression of the RXFP1 protein by immunohistochemistry. Pregnant Rln−/− mice had lower Has2 and Aqp3 expression on d 18.5 of pregnancy and decreased cervical HA compared with wild-type Rln+/+ mice. Chronic infusion of relaxin for 4 or 6 d in pregnant Rln−/− mice reversed these phenotypes and increased Has2 and Aqp3 compared with placebo controls. Relaxin-treated mice also had lower Has1 and Aqp5. Changes in gene expression were paralleled by increases in cervical HA and variations in AQP3 and AQP5 protein localization in epithelial cells of Rln−/− cervices. Our findings demonstrate that relaxin alters AQP expression in the cervix and initiates changes in glycosaminoglycan composition through increased HA synthesis. These effects are likely mediated through RXFP1 localized to subepithelial stromal cells and epithelial cells. We suggest these actions of relaxin collectively promote water recruitment into the extracellular matrix to loosen the dense collagen fiber network. PMID:23087172

  12. [Modification of seaweed polysaccharide-agarose and its application as skin dressing (III)--skin regeneration with agarose grafting hyaluronic acid sponge].

    PubMed

    Huang, Jianyan; Zhang, Lingmin; Chu, Bin; Chen, Peng; Tang, Shunqing

    2011-02-01

    In this paper, a kind of skin dressing, agarose- grafting- hyaluronic acid (Ag-g-HA) sponge was applied to test the modified agarose based scaffold for skin regeneration. The bFGF loading agarose-grafting hyaluronan scaffold had homogenous porosities, and the loaded bFGF was bioactive in 2 weeks. The Ag-g-HA sponge was applied into skin of mice, and it was found that the dressing promoted skin regeneration and no infection and leakage in lesion site took place. H&E staining results showed that the repaired skin was similar to autologous skin. These demonstrate that Ag-g-HA sponge has a promise in skin regeneration.

  13. Evaluation of hyaluronan from different sources: Streptococcus zooepidemicus, rooster comb, bovine vitreous, and human umbilical cord.

    PubMed

    Shiedlin, Aviva; Bigelow, Russell; Christopher, William; Arbabi, Saman; Yang, Laura; Maier, Ronald V; Wainwright, Norman; Childs, Alice; Miller, Robert J

    2004-01-01

    Sodium hyaluronate (HA) is widely distributed in extracellular matrixes and can play a role in orchestrating cell function. Consequently, many investigators have looked at the effect of exogenous HA on cell behavior in vitro. HA can be isolated from several sources (e.g., bacterial, rooster comb, umbilical cord) and therefore can possess diverse impurities. This current study compares the measured impurities and the differences in biological activity between HA preparations from these sources. It was demonstrated that nucleic acid and protein content was highest in human umbilical cord and bovine vitreous HA and was low in bacterial and rooster comb HA. Macrophages exposed to human umbilical cord HA produced significantly higher amounts of TNF-alpha relative to control or bacterial-derived HA. These results indicate that the source of HA should be considered due to differences in the amounts and types of contaminants that could lead to widely different behaviors in vitro and in vivo. PMID:15530025

  14. Chemically modified N-acylated hyaluronan fragments modulate proinflammatory cytokine production by stimulated human macrophages.

    PubMed

    Babasola, Oladunni; Rees-Milton, Karen J; Bebe, Siziwe; Wang, Jiaxi; Anastassiades, Tassos P

    2014-09-01

    Low molecular mass hyaluronans are known to induce inflammation. To determine the role of the acetyl groups of low molecular mass hyaluronan in stimulating the production of proinflammatory cytokines, partial N-deacetylation was carried out by hydrazinolysis. This resulted in 19.7 ± 3.5% free NH2 functional groups, which were then acylated by reacting with an acyl anhydride, including acetic anhydride. Hydrazinolysis resulted in bond cleavage of the hyaluronan chain causing a reduction of the molecular mass to 30-214 kDa. The total NH2 and N-acetyl moieties in the reacetylated hyaluronan were 0% and 98.7 ± 1.5% respectively, whereas for butyrylated hyaluronan, the total NH2, N-acetyl, and N-butyryl moieties were 0, 82.2 ± 4.6, and 22.7 ± 3.8%, respectively, based on (1)H NMR. We studied the effect of these polymers on cytokine production by cultured human macrophages (THP-1 cells). The reacetylated hyaluronan stimulated proinflammatory cytokine production to levels similar to LPS, whereas partially deacetylated hyaluronan had no stimulatory effect, indicating the critical role of the N-acetyl groups in the stimulation of proinflammatory cytokine production. Butyrylated hyaluronan significantly reduced the stimulatory effect on cytokine production by the reacetylated hyaluronan or LPS but had no stimulatory effect of its own. The other partially N-acylated hyaluronan derivatives tested showed smaller stimulatory effects than reacetylated hyaluronan. Antibody and antagonist experiments suggest that the acetylated and partially butyrylated lower molecular mass hyaluronans exert their effects through the TLR-4 receptor system. Selectively N-butyrylated lower molecular mass hyaluronan shows promise as an example of a novel semisynthetic anti-inflammatory molecule.

  15. Chemically Modified N-Acylated Hyaluronan Fragments Modulate Proinflammatory Cytokine Production by Stimulated Human Macrophages*

    PubMed Central

    Babasola, Oladunni; Rees-Milton, Karen J.; Bebe, Siziwe; Wang, Jiaxi; Anastassiades, Tassos P.

    2014-01-01

    Low molecular mass hyaluronans are known to induce inflammation. To determine the role of the acetyl groups of low molecular mass hyaluronan in stimulating the production of proinflammatory cytokines, partial N-deacetylation was carried out by hydrazinolysis. This resulted in 19.7 ± 3.5% free NH2 functional groups, which were then acylated by reacting with an acyl anhydride, including acetic anhydride. Hydrazinolysis resulted in bond cleavage of the hyaluronan chain causing a reduction of the molecular mass to 30–214 kDa. The total NH2 and N-acetyl moieties in the reacetylated hyaluronan were 0% and 98.7 ± 1.5% respectively, whereas for butyrylated hyaluronan, the total NH2, N-acetyl, and N-butyryl moieties were 0, 82.2 ± 4.6, and 22.7 ± 3.8%, respectively, based on 1H NMR. We studied the effect of these polymers on cytokine production by cultured human macrophages (THP-1 cells). The reacetylated hyaluronan stimulated proinflammatory cytokine production to levels similar to LPS, whereas partially deacetylated hyaluronan had no stimulatory effect, indicating the critical role of the N-acetyl groups in the stimulation of proinflammatory cytokine production. Butyrylated hyaluronan significantly reduced the stimulatory effect on cytokine production by the reacetylated hyaluronan or LPS but had no stimulatory effect of its own. The other partially N-acylated hyaluronan derivatives tested showed smaller stimulatory effects than reacetylated hyaluronan. Antibody and antagonist experiments suggest that the acetylated and partially butyrylated lower molecular mass hyaluronans exert their effects through the TLR-4 receptor system. Selectively N-butyrylated lower molecular mass hyaluronan shows promise as an example of a novel semisynthetic anti-inflammatory molecule. PMID:25053413

  16. Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior.

    PubMed

    Weigel, Paul H

    2015-01-01

    Hyaluronan (HA) biosynthesis has been studied for over six decades, but our understanding of the biochemical details of how HA synthase (HAS) assembles HA is still incomplete. Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains. HA-producing cells typically create extracellular HA coats (capsules) and also secrete HA into the surrounding space. Since HAS contains multiple transmembrane domains and is lipid-dependent, we proposed in 1999 that it creates an intraprotein HAS-lipid pore through which a growing HA-UDP chain is translocated continuously across the cell membrane to the exterior. We review here the evidence for a synthase pore-mediated polysaccharide translocation process and describe a possible mechanism (the Pendulum Model) and potential energy sources to drive this ATP-independent process. HA synthases also synthesize chitin oligosaccharides, which are created by cleavage of novel oligo-chitosyl-UDP products. The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes. These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.

  17. Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior

    PubMed Central

    Weigel, Paul H.

    2015-01-01

    Hyaluronan (HA) biosynthesis has been studied for over six decades, but our understanding of the biochemical details of how HA synthase (HAS) assembles HA is still incomplete. Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains. HA-producing cells typically create extracellular HA coats (capsules) and also secrete HA into the surrounding space. Since HAS contains multiple transmembrane domains and is lipid-dependent, we proposed in 1999 that it creates an intraprotein HAS-lipid pore through which a growing HA-UDP chain is translocated continuously across the cell membrane to the exterior. We review here the evidence for a synthase pore-mediated polysaccharide translocation process and describe a possible mechanism (the Pendulum Model) and potential energy sources to drive this ATP-independent process. HA synthases also synthesize chitin oligosaccharides, which are created by cleavage of novel oligo-chitosyl-UDP products. The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes. These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers. PMID:26472958

  18. Modulation of vesicle adhesion and spreading kinetics by hyaluronan cushions.

    PubMed

    Limozin, Laurent; Sengupta, Kheya

    2007-11-01

    The adhesion of giant unilamellar phospholipid vesicles to planar substrates coated with extracellular matrix mimetic cushions of hyaluronan is studied using quantitative reflection interference contrast microscopy. The absolute height of the vesicle membrane at the vicinity of the substrate is measured by considering, for the first time, the refractive indices of the reflecting media. The thickness of the cushion is varied in the range of approximately 50-100 nm, by designing various coupling strategies. On bare protein-coated substrates, the vesicles spread fast (0.5 s) and form a uniform adhesion disk, with the average membrane height approximately 4 nm. On thick hyaluronan cushions (>80 nm), the membrane height is approximately the same as the thickness of the cushion, implying that the vesicle lies on top of the cushion. On a thin and inhomogeneous hyaluronan cushion, the adhesion is modified but not prevented. The spreading is slow ( approximately 20 s) compared to the no-cushion case. The average membrane height is approximately 10 nm and the adhesion disk is studded with blisterlike structures. Observations with fluorescent hyaluronan indicate that the polymer is compressed under, rather than expelled from, the adhesion disk. The adhesion energy density is approximately threefold higher in the no-cushion case (1.2 microJ/m(2)) as compared to the thin-cushion case (0.54 microJ/m(2)). In the thin-cushion case, the presence of short ( approximately 4 nm) glyco-polymers on the vesicles results in a hitherto unreported stable partial adhesion state--the membrane height ranges from zero to approximately 250 nm. The minimal model system presented here mimics in vitro the hyaluronan-modulated early stages of cell adhesion, and demonstrates that the presence of a polymer cushion influences both the final equilibrium adhesion-state and the spreading kinetics. PMID:17631530

  19. 1- and 2-particle Microrheology of Hyaluronic Acid

    NASA Astrophysics Data System (ADS)

    Sagan, Austin; Kearns, Sarah; Ross, David; Das, Moumita; Thurston, George; Franklin, Scott

    2015-03-01

    Hyaluronic acid (also called HA or Hyaluronan) is a high molecular weight polysaccaride ubiquitous in the extracellular matrix of soft tissue such as cartilage, skin, the eye's vitreous gel and synovial fluid. It has been shown to play an important role in mechanotransduction, cell migration and proliferation, and in tissue morphodynamics. We present a confocal microrheology study of hyaluronic acid of varying concentrations. The mean squared displacement (MSD) of sub-micron colloidal tracer particles is tracked in two dimensions and shows a transition from diffusive motion at low concentrations to small-time trapping by the protein network as the concentration increases. Correlations between particle motion can be used to determine an effective mean-squared displacement which deviates from the single-particle MSD as the fluid becomes less homogeneous. The real and effective mean-squared displacements are used to probe the local and space-averaged frequency dependent rheological properties of the fluid as the concentration changes.

  20. Biological effects of hyaluronan in connective tissues, eye, skin, venous wall. Role in aging.

    PubMed

    Robert, L; Robert, A-M; Renard, G

    2010-06-01

    Hyaluronan, as most macromolecules of the extracellular matrix, are produced by the differentiated mesenchymal cells. These cells produce also enzymes degrading hyaluronan. This results in the presence of several hyaluronan pools of different molecular weights, all capable of interacting with surrounding cells, mediated by hyaluronan binding proteins and receptors. These interactions modulate cell phenotype and produce a variety of effects conditioning the specific functions of tissues. We shall discuss here several examples studied in our laboratory, concerning skin, cornea and the venous wall. Some of these actions might even be harmful, and could play an important role in aging of connective tissues with loss of function. Some of these age-dependent modifications mediated by hyaluronan will be reviewed and commented, especially the upregulation of matrix degrading enzymes as MMP-2 and MMP-9. We shall also mention some of our experiments for finding molecules capable of counteracting the harmful effects mediated by hyaluronan.

  1. The role of hyaluronan in the pathobiology and treatment of respiratory disease.

    PubMed

    Garantziotis, Stavros; Brezina, Martin; Castelnuovo, Paolo; Drago, Lorenzo

    2016-05-01

    Hyaluronan, a ubiquitous naturally occurring glycosaminoglycan, is a major component of the extracellular matrix, where it participates in biological processes that include water homeostasis, cell-matrix signaling, tissue healing, inflammation, angiogenesis, and cell proliferation and migration. There are emerging data that hyaluronan and its degradation products have an important role in the pathobiology of the respiratory tract. We review the role of hyaluronan in respiratory diseases and present evidence from published literature and from clinical practice supporting hyaluronan as a novel treatment for respiratory diseases. Preliminary data show that aerosolized exogenous hyaluronan has beneficial activity against airway inflammation, protects against bronchial hyperreactivity and remodeling, and disrupts the biofilm associated with chronic infection. This suggests a role in airway diseases with a predominant inflammatory component such as rhinosinusitis, asthma, chronic obstructive pulmonary disease, cystic fibrosis, and primary ciliary dyskinesia. The potential for hyaluronan to complement conventional therapy will become clearer when data are available from controlled trials in larger patient populations.

  2. Small synthetic hyaluronan disaccharides afford neuroprotection in brain ischemia-related models.

    PubMed

    Egea, J; Parada, E; Gómez-Rangel, V; Buendia, I; Negredo, P; Montell, E; Ruhí, R; Vergés, J; Roda, J M; García, A G; López, M G

    2014-04-18

    High molecular weight (HMW) glycosaminoglycanes of the extracellular matrix have been implicated in tissue repair. The aim of this study was to evaluate if small synthetic hyaluronan disaccharides with different degrees of sulfation (methyl 2-acetamido-2-deoxy-3-O-(β-d-glucopyranosyluronic acid)-O-sulfo-α-d-glucopyranoside, sodium salt (di0S), methyl 2-acetamido-2-deoxy-3-O-(β-d-glucopyranosyluronic acid)-6-di-O-sulfo-α-d-glucopyranoside, disodium salt (di6S) and methyl 2-acetamido-2-deoxy-3-O-(β-d-glucopyranosyluronic acid)-4,6-di-O-sulfo-α-d-glucopyranoside, trisodium salt (di4,6S)) could improve cell survival in in vitro and in vivo brain ischemia-related models. Rat hippocampal slices subjected to oxygen and glucose deprivation and a photothrombotic stroke model in mice were used. The three hyaluran disaccharides, incubated during the oxygen and glucose deprivation (15min) and re-oxygenation periods (120min), reduced cell death of hippocampal slices measured as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, being the most potent di4,6S; in contrast, high molecular hyaluronan was ineffective. The protective actions of di4,6S against oxygen and glucose deprivation were related to activation of the PI3K/Akt survival pathway, reduction of p65 translocation to the nucleus, inhibition of inducible nitric oxide oxidase induction and reactive oxygen species production, and to an increase in glutathione levels. Administered 1h post-stroke, di4,6S reduced cerebral infarct size and improved motor activity in the beam walk test. In conclusion, di4,6S affords neuroprotection in in vitro and in vivo models of ischemic neuronal damage. Our results suggest that its neuroprotective effect could be exerted through its capability to reduce oxidative stress during ischemia. Its small molecular size makes it a more potential druggable drug to target the brain as compared with its HMW parent compound hyaluronan. PMID:24486437

  3. Small synthetic hyaluronan disaccharides afford neuroprotection in brain ischemia-related models.

    PubMed

    Egea, J; Parada, E; Gómez-Rangel, V; Buendia, I; Negredo, P; Montell, E; Ruhí, R; Vergés, J; Roda, J M; García, A G; López, M G

    2014-04-18

    High molecular weight (HMW) glycosaminoglycanes of the extracellular matrix have been implicated in tissue repair. The aim of this study was to evaluate if small synthetic hyaluronan disaccharides with different degrees of sulfation (methyl 2-acetamido-2-deoxy-3-O-(β-d-glucopyranosyluronic acid)-O-sulfo-α-d-glucopyranoside, sodium salt (di0S), methyl 2-acetamido-2-deoxy-3-O-(β-d-glucopyranosyluronic acid)-6-di-O-sulfo-α-d-glucopyranoside, disodium salt (di6S) and methyl 2-acetamido-2-deoxy-3-O-(β-d-glucopyranosyluronic acid)-4,6-di-O-sulfo-α-d-glucopyranoside, trisodium salt (di4,6S)) could improve cell survival in in vitro and in vivo brain ischemia-related models. Rat hippocampal slices subjected to oxygen and glucose deprivation and a photothrombotic stroke model in mice were used. The three hyaluran disaccharides, incubated during the oxygen and glucose deprivation (15min) and re-oxygenation periods (120min), reduced cell death of hippocampal slices measured as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, being the most potent di4,6S; in contrast, high molecular hyaluronan was ineffective. The protective actions of di4,6S against oxygen and glucose deprivation were related to activation of the PI3K/Akt survival pathway, reduction of p65 translocation to the nucleus, inhibition of inducible nitric oxide oxidase induction and reactive oxygen species production, and to an increase in glutathione levels. Administered 1h post-stroke, di4,6S reduced cerebral infarct size and improved motor activity in the beam walk test. In conclusion, di4,6S affords neuroprotection in in vitro and in vivo models of ischemic neuronal damage. Our results suggest that its neuroprotective effect could be exerted through its capability to reduce oxidative stress during ischemia. Its small molecular size makes it a more potential druggable drug to target the brain as compared with its HMW parent compound hyaluronan.

  4. Hyaluronan nanoparticles bearing γ-secretase inhibitor: in vivo therapeutic effects on rheumatoid arthritis.

    PubMed

    Heo, Roun; Park, Jong-Sung; Jang, Hye Jin; Kim, Seol-Hee; Shin, Jung Min; Suh, Yung Doug; Jeong, Ji Hoon; Jo, Dong-Gyu; Park, Jae Hyung

    2014-10-28

    γ-Secretase inhibitors which prevent Notch activation are emerging as potent therapeutics for various inflammatory diseases, including ischemic stroke and rheumatoid arthritis. However, their indiscriminate distribution in the body causes serious side effects after systemic administration, since Notch proteins are ubiquitous receptors that play an important role in cellular functions such as differentiation, proliferation, and apoptosis. In this study, hyaluronan nanoparticles (HA-NPs) bearing a γ-secretase inhibitor (DAPT) were prepared as potential therapeutics for rheumatoid arthritis. In vivo biodistribution of the DAPT-loaded HA-NPs (DNPs), labeled with near-infrared dye, were observed using a non-invasive optical imaging system after systemic administration to a collagen-induced arthritis (CIA) mouse model. The results demonstrated that DNPs were effectively accumulated at the inflamed joint of the CIA mice. From the in vivo therapeutic efficacy tests, DNPs (1mg DAPT/kg) significantly attenuated the severity of RA induction compared to DAPT alone (2mg/kg), which was judged from clinical scores, tissue damage, and neutrophil infiltration. In addition, DNPs dramatically reduced the production of pro-inflammatory cytokines (TNF-α, IFN-γ, MCP-1, and IL-6, -12, -17) and collagen-specific auto-antibodies (IgG1 and IgG2a) in the serum of the CIA mice. These results suggest that DNPs have potential as therapeutics for rheumatoid arthritis.

  5. Clinical Evaluation of Hyaluronic Acid Sponge with Zinc versus Placebo for Scar Reduction after Breast Surgery

    PubMed Central

    Mahedia, Monali; Shah, Nilay

    2016-01-01

    Background: Scar formation is a major source of dissatisfaction among patients and surgeons. Individually, hyaluronan, or hyaluronic acid (HA), and zinc have been shown to reduce scarring. The authors evaluated the safety and efficacy of an HA sponge with zinc compared with placebo when applied to bilateral breast surgery scars; specifically, they evaluated whether the use of this product modulates inflammation and immediate scarring in treated patients after bilateral breast surgery. Methods: This double-blind, randomized, prospective study was approved by the local institutional review board. Bilateral breast surgery patients with right and left incision lines were randomly assigned to receive HA sponge with zinc or placebo within 2 to 4 days after their procedure. Participants were followed up at 6 weeks, 12 weeks, and 1 year and evaluated at 12 weeks. Three blinded evaluators reviewed photographs of the incision lines and assessed the scars using a visual analog scale, new scale, and a patient satisfaction survey. Results: Nineteen bilateral breast surgery patients were enrolled in the study. Statistical analysis was performed on 14 patients who completed the follow-up. The mean visual analog scale score was lower for the side receiving the HA sponge with zinc (2.6) than for the side receiving placebo (3.0), indicating a better outcome (t test; P = 0.08). The HA sponge with zinc was found to have significant positive findings on a patient satisfaction survey (P = 0.01). Conclusions: This is a preliminary study that shows zinc hyaluronan was associated with high patient satisfaction in achieving a better scar after bilateral breast surgery, irrespective of skin color. It seems to be safe and effective for early scars. PMID:27536470

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

    PubMed

    Winkler, Clayton W; Foster, Scott C; Itakura, Asako; Matsumoto, Steven G; Asari, Akira; McCarty, Owen J T; Sherman, Larry S

    2013-04-24

    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.

  7. Key Roles of Hyaluronan and Its CD44 Receptor in the Stemness and Survival of Cancer Stem Cells

    PubMed Central

    Chanmee, Theerawut; Ontong, Pawared; Kimata, Koji; Itano, Naoki

    2015-01-01

    Cancer stem cells (CSCs) represent a unique subpopulation of self-renewing oncogenic cells that drive cancer initiation and progression. CSCs often acquire multidrug and oxidative stress resistance and are thereby thought to be responsible for tumor recurrence following treatment and remission. Although the mechanisms responsible for CSC generation, maintenance, and expansion have become a major focus in cancer research, the molecular characteristics of CSCs remain poorly understood. The stemness and subsequent expansion of CSCs are believed to be highly influenced by changes in microenvironmental signals as well as genetic and epigenetic alterations. Hyaluronan (HA), a major component of the extracellular matrix, has recently been demonstrated to provide a favorable microenvironment for the self-renewal and maintenance of stem cells. HA directly and indirectly affects CSC self-renewal by influencing the behavior of both cancer and stromal cells. For instance, HA in the tumor microenvironment modulates the function of tumor-associated macrophages to support CSC self-renewal, and excessive HA production promotes the acquisition of CSC signatures through epithelial-to-mesenchymal transition. The importance of HA in mediating CSC self-renewal has been strengthened by the finding that interactions between HA and its receptor, CD44, propagate the stemness of CSCs. HA–CD44 interactions evoke a wide range of signals required for CSC self-renewal and maintenance. CD44 also plays a critical role in the preservation and multidrug resistance (MDR) of CSCs by transmitting survival and anti-apoptotic signals. Thus, a better understanding of the molecular mechanisms involved in HA and CD44 control of CSC stemness may help in the design of more effective therapies for cancer patients. In this review, we address the key roles of HA and CD44 in CSC self-renewal and maintenance. We also discuss the involvement of CD44 in the oxidative stress and MDR of CSCs. PMID:26322272

  8. Evaluation of the ability of xanthan gum/gellan gum/hyaluronan hydrogel membranes to prevent the adhesion of postrepaired tendons.

    PubMed

    Kuo, Shyh Ming; Chang, Shwu Jen; Wang, Hung-Yi; Tang, Shu Ching; Yang, Shan-Wei

    2014-12-19

    After tendon-repair surgery, adhesion between the surgical tendon and the synovial sheath is often presented resulting in poor functional repair of the tendon. This may be prevented using a commercially available mechanical barrier implant, Seprafilm, which is composed of hyaluronan (HA) and carboxymethyl cellulose hydrogels. In a rat model, prepared membranes of various compositions of gellan gum (GG), xanthan gum (XG) and HA as well as Seprafilm were wrapped around repaired tendons and the adhesion of the tendons was examined grossly and histologically after 3 weeks of healing. Certain formulations of the XG/GG/HA hydrogel membranes reduced tendon adhesion with equal efficacy but without reducing the tendon strength compared to Seprafilm. The designed membranes swelled rapidly and blanketed onto the tendon tissue more readily and closely than Seprafilm. Also they degraded slowly, which allowed the membranes to function as barriers for extended periods.

  9. Insulin-like growth factor-1 suspended in hyaluronan improves cartilage and subchondral cancellous bone repair in osteoarthritis of temporomandibular joint.

    PubMed

    Liu, X-W; Hu, J; Man, C; Zhang, B; Ma, Y-Q; Zhu, S-S

    2011-02-01

    This study sought to evaluate the effects of intra-articular injection of insulin-like growth factor-1 (IGF-1) suspended in hyaluronan (HA) on the cartilage and subchondral cancellous bone repair in osteoarthritis (OA) of the temporomandibular joint (TMJ). Disc perforation was performed bilaterally in rabbit TMJs to induce OA. Four groups of animals (n=12) received OA induction only, and either intra-articular HA injection alone, intra-articular IGF-1 injection alone, or a combination of HA and IGF-1 injection. All therapy was begun 4 weeks after OA induction. The animals were killed 12 or 24 weeks after the first injection, for histology and micro-CT examinations. Two additional animals were used as normal controls. Typical cartilage and subchondral cancellous bone lesions were observed in the OA group. No protective effect on cartilage and subchondral cancellous bone was found in the HA or IGF-1 alone groups. Better histological repair and nearly normal micro-architectural properties of the subchondral cancellous bone were observed in the HA+IGF-1 group compared with the HA or IGF-1 alone groups. HA may be used as an effective carrier for intra-articular injection of IGF-1 and the combination of HA/IGF-1 shows promise as a new rational approach to therapy of TMJ OA. PMID:21055904

  10. Phase I-II clinical trial of hyaluronan-cisplatin nanoconjugate in dogs with naturally occurring malignant tumors.

    PubMed

    Cai, Shuang; Zhang, Ti; Forrest, W C; Yang, Qiuhong; Groer, Chad; Mohr, Eva; Aires, Daniel J; Axiak-Bechtel, Sandra M; Flesner, Brian K; Henry, Carolyn J; Selting, Kimberly A; Tate, Deborah; Swarz, Jeffrey A; Bryan, Jeffrey N; Forrest, M Laird

    2016-09-01

    OBJECTIVE To conduct a phase I-II clinical trial of hyaluronan-cisplatin nanoconjugate (HA-Pt) in dogs with naturally occurring malignant tumors. ANIMALS 18 healthy rats, 9 healthy mice, and 16 dogs with cancer. PROCEDURES HA-Pt was prepared and tested by inductively coupled plasma mass spectrometry; DNA-platinum adduct formation and antiproliferation effects of cisplatin and HA-Pt were compared in vitro. Effects of cisplatin (IV) and HA-Pt (SC) in rodents were tested by clinicopathologic assays. In the clinical trial, dogs with cancer received 1 to 4 injections of HA-Pt (10 to 30 mg/m(2), intratumoral or peritumoral, q 3 wk). Blood samples were collected for pharmacokinetic analysis; CBC, serum BUN and creatinine concentration measurement, and urinalysis were conducted before and 1 week after each treatment. Some dogs underwent hepatic enzyme testing. Tumors were measured before the first treatment and 3 weeks after each treatment to assess response. RESULTS No adverse drug effects were detected in pretrial assessments in rodents. Seven of 16 dogs completed the study; 3 had complete tumor responses, 3 had stable disease, and 1 had progressive disease. Three of 7 dogs with oral and nasal squamous cell carcinoma (SCC) that completed the study had complete responses. Myelosuppression and cardiotoxicosis were identified in 6 and 2 dogs, respectively; none had nephrotoxicosis. Four of 5 dogs with hepatic enzymes assessed had increased ALT activities, attributed to diaquated cisplatin products in the HA-Pt. Pharmacokinetic data fit a 3-compartment model. CONCLUSIONS AND CLINICAL RELEVANCE HA-Pt treatment resulted in positive tumor responses in some dogs, primarily those with SCC. The adverse effect rate was high. IMPACT FOR HUMAN MEDICINE Oral SCC in dogs has characteristics similar to human head and neck SCC; these results could be useful in developing human treatments. PMID:27580113

  11. Development of a complex hydrogel of hyaluronan and PVA embedded with silver nanoparticles and its facile studies on Escherichia coli.

    PubMed

    Zhang, Fei; Wu, Juan; Kang, Ding; Zhang, Hongbin

    2013-01-01

    Novel nanocomposite hydrogels composed of hyaluronan (HA), poly(vinyl alcohol) (PVA) and silver nanoparticles were prepared by several cycles of freezing and thawing. The nanocomposite was then characterised using Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), wide-angle X-ray diffraction (XRD) and scanning electron microscopy (SEM). The complex hydrogels consisted of semi-interpenetrating network structures, with PVA microcrystallines as junction zones. By increasing the HA content, the crystallinity and melting temperature of the complex hydrogels decreased, whereas the glass transition temperatures of these materials increased because of the steric hindrance of HA and the occurrence of intermolecular interactions through hydrogen bonding between HA and PVA in the complex hydrogels. Swelling studies showed that in comparison with the swelling properties of the cryogels from PVA alone, those of the complex hydrogels can be significantly improved and presented in a pH-sensitive manner. In addition, silver nanoparticles were synthesised through UV-initiated photoreduction with HA functioning as a reducing agent and stabiliser. The silver nanoparticles were then incorporated in situ into the HA/PVA complex hydrogel matrix. The size and morphology of the as-prepared Ag nanoparticles were investigated through ultraviolet-visible light spectroscopy, transmission electron microscopy, XRD and thermogravimetric analysis. The experimental results indicated that silver nanoparticles 20-50 nm in size were uniformly dispersed in the hydrogel matrix. The antibacterial effects of the HA/PVA/Ag nanocomposite hydrogel against Escherichia coli were evaluated. The results show that this nanocomposite hydrogel possesses high antibacterial property and has a potential application as a wound dressing material. PMID:23829455

  12. The pharmacokinetics and dosing of oral 4-methylumbelliferone for inhibition of hyaluronan synthesis in mice.

    PubMed

    Kuipers, H F; Nagy, N; Ruppert, S M; Sunkari, V G; Marshall, P L; Gebe, J A; Ishak, H D; Keswani, S G; Bollyky, J; Frymoyer, A R; Wight, T N; Steinman, L; Bollyky, P L

    2016-09-01

    Recently, there has been considerable interest in using 4-methylumbelliferone (4-MU) to inhibit hyaluronan (HA) synthesis in mouse models of cancer, autoimmunity and a variety of other inflammatory disorders where HA has been implicated in disease pathogenesis. In order to facilitate future studies in this area, we have examined the dosing, treatment route, treatment duration and metabolism of 4-MU in both C57BL/6 and BALB/c mice. Mice fed chow containing 5% 4-MU, a dose calculated to deliver 250 mg/mouse/day, initially lose substantial weight but typically resume normal weight gain after 1 week. It also takes up to a week to see a reduction in serum HA in these animals, indicating that at least a 1-week loading period on the drug is required for most protocols. At steady state, more than 90% of the drug is present in plasma as the glucuronidated metabolite 4-methylumbelliferyl glucuronide (4-MUG), with the sulphated metabolite, 4-methylumbelliferyl sulphate (4-MUS) comprising most of the remainder. Chow containing 5% but not 0·65% 4-MU was effective at preventing disease in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, as well as in the DORmO mouse model of autoimmune diabetes. While oral 4-MU was effective at preventing EAE, daily intraperitoneal injections of 4-MU were not. Factors potentially affecting 4-MU uptake and plasma concentrations in mice include its taste, short half-life and low bioavailability. These studies provide a practical resource for implementing oral 4-MU treatment protocols in mice. PMID:27218304

  13. Systemic delivery of siRNA by hyaluronan-functionalized calcium phosphate nanoparticles for tumor-targeted therapy

    NASA Astrophysics Data System (ADS)

    Qiu, Chong; Wei, Wei; Sun, Jing; Zhang, Hai-Tao; Ding, Jing-Song; Wang, Jian-Cheng; Zhang, Qiang

    2016-06-01

    In this study, hyaluronan (HA)-functionalized calcium phosphate nanoparticles (CaP-AHA/siRNA NPs) were developed for an injectable and targetable delivery of siRNA, which were prepared by coating the alendronate-hyaluronan graft polymer (AHA) around the surface of calcium phosphate-siRNA co-precipitates. The prepared CaP-AHA/siRNA NPs had a uniform spherical core-shell morphology with an approximate size of 170 nm and zeta potential of -12 mV. The coating of hydrophilic HA improved the physical stability of nanoparticles over one month due to the strong interactions between phosphonate and calcium. In vitro experiments demonstrated that the negatively charged CaP-AHA/siRNA NPs could effectively deliver EGFR-targeted siRNA into A549 cells through CD44-mediated endocytosis and significantly down-regulate the level of EGFR expression. Also, the internalized CaP-AHA/siRNA NPs exhibited a pH-responsive release of siRNA, indicating that the acidification of lysosomes probably facilitated the disassembling of nanoparticles and the resultant ions sharply increased the inner osmotic pressure and thus expedited the release of siRNA from late lysosomes into the cytoplasm. Furthermore, in vivo tumor therapy demonstrated that high accumulation of CaP-AHA/siEGFR NPs in tumor led to a significant tumor growth inhibition with a specific EGFR gene silencing effect after intravenous administration in nude mice xenografted with A549 tumor, along with a negligible body weight loss. These results suggested that the CaP-AHA/siRNA NPs could be an effective and safe systemic siRNA delivery system for a RNAi-based tumor targeted therapy strategy.In this study, hyaluronan (HA)-functionalized calcium phosphate nanoparticles (CaP-AHA/siRNA NPs) were developed for an injectable and targetable delivery of siRNA, which were prepared by coating the alendronate-hyaluronan graft polymer (AHA) around the surface of calcium phosphate-siRNA co-precipitates. The prepared CaP-AHA/siRNA NPs had a uniform

  14. Hyaluronan-decorated polymer nanoparticles targeting the CD44 receptor for the combined photo/chemo-therapy of cancer

    NASA Astrophysics Data System (ADS)

    Maiolino, Sara; Moret, Francesca; Conte, Claudia; Fraix, Aurore; Tirino, Pasquale; Ungaro, Francesca; Sortino, Salvatore; Reddi, Elena; Quaglia, Fabiana

    2015-03-01

    In the attempt to develop novel concepts in designing targeted nanoparticles for combination therapy of cancer, we propose here CD44-targeted hyaluronan-decorated double-coated nanoparticles (dcNPs) delivering the lipophilic chemotherapeutic docetaxel (DTX) and an anionic porphyrin (TPPS4). dcNPs are based on electrostatic interactions between a negative DTX-loaded nanoscaffold of poly(lactide-co-glycolide), a polycationic shell of polyethyleneimine entangling negatively-charged TPPS4 and finally decorated with hyaluronan (HA) to promote internalization through CD44 receptor-mediated endocytosis. DTX/TPPS4-dcNPs, prepared through layer-by-layer deposition, showed a hydrodynamic diameter of around 180 nm, negative zeta potential and efficient loading of both DTX and TPPS4. DTX/TPPS4-dcNPs were freeze-dried with trehalose giving a powder that could be easily dispersed in different media. Excellent stability of dcNPs in specific salt- and protein-containing media was found. Spectroscopic behavior of DTX/TPPS4-dcNPs demonstrated a face-to-face arrangement of the TPPS4 units in non-photoresponsive H-type aggregates accounting for an extensive aggregation of the porphyrin embedded in the shell. Experiments in MDA-MB-231 cells overexpressing the CD44 receptor demonstrated a 9.4-fold increase in the intracellular level of TPPS4 delivered from dcNPs as compared to free TPPS4. Light-induced death increased tremendously in cells that had been treated with a combination of TPPS4 and DTX delivered through dcNPs as compared with free drugs, presumably due to efficient uptake and co-localization inside the cells. In perspective, the strategy proposed here to target synergistic drug combinations through HA-decorated nanoparticles seems very attractive to improve the specificity and efficacy of cancer treatment.In the attempt to develop novel concepts in designing targeted nanoparticles for combination therapy of cancer, we propose here CD44-targeted hyaluronan-decorated double

  15. A Hyaluronan-Based Injectable Hydrogel Improves the Survival and Integration of Stem Cell Progeny following Transplantation

    PubMed Central

    Ballios, Brian G.; Cooke, Michael J.; Donaldson, Laura; Coles, Brenda L.K.; Morshead, Cindi M.; van der Kooy, Derek; Shoichet, Molly S.

    2015-01-01

    Summary The utility of stem cells and their progeny in adult transplantation models has been limited by poor survival and integration. We designed an injectable and bioresorbable hydrogel blend of hyaluronan and methylcellulose (HAMC) and tested it with two cell types in two animal models, thereby gaining an understanding of its general applicability for enhanced cell distribution, survival, integration, and functional repair relative to conventional cell delivery in saline. HAMC improves cell survival and integration of retinal stem cell (RSC)-derived rods in the retina. The pro-survival mechanism of HAMC is ascribed to the interaction of the CD44 receptor with HA. Transient disruption of the retinal outer limiting membrane, combined with HAMC delivery, results in significantly improved rod survival and visual function. HAMC also improves the distribution, viability, and functional repair of neural stem and progenitor cells (NSCs). The HAMC delivery system improves cell transplantation efficacy in two CNS models, suggesting broad applicability. PMID:25981414

  16. Hyaluronan microspheres for sustained gene delivery and site-specific targeting.

    PubMed

    Yun, Yang H; Goetz, Douglas J; Yellen, Paige; Chen, Weiliam

    2004-01-01

    Hyaluronan is a naturally occurring polymer that has enjoyed wide successes in biomedical and cosmetic applications as coatings, matrices, and hydrogels. For controlled delivery applications, formulating native hyaluronan into microspheres could be advantageous but has been difficult to process unless organic solvents are used or hyaluronan has been modified by etherification. Therefore, we present a novel method of preparing hyaluronan microspheres using adipic dihydrazide mediated crosslinking chemistry. To evaluate their potential for medical applications, hyaluronan microspheres are incorporated with DNA for gene delivery or conjugated with an antigen for cell-specific targeting. The results show that our method, originally developed for preparing hyaluronan hydrogels, generates robust microspheres with a size distribution of 5-20mum. The release of the encapsulated plasmid DNA can be sustained for months and is capable of transfection in vitro and in vivo. Hyaluronan microspheres, conjugated with monoclonal antibodies to E- and P-selectin, demonstrate selective binding to cells expressing these receptors. In conclusion, we have developed a novel microsphere preparation using native hyaluronan that delivers DNA at a controlled rate and adaptable for site-specific targeting. PMID:14580918

  17. Hyaluronan Is Not a Ligand but a Regulator of Toll-Like Receptor Signaling in Mesangial Cells: Role of Extracellular Matrix in Innate Immunity

    PubMed Central

    Ebid, Rainer; Anders, Hans-Joachim

    2014-01-01

    Glomerular mesangial cells (MC), like most cell types secrete hyaluronan (HA), which attached to the cell surface via CD44, is the backbone of a hydrophilic gel matrix around these cells. Reduced extracellular matrix thickness and viscosity result from HA cleavage during inflammation. HA fragments were reported to trigger innate immunity via Toll-like receptor-(TLR-) 2 and/or TLR4 in immune cells. We questioned whether HA fragments also regulate the immunostimulatory capacity of smooth muscle cell-like MC. LPS (TLR4-ligand) and PAM3CysSK4 (TLR2-ligand) induced IL-6 secretion in MC; highly purified endotoxin-free HA < 3000 Da up to 50 μg/mL did not. Bovine-testis-hyaluronidase from was used to digest MC-HA into HA fragments of different size directly in the cell culture. Resultant HA fragments did not activate TLR4-deficient MC, while TLR2-deficient MC responded to LPS-contamination of hyaluronidase, not to produced HA fragments. Hyaluronidase increased the stimulatory effect of TLR2-/-3/-5 ligands on their TLR-receptors in TLR4-deficient MC, excluding any effect by LPS-contamination. Supplemented heparin suppressed every stimulatory effect in a dose-dependent manner. We conclude that the glycosaminoglycan HA creates a pericellular jelly barrier, which covers surface receptors like the TLRs. Barrier-thickness and viscosity balanced by HA-synthesis and degradation and the amount of HA-receptors on the cell surface regulate innate immunity via the accessibility of the receptors. PMID:24967246

  18. Clustered Conserved Cysteines in Hyaluronan Synthase Mediate Cooperative Activation by Mg(2+) Ions and Severe Inhibitory Effects of Divalent Cations.

    PubMed

    Tlapak-Simmons, Valarie L; Medina, Andria P; Baggenstoss, Bruce A; Nguyen, Long; Baron, Christina A; Weigel, Paul H

    2011-11-15

    Hyaluronan synthase (HAS) uses UDP-GlcUA and UDP-GlcNAc to make hyaluronan (HA). Streptococcus equisimilis HAS (SeHAS) contains four conserved cysteines clustered near the membrane, and requires phospholipids and Mg(2+) for activity. Activity of membrane-bound or purified enzyme displayed a sigmoidal saturation profile for Mg(2+) with a Hill coefficient of 2. To assess if Cys residues are important for cooperativity we examined the Mg(2+) dependence of mutants with various combinations of Cys-to-Ala mutations. All Cys-mutants lost the cooperative response to Mg(2+). In the presence of Mg(2+), other divalent cations inhibited SeHAS with different potencies (Cu(2+)~Zn(2+) >Co(2+) >Ni(2+) >Mn(2+) >Ba(2+) Sr(2+) Ca(2+)). Some divalent metal ions likely inhibit by displacement of Mg(2+)-UDP-Sugar complexes (e.g. Ca(2+), Sr(2+) and Ba(2+) had apparent Ki values of 2-5 mM). In contrast, Zn(2+) and Cu(2+) inhibited more potently (apparent Ki ≤ 0.2 mM). Inhibition of Cys-null SeHAS by Cu(2+), but not Zn(2+), was greatly attenuated compared to wildtype. Double and triple Cys-mutants showed differing sensitivities to Zn(2+) or Cu(2+). Wildtype SeHAS allowed to make HA prior to exposure to Zn(2+) or Cu(2+) was protected from inhibition, indicating that access of metal ions to sensitive functional groups was hindered in processively acting HA•HAS complexes. We conclude that clustered Cys residues mediate cooperative interactions with Mg(2+) and that transition metal ions inhibit SeHAS very potently by interacting with one or more of these -SH groups.

  19. Systemic delivery of siRNA by hyaluronan-functionalized calcium phosphate nanoparticles for tumor-targeted therapy.

    PubMed

    Qiu, Chong; Wei, Wei; Sun, Jing; Zhang, Hai-Tao; Ding, Jing-Song; Wang, Jian-Cheng; Zhang, Qiang

    2016-07-14

    In this study, hyaluronan (HA)-functionalized calcium phosphate nanoparticles (CaP-AHA/siRNA NPs) were developed for an injectable and targetable delivery of siRNA, which were prepared by coating the alendronate-hyaluronan graft polymer (AHA) around the surface of calcium phosphate-siRNA co-precipitates. The prepared CaP-AHA/siRNA NPs had a uniform spherical core-shell morphology with an approximate size of 170 nm and zeta potential of -12 mV. The coating of hydrophilic HA improved the physical stability of nanoparticles over one month due to the strong interactions between phosphonate and calcium. In vitro experiments demonstrated that the negatively charged CaP-AHA/siRNA NPs could effectively deliver EGFR-targeted siRNA into A549 cells through CD44-mediated endocytosis and significantly down-regulate the level of EGFR expression. Also, the internalized CaP-AHA/siRNA NPs exhibited a pH-responsive release of siRNA, indicating that the acidification of lysosomes probably facilitated the disassembling of nanoparticles and the resultant ions sharply increased the inner osmotic pressure and thus expedited the release of siRNA from late lysosomes into the cytoplasm. Furthermore, in vivo tumor therapy demonstrated that high accumulation of CaP-AHA/siEGFR NPs in tumor led to a significant tumor growth inhibition with a specific EGFR gene silencing effect after intravenous administration in nude mice xenografted with A549 tumor, along with a negligible body weight loss. These results suggested that the CaP-AHA/siRNA NPs could be an effective and safe systemic siRNA delivery system for a RNAi-based tumor targeted therapy strategy. PMID:27314204

  20. High-molar-mass hyaluronan behavior during testing its radical scavenging capacity in organic and aqueous media: effects of the presence of manganese(II) ions.

    PubMed

    Rapta, Peter; Valachová, Katarína; Gemeiner, Peter; Soltés, Ladislav

    2009-02-01

    This study compares the radical scavenging capacity of high-molar-mass hyaluronan (HA) using standardized methods applying 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and 2,2'-azinobis[3-ethylbenzthiazoline sulfonate] (ABTS) radical cations as oxidants. Additionally, spin-trapping technique combined with electron paramagnetic resonance (EPR) was used to evaluate the ability of HA to scavenge reactive radicals. The thermal decomposition of K2S2O8 in pure H2O or in a H2O/dimethyl sulfoxide (DMSO) mixture at 333 K was used as a source of reactive paramagnetic species. We found that HA does not exhibit radical-scavenging activity when DPPH radicals or ABTS(.+) radical cations are used as oxidant, but that HA is an effective radical scavenger at low concentrations, if the oxidation reactions are initiated by the decomposition of K2S2O8. At higher HA concentrations, a more complex behavior and prooxidant HA action was observed. The influence of Mn(II) ions on the reaction mechanisms of radical generation and termination in the K2S2O8/H2O/DMSO system in the presence of HA was studied in detail. PMID:19235158

  1. Modeling of hyaluronic acid containing anti-cancer drugs-loaded polylactic-co-glycolic acid bioconjugates for targeted delivery to cancer cells

    NASA Astrophysics Data System (ADS)

    Gul-e-Saba, Adulphakdee, A.; Madthing, A.; Zafar, M. N.; Abdullah, M. A.

    2012-09-01

    Molecular modeling of hyaluronan (HA), polylactic-co-glycolic acid (PLGA), polyethylene glycol-bis-amine (PEG-bis-amine), Curcumin, Cisplatin and the conjugate HA-PEG-PLGA containing Curcumin/Cisplatin were performed using Discovery Studio 2.5 to better understand issues and constraints related to targeted delivery of potent anticancer drugs to cancer cells. HA, a versatile biopolymer is a ligand of cancer cell receptor, CD44 that can be particularly useful in a receptor-mediated cellular uptake of drug-incorporated nanoparticles. Biocompatible and biodegradable polymers, PLGA and PEG, serve as polymeric micelles for controlled-release of drug. Curcumin as a natural anticancer agent has poor solubility that limits its use in drug therapeutics, while platinum-based Cisplatin exhibits systemic cytotoxicity. These can be overcome via drug delivery in polymeric biocompatible vehicles. The PLGA-PEG-HA conjugate shows the total measurement of 105 bond length with average bond length of 1.274163 Å. The conjugation between PEG and HA occurs at C8-O1 atoms and can be manipulated to improve properties.

  2. Sevoflurane mitigates shedding of hyaluronan from the coronary endothelium, also during ischemia/reperfusion: an ex vivo animal study

    PubMed Central

    Chen, Congcong; Chappell, Daniel; Annecke, Thorsten; Conzen, Peter; Jacob, Matthias; Welsch, Ulrich; Zwissler, Bernhard; Becker, Bernhard F

    2016-01-01

    Glycosaminoglycan hyaluronan (HA), a major constituent of the endothelial glycocalyx, helps to maintain vascular integrity. Preconditioning the heart with volatile anesthetic agents protects against ischemia/reperfusion injury. We investigated a possible protective effect of sevoflurane on the glycocalyx, especially on HA. The effect of pre-ischemic treatment with sevoflurane (15 minutes at 2% vol/vol gas) on shedding of HA was evaluated in 28 isolated, beating guinea pig hearts, subjected to warm ischemia (20 minutes at 37°C) followed by reperfusion (40 minutes), half with and half without preconditioning by sevoflurane. HA concentration was measured in the coronary effluent. Over the last 20 minutes of reperfusion hydroxyethyl starch (1 g%) was continuously infused and the epicardial transudate collected over the last 5 minutes for measuring the colloid extravasation. Additional hearts were fixed by perfusion after the end of reperfusion for immunohistology and electron microscopy. Sevoflurane did not significantly affect post-ischemic oxidative stress, but strongly inhibited shedding of HA during the whole period, surprisingly even prior to ischemia. Immunohistology demonstrated that heparan sulfates and SDC1 of the glycocalyx were also preserved by sevoflurane. Electron microscopy revealed shedding of glycocalyx caused by ischemia and a mostly intact glycocalyx in hearts exposed to sevoflurane. Coronary vascular permeability of the colloid hydroxyethyl starch was significantly decreased by sevoflurane vs the control. We conclude that application of sevoflurane preserves the coronary endothelial glycocalyx, especially HA, sustaining the vascular barrier against ischemic damage. This may explain beneficial effects associated with clinical use of volatile anesthetics against ischemia/reperfusion injury. PMID:27800510

  3. TGF{beta}2-mediated production of hyaluronan is important for the induction of epicardial cell differentiation and invasion

    SciTech Connect

    Craig, Evisabel A.; Austin, Anita F.; Vaillancourt, Richard R.; Barnett, Joey V.; Camenisch, Todd D.

    2010-12-10

    In the developing heart, the epicardium is a major source of progenitor cells that contribute to the formation of the coronary vessel system. These epicardial progenitors give rise to the different cellular components of the coronary vasculature by undergoing a number of morphological and physiological changes collectively known as epithelial to mesenchymal transformation (EMT). However, the specific signaling mechanisms that regulate epicardial EMT are yet to be delineated. In this study we investigated the role of TGF{beta}2 and hyaluronan (HA) during epicardial EMT and how signals from these two molecules are integrated during this important process. Here we show that TGF{beta}2 induces MEKK3 activation, which in turn promotes ERK1/2 and ERK5 phosphorylation. TGF{beta}2 also increases Has2 expression and subsequent HA production. Nevertheless, inhibition of MEKK3 kinase activity, silencing of ERK5 or pharmacological disruption of ERK1/2 activation significantly abrogates this response. Thus, TGF{beta}2 promotes Has2 expression and HA production through a MEKK3/ERK1/2/5-dependent cascade. Furthermore, TGF{beta}2 is able to induce epicardial cell invasion and differentiation but not proliferation. However, inhibition of MEKK3-dependent pathways, degradation of HA by hyaluronidases or blockade of CD44, significantly impairs the biological response to TGF{beta}2. Taken together, these findings demonstrate that TGF{beta}2 activation of MEKK3/ERK1/2/5 signaling modulates Has2 expression and HA production leading to the induction of EMT events. This is an important and novel mechanism showing how TGF{beta}2 and HA signals are integrated to regulate changes in epicardial cell behavior.

  4. miR-23a-3p causes cellular senescence by targeting hyaluronan synthase 2: possible implication for skin aging.

    PubMed

    Röck, Katharina; Tigges, Julia; Sass, Steffen; Schütze, Alexandra; Florea, Ana-Maria; Fender, Anke C; Theis, Florian J; Krutmann, Jean; Boege, Fritz; Fritsche, Ellen; Reifenberger, Guido; Fischer, Jens W

    2015-02-01

    Even though aging and cellular senescence appear to be linked, the biological mechanisms interconnecting these two processes remain to be unravelled. Therefore, microRNA (miRNA/miR) profiles were analyzed ex vivo by means of gene array in fibroblasts isolated from young and old human donors. Expression of several miRNAs was positively correlated with donor age. Among them, miR-23a-3p was shown to target hyaluronan synthase 2 (HAS2). HA is a polysaccharide of the extracellular matrix that critically regulates the phenotype of fibroblasts. Indeed, both aged and senescent fibroblasts showed increased miR-23a-3p expression and secreted significantly lower amounts of HA compared with young and non-senescent fibroblasts. Ectopic overexpression of miR-23a-3p in non-senescent fibroblasts led to decreased HAS2-mediated HA synthesis, upregulation of senescence-associated markers, and decreased proliferation. In addition, siRNA-mediated downregulation of HAS2 and pharmacological inhibition of HA synthesis by 4-methylumbelliferone mimicked the effects of miR-23a-3p. In vivo, miR-23a-3p was upregulated and HAS2 was downregulated in the skin of old mice compared with young mice. Inhibition of HA synthesis by 4-methylumbelliferone in mice reduced dermal hydration and viscoelasticity, thereby mimicking an aged skin phenotype. Taken together, these findings appear to link miR-23a-3p and the HA microenvironment as effector mechanisms in both dermal aging and senescence.

  5. Fabrication of tubular tissue constructs by centrifugal casting of cells suspended in an in situ crosslinkable hyaluronan-gelatin hydrogel.

    PubMed

    Mironov, Vladimir; Kasyanov, Vladimir; Zheng Shu, Xiao; Eisenberg, Carol; Eisenberg, Leonard; Gonda, Steve; Trusk, Thomas; Markwald, Roger R; Prestwich, Glenn D

    2005-12-01

    Achieving the optimal cell density and desired cell distribution in scaffolds is a major goal of cell seeding technologies in tissue engineering. In order to reach this goal, a novel centrifugal casting technology was developed using in situ crosslinkable hyaluronan-based (HA) synthetic extracellular matrix (sECM). Living cells were suspended in a viscous solution of thiol-modified HA and thiol-modified gelatin, a polyethyleneglycol diacrylate crosslinker was added, and a hydrogel was formed during rotation. The tubular tissue constructs consisting of a densely packed cell layer were fabricated with the rotation device operating at 2000 rpm for 10 min. The majority of cells suspended in the HA mixture before rotation were located inside the layer after centrifugal casting. Cells survived the effect of the centrifugal forces experienced under the rotational regime employed. The volume cell density (65.6%) approached the maximal possible volume density based on theoretical sphere packing models. Thus, centrifugal casting allows the fabrication of tubular constructs with the desired redistribution, composition and thickness of cell layers that makes the maximum efficient use of available cells. Centrifugal casting in this sECM would enable rapid fabrication of tissue-engineered vascular grafts, as well as other tubular and planar tissue-engineered constructs.

  6. Hyaluronan preserves the proliferation and differentiation potentials of long-term cultured murine adipose-derived stromal cells

    SciTech Connect

    Chen, P.-Y.; Huang, Lynn L.H. . E-mail: lynn@mail.ncku.edu.tw; Hsieh, H.-J. . E-mail: hjhsieh@ntu.edu.tw

    2007-08-17

    For long-term culture, murine adipose-derived stromal cells (mADSCs) at latter passages demonstrated a marked decline in proliferative activity, exhibited senescent morphology and reduced differentiation potentials, particularly osteogenesis. To extend the lifespan of mADSCs, two culture conditions containing hyaluronan (HA) was compared in our study, one as a culture medium supplement (SHA), and the other where HA was pre-coated on culture surface (CHA). mADSCs cultivated with SHA exhibited a prolonged lifespan, reduced cellular senescence, and enhanced osteogenic potential compared to regular culture condition (control). Upon CHA treatment, mADSCs tended to form cell aggregates with gradual growth profiles, while their differentiation activities remained similar to SHA groups. After transferring mADSCs from CHA to control surface, they were shown to have an extended lifespan and an increase of osteogenic potential. Our results suggested that HA can be useful for preserving the proliferation and differentiation potentials of long-term cultured mADSCs.

  7. Synthesis of photo-crosslinkable hyaluronan with tailored degree of substitution suitable for production of water resistant nanofibers.

    PubMed

    Huerta-Angeles, Gloria; Brandejsová, Martina; Knotková, Kateřina; Hermannová, Martina; Moravcová, Martina; Šmejkalová, Daniela; Velebný, Vladimír

    2016-02-10

    In this work, hyaluronan (HA) was grafted by a novel and an efficient mixed anhydrides methodology with (hetero)-aryl and aliphatic acrylic moieties suitable for cross-linking. A precise control of stoichiometry was achieved. Derivatives with degree of substitution (DS) below 20% did not show self-crosslinking. Due to mild reaction conditions, a negligible degradation of the polysaccharide was obtained. The influence of the feed components on the reaction efficiency and DS were studied up to 200 g/batch. The structure of the modified HA was characterized by Infrared Spectroscopy, Nuclear Magnetic Resonance, SEC-MALS and chromatographic analyses. Enzymatic degradation of derivatives was performed and isolated dimers demonstrated to be non-cytotoxic. The feasibility of the grafted HA for electrospinning with subsequent photo-crosslinking to avoid nanofibers water dissolution was demonstrated. The biocompatibility of the material, its degradation products, and the formation of honeycomb porous structures also proved the potential of the material for future in vivo applications. PMID:26686128

  8. Self-assembled nanoparticles based on amphiphilic chitosan derivative and hyaluronic acid for gene delivery.

    PubMed

    Liu, Ya; Kong, Ming; Cheng, Xiao Jie; Wang, Qian Qian; Jiang, Li Ming; Chen, Xi Guang

    2013-04-15

    The present work described nanoparticles (NPs) made of oleoyl-carboxymethy-chitosan (OCMCS)/hyaluronic acid (HA) using coacervation process as novel potential carriers for gene delivery. An N/P ratio of 5 and OCMCS/HA weight ratio of 4 were the optimal conditions leading to the smallest (164.94 nm), positive charged (+14.2 mV) and monodispersed NPs. OCMCS-HA/DNA (OHD) NPs showed higher in vitro DNA release rates and increased cellular uptake by Caco-2 cells due to the HA involved in NPs. The MTT survival assay indicated no significant cytotoxicity. The transfection efficiency of OHD NPs was 5-fold higher than OCMCS/DNA (OD) NPs; however, it decreased significantly in the presence of excess free HA. The results indicated that OHD NPs internalized in Caco-2 cells were mediated by the hyaluronan receptor CD44. The data obtained in the present research gave evidence of the potential of OHD NPs for the targeting and further transfer of genes to the epithelial cells. PMID:23544543

  9. α,β-Unsaturated aldehyde of hyaluronan--Synthesis, analysis and applications.

    PubMed

    Buffa, Radovan; Šedová, Petra; Basarabová, Ivana; Moravcová, Martina; Wolfová, Lucie; Bobula, Tomáš; Velebný, Vladimír

    2015-12-10

    Hyaluronic acid (HA) modified with an aldehyde group (HA-CHO or HA-aldehyde) has been extensively used for various biomedical applications. The main advantage of the aldehyde moieties is the ability to react with a wide range of amino compounds under physiological conditions. Reactions of aldehydes with primary amines in water are reversible and equilibrium is thoroughly shifted towards starting aldehyde and amine. This work presents an unique modification of HA: α,β-unsaturated aldehyde of HA (4,5-anhydro-6(GlcNAc)-oxo HA or ΔHA-CHO), which allows the primary amines to be attached to HA more effectively in comparison to the saturated HA-CHO. Higher hydrolytic stability is caused by the conjugation of imine with an adjacent --C=C-- double bond. Two strategies for the preparation of unsaturated HA-aldehyde were developed and chemical structures were studied in details. Cross-linked materials prepared from this precursor are biocompatible and suitable for applications in drug delivery and regenerative medicine. PMID:26428127

  10. Homeostasis of Hyaluronic Acid in Normal and Scarred Vocal Folds

    PubMed Central

    Tateya, Ichiro; Tateya, Tomoko; Watanuki, Makoto; Bless, Diane M.

    2015-01-01

    Summary Objectives/Hypothesis Vocal fold scarring is one of the most challenging laryngeal disorders to treat. Hyaluronic acid (HA) is the main component of lamina propria, and it plays an important role in proper vocal fold vibration and is also thought to be important in fetal wound healing without scarring. Although several animal models of vocal fold scarring have been reported, little is known about the way in which HA is maintained in vocal folds. The purpose of this study was to clarify the homeostasis of HA by examining the expression of hyaluronan synthase (Has) and hyaluronidase (Hyal), which produce and digest HA, respectively. Study Design Experimental prospective animal study. Methods Vocal fold stripping was performed on 38 Sprague-Dawley rats. Vocal fold tissue was collected at five time points (3 days–2 months). Expression of HA was examined by immunohistochemistry, and messenger RNA (mRNA) expression of Has and Hyal was examined by real-time polymerase chain reaction and in-situ hybridization. Results In scarred vocal folds, expression of Has1 and Has2 increased at day 3 together with expression of HA and returned to normal at 2 weeks. At 2 months, Has3 and Hyal3 mRNA showed higher expressions than normal. Conclusions Expression patterns of Has and Hyal genes differed between normal, acute-scarred, and chronic-scarred vocal folds, indicating the distinct roles of each enzyme in maintaining HA. Continuous upregulation of Has genes in the acute phase may be necessary to achieve scarless healing of vocal folds. PMID:25499520

  11. Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion

    SciTech Connect

    Takabe, Piia; Bart, Geneviève; Ropponen, Antti; Rilla, Kirsi; Tammi, Markku; Tammi, Raija; Pasonen-Seppänen, Sanna

    2015-09-10

    Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells. - Highlights: • Inducible HAS3-MV3 melanoma cell line was generated using Lentiviral transduction. • HAS3 overexpression inhibits MV3 cell migration via hyaluronan–receptor interaction. • HAS3 overexpression decreases MV3 melanoma cell proliferation and adhesion. • ERK1/2 phosphorylation is downregulated by 50% in HAS3 overexpressing cells. • The results suggest that hyaluronan has anti-cancer like effects in melanoma.

  12. The Content and Size of Hyaluronan in Biological Fluids and Tissues

    PubMed Central

    Cowman, Mary K.; Lee, Hong-Gee; Schwertfeger, Kathryn L.; McCarthy, James B.; Turley, Eva A.

    2015-01-01

    Hyaluronan is a simple repeating disaccharide polymer, synthesized at the cell surface by integral membrane synthases. The repeating sequence is perfectly homogeneous, and is the same in all vertebrate tissues and fluids. The polymer molecular mass is more variable. Most commonly, hyaluronan is synthesized as a high-molecular mass polymer, with an average molecular mass of approximately 1000–8000 kDa. There are a number of studies showing increased hyaluronan content, but reduced average molecular mass with a broader range of sizes present, in tissues or fluids when inflammatory or tissue-remodeling processes occur. In parallel studies, exogenous hyaluronan fragments of low-molecular mass (generally, <200 kDa) have been shown to affect cell behavior through binding to receptor proteins such as CD44 and RHAMM (gene name HMMR), and to signal either directly or indirectly through toll-like receptors. These data suggest that receptor sensitivity to hyaluronan size provides a biosensor of the state of the microenvironment surrounding the cell. Sensitive methods for isolation and characterization of hyaluronan and its fragments have been developed and continue to improve. This review provides an overview of the methods and our current state of knowledge of hyaluronan content and size distribution in biological fluids and tissues. PMID:26082778

  13. Growth as a solid tumor or reduced glucose concentrations in culture reversibly induce CD44-mediated hyaluronan recognition by Chinese hamster ovary cells.

    PubMed Central

    Zheng, Z; Cummings, R D; Pummill, P E; Kincade, P W

    1997-01-01

    The density, molecular isoform, and posttranslational modifications of CD44 can markedly influence growth and metastatic behavior of tumors. Many CD44 functions, including some involving tumors, have been attributed to its ability to recognize hyaluronan (HA). However, only certain CD44-bearing cells bind soluble or immobilized HA. We now show that CD44 made by wild-type Chinese hamster ovary (CHO-K1) cells and a ligand-binding subclone differ with respect to N-linked glycosylation. While both bear CD44 with highly branched, complex-type glycoforms, CD44 expressed by the wild type was more extensively sialylated. CHO-K1 cells which failed to recognize HA when grown in culture gained this ability when grown as a solid tumor and reverted to a non-HA-binding state when returned to culture. The ability of CHO-K1 cells to recognize HA was also reversibly induced when glucose concentrations in the medium were reduced. Glucose restriction influenced CD44-mediated HA binding by many but not all, of a series of murine tumors. Glucose concentrations and glycosylation inhibitors only partially influenced CD44 receptor function on resting murine B lymphocytes. These observations suggest that glucose levels or other local environmental conditions may markedly influence glycosylation pathways used by some tumor cells, resulting in dramatic alteration of CD44-mediated functions. PMID:9276740

  14. Enhancing hyaluronan pseudoplasticity via 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride-mediated conjugation with short alkyl moieties.

    PubMed

    Petta, Dalila; Eglin, David; Grijpma, Dirk W; D'Este, Matteo

    2016-10-20

    Hyaluronan (HA) is widely used in the clinical practice and in biomedical research. Through chemical modification, HA shear-thinning properties, essential for injectability and additive manufacturing, can be optimized. In this study, we employed 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) for grafting propylamine and butylamine to HA. A parametric study was performed to identify the optimal reaction conditions. Results showed that DMTMM amidation gives reproducible and accurate control over a range of degrees of substitution (DS) from 1% to 50% and proved reliable to tune viscoelasticity. At DS=3.0% for HA-propylamine and 3.7% for HA-butylamine a maximum for storage modulus and pseudoplasticity was found, whereas above or below this DS, rheological features go back to baseline values of pristine HA. Due to their singular rheological profiles, these derivatives are valuable biomaterials candidates for preparing bioinks and hydrogels for drug delivery and regenerative medicine. PMID:27474602

  15. A case of cervical cancer expressed three mRNA variant of Hyaluronan-mediated motility receptor

    PubMed Central

    Villegas-Ruíz, Vanessa; Salcedo, Mauricio; Zentella-Dehesa, Alejandro; de Oca, Edén V Montes; Román-Basaure, Edgar; Mantilla-Morales, Alejandra; Dávila-Borja, Víctor M; Juárez-Méndez, Sergio

    2014-01-01

    Cervical cancer is the second malignancy in Mexico, little is known about the prognostic factors associated with this disease. Several cellular components are important in their transformation and progression. Alternative mRNA splice is an important mechanism for generating protein diversity, nevertheless, in cancer unknown mRNA diversity is expressed. Hyaluronan-mediated motility receptor (HMMR, RHAMM, CD168) is a family member of proteins, hyaluronan acid dependent, and has been associated with different malignant processes such as: angiogenesis, cell invasiveness, proliferation, metastasis and poor outcome in some tumors. In the present study we identified expression of HMMR in cervical cancer by means of RT-PCR and sequencing. Our results indicate co-expression of two HMMR variants in all samples, and one case expressed three alternative HMMR splice transcripts. These results showed the heterogeneity of mRNA transcripts of HMMR that could express in cancer and the expression of HMMR could be marker of malignancy in CC. PMID:24966934

  16. Use of novel silver nanoparticles with Hyaluronan as potential biological labels for determining the quality of embryos development

    NASA Astrophysics Data System (ADS)

    Syrvatka, Vasyl J.; Slyvchuk, Yurij I.; Rozgoni, Ivan I.; Hevkan, Ivan I.; Bilyi, Oleksandr I.; Osypchuk, Oleksandr S.; Zyuzyun, Aza B.

    2013-09-01

    In reproductive medicine it is important to determine the quality of embryo development without interference in their function and viability. The surface plasmon resonance of silver nanoparticles makes them promising candidates for optical sensing, molecular labeling and imaging applications. Furthermore unique optical properties of silver nanoparticles provide an opportunity to use them as real time analytic tools in living state especially for observation of dynamic processes in gametes and embryos. The main aim of the study was to investigate the physicochemical properties and biological activities of novel silver nanoparticles with prospect of their use for the determining the quality of embryo development. For this purpose, we investigated the optical properties of new silver nanoparticles in biological mediums during cultivation and their influence on rabbit's embryos development in vitro. The physicochemical and biological properties of novel silver nanoparticles were compared with silver nanoparticles identical in size and shapes but with different chemical surfaces modifications by polyvinylpyrrolidone and bovine serum albumin. The results suggest that silver nanoparticles with hyaluronic acid were disintegrated with the formation of new complexes with proteins in biological mediums. This property with strong optical surface plasmon resonance of novel silver nanoparticles with hyaluronan makes them promising candidates in diagnostic area and gives reasons to explore them as biomarkers of target molecules. Nevertheless novel silver nanoparticles with hyaluronan at the concentrations of 0.1-1 μg/ml have no toxic effect on rabbit's embryos development and can be successfully applied in reproductive medicine.

  17. Hyaluronan deficiency due to Has3 knock-out causes altered neuronal activity and seizures via reduction in brain extracellular space.

    PubMed

    Arranz, Amaia M; Perkins, Katherine L; Irie, Fumitoshi; Lewis, David P; Hrabe, Jan; Xiao, Fanrong; Itano, Naoki; Kimata, Koji; Hrabetova, Sabina; Yamaguchi, Yu

    2014-04-30

    Hyaluronan (HA), a large anionic polysaccharide (glycosaminoglycan), is a major constituent of the extracellular matrix of the adult brain. To address its function, we examined the neurophysiology of knock-out mice deficient in hyaluronan synthase (Has) genes. Here we report that these Has mutant mice are prone to epileptic seizures, and that in Has3(-/-) mice, this phenotype is likely derived from a reduction in the size of the brain extracellular space (ECS). Among the three Has knock-out models, namely Has3(-/-), Has1(-/-), and Has2(CKO), the seizures were most prevalent in Has3(-/-) mice, which also showed the greatest HA reduction in the hippocampus. Electrophysiology in Has3(-/-) brain slices demonstrated spontaneous epileptiform activity in CA1 pyramidal neurons, while histological analysis revealed an increase in cell packing in the CA1 stratum pyramidale. Imaging of the diffusion of a fluorescent marker revealed that the transit of molecules through the ECS of this layer was reduced. Quantitative analysis of ECS by the real-time iontophoretic method demonstrated that ECS volume was selectively reduced in the stratum pyramidale by ∼ 40% in Has3(-/-) mice. Finally, osmotic manipulation experiments in brain slices from Has3(-/-) and wild-type mice provided evidence for a causal link between ECS volume and epileptiform activity. Our results provide the first direct evidence for the physiological role of HA in the regulation of ECS volume, and suggest that HA-based preservation of ECS volume may offer a novel avenue for development of antiepileptogenic treatments.

  18. Uncovering the dual role of RHAMM as an HA receptor and a regulator of CD44 expression in RHAMM-expressing mesenchymal progenitor cells

    PubMed Central

    Veiseh, Mandana; Leith, Sean J.; Tolg, Cornelia; Elhayek, Sallie S.; Bahrami, S. Bahram; Collis, Lisa; Hamilton, Sara; McCarthy, James B.; Bissell, Mina J.; Turley, Eva

    2015-01-01

    The interaction of hyaluronan (HA) with mesenchymal progenitor cells impacts trafficking and fate after tissue colonization during wound repair and these events contribute to diseases such as cancer. How this interaction occurs is poorly understood. Using 10T½ cells as a mesenchymal progenitor model and fluorescent (F-HA) or gold-labeled HA (G-HA) polymers, we studied the role of two HA receptors, RHAMM and CD44, in HA binding and uptake in non-adherent and adherent mesenchymal progenitor (10T½) cells to mimic aspects of cell trafficking and tissue colonization. We show that fluorescent labeled HA (F-HA) binding/uptake was high in non-adherent cells but dropped over time as cells became increasingly adherent. Non-adherent cells displayed both CD44 and RHAMM but only function-blocking anti-RHAMM and not anti-CD44 antibodies significantly reduced F-HA binding/uptake. Adherent cells, which also expressed CD44 and RHAMM, primarily utilized CD44 to bind to F-HA since anti-CD44 but not anti-RHAMM antibodies blocked F-HA uptake. RHAMM overexpression in adherent 10T½ cells led to increased F-HA uptake but this increased binding remained CD44 dependent. Further studies showed that RHAMM-transfection increased CD44 mRNA and protein expression while blocking RHAMM function reduced expression. Collectively, these results suggest that cellular microenvironments in which these receptors function as HA binding proteins differ significantly, and that RHAMM plays at least two roles in F-HA binding by acting as an HA receptor in non-attached cells and by regulating CD44 expression and display in attached cells. Our findings demonstrate adhesion-dependent mechanisms governing HA binding/ uptake that may impact development of new mesenchymal cell-based therapies. PMID:26528478

  19. Beta-arrestin-2 negatively modulates inflammation response in mouse chondrocytes induced by 4-mer hyaluronan oligosaccharide.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Calatroni, Alberto; Campo, Salvatore

    2015-01-01

    Beta-arrestin-2 is an adaptor protein that terminates G protein activation and seems to be involved in the modulation of the inflammatory response. Small hyaluronan (HA) fragments, such as 4-mer HA oligosaccharides, are known to interact with the toll-like receptor-4 (TLR-4) with consequent activation of the nuclear factor kappaB (NF-kB) that in turn stimulates the inflammation response. NF-kB activation is mediated by different pathways, in particular by the transforming growth factor-activated kinase-1 (TAK-1). Conversely, increased levels of protein kinase A (PKA), induced by cyclic adenosine monophosphate (cAMP), seem to inhibit NF-kB activation. We studied the involvement and role of beta-arrestin-2 in mouse chondrocytes stimulated with 4-mer HA fragments. The exposure of chondrocytes to 4-mer HA produced a significant up-regulation in TLR-4, cAMP, beta-arrestin-2, TAK-1, protein 38 mitogen-activated protein kinase (p38MAPK), and PKA, both in terms of mRNA expression and of the related protein levels. NF-kB was significantly activated, thereby producing the transcription of pro-inflammatory mediators, including tumor necrosis factor alpha, interleukin-6, and interleukin-17. The treatment of 4-mer HA-stimulated chondrocytes with antibodies against beta-arrestin-2 and/or a specific PKA inhibitor, significantly increased the inflammatory response, while the treatment with a specific p38MAPK inhibitor significantly reduced the inflammatory response. Interestingly, the anti-inflammatory action exerted by beta-arrestin-2 appeared to be mediated in part through the direct inhibition of p38MAPK, preventing NF-kB activation, and in part through cAMP and PKA activation primed by G protein signaling, which exerted an inhibitory effect on NF-kB. Taken together, these results could be useful for future anti-inflammatory strategies. PMID:25318610

  20. Beta-arrestin-2 negatively modulates inflammation response in mouse chondrocytes induced by 4-mer hyaluronan oligosaccharide.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Calatroni, Alberto; Campo, Salvatore

    2015-01-01

    Beta-arrestin-2 is an adaptor protein that terminates G protein activation and seems to be involved in the modulation of the inflammatory response. Small hyaluronan (HA) fragments, such as 4-mer HA oligosaccharides, are known to interact with the toll-like receptor-4 (TLR-4) with consequent activation of the nuclear factor kappaB (NF-kB) that in turn stimulates the inflammation response. NF-kB activation is mediated by different pathways, in particular by the transforming growth factor-activated kinase-1 (TAK-1). Conversely, increased levels of protein kinase A (PKA), induced by cyclic adenosine monophosphate (cAMP), seem to inhibit NF-kB activation. We studied the involvement and role of beta-arrestin-2 in mouse chondrocytes stimulated with 4-mer HA fragments. The exposure of chondrocytes to 4-mer HA produced a significant up-regulation in TLR-4, cAMP, beta-arrestin-2, TAK-1, protein 38 mitogen-activated protein kinase (p38MAPK), and PKA, both in terms of mRNA expression and of the related protein levels. NF-kB was significantly activated, thereby producing the transcription of pro-inflammatory mediators, including tumor necrosis factor alpha, interleukin-6, and interleukin-17. The treatment of 4-mer HA-stimulated chondrocytes with antibodies against beta-arrestin-2 and/or a specific PKA inhibitor, significantly increased the inflammatory response, while the treatment with a specific p38MAPK inhibitor significantly reduced the inflammatory response. Interestingly, the anti-inflammatory action exerted by beta-arrestin-2 appeared to be mediated in part through the direct inhibition of p38MAPK, preventing NF-kB activation, and in part through cAMP and PKA activation primed by G protein signaling, which exerted an inhibitory effect on NF-kB. Taken together, these results could be useful for future anti-inflammatory strategies.

  1. Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes.

    PubMed

    Huang, Guo-Shiang; Dai, Lien-Guo; Yen, Betty L; Hsu, Shan-hui

    2011-10-01

    Stem cells can lose their primitive properties during in vitro culture. The culture substrate may affect the behavior of stem cells as a result of cell-substrate interaction. The maintenance of self-renewal for adult human mesenchymal stem cells (MSCs) by a biomaterial substrate, however, has not been reported in literature. In this study, MSCs isolated from human adipose (hADAS) and placenta (hPDMC) were cultured on chitosan membranes and those further modified by hyaluronan (chitosan-HA). It was observed that the MSCs of either origin formed three-dimensional spheroids that kept attached on the membranes. Spheroid formation was associated with the increased MMP-2 expression. Cells on chitosan-HA formed spheroids more quickly and the size of spheroids were larger than on chitosan alone. The expression of stemness marker genes (Oct4, Sox2, and Nanog) for MSCs on the materials was analyzed by the real-time RT-PCR. It was found that formation of spheroids on chitosan and chitosan-HA membranes helped to maintain the expression of stemness marker genes of MSCs compared to culturing cells on polystyrene dish. The maintenance of stemness marker gene expression was especially remarkable in hPDMC spheroids (vs. hADAS spheroids). Blocking CD44 by antibodies prevented the spheroid formation and decreased the stemness gene expression moderately; while treatment by Y-27632 compound inhibited the spheroid formation and significantly decreased the stemness gene expression. Upon chondrogenic induction, the MSC spheroids showed higher levels of Sox9, aggrecan, and collagen type II gene expression and were stained positive for glycosaminoglycan and collagen type II. hPDMC had better chondrogenic differentiation potential than hADAS upon induction. Our study suggested that the formation of adhered spheroids on chitosan and chitosan-HA membranes may sustain the expression of stemness marker genes of MSCs and increase their chondrogenic differentiation capacity. The Rho

  2. Airway smooth muscle cells synthesize hyaluronan cable structures independent of inter-alpha-inhibitor heavy chain attachment.

    PubMed

    Lauer, Mark E; Fulop, Csaba; Mukhopadhyay, Durba; Comhair, Suzy; Erzurum, Serpil C; Hascall, Vincent C

    2009-02-20

    The covalent association of inter-alpha-inhibitor-derived heavy chains (HCs) with hyaluronan was first described in synovial fluid from arthritic patients and later described as a structural and functional component of hyaluronan "cable" structures produced by many different cells and stimuli. HC transfer has been shown to be mediated by the protein product of TSG-6 (tumor necrosis factor-stimulated gene 6). Considering the accumulation of hyaluronan in airways following asthmatic attacks and the subsequent infiltration of leukocytes, we sought to characterize HC substitution of hyaluronan "cables" in primary mouse airway smooth muscle cells (MASM) and primary human airway smooth muscle cells (HASM). We found that cells derived from mice lacking TSG-6 had no defect in hyaluronan production or hyaluronan-mediated leukocyte adhesion when treated with the viral mimic poly(I,C). Functional hyaluronan cables were induced by cycloheximide in the confirmed absence of protein synthesis, with or without simultaneous treatment with poly(I,C). We characterized the species specificity of the antibody other investigators used to describe the HC-hyaluronan complex of hyaluronan cables and found minimal affinity to bovine-derived HCs in contrast to HCs from mouse and human sera. Thus, we cultured MASM and HASM cells in serum from these three sources and analyzed hyaluronan extracts for HCs and other hyaluronan-binding proteins, using parallel cumulus cell-oocyte complex (COC) extracts as positive controls. We conclude that, if hyaluronan cables derived from MASM and HASM cells are substituted with HCs, the amount of substitution is significantly below the limit of detection when compared with COC extracts of similar hyaluronan mass.

  3. [Src kinase-MAPK signal pathway plays a role in proliferation of endothelial cells induced by o-HA].

    PubMed

    Yang, Cui Xia; Liu, Yi Wen; He, Yi Qing; Gao, Feng

    2006-12-01

    To investigate the effect of hyaluronan oligosaccharides (o-HA) on endothelial cell (EC) proliferation and the possible mechanism involved. The cell proliferation was determined by cell counting and flow cytometer, and the phosphorylation of Src kinase and ERK-1/2 as well as the expression of cyclin D1 were assayed by western blot. o-HA at concentration of 10 microg/ml caused a significantly increase in both cell cycle and cell number of EC. With increasing time and amount of o-HA of exposure, there was no further increase in the growth of cells. The cell proliferation started to be significant at 12 hr and reached peak at 72 hr. At the same time,the phosphorylation of Src kinase and ERK-1/2 was enhanced after treated with l microg/ml of o-HA at 5 min and the expression of cyclin D1 was enhanced by treating PIEC cells with o-HA at 3 hr. o-HA may increase EC growth by stimulating the Src kinase and MAPK signal pathway and thus promote the proliferation of PIEC cells,in which the regulation of cyclin D1 expression may be involved. PMID:17348201

  4. Effect of Carboxymethylation on the Rheological Properties of Hyaluronan

    PubMed Central

    Wendling, Rian J.; Christensen, Amanda M.; Quast, Arthur D.; Atzet, Sarah K.; Mann, Brenda K.

    2016-01-01

    Chemical modifications made to hyaluronan to enable covalent crosslinking to form a hydrogel or to attach other molecules may alter the physical properties as well, which have physiological importance. Here we created carboxymethyl hyaluronan (CMHA) with varied degree of modification and investigated the effect on the viscosity of CMHA solutions. Viscosity decreased initially as modification increased, with a minimum viscosity for about 30–40% modification. This was followed by an increase in viscosity around 45–50% modification. The pH of the solution had a variable effect on viscosity, depending on the degree of carboxymethyl modification and buffer. The presence of phosphates in the buffer led to decreased viscosity. We also compared large-scale production lots of CMHA to lab-scale and found that large-scale required extended reaction times to achieve the same degree of modification. Finally, thiolated CMHA was disulfide crosslinked to create hydrogels with increased viscosity and shear-thinning aspects compared to CMHA solutions. PMID:27611817

  5. Drug conjugation to hyaluronan widens therapeutic indications for ovarian cancer

    PubMed Central

    Montagner, Isabella Monia; Merlo, Anna; Carpanese, Debora; Zuccolotto, Gaia; Renier, Davide; Campisi, Monica; Pasut, Gianfranco; Zanovello, Paola; Rosato, Antonio

    2015-01-01

    Management of ovarian cancer still requires improvements in therapeutic options. A drug delivery strategy was tested that allows specific targeting of tumor cells in combination with a controlled release of a cytotoxic molecule. To this aim, the efficacy of a loco-regional intraperitoneal treatment with a bioconjugate (ONCOFID-S) derived by chemical linking of SN-38, the active metabolite of irinotecan (CPT-11), to hyaluronan was assessed in a mouse model of ovarian carcinomatosis. In vitro, the bioconjugate selectively interacted with ovarian cancer cells through the CD44 receptor, disclosed a dose-dependent tumor growth inhibition efficacy comparable to that of free SN-38 drug, and inhibited Topoisomerase I function leading to apoptosis by a mechanism involving caspase-3 and -7 activation and PARP cleavage. In vivo, the intraperitoneal administration of ONCOFID-S in tumor-bearing mice did not induce inflammation, and evidenced an improved therapeutic efficacy compared with CPT-11. In conclusion, SN-38 conjugation to hyaluronan significantly improved the profile of in vivo tolerability and widened the field of application of irinotecan. Therefore, this approach can be envisaged as a promising therapeutic strategy for loco-regional treatment of ovarian cancer. PMID:26097871

  6. Effect of Carboxymethylation on the Rheological Properties of Hyaluronan.

    PubMed

    Wendling, Rian J; Christensen, Amanda M; Quast, Arthur D; Atzet, Sarah K; Mann, Brenda K

    2016-01-01

    Chemical modifications made to hyaluronan to enable covalent crosslinking to form a hydrogel or to attach other molecules may alter the physical properties as well, which have physiological importance. Here we created carboxymethyl hyaluronan (CMHA) with varied degree of modification and investigated the effect on the viscosity of CMHA solutions. Viscosity decreased initially as modification increased, with a minimum viscosity for about 30-40% modification. This was followed by an increase in viscosity around 45-50% modification. The pH of the solution had a variable effect on viscosity, depending on the degree of carboxymethyl modification and buffer. The presence of phosphates in the buffer led to decreased viscosity. We also compared large-scale production lots of CMHA to lab-scale and found that large-scale required extended reaction times to achieve the same degree of modification. Finally, thiolated CMHA was disulfide crosslinked to create hydrogels with increased viscosity and shear-thinning aspects compared to CMHA solutions. PMID:27611817

  7. Effect of hyaluronan on periodontitis: A clinical and histological study

    PubMed Central

    Gontiya, Gauri; Galgali, Sushama R.

    2012-01-01

    Background: Conventional, non-surgical periodontal therapy consists of supra- and subgingival tooth debridement. However, it is a technically demanding procedure and is not always efficient at eradicating all periodontal pathogens and in reducing inflammation. Therefore, local subgingival application of other chemotherapeutic agents may be used as an adjunct to non-surgical therapy. The aim of this study was to investigate the clinical and histological outcomes of local subgingival application of 0.2% hyaluronic acid gel (GENGIGEL®) as an adjunct to scaling and root planing (SRP) in chronic periodontitis patients. Materials and Methods: One hundred and twenty sites were chosen from 26 patients with chronic periodontitis (criteria being periodontal pockets ≥5mm). Experimental sites additionally received HA gel subgingivally at baseline, 1st, 2nd, and 3rd week. Clinical parameters were re-assessed at 4th, 6th, and 12th week. At 4th week recall, a gingival biopsy was obtained from test and control site for histologic examination. Results: Intra-group analysis of all the clinical parameters at all sites from baseline to 4th, 6th, and 12th week showed statistically significant changes. Experimental sites showed statistically significant improvement in Gingival index and Bleeding index at 6th and 12th week when compared with control sites. However, no statistically significant differences were observed in the PPD and RAL between control and experimental sites at 4th, 6th, and 12th week time interval. No statistically significant association was found between the histological grading of the sites that received HA treatment. Conclusion: Subgingival placement of 0.2% HA gel along with SRP provided a significant improvement in gingival parameters. However, no additional benefit was found in periodontal parameters. Histologically, experimental sites showed reduced inflammatory infiltrate, but it was not statistically significant. PMID:23055583

  8. [Hypoallergenic milks (HA formulas) in infant nutrition].

    PubMed

    Zoppi, G

    1993-01-01

    According to the definition of the European Scientific Committee for Food, hypoallergenic or hypoantigenic formulas (HA-formulas) are those which contain hydrolysed protein derived both from casein or whey. Soy-based formulas are not comprised in this definition since it has been demonstrated from several years that soy-protein, in several circumstances, may be highly allergenic. Hypoallergenic formulas contain besides hydrolysed protein, carbohydrate and lipid in amount and proportion similar to those indicated by ESPGAN recommendations on adapted formulas. As far as it concerns composition in lipid, recently great attention has been given to optimal supply and ratio of omega 3 and omega 6 fatty acids. Hypoallergenic formulas are therefore suitable for balanced nutrition of suckling infants. Specific indications on prevention of atopic diseases are not treated.

  9. Visualization of a hyaluronan network on the surface of silicone-hydrogel materials.

    PubMed

    Wygladacz, Katarzyna A; Hook, Daniel J

    2016-01-01

    Biotrue multipurpose solution (MPS) is a bioinspired disinfecting and conditioning solution that includes hyaluronic acid (HA) as a natural wetting agent. Previous studies demonstrated that HA sorbed from Biotrue MPS on both conventional and silicone hydrogel (SiHy) contact lens materials; an in vitro simulated-wear test validated the presence of HA on the lens surfaces for as long as 20 hours. In this study, the morphology and distribution of HA sorbed from both Biotrue and pure HA solution on SiHy contact lens surfaces was examined. Atomic force microscopy imaging was used to illustrate the topography of fresh SiHy contact lens materials before and after incubation with 0.1% (w/v) HA solution. The distribution, as well as fine details of the HA network, were resolved by first staining HA with Gram's safranin, then imaging with confocal laser-scanning microscopy and differential interference-contrast microscopy. In this approach, SiHy materials take up the dye (safranin) nonspecifically, such that the resultant safranin-HA complex appears dim against the fluorescent lens background. Balafilcon A was chosen as the representative of glassy SiHy lenses that require postpolymerization plasma treatment to increase wettability. Senofilcon A and samfilcon A were chosen as representatives of SiHy materials fabricated with an internal wetting agent. A confluent and dim HA-safranin network was observed adhered to balafilcon A, senofilcon A, and samfilcon A lens surfaces incubated with either 0.1% (w/v) HA solution or Biotrue MPS. Therefore, the conditioning function provided by Biotrue MPS may be in part explained by the presence of the HA humectant layer that readily sorbs on the various types of SiHy contact lens materials. PMID:27555749

  10. Hyaluronan is organized into fiber-like structures along migratory pathways in the developing mouse cerebellum.

    PubMed

    Baier, Claudia; Baader, Stephan L; Jankowski, Jakob; Gieselmann, Volkmar; Schilling, Karl; Rauch, Uwe; Kappler, Joachim

    2007-06-01

    Hyaluronan is a free glycosaminoglycan which is abundant in the extracellular matrix of the developing brain. Although not covalently linked to any protein it can act as a backbone molecule forming aggregates with chondroitin sulfate proteoglycans of the lectican family and link proteins. Using neurocan-GFP as a direct histochemical probe we analyzed the distribution and organization of hyaluronan in the developing mouse cerebellum, and related its fine structure to cell types of specified developmental stages. We observed a high affinity of this probe to fiber-like structures in the prospective white matter which are preferentially oriented parallel to the cerebellar cortex during postnatal development suggesting a specially organized form of hyaluronan. In other layers of the cerebellar cortex, the hyaluronan organization seemed to be more diffuse. During the second postnatal week, the overall staining intensity of hyaluronan in the white matter declined but fiber-like structures were still present at the adult stage. This type of hyaluronan organization is different from perineuronal nets e.g. found in deep cerebellar nuclei. Double staining experiments with cell type specific markers indicated that these fiber-like structures are predominantly situated in regions where motile cells such as Pax2-positive inhibitory interneuron precursors and MBP-positive oligodendroglial cells are located. In contrast, more stationary cells such as mature granule cells and Purkinje cells are associated with lower levels of hyaluronan in their environment. Thus, hyaluronan-rich fibers are concentrated at sites where specific neural precursor cell types migrate, and the anisotropic orientation of these fibers suggests that they may support guided neural migration during brain development.

  11. The Role of Hyaluronan in Innate Defense Responses of the Intestine

    PubMed Central

    de la Motte, Carol A.; Kessler, Sean P.

    2015-01-01

    Hyaluronan is an abundant extracellular matrix component prevalent in the vertebrate intestinal tract. Here we discuss what is known about hyaluronan distribution during homeostasis and inflammatory diseases of the gut and discuss ways in which this glycosaminoglycan can participate in regulating innate host defense mechanisms. These natural responses include mechanisms promoting rapid leukocyte recruitment after bacterial challenge/colon tissue damage as well as promoting epithelial defense mechanisms in the intestine. PMID:25922605

  12. Prevention of peritendinous adhesions using a hyaluronan-derived hydrogel film following partial-thickness flexor tendon injury.

    PubMed

    Liu, Yanchun; Skardal, Aleksander; Shu, Xiao Zheng; Prestwich, Glenn D

    2008-04-01

    Peritendinous adhesions are an important complication of flexor tendon injury. Three hyaluronan (HA)-derived biomaterials were evaluated for the reduction of peritendinous adhesions following partial-thickness tendon injury in rabbits. Rabbits (n = 24) were divided into three groups (n = 8 per group), which were used for gross evaluation, histologic assessment, or biomechanical testing. The fourth and third toes from both hindpaws of each rabbit were randomly assigned to one of four treatments: (i) untreated control, (ii) Seprafilm, (iii) Carbylan-SX in situ crosslinked hydrogel, and (iv) preformed Carbylan-SX film. Rabbits were sacrificed at 3 weeks postsurgery and evaluated anatomically, histologically, and mechanically. All materials used reduced adhesions relative to untreated controls for all three evaluations. Both the gross anatomic and histologic results revealed that Carbylan-SX film was statistically superior to Seprafilm and Carbylan-SX gel in preventing tendon adhesion formation. In biomechanical tests, the Carbylan-SX film-treated hindpaws required the least force to pull the tendon from the sheath. This force was statistically indistinguishable from that required to extrude an unoperated tendon (n = 8). Carbylan-SX gel was less effective than Carbylan-SX film but superior to Seprafilm for all evaluations. A crosslinked HA-derived film promoted healing of a flexor tendon injury without the formation of fibrosis at 3 weeks postoperatively.

  13. An infection-preventing bilayered collagen membrane containing antibiotic-loaded hyaluronan microparticles: physical and biological properties.

    PubMed

    Lee, Jong-Eun; Park, Jong-Chul; Lee, Kwang Hoon; Oh, Sang Ho; Kim, Joong-Gon; Suh, Hwal

    2002-07-01

    An infection-preventing bilayered membrane consisting of a dense and porous collagen membrane has been developed. The membrane was fabricated using a combined freeze-drying/air-drying method. Hyaluronan (HA) microparticles containing silver sulfadiazine (AgSD) were fabricated by gelling an HA solution with calcium chloride and were incorporated into collagen layers to allow the sustained release of AgSD. In vitro biodegradability of the membrane and the release of AgSD from the membrane could be controlled by cross-linking the membrane with ultraviolet (UV) irradiation. In a cytotoxicity test, cellular damage was minimized by the sustained release of AgSD from dressings. The antibacterial capacity of the material against Pseudomonas aeruginosa was investigated using the Bauer-Kirby disk diffusion test, and bacterial growth was found to be inhibited for 4 days. In vivo tests showed that the bilayered membrane was associated with greater tissue regeneration than a polymeric membrane and with no infection-related biological signs.

  14. Hydration of hyaluronan polysaccharide observed by IR spectrometry. II. Definition and quantitative analysis of elementary hydration spectra and water uptake.

    PubMed

    Haxaire, K; Maréchal, Y; Milas, M; Rinaudo, M

    2003-01-01

    We recorded a series of spectra of sodium hyaluronan (HA) films that were in equilibrium with their surrounding humid atmosphere. The hygrometry of this atmosphere extended from 0 to 0.97% relative humidity. We performed a quantitative analysis of the corresponding series of hydration spectra that are the difference spectra of the film at a defined hygrometry minus the spectrum of the dried film (hygrometry = 0). The principle of this analysis is to use this series of hydration spectra to define a limited number (four) of "elementary hydration spectra" over which we can decompose all hydration spectra with good accuracy. This decomposition, combined with the measurements of the numbers of H(2)O molecules at the origin in these elementary hydration spectra of the three characteristic vibrational bands of H(2)O, allowed us to calculate the hydration number under different relative humidity conditions. This number compares well with that determined by thermogravimetry. Furthermore, the decomposition defines for each hygrometry value which chemical mechanisms represented by elementary hydration spectra are active. This analysis is pursued by determining for the elementary hydration spectra the number of hydrogen bonds established by each of the four alcohol groups found in each disaccharide repeat unit before performing the same analysis for amide and carboxylate groups. These results are later utilized to discuss the structure of HA at various stages of hydration. PMID:12722111

  15. Osteogenic Properties of PBLG-g-HA/PLLA Nanocomposites

    PubMed Central

    Liao, Lan; Yang, Shuang; Miron, Richard J.; Wei, Junchao; Zhang, Yufeng; Zhang, Meng

    2014-01-01

    New development of biomaterial scaffolds remains a prominent issue for the regeneration of lost or fractured bone. Of these scaffolds, a number of bioactive polymers have been synthesized and fabricated for diverse biological roles. Although recent evidence has demonstrated that composite scaffolds such as HA/PLLA have improved properties when compared to either HA or PLLA alone, recent investigations have demonstrated that the phase compatibility between HA and PLLA layers is weak preventing optimal enhancement of the mechanical properties and making the composites prone to breakdown. In the present study, poly (γ-benzyl-L-glutamate) modified hydroxyapatite/(poly (L-lactic acid)) (PBLG-g-HA/PLLA) composite scaffolds were fabricated with improved phase compatibility and tested for their osteogenic properties in 18 Wistar female rats by analyzing new bone formation in 3 mm bilateral femur defects in vivo. At time points, 2, 4 and 8 weeks post surgery, bone formation was evaluated by µ-CT and histological analysis by comparing 4 treatment groups; 1) blank defect, 2) PLLA, 3) HA/PLLA and 4) PBLG-g-HA/PLLA scaffolds. The in vivo analysis demonstrated that new bone formation was much more prominent in HA/PLLA and PBLG-g-HA/PLLA groups as depicted by µ-CT, H&E staining and immunohistochemistry for collagen I. TRAP staining was also utilized to determine the influence of osteoclast cell number and staining intensity to the various scaffolds. No significant differences in either staining intensity or osteoclast numbers between all treatment modalities was observed, however blank defects did contain a higher number of osteoclast-like cells. The results from the present study illustrate the potential of PBLG-g-HA/PLLA scaffolds for bone tissue engineering applications by demonstrating favorable osteogenic properties. PMID:25184285

  16. Oral administration of hyaluronan prevents skin dryness and epidermal thickening in ultraviolet irradiated hairless mice.

    PubMed

    Kawada, Chinatsu; Kimura, Mamoru; Masuda, Yasunobu; Nomura, Yoshihiro

    2015-12-01

    Hyaluronan is a component of the extracellular matrix that plays a role in water retention in tissues. In this study, we orally administered hyaluronans of varying molecular weights (300k and less than 10k) repeatedly to hairless mice exposed to ultraviolet (UV) irradiation and examined their effects on the skin of these mice. UV irradiation induces a marked increase in the epidermal thickness of the dorsal skin and a marked decrease in the skin moisture content; however, orally administered hyaluronan, particularly that with a molecular weight of less than 10k, markedly reversed the increase and decrease in the epidermal thickness and skin moisture content, respectively. Furthermore, on analyzing the mice skin, orally administered hyaluronan with a molecular weight of less than 10k increased the levels of the HAS2 gene expression in the skin. Based on these findings, it is assumed that orally administered hyaluronans, with molecular weight of 300k and less than 10k, reversed UV irradiation-induced skin disturbance. In particular, it was considered that the increase in the skin moisture content by orally administered hyaluronan, with a molecular weight of less than 10k, was related to the effect on skin cells.

  17. A hyaluronan-based scaffold for the in vitro construction of dental pulp-like tissue.

    PubMed

    Ferroni, Letizia; Gardin, Chiara; Sivolella, Stefano; Brunello, Giulia; Berengo, Mario; Piattelli, Adriano; Bressan, Eriberto; Zavan, Barbara

    2015-01-01

    Dental pulp tissue supports the vitality of the tooth, but it is particularly vulnerable to external insults, such as mechanical trauma, chemical irritation or microbial invasion, which can lead to tissue necrosis. In the present work, we present an endodontic regeneration method based on the use of a tridimensional (3D) hyaluronan scaffold and human dental pulp stem cells (DPSCs) to produce a functional dental pulp-like tissue in vitro. An enriched population of DPSCs was seeded onto hyaluronan-based non-woven meshes in the presence of differentiation factors to induce the commitment of stem cells to neuronal, glial, endothelial and osteogenic phenotypes. In vitro experiments, among which were gene expression profiling and immunofluorescence (IF) staining, proved the commitment of DPSCs to the main components of dental pulp tissue. In particular, the hyaluronan-DPSCs construct showed a dental pulp-like morphology consisting of several specialized cells growing inside the hyaluronan fibers. Furthermore, these constructs were implanted into rat calvarial critical-size defects. Histological analyses and gene expression profiling performed on hyaluronan-DPSCs grafts showed the regeneration of osteodentin-like tissue. Altogether, these data suggest the regenerative potential of the hyaluronan-DPSC engineered tissue. PMID:25739081

  18. Preventing effects of joint contracture by high molecular weight hyaluronan injections in a rat immobilized knee model

    PubMed Central

    Kanazawa, Kenji; Hagiwara, Yoshihiro; Tsuchiya, Masahiro; Yabe, Yutaka; Sonofuchi, Kazuaki; Koide, Masashi; Sekiguchi, Takuya; Itaya, Nobuyuki; Ando, Akira; Saijo, Yoshifumi; Itoi, Eiji

    2015-01-01

    Purpose: To elucidate preventive effects of high molecular weight hyaluronan (HMWHA) on the joint capsule of immobilized knees in rats. Materials and Methods: Unilateral knee joints of rats were immobilized with an internal fixator. Either 50 μl of HMWHA (Im-HA group) or 50 μl of saline (control group) was administered intra-articularly once a week after surgery. Sagittal sections were prepared from the medial midcondylar region of the knee joints and assessed by histological, histomorphometric, and immunohistochemical methods. Gene expressions related to inflammation, fibrotic conditions, and hypoxia were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Tissue elasticity of the capsule from both groups was examined using a scanning acoustic microscope (SAM). Results: CD68 positive cells decreased in adhesion areas of the synovial membrane after 1 week in both groups. The length of the superficial layer in the synovial membrane of the Im-HA group was significantly longer than those in the control group over a period of 4 to 8 weeks with significantly small numbers of CD68 positive cells. The gene expressions of IL-6, IL-1β, TGF-β, CTGF, COL1a1, COL3a1, SPARC, and HIF1-α were significantly lower in the Im-HA group compared to those in the control group. The sound speed of the anterior and posterior synovial membrane increased significantly (a reduction in elasticity) in the control group compared to those in the Im-HA group during weeks 1 to 4. Conclusions: This study demonstrated that HMWHA injections suppressed inflammatory, fibrotic, and hypoxic conditions observed in the immobilized joint capsule. PMID:26097527

  19. Effect of vasopressin on the expression of genes for key enzymes of hyaluronan turnover in Wistar Albino Glaxo and Brattleboro rat kidneys.

    PubMed

    Ivanova, Lyudmila N; Babina, Alina V; Baturina, Galina S; Katkova, Lyubov E

    2013-11-01

    Hyaluronan (HA), the major glycosaminoglycan of the interstitial matrix, is heterogeneously distributed within the kidney. Using real-time RT-PCR, we tested the assumption that renal HA may be involved in the long-term effect of vasopressin on water reabsorption. The expression of the genes encoding hyaluronan synthase-2 (Has2), hyaluronidase-1 and hyaluronidase-2 (Hyal1 and Hyal2) was studied in the kidneys of Wistar Albino Glaxo (WAG) and homozygous vasopressin-deficient Brattleboro rats treated with the V2 receptor-selective vasopressin analogue dDAVP (100 μg (kg body wt)(-1), i.p., twice a day for 2 days). The Has2 mRNA content was the highest in the kidney papilla of the hydrated WAG and control Brattleboro rats, devoid of vasopressin. In WAG rats, dDAVP induced a considerable decrease in Has2 mRNA content in the papilla, with less pronounced changes in the cortex. The changes elicited by dDAVP in Brattleboro rats tended to be the same as in WAG rats, but weaker. In contrast to Has2, dDAVP treatment caused a significant increase in the Hyal1 and Hyal2 mRNA content in the renal papilla of WAG and Brattleboro rats. In rats of both strains, there was a good fit between Hyal1 and Hyal2 transcriptional levels and changes in hyaluronidase activity in the renal tissue. It is suggested that vasopressin is able to inhibit the synthesis of HA and concomitantly promote its degradation in the interstitium of the renal papilla, thereby facilitating water flow between elements of the renal countercurrent system. The implications for this effect are discussed in the context of the data in the literature.

  20. The Human Hyaluronan Synthase 2 (HAS2) Gene and Its Natural Antisense RNA Exhibit Coordinated Expression in the Renal Proximal Tubular Epithelial Cell♦

    PubMed Central

    Michael, Daryn R.; Phillips, Aled O.; Krupa, Aleksandra; Martin, John; Redman, James E.; Altaher, Abdalsamed; Neville, Rachel D.; Webber, Jason; Kim, Min-young; Bowen, Timothy

    2011-01-01

    Aberrant expression of the human hyaluronan synthase 2 (HAS2) gene has been implicated in the pathology of malignancy, pulmonary arterial hypertension, osteoarthritis, asthma, thyroid dysfunction, and large organ fibrosis. Renal fibrosis is associated with increased cortical synthesis of hyaluronan (HA), an extracellular matrix glycosaminoglycan, and we have shown that HA is a correlate of interstitial fibrosis in vivo. Our previous in vitro data have suggested that both HAS2 transcriptional induction and subsequent HAS2-driven HA synthesis may contribute to kidney fibrosis via phenotypic modulation of the renal proximal tubular epithelial cell (PTC). Post-transcriptional regulation of HAS2 mRNA synthesis by the natural antisense RNA HAS2-AS1 has recently been described in osteosarcoma cells, but the antisense transcript was not detected in kidney. In this study, PTC stimulation with IL-1β or TGF-β1 induced coordinated temporal profiles of HAS2-AS1 and HAS2 transcription. Constitutive activity of the putative HAS2-AS1 promoter was demonstrated, and transcription factor-binding sequence motifs were identified. Knockdown of Sp1/Sp3 expression by siRNA blunted IL-1β induction of both HAS2-AS1 and HAS2, and Smad2/Smad3 knockdown similarly attenuated TGF-β1 stimulation. Inhibition of IL-1β-stimulated HAS2-AS1 RNA induction using HAS2-AS1-specific siRNAs also suppressed up-regulation of HAS2 mRNA transcription. The thermodynamic feasibility of HAS2-AS1/HAS2 heterodimer formation was demonstrated in silico, and locus-specific cytoplasmic double-stranded RNA was detected in vitro. In summary, our data show that transcriptional induction of HAS2-AS1 and HAS2 occurs simultaneously in PTCs and suggest that transcription of the antisense RNA stabilizes or augments HAS2 mRNA expression in these cells via RNA/mRNA heteroduplex formation. PMID:21357421

  1. Priming Adipose-Derived Mesenchymal Stem Cells with Hyaluronan Alters Growth Kinetics and Increases Attachment to Articular Cartilage

    PubMed Central

    Succar, Peter; Medynskyj, Michael; Breen, Edmond J.; Batterham, Tony; Molloy, Mark P.; Herbert, Benjamin R.

    2016-01-01

    Background. Biological therapeutics such as adipose-derived mesenchymal stem cell (MSC) therapy are gaining acceptance for knee-osteoarthritis (OA) treatment. Reports of OA-patients show reductions in cartilage defects and regeneration of hyaline-like-cartilage with MSC-therapy. Suspending MSCs in hyaluronan commonly occurs in animals and humans, usually without supporting data. Objective. To elucidate the effects of different concentrations of hyaluronan on MSC growth kinetics. Methods. Using a range of hyaluronan concentrations, we measured MSC adherence and proliferation on culture plastic surfaces and a novel cartilage-adhesion assay. We employed time-course and dispersion imaging to assess MSC binding to cartilage. Cytokine profiling was also conducted on the MSC-secretome. Results. Hyaluronan had dose-dependent effects on growth kinetics of MSCs at concentrations of entanglement point (1 mg/mL). At higher concentrations, viscosity effects outweighed benefits of additional hyaluronan. The cartilage-adhesion assay highlighted for the first time that hyaluronan-primed MSCs increased cell attachment to cartilage whilst the presence of hyaluronan did not. Our time-course suggested patients undergoing MSC-therapy for OA could benefit from joint-immobilisation for up to 8 hours. Hyaluronan also greatly affected dispersion of MSCs on cartilage. Conclusion. Our results should be considered in future trials with MSC-therapy using hyaluronan as a vehicle, for the treatment of OA. PMID:26981136

  2. Priming Adipose-Derived Mesenchymal Stem Cells with Hyaluronan Alters Growth Kinetics and Increases Attachment to Articular Cartilage.

    PubMed

    Succar, Peter; Medynskyj, Michael; Breen, Edmond J; Batterham, Tony; Molloy, Mark P; Herbert, Benjamin R

    2016-01-01

    Background. Biological therapeutics such as adipose-derived mesenchymal stem cell (MSC) therapy are gaining acceptance for knee-osteoarthritis (OA) treatment. Reports of OA-patients show reductions in cartilage defects and regeneration of hyaline-like-cartilage with MSC-therapy. Suspending MSCs in hyaluronan commonly occurs in animals and humans, usually without supporting data. Objective. To elucidate the effects of different concentrations of hyaluronan on MSC growth kinetics. Methods. Using a range of hyaluronan concentrations, we measured MSC adherence and proliferation on culture plastic surfaces and a novel cartilage-adhesion assay. We employed time-course and dispersion imaging to assess MSC binding to cartilage. Cytokine profiling was also conducted on the MSC-secretome. Results. Hyaluronan had dose-dependent effects on growth kinetics of MSCs at concentrations of entanglement point (1 mg/mL). At higher concentrations, viscosity effects outweighed benefits of additional hyaluronan. The cartilage-adhesion assay highlighted for the first time that hyaluronan-primed MSCs increased cell attachment to cartilage whilst the presence of hyaluronan did not. Our time-course suggested patients undergoing MSC-therapy for OA could benefit from joint-immobilisation for up to 8 hours. Hyaluronan also greatly affected dispersion of MSCs on cartilage. Conclusion. Our results should be considered in future trials with MSC-therapy using hyaluronan as a vehicle, for the treatment of OA. PMID:26981136

  3. Visualization of a hyaluronan network on the surface of silicone-hydrogel materials

    PubMed Central

    Wygladacz, Katarzyna A; Hook, Daniel J

    2016-01-01

    Biotrue multipurpose solution (MPS) is a bioinspired disinfecting and conditioning solution that includes hyaluronic acid (HA) as a natural wetting agent. Previous studies demonstrated that HA sorbed from Biotrue MPS on both conventional and silicone hydrogel (SiHy) contact lens materials; an in vitro simulated-wear test validated the presence of HA on the lens surfaces for as long as 20 hours. In this study, the morphology and distribution of HA sorbed from both Biotrue and pure HA solution on SiHy contact lens surfaces was examined. Atomic force microscopy imaging was used to illustrate the topography of fresh SiHy contact lens materials before and after incubation with 0.1% (w/v) HA solution. The distribution, as well as fine details of the HA network, were resolved by first staining HA with Gram’s safranin, then imaging with confocal laser-scanning microscopy and differential interference-contrast microscopy. In this approach, SiHy materials take up the dye (safranin) nonspecifically, such that the resultant safranin–HA complex appears dim against the fluorescent lens background. Balafilcon A was chosen as the representative of glassy SiHy lenses that require postpolymerization plasma treatment to increase wettability. Senofilcon A and samfilcon A were chosen as representatives of SiHy materials fabricated with an internal wetting agent. A confluent and dim HA–safranin network was observed adhered to balafilcon A, senofilcon A, and samfilcon A lens surfaces incubated with either 0.1% (w/v) HA solution or Biotrue MPS. Therefore, the conditioning function provided by Biotrue MPS may be in part explained by the presence of the HA humectant layer that readily sorbs on the various types of SiHy contact lens materials. PMID:27555749

  4. Selective Hyaluronan-CD44 Signaling Promotes miRNA-21 Expression and Interacts with Vitamin D Function during Cutaneous Squamous Cell Carcinomas Progression Following UV Irradiation.

    PubMed

    Bourguignon, Lilly Y W; Bikle, Daniel

    2015-01-01

    Hyaluronan (HA), the major extracellular matrix component, is often anchored to CD44, a family of structurally/functionally important cell surface receptors. Recent results indicate that UV irradiation (UVR)-induced cutaneous squamous cell carcinomas (SCC) overexpress a variety of CD44 variant isoforms (CD44v), with different CD44v isoforms appear to confer malignant SCC properties. UVR also stimulates HA degradation in epidermal keratinocytes. Both large HA polymers and their UVR-induced catabolic products (small HA) selectively activate CD44-mediated cellular signaling in normal keratinocytes and SCC cells, with all of the downstream processes being mediated by RhoGTPases (e.g., Rac1 and Rho). Importantly, we found that the hormonally active form of vitamin D 1,25(OH)2D3 not only prevents the UVR-induced small HA activation of abnormal keratinocyte behavior and SCC progression, but also enhances large HA stimulation of normal keratinocyte activities and epidermal function(s). The aim of this hypothesis and theory article is to question whether matrix HA and its UVR-induced catabolic products (e.g., large and small HA) can selectively activate CD44-mediated cellular signaling such as GTPase (Rac and RhA) activation. We suggested that large HA-CD44 interaction promotes Rac-signaling and normal keratinocyte differentiation (lipid synthesis), DNA repair, and keratinocyte survival function. Conversely, small HA-CD44 interaction stimulates RhoA activation, NFκB/Stat-3 signaling, and miR-21 production, resulting in inflammation and proliferation as well as SCC progression. We also question whether vitamin D treatment displays any effect on small HA-CD44v-mediated RhoA signaling, inflammation, and SCC progression, as well as large HA-CD44-mediated differentiation, DNA repair, keratinocyte survival, and normal keratinocyte function. In addition, we discussed that the topical application of signaling perturbation agents (e.g., Y27623, a ROK inhibitor) may be used to treat

  5. The effects of Musk T on peroxisome proliferator-activated receptor [PPAR]-alpha activation, epidermal skin homeostasis and dermal hyaluronic acid synthesis.

    PubMed

    Kim, Seung Hun; Nam, Gae Won; Lee, Hae Kwang; Moon, Seong Joon; Chang, Ih Seop

    2006-11-01

    Peroxisome proliferators activated receptors (PPARs) are a family of nuclear hormone receptors that heterodimer with the retinoid X receptor and function as transcriptional regulators of genes. Topically Applied PPAR-alpha agonists possess receptor mediated, pro-differentiating/anti-proliferative effects, lipid metabolism stimulation, and anti-inflammatory activity, which suggest that they could be beneficial for the treatment of a variety of cutaneous diseases. Hyaluronan (HA), a high-molecular-weight linear glycosaminoglycan consisting of alternating D: -glucuronic acid and N-acetyl-D: -glucosamine residues, is one of the major extracellular matrix components in skin. Among the family of HA synthase genes (HAS1, 2, 3) so far identified, one group has demonstrated that the expressions of HAS2 and HAS3 play crucial roles in the regulation of HA synthesis in human skin fibroblasts and keratinocytes, respectively, but the precise regulatory mechanisms are still unknown. We examine Musk T called Ethylene brassylate, Astratone or 1,4-Dioxacycloheptadecane-5,17-dione, which used as just a perfume ingredient, plays a role as PPAR-alpha ligand in vitro and stimulates skin barrier recovery, ceramide synthesis, beta-Glucocerebrosidase, involucrin expression in epidermis in vivo; and examine that Musk T stimulates HAS expression and HA synthesis in human skin fibroblast. Through these experiments, we conclude that Musk T is PPAR-alpha ligand, effects on keratinocyte differentiation, intercellular lipid synthesis in epidermis, HA synthesis stimulation in dermis.

  6. Hydrogels of collagen/chondroitin sulfate/hyaluronan interpenetrating polymer network for cartilage tissue engineering.

    PubMed

    Guo, Yan; Yuan, Tun; Xiao, Zhanwen; Tang, Pingping; Xiao, Yumei; Fan, Yujiang; Zhang, Xingdong

    2012-09-01

    The network structure of a three-dimensional hydrogel scaffold dominates its performance such as mechanical strength, mass transport capacity, degradation rate and subsequent cellular behavior. The hydrogels scaffolds with interpenetrating polymeric network (IPN) structure have an advantage over the individual component gels and could simulate partly the structure of native extracellular matrix of cartilage tissue. In this study, to develop perfect cartilage tissue engineering scaffolds, IPN hydrogels of collagen/chondroitin sulfate/hyaluronan were prepared via two simultaneous processes of collagen self-assembly and cross linking polymerization of chondroitin sulfate-methacrylate (CSMA) and hyaluronic acid-methacrylate. The degradation rate, swelling performance and compressive modulus of IPN hydrogels could be adjusted by varying the degree of methacrylation of CSMA. The results of proliferation and fluorescence staining of rabbit articular chondrocytes in vitro culture demonstrated that the IPN hydrogels possessed good cytocompatibility. Furthermore, the IPN hydrogels could upregulate cartilage-specific gene expression and promote the chondrocytes secreting glycosaminoglycan and collagen II. These results suggested that IPN hydrogels might serve as promising hydrogel scaffolds for cartilage tissue engineering. PMID:22639153

  7. Efficacy of a single intramuscular injection of porcine FSH in hyaluronan prior to ovum pick-up in Holstein cattle.

    PubMed

    Vieira, L M; Rodrigues, C A; Netto, A Castro; Guerreiro, B M; Silveira, C R A; Freitas, B G; Bragança, L G M; Marques, K N G; Sá Filho, M F; Bó, G A; Mapletoft, R J; Baruselli, P S

    2016-03-15

    Plasma FSH profiles, in vitro embryo production (IVP) after ovum pickup (OPU), and establishment of pregnancy with IVP embryos were compared in untreated Holstein oocyte donors and those superstimulated with multiple injections or a single intramuscular (IM) injection of porcine FSH (pFSH) in hyaluronan (HA). Plasma FSH profiles were determined in 23 heifers randomly allocated to one of four groups. Controls received no treatment, whereas the F200 group received 200 mg of pFSH in four doses, 12 hours apart. The F200HA and F300HA groups received 200- or 300-mg pFSH in 5 mL or 7.5 mL, respectively of a 0.5% HA solution by a single IM injection. Plasma FSH levels were determined before the first pFSH treatment and every 6 hours over 96 hours. All data were analyzed by orthogonal contrasts. Circulating FSH area under curve (AUC) in pFSH-treated animals was greater than that in the control group (P = 0.02). Although the AUC did not differ among FSH-treated groups (P = 0.56), the total period with elevated plasma FSH was greater in the F200 group than in the HA groups (P < 0.0001). However, the F300HA group had a greater AUC than the F200HA group (P = 0.006), with a similar total period with elevated plasma FSH (P = 0.17). The IVP was performed in 90 nonlactating Holstein cows randomly allocated to one of the four treatment groups as in the first experiment. A greater proportion of medium-sized (6-10 mm) follicles was observed in cows receiving pFSH, regardless of the treatment group (P < 0.0001). Also, numbers of follicles (P = 0.01), cumulus-oocyte complexes (COCs) retrieved (P = 0.01) and matured (P = 0.02), cleavage rates (P = 0.002), and blastocysts produced per OPU session (P = 0.06) were greater in cows receiving pFSH, regardless of the treatment group. Cows in the F200HA group had a greater recovery rate (P = 0.009), number of COCs cultured (P = 0.04), and blastocysts produced per OPU session (P = 0.06) than cows in the F300HA group. Similar pregnancy rates were

  8. Both hyaluronan and collagen type II keep proteoglycan 4 (lubricin) at the cartilage surface in a condition that provides low friction during boundary lubrication.

    PubMed

    Majd, Sara Ehsani; Kuijer, Roel; Köwitsch, Alexander; Groth, Thomas; Schmidt, Tannin A; Sharma, Prashant K

    2014-12-01

    Wear resistant and ultralow friction in synovial joints is the outcome of a sophisticated synergy between the major macromolecules of the synovial fluid, e.g., hyaluronan (HA) and proteoglycan 4 (PRG4), with collagen type II fibrils and other non-collagenous macromolecules of the cartilage superficial zone (SZ). This study aimed at better understanding the mechanism of PRG4 localization at the cartilage surface. We show direct interactions between surface bound HA and freely floating PRG4 using the quartz crystal microbalance with dissipation (QCM-D). Freely floating PRG4 was also shown to bind with surface bound collagen type II fibrils. Albumin, the most abundant protein of the synovial fluid, effectively blocked the adsorption of PRG4 with HA, through interaction with C and N terminals on PRG4, but not that of PRG4 with collagen type II fibrils. The above results indicate that collagen type II fibrils strongly contribute in keeping PRG4 in the SZ during cartilage articulation in situ. Furthermore, PRG4 molecules adsorbed very well on mimicked SZ of absorbed HA molecules with entangled collagen type II fibrils and albumin was not able to block this interaction. In this last condition PRG4 adsorption resulted in a coefficient of friction (COF) of the same order of magnitude as the COF of natural cartilage, measured with an atomic force microscope in lateral mode.

  9. Hyaluronan Oligosaccharides Induce MMP-1 and -3 via Transcriptional Activation of NF-κB and p38 MAPK in Rheumatoid Synovial Fibroblasts

    PubMed Central

    Hanabayashi, Masahiro; Takahashi, Nobunori; Sobue, Yasumori; Hirabara, Shinya; Ishiguro, Naoki; Kojima, Toshihisa

    2016-01-01

    Objective To explore the effect of hyaluronan oligosaccharides (HAoligos) on interactions between HA and its principal receptor, CD44, in rheumatoid synovial fibroblasts (RSFs) and matrix metalloproteinase (MMP) production. Methods RSFs were isolated from rheumatoid synovial tissue. HA distribution was visualized by immunocytochemistry. MMP-1 and MMP-3 induction was analyzed by real-time RT-PCR and immunoblotting. The interaction between HAoligos and their MMP-producing receptors was tested by blocking with anti-CD44 and anti-Toll-like receptor 4 (TLR-4). Phosphorylation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) was analyzed by immunoblotting. Results Endogenous HA decreased after treatment with HAoligos, while MMP-1 and MMP-3 expression increased in a dose-dependent manner. Pretreatment with anti-CD44 or anti-TLR-4 antibody significantly reduced the effect of HAoligos on MMP-1 and MMP-3 mRNA expression. NF-κB and p38 MAPK phosphorylation was enhanced by HAoligos pretreated with anti-TLR-4, and HAoligo-induced MMP production was blocked with an inhibitor of NF-κB and p38 MAPK pathways. Conclusions Disruptive changes in CD44-HA interactions by HAoligos enhanced MMP-1 and MMP-3 production via activation of NF-κB and p38 MAPK signaling pathways in RSFs. PMID:27564851

  10. Growth behaviour and mechanical properties of PLL/HA multilayer films studied by AFM.

    PubMed

    Uzüm, Cagri; Hellwig, Johannes; Madaboosi, Narayanan; Volodkin, Dmitry; von Klitzing, Regine

    2012-01-01

    Scanning- and colloidal-probe atomic force microscopy were used to study the mechanical properties of poly(L-lysine)/hyaluronan (PLL/HA)(n) films as a function of indentation velocity and the number of polymer deposition steps n. The film thickness was determined by two independent AFM-based methods: scratch-and-scan and newly developed full-indentation. The advantages and disadvantages of both methods are highlighted, and error minimization techniques in elasticity measurements are addressed. It was found that the film thickness increases linearly with the bilayer number n, ranging between 400 and 7500 nm for n = 12 and 96, respectively. The apparent Young's modulus E ranges between 15 and 40 kPa and does not depend on the indenter size or the film bilayer number n. Stress relaxation measurements show that PLL/HA films have a viscoelastic behaviour, regardless of their thickness. If indentation is performed several times at the same lateral position on the film, a viscous/plastic deformation takes place.

  11. Transactivation of the Receptor-tyrosine Kinase Ephrin Receptor A2 Is Required for the Low Molecular Weight Hyaluronan-mediated Angiogenesis That Is implicated in Tumor Progression*

    PubMed Central

    Lennon, Frances E; Mirzapoiazova, Tamara; Mambetsariev, Nurbek; Mambetsariev, Bolot; Salgia, Ravi; Singleton, Patrick A.

    2014-01-01

    Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, understanding the mechanism(s) by which angiogenesis occurs can have important therapeutic implications in numerous malignancies. We and others have demonstrated that low molecular weight hyaluronan (LMW-HA, ∼2500 Da) promotes endothelial cell (EC) barrier disruption and angiogenesis. However, the mechanism(s) by which this occurs is poorly defined. Our data indicate that treatment of human EC with LMW-HA induced CD44v10 association with the receptor-tyrosine kinase, EphA2, transactivation (tyrosine phosphorylation) of EphA2, and recruitment of the PDZ domain scaffolding protein, PATJ, to the cell periphery. Silencing (siRNA) CD44, EphA2, PATJ, or Dbs (RhoGEF) expression blocked LMW-HA-mediated angiogenesis (EC proliferation, migration, and tubule formation). In addition, silencing EphA2, PATJ, Src, or Dbs expression blocked LMW-HA-mediated RhoA activation. To translate our in vitro findings, we utilized a novel anginex/liposomal targeting of murine angiogenic endothelium with either CD44 or EphA2 siRNA and observed inhibition of LMW-HA-induced angiogenesis in implanted Matrigel plugs. Taken together, these results indicate LMW-HA-mediated transactivation of EphA2 is required for PATJ and Dbs membrane recruitment and subsequent RhoA activation required for angiogenesis. These results suggest that targeting downstream effectors of LMW-HA could be a useful therapeutic intervention for angiogenesis-associated diseases including tumor progression. PMID:25023279

  12. Beta-arrestin 1 is involved in the catabolic response stimulated by hyaluronan degradation in mouse chondrocytes.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Calatroni, Alberto; Campo, Salvatore

    2015-08-01

    Beta-arrestin-1 (β-arrestin-1) is an adaptor protein that functions in the termination of G-protein activation and seems to be involved in the mediation of the inflammatory response. Interleukin-1β (IL-1β) elicits the expression of inflammatory mediators through a mechanism involving hyaluronan (HA) degradation, thereby contributing to toll-like receptor 4 (TLR-4) and CD44 activation. Stimulation of both receptors induces nuclear factor kappaB (NF-kB) activation that, through transforming-growth-factor-activated-kinase-1 (TAK-1), in turn stimulates the inflammatory mediators of transcription. As β-arrestin-1 seems to play an inflammatory role in arthritis, we have investigated the involvement of β-arrestin-1 in a model of IL-1β-induced inflammatory response in mouse chondrocytes. IL-1β treatment significantly increases chondrocytes TLR-4, CD44, β-arrestin-1, TAK-1, and serine/threonine kinase (AKT) mRNA expression and related protein levels. NF-kB is also markedly activated with consequent tumor-necrosis-factor-alpha, interleukin-6, and inducible-nitric-oxide-synthase up-regulation. Treatment of IL-1β-stimulated chondrocytes with β-arrestin-1 and/or AKT and/or TAK-1-specific inhibitors significantly reduces all parameters, although the inhibitory effect exerted by TAK-1-mediated pathways is more effective than that of β-arrestin-1. β-Arrestin-1-induced NF-kB activation is mediated by the AKT pathway as shown by IL-1β-stimulated chondrocytes treated with AKT inhibitor. Finally, a specific HA-blocking peptide (Pep-1) has confirmed the inflammatory role of degraded HA as a mediator of the IL-1β-induced activation of β-arrestin-1. PMID:25673209

  13. Beta-arrestin 1 is involved in the catabolic response stimulated by hyaluronan degradation in mouse chondrocytes.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Calatroni, Alberto; Campo, Salvatore

    2015-08-01

    Beta-arrestin-1 (β-arrestin-1) is an adaptor protein that functions in the termination of G-protein activation and seems to be involved in the mediation of the inflammatory response. Interleukin-1β (IL-1β) elicits the expression of inflammatory mediators through a mechanism involving hyaluronan (HA) degradation, thereby contributing to toll-like receptor 4 (TLR-4) and CD44 activation. Stimulation of both receptors induces nuclear factor kappaB (NF-kB) activation that, through transforming-growth-factor-activated-kinase-1 (TAK-1), in turn stimulates the inflammatory mediators of transcription. As β-arrestin-1 seems to play an inflammatory role in arthritis, we have investigated the involvement of β-arrestin-1 in a model of IL-1β-induced inflammatory response in mouse chondrocytes. IL-1β treatment significantly increases chondrocytes TLR-4, CD44, β-arrestin-1, TAK-1, and serine/threonine kinase (AKT) mRNA expression and related protein levels. NF-kB is also markedly activated with consequent tumor-necrosis-factor-alpha, interleukin-6, and inducible-nitric-oxide-synthase up-regulation. Treatment of IL-1β-stimulated chondrocytes with β-arrestin-1 and/or AKT and/or TAK-1-specific inhibitors significantly reduces all parameters, although the inhibitory effect exerted by TAK-1-mediated pathways is more effective than that of β-arrestin-1. β-Arrestin-1-induced NF-kB activation is mediated by the AKT pathway as shown by IL-1β-stimulated chondrocytes treated with AKT inhibitor. Finally, a specific HA-blocking peptide (Pep-1) has confirmed the inflammatory role of degraded HA as a mediator of the IL-1β-induced activation of β-arrestin-1.

  14. Generation of recombinant pandemic H1N1 influenza virus with the HA cleavable by bromelain and identification of the residues influencing HA bromelain cleavage.

    PubMed

    Wang, Weijia; Suguitan, Amorsolo L; Zengel, James; Chen, Zhongying; Jin, Hong

    2012-01-20

    The proteolytic enzyme bromelain has been traditionally used to cleave the hemagglutinin (HA) protein at the C-terminus of the HA2 region to release the HA proteins from influenza virions. The bromelain cleaved HA (BHA) has been routinely used as an antigen to generate antiserum that is essential for influenza vaccine product release. The HA of the 2009 pandemic H1N1 influenza A/California/7/2009 (CA09) virus could not be cleaved efficiently by bromelain. To ensure timely delivery of BHA for antiserum production, we generated a chimeric virus that contained the HA1 region from CA09 and the HA2 region from the seasonal H1N1 A/South Dakota/6/2007 (SD07) virus that is cleavable by bromelain. The BHA from this chimeric virus was antigenically identical to CA09 and induced high levels of HA-specific antibodies and protected ferrets from wild-type H1N1 CA09 virus challenge. To determine the molecular basis of inefficient cleavage of CA09 HA by bromelain, the amino acids that differed between the HA2 of CA09 and SD07 were introduced into recombinant CA09 virus to assess their effect on bromelain cleavage. The D373N or E374G substitution in the HA2 stalk region of CA09 HA enabled efficient cleavage of CA09 HA by bromelain. Sequence analysis of the pandemic H1N1-like viruses isolated from 2010 revealed emergence of the E374K change. We found that K374 enabled the HA to be cleaved by bromelain and confirmed that the 374 residue is critical for HA bromelain cleavage.

  15. In situ cross-linkable hydrogel of hyaluronan produced via copper-free click chemistry.

    PubMed

    Takahashi, Akira; Suzuki, Yukimitsu; Suhara, Takashi; Omichi, Kiyohiko; Shimizu, Atsushi; Hasegawa, Kiyoshi; Kokudo, Norihiro; Ohta, Seiichi; Ito, Taichi

    2013-10-14

    Injectable hydrogels are useful in biomedical applications. We have synthesized hyaluronic acids chemically modified with azide groups (HA-A) and cyclooctyne groups (HA-C), respectively. Aqueous HA-A and HA-C solutions were mixed using a double-barreled syringe to form a hydrogel via strain-promoted [3 + 2] cycloaddition without any catalyst at physiological conditions. The hydrogel slowly degraded in PBS over 2 weeks, which was accelerated to 9 days by hyaluronidase, while it rapidly degraded in a cell culture media with fetal bovine serum within 4 days. Both HA-A and HA-C showed good biocompatibility with fibroblast cells in vitro. They were administered using the double-barreled syringe into mice subcutaneously and intraperitoneally. Residue of the hydrogel was cleared 21 days after subcutaneous administration, while it was cleared 7 days after intraperitoneal administration. This injectable HA hydrogel is expected to be useful for tissue engineering and drug delivery systems utilizing its orthogonality. PMID:24004342

  16. Hyaluronan Hybrid Cooperative Complexes as a Novel Frontier for Cellular Bioprocesses Re-Activation

    PubMed Central

    Stellavato, Antonietta; Corsuto, Luisana; D’Agostino, Antonella; La Gatta, Annalisa; Diana, Paola; Bernini, Patrizia; De Rosa, Mario

    2016-01-01

    Hyaluronic Acid (HA)-based dermal formulations have rapidly gained a large consensus in aesthetic medicine and dermatology. HA, highly expressed in the Extracellular Matrix (ECM), acts as an activator of biological cascades, stimulating cell migration and proliferation, and operating as a regulator of the skin immune surveillance, through specific interactions with its receptors. HA may be used in topical formulations, as dermal inducer, for wound healing. Moreover, intradermal HA formulations (injectable HA) provide an attractive tool to counteract skin aging (e.g., facial wrinkles, dryness, and loss of elasticity) and restore normal dermal functions, through simple and minimally invasive procedures. Biological activity of a commercially available hyaluronic acid, Profhilo®, based on NAHYCO™ technology, was compared to H-HA or L-HA alone. The formation of hybrid cooperative complexes was confirmed by the sudden drop in η0 values in the rheological measurements. Besides, hybrid cooperative complexes proved stable to hyaluronidase (BTH) digestion. Using in vitro assays, based on keratinocytes, fibroblasts cells and on the Phenion® Full Thickness Skin Model 3D, hybrid cooperative complexes were compared to H-HA, widely used in biorevitalization procedures, and to L-HA, recently proposed as the most active fraction modulating the inflammatory response. Quantitative real-time PCR analyses were accomplished for the transcript quantification of collagens and elastin. Finally immunofluorescence staining permitted to evaluate the complete biosynthesis of all the molecules investigated. An increase in the expression levels of type I and type III collagen in fibroblasts and type IV and VII collagen in keratinocytes were found with the hybrid cooperative complexes, compared to untreated cells (CTR) and to the H-HA and L-HA treatments. The increase in elastin expression found in both cellular model and in the Phenion® Full Thickness Skin Model 3D also at longer time (up

  17. Hyaluronan secretion into the synovial cavity of rabbit knees and comparison with albumin turnover.

    PubMed Central

    Coleman, P J; Scott, D; Ray, J; Mason, R M; Levick, J R

    1997-01-01

    1. Hyaluronan is not only a lubricant but also enhances the synovial lining's resistance to fluid outflow. This finding led to the proposal that hyaluronan (> 2 x 10(6) Da, approximately 210 nm radius) may escape across the synovial lining less freely than smaller solutes (e.g. albumin, 6.7 x 10(4) Da, 3.6 nm radius) or water. Here multiple washouts were used to measure intraarticular hyaluronan mass and secretion rate in rabbit knees, leading to an estimate of hyaluronan turnover time. Plasma albumin permeation into the joint cavity was also measured to enable comparison of turnover times between molecules of very disparate size. 2. Endogenous hyaluronan mass in the joint cavity, analysed by high performance liquid chromatography of joint washes, was 182 +/- 9.9 micrograms (mean +/- S.E.M; n = 21). Since hyaluronan concentration in synovial fluid averages 3.62 +/- 0.19 micrograms microliters-1, the endogenous synovial fluid volume was calculated to be 50 microliters (mass/concentration), about double the aspiratable volume. 3. The hyaluronan secretion rate over 4 h was 4.80 +/- 0.77 micrograms h-1 (n = 5). The rate was significantly higher in contralateral joints expanded by 2 ml Ringer solution (5.80 +/- 0.84 micrograms h-1, n = 5, P = 0.01, Student's paired t test), indicating a stretch/hydration sensitive secretory mechanism. The newly secreted chains ((2.05-2.48) x 10(6) Da) were not significantly different in length from the endogenous chains (2.95 x 10(6) Da). 4. Hyaluronan turnover time, calculated as mass/secretion rate, was 31.4-37.9 h. This is more than an order of magnitude longer than turnover time for intra-articular albumin. The latter, determined from the intra-articular albumin mass and plasma-to-cavity permeation rate was 1.8 h (95% confidence intervals 1.2-3.5 h, n = 9). The big difference in turnover times support the view that, relative to albumin and water, hyaluronan is partially sieved out and retained in the joint cavity by the synovial

  18. Synergistic interaction of platelet derived growth factor (PDGF) with the surface of PLLA/Col/HA and PLLA/HA scaffolds produces rapid osteogenic differentiation.

    PubMed

    Raghavendran, Hanumantha Rao Balaji; Mohan, Saktiswaren; Genasan, Krishnamurithy; Murali, Malliga Raman; Naveen, Sangeetha Vasudevaraj; Talebian, Sepehr; McKean, Robert; Kamarul, Tunku

    2016-03-01

    Scaffolds with structural features similar to the extracellular matrix stimulate rapid osteogenic differentiation in favorable microenvironment and with growth factor supplementation. In this study, the osteogenic potential of electrospun poly-l-lactide/hydroxyapatite/collagen (PLLA/Col/HA, PLLA/HA and PLLA/Col) scaffolds were tested in vitro with the supplementation of platelet derived growth factor-BB (PDGF-BB). Cell attachment and topography, mineralization, extracellular matrix protein localization, and gene expression of the human mesenchymal stromal cells were compared between the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA. The levels of osteocalcin, calcium, and mineralization were significantly greater in the PLLA/Col/HA and PLLA/HA compared with PLLA/Col. High expression of fibronectin, intracellular adhesion molecule, cadherin, and collagen 1 (Col1) suggests that PLLA/Col/HA and PLLA/HA scaffolds had superior osteoinductivity than PLLA/Col. Additionally, osteopontin, osteocalcin, osterix, Runt-related transcription factor 2 (Runx2), and bone morphogenic protein (BMP2) expression were higher in PLLA/Col/HA and PLLA/HA compared with PLLA/Col. In comparison with PLLA/Col, the PLLA/Col/HA and PLLA/HA scaffolds presented a significant upregulation of the genes Runx2, Col 1, Integrin, osteonectin (ON), bone gamma-carboxyglutamic acid-containing protein (BGALP), osteopontin (OPN), and BMP2. The upregulation of these genes was further increased with PDGF-BB supplementation. These results show that PDGF-BB acts synergistically with PLLA/Col/HA and PLLA/HA to enhance the osteogenic differentiation potential. Therefore, this combination can be used for the rapid expansion of bone marrow stromal cells into bone-forming cells for tissue engineering.

  19. Synergistic interaction of platelet derived growth factor (PDGF) with the surface of PLLA/Col/HA and PLLA/HA scaffolds produces rapid osteogenic differentiation.

    PubMed

    Raghavendran, Hanumantha Rao Balaji; Mohan, Saktiswaren; Genasan, Krishnamurithy; Murali, Malliga Raman; Naveen, Sangeetha Vasudevaraj; Talebian, Sepehr; McKean, Robert; Kamarul, Tunku

    2016-03-01

    Scaffolds with structural features similar to the extracellular matrix stimulate rapid osteogenic differentiation in favorable microenvironment and with growth factor supplementation. In this study, the osteogenic potential of electrospun poly-l-lactide/hydroxyapatite/collagen (PLLA/Col/HA, PLLA/HA and PLLA/Col) scaffolds were tested in vitro with the supplementation of platelet derived growth factor-BB (PDGF-BB). Cell attachment and topography, mineralization, extracellular matrix protein localization, and gene expression of the human mesenchymal stromal cells were compared between the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA. The levels of osteocalcin, calcium, and mineralization were significantly greater in the PLLA/Col/HA and PLLA/HA compared with PLLA/Col. High expression of fibronectin, intracellular adhesion molecule, cadherin, and collagen 1 (Col1) suggests that PLLA/Col/HA and PLLA/HA scaffolds had superior osteoinductivity than PLLA/Col. Additionally, osteopontin, osteocalcin, osterix, Runt-related transcription factor 2 (Runx2), and bone morphogenic protein (BMP2) expression were higher in PLLA/Col/HA and PLLA/HA compared with PLLA/Col. In comparison with PLLA/Col, the PLLA/Col/HA and PLLA/HA scaffolds presented a significant upregulation of the genes Runx2, Col 1, Integrin, osteonectin (ON), bone gamma-carboxyglutamic acid-containing protein (BGALP), osteopontin (OPN), and BMP2. The upregulation of these genes was further increased with PDGF-BB supplementation. These results show that PDGF-BB acts synergistically with PLLA/Col/HA and PLLA/HA to enhance the osteogenic differentiation potential. Therefore, this combination can be used for the rapid expansion of bone marrow stromal cells into bone-forming cells for tissue engineering. PMID:26700235

  20. Wear effects on microscopic morphology and hyaluronan uptake in siloxane-hydrogel contact lenses.

    PubMed

    Tavazzi, Silvia; Tonveronachi, Martina; Fagnola, Matteo; Cozza, Federica; Ferraro, Lorenzo; Borghesi, Alessandro; Ascagni, Miriam; Farris, Stefano

    2015-07-01

    The purpose of this study was a comparison between new and worn siloxane-hydrogel contact lenses in terms of microscopic structure, surface morphology, and loading of hyaluronan. The analyses were performed by scanning electron microscopy, with the support of the freeze-drying technique, and by fluorescence confocal microscopy. Along the depth profile of new lenses, a thin porous top layer was observed, which corresponds to the region of hyaluronan penetration inside well-defined channels. The time evolution was followed from one day to two weeks of daily wear, when a completely different scenario was found. Clear experimental evidence of a buggy surface was observed with several crests and regions of swelling, which could be filled by the hyaluronan solution. The modifications are attributed to the progressive relaxation of the structure of the polymeric network.

  1. Wear effects on microscopic morphology and hyaluronan uptake in siloxane-hydrogel contact lenses.

    PubMed

    Tavazzi, Silvia; Tonveronachi, Martina; Fagnola, Matteo; Cozza, Federica; Ferraro, Lorenzo; Borghesi, Alessandro; Ascagni, Miriam; Farris, Stefano

    2015-07-01

    The purpose of this study was a comparison between new and worn siloxane-hydrogel contact lenses in terms of microscopic structure, surface morphology, and loading of hyaluronan. The analyses were performed by scanning electron microscopy, with the support of the freeze-drying technique, and by fluorescence confocal microscopy. Along the depth profile of new lenses, a thin porous top layer was observed, which corresponds to the region of hyaluronan penetration inside well-defined channels. The time evolution was followed from one day to two weeks of daily wear, when a completely different scenario was found. Clear experimental evidence of a buggy surface was observed with several crests and regions of swelling, which could be filled by the hyaluronan solution. The modifications are attributed to the progressive relaxation of the structure of the polymeric network. PMID:25251841

  2. Effect of molecular weight on the exponential growth and morphology of hyaluronan/chitosan multilayers: a surface plasmon resonance spectroscopy and atomic force microscopy investigation.

    PubMed

    Kujawa, Piotr; Moraille, Patricia; Sanchez, Jacqueline; Badia, Antonella; Winnik, Françoise M

    2005-06-29

    The layer-by-layer growth of multilayer assemblies of two polysaccharides, the polyanion hyaluronan (HA) and the polycation chitosan (CH), was investigated using atomic force microscopy (AFM) and surface plasmon resonance (SPR) spectroscopy, with primary emphasis on the effect of the polysaccharide molecular weights on the film thickness and surface morphology. The HA/CH multilayers exhibit an exponential increase of the optical film thickness with the number of deposited bilayers. We show that the multilayer thickness at a given stage depends on the size of both CH, the diffusing polyelectrolyte, and HA, the non-diffusing species. Assemblies (12 bilayers) of high molecular weight polysaccharides (HA, 360,000; CH, 160,000) were twice as thick (approximately 900 nm vs approximately 450 nm) as those obtained with low molecular weight polymers (HA, 30,000; CH, 31,000), as assessed by AFM scratch tests. The exponential growth rate is the same for the high and low molecular weight pairs; the larger film thicknesses observed by SPR and by AFM arising from an earlier onset of the steep exponential growth phase in the case of the high molecular weight pair. In all cases, isolated islets form during the deposition of the first CH layer onto the underlying HA. Upon further film growth, individual islets coalesce into larger vermiculate features. The transition from distinct islands to vermiculate structures depends on the molecular weights of the polysaccharides and the lower molecular weight construct presents larger worm-like surface domains than the high molecular weight pair.

  3. Synergistic effect of targeting the epidermal growth factor receptor and hyaluronan synthesis in oesophageal squamous cell carcinoma cells

    PubMed Central

    Kretschmer, I; Freudenberger, T; Twarock, S; Fischer, J W

    2015-01-01

    Background and Purpose Worldwide, oesophageal cancer is the eighth most common cancer and has a very poor survival rate. In order to identify new tolerable treatment options for oesophageal squamous cell carcinoma (ESCC), erlotinib was tested with moderate efficacy in phase I and II studies. As 4-methylumbelliferone (4-MU), an hyaluronan (HA) synthesis inhibitor showed anti-cancer effects in vitro, and in ESCC xenograft tumours, we investigated whether the anti-cancer effects of erlotinib could be augmented by combining it with 4-MU. Experimental Approach ESCC cell lines were treated with erlotinib or gefitinib (1 μmol·L−1) and 4-MU (300 μmol·L−1), and the cell count, cell cycle progression and migration were determined as compared to the single agents and the solvent-control. Key Results The combination of erlotinib and 4-MU synergistically inhibited the proliferation of ESCC cell lines. Furthermore, the migration speed of ESCC cell line KYSE-410 in gap closure assays was significantly reduced by the combination of erlotinib and 4-MU. Decreased ERK phosphorylation could explain the anti-proliferative and anti-migratory effects in the combined treatment group. Finally, the combination was additionally able to decrease the growth of multicellular tumour spheroids, a three-dimensional cell culture model that was associated with sustained inhibition of ERK1/2 phosphorylation. Conclusions and Implications The combination of 4-MU and erlotinib showed promising anti-cancer efficacies in the ESCC cell lines. PMID:26140525

  4. Alterations in the Secretome of Clinically Relevant Preparations of Adipose-Derived Mesenchymal Stem Cells Cocultured with Hyaluronan

    PubMed Central

    Succar, Peter; Breen, Edmond J.; Kuah, Donald; Herbert, Benjamin R.

    2015-01-01

    Osteoarthritis (OA) can be a debilitating degenerative disease and is the most common form of arthritic disease. There is a general consensus that current nonsurgical therapies are insufficient for younger OA sufferers who are not candidates for knee arthroplasties. Adipose-derived mesenchymal stem cells (MSCs) therapy for the treatment of OA can slow disease progression and lead to neocartilage formation. The mechanism of action is secretion driven. Current clinical preparations from adipose tissue for the treatment of OA include autologous stromal vascular fraction (SVF), SVF plus mature adipocytes, and culture-purified MSCs. Herein we have combined these human adipose-derived preparations with Hyaluronan (Hylan G-F 20: Synvisc) in vitro and measured alterations in cytokine profile. SVF plus mature adipocytes showed the greatest decreased in the proinflammatory cytokines IL-1β, IFN-γ, and VEGF. MCP-1 and MIP-1α decreased substantially in the SVF preparations but not the purified MSCs. The purified MSC preparation was the only one to show increase in MIF. Overall the SVF plus mature adipocytes preparation may be most suited of all the preparations for combination with HA for the treatment of OA, based on the alterations of heavily implicated cytokines in OA disease progression. This will require further validation using in vivo models. PMID:26257790

  5. Hyaluronan scaffold supports osteogenic differentiation of bone marrow concentrate cells.

    PubMed

    Cavallo, C; Desando, G; Ferrari, A; Zini, N; Mariani, E; Grigolo, B

    2016-01-01

    Osteochondral lesions are considered a challenge for orthopedic surgeons. Currently, the treatments available are often unsatisfactory and unable to stimulate tissue regeneration. Tissue engineering offers a new therapeutic strategy, taking into account the role exerted by cells, biomaterial and growth factors in restoring tissue damage. In this light, Mesenchymal Stem Cells (MSCs) have been indicated as a fascinating tool for regenerative medicine thanks to their ability to differentiate into bone, cartilage and adipose tissue. However, in vitro-cultivation of MSCs could be associated with some risks such as de-differentiation/reprogramming, infection and contaminations of the cells. To overcome these shortcomings, a new approach is represented by the use of Bone Marrow Concentrate (BMC), that could allow the delivery of cells surrounded by their microenvironment in injured tissue. For this purpose, cells require a tridimensional scaffold that can support their adhesion, proliferation and differentiation. This study is focused on the potentiality of BMC seeded onto a hyaluronan-based scaffold (Hyaff-11) to differentiate into osteogenic lineage. This process depends on the specific interaction between cells derived from bone marrow (surrounded by their niche) and scaffold, that create an environment able to support the regeneration of damaged tissue. The data obtained from the present study demonstrate that BMC grown onto Hyaff-11 are able to differentiate toward osteogenic sense, producing specific osteogenic genes and matrix proteins.

  6. Hyaluronan scaffold supports osteogenic differentiation of bone marrow concentrate cells.

    PubMed

    Cavallo, C; Desando, G; Ferrari, A; Zini, N; Mariani, E; Grigolo, B

    2016-01-01

    Osteochondral lesions are considered a challenge for orthopedic surgeons. Currently, the treatments available are often unsatisfactory and unable to stimulate tissue regeneration. Tissue engineering offers a new therapeutic strategy, taking into account the role exerted by cells, biomaterial and growth factors in restoring tissue damage. In this light, Mesenchymal Stem Cells (MSCs) have been indicated as a fascinating tool for regenerative medicine thanks to their ability to differentiate into bone, cartilage and adipose tissue. However, in vitro-cultivation of MSCs could be associated with some risks such as de-differentiation/reprogramming, infection and contaminations of the cells. To overcome these shortcomings, a new approach is represented by the use of Bone Marrow Concentrate (BMC), that could allow the delivery of cells surrounded by their microenvironment in injured tissue. For this purpose, cells require a tridimensional scaffold that can support their adhesion, proliferation and differentiation. This study is focused on the potentiality of BMC seeded onto a hyaluronan-based scaffold (Hyaff-11) to differentiate into osteogenic lineage. This process depends on the specific interaction between cells derived from bone marrow (surrounded by their niche) and scaffold, that create an environment able to support the regeneration of damaged tissue. The data obtained from the present study demonstrate that BMC grown onto Hyaff-11 are able to differentiate toward osteogenic sense, producing specific osteogenic genes and matrix proteins. PMID:27358127

  7. Mechanical force effect on the two-state equilibrium of the hyaluronan-binding domain of CD44 in cell rolling

    PubMed Central

    Suzuki, Takashi; Suzuki, Miho; Ogino, Shinji; Umemoto, Ryo; Nishida, Noritaka; Shimada, Ichio

    2015-01-01

    CD44 is the receptor for hyaluronan (HA) and mediates cell rolling under fluid shear stress. The HA-binding domain (HABD) of CD44 interconverts between a low-affinity, ordered (O) state and a high-affinity, partially disordered (PD) state, by the conformational change of the C-terminal region, which is connected to the plasma membrane. To examine the role of tensile force on CD44-mediated rolling, we used a cell-free rolling system, in which recombinant HABDs were attached to beads through a C-terminal or N-terminal tag. We found that the rolling behavior was stabilized only at high shear stress, when the HABD was attached through the C-terminal tag. In contrast, no difference was observed for the beads coated with HABD mutants that constitutively adopt either the O state or the PD state. Steered molecular dynamics simulations suggested that the force from the C terminus disrupts the interaction between the C-terminal region and the core of the domain, thus providing structural insights into how the mechanical force triggers the allosteric O-to-PD transition. Based on these results, we propose that the force applied from the C terminus enhances the HABD–HA interactions by inducing the conformational change to the high-affinity PD transition more rapidly, thereby enabling CD44 to mediate lymphocyte trafficking and hematopoietic progenitor cell homing under high-shear conditions. PMID:26038553

  8. A single molecule assay to probe monovalent and multivalent bonds between hyaluronan and its key leukocyte receptor CD44 under force

    PubMed Central

    Bano, Fouzia; Banerji, Suneale; Howarth, Mark; Jackson, David G.; Richter, Ralf P.

    2016-01-01

    Glycosaminoglycans (GAGs), a category of linear, anionic polysaccharides, are ubiquitous in the extracellular space, and important extrinsic regulators of cell function. Despite the recognized significance of mechanical stimuli in cellular communication, however, only few single molecule methods are currently available to study how monovalent and multivalent GAG·protein bonds respond to directed mechanical forces. Here, we have devised such a method, by combining purpose-designed surfaces that afford immobilization of GAGs and receptors at controlled nanoscale organizations with single molecule force spectroscopy (SMFS). We apply the method to study the interaction of the GAG polymer hyaluronan (HA) with CD44, its receptor in vascular endothelium. Individual bonds between HA and CD44 are remarkably resistant to rupture under force in comparison to their low binding affinity. Multiple bonds along a single HA chain rupture sequentially and independently under load. We also demonstrate how strong non-covalent bonds, which are versatile for controlled protein and GAG immobilization, can be effectively used as molecular anchors in SMFS. We thus establish a versatile method for analyzing the nanomechanics of GAG·protein interactions at the level of single GAG chains, which provides new molecular-level insight into the role of mechanical forces in the assembly and function of GAG-rich extracellular matrices. PMID:27679982

  9. Oligosaccharides of Hyaluronan Activate Dendritic Cells via Toll-like Receptor 4

    PubMed Central

    Termeer, Christian; Benedix, Frauke; Sleeman, Jonathon; Fieber, Christina; Voith, Ursula; Ahrens, Thomas; Miyake, Kensuke; Freudenberg, Marina; Galanos, Christopher; Simon, Jan Christoph

    2002-01-01

    Low molecular weight fragmentation products of the polysaccharide of Hyaluronic acid (sHA) produced during inflammation have been shown to be potent activators of immunocompetent cells such as dendritic cells (DCs) and macrophages. Here we report that sHA induces maturation of DCs via the Toll-like receptor (TLR)-4, a receptor complex associated with innate immunity and host defense against bacterial infection. Bone marrow–derived DCs from C3H/HeJ and C57BL/10ScCr mice carrying mutant TLR-4 alleles were nonresponsive to sHA-induced phenotypic and functional maturation. Conversely, DCs from TLR-2–deficient mice were still susceptible to sHA. In accordance, addition of an anti–TLR-4 mAb to human monocyte–derived DCs blocked sHA-induced tumor necrosis factor α production. Western blot analysis revealed that sHA treatment resulted in distinct phosphorylation of p38/p42/44 MAP-kinases and nuclear translocation of nuclear factor (NF)-κB, all components of the TLR-4 signaling pathway. Blockade of this pathway by specific inhibitors completely abrogated the sHA-induced DC maturation. Finally, intravenous injection of sHA-induced DC emigration from the skin and their phenotypic and functional maturation in the spleen, again depending on the expression of TLR-4. In conclusion, this is the first report that polysaccharide degradation products of the extracellular matrix produced during inflammation might serve as an endogenous ligand for the TLR-4 complex on DCs. PMID:11781369

  10. HC-HA/PTX3 Purified From Amniotic Membrane as Novel Regenerative Matrix: Insight Into Relationship Between Inflammation and Regeneration

    PubMed Central

    Tseng, Scheffer C. G.

    2016-01-01

    Purpose Human limbal palisade of Vogt is an ideal model for studying and practicing regenerative medicine due to their accessibility. Nonresolving inflammation is a common manifestation of limbal stem cell deficiency, which is the major cause of corneal blindness, and presents as a threat to the success of transplanted limbal epithelial stem cells. Clinical studies have shown that the efficacy of transplantation of limbal epithelial stem cells can be augmented by transplantation of cryopreserved human amniotic membrane (AM), which exerts anti-inflammatory, antiscarring, and antiangiogenic action to promote wound healing. Methods Review of published data to determine the molecular action mechanism explaining how AM exerts the aforementioned therapeutic actions. Results From the water-soluble extract of cryopreserved AM, we have biochemically purified one novel matrix component termed heavy chain (HC)-hyaluronan (HA)/pentraxin 3 (PTX3) as the key relevant tissue characteristic responsible for the aforementioned AM's efficacy. Heavy chain–HA is a complex formed by a covalent linkage between HA and HC1 of inter-α-trypsin inhibitor (IαI) by tumor necrosis factor-stimulated gene-6 (TSG-6). This complex may then be tightly associated with PTX3 to form HC-HA/PTX3 complex. Besides exerting an anti-inflammatory, antiscarring, and antiangiogenic effects, HC-HA/PTX3 complex also uniquely maintains limbal niche cells to support the quiescence of limbal epithelial stem cells. Conclusions We envision that HC-HA/PTX3 purified from AM can be used as a unique substrate to refine ex vivo expansion of limbal epithelial stem cells by maintaining stem cell quiescence, self-renewal and fate decision. Furthermore, it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche. PMID:27116665

  11. Hyaluronan injection in murine osteoarthritis prevents TGFbeta 1-induced synovial neovascularization and fibrosis and maintains articular cartilage integrity by a CD44-dependent mechanism

    PubMed Central

    2012-01-01

    Introduction The mechanism by which intra-articular injection of hyaluronan (HA) ameliorates joint pathology is unknown. Animal studies have shown that HA can reduce synovial activation, periarticular fibrosis and cartilage erosion; however, its specific effects on the different cell types involved remain unclear. We have used the TTR (TGFbeta1 injection and Treadmill Running) model of murine osteoarthritis (OA), which exhibits many OA-like changes, including synovial activation, to examine in vivo tissue-specific effects of intra-articular HA. Methods The kinetics of clearance of fluorotagged HA from joints was examined with whole-body imaging. Naïve and treated knee joints were examined macroscopically for cartilage erosion, meniscal damage and fibrosis. Quantitative histopathology was done with Safranin O for cartilage and with Hematoxylin & Eosin for synovium. Gene expression in joint tissues for Acan, Col1a1, Col2a1, Col3a1, Col5a1, Col10a1, Adamts5 and Mmp13 was done by quantitative PCR. The abundance and distribution of aggrecan, collagen types I, II, III, V and X, ADAMTS5 and MMP13 were examined by immunohistochemistry. Results Injected HA showed a half-life of less than 2 h in the murine knee joint. At the tissue level, HA protected against neovascularization and fibrosis of the meniscus/synovium and maintained articular cartilage integrity in wild-type but not in Cd44 knockout mice. HA injection enhanced the expression of chondrogenic genes and proteins and blocked that of fibrogenic/degradative genes and proteins in cartilage/subchondral bone, whereas it blocked activation of both groups in meniscus/synovium. In all locations it reduced the expression/protein for Mmp13 and blocked Adamts5 expression but not its protein abundance in the synovial lining. Conclusions The injection of HA, 24 h after TGFbeta1 injection, inhibited the cascade of OA-like joint changes seen after treadmill use in the TTR model of OA. In terms of mechanism, tissue protection by

  12. [Ol'ha Petrivna Chepinoha--a founder of investigations of nucleic acids in biochemistry in Ukraine. To the 100th anniversary of birthday, 1.07.1907--27.04.1983].

    PubMed

    Vynohradova, R P

    2008-01-01

    Olga Petrivna Chepinoga, doctor of science (biology), senior scientific worker, was born on July 1, 1907, in Kyiv. She graduated from the 1st Kyiv Medical Institute (1927-1931). In 1931-1935 she worked at various medical institutions of Ukraine. In 1935 O. P. Chepinoga was employed by the Institute of Biochemistry of the National Academy of Sciences of the Ukr.SSR as a laborant, then as an assistant, junior and senior scientific worker. In 1940 O. P. Chepinoga defended a thesis for a Candidate's degree, and from 1941 she was given a rank of the senior scientific worker. During the Great Patriotic War she served in the armed forces of the Soviet Army (1941-1945) as a medical officer in the rank of captain. In 1944-1963 she worked at the Instutute of Biochemistry of the AS of the Ukr.SSR as a senior scientific worker, and in 1963-1965 headed the Laboratory of Nucleic Acids. In 1952 O. P. Chepinoga defended a thesis for Doctor's degree in biology On Biologic Role of Nucleic Acids. Investigations of O. P. Chepinoga were first devoted to oxidation processes in muscles in various physiologic conditions, physico-chemical properties of myosin and its ATPase activity. Since 1948 her scientific interests had been concentrating on studying the biologic role and metabolism of nucleic acids, their transformation in the organism in norm and in pathological states. She was the first to find that various proteins interacted with DNA molecule. The highest activity of DNAse and RNAse was revealed in the organs which permanently synthesize proteins (liver, spleen, pancreas). Under quantitative undifferentiated growth of malignant tumors (Brown-Pierse carcinoma and Crocker sarcoma) the great part belongs to the process of DNA disintegrations; DNAse activity increases considerably in the animal and human blood that is not observed at other somatic diseases and is of great diagnostic value. Considerable shifts in DNAse activity at various pathologies were not found. The enrichment of

  13. [Ol'ha Petrivna Chepinoha--a founder of investigations of nucleic acids in biochemistry in Ukraine. To the 100th anniversary of birthday, 1.07.1907--27.04.1983].

    PubMed

    Vynohradova, R P

    2008-01-01

    Olga Petrivna Chepinoga, doctor of science (biology), senior scientific worker, was born on July 1, 1907, in Kyiv. She graduated from the 1st Kyiv Medical Institute (1927-1931). In 1931-1935 she worked at various medical institutions of Ukraine. In 1935 O. P. Chepinoga was employed by the Institute of Biochemistry of the National Academy of Sciences of the Ukr.SSR as a laborant, then as an assistant, junior and senior scientific worker. In 1940 O. P. Chepinoga defended a thesis for a Candidate's degree, and from 1941 she was given a rank of the senior scientific worker. During the Great Patriotic War she served in the armed forces of the Soviet Army (1941-1945) as a medical officer in the rank of captain. In 1944-1963 she worked at the Instutute of Biochemistry of the AS of the Ukr.SSR as a senior scientific worker, and in 1963-1965 headed the Laboratory of Nucleic Acids. In 1952 O. P. Chepinoga defended a thesis for Doctor's degree in biology On Biologic Role of Nucleic Acids. Investigations of O. P. Chepinoga were first devoted to oxidation processes in muscles in various physiologic conditions, physico-chemical properties of myosin and its ATPase activity. Since 1948 her scientific interests had been concentrating on studying the biologic role and metabolism of nucleic acids, their transformation in the organism in norm and in pathological states. She was the first to find that various proteins interacted with DNA molecule. The highest activity of DNAse and RNAse was revealed in the organs which permanently synthesize proteins (liver, spleen, pancreas). Under quantitative undifferentiated growth of malignant tumors (Brown-Pierse carcinoma and Crocker sarcoma) the great part belongs to the process of DNA disintegrations; DNAse activity increases considerably in the animal and human blood that is not observed at other somatic diseases and is of great diagnostic value. Considerable shifts in DNAse activity at various pathologies were not found. The enrichment of

  14. Histomorphometric Evaluation of Cartilage Degradation using Rabbit Articular Chondrocytes Cultured in Alginate Beads − Effects of Hyaluronan

    PubMed Central

    Nakatsuka, K.; Kurita, K.; Hayakawa, Taro; Nakashima, Katsuhito; Yamashita, Kyoko; Hoshino, Takeshi; Miyazaki, Kyosuke

    2010-01-01

    Objective: A 3-dimensional alginate bead culturing method using rabbit articular chondrocytes was studied for the screening of the effectiveness of drugs for articular diseases. Design: The beads cultured with IL-1β, TGF-β, and Hyaluronan (HA) were evaluated histochemically with Alecian blue and immunohistochemically with CS-56 antibody. Chondrocytes in alginate beads were arbitrarily classified into four groups: 1) chodrocyte surrounded with cell-associated matrix (CAM) in which proteoglycan (PG) was positively stained (PG-possitive chondrocyte); 2) chondrocyte with PG-negative CAM; 3) PG-positive CAM alone, and 4) PG-negative CAM alone. Total sulfated GAG concentrations in the culture media were quantitated by dimethylmethylene blue (DMMB) assay. ProMMP-3, TIMP-1 and –2 concentrations in the culture media were determined by sandwich enzyme immunoassays. Results: Significant increase of PG-nagative cells were immunohistochemically found by IL-1β stimulation. The pretreatment with TGF-β almost fully suppressed those increase of PG-negative cells by IL-1β. Both GAG and proMMP-3 concentrations in the culture media were significantly increased after IL-1β stimulation. There were no significant differences in both TIMP-1 and TIMP-2 concentrations in the culture media with or without IL-1β stimulation. 800-kDa HA reduced significantly the number of PG-negative cells and proMMP-3 concentration in the culture media, but showed no effects on the concentrations of both TIMPs. Conclusions: Because this 3-dimensional chondrocyte culture in alginate beads is close to in vivo conditions, this method can be used for evaluation of the effectiveness of novel drugs for articular diseases. PMID:23675183

  15. Transforming growth factor-β1 induces EMT by the transactivation of epidermal growth factor signaling through HA/CD44 in lung and breast cancer cells.

    PubMed

    Li, Lingmei; Qi, Lisha; Liang, Zhijie; Song, Wangzhao; Liu, Yanxue; Wang, Yalei; Sun, Baocun; Zhang, Bin; Cao, Wenfeng

    2015-07-01

    Epithelial-mesenchymal transition (EMT), a process closely related to tumor development, is regulated by a variety of signaling pathways and growth factors, such as transforming growth factor-β1 (TGF-β1) and epidermal growth factor (EGF). Hyaluronan (HA) has been shown to induce EMT through either TGF-β1 or EGF signaling and to be a regulator of the crosstalk between these two pathways in fibroblasts. In this study, in order to clarify whether HA has the same effect in tumor cells, we utilized the lung cancer cell line, A549, and the breast cancer cell line, MCF-7, and found that the effects of stimulation with TGF-β1 were more potent than those of EGF in regulating the expression of EMT-associated proteins and in enhancing cell migration and invasion. In addition, we observed that TGF-β1 activated EGF receptor (EGFR) and its downstream AKT and extracellular signal-regulated kinase (ERK) pathways. Furthermore, we found that TGF-β1 upregulated the expression of hyaluronan synthases (HAS1, HAS2 and HAS3) and promoted the expression of CD44, a cell surface receptor for HA, which interacts with EGFR, resulting in the activation of the downstream AKT and ERK pathways. Conversely, treatment with 4-methylumbelliferone (4-MU; an inhibitor of HAS) prior to stimulation with TGF-β1, inhibited the expression of CD44 and EGFR, abolished the interaction between CD44 and EGFR. Furthermore, the use of shRNA targeting CD44 impaired the expression of EGFR, deactivated the AKT and ERK pathways, reversed EMT and decreased the migration and invasion ability of cells. In conclusion, our data demonstrate that TGF-β1 induces EMT by the transactivation of EGF signaling through HA/CD44 in lung and breast cancer cells.

  16. Morphological Effects of HA on the Cell Compatibility of Electrospun HA/PLGA Composite Nanofiber Scaffolds

    PubMed Central

    Haider, Adnan; Gupta, Kailash Chandra; Kang, Inn-Kyu

    2014-01-01

    Tissue engineering is faced with an uphill challenge to design a platform with appropriate topography and suitable surface chemistry, which could encourage desired cellular activities and guide bone tissue regeneration. To develop such scaffolds, composite nanofiber scaffolds of nHA and sHA with PLGA were fabricated using electrospinning technique. nHA was synthesized using precipitation method, whereas sHA was purchased. The nHA and sHA were suspended in PLGA solution separately and electrospun at optimized electrospinning parameters. The composite nanofiber scaffolds were characterized by FE-SEM, EDX analysis, TEM, XRD analysis, FTIR, and X-ray photoelectron. The potential of the HA/PLGA composite nanofiber as bone scaffolds in terms of their bioactivity and biocompatibility was assessed by culturing the osteoblastic cells onto the composite nanofiber scaffolds. The results from in vitro studies revealed that the nHA/PLGA composite nanofiber scaffolds showed higher cellular adhesion, proliferation, and enhanced osteogenesis performance, along with increased Ca+2 ions release compared to the sHA/PLGA composite nanofiber scaffolds and pristine PLGA nanofiber scaffold. The results show that the structural dependent property of HA might affect its potential as bone scaffold and implantable materials in regenerative medicine and clinical tissue engineering. PMID:24719853

  17. Morphological effects of HA on the cell compatibility of electrospun HA/PLGA composite nanofiber scaffolds.

    PubMed

    Haider, Adnan; Gupta, Kailash Chandra; Kang, Inn-Kyu

    2014-01-01

    Tissue engineering is faced with an uphill challenge to design a platform with appropriate topography and suitable surface chemistry, which could encourage desired cellular activities and guide bone tissue regeneration. To develop such scaffolds, composite nanofiber scaffolds of nHA and sHA with PLGA were fabricated using electrospinning technique. nHA was synthesized using precipitation method, whereas sHA was purchased. The nHA and sHA were suspended in PLGA solution separately and electrospun at optimized electrospinning parameters. The composite nanofiber scaffolds were characterized by FE-SEM, EDX analysis, TEM, XRD analysis, FTIR, and X-ray photoelectron. The potential of the HA/PLGA composite nanofiber as bone scaffolds in terms of their bioactivity and biocompatibility was assessed by culturing the osteoblastic cells onto the composite nanofiber scaffolds. The results from in vitro studies revealed that the nHA/PLGA composite nanofiber scaffolds showed higher cellular adhesion, proliferation, and enhanced osteogenesis performance, along with increased Ca(+2) ions release compared to the sHA/PLGA composite nanofiber scaffolds and pristine PLGA nanofiber scaffold. The results show that the structural dependent property of HA might affect its potential as bone scaffold and implantable materials in regenerative medicine and clinical tissue engineering. PMID:24719853

  18. Comparative chemical evaluation of two commercially available derivatives of hyaluronic acid (hylaform from rooster combs and restylane from streptococcus) used for soft tissue augmentation.

    PubMed

    Manna, F; Dentini, M; Desideri, P; De Pità, O; Mortilla, E; Maras, B

    1999-11-01

    Hyaluronic acid (HA) derivatives have been developed to try to enhance rheological properties of this molecule to make it suitable for various medical applications. The main dermatological application of HA derivatives is the augmentation of soft tissues, via injection into the dermis. HA derivatives are indicated for the correction of cutaneous contour deficiencies of the skin, particularly in cases of ageing or degenerative lesions or to increase lips. Two HA derivatives have been evaluated: Hylaform Viscoelastic Gel (Hylan B), derived from rooster combs and subjected to cross-linking, and Restylane, produced through bacterial fermentation (streptococci) and stabilized, as declared by the producer. In both cases the purpose is to improve HA theological characteristics and slow down its degradation once it is in contact with biological structures. Distribution of particle dimensions, pH, protein concentration and rheological properties have been investigated in order to evaluate their reliability as fillers for soft tissue augmentation. The results of the analyses showed that there are differences between Restylane and Hylaform. Especially as far as rheological characteristics are concerned, the results outline different structures of the products: Hylaform behaves as a strong hydrogel, Restylane as a weak hydrogel; rheologically Hylaform is clearly superior to Restylane. Hylaform contains a definitely minor quantity (about a quarter) of cross-linked hyaluronic acid than Restylane. Furthermore, although not declared by the manufacturer, Restylane contains protein, resulting from bacterial fermentation or added to enable cross-linking reaction; the quantity of proteins contained by Restylane can be as much as four times the quantity contained by Hylaform, for the same volume (1 ml). It is evident that Hylaform offers higher safety margin than Restylane. Furthermore, wide literature and 20 years of clinical experience on hyaluronan derived from rooster combs confirm

  19. Stimulation of small proteoglycan synthesis by the hyaluronan synthesis inhibitor 4-methylumbelliferone in human skin fibroblasts.

    PubMed

    Funahashi, Masaru; Nakamura, Toshiya; Kakizaki, Ikuko; Mizunuma, Hideki; Endo, Masahiko

    2009-01-01

    Human skin fibroblasts cultured with 4-methylumbelliferone (MU), a hyaluronan synthesis inhibitor, produce a hyaluronan-deficient extracellular matrix (See [9]). Our present study investigated the effects of MU on proteoglycan, which is the other main component of the extracellular matrix, and interacts with hyaluronan. Proteoglycans isolated from culture medium in the presence or absence of MU were characterized by gel-filtration chromatography, ion-exchange HPLC, electrophoresis, and immunoblotting. We found that MU had only a negligible effect on the synthesis of large proteoglycan but increased the production of small proteoglycan in comparison with cultures lacking MU. This small proteoglycan was identified by immunoblotting as decorin. The structures of decorin synthesized in the presence and absence of MU were compared by gel-filtration chromatography, and the data indicated that cells incubated with MU produced a larger decorin molecule than cells incubated without MU. Furthermore, the two decorins had galactosaminoglycan chains of different sizes. These results suggest that MU inhibits the synthesis of hyaluronan and accelerates production of the larger decorin in the extracellular matrix.

  20. Chondroitin sulfate addition to CD44H negatively regulates hyaluronan binding

    SciTech Connect

    Ruffell, Brian; Johnson, Pauline . E-mail: pauline@interchange.ubc.ca

    2005-08-26

    CD44 is a widely expressed cell adhesion molecule that binds hyaluronan, an extracellular matrix glycosaminoglycan, in a tightly regulated manner. This regulated interaction has been implicated in inflammation and tumor metastasis. CD44 exists in the standard form, CD44H, or as higher molecular mass isoforms due to alternative splicing. Here, we identify serine 180 in human CD44H as the site of chondroitin sulfate addition and show that lack of chondroitin sulfate addition at this site enhances hyaluronan binding by CD44. A CD44H-immunoglobulin fusion protein expressed in HEK293 cells, and CD44H expressed in murine L fibroblast cells were modified by chondroitin sulfate, as determined by reduced sulfate incorporation after chondroitinase ABC treatment. Mutation of serine 180 or glycine 181 in CD44H reduced chondroitin sulfate addition and increased hyaluronan binding, indicating that serine 180 is the site for chondroitin sulfate addition in CD44H and that this negatively regulates hyaluronan binding.

  1. Development and characterization of chitosan/hyaluronan film for transdermal delivery of thiocolchicoside.

    PubMed

    Bigucci, Federica; Abruzzo, Angela; Saladini, Bruno; Gallucci, Maria Caterina; Cerchiara, Teresa; Luppi, Barbara

    2015-10-01

    The objective of this study was the development of chitosan/hyaluronan transdermal films to improve bioavailability of thiocolchicoside. This approach offers the possibility to elude the first-pass metabolism and at the same time it is able to provide a predictable and extended duration of activity. Films were prepared by casting and drying of aqueous solutions containing different weight ratios of chitosan and hyaluronan and characterized for their physico-chemical and functional properties. In accordance with polymeric composition of films and, therefore, with the amount of the net charge after the complexation, films containing the same weight ratio of chitosan and hyaluronan showed lower water uptake ability with respect to films containing only one polymeric species or an excess of chitosan or hyaluronan. Moreover, the lower the hydration of the polymeric network, the lower is the drug diffusion through the films and its permeation through the skin. This study clearly confirmed that the selection of a suitable polymeric weight ratio and appropriate preparative conditions allows the modulation of film functional properties, suggesting that these formulations could be used as a novel technological platform for transdermal drug delivery.

  2. Effect of Fetal Size on Fetal Placental Hyaluronan and Hyaluronoglucosaminidases Throughout Gestation in the Pig

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous results indicated that the trophoblast-endometrial epithelial cell bilayer of porcine placenta undergoes microscopic folding during gestation, and the folded bilayer is embedded in placental stroma. We hypothesized that hyaluronan was a component of placental stroma, and that hyaluronidases...

  3. Reduction of sensory responses to passive movements of inflamed knee joints by hylan, a hyaluronan derivative.

    PubMed

    Pozo, M A; Balazs, E A; Belmonte, C

    1997-08-01

    Hyaluronan (sodium hyaluronate) is a glycosaminoglycan that is present in all joint tissues. Painful arthritic joints have been characterized by hyaluronan of reduced elastoviscosity. The purpose of this investigation was to determine whether hyaluronan has an influence on joint nociceptor sensitivity and whether restoration of elastoviscosity would decrease nerve responses from nociceptive afferent fibers in arthritic joints. Nerve impulse activity was recorded from nociceptive afferent fibers of the medial articular nerve in anesthetized cats. An acute experimental arthritis was produced by intra-articular injection of kaolin and carrageenan. This caused, within 3 h, the development of ongoing nerve activity and enhancement of nerve impulse responses to passive movements in the normal range of the joint. Intra-articular injection of an elastoviscous solution of hylan, a hyaluronan derivative, significantly reduced both the ongoing activity and the movement-evoked responses in 1-2 h. This effect was not obtained when a nonelastoviscous solution of hylan was injected into the inflamed joint. The results indicate that intra-articularly injected elastoviscous solutions of hylan reduced nociceptive activity in inflamed joints through an elastoviscous, rheological effect on nociceptive afferent fibers through the intercellular matrix in which these fibers are embedded.

  4. Influence of tiopronin, captopril and levamisole therapeutics on the oxidative degradation of hyaluronan.

    PubMed

    Valachová, Katarína; Baňasová, Mária; Topoľská, Dominika; Sasinková, Vlasta; Juránek, Ivo; Collins, Maurice N; Šoltés, Ladislav

    2015-12-10

    The ability to protect hyaluronic acid (HA) from oxidative degradation by cupric ions and ascorbate (production of (•)OH and peroxy-type radicals) during acute phase joint inflammation has been investigated using the following drugs: tiopronin, captopril, and levamisole. Radical scavenging activity, i.e. the propensity for donation of electrons was assessed for the drugs by ABTS and DPPH assays. The kinetics of HA degradation have been measured in the presence of each drug using rotational viscometry. The results of ABTS and DPPH assays show the highest radical scavenging activity for captopril, followed by tiopronin. For levamisole, no effect was observed. Captopril and tiopronin prevented HA degradation induced by (•)OH radicals in a similar manner, while tiopronin was more effective in scavenging peroxy-type radicals. On the other hand, levamisole was shown to be a pro-oxidant. Recovered HA fragments were characterized using FT-IR analysis, the incorporation of a sulphur atom from captopril and tiopronin but not from levamisole into the HA molecule was demonstrated.

  5. Stabilin-1 and -2 constitute a novel family of fasciclin-like hyaluronan receptor homologues.

    PubMed Central

    Politz, Oliver; Gratchev, Alexei; McCourt, Peter A G; Schledzewski, Kai; Guillot, Pierre; Johansson, Sophie; Svineng, Gunbjorg; Franke, Peter; Kannicht, Christoph; Kzhyshkowska, Julia; Longati, Paola; Velten, Florian W; Johansson, Staffan; Goerdt, Sergij

    2002-01-01

    MS-1, a high-molecular-mass protein expressed by non-continuous and angiogenic endothelial cells and by alternatively activated macrophages (Mphi2), and the hepatic sinusoidal endothelial hyaluronan clearance receptor are similar with respect to tissue distribution and biochemical characteristics. In the present study we purified these proteins by immuno- and hyaluronan-affinity chromatography respectively, sequenced tryptic peptides and generated full-length cDNA sequences in both mouse and human. The novel genes, i.e. stabilin-1 and stabilin-2, code for homologous transmembrane proteins featuring seven fasciclin-like adhesion domains, 18-20 epidermal-growth-factor domains, one X-link domain and three to six B-(X(7))-B hyaluronan-binding motifs. Northern-blotting experiments revealed the presence of both stabilins in organs with predominant endothelial sinuses such as liver, spleen and lymph node: stabilin-1 mRNA was also detected in organs with predominant Mphi2 cells, such as placenta, and in interleukin-4/glucocorticoid-stimulated Mphi2 cells in vitro. A polyclonal antibody made against human recombinant stabilin-1 confirmed the expression of stabilin-1 protein in splenic sinus endothelial cells in vivo and in Mphi2 in vitro. On the basis of high similarity at the protein level and the unique domain composition, which differs from that of all other known fasciclin-like proteins and hyaluronan receptors, stabilin-1 and stabilin-2 define a novel family of fasciclin-like hyaluronan receptor homologues that might play a role in cell-cell and cell-matrix interactions in vascular function and inflammatory processes. PMID:11829752

  6. Regulation of glioma cell phenotype in 3D matrices by hyaluronic acid.

    PubMed

    Pedron, Sara; Becka, Eftalda; Harley, Brendan A C

    2013-10-01

    Human glioblastoma multiforme (hGBM) is the most common, aggressive, and deadly form of brain cancer. A major obstacle to understanding the impact of extracellular cues on glioblastoma invasion is the absence of model matrix systems able to replicate compositional and structural elements of the glioma mass as well as the surrounding brain tissue. Contact with a primary extracellular matrix component in the brain, hyaluronan, is believed to play a pivotal role in glioma cell invasion and malignancy. In this study we report use of gelatin and poly(ethylene glycol) (PEG) based hydrogel platforms to evaluate the effect of extracellular (composition, mechanics, HA incorporation) and intracellular (epidermal growth factor receptor overexpression) factors on the malignant transformation of U87MG glioma cells. Three-dimensional culture platforms elicit significantly different responses of U87MG glioma cells versus standard 2D culture. Critically, grafting brain-mimetic hyaluronic acid (HA) into the hydrogel network was found to induce significant, dose-dependent alterations of markers of glioma malignancy versus non-grafted 3D gelatin or PEG hydrogels. Clustering of glioma cells was observed exclusively in HA containing gels and expression profiles of malignancy-associated genes were found to vary biphasically with incorporated HA content. We also found HA-induced expression of MMP-2 is blocked by +EGFR signaling, suggesting a connection between CD44 and EGFR in glioma malignancy. Together, this work describes an adaptable platform for manipulating the local extracellular microenvironment surrounding glioma cells and highlights the importance of developing such systems for investigating the etiology and early growth of glioblastoma multiforme tumors. PMID:23827186

  7. The SOD mimic MnTM-2-PyP(5+) reduces hyaluronan degradation-induced inflammation in mouse articular chondrocytes stimulated with Fe (II) plus ascorbate.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Nastasi, Giancarlo; Micali, Antonio; Puzzolo, Domenico; Pisani, Antonina; Calatroni, Alberto; Campo, Salvatore

    2013-08-01

    In pathological conditions, oxidative burst generates hyaluronan (HA) fragmentation with a consequent increase in the number of small HA oligosaccharides. These fragments are able to stimulate an inflammatory response in different cell types by activating the CD44 and the toll-like receptors 4 (TLR-4) and 2 (TLR-2). The stimulation of CD44 and TLRs in turn activates the NF-kB which induces the production of several pro-inflammatory mediators that amplify and perpetuate inflammation. We aimed to study the antioxidant effect of the SOD mimic, synthetic manganese porphyrin, Mn(III) 5,10,15,20-tetrakis(N-methylpyridinium-2-yl)porphyrin (MnTM-2-PyP(5+)) on preventing HA degradation in mouse articular chondrocytes stimulated with Fe (II) plus ascorbate. Fe (II) plus ascorbate stimulation induced oxidative burst confirmed by high levels of hydroxyl radical/peroxynitrite production, increased lipid peroxidation and HA degradation. HA fragments highly induced mRNA expression and the related protein production of CD44, TLR-4 and TLR-2, NF-kB activation and significantly up-regulated the inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and other pro-inflammatory mediators, i.e. matrix metalloprotease 13 (MMP-13) and inducible nitric oxide synthase (iNOS). Treatment of cells with MnTM-2-PyP(5+)was able to attenuate oxidative burst, HA degradation and NF-kB activation, and markedly decreased mRNA expression of CD44, and TLRs and the related protein synthesis, as well as the levels of up-regulated inflammatory mediators. Adding a specific HA-blocking peptide (PEP-1) to cells significantly reduced all the inflammatory parameters up-regulated by Fe (II) plus ascorbate, and increased MnTM-2-PyP(5+) activity. These findings suggest that HA degradation plays a key role in the initial inflammatory response of cartilage and antioxidants and could be a useful tool to prevent the propagation of this mechanism. PMID:23692848

  8. Advances and advantages of nanomedicine in the pharmacological targeting of hyaluronan-CD44 interactions and signaling in cancer.

    PubMed

    Skandalis, Spyros S; Gialeli, Chrisostomi; Theocharis, Achilleas D; Karamanos, Nikos K

    2014-01-01

    Extensive experimental evidence in cell and animal tumor models show that hyaluronan-CD44 interactions are crucial in both malignancy and resistance to cancer therapy. Because of the intimate relationship between the hyaluronan-CD44 system and tumor cell survival and growth, it is an increasingly investigated area for applications to anticancer chemotherapeutics. Interference with the hyaluronan-CD44 interaction by targeting drugs to CD44, targeting drugs to the hyaluronan matrix, or interfering with hyaluronan matrix/tumor cell-associated CD44 interactions is a viable strategy for cancer treatment. Many of these methods can decrease tumor burden in animal models but have yet to show significant clinical utility. Recent advances in nanomedicine have offered new valuable tools for cancer detection, prevention, and treatment. The enhanced permeability and retention effect has served as key rationale for using nanoparticles to treat solid tumors. However, the targeted and uniform delivery of these particles to all regions of tumors in sufficient quantities requires optimization. An ideal nanocarrier should be equipped with selective ligands that are highly or exclusively expressed on target cells and thus endow the carriers with specific targeting capabilities. In this review, we describe how the hyaluronan-CD44 system may provide such an alternative in tumors expressing specific CD44 variants.

  9. [Hyaluronic acid].

    PubMed

    Pomarede, N

    2008-01-01

    Hyaluronic Acid (HA) is now a leader product in esthetic procedures for the treatment of wrinkles and volumes. The structure of HA, its metabolism, its physiological function are foremost breaking down then its use in aesthetic dermatology: steps of injection, possible side effects, benefits and downsides of the use of HA in aesthetic dermatology.

  10. Arrival time distributions of product ions reveal isomeric ratio of deprotonated molecules in ion mobility-mass spectrometry of hyaluronan-derived oligosaccharides.

    PubMed

    Hermannová, Martina; Iordache, Andreea-Maria; Slováková, Kristína; Havlíček, Vladimír; Pelantová, Helena; Lemr, Karel

    2015-06-01

    Hyaluronic acid is a naturally occurring linear polysaccharide with substantial medical potential. In this work, discrimination of tyramine-based hyaluronan derivatives was accessed by ion mobility-mass spectrometry of deprotonated molecules and nuclear magnetic resonance spectroscopy. As the product ion mass spectra did not allow for direct isomer discrimination in mixture, the reductive labeling of oligosaccharides as well as stable isotope labeling was performed. The ion mobility separation of parent ions together with the characteristic fragmentation for reduced isomers providing unique product ions allowed us to identify isomers present in a mixture and determine their mutual isomeric ratio. The determination used simple recalculation of arrival time distribution areas of unique ions to areas of deprotonated molecules. Mass spectrometry data were confirmed by nuclear magnetic resonance spectroscopy.

  11. Cationic Conjugated Polymer/Hyaluronan-Doxorubicin Complex for Sensitive Fluorescence Detection of Hyaluronidase and Tumor-Targeting Drug Delivery and Imaging.

    PubMed

    Huang, Yanqin; Song, Caixia; Li, Huichang; Zhang, Rui; Jiang, Rongcui; Liu, Xingfen; Zhang, Guangwei; Fan, Quli; Wang, Lianhui; Huang, Wei

    2015-09-30

    Hyaluronidase (HAase) is becoming a new type of tumor marker since it has been demonstrated to be overexpressed in various kinds of cancer cells. In this study, we described a novel fluorescence method for sensitive, rapid, and convenient HAase detection and tumor-targeting drug delivery and imaging, using a probe prepared by electrostatic assembly of a cationic conjugated polymer (CCP) and anionic hyaluronan (HA) conjugated with the anticancer drug doxorubicin (Dox). The CCP we used was poly{[9,9-bis(6'-(N,N,N-diethylmethylammonium)hexyl)-2,7-fluorenylene ethynylene]-alt-co-[2,5-bis(3'-(N,N,N-diethylmethylammonium)-1'-oxapropyl)-1,4-phenylene]} tetraiodide (PFEP). HA is a natural mucopolysaccharide that can be hydrolyzed by HAase into fragments with low molecular weights. In the PFEP/HA-Dox complex, the fluorescence of PFEP was efficiently quenched due to electron transfer from PFEP to Dox. After the PFEP/HA-Dox complex was exposed to HAase or was taken up by cancer cells through the specific binding between HA and CD44 receptor, HA was degraded by HAase to release the Dox, leading to the recovery of PFEP fluorescence to the "turn-on" state. Moreover, the degree of fluorescence recovery was quantitatively correlated with the concentrations of HAase. Compared with many previously reported methods, our work did not require laborious multiple modifications of HA that may affect the activity of HAase. This point, combined with the excellent optoelectronic property of conjugated polymer, endowed this method with high sensitivity (detection limit: 0.075 U/mL), high specificity, and rapid response, making it applicable for reliable and routine detection of HAase. This fluorescent probe was successfully utilized to detect HAase levels in human urine samples; furthermore, it can also be employed as a multifunctional system by realizing tumor-targeting drug delivery and cell imaging simultaneously. The development of this fluorescence method showed promising potential for

  12. The Human Hyaluronan Receptor for Endocytosis (HARE/Stabilin-2) Is a Systemic Clearance Receptor for Heparin*

    PubMed Central

    Harris, Edward N.; Weigel, Janet A.; Weigel, Paul H.

    2008-01-01

    The hyaluronic acid receptor for endocytosis (HARE; also designated Stabilin-2) mediates systemic clearance of hyaluronan and chondroitin sulfates from the vascular and lymphatic circulations. The internalized glycosaminoglycans are degraded in lysosomes, thus completing their normal turnover process. Sinusoidal endothelial cells of human liver, lymph node, and spleen express two HARE isoforms of 315 and 190 kDa. Here we report that the 190- and 315-kDa HARE isoforms, expressed stably either in Flp-In 293 cell lines or as soluble ectodomains, specifically bind heparin (Hep). The Kd for Hep binding to purified 190- and 315-kDa HARE ectodomains was 17.2 ± 4.9 and 23.4 ± 5.3 nm, respectively. Cells expressing HARE readily and specifically internalized 125I-streptavidin-biotin-Hep complexes, which was inhibited >70% by hyperosmolar conditions, confirming that uptake is mediated by the clathrin-coated pit pathway. Internalization of Hep occurred for many hours with an estimated HARE recycling time of ∼12 min. Internalized fluorescent streptavidin-biotin-Hep was present in a typical endocytic vesicular pattern and was delivered to lysosomes. We conclude that HARE in the sinusoidal endothelial cells of lymph nodes and liver likely mediates the efficient systemic clearance of Hep and many different Hep-binding protein complexes from the lymphatic and vascular circulations. PMID:18434317

  13. Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-{alpha}-dependent pathway in human dermal fibroblasts

    SciTech Connect

    Yamane, Takumi; Kobayashi-Hattori, Kazuo; Oishi, Yuichi

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. Black-Right-Pointing-Pointer Adiponectin also increases the phosphorylation of AMPK. Black-Right-Pointing-Pointer A pharmacological activator of AMPK increases mRNA levels of PPAR{alpha} and HAS2. Black-Right-Pointing-Pointer Adiponectin-induced HAS2 mRNA expression is blocked by a PPAR{alpha} antagonist. Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis via an AMPK/PPAR{alpha}-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis along with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1{beta}-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPAR{alpha} antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPAR{alpha}-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.

  14. Characterization of polyelectrolyte behavior of the polysaccharides chitosan, heparin, and hyaluronan, by light scattering and viscometry.

    NASA Astrophysics Data System (ADS)

    Boddohi, Soheil; Yonemura, Susan; Kipper, Matt

    2008-03-01

    This study on the polyelectrolyte behavior of polysaccharides in solution is motivated by our recent work in development of nanostructured polysaccharide-based surface coatings. Chitosan behaves as a weak polycation, and hyaluronan behaves as a weak polyanion, while heparin behaves as a strong polyanion. The ability to control the conformation of these polysaccharides in solution, by changing the solution ionic strength and pH may offer the opportunity to further tune the nanoscale features of polysaccharide-based surface coatings assembled from solution. In the work reported here, the solution conformation of these polymers is determined from gel permeation chromatography coupled to differential refractive index, light scattering, and viscometry detection. These results are related to the nanostructure of chitosan-heparin and chitosan-hyaluronan surface coatings based on polyelectrolyte multilayers.

  15. X-ray ablation of hyaluronan hydrogels: Fabrication of three-dimensional microchannel networks

    SciTech Connect

    Weon, B. M.; Chang, S.; Je, J. H.; Yeom, J.; Hahn, S. K.; Hwu, Y.; Margaritondo, G.

    2009-09-01

    We present a simple and highly versatile protocol for polymer ablation: hard x-ray irradiation makes it possible to rapidly depolymerize hyaluronan hydrogels and fabricate three-dimensional network of microchannels. Photodynamic and photochemical analyses show that x-ray irradiation directly cleaves the polymer backbone and the total dose controls the degradation kinetics. This nonthermal ablation protocol may offer opportunities for processing organic polymers and biological materials.

  16. Freeze-dried eudragit-hyaluronan multicompartment liposomes to improve the intestinal bioavailability of curcumin.

    PubMed

    Catalan-Latorre, Ana; Ravaghi, Maryam; Manca, Maria Letizia; Caddeo, Carla; Marongiu, Francesca; Ennas, Guido; Escribano-Ferrer, Elvira; Peris, José Esteban; Diez-Sales, Octavio; Fadda, Anna Maria; Manconi, Maria

    2016-10-01

    This work aimed at finding an innovative vesicle-type formulation able to improve the bioavailability of curcumin upon oral administration. To this purpose, phospholipid, Eudragit® S100 and hyaluronan sodium salt were combined to obtain eudragit-hyaluronan immobilized vesicles using an easy and environmentally-friendly method. For the first time, the two polymers were combined in a system intended for oral delivery, to enhance curcumin stability when facing the harsh environment of the gastrointestinal tract. Four different formulations were prepared, keeping constant the amount of the phospholipid and varying the eudragit-hyaluronan ratio. The freeze-drying of the samples, performed to increase their stability, led to a reduction of vesicle size and a good homogeneity of the systems, after simple rehydration with water. X-ray diffraction study demonstrated that after the freeze-drying process, curcumin remained successfully incorporated within the vesicles. All the vesicles displayed similar features: size ranging from 220 to 287nm, spherical or oval shape, multilamellar or large unilamellar morphology with a peculiar multicompartment organization involving 1-4 smaller vesicles inside. In vitro studies demonstrated the ability of the combined polymers to protect the vesicles from the harsh conditions of the gastro-intestinal tract (i.e., ionic strength and pH variation), which was confirmed in vivo by the greater deposition of curcumin in the intestinal region, as compared to the free drug in dispersion. This enhanced accumulation of curcumin provided by the eudragit-hyaluronan immobilized vesicles, together with an increase in Caco-2 cell viability exposed to hydrogen peroxide, indicated that vesicles can ensure a local protection against oxidative stress and an increase in its intestinal absorption. PMID:27349806

  17. Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro by inhibiting the p53-dependent mitochondrial apoptotic pathway

    PubMed Central

    Lee, Yoon-Jin; Kim, Soo A; Lee, Sang-Han

    2016-01-01

    Aim: Intra-articular injection of local anesthetics (LAs) is a common procedure for therapeutic purposes. However, LAs have been found toxic to articular cartilage, and hyaluronan may attenuate this toxicity. In this study we investigated whether hyaluronan attenuated lidocaine-induced chondrotoxicity, and if so, to elucidate the underlying mechanisms. Methods: Human chondrocyte cell line SW1353 and newly isolated murine chondrocytes were incubated in culture medium containing hyaluronan and/or lidocaine for 72 h. Cell viability was evaluated using MTT assay. Cell apoptosis was detected with DAPI staining, caspase 3/7 activity assay and flow cytometry. Cell cycle distributions, ROS levels and mitochondrial membrane potential (ΔΨm) were determined using flow cytometry. The expression of p53 and p53-regulated gene products was measured with Western blotting. Results: Lidocaine (0.005%−0.03%) dose-dependently decreased the viability of SW1353 cells. This local anesthetic (0.015%, 0.025%) induced apoptosis, G2/M phase arrest and loss of ΔΨm, and markedly increased ROS production in SW1353 cells. Hyaluronan (50−800 μg/mL) alone did not affect the cell viability, but co-treatment with hyaluronan (200 μg/mL) significantly attenuated lidocaine-induced apoptosis and other abnormalities in SW1353 cells. Furthermore, co-treatment with lidocaine and hyaluronan significantly decreased the levels of p53 and its transcription targets Bax and p21 in SW1353 cells, although treatment with lidocaine alone did not significantly change these proteins. Similar results were obtained in ex vivo cultured murine chondrocytes. Conclusion: Hyaluronan suppresses lidocaine-induced apoptosis of human chondrocytes in vitro through inhibiting the p53-dependent mitochondrial apoptotic pathway. PMID:27041463

  18. Bimodal Tumor-Targeting from Microenvironment Responsive Hyaluronan Layer-by-Layer (LbL) Nanoparticles

    PubMed Central

    2015-01-01

    Active targeting of nanoscale drug carriers can improve tumor-specific delivery; however, cellular heterogeneity both within and among tumor sites is a fundamental barrier to their success. Here, we describe a tumor microenvironment-responsive layer-by-layer (LbL) polymer drug carrier that actively targets tumors based on two independent mechanisms: pH-dependent cellular uptake at hypoxic tumor pH and hyaluronan-directed targeting of cell-surface CD44 receptor, a well-characterized biomarker for breast and ovarian cancer stem cells. Hypoxic pH-induced structural reorganization of hyaluronan-LbL nanoparticles was a direct result of the nature of the LbL electrostatic complex, and led to targeted cellular delivery in vitro and in vivo, with effective tumor penetration and uptake. The nanoscale drug carriers selectively bound CD44 and diminished cancer cell migration in vitro, while co-localizing with the CD44 receptor in vivo. Multimodal targeting of LbL nanoparticles is a powerful strategy for tumor-specific cancer diagnostics and therapy that can be accomplished using a single bilayer of polyamine and hyaluronan that, when assembled, produce a dynamic and responsive cell–particle interface. PMID:25100313

  19. Natural Antisense Transcript for Hyaluronan Synthase 2 (HAS2-AS1) Induces Transcription of HAS2 via Protein O-GlcNAcylation*

    PubMed Central

    Vigetti, Davide; Deleonibus, Sara; Moretto, Paola; Bowen, Timothy; Fischer, Jens W.; Grandoch, Maria; Oberhuber, Alexander; Love, Dona C.; Hanover, John A.; Cinquetti, Raffaella; Karousou, Eugenia; Viola, Manuela; D'Angelo, Maria Luisa; Hascall, Vincent C.; De Luca, Giancarlo; Passi, Alberto

    2014-01-01

    Changes in the microenvironment organization within vascular walls are critical events in the pathogenesis of vascular pathologies, including atherosclerosis and restenosis. Hyaluronan (HA) accumulation into artery walls supports vessel thickening and is involved in many cardiocirculatory diseases. Excessive cytosolic glucose can enter the hexosamine biosynthetic pathway, increase UDP-N-acetylglucosamine (UDP-GlcNAc) availability, and lead to modification of cytosolic proteins via O-linked attachment of the monosaccharide β-N-GlcNAc (O-GlcNAcylation) from UDP-GlcNAc by the enzyme O-GlcNAc transferase. As many cytoplasmic and nuclear proteins can be glycosylated by O-GlcNAc, we studied whether the expression of the HA synthases that synthesize HA could be controlled by O-GlcNAcylation in human aortic smooth muscle cells. Among the three HAS isoenzymes, only HAS2 mRNA increased after O-GlcNAcylation induced by glucosamine treatments or by inhibiting O-GlcNAc transferase with PUGNAC (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate). We found that the natural antisense transcript of HAS2 (HAS2-AS1) was absolutely necessary to induce the transcription of the HAS2 gene. Moreover, we found that O-GlcNAcylation modulated HAS2-AS1 promoter activation by recruiting the NF-κB subunit p65, but not the HAS2 promoter, whereas HAS2-AS1 natural antisense transcript, working in cis, regulated HAS2 transcription by altering the chromatin structure around the HAS2 proximal promoter via O-GlcNAcylation and acetylation. These results indicate that HAS2 transcription can be finely regulated not only by recruiting transcription factors to the promoter as previously described but also by modulating chromatin accessibility by epigenetic modifications. PMID:25183006

  20. High Sensitivity Method to Estimate Distribution of Hyaluronan Molecular Sizes in Small Biological Samples Using Gas-Phase Electrophoretic Mobility Molecular Analysis

    PubMed Central

    Do, Lan; Dahl, Christen P.; Kerje, Susanne; Hansell, Peter; Mörner, Stellan; Lindqvist, Ulla; Engström-Laurent, Anna; Larsson, Göran; Hellman, Urban

    2015-01-01

    Hyaluronan is a negatively charged polydisperse polysaccharide where both its size and tissue concentration play an important role in many physiological and pathological processes. The various functions of hyaluronan depend on its molecular size. Up to now, it has been difficult to study the role of hyaluronan in diseases with pathological changes in the extracellular matrix where availability is low or tissue samples are small. Difficulty to obtain large enough biopsies from human diseased tissue or tissue from animal models has also restricted the study of hyaluronan. In this paper, we demonstrate that gas-phase electrophoretic molecular mobility analyzer (GEMMA) can be used to estimate the distribution of hyaluronan molecular sizes in biological samples with a limited amount of hyaluronan. The low detection level of the GEMMA method allows for estimation of hyaluronan molecular sizes from different parts of small organs. Hence, the GEMMA method opens opportunity to attain a profile over the distribution of hyaluronan molecular sizes and estimate changes caused by disease or experimental conditions that has not been possible to obtain before. PMID:26448761

  1. Effect of bioactive extruded PLA/HA composite films on focal adhesion formation of preosteoblastic cells.

    PubMed

    Persson, Maria; Lorite, Gabriela S; Kokkonen, Hanna E; Cho, Sung-Woo; Lehenkari, Petri P; Skrifvars, Mikael; Tuukkanen, Juha

    2014-09-01

    The quality of the initial cell attachment to a biomaterial will influence any further cell function, including spreading, proliferation, differentiation and viability. Cell attachment is influenced by the material's ability to adsorb proteins, which is related to the surface chemistry and topography of the material. In this study, we incorporated hydroxyapatite (HA) particles into a poly(lactic acid) (PLA) composite and evaluated the surface structure and the effects of HA density on the initial cell attachment in vitro of murine calvarial preosteoblasts (MC3T3-EI). Scanning electron microscopy (SEM), atomic force microscopy (AFM) and infrared spectroscopy (FTIR) showed that the HA particles were successfully incorporated into the PLA matrix and located at the surface which is of importance in order to maintain the bioactive effect of the HA particles. SEM and AFM investigation revealed that the HA density (particles/area) as well as surface roughness increased with HA loading concentration (i.e. 5, 10, 15 and 20wt%), which promoted protein adsorption. Furthermore, the presence of HA on the surface enhanced cell spreading, increased the formation of actin stress fibers and significantly improved the expression of vinculin in MC3T3-E1 cells which is a key player in the regulation of cell adhesion. These results suggest the potential utility of PLA/HA composites as biomaterials for use as a bone substitute material and in tissue engineering applications. PMID:24986753

  2. Characterization of New PEEK/HA Composites with 3D HA Network Fabricated by Extrusion Freeforming.

    PubMed

    Vaezi, Mohammad; Black, Cameron; Gibbs, David M R; Oreffo, Richard O C; Brady, Mark; Moshrefi-Torbati, Mohamed; Yang, Shoufeng

    2016-05-26

    Addition of bioactive materials such as calcium phosphates or Bioglass, and incorporation of porosity into polyetheretherketone (PEEK) has been identified as an effective approach to improve bone-implant interfaces and osseointegration of PEEK-based devices. In this paper, a novel production technique based on the extrusion freeforming method is proposed that yields a bioactive PEEK/hydroxyapatite (PEEK/HA) composite with a unique configuration in which the bioactive phase (i.e., HA) distribution is computer-controlled within a PEEK matrix. The 100% interconnectivity of the HA network in the biocomposite confers an advantage over alternative forms of other microstructural configurations. Moreover, the technique can be employed to produce porous PEEK structures with controlled pore size and distribution, facilitating greater cellular infiltration and biological integration of PEEK composites within patient tissue. The results of unconfined, uniaxial compressive tests on these new PEEK/HA biocomposites with 40% HA under both static and cyclic mode were promising, showing the composites possess yield and compressive strength within the range of human cortical bone suitable for load bearing applications. In addition, preliminary evidence supporting initial biological safety of the new technique developed is demonstrated in this paper. Sufficient cell attachment, sustained viability in contact with the sample over a seven-day period, evidence of cell bridging and matrix deposition all confirmed excellent biocompatibility.

  3. Characterization of New PEEK/HA Composites with 3D HA Network Fabricated by Extrusion Freeforming.

    PubMed

    Vaezi, Mohammad; Black, Cameron; Gibbs, David M R; Oreffo, Richard O C; Brady, Mark; Moshrefi-Torbati, Mohamed; Yang, Shoufeng

    2016-01-01

    Addition of bioactive materials such as calcium phosphates or Bioglass, and incorporation of porosity into polyetheretherketone (PEEK) has been identified as an effective approach to improve bone-implant interfaces and osseointegration of PEEK-based devices. In this paper, a novel production technique based on the extrusion freeforming method is proposed that yields a bioactive PEEK/hydroxyapatite (PEEK/HA) composite with a unique configuration in which the bioactive phase (i.e., HA) distribution is computer-controlled within a PEEK matrix. The 100% interconnectivity of the HA network in the biocomposite confers an advantage over alternative forms of other microstructural configurations. Moreover, the technique can be employed to produce porous PEEK structures with controlled pore size and distribution, facilitating greater cellular infiltration and biological integration of PEEK composites within patient tissue. The results of unconfined, uniaxial compressive tests on these new PEEK/HA biocomposites with 40% HA under both static and cyclic mode were promising, showing the composites possess yield and compressive strength within the range of human cortical bone suitable for load bearing applications. In addition, preliminary evidence supporting initial biological safety of the new technique developed is demonstrated in this paper. Sufficient cell attachment, sustained viability in contact with the sample over a seven-day period, evidence of cell bridging and matrix deposition all confirmed excellent biocompatibility. PMID:27240326

  4. Chimeric proteins define variable and essential regions of Ha-ras-encoded protein

    SciTech Connect

    Lowe, D.G.; Ricketts, M.; Levinson, A.D.; Goeddel, D.V.

    1988-02-01

    The biological role of amino acid differences between the human 21-kDa Ha-ras protein (p21) and the human 23-kDa R-ras protein (p23) was investigated by engineering mutant Ha-ras p21 molecules containing divergent amino acid sequences from R-ras p23. Variant amino acids from R-ras p23 regions 1-30, 52-57, 67-78, 1-30 and 67-78 together, and 112-124 were substituted for the corresponding Ha-ras p21 amino acid regions 1-4, 26-31, 41-52, 1-4 and 41-52 together, and 86-98, respectively. Rat fibroblasts transfected with genes encoding these position-12 valine-substituted chimeric Ha-ras proteins displayed the same properties of morphological transformation and anchorage-independent growth as Ha-ras T24 oncogene-transformed fibroblasts. However, substitution of variant amino acids from the 80 C-terminal residues (amino acids 138-218) of R-ras p23 for the corresponding p21 amino acids (residues 112-189) inactivated the transforming activity of position-12 valine-substituted p21. The converse substitution of Ha-ras p21 C-terminal residues into R-ras p23 did not result in transformation by position-38 valine-substituted p232. These data are discussed in terms of the structure of ras proteins and the nature of interactions determining the specificity of effector function.

  5. Radio Observations of SN 2008ha

    NASA Astrophysics Data System (ADS)

    Soderberg, Alicia

    2009-03-01

    I observed the peculiar SN 2008ha (CBET #1567) with the Very Large Array on 2008 Nov 21.99 UT at a frequency of 8.46 GHz. No radio source is detected at the optical SN position to a limit of 93 microJy (3 sigma). At a distance of 21 Mpc, this corresponds to a radio luminosity limit similar to those of nearby Type Ia supernovae (Panagia et al. 2006). It is also consistent with the observed radio luminosities for the nearest Type Ibc supernovae (e.g., SN 2002ap; Berger, Kulkarni & Chevalier 2002), but a factor of 10^3 and 10^5 below the radio luminosities of sub-energetic GRBs (Soderberg et al.

  6. The hyaluronan receptor RHAMM in noradrenergic fibers contributes to axon growth capacity of locus coeruleus neurons in an intraocular transplant model.

    PubMed

    Nagy, J I; Price, M L; Staines, W A; Lynn, B D; Granholm, A C

    1998-09-01

    The hyaluronan receptor for hyaluronic acid-mediated motility (RHAMM) plays a role in cell migration and motility in many systems. Recent observations on the involvement of RHAMM in neurite motility in vitro suggest that it might also be important in axon outgrowth in situ. This was addressed directly by investigating both RHAMM expression in the rat CNS and the ability of anti-RHAMM reagents to interfere with tissue growth and axon outgrowth in intraocular brainstem transplants. By western blotting, anti-RHAMM antibody detected a RHAMM isoform of 75,000 mol. wt in both whole brain homogenate and synaptosome preparations, and a 65,000 mol. wt isoform in synaptosomes. Immunofluorescence of adult brain sections revealed RHAMM-like immunoreactivity in varicose fibers that were also positive for the noradrenergic marker dopamine-beta-hydroxylase. Not all noradrenergic fibers contained RHAMM, nor was RHAMM detected in other monoaminergic fiber types. Lesions of noradrenergic fiber systems with beta-halobenzylamine-N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) eliminated RHAMM-positive fibers, but noradrenergic axons that sprouted extensively after this treatment were strongly RHAMM-positive. To assess RHAMM's role in fiber outgrowth, fetal brainstem tissue containing noradrenergic neurons was grafted into the anterior chamber of the eye. Treatment of grafts with anti-RHAMM antibody caused significant inhibition of tissue growth and axon outgrowth, as did a peptide corresponding to a hyaluronan binding domain of RHAMM. These agents had no such effects on transplants containing serotonergic and dopaminergic neurons. These results suggest that RHAMM, an extracellular matrix receptor previously shown to contribute to migratory and contact behavior of cells, may also be important in the growth and/or regenerative capacity of central noradrenergic fibers originating from the locus coeruleus. PMID:9692758

  7. Lac color inhibits development of rat thyroid carcinomas through targeting activation of plasma hyaluronan-binding protein.

    PubMed

    Kemmochi, Sayaka; Yamamichi, Shingo; Shimamoto, Keisuke; Onda, Nobuhiko; Hasumi, Keiji; Suzuki, Kazuhiko; Mitsumori, Kunitoshi; Shibutani, Makoto

    2012-06-01

    Coccid-derived natural food colorants contain active ingredients that potentiate inhibition of tissue proteolysis mediated by activation of plasma hyaluronan-binding protein (PHBP). In the present study, we examined the effect of lac color (LC) and cochineal extract (CE), representative coccid-derived colorants containing laccaic acid and carminic acid as active ingredients, in an intracapsular invasion model of experimental thyroid cancers using rats. One week after initiation with N-bis(hydroxypropyl)nitrosamine, male F344/NSIc rats were fed a powdered diet containing 5.0% LC or 3.0% CE during promotion with 0.15% sulfadimethoxine (SDM) in the drinking water for 13 weeks. Capsular invasive carcinomas (CICs) and lung metastases were decreased by LC treatment and accompanied by transcript downregulation on angiogenesis and PHBP-related tissue proteolysis in CICs. In contrast, CE upregulated angiogenesis-related genes in CICs. PHBP was expressed in capsular macrophages and thyroid proliferative lesions with increased intensity in CICs, and LC decreased PHBP-expressing CICs. The size of CICs and their proliferation activity, however, were unchanged compared with those treated with SDM alone. Suppression of cancer by invasion by LC was more evident after an eight-week treatment, exhibiting a profound decrease in tenascin-C-positive early invasive foci and marked reductions in capsular inflammation and fibrosis. These results suggest that LC and CE exerted dissimilar effects on CIC development, the former suppressing the initial step of neoplastic cell invasion into the capsule by targeting PHBP activity of macrophages and neoplastic cells on tissue proteolysis involving inflammatory responses and angiogenesis, and the latter promoting angiogenesis of developed CICs at later stages. PMID:22715430

  8. Silk-hyaluronan-based composite hydrogels: a novel, securable vehicle for drug delivery.

    PubMed

    Elia, Roberto; Newhide, Danny R; Pedevillano, Paul D; Reiss, G Russell; Firpo, Matthew A; Hsu, Edward W; Kaplan, David L; Prestwich, Glenn D; Peattie, Robert A

    2013-02-01

    A new, biocompatible hyaluronic acid (HA)-silk hydrogel composite was fabricated and tested for use as a securable drug delivery vehicle. The composite consisted of a hydrogel formed by cross-linking thiol-modified HA with poly(ethylene glycol)-diacrylate, within which was embedded a reinforcing mat composed of electrospun silk fibroin protein. Both HA and silk are biocompatible, selectively degradable biomaterials with independently controllable material properties. Mechanical characterization showed the composite tensile strength as fabricated to be 4.43 ± 2.87 kPa, two orders of magnitude above estimated tensions found around potential target organs. In the presence of hyaluronidase (HAse) in vitro, the rate of gel degradation increased with enzyme concentration although the reinforcing silk mesh was not digested. Composite gels demonstrated the ability to store and sustainably deliver therapeutic agents. Time constants for in vitro release of selected representative antibacterial and anti-inflammatory drugs varied from 46.7 min for cortisone to 418 min for hydrocortisone. This biocomposite showed promising mechanical characteristics for direct fastening to tissue and organs, as well as controllable degradation properties suitable for storage and release of therapeutically relevant drugs.

  9. Silk-hyaluronan-based composite hydrogels: a novel, securable vehicle for drug delivery.

    PubMed

    Elia, Roberto; Newhide, Danny R; Pedevillano, Paul D; Reiss, G Russell; Firpo, Matthew A; Hsu, Edward W; Kaplan, David L; Prestwich, Glenn D; Peattie, Robert A

    2013-02-01

    A new, biocompatible hyaluronic acid (HA)-silk hydrogel composite was fabricated and tested for use as a securable drug delivery vehicle. The composite consisted of a hydrogel formed by cross-linking thiol-modified HA with poly(ethylene glycol)-diacrylate, within which was embedded a reinforcing mat composed of electrospun silk fibroin protein. Both HA and silk are biocompatible, selectively degradable biomaterials with independently controllable material properties. Mechanical characterization showed the composite tensile strength as fabricated to be 4.43 ± 2.87 kPa, two orders of magnitude above estimated tensions found around potential target organs. In the presence of hyaluronidase (HAse) in vitro, the rate of gel degradation increased with enzyme concentration although the reinforcing silk mesh was not digested. Composite gels demonstrated the ability to store and sustainably deliver therapeutic agents. Time constants for in vitro release of selected representative antibacterial and anti-inflammatory drugs varied from 46.7 min for cortisone to 418 min for hydrocortisone. This biocomposite showed promising mechanical characteristics for direct fastening to tissue and organs, as well as controllable degradation properties suitable for storage and release of therapeutically relevant drugs. PMID:22090427

  10. Combining colloidal probe atomic force and reflection interference contrast microscopy to study the compressive mechanics of hyaluronan brushes.

    PubMed

    Attili, Seetharamaiah; Richter, Ralf P

    2012-02-14

    We describe a method that combines colloidal probe atomic force microscopy (AFM) and reflection interference contrast microscopy (RICM) to characterize the mechanical properties of thin and solvated polymer films. When analyzing polymer films, a fundamental problem in colloidal probe AFM experiments is to determine the distance at closest approach between the probe and the substrate on which the film is deposited. By combining AFM and RICM in situ, forces and absolute distances can be measured simultaneously. Using the combined setup, we quantify the compressive mechanics of films of the polysaccharide hyaluronan that is end-grafted to a supported lipid bilayer. The experimental data, and comparison with polymer theory, show that hyaluronan films are well-described as elastic, very soft and highly solvated polymer brushes. The data on these well-defined films should be a useful reference for the investigation of the more complex hyaluronan-rich coats that surround many living cells.

  11. Salmonella-Based Therapy Targeting Indoleamine 2,3-Dioxygenase Coupled with Enzymatic Depletion of Tumor Hyaluronan Induces Complete Regression of Aggressive Pancreatic Tumors.

    PubMed

    Manuel, Edwin R; Chen, Jeremy; D'Apuzzo, Massimo; Lampa, Melanie G; Kaltcheva, Teodora I; Thompson, Curtis B; Ludwig, Thomas; Chung, Vincent; Diamond, Don J

    2015-09-01

    Bacterial-based therapies are emerging as effective cancer treatments and hold promise for refractory neoplasms, such as pancreatic ductal adenocarcinoma (PDAC), which has not shown significant improvement in therapy for more than 25 years. Using a novel combination of shIDO-ST, a Salmonella-based therapy targeting the immunosuppressive molecule indoleamine 2,3-dioxygenase (IDO), with an enzyme, PEGPH20, which depletes extracellular matrix hyaluronan, we observed extended survival with frequent total regression of autochthonous and orthotopic PDAC tumors. This observation was associated with migration and accumulation of activated polymorphonuclear neutrophils (PMN) from spleens into tumors, which was not seen using a scrambled control (shScr-ST). Purified splenic PMNs from PEGPH20/shIDO-ST-treated mice exhibited significant IDO knockdown and were able to kill tumor targets ex vivo through mechanisms involving FasL and serine proteases. In addition, CD8(+) T cells were observed to contribute to late control of pancreatic tumors. Collectively, our data demonstrate that entry of shIDO-ST and PMNs into otherwise impermeable desmoplastic tumors is facilitated by PEGPH20-mediated HA removal, further highlighting an important component of effective treatment for PDAC.

  12. Salmonella-Based Therapy Targeting Indoleamine 2,3-Dioxygenase Coupled with Enzymatic Depletion of Tumor Hyaluronan Induces Complete Regression of Aggressive Pancreatic Tumors

    PubMed Central

    Manuel, Edwin R.; Chen, Jeremy; D'Apuzzo, Massimo; Lampa, Melanie G.; Kaltcheva, Teodora I.; Thompson, Curtis B.; Ludwig, Thomas; Chung, Vincent; Diamond, Don J.

    2015-01-01

    Bacterial-based therapies are emerging as effective cancer treatments and hold promise for refractory neoplasms such as pancreatic ductal adenocarcinoma (PDAC), which has not shown significant improvement in therapy for over twenty-five years. Using a novel combination of shIDO-ST, a Salmonella-based therapy targeting the immunosuppressive molecule indoleamine 2,3-dioxygenase (IDO), with an enzyme, PEGPH20, which depletes extracellular matrix hyaluronan, we observed extended survival with frequent total regression of autochthonous and orthotopic PDAC tumors. This was associated with migration and accumulation of activated polymorphonuclear neutrophils (PMN) from spleens into tumors, which was not observed using a scrambled control (shScr-ST). Purified splenic PMNs from PEGPH20/shIDO-ST-treated mice exhibited significant IDO knockdown and were able to kill tumor targets ex-vivo through mechanisms involving FasL and serine proteases. In addition, CD8+ T cells were observed to contribute to late control of pancreatic tumors. Collectively, our data demonstrate that entry of shIDO-ST and PMNs into otherwise impermeable desmoplastic tumors is facilitated by PEGPH20-mediated HA removal, further highlighting an important component of effective treatment for PDAC. PMID:26134178

  13. Salmonella-Based Therapy Targeting Indoleamine 2,3-Dioxygenase Coupled with Enzymatic Depletion of Tumor Hyaluronan Induces Complete Regression of Aggressive Pancreatic Tumors.

    PubMed

    Manuel, Edwin R; Chen, Jeremy; D'Apuzzo, Massimo; Lampa, Melanie G; Kaltcheva, Teodora I; Thompson, Curtis B; Ludwig, Thomas; Chung, Vincent; Diamond, Don J

    2015-09-01

    Bacterial-based therapies are emerging as effective cancer treatments and hold promise for refractory neoplasms, such as pancreatic ductal adenocarcinoma (PDAC), which has not shown significant improvement in therapy for more than 25 years. Using a novel combination of shIDO-ST, a Salmonella-based therapy targeting the immunosuppressive molecule indoleamine 2,3-dioxygenase (IDO), with an enzyme, PEGPH20, which depletes extracellular matrix hyaluronan, we observed extended survival with frequent total regression of autochthonous and orthotopic PDAC tumors. This observation was associated with migration and accumulation of activated polymorphonuclear neutrophils (PMN) from spleens into tumors, which was not seen using a scrambled control (shScr-ST). Purified splenic PMNs from PEGPH20/shIDO-ST-treated mice exhibited significant IDO knockdown and were able to kill tumor targets ex vivo through mechanisms involving FasL and serine proteases. In addition, CD8(+) T cells were observed to contribute to late control of pancreatic tumors. Collectively, our data demonstrate that entry of shIDO-ST and PMNs into otherwise impermeable desmoplastic tumors is facilitated by PEGPH20-mediated HA removal, further highlighting an important component of effective treatment for PDAC. PMID:26134178

  14. Cell protrusions induced by hyaluronan synthase 3 (HAS3) resemble mesothelial microvilli and share cytoskeletal features of filopodia.

    PubMed

    Koistinen, Ville; Kärnä, Riikka; Koistinen, Arto; Arjonen, Antti; Tammi, Markku; Rilla, Kirsi

    2015-10-01

    Previous studies have shown that overexpression of enzymatically active GFP-HAS induces the growth of long, slender protrusions that share many features of both filopodia and microvilli. These protrusions are dependent on continuing hyaluronan synthesis, and disrupt upon digestion of hyaluronan by hyaluronidase. However, complete understanding of their nature is still missing. This work shows that the protrusions on rat peritoneal surface are ultrastructurally indistinguishable from those induced by GFP-HAS3 in MCF-7 cells. Analysis of the actin-associated proteins villin, ezrin, espin, fascin, and Myo10 indicated that the HAS3-induced protrusions share most cytoskeletal features with filopodia, but they do not require adherence to the substratum like traditional filopodia. GFP-HAS3 overexpression was found to markedly enhance filamentous actin in the protrusions and their cortical basis. Analysis of the protrusion dynamics after enzymatic digestion of hyaluronan revealed that while GFP-HAS3 escape from the protrusions and the protrusion collapse takes place immediately, the complete retraction of the protrusions occurs more slowly. This finding also suggests that hyaluronan chain maintains HAS3 in the plasma membrane. The results of this work suggest that protrusions similar to those of HAS3 overexpressing cells in vitro exist also in cells with active hyaluronan synthesis in vivo. These protrusions are similar to common filopodia but are independent of substratum attachment due to the extracellular scaffolding by the hyaluronan coat that accounts for the growth and maintenance of these structures, previously associated to invasion, adhesion and multidrug resistance. PMID:26162854

  15. Extracellular matrix hyaluronan signals via its CD44 receptor in the increased responsiveness to mechanical stimulation.

    PubMed

    Ferrari, L F; Araldi, D; Bogen, O; Levine, J D

    2016-06-01

    We propose that the extracellular matrix (ECM) signals CD44, a hyaluronan receptor, to increase the responsiveness to mechanical stimulation in the rat hind paw. We report that intradermal injection of hyaluronidase induces mechanical hyperalgesia, that is inhibited by co-administration of a CD44 receptor antagonist, A5G27. The intradermal injection of low (LMWH) but not high (HMWH) molecular weight hyaluronan also induces mechanical hyperalgesia, an effect that was attenuated by pretreatment with HMWH or A5G27. Pretreatment with HMWH also attenuated the hyperalgesia induced by hyaluronidase. Similarly, intradermal injection of A6, a CD44 receptor agonist, produced hyperalgesia that was inhibited by HMWH and A5G27. Inhibitors of protein kinase A (PKA) and Src, but not protein kinase C (PKC), significantly attenuated the hyperalgesia induced by both A6 and LMWH. Finally, to determine if CD44 receptor signaling is involved in a preclinical model of inflammatory pain, we evaluated the effect of A5G27 and HMWH on the mechanical hyperalgesia associated with the inflammation induced by carrageenan. Both A5G27 and HMWH attenuated carrageenan-induced mechanical hyperalgesia. Thus, while LMWH acts at its cognate receptor, CD44, to induce mechanical hyperalgesia, HMWH acts at the same receptor as an antagonist. That the local administration of HMWH or A5G27 inhibits carrageenan-induced hyperalgesia supports the suggestion that carrageenan produces changes in the ECM that contributes to inflammatory pain. These studies define a clinically relevant role for signaling by the hyaluronan receptor, CD44, in increased responsiveness to mechanical stimulation. PMID:26996509

  16. Cleavage of Hyaluronan and CD44 Adhesion Molecule Regulate Astrocyte Morphology via Rac1 Signalling

    PubMed Central

    Konopka, Anna; Zeug, Andre; Skupien, Anna; Kaza, Beata; Mueller, Franziska; Chwedorowicz, Agnieszka; Ponimaskin, Evgeni; Wilczynski, Grzegorz M.; Dzwonek, Joanna

    2016-01-01

    Communication of cells with their extracellular environment is crucial to fulfill their function in physiological and pathophysiological conditions. The literature data provide evidence that such a communication is also important in case of astrocytes. Mechanisms that contribute to the interaction between astrocytes and extracellular matrix (ECM) proteins are still poorly understood. Hyaluronan is the main component of ECM in the brain, where its major receptor protein CD44 is expressed by a subset of astrocytes. Considering the fact that functions of astrocytes are tightly coupled with changes in their morphology (e.g.: glutamate clearance in the synaptic cleft, migration, astrogliosis), we investigated the influence of hyaluronan cleavage by hyaluronidase, knockdown of CD44 by specific shRNA and CD44 overexpression on astrocyte morphology. Our results show that hyaluronidase treatment, as well as knockdown of CD44, in astrocytes result in a “stellate”-like morphology, whereas overexpression of CD44 causes an increase in cell body size and changes the shape of astrocytes into flattened cells. Moreover, as a dynamic reorganization of the actin cytoskeleton is supposed to be responsible for morphological changes of cells, and this reorganization is controlled by small GTPases of the Rho family, we hypothesized that GTPase Rac1 acts as a downstream effector for hyaluronan and CD44 in astrocytes. We used FRET-based biosensor and a dominant negative mutant of Rac1 to investigate the involvement of Rac1 activity in hyaluronidase- and CD44-dependent morphological changes of astrocytes. Both, hyaluronidase treatment and knockdown of CD44, enhances Rac1 activity while overexpression of CD44 reduces the activity state in astrocytes. Furthermore, morphological changes were blocked by specific inhibition of Rac1 activity. These findings indicate for the first time that regulation of Rac1 activity is responsible for hyaluronidase and CD44-driven morphological changes of

  17. Clinical Performance and Biocompatibility of Novel Hyaluronan-Based Heparin-Bonded Extracorporeal Circuits

    PubMed Central

    Gunaydin, Serdar; Mccusker, Kevin; Vijay, Venkataramana

    2005-01-01

    Abstract: We tested documented in vitro and ex vivo advantages of novel hyaluronan based heparin bonded extracorporeal circuits in a prospective randomized study. During the period from June until September 2005, 40 patients undergoing reoperation for coronary artery bypass grafting were allocated into two equal groups (n = 20): Group 1 was treated with hyaluronan-based heparin-bonded circuits and group 2 was treated with uncoated control circuits. Complete blood count, fibrinogen, albumin, C3a, interleukin-2 levels, and thromboelastographic data were documented after induction of anesthesia (T1) and heparin administration before cardiopulmonary bypass (CPB) (T2), 15 minutes after initiation of CPB (T3), before cessation of CPB (T4), 15 minutes after reversal with protamine (T5), and the first postoperative day at 8:00 a.m. (T6). Hollow fibers were collected for consecutive biomaterial analysis by optical and scanning electron microscopy (SEM). Desorbed protein deposition on fibers was compared by spectrophotometry. Leukocyte counts were lower in T4-T6 in group 1 (p < .05). Platelet counts demonstrated significant differences at T4 and T5 in coated group (p < .05). Albumin and fibrinogen levels were better preserved in Group 1 at T4, T5 and T4, T6, consecutively (p < .05). C3a and IL-2 levels were lower at T3-T5 and T4-T5 in intervention group (p < .05). Postoperative hemorrhage was 412 ± 50 mL in group 1 and 684 ± 50 ml in group 2 (p < .05). Respiratory support time was shorter in group 1 versus control (p < .05). Platelet adhesion was significantly lower in intervention group. Amount of desorbed protein was 1.44 ± 0.01 mg/dL in group 1 and 1.94 ± 0.01 mg/dL in control (p < .05). SEM and spectrophotometry demonstrated better surface preservation in the hyaluronan coated group. Novel hyaluronan-based heparin-bonded circuits reduce platelet adhesion-aggregation and protein adsorption and provide better perioperative clinical parameters through platelet, albumin

  18. Synthesis and characterization of nano-HA/PA66 composites.

    PubMed

    Huang, Mei; Feng, Jianqing; Wang, Jianxin; Zhang, Xingdong; Li, Yubao; Yan, Yonggang

    2003-07-01

    Based on the bioactivity and biocompatibility of hydroxyapatite (HA) and the excellent mechanical performance of polyamide 66 (PA66), a composite of nanograde HA with PA66 was designed and fabricated to mimic the structure of biological bone which exhibits a composite of nanograde apatite crystals and natural polymer. The HA/PA66 composite combines the bioactivity of HA and the mechanical property of PA66. This study focused on the preparation method of HA/PA66 composite and the influence of HA crystals on the characterization of the composite. HA slurry was used directly to prepare HA/PA66 composite by a solution method, in which HA is able to form hydrogen bond, i.e. chemical bonding with PA66. The nano-HA needle-like crystals treated by hydrothermal method are better in the particle size distribution and the particle dispersion. The morphology, crystal structure and crystallinity as well as crystal size of these needle-like crystals are similar to bone apatite. The nano-HA needle-like crystals dispersed uniformly in PA66 matrix with reinforcement effect and can prevent the micro-crackle spreading into cleft and fracture during the deformation process. The mechanical testing shows that the nano-HA/PA66 composite has a good mechanical property, and may be a promising bone replacement material.

  19. Ha83, a Chitin Binding Domain Encoding Gene, Is Important to Helicoverpa armigera Nucleopolyhedrovirus Budded Virus Production and Occlusion Body Assembling.

    PubMed

    Yu, Huan; Xu, Jian; Liu, Qiang; Liu, Tong-Xian; Wang, Dun

    2015-01-01

    Helicoerpa armigera nucleopolyhedrovirus (HearNPV) ha83 is a late expressed gene that encodes a chitin binding protein. Chitin domain truncation studies revealed that the cysteine at the 128 amino acid position probably played an important role in both chitin binding ability and protein transmission of Ha83. In order to study the function of ha83 in the HearNPV infection cycle, an ha83 knockout HearNPV (Ha83KO) was constructed via homologous recombination. Viral growth and viral DNA replication curves showed that fewer budded virions were produced in Ha83KO transfected cells, while viral DNA replication was increased. Electron microscopy revealed that fewer nucleocapsids were transmitted from virogenic stroma in the Ha83KO transfected cell nucleus, and the morphology of occlusion bodies was prominently larger and cube-shaped. Furthermore, DNA quantity in occlusion bodies of Ha83KO was significantly lower than the occlusion bodies of HaWT. The transcription analysis indicated that these changes may be due to the decreased expression level of viral structural associated genes, such as polyhedrin, p10, pif-2, or cg30 in Ha83KO infected cells. Above results demonstrated that the cysteine at the 128 amino acid position in Ha83 might be the key amino acid, and Ha83 plays an important role in BVs production and OBs assembling. PMID:26057202

  20. Ha83, a Chitin Binding Domain Encoding Gene, Is Important to Helicoverpa armigera Nucleopolyhedrovirus Budded Virus Production and Occlusion Body Assembling

    PubMed Central

    Yu, Huan; Xu, Jian; Liu, Qiang; Liu, Tong-Xian; Wang, Dun

    2015-01-01

    Helicoerpa armigera nucleopolyhedrovirus (HearNPV) ha83 is a late expressed gene that encodes a chitin binding protein. Chitin domain truncation studies revealed that the cysteine at the 128 amino acid position probably played an important role in both chitin binding ability and protein transmission of Ha83. In order to study the function of ha83 in the HearNPV infection cycle, an ha83 knockout HearNPV (Ha83KO) was constructed via homologous recombination. Viral growth and viral DNA replication curves showed that fewer budded virions were produced in Ha83KO transfected cells, while viral DNA replication was increased. Electron microscopy revealed that fewer nucleocapsids were transmitted from virogenic stroma in the Ha83KO transfected cell nucleus, and the morphology of occlusion bodies was prominently larger and cube-shaped. Furthermore, DNA quantity in occlusion bodies of Ha83KO was significantly lower than the occlusion bodies of HaWT. The transcription analysis indicated that these changes may be due to the decreased expression level of viral structural associated genes, such as polyhedrin, p10, pif-2, or cg30 in Ha83KO infected cells. Above results demonstrated that the cysteine at the 128 amino acid position in Ha83 might be the key amino acid, and Ha83 plays an important role in BVs production and OBs assembling. PMID:26057202

  1. Long-term degradation study of hyaluronic acid in aqueous solutions without protection against microorganisms.

    PubMed

    Simulescu, Vasile; Kalina, Michal; Mondek, Jakub; Pekař, Miloslav

    2016-02-10

    The degradation of hyaluronan (HA) of different molecular weights (Mw 14.3, 267.2 and 1160.6 kDa, measured for fresh solutions, before degradation) was studied in aqueous solutions by SEC-MALLS determination of molecular mass, polydispersity and conformation parameters. The solutions were stored either at laboratory or refrigerator temperatures for two months. After this period the weight average molecular weight decreased by 90% for 14.3 kDa, 95% for 267.2 kDa and 71% for 1160.6 kDa hyaluronan (room temperature) or 5.6% for 14.3 kDa, 6.2% for 267.2 kDa and 7.7% for 1160.6 kDa hyaluronan (refrigerator temperature). The hyaluronan aqueous solutions studied did not contain sodium azide or other protectants against microorganisms, because the aim of our study was to assess the degradation in solutions to be used in medicine or cosmetics (without any compounds that are poisonous or toxic for the human body). The solvent used to prepare the samples was pure water. The polydispersity of all the samples remained unaltered during the entire degradation at both temperatures. This indicates a non-random mechanism of degradation. PMID:26686177

  2. Initial stages of cell-matrix adhesion can be mediated and modulated by cell-surface hyaluronan.

    PubMed Central

    Zimmerman, Ella; Geiger, Benjamin; Addadi, Lia

    2002-01-01

    A conceptual temporal and spatial gap exists between the first encounter of a cell with an adhesive substrate and the advanced stages of focal adhesion formation. Although ample information is available on focal adhesions structure and function, the mechanism of the first interaction events and the nature of the molecules mediating them are largely unknown. In this paper we identify cell-surface-associated hyaluronan as a mediator and modulator of the first steps of adhesion of A6 and other cells to conventional tissue culture substrates as well as to the surfaces of calcium-(R,R)-tartrate tetrahydrate crystals. Treatment of A6 cells with hyaluronidase suppresses their rapid interactions with these adhesive substrates, and incubation of either the hyaluronidase-treated cells or the substrate with hyaluronan restores cell adhesion. In contrast, excess hyaluronan on both the cells and the substrate strongly inhibits adhesion. We thus propose that cell-surface-associated hyaluronan can mediate and modulate cell-matrix adhesion at the very first encounter with the substrate. It may promote it through the establishment of exquisitely stereospecific chemical interactions or inhibit it by virtue of steric exclusion and/or electrostatic repulsion. PMID:11916844

  3. Peritoneal Tumor Carcinomatosis: Pharmacological Targeting with Hyaluronan-Based Bioconjugates Overcomes Therapeutic Indications of Current Drugs

    PubMed Central

    Montagner, Isabella Monia; Merlo, Anna; Zuccolotto, Gaia; Renier, Davide; Campisi, Monica; Pasut, Gianfranco; Zanovello, Paola; Rosato, Antonio

    2014-01-01

    Peritoneal carcinomatosis still lacks reliable therapeutic options. We aimed at testing a drug delivery strategy allowing a controlled release of cytotoxic molecules and selective targeting of tumor cells. We comparatively assessed the efficacy of a loco-regional intraperitoneal treatment in immunocompromised mice with bioconjugates formed by chemical linking of paclitaxel or SN-38 to hyaluronan, against three models of peritoneal carcinomatosis derived from human colorectal, gastric and esophageal tumor cell xenografts. In vitro, bioconjugates were selectively internalized through mechanisms largely dependent on interaction with the CD44 receptor and caveolin-mediated endocytosis, which led to accumulation of compounds into lysosomes of tumor cells. Moreover, they inhibited tumor growth comparably to free drugs. In vivo, efficacy of bioconjugates or free drugs against luciferase-transduced tumor cells was assessed by bioluminescence optical imaging, and by recording mice survival. The intraperitoneal administration of bioconjugates in tumor-bearing mice exerted overlapping or improved therapeutic efficacy compared with unconjugated drugs. Overall, drug conjugation to hyaluronan significantly improved the profiles of in vivo tolerability and widened the field of application of existing drugs, over their formal approval or current use. Therefore, this approach can be envisaged as a promising therapeutic strategy for loco-regional treatment of peritoneal carcinomatosis. PMID:25383653

  4. Treatment of partial thickness burns with Zn-hyaluronan: lessons of a clinical pilot study

    PubMed Central

    Juhász, I.; Zoltán, P.; Erdei, I.

    2012-01-01

    Summary A clinical investigation to determine the effectiveness of Zn-hyaluronan gel for the treatment of partial thickness burns was carried out. 60 patients were enrolled in the study with an average of 3% TBSA burn. Exudation lasted 3 days, no infectious complications were observed. By day 14 the wounds of 52 patients have healed, average complete healing time was 10,5 days. An overall 93,3% healing rate was achieved within the planned observation period. Reduction of spontaneous and movementrelated pain was reduced to less than half of the initial values by day 5,5 and 6,3 respectively. Development of a thin, elastic, well tolerable and protective membrane-like layer was noted. This kept the wounds moist while clean during wound-healing, and was spontaneously shed as epithelisation proceeded. Zn-hyaluronan gel is a novel topical wound care product that has proven to be suitable for the treatment of partial thickness burns. PMID:23233826

  5. Design of benzimidazole- and benzoxazole-2-thione derivatives as inhibitors of bacterial hyaluronan lyase.

    PubMed

    Braun, Stephan; Botzki, Alexander; Salmen, Sunnhild; Textor, Christian; Bernhardt, Günther; Dove, Stefan; Buschauer, Armin

    2011-09-01

    Bacterial hyaluronan lyases (Hyal) degrade hyaluronan, an important component of the extracellular matrix, and are involved in microbial spread. Hyal inhibitors may serve as tools to study the role of the enzyme, its substrates and products in the course of bacterial infections. Moreover, such enzyme inhibitors are potential candidates for antibacterial combination therapy. Based on crystal structures of Streptococcus pneumoniae Hyal in complex with a hexasaccharide substrate and with different inhibitors, 1-acylated benzimidazole-2-thiones and benzoxazole-2-thiones were derived as new leads for the inhibition of Streptococcus agalactiae strain 4755 Hyal. Structure-based optimization led to N-(3-phenylpropionyl)benzoxazole-2-thione, one of the most potent compounds known to date (IC(50) values: 24 μM at pH 7.4, 15 μM at pH 5). Among the 27 new derivatives, other N-acylated benzimidazoles and benzoxazoles are just as active at pH 7.4, but not at pH 5. The results support a binding mode characterized by interactions with residues in the catalytic site and with a hydrophobic patch.

  6. A site-selective hyaluronan-interferonα2a conjugate for the treatment of ovarian cancer.

    PubMed

    Montagner, Isabella Monia; Merlo, Anna; Carpanese, Debora; Dalla Pietà, Anna; Mero, Anna; Grigoletto, Antonella; Loregian, Arianna; Renier, Davide; Campisi, Monica; Zanovello, Paola; Pasut, Gianfranco; Rosato, Antonio

    2016-08-28

    While interferon alpha (IFNα) is used in several viral and cancer contexts, its efficacy against ovarian cancer (OC) is far from being incontrovertibly demonstrated and, more importantly, is hindered by heavy systemic side effects. To overcome these issues, here we propose a strategy that allows a targeted delivery of the cytokine, by conjugating IFNα2a with an aldehyde-modified form of hyaluronic acid (HA). The resulting HA-IFNα2a bioconjugate was biochemically and biologically characterized. The conjugation with HA did not substantially modified both the antiviral function and the anti-proliferative activity of the cytokine. Moreover, the induction of STAT1 phosphorylation and of a specific gene expression signature in different targets was retained. In vivo optical imaging biodistribution showed that the i.p.-injected HA-IFNα2a persisted into the peritoneal cavity longer than IFNα2a without being toxic for intraperitoneal organs, thus potentially enhancing the loco-regional therapeutic effect. Indeed, in OC xenograft mouse models bioconjugate significantly improved survival as compared to the free cytokine. Overall, HA-IFNα2a bioconjugate disclosed an improved anticancer efficacy, and can be envisaged as a promising loco-regional treatment for OC. PMID:27356018

  7. Enhanced chondrogenic differentiation of dental pulp stem cells using nanopatterned PEG-GelMA-HA hydrogels.

    PubMed

    Nemeth, Cameron L; Janebodin, Kajohnkiart; Yuan, Alex E; Dennis, James E; Reyes, Morayma; Kim, Deok-Ho

    2014-11-01

    We have examined the effects of surface nanotopography and hyaluronic acid (HA) on in vitro chondrogenesis of dental pulp stem cells (DPSCs). Ultraviolet-assisted capillary force lithography was employed to fabricate well-defined nanostructured scaffolds of composite PEG-GelMA-HA hydrogels that consist of poly(ethylene glycol) dimethacrylate (PEGDMA), methacrylated gelatin (GelMA), and HA. Using this microengineered platform, we first demonstrated that DPSCs formed three-dimensional spheroids, which provide an appropriate environment for in vitro chondrogenic differentiation. We also found that DPSCs cultured on nanopatterned PEG-GelMA-HA scaffolds showed a significant upregulation of the chondrogenic gene markers (Sox9, Alkaline phosphatase, Aggrecan, Procollagen type II, and Procollagen type X), while downregulating the pluripotent stem cell gene, Nanog, and epithelial-mesenchymal genes (Twist, Snail, Slug) compared with tissue culture polystyrene-cultured DPSCs. Immunocytochemistry showed more extensive deposition of collagen type II in DPSCs cultured on the nanopatterned PEG-GelMA-HA scaffolds. These findings suggest that nanotopography and HA provide important cues for promoting chondrogenic differentiation of DPSCs.

  8. Enhanced Chondrogenic Differentiation of Dental Pulp Stem Cells Using Nanopatterned PEG-GelMA-HA Hydrogels

    PubMed Central

    Nemeth, Cameron L.; Janebodin, Kajohnkiart; Yuan, Alex E.; Dennis, James E.

    2014-01-01

    We have examined the effects of surface nanotopography and hyaluronic acid (HA) on in vitro chondrogenesis of dental pulp stem cells (DPSCs). Ultraviolet-assisted capillary force lithography was employed to fabricate well-defined nanostructured scaffolds of composite PEG-GelMA-HA hydrogels that consist of poly(ethylene glycol) dimethacrylate (PEGDMA), methacrylated gelatin (GelMA), and HA. Using this microengineered platform, we first demonstrated that DPSCs formed three-dimensional spheroids, which provide an appropriate environment for in vitro chondrogenic differentiation. We also found that DPSCs cultured on nanopatterned PEG-GelMA-HA scaffolds showed a significant upregulation of the chondrogenic gene markers (Sox9, Alkaline phosphatase, Aggrecan, Procollagen type II, and Procollagen type X), while downregulating the pluripotent stem cell gene, Nanog, and epithelial–mesenchymal genes (Twist, Snail, Slug) compared with tissue culture polystyrene-cultured DPSCs. Immunocytochemistry showed more extensive deposition of collagen type II in DPSCs cultured on the nanopatterned PEG-GelMA-HA scaffolds. These findings suggest that nanotopography and HA provide important cues for promoting chondrogenic differentiation of DPSCs. PMID:24749806

  9. Complexation and Sequestration of BMP-2 from an ECM Mimetic Hyaluronan Gel for Improved Bone Formation

    PubMed Central

    Kisiel, Marta; Klar, Agnieszka S.; Ventura, Manuela; Buijs, Jos; Mafina, Marc-Krystelle; Cool, Simon M.; Hilborn, Jöns

    2013-01-01

    Bone morphogenetic protein-2 (BMP-2) is considered a promising adjuvant for the treatment of skeletal non-union and spinal fusion. However, BMP-2 delivery in a conventional collagen scaffold necessitates a high dose to achieve an efficacious outcome. To lower its effective dose, we precomplexed BMP-2 with the glycosaminoglycans (GAGs) dermatan sulfate (DS) or heparin (HP), prior to loading it into a hyaluronic acid (HA) hydrogel. In vitro release studies showed that BMP-2 precomplexed with DS or HP had a prolonged delivery compared to without GAG. BMP-2-DS complexes achieved a slightly faster release in the first 24 h than HP; however, both delivered BMP-2 for an equal duration. Analysis of the kinetic interaction between BMP-2 and DS or HP showed that HP had approximately 10 times higher affinity for BMP-2 than DS, yet it equally stabilized the protein, as determined by alkaline phosphatase activity. Ectopic bone formation assays at subcutaneous sites in rats demonstrated that HA hydrogel-delivered BMP-2 precomplexed with GAG induced twice the volume of bone compared with BMP-2 delivered uncomplexed to GAG. PMID:24167632

  10. Desmosine-Inspired Cross-Linkers for Hyaluronan Hydrogels

    NASA Astrophysics Data System (ADS)

    Hagel, Valentin; Mateescu, Markus; Southan, Alexander; Wegner, Seraphine V.; Nuss, Isabell; Haraszti, Tamás; Kleinhans, Claudia; Schuh, Christian; Spatz, Joachim P.; Kluger, Petra J.; Bach, Monika; Tussetschläger, Stefan; Tovar, Günter E. M.; Laschat, Sabine; Boehm, Heike

    2013-06-01

    We designed bioinspired cross-linkers based on desmosine, the cross-linker in natural elastin, to prepare hydrogels with thiolated hyaluronic acid. These short, rigid cross-linkers are based on pyridinium salts (as in desmosine) and can connect two polymer backbones. Generally, the obtained semi-synthetic hydrogels are form-stable, can withstand repeated stress, have a large linear-elastic range, and show strain stiffening behavior typical for biopolymer networks. In addition, it is possible to introduce a positive charge to the core of the cross-linker without affecting the gelation efficiency, or consequently the network connectivity. However, the mechanical properties strongly depend on the charge of the cross-linker. The properties of the presented hydrogels can thus be tuned in a range important for engineering of soft tissues by controlling the cross-linking density and the charge of the cross-linker.

  11. HA95 and LAP2 beta mediate a novel chromatin-nuclear envelope interaction implicated in initiation of DNA replication.

    PubMed

    Martins, Sandra; Eikvar, Sissel; Furukawa, Kazuhiro; Collas, Philippe

    2003-01-20

    HA95 is a chromatin-associated protein that interfaces the nuclear envelope (NE) and chromatin. We report an interaction between HA95 and the inner nuclear membrane protein lamina-associated polypeptide (LAP) 2 beta, and a role of this association in initiation of DNA replication. Precipitation of GST-LAP2 beta fusion proteins and overlays of immobilized HA95 indicate that a first HA95-binding region lies within amino acids 137-242 of LAP2 beta. A second domain sufficient to bind HA95 colocalizes with the lamin B-binding domain of LAP2beta at residues 299-373. HA95-LAP2 beta interaction is not required for NE formation. However, disruption of the association of HA95 with the NH2-terminal HA95-binding domain of LAP2 beta abolishes the initiation, but not elongation, of DNA replication in purified G1 phase nuclei incubated in S-phase extract. Inhibition of replication initiation correlates with proteasome-mediated proteolysis of Cdc6, a component of the prereplication complex. Rescue of Cdc6 degradation with proteasome inhibitors restores replication. We propose that an interaction of LAP2beta, or LAP2 proteins, with HA95 is involved in the control of initiation of DNA replication. PMID:12538639

  12. Multiresponsive hyaluronan-p(NiPAAm) "click"-linked hydrogels.

    PubMed

    Pasale, Sharad K; Cerroni, Barbara; Ghugare, Shivkumar V; Paradossi, Gaio

    2014-07-01

    Combined reversible addition-fragmentation chain transfer (RAFT) and chemoselective "click" chemistry are used for assembling two polymeric chains into a hybrid network capable to respond simultaneously or separately to different external stimuli. An azido-derivative of hyaluronate is clicked together with a new telechelic RAFT-generated p(NiPAAm), carrying a propargyl function at both ends, suitable as macromolecular "clickable" cross-linker with controlled molecular weight. This hybrid system displays a multiresponsive behavior versus temperature, pH, and ionic strength, maintaining cumulative as well as separate sensitivities to the external stimuli. Hyaluronidase catalyzed degradation of the hydrogels, mimicking the extracellular matrix degradation process, is an additional asset for the use of this class of hydrogels as scaffold. Tumor cells, HT-29, grow on the surface of these hybrid hydrogels more than the healthy ones, as NIH3T3. This finding opens a road to micro- and nano-devices based on hyaluronic acid as a promising biopolymer to pursue localized drug delivery.

  13. Precision Landing and Hazard Avoidance (PL&HA) Domain

    NASA Technical Reports Server (NTRS)

    Robertson, Edward A.; Carson, John M., III

    2016-01-01

    The Precision Landing and Hazard Avoidance (PL&HA) domain addresses the development, integration, testing, and spaceflight infusion of sensing, processing, and GN&C (Guidance, Navigation and Control) functions critical to the success and safety of future human and robotic exploration missions. PL&HA sensors also have applications to other mission events, such as rendezvous and docking.

  14. Vibrio cholerae hemagglutinin(HA)/protease: An extracellular metalloprotease with multiple pathogenic activities.

    PubMed

    Benitez, Jorge A; Silva, Anisia J

    2016-06-01

    Vibrio cholerae of serogroup O1 and O139, the etiological agent of the diarrheal disease cholera, expresses the extracellular Zn-dependent metalloprotease hemagglutinin (HA)/protease also reported as vibriolysin. This enzyme is also produced by non-O1/O139 (non-cholera) strains that cause mild, sporadic illness (i.e. gastroenteritis, wound or ear infections). Orthologs of HA/protease are present in other members of the Vibrionaceae family pathogenic to humans and fish. HA/protease belongs to the M4 neutral peptidase family and displays significant amino acid sequence homology to Pseudomonas aeruginosa elastase (LasB) and Bacillus thermoproteolyticus thermolysin. It exhibits a broad range of potentially pathogenic activities in cell culture and animal models. These activities range from the covalent modification of other toxins, the degradation of the protective mucus barrier and disruption of intestinal tight junctions. Here we review (i) the structure and regulation of HA/protease expression, (ii) its interaction with other toxins and the intestinal mucosa and (iii) discuss the possible role(s) of HA/protease in the pathogenesis of cholera. PMID:26952544

  15. Cross-Linked Hyaluronan Gel Reduces the Acute Rectal Toxicity of Radiotherapy for Prostate Cancer

    SciTech Connect

    Wilder, Richard B.; Barme, Greg A.; Gilbert, Ronald F.; Holevas, Richard E.; Kobashi, Luis I.; Reed, Richard R.; Solomon, Ronald S.; Walter, Nancy L.; Chittenden, Lucy; Mesa, Albert V.; Agustin, Jeffrey; Lizarde, Jessica; Macedo, Jorge; Ravera, John; Tokita, Kenneth M.

    2010-07-01

    Purpose: To prospectively analyze whether cross-linked hyaluronan gel reduces the mean rectal dose and acute rectal toxicity of radiotherapy for prostate cancer. Methods and Materials: Between September 2008 and March 2009, we transperitoneally injected 9mL of cross-linked hyaluronan gel (Hylaform; Genzyme Corporation, Cambridge, MA) into the anterior perirectal fat of 10 early-stage prostate cancer patients to increase the separation between the prostate and rectum by 8 to 18mm at the start of radiotherapy. Patients then underwent high-dose rate brachytherapy to 2,200cGy followed by intensity-modulated radiation therapy to 5,040cGy. We assessed acute rectal toxicity using the National Cancer Institute Common Terminology Criteria for Adverse Events v3.0 grading scheme. Results: Median follow-up was 3 months. The anteroposterior dimensions of Hylaform at the start and end of radiotherapy were 13 {+-} 3mm (mean {+-} SD) and 10 {+-} 4mm, respectively. At the start of intensity-modulated radiation therapy, daily mean rectal doses were 73 {+-} 13cGy with Hylaform vs. 106 {+-} 20cGy without Hylaform (p = 0.005). There was a 0% incidence of National Cancer Institute Common Terminology Criteria for Adverse Events v3.0 Grade 1, 2, or 3 acute diarrhea in 10 patients who received Hylaform vs. a 29.7% incidence (n = 71) in 239 historical controls who did not receive Hylaform (p = 0.04). Conclusions: By increasing the separation between the prostate and rectum, Hylaform decreased the mean rectal dose. This led to a significant reduction in the acute rectal toxicity of radiotherapy for prostate cancer.

  16. Influenza A HA's conserved epitopes and broadly neutralizing antibodies: a prediction method.

    PubMed

    Ren, Jing; Ellis, John; Li, Jinyan

    2014-10-01

    A conserved epitope is an epitope retained by multiple strains of influenza as the key target of a broadly neutralizing antibody. Identification of conserved epitopes is of strong interest to help design broad-spectrum vaccines against influenza. Conservation score measures the evolutionary conservation of an amino acid position in a protein based on the phylogenetic relationships observed amongst homologous sequences. Here, Average Amino Acid Conservation Score (AAACS) is proposed as a method to identify HA's conserved epitopes. Our analysis shows that there is a clear distinction between conserved epitopes and nonconserved epitopes in terms of AAACS. This method also provides an excellent classification performance on an independent dataset. In contrast, alignment-based comparison methods do not work well for this problem, because conserved epitopes to the same broadly neutralizing antibody are usually not identical or similar. Location-based methods are not successful either, because conserved epitopes are located at both the less-conserved globular head (HA1) and the more-conserved stem (HA2). As a case study, two conserved epitopes on HA are predicted for the influenza A virus H7N9: One should match the broadly neutralizing antibodies CR9114 or FI6v3, while the other is new and requires validation by wet-lab experiments.

  17. Up-regulation of Histone Methyltransferase, DOT1L, by Matrix Hyaluronan Promotes MicroRNA-10 Expression Leading to Tumor Cell Invasion and Chemoresistance in Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma.

    PubMed

    Bourguignon, Lilly Y W; Wong, Gabriel; Shiina, Marisa

    2016-05-13

    Human head and neck squamous cell carcinoma is a solid tumor malignancy associated with major morbidity and mortality. In this study, we determined that human head and neck squamous cell carcinoma-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by a high level of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. Importantly, matrix hyaluronan (HA) induces the up-regulation of stem cell markers that display the hallmark CSC properties. Histone methyltransferase, DOT1L, is also up-regulated by HA in CSCs (isolated from HSC-3 cells). Further analyses indicate that the stimulation of microRNA-10b (miR-10b) expression is DOT1L-specific and HA/CD44-dependent in CSCs. This process subsequently results in the overexpression of RhoGTPases and survival proteins leading to tumor cell invasion and cisplatin resistance. Treatment of CSCs with DOT1L-specific small interfering RNAs (siRNAs) effectively blocks HA/CD44-mediated expression of DOT1L, miR-10b production, and RhoGTPase/survival protein up-regulation as well as reduces tumor cell invasion and enhances chemosensitivity. CSCs were also transfected with a specific anti-miR-10b inhibitor to silence miR-10b expression and block its target functions. Our results demonstrate that the anti-miR-10 inhibitor not only decreases RhoGTPase/survival protein expression and tumor cell invasion, but also increases chemosensitivity in HA-treated CSCs. Taken together, these findings strongly support the contention that histone methyltransferase, DOT1L-associated epigenetic changes induced by HA play pivotal roles in miR-10 production leading to up-regulation of RhoGTPase and survival proteins. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, tumor cell invasion, and chemotherapy resistance in HA/CD44-activated head and neck cancer.

  18. Registration of two confection sunflower germplasm Lines, HA-R10 and HA-R11, Resistant to sunflower rust

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two confection sunflower (Helianthus annuus L.) germplasm lines, HA-R10 (Reg. No.xxx, PI670043) and HA-R11 (Reg. No.xxx, PI670044) were developed by the USDA-ARS Sunflower and Plant Biology Research Unit in collaboration with the North Dakota Agricultural Experiment Station and released December, 20...

  19. Registration of two double rust resistant germplasms, HA-R12 and HA-R13 for confection sunflower

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The confection sunflower (Helianthus annuus L.) germplasms HA-R12 (Reg. No. ______, PI 673104) and HA-R13 (Reg. No. ______, PI 673105) were developed by the USDA-ARS, Sunflower and Plant Biology Research Unit in collaboration with the North Dakota Agricultural Experiment Station, and released in Jul...

  20. Glucose regulated proteins 78 and 75 bind to the receptor for hyaluronan mediated motility in interphase microtubules

    SciTech Connect

    Kuwabara, Hiroko . E-mail: pa2020@art.osaka-med.ac.jp; Yoneda, Masahiko; Hayasaki, Hana; Nakamura, Toshiya; Mori, Hiroshi

    2006-01-20

    The receptor for hyaluronan mediated motility (RHAMM), which is a hyaluronan-binding protein, is a centrosomal and microtubal protein. Here, we have identified two RHAMM-binding proteins, glucose regulated protein (GRP) 78 and GRP75, using co-immunoprecipitation analysis. These two proteins directly bound to glutathione-S-transferase-RHAMM fusion proteins. By double immunostaining, GRP78 and GRP75 colocalized with RHAMM in interphase microtubules, but were separated in mitotic spindles. Prevention of microtubule polymerization by TN-16 and vincristine sulfate induced RHAMM overexpression without a significant change in GRP78/75. Taken together, GRP78/75 and RHAMM complexes may stabilize microtubules in the interphase, associated with a downregulation of RHAMM. These results reveal a new biochemical activity of RHAMM.

  1. Preliminary study on HA coating percutaneously implanted in bone.

    PubMed

    Yang, B C; Weng, J; Li, X D; Yang, Z J; Feng, J M; Chen, J Y; Zhang, X D

    1999-10-01

    A comparative investigation on the possibility of hydroxyapatite (HA) coating and pure Ti column to form biological sealing with skin tissue was completed in this study. HA coating and pure Ti column were percutaneously implanted in the tibia of rabbits. Compared with titanium (Ti) implant, HA coating forms epithelial sealing with skin tissue at 6 weeks postoperatively, while the Ti implant may loosen from the implanted site and be lost. The Ti column loosing rate at this time was 50%. However, once the Ti implant becomes fixed with the bone tissue, it can form epithelial sealing with skin tissue just like the HA coating, at 8 weeks postoperatively. At 8 weeks postoperatively, the epithelial sealing is not destroyed in spite of the fact that the HA coating is biodegraded. Our results show that the HA coating can become fixed with the bone faster than the Ti, which is beneficial for epithelial sealing formation. The main role of HA coating for epithelial sealing is beneficial for sealing at the initial period after it is implanted.

  2. Cytocompatible in situ forming chitosan/hyaluronan hydrogels via a metal-free click chemistry for soft tissue engineering.

    PubMed

    Fan, Ming; Ma, Ye; Mao, Jiahui; Zhang, Ziwei; Tan, Huaping

    2015-07-01

    Injectable hydrogels are important cell scaffolding materials for tissue engineering and regenerative medicine. Here, we report a new class of biocompatible and biodegradable polysaccharide hydrogels derived from chitosan and hyaluronan via a metal-free click chemistry, without the addition of copper catalyst. For the metal-free click reaction, chitosan and hyaluronan were modified with oxanorbornadiene (OB) and 11-azido-3,6,9-trioxaundecan-1-amine (AA), respectively. The gelation is attributed to the triazole ring formation between OB and azido groups of polysaccharide derivatives. The molecular structures were verified by FT-IR spectroscopy and elemental analysis, giving substitution degrees of 58% and 47% for chitosan-OB and hyaluronan-AA, respectively. The in vitro gelation, morphologies, equilibrium swelling, compressive modulus and degradation of the composite hydrogels were examined. The potential of the metal-free hydrogel as a cell scaffold was demonstrated by encapsulation of human adipose-derived stem cells (ASCs) within the gel matrix in vitro. Cell culture showed that this metal-free hydrogel could support survival and proliferation of ASCs. A preliminary in vivo study demonstrated the usefulness of the hydrogel as an injectable scaffold for adipose tissue engineering. These characteristics provide a potential opportunity to use the metal-free click chemistry in preparation of biocompatible hydrogels for soft tissue engineering applications.

  3. Single Molecule Microscopy Reveals an Increased Hyaluronan Diffusion Rate in Synovial Fluid from Knees Affected by Osteoarthritis.

    PubMed

    Kohlhof, Hendrik; Gravius, Sascha; Kohl, Sandro; Ahmad, Sufian S; Randau, Thomas; Schmolders, Jan; Rommelspacher, Yorck; Friedrich, Max; Kaminski, Tim P

    2016-02-12

    Osteoarthritis is a common and progressive joint disorder. Despite its widespread, in clinical practice only late phases of osteoarthritis that are characterized by severe joint damage are routinely detected. Since osteoarthritis cannot be cured but relatively well managed, an early diagnosis and thereby early onset of disease management would lower the burden of osteoarthritis. Here we evaluated if biophysical parameters of small synovial fluid samples extracted by single molecule microscopy can be linked to joint damage. In healthy synovial fluid (ICRS-score < 1) hyaluronan showed a slower diffusion (2.2 μm(2)/s, N = 5) than in samples from patients with joint damage (ICRS-score > 2) (4.5 μm(2)/s, N = 16). More strikingly, the diffusion coefficient of hyaluronan in healthy synovial fluid was on average 30% slower than expected by sample viscosity. This effect was diminished or missing in samples from patients with joint damage. Since single molecule microscopy needs only microliters of synovial fluid to extract the viscosity and the specific diffusion coefficient of hyaluronan this method could be of use as diagnostic tool for osteoarthritis.

  4. Single Molecule Microscopy Reveals an Increased Hyaluronan Diffusion Rate in Synovial Fluid from Knees Affected by Osteoarthritis

    PubMed Central

    Kohlhof, Hendrik; Gravius, Sascha; Kohl, Sandro; Ahmad, Sufian S.; Randau, Thomas; Schmolders, Jan; Rommelspacher, Yorck; Friedrich, Max; Kaminski, Tim P.

    2016-01-01

    Osteoarthritis is a common and progressive joint disorder. Despite its widespread, in clinical practice only late phases of osteoarthritis that are characterized by severe joint damage are routinely detected. Since osteoarthritis cannot be cured but relatively well managed, an early diagnosis and thereby early onset of disease management would lower the burden of osteoarthritis. Here we evaluated if biophysical parameters of small synovial fluid samples extracted by single molecule microscopy can be linked to joint damage. In healthy synovial fluid (ICRS-score < 1) hyaluronan showed a slower diffusion (2.2 μm2/s, N = 5) than in samples from patients with joint damage (ICRS-score > 2) (4.5 μm2/s, N = 16). More strikingly, the diffusion coefficient of hyaluronan in healthy synovial fluid was on average 30% slower than expected by sample viscosity. This effect was diminished or missing in samples from patients with joint damage. Since single molecule microscopy needs only microliters of synovial fluid to extract the viscosity and the specific diffusion coefficient of hyaluronan this method could be of use as diagnostic tool for osteoarthritis. PMID:26868769

  5. Understanding the Underlying Mechanism of HA-Subtyping in the Level of Physic-Chemical Characteristics of Protein

    PubMed Central

    Ebrahimi, Mansour; Aghagolzadeh, Parisa; Shamabadi, Narges; Tahmasebi, Ahmad; Alsharifi, Mohammed; Adelson, David L.

    2014-01-01

    The evolution of the influenza A virus to increase its host range is a major concern worldwide. Molecular mechanisms of increasing host range are largely unknown. Influenza surface proteins play determining roles in reorganization of host-sialic acid receptors and host range. In an attempt to uncover the physic-chemical attributes which govern HA subtyping, we performed a large scale functional analysis of over 7000 sequences of 16 different HA subtypes. Large number (896) of physic-chemical protein characteristics were calculated for each HA sequence. Then, 10 different attribute weighting algorithms were used to find the key characteristics distinguishing HA subtypes. Furthermore, to discover machine leaning models which can predict HA subtypes, various Decision Tree, Support Vector Machine, Naïve Bayes, and Neural Network models were trained on calculated protein characteristics dataset as well as 10 trimmed datasets generated by attribute weighting algorithms. The prediction accuracies of the machine learning methods were evaluated by 10-fold cross validation. The results highlighted the frequency of Gln (selected by 80% of attribute weighting algorithms), percentage/frequency of Tyr, percentage of Cys, and frequencies of Try and Glu (selected by 70% of attribute weighting algorithms) as the key features that are associated with HA subtyping. Random Forest tree induction algorithm and RBF kernel function of SVM (scaled by grid search) showed high accuracy of 98% in clustering and predicting HA subtypes based on protein attributes. Decision tree models were successful in monitoring the short mutation/reassortment paths by which influenza virus can gain the key protein structure of another HA subtype and increase its host range in a short period of time with less energy consumption. Extracting and mining a large number of amino acid attributes of HA subtypes of influenza A virus through supervised algorithms represent a new avenue for understanding and

  6. Understanding the undelaying mechanism of HA-subtyping in the level of physic-chemical characteristics of protein.

    PubMed

    Ebrahimi, Mansour; Aghagolzadeh, Parisa; Shamabadi, Narges; Tahmasebi, Ahmad; Alsharifi, Mohammed; Adelson, David L; Hemmatzadeh, Farhid; Ebrahimie, Esmaeil

    2014-01-01

    The evolution of the influenza A virus to increase its host range is a major concern worldwide. Molecular mechanisms of increasing host range are largely unknown. Influenza surface proteins play determining roles in reorganization of host-sialic acid receptors and host range. In an attempt to uncover the physic-chemical attributes which govern HA subtyping, we performed a large scale functional analysis of over 7000 sequences of 16 different HA subtypes. Large number (896) of physic-chemical protein characteristics were calculated for each HA sequence. Then, 10 different attribute weighting algorithms were used to find the key characteristics distinguishing HA subtypes. Furthermore, to discover machine leaning models which can predict HA subtypes, various Decision Tree, Support Vector Machine, Naïve Bayes, and Neural Network models were trained on calculated protein characteristics dataset as well as 10 trimmed datasets generated by attribute weighting algorithms. The prediction accuracies of the machine learning methods were evaluated by 10-fold cross validation. The results highlighted the frequency of Gln (selected by 80% of attribute weighting algorithms), percentage/frequency of Tyr, percentage of Cys, and frequencies of Try and Glu (selected by 70% of attribute weighting algorithms) as the key features that are associated with HA subtyping. Random Forest tree induction algorithm and RBF kernel function of SVM (scaled by grid search) showed high accuracy of 98% in clustering and predicting HA subtypes based on protein attributes. Decision tree models were successful in monitoring the short mutation/reassortment paths by which influenza virus can gain the key protein structure of another HA subtype and increase its host range in a short period of time with less energy consumption. Extracting and mining a large number of amino acid attributes of HA subtypes of influenza A virus through supervised algorithms represent a new avenue for understanding and

  7. Selective Targeting and Restrictive Damage for Nonspecific Cells by Pulsed Laser-Activated Hyaluronan-Gold Nanoparticles.

    PubMed

    Rau, Lih-Rou; Tsao, Shu-Wei; Liaw, Jiunn-Woei; Tsai, Shiao-Wen

    2016-08-01

    Herein, we describe an approach that immobilizes low-molecular-weight hyaluronic acid (low-MW HA) on the surface of gold nanoparticles (GNPs), which can serve as a cellular probe and photodamage media, to evaluate the selectivity and efficiency of HA-based GNPs (HGNPs) as a mediator of laser-induced photothermal cell damage. In addition, it is known that solid tumors contain a higher content of low-MW HA than normal tissues. Thus, we used low-MW HA rather than high-MW HA used in other studies. In the present study, we conjugated low-MW HA, which is a linear polysaccharide with a disaccharide repeat unit, to prevent a reduction of the ligand-receptor binding efficiency in contrast to the conjugation of protein or peptides, which have unique three-dimensional structures. Three cell lines-MDA-MB-435 S (with CD44), MDA-MB-453 and NIH/3T3 (both are without CD44)-were investigated in the study, and qualitative observations were conducted by dark-field microscopy and laser scanning confocal microscopy (LSCM). In addition, quantitative measurements calculated using inductively coupled plasma emissions were taken for comparison. Our results showed that within the same treatment time, the uptake dosage of HGNPs by the MDA-MB-435 S cells was higher than that by the MDA-MB-453 and NIH 3T3 cells. Meanwhile, HGNPs uptake by the untreated MDA-MB-435 S cells was higher than that of MDA-MB-435 S cells with CD44 blocked by antibodies or silencing CD44 expression. This result implies that receptor-mediated endocytosis can enhance the cellular uptake of HGNPs. In addition, when exposed to a low-power pulsed laser, the former cell morphologies showed a more laser-induced giant plasma membrane vesicles (GPMV) than the latter morphologies. Therefore, this study utilized the specific photothermal property of HA-modified GNPs with laser-induced blebs to create a possible new method for medical applications. PMID:27439142

  8. The linker domain of the Ha-Ras hypervariable region regulates interactions with exchange factors, Raf-1 and phosphoinositide 3-kinase.

    PubMed

    Jaumot, Montserrat; Yan, Jun; Clyde-Smith, Jodi; Sluimer, Judith; Hancock, John F

    2002-01-01

    Ha-Ras and Ki-Ras have different distributions across plasma membrane microdomains. The Ras C-terminal anchors are primarily responsible for membrane micro-localization, but recent work has shown that the interaction of Ha-Ras with lipid rafts is modulated by GTP loading via a mechanism that requires the hypervariable region (HVR). We have now identified two regions in the HVR linker domain that regulate Ha-Ras raft association. Release of activated Ha-Ras from lipid rafts is blocked by deleting amino acids 173-179 or 166-172. Alanine replacement of amino acids 173-179 but not 166-172 restores wild type micro-localization, indicating that specific N-terminal sequences of the linker domain operate in concert with a more C-terminal spacer domain to regulate Ha-Ras raft association. Mutations in the linker domain that confine activated Ha-RasG12V to lipid rafts abrogate Raf-1, phosphoinositide 3-kinase, and Akt activation and inhibit PC12 cell differentiation. N-Myristoylation also prevents the release of activated Ha-Ras from lipid rafts and inhibits Raf-1 activation. These results demonstrate that the correct modulation of Ha-Ras lateral segregation is critical for downstream signaling. Mutations in the linker domain also suppress the dominant negative phenotype of Ha-RasS17N, indicating that HVR sequences are essential for efficient interaction of Ha-Ras with exchange factors in intact cells.

  9. Dynamic mechanical and swelling properties of maleated hyaluronic acid hydrogels.

    PubMed

    Lin, Hai; Liu, Jun; Zhang, Kai; Fan, Yujiang; Zhang, Xingdong

    2015-06-01

    A series of maleated hyaluronan (MaHA) are developed by modification with maleic anhydride. The degrees of substitution (DS) of MaHA vary between 7% and 75%. The DS of MaHA is both higher and wider than methacrylated HA derivatives (MeHA) reported in the literature. MaHA hydrogels are then prepared by photopolymerization and their dynamic mechanical and swelling properties of the hydrogels are investigated. The results showed that MaHA hydrogels with moderate DS (25%, 50% and 65%) have higher storage modulus and lower equilibrium swelling ratios than those with either low or high DS (7%, 15% and 75%). Theoretical analyses also suggest a similar pattern among hydrogels with different DS. The results confirm that the increased cross-linking density enhances the strength of hydrogels. Meanwhile, the hydrophilicity of introduced groups during modification and the degree of incomplete crosslinking reaction might have negative impact on the mechanical and swelling properties of MaHA hydrogels.

  10. BMP-2 Grafted nHA/PLGA Hybrid Nanofiber Scaffold Stimulates Osteoblastic Cells Growth.

    PubMed

    Haider, Adnan; Kim, Sukyoung; Huh, Man-Woo; Kang, Inn-Kyu

    2015-01-01

    Biomaterials play a pivotal role in regenerative medicine, which aims to regenerate and replace lost/degenerated tissues or organs. Natural bone is a hierarchical structure, comprised of various cells having specific functions that are regulated by sophisticated mechanisms. However, the regulation of the normal functions in damaged or injured cells is disrupted. In order to address this problem, we attempted to artificially generate a scaffold for mimicking the characteristics of the extracellular matrix at the nanoscale level to trigger osteoblastic cell growth. For this purpose, we have chemically grafted bone morphogenetic protein (BMP-2) onto the surface of L-glutamic acid modified hydroxyapatite incorporated into the PLGA nanofiber matrix. After extensive characterization using various spectroscopic techniques, the BMP-g-nHA/PLGA hybrid nanofiber scaffolds were subjected to various in vitro cytocompatibility tests. The results indicated that BMP-2 on BMP-g-nHA/PLGA hybrid nanofiber scaffolds greatly stimulated osteoblastic cells growth, contrary to the nHA/PLGA and pristine PLGA nanofiber scaffold, which are used as control. These results suggest that BMP-g-nHA/PLGA hybrid nanofiber scaffold can be used as a nanodrug carrier for the controlled and targeted delivery of BMP-2, which will open new possibilities for enhancing bone tissue regeneration and will help in the treatment of various bone-related diseases in the future. PMID:26539477

  11. BMP-2 Grafted nHA/PLGA Hybrid Nanofiber Scaffold Stimulates Osteoblastic Cells Growth

    PubMed Central

    Haider, Adnan; Kim, Sukyoung; Huh, Man-Woo; Kang, Inn-Kyu

    2015-01-01

    Biomaterials play a pivotal role in regenerative medicine, which aims to regenerate and replace lost/degenerated tissues or organs. Natural bone is a hierarchical structure, comprised of various cells having specific functions that are regulated by sophisticated mechanisms. However, the regulation of the normal functions in damaged or injured cells is disrupted. In order to address this problem, we attempted to artificially generate a scaffold for mimicking the characteristics of the extracellular matrix at the nanoscale level to trigger osteoblastic cell growth. For this purpose, we have chemically grafted bone morphogenetic protein (BMP-2) onto the surface of L-glutamic acid modified hydroxyapatite incorporated into the PLGA nanofiber matrix. After extensive characterization using various spectroscopic techniques, the BMP-g-nHA/PLGA hybrid nanofiber scaffolds were subjected to various in vitro cytocompatibility tests. The results indicated that BMP-2 on BMP-g-nHA/PLGA hybrid nanofiber scaffolds greatly stimulated osteoblastic cells growth, contrary to the nHA/PLGA and pristine PLGA nanofiber scaffold, which are used as control. These results suggest that BMP-g-nHA/PLGA hybrid nanofiber scaffold can be used as a nanodrug carrier for the controlled and targeted delivery of BMP-2, which will open new possibilities for enhancing bone tissue regeneration and will help in the treatment of various bone-related diseases in the future. PMID:26539477

  12. Biodegradable HA-PLA 3-D porous scaffolds: effect of nano-sized filler content on scaffold properties.

    PubMed

    Kothapalli, Chandrasekhar R; Shaw, Montgomery T; Wei, Mei

    2005-11-01

    Scaffolds comprising poly(lactic acid) and nano-hydroxyapatite (HA) were prepared using the solvent-casting/salt-leaching technique. NaCl was used as the leaching agent. Nano-sized HA was synthesized by a hydrothermal method at 170 degrees C and autogenous pressure. High-resolution TEM imaging revealed that the HA particles were ellipsoidal-shaped with needle-like morphologies. The particles had an average size of approximately 25 nm in width and 150 nm in length with aspect ratios ranging from 6 to 8. As the HA content increased in the scaffold from 0 to 50 wt%, the compression modulus of the scaffolds increased from 4.72+/-1.2 to 9.87+/-1.8 MPa, while the yield strength from 0.29+/-0.03 to 0.44+/-0.01 MPa. Such polymeric scaffolds should be suitable materials for non-load sharing tissue-engineering applications. PMID:16701846

  13. Prospects of HA-Based Universal Influenza Vaccine

    PubMed Central

    Hashem, Anwar M.

    2015-01-01

    Current influenza vaccines afford substantial protection in humans by inducing strain-specific neutralizing antibodies (Abs). Most of these Abs target highly variable immunodominant epitopes in the globular domain of the viral hemagglutinin (HA). Therefore, current vaccines may not be able to induce heterosubtypic immunity against the divergent influenza subtypes. The identification of broadly neutralizing Abs (BnAbs) against influenza HA using recent technological advancements in antibody libraries, hybridoma, and isolation of single Ab-secreting plasma cells has increased the interest in developing a universal influenza vaccine as it could provide life-long protection. While these BnAbs can serve as a source for passive immunotherapy, their identification represents an important step towards the design of such a universal vaccine. This review describes the recent advances and approaches used in the development of universal influenza vaccine based on highly conserved HA regions identified by BnAbs. PMID:25785268

  14. The Muon Collider as a $H/A$ factory

    SciTech Connect

    Eichten, Estia; Martin, Adam

    2013-11-22

    We show that a muon collider is ideally suited for the study of heavy H/A scalars, cousins of the Higgs boson found in two-Higgs doublet models and required in supersymmetric models. The key aspects of H/A are: (1) they are narrow, yet have a width-to-mass ratio far larger than the expected muon collider beam-energy resolution, and (2) the larger muon Yukawa allows efficient s-channel production. We study in detail a representative Natural Supersymmetry model which has a 1.5 Tev H/A with $m_H$- $m_A$ = 10 Gev. The large event rates at resonant peak allow the determination of the individual H and A resonance parameters (including CP) and the decays into electroweakinos provides a wealth of information unavailable to any other present or planned collider.

  15. The Muon Collider as a $H/A$ factory

    DOE PAGES

    Eichten, Estia; Martin, Adam; Univ. of Notre Dame, IN

    2013-11-22

    We show that a muon collider is ideally suited for the study of heavy H/A scalars, cousins of the Higgs boson found in two-Higgs doublet models and required in supersymmetric models. The key aspects of H/A are: (1) they are narrow, yet have a width-to-mass ratio far larger than the expected muon collider beam-energy resolution, and (2) the larger muon Yukawa allows efficient s-channel production. We study in detail a representative Natural Supersymmetry model which has a 1.5 Tev H/A with $m_H$- $m_A$ = 10 Gev. The large event rates at resonant peak allow the determination of the individual Hmore » and A resonance parameters (including CP) and the decays into electroweakinos provides a wealth of information unavailable to any other present or planned collider.« less

  16. CILogon-HA. Higher Assurance Federated Identities for DOE Science

    SciTech Connect

    Basney, James

    2015-08-01

    The CILogon-HA project extended the existing open source CILogon service (initially developed with funding from the National Science Foundation) to provide credentials at multiple levels of assurance to users of DOE facilities for collaborative science. CILogon translates mechanism and policy across higher education and grid trust federations, bridging from the InCommon identity federation (which federates university and DOE lab identities) to the Interoperable Global Trust Federation (which defines standards across the Worldwide LHC Computing Grid, the Open Science Grid, and other cyberinfrastructure). The CILogon-HA project expanded the CILogon service to support over 160 identity providers (including 6 DOE facilities) and 3 internationally accredited certification authorities. To provide continuity of operations upon the end of the CILogon-HA project period, project staff transitioned the CILogon service to operation by XSEDE.

  17. The hyaluronan receptors CD44 and RHAMM (CD168) form complexeswith ERK1,2, which sustain high basal motility in breast cancercells

    SciTech Connect

    Hamilton, Sara R.; Fard, Shireen F.; Paiwand, Frouz F.; Tolg,Cornelia; Veiseh, Mandana; Wang, Chao; McCarthy, James B.; Bissell, MinaJ.; Koropatnick, James; Turley, Eva A.

    2007-03-28

    CD44 is an integral hyaluronan receptor that can promote or inhibit motogenic signaling in tumor cells. Rhamm is a non-integral cell surface hyaluronan receptor (CD168) and intracellular protein that promotes cell motility in culture and its expression is strongly upregulated in diseases like arthritis and aggressive cancers. Here we describe an autocrine mechanism utilizing cell surface Rhamm/CD44 interactions to sustain rapid basal motility in invasive breast cancer cell lines. This mechanism requires endogenous hyaluronan synthesis and the formation of Rhamm/CD44/ERK1, 2 complexes. Motile/ invasive MDA-MB-231 and Ras-MCF10A cells produce more endogenous hyaluronan, cell surface CD44 and Rhamm, an oncogenic Rhamm isoform, and exhibit elevated basal activation of ERK1, 2 than less invasive MCF7 and MCF10A breast cancer cells. Furthermore, CD44, Rhamm and ERK1, 2 uniquely co-immunoprecipitate and co-localize in MDA-MB-231 and Ras-MCF10A cells. Rapid motility of the invasive cell lines requires interaction of hyaluronan with cells, activation of ERK1, 2 and the participation of both cell surface CD44 and Rhamm. Combinations of anti-CD44, anti-Rhamm antibodies and a MEK1 inhibitor (PD098059) have less-than-additive blocking effects, suggesting action of all three proteins on a common motogenic signaling pathway. Collectively, these results show that cell surface Rhamm and CD44 act together in a hyaluronan-dependent, autocrine mechanism to coordinate sustained signaling through ERK1, 2 leading to high basal motility of invasive breast cancer cells. Since CD44/Rhamm complexes are not evident in less motile cells, an effect of CD44 on tumor cell motility may depend in part on its ability to partner with additional proteins, in this case cell surface Rhamm.

  18. AP-1-mediated invasion requires increased expression of the hyaluronan receptor CD44.

    PubMed Central

    Lamb, R F; Hennigan, R F; Turnbull, K; Katsanakis, K D; MacKenzie, E D; Birnie, G D; Ozanne, B W

    1997-01-01

    Fibroblasts transformed by Fos oncogenes display increased expression of a number of genes implicated in tumor cell invasion and metastasis. In contrast to normal 208F rat fibroblasts, Fos-transformed 208F fibroblasts are growth factor independent for invasion. We demonstrate that invasion of v-Fos- or epidermal growth factor (EGF)-transformed cells requires AP-1 activity. v-Fos-transformed cell invasion is inhibited by c-jun antisense oligonucleotides and by expression of a c-jun dominant negative mutant, TAM-67. EGF-induced invasion is inhibited by both c-fos and c-jun antisense oligonucleotides. CD44s, the standard form of a transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. We demonstrate that increased expression of CD44 in Fos- and EGF-transformed cells is dependent upon AP-1. CD44 antisense oligonucleotides reduce expression of CD44 in v-Fos- or EGF-transformed cells and inhibit invasion but not migration. Expression of a fusion protein between human CD44s and Aequorea victoria green fluorescent protein (GFP) in 208F cells complements the inhibition of invasion by the rat-specific CD44 antisense oligonucleotide. We further show that both v-Fos and EGF transformations result in a concentration of endogenous CD44 or exogenous CD44-GFP at the ends of pseudopodial cell extensions. These results support the hypothesis that one role of AP-1 in transformation is to activate a multigenic invasion program. PMID:9001250

  19. Laminin modified infection-preventing collagen membrane containing silver sulfadiazine-hyaluronan microparticles.

    PubMed

    Lee, Jong-Eun; Park, Jong-Chul; Lee, Kwang Hoon; Oh, Sang Ho; Suh, Hwal

    2002-06-01

    The newly developed laminin modified infection-preventing collagen membrane consists of a 3 component laminate, comprising 2 outer collagen layers and a central laminin layer. The 2 outer collagen layers (dense and porous layers) were fabricated by air-drying and freeze drying, respectively, and the laminin layer was formed by a straightforward liquid coating method. In addition, hyaluronan based microparticles containing silver sulfadiazine (AgSD) were incorporated into the 2 collagen layers (AgSD content 50 microg/cm2). Laminin coated collagen surfaces did not promote fibroblast attachment but showed a retarded fibroblast proliferation rate and an increased rate of collagen synthesis versus pure collagen surfaces. In an animal study, a laminin coating on a nonmedicated collagen membrane significantly increased both wound size reduction and vessel proliferation 7 days after application versus polyurethane film. Interestingly, the laminin coated AgSD medicated collagen membrane demonstrated higher wound size reduction and vessel proliferation and lower inflammation than the polyurethane control, suggesting that the laminin AgSD medicated collagen membrane substantially improves dermal wound healing.

  20. In vivo carotid artery closure by laser activation of hyaluronan-embedded gold nanorods

    NASA Astrophysics Data System (ADS)

    Matteini, Paolo; Ratto, Fulvio; Rossi, Francesca; Rossi, Giacomo; Esposito, Giuseppe; Puca, Alfredo; Albanese, Alessio; Maira, Giulio; Pini, Roberto

    2010-07-01

    We prove the first application of near-infrared-absorbing gold nanorods (GNRs) for in vivo laser closure of a rabbit carotid artery. GNRs are first functionalized with a biopolymeric shell and then embedded in hyaluronan, which gives a stabilized and handy laser-activable formulation. Four rabbits undergo closure of a 3-mm longitudinal incision performed on the carotid artery by means of a 810-nm diode laser in conjunction with the topical application of the GNRs composite. An effective surgery is obtained by using a 40-W/cm2 laser power density. The histological and electron microscopy evaluation after a 30-day follow-up demonstrates complete healing of the treated arteries with full re-endothelization at the site of GNRs application. The absence of microgranuloma formation and/or dystrophic calcification is evidence that no host reaction to nanoparticles interspersed through the vascular tissue occurred. The observation of a reshaping and associated blue shift of the NIR absorption band of GNRs after laser treatment supports the occurrence of a self-terminating process, and thus of additional safety of the minimally invasive laser procedure. This study underlines the feasibility of using GNRs for in vivo laser soldering applications, which represents a step forward toward the introduction of nanotechnology-based therapies in minimally invasive clinical practices.

  1. Effect of Water-Glass Coating on HA and HA-TCP Samples for MSCs Adhesion, Proliferation, and Differentiation.

    PubMed

    Bajpai, Indu; Kim, Duk Yeon; Kyong-Jin, Jung; Song, In-Hwan; Kim, Sukyoung

    2016-01-01

    Ca-P and silicon based materials have become very popular as bone tissue engineering materials. In this study, water-glass (also known as sodium silicate glass) was coated on sintered hydroxyapatite (HA) and HA-TCP (TCP stands for tricalcium phosphate) samples and subsequently heat-treated at 600°C for 2 hrs. X-rays diffraction showed the presence of β- and α-TCP phases along with HA in the HA-TCP samples. Samples without coating, with water-glass coating, and heat-treated after water-glass coating were used to observe the adhesion and proliferation response of bone marrow derived-mesenchymal stem cells (MSCs). Cell culture was carried out for 4 hrs, 1 day, and 7 days. Interestingly, all samples showed similar response for cell adhesion and proliferation up to 7-day culture but fibronectin, E-cadherin, and osteogenic differentiation related genes (osteocalcin and osteopontin) were significantly induced in heat-treated water-glass coated HA-TCP samples. A water-glass coating on Ca-P samples was not found to influence the cell proliferation response significantly but activated some extracellular matrix genes and induced osteogenic differentiation in the MSCs. PMID:27429988

  2. Effect of Water-Glass Coating on HA and HA-TCP Samples for MSCs Adhesion, Proliferation, and Differentiation

    PubMed Central

    Bajpai, Indu; Kim, Duk Yeon; Kyong-Jin, Jung; Song, In-Hwan

    2016-01-01

    Ca-P and silicon based materials have become very popular as bone tissue engineering materials. In this study, water-glass (also known as sodium silicate glass) was coated on sintered hydroxyapatite (HA) and HA-TCP (TCP stands for tricalcium phosphate) samples and subsequently heat-treated at 600°C for 2 hrs. X-rays diffraction showed the presence of β- and α-TCP phases along with HA in the HA-TCP samples. Samples without coating, with water-glass coating, and heat-treated after water-glass coating were used to observe the adhesion and proliferation response of bone marrow derived-mesenchymal stem cells (MSCs). Cell culture was carried out for 4 hrs, 1 day, and 7 days. Interestingly, all samples showed similar response for cell adhesion and proliferation up to 7-day culture but fibronectin, E-cadherin, and osteogenic differentiation related genes (osteocalcin and osteopontin) were significantly induced in heat-treated water-glass coated HA-TCP samples. A water-glass coating on Ca-P samples was not found to influence the cell proliferation response significantly but activated some extracellular matrix genes and induced osteogenic differentiation in the MSCs. PMID:27429988

  3. Bilingual Creativity in Chinese English: Ha Jin's "In the Pond."

    ERIC Educational Resources Information Center

    Zhang, Hang

    2002-01-01

    Addresses issues related to bilingual creativity in Chinese English and their implications for world Englishes in the Chinese context. The language examined is drawn from Ha Jin's novella, "In the Pond, in which the author's use of English is nativized in the Chinese context in order to recast the cultural meanings of the language. (Author/VWL)

  4. The Te-ni-wo-ha: An Etymological Study.

    ERIC Educational Resources Information Center

    Jolly, Yukiko S.

    1972-01-01

    The designation of the Japanese word class "joshi" (in English known as particles, post-positional case markers, or relationals) by the term te-ni-wo-ha can be traced to the early superimposition of the Chinese writing system on Japanese speech. Because of the structural differences between the two languages and the existence of elements in…

  5. Bisphosphonate-linked hyaluronic acid hydrogel sequesters and enzymatically releases active bone morphogenetic protein-2 for induction of osteogenic differentiation.

    PubMed

    Hulsart-Billström, Gry; Yuen, Pik Kwan; Marsell, Richard; Hilborn, Jöns; Larsson, Sune; Ossipov, Dmitri

    2013-09-01

    Regeneration of bone by delivery of bone morphogenetic proteins (BMPs) from implantable scaffolds is a promising alternative to the existing autologous bone grafting procedures. Hydrogels are used extensively in biomaterials as delivery systems for different growth factors. However, a controlled release of the growth factors is necessary to induce bone formation, which can be accomplished by various chemical functionalities. Herein we demonstrate that functionalization of a hyaluronan (HA) hydrogel with covalently linked bisphosphonate (BP) ligands provides efficient sequestering of BMP-2 in the resulting HA-BP hydrogel. The HA-BP hydrogel was investigated in comparison with its analogue lacking BP groups (HA hydrogel). While HA hydrogel released 100% of BMP-2 over two weeks, less than 10% of BMP-2 was released from the HA-BP hydrogel for the same time. We demonstrate that the sequestered growth factor can still be released by enzymatic degradation of the HA-BP hydrogel. Most importantly, entrapment of BMP-2 in HA-BP hydrogel preserves the growth factor bioactivity, which was confirmed by induction of osteogenic differentiation of mesenchymal stem cells (MSCs) after the cells incubation with the enzymatic digest of the hydrogel. At the same time, the hydrogels degradation products were not toxic to MSCs and osteoblasts. Furthermore, BP-functionalization of HA hydrogels promotes adhesion of the cells to the surface of HA hydrogel. Altogether, the present findings indicate that covalent grafting of HA hydrogel with BP groups can alter the clinical effects of BMPs in bone tissue regeneration.

  6. Kinetic investigation of the action of hyaluronidase on hyaluronan using the Morgan-Elson and neocuproine assays.

    PubMed

    Vercruysse, K P; Lauwers, A R; Demeester, J M

    1995-08-15

    In this paper we describe kinetic investigations of the action of testicular hyaluronidase on hyaluronan. We have compared the use of two spectrophotometric assays, the first based on the Morgan-Elson reaction and the second on the neocuproine reaction. With the neocuproine reaction Km was found to be 0.46 mg/ml and Vmax to be 126 nmol l-1 s-1. Because of a low sensitivity and the production of interfering precipitates, the Morgan-Elson assay cannot be used for kinetic investigation of the enzyme. Furthermore this assay is prone to interference from compounds such as disodium cromoglycate, (+)-catechine, penicillamine, CaCl2 and acetate buffer.

  7. Fabrication and mechanical properties of PLLA/PCL/HA composites via a biomimetic, dip coating, and hot compression procedure.

    PubMed

    Charles, L F; Shaw, M T; Olson, J R; Wei, M

    2010-06-01

    Currently, the bone-repair biomaterials market is dominated by high modulus metals and their alloys. The problem of stress-shielding, which results from elastic modulus mismatch between these metallic materials and natural bone, has stimulated increasing research into the development of polymer-ceramic composite materials that can more closely match the modulus of bone. In this study, we prepared poly(L: -lactic acid)/hydroxyapatite/poly(epsilon-caprolactone) (PLLA/HA/PCL) composites via a four-step process, which includes surface etching of the fiber, the deposition of the HA coating onto the PLLA fibers through immersion in simulated body fluid (SBF), PCL coating through a dip-coating process, and hot compression molding. The initial HA-coated PLLA fiber had a homogeneous and continuous coating with a gradient structure. The effects of HA: PCL ratio and molding temperature on flexural mechanical properties were studied and both were shown to be important to mechanical properties. Mechanical results showed that at low molding temperatures and up to an HA: PCL volume ratio of 1, the flexural strain decreased while the flexural modulus and strength increased. At higher mold temperatures with a lower viscosity of the PCL a HA: PCL ratio of 1.6 gave similar properties. The process successfully produced composites with flexural moduli near the lower range of bone. Such composites may have clinical use for load bearing bone fixation.

  8. The Hematopoietic System–specific Minor Histocompatibility Antigen HA-1 Shows Aberrant Expression in Epithelial Cancer Cells

    PubMed Central

    Klein, Christoph A.; Wilke, Martina; Pool, Jos; Vermeulen, Corine; Blokland, Els; Burghart, Elke; Krostina, Sabine; Wendler, Nicole; Passlick, Bernward; Riethmüeller, Gert; Goulmy, Els

    2002-01-01

    Allogeneic stem cell transplantation (SCT) can induce curative graft-versus-tumor reactions in patients with hematological malignancies and solid tumors. The graft-versus-tumor reaction after human histocompatibility leukocyte antigen (HLA)-identical SCT is mediated by alloimmune donor T cells specific for polymorphic minor histocompatibility antigens (mHags). Among these, the mHag HA-1 was found to be restricted to the hematopoietic system. Here, we report on the HA-1 ribonucleic acid expression by microdissected carcinoma tissues and by single disseminated tumor cells isolated from patients with various epithelial tumors. The HA-1 peptide is molecularly defined, as it forms an immunogenic peptide ligand with HLA-A2 on the cell membrane of carcinoma cell lines. HA-1–specific cytotoxic T cells lyse epithelial tumor cell lines in vitro, whereas normal epithelial cells are not recognized. Thus, HA-1–specific immunotherapy combined with HLA-identical allogeneic SCT may now be feasible for patients with HA-1+ carcinomas. PMID:12163564

  9. In vitro study of RRS HA injectable mesotherapy/biorevitalization product on human skin fibroblasts and its clinical utilization.

    PubMed

    Deglesne, Pierre-Antoine; Arroyo, Rodrigo; Ranneva, Evgeniya; Deprez, Philippe

    2016-01-01

    Mesotherapy/biorevitalization with hyaluronic acid (HA) is a treatment approach currently used for skin rejuvenation. Various products with a wide range of polycomponent formulations are available on the market. Most of these formulations contain noncross-linked HA in combination with a biorevitalization cocktail, formed by various amounts of vitamins, minerals, amino acids, nucleotides, coenzymes, and antioxidants. Although ingredients are very similar among the different products, in vitro and clinical effects may vary substantially. There is a real need for better characterization of these products in terms of their action on human skin or in vitro skin models. In this study, we analyzed the effect of the RRS(®) (Repairs, Refills, Stimulates) HA injectable medical device on human skin fibroblasts in vitro. Skin fibroblast viability and its capacity to induce the production of key extracellular matrix were evaluated in the presence of different concentrations of RRS HA injectable. Viability was evaluated through colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, and key extracellular matrix genes, type I collagen and elastin, were quantified by quantitative polymerase chain reaction. Results demonstrated that RRS HA injectable could promote human skin fibroblast viability (+15%) and increase fibroblast gene expression of type I collagen and elastin by 9.7-fold and 14-fold in vitro, respectively. These results demonstrate that mesotherapy/biorevitalization products can, at least in vitro, effectively modulate human skin fibroblasts. PMID:26966384

  10. In vitro study of RRS HA injectable mesotherapy/biorevitalization product on human skin fibroblasts and its clinical utilization.

    PubMed

    Deglesne, Pierre-Antoine; Arroyo, Rodrigo; Ranneva, Evgeniya; Deprez, Philippe

    2016-01-01

    Mesotherapy/biorevitalization with hyaluronic acid (HA) is a treatment approach currently used for skin rejuvenation. Various products with a wide range of polycomponent formulations are available on the market. Most of these formulations contain noncross-linked HA in combination with a biorevitalization cocktail, formed by various amounts of vitamins, minerals, amino acids, nucleotides, coenzymes, and antioxidants. Although ingredients are very similar among the different products, in vitro and clinical effects may vary substantially. There is a real need for better characterization of these products in terms of their action on human skin or in vitro skin models. In this study, we analyzed the effect of the RRS(®) (Repairs, Refills, Stimulates) HA injectable medical device on human skin fibroblasts in vitro. Skin fibroblast viability and its capacity to induce the production of key extracellular matrix were evaluated in the presence of different concentrations of RRS HA injectable. Viability was evaluated through colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, and key extracellular matrix genes, type I collagen and elastin, were quantified by quantitative polymerase chain reaction. Results demonstrated that RRS HA injectable could promote human skin fibroblast viability (+15%) and increase fibroblast gene expression of type I collagen and elastin by 9.7-fold and 14-fold in vitro, respectively. These results demonstrate that mesotherapy/biorevitalization products can, at least in vitro, effectively modulate human skin fibroblasts.

  11. In vitro study of RRS HA injectable mesotherapy/biorevitalization product on human skin fibroblasts and its clinical utilization

    PubMed Central

    Deglesne, Pierre-Antoine; Arroyo, Rodrigo; Ranneva, Evgeniya; Deprez, Philippe

    2016-01-01

    Mesotherapy/biorevitalization with hyaluronic acid (HA) is a treatment approach currently used for skin rejuvenation. Various products with a wide range of polycomponent formulations are available on the market. Most of these formulations contain noncross-linked HA in combination with a biorevitalization cocktail, formed by various amounts of vitamins, minerals, amino acids, nucleotides, coenzymes, and antioxidants. Although ingredients are very similar among the different products, in vitro and clinical effects may vary substantially. There is a real need for better characterization of these products in terms of their action on human skin or in vitro skin models. In this study, we analyzed the effect of the RRS® (Repairs, Refills, Stimulates) HA injectable medical device on human skin fibroblasts in vitro. Skin fibroblast viability and its capacity to induce the production of key extracellular matrix were evaluated in the presence of different concentrations of RRS HA injectable. Viability was evaluated through colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, and key extracellular matrix genes, type I collagen and elastin, were quantified by quantitative polymerase chain reaction. Results demonstrated that RRS HA injectable could promote human skin fibroblast viability (+15%) and increase fibroblast gene expression of type I collagen and elastin by 9.7-fold and 14-fold in vitro, respectively. These results demonstrate that mesotherapy/biorevitalization products can, at least in vitro, effectively modulate human skin fibroblasts. PMID:26966384

  12. An in vivo evaluation of PLLA/PLLA-gHA nano-composite for internal fixation of mandibular bone fractures.

    PubMed

    Peng, Weihai; Zheng, Wei; Shi, Kai; Wang, Wangshu; Shao, Ying; Zhang, Duo

    2015-11-09

    Internal fixation of bone fractures using biodegradable poly(L-lactic-acid) (PLLA)-based materials has attracted the attention of many researchers. In the present study, 36 male beagle dogs were randomly assigned to two groups: PLLA/PLLA-gHA (PLLA-grafted hydroxyapatite) group and PLLA group. PLLA/PLLA-gHA and PLLA plates were embedded in the muscular bags of the erector spinae and also implanted to fix mandibular bone fractures in respective groups. At 1, 2, 3, 6, 9, and 12 months postoperatively, the PLLA/PLLA-gHA and PLLA plates were evaluated by adsorption and degradation tests, and the mandibles were examined through radiographic analysis, biomechanical testing, and histological analysis. The PLLA/PLLA-gHA plates were non-transparent and showed a creamy white color, and the PLLA plates were transparent and faint yellow in color. At all time points following surgery, adsorption and degradation of the PLLA/PLLA-gHA plates were significantly less than those of the PLLA plates, and the lateral and longitudinal bending strengths of the surgically treated mandibles of the beagle dogs in the PLLA/PLLA-gHA group were significantly greater than those of the PLLA group and reached almost the value of intact mandibles at 12 months postoperatively. Additionally, relatively rapid bone healing was observed in the PLLA/PLLA-gHA group with the formation of new lamellar bone tissues at 12 months after the surgery. The PLLA/PLLA-gHA nano-composite can be employed as a biodegradable material for internal fixation of mandibular bone fractures.

  13. An in vivo evaluation of PLLA/PLLA-gHA nano-composite for internal fixation of mandibular bone fractures.

    PubMed

    Peng, Weihai; Zheng, Wei; Shi, Kai; Wang, Wangshu; Shao, Ying; Zhang, Duo

    2015-12-01

    Internal fixation of bone fractures using biodegradable poly(L-lactic-acid) (PLLA)-based materials has attracted the attention of many researchers. In the present study, 36 male beagle dogs were randomly assigned to two groups: PLLA/PLLA-gHA (PLLA-grafted hydroxyapatite) group and PLLA group. PLLA/PLLA-gHA and PLLA plates were embedded in the muscular bags of the erector spinae and also implanted to fix mandibular bone fractures in respective groups. At 1, 2, 3, 6, 9, and 12 months postoperatively, the PLLA/PLLA-gHA and PLLA plates were evaluated by adsorption and degradation tests, and the mandibles were examined through radiographic analysis, biomechanical testing, and histological analysis. The PLLA/PLLA-gHA plates were non-transparent and showed a creamy white color, and the PLLA plates were transparent and faint yellow in color. At all time points following surgery, adsorption and degradation of the PLLA/PLLA-gHA plates were significantly less than those of the PLLA plates, and the lateral and longitudinal bending strengths of the surgically treated mandibles of the beagle dogs in the PLLA/PLLA-gHA group were significantly greater than those of the PLLA group and reached almost the value of intact mandibles at 12 months postoperatively. Additionally, relatively rapid bone healing was observed in the PLLA/PLLA-gHA group with the formation of new lamellar bone tissues at 12 months after the surgery. The PLLA/PLLA-gHA nano-composite can be employed as a biodegradable material for internal fixation of mandibular bone fractures. PMID:26551378

  14. Generation of New M2e-HA2 Fusion Chimeric Peptide to Development of a Recombinant Fusion Protein Vaccine

    PubMed Central

    Ameghi, Ali; Baradaran, Behzad; Aghaiypour, Khosrow; Barzegar, Abolfazl; Pilehvar-Soltanahmadi, Yones; Moghadampour, Masood; Taghizadeh, Morteza; Zarghami, Nosratollah

    2015-01-01

    Purpose: The purpose was to design a new construction containing influenza virus (H1N1) M2e gene and HA2 gene by bioinformatics approach, cloning the construct in to Escherichia coli and produce M2e-HA2 peptide. Methods: The procedure was done by virus cultivation in SPF eggs, hemagglutination assay (HA), RNA isolation, RT-PCR, primers designed (DNAMAN 4 and Oligo7), virtual fusion construction translation (ExPASy), N-Glycosylated sites prediction (Ensemblegly-Iowa), complete open reading frame (ORF), stop codon studied (NCBI ORF Finder), rare codon determination (GenScript), Solvent accessibility of epitopes (Swiss-PdbViewer), antigenic sites prediction (Protean), fusion PCR of M2e-HA2 gene, sequence analysis, nested PCR, gel electrophoresis, double digestion of pET22b(+) plasmid and the fusion construct, ligation of them, transformation of the ligated vector (pET22b-M2e-HA2) to E.coli (BL21), mass culture the cloned bacterium ,induction the expression by isopropyl-beta-D-thiogalactopyranoside (IPTG), sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), purification the fusion peptide by Ni-NTA column, western blot to verify the purification. Results: In this study we developed a new approach for fusion of Influenza virus M2e (96 nucleotides) and HA2 (663 nucleotides) genes based on fusion PCR strategy and produced a fused fragment with 793 nucleotides. The construct was successfully cloned and expressed. Conclusion: This construct is a 261 amino acid chimeric fusion peptide with about 30 KD molecular weight. According on the latest information; this is the first case of expression and purification M2e-HA2 fusion chimeric peptide, which could be used for development of a recombinant M2e-HA2 fusion protein vaccine. PMID:26793615

  15. Effects of the Q223R mutation in the hemagglutinin (HA) of egg-adapted pandemic 2009 (H1N1) influenza A virus on virus growth and binding of HA to human- and avian-type cell receptors.

    PubMed

    Suptawiwat, O; Jeamtua, W; Boonarkart, Ch; Kongchanagul, A; Puthawathana, P; Auewarakul, P

    2013-01-01

    The 2009 swine-origin influenza A virus (H1N1) and its initial reassortant vaccine strains did not grow well in embryonated eggs. The glutamine to arginine mutation at the amino acid position 223 (Q223R) of the hemagglutinin (HA) gene is the major mutation previously found in egg-adapted 2009 H1N1 strains and shown to enhance viral growth in embryonated eggs. However, the effect of this mutation on the receptor-binding preference had not been directly demonstrated. In this study, the Q223R mutation was shown to change the viral HA binding preference from the human-type receptor, α2,6-linked sialic acid, to the avian-type receptor, α2,3-linked sialic acid; and to enhance the viral growth in embryonated eggs but not in cell culture.

  16. CD44, Hyaluronan, the Hematopoietic Stem Cell, and Leukemia-Initiating Cells

    PubMed Central

    Zöller, Margot

    2015-01-01

    CD44 is an adhesion molecule that varies in size due to glycosylation and insertion of so-called variant exon products. The CD44 standard isoform (CD44s) is highly expressed in many cells and most abundantly in cells of the hematopoietic system, whereas expression of CD44 variant isoforms (CD44v) is more restricted. CD44s and CD44v are known as stem cell markers, first described for hematopoietic stem cells and later on confirmed for cancer- and leukemia-initiating cells. Importantly, both abundantly expressed CD44s as well as CD44v actively contribute to the maintenance of stem cell features, like generating and embedding in a niche, homing into the niche, maintenance of quiescence, and relative apoptosis resistance. This is surprising, as CD44 is not a master stem cell gene. I here will discuss that the functional contribution of CD44 relies on its particular communication skills with neighboring molecules, adjacent cells and, last not least, the surrounding matrix. In fact, it is the interaction of the hyaluronan receptor CD44 with its prime ligand, which strongly assists stem cells to fulfill their special and demanding tasks. Recent fundamental progress in support of this “old” hypothesis, which may soon pave the way for most promising new therapeutics, is presented for both hematopoietic stem cell and leukemia-initiating cell. The contribution of CD44 to the generation of a stem cell niche, to homing of stem cells in their niche, to stem cell quiescence and apoptosis resistance will be in focus. PMID:26074915

  17. Effects of bound versus soluble pentosan polysulphate in PEG/HA-based hydrogels tailored for intervertebral disc regeneration.

    PubMed

    Frith, Jessica E; Menzies, Donna J; Cameron, Andrew R; Ghosh, P; Whitehead, Darryl L; Gronthos, S; Zannettino, Andrew C W; Cooper-White, Justin J

    2014-01-01

    Previous reports in the literature investigating chondrogenesis in mesenchymal progenitor cell (MPC) cultures have confirmed the chondro-inductive potential of pentosan polysulphate (PPS), a highly sulphated semi-synthetic polysaccharide, when added as a soluble component to culture media under standard aggregate-assay conditions or to poly(ethylene glycol)/hyaluronic acid (PEG/HA)-based hydrogels, even in the absence of inductive factors (e.g. TGFβ). In this present study, we aimed to assess whether a 'bound' PPS would have greater activity and availability over a soluble PPS, as a media additive or when incorporated into PEG/HA-based hydrogels. We achieved this by covalently pre-binding the PPS to the HA component of the gel (forming a new molecule, HA-PPS). We firstly investigated the activity of HA-PPS compared to free PPS, when added as a soluble factor to culture media. Cell proliferation, as determined by CCK8 and EdU assay, was decreased in the presence of either bound or free PPS whilst chondrogenic differentiation, as determined by DMMB assay and histology, was enhanced. In all cases, the effect of the bound PPS (HA-PPS) was more potent than that of the unbound form. These results alone suggest wider applications for this new molecule, either as a culture supplement or as a coating for scaffolds targeted at chondrogenic differentiation or maturation. We then investigated the incorporation of HA-PPS into a PEG/HA-based hydrogel system, by simply substituting some of the HA for HA-PPS. Rheological testing confirmed that incorporation of either HA-PPS or PPS did not significantly affect gelation kinetics, final hydrogel modulus or degradation rate but had a small, but significant, effect on swelling. When encapsulated in the hydrogels, MPCs retained good viability and rapidly adopted a rounded morphology. Histological analysis of both GAG and collagen deposition after 21 days showed that the incorporation of the bound-PPS into the hydrogel resulted in

  18. Hyperglycemia Diverts Dividing Osteoblastic Precursor Cells to an Adipogenic Pathway and Induces Synthesis of a Hyaluronan Matrix That Is Adhesive for Monocytes*

    PubMed Central

    Wang, Aimin; Midura, Ronald J.; Vasanji, Amit; Wang, Andrew J.; Hascall, Vincent C.

    2014-01-01

    Isolated rat bone marrow stromal cells cultured in osteogenic medium in which the normal 5.6 mm glucose is changed to hyperglycemic 25.6 mm glucose greatly increase lipid formation between 21–31 days of culture that is associated with decreased biomineralization, up-regulate expression of cyclin D3 and two adipogenic markers (CCAAT/enhancer binding protein α and peroxisome proliferator-activated receptor γ) within 5 days of culture, increase neutral and polar lipid synthesis within 5 days of culture, and form a monocyte-adhesive hyaluronan matrix through an endoplasmic reticulum stress-induced autophagic mechanism. Evidence is also provided that, by 4 weeks after diabetes onset in the streptozotocin-induced diabetic rat model, there is a large loss of trabecular bone mineral density without apparent proportional changes in underlying collagen matrices, a large accumulation of a hyaluronan matrix within the trabecular bone marrow, and adipocytes and macrophages embedded in this hyaluronan matrix. These results support the hypothesis that hyperglycemia in bone marrow diverts dividing osteoblastic precursor cells (bone marrow stromal cells) to a metabolically stressed adipogenic pathway that induces synthesis of a hyaluronan matrix that recruits inflammatory cells and establishes a chronic inflammatory process that demineralizes trabecular cancellous bone. PMID:24569987

  19. Synovial fluid hyaluronan mediates MSC attachment to cartilage, a potential novel mechanism contributing to cartilage repair in osteoarthritis using knee joint distraction

    PubMed Central

    Mastbergen, Simon C; Jones, Elena; Calder, Stuart J; Lafeber, Floris P J G; McGonagle, Dennis

    2016-01-01

    Objectives Knee joint distraction (KJD) is a novel, but poorly understood, treatment for osteoarthritis (OA) associated with remarkable ‘spontaneous’ cartilage repair in which resident synovial fluid (SF) multipotential mesenchymal stromal cells (MSCs) may play a role. We hypothesised that SF hyaluronic acid (HA) inhibited the initial interaction between MSCs and cartilage, a key first step to integration, and postulate that KJD environment favoured MSC/cartilage interactions. Methods Attachment of dual-labelled SF-MSCs were assessed in a novel in vitro human cartilage model using OA and rheumatoid arthritic (RA) SF. SF was digested with hyaluronidase (hyase) and its effect on adhesion was observed using confocal microscopy. MRI and microscopy were used to image autologous dual-labelled MSCs in an in vivo canine model of KJD. SF-HA was investigated using gel electrophoresis and densitometry. Results Osteoarthritic-synovial fluid (OA-SF) and purified high molecular weight (MW) HA inhibited SF-MSC adhesion to plastic, while hyase treatment of OA-SF but not RA-SF significantly increased MSC adhesion to cartilage (3.7-fold, p<0.05) These differences were linked to the SF mediated HA-coat which was larger in OA-SF than in RA-SF. OA-SF contained >9 MDa HA and this correlated with increases in adhesion (r=0.880). In the canine KJD model, MSC adhesion to cartilage was evident and also dependent on HA MW. Conclusions These findings highlight an unappreciated role of SF-HA on MSC interactions and provide proof of concept that endogenous SF-MSCs are capable of adhering to cartilage in a favourable biochemical and biomechanical environment in OA distracted joints, offering novel one-stage strategies towards joint repair. PMID:25948596

  20. Property peculiarities of the atelocollagen-hyaluronan conjugates crosslinked with a short chain di-oxirane compound.

    PubMed

    Maier, Vasilica; Lefter, Cristina M; Maier, Stelian S; Butnaru, Maria; Danu, Maricel; Ibanescu, Constanta; Popa, Marcel; Desbrieres, Jacques

    2014-09-01

    Minimal amounts of a short-chain bifunctional crosslinker of about 1.3 nm length, the 1,4-butanediol-diglycidyl ether (BDDGE), were used to generate atelocollagen-hyaluronan conjugates in hydrogel state. Two a priori constraints were considered in recipe/procedure developing: (i) working in nondenaturing conditions, and (ii) ensuring a low cytotoxicity of the final product. Both atelocollagen (aK) and hyaluronan (NaHyal) were accurately purified to reduce their molecular-weight dispersity, in order to ensure the reproducibility of hydrogels characteristics. 1:5 aK:NaHyal weight ratios and 1:2.5 to 1:5 α-NH2:BDDGE molar ratios were found to be the most favorable recipe prescriptions that allow the obtaining of rheo-mechanically stable hydrogels, able to be manipulated during cell culturing protocols. Experiments revealed two unexpected effects due to the crosslinking reactions mediated by a short-chain molecule: (i) the occurrence of two thresholds in the rheological behavior of the hydrogels, related with the amount of added crosslinker, and (ii) a quasi-denaturation side-effect induced over the protein component by large or in excess amounts of crosslinker. PMID:25063116

  1. Effect of Thickness of HA-Coating on Microporous Silk Scaffolds Using Alternate Soaking Technology

    PubMed Central

    Zhu, Rui; Xue, Yingsen; Hao, Zhangying; Xie, Zhenghong; Fan, Xiangli; Fan, Hongbin

    2014-01-01

    Hydroxyapatite (HA) can be coated on various materials surface and has the function of osteogenicity. Microporous silk scaffold has excellent biocompatibility. In this study, alternate soaking technology was used to coat HA on microporous silk scaffolds. However, the cell proliferation was found to decrease with the increasing thickness (cycles of soaking) of HA-coating. This study aims to determine the best thickness (cycles of