Shear Stress Regulates Adhesion and Rolling of CD44+ Leukemic and Hematopoietic Progenitor Cells on Hyaluronan

PubMed Central

Christophis, Christof; Taubert, Isabel; Meseck, Georg R.; Schubert, Mario; Grunze, Michael; Ho, Anthony D.; Rosenhahn, Axel

2011-01-01

Leukemic cells and human hematopoietic progenitor cells expressing CD44 receptors have the ability to attach and roll on hyaluronan. We investigated quantitatively the adhesion behavior of leukemic cell lines and hematopoietic progenitor cells on thin films of the polysaccharides hyaluronan and alginate in a microfluidic system. An applied flow enhances the interaction between CD44-positive cells and hyaluronan if a threshold shear stress of 0.2 dyn/cm2 is exceeded. At shear stress ∼1 dyn/cm2, the cell rolling speed reaches a maximum of 15 μm/s. Leukemic Jurkat and Kasumi-1 cells lacking CD44-expression showed no adhesion or rolling on the polysaccharides whereas the CD44-expressing leukemic cells KG-1a, HL-60, K-562, and hematopoietic progenitor cells attached and rolled on hyaluronan. Interestingly, the observations of flow-induced cell rolling are related to those found in the recruitment of leukocytes to inflammatory sites and the mechanisms of stem-cell homing into the bone marrow. PMID:21806926

TMEM2: A missing link in hyaluronan catabolism identified?

PubMed

Yamaguchi, Yu; Yamamoto, Hayato; Tobisawa, Yuki; Irie, Fumitoshi

2018-03-27

Hyaluronan (HA) is a glycosaminoglycan (GAG) composed of repeating disaccharide units of glucuronic acid and N-acetylglucosamine. HA is an extremely long, unbranched polymer, which often exceeds 10 6  Da and sometimes reaches 10 7  Da. A feature that epitomizes HA is its rapid turnover; one-third of the total body HA is turned over daily. The current model of HA catabolism postulates that high-molecular weight HA in the extracellular space is first cleaved into smaller fragments by a hyaluronidase(s) that resides at the cell surface, followed by internalization of fragments and their degradation into monosaccharides in lysosomes. Over the last decade, considerable research has shown that the HYAL family of hyaluronidases plays significant roles in HA catabolism. Nonetheless, the identity of a hyaluronidase responsible for the initial step of HA cleavage on the cell surface remains elusive, as biochemical and enzymological properties of HYAL proteins are not entirely consistent with those expected of cell surface hyaluronidases. Recent identification of transmembrane 2 (TMEM2) as a cell surface protein that possesses potent hyaluronidase activity suggests that it may be the "missing" cell surface hyaluronidase, and that novel models of HA catabolism should include this protein. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Repair of osteochondral defects with hyaluronan- and polyester-based scaffolds.

PubMed

Solchaga, Luis A; Temenoff, Johnna S; Gao, Jizong; Mikos, Antonios G; Caplan, Arnold I; Goldberg, Victor M

2005-04-01

The natural repair of osteochondral defects can be enhanced with biocompatible, biodegradable materials that support the repair process. It is our hypothesis that hyaluronan-based scaffolds are superior to synthetic scaffolds because they provide biological cues. We tested this thesis by comparing two hyaluronan-based scaffolds [auto cross-linked polysaccharide polymer (ACP) and HYAFF-11] to polyester-based scaffolds [poly(DL-lactic-co-glycolic acid) (PLGA) and poly(L-lactic acid) (PLLA)] with similar pore size, porosity and degradation times. Fifty-four rabbits received bilateral osteochondral defects. One defect received a hyaluronan-based scaffold and the contralateral defect received the corresponding polyester-based scaffold. Rabbits were euthanized 4, 12 and 20 weeks after surgery and the condyles dissected and processed for histology. Only ACP-treated defects presented bone at the base of the defect at 4 weeks. At 12 weeks, only defects treated with rapidly dissolving implants (ACP and PLGA) presented bone reconstitution consistently, while bone was present in only one third of those treated with slowly dissolving scaffolds (HYAFF-11 and PLLA). After 20 weeks, the articular surface of PLGA-treated defects presented fibrillation more frequently than in ACP-treated defects. The surface of defects treated with slowly dissolving scaffolds presented more cracks and fissures. The degradation rate of the scaffolds is critical for the repair process. Slowly dissolving scaffolds sustain thicker cartilage at the surface but, it frequently presents cracks and discontinuities. These scaffolds also delay bone formation at the base of the defects. Hyaluronan-based scaffolds appear to allow faster cell infiltration leading to faster tissue formation. The degradation of ACP leads to rapid bone formation while the slow degradation of HYAFF-11 prolongs the presence of cartilage and delays endochondral bone formation.

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.

MDA-MB-231 breast cancer cell viability, motility and matrix adhesion are regulated by a complex interplay of heparan sulfate, chondroitin-/dermatan sulfate and hyaluronan biosynthesis.

PubMed

Viola, Manuela; Brüggemann, Kathrin; Karousou, Evgenia; Caon, Ilaria; Caravà, Elena; Vigetti, Davide; Greve, Burkhard; Stock, Christian; De Luca, Giancarlo; Passi, Alberto; Götte, Martin

2017-06-01

Proteoglycans and glycosaminoglycans modulate numerous cellular processes relevant to tumour progression, including cell proliferation, cell-matrix interactions, cell motility and invasive growth. Among the glycosaminoglycans with a well-documented role in tumour progression are heparan sulphate, chondroitin/dermatan sulphate and hyaluronic acid/hyaluronan. While the mode of biosynthesis differs for sulphated glycosaminoglycans, which are synthesised in the ER and Golgi compartments, and hyaluronan, which is synthesized at the plasma membrane, these polysaccharides partially compete for common substrates. In this study, we employed a siRNA knockdown approach for heparan sulphate (EXT1) and heparan/chondroitin/dermatan sulphate-biosynthetic enzymes (β4GalT7) in the aggressive human breast cancer cell line MDA-MB-231 to study the impact on cell behaviour and hyaluronan biosynthesis. Knockdown of β4GalT7 expression resulted in a decrease in cell viability, motility and adhesion to fibronectin, while these parameters were unchanged in EXT1-silenced cells. Importantly, these changes were associated with a decreased expression of syndecan-1, decreased signalling response to HGF and an increase in the synthesis of hyaluronan, due to an upregulation of the hyaluronan synthases HAS2 and HAS3. Interestingly, EXT1-depleted cells showed a downregulation of the UDP-sugar transporter SLC35D1, whereas SLC35D2 was downregulated in β4GalT7-depleted cells, indicating an intricate regulatory network that connects all glycosaminoglycans synthesis. The results of our in vitro study suggest that a modulation of breast cancer cell behaviour via interference with heparan sulphate biosynthesis may result in a compensatory upregulation of hyaluronan biosynthesis. These findings have important implications for the development of glycosaminoglycan-targeted therapeutic approaches for malignant diseases.

Regulation of cell volume by glycosaminoglycans.

PubMed

Joerges, Jelena; Schulz, Tobias; Wegner, Jeannine; Schumacher, Udo; Prehm, Peter

2012-01-01

Cell volume is regulated by a delicate balance between ion distribution across the plasma membrane and the osmotic properties of intra- and extracellular components. Using a fluorescent calcein indicator, we analysed the effects of glycosaminoglycans on the cell volume of hyaluronan producing fibroblasts and hyaluronan deficient HEK cells over a time period of 30 h. Exogenous glycosaminoglycans induced cell blebbing after 2 min and swelling of fibroblasts to about 110% of untreated cell volume at low concentrations which decreased at higher concentrations. HEK cells did not show cell blebbing and responded by shrinking to 65% of untreated cell volume. Heparin induced swelling of both fibroblasts and HEK cells. Hyaluronidase treatment or inhibition of hyaluronan export led to cell shrinkage indicating that the hyaluronan coat maintained fibroblasts in a swollen state. These observations were explained by the combined action of the Donnan effect and molecular crowding. Copyright © 2011 Wiley Periodicals, Inc.

Hyaluronan Protects Bovine Articular Chondrocytes against Cell Death Induced by Bupivacaine under Supraphysiologic Temperatures

PubMed Central

Liu, Sen; Zhang, Qing-Song; Hester, William; O’Brien, Michael J.; Savoie, Felix H.; You, Zongbing

2013-01-01

Background Bupivacaine and supraphysiologic temperature can independently reduce cell viability of articular chondrocytes. In combination these two deleterious factors could further impair cell viability. Hypothesis Hyaluronan may protect chondrocytes from death induced by bupivacaine at supraphysiologic temperatures. Study Design Controlled laboratory study. Methods Bovine articular chondrocytes were treated with hyaluronan at physiologic (37°C) and supraphysiologic temperatures (45°C and 50°C) for one hour, and then exposed to bupivacaine for one hour at room temperature. Cell viability was assessed at three time points: immediately after treatment, six hours later, and twenty-four hours later using flow cytometry and fluorescence microscopy. The effects of hyaluronan on the levels of sulfated glycosaminoglycan in the chondrocytes were determined using Alcian blue staining. Results (1) Bupivacaine alone did not induce noticeable chondrocyte death at 37°C; (2) bupivacaine and temperature synergistically increased chondrocyte death, that is, when the chondrocytes were conditioned to 45°C and 50°C, 0.25% and 0.5% bupivacaine increased the cell death rate by 131% to 383% in comparison to the phosphate-buffered saline control group; and, (3) addition of hyaluronan reduced chondrocyte death rates to approximately 14% and 25% at 45°C and 50°C, respectively. Hyaluronan’s protective effects were still observed at six and twenty-four hours after bupivacaine treatment at 45°C. However, at 50°C, hyaluronan delayed but did not prevent the cell death caused by bupivacaine. One-hour treatment with hyaluronan significantly increased sulfated glycosaminoglycan levels in the chondrocytes. Conclusions Bupivacaine and supraphysiologic temperature synergistically increase chondrocyte death and hyaluronan may protect articular chondrocytes from death caused by bupivacaine. Clinical Relevance This study provides a rationale to perform pre-clinical and clinical studies to evaluate whether intra-articular injection of a mixture of bupivacaine and hyaluronan after arthroscopic surgery may protect against bupivacaine’s chondrotoxicity. PMID:22427617

Hyaluronan and calcium carbonate hybrid nanoparticles for colorectal cancer chemotherapy

NASA Astrophysics Data System (ADS)

Bai, Jinghui; Xu, Jian; Zhao, Jian; Zhang, Rui

2017-09-01

A hybrid drug delivery system (DDS) composed of hyaluronan and calcium carbonate (CC) was developed. By taking advantage of the tumor-targeting ability of hyaluronan and the drug-loading property of CC, the well-formed hyaluronan-CC nanoparticles were able to serve as a DDS targeting colorectal cancer with a decent drug loading content, which is beneficial in the chemotherapy of colorectal cancer. In this study, hyaluronan-CC nanoparticles smaller than 100 nm were successfully developed to load the wide-range anti-cancer drug adriamycin (Adr) to construct hyaluronan-CC/Adr nanoparticles. On the other hand, we also found that hyaluronan-CC/Adr nanoparticles can possibly increase the uptake ratio of Adr into HT29 colorectal cancer cells when compared with hyaluronan-free nanoparticles (CC/Adr) via the CD44 receptor-mediated endocytosis via competitive uptake and in vivo imaging assays. Note that both in vitro (CCK-8 assay on HT29 cells) and in vivo (anti-cancer assay on HT-29 tumor-bearing nude mice model) experiments revealed that hyaluronan-CC/Adr nanoparticles exhibited stronger anti-cancer activity than free Adr or CC/Adr nanoparticles with minimized toxic side effects and preferable cancer-suppression potential.

Fluorescent molecularly imprinted polymers as plastic antibodies for selective labeling and imaging of hyaluronan and sialic acid on fixed and living cells.

PubMed

Panagiotopoulou, Maria; Kunath, Stephanie; Medina-Rangel, Paulina Ximena; Haupt, Karsten; Tse Sum Bui, Bernadette

2017-02-15

Altered glycosylation levels or distribution of sialic acids (SA) or hyaluronan in animal cells are indicators of pathological conditions like infection or malignancy. We applied fluorescently-labeled molecularly imprinted polymer (MIP) particles for bioimaging of fixed and living human keratinocytes, to localize hyaluronan and sialylation sites. MIPs were prepared with the templates D-glucuronic acid (GlcA), a substructure of hyaluronan, and N-acetylneuraminic acid (NANA), the most common member of SA. Both MIPs were found to be highly selective towards their target monosaccharides, as no cross-reactivity was observed with other sugars like N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-glucose and D-galactose, present on the cell surface. The dye rhodamine and two InP/ZnS quantum dots (QDs) emitting in the green and in the red regions were used as fluorescent probes. Rhodamine-MIPGlcA and rhodamine-MIPNANA were synthesized as monodispersed 400nm sized particles and were found to bind selectively their targets located in the extracellular region, as imaged by epifluorescence and confocal microscopy. In contrast, when MIP-GlcA and MIP-NANA particles with a smaller size (125nm) were used, the MIPs being synthesized as thin shells around green and red emitting QDs respectively, it was possible to stain the intracellular and pericellular regions as well. In addition, simultaneous dual-color imaging with the two different colored QDs-MIPs was demonstrated. Importantly, the MIPs were not cytotoxic and did not affect cell viability; neither was the cells morphology affected as demonstrated by live cell imaging. These synthetic receptors could offer a new and promising imaging tool to monitor disease progression. Copyright © 2016 Elsevier B.V. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, P.-Y.; Huang, Lynn L.H.; Hsieh, H.-J.

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 activitiesmore » 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.« less

Analysis of CD44-Hyaluronan Interactions in an Artificial Membrane System

PubMed Central

Wolny, Patricia M.; Banerji, Suneale; Gounou, Céline; Brisson, Alain R.; Day, Anthony J.; Jackson, David G.; Richter, Ralf P.

2010-01-01

CD44 is a major cell surface receptor for the large polydisperse glycosaminoglycan hyaluronan (HA). Binding of the long and flexible HA chains is thought to be stabilized by the multivalent nature of the sugar molecule. In addition, high and low molecular weight forms of HA provoke distinct proinflammatory and anti-inflammatory effects upon binding to CD44 and can deliver either proliferative or antiproliferative signals in appropriate cell types. Despite the importance of such interactions, however, neither the stoichiometry of multivalent HA binding at the cell surface nor the molecular basis for functional distinction between different HA size categories is understood. Here we report on the design of a supported lipid bilayer system that permits quantitative analysis of multivalent binding through presentation of CD44 in a stable, natively oriented manner and at controlled density. Using this system in combination with biophysical techniques, we show that the amount of HA binding to bilayers that are densely coated with CD44 increases as a function of HA size, with half-maximal saturation at ∼30 kDa. Moreover, reversible binding was confined to the smaller HA species (molecular weight of ≤10 kDa), whereas the interaction was essentially irreversible with larger polymers. The amount of bound HA decreased with decreasing receptor surface density, but the stability of binding was not affected. From a physico-chemical perspective, the binding properties of HA share many similarities with the typical behavior of a flexible polymer as it adsorbs onto a homogeneously attractive surface. These findings provide new insight into the multivalent nature of CD44-HA interactions and suggest a molecular basis for the distinct biological properties of different size fractions of hyaluronan. PMID:20663884

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Ningbo, E-mail: curl-zhao@163.com; Wang, Xin, E-mail: 394041230@qq.com; Qin, Lei, E-mail: qinlei30@126.com

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 bymore » 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.« less

4-Methylumbelliferone Diminishes Catabolically Activated Articular Chondrocytes and Cartilage Explants via a Mechanism Independent of Hyaluronan Inhibition*

PubMed Central

Ishizuka, Shinya; Askew, Emily B.; Ishizuka, Naoko; Knudson, Cheryl B.; Knudson, Warren

2016-01-01

Depletion of the cartilage proteoglycan aggrecan is one of the earliest events that occurs in association with osteoarthritis. This loss is often accompanied by a coordinate loss in another glycosaminoglycan, hyaluronan. Chondrocytes experimentally depleted of cell-associated hyaluronan respond by switching to a pro-catabolic metabolism that includes enhanced production of endogenous inflammatory mediators and increased synthesis of matrix metalloproteinases. Hyaluronan turnover is also increased. Together, such a response provides for possible establishment of a self-perpetuating spiral of events that maintains or prolongs the pro-catabolic state. Chondrocytes or cartilage can also be activated by treatment with pro-inflammatory cytokines and mediators such as IL-1β, TNFα, LPS, fibronectin fragments, and hyaluronan oligosaccharides. To determine the mechanism of chondrocyte activation due to hyaluronan loss, a depletion method was required that did not include degrading the hyaluronan. In recent years, several laboratories have used the coumarin derivative, 4-methylumbelliferone, as a potent inhibitor of hyaluronan biosynthesis, due in part to its ability to sequester intracellular UDP-glucuronic acid and inhibition of hyaluronan synthase transcription. However, contrary to our expectation, although 4-methylumbelliferone was indeed an inhibitor of hyaluronan biosynthesis, this depletion did not give rise to an activation of chondrocytes or cartilage. Rather, 4-methylumbelliferone directly and selectively blocked gene products associated with the pro-catabolic metabolic state of chondrocytes and did so through a mechanism preceding and independent of hyaluronan inhibition. These data suggest that 4-methylumbelliferone has additional useful applications to block pro-inflammatory cell activation events but complicates how it is used for defining functions related to hyaluronan. PMID:27129266

Controlling adsorption of albumin with hyaluronan on silica surfaces and sulfonated latex particles.

PubMed

Berts, Ida; Fragneto, Giovanna; Porcar, Lionel; Hellsing, Maja S; Rennie, Adrian R

2017-10-15

Polysaccharides are known to modify binding of proteins at interfaces and this paper describes studies of these interactions and how they are modified by pH. Specifically, the adsorption of human serum albumin on to polystyrene latex and to silica is described, focusing on how this is affected by hyaluronan. Experiments were designed to test how such binding might be modified under relevant physiological conditions. Changes in adsorption of albumin alone and the co-adsorption of albumin and hyaluronan are driven by electrostatic interactions. Multilayer binding is found to be regulated by the pH of the solution and the molecular mass and concentration of hyaluronan. Highest adsorption was observed at pH below 4.8 and for low molecular mass hyaluronan (≤150kDa) at concentrations above 2mgml -1 . On silica with grafted hyaluronan, albumin absorption is reversed by changes in solvent pH due to their strong electrostatic attraction. Albumin physisorbed on silica surfaces is also rinsed away with dilute hyaluronan solution at pH 4.8. The results demonstrate that the protein adsorption can be controlled both by changes of pH and by interaction with other biological macromolecules. Copyright © 2017 Elsevier Inc. All rights reserved.

The interaction between LYVE-1 with hyaluronan on the cell surface may play a role in the diversity of adhesion to cancer cells.

PubMed

Du, Yan; Liu, Hua; He, Yiqing; Liu, Yiwen; Yang, Cuixia; Zhou, Muqing; Wang, Wenjuan; Cui, Lian; Hu, Jiajie; Gao, Feng

2013-01-01

Hyaluronan (HA), a simple disaccharide unit, can polymerize and is considered a primary component of the extracellular matrix, which has a wide range of biological functions. In recent years, HA was found on the surface of tumor cells. According to previous reports, differing HA content on the cell surface of tumor cells is closely related to lymph node metastases, but the mechanisms mediating this process remained unclear. This research intended to study the surface content of HA on tumor cells and analyze cell adhesive changes caused by the interaction between HA and its lymphatic endothelial receptor (LYVE-1). We screened and observed high HA content on HS-578T breast cells and low HA content on MCF-7 breast cells through particle exclusion, immunofluorescence and flow cytometry experiments. The expression of LYVE-1, the lymph-vessel specific HA receptor, was consistent with our previous report and enhanced the adhesion of HA(high)-HS-578T cells to COS-7(LYVE-1(+)) through HA in cell static adhesion and dynamic parallel plate flow chamber experiments. MCF-7 breast cells contain little HA on the surface; however, our results showed little adhesion difference between MCF-7 cells and COS-7(LYVE-1(+)) and COS-7(LYVE-1(-)) cells. Similar results were observed concerning the adhesion of HS-578T cells or MCF-7 cells to SVEC4-10 cells. Furthermore, we observed for the first time that the cell surface HA content of high transfer tumor cells was rich, and we visualized the cross-linking of HA cable structures, which may activate LYVE-1 on lymphatic endothelial cells, promoting tumor adhesion. In summary, high-low cell surface HA content of tumor cells through the interaction with LYVE-1 leads to adhesion differences.

Hyaluronan synthesis in cultured tobacco cells (BY-2) expressing a chlorovirus enzyme: cytological studies.

PubMed

Rakkhumkaew, Numfon; Shibatani, Shigeo; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

2013-04-01

Extraction of hyaluronan from animals or microbial fermentation has risks including contamination with pathogens and microbial toxins. In this work, tobacco cultured-cells (BY-2) were successfully transformed with a chloroviral hyaluronan synthase (cvHAS) gene to produce hyaluronan. Cytological studies revealed accumulation of HA on the cells, and also in subcellular fractions (protoplasts, miniplasts, vacuoplasts, and vacuoles). Transgenic BY-2 cells harboring a vSPO-cvHAS construct containing the vacuolar targeting signal of sporamin connected to the N-terminus of cvHAS accumulated significant amounts of HA in vacuoles. These results suggested that cvHAS successfully functions on the vacuolar membrane and synthesizes/transports HA into vacuoles. Efficient synthesis of HA using this system provides a new method for practical production of HA. Copyright © 2012 Wiley Periodicals, Inc.

Intracellular trafficking of hyaluronic acid-chitosan oligomer-based nanoparticles in cultured human ocular surface cells.

PubMed

Contreras-Ruiz, Laura; de la Fuente, María; Párraga, Jenny E; López-García, Antonio; Fernández, Itziar; Seijo, Begoña; Sánchez, Alejandro; Calonge, Margarita; Diebold, Yolanda

2011-01-27

Nanoparticles are a promising alternative for ocular drug delivery, and our group has proposed that they are especially suited for ocular mucosal disorders. The goal of the present study was to determine which internalization pathway is used by cornea-derived and conjunctiva-derived cell lines to take up hyaluronic acid (HA)-chitosan oligomer (CSO)-based nanoparticles (HA-CSO NPs). We also determined if plasmids loaded onto the NPs reached the cell nucleus. HA-CSO NPs were made of fluoresceinamine labeled HA and CSO by ionotropic gelation and were conjugated with a model plasmid DNA for secreted alkaline phosphatase. Human epithelial cell lines derived from the conjunctiva and the cornea were exposed to HA-CSO NPs for 1 h and the uptake was investigated in living cells by fluorescence microscopy. The influence of temperature and metabolic inhibition, the effect of blocking hyaluronan receptors, and the inhibition of main endocytic pathways were studied by fluorometry. Additionally, the metabolic pathways implicated in the degradation of HA-CSO NPs were evaluated by lysosome identification. There was intracellular localization of plasmid-loaded HACSO NPs in both corneal and conjunctival cells. The intracellular presence of NPs diminished with time. HA-CSO NP uptake was significantly reduced by inhibition of active transport at 4 °C and by sodium azide. Uptake was also inhibited by blocking hyaluronan receptors with anti-CD44 Hermes-1 antibody, by excess HA, and by filipin, an inhibitor of caveolin-dependent endocytosis. HA-CSO NPs had no effect on cell viability. The transfection efficiency of the model plasmid was significantly higher in NP treated cells than in controls. HA-CSO NPs were internalized by two different ocular surface cell lines by an active transport mechanism. The uptake was mediated by hyaluronan receptors through a caveolin-dependent endocytic pathway, yielding remarkable transfection efficiency. Most of HA-CSO NPs were metabolized within 48 h. This uptake did not compromise cell viability. These findings further support the potential use of HA-CSO NPs to deliver genetic material to the ocular surface.

Reinjury risk of nano-calcium oxalate monohydrate and calcium oxalate dihydrate crystals on injured renal epithelial cells: aggravation of crystal adhesion and aggregation

PubMed Central

Gan, Qiong-Zhi; Sun, Xin-Yuan; Bhadja, Poonam; Yao, Xiu-Qiong; Ouyang, Jian-Ming

2016-01-01

Background Renal epithelial cell injury facilitates crystal adhesion to cell surface and serves as a key step in renal stone formation. However, the effects of cell injury on the adhesion of nano-calcium oxalate crystals and the nano-crystal-induced reinjury risk of injured cells remain unclear. Methods African green monkey renal epithelial (Vero) cells were injured with H2O2 to establish a cell injury model. Cell viability, superoxide dismutase (SOD) activity, malonaldehyde (MDA) content, propidium iodide staining, hematoxylin–eosin staining, reactive oxygen species production, and mitochondrial membrane potential (Δψm) were determined to examine cell injury during adhesion. Changes in the surface structure of H2O2-injured cells were assessed through atomic force microscopy. The altered expression of hyaluronan during adhesion was examined through laser scanning confocal microscopy. The adhesion of nano-calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals to Vero cells was observed through scanning electron microscopy. Nano-COM and COD binding was quantitatively determined through inductively coupled plasma emission spectrometry. Results The expression of hyaluronan on the cell surface was increased during wound healing because of Vero cell injury. The structure and function of the cell membrane were also altered by cell injury; thus, nano-crystal adhesion occurred. The ability of nano-COM to adhere to the injured Vero cells was higher than that of nano-COD crystals. The cell viability, SOD activity, and Δψm decreased when nano-crystals attached to the cell surface. By contrast, the MDA content, reactive oxygen species production, and cell death rate increased. Conclusion Cell injury contributes to crystal adhesion to Vero cell surface. The attached nano-COM and COD crystals can aggravate Vero cell injury. As a consequence, crystal adhesion and aggregation are enhanced. These findings provide further insights into kidney stone formation. PMID:27382277

Provisional matrix: A role for versican and hyaluronan.

PubMed

Wight, Thomas N

2017-07-01

Hyaluronan and versican are extracellular matrix (ECM) components that are enriched in the provisional matrices that form during the early stages of development and disease. These two molecules interact to create pericellular "coats" and "open space" that facilitate cell sorting, proliferation, migration, and survival. Such complexes also impact the recruitment of leukocytes during development and in the early stages of disease. Once thought to be inert components of the ECM that help hold cells together, it is now quite clear that they play important roles in controlling cell phenotype, shaping tissue response to injury and maintaining tissue homeostasis. Conversion of hyaluronan-/versican-enriched provisional matrix to collagen-rich matrix is a "hallmark" of tissue fibrosis. Targeting the hyaluronan and versican content of provisional matrices in a variety of diseases including, cardiovascular disease and cancer, is becoming an attractive strategy for intervention. Copyright © 2016 Elsevier B.V. All rights reserved.

Pleural tissue hyaluronan produced by postmortem ventilation in rabbits.

PubMed

Wang, P M; Lai-Fook, S J

2000-01-01

We developed a method that used Alcian blue bound to hyaluronan to measure pleural hyaluronan in rabbits postmortem. Rabbits were killed, then ventilated with 21% O2--5% CO2--74% N2 for 3 h. The pleural liquid was removed by suction and 5 ml Alcian blue stock solution (0.33 mg/ml, 3.3 pH) was injected into each chest cavity. After 10 min, the Alcian blue solution was removed and the unbound Alcian blue solution (supernatant) separated by centrifugation and filtration. The supernatant transmissibility (T) was measured spectrophotometrically at 613 nm. Supernatant Alcian blue concentration (Cab) was obtained from a calibration curve of T versus dilutions of stock solution Cab. Alcian blue bound to pleural tissue hyaluronan was obtained by subtracting supernatant Cab from stock solution Cab. Pleural tissue hyaluronan was obtained from a calibration curve of hyaluronan versus Alcian blue bound to hyaluronan. Compared with control rabbits, pleural tissue hyaluronan (0.21 +/- 0.04 mg/kg) increased twofold, whereas pleural liquid volume decreased by 30% after 3 h of ventilation. Pleural effusions present 3 h postmortem without ventilation did not change pleural tissue hyaluronan from control values. Thus ventilation-induced pleural liquid shear stress, not increased filtration, was the stimulus for the increased hyaluronan produced from pleural mesothelial cells.

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

Post-secretory fate of host defence components in mucus.

PubMed

Salathe, Matthias; Forteza, Rosanna; Conner, Gregory E

2002-01-01

Airway mucus is a complex mixture of secretory products that provide a multifaceted defence against infection. Among many antimicrobial substances, mucus contains a peroxidase identical to milk lactoperoxidase (LPO) that is produced by goblet cells and submucosal glands. Airway secretions contain the substrates for LPO, namely thiocyanate and hydrogen peroxide, at concentrations sufficient for production of the biocidal compound hypothiocyanite, a fact confirmed by us in vitro. In vivo, inhibition of airway LPO in sheep significantly inhibits bacterial clearance, suggesting that the LPO system is a major contributor to host defences. Since secretory products including LPO are believed to be steadily removed by mucociliary clearance, their amount and availability on the surface is thought to be controlled solely by secretion. In contrast to this paradigm, new data suggest that LPO and other substances are retained at the ciliary border of the airway epithelium by binding to surface-associated hyaluronan, thereby providing an apical, fully active enzyme pool. Thus, hyaluronan, secreted from submucosal gland cells, plays a previously unrecognized pivotal role in mucosal host defence by retaining LPO and possibly other substances important for first line host defence at the apical surface 'ready for use' and protected from ciliary clearance.

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.

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. © 2014 Wiley Periodicals, Inc.

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

PubMed

Seino, Satoshi; Takeshita, Fumitaka; Asari, Akira; Masuda, Yasunobu; Kunou, Masaaki; Ochiya, Takahiro

2014-07-01

Hyaluronan (HA), a type of glycosaminoglycan used to construct the extracellular matrix, is involved in the proliferation and motility of cells, including cancer cells. The aim of this study was to determine whether exogenous HA has an influence on cancer in vitro and in vivo. High-molecular-weight HA (900 kDa) and low-molecular-weight HA (10 kDa) were added to several types of cancer cell lines in vitro, and proliferation and invasion were assessed. The effect of HA on capillary formation by human umbilical vein endothelial cells was also analyzed. The results showed that both types of HA had no apparent effect on cellular proliferation, invasion, or capillary formation. In an animal study, the 2 types of HA were orally administered to tumor-bearing mice at a dosage of 200 mg/kg/d for 4 wk. Analysis using an in vivo imaging system revealed that tumor proliferation and metastasis were not greatly altered by HA administration. Furthermore, CD31 immunohistochemical staining revealed no obvious change in tumor microvessels. Taken together, these results demonstrate that exogenously administered HA has little effect on cancer. This study may support the safety of various forms of HA administration, including oral intake. Orally administered hyaluronan was recently found to have beneficial effects. However, the effect of exogenous hyaluronan on cancer remains unclear. Our findings support the safety of orally administered hyaluronan and its use as a functional food ingredient. © 2014 Institute of Food Technologists®

Mucin-like Region of Herpes Simplex Virus Type 1 Attachment Protein Glycoprotein C (gC) Modulates the Virus-Glycosaminoglycan Interaction*

PubMed Central

Altgärde, Noomi; Eriksson, Charlotta; Peerboom, Nadia; Phan-Xuan, Tuan; Moeller, Stephanie; Schnabelrauch, Matthias; Svedhem, Sofia; Trybala, Edward; Bergström, Tomas; Bally, Marta

2015-01-01

Glycoprotein C (gC) mediates the attachment of HSV-1 to susceptible host cells by interacting with glycosaminoglycans (GAGs) on the cell surface. gC contains a mucin-like region located near the GAG-binding site, which may affect the binding activity. Here, we address this issue by studying a HSV-1 mutant lacking the mucin-like domain in gC and the corresponding purified mutant protein (gCΔmuc) in cell culture and GAG-binding assays, respectively. The mutant virus exhibited two functional alterations as compared with native HSV-1 (i.e. decreased sensitivity to GAG-based inhibitors of virus attachment to cells and reduced release of viral particles from the surface of infected cells). Kinetic and equilibrium binding characteristics of purified gC were assessed using surface plasmon resonance-based sensing together with a surface platform consisting of end-on immobilized GAGs. Both native gC and gCΔmuc bound via the expected binding region to chondroitin sulfate and sulfated hyaluronan but not to the non-sulfated hyaluronan, confirming binding specificity. In contrast to native gC, gCΔmuc exhibited a decreased affinity for GAGs and a slower dissociation, indicating that once formed, the gCΔmuc-GAG complex is more stable. It was also found that a larger number of gCΔmuc bound to a single GAG chain, compared with native gC. Taken together, our data suggest that the mucin-like region of HSV-1 gC is involved in the modulation of the GAG-binding activity, a feature of importance both for unrestricted virus entry into the cells and release of newly produced viral particles from infected cells. PMID:26160171

CD147, CD44, and the epidermal growth factor receptor (EGFR) signaling pathway cooperate to regulate breast epithelial cell invasiveness.

PubMed

Grass, G Daniel; Tolliver, Lauren B; Bratoeva, Momka; Toole, Bryan P

2013-09-06

The immunoglobulin superfamily glycoprotein CD147 (emmprin; basigin) is associated with an invasive phenotype in various types of cancers, including malignant breast cancer. We showed recently that up-regulation of CD147 in non-transformed, non-invasive breast epithelial cells is sufficient to induce an invasive phenotype characterized by membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invadopodia activity (Grass, G. D., Bratoeva, M., and Toole, B. P. (2012) Regulation of invadopodia formation and activity by CD147. J. Cell Sci. 125, 777-788). Here we found that CD147 induces breast epithelial cell invasiveness by promoting epidermal growth factor receptor (EGFR)-Ras-ERK signaling in a manner dependent on hyaluronan-CD44 interaction. Furthermore, CD147 promotes assembly of signaling complexes containing CD147, CD44, and EGFR in lipid raftlike domains. We also found that oncogenic Ras regulates CD147 expression, hyaluronan synthesis, and formation of CD147-CD44-EGFR complexes, thus forming a positive feedback loop that may amplify invasiveness. Last, we showed that malignant breast cancer cells are heterogeneous in their expression of surface-associated CD147 and that high levels of membrane CD147 correlate with cell surface EGFR and CD44 levels, activated EGFR and ERK1, and activated invadopodia. Future studies should evaluate CD147 as a potential therapeutic target and disease stratification marker in breast cancer.

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

Tuning the density profile of surface-grafted hyaluronan and the effect of counter-ions.

PubMed

Berts, Ida; Fragneto, Giovanna; Hilborn, Jöns; Rennie, Adrian R

2013-07-01

The present paper investigates the structure and composition of grafted sodium hyaluronan at a solid-liquid interface using neutron reflection. The solvated polymer at the surface could be described with a density profile that decays exponentially towards the bulk solution. The density profile of the polymer varied depending on the deposition protocol. A single-stage deposition resulted in denser polymer layers, while layers created with a two-stage deposition process were more diffuse and had an overall lower density. Despite the diffuse density profile, two-stage deposition leads to a higher surface excess. Addition of calcium ions causes a strong collapse of the sodium hyaluronan chains, increasing the polymer density near the surface. This effect is more pronounced on the sample prepared by two-stage deposition due to the initial less dense profile. This study provides an understanding at a molecular level of how surface functionalization alters the structure and how surface layers respond to changes in calcium ions in the solvent.

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

Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

PubMed Central

Schmelzer, Eva; Over, Patrick; Nettleship, Ian; Gerlach, Joerg C.

2016-01-01

Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications. PMID:27403430

Atomic force microscopy evaluation of aqueous interfaces of immobilized hyaluronan.

PubMed

Morra, Marco; Cassinelli, Clara; Pavesio, Alessandra; Renier, Davide

2003-03-15

Hyaluronan (HA) was immobilized on aminated glass surfaces in three different ways: by simple ionic interaction and by covalent linking at low density and at full density. In agreement with previous reports, in vitro experiments show that the outcome of fibroblast adhesion tests is markedly affected by the details of the coupling procedure, suggesting that different interfacial forces are operating at the aqueous/HA interface in the three cases investigated. The interfacial properties of the HA-coated surfaces were probed by force-distance curves obtained with the atomic force microscope (AFM). This approach readily shows significant differences among the tested samples, which are directly related to the coupling strategy and to results of cell adhesion tests. In particular, the range of interaction between the tip and the surface is much lower when HA is covalently linked than when it is ionically coupled, suggesting a more compact surface structure in the former case. Increasing HA surface density minimizes the interaction force between the surface and the AFM tip, likely reflecting more complete shielding by the HA chains of the underlying substrate. In summary, these measurements clearly show the different nature of the aqueous interfaces tested, and underline the role of this analytical approach in the development and control of finely tuned biomaterial surfaces.

CD147, CD44, and the Epidermal Growth Factor Receptor (EGFR) Signaling Pathway Cooperate to Regulate Breast Epithelial Cell Invasiveness*

PubMed Central

Grass, G. Daniel; Tolliver, Lauren B.; Bratoeva, Momka; Toole, Bryan P.

2013-01-01

The immunoglobulin superfamily glycoprotein CD147 (emmprin; basigin) is associated with an invasive phenotype in various types of cancers, including malignant breast cancer. We showed recently that up-regulation of CD147 in non-transformed, non-invasive breast epithelial cells is sufficient to induce an invasive phenotype characterized by membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invadopodia activity (Grass, G. D., Bratoeva, M., and Toole, B. P. (2012) Regulation of invadopodia formation and activity by CD147. J. Cell Sci. 125, 777–788). Here we found that CD147 induces breast epithelial cell invasiveness by promoting epidermal growth factor receptor (EGFR)-Ras-ERK signaling in a manner dependent on hyaluronan-CD44 interaction. Furthermore, CD147 promotes assembly of signaling complexes containing CD147, CD44, and EGFR in lipid raftlike domains. We also found that oncogenic Ras regulates CD147 expression, hyaluronan synthesis, and formation of CD147-CD44-EGFR complexes, thus forming a positive feedback loop that may amplify invasiveness. Last, we showed that malignant breast cancer cells are heterogeneous in their expression of surface-associated CD147 and that high levels of membrane CD147 correlate with cell surface EGFR and CD44 levels, activated EGFR and ERK1, and activated invadopodia. Future studies should evaluate CD147 as a potential therapeutic target and disease stratification marker in breast cancer. PMID:23888049

Construction of Hyaluronic Tetrasaccharide Clusters Modified Polyamidoamine siRNA Delivery System.

PubMed

Ma, Yingcong; Sha, Meng; Cheng, Shixuan; Yao, Wang; Li, Zhongjun; Qi, Xian-Rong

2018-06-14

The CD44 protein, as a predominant receptor for hyaluronan (HA), is highly expressed on the surface of multiple tumor cells. HA, as a targeting molecule for a CD44-contained delivery system, increases intracellular drug concentration in tumor tissue. However, due to the weak binding ability of hyaluronan oligosaccharide to CD44, targeting for tumor drug delivery has been restricted. In this study, we first use a HA tetrasaccharide cluster as the target ligand to enhance the binding ability to CD44. A polyamidoamine (PAMAM) dendrimer was modified by a HA tetrasaccharide cluster as a nonviral vector for small interfering RNA (siRNA) delivery. The dendrimer/siRNA nanocomplexes increased the cellular uptake capacity of siRNA through the CD44 receptor-mediated endocytosis pathway, allowing the siRNA to successfully escape the endosome/lysosome. Compared with the control group, nanocomplexes effectively reduced the expression of GFP protein and mRNA in MDA-MB-231-GFP cells. This delivery system provides a foundation to increase the clinical applications of PAMAM nanomaterials.

CD44 mediated hyaluronan adhesion of Toxoplasma gondii-infected leukocytes.

PubMed

Hayashi, Takeshi; Unno, Akihiro; Baba, Minami; Ohno, Tamio; Kitoh, Katsuya; Takashima, Yasuhiro

2014-04-01

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects humans and animals. Ingested parasites cross the intestinal epithelium, invade leukocytes and are then disseminated to peripheral organs. However, the mechanism of extravasation of the infected leukocytes remains poorly understood. In this study, we demonstrate that T. gondii-invaded human and mouse leukocytes express higher level of CD44, a ligand of hyaluronan (HA), and its expression on myeloid and non-myeloid leukocytes causes T. gondii-invaded human and mouse leukocyte to adhere to HA more effectively than non-invaded leukocytes. The specific adherence of parasite-invaded leukocytes was inhibited by anti CD44 antibody. Leukocytes of CD44 knockout mice did not show parasite-invaded leukocyte specific adhesion. Our results indicate that parasite-invaded leukocytes, regardless of whether myeloid or not, gain higher ability to adhere to HA than non-invaded leukocytes, via upregulation of CD44 expression and/or selective invasion to CD44 highly expressing cells. The difference in ability to adhere to HA between parasite-invaded cells and non-invaded neighboring cells might facilitate effective delivery of parasite-invaded leukocytes to the HA-producing endothelial cell surface and/or HA-rich extra cellular matrix. © 2013.

Pursuing Intracellular Pathogens with Hyaluronan. From a 'Pro-Infection' Polymer to a Biomaterial for 'Trojan Horse' Systems.

PubMed

Montanari, Elita; Di Meo, Chiara; Oates, Angela; Coviello, Tommasina; Matricardi, Pietro

2018-04-18

Hyaluronan (HA) is among the most important bioactive polymers in mammals, playing a key role in a number of biological functions. In the last decades, it has been increasingly studied as a biomaterial for drug delivery systems, thanks to its physico-chemical features and ability to target and enter certain cells. The most important receptor of HA is ‘Cluster of Differentiation 44’ (CD44), a cell surface glycoprotein over-expressed by a number of cancers and heavily involved in HA endocytosis. Moreover, CD44 is highly expressed by keratinocytes, activated macrophages and fibroblasts, all of which can act as ‘reservoirs’ for intracellular pathogens. Interestingly, both CD44 and HA appear to play a key role for the invasion and persistence of such microorganisms within the cells. As such, HA is increasingly recognised as a potential target for nano-carriers development, to pursuit and target intracellular pathogens, acting as a ‘Trojan Horse’. This review describes the biological relationship between HA, CD44 and the entry and survival of a number of pathogens within the cells and the subsequent development of HA-based nano-carriers for enhancing the intracellular activity of antimicrobials.

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. Copyright © 2015 Elsevier B.V. All rights reserved.

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Role Of Cathepsin C During Breast Cancer Metastasis

DTIC Science & Technology

2010-09-01

tumor histopathology is regulated by CSTC; however, in cathepsin C (Ctsc)-deficient mice, there is a significant reduction in the number of...hyaluronan. Glycobiology 14(11): 1108. 2. Ruffell B, Johnson P. (2006) Hyaluronan induces apoptosis through CD44 in activated lymphoma cells. EJC...tumors and lymphomas [16,17]. The ability of immune-deficient mice to reject and/or inhibit the growth of many, but not all cell lines is also impaired

Hyaluronan and hyaluronectin in the extracellular matrix of human brain tumour stroma.

PubMed

Delpech, B; Maingonnat, C; Girard, N; Chauzy, C; Maunoury, R; Olivier, A; Tayot, J; Creissard, P

1993-01-01

Hyaluronan (HA) and the hyaluronan-binding glycoprotein hyaluronectin (HN) were measured in 23 gliomas and 8 meningiomas and their location was revisited in 35 tumours. A clear-cut difference was found in the HN/HA ratio values of glioblastomas (below 0.5) and that of astrocytomas (above 0.5 P < 0.001). Besides their location in the intercellular part of gliomas, HA and HN displayed a perivascular location in 1/3 astrocytomas, 17/24 glioblastomas, and 3/7 meningiomas, suggesting they could be produced also by the vascular stroma of tumours and that they would characterise the neoangiogenesis. All cultivated glioma cells tested produced HA in vitro, whereas only 1/11 cell lines produced HN, at a low level. The results obtained suggest that glioma HA and HN are produced by both cancer cells and vascular stroma cells, which contribute to the edification of the extracellular matrix. In meningiomas only the stroma would be responsible for HA and HN production.

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. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Hyaluronan/Tween 80-assisted synthesis of silver nanoparticles for biological application

NASA Astrophysics Data System (ADS)

Li, Hui-Jun; Zhang, An-Qi; Sui, Li; Qian, Dong-Jin; Chen, Meng

2015-02-01

Water-soluble and well-stabilized silver nanoparticles (NPs) of small size have been synthesized using hyaluronan (HA) and Tween 80 as reducing and stabilizing agents. The effect of reaction conditions on the formation process of silver NPs was studied, and an aggregative growth mechanism of the silver NPs dominated in HA/Tween 80 system at pH 12 has been proposed. The obtained Ag NPs were characterized by UV-Vis spectroscopy, transmission electron microscopy, X-ray powder diffraction, and X-ray photoelectron spectroscopy. Moreover, the stability of the HA-Tween 80-silver NPs in normal saline was also studied, and a flexible blend membrane containing chitosan, gelatin, and the HA-Tween 80-silver NPs was prepared for further biological applications. Due to the high specific surface area and improved stability of silver NPs, the chitosan-gelatin-silver membrane has shown high antibacterial activity for strains of Escherichia coli. The cell viability tests indicate that the polymer membrane is non-cytotoxic to HepG2 cells, which might be attributed to its good biocompatibility.

Hyaluronan Benzyl Ester as a Scaffold for Tissue Engineering

PubMed Central

Vindigni, Vincenzo; Cortivo, Roberta; Iacobellis, Laura; Abatangelo, Giovanni; Zavan, Barbara

2009-01-01

Tissue engineering is a multidisciplinary field focused on in vitro reconstruction of mammalian tissues. In order to allow a similar three-dimensional organization of in vitro cultured cells, biocompatible scaffolds are needed. This need has provided immense momentum for research on “smart scaffolds” for use in cell culture. One of the most promising materials for tissue engineering and regenerative medicine is a hyaluronan derivative: a benzyl ester of hyaluronan (HYAFF®). HYAFF® can be processed to obtain several types of devices such as tubes, membranes, non-woven fabrics, gauzes, and sponges. All these scaffolds are highly biocompatible. In the human body they do not elicit any adverse reactions and are resorbed by the host tissues. Human hepatocytes, dermal fibroblasts and keratinocytes, chondrocytes, Schwann cells, bone marrow derived mesenchymal stem cells and adipose tissue derived mesenchymal stem cells have been successfully cultured in these meshes. The same scaffolds, in tube meshes, has been applied for vascular tissue engineering that has emerged as a promising technology for the design of an ideal, responsive, living conduit with properties similar to that of native tissue. PMID:19742179

Targeting of adhesion molecules as a therapeutic strategy in multiple myeloma.

PubMed

Neri, Paola; Bahlis, Nizar J

2012-09-01

Multiple myeloma (MM) is a clonal disorder of plasma cells that remains, for the most part, incurable despite the advent of several novel therapeutic agents. Tumor cells in this disease are cradled within the bone marrow (BM) microenvironment by an array of adhesive interactions between the BM cellular residents, the surrounding extracellular matrix (ECM) components such as fibronectin (FN), laminin, vascular cell adhesion molecule-1 (VCAM-1), proteoglycans, collagens and hyaluronan, and a variety of adhesion molecules on the surface of MM cells including integrins, hyaluronan receptors (CD44 and RHAMM) and heparan sulfate proteoglycans. Several signaling responses are activated by these interactions, affecting the survival, proliferation and migration of MM cells. An important consequence of these direct adhesive interactions between the BM/ECM and MM cells is the development of drug resistance. This phenomenon is termed "cell adhesion-mediated drug resistance" (CAM-DR) and it is thought to be one of the major mechanisms by which MM cells escape the cytotoxic effects of therapeutic agents. This review will focus on the adhesion molecules involved in the cross-talk between MM cells and components of the BM microenvironment. The complex signaling networks downstream of these adhesive molecules mediated by direct ligand binding or inside-out soluble factors signaling will also be reviewed. Finally, novel therapeutic strategies targeting these molecules will be discussed. Identification of the mediators of MM-BM interaction is essential to understand MM biology and to elucidate novel therapeutic targets for this disease.

Targeting of Prostate Cancer with Hyaluronan-Binding Proteins

DTIC Science & Technology

2005-06-01

16). Briefly, bovine nasal cartilage (Pel-Freez, Rogers, AR) was shredded with a Sure-Form blade (Stanley). extracted overnight with 4 M guanidine... nasal cartilage by affinity chromatography on hyaluronan- E Sepharose. As shown in Fig. 1, Metastatin consisted of two molecular C 40 factions as...biotinylated 490 pg/ml.) was isolated from the medium of rat fibrosarcoma cells grown tachvplesin (Fig. IB). However, this interaction was

Elasticity, biodegradability and cell adhesive properties of chitosan/hyaluronan multilayer films

NASA Astrophysics Data System (ADS)

Schneider, Aurore; Richert, Ludovic; Francius, Gregory; Voegel, Jean-Claude; Picart, Catherine

2007-03-01

In the bioengineering field, a recent and promising approach to modifying biomaterial surfaces is the layer-by-layer (LbL) technique used to build thin polyelectrolyte multilayer films. In this work, we focused on polyelectrolyte multilayer films made of two polysaccharides, chitosan (CHI) and hyaluronan (HA), and on the control of their physico-chemical and cell adhesive properties by chemical cross-linking. CHI/HA films were cross-linked using a water soluble carbodiimide and observed by confocal laser scanning microscopy (CLSM) with a fluorescently labeled CHI. Film thicknesses were similar for native and cross-linked films. The film nanometer roughness was measured by atomic force microscopy and was found to be higher for cross-linked films. Cross-linking the films also leads to a drastic change in film stiffness. The elastic modulus of the films (Young's modulus) as measured by AFM nano-indentation was about tenfold increased for cross-linked films as compared to native ones. From a biological point of view, cross-liked films are more resistant to enzymatic degradation by hyaluronidase. Furthermore, the increase in film stiffness has a favorable effect on the adhesion and spreading of chondrosarcoma cells. Thus, the CHI/HA cross-linked films could be used for various applications due to their adhesive properties and to their mechanical properties (including stability in enzymatic media).

Multifunctional Role of 35 Kilodalton Hyaluronan in Promoting Defense of the Intestinal Epithelium.

PubMed

Kessler, Sean P; Obery, Dana R; Nickerson, Kourtney P; Petrey, Aaron C; McDonald, Christine; de la Motte, Carol A

2018-04-01

Intestinal epithelium plays a critical role in host defense against orally acquired pathogens. Dysregulation of this protective barrier is a primary driver of inflammatory bowel diseases (Crohn's and ulcerative colitis) and also infant gastrointestinal infections. Previously, our lab reported that hyaluronan (HA) isolated from human milk induces the expression of the antimicrobial peptide β-defensin in vivo and protects against Salmonella Typhimurium infection of epithelial cells in vitro. In addition, we demonstrated that commercially available 35 kDa size HA induces the expression of β-defensin, upregulates the expression of tight junction protein zonula occludens-1 (ZO-1), and attenuates murine Citrobacter rodentium infection in vivo. In this current study, we report that HA35 remains largely intact and biologically active during transit through the digestive tract where it directly induces β-defensin expression upon epithelial cell contact. We also demonstrate HA35 abrogation of murine Salmonella Typhimurium infection as well as downregulation of leaky tight junction protein claudin-2 expression. Taken together, we propose a dual role for HA in host innate immune defense at the epithelial cell surface, acting to induce antimicrobial peptide production and also block pathogen-induced leaky gut. HA35 is therefore a promising therapeutic in the defense against bacterially induced colitis in compromised adults and vulnerable newborns.

Stem cell-based tissue-engineering for treatment of meniscal tears in the avascular zone.

PubMed

Zellner, Johannes; Hierl, Katja; Mueller, Michael; Pfeifer, Christian; Berner, Arne; Dienstknecht, Thomas; Krutsch, Werner; Geis, Sebastian; Gehmert, Sebastian; Kujat, Richard; Dendorfer, Sebastian; Prantl, Lukas; Nerlich, Michael; Angele, Peter

2013-10-01

Meniscal tears in the avascular zone have a poor self-healing potential, however partial meniscectomy predisposes the knee for early osteoarthritis. Tissue engineering with mesenchymal stem cells and a hyaluronan collagen based scaffold is a promising approach to repair meniscal tears in the avascular zone. 4 mm longitudinal meniscal tears in the avascular zone of lateral menisci of New Zealand White Rabbits were performed. The defect was left empty, sutured with a 5-0 suture or filled with a hyaluronan/collagen composite matrix without cells, with platelet rich plasma or with autologous mesenchymal stem cells. Matrices with stem cells were in part precultured in chondrogenic medium for 14 days prior to the implantation. Menisci were harvested at 6 and 12 weeks. The developed repair tissue was analyzed macroscopically, histologically and biomechanically. Untreated defects, defects treated with suture alone, with cell-free or with platelet rich plasma seeded implants showed a muted fibrous healing response. The implantation of stem cell-matrix constructs initiated fibrocartilage-like repair tissue, with better integration and biomechanical properties in the precultured stem cell-matrix group. A hyaluronan-collagen based composite scaffold seeded with mesenchymal stem cells is more effective in the repair avascular meniscal tear with stable meniscus-like tissue and to restore the native meniscus. Copyright © 2013 Wiley Periodicals, Inc., a Wiley Company.

Cell and Tissue Imaging with Molecularly Imprinted Polymers.

PubMed

Panagiotopoulou, Maria; Kunath, Stephanie; Haupt, Karsten; Tse Sum Bui, Bernadette

2017-01-01

Advanced tools for cell imaging are of particular interest as they can detect, localize and quantify molecular targets like abnormal glycosylation sites that are biomarkers of cancer and infection. Targeting these biomarkers is often challenging due to a lack of receptor materials. Molecularly imprinted polymers (MIPs) are promising artificial receptors; they can be tailored to bind targets specifically, be labeled easily, and are physically and chemically stable. Herein, we demonstrate the application of MIPs as artificial antibodies for selective labeling and imaging of cellular targets, on the example of hyaluronan and sialylation moieties on fixated human skin cells and tissues. Thus, fluorescently labeled MIP nanoparticles templated with glucuronic acid (MIPGlcA) and N-acetylneuraminic acid (MIPNANA) are respectively applied. Two different fluorescent probes are used: (1) MIPGlcA particles, ~400 nm in size are labeled with the dye rhodamine that target the extracellular hyaluronan on cells and tissue specimens and (2) MIP-coated InP/ZnS quantum dots (QDs) of two different colors, ~125 nm in size that target the extracellular and intracellular hyaluronan and sialylation sites. Green and red emitting QDs are functionalized with MIPGlcA and MIPNANA respectively, enabling multiplexed cell imaging. This is a general approach that can also be adapted to other target molecules on and in cells.

Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels

PubMed Central

Chauhan, Vikash P.; Martin, John D.; Liu, Hao; Lacorre, Delphine A.; Jain, Saloni R.; Kozin, Sergey V.; Stylianopoulos, Triantafyllos; Mousa, Ahmed S.; Han, Xiaoxing; Adstamongkonkul, Pichet; Popović, Zoran; Huang, Peigen; Bawendi, Moungi G.; Boucher, Yves; Jain, Rakesh K.

2013-01-01

Cancer and stromal cells actively exert physical forces (solid stress) to compress tumour blood vessels, thus reducing vascular perfusion. Tumour interstitial matrix also contributes to solid stress, with hyaluronan implicated as the primary matrix molecule responsible for vessel compression because of its swelling behaviour. Here we show, unexpectedly, that hyaluronan compresses vessels only in collagen-rich tumours, suggesting that collagen and hyaluronan together are critical targets for decompressing tumour vessels. We demonstrate that the angiotensin inhibitor losartan reduces stromal collagen and hyaluronan production, associated with decreased expression of profibrotic signals TGF-β1, CCN2 and ET-1, downstream of angiotensin-II-receptor-1 inhibition. Consequently, losartan reduces solid stress in tumours resulting in increased vascular perfusion. Through this physical mechanism, losartan improves drug and oxygen delivery to tumours, thereby potentiating chemotherapy and reducing hypoxia in breast and pancreatic cancer models. Thus, angiotensin inhibitors —inexpensive drugs with decades of safe use — could be rapidly repurposed as cancer therapeutics. PMID:24084631

Direct evidence for the partial reflection of hyaluronan molecules by the lining of rabbit knee joints during trans-synovial flow

PubMed Central

Scott, D; Coleman, P J; Mason, R M; Levick, J R

1998-01-01

In synovial joints hydraulic and turnover studies indicate that the synovial lining may partially reflect large macromolecules like hyaluronan, despite discontinuities in the lining cell layer. The reflection hypothesis was tested directly in the present study. Solutions of high molecular weight hyaluronan were infused at controlled pressures into the cavity of rabbit knees under anaesthesia, at concentrations of 0.2 g l−1 (n = 5), 2 g l−1 (n = 5) and 4 g l−1 (n = 6). Time-averaged trans-synovial flows were 9.6, 4.8 and 2.9 μl min−1, respectively. After 5 h infusion the intra-articular fluid was mixed and sampled. Hyaluronan concentration was determined by size-exclusion chromatography. In all sixteen experiments the hyaluronan concentration in the aspirate was greater than that in the infusate (P = 0.0001, Student's paired t test). The increases averaged 2.28 ± 0.04 times at high filtration rates (0.2 g l−1 infusates; mean ± s.e.m.), 1.60 ± 0.09 times at intermediate filtration rates (2 g l−1 infusates) and 1.26 ± 0.08 times at low filtration rates (4 g l−1 infusates). Between 48 and 95% of the hyaluronan in the filtrand was retained in the joint cavity. The greater retention at 2 g l−1, viz.95%, than at 0.2 g l−1, viz.48%, was attributed to interactions between overlapping molecular domains in the more concentrated solution. It is concluded that synovial interstitial matrix can partially reflect hyaluronan molecules, and thus conserve intra-articular lubricant. PMID:9508822

Investigating the importance of flow when utilizing hyaluronan scaffolds for tissue engineering.

PubMed

Donegan, Gail C; Hunt, John A; Rhodes, Nicholas

2010-02-01

Esterified hyaluronan scaffolds offer significant advantages for tissue engineering. They are recognized by cellular receptors, interact with many other extracellular matrix proteins and their metabolism is mediated by intrinsic cellular pathways. In this study differences in the viability and structural integrity of vascular tissue models cultured on hyaluronan scaffolds under laminar flow conditions highlighted potential differences in the biodegradation kinetics, processes and end-products, depending on the culture environment. Critical factors are likely to include seeding densities and the duration and magnitude of applied biomechanical stress. Proteomic evaluation of the timing and amount of remodelling protein expression, the resulting biomechanical changes arising from this response and metabolic cell viability assay, together with examination of tissue morphology, were conducted in vascular tissue models cultured on esterified hyaluronan felt and PTFE mesh scaffolds. The vascular tissue models were derived using complete cell sheets derived from harvested and expanded umbilical cord vein cells. This seeding method utilizes high-density cell populations from the outset, while the cells are already supported by their own abundant extracellular matrix. Type I and type IV collagen expression in parallel with MMP-1 and MMP-2 expression were monitored in the tissue models over a 10 day culture period under laminar flow regimes using protein immobilization technologies. Uniaxial tensile testing and scanning electron microscopy were used to compare the resulting effects of hydrodynamic stimulation upon structural integrity, while viability assays were conducted to evaluate the effects of shear on metabolic function. The proteomic results showed that the hyaluronan felt-supported tissues expressed higher levels of all remodelling proteins than those cultured on PTFE mesh. Overall, a 21% greater expression of type I collagen, 24% higher levels of type IV collagen, 24% higher levels of MMP-1 and 34% more MMP-2 were observed during hydrodynamic stress. This was coupled with a loss of structural integrity in these models after the introduction of laminar flow, as compared to the increases in all mechanical properties observed in the PTFE mesh-supported tissues. However, under flow conditions, the hyaluronan-supported tissues showed some recovery of the viability originally lost during static culture conditions, in contrast to PTFE mesh-based models, where initial gains were followed by a decline in metabolic viability after applied shear stress. Proteomic, cell viability and mechanical testing data emphasized the need for extended in vitro evaluations to enable better understanding of multi-stage remodelling and reparative processes in tissues cultured on biodegradable scaffolds. This study also highlighted the possibility that in high-density tissue culture with a biodegradable component, dynamic conditions may be more conducive to optimal tissue development than the static environment because they facilitate the efficient removal of high concentrations of degradation end-products accumulating in the pericellular space.

Hyaluronan (HA) interacting proteins RHAMM and hyaluronidase impact prostate cancer cell behavior and invadopodia formation in 3D HA-based hydrogels.

PubMed

Gurski, Lisa A; Xu, Xian; Labrada, Lyana N; 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 biological processes associated with cancer cell motility through HA-rich connective tissues.

Acute and chronic rejection: compartmentalization and kinetics of counterbalancing signals in cardiac transplants.

PubMed

Kaul, A M K; Goparaju, S; Dvorina, N; Iida, S; Keslar, K S; de la Motte, C A; Valujskikh, A; Fairchild, R L; Baldwin, W M

2015-02-01

Acute and chronic rejection impact distinct compartments of cardiac allografts. Intramyocardial mononuclear cell infiltrates define acute rejection, whereas chronic rejection affects large arteries. Hearts transplanted from male to female C57BL/6 mice undergo acute rejection with interstitial infiltrates at 2 weeks that resolve by 6 weeks when large arteries develop arteriopathy. These processes are dependent on T cells because no infiltrates developed in T cell-deficient mice and transfer of CD4 T cells restored T cell as well as macrophage infiltrates and ultimately neointima formation. Markers of inflammatory macrophages were up-regulated in the interstitium acutely and decreased as markers of wound healing macrophages increased chronically. Programmed cell death protein, a negative costimulator, and its ligand PDL1 were up-regulated in the interstitium during resolution of acute rejection. Blocking PDL1:PD1 interactions in the acute phase increased interstitial T cell infiltrates. Toll-like receptor (TLR) 4 and its endogenous ligand hyaluronan were increased in arteries with neointimal expansion. Injection of hyaluronan fragments increased intragraft production of chemokines. Our data indicate that negative costimulatory pathways are critical for the resolution of acute interstitial infiltrates. In the arterial compartment recognition of endogenous ligands including hyaluronan by the innate TLRs may support the progression of arteriopathy. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

Methacrylated gelatin/hyaluronan-based hydrogels for soft tissue engineering

PubMed Central

Kessler, Lukas; Gehrke, Sandra; Winnefeld, Marc; Huber, Birgit; Hoch, Eva; Walter, Torsten; Wyrwa, Ralf; Schnabelrauch, Matthias; Schmidt, Malte; Kückelhaus, Maximilian; Lehnhardt, Marcus; Hirsch, Tobias; Jacobsen, Frank

2017-01-01

In vitro–generated soft tissue could provide alternate therapies for soft tissue defects. The aim of this study was to evaluate methacrylated gelatin/hyaluronan as scaffolds for soft tissue engineering and their interaction with human adipose–derived stem cells (hASCs). ASCs were incorporated into methacrylated gelatin/hyaluronan hydrogels. The gels were photocrosslinked with a lithium phenyl-2,4,6-trimethylbenzoylphosphinate photoinitiator and analyzed for cell viability and adipogenic differentiation of ASCs over a period of 30 days. Additionally, an angiogenesis assay was performed to assess their angiogenic potential. After 24 h, ASCs showed increased viability on composite hydrogels. These results were consistent over 21 days of culture. By induction of adipogenic differentiation, the mature adipocytes were observed after 7 days of culture, their number significantly increased until day 28 as well as expression of fatty acid binding protein 4 and adiponectin. Our scaffolds are promising as building blocks for adipose tissue engineering and allowed long viability, proliferation, and differentiation of ASCs. PMID:29318000

Hyaluronic Acid as a Target for Intervention in Prostate Cancer Metastases

DTIC Science & Technology

2011-06-01

Coumarin (HMC) is an inhibitor of hyaluronan synthase. It is commonly available in herbal supplements and, up to now, has been utilized mainly for...commonly available in herbal supplements and, up to now, has been utilized mainly for digestion complaints. We propose that it may be efficacious in the...metastatic prostate cancer cells. 7-Hydroxy-4-Methyl Coumarin (HMC) is an inhibitor of hyaluronan synthase. It is commonly available in herbal supplements

Effects of ventilation on hyaluronan and protein concentration in pleural liquid of anesthetized and conscious rabbits.

PubMed

Wang, P M; Lai-Fook, S J

1998-01-01

The hypothesis of this study is that pleural lubrication is enhanced by hyaluronan acting as a boundary lubricant in pleural liquid and by pleural filtration as reflected in changes in protein concentration with ventilation. Anesthetized rabbits were injected intravenously with Evans blue dye and ventilated with 100% O2 at either of two levels of ventilation for 6 h. Postmortem values of hyaluronan, total protein, and Evans blue-dyed albumin (EBA) concentrations in pleural liquid were greater at the higher ventilation, consistent with increases in boundary lubrication, pleural membrane permeability, and pleural filtration. To determine whether these effects were caused by hyperoxia or anesthesia, conscious rabbits were ventilated with either 3% CO2 or room air in a box for 6, 12, or 24 h. Similar to the anesthetized rabbits, pleural liquid hyaluronan concentration after 24 h was higher in the conscious rabbits with the hypercapnic-induced greater ventilation. By contrast, the time course of total protein and EBA in pleural liquid was similar in both groups of conscious rabbits, indicating no effect of ventilation on pleural permeability. The increase in pleural liquid hyaluronan concentration might be the result of mesothelial cell stimulation by a ventilation-induced increase in pleural liquid shear stress.

Utilization of Glycosaminoglycans/Proteoglycans as Carriers for Targeted Therapy Delivery

PubMed Central

Misra, Suniti; Hascall, Vincent C.; Atanelishvili, Ilia; Moreno Rodriguez, Ricardo; Markwald, Roger R.; Ghatak, Shibnath

2015-01-01

The outcome of patients with cancer has improved significantly in the past decade with the incorporation of drugs targeting cell surface adhesive receptors, receptor tyrosine kinases, and modulation of several molecules of extracellular matrices (ECMs), the complex composite of collagens, glycoproteins, proteoglycans, and glycosaminoglycans that dictates tissue architecture. Cancer tissue invasive processes progress by various oncogenic strategies, including interfering with ECM molecules and their interactions with invasive cells. In this review, we describe how the ECM components, proteoglycans and glycosaminoglycans, influence tumor cell signaling. In particular this review describes how the glycosaminoglycan hyaluronan (HA) and its major receptor CD44 impact invasive behavior of tumor cells, and provides useful insight when designing new therapeutic strategies in the treatment of cancer. PMID:26448753

Contribution of the 37-kDa laminin receptor precursor in the anti-metastatic PSP94-derived peptide PCK3145 cell surface binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Annabi, Borhane; Currie, Jean-Christophe; Bouzeghrane, Mounia

Purpose: PCK3145 is an anti-metastatic synthetic peptide with promising therapeutic efficacy against hormone-refractory prostate cancer. The characterization of the PCK3145 peptide cell surface binding/internalization mechanisms and of the receptors involved remained to be explored. Results: [{sup 14}C]PCK3145 cell surface binding assays showed rapid and transient kinetic profile, that was inhibited by RGD peptides, laminin, hyaluronan, and type-I collagen. RGD peptides were however unable to inhibit PCK3145 intracellular uptake. Far-Western ligand binding studies enabled the identification of the 37-kDa laminin receptor precursor (37LRP) as a potential ligand for PCK3145. Overexpression of the recombinant 37LRP indeed led to an increase in PCK3145more » binding but unexpectedly not to its uptake. Conclusions: Our data support the implication of laminin receptors in cell surface binding and in transducing PCK3145 anti-metastatic effects, and provide a rational for targeting cancers that express high levels of such laminin receptors.« less

Chitosan-hyaluronan based 3D co-culture platform for studying the crosstalk of lung cancer cells and mesenchymal stem cells.

PubMed

Han, Hao-Wei; Hsu, Shan-Hui

2016-09-15

The controversial roles of mesenchymal stem cells (MSCs) in lung cancer development are not yet resolved because of the lack of an extracellular environment that mimics the tumor microenvironment. Three-dimensional (3D) culture system is an emerging research tool for biomedical applications such as drug screening. In this study, MSCs and human non-small cell lung carcinoma cells (A549) were co-cultured on a thin biomaterial-based substratum (hyaluronan-grafted chitosan, CS-HA; ∼2μm), and they were self-organized into the 3D tumor co-spheroids with core-shell structure. The gene expression levels of tumorigenicity markers in cancer cells associated with cancer stemness, epithelial-mesenchymal transition (EMT) property, and cell mobility were up-regulated for more than twofold in the MSC-tumor co-spheroids, through the promoted expression of certain tumor enhancers and the direct cell-cell interaction. To verify the different extents of tumorigenicity, A549 cells or those co-cultured with MSCs were transplanted into zebrafish embryos for evaluation in vivo. The tumorigenicity obtained from the zebrafish xenotransplantation model was consistent with that observed in vitro. These evidences suggest that the CS-HA substrate-based 3D co-culture platform for cancer cells and MSCs may be a convenient tool for studying the cell-cell interaction in a tumor-like microenvironment and potentially for cancer drug testing. Mesenchymal stem cells (MSCs) have been found in several types of tumor tissues. However, the controversial roles of MSCs in cancer development are still unsolved. Chitosan and hyaluronan are commonly used materials in the biomedical field. In the current study, we co-cultured lung cancer cells and MSCs on the planar hyaluronan-grafted chitosan (CS-HA) hybrid substrates, and discovered that lung cancer cells and MSCs were rapidly self-assembled into 3D tumor spheroids with core-shell structure on the substrates after only two days in culture. Therefore, CS-HA based 3D co-culture platform can be applied to exploration of the relationship between cancer cells and MSCs and other cancer-related medical applications such as drug screening. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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

Combined Cancer Therapy with Hyaluronan-Decorated Fullerene-Silica Multifunctional Nanoparticles to Target Cancer Stem-Like Cells

PubMed Central

Wang, Hai; Agarwal, Pranay; Zhao, Shuting; Yu, Jianhua; Lu, Xiongbin; He, Xiaoming

2016-01-01

Cancer stem-like cells (CSCs) are resistant to chemotherapy and highly tumorigenic, which contributes to tumor occurrence and post-treatment relapse. We developed a novel C60 fullerene-silica nanoparticle system surface-decorated with hyaluronan (HA) to target the variant CD44 overexpressed on breast CSCs. Furthermore, doxorubicin hydrochloride (DOX) and indocyanine green (ICG) can be encapsulated in the nanoparticles with ultrahigh encapsulation efficiency (> 90%) and loading content (e.g., 48.5% at a drug-to-nanoparticle feeding ratio of 1:1, compared to the commonly used drug-to-nanoparticle feeding ratio of 1:20 with a drug loading content of less than 5%). As a result, the DOX and ICG-laden nanoparticles can be used as a single nanoplatform to achieve combined chemo, photodynamic, and photothermal therapy under near infrared laser irradiation for effective destruction of the breast CSCs both in vitro and in vivo, with no evident systemic toxicity. Moreover, we found the nanoparticles with a higher drug loading content (e.g., 48.5 versus 4.6%) also have significantly higher antitumor efficacy, given the same total drug dose. These results demonstrate the great potential of the multifunctional hybrid nanoparticle system for augmenting cancer therapy by eliminating the CSCs. PMID:27162075

Stimulating effects of Bacillus subtilis natto-fermented Radix astragali on hyaluronic acid production in human skin cells.

PubMed

Hsu, Mei-Fang; Chiang, Been-Huang

2009-09-25

Radix astragali, a well-known Chinese herb, which has been traditionally used for skincare, and microbial fermentation is one of the conventional methods for processing Chinese herbs. This research studied the effects of non-fermented (HQNB) and fermented preparations (HQB) of Radix astragali on hyaluronic acid (HA) production in primary human skin cells. HQB and HQNB were prepared and added to the cultures of primary human skin cells. Hyaluronic acid content was determined using ELISA. Real-time RT-PCR was used to evaluate hyaluronan synthase gene expression. The bioactive compounds were analyzed by HPLC. The growth-stimulating effect of HQNB on both of keratinocytes and fibroblasts were significantly higher than that of HQB. Conversely, HQB, but not HQNB significantly stimulated HA production in both cultured primary human epidermal keratinocytes and human dermal fibroblasts in dose-dependent manners. In addition, HQB markedly and dose-dependently increased the expression of hyaluronan synthase 3 and hyaluronan synthase 2 mRNA in HaCaT cells and human fibroblasts, respectively. Therefore, HQB might be a promising candidate for preventing the age-dependent loss of HA content in aged human skin, and its effect on the enhancement of HA synthesis in skin cells is highly related to its effect on the expression of hyaluronan synthase genes. The three major active isoflavonoids in Radix astragali were identified as ononin, calycosin, and formononetin. After fermentation, all of these three compounds in HQB were significantly reduced. However, HQB still had significantly higher enhancement effect on the production of HA than HQNB. It appeared that isoflavonoid aglycones or other metabolites, converted from their primary isoflavones during fermentation, might be responsible for the skincare functions found in this study. This study demonstrated the low toxicity and the stimulating effects of HQB on HA synthesis, and suggests that HQB may play a promising role in anti-aging cosmetic applications.

Synthesis and Characterization of Rhodamine B-ethylenediamine-hyaluronan Acid as Potential Biological Functional Materials

NASA Astrophysics Data System (ADS)

Li, Y. L.; Wang, W. X.; Wang, Y.; Zhang, W. B.; Gong, H. M.; Liu, M. X.

2018-05-01

The purpose of this study is to synthesize and characterize fluorescent polymers, rhodamine B-ethylenediamine-hyaluronan acid (RhB-EA-HA). RhB-EA-HA was successfully synthesized by ester ammonolysis reaction and amidation reaction. Moreover, the structural properties of RhB-EA-HA were characterized by 1H-NMR spectra, UV-vis spectrometry and Fourier transform infrared spectroscopy (FT-IR). RhB-EA-HA can be grafted on the surface of silica nanomaterials, which may be potential biological functional materials for drug delivery system.

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

PubMed

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

2015-06-09

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. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Roles of the multifunctional glycoprotein, emmprin (basigin; CD147), in tumour progression.

PubMed

Yan, Li; Zucker, Stanley; Toole, Bryan P

2005-02-01

Emmprin (basigin;CD147) is a widely distributed cell surface glycoprotein that belongs to the Ig superfamily and is highly enriched on the surface of malignant tumour cells. Emmprin is involved in numerous physiological and pathological systems and exhibits several molecular and cellular characteristics, but a major function of emmprin is stimulation of synthesis of several matrix metalloproteinases. In tumours, emmprin most likely stimulates matrix metalloproteinase production in stromal fibroblasts and endothelial cells as well as in tumour cells themselves by a mechanism involving homophilic interactions between emmprin molecules on apposing cells or on neighbouring cells after membrane vesicle shedding. Membrane-associated cofactors, including caveolin-1 and annexin II, regulate emmprin activity. Emmprin induces angiogenesis via stimulation of VEGF production, invasiveness via stimulation of matrix metalloproteinase production and multidrug resistance via hyaluronan-mediated up-regulation of ErbB2 signaling and cell survival pathway activities. Although the detailed mechanisms whereby it regulates these numerous phenomena are not yet known, it is clear that emmprin is a major mediator of malignant cell behavior.

A cell-free scaffold-based cartilage repair provides improved function hyaline-like repair at one year.

PubMed

Siclari, Alberto; Mascaro, Gennaro; Gentili, Chiara; Cancedda, Ranieri; Boux, Eugenio

2012-03-01

Bone marrow stimulation techniques in cartilage repair such as drilling are limited by the formation of fibrous to hyaline-like repair tissue. It has been suggested such techniques can be enhanced by covering the defect with scaffolds. We present an innovative approach using a polyglycolic acid (PGA)-hyaluronan scaffold with platelet-rich-plasma (PRP) in drilling. We asked whether (1) PRP immersed in a cell-free PGA-hyaluronan scaffold improves patient-reported 1-year outcomes for the Knee injury and Osteoarthritis Score (KOOS), and (2) implantation of the scaffold in combination with bone marrow stimulation leads to the formation of hyaline-like cartilage repair tissue. We reviewed 52 patients who had arthroscopic implantation of the PGA-hyaluronan scaffold immersed with PRP in articular cartilage defects of the knee pretreated with Pridie drilling. Patients were assessed by KOOS. At 9 months followup, histologic staining was performed in specimens obtained from five patients to assess the repair tissue quality. The KOOS subscores improved for pain (55 to 91), symptoms (57 to 88), activities of daily living (69 to 86), sports and recreation (36 to 70), and quality of life (38 to 73). The histologic evaluation showed a homogeneous hyaline-like cartilage repair tissue. The cell-free PGA-hyaluronan scaffold combined with PRP leads to cartilage repair and improved patient-reported outcomes (KOOS) during 12 months of followup. Histologic sections showed morphologic features of hyaline-like repair tissue. Long-term followup is needed to determine if the cartilage repair tissue is durable. Level IV, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.

Mesenchymal stem cells display hepato-protective activity in lymphoma bearing xenografts.

PubMed

Secchiero, Paola; Corallini, Federica; Zavan, Barbara; Tripodo, Claudio; Vindigni, Vincenzo; Zauli, Giorgio

2012-04-01

A disseminated model of non-Hodgkin's lymphoma with prevalent liver metastasis was generated by intraperitoneal (i.p.) injection of EBV(+) B lymphoblastoid SKW6.4 in nude-SCID mice. The survival of SKW6.4 xenografts (median survival = 27 days) was significantly improved when hyaluronan scaffolds embedded with mesenchimal stem cells (MSC) were implanted in the abdominal area 4 days after SKW6.4 injection (median survival = 39.5 days). Mice implanted with MSC showed a significant improvement of hepatic functionality in lymphoma xenografts, as demonstrated by measurement of serum ALT/AST levels. Co-culture of MSC with lymphoma cells enhanced the release of hepatocyte growth factor (HGF) by MSC. These data suggest that hyaluronan-embedded MSC exert anti-lymphoma activity by ameliorating hepatic functionality.

A hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo expansion of human corneal epithelial stem cells.

PubMed

Chen, D; Qu, Y; Hua, X; Zhang, L; Liu, Z; Pflugfelder, S C; Li, D-Q

2017-06-01

PurposeTo develop a hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo cultivation of human corneal epithelial stem cells (CESCs).Patients and MethodsCESCs were cultivated from donor limbal explants on the HyStem-C Hydrogel bio-scaffold in 12-well plates for 3 weeks. Group A used the traditional supplemented hormonal epidermal medium (SHEM) and group B used the defined SHEM (without fetal bovine serum and toxin A, adding 20% serum replacement). The growth and morphology of the cultured cells were assessed by phase contrast microscope. The expressions of specific cell markers were assessed by immunofluorescence staining and quantitative real-time PCR (qRT-PCR).ResultsSuccessful cultures of CESCs were obtained in both groups, resulting in multilayered stratified epithelia. Comparing to group A, the cells in group B was grown slightly slower and formed less cellular layers at the end of culture. The corneal specific cytokeratin (K) 12 and differentiation markers, involucrin, and connexin 43, were mainly expressed in the superficial cellular layers in both groups. Interestingly, certain basal cells were immune-positive to proposed stem cell markers such as K19, ABCG2, and integrin β1 in both groups. There was no significant difference between the two groups with regard to the gene expression levels of all these selected corneal markers (all P>0.05).ConclusionsThe hyaluronan hydrogel scaffold-based xeno-free culture system may support the expansion of regenerative CESCs without the risk of xeno component contamination. The regenerated epithelium maintains similar characteristics of native corneal epithelium.

Hyaluronan as a propellant for epithelial movement: the development of semicircular canals in the inner ear of Xenopus.

PubMed

Haddon, C M; Lewis, J H

1991-06-01

The membranous labyrinth of the inner ear, with its three semicircular canals, originates from a simple spheroidal otic vesicle. The process is easily observed in Xenopus. The vesicle develops three dorsal outpocketings; from the two opposite faces of each outpocketing pillars of tissue are protruded into the lumen; and these paired 'axial protrusions' eventually meet and fuse, to form a column of tissue spanning the lumen of the outpocketing like the hub of a wheel, with a tube of epithelium forming the semicircular canal around the periphery. Each axial protrusion consists of epithelium encasing a core of largely cell-free extracellular matrix that stains strongly with alcian blue. In sections, at least 60% of the stainable material is removed by treatment with Streptomyces hyaluronidase. When Streptomyces hyaluronidase is microinjected into the core of a protrusion in vivo, the protrusion collapses and the corresponding semicircular canal fails to form. Hyaluronan (hyaluronic acid) in the core of the protrusion therefore seems to be essential in driving the extension of the protrusion. Autoradiography with tritiated glucosamine indicates that the hyaluronan-rich matrix is synthesised by the epithelium covering the tip of the protrusion; the basal lamina here appears to be discontinuous. These findings indicate that the epithelium of the axial protrusion propels itself into the lumen of the otocyst by localised synthesis of hyaluronan. Hyaluronan may be used in a similar way in the development of other organs, such as the heart and the secondary palate.

Cyclic AMP-elevating Agents Promote Cumulus Cell Survival and Hyaluronan Matrix Stability, Thereby Prolonging the Time of Mouse Oocyte Fertilizability*

PubMed Central

Di Giacomo, Monica; Camaioni, Antonella; Klinger, Francesca G.; Bonfiglio, Rita; Salustri, Antonietta

2016-01-01

Cumulus cells sustain the development and fertilization of the mammalian oocyte. These cells are retained around the oocyte by a hyaluronan-rich extracellular matrix synthesized before ovulation, a process called cumulus cell-oocyte complex (COC) expansion. Hyaluronan release and dispersion of the cumulus cells progressively occur after ovulation, paralleling the decline of oocyte fertilization. We show here that, in mice, postovulatory changes of matrix are temporally correlated to cumulus cell death. Cumulus cell apoptosis and matrix disassembly also occurred in ovulated COCs cultured in vitro. COCs expanded in vitro with FSH or EGF underwent the same changes, whereas those expanded with 8-bromo-adenosine-3′,5′-cyclic monophosphate (8-Br-cAMP) maintained integrity for a longer time. It is noteworthy that 8-Br-cAMP treatment was also effective on ovulated COCs cultured in vitro, prolonging the vitality of the cumulus cells and the stability of the matrix from a few hours to >2 days. Stimulation of endogenous adenylate cyclase with forskolin or inhibition of phosphodiesterase with rolipram produced similar effects. The treatment with selective cAMP analogues suggests that the effects of cAMP elevation are exerted through an EPAC-independent, PKA type II-dependent signaling pathway, probably acting at the post-transcriptional level. Finally, overnight culture of ovulated COCs with 8-Br-cAMP significantly counteracted the decrease of fertilization rate, doubling the number of fertilized oocytes compared with control conditions. In conclusion, these studies suggest that cAMP-elevating agents prevent cumulus cell senescence and allow them to continue to exert beneficial effects on oocyte and sperm, thereby extending in vitro the time frame of oocyte fertilizability. PMID:26694612

Interaction between hyaluronan and CD44 in the development of dimethylnitrosamine-induced liver cirrhosis.

PubMed

Satoh, T; Ichida, T; Matsuda, Y; Sugiyama, M; Yonekura, K; Ishikawa, T; Asakura, H

2000-04-01

A significant increase in serum hyaluronan (HA) levels has been reported in patients with liver cirrhosis. This mechanism is not yet clear, and receptors for HA have not been characterized. In this study, we examined the expression of both HA and its receptors, CD44 and intercellular adhesion molecule-1 (ICAM-1), in dimethylnitrosamine-induced liver cirrhosis. Using biotinylated HA binding protein, HA was detected in the area of periportal fibrosis and around the sinusoidal wall where hepatic fibrosis was developing. Electron microscopy revealed that HA was localized on Ito cells and sinusoidal endothelial cells (SEC). Conversely, CD44, which was only expressed weakly in normal liver, was present in large amounts in cirrhotic liver. The distribution pattern of CD44 was similar to that of HA, however, CD44 was mainly localized on the infiltrating lymphocytes and Kupffer cells. Moreover, CD44 was detected on part of factor VIII-positive SEC. Intercellular adhesion molecule-1, another receptor for HA, was detected on the surface of hepatocytes and around the sinusoidal wall in cirrhotic liver, but its distribution was not accompanied by expression of HA. With respect to CD44 isoforms, the standard form m-RNA predominated in both normal and cirrhotic liver. Variant pMeta-1 mRNA was detected at low levels. An interaction between HA and CD44 may play a role in the recruitment of numerous infiltrating cells and HA accumulation in hepatic sinusoids. Together with phenotypic changes in the SEC, these results may lead to a disturbance in the elimination of HA during the progression of liver cirrhosis.

Role of hyaluronan chain length in buffering interstitial flow across synovium in rabbits

PubMed Central

Coleman, P J; Scott, D; Mason, R M; Levick, J R

2000-01-01

Synovial fluid drains out of joints through an interstitial pathway. Hyaluronan, the major polysaccharide of synovial fluid, attenuates this fluid drainage; it creates a graded opposition to outflow that increases with pressure (outflow ‘buffering’). This has been attributed to size-related molecular reflection at the interstitium-fluid interface. Chain length is reduced in inflammatory arthritis. We therefore investigated the dependence of outflow buffering on hyaluronan chain length.Hyaluronan molecules of mean molecular mass ≈2200, 530, 300 and 90 kDa and concentration 3.6 mg ml−1 were infused into the knees of anaesthetized rabbits, with Ringer solution as control in the contralateral joint. Trans-synovial drainage rate was recorded at known joint pressures. Pressure was raised in steps every 30–60 min (range 2–24 cmH2O).With hyaluronan-90 and hyaluronan-300 the fluid drainage rate was reduced relative to Ringer solution (P < 0.001, ANOVA) but increased steeply with pressure. The opposition to outflow, defined as the pressure required to drive unit outflow, did not increase with pressure, i.e. there was no outflow buffering.With hyaluronan-530 and hyaluronan-2000 the fluid drainage rate became relatively insensitive to pressure, causing a near plateau of flow. Opposition to outflow increased markedly with pressure, by up to 3.3 times over the explored pressures.Hyaluronan concentration in the joint cavity increased over the drainage period, indicating partial reflection of hyaluronan by synovial interstitium. Reflected fractions were 0.12, 0.33, 0.25 and 0.79 for hyaluronan-90, -300, -530 and -2200, respectively.Thus the flow-buffering effect of hyaluronan depended on chain length, and shortening the chains reduced the degree of molecular reflection. The latter should reduce the concentration polarization at the tissue interface, and hence the local osmotic pressure opposing fluid drainage. In rheumatoid arthritis the reduced chain length will facilitate the escape of hyaluronan and fluid. PMID:10896731

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 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. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr04034a

Intracellular Domain Fragment of CD44 Alters CD44 Function in Chondrocytes*

PubMed Central

Mellor, Liliana; Knudson, Cheryl B.; Hida, Daisuke; Askew, Emily B.; Knudson, Warren

2013-01-01

The hyaluronan receptor CD44 undergoes sequential proteolytic cleavage at the cell surface. The initial cleavage of the CD44 extracellular domain is followed by a second intramembranous cleavage of the residual CD44 fragment, liberating the C-terminal cytoplasmic tail of CD44. In this study conditions that promote CD44 cleavage resulted in a diminished capacity to assemble and retain pericellular matrices even though sufficient non-degraded full-length CD44 remained. Using stable and transient overexpression of the cytoplasmic domain of CD44, we determined that the intracellular domain interfered with anchoring of the full-length CD44 to the cytoskeleton and disrupted the ability of the cells to bind hyaluronan and assemble a pericellular matrix. Co-immunoprecipitation assays were used to determine whether the mechanism of this interference was due to competition with actin adaptor proteins. CD44 of control chondrocytes was found to interact and co-immunoprecipitate with both the 65- and 130-kDa isoforms of ankyrin-3. Moreover, this interaction with ankyrin-3 proteins was diminished in cells overexpressing the CD44 intracellular domain. Mutating the putative ankyrin binding site of the transiently transfected CD44 intracellular domain diminished the inhibitory effects of this protein on matrix retention. Although CD44 in other cells types has been shown to interact with members of the ezrin/radixin/moesin (ERM) family of adaptor proteins, only modest interactions between CD44 and moesin could be demonstrated in chondrocytes. The data suggest that release of the CD44 intracellular domain into the cytoplasm of cells such as chondrocytes exerts a competitive or dominant-negative effect on the function of full-length CD44. PMID:23884413

Emmprin (basigin/CD147): matrix metalloproteinase modulator and multifunctional cell recognition molecule that plays a critical role in cancer progression.

PubMed

Nabeshima, Kazuki; Iwasaki, Hiroshi; Koga, Kaori; Hojo, Hironobu; Suzumiya, Junji; Kikuchi, Masahiro

2006-07-01

Emmprin (basigin, CD147) is a cell surface glycoprotein that belongs to the immunoglobulin superfamily. It is highly expressed on the surface of tumor cells and stimulates adjacent fibroblasts or tumor cells to produce matrix metalloproteinases. Moreover, it has recently been shown that emmprin also stimulates expression of vascular endothelial growth factor and hyaluronan, which leads to angiogenesis and anchorage-independent growth/multidrug resistance, respectively. These findings have made emmprin an important molecule in tumor progression and, thus, more attractive as a target for antitumor treatment. However, other functions of emmprin, including as an activator of T cells, a chaperone for monocarboxylate transporters, a receptor for cyclophilin A and a neural recognition molecule, are also being identified in physiological and pathological conditions. Therefore, it is essential to develop specific means to control particular functions of emmprin, for which elucidation of each mechanism is crucial. This review will discuss the role of emmprin in tumor progression and recent advances in the molecular mechanisms of diverse phenomena regulated by emmprin.

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

2015-10-07

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.

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

Hyaluronan and Its Binding Proteins during Cervical Ripening and Parturition: Dynamic Changes in Size, Distribution and Temporal Sequence

PubMed Central

Ruscheinsky, Monika; De la Motte, Carol; Mahendroo, Mala

2008-01-01

The uterine cervix undergoes changes during pregnancy and labor that transform it from a closed, rigid, collagen dense structure to one that is distensible, has a disorganized collagen matrix, and dilates sufficiently to allow birth. To protect the reproductive tract from exposure to the external environment, the cervix must be rapidly altered to a closed, undistensible structure after birth. Preparturition remodeling is characterized by increased synthesis of hyaluronan, decreased expression of collagen assembly genes and increased distribution of inflammatory cells into the cervical matrix. Postpartum remodeling is characterized by decreased hyaluronan (HA) content, increased expression of genes involved in assembly of mature collagen and inflammation. The focus of this study is to advance our understanding of functions HA plays in this dynamic process through characterization of HA size, structure and binding proteins in the mouse cervix. Changes in size and structure of HA before and after birth were observed as well as cell specific expression of HA binding proteins. CD44 expression is localized to the pericellular matrix surrounding the basal epithelia and on immune cells while inter α trypsin inhibitor (IαI) and versican are localized to the stromal matrix. Co-localization of HA and IαI is most pronounced after birth. Upregulation of the versican degrading protease, ADAMTS1 occurs in the cervix prior to birth. These studies suggest that HA has multiple, cell specific functions in the cervix that may include modulation of tissue structure and integrity, epithelial cell migration and differentiation, and inflammatory responses. PMID:18353623

Biocompatible blood pool MRI contrast agents based on hyaluronan

PubMed Central

Zhu, Wenlian; Artemov, Dmitri

2010-01-01

Biocompatible gadolinium blood pool contrast agents based on a biopolymer, hyaluronan, were investigated for magnetic resonance angiography application. Hyaluronan, a non-sulfated linear glucosaminoglycan composed of 2000–25,000 repeating disaccharide subunits of D-glucuronic acid and N-acetylglucosamine with molecular weight up to 20 MDa, is a major component of the extracellular matrix. Two gadolinium contrast agents based on 16 and 74 kDa hyaluronan were synthesized, both with R1 relaxivity around 5 mM−1 s−1 per gadolinium at 9.4 T at 25°C. These two hyaluronan based agents show significant enhancement of the vasculature for an extended period of time. Initial excretion was primarily through the renal system. Later uptake was observed in the stomach and lower gastrointestinal tract. Macromolecular hyaluronan-based gadolinium agents have a high clinical translation potential as hyaluronan is already approved by FDA for a variety of medical applications. PMID:21504061

A RHAMM Mimetic Peptide Blocks Hyaluronan Signaling and Reduces Inflammation and Fibrogenesis in Excisional Skin Wounds

PubMed Central

Tolg, Cornelia; Hamilton, Sara R.; Zalinska, Ewa; McCulloch, Lori; Amin, Ripal; Akentieva, Natalia; Winnik, Francoise; Savani, Rashmin; Bagli, Darius J.; Luyt, Len G.; Cowman, Mary K.; McCarthy, Jim B.; Turley, Eva A.

2013-01-01

Hyaluronan is activated by fragmentation and controls inflammation and fibroplasia during wound repair and diseases (eg, cancer). Hyaluronan-binding peptides were identified that modify fibrogenesis during skin wound repair. Peptides were selected from 7- to 15mer phage display libraries by panning with hyaluronan-Sepharose beads and assayed for their ability to block fibroblast migration in response to hyaluronan oligosaccharides (10 kDa). A 15mer peptide (P15-1), with homology to receptor for hyaluronan mediated motility (RHAMM) hyaluronan binding sequences, was the most effective inhibitor. P15-1 bound to 10-kDa hyaluronan with an affinity of Kd = 10−7 and appeared to specifically mimic RHAMM since it significantly reduced binding of hyaluronan oligosaccharides to recombinant RHAMM but not to recombinant CD44 or TLR2,4, and altered wound repair in wild-type but not RHAMM−/− mice. One topical application of P15-1 to full-thickness excisional rat wounds significantly reduced wound macrophage number, fibroblast number, and blood vessel density compared to scrambled, negative control peptides. Wound collagen 1, transforming growth factor β-1, and α-smooth muscle actin were reduced, whereas tenascin C was increased, suggesting that P15-1 promoted a form of scarless healing. Signaling/microarray analyses showed that P15-1 blocks RHAMM-regulated focal adhesion kinase pathways in fibroblasts. These results identify a new class of reagents that attenuate proinflammatory, fibrotic repair by blocking hyaluronan oligosaccharide signaling. PMID:22889846

Mechanisms involved in enhancement of the expression and function of aggrecanases by hyaluronan oligosaccharides

PubMed Central

Ariyoshi, Wataru; Takahashi, Nobunori; Hida, Daisuke; Knudson, Cheryl B.; Knudson, Warren

2011-01-01

Objective Small hyaluronan (HA) oligosaccharides serve as competitive receptor antagonists to displace HA from the cell surface and induce cell signaling events. In articular chondrocytes this cell signaling is mediated by the HA receptor CD44 and induces stimulation of genes involved in matrix degradation such as matrix metalloproteinases as well as matrix repair genes including collagen type II, aggrecan and HA synthase-2. The objective of this study was to determine changes in the expression and function of aggrecanases after disruption of chondrocyte CD44-HA interactions. Methods Bovine articular chondrocytes or bovine cartilage tissue were pre-treated with a variety of inhibitors of major signaling pathways prior to the addition of HA oligosaccharides. Changes in aggrecanase were monitored by real time reverse transcriptase-polymerase chain reaction and western blot analysis of ADAMTS4, ADAMTS5 and aggrecan proteolytic fragments. To test the interactions between ADAMTS4 and MT4-MMP, protein lysates purified from stimulated chondrocytes were subjected to co-immunoprecipitation. Results Disruption of chondrocyte CD44-HA interactions with HA oligosaccharides induced the transcription of ADAMTS4 and ADAMTS5 in time- and dose-dependent manner. The association of GPI-anchored MT4-MMP with ADAMTS4 was also induced in articular chondrocytes by HA oligosaccharides. Inhibition of the NF-κB pathway blocked HA oligosaccharides-mediated stimulation of aggrecanases. Conclusions Disruptive changes in chondrocyte-matrix interactions by HA oligosaccharides induce matrix degradation and elevate aggrecanases via the activation of the NF-κB signaling pathway. PMID:21905012

Carboxymethyl Hyaluronan-Stabilized Nanoparticles for Anticancer Drug Delivery.

PubMed

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 (nCaP(CMHA)) were loaded with the chemotherapy drug cis-diamminedichloroplatinum(II) (CDDP) to form nCaP(CMHA)CDDP. nCaP(CMHA)CDDP was determined to be poorly crystalline hydroxyapatite, 200 nm in diameter with a -43 mV zeta potential. nCaP(CMHA)CDDP exhibited a two-day burst release of CDDP that tapered resulting in 86% release by 7 days. Surface plasmon resonance showed that nCaP(CMHA)CDDP binds to CD44, but less effectively than CMHA or hyaluronan. nCaP(CMHA-AF488) was taken up by CD44(+)/CD24(-) BT-474EMT breast cancer cells within 18 hours. nCaP(CMHA)CDDP 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 nCaP(CMHA)CDDP. This was likely due to a lack of distribution of nCaP(CMHA)CDDP throughout the dense tumor tissue that limited drug diffusion.

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

A RHAMM mimetic peptide blocks hyaluronan signaling and reduces inflammation and fibrogenesis in excisional skin wounds.

PubMed

Tolg, Cornelia; Hamilton, Sara R; Zalinska, Ewa; McCulloch, Lori; Amin, Ripal; Akentieva, Natalia; Winnik, Francoise; Savani, Rashmin; Bagli, Darius J; Luyt, Len G; Cowman, Mary K; McCarthy, Jim B; Turley, Eva A

2012-10-01

Hyaluronan is activated by fragmentation and controls inflammation and fibroplasia during wound repair and diseases (eg, cancer). Hyaluronan-binding peptides were identified that modify fibrogenesis during skin wound repair. Peptides were selected from 7- to 15mer phage display libraries by panning with hyaluronan-Sepharose beads and assayed for their ability to block fibroblast migration in response to hyaluronan oligosaccharides (10 kDa). A 15mer peptide (P15-1), with homology to receptor for hyaluronan mediated motility (RHAMM) hyaluronan binding sequences, was the most effective inhibitor. P15-1 bound to 10-kDa hyaluronan with an affinity of K(d) = 10(-7) and appeared to specifically mimic RHAMM since it significantly reduced binding of hyaluronan oligosaccharides to recombinant RHAMM but not to recombinant CD44 or TLR2,4, and altered wound repair in wild-type but not RHAMM(-/-) mice. One topical application of P15-1 to full-thickness excisional rat wounds significantly reduced wound macrophage number, fibroblast number, and blood vessel density compared to scrambled, negative control peptides. Wound collagen 1, transforming growth factor β-1, and α-smooth muscle actin were reduced, whereas tenascin C was increased, suggesting that P15-1 promoted a form of scarless healing. Signaling/microarray analyses showed that P15-1 blocks RHAMM-regulated focal adhesion kinase pathways in fibroblasts. These results identify a new class of reagents that attenuate proinflammatory, fibrotic repair by blocking hyaluronan oligosaccharide signaling. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Natural Arctium lappa fruit extract improves the clinical signs of aging skin.

PubMed

Knott, Anja; Reuschlein, Katja; Mielke, Heiko; Wensorra, Ursula; Mummert, Christopher; Koop, Urte; Kausch, Martina; Kolbe, Ludger; Peters, Nils; Stäb, Franz; Wenck, Horst; Gallinat, Stefan

2008-12-01

Subclinical, chronic tissue inflammation involving the generation of cytokines (e.g., interleukin-6 and tumor necrosis factor-alpha) might contribute to the cutaneous aging process. This study aims to screen for an active ingredient with anti-inflammatory (i.e., reduction of interleukin-6 and tumor necrosis factor-alpha) and matrix-stimulating efficacy which improves the clinical signs of skin aging in vivo. In vitro studies with pure Arctiin were performed investigating the inhibition of cytokine induction and stimulation of collagen neo-synthesis. In vivo home-in-use studies using an Arctium lappa fruit extract-containing formulation were carried out to determine procollagen and hyaluronan synthesis, hyaluronan synthase-2 gene expression, and reduction of wrinkle volume after treatment. In vitro studies on human dermal fibroblasts and monocyte-derived dendritic cells supplemented with pure Arctiin showed relative to untreated control cells a stimulation of collagen synthesis and a decrease in interleukin-6 and tumor necrosis factor-alpha concentration, respectively. In addition, topical in vivo application of an A. lappa fruit extract-containing formulation for 12 weeks significantly stimulated procollagen synthesis and increased hyaluronan synthase-2 expression as well as hyaluronan levels compared to vehicle-treated control areas. Similarly, after a 4-week treatment with an A. lappa fruit extract-containing formulation, wrinkle volume in the crow's feet area was significantly reduced as compared to treatment with the vehicle. Our data show that topical treatment with a natural A. lappa fruit extract significantly improves the metabolism of the dermal extracellular matrix and leads to a visible wrinkle reduction in vivo. In conclusion, A. lappa fruit extract represents a targeted means to regenerate dermal structures and, thus, offers an effective treatment option for mature skin.

Hyaluronan Enhances Bone Marrow Cell Therapy for Myocardial Repair After Infarction

PubMed Central

Chen, Chien-Hsi; Wang, Shoei-Shen; Wei, Erika IH; Chu, Ting-Yu; Hsieh, Patrick CH

2013-01-01

Hyaluronan (HA) has been shown to play an important role during early heart development and promote angiogenesis under various physiological and pathological conditions. In recent years, stem cell therapy, which may reduce cardiomyocyte apoptosis, increase neovascularization, and prevent cardiac fibrosis, has emerged as a promising approach to treat myocardial infarction (MI). However, effective delivery of stem cells for cardiac therapy remains a major challenge. In this study, we tested whether transplanting a combination of HA and allogeneic bone marrow mononuclear cells (MNCs) promotes cell therapy efficacy and thus improves cardiac performance after MI in rats. We showed that HA provided a favorable microenvironment for cell adhesion, proliferation, and vascular differentiation in MNC culture. Following MI in rats, compared with the injection of HA alone or MNC alone, injection of both HA and MNCs significantly reduced inflammatory cell infiltration, cardiomyocyte apoptosis, and infarct size and also improved cell retention, angiogenesis, and arteriogenesis, and thus the overall cardiac performance. Ultimately, HA/MNC treatment improved vasculature engraftment of transplanted cells in the infarcted region. Together, our results indicate that combining the biocompatible material HA with bone marrow stem cells exerts a therapeutic effect on heart repair and may further provide potential treatment for ischemic diseases. PMID:23295948

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

MitoQ Loaded Chitosan-Hyaluronan Composite Membranes for Wound Healing

PubMed Central

Tamer, Tamer M.; Collins, Maurice N.; Valachová, Katarina; Hassan, Mohamed A.; Omer, Ahmed M.; Mohy-Eldin, Mohamed S.; Švík, Karol; Jurčík, Rastislav; Ondruška, Ľubomír; Biró, Csaba; Albadarin, Ahmad B.; Šoltés, Ladislav

2018-01-01

Two self-associating biopolymers, namely chitosan (Ch) and a high-molar-mass hyaluronan (HA), were used to prepare membranes with the aim to protect and to enhance the healing of injured skin. A mitochondrially-targeted antioxidant—MitoQ—was incorporated into the mixture of biopolymers prior to their self-association. These three-component membranes were evaluated in detail utilising surface roughness measurements, contact angle measurements, hemocompatibility, and thrombogenicity analyses. Furthermore, in vivo application of Ch/HA/MitoQ membranes was assessed on injured rabbit and rat skin utilizing histological methods. The results showed that the prepared thrombogenic Ch/HA/MitoQ membranes had higher roughness, which allowed for greater surface area for tissue membrane interaction during the healing processes, and lower cytotoxicity levels than controls. MitoQ-loaded composite membranes displayed superior healing properties in these animal models compared to control membranes. PMID:29642447

MitoQ Loaded Chitosan-Hyaluronan Composite Membranes for Wound Healing.

PubMed

Tamer, Tamer M; Collins, Maurice N; Valachová, Katarina; Hassan, Mohamed A; Omer, Ahmed M; Mohy-Eldin, Mohamed S; Švík, Karol; Jurčík, Rastislav; Ondruška, Ľubomír; Biró, Csaba; Albadarin, Ahmad B; Šoltés, Ladislav

2018-04-07

Two self-associating biopolymers, namely chitosan (Ch) and a high-molar-mass hyaluronan (HA), were used to prepare membranes with the aim to protect and to enhance the healing of injured skin. A mitochondrially-targeted antioxidant-MitoQ-was incorporated into the mixture of biopolymers prior to their self-association. These three-component membranes were evaluated in detail utilising surface roughness measurements, contact angle measurements, hemocompatibility, and thrombogenicity analyses. Furthermore, in vivo application of Ch/HA/MitoQ membranes was assessed on injured rabbit and rat skin utilizing histological methods. The results showed that the prepared thrombogenic Ch/HA/MitoQ membranes had higher roughness, which allowed for greater surface area for tissue membrane interaction during the healing processes, and lower cytotoxicity levels than controls. MitoQ-loaded composite membranes displayed superior healing properties in these animal models compared to control membranes.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease.

PubMed

Chang, Mary Y; Tanino, Yoshinori; Vidova, Veronika; Kinsella, Michael G; Chan, Christina K; Johnson, Pamela Y; Wight, Thomas N; Frevert, Charles W

2014-02-01

The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4(-/-) mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection. Copyright © 2014 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

In situ cross-linkable high molecular weight hyaluronan-bisphosphonate conjugate for localized delivery and cell-specific targeting: a hydrogel linked prodrug approach.

PubMed

Varghese, Oommen P; Sun, Weilun; Hilborn, Jöns; Ossipov, Dmitri A

2009-07-01

We present here a novel synthesis route to functionalize high molecular weight hyaluronan (HMW-HA) with a hydrazide group and a bioactive ligand, namely bisphosphonate (BP). For this purpose, a new symmetrical self-immolative biscarbazate linker has been devised. The hydrazide group was used to form hydrazone cross-linked hydrogel upon treating with previously described aldehyde modified hyaluronan. The 1:1 weight ratio of these two polymers gave hydrogel in less than 30 s. In this communication we present the first in vitro results showing that even though HA can target CD44 positive cancer cells (HCT-116), receptor mediated endocytosis could only occur by cleavage of high molecular weight HA with an ubiquitous enzyme, hyaluronidase (Hase). The cancer cells are known to overexpress CD44 receptors and also increase the hyaluronidase activity in vivo. Thus the pro-drug design, based on drug conjugation to HMW-HA, represents a new drug delivery platform where the drug potency is triggered by Hase mediated degradation of the HA-drug conjugate. We have successfully demonstrated that the cross-linkable HA-BP conjugate first undergoes Hase-mediated scission to the fragments of suitable sizes so as to be internalized by CD44 positive cells. The specificity of this targeting was proven by comparing the results with less CD44 positive HEK-293T cells. The localized delivery of such drugs at the surgical resection site opens up avenues to control tumor recurrence after removal of the tumor. In the form of hydrogel it would prevent systemic exposure of the drug and would allow its controlled release.

Factors that influence serum hyaluronan levels in hemodialysis patients.

PubMed

de Medina, M; Ashby, M; Diego, J; Pennell, J P; Hill, M; Schiff, E R; Perez, G O

1999-01-01

Serum hyaluronan levels are increased in dialysis patients. We evaluated several factors that influence serum hyaluronan levels in 184 patients on chronic hemodialysis (duration 2.3 +/- 2.3 [SD] years). The levels were higher than normal in the whole group and in a subgroup of 133 patients without chronic infection, liver disease, or rheumatoid arthritis (215 +/- 19 and 205 +/- 22 microg/L, respectively). There was a tendency for the levels to be higher in a subgroup of patients with hepatitis c virus (HCV) infection. There was no correlation between hyaluronan levels, alanine aminotransferase (ALT), and duration or dose of dialysis. A weak but highly significant negative correlation between serum albumin levels and serum hyaluronan and ferritin levels was seen. The data suggest that chronic inflammation may explain, at least in part, the increased hyaluronan levels found in chronic dialysis patients.

Evaluation of hyaluronan gel (Gengigel(®) ) as a topical applicant in the treatment of gingivitis.

PubMed

Sapna, Nadiger; Vandana, Kharidi Laxman

2011-08-01

  To clinically and histopathologically evaluate the anti-inflammatory effect of 0.2% hyaluronan gel alone and with mechanical therapy on gingivitis. The argyrophilic nucleolar organizer region staining technique was attempted to routinely determine its diagnostic and prognostic dependability for periodontal lesions.   In each of the 28 gingivitis patients, the four quadrants were subjected to different treatments: scaling, scaling + topical hyaluronan gel, only topical hyaluronan gel, and topical + intrasulcular hyaluronan gel. Clinical parameters were recorded at baseline, and on days 7, 14, and 21. Biopsies were taken from each quadrant, inflammatory infiltrates were graded, and the argyrophilic nucleolar organizer region count was measured before and after treatment.   A significant reduction was seen in clinical parameters, inflammatory infiltrates, and the argyrophilic nucleolar organizer region count within the groups. The effect of topical + intrasulcular gel was equivalent to scaling (P > 0.05). Topical + intrasulcular hyaluronan gel application demonstrated a better reduction than topical hyaluronan gel alone.   Hyaluronan gel is an effective topical agent for treating gingivitis, along with scaling and intrasulcular application. The argyrophilic nucleolar organizer region count can be used as a histopathological indicator in cases of non-responsive gingivitis to assess the severity of gingival inflammation. © 2011 Blackwell Publishing Asia Pty Ltd.

Novel Hyaluronan Formulation Enhances the Efficacy of Boron Neutron Capture Therapy for Murine Mesothelioma.

PubMed

Sasai, Masao; Nakamura, Hiroyuki; Sougawa, Nagako; Sakurai, Yoshinori; Suzuki, Minoru; Lee, Chun Man

2016-03-01

Malignant pleural mesothelioma (MPM) is a refractory cancer of the pleura caused by asbestos exposure. MPM is difficult to treat because it easily disseminates. Boron neutron capture therapy (BNCT) is a radiotherapy in which cancer cells that selectively take up (10)Boron-containing compounds are destroyed, and normal cells are uninjured. Hyaluronan (HA) is a ligand of cluster of differentiation 44 (CD44), that is expressed on MPM cells. In order to enhance BNCT for MPM tumors, we developed a novel HA-containing (10)B (sodium borocaptate: BSH) formulation (HA-BND-S). We examined the efficacy of HA-BND-S using MPM cells and a mouse MPM model. HA-BND-S preferentially bound MPM cells dose-dependently, and increased the cytotoxicity of BNCT compared to BSH in vitro. HA-BND-S administration significantly increased the survival of MPM tumor-bearing mice compared to BSH at the same (10)B dosage in BNCT. Modifying BSH with HA is a promising strategy for enhancing the efficacy of BNCT for therapy of MPM. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines.

PubMed

Katona, Éva; Juhász, Tamás; Somogyi, Csilla Szűcs; Hajdú, Tibor; Szász, Csaba; Rácz, Kálmán; Kókai, Endre; Gergely, Pál; Zákány, Róza

2016-03-01

Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment.

PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines

PubMed Central

KATONA, ÉVA; JUHÁSZ, TAMÁS; SOMOGYI, CSILLA SZŰCS; HAJDÚ, TIBOR; SZÁSZ, CSABA; RÁCZ, KÁLMÁN; KÓKAI, ENDRE; GERGELY, PÁL; ZÁKÁNY, RÓZA

2016-01-01

Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment. PMID:26717964

On-line separation and characterization of hyaluronan oligosaccharides derived from radical depolymerization

PubMed Central

Zhao, Xue; Yang, Bo; Li, Lingyun; Zhang, Fuming; Linhardt, Robert J.

2013-01-01

Hydroxyl radicals are widely implicated in the oxidation of carbohydrates in biological and industrial processes and are often responsible for their structural modification resulting in functional damage. In this study, the radical depolymerization of the polysaccharide hyaluronan was studied in a reaction with hydroxyl radicals generated by Fenton Chemistry. A simple method for isolation and identification of the resulting non-sulfated oligosaccharide products of oxidative depolymerization was established. Hyaluronan oligosaccharides were analyzed using ion-pairing reversed phase high performance liquid chromotography coupled with tandem electrospray mass spectrometry. The sequence of saturated hyaluronan oligosaccharides having even- and odd-numbers of saccharide units, afforded through oxidative depolymerization, were identified. This study represents a simple, effective ‘fingerprinting’ protocol for detecting the damage done to hyaluronan by oxidative radicals. This study should help reveal the potential biological outcome of reactive-oxygen radical-mediated depolymerization of hyaluronan. PMID:23768593

A single molecule assay to probe monovalent and multivalent bonds between hyaluronan and its key leukocyte receptor CD44 under force

NASA Astrophysics Data System (ADS)

Bano, Fouzia; Banerji, Suneale; Howarth, Mark; Jackson, David G.; Richter, Ralf P.

2016-09-01

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.

Hyaluronan in vaginal secretions: association with recurrent vulvovaginal candidiasis.

PubMed

Lev-Sagie, Ahinoam; Nyirjesy, Paul; Tarangelo, Nicholas; Bongiovanni, Ann Marie; Bayer, Cynthia; Linhares, Iara M; Giraldo, Paulo C; Ledger, William J; Witkin, Steven S

2009-08-01

We evaluated whether vaginal concentrations of hyaluronan were altered in women with recurrent vulvovaginal candidiasis (RVVC). Lavage samples from 17 women with acute RVVC, 27 women who were receiving a maintenance antifungal regimen, and 24 control women were tested for hyaluronan and interleukin (IL)-6, IL-12, and IL-23 by enzyme-linked immunosorbent assay. Median vaginal hyaluronan concentrations were 33.8 ng/mL (range, 21.6-66.3 ng/mL) in women with acute RVVC, 15.0 ng/mL (range, 11.2-50.6 ng/mL) in women who were receiving maintenance therapy, and 4.2 ng/mL (range, 3.6-12.0 ng/mL) in control subjects (P

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

Chitosan-hyaluronan/nano chondroitin sulfate ternary composite sponges for medical use.

PubMed

Anisha, B S; Sankar, Deepthi; Mohandas, Annapoorna; Chennazhi, K P; Nair, Shantikumar V; Jayakumar, R

2013-02-15

In this work chitosan-hyaluronan composite sponge incorporated with chondroitin sulfate nanoparticle (nCS) was developed. The fabrication of hydrogel was based on simple ionic cross-linking using EDC, followed by lyophilization to obtain the composite sponge. nCS suspension was characterized using DLS and SEM and showed a size range of 100-150 nm. The composite sponges were characterized using SEM, FT-IR and TG-DTA. Porosity, swelling, biodegradation, blood clotting and platelet activation of the prepared sponges were also evaluated. Nanocomposites showed a porosity of 67% and showed enhanced swelling and blood clotting ability. Cytocompatibility and cell adhesion studies of the sponges were done using human dermal fibroblast (HDF) cells and the nanocomposite sponges showed more than 90% viability. Nanocomposite sponges also showed enhanced proliferation of HDF cells within two days of study. These results indicated that this nanocomposite sponges would be a potential candidate for wound dressing. Copyright © 2012 Elsevier Ltd. All rights reserved.

Design and development of hyaluronan-functionalized polybenzofulvene nanoparticles as CD44 receptor mediated drug delivery system

NASA Astrophysics Data System (ADS)

Licciardi, Mariano; Scialabba, Cinzia; Giammona, Gaetano; Paolino, Marco; Razzano, Vincenzo; Grisci, Giorgio; Giuliani, Germano; Makovec, Francesco; Cappelli, Andrea

2017-06-01

A tri-component polymer brush (TCPB ), composed of a polybenzofulvene copolymer bearing low molecular weight hyaluronic acid (HA) on the surface of its cylindrical brush-like backbone and oligo-PEG fractions, was employed in the preparation of 350 nm nanostructured drug delivery systems capable of delivering the anticancer drug doxorubicin. The obtained drug delivery systems were characterized on the basis of drug loading and release, dimensions and zeta potential, morphology and in vitro cell activity, and uptake on three different human cell lines, namely the bronchial epithelial 16HBE, the breast adenocarcinoma MCF-7, and the colon cancer HCT116 cells. Finally, the ability of doxorubicin-loaded TCPB nanoparticles (DOXO-TCPB) to be internalized into cancer cells by CD44 receptor mediated uptake was assessed by means of uptake studies in HCT cells. These data were supported by anti-CD44-FITC staining assay. The proposed TCPB nanostructured drug delivery systems have many potential applications in nanomedicine, including cancer targeted drug delivery.

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

PubMed

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

2014-04-07

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.

Role of UDP-N-Acetylglucosamine (GlcNAc) and O-GlcNAcylation of Hyaluronan Synthase 2 in the Control of Chondroitin Sulfate and Hyaluronan Synthesis*

PubMed Central

Vigetti, Davide; Deleonibus, Sara; Moretto, Paola; Karousou, Eugenia; Viola, Manuela; Bartolini, Barbara; Hascall, Vincent C.; Tammi, Markku; De Luca, Giancarlo; Passi, Alberto

2012-01-01

Hyaluronan (HA) is a glycosaminoglycan present in most tissue microenvironments that can modulate many cell behaviors, including proliferation, migration, and adhesive proprieties. In contrast with other glycosaminoglycans, which are synthesized in the Golgi, HA is synthesized at the plasma membrane by one or more of the three HA synthases (HAS1–3), which use cytoplasmic UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. Previous studies revealed the importance of UDP-sugars for regulating HA synthesis. Therefore, we analyzed the effect of UDP-GlcNAc availability and protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAcylation) on HA and chondroitin sulfate synthesis in primary human aortic smooth muscle cells. Glucosamine treatment, which increases UDP-GlcNAc availability and protein O-GlcNAcylation, increased synthesis of both HA and chondroitin sulfate. However, increasing O-GlcNAcylation by stimulation with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate without a concomitant increase of UDP-GlcNAc increased only HA synthesis. We found that HAS2, the main synthase in aortic smooth muscle cells, can be O-GlcNAcylated on serine 221, which strongly increased its activity and its stability (t½ >5 h versus ∼17 min without O-GlcNAcylation). S221A mutation prevented HAS2 O-GlcNAcylation, which maintained the rapid turnover rate even in the presence of GlcN and increased UDP-GlcNAc. These findings could explain the elevated matrix HA observed in diabetic vessels that, in turn, could mediate cell dedifferentiation processes critical in vascular pathologies. PMID:22887999

Hyaluronan-Inorganic Nanohybrid Materials for Biomedical Applications.

PubMed

Cai, Zhixiang; Zhang, Hongbin; Wei, Yue; Cong, Fengsong

2017-06-12

Nanomaterials, including gold, silver, and magnetic nanoparticles, carbon, and mesoporous materials, possess unique physiochemical and biological properties, thus offering promising applications in biomedicine, such as in drug delivery, biosensing, molecular imaging, and therapy. Recent advances in nanotechnology have improved the features and properties of nanomaterials. However, these nanomaterials are potentially cytotoxic and demonstrate a lack of cell-specific function. Thus, they have been functionalized with various polymers, especially polysaccharides, to reduce toxicity and improve biocompatibility and stability under physiological conditions. In particular, nanomaterials have been widely functionalized with hyaluronan (HA) to enhance their distribution in specific cells and tissues. This review highlights the most recent advances on HA-functionalized nanomaterials for biotechnological and biomedical applications, as nanocarriers in drug delivery, contrast agents in molecular imaging, and diagnostic agents in cancer therapy. A critical evaluation of barriers affecting the use of HA-functionalized nanomaterials is also discussed, and insights into the outlook of the field are explored.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamane, Takumi; Kobayashi-Hattori, Kazuo; Oishi, Yuichi, E-mail: y3oishi@nodai.ac.jp

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 alongmore » 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.« less

Thermo-and pH-sensitive hydrogel membranes composed of poly(N-isopropylacrylamide)-hyaluronan for biomedical applications: Influence of hyaluronan incorporation on the membrane properties.

PubMed

Kamoun, Elbadawy A; Fahmy, Alaa; Taha, Tarek H; El-Fakharany, Esmail M; Makram, Mohamed; Soliman, Hesham M A; Shehata, Hassan

2018-01-01

Interpenetrating hydrogel membranes consisting of pH-sensitive hyaluronan (HA) and thermo-sensitive poly(N-isopropylacrylamide) (PNIPAAM) were synthesized using redox polymerization, followed by N,N-methylenebisacrylamide (BIS) and epichlorohydrin (EPI) were added as chemical crosslinkers. The interaction between membrane compositions has been characterized by FTIR spectroscopy and discussed intensively. The result indicates that HA incorporation in membranes increase the gel fraction, swelling uptake, and the flexibility/elasticity of crosslinked membranes, however it reduced oppositely the mechanical elongation of membranes. PNIPAAm-HA hydrogels responded to both temperature and pH changes and the stimuli-responsiveness was reversible. However, in vitro bioevaluation results revealed that the released ampicillin during the burst release time was sharply influenced and increased with increasing HA contents in membranes; afterwards it became sustainable. Whereas, high HA contents in hydrogels unexpectedly impacted negatively on the cells viability, owing to the viscosity of cell culture media changed. A big resistance was observed against microbial growth of Staphylococcus aureus, Salmonella typhi, and Candida albicans in case of pure PNIPAAm hydrogel membranes without HA or ampicillin. However, HA incorporation or the loaded ampicillin in membranes showed unexpected easily microbial growth. The fast release performance with dual pH-thermo-sensitive hydrogels were suggested as promising materials for quick drug carrier in the biomedical field. Copyright © 2017 Elsevier B.V. All rights reserved.

Study of interactions between hyaluronan and cationic surfactants by means of calorimetry, turbidimetry, potentiometry and conductometry.

PubMed

Krouská, J; Pekař, M; Klučáková, M; Šarac, B; Bešter-Rogač, M

2017-02-10

The thermodynamics of the micelle formation of the cationic surfactants tetradecyltrimethylammonium bromide (TTAB) and cetyltrimethylammonium bromide (CTAB) with and without the addition of hyaluronan of two molecular weights was studied in aqueous solution by titration calorimetry. Macroscopic phase separation, which was detected by calorimetry and also by conductometry, occurs when charges on the surfactant and hyaluronan are balanced. In contrast, turbidimetry and potentiometry showed hyaluronan-surfactant interactions at very low surfactant concentrations. The observed differences between systems prepared with CTAB and TTAB indicate that besides the electrostatic interactions, which probably predominate, hydrophobic effects also play a significant role in hyaluronan interactions with cationic surfactants. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cancer Cell Glycocalyx Mediates Mechanostransduction and Flow-Regulated Invasion

PubMed Central

Qazi, Henry; Palomino, Rocio; Shi, Zhong-Dong; Munn, Lance L.; Tarbell, John M.

2014-01-01

Mammalian cells are covered by a surface proteoglycan (glycocalyx) layer, and it is known that blood vessel-lining endothelial cells use the glycocalyx to sense and transduce the shearing forces of blood flow into intracellular signals. Tumor cells in vivo are exposed to forces from interstitial fluid flow that may affect metastatic potential but are not reproduced by most in vitro cell motility assays. We hypothesized that glycocalyx-mediated mechanotransduction of interstitial flow shear stress is an un-recognized factor that can significantly enhance metastatic cell motility and play a role in augmentation of invasion. Involvement of MMP levels, cell adhesion molecules (CD44, α3 integrin), and glycocalyx components (heparan sulfate and hyaluronan) were investigated in a cell/collagen gel suspension model designed to mimic the interstitial flow microenvironment. Physiologic levels of flow upregulated MMP levels and enhanced the motility of metastatic cells. Blocking the flow-enhanced expression of MMP actvity or adhesion molecules (CD44 and integrins) resulted in blocking the flow-enhanced migratory activity. The presence of a glycocalyx-like layer was verified around tumor cells, and the degradation of this layer by hyaluronidase and heparinase blocked the flow-regulated invasion. This study shows for the first time that interstitial flow enhancement of metastatic cell motility can be mediated by the cell surface glycocalyx – a potential target for therapeutics. PMID:24077103

Hydration Dynamics of Hyaluronan and Dextran

PubMed Central

Hunger, Johannes; Bernecker, Anja; Bakker, Huib J.; Bonn, Mischa; Richter, Ralf P.

2012-01-01

Hyaluronan is a polysaccharide, which is ubiquitous in vertebrates and has been reported to be strongly hydrated in a biological environment. We study the hydration of hyaluronan in solution using the rotational dynamics of water as a probe. We measure these dynamics with polarization-resolved femtosecond-infrared and terahertz time-domain spectroscopies. Both experiments reveal that a subensemble of water molecules is slowed down in aqueous solutions of hyaluronan amounting to ∼15 water molecules per disaccharide unit. This quantity is consistent with what would be expected for the first hydration shell. Comparison of these results to the water dynamics in aqueous dextran solution, a structurally similar polysaccharide, yields remarkably similar results. This suggests that the observed interaction with water is a common feature for hydrophilic polysaccharides and is not specific to hyaluronan. PMID:22828349

Inhibitory effect of chondroitin sulfate oligosaccharides on bovine testicular hyaluronidase.

PubMed

Kakizaki, Ikuko; Koizumi, Hideyo; Chen, Fengchao; Endo, Masahiko

2015-05-05

Hyaluronan and chondroitin sulfates are prominent components of the extracellular matrices of animal tissues; however, their functions in relation to their oligosaccharide structures have not yet been fully elucidated. The oligosaccharides of hyaluronan and chondroitin sulfate were prepared and used to investigate their effects on the hydrolysis and transglycosylation reactions of bovine testicular hyaluronidase when hyaluronan was used as a substrate. Hydrolysis and transglycosylation activities were assessed in independent reaction systems by analyzing the products by HPLC. The hydrolysis and transglycosylation reactions of bovine testicular hyaluronidase were dose-dependently inhibited by chondroitin sulfate oligosaccharides, but not by hyaluronan or chondroitin oligosaccharides. A kinetic analysis of the hydrolysis reaction using hyaluronan octasaccharide revealed that the inhibition mode by chondroitin sulfate oligosaccharides was competitive. Copyright © 2015 Elsevier Ltd. All rights reserved.

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. Copyright 2003 Elsevier Science B.V.

Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb.

PubMed

Matsumoto, Kazu; Li, Yingcui; Jakuba, Caroline; Sugiyama, Yoshinori; Sayo, Tetsuya; Okuno, Misako; Dealy, Caroline N; Toole, Bryan P; Takeda, Junji; Yamaguchi, Yu; Kosher, Robert A

2009-08-01

The glycosaminoglycan hyaluronan (HA) is a structural component of extracellular matrices and also interacts with cell surface receptors to directly influence cell behavior. To explore functions of HA in limb skeletal development, we conditionally inactivated the gene for HA synthase 2, Has2, in limb bud mesoderm using mice that harbor a floxed allele of Has2 and mice carrying a limb mesoderm-specific Prx1-Cre transgene. The skeletal elements of Has2-deficient limbs are severely shortened, indicating that HA is essential for normal longitudinal growth of all limb skeletal elements. Proximal phalanges are duplicated in Has2 mutant limbs indicating an involvement of HA in patterning specific portions of the digits. The growth plates of Has2-deficient skeletal elements are severely abnormal and disorganized, with a decrease in the deposition of aggrecan in the matrix and a disruption in normal columnar cellular relationships. Furthermore, there is a striking reduction in the number of hypertrophic chondrocytes and in the expression domains of markers of hypertrophic differentiation in the mutant growth plates, indicating that HA is necessary for the normal progression of chondrocyte maturation. In addition, secondary ossification centers do not form in the central regions of Has2 mutant growth plates owing to a failure of hypertrophic differentiation. In addition to skeletal defects, the formation of synovial joint cavities is defective in Has2-deficient limbs. Taken together, our results demonstrate that HA has a crucial role in skeletal growth, patterning, chondrocyte maturation and synovial joint formation in the developing limb.

Hyaluronan Oligosaccharides for the Promotion of Remyelination (Revised)

DTIC Science & Technology

2012-10-01

AD_________________ Award Number: W81XWH-10-1-0967 TITLE: Hyaluronan oligosaccharides for the...REPORT TYPE FINAL (revised) 3. DATES COVERED 30 2010- 2012 4. TITLE AND SUBTITLE Hyaluronan oligosaccharides for the promotion of...differentiation and remyelination; and if particular formulations of HA oligosaccharides could block the activities of these digestion products

Hyaluronan Production Regulates Metabolic and Cancer Stem-like Properties of Breast Cancer Cells via Hexosamine Biosynthetic Pathway-coupled HIF-1 Signaling*

PubMed Central

Chanmee, Theerawut; Ontong, Pawared; Izumikawa, Tomomi; Higashide, Miho; Mochizuki, Nobutoshi; Chokchaitaweesuk, Chatchadawalai; Khansai, Manatsanan; Nakajima, Kazuki; Kakizaki, Ikuko; Kongtawelert, Prachya; Taniguchi, Naoyuki; Itano, Naoki

2016-01-01

Cancer stem cells (CSCs) represent a small subpopulation of self-renewing oncogenic cells. As in many other stem cells, metabolic reprogramming has been implicated to be a key characteristic of CSCs. However, little is known about how the metabolic features of cancer cells are controlled to orchestrate their CSC-like properties. We recently demonstrated that hyaluronan (HA) overproduction allowed plastic cancer cells to revert to stem cell states. Here, we adopted stable isotope-assisted tracing and mass spectrometry profiling to elucidate the metabolic features of HA-overproducing breast cancer cells. These integrated approaches disclosed an acceleration of metabolic flux in the hexosamine biosynthetic pathway (HBP). A metabolic shift toward glycolysis was also evident by quantitative targeted metabolomics, which was validated by the expression profiles of key glycolytic enzymes. Forced expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), an HBP rate-limiting enzyme, resembled the results of HA overproduction with regard to HIF-1α accumulation and glycolytic program, whereas GFAT1 inhibition significantly decreased HIF-1α protein level in HA-overproducing cancer cells. Moreover, inhibition of the HBP-HIF-1 axis abrogated HA-driven glycolytic enhancement and reduced the CSC-like subpopulation. Taken together, our results provide compelling evidence that HA production regulates the metabolic and CSC-like properties of breast cancer cells via HBP-coupled HIF-1 signaling. PMID:27758869

Identification of Metastatic Tumor Stem Cell

DTIC Science & Technology

2011-09-01

Aya Kobayashi, Bo Xia, Misako Watabe, Sudha K Pai, Shigeru Hirota, Fei Xing, Wen Liu, Puspa R Pandey, Koji Fukuda, Vishnu Modur, Arnab Ghosh, Andrew...Xing, Puspa R. Pandey, Koji Fukuda, Megumi Iiizumi, Kounosuke Watabe. Hyaluronan synthase 2 promotes tumor progression by stimulating interaction of

Simultaneous analysis of heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan disaccharides by glycoblotting-assisted sample preparation followed by single-step zwitter-ionic-hydrophilic interaction chromatography.

PubMed

Takegawa, Yasuhiro; Araki, Kayo; Fujitani, Naoki; Furukawa, Jun-ichi; Sugiyama, Hiroaki; Sakai, Hideaki; Shinohara, Yasuro

2011-12-15

Glycosaminoglycans (GAGs) play important roles in cell adhesion and growth, maintenance of extracellular matrix (ECM) integrity, and signal transduction. To fully understand the biological functions of GAGs, there is a growing need for sensitive, rapid, and quantitative analysis of GAGs. The present work describes a novel analytical technique that enables high throughput cellular/tissue glycosaminoglycomics for all three families of uronic acid-containing GAGs, hyaluronan (HA), chondroitin sulfate (CS)/dermatan sulfate (DS), and heparan sulfate (HS). A one-pot purification and labeling procedure for GAG Δ-disaccharides was established by chemo-selective ligation of disaccharides onto high density hydrazide beads (glycoblotting) and subsequent labeling by fluorescence. The 17 most common disaccharides (eight comprising HS, eight CS/DS, and one comprising HA) could be separated with a single chromatography for the first time by employing a zwitter-ionic type of hydrophilic-interaction chromatography column. These novel analytical techniques were able to precisely characterize the glycosaminoglycome in various cell types including embryonal carcinoma cells and ocular epithelial tissues (cornea, conjunctiva, and limbus).

Hyaluronan synthase 3 mediated oncogenic action through forming inter-regulation loop with tumor necrosis factor alpha in oral cancer

PubMed Central

Kuo, Yi-Zih; Fang, Wei-Yu; Huang, Cheng-Chih; Tsai, Sen-Tien; Wang, Yi-Ching; Yang, Chih-Li; Wu, Li-Wha

2017-01-01

Hyaluronan (HA) is a major extracellular matrix component. However, its role and mediation in oral cancer remains elusive. Hyaluronan synthase 3 (HAS3), involved in pro-inflammatory short chain HA synthesis, was the predominant synthase in oral cancer cells and tissues. HAS3 overexpression significantly increased oral cancer cell migration, invasion and xenograft tumorigenesis accompanied with the increased expression of tumor necrosis factor alpha (TNF-α) and monocyte chemoattractant protein 1 (MCP-1). Conversely, HAS3 depletion abrogated HAS3-mediated stimulation. HAS3 induced oncogenic actions partly through activating EGFR-SRC signaling. HAS3-derived HA release into extracellular milieu enhanced transendothelial monocyte migration and MCP-1 expression, which was attenuated by anti-HAS3 antibodies or a HAS inhibitor, 4-Methylumbelliferone (4-MU). The NF-κB-binding site III at -1692 to -1682 bp upstream from the transcript 1 start site in HAS3 proximal promoter was the most responsive to TNF-α-stimulated transcription. ChIP-qPCR analysis confirmed the highest NF-κB-p65 enrichment on site III. Increased HAS3 mRNA expression was negatively correlated with the overall survival of oral cancer patients. A concomitant increase of TNF-α, a stimulus for HAS3 expression, with HAS3 expression was not only associated with lymph node metastasis but also negated clinical outcome. Together, HAS3 and TNF-α formed an inter-regulation loop to enhance tumorigenesis in oral cancer. PMID:28107185

Endocytosis of hyaluronan in rat Kupffer cells.

PubMed

Alston-Smith, J; Pertoft, H; Laurent, T C

1992-09-01

The binding, uptake and degradation of hyaluronan (HA) labelled with 3H in its acetyl group were studied in cultured rat Kupffer cells (KC). At 4 degrees C the binding increased with increasing concentrations of HA in the culture medium up to at least 1 microgram/ml, when saturation occurred. Binding could be prevented efficiently by the addition of an excess of unlabelled HA, and to a lesser extent by chondroitin sulphate and oligosaccharide fragments of HA, consisting of four sugars or more. The labelled HA bound to the cells could be removed by incubating the cells with Streptomyces hyaluronidase, or trypsin, indicating that the HA-binding sites are located on the cell surface. At 37 degrees C HA was internalized in a concentration-dependent manner, and degradation products appeared in the supernatant after 1-5 h, depending on the concentration applied. At 50 ng of free HA/ml, each KC accumulated 60 ag of the polysaccharide/min in the first 1 h, and degraded a total amount of 10 fg of HA during an 8 h period. Addition of the negatively charged polysaccharide dextran sulphate reduced binding, and to an even greater extent internalization, of HA in KC, while no effect was observed with dextran. Depletion of intracellular potassium caused a marked reduction in the rate of endocytosis of cell-membrane-associated HA into KC, without affecting binding. Addition of KCl to the culture medium returned endocytosis of [3H]HA to normal levels. There was no effect on binding and a partial effect on internalization by depletion of bivalent cations or in the presence of EDTA. The degradation of [3H]HA by KC cultures was abolished in the presence of weak bases, NH4Cl and chloroquine, supporting the idea that HA is endocytosed into lysosomes prior to degradation. The fluid-phase marker [14C]sucrose was internalized in the cells at much lower rate than was HA. Rates of binding, internalization and degradation of HA in KC point therefore to a specific endocytosis followed by an intracellular degradation to low-M(r) compounds. It was estimated that, under physiological conditions, KC only clear a minor proportion of circulating HA.

Effect of depletion of interstitial hyaluronan on hydraulic conductance in rabbit knee synovium

PubMed Central

Coleman, P J; Scott, D; Abiona, A; Ashhurst, D E; Mason, R M; Levick, J R

1998-01-01

The hydraulic resistance of the synovial lining to fluid outflow from a joint cavity () is important for the retention of intra-articular lubricant. The resistance has been attributed in part to extracellular glycosaminoglycans, including hyaluronan and chondroitin sulphates. Increased permeability in joints infused with testicular hyaluronidase, which digests both chondroitin sulphates and hyaluronan, supports this view. In this study the importance of interstitial hyaluronan per se was assessed using leech and Streptomyces hyaluronidases, which degrade only hyaluronan. Ringer solution was infused into the knee joint cavity of anaesthetized rabbits for 30 min, with or without hyaluronidase, after which intra-articular pressure (Pj) was raised and the relation between pressure and outflow determined. Treatment with Streptomyces, leech or testicular hyaluronidases increased the fluid escape rates by similar factors, namely 4- to 6-fold. After Streptomyces hyaluronidase treatment the slope d/dPj, which at low pressures represents synovial hydraulic conductance, increased from a control of 0.90 ± 0.20 μl min−1 cmH2O−1 (mean ± s.e.m., n = 6) to 4.52 ± 0.70 μl min−1 cmH2O−1. The slope d/dPj increased to a similar level after testicular hyaluronidase, namely to 4.14 ± 1.06 μl min−1 cmH2O−1 (control, 0.54 ± 0.24 μl min−1 cmH2O−1). Streptomyces and leech hyaluronidases were as effective as testicular hyaluronidase (no statistically significant differences) despite differences in substrate specificity. It was shown using histochemical and immunohistochemical techniques that hyaluronan was removed from the synovium by leech, Streptomyces and testicular hyaluronidases. The binding of antibodies 2-B-6 and 3-B-3 showed that the core proteins of the chondroitin sulphate proteoglycans remained intact after treatment with hyaluronidases, and the binding of 5-D-4 showed that keratan sulphate was unaffected. An azocasein digestion assay confirmed that the hyaluronidase preparations had no significant proteolytic activity. The effect of the hyaluronidases was four times greater than predicted from the low concentration of interstitial hyaluronan and its resistivity. Factors that might amplify the effect of hyaluronan depletion include the matrix-organizing role of hyaluronan, and/or non-uniformity of hyaluronan distribution. It is concluded that interstitial hyaluronan makes a major contribution to synovial hydraulic resistance, but the mechanisms are as yet poorly understood. PMID:9596792

Viscosupplementation for treatment of osteoarthritis: from initial discovery to current status and results.

PubMed

Balazs, Endre A

2004-01-01

Viscosupplementation is a therapeutic modality based on the replacement of synovial fluid or exudates with an elastoviscous hyaluronan solution. The first clinical trials were carried out on race horses with painful osteoarthritis of traumatic origin. In the early 1970s, the clinical trials were extended to painful osteoarthritis in humans. Analgesic effects lasting longer than the residence time of the injected hyaluronan in joints were reported both in horses and humans. The hyaluronan used was a noninflammatory fraction of the molecule with an average molecular weight of 2-3 million at a 1% concentration. The analgesic effect of this elastoviscous hyaluronan solution was demonstrated in behavioral animal pain models. Later it was shown that the elastoviscous properties of hyaluronan solutions are the determining factors in reducing pain-elicited nerve activity in both normal and inflamed cat and rat joints. It also was demonstrated in animal arthritis models that elastoviscous hyaluronan solutions promote the healing of traumatic intra-articular wounds. From the mid-1980s, several hyaluronan preparations of greatly varying average molecular weight but with the same concentration were introduced as viscosupplementation-based therapeutic agents. The elastoviscous properties of these solutions varied also, because of the greatly varying average molecular weights (0.5-6.0 million), imitating the rheological properties of either healthy or pathological synovial fluid. Currently, viscosupplementation products available worldwide vary greatly in their elastoviscous properties, and their dosage is not standardized in terms of frequency of injections required or in regard to the removal of exudates before injection. The question of which patient at what stage of the disease responds best with long-lasting pain relief to the many therapeutic products marketed with greatly varying elastoviscous properties has not yet been answered. At the same time, viscosupplementation was introduced, the same highly elastoviscous hyaluronan solutions also were applied in ophthalmic surgery as viscosurgical tools to protect sensitive tissues in the eye during surgery and to be used as soft instruments for tissue manipulation. Modified hyaluronan products (gels) also were introduced for augmentation of the intercellular matrix in tissues (viscoaugmentation) and for separating tissues to prevent adhesions and excessive scar formation (viscoseparation). Hyaluronan and its derivatives (gels) also have been used for drug delivery. The therapeutic use of highly elastoviscous solutions and gels of hyaluronan and its derivatives to build intercellular matrices for supplementation, regeneration, and developing new tissues introduced the concept of matrix engineering into medical practice.

Beneficial effects of dried pomegranate juice concentrated powder on ultraviolet B-induced skin photoaging in hairless mice.

PubMed

Kang, Su-Jin; Choi, Beom-Rak; Kim, Seung-Hee; Yi, Hae-Yeon; Park, Hye-Rim; Song, Chang-Hyun; Ku, Sae-Kwang; Lee, Young-Joon

2017-08-01

The present study investigated the anti-aging effects of pomegranate juice concentrated powder (PCP) in hairless mice following 15 weeks of UVB irradiation (three times a week; 0.18 J/cm 2 ). Skin moisturizing effects were evaluated through skin water, collagen type I and hyaluronan contents, as well as collagen type I and hyaluronan synthesis-related transcript levels. Wrinkle formation and edema scores (skin weights) were also assessed, along with skin matrix metalloproteinase (MMP)-1, MMP-9 and MMP-13 transcript levels. To determine the anti-inflammatory effects of PCP, myeloperoxidase (MPO) activity, interleukin (IL)-1β and IL-10 contents were observed. Caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) were used as an apoptotic index in epidermal keratinocytes. To determine the anti-oxidative effects of PCP, nitrotyrosine and 4-hydroxynonenal immunoreactive cells were detected and glutathione (GSH) content, malondialdehyde levels, superoxide anion production, Nox2, and GSH reductase mRNA expression were all measured. The results indicated that skin wrinkles induced by photoaging were significantly reduced by PCP, whereas skin water contents, collagen type I and hyaluronan contents all increased. Furthermore, IL-1β levels in the PCP-treated groups were lower than those in the UVB-exposed control group. UVB-induced GSH depletion was also inhibited by PCP. Taken together, the results of the current study suggest that PCP has favorable protective effects against UVB-induced photoaging through anti-apoptotic effects, MMP activity inhibition and ECM (COL1 and hyaluronan) synthesis-related moisturizing, anti-inflammatory and anti-oxidative effects.

Beneficial effects of dried pomegranate juice concentrated powder on ultraviolet B-induced skin photoaging in hairless mice

PubMed Central

Kang, Su-Jin; Choi, Beom-Rak; Kim, Seung-Hee; Yi, Hae-Yeon; Park, Hye-Rim; Song, Chang-Hyun; Ku, Sae-Kwang; Lee, Young-Joon

2017-01-01

The present study investigated the anti-aging effects of pomegranate juice concentrated powder (PCP) in hairless mice following 15 weeks of UVB irradiation (three times a week; 0.18 J/cm2). Skin moisturizing effects were evaluated through skin water, collagen type I and hyaluronan contents, as well as collagen type I and hyaluronan synthesis-related transcript levels. Wrinkle formation and edema scores (skin weights) were also assessed, along with skin matrix metalloproteinase (MMP)-1, MMP-9 and MMP-13 transcript levels. To determine the anti-inflammatory effects of PCP, myeloperoxidase (MPO) activity, interleukin (IL)-1β and IL-10 contents were observed. Caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) were used as an apoptotic index in epidermal keratinocytes. To determine the anti-oxidative effects of PCP, nitrotyrosine and 4-hydroxynonenal immunoreactive cells were detected and glutathione (GSH) content, malondialdehyde levels, superoxide anion production, Nox2, and GSH reductase mRNA expression were all measured. The results indicated that skin wrinkles induced by photoaging were significantly reduced by PCP, whereas skin water contents, collagen type I and hyaluronan contents all increased. Furthermore, IL-1β levels in the PCP-treated groups were lower than those in the UVB-exposed control group. UVB-induced GSH depletion was also inhibited by PCP. Taken together, the results of the current study suggest that PCP has favorable protective effects against UVB-induced photoaging through anti-apoptotic effects, MMP activity inhibition and ECM (COL1 and hyaluronan) synthesis-related moisturizing, anti-inflammatory and anti-oxidative effects. PMID:28810554

MicroRNA miR-328 Regulates Zonation Morphogenesis by Targeting CD44 Expression

PubMed Central

Wang, Chia-Hui; Lee, Daniel Y.; Deng, Zhaoqun; Jeyapalan, Zina; Lee, Shao-Chen; Kahai, Shireen; Lu, Wei-Yang; Zhang, Yaou; Yang, Burton B.

2008-01-01

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion. PMID:18560585

MicroRNA miR-328 regulates zonation morphogenesis by targeting CD44 expression.

PubMed

Wang, Chia-Hui; Lee, Daniel Y; Deng, Zhaoqun; Jeyapalan, Zina; Lee, Shao-Chen; Kahai, Shireen; Lu, Wei-Yang; Zhang, Yaou; Yang, Burton B

2008-06-18

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion.

Hyaluronan based materials with catanionic sugar-derived surfactants as drug delivery systems.

PubMed

Roig, F; Blanzat, M; Solans, C; Esquena, J; García-Celma, M J

2018-04-01

In the present work novel drug delivery systems consisting in highly porous Hyaluronan foams for the administration of a non-steroidal anti-inflammatory drug (NSAID), ketoprofen, have been obtained. A sugar-derived surfactant associated with ketoprofen was prepared and incorporated into the porous hyaluronan materials. The association between a lactose derived surfactant, Lhyd 12 , and ketoprofen was obtained by acid-base reaction and its physicochemical properties were studied. Tensiometric and dynamic light scattering (DLS) determinations showed the formation of catanionic surfactant aggregates, Lhyd 12 /ketoprofen, in aqueous solution. Furthermore, the catanionic surfactants allowed greater solubilisation of ketoprofen. Hyaluronan porous materials were developed using butanediol diglycidyl ether as crosslinking agent. The profile release of Lhyd 12 /ketoprofen from hyaluronan based materials shows differences as a function of the aggregation state of catanionic surfactant. Copyright © 2018 Elsevier B.V. All rights reserved.

High extent of O-GlcNAcylation in breast cancer cells correlates with the levels of HAS enzymes, accumulation of hyaluronan, and poor outcome.

PubMed

Tiainen, Satu; Oikari, Sanna; Tammi, Markku; Rilla, Kirsi; Hämäläinen, Kirsi; Tammi, Raija; Kosma, Veli-Matti; Auvinen, Päivi

2016-11-01

Obesity and oversupply of glucose, e.g., due to nutritional factors may shape the tumor microenvironment favorable for tumor progression. O-GlcNAcylation, a reversible modification of intracellular proteins, influences on several cellular functions and is connected to many diseases including cancer. Glycosaminoglycan hyaluronan (HA) enhances tumor progression and in breast cancer HA accumulation associates strongly with poor outcome. In vitro studies have suggested that O-GlcNAcylation may enhance HA synthesis. The aim of this study was to investigate the correlations between O-GlcNAcylation, HA-related parameters, and disease outcome in a clinical breast cancer material consisting of 278 breast cancer cases. In microscopic analyses, O-GlcNAc staining of the breast carcinoma cells was evaluated in several randomly picked high-power fields of each section. The extent of cytoplasmic O-GlcNAc staining was graded as either low or high according to the intensity of the staining and the percentage of stained cells. The extent of nuclear O-GlcNAc staining was categorized as either low or high according to the percentage of stained nuclei. A high extent of both cytoplasmic and nuclear O-GlcNAcylation correlated with an increased relapse rate, development of distant metastases, and poor outcome. A high extent of cytoplasmic O-GlcNAcylation correlated also with the accumulation of all hyaluronan synthase (HAS1-3) proteins and with a large amount of HA in the tumor stroma. In addition, a high extent of nuclear O-GlcNAcylation associated with obesity. The results suggest a mechanistic association between increased O-GlcNAcylation and HA synthesis, leading to a HA-rich microenvironment favorable for breast cancer progression.

Hyaluronan and Layilin Mediate Loss of Airway Epithelial Barrier Function Induced by Cigarette Smoke by Decreasing E-cadherin*

PubMed Central

Forteza, Rosanna Malbran; Casalino-Matsuda, S. Marina; Falcon, Nieves S.; Valencia Gattas, Monica; Monzon, Maria E.

2012-01-01

Cigarette smoke (CigS) exposure is associated with increased bronchial epithelial permeability and impaired barrier function. Primary cultures of normal human bronchial epithelial cells exposed to CigS exhibit decreased E-cadherin expression and reduced transepithelial electrical resistance. These effects were mediated by hyaluronan (HA) because inhibition of its synthesis with 4-methylumbelliferone prevented these effects, and exposure to HA fragments of <70 kDa mimicked these effects. We show that the HA receptor layilin is expressed apically in human airway epithelium and that cells infected with lentivirus expressing layilin siRNAs were protected against increased permeability triggered by both CigS and HA. We identified RhoA/Rho-associated protein kinase (ROCK) as the signaling effectors downstream layilin. We conclude that HA fragments generated by CigS bind to layilin and signal through Rho/ROCK to inhibit the E-cadherin gene and protein expression, leading to a loss of epithelial cell-cell contact. These studies suggest that HA functions as a master switch protecting or disrupting the epithelial barrier in its high versus low molecular weight form and that its depolymerization is a first and necessary step triggering the inflammatory response to CigS. PMID:23048036

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. 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. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr06102g

Gait patterns after intraarticular treatment of patients with osteoarthritis of the Knee - Hyaluronan versus triamcinolone: a prospective, randomized, doubleblind, monocentric study

PubMed Central

2009-01-01

Objective Evaluation of gait performance and muscle activity patterns as well as clinical efficacy and safety after single intraarticular injection with hyaluronan compared with triamcinolone in patients with knee osteoarthritis. Materials and Methods This trial evaluated the influence of a single injection of hyaluronan or triamcinolone on gait pattern and muscle activity. For clinical evaluation a visual analogue scale for pain, Lequesne index, and Knee Society Score were used. Quality of life was assessed with the SF-36. Results The complete analysis was performed in 50 of 60 patients. 26 patients were treated with triamcinolone and 24 with hyaluronan. Hyaluronan treatment led to significant improvement of range of motion at hip and knee. Significant improvement could be either demonstrated for the pain scale, Lequesne and Knee Society score in both groups. Quality of life showed greater improvement in the triamcinolone group. Conclusion Single application of high-viscosity hyaluronan shows superior range of motion and pain reduction as well as improvement in clinical results. Even if there was a lack of significant differences compared to triamcinolone, this therapy classified as safe and effective in the short follow up. PMID:19380288

Immunohistochemical analysis of EGFR and hyaluronan in tongue cancer and the development of regional recurrence in patients initially diagnosed N0.

PubMed

Lindell Jonsson, Eva; Nylander, Karin; Hallén, Lars; Laurell, Göran

2017-08-01

To investigate whether the extent of expression of hyaluronan (HA) and epidermal growth factor receptor (EGFR) in squamous cell carcinoma of the mobile tongue can predict the risk of cervical metastasis and survival. Retrospective histopathologic study. Surgical specimens from 64 patients who had undergone surgery for squamous cell carcinoma of the mobile tongue were assessed using immunohistochemistry to investigate the expression of HA and EGFR in the primary tumours, and the data were then correlated to cervical metastasis and survival. There was a significant correlation between the intensity of HA staining and patient survival (p .024), and a weak correlation between the staining proportion of EGFR and the risk for regional recurrence (AUC 66). This study indicates that immunoscoring using HA could be used to provide prognostic tools for tongue cancer, and that it might be of interest to study the prognostic properties of EGFR further concerning the risk for regional recurrence after the primary treatment.

Muscle stem cell intramuscular delivery within hyaluronan methylcellulose improves engraftment efficiency and dispersion.

PubMed

Davoudi, Sadegh; Chin, Chih-Ying; Cooke, Michael J; Tam, Roger Y; Shoichet, Molly S; Gilbert, Penney M

2018-04-26

Adult skeletal muscle tissue harbors the capacity for self-repair due to the presence of tissue resident muscle stem cells (MuSCs). Advances in the area of prospective MuSC isolation demonstrated the potential of cell transplantation therapy as a regenerative medicine strategy to restore strength and long-term regenerative capacity to aged, injured, or diseased skeletal muscle tissue. However, cell loss during ejection, limits to post-injection proliferation, and poor donor cell dispersion distal to the injection site are amongst hurdles to overcome to maximize MuSC transplant impact. Here, we assess a physical blend of hyaluronan and methylcellulose (HAMC) as a bioactive, shear thinning hydrogel cell delivery system to improve MuSC transplantation efficiency. Using in vivo transplantation studies, we found that the HAMC delivery system results in a >45% increase in the number of donor-derived fibers as compared to saline delivery. We demonstrate that increases in donor-derived fibers when using HAMC are attributed to increased MuSC proliferation via a CD44-independent mechanism, preventing injected cell active clearance, and supporting in vivo expansion by delaying differentiation. Furthermore, we observed a significant improvement in donor fiber dispersion when MuSCs were delivered in HAMC. Our study results suggest that HAMC is a promising muscle stem cell delivery vehicle. Copyright © 2018 Elsevier Ltd. All rights reserved.

Self-Renewing Microns-Thick Biopolymer Brush Made of Hyaluronan under Active Synthesis

NASA Astrophysics Data System (ADS)

Wei, W.; Washburn, J.; Weigel, P.; Curtis, J. E.

Hyaluronan (HA) is a large anionic polysaccharide distributed throughout many vertebrate tissues. We introduce a technology to produce dynamic HA polymer brush interfaces. The strategy relies on the enzyme hyaluronan synthase (HA synthase), which synthesizes and extrudes HA polymers up to 20 microns in length. We show that interfaces decorated by HA synthase-rich membrane fragments robustly produce polymer brushes of predictable heights and concentration profiles. The brush thickness can be tuned by the duration of growth or the enzyme density in the membranes. The system is self-renewing in that old polymers desorb and new polymers are produced. The brush can also be replenished after enzymatic removal multiple times. The large extent of the polymer interface allows for characterization of the brush architecture and for studying dynamic processes inside the brush using optical microscopy. At low ionic strengths (1 mM), we measure one of the largest polymer brushes yet reported, an average of 7.8 microns thick. For applications that require a stable brush interface, we have covalently reinforced the HA to the surfaces, and demonstrated that the brush is stable for at least two months. This self-renewing, dynamic biopolymer brush has great potential as a new biomaterial for implants, antifouling, tissue engineering and drug delivery.

Selective in vitro anticancer effect of superparamagnetic iron oxide nanoparticles loaded in hyaluronan polymeric micelles.

PubMed

Smejkalová, Daniela; Nešporová, Kristina; Huerta-Angeles, Gloria; Syrovátka, Jakub; Jirák, Daniel; Gálisová, Andrea; Velebný, Vladimír

2014-11-10

Due to its native origin, excellent biocompatibility and biodegradability, hyaluronan (HA) represents an attractive polymer for superparamagnetic iron oxide nanoparticles (SPION) coating. Herein, we report HA polymeric micelles encapsulating oleic acid coated SPIONs, having a hydrodynamic size of about 100 nm and SPION loading capacity of 1-2 wt %. The HA-SPION polymeric micelles were found to be selectively cytotoxic toward a number of human cancer cell lines, mainly those of colon adenocarcinoma (HT-29). The selective inhibition of cell growth was even observed when the SPION loaded HA polymeric micelles were incubated with a mixture of control and cancer cells. The selective in vitro inhibition could not be connected with an enhanced CD44 uptake or radical oxygen species formation and was rather connected with a different way of SPION intracellular release. While aggregated iron particles were visualized in control cells, nonaggregated solubilized iron oxide particles were detected in cancer cells. In vivo SPION accumulation in intramuscular tumor following an intravenous micelle administration was confirmed by magnetic resonance (MR) imaging and histological analysis. Having a suitable hydrodynamic size, high magnetic relaxivity, and being cancer specific and able to accumulate in vivo in tumors, SPION-loaded HA micelles represent a promising platform for theranostic applications.

Unique CD44 intronic SNP is associated with tumor grade in breast cancer: a case control study and in silico analysis.

PubMed

Esmaeili, Rezvan; Abdoli, Nasrin; Yadegari, Fatemeh; Neishaboury, Mohamadreza; Farahmand, Leila; Kaviani, Ahmad; Majidzadeh-A, Keivan

2018-01-01

CD44 encoded by a single gene is a cell surface transmembrane glycoprotein. Exon 2 is one of the important exons to bind CD44 protein to hyaluronan. Experimental evidences show that hyaluronan-CD44 interaction intensifies the proliferation, migration, and invasion of breast cancer cells. Therefore, the current study aimed at investigating the association between specific polymorphisms in exon 2 and its flanking region of CD44 with predisposition to breast cancer. In the current study, 175 Iranian female patients with breast cancer and 175 age-matched healthy controls were recruited in biobank, Breast Cancer Research Center, Tehran, Iran. Single nucleotide polymorphisms of CD44 exon 2 and its flanking were analyzed via polymerase chain reaction and gene sequencing techniques. Association between the observed variation with breast cancer risk and clinico-pathological characteristics were studied. Subsequently, bioinformatics analysis was conducted to predict potential exonic splicing enhancer (ESE) motifs changed as the result of a mutation. A unique polymorphism of the gene encoding CD44 was identified at position 14 nucleotide upstream of exon 2 (A37692→G) by the sequencing method. The A > G polymorphism exhibited a significant association with higher-grades of breast cancer, although no significant relation was found between this polymorphism and breast cancer risk. Finally, computational analysis revealed that the intronic mutation generated a new consensus-binding motif for the splicing factor, SC35, within intron 1. The current study results indicated that A > G polymorphism was associated with breast cancer development; in addition, in silico analysis with ESE finder prediction software showed that the change created a new SC35 binding site.

Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb

PubMed Central

Matsumoto, Kazu; Li, Yingcui; Jakuba, Caroline; Sugiyama, Yoshinori; Sayo, Tetsuya; Okuno, Misako; Dealy, Caroline N.; Toole, Bryan P.; Takeda, Junji; Yamaguchi, Yu; Kosher, Robert A.

2009-01-01

Summary The glycosaminoglycan hyaluronan (HA) is a structural component of extracellular matrices and also interacts with cell surface receptors to directly influence cell behavior. To explore functions of HA in limb skeletal development, we conditionally inactivated the gene for HA synthase 2, Has2, in limb bud mesoderm using mice that harbor a floxed allele of Has2 and mice carrying a limb mesoderm-specific Prx1-Cre transgene. The skeletal elements of Has2-deficient limbs are severely shortened, indicating that HA is essential for normal longitudinal growth of all limb skeletal elements. Proximal phalanges are duplicated in Has2 mutant limbs indicating an involvement of HA in patterning specific portions of the digits. The growth plates of Has2-deficient skeletal elements are severely abnormal and disorganized, with a decrease in the deposition of aggrecan in the matrix and a disruption in normal columnar cellular relationships. Furthermore, there is a striking reduction in the number of hypertrophic chondrocytes and in the expression domains of markers of hypertrophic differentiation in the mutant growth plates, indicating that HA is necessary for the normal progression of chondrocyte maturation. In addition, secondary ossification centers do not form in the central regions of Has2 mutant growth plates owing to a failure of hypertrophic differentiation. In addition to skeletal defects, the formation of synovial joint cavities is defective in Has2-deficient limbs. Taken together, our results demonstrate that HA has a crucial role in skeletal growth, patterning, chondrocyte maturation and synovial joint formation in the developing limb. PMID:19633173

Endothelial Glycocalyx as a Shield Against Diabetic Vascular Complications: Involvement of Hyaluronan and Hyaluronidases.

PubMed

Dogné, Sophie; Flamion, Bruno; Caron, Nathalie

2018-07-01

The endothelial glycocalyx (EG), which covers the apical surface of the endothelial cells and floats into the lumen of the vessels, is a key player in vascular integrity and cardiovascular homeostasis. The EG is composed of PGs (proteoglycans), glycoproteins, glycolipids, and glycosaminoglycans, in particular hyaluronan (HA). HA seems to be implicated in most of the functions described for EG such as creating a space between blood and the endothelium, controlling vessel permeability, restricting leukocyte and platelet adhesion, and allowing an appropriate endothelial response to flow variation through mechanosensing. The amount of HA in the EG may be regulated by HYAL (hyaluronidase) 1, the most active somatic hyaluronidase. HYAL1 seems enriched in endothelial cells through endocytosis from the bloodstream. The role of the other main somatic hyaluronidase, HYAL2, in the EG is uncertain. Damage to the EG, accompanied by shedding of one or more of its components, is an early sign of various pathologies including diabetes mellitus. Shedding increases the blood or plasma concentration of several EG components, such as HA, heparan sulfate, and syndecan. The plasma levels of these molecules can then be used as sensitive markers of EG degradation. This has been shown in type 1 and type 2 diabetic patients. Recent experimental studies suggest that preserving the size and amount of EG HA in the face of diabetic insults could be a useful novel therapeutic strategy to slow diabetic complications. One way to achieve this goal, as suggested by a murine model of HYAL1 deficiency, may be to inhibit the function of HYAL1. The same approach may succeed in other pathological situations involving endothelial dysfunction and EG damage. © 2018 American Heart Association, Inc.

Hyaluronan hydrogels with a low degree of modification as scaffolds for cartilage engineering.

PubMed

La Gatta, Annalisa; Ricci, Giulia; Stellavato, Antonietta; Cammarota, Marcella; Filosa, Rosanna; Papa, Agata; D'Agostino, Antonella; Portaccio, Marianna; Delfino, Ines; De Rosa, Mario; Schiraldi, Chiara

2017-10-01

In the field of cartilage engineering, continuing efforts have focused on fabricating scaffolds that favor maintenance of the chondrocytic phenotype and matrix formation, in addition to providing a permeable, hydrated, microporous structure and mechanical support. The potential of hyaluronan-based hydrogels has been well established, but the ideal matrix remains to be developed. This study describes the development of hyaluronan sponges-based scaffolds obtained by lysine methyl-ester crosslinking. The reaction conditions are optimized with minimal chemical modifications to obtain materials that closely resemble elements in physiological cellular environments. Three hydrogels with different amounts of crosslinkers were produced that show morphological, water-uptake, mechanical, and stability properties comparable or superior to those of currently available hyaluronan-scaffolds, but with significantly fewer hyaluronan modifications. Primary human chondrocytes cultured with the most promising hydrogel were viable and maintained lineage identity for 3 weeks. They also secreted cartilage-specific matrix proteins. These scaffolds represent promising candidates for cartilage engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Examination of synovial fluid hyaluronan quantity and quality in stifle joints of dogs with osteoarthritis.

PubMed

Venable, Rachel O; Stoker, Aaron M; Cook, Cristi R; Cockrell, Mary K; Cook, James L

2008-12-01

To determine the quantity (concentration) and quality (molecular weight) of synovial fluid hyaluronan with respect to presence and severity of osteoarthritis in stifle joints of dogs. 21 purpose-bred dogs and 6 clinically affected large-breed dogs (cranial cruciate ligament [CrCL] disease with secondary osteoarthritis). Research dogs underwent arthroscopic surgery in 1 stifle joint to induce osteoarthritis via CrCL transection (CrCLt; n=5 stifle joints), femoral condylar articular cartilage groove creation (GR; 6), or meniscal release (MR; 5); 5 had sham surgery (SH) performed. Contralateral stifle joints (n=21) were used as unoperated control joints. Synovial fluid was obtained from research dogs at time 0 and 12 weeks after surgery and from clinically affected dogs prior to surgery. All dogs were assessed for lameness, radiographic signs of osteoarthritis, and pathologic findings on arthroscopy as well as for quantity and quality of hyaluronan. Clinically affected dogs had significantly greater degrees of pathologic findings, compared with dogs with surgically induced osteoarthritis (ie, those with CrCLt, GR, and MR stifle joints), and with respect to lameness scores, radiographic signs of osteoarthritis, pathologic findings on arthroscopy, and synovial fluid hyaluronan concentration. Synovial fluid from stifle joints of dogs with surgically induced osteoarthritis had hyaluronan bands at 35 kd on western blots that synovial fluid from SH and clinically affected stifle joints did not. Synovial fluid hyaluronan quantity and quality were altered in stifle joints of dogs with osteoarthritis, compared with control stifle joints. A specific hyaluronan protein fragment may be associated with early pathologic changes in affected joints.

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

2013-10-04

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.

Human Milk Hyaluronan Enhances Innate Defense of the Intestinal Epithelium*

PubMed Central

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

2013-01-01

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. PMID:23950179

Hyaluronan Tumor Cell Interactions in Prostate Cancer Growth and Survival

DTIC Science & Technology

2008-12-01

different outcomes. For example, colo- rectal cancers can be grouped into DNA mismatch repair-proficient, MLH1 negative and presumed Lynch syndrome. Although...a prognostic factor in DNA-mismatch repair-proficient (MMR-proficient) and presumed Lynch syndrome forms of colorectal cancer but not in MLH1 negative

Cervical hyaluronan biology in pregnancy, parturition and preterm birth.

PubMed

Mahendroo, Mala

2018-03-03

Cervical hyaluronan (HA) synthesis is robustly induced in late pregnancy in numerous species including women and mice. Recent evidence highlights the diverse and dynamic functions of HA in cervical biology that stem from its expression in the cervical stroma, epithelia and immune cells, changes in HA molecular weight and cell specific expression of HA binding partners. Mice deficient in HA in the lower reproductive tract confirm a structural role of HA to increase spacing and disorganization of fibrillar collagen, though this function is not critical for pregnancy and parturition. In addition, cervical HA depletion via targeted deletion of HA synthase genes, disrupts cell signaling required for the differentiation of epithelia and their mucosal and junctional barrier, resulting in increased susceptibility to ascending infection-mediated preterm birth. Finally the generation of HA disaccharides by bacterial hyaluronidases as made by Group B streptococcus can ligate toll like receptors TLR2/4 thus preventing appropriate inflammatory responses as needed to fight ascending infection and preterm birth. This review summarizes our current understanding of HA's novel and unique roles in cervical remodeling in the process of birth. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Development of drug loaded nanoparticles for tumor targeting. Part 1: Synthesis, characterization, and biological evaluation in 2D cell cultures.

PubMed

El-Dakdouki, Mohammad H; Puré, Ellen; Huang, Xuefei

2013-05-07

Nanoparticles (NPs) are being extensively studied as carriers for drug delivery, but they often have limited penetration inside tumors. We envision that by targeting an endocytic receptor on the cell surface, the uptake of NPs can be significantly enhanced through receptor mediated endocytosis. In addition, if the receptor is recycled to the cell surface, the NP cargo can be transported out of the cells, which is then taken up by neighboring cells thus enhancing solid tumor penetration. To validate our hypothesis, in the first of two articles, we report the synthesis of doxorubicin (DOX)-loaded, hyaluronan (HA) coated silica nanoparticles (SNPs) containing a highly fluorescent core to target CD44, a receptor expressed on the cancer cell surface. HA was conjugated onto amine-functionalized SNPs prepared through an oil-water microemulsion method. The immobilization of the cytotoxic drug DOX was achieved through an acid sensitive hydrazone linkage. The NPs were fully characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements, thermogravimetric analysis (TGA), UV-vis absorbance, and nuclear magnetic resonance (NMR). Initial biological evaluation experiments demonstrated that compared to ligand-free SNPs, the uptake of HA-SNPs by the CD44-expressing SKOV-3 ovarian cancer cells was significantly enhanced when evaluated in the 2D monolayer cell culture. Mechanistic studies suggested that cellular uptake of HA-SNPs was mainly through CD44 mediated endocytosis. HA-SNPs with immobilized DOX were endocytosed efficiently by the SKOV-3 cells as well. The enhanced tumor penetration and drug delivery properties of HA-SNPs will be evaluated in 3D tumor models in the subsequent paper.

Characterization of the effect of high molecular weight hyaluronan on trans-synovial flow in rabbit knees.

PubMed

Coleman, P J; Scott, D; Mason, R M; Levick, J R

1999-01-01

1. The effect of a rooster comb hyaluronan (3.6-4.0 g l-1) of similar chain length to rabbit synovial fluid hyaluronan, on the trans-synovial escape of fluid from the joint cavity in the steady state ( 8d s) was studied in 29 rabbit knees at controlled intra-articular pressures (Pj). 2. Rooster hyaluronan caused the pressure-flow relation to flatten out as pressure was raised. At 10-20 cmH2O the slope of the quasi-plateau, 0.05 +/- 0.01 microliter min-1 cmH2O-1 (mean +/- s.e.m.), was 1/39th that for Ringer solution (1.94 +/- 0.01 microliter 2O-1 ). 3. Bovine synovial fluid had a similar effect to hyaluronan in Ringer solution. 4. The quasi-plateau was caused by increasing opposition to outflow; the pressure required to drive unit outflow increased 4.4-fold between 5 and 20 cmH2O. The increased opposition to outflow at 20 cmH2O was equivalent to an effective osmotic pressure of 13-17 cmH2O at the interface. Since the infusate's osmotic pressure was only 0.9 cmH2O, this implied concentration polarization to 15-18 g l-1 hyaluronan at the interface. 5. Mechanical perforation of the lining, or enzymatic degradation of the interstitial matrix by chymopapain, abolished the quasi-plateau. Hydrational expansion of the matrix by approximately 2-fold did not. The increased opposition to outflow was reversible by washing out the hyaluronan, or by reducing Pj. It was unaffected by interruption of tissue blood flow or synoviocyte oxidative metabolism. These properties are compatible with a concentration polarization mechanism, i.e. flow-induced concentration of hyaluronan at the synovial interface due to molecular reflection. 6. A concentration polarization theory was developed for a partially reflected solute. Numerical solutions supported the feasibility of this osmotic explanation of the quasi-plateau. Additional mechanisms may also be involved. 7. It is concluded that native-size hyaluronan helps to retain synovial fluid in the joint cavity when pressure is raised and acts, at least in part, by exerting osmotic pressure at the interface between synovial matrix and a concentration polarization layer.

Characterization of the effect of high molecular weight hyaluronan on trans-synovial flow in rabbit knees

PubMed Central

Coleman, P J; Scott, D; Mason, R M; Levick, J R

1999-01-01

The effect of a rooster comb hyaluronan (3.6–4.0 g l−1) of similar chain length to rabbit synovial fluid hyaluronan, on the trans-synovial escape of fluid from the joint cavity in the steady state (Q̇s) was studied in 29 rabbit knees at controlled intra-articular pressures (Pj).Rooster hyaluronan caused the pressure-flow relation to flatten out as pressure was raised. At 10–20 cmH2O the slope of the quasi-plateau, 0.05 ± 0.01 μl min−1 cmH2O−1 (mean ±s.e.m.), was 1/39th that for Ringer solution (1.94 ± 0.01 μl min−1 cmH2O−1).Bovine synovial fluid had a similar effect to hyaluronan in Ringer solution.The quasi-plateau was caused by increasing opposition to outflow; the pressure required to drive unit outflow increased 4.4-fold between 5 and 20 cmH2O. The increased opposition to outflow at 20 cmH2O was equivalent to an effective osmotic pressure of 13–17 cmH2O at the interface. Since the infusate's osmotic pressure was only 0.9 cmH2O, this implied concentration polarization to 15–18 g l−1 hyaluronan at the interface.Mechanical perforation of the lining, or enzymatic degradation of the interstitial matrix by chymopapain, abolished the quasi-plateau. Hydrational expansion of the matrix by /2-fold did not. The increased opposition to outflow was reversible by washing out the hyaluronan, or by reducing Pj. It was unaffected by interruption of tissue blood flow or synoviocyte oxidative metabolism. These properties are compatible with a concentration polarization mechanism, i.e. flow-induced concentration of hyaluronan at the synovial interface due to molecular reflection.A concentration polarization theory was developed for a partially reflected solute. Numerical solutions supported the feasibility of this osmotic explanation of the quasi-plateau. Additional mechanisms may also be involved.It is concluded that native-size hyaluronan helps to retain synovial fluid in the joint cavity when pressure is raised and acts, at least in part, by exerting osmotic pressure at the interface between synovial matrix and a concentration polarization layer. PMID:9831732

Inhibition of Ovarian Cancer Chemoresistance and Metastasis with Antagonists of Hyaluronan-CD44-CD147 Interactions

DTIC Science & Technology

2014-07-01

attached but most leucocytes did not. The attached cells were washed and used for FACS analysis as described previously (Slomiany et al., 2009b...SKOV3 cells, Hey-A8-MDR cells and ascites-derived cells were subject to FACS analysis in the same manner, as follows. We first separated live cells...Cancer 11, 254-267. Zucker, S., Hymowitz, M., Rollo, E. E., Mann, R., Conner, C. E., Cao, J., Foda , H. D., Tompkins, D. C. and Toole, B. P. (2001

Hyaluronan Tumor Cell Interactions in Prostate Cancer Growth and Survival

DTIC Science & Technology

2009-12-01

is a high molecular weight polyanionic polysaccharide that is increased in more advanced prostate cancers. Tumor cell interaction with this... polysaccharide by specific receptors CD44 and RHAMM promote tumor growth, survival and invasion. Work during the last funding period have further defined the... polysaccharide to its cognate receptors. These peptides inhibit tumor growth both in vitro and in vivo and the residues important for the activity

Hyaluronan-Based Therapy for Metastatic Prostate Cancer

DTIC Science & Technology

2015-07-01

8217-AAG ACC CTT GTG CTC GTT GT-3’ and re- verse 5’-AGG TGG ACA CAA TCC CTC TG-3’ actin, forward 5’-TCC CTG GAG AAG AGC TAC GA-3’ and reverse 5’-AGC ACT... GTG TTG GCG TAC AG-3’. 2.2.5. Death-inducing signaling complex immunoprecipitation After HT-29 cells achieved 80% confluence, the cells were pretreated

Hyaluronan Induces the Selective Accumulation of Matrix- and Cell-Associated Proteoglycans by Mesangial Cells

PubMed Central

Kastner, Sabine; Thomas, Gareth J.; Jenkins, Robert H.; Davies, Malcolm; Steadman, Robert

2007-01-01

Mesangial cells (MCs) are essential for normal renal function through the synthesis of their own extracellular matrix, which forms the structural support of the renal glomerulus. In many renal diseases this matrix is reorganized in response to a variety of cytokines and growth factors. This study examines proteoglycan and hyaluronan (HA) synthesis by MCs triggered by proinflammatory agents and investigates the effect of an exogenous HA matrix on matrix synthesis by MCs. Metabolic labeling, ion exchange and size exclusion chromatography, Western blotting, and immunocytochemistry were used to identify changes in matrix accumulation. When incubated with interleukin-1, platelet-derived growth factor, or fetal calf serum, MCs initiated rapid HA synthesis associated with the up-regulation of HA synthase-2 and increased the synthesis of versican, perlecan, and decorin/biglycan. HA was both released into the medium and incorporated into extensive pericellular coats. Adding exogenous HA to unstimulated cells that had undetectable pericellular coats of HA selectively reduced perlecan and versican turnover, whereas other proteoglycans were unaffected. These results suggest that high levels of HA in the mesangium in disease is a mechanism controlling the accumulation of specific mesangial matrix components. HA may thus be an attractive target for therapeutic intervention. PMID:17974600

Inhibition of Ovarian Cancer Chemoresistance and Metastasis with Antagonists of Hyaluronan-CD44-CD147 Interactions

DTIC Science & Technology

2015-09-01

malignant and drug- resistant properties. This most likely occurs through assembly and/or stabilization of plasma membrane signaling complexes ...interactions of hyaluronan polymer with CD44 are necessary for stabilizing CD44-CD147 signaling complexes , and that small, monovalent, hyaluronan...transporter complexes (Ghatak et al., 2005; Grass et al., 2012; Grass et al., 2013; Qin et al., 2011; Slomiany et al., 2009a; Slomiany et al., 2009b

The molecular basis of the solution properties of hyaluronan investigated by confocal fluorescence recovery after photobleaching.

PubMed Central

Gribbon, P; Heng, B C; Hardingham, T E

1999-01-01

Hyaluronan (HA) is a highly hydrated polyanion, which is a network-forming and space-filling component in the extracellular matrix of animal tissues. Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to investigate intramolecular hydrogen bonding and electrostatic interactions in hyaluronan solutions. Self and tracer lateral diffusion coefficients within hyaluronan solutions were measured over a wide range of concentrations (c), with varying electrolyte and at neutral and alkaline pH. The free diffusion coefficient of fluoresceinamine-labeled HA of 500 kDa in PBS was 7.9 x 10(-8) cm(2) s(-1) and of 830 kDa HA was 5.6 x 10(-8) cm(2) s(-1). Reductions in self- and tracer-diffusion with c followed a stretched exponential model. Electrolyte-induced polyanion coil contraction and destiffening resulted in a 2.8-fold increase in self-diffusion between 0 and 100 mM NaCl. Disruption of hydrogen bonds by strong alkali (0.5 M NaOH) resulted in further larger increases in self- and tracer-diffusion coefficients, consistent with a more dynamic and permeable network. Concentrated hyaluronan solution properties were attributed to hydrodynamic and entanglement interactions between domains. There was no evidence of chain-chain associations. At physiological electrolyte concentration and pH, the greatest contribution to the intrinsic stiffness of hyaluronan appeared to be due to hydrogen bonds between adjacent saccharides. PMID:10512840

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

Self-assembled adult adipose-derived stem cell spheroids combined with biomaterials promote wound healing in a rat skin repair model.

PubMed

Hsu, Shan-Hui; Hsieh, Pai-Shan

2015-01-01

Adult adipose-derived stem cells (ASCs) are a type of multipotent mesenchymal stem cells (MSCs) with easy availability and serve as a potential cell source for cell-based therapy. Three-dimensional MSC spheroids may be derived from the self-assembly of individual MSCs grown on certain polymer membranes. In this study, we demonstrated that the self-assembled ASC spheroids on chitosan-hyaluronan membranes expressed more cytokine genes (fibroblast growth factor 1, vascular endothelial growth factor, and chemokine [C-C motif] ligand 2) as well as migration-associated genes (chemokine [C-X-C motif] receptor type 4 and matrix metalloprotease 1) compared with ASC dispersed single cells grown on culture dish. To evaluate the in vivo effects of these spheroids, we applied ASC single cells and ASC spheroids in a designed rat skin repair model. Wounds of 15 × 15 mm were created on rat dorsal skin, where ASCs were administered and covered with hyaluronan gel/chitosan sponge to maintain a moist environment. Results showed that skin wounds treated with ASC spheroids had faster wound closure and a significantly higher ratio of angiogenesis. Tracking of fluorescently labeled ASCs showed close localization of ASC spheroids to microvessels, suggesting enhanced angiogenesis through paracrine effects. Based on the in vitro and in vivo results, the self-assembled ASC spheroids may be a promising cellular source for skin tissue engineering and wound regeneration. © 2014 by the Wound Healing Society.

Lubricin is Required for the Structural Integrity and Post-natal Maintenance of TMJ

PubMed Central

Koyama, E.; Saunders, C.; Salhab, I.; Decker, R.S.; Chen, I.; Um, H.; Pacifici, M.; Nah, H.D.

2014-01-01

The Proteoglycan 4 (Prg4) product lubricin plays essential roles in boundary lubrication and movement in limb synovial joints, but its roles in temporomandibular joint (TMJ) are unclear. Thus, we characterized the TMJ phenotype in wild-type and Prg4 –/– mouse littermates over age. As early as 2 weeks of age, mutant mice exhibited hyperplasia in the glenoid fossa articular cartilage, articular disc, and synovial membrane. By 1 month of age, there were fewer condylar superficial tenascin-C/Col1-positive cells and more numerous apoptotic condylar apical cells, while chondroprogenitors displayed higher mitotic activity, and Sox9-, Col2-, and ColX-expressing chondrocyte zones were significantly expanded. Mutant subchondral bone contained numerous Catepsin K- expressing osteoclasts at the chondro-osseous junction, increased invasive marrow cavities, and suboptimal subchondral bone. Mutant glenoid fossa, disc, synovial cells, and condyles displayed higher Hyaluronan synthase 2 expression. Mutant discs also lost their characteristic concave shape, exhibited ectopic chondrocyte differentiation, and occasionally adhered to condylar surfaces. A fibrinoid substance of unclear origin often covered the condylar surface. By 6 months of age, mutant condyles displayed osteoarthritic degradation with apical/mid-zone separation. In sum, lubricin exerts multiple essential direct and indirect roles to preserve TMJ structural and cellular integrity over post-natal life. PMID:24834922

Low-molecular-weight hyaluronan (LMW-HA) accelerates lymph node metastasis of melanoma cells by inducing disruption of lymphatic intercellular adhesion.

PubMed

Du, Yan; Cao, Manlin; Liu, Yiwen; He, Yiqing; Yang, Cuixia; Wu, Man; Zhang, Guoliang; Gao, Feng

2016-01-01

Endothelial integrity defects initiate lymphatic metastasis of tumor cells. Low-molecular-weight hyaluronan (LMW-HA) derived from plasma and interstitial fluid was reported to be associated with tumor lymphatic metastasis. In addition, LMW-HA was proved to disrupt lymphatic vessel endothelium integrity, thus promoting lymphatic metastasis of tumor cells. Until now, there are few reports on how LMW-HA modulates lymphatic endothelial cells adhesion junctions and affects cancer cells metastasizing into lymph vessels. The aim of our study is to unravel the novel mechanism of LMW-HA in mediating tumor lymphatic metastasis. Here, we employed a melanoma metastasis model to investigate whether LMW-HA facilitates tumor cells transferring from foci to remote lymph nodes by disrupting the lymphatic endothelial integrity. Our data indicate that LMW-HA significantly induces metastasis of melanoma cells to lymph nodes and accelerates interstitial-lymphatic flow in vivo . Further experiments show that increased migration of melanoma cells across human dermal lymphatic endothelial cell (HDLEC) monolayers is accompanied by impaired lymphatic endothelial barrier function and increased permeability. The mechanism study reveals that VE-cadherin-β-catenin pathway and relevant signals are involved in modulating the interactions between endothelial cells and that a significant inhibition of lymphatic endothelium disruption is observed when antibodies to the LMW-HA receptor (LYVE-1) are present. Thus, our findings demonstrate a disruptive effect of LMW-HA on lymphatic endothelium continuity which leads to a promotion on melanoma lymphatic metastasis and also suggest a cellular signaling mechanism associated with VE-cadherin-mediated lymphatic intercellular junctions.

Acceleration of Ligament Healing with Cellular Attractants

DTIC Science & Technology

2008-07-01

major cause of morbidity in the armed forces. type VI collagen is a haptotactic cell attractant. We have shown that type VI collagen with bound...heparin/FGF-2 or hyaluronan or fibronectin promotes migration of canine ACL and DET cells. Insertion of type VI collagen into a wound in the canine...1984). Type I collagen is known to be the predominant fibrillar collagen in the meniscus. Smaller amounts of type II collagen are also present. In

Hyaluronan Production by Renomedullary Interstitial Cells: Influence of Endothelin, Angiotensin II and Vasopressin

PubMed Central

Palm, Fredrik; Takahashi, Tomoko; Ikegami-Kawai, Mayumi; Friederich-Persson, Malou; Hansell, Peter

2017-01-01

The content of hyaluronan (HA) in the interstitium of the renal medulla changes in relation to body hydration status. We investigated if hormones of central importance for body fluid homeostasis affect HA production by renomedullary interstitial cells in culture (RMICs). Simultaneous treatment with vasopressin and angiotensin II (Ang II) reduced HA by 69%. No change occurred in the mRNA expressions of hyaluronan synthase 2 (HAS2) or hyaluronidases (Hyals), while Hyal activity in the supernatant increased by 67% and CD44 expression reduced by 42%. The autocoid endothelin (ET-1) at low concentrations (10−10 and 10−8 M) increased HA 3-fold. On the contrary, at a high concentration (10−6 M) ET-1 reduced HA by 47%. The ET-A receptor antagonist BQ123 not only reversed the reducing effect of high ET-1 on HA, but elevated it to the same level as low concentration ET-1, suggesting separate regulating roles for ET-A and ET-B receptors. This was corroborated by the addition of ET-B receptor antagonist BQ788 to low concentration ET-1, which abolished the HA increase. HAS2 and Hyal2 mRNA did not alter, while Hyal1 mRNA was increased at all ET-1 concentrations tested. Hyal activity was elevated the most by high ET-1 concentration, and blockade of ET-A receptors by BQ123 prevented about 30% of this response. The present study demonstrates an important regulatory influence of hormones involved in body fluid balance on HA handling by RMICs, thereby supporting the concept of a dynamic involvement of interstitial HA in renal fluid handling. PMID:29236055

Local delivery of hyaluronan as an adjunct to scaling and root planing in the treatment of chronic periodontitis.

PubMed

Johannsen, Annsofi; Tellefsen, Monica; Wikesjö, Ulf; Johannsen, Gunnar

2009-09-01

The aim of the present study was to evaluate the adjunctive effect of the local application of a hyaluronan gel to scaling and root planing in the treatment of chronic periodontitis. Twelve patients with chronic periodontitis were recruited to participate in a study with a split-mouth design and provided informed consent. Plaque formation and bleeding on probing were evaluated pretreatment (baseline) and at 1, 4, and 12 weeks post-treatment. Probing depths and attachment levels were evaluated at baseline and at 12 weeks. The patients received full-mouth scaling and root planing. A hyaluronan gel was administered subgingivally in the test sites at baseline and after 1 week. Significant differences between test and control were evaluated using the paired t test, repeated-measures analysis of variance (Wilks lambda), and a non-parametric Wilcoxon signed-rank test. A significant reduction in bleeding on probing scores and probing depths was observed in both groups at 12 weeks (P <0.05). Significantly lower bleeding on probing scores were observed in the hyaluronan group compared to control at 12 weeks (P <0.05). Mean probing depth reductions between baseline and 12 weeks were 1.0 +/- 0.3 mm and 0.8 +/- 0.2 mm for the hyaluronan and control groups, respectively. The difference between the groups was statistically significant (P <0.05). The local application of hyaluronan gel in conjunction with scaling and root planing may have a beneficial effect on periodontal health in patients with chronic periodontitis.

Effect of long-term culture of mouse embryonic stem cells under low oxygen concentration as well as on glycosaminoglycan hyaluronan on cell proliferation and differentiation.

PubMed

Ramírez, M Á; Pericuesta, E; Yáñez-Mó, M; Palasz, A; Gutiérrez-Adán, A

2011-02-01

Maintaining undifferentiated stem cells in defined conditions is of critical importance to improve their in vitro culture. We have evaluated the effects of culturing mouse stem (mES) cells under physiological oxygen concentration as well as by replacing fibroblast feeder layer (mEF) with gelatin or glycosaminoglycan hyaluronan (HA), on cell proliferation and differentiation. After 3 days culture or after long-term cell culture under different conditions, levels of apoptotic cell death were determined by cell cycle and TUNEL (TdT-mediated dUTP nick end labelling) assays and levels of cell proliferation by CFSE (5-(and-6)-carboxyfluorescein diacetate succinimidyl ester) labelling. We assessed spontaneous differentiation into cardiomyocytes and mRNA expression of pluripotency and differentiation biomarkers. After 3 days culture under hypoxic conditions, levels of proliferation and apoptosis of mES cells were higher, in correlation with increase in intracellular reactive oxygen species. However, when cells were continuously grown for 1 month under those conditions, the level of apoptosis was, in all cases, under 4%. Hypoxia reduced spontaneous differentiation of mES into cardiomyocytes. Long-term culture on HA was more effective in maintaining the pluripotent state of the mES cells when compared to that on gelatin. Level of terminal differentiation was highest on mEF, intermediate on HA and lowest on gelatin. Our data suggest that hypoxia is not necessary for maintaining pluripotency of mES cells and appeared to be detrimental during ES differentiation. Moreover, HA may offer a valuable alternative for long-term culture of mES cells in vitro. © 2010 Blackwell Publishing Ltd.

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-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. Electronic supplementary information (ESI) available: Synthetic and experimental procedures. See DOI: 10.1039/c4nr06910b

A Modified ELISA Accurately Measures Secretion of High Molecular Weight Hyaluronan (HA) by Graves' Disease Orbital Cells

PubMed Central

Krieger, Christine C.

2014-01-01

Excess production of hyaluronan (hyaluronic acid [HA]) in the retro-orbital space is a major component of Graves' ophthalmopathy, and regulation of HA production by orbital cells is a major research area. In most previous studies, HA was measured by ELISAs that used HA-binding proteins for detection and rooster comb HA as standards. We show that the binding efficiency of HA-binding protein in the ELISA is a function of HA polymer size. Using gel electrophoresis, we show that HA secreted from orbital cells is primarily comprised of polymers more than 500 000. We modified a commercially available ELISA by using 1 million molecular weight HA as standard to accurately measure HA of this size. We demonstrated that IL-1β-stimulated HA secretion is at least 2-fold greater than previously reported, and activation of the TSH receptor by an activating antibody M22 from a patient with Graves' disease led to more than 3-fold increase in HA production in both fibroblasts/preadipocytes and adipocytes. These effects were not consistently detected with the commercial ELISA using rooster comb HA as standard and suggest that fibroblasts/preadipocytes may play a more prominent role in HA remodeling in Graves' ophthalmopathy than previously appreciated. PMID:24302624

Hyaluronan Tumor Cell Interactions in Prostate Cancer Growth and Survival

DTIC Science & Technology

2006-12-01

prognostic value of CD44 standard and variant v3 and v6 isoforms in prostate cancer. Eur Urol, 2001. 39(2): p. 138-44. 32. De Marzo , A.M., et al., CD44...subcutaneous injection model [ 24 ]and in orthotopic or intrafemoral bone injection models (see progress report below). Importantly, the addition of...expression from these cells, completely reverses growth inhibition[ 24 ]. CD44 and Rhamm – Two Hyaladherins with Overlapping Function: The two most

Inhibition of hyaluronan synthesis by vesnarinone in cultured human myofibroblasts.

PubMed

Ueki, N; Taguchi, T; Takahashi, M; Adachi, M; Ohkawa, T; Amuro, Y; Hada, T; Higashino, K

2000-02-02

Hyaluronan (HA), which is a major component of the extracellular matrix (ECM), is regulated during myofibroproliferative responses to numerous forms of inflammatory stimuli. It is a key factor involved in cellular migration and adherence. The development of a potent and non-toxic inhibitor of HA synthesis would open up a new avenue for the treatment of fibrocontractive diseases such as pulmonary fibrosis and liver cirrhosis. In this study, the effects of vesnarinone (OPC-8212: 3,4-dihydro-6-[4-(3, 4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone) on the secretion of HA in human myofibroblast cell lines (MRC-5 and LI90 cells, referred to as pulmonary and hepatic myofibroblasts, respectively) were examined. Vesnarinone specifically and dose-dependently inhibited HA secretion by myofibroblasts up-regulated by fetal calf serum (FCS). The treatment of vesnarinone did not modify the phenotype of myofibroblast cells in culture. Vesnarinone also potently inhibited the HA secretion by the two myofibroblast cell lines up-regulated by transforming growth factor-beta1 (TGF-beta1) or tumor necrosis factor-alpha (TNF-alpha). The addition of vesnarinone to myofibroblasts resulted in a significant decrease of HA synthase (HAS) activity, with or without the addition of FCS or either cytokine. These findings suggest that vesnarinone inhibits the secretion of HA in myofibroblasts by specifically suppressing HAS activity, and may therefore prove useful for the treatment of chronic inflammation and tissue fibrosis.

Polyglycolic acid-hyaluronan scaffolds loaded with bone marrow-derived mesenchymal stem cells show chondrogenic differentiation in vitro and cartilage repair in the rabbit model.

PubMed

Patrascu, Jenel M; Krüger, Jan Philipp; Böss, Hademar G; Ketzmar, Anna-Katharina; Freymann, Undine; Sittinger, Michael; Notter, Michael; Endres, Michaela; Kaps, Christian

2013-10-01

In cartilage repair, scaffold-assisted one-step approaches are used to improve the microfracture (Mfx) technique. Since the number of progenitors in Mfx is low and may further decrease with age, aim of our study was to analyze the chondrogenic potential of freeze-dried polyglycolic acid-hyaluronan (PGA-HA) implants preloaded with mesenchymal stem cells (MSCs) in vitro and in a rabbit articular cartilage defect model. Human bone marrow-derived MSC from iliac crest were cultured in freeze-dried PGA-HA implants for chondrogenic differentiation. In a pilot study, implants were loaded with autologous rabbit MSC and used to cover 5 mm × 6 mm full-thickness femoral articular cartilage defects (n = 4). Untreated defects (n = 3) served as controls. Gene expression analysis and histology showed induction of typical chondrogenic marker genes like type II collagen and formation of hyaline-like cartilaginous tissue in MSC-laden PGA-HA implants. Histological evaluation of rabbit repair tissue formation after 30 and 45 days showed formation of repair tissue, rich in chondrocytic cells and of a hyaline-like appearance. Controls showed no articular resurfacing, tissue repair in the subchondral zone and fibrin formation. These results suggest that MSC-laden PGA-HA scaffolds have chondrogenic potential and are a promising option for stem cell-mediated cartilage regeneration. Copyright © 2013 Wiley Periodicals, Inc.

Hyaluronan synthase assembles hyaluronan on a [GlcNAc(β1,4)]n-GlcNAc(α1→)UDP primer and hyaluronan retains this residual chitin oligomer as a cap at the nonreducing end

PubMed Central

Baggenstoss, Bruce A; Washburn, Jennifer L

2017-01-01

Abstract Class I hyaluronan synthases (HAS) assemble [GlcNAc(β1,4)GlcUA(β1,3)]n-UDP at the reducing end and also make chitin. Streptococcus equisimilis HAS (SeHAS) also synthesizes chitin-UDP oligosaccharides, (GlcNAc-β1,4)n-GlcNAc(α1→)UDP (Weigel et al. 2015). Here we determined if HAS uses chitin-UDPs as primers to initiate HA synthesis, leaving the non-HA primer at the nonreducing (NR) end. HA made by SeHAS membranes was purified, digested with streptomyces lyase, and hydrophobic oligomers were enriched by solid phase extraction and analyzed by MALDI-TOF MS. Jack bean hexosaminidase (JBH) and MS/MS were used to analyze 19 m/z species of possible GnHn ions with clustered GlcNAc (G) residues attached to disaccharide units (H): (GlcNAcβ1,4)2–5[GlcUA(β1,3)GlcNAc]2–6. JBH digestion sequentially removed GlcNAc from the NR-end of GnHn oligomers, producing successively smaller GnH2–3 series members. Since lyase releases dehydro-oligos (dHn; M−18), only the unique NR-end oligo lacks dehydro-GlcUA. Hn oligomers were undetectable in lyase digests, whereas JBH treatment created new H2–6m/z peaks (i.e. HA tetra- through dodeca-oligomers). MS/MS of larger GnHn species produced chitin (2–5 GlcNAcs), HA oligomers and multiple smaller series members with fewer GlcNAcs. All NR-ends (97%) started with GlcNAc, as a chitin trimer (three GlcNAcs), indicating that GlcNAc(β1,4)2GlcNAc(α1→)-UDP may be optimal for initiation of HA synthesis. Also, HA made by live S. pyogenes cells had G4Hn chitin-oligo NR-ends. We conclude that chitin-UDP functions in vitro and in live cells as a primer to initiate synthesis of all HA chains and these primers remain at the NR-ends of HA chains as residual chitin caps [(GlcNAc-β1,4)3–4]. PMID:28138013

Hyaluronan synthase assembles hyaluronan on a [GlcNAc(β1,4)]n-GlcNAc(α1→)UDP primer and hyaluronan retains this residual chitin oligomer as a cap at the nonreducing end.

PubMed

Weigel, Paul H; Baggenstoss, Bruce A; Washburn, Jennifer L

2017-06-01

Class I hyaluronan synthases (HAS) assemble [GlcNAc(β1,4)GlcUA(β1,3)]n-UDP at the reducing end and also make chitin. Streptococcus equisimilis HAS (SeHAS) also synthesizes chitin-UDP oligosaccharides, (GlcNAc-β1,4)n-GlcNAc(α1→)UDP (Weigel et al. 2015). Here we determined if HAS uses chitin-UDPs as primers to initiate HA synthesis, leaving the non-HA primer at the nonreducing (NR) end. HA made by SeHAS membranes was purified, digested with streptomyces lyase, and hydrophobic oligomers were enriched by solid phase extraction and analyzed by MALDI-TOF MS. Jack bean hexosaminidase (JBH) and MS/MS were used to analyze 19 m/z species of possible GnHn ions with clustered GlcNAc (G) residues attached to disaccharide units (H): (GlcNAcβ1,4)2-5[GlcUA(β1,3)GlcNAc]2-6. JBH digestion sequentially removed GlcNAc from the NR-end of GnHn oligomers, producing successively smaller GnH2-3 series members. Since lyase releases dehydro-oligos (dHn; M-18), only the unique NR-end oligo lacks dehydro-GlcUA. Hn oligomers were undetectable in lyase digests, whereas JBH treatment created new H2-6m/z peaks (i.e. HA tetra- through dodeca-oligomers). MS/MS of larger GnHn species produced chitin (2-5 GlcNAcs), HA oligomers and multiple smaller series members with fewer GlcNAcs. All NR-ends (97%) started with GlcNAc, as a chitin trimer (three GlcNAcs), indicating that GlcNAc(β1,4)2GlcNAc(α1→)-UDP may be optimal for initiation of HA synthesis. Also, HA made by live S. pyogenes cells had G4Hn chitin-oligo NR-ends. We conclude that chitin-UDP functions in vitro and in live cells as a primer to initiate synthesis of all HA chains and these primers remain at the NR-ends of HA chains as residual chitin caps [(GlcNAc-β1,4)3-4]. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Role of Hyaluronan in Schwannoma Growth

DTIC Science & Technology

2008-06-01

schwannomatosis . Although schwannomas occur due to mutations in the neurofibromatosis 2 gene, which encodes the merlin tumor suppressor protein, recent...pre-disposition syndromes neurofibromatosis 2 (NF2) and schwannomatosis . We and others found that the glycosaminoglycan hyaluronan (HA) is present

Ultra-low friction between boundary layers of hyaluronan-phosphatidylcholine complexes.

PubMed

Zhu, Linyi; Seror, Jasmine; Day, Anthony J; Kampf, Nir; Klein, Jacob

2017-09-01

The boundary layers coating articular cartilage in synovial joints constitute unique biomaterials, providing lubricity at levels unmatched by any human-made materials. The underlying molecular mechanism of this lubricity, essential to joint function, is not well understood. Here we study the interactions between surfaces bearing attached hyaluronan (hyaluronic acid, or HA) to which different phosphatidylcholine (PC) lipids had been added, in the form of small unilamellar vesicles (SUVs or liposomes), using a surface force balance, to shed light on possible cartilage boundary lubrication by such complexes. Surface-attached HA was complexed with different PC lipids (hydrogenated soy PC (HSPC), 1,2-dimyristoyl-sn-glycero-3-PC (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-PC (POPC)), followed by rinsing. Atomic force microscopy (AFM) and cryo-scanning electron microscopy (Cryo-SEM) were used to image the HA-PC surface complexes following addition of the SUVs. HA-HSPC complexes provide very efficient lubrication, with friction coefficients as low as μ∼0.001 at physiological pressures P≈150atm, while HA-DMPC and HA-POPC complexes are efficient only at low P (up to 10-20atm). The friction reduction in all cases is attributed to hydration lubrication by highly-hydrated phosphocholine groups exposed by the PC-HA complexes. The greater robustness at high P of the HSPC (C 16(15%) ,C 18(85%) ) complexes relative to the DMPC ((C 14 ) 2 ) or POPC (C 16 , C 18:1 ) complexes is attributed to the stronger van der Waals attraction between the HSPC acyl tails, relative to the shorter or un-saturated tails of the other two lipids. Our results shed light on possible lubrication mechanisms at the articular cartilage surface in joints. Can designed biomaterials emulate the unique lubrication ability of articular cartilage, and thus provide potential alleviation to friction-related joint diseases? This is the motivation behind the present study. The principles of cartilage lubrication have attracted considerable attention for decades, and several models have been proposed to elucidate it, however, the mechanism of this ultralow friction is still not clear. In this paper we explore the recent suggestion that its efficient lubrication arises from boundary layers of hyaluronan-lipid complexes at its surface, in particular exploring a range of different phosphatidylcholines (PCs) mimicking the wide range of PCs in synovial joints. The present study suggests a synergistic lubricating behavior of the different lipids in living joints, and potential treatment directions using such biomaterial complexes for widespread cartilage-friction-related diseases such as osteoarthritis. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Biguanides Metformin and Phenformin Generate Therapeutic Effects via AMP-Activated Protein Kinase/Extracellular-Regulated Kinase Pathways in an In Vitro Model of Graves' Orbitopathy.

PubMed

Han, Ye Eon; Hwang, Sena; Kim, Jin Hee; Byun, Jung Woo; Yoon, Jin Sook; Lee, Eun Jig

2018-04-01

It was hypothesized that the biguanides metformin and phenformin, which are anti-hyperglycemic drugs used for diabetes mellitus, would have therapeutic effects in an in vitro model of Graves' orbitopathy (GO). Because adipogenesis, hyaluronan production, and inflammation are considered important in the pathogenesis of GO, this study aimed to determine the therapeutic effects and underlying mechanisms of biguanides on these parameters. In vitro experiments were performed using primary cultured orbital fibroblasts from patients with GO. Orbital preadipocyte fibroblasts were allowed to differentiate into adipocytes and were treated with various concentrations of metformin or phenformin. Oil Red O staining was performed to evaluate lipid accumulation within the cells. Western blot analysis was used to measure the expression of adipogenic transcription factors and the phosphorylation of AMP-activated protein kinase and mitogen-activated protein kinase signaling proteins. Hyaluronan production was measured using enzyme-linked immunosorbent assay, and mRNA levels of proinflammatory molecules were determined using real-time polymerase chain reaction after interleukin (IL)-1β stimulation with or without biguanide treatment. Lipid accumulation during adipogenesis in GO orbital fibroblasts was dose-dependently suppressed by both metformin and phenformin. Adipocyte differentiation was attenuated, and the adipogenic transcription factors peroxisome proliferator-activated receptor γ and CCAAT-enhancer-binding proteins-α/β were downregulated. Furthermore, metformin and phenformin increased the phosphorylation of AMP-activated protein kinase and suppressed extracellular-regulated kinase activation. The IL-1β-induced hyaluronan production and mRNA expression of IL-6, cyclooxygenase-2, and intercellular adhesion molecule-1 were also significantly suppressed after metformin or phenformin co-treatment. The present study indicates that the biguanides metformin and phenformin exert an anti-adipogenic and inhibitory effect on hyaluronan production and expression of pro-inflammatory molecules in GO orbital fibroblasts, suggesting that they could potentially be used for the treatment of GO.

Acquisition of epithelial-mesenchymal transition and cancer stem-like phenotypes within chitosan-hyaluronan membrane-derived 3D tumor spheroids.

PubMed

Huang, Yen-Jang; Hsu, Shan-Hui

2014-12-01

Cancer drug development has to go through rigorous testing and evaluation processes during pre-clinical in vitro studies. However, the conventional two-dimensional (2D) in vitro culture is often discounted by the insufficiency to present a more typical tumor microenvironment. The multicellular tumor spheroids have been a valuable model to provide more comprehensive assessment of tumor in response to therapeutic strategies. Here, we applied chitosan-hyaluronan (HA) membranes as a platform to promote three-dimensional (3D) tumor spheroid formation. The biological features of tumor spheroids of human non-small cell lung cancer (NSCLC) cells on chitosan-HA membranes were compared to those of 2D cultured cells in vitro. The cells in tumor spheroids cultured on chitosan-HA membranes showed higher levels of stem-like properties and epithelial-mesenchymal transition (EMT) markers, such as NANOG, SOX2, CD44, CD133, N-cadherin, and vimentin, than 2D cultured cells. Moreover, they exhibited enhanced invasive activities and multidrug resistance by the upregulation of MMP2, MMP9, BCRC5, BCL2, MDR1, and ABCG2 as compared with 2D cultured cells. The grafting densities of HA affected the tumor sphere size and mRNA levels of genes on the substrates. These evidences suggest that chitosan-HA membranes may offer a simple and valuable biomaterial platform for rapid generation of tumor spheroids in vitro as well as for further applications in cancer stem cell research and cancer drug screening. Copyright © 2014 Elsevier Ltd. All rights reserved.

BMP-2 Induces Versican and Hyaluronan That Contribute to Post-EMT AV Cushion Cell Migration

PubMed Central

Inai, Kei; Burnside, Jessica L.; Hoffman, Stanley; Toole, Bryan P.; Sugi, Yukiko

2013-01-01

Distal outgrowth and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT atrioventricular (AV) valvuloseptal morphogenesis. We explored the role of BMP-2 in the regulation of valvulogenic extracellular matrix (ECM) components, versican and hyaluronan (HA), and cell migration during post-EMT AV cushion distal outgrowth/expansion. We observed intense staining of versican and HA in AV cushion mesenchyme from the early cushion expansion stage, Hamburger and Hamilton (HH) stage-17 to the cushion maturation stage, HH stage-29 in the chick. Based on this expression pattern we examined the role of BMP-2 in regulating versican and HA using 3D AV cushion mesenchymal cell (CMC) aggregate cultures on hydrated collagen gels. BMP-2 induced versican expression and HA deposition as well as mRNA expression of versican and Has2 by CMCs in a dose dependent manner. Noggin, an antagonist of BMP, abolished BMP-2-induced versican and HA as well as mRNA expression of versican and Has2. We further examined whether BMP-2-promoted cell migration was associated with expression of versican and HA. BMP-2- promoted cell migration was significantly impaired by treatments with versican siRNA and HA oligomer. In conclusion, we provide evidence that BMP-2 induces expression of versican and HA by AV CMCs and that these ECM components contribute to BMP-2-induced CMC migration, indicating critical roles for BMP-2 in distal outgrowth/expansion of mesenchymalized AV cushions. PMID:24147033

Quantification of hyaluronan (HA) using a simplified fluorophore-assisted carbohydrate electrophoresis (FACE) procedure.

PubMed

Midura, Ronald J; Cali, Valbona; Lauer, Mark E; Calabro, Anthony; Hascall, Vincent C

2018-01-01

Hyaluronan (HA) exhibits numerous important roles in physiology and pathologies, and these facts necessitate an ability to accurately and reproducibly measure its quantities in tissues and cell cultures. Our group previously reported a rigorous and analytical procedure to quantify HA (and chondroitin sulfate, CS) using a reductive amination chemistry and separation of the fluorophore-conjugated, unsaturated disaccharides unique to HA and CS on high concentration acrylamide gels. This procedure is known as fluorophore-assisted carbohydrate electrophoresis (FACE) and has been adapted for the detection and quantification of all glycosaminoglycan types. While this previous FACE procedure is relatively straightforward to implement by carbohydrate research investigators, many nonglycoscience laboratories now studying HA biology might have difficulties establishing this prior FACE procedure as a routine assay for HA. To address this need, we have greatly simplified our prior FACE procedure for accurate and reproducible assessment of HA in tissues and cell cultures. This chapter describes in detail this simplified FACE procedure and, because it uses an enzyme that degrades both HA and CS, investigators will also gain additional insight into the quantities of CS in the same samples dedicated for HA analysis. © 2018 Elsevier Inc. All rights reserved.

Biomechanical and histologic evaluation of tissue engineered ligaments using chitosan and hyaluronan hybrid polymer fibers: a rabbit medial collateral ligament reconstruction model.

PubMed

Irie, Toru; Majima, Tokifumi; Sawaguchi, Naohiro; Funakoshi, Tadanao; Nishimura, Shin-Ichiro; Minami, Akio

2011-05-01

In this study, we used a rabbit medial collateral ligament reconstruction model to evaluate a novel chitosan-based hyaluronan hybrid polymer fiber scaffold for ligament tissue engineering and to examine whether mechanical forces exerted in an in vivo model increased extracellular matrix production by seeded fibroblasts. Scaffolds were used 2 weeks after incubation with fibroblasts obtained from the same rabbit in a cell-seeded scaffold (CSS) group and without cells in a noncell-seeded scaffold (NCSS) group. At 3, 6, and 12 weeks after surgery, the failure loads of the engineered ligaments in the CSS groups were significantly greater than those in the NCSS groups. At 6 weeks after surgery, the reconstructed tissue of the CSS group was positive for type I collagen, whereas that in the NCSS group was negative for type I collagen. At 12 weeks after surgery, the reconstructed tissue stained positive for type I collagen in the CSS group, but negative in the NCSS group. Our results indicate that the scaffold material enhanced the production of type I collagen and led to improved mechanical strength in the engineered ligament in vivo. Copyright © 2011 Wiley Periodicals, Inc.

Glucosamine supplementation during late gestation alters placental development and increases litter size

USDA-ARS?s Scientific Manuscript database

Background: During late gestation the placental epithelial interface becomes highly folded, which involves changes in stromal hyaluronan. Hyaluronan is composed of glucoronate and N-acetyl-glucosamine. We hypothesized that supplementing gestating dams with glucosamine during this time would support ...

CYTOKINE - The Official Journal of the International Cytokine Society. Volume 11, Number 11

DTIC Science & Technology

1999-12-09

in the host. Two herpes viruses capable of infecting humans harbor homologs of IL-10. Epstein Barr virus (EBV) encoded IL-10 was the first viral DL...tumor metastasis. We have previously shown that IL4 enhances CD44 expression and hyaluronan (HA) binding ability in an Epstein Barr -vinis (EBV) positive... viruses utilize the same strategy to protect virus -infected cells from immune defenses and thus enhance, survival of virus and virus -infected cells

Yin and Yang of Heparanase in Breast Tumor Initiation

DTIC Science & Technology

2013-04-01

of heparanase in cancer metastasis and angiogenesis. Int J Biochem Cell Biol 38: 2018 -2039, 2006 2. Gotte M, Yip GW: Heparanase, hyaluronan, and...into a RCAS vector digested with a PacI and Cla I. The plasmid was designated RCAS-C. An oligonucleotide containing a Myc tag sequence (atg gaa...ligated into Not I/PacI- digested RCAS-C. The following plasmid designated as RCAS-8C was used to transfected DF-1 cells to generate RCAS-8C virus

Absence of skin rash in Goodpasture's syndrome: the hyaluronan effect.

PubMed

Stern, A; Stern, R

2014-12-01

Goodpasture's syndrome is a rare disease that involves rapidly progressive kidney failure as well as hemorrhagic lung disease. It is a form of autoimmune disorder with unusual features; marked male preponderance, in contrast with other autoimmune disease in which females are at far greater risk. The autoantibodies are directed again the carboxy-extension non-collagenous (NC1) portion of one of the basement membrane-specific collagen IV alpha 3 chains. Basal laminas throughout the body share this structure, including those in kidney, lung and skin. But curiously, skin is rarely involved in Goodpasture's syndrome. Hyaluronan is a large extracellular matrix carbohydrate polymer. Half of total body hyaluronan occurs in skin. High molecular weight hyaluronan, a potent immunosuppressive polymer, might be functioning as an immune shield for skin in Goodpasture's syndrome, and be the basis for the anomaly. A summary of this putative effect is described, including possible molecular mechanisms involved, and suggestions for testing this hypothesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

High molecular weight hyaluronan decreases oxidative DNA damage induced by EDTA in human corneal epithelial cells

PubMed Central

Ye, J; Wu, H; Wu, Y; Wang, C; Zhang, H; Shi, X; Yang, J

2012-01-01

Purpose To investigate the toxic effects of ethylenediaminetetraacetic acid disodium salt (EDTA), a corneal penetration enhancer in topical ophthalmic formulations, on DNA in human corneal epithelial cells (HCEs), and to investigate whether the effect induced by EDTA can be inhibited by high molecular weight hyaluronan (HA). Methods Cells were exposed to EDTA in concentrations ranging from 0.00001 to 0.01% for 60 min, or 30 min high molecular weight HA pretreatment followed by EDTA treatment. The cell viability was measured by the MTT test. Cell apoptosis was determined with annexin V staining by flow cytometry. The DNA single- and double-strand breaks of HCEs were examined by alkaline comet assay and by immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (γH2AX) foci, respectively. Reactive oxygen species (ROS) production was assessed by the fluorescent probe, 2′, 7′-dichlorodihydrofluorescein diacetate. Results EDTA exhibited no adverse effect on cell viability and did not induce cell apoptosis in human corneal epithelial cells at concentrations lower than 0.01%. However, a significant increase of DNA single- and double-strand breaks was observed in a dose-dependent manner with all the concentrations of EDTA tested in HCEs. In addition, EDTA treatment led to elevated ROS generation. Moreover, 30 min preincubation with high molecular weight HA significantly decreased EDTA-induced ROS generation and DNA damage. Conclusions EDTA could induce DNA damage in HCEs, probably through oxidative stress. Furthermore, high molecular weight HA was an effective protective agent that had antioxidant properties and decreased DNA damage induced by EDTA. PMID:22595911

Hyaluronan in aged collagen matrix increases prostate epithelial cell proliferation

PubMed Central

Damodarasamy, Mamatha; Vernon, Robert B.; Chan, Christina K.; Plymate, Stephen R.; Wight, Thomas N.

2015-01-01

The extracellular matrix (ECM) of the prostate, which is comprised primarily of collagen, becomes increasingly disorganized with age, a property that may influence the development of hyperplasia and cancer. Collageous ECM extracted from the tails of aged mice exhibits many characteristics of collagen in aged tissues, including the prostate. When polymerized into a 3-dimensional (3D) gel, these collagen extracts can serve as models for the study of specific cell-ECM interactions. In the present study, we examined the behaviors of human prostatic epithelial cell lines representing normal prostate epithelial cells (PEC), benign prostatic hyperplasia (BPH-1), and adenocarcinoma (LNCaP) cultured in contact with 3D gels made from collagen extracts of young and aged mice. We found that proliferation of PEC, BPH-1, and LNCaP cells were all increased by culture on aged collagen gels relative to young collagen gels. In examining age-associated differences in the composition of the collagen extracts, we found that aged and young collagen had a similar amount of several collagen-associated ECM components, but aged collagen had a much greater content of the glycosaminoglycan hyaluronan (HA) than young collagen. The addition of HA (of similar size and concentration to that found in aged collagen extracts) to cells placed in young collagen elicited significantly increased proliferation in BPH-1 cells, but not in PEC or LNCaP cells, relative to controls not exposed to HA. Of note, histochemical analyses of human prostatic tissues showed significantly higher expression of HA in BPH and prostate cancer stroma relative to stroma of normal prostate. Collectively, these results suggest that changes in ECM involving increased levels of HA contribute to the growth of prostatic epithelium with aging. PMID:25124870

Viscoelastic measurements after vocal fold scarring in rabbits--short-term results after hyaluronan injection.

PubMed

Hertegård, S; Dahlqvist, A; Goodyer, E

2006-07-01

The scarring model resulted in significant damage and elevated viscoelasticity of the lamina propria. Hyaluronan preparations may alter viscoelasticity in scarred rabbit vocal folds. Vocal fold scarring results in stiffness of the lamina propria and severe voice problems. The aims of this study were to examine the degree of scarring achieved in the experiment and to measure the viscoelastic properties after injection of hyaluronan in rabbit vocal folds. Twenty-two vocal folds from 15 New Zealand rabbits were scarred, 8 vocal folds were controls. After 8 weeks 12 of the scarred vocal folds received injections with 2 types of cross-linked hyaluronan products and 10 scarred folds were injected with saline. After 11 more weeks the animals were sacrificed. After dissection, 15 vocal folds were frozen for viscoelastic measurements, whereas 14 vocal folds were prepared and stained. Measurements were made of the lamina propria thickness. Viscoelasticity was measured on intact vocal folds with a linear skin rheometer (LSR) adapted to laryngeal measurements. Measurements on the digitized slides showed a thickened lamina propria in the scarred samples as compared with the normal vocal folds (p<0.05). The viscoelastic analysis showed a tendency to stiffening of the scarred vocal folds as compared with the normal controls (p=0.05). There was large variation in stiffness between the two injected hyaluronan products.

Corneal protection with high-molecular-weight hyaluronan against in vitro and in vivo sodium lauryl sulfate-induced toxic effects.

PubMed

Pauloin, Thierry; Dutot, Mélody; Liang, Hong; Chavinier, Emilie; Warnet, Jean-Michel; Rat, Patrice

2009-10-01

The aim of this study was to investigate high-molecular-weight hyaluronan (HA-HMW) corneal protection against sodium lauryl sulfate (SLS)-induced toxic effects with in vitro and in vivo experimental approaches. In vitro experiments consisted of a human corneal epithelial cell line incubated with HA-HMW, rinsed, and incubated with SLS. Cell viability, oxidative stress, chromatin condensation, caspase-3, -8, -9, and P2X7 cell death receptor activation, interleukin-6, and interleukin-8 production were investigated. In vivo experiments consisted of 36 New Zealand white rabbits treated for 3 days, 3 times per day, with HA-HMW or phosphate-buffered salt solution. At day 4, eyes were treated with SLS. Clinical observation and in vivo confocal microscopy using the Rostock Cornea Module of the Heidelberg Retina Tomograph-II were performed to evaluate and to compare SLS-induced toxicity between eyes treated with HA-HMW and eyes treated with phosphate-buffered salt solution. In vitro data indicate that exposure of human corneal epithelial cells to HA-HMW significantly decreased SLS-induced oxidative stress, apoptosis, and inflammation cytokine production. In vivo data indicate that SLS cornea injuries, characterized by damaged corneal epithelium, damaged anterior stroma, and inflammatory infiltrations, were attenuated with HA-HMW treatment. A good correlation was seen between in vitro and in vivo findings showing that HA-HMW decreases SLS-induced toxic effects and protects cornea.

Low molecular weight hyaluronan induces migration of human choriocarcinoma JEG-3 cells mediated by RHAMM as well as by PI3K and MAPK pathways.

PubMed

Mascaró, Marilina; Pibuel, Matías A; Lompardía, Silvina L; Díaz, Mariangeles; Zotta, Elsa; Bianconi, Maria I; Lago, Néstor; Otero, Silvina; Jankilevich, Gustavo; Alvarez, Elida; Hajos, Silvia E

2017-08-01

Hyaluronan (HA) is the major glycosaminoglycan present in the extracellular matrix. It is produced by some tumours and promotes proliferation, differentiation and migration among others cellular processes. Gestational trophoblastic disease (GTD) is composed by non-tumour entities, such as hydatidiform mole (HM), which is the most common type of GTD and also malignant entities such as choriocarcinoma (CC) and placental site trophoblastic tumour (PSTT), being CC the most aggressive tumour. Although there is a growing understanding of GTD biology, the role of HA in the pathogenesis of this group of diseases remains largely unknown. The aim of this work was to study the role of HA in the pathogenesis of GTD by defining the expression pattern of HA and its receptors CD44 and RHAMM, as well as to determine if HA can modulate proliferation, differentiation and migration of CC cells. Receptors and signalling pathways involved were also analyzed. We demonstrated that HA and RHAMM are differently expressed among GTD entities and even among trophoblast subtypes. We also showed that HA is able to enhance the expression of extravillous trophoblast markers and also to induce migration of JEG-3 cells, the latter mediated by RHAMM as well as PI3K and MAPK pathways. These findings indicate a novel regulatory mechanism for CC cell biology and also contribute to the understanding of GTD pathophysiology.

Ingested hyaluronan moisturizes dry skin.

PubMed

Kawada, Chinatsu; Yoshida, Takushi; Yoshida, Hideto; Matsuoka, Ryosuke; Sakamoto, Wakako; Odanaka, Wataru; Sato, Toshihide; Yamasaki, Takeshi; Kanemitsu, Tomoyuki; Masuda, Yasunobu; Urushibata, Osamu

2014-07-11

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.

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

Absence of differences among low, middle, and high molecular weight hyaluronan in activating murine immune cells in vitro.

PubMed

Šafránková, Barbora; Hermannová, Martina; Nešporová, Kristina; Velebný, Vladimír; Kubala, Lukáš

2018-02-01

Hyaluronan (HA) effects on immune response are suggested to be dependent on HA molecular weight (MW), as low MW HA should activate immune cells in contrast to high MW HA. However, some current studies do not support this conception and emphasize the importance of the form of preparation of HA, particularly with respect to its purity and origin. We compared the activation of mouse immune cells by HA samples (100kDa, 500kDa, and 997kDa) prepared from HA originating from rooster comb, and HA samples (71kDa, 500kDa, and 1000kDa) prepared from pharmacological grade HA originating from Streptococcus equi. Interestingly, in contrast to established theory, only middle and high MW HA originating from rooster comb induced the production of tumor necrosis factor-α by macrophages and in whole blood. Further, all tested preparations of HA failed to induce the expression of inducible nitric oxide synthase, the production of nitric oxide, or the expression of cyclooxygenase 2 in macrophages and splenocytes. Importantly, all HA samples originating from rooster comb were found to be contaminated by endotoxin (up to 1.23EU/ml). Hence, low MW HA did not reveal itself to have significantly higher immunostimulatory activity compared to HA of higher MW. Copyright © 2017 Elsevier B.V. All rights reserved.

LIF inhibits osteoblast differentiation at least in part by regulation of HAS2 and its product hyaluronan.

PubMed

Falconi, Dominic; Aubin, Jane E

2007-08-01

LIF arrests osteogenesis in fetal rat calvaria cells in a differentiation stage-specific manner. Differential display identified HAS2 as a LIF-induced gene and its product, HA, modulated osteoblast differentiation similarly to LIF. Our data suggest that LIF arrests osteoblast differentiation by altering HA content of the extracellular matrix. Leukemia inhibitory factor (LIF) elicits both anabolic and catabolic effects on bone. We previously showed in the fetal rat calvaria (RC) cell system that LIF inhibits osteoblast differentiation at the late osteoprogenitor/early osteoblast stage. To uncover potential molecular mediators of this inhibitory activity, we used a positive-negative genome-wide differential display screen to identify LIF-induced changes in the developing osteoblast transcriptome. Although LIF signaling is active throughout the RC cell proliferation-differentiation sequence, only a relatively small number of genes, in several different functional clusters, are modulated by LIF specifically during the LIF-sensitive inhibitory time window. Based on their known and predicted functions, most of the LIF-regulated genes identified are plausible candidates to be involved in the LIF-induced arrest of osteoprogenitor differentiation. To test this hypothesis, we further analyzed the function of one of the genes identified, hyaluronan synthase 2 (HAS2), in the LIF-induced inhibition. Synthesis of hyaluronan (HA), the product of HAS enzymatic activity, was stimulated by LIF and mimicked the HAS2 expression profile, with highest expression in early/proliferative and late/maturing cultures and lowest levels in intermediate/late osteoprogenitor-early osteoblast cultures. Exogenously added high molecular weight HA, the product of HAS2, dose-dependently inhibited osteoblast differentiation, with pulse-treatment effective in the same differentiation stage-specific inhibitory window as seen with LIF. In addition, however, pulse treatment with HA in early cultures slightly increased bone nodule formation. Treatment with hyaluronidase, on the other hand, stimulated bone nodule formation in early cultures but caused a small dose-dependent inhibition of osteoblast differentiation in the LIF- and HA-sensitive late time window. Together the data suggest that osteoblast differentiation is acutely sensitive to HA levels and that LIF inhibits osteoblast development at least in part by stimulating high molecular weight HA synthesis through HAS2.

Hyaluronan activates Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed

PubMed Central

Hsu, Li-Jin; Hong, Qunying; Chen, Shur-Tzu; Kuo, Hsiang-Lin; Schultz, Lori; Heath, John; Lin, Sing-Ru; Lee, Ming-Hui; Li, Dong-Zhang; Li, Zih-Ling; Cheng, Hui-Ching; Armand, Gerard; Chang, Nan-Shan

2017-01-01

Malignant cancer cells frequently secrete significant amounts of transforming growth factor beta (TGF-β), hyaluronan (HA) and hyaluronidases to facilitate metastasizing to target organs. In a non-canonical signaling, TGF-β binds membrane hyaluronidase Hyal-2 for recruiting tumor suppressors WWOX and Smad4, and the resulting Hyal-2/WWOX/Smad4 complex is accumulated in the nucleus to enhance SMAD-promoter dependent transcriptional activity. Yeast two-hybrid analysis showed that WWOX acts as a bridge to bind both Hyal-2 and Smad4. When WWOX-expressing cells were stimulated with high molecular weight HA, an increased formation of endogenous Hyal-2/WWOX/Smad4 complex occurred rapidly, followed by relocating to the nuclei in 20-40 min. In WWOX-deficient cells, HA failed to induce Smad2/3/4 relocation to the nucleus. To prove the signaling event, we designed a real time tri-molecular FRET analysis and revealed that HA induces the signaling pathway from ectopic Smad4 to WWOX and finally to p53, as well as from Smad4 to Hyal-2 and then to WWOX. An increased binding of the Smad4/Hyal-2/WWOX complex occurs with time in the nucleus that leads to bubbling cell death. In contrast, HA increases the binding of Smad4/WWOX/p53, which causes membrane blebbing but without cell death. In traumatic brain injury-induced neuronal death, the Hyal-2/WWOX complex was accumulated in the apoptotic nuclei of neurons in the rat brains in 24 hr post injury, as determined by immunoelectron microscopy. Together, HA activates the Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed. PMID:27845895

Hyaluronan activates Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.

PubMed

Hsu, Li-Jin; Hong, Qunying; Chen, Shur-Tzu; Kuo, Hsiang-Lin; Schultz, Lori; Heath, John; Lin, Sing-Ru; Lee, Ming-Hui; Li, Dong-Zhang; Li, Zih-Ling; Cheng, Hui-Ching; Armand, Gerard; Chang, Nan-Shan

2017-03-21

Malignant cancer cells frequently secrete significant amounts of transforming growth factor beta (TGF-β), hyaluronan (HA) and hyaluronidases to facilitate metastasizing to target organs. In a non-canonical signaling, TGF-β binds membrane hyaluronidase Hyal-2 for recruiting tumor suppressors WWOX and Smad4, and the resulting Hyal-2/WWOX/Smad4 complex is accumulated in the nucleus to enhance SMAD-promoter dependent transcriptional activity. Yeast two-hybrid analysis showed that WWOX acts as a bridge to bind both Hyal-2 and Smad4. When WWOX-expressing cells were stimulated with high molecular weight HA, an increased formation of endogenous Hyal-2/WWOX/Smad4 complex occurred rapidly, followed by relocating to the nuclei in 20-40 min. In WWOX-deficient cells, HA failed to induce Smad2/3/4 relocation to the nucleus. To prove the signaling event, we designed a real time tri-molecular FRET analysis and revealed that HA induces the signaling pathway from ectopic Smad4 to WWOX and finally to p53, as well as from Smad4 to Hyal-2 and then to WWOX. An increased binding of the Smad4/Hyal-2/WWOX complex occurs with time in the nucleus that leads to bubbling cell death. In contrast, HA increases the binding of Smad4/WWOX/p53, which causes membrane blebbing but without cell death. In traumatic brain injury-induced neuronal death, the Hyal-2/WWOX complex was accumulated in the apoptotic nuclei of neurons in the rat brains in 24 hr post injury, as determined by immunoelectron microscopy. Together, HA activates the Hyal-2/WWOX/Smad4 signaling and causes bubbling cell death when the signaling complex is overexpressed.

Activation of Beta-Catenin Signaling in Androgen Receptor–Negative Prostate Cancer Cells

PubMed Central

Wan, Xinhai; Liu, Jie; Lu, Jing-Fang; Tzelepi, Vassiliki; Yang, Jun; Starbuck, Michael W.; Diao, Lixia; Wang, Jing; Efstathiou, Eleni; Vazquez, Elba S.; Troncoso, Patricia; Maity, Sankar N.; Navone, Nora M.

2012-01-01

Purpose To study Wnt/beta-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the beta-catenin–androgen receptor (AR) interaction. Experimental Design We performed beta-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of beta-catenin–mediated transcription), and sequenced the beta-catenin gene in MDA PCa 118a, MDA PCa 118b, MDA PCa 2b, and PC-3 prostate cancer (PCa) cells. We knocked down beta-catenin in AR-negative MDA PCa 118b cells and performed comparative gene-array analysis. We also immunohistochemically analyzed beta-catenin and AR in 27 bone metastases of human CRPCs. Results Beta-catenin nuclear accumulation and TOP-flash reporter activity were high in MDA PCa 118b but not in MDA PCa 2b or PC-3 cells. MDA PCa 118a and 118b cells carry a mutated beta-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA PCa 118b cells with downregulated beta-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant beta-catenin. Finally, we found nuclear localization of beta-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher’s exact test), suggesting that reduced AR expression enables Wnt/beta-catenin signaling. Conclusion We identified a previously unknown downstream target of beta-catenin, HAS2, in PCa, and found that high beta-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone-metastatic PCa. These findings may guide physicians in managing these patients. PMID:22298898

Mucoadhesive nanoparticles made of thiolated quaternary chitosan crosslinked with hyaluronan.

PubMed

Zambito, Ylenia; Felice, Francesca; Fabiano, Angela; Di Stefano, Rossella; Di Colo, Giacomo

2013-01-30

Mucoadhesive polymeric nanoparticles intended for drug transport across the gastrointestinal mucosa were prepared from quaternary ammonium-chitosan conjugates synthesised from reduced-MW chitosan (32 kDa). Conjugates contained pendant moieties of 2-4 adjacent diethyl-dimethylene-ammonium groups substituted on repeating units (26-55%). Conjugates were thiolated via amide bonds with thioglycolic acid to yield products with thiol content in the 35-87 μmol/g range. Nanoparticles with mean size in the 270-370 nm range and positive zeta-potential (+3.7 to +12.5 mV) resulted from ionotropic gelation of the thiolated conjugates with de-polymerised hyaluronic acid (470 kDa). The nanoparticles were fairly stable in size and thiol content and showed a significant mucoadhesivity, matching and even exceeding that of the constituent polymers. Nanoparticles were internalised by endothelial progenitor cells in direct relation to their surface charge intensity. Nanoparticle uptake significantly improved cell viability and resistance to oxidation. The lyophilised nanoparticles were re-dispersible and could make a manageable formulation for oral use. Copyright © 2012 Elsevier Ltd. All rights reserved.

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.

Functional role of R462 in the degradation of hyaluronan catalyzed by hyaluronate lyase from Streptococcus pneumoniae.

PubMed

Li, Fengxue; Xu, Dingguo

2015-08-01

Hyaluronan lyase from Streptococcus pneumoniae can degrade hyaluronic acid, which is one of the major components in the extracellular matrix. Hyaluronan can regulate water balance, osmotic pressure, and act as an ion exchange resin. Followed by our recent work on the catalytic reaction mechanism and substrate binding mode, we in this work further investigate the functional role of active site arginine residue, R462, in the degradation of hyaluronan. The site directed mutagenesis simulation of R462A and R462Q were modeled using a combined quantum mechanical and molecular mechanical method. The overall substrate binding features upon mutations do not have significant changes. The energetic profiles for the reaction processes are essentially the same as that in wild type enzyme, but significant activation barrier height changes can be observed. Both mutants were shown to accelerate the overall enzymatic activity, e.g., R462A can reduce the barrier height by about 2.8 kcal mol(-1), while R462Q reduces the activation energy by about 2.9 kcal mol(-1). Consistent with the active site model calculated using density functional theory, our results can support that the positive charge on R462 guanidino side chain group plays a negative role in the catalysis. Finally, the functional role of R462 was proposed to facilitate the formation of initial enzyme-substrate complex, but not in the subsequent catalytic degradation reaction. Graphical Abstract Degradation of hyaluronan catalyzed by hyaluronate lyase from Streptococcus pneumoniae.

Hyaluronan and N-ERC/mesothelin as key biomarkers in a specific two-step model to predict pleural malignant mesothelioma.

PubMed

Mundt, Filip; Nilsonne, Gustav; Arslan, Sertaç; Csürös, Karola; Hillerdal, Gunnar; Yildirim, Huseyin; Metintas, Muzaffer; Dobra, Katalin; Hjerpe, Anders

2013-01-01

Diagnosis of malignant mesothelioma is challenging. The first available diagnostic material is often an effusion and biochemical analysis of soluble markers may provide additional diagnostic information. This study aimed to establish a predictive model using biomarkers from pleural effusions, to allow early and accurate diagnosis. Effusions were collected prospectively from 190 consecutive patients at a regional referral centre. Hyaluronan, N-ERC/mesothelin, C-ERC/mesothelin, osteopontin, syndecan-1, syndecan-2, and thioredoxin were measured using ELISA and HPLC. A predictive model was generated and validated using a second prospective set of 375 effusions collected consecutively at a different referral centre. Biochemical markers significantly associated with mesothelioma were hyaluronan (odds ratio, 95% CI: 8.82, 4.82-20.39), N-ERC/mesothelin (4.81, 3.19-7.93), CERC/mesothelin (3.58, 2.43-5.59) and syndecan-1 (1.34, 1.03-1.77). A two-step model using hyaluronan and N-ERC/mesothelin, and combining a threshold decision rule with logistic regression, yielded good discrimination with an area under the ROC curve of 0.99 (95% CI: 0.97-1.00) in the model generation dataset and 0.83 (0.74-0.91) in the validation dataset, respectively. A two-step model using hyaluronan and N-ERC/mesothelin predicts mesothelioma with high specificity. This method can be performed on the first available effusion and could be a useful adjunct to the morphological diagnosis of mesothelioma.

Pericellular Versican Regulates the Fibroblast-Myofibroblast Transition

PubMed Central

Hattori, Noriko; Carrino, David A.; Lauer, Mark E.; Vasanji, Amit; Wylie, James D.; Nelson, Courtney M.; Apte, Suneel S.

2011-01-01

The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5−/− mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFβ signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5−/− cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcanhdf/+) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5−/− cells resulted from increased PCM versican content, we generated Adamts5−/−;Vcanhdf/+ mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5−/− mice. In Adamts5−/− fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates. PMID:21828051

CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention

PubMed Central

Huang, Yi; Askew, Emily B.; Knudson, Cheryl B.; Knudson, Warren

2016-01-01

Hyaluronan (HA) plays an essential role in cartilage where it functions to retain aggrecan. Previous studies have suggested that aggrecan is anchored indirectly to the plasma membrane of chondrocytes via its binding to cell-associated HA. However, reagents used to test these observations such as hyaluronidase and HA oligosaccharides are short term and may have side activities that complicate interpretation. Using the CRISPR/Cas9 gene editing approach, a model system was developed by generating HA-deficient chondrocyte cell lines. HA synthase-2 (Has2)-specific single guide RNA was introduced into two different variant lines of rat chondrosarcoma chondrocytes; knockout clones were isolated and characterized. Two other members of the HA synthase gene family were expressed at very low relative copy number but showed no compensatory response in the Has2 knockouts. Wild type chondrocytes of both variants exhibited large pericellular matrices or coats extending from the plasma membrane. Addition of purified aggrecan monomer expanded the size of these coats as the proteoglycan became retained within the pericellular matrix. Has2 knockout chondrocytes lost all capacity to assemble a particle-excluding pericellular matrix and more importantly, no matrices formed around the knockout cells following the addition of purified aggrecan. When grown as pellet cultures so as to generate a bioengineered neocartilage tissue, the Has2 knockout chondrocytes assumed a tightly-compacted morphology as compared to the wild type cells. When knockout chondrocytes were transduced with Adeno-ZsGreen1-mycHas2, the cell-associated pericellular matrices were restored including the capacity to bind and incorporate additional exogenous aggrecan into the matrix. These results suggest that HA is essential for aggrecan retention and maintaining cell separation during tissue formation. PMID:27094859

Sperm DNA Fragmentation Index and Hyaluronan Binding Ability in Men from Infertile Couples and Men with Testicular Germ Cell Tumor

PubMed Central

Filipiak, Eliza; Walczak-Jedrzejowska, Renata; Oszukowska, Elzbieta; Sobkiewicz, Slawomir; Wojt, Malgorzata; Chmiel, Jacek; Kula, Krzysztof; Slowikowska-Hilczer, Jolanta

2016-01-01

Objective. To investigate sperm DNA fragmentation and sperm functional maturity in men from infertile couples (IC) and men with testicular germ cell tumor (TGCT). Materials and Methods. Semen samples were collected from 312 IC men and 23 men with TGCT before unilateral orchiectomy and oncological treatment. The sperm chromatin dispersion test was performed to determine DNA fragmentation index (DFI) and the ability of sperm to bind with hyaluronan (HA) was assessed. Results. In comparison with the IC men, the men with TGCT had a higher percentage of sperm with fragmented DNA (median 28% versus 21%; p < 0.01) and a lower percentage of HA-bound sperm (24% versus 66%; p < 0.001). Normal results of both analyses were observed in 24% of IC men and 4% of men with TGCT. Negative Spearman's correlations were found between DFI and the percentage of HA-bound sperm in the whole group and in IC subjects and those with TGCT analyzed separately. Conclusions. Approximately 76% of IC men and 96% with TGCT awaiting orchiectomy demonstrated DNA fragmentation and/or sperm immaturity. We therefore recommend sperm banking after unilateral orchiectomy, but before irradiation and chemotherapy; the use of such a deposit appears to be a better strategy to obtain functionally efficient sperms. PMID:27999814

Modeling the growth processes of polyelectrolyte multilayers using a quartz crystal resonator.

PubMed

Salomäki, Mikko; Kankare, Jouko

2007-07-26

The layer-by-layer buildup of chitosan/hyaluronan (CH/HA) and poly(l-lysine)/hyaluronan (PLL/HA) multilayers was followed on a quartz crystal resonator (QCR) in different ionic strengths and at different temperatures. These polyelectrolytes were chosen to demonstrate the method whereby useful information is retrieved from acoustically thick polymer layers during their buildup. Surface acoustic impedance recorded in these measurements gives a single or double spiral when plotted in the complex plane. The shape of this spiral depends on the viscoelasticity of the layer material and regularity of the growth process. The polymer layer is assumed to consist of one or two zones. A mathematical model was devised to represent the separation of the layer to two zones with different viscoelastic properties. Viscoelastic quantities of the layer material and the mode and parameters of the growth process were acquired by fitting a spiral to the experimental data. In all the cases the growth process was mainly exponential as a function of deposition cycles, the growth exponent being between 0.250 and 0.275.

Surface functionalization of liposomes with proteins and carbohydrates for use in anti-cancer applications

NASA Astrophysics Data System (ADS)

Platt, Virginia M.

Liposomes can be used to exploit the altered biology of cancer thereby increasing delivery of liposome-associated anti-cancer drugs. In this dissertation, I explore methods that utilize the unique cancer expression of the polymeric glycosaminoglycan hyaluronan (HA) and the HA receptor CD44 to target liposomes to tumors, using liposomes functionalized with proteins or oligosaccharides on their surface. To make it easier to prepare protein-functionalized liposomes, a non-covalent protein/liposome association method based upon metal chelation/his 6 interaction was devised and characterized. I evaluated non-covalent attachment of the prodrug converting enzyme yeast cytosine deaminase, the far-red fluorescent protein mKate, two antigens ovalbumin and the membrane proximal region of an HIV GAG and hyaluronidase, a HA-degrading enzyme. In Chapter 2, I describe the synthesis of hyaluronan-oligosaccharide (HA-O) lipid conjugates and their incorporation into liposomes to target CD44-overexpressing cancer cells. HA-O ligands of defined-length, up to 10 monosaccharides, were attached to lipids via various linkers by reductive amination. The HA-lipids were easily incorporated into liposomes but did not mediate binding of liposomes to CD44 overexpressing cells. In Chapter 3, I evaluate the capacity of tris-NTA-Ni-lipids incorporated within a liposome bilayer to associate with his6-tagged proteins. Tris-NTA-lipids of differing structures and avidities were used to associate yeast cytosine deaminase and mKate to the surface of liposomes. Two tris-NTA-lipids and a mono-NTA lipid associated his-tagged proteins to a 1:1 molar ratio in solution. The proteins remained active while associated with the liposome surface. When challenged in vitro with fetal calf serum, tris-NTA-containing liposomes retained his-tagged proteins longer than mono-NTA. However, the tris-NTA/his6 interaction was found to be in a dynamic state; free yeast cytosine deaminase rapidly competed with pre-bound mKate for NTA occupancy. In the circulation of mice, his-tagged proteins associated with NTA-liposomes were cleared as rapidly as free protein. In Chapter 4, I study the effect of NTA/his-tag avidity on immune response when NTA-containing liposomes are used as non-covalent, particulate adjuvants. Two his-tagged antigens, ovalbumin and the membrane proximal portion of HIV Gag, were associated with NTA-liposomes containing either mono-NTA or tris-NTA lipids. The immune response to each antigen was compared to control adjuvant formulations in which antigens were admixed with or covalently-conjugated to liposomes. The weaker antigen, the HIV Gag peptide, induced a stronger immune response when associated with NTA-containing liposomes than when admixed with liposomes. Ovalbumin preparations in which the protein was admixed with particles or non-covalently associated with NTA-liposomes elicited a higher immune response than free ovalbumin or ovalbumin admixed with the control adjuvant alum. For both antigens, NTA-liposome responses were less than the response to antigens covalently linked to the liposome. In Chapter 5, I evaluate the potential for hyaluronidase to target conjugated liposomes to tumors or improve liposome motility within hyaluronan-rich tumors. Ovine hyaluronidase was modified using iminothiolane to introduce sulfhydryl groups into the enzyme. The enzyme was attached to liposomes via maleimide lipids or to maleimidehis10 in order to engineer non-covalent NTA-liposome association. Enzyme activity was retained after sulfhydryl addition and after attachment to liposomes. Liposome-conjugated hyaluronidase degraded an HA-gel at the same rate as admixed liposomes. When hyaluronidase-liposomes were injected intravenously in mice, the hyaluronidase conjugated-liposomes experienced faster clearance than control liposomes but slower clearance than free hyaluronidase. As a whole, these studies may help develop universal methods for a range of protein therapeutics and anti-cancer targeting agents.

A 3D-structural model of unsulfated chondroitin from high-field NMR: 4-sulfation has little effect on backbone conformation

PubMed Central

Sattelle, Benedict M.; Shakeri, Javad; Roberts, Ian S.; Almond, Andrew

2010-01-01

The glycosaminoglycan chondroitin sulfate is essential in human health and disease but exactly how sulfation dictates its 3D-strucutre at the atomic level is unclear. To address this, we have purified homogenous oligosaccharides of unsulfated chondroitin (with and without 15N-enrichment) and analysed them by high-field NMR to make a comparison published chondroitin sulfate and hyaluronan 3D-structures. The result is the first full assignment of the tetrasaccharide and an experimental 3D-model of the hexasaccharide (PDB code 2KQO). In common with hyaluronan, we confirm that the amide proton is not involved in strong, persistent inter-residue hydrogen bonds. However, in contrast to hyaluronan, a hydrogen bond is not inferred between the hexosamine OH-4 and the glucuronic acid O5 atoms across the β(1→3) glycosidic linkage. The unsulfated chondroitin bond geometry differs slightly from hyaluronan by rotation about the β(1→3) ψ dihedral (as previously predicted by simulation), while the β(1→4) linkage is unaffected. Furthermore, comparison shows that this glycosidic linkage geometry is similar in chondroitin-4-sulfate. We therefore hypothesise that both hexosamine OH-4 and OH-6 atoms are solvent exposed in chondroitin, explaining why it is amenable to sulfation and hyaluronan is not, and also that 4-sulfation has little effect on backbone conformation. Our conclusions exemplify the value of the 3D-model presented here and progress our understanding of glycosaminoglycan molecular properties. PMID:20022001

Esophageal Squamous Cell Carcinoma Cells Modulate Chemokine Expression and Hyaluronan Synthesis in Fibroblasts.

PubMed

Kretschmer, Inga; Freudenberger, Till; Twarock, Sören; Yamaguchi, Yu; Grandoch, Maria; Fischer, Jens W

2016-02-19

The aim of this study was to characterize the interaction of KYSE-410, an esophageal squamous cell carcinoma cell line, and fibroblasts with respect to the extracellular matrix component hyaluronan (HA) and chemokine expression. KYSE-410 cells induced the mRNA expression of HA synthase 2 (Has2) in normal skin fibroblasts (SF) only in direct co-cultures. Parallel to Has2 mRNA, Has2 antisense RNA (Has2os2) was up-regulated in co-cultures. Knockdown of LEF1, a downstream target of Wnt signaling, abrogated Has2 and Has2os2 induction. After knockdown of Has2 in SF, significantly less α-smooth muscle actin expression was detected in co-cultures. Moreover, it was investigated whether the phenotype of KYSE-410 was affected in co-culture with SF and whether Has2 knockdown in SF had an impact on KYSE-410 cells in co-culture. However, no effects on epithelial-mesenchymal transition markers, proliferation, and migration were detected. In addition to Has2 mRNA, the chemokine CCL5 was up-regulated and CCL11 was down-regulated in SF in co-culture. Furthermore, co-cultures of KYSE-410 cells and cancer-associated fibroblasts (CAF) were investigated. Similar to SF, Has2 and Ccl5 were up-regulated and Ccl11 was down-regulated in CAF in co-culture. Importantly and in contrast to SF, inhibiting HA synthesis by 4-methylumbelliferone abrogated the effect of co-culture on Ccl5 in CAF. Moreover, HA was found to promote adhesion of CD4(+) but not CD8(+) cells to xenogaft tumor tissues. In conclusion, direct co-culture of esophageal squamous cell carcinoma and fibroblasts induced stromal HA synthesis via Wnt/LEF1 and altered the chemokine profile of stromal fibroblasts, which in turn may affect the tumor immune response. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Esophageal Squamous Cell Carcinoma Cells Modulate Chemokine Expression and Hyaluronan Synthesis in Fibroblasts*

PubMed Central

Kretschmer, Inga; Freudenberger, Till; Twarock, Sören; Yamaguchi, Yu; Grandoch, Maria; Fischer, Jens W.

2016-01-01

The aim of this study was to characterize the interaction of KYSE-410, an esophageal squamous cell carcinoma cell line, and fibroblasts with respect to the extracellular matrix component hyaluronan (HA) and chemokine expression. KYSE-410 cells induced the mRNA expression of HA synthase 2 (Has2) in normal skin fibroblasts (SF) only in direct co-cultures. Parallel to Has2 mRNA, Has2 antisense RNA (Has2os2) was up-regulated in co-cultures. Knockdown of LEF1, a downstream target of Wnt signaling, abrogated Has2 and Has2os2 induction. After knockdown of Has2 in SF, significantly less α-smooth muscle actin expression was detected in co-cultures. Moreover, it was investigated whether the phenotype of KYSE-410 was affected in co-culture with SF and whether Has2 knockdown in SF had an impact on KYSE-410 cells in co-culture. However, no effects on epithelial-mesenchymal transition markers, proliferation, and migration were detected. In addition to Has2 mRNA, the chemokine CCL5 was up-regulated and CCL11 was down-regulated in SF in co-culture. Furthermore, co-cultures of KYSE-410 cells and cancer-associated fibroblasts (CAF) were investigated. Similar to SF, Has2 and Ccl5 were up-regulated and Ccl11 was down-regulated in CAF in co-culture. Importantly and in contrast to SF, inhibiting HA synthesis by 4-methylumbelliferone abrogated the effect of co-culture on Ccl5 in CAF. Moreover, HA was found to promote adhesion of CD4+ but not CD8+ cells to xenogaft tumor tissues. In conclusion, direct co-culture of esophageal squamous cell carcinoma and fibroblasts induced stromal HA synthesis via Wnt/LEF1 and altered the chemokine profile of stromal fibroblasts, which in turn may affect the tumor immune response. PMID:26699196

Interaction of Low Molecular Weight Hyaluronan (LMW-HA) with CD44 and Toll-Like Receptors Promotes the Actin Filament-Associated Protein (AFAP-110)-Actin Binding and MyD88-NFκB Signaling Leading to Pro-inflammatory Cytokine/Chemokine Production and Breast Tumor Invasion

PubMed Central

Bourguignon, Lilly Y.W.; Wong, Gabriel; Earle, Christine A.; Xia, Weiliang

2011-01-01

Both high and low molecular weight hyaluronan (HMW-HA vs. LMW-HA) exist in various tissues and cells. In this study we investigated LMW-HA-mediated CD44 interaction with Toll-like receptors (TLRs), the actin filament-associated protein (AFAP-110) and a myeloid differentiation factor (MyD88) in breast tumor cells (MDA-MB-231 cells). Our data indicate that LMW-HA (but not HMW-HA) preferentially stimulates a physical association between CD44 and TLRs followed by a concomitant recruitment of AFAP-110 and MyD88 into receptor-containing complexes in breast tumor cells. LMW-HA-activated AFAP-110 then binds to F-actin resulting in MyD88/NF-κB nuclear translocation, NF-κB-specific transcription and target gene (IL-1β and IL-8) expression. These signaling events lead to pro-inflammatory cytokine/chemokine production in the breast tumor cells. AFAP-110-F-actin (activated by LMW-HA) also promotes tumor cell invasion. Downregulation of AFAP-110 or MyD88 by transfecting breast tumor cells with AFAP-110 siRNA or MyD88 siRNA, respectively not only blocks the ability of LMW-HA to stimulate AFAP-110-actin function, but also impairs MyD88-NF-κB nuclear translocation and NF-κB transcriptional activation. Consequently, both IL-1β/IL-8 production and tumor cell invasion are impaired. Taken together, these findings suggest that LMW-HA plays an important role in CD44-TLR-associated AFAP-110-actin interaction and MyD88-NF-κB signaling required for tumor cell behaviors which may contribute to the progression of breast cancer. PMID:22031535

Influence of freeze-thawing on hyaluronic acid binding of human spermatozoa.

PubMed

Nijs, Martine; Creemers, Eva; Cox, Annemie; Janssen, Mia; Vanheusden, Elke; Castro-Sanchez, Yovanna; Thijs, Herbert; Ombelet, Willem

2009-08-01

Mature human spermatozoa have at least three specific hyaluronic acid (HA) binding proteins present on their sperm membrane. These receptors play a role in the acrosome reaction, hyaluronidase activity, hyaluronan-mediated motility and sperm-zona and sperm-oolemmal binding. Cryopreservation of spermatozoa can cause ultrastructural and even molecular damage. The aim of this study was to investigate if HA binding receptors of human spermatozoa remain functional after freeze-thawing. Forty patients were enrolled in the study. Semen samples were analysed before and after cryopreservation. Parameters analysed included concentration, motility, morphology and hyaluronan binding. Samples were frozen in CBS straws using a glycerol-glucose-based cryoprotectant. HA binding was studied using the sperm-hyaluronan binding assay. Freeze-thawing resulted in a significant decline in motility: the percentage of motile spermatozoa reduced from 50.6 to 30.3% (P < 0.001). HA binding properties of frozen-thawed spermatozoa remained unchanged after the freeze-thawing process: 68.5 +/- 17.1% spermatozoa of the neat sample were bound to HA, as were 71.3 +/- 20.4 of the frozen-thawed sample. This study indicates that freeze-thawing did not alter the functional hyaluronan binding sites of mature motile spermatozoa, and therefore will not alter their fertilizing potential.

Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix.

PubMed

Müller, Stephan A; van der Smissen, Anja; von Feilitzsch, Margarete; Anderegg, Ulf; Kalkhof, Stefan; von Bergen, Martin

2012-12-01

Fibroblasts are the main matrix producing cells of the dermis and are also strongly regulated by their matrix environment which can be used to improve and guide skin wound healing processes. Here, we systematically investigated the molecular effects on primary dermal fibroblasts in response to high-sulfated hyaluronan [HA] (hsHA) by quantitative proteomics. The comparison of non- and high-sulfated HA revealed regulation of 84 of more than 1,200 quantified proteins. Based on gene enrichment we found that sulfation of HA alters extracellular matrix remodeling. The collagen degrading enzymes cathepsin K, matrix metalloproteinases-2 and -14 were found to be down-regulated on hsHA. Additionally protein expression of thrombospondin-1, decorin, collagen types I and XII were reduced, whereas the expression of trophoblast glycoprotein and collagen type VI were slightly increased. This study demonstrates that global proteomics provides a valuable tool for revealing proteins involved in molecular effects of growth substrates for further material optimization.

Basigin/CD147 promotes renal fibrosis after unilateral ureteral obstruction.

PubMed

Kato, Noritoshi; Kosugi, Tomoki; Sato, Waichi; Ishimoto, Takuji; Kojima, Hiroshi; Sato, Yuka; Sakamoto, Kazuma; Maruyama, Shoichi; Yuzawa, Yukio; Matsuo, Seiichi; Kadomatsu, Kenji

2011-02-01

Regardless of their primary causes, progressive renal fibrosis and tubular atrophy are the main predictors of progression to end-stage renal disease. Basigin/CD147 is a multifunctional molecule-it induces matrix metalloproteinases and hyaluronan, for example-and has been implicated in organ fibrosis. However, the relationship between basigin and organ fibrosis has been poorly studied. We investigated basigin's role in renal fibrosis using a unilateral ureteral obstruction model. Basigin-deficient mice (Bsg(-/-)) demonstrated significantly less fibrosis after surgery than Bsg(+/+) mice. Fewer macrophages had infiltrated in Bsg(-/-) kidneys. Consistent with these in vivo data, primary cultured tubular epithelial cells from Bsg(-/-) mice produced less matrix metalloproteinase and exhibited less motility on stimulation with transforming growth factor β. Furthermore, Bsg(-/-) embryonic fibro blasts produced less hyaluronan and α-smooth muscle actin after transforming growth factor β stimulation. Together, these results demonstrate for the first time that basigin is a key regulator of renal fibrosis. Basigin could be a candidate target molecule for the prevention of organ fibrosis. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Orally administered hyaluronan affects skin dryness and epidermal thickening in photoaged hairless mice.

PubMed

Kawada, Chinatsu; Kimura, Mamoru; Masuda, Yasunobu; Nomura, Yoshihiro

2016-06-01

The oral administration of hyaluronans (HAs) (molecular weight, 300k and less than 10k) to photoaged hairless mice increased the moisture content of the stratum corneum and decreased the epidermal thickness, respectively. Furthermore, orally administered HAs suppressed the low-molecular weight of HA content of the skin. This study indicates oral administered HAs may ameliorate the skin condition resulting from photoaging.

Role of Recurrent Hypoxia-Ischemia in Preterm White Matter Injury Severity

PubMed Central

Hagen, Matthew W.; Riddle, Art; McClendon, Evelyn; Gong, Xi; Shaver, Daniel; Srivastava, Taasin; Dean, Justin M.; Bai, Ji-Zhong; Fowke, Tania M.; Gunn, Alistair J.; Jones, Daniel F.; Sherman, Larry S.; Grafe, Marjorie R.; Hohimer, A. Roger; Back, Stephen A.

2014-01-01

Objective Although the spectrum of white matter injury (WMI) in preterm infants is shifting from cystic necrotic lesions to milder forms, the factors that contribute to this changing spectrum are unclear. We hypothesized that recurrent hypoxia-ischemia (rHI) will exacerbate the spectrum of WMI defined by markers of inflammation and molecules related to the extracellular matrix (hyaluronan (HA) and the PH20 hyaluronidase) that regulate maturation of the oligodendrocyte (OL) lineage after WMI. Methods We employed a preterm fetal sheep model of in utero moderate hypoxemia and global severe but not complete cerebral ischemia that reproduces the spectrum of human WMI. The response to rHI was compared against corresponding early or later single episodes of HI. An ordinal rating scale of WMI was compared against an unbiased quantitative image analysis protocol that provided continuous histo-pathological outcome measures for astrogliosis and microglial activation. Late oligodendrocyte progenitors (preOLs) were quantified by stereology. Analysis of hyaluronan and the hyaluronidase PH20 defined the progressive response of the extracellular matrix to WMI. Results rHI resulted in a more severe spectrum of WMI with a greater burden of necrosis, but an expanded population of preOLs that displayed reduced susceptibility to cell death. WMI from single episodes of HI or rHI was accompanied by elevated HA levels and increased labeling for PH20. Expression of PH20 in fetal ovine WMI was confirmed by RT-PCR and RNA-sequencing. Conclusions rHI is associated with an increased risk for more severe WMI with necrosis, but reduced risk for preOL degeneration compared to single episodes of HI. Expansion of the preOL pool may be linked to elevated hyaluronan and PH20. PMID:25390897

Altered expression of hyaluronan, HAS1-2, and HYAL1-2 in oral lichen planus.

PubMed

Siponen, Maria; Kullaa, Arja; Nieminen, Pentti; Salo, Tuula; Pasonen-Seppänen, Sanna

2015-07-01

Oral lichen planus (OLP) is an immune-mediated mucosal disease of unclear etiology and of unresolved pathogenesis. Hyaluronan (HA) is an extracellular matrix glycosaminoglycan involved in inflammation and tumor progression. However, its presence in OLP has not been reported. We therefore aimed to study the immunohistochemical expression of HA, its receptor CD44, hyaluronan synthases (HAS1-3), and hyaluronidases (HYAL1-2) in OLP. The presence of HA, CD44, HAS1-3, and HYAL1-2 was studied by immunohistochemical methods in 55 OLP and 23 control oral mucosal specimens (CTR). The localization, intensity, and differences of the epithelial expression between OLP and CTRs were analyzed. HA and CD44 were found on cell membranes in the epithelial basal and intermediate layers in CTR and OLP specimens. The HA staining intensity was stronger in the basal layer of the epithelium in OLP than in CTRs (P < 0.001). HAS1 (P = 0.001) and HAS2 (P < 0.001) showed stronger staining in the basal and weaker staining in the superficial (P < 0.001) epithelial layers in OLP than in CTRs. The immunostaining of HAS3 was low in both OLP and CTRs. Positive HYAL1 and HYAL2 staining were mainly found in the basal and intermediate epithelial layers, and their intensities were significantly increased in OLP, except HYAL 2 in the intermediate epithelial layer. HA, HAS1-2, and HYAL1-2 have altered expression in OLP compared to CTRs and may therefore have a role in OLP pathogenesis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effects of low molecular weight hyaluronan combined with carprofen on canine osteoarthritis articular chondrocytes and cartilage explants in vitro.

PubMed

Euppayo, Thippaporn; Siengdee, Puntita; Buddhachat, Kittisak; Pradit, Waranee; Viriyakhasem, Nawarat; Chomdej, Siriwadee; Ongchai, Siriwan; Harada, Yasuji; Nganvongpanit, Korakot

2015-09-01

Intra-articular injection with non-steroidal anti-inflammatory drugs (NSAIDs) is used to treat inflammatory joint disease, but the side effects of NSAIDs include chondrotoxicity. Hyaluronan has shown positive effects on chondrocytes by reducing apoptosis and increasing proteoglycan synthesis. The purposes of this study were to evaluate the effects of low molecular weight hyaluronan (low MW HA), carprofen 25 mg/ml, carprofen 12.5 mg/ml, and a combination of HA and carprofen on canine osteoarthritis (OA) articular chondrocytes and a cartilage explant model in terms of cell viability, extracellular matrix remaining, and gene expression after exposure. In chondrocyte culture, MTT assay was used to evaluate the chondrotoxicity of IC50 and IC80 of carprofen with HA. In cartilage explant culture, two kinds of extracellular matrix (uronic acid and collagen) remaining in cartilage were used to evaluate cartilage damage for 14 d after treatment. Expression of COL2A1, AGG, and MMP3 was used to evaluate the synthesis and degradation of the matrix for 7 d after treatment. In chondrocyte culture, low MW HA could preserve OA chondrocyte viability but could not reduce the chondrotoxicity level of carprofen (P < 0.05). In explant culture, low MW HA combined with 12.5 mg/ml carprofen caused less destruction of uronic acid and collagen structure when compared with the control (P < 0.05). Low MW HA caused high expression levels of COL2A1 and AGG in OA cartilage (P < 0.05); HA combined with carprofen resulted in higher COL2A1 and AGG expression levels than carprofen alone.

Hyaluronan Deficiency Due to Has3 Knock-Out Causes Altered Neuronal Activity and Seizures via Reduction in Brain Extracellular Space

PubMed Central

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

2014-01-01

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 Has2CKO, 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. PMID:24790187

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

Black rice (Oryza sativa L. var. japonica) hydrolyzed peptides induce expression of hyaluronan synthase 2 gene in HaCaT keratinocytes.

PubMed

Sim, Gwan Sub; Lee, Dong-Hwan; Kim, Jin-Hwa; An, Sung-Kwan; Choe, Tae-Boo; Kwon, Tae-Jong; Pyo, Hyeong-Bae; Lee, Bum-Chun

2007-02-01

Black rice (Oryza sativa L. var. japonica) has been used in folk medicine in Asia. To understand the effects of black rice hydrolyzed peptides (BRP) from germinated black rice, we assessed the expression levels of about 20,000 transcripts in BRP-treated HaCaT keratinocytes using human 1A oligo microarray analysis. As a result, the BRP treatment showed a differential expression ratio of more than 2-fold: 745 were activated and 1,011 were repressed. One of the most interesting findings was a 2-fold increase in hyaluronan synthase 2 (HAS2) gene expression by BRP. Semiquantitative RT-PCR showed that BRP increased HAS2 mRNA in dose-dependent manners. ELISA showed that BRP effectively increased hyaluronan (HA) production in HaCaT keratinocytes.

Formulation and Evaluation of Organogels Containing Hyaluronan Microparticles for Topical Delivery of Caffeine.

PubMed

Simsolo, Erol Eli; Eroğlu, İpek; Tanrıverdi, Sakine Tuncay; Özer, Özgen

2018-04-01

Cellulite is a dermal disorder including the extracellular matrix, the lymphatic and microcirculatory systems and the adipose tissue. Caffeine is used as the active moiety depending its preventive effect on localization of fat in the cellular structure. Hyaluronic acid (hyaluronan-HA) is a natural constituent of skin that generates formation and poliferation of new cells having a remarkable moisturizing ability. The aim of this study is to formulate HA microparticles loaded with caffeine via spray-drying method. Resulting microparticle formulations (33.97 ± 0.3 μm, span < 2, 88.56 ± 0.42% encapsulation efficiency) were distributed in lecithin organogels to maintain the proper viscosity for topical application. Following the characterization and cell culture studies, in vitro drug release and ex vivo permeation studies were performed. The accumulated amount of caffeine was twice higher than the aqueous solution for the microparticle-loaded organogels at 24 h (8262,673 μg/cm 2 versus 4676,691 μg/cm 2 ). It was related to the sustained behaviour of caffeine release from the microparticles. As a result, lecithin organogel containing HA-encapsulated microparticles could be considered as suitable candidate formulations for efficient topical drug delivery system of caffeine. In addition to that, synergistic effect of this combination appears as a promising approach for long-acting treatment of cellulite.

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

Sulfated hyaluronan alters fibronectin matrix assembly and promotes osteogenic differentiation of human bone marrow stromal cells

NASA Astrophysics Data System (ADS)

Vogel, Sarah; Arnoldini, Simon; Möller, Stephanie; Schnabelrauch, Matthias; Hempel, Ute

2016-11-01

Extracellular matrix (ECM) composition and structural integrity is one of many factors that influence cellular differentiation. Fibronectin (FN) which is in many tissues the most abundant ECM protein forms a unique fibrillary network. FN homes several binding sites for sulfated glycosaminoglycans (sGAG), such as heparin (Hep), which was previously shown to influence FN conformation and protein binding. Synthetically sulfated hyaluronan derivatives (sHA) can serve as model molecules with a well characterized sulfation pattern to study sGAG-FN interaction. Here is shown that the low-sulfated sHA (sHA1) interacts with FN and influences fibril assembly. The interaction of FN fibrils with sHA1 and Hep, but not with non-sulfated HA was visualized by immunofluorescent co-staining. FRET analysis of FN confirmed the presence of more extended fibrils in human bone marrow stromal cells (hBMSC)-derived ECM in response to sHA1 and Hep. Although both sHA1 and Hep affected FN conformation, exclusively sHA1 increased FN protein level and led to thinner fibrils. Further, only sHA1 had a pro-osteogenic effect and enhanced the activity of tissue non-specific alkaline phosphatase. We hypothesize that the sHA1-triggered change in FN assembly influences the entire ECM network and could be the underlying mechanism for the pro-osteogenic effect of sHA1 on hBMSC.

Atomic force microscopy of adsorbed proteoglycan mimetic nanoparticles: Toward new glycocalyx-mimetic model surfaces.

PubMed

Hedayati, Mohammadhasan; Kipper, Matt J

2018-06-15

Blood vessels present a dense, non-uniform, polysaccharide-rich layer, called the endothelial glycocalyx. The polysaccharides in the glycocalyx include polyanionic glycosaminoglycans (GAGs). This polysaccharide-rich surface has excellent and unique blood compatibility. We report new methods for preparing and characterizing dense GAG surfaces that can serve as models of the vascular endothelial glycocalyx. The GAG-rich surfaces are prepared by adsorbing heparin or chondroitin sulfate-containing polyelectrolyte complex nanoparticles (PCNs) to chitosan-hyaluronan polyelectrolyte multilayers (PEMs). The surfaces are characterized by PeakForce tapping atomic force microscopy, both in air and in aqueous pH 7.4 buffer, and by PeakForce quantitative nanomechanics (PF-QNM) mode with high spatial resolution. These new surfaces provide access to heparin-rich or chondroitin sulfate-rich coatings that mimic both composition and nanoscale structural features of the vascular endothelial glycocalyx. Copyright © 2018. Published by Elsevier Ltd.

Activation of β-catenin signaling in androgen receptor-negative prostate cancer cells.

PubMed

Wan, Xinhai; Liu, Jie; Lu, Jing-Fang; Tzelepi, Vassiliki; Yang, Jun; Starbuck, Michael W; Diao, Lixia; Wang, Jing; Efstathiou, Eleni; Vazquez, Elba S; Troncoso, Patricia; Maity, Sankar N; Navone, Nora M

2012-02-01

To study Wnt/β-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the β-catenin-androgen receptor (AR) interaction. We carried out β-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of β-catenin-mediated transcription), and sequenced the β-catenin gene in MDA prostate cancer 118a, MDA prostate cancer 118b, MDA prostate cancer 2b, and PC-3 prostate cancer cells. We knocked down β-catenin in AR-negative MDA prostate cancer 118b cells and carried out comparative gene-array analysis. We also immunohistochemically analyzed β-catenin and AR in 27 bone metastases of human CRPCs. β-Catenin nuclear accumulation and TOP-flash reporter activity were high in MDA prostate cancer 118b but not in MDA prostate cancer 2b or PC-3 cells. MDA prostate cancer 118a and MDA prostate cancer 118b cells carry a mutated β-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA prostate cancer 118b cells with downregulated β-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant β-catenin. Finally, we found nuclear localization of β-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher's exact test), suggesting that reduced AR expression enables Wnt/β-catenin signaling. We identified a previously unknown downstream target of β-catenin, HAS2, in prostate cancer, and found that high β-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone metastatic prostate cancer. These findings may guide physicians in managing these patients.

Insights into the structure and function of membrane-integrated processive glycosyltransferases

DOE PAGES

Bi, Yunchen; Hubbard, Caitlin; Purushotham, Pallinti; ...

2015-09-02

Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. In this paper, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin,more » alginate, hyaluronan and poly-N-acetylglucosamine (PNAG).« less

Insights into the structure and function of membrane-integrated processive glycosyltransferases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bi, Yunchen; Hubbard, Caitlin; Purushotham, Pallinti

Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. In this paper, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin,more » alginate, hyaluronan and poly-N-acetylglucosamine (PNAG).« less

The distribution of renal hyaluronan and the expression of hyaluronan synthases during water deprivation in the Spinifex hopping mouse, Notomys alexis.

PubMed

Bartolo, Ray C; Donald, John A

2007-12-01

Hyaluronan (HA) is a glycosaminoglycan that is synthesized by a family of enzymes called hyaluronan synthases (HASs), of which there are three isoforms (HAS1, 2 and 3) in mammals. The HASs have different tissue expression patterns and function, indicating that synthesis of HA and formation of the HA matrix may be regulated by various factors. The HA matrix has an important role in renal water handling and the production of a concentrated urine. We investigated the distribution of HA and the expression of HAS1, HAS2 and HAS3 mRNAs in the kidney of the Spinifex hopping mouse, Notomys alexis, a native Australian desert rodent that is reported to produce the most concentrated urine of any mammal. After periods of three, seven and fourteen days of water deprivation, the distribution of renal HA changed considerably, and there was a general down-regulation of HAS mRNA expression. It is proposed that the regulation of HA synthesis by the different HAS isoforms during water deprivation in N. alexis, could be influenced by the molecular mass of the HA chains produced by each isoform, followed by the rate at which the individual HAS produces HA.

Glycosaminoglycan in cerebrum, cerebellum and brainstem of young sheep brain with particular reference to compositional and structural variations of chondroitin-dermatan sulfate and hyaluronan.

PubMed

Kilia, Virginia; Skandalis, Spyros S; Theocharis, Achilleas D; Theocharis, Dimitrios A; Karamanos, Nikos K; Papageorgakopoulou, Nickoletta

2008-09-01

Recent advances in the structural biology of chondroitin sulfate chains have suggested important biological functions in the development of the brain. Several studies have demonstrated that the composition of chondroitin sulfate chains changes with aging and normal brain maturation. In this study, we determined the concentration of all glycosaminoglycan types, i.e. chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, hyaluronan and chondroitin in cerebrum, cerebellum and brainstem of young sheep brain. In all cases, chondroitin sulfate was the predominant glycosaminoglycan type, comprising about 54-58% of total glycosaminoglycans, with hyaluronan being present also in significant amounts of about 19-28%. Of particular interest was the increased presence of the disulfated disaccharides and dermatan sulfate in cerebellum and brainstem, respectively, as well as the detectable and measurable occurrence of chondroitin in young sheep brain. Among the three brain areas, cerebrum was found to be significantly richer in chondroitin sulfate and hyaluronan, two major extracellular matrix components. These findings imply that the extracellular matrix of the cerebrum is different from those of cerebellum and brainstem, and probably this fact is related to the particular histological and functional characteristics of each anatomic area of the brain.

Autophagic Vacuolation Induced by Excess ROS Generation in HABP1/p32/gC1qR Overexpressing Fibroblasts and Its Reversal by Polymeric Hyaluronan

PubMed Central

Saha, Paramita; Chowdhury, Anindya Roy; Dutta, Shubhra; Chatterjee, Soumya; Ghosh, Ilora; Datta, Kasturi

2013-01-01

The ubiquitous hyaladherin, hyaluronan-binding protein 1 (HABP1/p32/gC1qR) upon stable overexpression in normal fibroblasts (F-HABP07) has been reported to induce mitochondrial dysfunction, growth retardation and apoptosis after 72 h of growth. HABP1 has been observed to accumulate in the mitochondria resulting in generation of excess Reactive Oxygen Species (ROS), mitochondrial Ca++ efflux and drop in mitochondrial membrane potential. In the present study, autophagic vacuolation was detected with monodansylcadaverin (MDC) staining from 36 h to 60 h of culture period along with elevated level of ROS in F-HABP07 cells. Increased expression of autophagic markers like MAP-LC3-II, Beclin 1 and autophagic modulator, DRAM confirmed the occurrence of the phenomenon. Reduced vacuole formation was observed upon treatment with 3-MA, a known PI3 kinase inhibitor, only at 32 h and was ineffective if treated later, as high ROS level was already attained. Treatment of F111 and F-HABP07 cells with bafilomycin A1 further indicated an increase in autophagosome formation along with autophagic degradation in HABP1 overexpressed fibroblasts. Comparison between normal fibroblast (F111) and F-HABP07 cells indicate reduced level of polymeric HA, its depolymerization and perturbed HA-HABP1 interaction in F-HABP07. Interestingly, supplementation of polymeric HA, an endogenous ROS scavenger, in the culture medium prompted reduction in number of vacuoles in F-HABP07 along with drop in ROS level, implying that excess ROS generation triggers initiation of autophagic vacuole formation prior to apoptosis due to overexpression of HABP1. Thus, the phenomenon of autophagy takes place prior to apoptosis induction in the HABP1 overexpressing cell line, F-HABP07. PMID:24205125

Overexpression of hyaluronan synthase 2 and gonadotropin receptors in cumulus cells of goats subjected to one-shot eCG/FSH hormonal treatment for ovarian stimulation.

PubMed

Santos, Juliana D R; Batista, Ribrio I T P; Magalhães, Livia C; Paula, Alexandre R; Souza, Samara S; Salamone, Daniel F; Bhat, Maajid H; Teixeira, Dárcio I A; Freitas, Vicente J F; Melo, Luciana M

2016-07-01

Hormonal ovarian stimulation may affect transcripts in somatic cells of cumulus-oocyte complexes (COCs) and affect the resulting oocyte quality. Here, in parallel with morphological classification and in vitro maturation (IVM) rate analysis, we investigated the expression of hyaluronan synthase 2 (HAS2), gonadotropic receptors (FSHR and LHR) and connexin 43 (GJA1) in cumulus cells (CCs) from goat COCs after multi-dose FSH (MD) or one-shot FSH/eCG (OS) treatments, using bovine COCs as control groups. The MD treatment produced more large follicles, and the resulting COCs had a better morphology and IVM rate than were obtained with OS. The OS treatment produced COCs with increased HAS2, FSHR, LHR and GJA1 expression. This gene expression pattern was also observed in the CCs of COCs that showed poor morphological characteristics. On the other hand, the mRNA levels were more similar between groups after IVM; FSHR and LHR were the main genes that showed decreased expression. Some events that occurred in bovine CCs during IVM, such as cell expansion, increased HAS2 expression and decreased GJA1 expression, were less evident or did not occur in goat COCs. In conclusion, increasing HAS2, FSHR, LHR and GJA1 expression in goat COCs does not confer greater meiotic competence to oocytes. Instead, it may result from poor regulation of gene expression in CCs by lower quality oocytes. Finally, cumulus expansion, together with HAS2 upregulation and GJA1 downregulation, seems to be more important for bovine COCs than for goat COCs. Additional studies are needed to investigate the importance of other HAS isoforms and connexins in goat COCs. Copyright © 2016 Elsevier B.V. All rights reserved.

β-Cyclodextrin grafted polypyrrole magnetic nanocomposites toward the targeted delivery and controlled release of doxorubicin

NASA Astrophysics Data System (ADS)

Hong, Shasha; Li, Zengbo; Li, Chenzhong; Dong, Chuan; Shuang, Shaomin

2018-01-01

The Fe3O4@PPy-HA-β-CD nanocomposites as the novel nanocarrier were prepared by grafting ethylenediamine derivative of​ β-​CD to the surface of polypyrrole-coated magnetic nanoparticles (Fe3O4@PPy) via using hyaluronan (HA) as the intermediate linker. HA was also the efficient target ligand for CD44. The as-prepared drug carrier was characterized by TEM, TGA, XRD, and VSM and used for the delivery of doxorubicin hydrochloride (DOX) with the high loading content of 447 mg/g. The multilayer Freundlich isotherm model was found to be a good fit for the loading of the drug carrier for DOX. Significant NIR-triggered release of DOX was observed in a weak acidic pH. And the release data in vitro was well described using the Retiger-Pepper kinetic model. Furthermore, MTT assay and confocal microscopy against Hep-G2 cells clearly illustrated that the drug carrier had no associated cytotoxicity and could easily enter the cells. The release and accumulation of DOX were observed in the cell nuclei. Thus, the DOX-loaded drug carrier killed the cancer cells efficaciously and minimized adverse side effects due to its target effect. These results suggested the as-prepared drug carrier would be of great potential for the controlled release and targeted delivery of DOX.

Variation in primary and culture-expanded cells derived from connective tissue progenitors in human bone marrow space, bone trabecular surface and adipose tissue.

PubMed

Qadan, Maha A; Piuzzi, Nicolas S; Boehm, Cynthia; Bova, Wesley; Moos, Malcolm; Midura, Ronald J; Hascall, Vincent C; Malcuit, Christopher; Muschler, George F

2018-03-01

Connective tissue progenitors (CTPs) embody the heterogeneous stem and progenitor cell populations present in native tissue. CTPs are essential to the formation and remodeling of connective tissue and represent key targets for tissue-engineering and cell-based therapies. To better understand and characterize CTPs, we aimed to compare the (i) concentration and prevalence, (ii) early in vitro biological behavior and (iii) expression of surface-markers and transcription factors among cells derived from marrow space (MS), trabecular surface (TS), and adipose tissues (AT). Cancellous-bone and subcutaneous-adipose tissues were collected from 8 patients. Cells were isolated and cultured. Colony formation was assayed using Colonyze software based on ASTM standards. Cell concentration ([Cell]), CTP concentration ([CTP]) and CTP prevalence (P CTP ) were determined. Attributes of culture-expanded cells were compared based on (i) effective proliferation rate and (ii) expression of surface-markers CD73, CD90, CD105, SSEA-4, SSEA-3, SSEA-1/CD15, Cripto-1, E-Cadherin/CD324, Ep-CAM/CD326, CD146, hyaluronan and transcription factors Oct3/4, Sox-2 and Nanog using flow cytometry. Mean [Cell], [CTP] and P CTP were significantly different between MS and TS samples (P = 0.03, P = 0.008 and P= 0.0003), respectively. AT-derived cells generated the highest mean total cell yield at day 6 of culture-4-fold greater than TS and more than 40-fold greater than MS per million cells plated. TS colonies grew with higher mean density than MS colonies (290 ± 11 versus 150 ± 11 cell per mm 2 ; P = 0.0002). Expression of classical-mesenchymal stromal cell (MSC) markers was consistently recorded (>95%) from all tissue sources, whereas all the other markers were highly variable. The prevalence and biological potential of CTPs are different between patients and tissue sources and lack variation in classical MSC markers. Other markers are more likely to discriminate differences between cell populations in biological performance. Understanding the underlying reasons for variation in the concentration, prevalence, marker expression and biological potential of CTPs between patients and source tissues and determining the means of managing this variation will contribute to the rational development of cell-based clinical diagnostics and targeted cell-based therapies. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Formation and remodeling of the brain extracellular matrix in neural plasticity: Roles of chondroitin sulfate and hyaluronan.

PubMed

Miyata, Shinji; Kitagawa, Hiroshi

2017-10-01

The extracellular matrix (ECM) of the brain is rich in glycosaminoglycans such as chondroitin sulfate (CS) and hyaluronan. These glycosaminoglycans are organized into either diffuse or condensed ECM. Diffuse ECM is distributed throughout the brain and fills perisynaptic spaces, whereas condensed ECM selectively surrounds parvalbumin-expressing inhibitory neurons (PV cells) in mesh-like structures called perineuronal nets (PNNs). The brain ECM acts as a non-specific physical barrier that modulates neural plasticity and axon regeneration. Here, we review recent progress in understanding of the molecular basis of organization and remodeling of the brain ECM, and the involvement of several types of experience-dependent neural plasticity, with a particular focus on the mechanism that regulates PV cell function through specific interactions between CS chains and their binding partners. We also discuss how the barrier function of the brain ECM restricts dendritic spine dynamics and limits axon regeneration after injury. The brain ECM not only forms physical barriers that modulate neural plasticity and axon regeneration, but also forms molecular brakes that actively controls maturation of PV cells and synapse plasticity in which sulfation patterns of CS chains play a key role. Structural remodeling of the brain ECM modulates neural function during development and pathogenesis. Genetic or enzymatic manipulation of the brain ECM may restore neural plasticity and enhance recovery from nerve injury. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa. Copyright © 2017 Elsevier B.V. All rights reserved.

Hyperglycaemia and aberrated insulin signalling stimulate tumour progression via induction of the extracellular matrix component hyaluronan.

PubMed

Twarock, Sören; Reichert, Christina; Peters, Ulrike; Gorski, Daniel J; Röck, Katharina; Fischer, Jens W

2017-08-15

Epidemiological studies have detected a higher incidence of various tumour entities in diabetic patients. However, the underlying mechanisms remain insufficiently understood. Glucose-derived pericellular and extracellular hyaluronan (HA) promotes tumour progression and development. In our study, we tested the hypothesis that a diabetic metabolic state, characterised by hyperglycaemia and concomitant aberrant insulin signalling, stimulates tumour progression via the induction of HA synthesis. In a streptozotocin-induced diabetic nude mouse tumour xenograft model, hyperglycaemia and lack of insulin caused an increased formation of tumour-associated HA-matrix, which in turn accelerated tumour progression and neoangiogenesis. This process was effectively attenuated by treatment with 4-methylumbelliferone, a pharmacological inhibitor of HA-synthesis. To define the mechanisms behind these in vivo observations, we investigated the impact of hyperglycaemia and insulin on the glucose metabolism in oesophageal squamous cell cancer cells (ESCC). Hyperglycaemia induced HA synthesis while insulin diminished HA production by directing glucose metabolites to glycolysis. Vice versa, inhibition of glycolysis, either by knockdown of the glycolytic key enzyme phosphofructokinase or by an experimental abrogation of insulin signalling (knockdown of the insulin receptor and long-term treatment with insulin) augmented HA synthesis. Consequently, these processes induced invasion, anchorage-independent growth and adhesion of ESCC to endothelial cells in vitro. Thus, the cellular shift in glucose usage from catabolism of glucose to anabolism of HA driven by hyperglycaemia and insulin resistance may represent an important link between diabetes and cancer progression. Hence, therapeutical inhibition of HA synthesis may represent a promising approach for tumour treatment in diabetic patients. © 2017 UICC.

Design and evaluation of dual CD44 receptor and folate receptor-targeting double-smart pH-response multifunctional nanocarrier

NASA Astrophysics Data System (ADS)

Chen, Daquan; Song, Xiaoyan; Wang, Kaili; Guo, Chunjing; Yu, Yueming; Fan, Huaying; Zhao, Feng

2017-12-01

In this article, in order to enhance the bioavailiability and tumor targeting of curcumin (Cur), the oligosaccharides of hyaluronan conjugates, folic acid-oligosaccharides of hyaluronan-acetal-menthone 1,2-glycerol ketal (FA-oHA-Ace-MGK) carried oHA as a ligand to CD44 receptor, double-pH-sensitive Ace-MGK as hydrophobic moieties, and FA as the target of folate receptor. The structure characteristics of this smart response multifunctional dual-targeting nano-sized carrier was measured by fourier-transform infrared (FT-IR) and nuclear magnetic resonance (1H-NMR). Cur, an anticancer drug, was successfully loaded in FA-oHA-Ace-MGK micelles by self-assembly. The measurement results of transmission electron microscopy (TEM) presented that the Cur-loaded micelles were spherical in shape with the average size of 166.3 ± 2.12 nm and zeta potential - 30.07 mV. Much more encapsulated Cur could be released at mildly acidic environments than at pH 7.4, from the Cur-FA-oHA-Ace-MGK micelles. Cytotoxicity assay indicated that non-Cur loaded micelles mostly had no cytotoxicity to MCF-7 cells and A549 cells, and Cur-loaded micelles had significantly lower survival rate than Cur suspension in the same concentration, which proved that the drug-loaded micelles can effectively inhibit tumor cell growth. The targeting of CD44 receptors and folate receptors was proved in vitro cellular uptake assay. These results showed the promising potential of FA-oHA-Ace-MGK as an effective nano-sized carrier for anti-tumor drug delivery.

Characterization of Hyaluronan-Degrading Enzymes from Yeasts.

PubMed

Smirnou, Dzianis; Krčmář, Martin; Kulhánek, Jaromír; Hermannová, Martina; Bobková, Lenka; Franke, Lukáš; Pepeliaev, Stanislav; Velebný, Vladimír

2015-10-01

Hyaluronidases (HAases) from yeasts were characterized for the first time. The study elucidated that hyaluronate 4-glycanohydrolase and hyaluronan (HA) lyase can be produced by yeasts. Six yeasts producing HAases were found through express screening of activities. The extracellular HAases from two of the yeast isolates, Pseudozyma aphidis and Cryptococcus laurentii, were characterized among them. P. aphidis HAase hydrolyzed β-1,4 glycosidic bonds of HA, yielding even-numbered oligosaccharides with N-acetyl-D-glucosamine at the reducing end. C. laurentii produced hyaluronan lyase, which cleaved β-1,4 glycosidic bonds of HA in β-elimination reaction, and the products of HA degradation were different-sized even-numbered oligosaccharides. The shortest detected HA oligomer was dimer. The enzymes' pH and temperature optima were pH 3.0 and 37-45 °C (P. aphidis) and pH 6.0 and 37 °C (C. laurentii), respectively. Both HAases showed good thermostability.

The Emerging Role of the Hippo Pathway in Lung Cancers: Clinical Implications.

PubMed

Teoh, Seong Lin; Das, Srijit

2017-11-30

The incidence of lung cancers has increased globally. Increased exposure to tobacco, passive smoking, less consumption of vegetables and fruits and occupational exposure to asbestos, arsenic and chromium are the main risk factors. The pathophysiology of lung cancer is complex and not well understood. Various microRNAs, genes and pathways are associated with lung cancers. The genes involved in lung cancers produce proteins involved in cell growth, differentiation, different cell cycles, apoptosis, immune modulation, tumor spread and progression. The Hippo pathway (also known as the Salvador-Warts-Hippo pathway) is the latest emerging concept in cancers. The Hippo pathway plays an important role in controlling the size of the tissue and organ by virtue of its action on cell proliferation and apoptosis. In the present review, we highlight the mammalian Hippo pathway, role of its core members, its upstream regulators, downstream effectors and the resistance cases in lung cancers. Specific interaction of Mer with cell surface hyaluronan receptor CD44 is vital in cell contact inhibition, thereby activating Hippo pathway. Both transcription co-activators YAP and TAZ (also known as WWTR1, being homologs of Drosophila Yki) are important regulators of proliferation and apoptosis, and serve as major downstream effectors of the Hippo pathway. Mutation of NF2, the upstream regulator of Hippo pathway is linked to the cancers. Targeting YAP and TAZ may be important for future drug delivery and treatment. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Qualitative and quantitative analysis of hyaluronan oligosaccharides with high performance thin layer chromatography using reagent-free derivatization on amino-modified silica and electrospray ionization-quadrupole time-of-flight mass spectrometry coupling on normal phase.

PubMed

Rothenhöfer, Martin; Scherübl, Rosmarie; Bernhardt, Günther; Heilmann, Jörg; Buschauer, Armin

2012-07-27

Purified oligomers of hyalobiuronic acid are indispensable tools to elucidate the physiological and pathophysiological role of hyaluronan degradation by various hyaluronidase isoenzymes. Therefore, we established and validated a novel sensitive, convenient, rapid, and cost-effective high performance thin layer chromatography (HPTLC) method for the qualitative and quantitative analysis of small saturated hyaluronan oligosaccharides consisting of 2-4 hyalobiuronic acid moieties. The use of amino-modified silica as stationary phase allows a simple reagent-free in situ derivatization by heating, resulting in a very low limit of detection (7-19 pmol per band, depending on the analyzed saturated oligosaccharide). By this derivatization procedure for the first time densitometric quantification of the analytes could be performed by HPTLC. The validated method showed a quantification limit of 37-71 pmol per band and was proven to be superior in comparison to conventional detection of hyaluronan oligosaccharides. The analytes were identified by hyphenation of normal phase planar chromatography to mass spectrometry (TLC-MS) using electrospray ionization. As an alternative to sequential techniques such as high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), the validated HPTLC quantification method can easily be automated and is applicable to the analysis of multiple samples in parallel. Copyright © 2012 Elsevier B.V. All rights reserved.

Synthesis and characterization of a Hyaluronan-polyethylene copolymer for biomedical applications.

PubMed

Oldinski, Rachael A; Cranson, Cody N; James, Susan P

2010-08-01

Hyaluronan (HA)-based biomaterials are of interest for bone and cartilage tissue engineering because HA plays an important role in orthopedic tissue development, function, and repair. The goal of this project was to develop a biomaterial that incorporated the constituents of both a hydrogel and a hydrophobic polymer for biomedical applications. A series of amphiphilic graft copolymers consisting of HA, a glycosaminoglycan, and high-density polyethylene (HDPE), that is, HA-co-HDPE, were fabricated. The chemical characteristics, physical and viscoelastic properties, and cytocompatibility of novel HA-co-HDPE materials were characterized via Fourier Transform infrared (FTIR) spectroscopy, solid state nuclear magnetic resonance (ssNMR) spectroscopy, differential scanning calorimetry (DSC), dynamic shear testing, and an in vitro human osteoblast cell study. The esterification reaction between HA and functionalized HDPE resulted in semicrystalline, insoluble powder. The dynamic shear properties of HA-co-HDPE concentrated solutions were more like natural proteoglycans than the HA control. HA-co-HDPE was successfully compression molded into disks that swelled upon hydration. Osteoblasts were viable and expressed the osteoblast phenotype after 7 days of culture on HA-co-HDPE materials. These HA-co-HDPE materials may have several biomaterial applications in saline suspension or molded form, including orthopedic tissue repair.

Hyaluronan–cisplatin conjugate nanoparticles embedded in Eudragit S100-coated pectin/alginate microbeads for colon drug delivery

PubMed Central

Tsai, Shiao-Wen; Yu, Ding-Syuan; Tsao, Shu-Wei; Hsu, Fu-Yin

2013-01-01

Hyaluronan–cisplatin conjugate nanoparticles (HCNPs) were chosen as colon-targeting drug-delivery carriers due to the observation that a variety of malignant tumors overexpress hyaluronan receptors. HCNPs were prepared by mixing cisplatin with a hyaluronan solution, followed by dialysis to remove trace elements. The cells treated with HCNPs showed significantly lower viability than those treated with cisplatin alone. HCNPs were entrapped in Eudragit S100-coated pectinate/alginate microbeads (PAMs) by using an electrospray method and a polyelectrolyte multilayer-coating technique in aqueous solution. The release profile of HCNPs from Eudragit S100-coated HCNP-PAMs was pH-dependent. The percentage of 24-hour drug release was approximately 25.1% and 39.7% in pH 1.2 and pH 4.5 media, respectively. However, the percentage of drug released quickly rose to 75.6% at pH 7.4. Moreover, the result of an in vivo nephrotoxicity study demonstrated that Eudragit S100-coated HCNP-PAMs treatment could mitigate the nephrotoxicity that resulted from cisplatin. From these results, it can be concluded that Eudragit S100-coated HCNP-PAMs are promising carriers for colonspecific drug delivery. PMID:23861585

Regulation and roles of the hyaluronan system in mammalian reproduction.

PubMed

Fouladi-Nashta, Ali A; Raheem, Kabir A; Marei, Waleed F; Ghafari, Fataneh; Hartshorne, Geraldine M

2017-02-01

Hyaluronan (HA) is a non-sulphated glycosaminoglycan polymer naturally occurring in many tissues and fluids of mammals, including the reproductive system. Its biosynthesis by HA synthase (HAS1-3) and catabolism by hyaluronidases (HYALs) are affected by ovarian steroid hormones. Depending upon its molecular size, HA functions both as a structural component of tissues in the form of high-molecular-weight HA or as a signalling molecule in the form of small HA molecules or HA fragments with effects mediated through interaction with its specific cell-membrane receptors. HA is produced by oocytes and embryos and in various segments of the reproductive system. This review provides information about the expression and function of members of the HA system, including HAS, HYALs and HA receptors. We examine their role in various processes from folliculogenesis through oocyte maturation, fertilisation and early embryo development, to pregnancy and cervical dilation, as well as its application in assisted reproduction technologies. Particular emphasis has been placed upon the role of the HA system in pre-implantation embryo development and embryo implantation, for which we propose a hypothetical sequential model. © 2017 Society for Reproduction and Fertility.

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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Large-Volume Crystalloid Fluid Is Associated with Increased Hyaluronan Shedding and Inflammation in a Canine Hemorrhagic Shock Model.

PubMed

Smart, Lisa; Boyd, C J; Claus, M A; Bosio, E; Hosgood, G; Raisis, A

2018-05-04

Shedding of the endothelial glycocalyx precedes leukocyte activation and adherence in acute inflammation. Rapid administration of crystalloid or colloid fluids for treating hemorrhagic shock may cause endothelial glycocalyx shedding, thereby increasing inflammation. This study aimed to compare the effect of different fluid treatments in a canine shock model on glycocalyx biomarker, hyaluronan, and inflammatory biomarkers. Greyhound dogs under general anesthesia subject to hemorrhage for 60 min were given 20 mL kg -1 of either fresh whole blood (FWB), hydroxyethyl starch (HES) 130/0.4, 4% succinylated gelatin (GELO), or 80 mL kg -1 of isotonic crystalloid (CRYST) over 20 min (n = 6 per group). Plasma biomarkers hyaluronan, interleukin (IL) 6, 8, 10, tumor necrosis factor-α, monocyte chemoattractant protein-1, keratinocyte chemokine-like, and atrial natriuretic peptide were measured at baseline, end of hemorrhage (Shock), end of fluid administration (T20), and then 40 (T60), 100 (T120), and 160 (T180) minutes later. Biomarker concentrations were compared between groups using the Kruskal-Wallis test or Fisher's exact test (measurable versus unmeasurable) (significance set at P < 0.05). Hyaluronan concentration peaked early in the CRYST group at T20, compared to HES (P = 0.005) and GELO (P = 0.018), and later in the GELO group at T60, compared to FWB (P < 0.001). The CRYST group had significantly more samples with measurable IL6 at T180 (P = 0.015), compared to GELO, and IL10 at T60, T120, and T180 (all P = 0.015), compared to FWB. There were no significant differences in other biomarker concentrations. In conclusion, rapid large-volume crystalloid administered for hemorrhagic shock was associated with increased hyaluronan and a greater inflammatory response.

Modulation of hyaluronan synthase activity in cellular membrane fractions.

PubMed

Vigetti, Davide; Genasetti, Anna; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Bartolini, Barbara; Moretto, Paola; De Luca, Giancarlo; Hascall, Vincent C; Passi, Alberto

2009-10-30

Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1beta, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.

Thiolated hyaluronan-based hydrogels crosslinked using oxidized glutathione: an injectable matrix designed for ophthalmic applications.

PubMed

Zarembinski, Thomas I; Doty, Nathaniel J; Erickson, Isaac E; Srinivas, Ramya; Wirostko, Barbara M; Tew, William P

2014-01-01

Future ophthalmic therapeutics will require the sustained delivery of bioactive proteins and nucleic acid-based macromolecules and/or provide a suitable microenvironment for the localization and sustenance of reparative progenitor cells after transplantation into or onto the eye. Water-rich hydrogels are ideal vehicles for such cargo, but few have all the qualities desired for novel ophthalmic use, namely in situ gelation speed, cytocompatibility, biocompatibility and capacity to functionalize. We describe here the development of an ophthalmic-compatible crosslinking system using oxidized glutathione (GSSG), a physiologically relevant molecule with a history of safe use in humans. When GSSG is used in conjunction with an existing hyaluronate-based, in situ crosslinkable hydrogel platform, gels form in less than 5 min using the thiol-disulfide exchange reaction. This GSSG hydrogel supports the 3-D culture of adipose-derived stem cells in vitro and shows biocompatibility in preliminary intracutaneous and subconjunctival experiments in vivo. In addition, the thiol-disulfide exchange reaction can also be used in conjunction with other thiol-compatible chemistries to covalently link peptides for more complex formulations. These data suggest that this hydrogel could be well suited for local ocular delivery, focusing initially on front of the eye therapies. Subsequent uses of the hydrogel include delivery of back of the eye treatments and eventually into other soft, hyaluronan-rich tissues such as those from the liver and brain. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Concentration polarization of hyaluronan on the surface of the synovial lining of infused joints

PubMed Central

Lu, Y; Levick, JR; Wang, W

2004-01-01

Hyaluronan (HA) in joints conserves the lubricating synovial fluid by making trans-synovial fluid escape almost insensitive to pressure elevation (e.g. effusions, joint flexion). This phenomenon, ‘outflow buffering’, was discovered during HA infusion into the rabbit knee joint cavity. It was also found that HA is partially reflected by the joint lining (molecular sieving), and that the reflected fraction R decreases as trans-synovial filtration rate Q is increased. It was postulated therefore that outflow buffering is mediated by HA reflection. Reflection creates a HA concentration polarization layer, the osmotic pressure of which opposes fluid loss. A steady-state, cross-flow ultrafiltration model was previously used to explain the outflow buffering and negative R-vs.-Q relation. However, the steady-state, cross-perfusion assumptions restricted the model's applicability for an infused, dead-end cavity or a non-infused joint during cyclical motion. We therefore developed a new, non-steady-state model which describes the time course of dead-end, partial HA ultrafiltration. The model describes the progressive build-up of a HA concentration polarization layer at the synovial surface over time. Using experimental parameter values, the model successfully accounts for the observed negative R-vs.-Q relation and shows that the HA reflected fraction (R) also depends on HA diffusivity, membrane area expansion and the synovial HA reflection coefficient. The non-steady-state model thus explains existing experimental work, and it is a key stage in understanding synovial fluid turnover in intact, moving, human joints or osteoarthritic joints treated by HA injections. PMID:15579541

Improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles

NASA Astrophysics Data System (ADS)

Syrvatka, Vasyl J.; Slyvchuk, Yurij I.; Rozgoni, Ivan I.; Gevkan, Ivan I.; Overchuk, Marta O.

2014-02-01

Modern routine enzyme immunoassays for detection and quantification of biomolecules have several disadvantages such as high cost, insufficient sensitivity, complexity and long-term execution. The surface plasmon resonance of silver nanoparticles gives reasons of creating new in the basis of simple, highly sensitive and low cost colorimetric assays that can be applied to the detection of small molecules, DNA, proteins and pollutants. The main aim of the study was the improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles. For this purpose we developed method for synthesis of silver nanoparticles with hyaluronic acid and studied possibility of use these nanoparticles in direct determination of target molecules concentration (in particular proteins) and for improving of enzyme immunoassay. As model we used conventional enzyme immunoassays for determination of progesterone and estradiol concentration. We obtained the possibility to produce silver nanoparticles with hyaluronan homogeneous in size between 10 and 12 nm, soluble and stable in water during long term of storage using modified procedure of silver nanoparticles synthesis. New method allows to obtain silver nanoparticles with strong optical properties at the higher concentrations - 60-90 μg/ml with the peak of absorbance at the wavelength 400 nm. Therefore surface plasmon resonance of silver nanoparticles with hyaluronan and ultraviolet-visible spectroscopy provide an opportunity for rapid determination of target molecules concentration (especial protein). We used silver nanoparticles as enzyme carriers and signal enhancers. Our preliminary data show that silver nanoparticles increased absorbance of samples that allows improving upper limit of determination of estradiol and progesterone concentration.

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

Effect of Shock-Induced Cavitation Bubble Collapse on the damage in the Simulated Perineuronal Net of the Brain.

PubMed

Wu, Yuan-Ting; Adnan, Ashfaq

2017-07-13

The purpose of this study is to conduct modeling and simulation to understand the effect of shock-induced mechanical loading, in the form of cavitation bubble collapse, on damage to the brain's perineuronal nets (PNNs). It is known that high-energy implosion due to cavitation collapse is responsible for corrosion or surface damage in many mechanical devices. In this case, cavitation refers to the bubble created by pressure drop. The presence of a similar damage mechanism in biophysical systems has long being suspected but not well-explored. In this paper, we use reactive molecular dynamics (MD) to simulate the scenario of a shock wave induced cavitation collapse within the perineuronal net (PNN), which is the near-neuron domain of a brain's extracellular matrix (ECM). Our model is focused on the damage in hyaluronan (HA), which is the main structural component of PNN. We have investigated the roles of cavitation bubble location, shockwave intensity and the size of a cavitation bubble on the structural evolution of PNN. Simulation results show that the localized supersonic water hammer created by an asymmetrical bubble collapse may break the hyaluronan. As such, the current study advances current knowledge and understanding of the connection between PNN damage and neurodegenerative disorders.

Capillary electrophoresis for the quality control of chondroitin sulfates in raw materials and formulations.

PubMed

Malavaki, Christina J; Asimakopoulou, Athanasia P; Lamari, Fotini N; Theocharis, Achilleas D; Tzanakakis, George N; Karamanos, Nikos K

2008-03-01

Exogenous administration of chondroitin sulfate (CS) is widely practiced for the treatment of osteoarthritis, although the efficacy of this treatment has not been completely established by clinical studies. A reason for the inconsistency of the results may be the quality of the CS preparations, which are commercially available as dietary supplements. In this article, we describe the development of a new method of capillary electrophoresis (CE) for the quantification of CS concentrations, screening for other glycosaminoglycan or DNA impurities and determination of hyaluronan impurities in CS raw materials, tablets, hard capsules, and liquid formulations. Analysis is performed within 12 min in bare fused silica capillaries using reversed polarity and an operating phosphate buffer of low pH. The method has high sensitivity (lower limit of quantitation [LLOQ] values of 30.0 microg/ml for CS and 5.0 microg/ml for hyaluronan), high precision, and accuracy. Analysis of 11 commercially available products showed the presence of hyaluronan impurities in most of them (up to 1.5%). CE analysis of the samples after treatment with chondroitinase ABC and ACII, which depolymerize the chains to unsaturated disaccharides, with a previously described method (Karamanos et al., J. Chromatogr. A 696 (1995) 295-305) confirmed the results of hyaluronan determination and showed that the structural characteristics (i.e., disaccharide composition) of CS are very different, showing the different species or tissue origin and possibly affecting the therapeutic outcome.

The ligand-binding profile of HARE: hyaluronan and chondroitin sulfates A, C, and D bind to overlapping sites distinct from the sites for heparin, acetylated low-density lipoprotein, dermatan sulfate, and CS-E.

PubMed

Harris, Edward N; Weigel, Paul H

2008-08-01

The hyaluronic acid receptor for endocytosis (HARE)/ Stabilin-2 is the primary systemic scavenger receptor for hyaluronan (HA), the chondroitin sulfates (CS), dermatan sulfate (DS), and nonglycosaminoglycan (GAG) ligands such as acetylated low-density lipoprotein (AcLDL), pro-collagen propeptides, and advanced glycation end products. We recently discovered that HARE is also a systemic scavenger receptor for heparin (Hep) (Harris EN, Weigel JA, Weigel PH. 2008. The human hyaluronan receptor for endocytosis [HARE/Stabilin-2] is a systemic clearance receptor for heparin. J Biol Chem. 283:17341-17350). Our goal was to map the binding sites of eight different ligands within HARE. We used biotinylated GAGs and radio-iodinated streptavidin or AcLDL to assess the binding activities of ligands directly or indirectly (by competition with unlabeled ligands) in endocytosis assays using stable cell lines expressing the 315 or 190 kDa HA receptor for endocytosis (315- or 190-HARE) isoforms, and ELISA-like assays, with purified recombinant soluble 190-HARE ecto-domain. For example, Hep binding to HARE was competed by DS, CS-E, AcLDL, and dextran sulfate, but not by other CS types, HA, dextran, or heparosan. (125)I-AcLDL binding to HARE was partially competed by Hep and dextran sulfate, but not competed by HA. Two ligands, DS and CS-E, competed with both Hep and HA to some degree. Hep and HA binding or endocytosis is mutually inclusive; binding of these two GAGs occurs with functionally separate, noncompetitive, and apparently noninteracting domains. Thus, HARE binds to HA and Hep simultaneously. Although the domain(s) responsible for Hep binding remains unknown, the Link domain was required for HARE binding to HA, CS-A, CS-C, and CS-D. These results enable us to outline, for the first time, a binding activity map for multiple ligands of HARE.

The ligand-binding profile of HARE: hyaluronan and chondroitin sulfates A, C, and D bind to overlapping sites distinct from the sites for heparin, acetylated low-density lipoprotein, dermatan sulfate, and CS-E

PubMed Central

Harris, Edward N.; Weigel, Paul H.

2008-01-01

The hyaluronic acid receptor for endocytosis (HARE)/ Stabilin-2 is the primary systemic scavenger receptor for hyaluronan (HA), the chondroitin sulfates (CS), dermatan sulfate (DS), and nonglycosaminoglycan (GAG) ligands such as acetylated low-density lipoprotein (AcLDL), pro-collagen propeptides, and advanced glycation end products. We recently discovered that HARE is also a systemic scavenger receptor for heparin (Hep) (Harris EN, Weigel JA, Weigel PH. 2008. The human hyaluronan receptor for endocytosis [HARE/Stabilin-2] is a systemic clearance receptor for heparin. J Biol Chem. 283:17341–17350). Our goal was to map the binding sites of eight different ligands within HARE. We used biotinylated GAGs and radio-iodinated streptavidin or AcLDL to assess the binding activities of ligands directly or indirectly (by competition with unlabeled ligands) in endocytosis assays using stable cell lines expressing the 315 or 190 kDa HA receptor for endocytosis (315- or 190-HARE) isoforms, and ELISA-like assays, with purified recombinant soluble 190-HARE ecto-domain. For example, Hep binding to HARE was competed by DS, CS-E, AcLDL, and dextran sulfate, but not by other CS types, HA, dextran, or heparosan. 125I-AcLDL binding to HARE was partially competed by Hep and dextran sulfate, but not competed by HA. Two ligands, DS and CS-E, competed with both Hep and HA to some degree. Hep and HA binding or endocytosis is mutually inclusive; binding of these two GAGs occurs with functionally separate, noncompetitive, and apparently noninteracting domains. Thus, HARE binds to HA and Hep simultaneously. Although the domain(s) responsible for Hep binding remains unknown, the Link domain was required for HARE binding to HA, CS-A, CS-C, and CS-D. These results enable us to outline, for the first time, a binding activity map for multiple ligands of HARE. PMID:18499864

Cell surface chondroitin sulfate glycosaminoglycan in melanoma: role in the activation of pro-MMP-2 (pro-gelatinase A)

PubMed Central

Iida, Joji; Wilhelmson, Krista L.; Ng, Janet; Lee, Peter; Morrison, Charlotte; Tam, Eric; Overall, Christopher M.; McCarthy, James B.

2007-01-01

We previously reported that CS (chondroitin sulfate) GAG (glycosaminoglycan), expressed on MCSP (melanoma-specific CS proteoglycan), is important for regulating MT3-MMP [membrane-type 3 MMP (matrix metalloproteinase)]-mediated human melanoma invasion and gelatinolytic activity in vitro. In the present study, we sought to determine if CS can directly enhance MT3-MMP-mediated activation of pro-MMP-2. Co-immunoprecipitation studies suggest that MCSP forms a complex with MT3-MMP and MMP-2 on melanoma cell surface. When melanoma cells were treated with βDX (p-nitro-β-D-xylopyranoside) to inhibit coupling of CS on the core protein, both active form and proform of MMP-2 were no longer co-immunoprecipitated with either MCSP or MT3-MMP, suggesting a model in which CS directly binds to MMP-2 and presents the gelatinase to MT3-MMP to be activated. By using recombinant proteins, we determined that MT3-MMP directly activates pro-MMP-2 and that this activation requires the interaction of the C-terminal domain of pro-MMP-2 with MT3-MMP. Activation of pro-MMP-2 by suboptimal concentrations of MT3-MMP is also significantly enhanced in the presence of excess C4S (chondroitin 4-sulfate), whereas C6S (chondroitin 6-sulfate) or low-molecular-mass hyaluronan was ineffective. Affinity chromatography studies using CS isolated from aggrecan indicate that the catalytic domain of MT3-MMP and the C-terminal domain of MMP-2 directly bind to the GAG. Thus the direct binding of pro-MMP-2 with CS through the C-domain would present the catalytic domain of pro-MMP-2 to MT3-MMP, which facilitates the generation of the active form of MMP-2. These results suggest that C4S, which is expressed on tumour cell surface, can function to bind to pro-MMP-2 and facilitate its activation by MT3-MMP-expressing tumour cells to enhance invasion and metastasis. PMID:17217338

Nebulized hyaluronan ameliorates lung inflammation in cystic fibrosis mice.

PubMed

Gavina, Manuela; Luciani, Alessandro; Villella, Valeria R; Esposito, Speranza; Ferrari, Eleonora; Bressani, Ilaria; Casale, Alida; Bruscia, Emanuela M; Maiuri, Luigi; Raia, Valeria

2013-08-01

Chronic lung inflammation with increased susceptibility to bacterial infections cause much of the morbidity and mortality in patients with cystic fibrosis (CF), the most common severe, autosomal recessively inherited disease in the Caucasian population. Exogenous inhaled hyaluronan (HA) can exert a protective effect against injury and beneficial effects of HA have been shown in experimental models of chronic respiratory diseases. Our objective was to examine whether exogenous administration of nebulized HA might interfere with lung inflammation in CF. F508del homozygous mice (Cftr(F508del) ) and transgenic mice overexpressing the ENaC channel β-subunit (Scnn1b-Tg) were treated with nebulized HA (0.5 mg/mouse/day for 7 days). Tumor necrosis factor-alpha (TNFα), macrophage inflammatory protein-2 (MIP-2), myeloperoxidase (MPO) levels, and macrophage infiltration were assessed on lung tissues. IB3-1 and CFBE41o-epithelial cell lines were cultured with HA (24 hr, 100 µg/ml) and Reactive Oxygen Species (ROS), Tissue Transglutaminase (TG2) SUMOylation and Peroxisome Proliferator Activated Receptor gamma (PPARγ) and phospho-p42/p44 levels were measured by dichlorodihydrofluorescein assay, or fluorescence resonance energy transfer (FRET) microscopy or immunoblots. Nebulized HA reduced TNFα expression (P < 0.005); TNFα, MIP-2, and MPO protein levels (P < 0.05); MPO activity (P < 0.05); and CD68+ cells counts (P < 0.005) in lung tissues of Cftr(F508del) and Scnn1b-Tg mice, compared with saline-treated mice. HA reduced ROS, TG2 SUMOylation, TG2 activity, phospho-p42-44, and increased PPARγ protein in both IB3-1 and CFBE41o cells (P < 0.05). Nebulized HA is effective in controlling inflammation in vivo in mice CF airways and in vitro in human airway epithelial cells. We provide the proof of concept for the use of inhaled HA as a potential anti-inflammatory drug in CF therapy. Copyright © 2012 Wiley Periodicals, Inc.

The Anti-Inflammatory Effects of Matrix Metalloproteinase-3 on Irreversible Pulpitis of Mature Erupted Teeth

PubMed Central

Eba, Hisanori; Murasawa, Yusuke; Iohara, Koichiro; Isogai, Zenzo; Nakamura, Hiroshi; Nakamura, Hiroyuki; Nakashima, Misako

2012-01-01

Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation and the modulation of cell behavior. These proteinases have also been implicated in tissue repair and regeneration. Our previous studies have demonstrated that MMP-3 elicits stimulatory effects on the proliferation and the migration of endothelial cells as well as anti-apoptotic effects on these cells in vitro. In addition, we found that MMP-3 enhanced the regeneration of lost pulp tissue in a rat incisor pulp injury model. However, continuously erupting rodent incisors exhibit significantly different pulp organization compared with mature erupted teeth. Therefore, we have further extended these studies using a canine irreversible pulpitis model to investigate the effects of MMP-3. In this study, the crowns of the canine mature premolars were removed and the pulp tissues were amputated. The amputated pulp tissues remained exposed for 24 or 72 hours to induce mild or severe irreversible pulpitis, respectively, followed by sealing of the cavities. In both models, the whole pulp tissues became necrotic by day 14. In this mild pulpitis model, the regeneration of pulp tissue with vasculature and nerves was observed until 14 days after sealing with MMP-3, followed by extracellular matrix formation in the regenerated pulp tissues until day 28. The treatment with MMP-3 resulted in a decrease in the number of macrophage and antigen-presenting cells and a significant inhibition of IL-6 expression on day 3. The inhibition of MMP-3 activity abolished these anti-inflammatory effects. Immunofluorescence staining demonstrated that MMP-3 was involved in the modification of serum-derived hyaluronan-associated proteins and hyaluronan (SHAP-HA) complexes possibly through the degradation of versican. These results demonstrate that MMP-3 can act as an anti-inflammatory agent and suggest that MMP-3 might represent a useful therapy for the treatment of mild irreversible pulpitis. PMID:23285075

Cellular interactions with tissue-engineered microenvironments and nanoparticles

NASA Astrophysics Data System (ADS)

Pan, Zhi

Tissue-engineered hydrogels composed of intermolecularlly crosslinked hyaluronan (HA-DTPH) and fibronectin functional domains (FNfds) were applied as a physiological relevant ECM mimic with controlled mechanical and biochemical properties. Cellular interactions with this tissue-engineered environment, especially physical interactions (cellular traction forces), were quantitatively measured by using the digital image speckle correlation (DISC) technique and finite element method (FEM). By correlating with other cell functions such as cell morphology and migration, a comprehensive structure-function relationship between cells and their environments was identified. Furthermore, spatiotemporal redistribution of cellular traction stresses was time-lapse measured during cell migration to better understand the dynamics of cell mobility. The results suggest that the reinforcement of the traction stresses around the nucleus, as well as the relaxation of nuclear deformation, are critical steps during cell migration, serving as a speed regulator, which must be considered in any dynamic molecular reconstruction model of tissue cell migration. Besides single cell migration, en masse cell migration was studied by using agarose droplet migration assay. Cell density was demonstrated to be another important parameter to influence cell behaviors besides substrate properties. Findings from these studies will provide fundamental design criteria to develop novel and effective tissue-engineered constructs. Cellular interactions with rutile and anatase TiO2 nanoparticles were also studied. These particles can penetrate easily through the cell membrane and impair cell function, with the latter being more damaging. The exposure to nanoparticles was found to decrease cell area, cell proliferation, motility, and contractility. To prevent this, a dense grafted polymer brush coating was applied onto the nanoparticle surface. These modified nanoparticles failed to adhere to and penetrate through the cell membrane. As a consequence, the coating effectively decreased reactive oxygen species (ROS) formation and protected the cells. Considering the broad applications of these nanoparticles in personal health care products, the functionalized polymer coating will likely play an important role in protecting cells and tissue from damage.

Induction of KIAA1199/CEMIP is associated with colon cancer phenotype and poor patient survival

PubMed Central

Fink, Stephen P.; Myeroff, Lois L.; Kariv, Revital; Platzer, Petra; Xin, Baozhong; Mikkola, Debra; Lawrence, Earl; Morris, Nathan; Nosrati, Arman; Willson, James K. V.; Willis, Joseph; Veigl, Martina; Barnholtz-Sloan, Jill S.; Wang, Zhenghe; Markowitz, Sanford D.

2015-01-01

Genes induced in colon cancer provide novel candidate biomarkers of tumor phenotype and aggressiveness. We originally identified KIAA1199 (now officially called CEMIP) as a transcript highly induced in colon cancer: initially designating the transcript as Colon Cancer Secreted Protein 1. We molecularly characterized CEMIP expression both at the mRNA and protein level and found it is a secreted protein induced an average of 54-fold in colon cancer. Knockout of CEMIPreduced the ability of human colon cancer cells to form xenograft tumors in athymic mice. Tumors that did grow had increased deposition of hyaluronan, linking CEMIP participation in hyaluronan degradation to the modulation of tumor phenotype. We find CEMIP mRNA overexpression correlates with poorer patient survival. In stage III only (n = 31) or in combined stage II plus stage III colon cancer cases (n = 73), 5-year overall survival was significantly better (p = 0.004 and p = 0.0003, respectively) among patients with low CEMIP expressing tumors than those with high CEMIP expressing tumors. These results demonstrate that CEMIP directly facilitates colon tumor growth, and high CEMIP expression correlates with poor outcome in stage III and in stages II+III combined cohorts. We present CEMIP as a candidate prognostic marker for colon cancer and a potential therapeutic target. PMID:26437221

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

Probing vocal fold fibroblast response to hyaluronan in 3D contexts.

PubMed

Munoz-Pinto, Dany J; Jimenez-Vergara, Andrea Carolina; Gelves, L Marcela; McMahon, Rebecca E; Guiza-Arguello, Viviana; Hahn, Mariah S

2009-11-01

A number of treatments are being investigated for vocal fold (VF) scar, including designer implants. The aim of the present study was to validate a 3D model system for probing the effects of various bioactive moieties on VF fibroblast (VFF) behavior toward rational implant design. We selected poly(ethylene glycol) diacrylate (PEGDA) hydrogels as our base-scaffold due to their broadly tunable material properties. However, since cells encapsulated in PEGDA hydrogels are generally forced to take on rounded/stellate morphologies, validation of PEGDA gels as a 3D VFF model system required that the present work directly parallel previous studies involving more permissive scaffolds. We therefore chose to focus on hyaluronan (HA), a polysaccharide that has been a particular focus of the VF community. Toward this end, porcine VFFs were encapsulated in PEGDA hydrogels containing consistent levels of high Mw HA (HA(HMW)), intermediate Mw HA (HA(IMW)), or the control polysaccharide, alginate, and cultured for 7 and 21 days. HA(HMW) promoted sustained increases in active ERK1/2 relative to HA(IMW). Furthermore, VFFs in HA(IMW) gels displayed a more myofibroblast-like phenotype, higher elastin production, and greater protein kinase C (PkC) levels at day 21 than VFFs in HA(HMW) and alginate gels. The present results are in agreement with a previous 3D study of VFF responses to HA(IMW) relative to alginate in collagen-based scaffolds permissive of cell elongation, indicating that PEGDA hydrogels may serve as an effective 3D model system for probing at least certain aspects of VFF behavior. Copyright 2009 Wiley Periodicals, Inc.

The effects of levofloxacin on rabbit fibroblast-like synoviocytes in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Yang; Lu, Kaihang; Deng, Yu

2012-12-01

It is widely accepted that tendon and cartilage are adversely affected with the toxic effects of quinolones. However, the effects of quinolones on synovium have not been deciphered completely. In this study, our main objective was to investigate the effects of levofloxacin, a typical quinolone antibiotic drug, on fibroblast-like synoviocytes (FLSs) in vitro. FLSs of rabbits were treated with levofloxacin at different concentrations (0, 14, 28, 56, 112 and 224 μM). The possible cytotoxic effects of levofloxacin on FLS were determined. Levofloxacin significantly reduced the cell viabilities, gene expression of hyaluronan synthase-2 (HAS-2), and the level of hyaluronan in FLSs.more » Moreover, levofloxacin-induced concentration-dependent increases of apoptosis and active caspase-3 were determined in this study. Ultrastructural damages of FLSs were observed by electron microscopy. The mRNA expression levels of matrix metalloproteinase (MMP)-3 and MMP-13 were increased in FLSs treated with levofloxacin. In addition, levofloxacin played a role in suppressing the expression of interleukin (IL)-1 and IL-6. Our data suggest that the cytotoxic effects of levofloxacin on FLS were shown to be able to affect cell viability and HA synthesis capacity. The potential mechanisms of the cytotoxic effects may be attributed to the apoptosis and increased expression of MMPs. -- Highlights: ► Levofloxacin decreases hyaluronic acid synthesis in fibroblast-like synoviocytes. ► Levofloxacin exerts pro-apoptosis effects on fibroblast-like synoviocytes. ► Levofloxacin increases gene expression of MMPs in fibroblast-like synoviocytes. ► Levofloxacin exerts anti-inflammatory effects on fibroblast-like synoviocytes.« less

Hyaluronan Does Not Regulate Human Epidermal Keratinocyte Proliferation and Differentiation*

PubMed Central

Malaisse, Jérémy; Pendaries, Valérie; Hontoir, Fanny; De Glas, Valérie; Van Vlaender, Daniel; Simon, Michel; Lambert de Rouvroit, Catherine; Poumay, Yves; Flamion, Bruno

2016-01-01

Hyaluronan (HA) is synthesized by three HA synthases (HAS1, HAS2, and HAS3) and secreted in the extracellular matrix. In human skin, large amounts of HA are found in the dermis. HA is also synthesized by keratinocytes in the epidermis, although its epidermal functions are not clearly identified yet. To investigate HA functions, we studied the effects of HA depletion on human keratinocyte physiology within in vitro reconstructed human epidermis. Inhibition of HA synthesis with 4-methylumbelliferone (4MU) did not modify the expression profile of the epidermal differentiation markers involucrin, keratin 10, and filaggrin during tissue reconstruction. In contrast, when keratinocytes were incubated with 4MU, cell proliferation was decreased. In an attempt to rescue the proliferation function, HA samples of various mean molecular masses were added to keratinocyte cultures treated with 4MU. These samples were unable to rescue the initial proliferation rate. Furthermore, treatments with HA-specific hyaluronidase, although removing almost all HA from keratinocyte cultures, did not alter the differentiation or proliferation processes. The differences between 4MU and hyaluronidase effects did not result from differences in intracellular HA, sulfated glycosaminoglycan concentration, apoptosis, or levels of HA receptors, all of which remained unchanged. Similarly, knockdown of UDP-glucose 6-dehydrogenase (UGDH) using lentiviral shRNA effectively decreased HA production but did not affect proliferation rate. Overall, these data suggest that HA levels in the human epidermis are not directly correlated with keratinocyte proliferation and differentiation and that incubation of cells with 4MU cannot equate with HA removal. PMID:26627828

Fetal development of the elastic-fiber-mediated enthesis in the human middle ear.

PubMed

Takanashi, Yoshitaka; Shibata, Shunichi; Katori, Yukio; Murakami, Gen; Abe, Shinichi; Rodríguez-Vázquez, Jose Francisco; Kawase, Tetsuaki

2013-10-01

In the human middle ear, the annular ligament of the incudostapedial joint and the insertions of the tensor tympani and stapedius muscles contain abundant elastic fibers; i.e., the elastic-fiber-mediated entheses. Hyaluronan also coexists with the elastic fibers. In the present study using immunohistochemistry, we demonstrated the distribution of elastin not only in the incudostapedial joint but also in the other two joints of the middle ear in adults and fetuses. In adults, the expression of elastin did not extend out of the annular ligament composed of mature elastic fibers but clearly overlapped with it. Electron microscopic observations of the annular ligament demonstrated a few microfibrils along the elastic fibers. Thus, in contrast to the vocal cord, the middle ear entheses seemed not to contain elaunin and oxytalan fibers. In mid-term fetuses (at approximately 15-16 weeks of gestation) before opening of the external acoustic meatus, the incudostapedial joint showed abundant elastic fibers, but the incudomalleolar and stapediovestibular joints did not. At this stage, hyaluronan was not colocalized, but distributed diffusely in loose mesenchymal tissues surrounding the ear ossicles. Therefore, fetal development of elastin and elastic fibers in the middle ear entheses is unlikely to require acoustic oscillation. In late-stage fetuses (25-30 weeks), whose ear ossicles were almost the same size as those in adults, we observed bundling and branching of elastic fibers. However, hyaluronan expression was not as strong as in adults. Colocalization between elastic fibers and hyaluronan appeared to be a result of postnatal maturation of the entheses. Copyright © 2013 Elsevier GmbH. All rights reserved.

Go Fly a Chitin: The Mystery of Chitin and Chitinases in Vertebrate Tissues.

PubMed

Stern, Robert

2017-01-01

A controversy arose decades ago whether the DG42 gene product expressed during frog embryogenesis synthesized hyaluronan or chitin. Both sets of investigators were correct. It is now possible to understand how prescient those findings were. Synthesis of a seven to nine chitin sugar chain fragment is required before hyaluronan synthesis begins. Thus, DG42 indeed synthesizes both hyaluronan and chitin. Hyaluronan turns over rapidly in vertebrate tissues, but chitin oligomers are difficult to degrade. They accumulate and can cause pathology. Chitin is a simple beta-linked repeating sugar homopolymer found prominently in the building block structures of fungi, molluscs, arthropods, and other forms of invertebrate life. It is a highly resistant insoluble material requiring chitin synthases for production and chitinases for degradation. Mysteriously, chitins and chitinases also occur in vertebrate tissues, while it had previously been assumed that no chitins were contained therein. That assumption is now challenged based on recent biochemical evidence. Chitin does accumulate in many tissues, but may be particularly toxic to neurons. Its accumulation in the brain may account for the cognitive decline found in patients with Alzheimer's disease. The DG42 observations together with the participation of chitins and chitinases in several human diseases, among which in addition to Alzheimer's disease include Gaucher's disease, asthma, and aspects of abnormal immune recognition justify a reexamination of these topics. The purpose of this review is to summarize data in order to place chitins and their attendant enzymes in a rational framework in an attempt to create a cohesive story.

Lectin histochemical aspects of mucus function in the oesophagus of the reticulated python (Python reticulatus).

PubMed

Meyer, W; Luz, S; Schnapper, A

2009-08-01

Using lectin histochemistry, the study characterizes basic functional aspects of the mucus produced by the oesophageal epithelium of the Reticulated python (Python reticulatus). Reaction staining varied as related to the two epithelium types present, containing goblet cells and ciliary cells. Remarkable intensities were achieved especially in the luminal mucus layer and the fine mucus covering the epithelial ciliary border for Con A (alpha-D-Man; alpha-D-Glc) as part of neutral glycoproteins, Limax flavus agglutinin (NeuNac = NeuNgc), emphasizing that water binding hyaluronan provides a hydrated interface conductive to the passage of material and UEA-I (alpha-L-Fuc), corroborating the view that fucose-rich highly viscous mucus is helpful against mechanical stress during prey transport.

PDGF Suppresses the Sulfation of CD44v and Potentiates CD44v-Mediated Binding of Colon Carcinoma Cells to Fibrin under Flow

PubMed Central

Alves, Christina S.; Konstantopoulos, Konstantinos

2012-01-01

Fibrin(ogen) mediates sustained tumor cell adhesion and survival in the pulmonary vasculature, thereby facilitating the metastatic dissemination of tumor cells. CD44 is the major functional fibrin receptor on colon carcinoma cells. Growth factors, such as platelet-derived growth factor (PDGF), induce post-translational protein modifications, which modulate ligand binding activity. In view of the roles of PDGF, fibrin(ogen) and CD44 in cancer metastasis, we aimed to delineate the effect of PDGF on CD44-fibrin recognition. By immunoprecipitating CD44 from PDGF-treated and untreated LS174T colon carcinoma cells, which express primarily CD44v, we demonstrate that PDGF enhances the adhesion of CD44v-coated beads to immobilized fibrin. Enzymatic inhibition studies coupled with flow-based adhesion assays and autoradiography reveal that PDGF augments the binding of CD44v to fibrin by significantly attenuating the extent of CD44 sulfation primarily on chondroitin and dermatan sulfate chains. Surface plasmon resonance assays confirm that PDGF enhances the affinity of CD44v-fibrin binding by markedly reducing its dissociation rate while modestly increasing the association rate. PDGF mildly reduces the affinity of CD44v-hyaluronan binding without affecting selectin-CD44v recognition. The latter is attributed to the fact that CD44v binds to selectins via sialofucosylated O-linked residues independent of heparan, dermatan and chondroitin sulfates. Interestingly, PDGF moderately reduces the sulfation of CD44s and CD44s-fibrin recognition. Collectively, these data offer a novel perspective into the mechanism by which PGDF regulates CD44-dependent binding of metastatic colon carcinoma cells to fibrin(ogen). PMID:23056168

Interaction of chitin/chitosan with salivary and other epithelial cells-An overview.

PubMed

Patil, Sharvari Vijaykumar; Nanduri, Lalitha S Y

2017-11-01

Chitin and its deacetylated form, chitosan, have been widely used for tissue engineering of both epithelial and mesenchymal tissues. Epithelial cells characterised by their sheet-like tight cellular arrangement and polarised nature, constitute a major component in various organs and play a variety of roles including protection, secretion and maintenance of tissue homeostasis. Regeneration of damaged epithelial tissues has been studied using biomaterials such as chitin, chitosan, hyaluronan, gelatin and alginate. Chitin and chitosan are known to promote proliferation of various embryonic and adult epithelial cells. However it is not clearly understood how this activity is achieved or what are the mechanisms involved in the chitin/chitosan driven proliferation of epithelial cells. Mechanistic understanding of influence of chitin/chitosan on epithelial cells will guide us to develop more targeted regenerative scaffold/hydrogel systems. Therefore, current review attempts to elicit a mechanistic insight into how chitin and chitosan interact with salivary, mammary, skin, nasal, lung, intestinal and bladder epithelial cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Albumin extravasation and tissue washout of hyaluronan after plasma volume expansion with crystalloid or hypooncotic colloid solutions.

PubMed

Berg, S; Golster, M; Lisander, B

2002-02-01

Intravascular volume expansion is followed by loss of fluid from the circulation. The extravasation of albumin in this readjustment is insufficiently known. Twelve male volunteers participated, each in three separate sessions, in a controlled, randomised, open fashion. They received one of the following: albumin 40 g/L,(7.1 mL/kg, i.e. 500 mL per 70 kg); Ringer's acetate (21.4 mL/kg), or dextran 30 g/L (7.1 mL/kg). The fluids were infused during 30 min and the subjects were followed for 180 min. ECG, arterial oxygen saturation and non-invasive arterial pressure were recorded. Haemoglobin, haematocrit, serum albumin and osmolality, plasma colloid osmotic pressure and hyaluronan concentration were determined in venous samples. The serum albumin concentration decreased (P < 0.05, anova) following Ringer's acetate or dextran, whereas serum osmolality was unchanged in all groups. The colloid osmotic pressure decreased (P < 0.05) after the Ringer solution. The blood volume increase was estimated from the decrease in haemoglobin concentration and did not differ between the three fluids. The cumulated extravasation of albumin was largest following albumin (10.4 +/- 5.4 g, mean +/- SD), less following dextran (5.6 +/- 5.0 g) and negligible in the Ringer group (0.5 +/- 10.0 g; P < 0.05 against albumin). However, the Ringer solution increased the plasma concentration of hyaluronan drastically. Infusion of hypotonic colloidal solutions entails net loss of albumin from the vascular space. This is not the case after Ringer's acetate. Increased interstitial hydration from the latter fluid is followed by lymphatic wash out of hyaluronan.

Expression of a monocarboxylate transporter in reticular cells of the mouse lymph node and its involvement in the uptake of exogenous particles.

PubMed

Zheng, Miao; Ishiguro-Oonuma, Toshina; Iwanaga, Toshihiko

2014-01-01

The monocarboxylate transporter (MCT)-1 plays an important role in the transfer of monocarboxylate metabolites such as lactate, ketone bodies, and acetic acid. The present study revealed the selective localization of MCT1 in reticular cells of the murine lymph node. An intense MCT1 immunoreactivity was found in the reticular cells forming a cellular network together with sinus-lining cells in the medullary sinuses and in cells covering the inside of subcapsular sinuses.Electron-microscopically, MCT1 was localized along the plasma membrane of the reticular cells.The medullary reticular cells vigorously ingested carboxylate-modified latex particles, but any reticular cells within the cortical lymphoid follicles and medullary cords neither expressed MCT1 nor incorporated latex particles. MCT1-immunoreactive reticular cells also expressed LYVE-1,which is a hyaluronan receptor abundant in both the lymphatic endothelium and hepatic sinusoidal epithelium. The selective localization of MCT1 and LYVE-1 suggests a high level of activity for lymphoid reticular cells in the uptake of carboxylate-modified and hyaluronate waste substances circulating in the body.

Oral hyaluronan relieves knee pain: a review.

PubMed

Oe, Mariko; Tashiro, Toshiyuki; Yoshida, Hideto; Nishiyama, Hiroshi; Masuda, Yasunobu; Maruyama, Koh; Koikeda, Takashi; Maruya, Reiko; Fukui, Naoshi

2016-01-27

Hyaluronan (HA) is a component that is particularly abundant in the synovial fluid. Randomized, double-blinded, placebo-controlled trials carried out between 2008 and 2015 have proven the effectiveness of HA for the treatment of symptoms associated with synovitis, and particularly, knee pain, relief of synovial effusion or inflammation, and improvement of muscular knee strength. The mechanism by which HA exerts its effects in the living body, specifically receptor binding in the intestinal epithelia, has gradually been clarified. This review examines the effects of HA upon knee pain as assessed in clinical trials, as well as the mechanism of these effects and the safety of HA.

Optical coherence tomography of lymphatic vessel endothelial hyaluronan receptors in vivo

NASA Astrophysics Data System (ADS)

Si, Peng; Sen, Debasish; Dutta, Rebecca; Yousefi, Siavash; Dalal, Roopa; Winetraub, Yonatan; Liba, Orly; de la Zerda, Adam

2018-02-01

Optical Coherence Tomography (OCT) imaging of living subjects offers millimeters depth of penetration into tissue while maintaining high spatial resolution. However, because most molecular biomarkers do not produce inherent OCT contrast signals, exogenous contrast agents must be employed to achieve molecular imaging. Here we demonstrate that microbeads (μBs) can be used as effective contrast agents to target cellular biomarkers in lymphatic vessels and can be detected by OCT using a phase variance algorithm. We applied this technique to image the molecular dynamics of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) in vivo, which showed significant down-regulation during tissue inflammation.

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

2016-06-05

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Antimalarial Activity of Orally Administered Curcumin Incorporated in Eudragit®-Containing Liposomes.

PubMed

Martí Coma-Cros, Elisabet; Biosca, Arnau; Lantero, Elena; Manca, Maria Letizia; Caddeo, Carla; Gutiérrez, Lucía; Ramírez, Miriam; Borgheti-Cardoso, Livia Neves; Manconi, Maria; Fernàndez-Busquets, Xavier

2018-05-04

Curcumin is an antimalarial compound easy to obtain and inexpensive, having shown little toxicity across a diverse population. However, the clinical use of this interesting polyphenol has been hampered by its poor oral absorption, extremely low aqueous solubility and rapid metabolism. In this study, we have used the anionic copolymer Eudragit ® S100 to assemble liposomes incorporating curcumin and containing either hyaluronan (Eudragit-hyaluronan liposomes) or the water-soluble dextrin Nutriose ® FM06 (Eudragit-nutriosomes). Upon oral administration of the rehydrated freeze-dried nanosystems administered at 25/75 mg curcumin·kg −1 ·day −1 , only Eudragit-nutriosomes improved the in vivo antimalarial activity of curcumin in a dose-dependent manner, by enhancing the survival of all Plasmodium yoelii -infected mice up to 11/11 days, as compared to 6/7 days upon administration of an equal dose of the free compound. On the other hand, animals treated with curcumin incorporated in Eudragit-hyaluronan liposomes did not live longer than the controls, a result consistent with the lower stability of this formulation after reconstitution. Polymer-lipid nanovesicles hold promise for their development into systems for the oral delivery of curcumin-based antimalarial therapies.

Occurrence and structural characterization of versican-like proteoglycan in human vitreous.

PubMed

Theocharis, Achilleas D; Papageorgakopoulou, Nickoletta; Feretis, Elias; Theocharis, Dimitrios A

2002-12-01

Human vitreous gel is a special type of extracellular matrix, in which interpenetrating networks of collagen fibrils and hyaluronan are found. In this study, we report that apart from significant amounts of collagen, hyaluronan and sialylated glycoproteins, it was found that the human vitreous gel also contained low amounts of versican-like proteoglycan. The concentration of versican-like proteoglycan in the whole vitreous is 0.06 mg protein/ml of vitreous gel and represents a small percentage (about 5%) of the total protein content. The versican-like proteoglycan has a molecular mass of 380 kDa, as estimated by gel chromatography. Its core protein is substituted by chondroitin sulphate side chains (average molecular weight 37 kDa), in which 6-sulphated disaccharides predominated. According to the physicochemical data, the number of chondroitin sulphate chains is likely to be 5-7 per molecule. These proteoglycan monomers form large aggregates with endogenous hyaluronan. Versican, which is able to bind lectins via its C-terminal region, may bridge or interconnect various constituents of the extracellular matrix via its terminal domains in order to stabilize large supramolecular complexes at the vitreous, contributing towards the integrity and specific properties of the tissue.

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. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Mounting Pressure in the Microenvironment: Fluids, Solids, and Cells in Pancreatic Ductal Adenocarcinoma.

PubMed

DuFort, Christopher C; DelGiorno, Kathleen E; Hingorani, Sunil R

2016-06-01

The microenvironment influences the pathogenesis of solid tumors and plays an outsized role in some. Our understanding of the stromal response to cancers, particularly pancreatic ductal adenocarcinoma, has evolved from that of host defense to tumor offense. We know that most, although not all, of the factors and processes in the microenvironment support tumor epithelial cells. This reappraisal of the roles of stromal elements has also revealed potential vulnerabilities and therapeutic opportunities to exploit. The high concentration in the stroma of the glycosaminoglycan hyaluronan, together with the large gel-fluid phase and pressures it generates, were recently identified as primary sources of treatment resistance in pancreas cancer. Whereas the relatively minor role of free interstitial fluid in the fluid mechanics and perfusion of tumors has been long appreciated, the less mobile, gel-fluid phase has been largely ignored for historical and technical reasons. The inability of classic methods of fluid pressure measurement to capture the gel-fluid phase, together with a dependence on xenograft and allograft systems that inaccurately model tumor vascular biology, has led to an undue emphasis on the role of free fluid in impeding perfusion and drug delivery and an almost complete oversight of the predominant role of the gel-fluid phase. We propose that a hyaluronan-rich, relatively immobile gel-fluid phase induces vascular collapse and hypoperfusion as a primary mechanism of treatment resistance in pancreas cancers. Similar properties may be operant in other solid tumors as well, so revisiting and characterizing fluid mechanics with modern techniques in other autochthonous cancers may be warranted. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

Bidirectional TSH and IGF-1 receptor cross talk mediates stimulation of hyaluronan secretion by Graves' disease immunoglobins.

PubMed

Krieger, Christine C; Neumann, Susanne; Place, Robert F; Marcus-Samuels, Bernice; Gershengorn, Marvin C

2015-03-01

There is no pathogenetically linked medical therapy for Graves' ophthalmopathy (GO). Lack of animal models and conflicting in vitro studies have hindered the development of such therapy. Recent reports propose that Graves' Igs bind to and activate thyrotropin receptors (TSHRs) and IGF-1 receptors (IGF-1Rs) on cells in orbital fat, stimulating hyaluronan (HA) secretion, a component of GO. The objective of the study was to investigate potential cross talk between TSHRs and IGF-1Rs in the pathogenesis of GO using a sensitive HA assay. Orbital fibroblasts from GO patients were collected in an academic clinical practice and cultured in a research laboratory. Cells were treated with TSH, IGF-1, and a monoclonal Graves' Ig M22. HA was measured by a modified ELISA. Simultaneous activation by TSH and IGF-1 synergistically increased HA secretion from 320 ± 52 for TSH and 430 ± 65 μg/mL for IGF-1 alone, to 1300 ± 95 μg/mL. IGF-1 shifted the TSH EC50 19-fold to higher potency. The dose response to M22 was biphasic. An IGF-1R antagonist inhibited the higher potency phase but had no effect on the lower potency phase. M22 did not cause IGF-1R autophosphorylation. A TSHR antagonist abolished both phases of M22-stimulated HA secretion. M22 stimulation of HA secretion by GO fibroblasts/preadipocytes involves cross talk between TSHR and IGF-1R. This cross talk relies on TSHR activation rather than direct activation of IGF-1R and leads to synergistic stimulation of HA secretion. These data propose a model for GO pathogenesis that explains previous contradictory results and argues for TSHR as the primary therapeutic target for GO.

Hyaluronan influence on the onset of chondrogenic differentiation of mesenchymal stem cells.

PubMed

Dvorakova, Jana; Velebny, Vladimir; Kubala, Lukas

2008-10-01

Hyaluronan (HA) is an abundant component of chondrogenic tissue hence it is often used as a fundamental constituent in cartilage tissue substitutes. However, effects of different molecular weight HA on chondrogenic differentiation are not clear. The aim was to evaluate modulation of mesenchymal stem cell (MSC) early chondrogenesis by HA of molecular weights 100, 600 and 1 500 kDa. HA was applied on MSCs cultured in a pellet system for one, two and three weeks. Chondrogenesis was evaluated by determinations of gene expression of transcription factor Sox-9 and extracellular matrix proteins collagen type II and XI, aggrecan, and COMP by Real-Time PCR and completed with histological analysis. Upon chondrogenic induction, the respective pellets revealed active transcription of the chondrogenic genes together with proceeding accumulation of glycosaminoglycan (GAG) rich extracellular matrix. Sox-9 was also expressed in non-chondrogenic MSC controls. HA treated pellets were not significantly influenced on day 7 of culture. However, on day 14, lowered expression in some of the extracellular matrix proteins appeared together with a moderately smaller amount of GAG content in pellet sections. Nevertheless, the analysis on day 21 has demonstrated that HA did not affect the outcome of the differentiation by the end of the culture. Any difference regarding the molecular weight of the HA was not found. It could be speculated that HA induced a time shift in the phase of the dominant matrix protein onset which was in full compensated by the end of the evaluated time period. Thus, data suggest that HA of any tested molecular weight does not significantly modulate chondrogenesis of MSCs in pellet system.

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

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Extracellular matrix--regulation of cancer invasion and metastasis].

PubMed

Watanabe, Hideto

2010-11-01

Cancer cell invasion comprises steps in the destruction of the basement membrane and migration of cells into the connective tissue. These cells further migrate into lymph ducts and small vessels to reach metastasis. The extracellular matrix (ECM) provides a microenvironment for cells, and its destruction is associated with cancer cell invasion. Among matrix metalloproteinases (MMPs), both MMP-2 and 9 digest type IV collagen, a major component of the basement membrane, and MMP-14/MT1-MMP, a membrane-type MMP, activates MMP-2. Thus, these MMPs play a central role in cancer cell invasion. MMPs also cleave latent forms of growth factors and signaling molecules, releasing and activating them, which influence neo-vascularization and cancer apoptosis. Like proteins, carbohydrates are known to be involved in cancer invasion. Hyaluronan is known to both stimulate and inhibit cancer invasion, depending on its molecular size. Heparanase, which digests heparan sulfate, is known to facilitate cancer invasion and metastasis. In summary, ECM provides a microenvironment that regulates cell behavior and its structure altered by MMPs affects cancer cell invasion.

Hyaluronan-CD44v3 interaction with Oct4-Sox2-Nanog promotes miR-302 expression leading to self-renewal, clonal formation, and cisplatin resistance in cancer stem cells from head and neck squamous cell carcinoma.

PubMed

Bourguignon, Lilly Y W; Wong, Gabriel; Earle, Christine; Chen, Liqun

2012-09-21

Human head and neck squamous cell carcinoma (HNSCC) is a highly malignant cancer associated with major morbidity and mortality. In this study, we determined that human HNSCC-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by high levels of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. These tumor cells also express several stem cell markers (the transcription factors Oct4, Sox2, and Nanog) and display the hallmark CSC properties of self-renewal/clonal formation and the ability to generate heterogeneous cell populations. Importantly, hyaluronan (HA) stimulates the CD44v3 (an HA receptor) interaction with Oct4-Sox2-Nanog leading to both a complex formation and the nuclear translocation of three CSC transcription factors. Further analysis reveals that microRNA-302 (miR-302) is controlled by an upstream promoter containing Oct4-Sox2-Nanog-binding sites, whereas chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-302 expression by HA-CD44 is Oct4-Sox2-Nanog-dependent in HNSCC-specific CSCs. This process results in suppression of several epigenetic regulators (AOF1/AOF2 and DNMT1) and the up-regulation of several survival proteins (cIAP-1, cIAP-2, and XIAP) leading to self-renewal, clonal formation, and cisplatin resistance. These CSCs were transfected with a specific anti-miR-302 inhibitor to silence miR-302 expression and block its target functions. Our results demonstrate that the anti-miR-302 inhibitor not only enhances the expression of AOF1/AOF2 and DNMT1 but also abrogates the production of cIAP-1, cIAP-2, and XIAP and HA-CD44v3-mediated cancer stem cell functions. Taken together, these findings strongly support the contention that the HA-induced CD44v3 interaction with Oct4-Sox2-Nanog signaling plays a pivotal role in miR-302 production leading to AOF1/AOF2/DNMT1 down-regulation and survival of protein activation. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, clonal formation, and chemotherapy resistance in HA-CD44v3-activated head and neck cancer.

Hyaluronan-CD44v3 Interaction with Oct4-Sox2-Nanog Promotes miR-302 Expression Leading to Self-renewal, Clonal Formation, and Cisplatin Resistance in Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma*

PubMed Central

Bourguignon, Lilly Y. W.; Wong, Gabriel; Earle, Christine; Chen, Liqun

2012-01-01

Human head and neck squamous cell carcinoma (HNSCC) is a highly malignant cancer associated with major morbidity and mortality. In this study, we determined that human HNSCC-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by high levels of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. These tumor cells also express several stem cell markers (the transcription factors Oct4, Sox2, and Nanog) and display the hallmark CSC properties of self-renewal/clonal formation and the ability to generate heterogeneous cell populations. Importantly, hyaluronan (HA) stimulates the CD44v3 (an HA receptor) interaction with Oct4-Sox2-Nanog leading to both a complex formation and the nuclear translocation of three CSC transcription factors. Further analysis reveals that microRNA-302 (miR-302) is controlled by an upstream promoter containing Oct4-Sox2-Nanog-binding sites, whereas chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-302 expression by HA-CD44 is Oct4-Sox2-Nanog-dependent in HNSCC-specific CSCs. This process results in suppression of several epigenetic regulators (AOF1/AOF2 and DNMT1) and the up-regulation of several survival proteins (cIAP-1, cIAP-2, and XIAP) leading to self-renewal, clonal formation, and cisplatin resistance. These CSCs were transfected with a specific anti-miR-302 inhibitor to silence miR-302 expression and block its target functions. Our results demonstrate that the anti-miR-302 inhibitor not only enhances the expression of AOF1/AOF2 and DNMT1 but also abrogates the production of cIAP-1, cIAP-2, and XIAP and HA-CD44v3-mediated cancer stem cell functions. Taken together, these findings strongly support the contention that the HA-induced CD44v3 interaction with Oct4-Sox2-Nanog signaling plays a pivotal role in miR-302 production leading to AOF1/AOF2/DNMT1 down-regulation and survival of protein activation. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, clonal formation, and chemotherapy resistance in HA-CD44v3-activated head and neck cancer. PMID:22847005

Covalent growth factor tethering to direct neural stem cell differentiation and self-organization.

PubMed

Ham, Trevor R; Farrag, Mahmoud; Leipzig, Nic D

2017-04-15

Tethered growth factors offer exciting new possibilities for guiding stem cell behavior. However, many of the current methods present substantial drawbacks which can limit their application and confound results. In this work, we developed a new method for the site-specific covalent immobilization of azide-tagged growth factors and investigated its utility in a model system for guiding neural stem cell (NSC) behavior. An engineered interferon-γ (IFN-γ) fusion protein was tagged with an N-terminal azide group, and immobilized to two different dibenzocyclooctyne-functionalized biomimetic polysaccharides (chitosan and hyaluronan). We successfully immobilized azide-tagged IFN-γ under a wide variety of reaction conditions, both in solution and to bulk hydrogels. To understand the interplay between surface chemistry and protein immobilization, we cultured primary rat NSCs on both materials and showed pronounced biological effects. Expectedly, immobilized IFN-γ increased neuronal differentiation on both materials. Expression of other lineage markers varied depending on the material, suggesting that the interplay of surface chemistry and protein immobilization plays a large role in nuanced cell behavior. We also investigated the bioactivity of immobilized IFN-γ in a 3D environment in vivo and found that it sparked the robust formation of neural tube-like structures from encapsulated NSCs. These findings support a wide range of potential uses for this approach and provide further evidence that adult NSCs are capable of self-organization when exposed to the proper microenvironment. For stem cells to be used effectively in regenerative medicine applications, they must be provided with the appropriate cues and microenvironment so that they integrate with existing tissue. This study explores a new method for guiding stem cell behavior: covalent growth factor tethering. We found that adding an N-terminal azide-tag to interferon-γ enabled stable and robust Cu-free 'click' immobilization under a variety of physiologic conditions. We showed that the tagged growth factors retained their bioactivity when immobilized and were able to guide neural stem cell lineage commitment in vitro. We also showed self-organization and neurulation from neural stem cells in vivo. This approach will provide another tool for the orchestration of the complex signaling events required to guide stem cell integration. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Hyaluronan--a review of the recent patent literature.

PubMed

Moore, A R

2000-02-01

A review of patent literature surrounding the use of hyaluronan (HA) and its derivatives, or agents capable of modulating endogenous levels, is presented. Indications are many and varied and include arthritis, abnormal wound healing, neoplasia, prevention of surgical adhesions and dermatological disorders. In many cases, it is the physicochemical and biological properties of HA itself that have been exploited. For example, HA and/or its derivatives have been suggested as a synovial fluid replacement, a vehicle for topical application of drugs, a matrix for sustained drug release and a putative drug delivery system (targeting HA receptors at sites of pathology). In each of these examples, the lack of toxicity of HA and its biocompatability are important considerations.

Functional expression of BMP7 receptors in oral epithelial cells. Interleukin-17F production in response to BMP7.

PubMed

Nishio, Kensuke; Ozawa, Yasumasa; Ito, Hisanori; Kifune, Takashi; Narita, Tatsuya; Iinuma, Toshimitsu; Gionhaku, Nobuhito; Asano, Masatake

2017-10-01

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily. Recently, BMP7 has been demonstrated to be produced by salivary glands and contribute to embryonic branching in mice. The BMP7 in saliva is thought to be delivered to the oral cavity and is expected to contact with stratified squamous epithelial cells which line the surface of oral mucosa. In this study, we attempted to investigate the effects of BMP7 on oral epithelial cells. The expression of BMP receptors was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). OSCCs were stimulated with human recombinant BMP7 (hrBMP7) and the phosphorylation status of Smad1/5/8 was examined by western blotting. For microarray analysis, Ca9-22 cells were stimulated with 100 ng/mL of hrBMP7 and total RNA was extracted and subjected to real-time PCR. The 5'-untranslated region (5'-UTR) of IL-17 F gene was cloned to pGL4-basic vector and used for luciferase assay. Ca9-22 cells were pre-incubated with DM3189, a specific inhibitor of Smad1/5/8, for inhibition assay. All isoforms of type I and type II BMP receptors were expressed in both Ca9-22 and HSC3 cells and BMP7 stimulation resulted in the phosphorylation of Smad1/5/8 in both cell lines. The microarray analysis revealed the induction of interleukin-17 F (IL-17 F), netrin G2 (NTNG2) and hyaluronan synthase 1 (HAS1). Luciferase assay using the 5'-UTR of the IL-17 F gene revealed transcriptional regulation. Induced IL-17 F production was further confirmed at the protein level by ELISA. Smad1/5/8 inhibitor pretreatment decreased IL-17 F expression levels in the cells.

Anti-bacterial factors secreted from cumulus cells of ovulated COCs enhance sperm capacitation during in vitro fertilization.

PubMed

Shimada, Masayuki; Mihara, Toshihiro; Kawashima, Ikko; Okazaki, Tetsuji

2013-02-01

The aim of this study was to find immune-related genes expressed in cumulus cells of ovulated cumulus oocyte complexes (COCs) and to clear the functional roles during fertilization process. Ovulated COCs were collected from oviduct 16 hr after the hCG injections followed by eCG priming. The cumulus cells were used for RT-PCR or western blotting study. COCs were also used for in vitro fertilization study. Cramp, Trf, Lyz2, S100a8, and S100a9 were expressed in cumulus cells during ovulation process. The protein levels of CRAMP or transferrin were detected in ovulated COCs and then secreted into hyaluronan-rich matrix. The high dose of these factors reduced the proliferative activity of E. coli; however, the lower levels of them significantly increased the rate of fertilization in in vitro via the induction of sperm capacitation. Cumulus-secreted anti-bacterial factors act on sperm to induce sperm capacitation. © 2012 John Wiley & Sons A/S.

Macroscopic and histological investigation of guanaco footpads (Lama guanicoe, Müller 1776).

PubMed

König, Horst Erich; Skewes, Oscar; Helmreich, Magda; Böck, Peter

2015-03-01

The surface of guanaco footpads is characterized by hairless skin with up to 4-mm-thick stratum corneum that protects from abrasion. The horny layer is pliable and elastic, and ensures firm contact with irregular ground. It is padded with a particular structure of the subcutaneous layer, the digital cushion. The flat cushions of each of the two digits are of elongated ovate shape, each about 45-mm long, up to 20-mm wide, and 8-mm thick. The cushions are lined by a 1-2-mm-thick capsule that resembles a tunica albuginea. The capsule consists of coarse collagen fibers, with elastic fibers absent. The cushion capsule and dermis approach each other, and fuse along a line that runs parallel to the longitudinal axes of cushion and digit. Loose connective tissue rich in elastic fibers and acidic glycosaminoglycans separates dermis and cushion capsule lateral to the narrow interconnecting zone. The cushion capsule encloses cloudy yellowish, gelatinous material. Microscopy shows bundles of elastic fibers in abundant mucinous matrix. Tightly gathered elastic bundles adjoin the inner surface of the capsule. Rough cords of elastic fibers branch out from there and traverse to the opposite side. The cushion is pressed flat, and elastic fibers are stretched when bearing weight. With relief of load, elastic fibers contract and reset the cushion's shape. Contractile cells are absent. A resistant capsule and easily malleable mucinous contents establish the functioning as a gel pad. Mucinous connective tissue between elastic fiber bundles contains abundant basophilic matrix. Hyaluronan, chondroitin sulfate, and dermatan sulfate are main matrix constituents. Spindle-shaped or stellate fibroblasts contain vimentin, S100 protein, and neuron specific enolase. Moprhology, staining characteristics and synthesis activities of these cells meet the criteria to be classified as myxoid cells. The connective tissue in guanaco digital cushions represents myxoid tissue. © 2014 Wiley Periodicals, Inc.

A simple and efficient feeder-free culture system to up-scale iPSCs on polymeric material surface for use in 3D bioprinting.

PubMed

Wong, Chui-Wei; Chen, You-Tzung; Chien, Chung-Liang; Yu, Tien-Yu; Rwei, Syang-Peng; Hsu, Shan-Hui

2018-01-01

The 3D bioprinting and cell/tissue printing techniques open new possibilities for future applications. To facilitate the 3D bioprinting process, a large amount of living cells are required. Induced pluripotent stem cells (iPSCs) represent a promising cell source for bioprinting. However, the maintenance and expansion of undifferentiated iPSCs are expensive and time consuming. Therefore, in this study a culture method to obtain a sufficient amount of healthy and undifferentiated iPSCs in a short-term period was established. The iPSCs could be passaged for twice on tissue culture polystyrene (TCPS) dish with the conditional medium and could adapt to the feeder-free environment. Feeder-free dishes were further prepared from chitosan, chitosan-hyaluronan, silk fibroin, and polyurethane (PU1 and PU2) two-dimensional substrates. The iPSCs cultured on the chitosan substrates showed a higher proliferation rate without losing the stemness feature. Among the different materials, PU2 could be prepared as a thermoresponsive hydrogel, which was a potential ink for 3D bioprinting. The iPSCs cultured on PU2 substrates well survived when further embedded in PU2 hydrogel. Moreover, PU2 hydrogel printed with iPSCs remained structural integrity. The use of PU2 hydrogel to embed iPSCs reduced the injury to iPSCs by shear stress. These results indicate that iPSCs could be expanded on chitosan or PU2 membranes without the feeder layer and then printed in PU2 hydrogel. The combination of these steps could offer a new possibility for future applications of iPSC-based 3D bioprinting in tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

In Vivo Engineering of the Vocal Fold ECM with Injectable HA Hydrogels -- Late Effects on Tissue Repair and Biomechanics in a Rabbit Model

PubMed Central

Klemuk, Sarah A.; Chen, Xia; Quinchia Johnson, Beatriz H.

2009-01-01

Objectives To determine if the utilization of injectable chemically-modified hyaluronan (HA) derivative at the time of intentional vocal fold resection may facilitate wound repair and preserve the unique viscoelastic properties of the extracellular matrix and lamina propria 6 months after treatment. Study Design Prospective, controlled animal study. Methods Twelve rabbit vocal folds were biopsied bilaterally, and the left side of vocal fold was treated with Extracel, an injectable, chemically-modified HA derivative, and the right side of vocal fold was injected with saline as control at the time of resection. Animals were sacrificed six months after biopsy and injection. Outcomes measured include transcription levels for procollagen, fibronectin, fibromodulin, TGF-β1, hyaluronan synthase and hyaluronidase and tissue biomechanics -- viscosity and elasticity. Results Extracel treated vocal folds were found to have significantly less fibrosis than saline treated controls. Extracel treated vocal folds had significantly improved biomechanical properties of elasticity and viscosity. Significantly decreased levels of fibronectin, fibromodulin, TGF-β1, procollagen I and hyaluronan synthase were measured. Conclusions Prophylactic in vivo manipulation of the extracellular matrix with an injectable HA hydrogel appears to induce vocal fold tissue regeneration to yield improved tissue composition and biomechanical properties at 6 months. PMID:20456912

Therapeutic efficacy of intra-articular hyaluronan derivative and platelet-rich plasma in mice following axial tibial loading

PubMed Central

Duan, Xin; Sandell, Linda J.; Chinzei, Nobuaki; Holguin, Nilsson; Silva, Matthew J.; Schiavinato, Antonella

2017-01-01

Objective To investigate the therapeutic potential of intra-articular hyaluronan-derivative HYADD® 4-G and/or platelet-rich plasma (PRP) in a mouse model of non-invasive joint injury. Methods Non-invasive axial tibial loading was used to induce joint injury in 10-week-old C57BL/6J mice (n = 86). Mice underwent a single loading of either 6 Newton (N) or 9N axial tibial compression. HYADD® 4-G was injected intra-articularly at 8 mg/mL or 15 mg/mL either before or after loading with or without PRP. Phosphate-buffered-saline was injected as control. Knee joints were harvested at 5 or 56 days post-loading and prepared for micro-computed tomography scanning and subsequently processed for histology. Immunostaining was performed for aggrecan to monitor its distribution, for CD44 to monitor chondrocyte reactive changes and for COMP (cartilage oligomeric matrix protein) as an index for cartilage matrix changes related to loading and cartilage injury. TUNEL assay was performed to identify chondrocyte apoptosis. Results Loading initiated cartilage proteoglycan loss and chondrocyte apoptosis within 5 days with slowly progressive post-traumatic osteoarthritis (no cartilage degeneration, but increased synovitis and ectopic calcification after 9N loading) at 56 days. Mice treated with repeated HYADD® 4-G (15 mg/mL) or HYADD® 4-G (8 mg/mL) ± PRP or PRP alone exhibited no significant improvement in the short-term (5 days) and long-term (56 days) consequences of joint loading except for a trend for improved bone changes compared to non-loaded joints. Conclusion While we failed to show an overall effect of intra-articular delivery of hyaluronan-derivative and/or PRP in reversing/protecting the pathological events in cartilage and synovium following joint injury, some bone alterations were relatively less severe with hyaluronan-derivative at higher concentration or in association with PRP. PMID:28406954

Unilateral dampening of Bmp activity by nodal generates cardiac left-right asymmetry.

PubMed

Veerkamp, Justus; Rudolph, Franziska; Cseresnyes, Zoltan; Priller, Florian; Otten, Cécile; Renz, Marc; Schaefer, Liliana; Abdelilah-Seyfried, Salim

2013-03-25

Signaling by Nodal and Bmp is essential for cardiac laterality. How activities of these pathways translate into left-right asymmetric organ morphogenesis is largely unknown. We show that, in zebrafish, Nodal locally reduces Bmp activity on the left side of the cardiac field. This effect is mediated by the extracellular matrix enzyme Hyaluronan synthase 2, expression of which is induced by Nodal. Unilateral reduction of Bmp signaling results in lower expression of nonmuscle myosin II and higher cell motility on the left, driving asymmetric displacement of the entire cardiac field. In silico modeling shows that left-right differences in cell motility are sufficient to induce a robust, directional migration of cardiac tissue. Thus, the mechanism underlying the formation of cardiac left-right asymmetry involves Nodal modulating an antimotogenic Bmp activity. Copyright © 2013 Elsevier Inc. All rights reserved.

Phase I/II Clinical Trial of Hyaluronan-Cisplatin Nanoconjugate for the Treatment of Spontaneous Canine Cancers

PubMed Central

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

2015-01-01

Objective To conduct an open label, multi-dose Phase I/II clinical study in spontaneous canine cancers and evaluate the pharmacokinetics, safety, and efficacy of the hyaluronan-based cisplatin formulation (HA-Pt). Animals 13 dogs with heterogeneous, naturally occurring cancers. Procedures The dogs received up to four injections of 10-30 mg/m2 HA-Pt into the tumor or peritumoral sub-mucosa at three-week intervals. Blood sample (2 mL) was collected from the jugular catheter at 0.5, 1, 2, 4, and 24 hours following drug administration. A complete blood count and renal profile with urinalysis were conducted prior to and one week after each treatment. Tumor measurements were collected three weeks following each administration to assess response. Results Of the 13 dogs with heterogeneous, naturally occurring cancers, 23% had complete response and 15% had partial response or stable disease. Among the dogs that received drug with low diaquated content, the complete response rate for SCC was 3/7 (43%). Myelosuppression and cardiac toxicity were observed for 38% and 19% of the dogs, respectively. The formulation did not cause nephrotoxicity, the dose-limiting toxicity of standard cisplatin, in any dogs. Conclusions and Clinical Relevance The HA-Pt formulation demonstrated positive response in spontaneous canine squamous cell carcinomas. It did not cause nephrotoxicity in any patients. Canine oral SCC is very homogenous in progression and drug response to human HNSCC, and these results could be useful in developing human treatments. PMID:27580113

Pulmonary vascular clearance of harmful endogenous macromolecules in a porcine model of acute liver failure.

PubMed

Nedredal, Geir I; Elvevold, Kjetil; Chedid, Marcio F; Ytrebø, Lars M; Rose, Christopher F; Sen, Sambit; Smedsrød, Bård; Jalan, Rajiv; Revhaug, Arthur

2016-01-01

Pulmonary complications are common in acute liver failure (ALF). The role of the lungs in the uptake of harmful soluble endogenous macromolecules was evaluated in a porcine model of ALF induced by hepatic devascularization (n = 8) vs. controls (n = 8). In additional experiments, pulmonary uptake was investigated in healthy pigs. Fluorochrome-labeled modified albumin (MA) was applied to investigate the cellular uptake. As compared to controls, the ALF group displayed a 4-fold net increased lung uptake of hyaluronan, and 5-fold net increased uptake of both tissue plasminogen activator and lysosomal enzymes. Anatomical distribution experiments in healthy animals revealed that radiolabeled MA uptake (taken up by the same receptor as hyaluronan) was 53% by the liver, and 24% by the lungs. The lung uptake of LPS was 14% whereas 60% remained in the blood. Both fluorescence and electron microscopy revealed initial uptake of MA by pulmonary endothelial cells (PECs) with later translocation to pulmonary intravascular macrophages (PIMs). Moreover, the presence of PIMs was evident 10 min after injection. Systemic inflammatory markers such as leukopenia and increased serum TNF-α levels were evident after 20 min in the MA and LPS groups. Significant lung uptake of harmful soluble macromolecules compensated for the defect liver scavenger function in the ALF-group. Infusion of MA induced increased TNF-α serum levels and leukopenia, similar to the effect of the known inflammatory mediator LPS. These observations suggest a potential mechanism that may contribute to lung damage secondary to liver disease.

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

PubMed Central

Dong, Yifei; Arif, Arif A.; Poon, Grace F. T.; Hardman, Blair; Dosanjh, Manisha; Johnson, Pauline

2016-01-01

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function. PMID:27404290

Plantar fascia anatomy and its relationship with Achilles tendon and paratenon

PubMed Central

Stecco, Carla; Corradin, Marco; Macchi, Veronica; Morra, Aldo; Porzionato, Andrea; Biz, Carlo; De Caro, Raffaele

2013-01-01

Although the plantar fascia (PF) has been studied quite well from a biomechanical viewpoint, its microscopic properties have been overlooked: nothing is known about its content of elastic fibers, the features of the extracellular matrix or the extent of innervation. From a functional and clinical standpoint, the PF is often correlated with the triceps surae muscle, but the anatomical grounds for this link are not clear. The aim of this work was to focus on the PF macroscopic and microscopic properties and study how Achilles tendon diseases might affect it. Twelve feet from unembalmed human cadavers were dissected to isolate the PF. Specimens from each PF were tested with various histological and immunohistochemical stains. In a second stage, 52 magnetic resonance images (MRI) obtained from patients complaining of aspecific ankle or foot pain were analyzed, dividing the cases into two groups based on the presence or absence of signs of degeneration and/or inflammation of the Achilles tendon. The thickness of PF and paratenon was assessed in the two groups and statistical analyses were conducted. The PF is a tissue firmly joined to plantar muscles and skin. Analyzing its possible connections to the sural structures showed that this fascia is more closely connected to the paratenon of Achilles tendon than to the Achilles tendon, through the periosteum of the heel. The PF extended medially and laterally, continuing into the deep fasciae enveloping the abductor hallucis and abductor digiti minimi muscles, respectively. The PF was rich in hyaluronan, probably produced by fibroblastic-like cells described as ‘fasciacytes’. Nerve endings and Pacini and Ruffini corpuscles were present, particularly in the medial and lateral portions, and on the surface of the muscles, suggesting a role for the PF in the proprioception of foot. In the radiological study, 27 of the 52 MRI showed signs of Achilles tendon inflammation and/or degeneration, and the PF was 3.43 ± 0.48 mm thick (99%CI and SD = 0.95), as opposed to 2.09 ± 0.24 mm (99%CI, SD = 0.47) in the patients in which the MRI revealed no Achilles tendon diseases; this difference in thickness of 1.29 ± 0.57 mm (99%CI) was statistically significant (P < 0.001). In the group of 27/52 patients with tendinopathies, the PF was more than 4.5 mm thick in 5, i.e. they exceeded the threshold for a diagnosis of plantar fasciitis. None of the other 25/52 paitents had a PF more than 4 mm thick. There was a statistically significant correlation between the thicknesses of the PF and the paratenon. These findings suggest that the plantar fascia has a role not only in supporting the longitudinal arch of the foot, but also in its proprioception and peripheral motor coordination. Its relationship with the paratenon of the Achilles tendon is consistent with the idea of triceps surae structures being involved in the PF pathology, so their rehabilitation can be considered appropriate. Finally, the high concentration of hyaluronan in the PF points to the feasibility of using hyaluronan injections in the fascia to treat plantar fasciitis. PMID:24028383

Polypeptide Grafted Hyaluronan: Synthesis and Characterization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xiaojun; Messman, Jamie M; Mays, Jimmy

2010-01-01

Poly(L-leucine) grafted hyaluronan (HA-g-PLeu) has been synthesized via a Michael addition reaction between primary amine terminated poly(L-leucine) and acrylate-functionalized HA (TBAHA-acrylate). The precursor hyaluronan was first functionalized with acrylate groups by reaction with acryloyl chloride in the presence of triethylamine in N,N-dimethylformamide. 1H NMR analysis of the resulting product indicated that an increase in the concentration of acryloylchoride with respect to hydroxyl groups on HA has only a moderate effect on functionalization efficiency, f. A precise control of stoichiometry was not achieved, which could be attributed to partial solubility of intermolecular aggregates and the hygroscopic nature of HA. Michael additionmore » at high [PLeu- NH2]/[acrylate]TBAHA ratios gave a molar grafting ratio of only 0.20 with respect to the repeat unit of HA, indicating grafting limitation due to insolubility of the grafted HA-g-PLeu. Soluble HA-g-PLeu graft copolymers were obtained for low grafting ratios (<0.039) with <8.6% by mass of PLeu and were characterized thoroughly using light scattering, 1H NMR, FT-IR, and AFM techniques. Light scattering experiments showed a strong hydrophobic interaction between PLeu chains, resulting in aggregates with segregated nongrafted HA segments. This yields local networks of aggregates, as demonstrated by atomic force microscopy. Circular dichroism spectroscopy showed a -sheet conformation for aggregates of poly(L-leucine).« less

Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis.

PubMed

Kooy, Floor K; Ma, Muyuan; Beeftink, Hendrik H; Eggink, Gerrit; Tramper, Johannes; Boeriu, Carmen G

2009-01-15

Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis.

The influence of hyaluronan on the structure of a DPPC-bilayer under high pressures.

PubMed

Zander, Thomas; Wieland, D C Florian; Raj, Akanksha; Wang, Min; Nowak, Benedikt; Krywka, Christina; Dėdinaitė, Andra; Claesson, Per Martin; Garamus, Vasil M; Schreyer, Andreas; Willumeit-Römer, Regine

2016-06-01

The superior lubrication properties of synovial joints have inspired many studies aiming at uncovering the molecular mechanisms which give rise to low friction and wear. However, the mechanisms are not fully understood yet, and, in particular, it has not been elucidated how the biolubricants present at the interface of cartilage respond to high pressures, which arise during high loads of joints. In this study we utilize a simple model system composed of two biomolecules that have been implied as being important for joint lubrication. It consists of a solid supported dipalmitoylphosphatidylcholin (DPPC) bilayer, which was formed via vesicles fusion on a flat Si wafer, and the anionic polysaccharide hyaluronan (HA). We first characterized the structure of the HA layer that adsorbed to the DPPC bilayers at ambient pressure and different temperatures using X-ray reflectivity (XRR) measurements. Next, XRR was utilized to evaluate the response of the system to high hydrostatic pressures, up to 2kbar (200MPa), at three different temperatures. By means of fluorescence microscopy images the distribution of DPPC and HA on the surface was visualized. Our data suggest that HA adsorbs to the headgroup region that is oriented towards the water side of the supported bilayer. Phase transitions of the bilayer in response to temperature and pressure changes were also observed in presence and absence of HA. Our results reveal a higher stability against high hydrostatic pressures for DPPC/HA composite layers compared to that of the DPPC bilayer in absence of HA. Copyright © 2016 Elsevier B.V. All rights reserved.

Imaging specific cellular glycan structures using glycosyltransferases via click chemistry.

PubMed

Wu, Zhengliang L; Person, Anthony D; Anderson, Matthew; Burroughs, Barbara; Tatge, Timothy; Khatri, Kshitij; Zou, Yonglong; Wang, Lianchun; Geders, Todd; Zaia, Joseph; Sackstein, Robert

2018-02-01

Heparan sulfate (HS) is a polysaccharide fundamentally important for biologically activities. T/Tn antigens are universal carbohydrate cancer markers. Here, we report the specific imaging of these carbohydrates using a mesenchymal stem cell line and human umbilical vein endothelial cells (HUVEC). The staining specificities were demonstrated by comparing imaging of different glycans and validated by either removal of target glycans, which results in loss of signal, or installation of target glycans, which results in gain of signal. As controls, representative key glycans including O-GlcNAc, lactosaminyl glycans and hyaluronan were also imaged. HS staining revealed novel architectural features of the extracellular matrix (ECM) of HUVEC cells. Results from T/Tn antigen staining suggest that O-GalNAcylation is a rate-limiting step for O-glycan synthesis. Overall, these highly specific approaches for HS and T/Tn antigen imaging should greatly facilitate the detection and functional characterization of these biologically important glycans. © The Author(s) 2017. Published by Oxford University Press.

Effects of inhaled high-molecular weight hyaluronan in inflammatory airway disease.

PubMed

Lamas, Adelaida; Marshburn, Jamie; Stober, Vandy P; Donaldson, Scott H; Garantziotis, Stavros

2016-10-03

Cystic fibrosis (CF) is a chronic inflammatory disease that is affecting thousands of patients worldwide. Adjuvant anti-inflammatory treatment is an important component of cystic fibrosis treatment, and has shown promise in preserving lung function and prolonging life expectancy. Inhaled high molecular weight hyaluronan (HMW-HA) is reported to improve tolerability of hypertonic saline and thus increase compliance, and has been approved in some European countries for use as an adjunct to hypertonic saline treatment in cystic fibrosis. However, there are theoretical concerns that HMW-HA breakdown products may be pro-inflammatory. In this clinical pilot study we show that sputum cytokines in CF patients receiving HMW-HA are not increased, and therefore HMW-HA does not appear to adversely affect inflammatory status in CF airways.

Hyaluronan- and heparin-reduced silver nanoparticles with antimicrobial properties

PubMed Central

Kemp, Melissa M; Kumar, Ashavani; Clement, Dylan; Ajayan, Pulickel; Mousa, Shaker

2009-01-01

Aims Silver nanoparticles exhibit unique antibacterial properties that make these ideal candidates for biological and medical applications. We utilized a clean method involving a single synthetic step to prepare silver nanoparticles that exhibit antimicrobial activity. Materials & methods These nanoparticles were prepared by reducing silver nitrate with diaminopyridinylated heparin (DAPHP) and hyaluronan (HA) polysaccharides and tested for their efficacy in inhibiting microbial growth. Results & discussion The resulting silver nanoparticles exhibit potent antimicrobial activity against Staphylococcus aureus and modest activity against Escherichia coli. Silver–HA showed greater antimicrobial activity than silver–DAPHP, while silver–glucose nanoparticles exhibited very weak antimicrobial activity. Neither HA nor DAPHP showed activity against S. aureus or E. coli. Conclusion These results suggest that DAPHP and HA silver nanoparticles have potential in antimicrobial therapeutic applications. PMID:19505245

Intra-articular Recombinant Human Proteoglycan 4 Mitigates Cartilage Damage Following Destabilization of the Medial Meniscus in the Yucatan Minipig

PubMed Central

Waller, Kimberly A.; Chin, Kaitlyn E.; Jay, Gregory D.; Zhang, Ling X.; Teeple, Erin; McAllister, Scott; Badger, Gary J.; Schmidt, Tannin A.; Fleming, Braden C.

2016-01-01

Background Lubricin, or proteoglycan 4 (PRG4), is a glycoprotein responsible for joint boundary lubrication. PRG4 has been previously shown to be down-regulated following traumatic joint injury such as a meniscal tear. There is preliminary evidence suggesting that intra-articular injection of PRG4 post-injury will reduce cartilage damage in rat models of surgically-induced post-traumatic osteoarthritis. Objective To determine the efficacy of intra-articular injection of full length recombinant human lubricin (rhPRG4) for reducing cartilage damage after medial meniscus destabilization (DMM) in a pre-clinical large animal model. Study Design Controlled laboratory study Methods Unilateral DMM was performed in 29 Yucutan minipigs. One week post-DMM, animals received 3 weekly intra-articular injections (3cc/injection): 1) rhPRG4 [1.3mg/ml; n=10], 2) rhPRG4+hyaluronan [1.3mg/ml rhPRG4 and 3mg/ml hyaluronan (~950 kDA); n=10], and 3) phosphate buffered saline [PBS; n=9]. Hind limbs were harvested 26 weeks post-surgery. Cartilage integrity was evaluated using macroscopic (India Ink) and microscopic (Safranin O-fast green and hematoxylin & eosin) scoring systems. Secondary outcomes evaluated using ELISA included PRG4 levels in synovial fluid, CTX-II concentrations in urine and serum, and IL-1β levels in synovial fluid and serum. Results The rhPRG4 group had significantly less macroscopic cartilage damage in the medial tibial plateau compared to the PBS group (p=.002). No difference was found between the rhPRG4+hyaluronan and PBS groups (p=.23). However, no differences in microscopic damage scores were observed between the three groups (p=.70). PRG4 production was elevated in the rhPRG4 group synovial fluid compared to the PBS group (p=.033). The rhPRG4 group presented significantly lower urinary CTX-II levels, but not serum levels, when compared to the PBS (p=.013) and rhPRG4+hyaluronan (p=.011) groups. In serum and synovial fluid, both rhPRG4 (p=.006; p=.017) and rhPRG4+hyaluronan groups (p=.009; p=.03) presented decreased IL-1β levels. Conclusion All groups exhibited significant cartilage degeneration following DMM surgery. However, animals treated with rhPRG4 had the least amount of cartilage damage and less inflammation, providing evidence that intra-articular injections of rhPRG4 may slow the progression of post-traumatic osteoarthritis. Clinical Relevance Patients with meniscal trauma are at high risk for post-traumatic osteoarthritis. This study demonstrates that an intra-articular injection regimen of rhPRG4 may attenuate cartilage damage following meniscal injury. PMID:28129516

Bromelain Reversibly Inhibits Invasive Properties of Glioma Cells

PubMed Central

Tysnes, Berit B; Maurer, H Rainer; Porwol, Torsten; Probst, Beatrice; Bjerkvig, Rolf; Hoover, Frank

2001-01-01

Abstract Bromelain is an aqueous extract from pineapple stem that contains proteinases and exhibits pleiotropic therapeutic effects, i.e., antiedematous, antiinflammatory, antimetastatic, antithrombotic, and fibrinolytic activities. In this study, we tested bromelain's effects on glioma cells to assess whether bromelain could be a potential contributor to new antiinvasive strategies for gliomas. Several complementary assays demonstrated that bromelain significantly and reversibly reduced glioma cell adhesion, migration, and invasion without affecting cell viability, even after treatment periods extending over several months. Immunohistochemistry and immunoblotting experiments demonstrated that α3 and β1 integrin subunits and hyaluronan receptor CD44 protein levels were reduced within 24 hours of bromelain treatment. These effects were not reflected at the RNA level because RNA profiling did not show any significant effects on gene expression. Interestingly, metabolic labelling with 35-S methionine demonstrated that de novo protein synthesis was greatly attenuated by bromelain, in a reversible manner. By using a trans-activating signaling assay, we found that CRE-mediated signaling processes were suppressed. These results indicate that bromelain exerts its antiinvasive effects by proteolysis, signaling cascades, and translational attenuation. PMID:11774029

Joint nociceptor nerve activity and pain in an animal model of acute gout and its modulation by intra-articular hyaluronan

PubMed Central

Marcotti, Aida; Miralles, Ana; Dominguez, Eduardo; Pascual, Eliseo; Gomis, Ana; Belmonte, Carlos; de la Peña, Elvira

2018-01-01

Abstract The mechanisms whereby deposition of monosodium urate (MSU) crystals in gout activates nociceptors to induce joint pain are incompletely understood. We tried to reproduce the signs of painful gouty arthritis, injecting into the knee joint of rats suspensions containing amorphous or triclinic, needle MSU crystals. The magnitude of MSU-induced inflammation and pain behavior signs were correlated with the changes in firing frequency of spontaneous and movement-evoked nerve impulse activity recorded in single knee joint nociceptor saphenous nerve fibers. Joint swelling, mechanical and cold allodynia, and hyperalgesia appeared 3 hours after joint injection of MSU crystals. In parallel, spontaneous and movement-evoked joint nociceptor impulse activity raised significantly. Solutions containing amorphous or needle-shaped MSU crystals had similar inflammatory and electrophysiological effects. Intra-articular injection of hyaluronan (HA, Synvisc), a high-MW glycosaminoglycan present in the synovial fluid with analgesic effects in osteoarthritis, significantly reduced MSU-induced behavioral signs of pain and decreased the enhanced joint nociceptor activity. Our results support the interpretation that pain and nociceptor activation are not triggered by direct mechanical stimulation of nociceptors by MSU crystals, but are primarily caused by the release of excitatory mediators by inflammatory cells activated by MSU crystals. Intra-articular HA decreased behavioral and electrophysiological signs of pain, possibly through its viscoelastic filtering effect on the mechanical forces acting over sensitized joint sensory endings and probably also by a direct interaction of HA molecules with the transducing channels expressed in joint nociceptor terminals. PMID:29319609

The effect of vestibular nerve section on the expression of the hyaluronan in the frog, Rana esculenta.

PubMed

Halasi, Gábor; Wolf, Ervin; Bácskai, Tímea; Székely, György; Módis, László; Szigeti, Zsuzsa M; Mészár, Zoltán; Felszeghy, Szabolcs; Matesz, Clara

2007-12-01

Following postganglionic lesion of the eighth cranial nerve, the changes in the expression of hyaluronan (HA), one of the extracellular matrix macromolecules, were examined in the medial (MVN) and lateral (LVN) vestibular nuclei and in the entry or transitional zone (TZ) of the nerve in the frog. HA was detected in different survival times by using a specific biotinylated hyaluronan-binding probe. HA expression was defined by the area-integrated optical density (AIOD), calculated from pixel intensities of digitally captured images. During the first postoperative days the perineuronal net (PN), a HA-rich area around the neurons, was not distinguishable from the surrounding neuropil in the MVN and LVN, characterized by a bilateral drop of AIOD specifically on the operated side. From postoperative day 14 onwards AIOD increased whilst the PN reorganized. In contrast, the AIOD wobbled up and down bilaterally without any trend in the TZ. Statistical analysis indicated that AIOD changes in the structures studied ran parallel bilaterally presumably because of the operation. Our results demonstrated for the first time that (1) the lesion of the eighth cranial nerve is accompanied by the modification of AIOD reflected HA expression in the MVN, LVN and TZ, (2) different tendencies exist in the time course of AIOD in the structures studied and (3) these tendencies are similar on the intact and operated sides. Our findings may suggest an area dependent molecular mechanism of HA in the restoration of vestibular function.

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells

PubMed Central

Aguirre-Alvarado, Charmina; Segura-Cabrera, Aldo; Velázquez-Quesada, Inés; Hernández-Esquivel, Miguel A.; García-Pérez, Carlos A.; Guerrero-Rodríguez, Sandra L.; Ruiz, Angel J.; Rodríguez-Moreno, Andrea; Pérez-Tapia, Sonia M.; Velasco-Velázquez, Marco A.

2016-01-01

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24− cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists. PMID:27009862

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells.

PubMed

Aguirre-Alvarado, Charmina; Segura-Cabrera, Aldo; Velázquez-Quesada, Inés; Hernández-Esquivel, Miguel A; García-Pérez, Carlos A; Guerrero-Rodríguez, Sandra L; Ruiz-Moreno, Angel J; Rodríguez-Moreno, Andrea; Pérez-Tapia, Sonia M; Velasco-Velázquez, Marco A

2016-04-26

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24- cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists.

Mechanisms of erosion of atherosclerotic plaques.

PubMed

Quillard, Thibaut; Franck, Grégory; Mawson, Thomas; Folco, Eduardo; Libby, Peter

2017-10-01

The present review explores the mechanisms of superficial intimal erosion, a common cause of thrombotic complications of atherosclerosis. Human coronary artery atheroma that give rise to thrombosis because of erosion differ diametrically from those associated with fibrous cap rupture. Eroded lesions characteristically contain few inflammatory cells, abundant extracellular matrix, and neutrophil extracellular traps (NETs). Innate immune mechanisms such as engagement of Toll-like receptor 2 (TLR2) on cultured endothelial cells can impair their viability, attachment, and ability to recover a wound. Hyaluronan fragments may serve as endogenous TLR2 ligands. Mouse experiments demonstrate that flow disturbance in arteries with neointimas tailored to resemble features of human eroded plaques disturbs endothelial cell barrier function, impairs endothelial cell viability, recruits neutrophils, and provokes endothelial cells desquamation, NET formation, and thrombosis in a TLR2-dependent manner. Mechanisms of erosion have received much less attention than those that provoke plaque rupture. Intensive statin treatment changes the characteristic of plaques that render them less susceptible to rupture. Thus, erosion may contribute importantly to the current residual burden of risk. Understanding the mechanisms of erosion may inform the development and deployment of novel therapies to combat the remaining atherothrombotic risk in the statin era.

A Refined Model for the TSG-6 Link Module in Complex with Hyaluronan

PubMed Central

Higman, Victoria A.; Briggs, David C.; Mahoney, David J.; Blundell, Charles D.; Sattelle, Benedict M.; Dyer, Douglas P.; Green, Dixy E.; DeAngelis, Paul L.; Almond, Andrew; Milner, Caroline M.; Day, Anthony J.

2014-01-01

Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this high molecular weight polysaccharide composed of repeating disaccharides of d-glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was 13C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a d-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation. PMID:24403066

Biomaterial Substrate-Mediated Multicellular Spheroid Formation and Their Applications in Tissue Engineering.

PubMed

Tseng, Ting-Chen; Wong, Chui-Wei; Hsieh, Fu-Yu; Hsu, Shan-Hui

2017-12-01

Three-dimentional (3D) multicellular aggregates (spheroids), compared to the traditional 2D monolayer cultured cells, are physiologically more similar to the cells in vivo. So far there are various techniques to generate 3D spheroids. Spheroids obtained from different methods have already been applied to regenerative medicine or cancer research. Among the cell spheroids created by different methods, the substrate-derived spheroids and their forming mechanism are unique. This review focuses on the formation of biomaterial substrate-mediated multicellular spheroids and their applications in tissue engineering and tumor models. First, the authors will describe the special chitosan substrate-derived mesenchymal stem cell (MSC) spheroids and their greater regenerative capacities in various tissues. Second, the authors will describe tumor spheroids derived on chitosan and hyaluronan substrates, which serve as a simple in vitro platform to study 3D tumor models or to perform cancer drug screening. Finally, the authors will mention the self-assembly process for substrate-derived multiple cell spheroids (co-spheroids), which may recapitulate the heterotypic cell-cell interaction for co-cultured cells or crosstalk between different types of cells. These unique multicellular mono-spheroids or co-spheroids represent a category of 3D cell culture with advantages of biomimetic cell-cell interaction, better functionalities, and imaging possibilities. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plantar fascia anatomy and its relationship with Achilles tendon and paratenon.

PubMed

Stecco, Carla; Corradin, Marco; Macchi, Veronica; Morra, Aldo; Porzionato, Andrea; Biz, Carlo; De Caro, Raffaele

2013-12-01

Although the plantar fascia (PF) has been studied quite well from a biomechanical viewpoint, its microscopic properties have been overlooked: nothing is known about its content of elastic fibers, the features of the extracellular matrix or the extent of innervation. From a functional and clinical standpoint, the PF is often correlated with the triceps surae muscle, but the anatomical grounds for this link are not clear. The aim of this work was to focus on the PF macroscopic and microscopic properties and study how Achilles tendon diseases might affect it. Twelve feet from unembalmed human cadavers were dissected to isolate the PF. Specimens from each PF were tested with various histological and immunohistochemical stains. In a second stage, 52 magnetic resonance images (MRI) obtained from patients complaining of aspecific ankle or foot pain were analyzed, dividing the cases into two groups based on the presence or absence of signs of degeneration and/or inflammation of the Achilles tendon. The thickness of PF and paratenon was assessed in the two groups and statistical analyses were conducted. The PF is a tissue firmly joined to plantar muscles and skin. Analyzing its possible connections to the sural structures showed that this fascia is more closely connected to the paratenon of Achilles tendon than to the Achilles tendon, through the periosteum of the heel. The PF extended medially and laterally, continuing into the deep fasciae enveloping the abductor hallucis and abductor digiti minimi muscles, respectively. The PF was rich in hyaluronan, probably produced by fibroblastic-like cells described as 'fasciacytes'. Nerve endings and Pacini and Ruffini corpuscles were present, particularly in the medial and lateral portions, and on the surface of the muscles, suggesting a role for the PF in the proprioception of foot. In the radiological study, 27 of the 52 MRI showed signs of Achilles tendon inflammation and/or degeneration, and the PF was 3.43 ± 0.48 mm thick (99%CI and SD = 0.95), as opposed to 2.09 ± 0.24 mm (99%CI, SD = 0.47) in the patients in which the MRI revealed no Achilles tendon diseases; this difference in thickness of 1.29 ± 0.57 mm (99%CI) was statistically significant (P < 0.001). In the group of 27/52 patients with tendinopathies, the PF was more than 4.5 mm thick in 5, i.e. they exceeded the threshold for a diagnosis of plantar fasciitis. None of the other 25/52 paitents had a PF more than 4 mm thick. There was a statistically significant correlation between the thicknesses of the PF and the paratenon. These findings suggest that the plantar fascia has a role not only in supporting the longitudinal arch of the foot, but also in its proprioception and peripheral motor coordination. Its relationship with the paratenon of the Achilles tendon is consistent with the idea of triceps surae structures being involved in the PF pathology, so their rehabilitation can be considered appropriate. Finally, the high concentration of hyaluronan in the PF points to the feasibility of using hyaluronan injections in the fascia to treat plantar fasciitis. © 2013 Anatomical Society.

Artificial Extracellular Matrices with Oversulfated Glycosaminoglycan Derivatives Promote the Differentiation of Osteoblast-Precursor Cells and Premature Osteoblasts

PubMed Central

Hempel, Ute; Preissler, Carolin; Möller, Stephanie; Becher, Jana; Rauner, Martina; Hofbauer, Lorenz C.; Dieter, Peter

2014-01-01

Sulfated glycosaminoglycans (GAG) are components of the bone marrow stem cell niche and to a minor extent of mature bone tissue with important functions in regulating stem cell lineage commitment and differentiation. We anticipated that artificial extracellular matrices (aECM) composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially the differentiation of osteoblast-precursor cells and early osteoblasts. A set of gradually sulfated chondroitin sulfate and hyaluronan derivatives was used for the preparation of aECM. All these matrices were analysed with human bone marrow stromal cells to identify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind of disaccharide units on the osteogenic differentiation. Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition. A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells. It was shown that the proosteogenic effect of aECM was most prominent in early osteoblasts. PMID:24864267

Radiation synthesis and characterization of hyaluronan capped gold nanoparticles.

PubMed

Hien, Nguyen Quoc; Van Phu, Dang; Duy, Nguyen Ngoc; Quoc, Le Anh

2012-06-20

Gold nanoparticles (AuNPs) with diameter from 4 to 10nm, capping by hyaluronan (HA) were synthesized using a γ-irradiation method. The maximum absorption wavelengths at 517-525 nm of colloidal AuNPs/HA solutions were measured by UV-vis spectroscopy. The size and size distribution of AuNPs were determined from TEM images. The influence of various factors on the size of AuNPs particularly the concentration of Au3+ and HA, and dose rate were also investigated. Results indicated that higher dose rate and HA concentration favor smaller sizes of AuNPs whereas the size increases with Au3+ concentration. The colloidal AuNPs/HA solution was fairly stable more than 6 months under storage at ambient condition. The AuNPs stabilized by biocompatible HA with the size less than 10nm as prepared can potentially be applied in biomedicines and cosmetics. Copyright © 2012 Elsevier Ltd. All rights reserved.

Absorption of Orally Administered Hyaluronan.

PubMed

Kimura, Mamoru; Maeshima, Takuya; Kubota, Takumi; Kurihara, Hitoshi; Masuda, Yasunobu; Nomura, Yoshihiro

2016-12-01

Hyaluronan (HA) has been utilized as a supplement. However, the absorption of orally administrated HA remains controversial. The degradation and absorption of HA in the intestine were investigated in this study. HA excretion into the feces, degradation in the intestinal tract, absorption through the large intestine, and translocation to the blood and skin were examined. HA administered orally was not detected in rat feces. HA was degraded by cecal content, but not by artificial gastric juice and intestinal juice. Oligosaccharide HA passed through excised large intestine sacs. Furthermore, disaccharides, tetrasaccharides, and polysaccharides HA were distributed to the skin of rats following oral administration of high molecular weight HA (300 kDa). The results of the study suggest that orally administered HA is degraded to oligosaccharides by intestinal bacteria, and oligosaccharide HA is absorbed in the large intestine and is subsequently distributed throughout the tissues, including the skin.

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.

Mechanically triggered solute uptake in soft contact lenses.

PubMed

Tavazzi, Silvia; Ferraro, Lorenzo; Fagnola, Matteo; Cozza, Federica; Farris, Stefano; Bonetti, Simone; Simonutti, Roberto; Borghesi, Alessandro

2015-06-01

Molecular arrangement plays a role in the diffusion of water and solutes across soft contact lenses. In particular, the uptake of solutes in hydrated contact lenses can occur as long as free water is available for diffusion. In this work, we investigated the effect of mechanical vibrations of low frequency (200 Hz) on the solute uptake. Hyaluronan, a polysaccharide of ophthalmic use, was taken as example of solute of interest. For a specific water-hydrated hydrogel material, differential scanning calorimetry experiments showed that a large fraction of the hydration water accounted for loosely-bound water, both before and after one week of daily-wear of the lenses. The size (of the order of magnitude of few hundreds of nanometers) of hyaluronan in aqueous solution was found to be less than the size of the pores of the lens observed by scanning electron microscopy. However, solute uptake in already-hydrated lenses was negligible by simple immersion, while a significant increase occurred under mechanical vibrations of 200 Hz, thus providing experimental evidence of mechanically triggered enhanced solute uptake, which is attributed to the release of interfacial loosely-bound water. Also other materials were taken into consideration. However, the effectiveness of mechanical vibrations for hyaluronan uptake is restricted to lenses containing interfacial loosely-bound water. Indeed, loosely-bound water is expected to be bound to the polymer with bonding energies of the order of magnitude of 10-100 J/g, which are compatible with the energy input supplied by the vibrations. Copyright © 2015 Elsevier B.V. All rights reserved.

Serum and synovial fluid concentrations of keratan sulfate and hyaluronan in dogs with induced stifle joint osteoarthritis following cranial cruciate ligament transection.

PubMed

Budsberg, Steven C; Lenz, Mary Ellen; Thonar, Eugene J-M A

2006-03-01

To examine longitudinal changes in serum and synovial fluid concentrations of keratan sulfate (KS) and hyaluronan (HA) after cranial cruciate ligament (CCL) transection in dogs. 12 clinically normal adult mixed-breed dogs. Following CCL transection in the right stifle joint, KS and HA concentrations were determined in serum and neat (undiluted) synovial fluid prior to and 1, 2, 3, and 12 months after surgery. Postsurgical dilution of synovial fluid was corrected by use of urea as a passive marker. Synovial fluid KS and HA concentrations decreased at 1, 2, and 3 months after surgery in operated stifle joints, compared with baseline values. Synovial fluid KS concentration decreased in unoperated stifle joints at 1 month. A decrease in synovial fluid KS concentration was found in operated stifle joints, compared with unoperated stifle joints, at 2 and 3 months, and a decrease in synovial fluid HA concentrations was also found in operated stifle joints, compared with unoperated stifle joints, at 1, 2, and 3 months. Serum KS concentrations increased from baseline values at 3 months after surgery. Hyaluronan concentrations in operated stifle joints were lower than baseline values at 1, 2, and 3 months. Urea-adjusted synovial fluid concentrations revealed that dilution did not account for the decline in biomarker concentrations. The initial decrease and subsequent increase in synovial fluid concentrations of HA and KS may be caused by an acute inflammatory response to surgical intervention that negatively affects cartilage metabolism or an increase in production of immature proteoglycans.

Noninvasive Transdermal Vaccination Using Hyaluronan Nanocarriers and Laser Adjuvant

PubMed Central

Kim, Ki Su; Kim, Hyemin; Park, Yunji; Kong, Won Ho; Lee, Seung Woo; Kwok, Sheldon J. J.

2016-01-01

Vaccines are commonly administered by injection using needles. Although transdermal microneedles are less-invasive promising alternatives, needle-free topical vaccination without involving physical damage to the natural skin barrier is still sought after as it can further reduce needle-induced anxiety and simply administration. However, this long-standing goal has been elusive since the intact skin is impermeable to most macromolecules. Here, we show an efficient, non-invasive transdermal vaccination in mice by employing two key innovations: first, the use of hyaluronan (HA) as vaccine carriers and, second, non-ablative laser adjuvants. Conjugates of a model vaccine ovalbumin (OVA) and HA—HA-OVA conjugates—induced more effective maturation of dendritic cells in vitro, compared to OVA or HA alone, through synergistic HA receptor-mediated effects. Following topical administration in the back skin, HA-OVA conjugates penetrated into the epidermis and dermis in murine and porcine skins up to 30% of the total applied quantity, as revealed by intravital microscopy and quantitative fluorescence assay. Topical administration of HA-OVA conjugates significantly elevated both anti-OVA IgG antibody levels in serum and IgA antibody levels in bronchioalveolar lavage, with peak levels at 4 weeks, while OVA alone had a negligible effect. An OVA challenge at week 8 elicited strong immune-recall humoral responses. With pre-treatment of the skin using non-ablative fractional laser beams (1410 nm wavelength, 10 ms pulse duration, 0.2 mJ/pulse) as laser adjuvant, strong immunization was achieved with much reduced doses of HA-OVA (1 mg/kg OVA). Our results demonstrate the potential of the non-invasive patch-type transdermal vaccination platform. PMID:27833475

Deficiency of Hyaluronan Synthase 1 (Has1) Results in Chronic Joint Inflammation and Widespread Intra-Articular Fibrosis in a Murine Model of Knee Joint Cartilage Damage

PubMed Central

Chan, Deva D.; Xiao, Wenfeng; Li, Jun; de la Motte, Carol A.; Sandy, John D.; Plaas, Anna

2015-01-01

Objective Articular cartilage defects commonly result from traumatic injury and predispose to degenerative joint diseases. To test the hypothesis that aberrant healing responses and chronic inflammation lead to osteoarthritis, we examined spatiotemporal changes in joint tissues after cartilage injury in murine knees. Since intra-articular injection of hyaluronan (HA) can attenuate injury-induced osteoarthritis in wild-type (WT) mice, we investigated a role for HA in the response to cartilage injury in mice lacking HA synthase 1 (Has1−/−). Design Femoral groove cartilage of WT and Has1−/− mice was debrided to generate a non-bleeding wound. Macroscopic imaging, histology, and gene expression were used to evaluate naïve, sham-operated, and injured joints. Results Acute responses (1–2 weeks) in injured joints from WT mice included synovial hyperplasia with HA deposition and joint-wide increases in expression of genes associated with inflammation, fibrosis, and extracellular matrix (ECM) production. By 4 weeks, some resurfacing of damaged cartilage occurred, and early cell responses were normalized. Cartilage damage in Has1−/− mice also induced early responses; however, at 4 weeks, inflammation and fibrosis genes remained elevated with widespread cartilage degeneration and fibrotic scarring in the synovium and joint capsule. Conclusions We conclude that the ineffective repair of injured cartilage in Has1−/− joints can be at least partly explained by the markedly enhanced expression of particular genes in pathways linked to ECM turnover, IL-17/IL-6 cytokine signaling, and apoptosis. Notably, Has1 ablation does not alter gross HA content in the ECM, suggesting that HAS1 has a unique function in the metabolism of inflammatory HA matrices. PMID:26521733

Adapted to Roar: Functional Morphology of Tiger and Lion Vocal Folds

PubMed Central

Klemuk, Sarah A.; Riede, Tobias; Walsh, Edward J.; Titze, Ingo R.

2011-01-01

Vocal production requires active control of the respiratory system, larynx and vocal tract. Vocal sounds in mammals are produced by flow-induced vocal fold oscillation, which requires vocal fold tissue that can sustain the mechanical stress during phonation. Our understanding of the relationship between morphology and vocal function of vocal folds is very limited. Here we tested the hypothesis that vocal fold morphology and viscoelastic properties allow a prediction of fundamental frequency range of sounds that can be produced, and minimal lung pressure necessary to initiate phonation. We tested the hypothesis in lions and tigers who are well-known for producing low frequency and very loud roaring sounds that expose vocal folds to large stresses. In histological sections, we found that the Panthera vocal fold lamina propria consists of a lateral region with adipocytes embedded in a network of collagen and elastin fibers and hyaluronan. There is also a medial region that contains only fibrous proteins and hyaluronan but no fat cells. Young's moduli range between 10 and 2000 kPa for strains up to 60%. Shear moduli ranged between 0.1 and 2 kPa and differed between layers. Biomechanical and morphological data were used to make predictions of fundamental frequency and subglottal pressure ranges. Such predictions agreed well with measurements from natural phonation and phonation of excised larynges, respectively. We assume that fat shapes Panthera vocal folds into an advantageous geometry for phonation and it protects vocal folds. Its primary function is probably not to increase vocal fold mass as suggested previously. The large square-shaped Panthera vocal fold eases phonation onset and thereby extends the dynamic range of the voice. PMID:22073246

Hyaluronan inhibits prostaglandin E2 production via CD44 in U937 human macrophages.

PubMed

Yasuda, Tadashi

2010-03-01

Prostaglandin E(2) (PGE(2)) is one of the key mediators of inflammation in affected joints of rheumatoid arthritis (RA). Intra-articular injection of high molecular weight hyaluronan (HA) into RA knee joints relieves arthritic pain. Although HA has been shown to inhibit PGE(2) production in cytokine-stimulated synovial fibroblasts, it remains unclear how HA suppresses PGE(2) production in activated cells. Furthermore, HA effect on macrophages has rarely been investigated in spite of their contribution to RA joint pathology. This study was aimed to investigate the inhibitory mechanism of HA on lipopolysaccharide (LPS)-stimulated PGE(2) production in U937 human macrophages. Stimulation of U937 macrophages with LPS enhanced PGE(2) production in association with increased protein levels of cyclooxygenase-2 (COX-2). Pretreatment with HA of 2,700 kDa resulted in suppression of the LPS-mediated induction of COX-2, leading to a decrease in PGE(2) production. Likewise, the LPS-stimulated PGE(2) production was inhibited by the pretreatment with a specific COX2 inhibitor, NS-398, or a specific inhibitor of nuclear factor (NF)-kappaB, BAY11-7085. HA also decreased the degree of phosphorylation and nuclear translocation of NF-kappaB enhanced by LPS. Fluorescence cytochemistry demonstrated that HA bound to CD44, the principal HA receptor, on U937 macrophages. Anti-CD44 antibody reversed the inhibitory effects of HA on the LPS-mediated increase in PGE(2) production, COX-2 induction, and activation of NF-kappaB. These results indicate that HA suppresses the LPS-stimulated PGE(2) production via CD44 through down-regulation of NF-kappaB. Administration of HA into RA joints may decrease PGE(2) production by activated macrophages, which could result in improvement of arthritic pain.

Potential role of fibroblast growth factor in enhancement of fracture healing.

PubMed

Radomsky, M L; Thompson, A Y; Spiro, R C; Poser, J W

1998-10-01

Fibroblast growth factors are present in significant amounts in bone and several studies have suggested that they may be involved in normal fracture healing. It is well established that fibroblast growth factors have mitogenic and angiogenic activity on mesoderm and neuroectoderm derived cells. Of particular interest as a member of the fibroblast growth factor family, basic fibroblast growth factor stimulates mitogenesis, chemotaxis, differentiation, and angiogenesis. It also plays an important role in the development of vascular, nervous, and skeletal systems, promotes the maintenance and survival of certain tissues, and stimulates wound healing and tissue repair. Animal studies have shown that the direct injection of fibroblast growth factor into fresh fractures stimulates callus formation, which provides mechanical stability to the fracture, accelerates healing, and restores competence. The matrix used to present the fibroblast growth factor at the fracture site plays a critical role in the effectiveness of the treatment. The evaluation of injectable basic fibroblast growth factor in a sodium hyaluronate gel for its effectiveness in stimulating fracture healing is described. When applied directly into a freshly created fracture in the rabbit fibula, a single injection of the basic fibroblast growth factor and hyaluronan results in the stimulation of callus formation, increased bone formation, and earlier restoration of mechanical strength at the fracture site. The hyaluronan gel serves as a reservoir that sequesters the basic fibroblast growth factor at the injection site for the length of time necessary to create an environment conducive to fracture healing. It is concluded that basic fibroblast growth factor and sodium hyaluronate act synergistically to accelerate fracture healing and that the combination is suitable for clinical evaluation as a therapy in fracture treatment.

Effects of Melatonin, Aluminum Oxide, and Polymethylsiloxane Complex on the Expression of LYVE-1 in the Liver of Mice with Obesity and Type 2 Diabetes Mellitus.

PubMed

Michurina, S V; Ishchenko, I Yu; Arkhipov, S A; Klimontov, V V; Rachkovskaya, L N; Konenkov, V I; Zavyalov, E L

2016-12-01

The effects of melatonin, aluminum oxide, and polymethylsiloxane complex on the expression of LYVE-1 (lymphatic vessel endothelial hyaluronan receptor) in the liver were studied in db/db mice with experimental obesity and type 2 diabetes mellitus. The complex or placebo was administered daily by gavage from week 8 to week 16 of life. The animals receiving the complex exhibited enhanced, in comparison with the placebo group, immunohistochemical LYVE-1+ staining of endothelial cells in sinusoids. Enhanced expression of LYVE-1 was associated with less pronounced dilatation of interlobular arteries, veins, and lymphatic vessels. Thee findings suggest a protective effect of the complex towards structural changes in the liver of mice with obesity and type 2 diabetes.

Oxidation of glycosaminoglycans by free radicals and reactive oxidative species: A review of investigative methods.

PubMed

Parsons, B J

2015-05-01

Glycosaminoglycans, in particular hyaluronan (HA), and proteoglycans are components of the extracellular matrix (ECM). The ECM plays a key role in the regulation of cellular behaviour and alterations to it can modulate both the development of human diseases as well as controlling normal biochemical processes such as cell signalling and pro-inflammatory responses. For these reasons, in vitro fragmentation studies of glycosaminoglycans by free radicals and oxidative species are seen to be relevant to the understanding of in vivo studies of damage to the ECM. A wide range of investigative techniques have therefore been applied to gain insights into the relative fragmentation effects of several reactive oxidative species with the ultimate goal of determining mechanisms of fragmentation at the molecular level. These methods are reviewed here.

FRMD4A upregulation in human squamous cell carcinoma promotes tumor growth and metastasis and is associated with poor prognosis.

PubMed

Goldie, Stephen J; Mulder, Klaas W; Tan, David Wei-Min; Lyons, Scott K; Sims, Andrew H; Watt, Fiona M

2012-07-01

New therapeutic strategies are needed to improve treatment of head and neck squamous cell carcinoma (HNSCC), an aggressive tumor with poor survival rates. FRMD4A is a human epidermal stem cell marker implicated previously in epithelial polarity that is upregulated in SCC cells. Here, we report that FRMD4A upregulation occurs in primary human HNSCCs where high expression levels correlate with increased risks of relapse. FRMD4A silencing decreased growth and metastasis of human SCC xenografts in skin and tongue, reduced SCC proliferation and intercellular adhesion, and stimulated caspase-3 activity and expression of terminal differentiation markers. Notably, FRMD4A attenuation caused nuclear accumulation of YAP, suggesting a potential role for FRMD4A in Hippo signaling. Treatment with the HSP90 inhibitor 17-DMAG or ligation of CD44 with hyaluronan caused nuclear depletion of FRMD4A, nuclear accumulation of YAP and reduced SCC growth and metastasis. Together, our findings suggest FRMD4A as a novel candidate therapeutic target in HNSCC based on the key role in metastatic growth we have identified. ©2012 AACR.

Viscoelastic response of a model endothelial glycocalyx

NASA Astrophysics Data System (ADS)

Nijenhuis, Nadja; Mizuno, Daisuke; Spaan, Jos A. E.; Schmidt, Christoph F.

2009-06-01

Many cells cover themselves with a multifunctional polymer coat, the pericellular matrix (PCM), to mediate mechanical interactions with the environment. A particular PCM, the endothelial glycocalyx (EG), is formed by vascular endothelial cells at their luminal side, forming a mechanical interface between the flowing blood and the endothelial cell layer. The glycosaminoglycan (GAG) hyaluronan (HA) is involved in the main functions of the EG, mechanotransduction of fluid shear stress and molecular sieving. HA, due to its length, is the only GAG in the EG or any other PCM able to form an entangled network. The mechanical functions of the EG are, however, impaired when any one of its components is removed. We here used microrheology to measure the effect of the EG constituents heparan sulfate, chondroitin sulfate, whole blood plasma and albumin on the high-bandwidth mechanical properties of a HA solution. Furthermore, we probed the effect of the hyaldherin aggrecan, a constituent of the PCM of chondrocytes, and very similar to versican (present in the PCM of various cells, and possibly in the EG). We show that components directly interacting with HA (chondroitin sulfate and aggrecan) can increase the viscoelastic shear modulus of the polymer composite.

Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in Mice: Implications for Superficial Erosion.

PubMed

Franck, Grégory; Mawson, Thomas; Sausen, Grasiele; Salinas, Manuel; Masson, Gustavo Santos; Cole, Andrew; Beltrami-Moreira, Marina; Chatzizisis, Yiannis; Quillard, Thibault; Tesmenitsky, Yevgenia; Shvartz, Eugenia; Sukhova, Galina K; Swirski, Filip K; Nahrendorf, Matthias; Aikawa, Elena; Croce, Kevin J; Libby, Peter

2017-06-23

Superficial erosion currently causes up to a third of acute coronary syndromes; yet, we lack understanding of its mechanisms. Thrombi because of superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. This study tested in vivo the involvement of disturbed flow and of neutrophils, hyaluronan, and Toll-like receptor 2 ligation in superficial intimal injury, a process implicated in superficial erosion. In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell activation, neutrophil accumulation, endothelial cell death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. Toll-like receptor 2 agonism activated luminal endothelial cells, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing endothelial cell injury and local thrombosis ( P <0.05). These results implicate flow disturbance, neutrophils, and Toll-like receptor 2 signaling as mechanisms that contribute to superficial erosion, a cause of acute coronary syndrome of likely growing importance in the statin era. © 2017 American Heart Association, Inc.

Electron microscopy of the amphibian model systems Xenopus laevis and Ambystoma mexicanum.

PubMed

Kurth, Thomas; Berger, Jürgen; Wilsch-Bräuninger, Michaela; Kretschmar, Susanne; Cerny, Robert; Schwarz, Heinz; Löfberg, Jan; Piendl, Thomas; Epperlein, Hans H

2010-01-01

In this chapter we provide a set of different protocols for the ultrastructural analysis of amphibian (Xenopus, axolotl) tissues, mostly of embryonic origin. For Xenopus these methods include: (1) embedding gastrulae and tailbud embryos into Spurr's resin for TEM, (2) post-embedding labeling of methacrylate (K4M) and cryosections through adult and embryonic epithelia for correlative LM and TEM, and (3) pre-embedding labeling of embryonic tissues with silver-enhanced nanogold. For the axolotl (Ambystoma mexicanum) we present the following methods: (1) SEM of migrating neural crest (NC) cells; (2) SEM and TEM of extracellular matrix (ECM) material; (3) Cryo-SEM of extracellular matrix (ECM) material after cryoimmobilization; and (4) TEM analysis of hyaluronan using high-pressure freezing and HABP labeling. These methods provide exemplary approaches for a variety of questions in the field of amphibian development and regeneration, and focus on cell biological issues that can only be answered with fine structural imaging methods, such as electron microscopy. Copyright © 2010 Elsevier Inc. All rights reserved.

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.

Imaging articular cartilage using second harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Mansfield, Jessica C.; Winlove, C. Peter; Knapp, Karen; Matcher, Stephen J.

2006-02-01

Sub cellular resolution images of equine articular cartilage have been obtained using both second harmonic generation microscopy (SHGM) and two-photon fluorescence microscopy (TPFM). The SHGM images clearly map the distribution of the collagen II fibers within the extracellular matrix while the TPFM images show the distribution of endogenous two-photon fluorophores in both the cells and the extracellular matrix, highlighting especially the pericellular matrix and bright 2-3μm diameter features within the cells. To investigate the source of TPF in the extracellular matrix experiments have been carried out to see if it may originate from the proteoglycans. Pure solutions of the following proteoglycans hyaluronan, chondroitin sulfate and aggrecan have been imaged, only the aggrecan produced any TPF and here the intensity was not great enough to account for the TPF in the extracellular matrix. Also cartilage samples were subjected to a process to remove proteoglycans and cellular components. After this process the TPF from the samples had decreased by a factor of two, with respect to the SHG intensity.

Improved quality of porcine embryos cultured with hyaluronan due to the modification of the mitochondrial membrane potential and reactive oxygen species level.

PubMed

Romek, Marek; Gajda, Barbara; Krzysztofowicz, Ewa; Kucia, Marcin; Uzarowska, Agnieszka; Smorag, Zdzislaw

2017-10-15

Although considerable progress has been made in pig embryo culture systems, the developmental competence and quality of the produced embryos are still lower than their in vivo-derived counterparts. Because hyaluronan (HA) regulates various cellular processes and possesses antioxidant properties, this glycosaminoglycan seems to be a promising supplement in culture media. However, until now, its beneficial influence on in vitro pig embryo development has been debatable. Hence, we aimed to investigate the effect of 0.25 mg/mL, 0.5 mg/mL and 1 mg/mL concentrations of HA on the developmental potential and quality of cultured porcine embryos. We found that 1 mg/mL HA supplementation significantly increased the obtained percentages of cleaved embryos to ∼95%, morulae to ∼87% and blastocysts to ∼77%. At 0.5 mg/mL and 1 mg/mL HA concentrations, we observed a significantly improved blastocyst quality, expressed as the total number of cells per blastocyst, number of cells in the inner cell mass, number of TUNEL-positive nuclei per blastocyst, the TUNEL index and the blastocyst diameter. Because the inner mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) level are important for proper embryo development, for the first time, we measured these two parameters in cultured embryos at various HA concentrations and during their development up to the expanded blastocyst stage. For blastocysts cultured with 1 mg/mL HA, the ΔΨm and ROS level were ∼1.6 and 2.7 times lower, respectively, than those of the control blastocysts. Both ΔΨm and the ROS level were increased in parallel during in vitro embryo development with and without HA, but this increase was less pronounced in the presence of HA. Hence, our quantitative data unequivocally show that supplementation of NCSU-23 culture medium with 1 mg/mL HA improves the developmental potential and quality of pig embryos. This effect results from a significant decrease in the ROS level induced by the HA-dependent ΔΨm reduction. Copyright © 2017 Elsevier Inc. All rights reserved.

The control and importance of hyaluronan synthase expression in palatogenesis

PubMed Central

Galloway, Jennifer L.; Jones, Sarah J.; Mossey, Peter A.; Ellis, Ian R.

2013-01-01

Development of the lip and palate involves a complex series of events that requires the close co-ordination of cell migration, growth, differentiation, and apoptosis. Palatal shelf elevation is considered to be driven by regional accumulation and hydration of glycosoaminoglycans, principally hyaluronan (HA), which provides an intrinsic shelf force, directed by components of the extracellular matrix (ECM). During embryogenesis, the extracellular and pericellular matrix surrounding migrating and proliferating cells is rich in HA. This would suggest that HA may be important in both shelf growth and fusion. TGFβ3 plays an important role in palatogenesis and the corresponding homozygous null (TGFβ3−/−) mouse, exhibits a defect in the fusion of the palatal shelves resulting in clefting of the secondary palate. TGFβ3 is expressed at the future medial edge epithelium (MEE) and at the actual edge epithelium during E14.5, suggesting a role for TGFβ3 in fusion. This is substantiated by experiments showing that addition of exogenous TGFβ3 can “rescue” the cleft palate phenotype in the null mouse. In addition, TGFβ1 and TGFβ2 can rescue the null mouse palate (in vitro) to near normal fusion. In vivo a TGFβ1 knock-in mouse, where the coding region of the TGFβ3 gene was replaced with the full-length TGFβ1 cDNA, displayed complete fusion at the mid portion of the secondary palate, whereas the anterior and posterior regions failed to fuse appropriately. We present experimental data indicating that the three HA synthase (Has) enzymes are differentially expressed during palatogenesis. Using immunohistochemistry (IHC) and embryo sections from the TGFβ3 null mouse at days E13.5 and E14.5, it was established that there was a decrease in expression of Has2 in the mesenchyme and an increase in expression of Has3 in comparison to the wild-type mouse. In vitro data indicate that HA synthesis is affected by addition of exogenous TGFβ3. Preliminary data suggests that this increase in HA synthesis, in response to TGFβ3, is under the control of the PI3kinase/Akt pathway. PMID:23382716

Depot-specific characteristics of adipose tissue-derived stromal cells in thyroid-associated orbitopathy.

PubMed

Wong, Janice Siu Chong; Chu, Wai Kit; Li, Benjamin Fuk-Loi; Pang, Chi-Pui; Chong, Kelvin Kam-Lung

2018-04-17

Thyroid-associated orbitopathy (TAO) causes inflammatory fibroproliferation of periocular connective tissues. We compared adipose tissue-derived stem/stromal cells (ADSCs) from three adipose depots of each patient with TAO on mesenchymal, myofibrogenic, adipogenic properties and associated hyaluronan (HA) synthesis. ADSCs were generated from periocular (eyelid, orbital) and subcutaneous (abdominal) adipose tissues of three patients with TAO. Mesenchymal markers were characterised by reverse transcription-PCR and immunofluorescent staining. A 3-week adipogenic induction was evaluated by Nile red staining and quantitative PCR (qPCR) of peroxisome proliferator-activated receptor (PPARγ), adiponectin and hyaluronan synthase (HAS)-2. A 7-day myofibrogenic induction was assayed by immunofluorescent staining and qPCR of α-smooth muscle actin (α-SMA). ADSCs from all depots expressed similar levels of mesenchymal markers CD44, CD90 and CD105 (p=0.288, p=0.43 and p=0.837, respectively). After adipogenic induction, intracellular lipid increased for more than 32% and PPARγ mRNA showed more than twofold increase from all three depots. However, adiponectin and HAS-2 mRNA levels were significantly higher in the eyelid and orbital ADSCs than those from the subcutaneous ADSCs after induction (2.4×10 7 , 3.9×10 6  folds vs below detection limit; 63.3-fold, 26.1-fold, vs 33% reduction, respectively; all p=0.002). Significantly more myofibroblasts and higher mRNA level of α-SMA were obtained from the orbital and eyelid compared with the subcutaneous ADSCs during myofibrogenic induction (80.2%, 70.6% vs 29.3%; 30.2-fold, 24.2-fold vs 1.7-fold, respectively; all p=0.002). ADSCs from different adipose depots of the same donors exhibited similar mesenchymal phenotypes but differed significantly in adipogenic, myofibrogenic potentials and associated HA synthesis. These depot-specific characteristics of ADSCs may contribute to site-specific adipose tissue involvement in TAO. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Mef2c Regulates Transcription of the Extracellular Matrix Protein Cartilage Link Protein 1 in the Developing Murine Heart

PubMed Central

Phelps, Aimee L.; Ghatnekar, Angela V.; Barth, Jeremy L.; Norris, Russell A.; Wessels, Andy

2013-01-01

Cartilage Link Protein 1 (Crtl1) is an extracellular matrix (ECM) protein that stabilizes the interaction between hyaluronan and versican and is expressed in endocardial and endocardially-derived cells in the developing heart, including cells in the atrioventricular (AV) and outflow tract (OFT) cushions. Previous investigations into the transcriptional regulation of the Crtl1 gene have shown that Sox9 regulates Crtl1 expression in both cartilage and the AV valves. The cardiac transcription factor Mef2c is involved in the regulation of gene expression in cardiac and skeletal muscle cell lineages. In this study we have investigated the potential role of Mef2c in the regulation of ECM production in the endocardial and mesenchymal cell lineages of the developing heart. We demonstrate that the Crtl1 5′ flanking region contains two highly conserved Mef2 binding sites and that Mef2c is able to bind to these sites in vivo during cardiovascular development. Additionally, we show that Crtl1 transcription is dependent on Mef2c expression in fetal mitral valve interstitial cells (VICs). Combined, these findings highlight a new role for Mef2c in cardiac development and the regulation of cardiac extracellular matrix protein expression. PMID:23468913

Hyaluronan Oligosaccharides for the Promotion of Remyelination (Revised)

DTIC Science & Technology

2012-10-01

treated with StrepH or with chondroitinase ABC (which degrades chondroitin sulfate into unsaturated disaccharides) at concentrations that were...optimal for the digestion of HA and chondroitin sulfate , respectively (see Fig 1H). This finding is consistent with the hypothesis that specific HA

Targeting CD44 with nanoparticles in head and neck squamous cell carcinoma: A novel therapeutic strategy against cancer stem cells

NASA Astrophysics Data System (ADS)

Thapa, Ranjeeta

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide and is associated with significant morbidity and mortality. Advances in multi-modality treatments have only minimally improved survival rates in the past several years. Recent attention has been focused on the hypothesis that cancer stem cells (CSCs) may be responsible for the failure of current treatments. In HNSCC, a CSC population is contained within the cell fraction that expresses high levels of CD44. CD44 is a cell surface glycoprotein and was the first CSC marker to be described in solid malignancies. in this study, hyaluronan conjugated, dextran-coated super paramagnetic iron-oxide nanoparticles (HA-DESPIONs) were used to target the CD44 population in CD44-overexpressed HNSCC cell lines for treatment by establishing the interaction of HA-DESPIONs with radiation and hyperthermia therapy. The first part of this dissertation studied the cytotoxic, radiosensitizing, and hyperthermic properties of the HA-DESPIONs using cell proliferation and clonogenic survival assays. Cells were grown, plated, treated with HA-DESPIONs, irradiated/exposed to local hyperthermia, and then analyzed for apoptosis. HA-DESPIONs proved to be relatively non-toxic and nonradiosensitizing. However, temperature-dependent cell survival reduction upon incubation with HA-DESPIONs was observed with evidence of apoptotic cell death. These results supported further development of an alternating magnetic field (AMF) approach to activate the HADESPIONs attached to CSCs. In the second part of the dissertation, an AMF generator was constructed and its heat generating effect was tested via kinetic and dose-dependent bulk heating experiments by exposing magnetic nanoparticles to AMF. For elimination of the CD44 population, cells were treated with HA-DESPIONs/DESPIONs, exposed to AMF, and processed for flow cytometrybased apoptosis analysis. Magnetic nanoparticles caused concentration-dependent bulk heating in response to AMF resulting in a significant temperature rise. Following exposure to AMF, DESPIONs were unable to induce targeted hyperthermia and hence had no effect on CD44 cell death in HNSCC cells. However, there was significant cell death in the CD44 population treated with HA-DESPIONs and exposed to AMF. This effect was observed only when the AMF was turned on. These results demonstrated that HA-DESPIONs caused targeted cell-death in CD44overexpressing cells. This may be a promising strategy to specifically target CSCs for the treatment of HNSCC.

Examination of synovial fluid and serum following intravenous injections of hyaluronan for the treatment of osteoarthritis in dogs.

PubMed

Canapp, S O; Cross, A R; Brown, M P; Lewis, D D; Hernandez, J; Merritt, K A; Tran-Son-Tay, R

2005-01-01

A randomized, blinded, prospective clinical trial was performed to determine the effects of intravenous (i.v.) administration of hyaluronan sodium (HA) on serum glycosaminoglycans (GAG) concentrations, synovial fluid (SF) hyaluronan concentrations and viscosity in dogs treated for unilateral rupture of the cranial cruciate ligament. Twenty-two dogs undergoing tibial plateau leveling osteotomy were used in this study. Synovial fluid from both stifles and serum were collected prior to surgery and at 2, 4, and 8 weeks following surgery. Dogs received either 1.0 ml (10 mg) of sodium hyaluronate (treatment group 1; n = 10) or equal volume of 0.9% NaCl (treatment group 2; n = 12), i.v. immediately, 2 and 4 weeks following surgery. Synovial fluid viscosity was evaluated using a magnetically driven, acoustically tracked, translating-ball rheometer. Synovial fluid HA disaccharide content was measured by fluorophore-assisted carbohydrate electrophoresis. Serum GAG concentrations were measured by alcian blue spectrophotometric assay. Data were analyzed using a Wilcoxon sign rank test (p < 0.05). Mean +/- SD viscosity (cP) was significantly higher (p = 0.011) in SF obtained from the intact stifle (450 +/- 604.1) than injured (54.8 +/- 60.8) prior to surgery. Mean +/- SD HA concentrations (ug/ml) were significantly higher (p = 0.02) in synovial fluid obtained from the injured stifles (281.4 +/- 145.9) than intact stifles (141.6 +/- 132.5). No significant difference was noted within or between treatment groups in SF viscosity, HA concentrations, or serum GAG concentrations at any time following surgery. Stifles with cranial cruciate ligament insufficiency had significant alterations in SF viscosity and HA concentrations.

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

Interplay of Substrate Conductivity, Cellular Microenvironment, and Pulsatile Electrical Stimulation toward Osteogenesis of Human Mesenchymal Stem Cells in Vitro.

PubMed

Thrivikraman, Greeshma; Lee, Poh S; Hess, Ricarda; Haenchen, Vanessa; Basu, Bikramjit; Scharnweber, Dieter

2015-10-21

The influences of physical stimuli such as surface elasticity, topography, and chemistry over mesenchymal stem cell proliferation and differentiation are well investigated. In this context, a fundamentally different approach was adopted, and we have demonstrated the interplay of inherent substrate conductivity, defined chemical composition of cellular microenvironment, and intermittent delivery of electric pulses to drive mesenchymal stem cell differentiation toward osteogenesis. For this, conducting polyaniline (PANI) substrates were coated with collagen type 1 (Coll) alone or in association with sulfated hyaluronan (sHya) to form artificial extracellular matrix (aECM), which mimics the native microenvironment of bone tissue. Further, bone marrow derived human mesenchymal stem cells (hMSCs) were cultured on these moderately conductive (10(-4)-10(-3) S/cm) aECM coated PANI substrates and exposed intermittently to pulsed electric field (PEF) generated through transformer-like coupling (TLC) approach over 28 days. On the basis of critical analysis over an array of end points, it was inferred that Coll/sHya coated PANI (PANI/Coll/sHya) substrates had enhanced proliferative capacity of hMSCs up to 28 days in culture, even in the absence of PEF stimulation. On the contrary, the adopted PEF stimulation protocol (7 ms rectangular pulses, 3.6 mV/cm, 10 Hz) is shown to enhance osteogenic differentiation potential of hMSCs. Additionally, PEF stimulated hMSCs had also displayed different morphological characteristics as their nonstimulated counterparts. Concomitantly, earlier onset of ALP activity was also observed on PANI/Coll/sHya substrates and resulted in more calcium deposition. Moreover, real-time polymerase chain reaction results indicated higher mRNA levels of alkaline phosphatase and osteocalcin, whereas the expression of other osteogenic markers such as Runt-related transcription factor 2, Col1A, and osteopontin exhibited a dynamic pattern similar to control cells that are cultured in osteogenic medium. Taken together, our experimental results illustrate the interplay of multiple parameters such as substrate conductivity, electric field stimulation, and aECM coating on the modulation of hMSC proliferation and differentiation in vitro.

The CD44-initiated pathway of T-cell extravasation uses VLA-4 but not LFA-1 for firm adhesion

PubMed Central

Siegelman, Mark H.; Stanescu, Diana; Estess, Pila

2000-01-01

Leukocytes extravasate from the blood in response to physiologic or pathologic demands by means of complementary ligand interactions between leukocytes and endothelial cells. The multistep model of leukocyte extravasation involves an initial transient interaction (“rolling” adhesion), followed by secondary (firm) adhesion. We recently showed that binding of CD44 on activated T lymphocytes to endothelial hyaluronan (HA) mediates a primary adhesive interaction under shear stress, permitting extravasation at sites of inflammation. The mechanism for subsequent firm adhesion has not been elucidated. Here we demonstrate that the integrin VLA-4 is used in secondary adhesion after CD44-mediated primary adhesion of human and mouse T cells in vitro, and by mouse T cells in an in vivo model. We show that clonal cell lines and polyclonally activated normal T cells roll under physiologic shear forces on hyaluronate and require VCAM-1, but not ICAM-1, as ligand for subsequent firm adhesion. This firm adhesion is also VLA-4 dependent, as shown by antibody inhibition. Moreover, in vivo short-term homing experiments in a model dependent on CD44 and HA demonstrate that superantigen-activated T cells require VLA-4, but not LFA-1, for entry into an inflamed peritoneal site. Thus, extravasation of activated T cells initiated by CD44 binding to HA depends upon VLA-4–mediated firm adhesion, which may explain the frequent association of these adhesion receptors with diverse chronic inflammatory processes. PMID:10712440

Simplifying the extracellular matrix for 3-D cell culture and tissue engineering: a pragmatic approach.

PubMed

Prestwich, Glenn D

2007-08-15

The common technique of growing cells on tissue culture plastic (TCP) is gradually being supplanted by methods for culturing cells in two-dimensions (2-D) on matrices with more appropriate physical and biological properties or by encapsulation of cells in three-dimensions (3-D). The universal acceptance of the new 3-D paradigm is currently constrained by the lack of a biocompatible material in the marketplace that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. In this Prospect, I argue that the standard for 3-D cell culture should be bio-inspired, biomimetic materials that can be used "as is" in drug discovery, toxicology, cell banking, and ultimately in medicine. Such biomaterials must therefore be highly reproducible, manufacturable, approvable, and affordable. To obtain integrated, functional, multicellular systems that recapitulate tissues and organs, the needs of the true end-users-physicians and patients-must dictate the key design criteria. Herein I describe the development of one such material that meets these requirements: a covalently crosslinked, biodegradable, simplified mimic of the extracellular matrix (ECM) that permits 3-D culture of cells in vitro and enables tissue formation in vivo. In contrast to materials that were designed for in vitro cell culture and then found unsuitable for clinical use, these semi-synthetic hyaluronan-derived materials were developed for in vivo tissue repair, and are now being re-engineered for in vitro applications in research.

Recapitulating in vivo-like plasticity of glioma cell invasion along blood vessels and in astrocyte-rich stroma.

PubMed

Gritsenko, Pavlo; Leenders, William; Friedl, Peter

2017-10-01

Diffuse invasion of glioma cells into the brain parenchyma leads to nonresectable brain tumors and poor prognosis of glioma disease. In vivo, glioma cells can adopt a range of invasion strategies and routes, by moving as single cells, collective strands and multicellular networks along perivascular, perineuronal and interstitial guidance cues. Current in vitro assays to probe glioma cell invasion, however, are limited in recapitulating the modes and adaptability of glioma invasion observed in brain parenchyma, including collective behaviours. To mimic in vivo-like glioma cell invasion in vitro, we here applied three tissue-inspired 3D environments combining multicellular glioma spheroids and reconstituted microanatomic features of vascular and interstitial brain structures. Radial migration from multicellular glioma spheroids of human cell lines and patient-derived xenograft cells was monitored using (1) reconstituted basement membrane/hyaluronan interfaces representing the space along brain vessels; (2) 3D scaffolds generated by multi-layered mouse astrocytes to reflect brain interstitium; and (3) freshly isolated mouse brain slice culture ex vivo. The invasion patterns in vitro were validated using histological analysis of brain sections from glioblastoma patients and glioma xenografts infiltrating the mouse brain. Each 3D assay recapitulated distinct aspects of major glioma invasion patterns identified in mouse xenografts and patient brain samples, including individually migrating cells, collective strands extending along blood vessels, and multicellular networks of interconnected glioma cells infiltrating the neuropil. In conjunction, these organotypic assays enable a range of invasion modes used by glioma cells and will be applicable for mechanistic analysis and targeting of glioma cell dissemination.

Synthesis and Characterization of a Lubricin Mimic (mLub) To Reduce Friction and Adhesion on the Articular Cartilage Surface

PubMed Central

Lawrence, Alexandra; Xu, Xin; Bible, Melissa D.; Calve, Sarah; Neu, Corey P.; Panitch, Alyssa

2015-01-01

The lubricating proteoglycan, lubricin, facilitates the remarkable low friction and wear properties of articular cartilage in the synovial joints of the body. Lubricin lines the joint surfaces and plays a protective role as a boundary lubricant in sliding contact; decreased expression of lubricin is associated with cartilage degradation and the pathogenesis of osteoarthritis. An unmet need for early osteoarthritis treatment is the development of therapeutic molecules that mimic lubricin function and yet are also resistant to enzymatic degradation common in the damaged joint. Here, we engineered a lubricin mimic (mLub) that is less susceptible to enzymatic degradation and binds to the articular surface to reduce friction. mLub was synthesized using a chondroitin sulfate backbone with type II collagen and hyaluronic acid (HA) binding peptides to promote interaction with the articular surface and synovial fluid constituents. In vitro and in vivo characterization confirmed the binding ability of mLub to isolated type II collagen and HA, and to the cartilage surface. Following trypsin treatment to the cartilage surface, application of mLub, in combination with purified or commercially available hyaluronan, reduced the coefficient of friction, and adhesion, to control levels as assessed over macro- to micro-scales by rheometry and atomic force microscopy. In vivo studies demonstrate an mLub residency time of less than 1 week. Enhanced lubrication by mLub reduces surface friction and adhesion, which may suppress the progression of degradation and cartilage loss in the joint. mLub therefore shows potential for treatment in early osteoarthritis following injury. PMID:26398308

pH effects on the hyaluronan hydrolysis catalysed by hyaluronidase in the presence of proteins: Part I. Dual aspect of the pH-dependence.

PubMed

Lenormand, Hélène; Deschrevel, Brigitte; Vincent, Jean-Claude

2010-05-01

Hyaluronan (HA) hydrolysis catalysed by hyaluronidase (HAase) is strongly inhibited when performed at a low ratio of HAase to HA concentrations and at low ionic strength. This is because long HA chains can form non-active complexes with HAase. Bovine serum albumin (BSA) is able to compete with HAase to form electrostatic complexes with HA so freeing HAase which then recovers its catalytic activity. This BSA-dependence is characterised by two main domains separated by the optimal BSA concentration: below this concentration the HAase activity increases when the BSA concentration is increased, above this concentration the HAase activity decreases. This occurs provided that HA is negatively charged and BSA is positively charged, i.e. in a pH range from 3 to 5.25. The higher the pH value the higher the optimal BSA concentration. Other proteins can also modulate HAase activity. Lysozyme, which has a pI higher than that of BSA, is also able to compete with HAase to form electrostatic complexes with HA and liberate HAase. This occurs over a wider pH range that extends from 3 to 9. These results mean that HAase can form complexes with HA and recover its enzymatic activity at pH as high as 9, consistent with HAase having either a high pI value or positively charged patches on its surface at high pH. Finally, the pH-dependence of HAase activity, which results from the influence of pH on both the intrinsic HAase activity and the formation of complexes between HAase and HA, shows a maximum at pH 4 and a significant activity up to pH 9. Copyright 2009 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers.

PubMed

Burleson, Kathryn M; Casey, Rachael C; Skubitz, Keith M; Pambuccian, Stephan E; Oegema, Theodore R; Skubitz, Amy P N

2004-04-01

Ovarian carcinoma cells form multicellular aggregates, or spheroids, in the peritoneal cavity of patients with advanced disease. The current paradigm that ascites spheroids are non-adhesive leaves their contribution to ovarian carcinoma dissemination undefined. Here, spheroids obtained from ovarian carcinoma patients' ascites were characterized for their ability to adhere to molecules encountered in the peritoneal cavity, with the goal of establishing their potential to contribute to ovarian cancer spread. Spheroids were recovered from the ascites fluid of 11 patients with stage III or stage IV ovarian carcinoma. Adhesion assays to extracellular matrix (ECM) proteins and human mesothelial cell monolayers were performed for each of the ascites spheroid samples. Subsequently, inhibition assays were performed to identify the cell receptors involved. Most ascites samples adhered moderately to fibronectin and type I collagen, with reduced adhesion to type IV collagen and laminin. Monoclonal antibodies against the beta1 integrin subunit partially inhibited this adhesion. Ascites spheroids also adhered to hyaluronan. Additionally, spheroids adhered to live, but not fixed, human mesothelial cell monolayers, and this adhesion was partially mediated by beta1 integrins. The cellular content of the ascites fluid has often been considered non-adhesive, but our findings are the first to suggest that patient-derived ascites spheroids can adhere to mesothelial extracellular matrix via beta1 integrins, indicating that spheroids should not be ignored in the dissemination of ovarian cancer.

CD44-mediated activation of α5β1-integrin, cortactin and paxillin signaling underpins adhesion of basal-like breast cancer cells to endothelium and Fibronectin-enriched matrices

PubMed Central

McFarlane, Suzanne; McFarlane, Cheryl; Montgomery, Nicola; Hill, Ashleigh; Waugh, David J.J.

2015-01-01

CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of β1-integrin receptors, and increased α5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an α5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the β1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with β1-integrin and repressed HA-induced, CD44-mediated activation of β1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and β1-integrin-dependent mechanism. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche. PMID:26447611

HA metabolism in skin homeostasis and inflammatory disease.

PubMed

Kavasi, Rafaela-Maria; Berdiaki, Aikaterini; Spyridaki, Ioanna; Corsini, Emanuela; Tsatsakis, Aristidis; Tzanakakis, George; Nikitovic, Dragana

2017-03-01

Hyaluronan (HA), an unsulfated glycosaminoglycan, is an important component of the complex extracellular matrix network which surrounds and supports cells in tissues. HA is detected in all vertebrate tissues, but the bulk of HA is produced and deposited in the skin. In this review we focus on the role of HA in skin-associated inflammatory disease and wound healing. Properties of HA are directly dependent on its molecular weight. Thus, high molecular weight HA (HMWHA) is deposited in normal tissues during homeostasis and promotes their stability whereas low molecular weight HA fragments (LMWHA), on the other hand, may arise from enzymatic or chemical activities. The degradation of HMWHA to LMWHA fragments, often leads to the generation of biologically active oligosaccharides with different properties and postulated functions in wound scar formation and inflammation. More detailed studies of HA involvement in skin-associated inflammatory disease may result in novel treatment modalities. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Viscosupplementation for grade II osteoarthritis of the ankle: a prospective study at 18 months' follow-up.

PubMed

Luciani, Deianira; Cadossi, Matteo; Tesei, Federico; Chiarello, Eugenio; Giannini, Sandro

2008-12-01

Viscosupplementation, with hyaluronan derivates injected into the intra-space of osteoarthritic joints, is now widely used for the treatment of knee osteoarthritis. This study evaluates the results in terms of pain and disability of intra-articular injections of hyaluronan derivates into the ankle joint in patients suffering from grade II primary or secondary osteoarthritis of the ankle. Twenty-one patients with a painful ankle and radiographic evidence of grade II osteoarthritis had three weekly intra-articular injections of 2 ml of hylan G-F 20 (10 mg/ml) into the ankle joint. The primary clinical outcome measurement was the ankle osteoarthritis score (AOS) at the baseline, and at 6, 12 and 18 months. Significant improvement of the AOS from baseline was seen after 6 months (p=0.0001). This improvement was maintained over time with no further changes at 12- and 18-month follow-ups. Regarding pain, the AOS improved over time from the baseline to the 18-month follow up and became statistically significant at the 12- and 18-month follow-ups (p<0.05).

Biosimulation of Inflammation and Healing in Surgically Injured Vocal Folds

PubMed Central

Li, Nicole Y. K.; Vodovotz, Yoram; Hebda, Patricia A.; Abbott, Katherine Verdolini

2010-01-01

Objectives The pathogenesis of vocal fold scarring is complex and remains to be deciphered. The current study is part of research endeavors aimed at applying systems biology approaches to address the complex biological processes involved in the pathogenesis of vocal fold scarring and other lesions affecting the larynx. Methods We developed a computational agent-based model (ABM) to quantitatively characterize multiple cellular and molecular interactions involved in inflammation and healing in vocal fold mucosa after surgical trauma. The ABM was calibrated with empirical data on inflammatory mediators (eg, tumor necrosis factor) and extracellular matrix components (eg, hyaluronan) from published studies on surgical vocal fold injury in the rat population. Results The simulation results reproduced and predicted trajectories seen in the empirical data from the animals. Moreover, the ABM studies suggested that hyaluronan fragments might be the clinical surrogate of tissue damage, a key variable that in these simulations both is enhanced by and further induces inflammation. Conclusions A relatively simple ABM such as the one reported in this study can provide new understanding of laryngeal wound healing and generate working hypotheses for further wet-lab studies. PMID:20583741

Study on mutual interactions and electronic structures of hyaluronan with Lysine, 6-Aminocaproic acid and Arginine.

PubMed

Chytil, Martin; Trojan, Martin; Kovalenko, Alexander

2016-05-20

Interactions between polyelectrolytes and oppositely charged surfactants have been in a great interest for several decades, yet the conventional surfactants may cause a problem in medical applications. Interactivity between polysaccharide hyaluronan (HA) and amino acids Lysine, 6-Aminocaproic acid (6-AcA), and Arginine as an alternative system is reported. The interactions were investigated by means of rheology and electric conductance and the electronic structures were explored by the density functional theory (DFT). Lysine exhibits the strongest interaction of all, which was manifested, e.g. by nearly 6-time drop of the initial viscosity comparing with only 1.3-time lower value in the case of 6-AcA. Arginine interaction with HA was surprisingly weaker in terms of viscosity than that of Lysine due to a lower and delocalized charge density on its guanidine group. According to the DFT calculations, the binding of Lysine to HA was found to be more flexible, while Arginine creates more rigid structure with HA. Copyright © 2016 Elsevier Ltd. All rights reserved.

Biosimulation of inflammation and healing in surgically injured vocal folds.

PubMed

Li, Nicole Y K; Vodovotz, Yoram; Hebda, Patricia A; Abbott, Katherine Verdolini

2010-06-01

The pathogenesis of vocal fold scarring is complex and remains to be deciphered. The current study is part of research endeavors aimed at applying systems biology approaches to address the complex biological processes involved in the pathogenesis of vocal fold scarring and other lesions affecting the larynx. We developed a computational agent-based model (ABM) to quantitatively characterize multiple cellular and molecular interactions involved in inflammation and healing in vocal fold mucosa after surgical trauma. The ABM was calibrated with empirical data on inflammatory mediators (eg, tumor necrosis factor) and extracellular matrix components (eg, hyaluronan) from published studies on surgical vocal fold injury in the rat population. The simulation results reproduced and predicted trajectories seen in the empirical data from the animals. Moreover, the ABM studies suggested that hyaluronan fragments might be the clinical surrogate of tissue damage, a key variable that in these simulations both is enhanced by and further induces inflammation. A relatively simple ABM such as the one reported in this study can provide new understanding of laryngeal wound healing and generate working hypotheses for further wet-lab studies.

Novel Gene Expression Profile of Women with Intrinsic Skin Youthfulness by Whole Transcriptome Sequencing

PubMed Central

Xu, Jin; Spitale, Robert C.; Guan, Linna; Flynn, Ryan A.; Torre, Eduardo A.; Li, Rui; Raber, Inbar; Qu, Kun; Kern, Dale; Knaggs, Helen E.; Chang, Howard Y.; Chang, Anne Lynn S.

2016-01-01

While much is known about genes that promote aging, little is known about genes that protect against or prevent aging, particularly in human skin. The main objective of this study was to perform an unbiased, whole transcriptome search for genes that associate with intrinsic skin youthfulness. To accomplish this, healthy women (n = 122) of European descent, ages 18–89 years with Fitzpatrick skin type I/II were examined for facial skin aging parameters and clinical covariates, including smoking and ultraviolet exposure. Skin youthfulness was defined as the top 10% of individuals whose assessed skin aging features were most discrepant with their chronological ages. Skin biopsies from sun-protected inner arm were subjected to 3’-end sequencing for expression quantification, with results verified by quantitative reverse transcriptase-polymerase chain reaction. Unbiased clustering revealed gene expression signatures characteristic of older women with skin youthfulness (n = 12) compared to older women without skin youthfulness (n = 33), after accounting for gene expression changes associated with chronological age alone. Gene set analysis was performed using Genomica open-access software. This study identified a novel set of candidate skin youthfulness genes demonstrating differences between SY and non-SY group, including pleckstrin homology like domain family A member 1 (PHLDA1) (p = 2.4x10-5), a follicle stem cell marker, and hyaluronan synthase 2-anti-sense 1 (HAS2-AS1) (p = 0.00105), a non-coding RNA that is part of the hyaluronan synthesis pathway. We show that immunologic gene sets are the most significantly altered in skin youthfulness (with the most significant gene set p = 2.4x10-5), suggesting the immune system plays an important role in skin youthfulness, a finding that has not previously been recognized. These results are a valuable resource from which multiple future studies may be undertaken to better understand the mechanisms that promote skin youthfulness in humans. PMID:27829007

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.

Selective Matrix (Hyaluronan) Interaction with CD44 and RhoGTPase Signaling Promotes Keratinocyte Functions and Overcomes Age-related Epidermal Dysfunction

PubMed Central

Bourguignon, Lilly Y.W.; Wong, Gabriel; Xia, Weiliang; Man, Mao-Qiang; Holleran, Walter M.; Elias, Peter M.

2013-01-01

Background Mouse epidermal chronologic aging is closely associated with aberrant matrix (hyaluronan, HA) -size distribution/production and impaired keratinocyte proliferation/differentiation, leading to a marked thinning of the epidermis with functional consequence that causes a slower recovery of permeability barrier function. Objective The goal of this study is to demonstrate mechanism-based, corrective therapeutic strategies using topical applications of small HA (HAS) and/or large HA (HAL) [or a sequential small HA (HAS) and large HA(HAL) (HAs-»HAL) treatment] as well as RhoGTPase signaling perturbation agents to regulate HA/CD44-mediated signaling, thereby restoring normal epidermal function, and permeability barrier homeostasis in aged mouse skin. Methods A number of biochemical, cell biological/molecular, pharmacological and physiological approaches were used to investigate matrix HA-CD44-mediated RhoGTPase signaling in regulating epidermal functions and skin aging. Results In this study we demonstrated that topical application of small HA (HAS) promotes keratinocyte proliferation and increases skin thickness, while it fails to upregulate keratinocyte differentiation or permeability barrier repair in aged mouse skin. In contrast, large HA (HAL) induces only minimal changes in keratinocyte proliferation and skin thickness, but restores keratinocyte differentiation and improves permeability barrier function in aged epidermis. Since neither HAS nor HAL corrects these epidermal defects in aged CD44 knock-out mice, CD44 likely mediates HA-associated epidermal functions in aged mouse skin. Finally, blockade of Rho-kinase activity with Y27632 or protein kinase-Nγ activity with Ro31-8220 significantly decreased the HA (HAS or HAL)-mediated changes in epidermal function in aged mouse skin. Conclusion The results of our study show first that HA application of different sizes regulates epidermal proliferation, differentiation and barrier function in aged mouse skin. Second, manipulation of matrix (HA) interaction with CD44 and RhoGTPase signaling could provide further novel therapeutic approaches that could be targeted for the treatment of various aging-related skin disorders. PMID:23790635

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

PubMed

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.

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

Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei.

PubMed

Biner, Olivier; Trachsel, Christian; Moser, Aline; Kopp, Lukas; Langenegger, Nicolas; Kämpfer, Urs; von Ballmoos, Christoph; Nentwig, Wolfgang; Schürch, Stefan; Schaller, Johann; Kuhn-Nentwig, Lucia

2015-01-01

Hyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40-60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis. Besides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-β-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae) without activity of hyaluronidase-like enzymes. This is interpreted as a loss of this enzyme and fits quite well the current phylogenetic idea on a more isolated position of these families and can perhaps be explained by specialized prey catching techniques.

Interactive effect of chondroitin sulphate C and hyaluronan on fluid movement across rabbit synovium

PubMed Central

Sabaratnam, S; Coleman, P J; Badrick, E; Mason, R M; Levick, J R

2002-01-01

The polysaccharide hyaluronan (HA) conserves synovial fluid by keeping outflow low and almost constant over a wide pressure range (‘buffering’), but only at concentrations associated with polymer domain overlap. We therefore tested whether polymer interactions can cause buffering, using HA-chondroitin sulphate C (CSC) mixtures. Also, since it has been found that capillary filtration is insensitive to the Starling force interstitial osmotic pressure in frog mesenteries, this was assessed in synovium. Hyaluronan at non-buffering concentrations (0.50–0.75 mg ml−1) and/or 25 mg ml−1 CSC (osmotic pressure 68 cmH2O) was infused into knees of anaesthetised rabbits in vivo. Viscometry and chromatography confirmed that HA interacts with CSC. Pressure (Pj) versus trans-synovial flow (Q̇s) relations were measured. Q̇s was outwards for HA alone (1.2 ± 0.9 μl min−1 at 3 cmH2O, mean ± s.e.m.; n = 6). CSC diffused into synovium and changed Q̇s to filtration at low Pj (−4.1 μl min−1, 3 cmH2O, n = 5, P < 0.02, t test). Filtration ceased upon circulatory arrest (n = 3). At higher Pj, 0.75 mg ml−1 HA plus CSC buffered Q̇s to ∼3 μl min−1 over a wide range of Pj, with an outflow increase of only 0.04 ± 0.02 μl min−1 cmH2O−1 (n = 4). With HA or CSC alone, buffering was absent (slopes 0.57 ± 0.04 μl min−1 cmH2O−1 (n = 4) and 0.86 ± 0.05 μl min−1 cmH2O−1 (n = 5), respectively). Therefore, polymer interactions can cause outflow buffering in joints. Also, interstitial osmotic pressure promoted filtration in fenestrated synovial capillaries, so the results for frog mesentery capillaries cannot be generalised. The difference is attributed to differences in pore ultrastructure. PMID:11927686

Angiotensin II AT1 receptor alters ACE2 activity, eNOS expression and CD44-hyaluronan interaction in rats with hypertension and myocardial fibrosis.

PubMed

Bai, Feng; Pang, Xue-Fen; Zhang, Li-Hui; Wang, Ning-Ping; McKallip, Robert J; Garner, Ronald E; Zhao, Zhi-Qing

2016-05-15

This study tested the hypothesis that angiotensin II (Ang II) AT1 receptor is involved in development of hypertension and cardiac fibrosis via modifying ACE2 activity, eNOS expression and CD44-hyaluronan interaction. Male Sprague-Dawley rats were subjected to Ang II infusion (500ng/kg/min) using osmotic minipumps up to 4weeks and the AT1 receptor blocker, telmisartan was administered by gastric gavage (10mg/kg/day) during Ang II infusion. Our results indicated that Ang II enhances AT1 receptor, downregulates AT2 receptor, ACE2 activity and eNOS expression, and increases CD44 expression and hyaluronidase activity, an enzyme for hyaluronan degradation. Further analyses revealed that Ang II increases blood pressure and augments vascular/interstitial fibrosis. Comparison of the Ang II group, treatment with telmisartan significantly increased ACE2 activity and eNOS expression in the intracardiac vessels and intermyocardium. These changes occurred in coincidence with decreased blood pressure. Furthermore, the locally-expressed AT1 receptor was downregulated, as evidenced by an increased ratio of the AT2 over AT1 receptor (1.4±0.4% vs. 0.4±0.1% in Ang II group, P<0.05). Along with these modulations, telmisartan inhibited membrane CD44 expression and hyaluronidase activity, decreased populations of macrophages and myofibroblasts, and reduced expression of TGFβ1 and Smads. Collagen I synthesis and tissue fibrosis were attenuated as demonstrated by the less extensive collagen-rich area. These results suggest that the AT1 receptor is involved in development of hypertension and cardiac fibrosis. Selective activating ACE2/eNOS and inhibiting CD44/HA interaction might be considered as the therapeutic targets for attenuating Ang II induced deleterious cardiovascular effects. Copyright © 2016 Elsevier Inc. All rights reserved.

Hyaluronan Synthase 3 Variant and Anthracycline-Related Cardiomyopathy: A Report From the Children's Oncology Group

PubMed Central

Wang, Xuexia; Liu, Wei; Sun, Can-Lan; Armenian, Saro H.; Hakonarson, Hakon; Hageman, Lindsey; Ding, Yan; Landier, Wendy; Blanco, Javier G.; Chen, Lu; Quiñones, Adolfo; Ferguson, Daniel; Winick, Naomi; Ginsberg, Jill P.; Keller, Frank; Neglia, Joseph P.; Desai, Sunil; Sklar, Charles A.; Castellino, Sharon M.; Cherrick, Irene; Dreyer, ZoAnn E.; Hudson, Melissa M.; Robison, Leslie L.; Yasui, Yutaka; Relling, Mary V.; Bhatia, Smita

2014-01-01

Purpose The strong dose-dependent association between anthracyclines and cardiomyopathy is further exacerbated by the co-occurrence of cardiovascular risk factors (diabetes and hypertension). The high morbidity associated with cardiomyopathy necessitates an understanding of the underlying pathogenesis so that targeted interventions can be developed. Patients and Methods By using a two-stage design, we investigated host susceptibility to anthracycline-related cardiomyopathy by using the ITMAT/Broad CARe cardiovascular single nucleotide polymorphism (SNP) array to profile common SNPs in 2,100 genes considered relevant to de novo cardiovascular disease. Results By using a matched case-control design (93 cases, 194 controls), we identified a common SNP, rs2232228, in the hyaluronan synthase 3 (HAS3) gene that exerts a modifying effect on anthracycline dose-dependent cardiomyopathy risk (P = 5.3 × 10−7). Among individuals with rs2232228 GG genotype, cardiomyopathy was infrequent and not dose related. However, in individuals exposed to high-dose (> 250 mg/m2) anthracyclines, the rs2232228 AA genotype conferred an 8.9-fold (95% CI, 2.1- to 37.5-fold; P = .003) increased cardiomyopathy risk compared with the GG genotype. This gene-environment interaction was successfully replicated in an independent set of 76 patients with anthracycline-related cardiomyopathy. Relative HAS3 mRNA levels measured in healthy hearts tended to be lower among individuals with AA compared with GA genotypes (P = .09). Conclusion Hyaluronan (HA) produced by HAS3 is a ubiquitous component of the extracellular matrix and plays an active role in tissue remodeling. In addition, HA is known to reduce reactive oxygen species (ROS) –induced cardiac injury. The high cardiomyopathy risk associated with AA genotype could be due to inadequate remodeling and/or inadequate protection of the heart from ROS-mediated injury on high anthracycline exposure. PMID:24470002

An anti-let-7 sponge decoys and decays endogenous let-7 functions

PubMed Central

Yang, Xiangling; Rutnam, Zina Jeyapalan; Jiao, Chunwei; Wei, Duo; Xie, Yizhen; Du, Jun; Zhong, Ling; Yang, Burton B.

2012-01-01

The let-7 family contains 12 members, which share identical seed regions, suggesting that they may target the same mRNAs. It is essential to develop a means that can regulate the functions of all members. Using a DNA synthesis technique, we have generated an anti-let-7 sponge aiming to modulate the function of all members. We found that products of the anti-let-7 construct could bind and inactivate all members of the let-7 family, producing decoy and decay effects. To test the role of the anti-let-7 sponge, we stably expressed the anti-let-7 construct in two types of cells, the breast carcinoma cells MT-1 and the oldest and most commonly used human cervical cancer cell line, HeLa cells. We found that expression of anti-let-7 increased cell survival, invasion and adhesion, which corroborate with known functions of let-7 family members. We further identified a novel target site across all species of the let-7 family in hyaluronan synthase 2 (HAS2). HAS2 overexpression produced similar effects as the anti-let-7 sponge. Silencing HAS2 expression by siRNAs produced opposite effects to anti-let-7 on cell survival and invasion. The ability of anti-let-7 to regulate multiple members of the let-7 family allows us to observe their multiple functions using a single reagent. This approach can be applied to other family members with conserved sequences. PMID:22871741

Rigidity-patterned polyelectrolyte films to control myoblast cell adhesion and spatial organization

PubMed Central

Monge, Claire; Saha, Naresh; Boudou, Thomas; Pózos-Vásquez, Cuauhtemoc; Dulong, Virginie; Glinel, Karine; Picart, Catherine

2014-01-01

In vivo, cells are sensitive to the stiffness of their micro-environment and especially to the spatial organization of the stiffness. In vitro studies of this phenomenon can help to better understand the mechanisms of the cell response to spatial variations of the matrix stiffness. In this work, we design polelyelectrolyte multilayer films made of poly(L-lysine) and a photo-reactive hyaluronan derivative. These films can be photo-crosslinked through a photomask to create spatial patterns of rigidity. Quartz substrates incorporating a chromium mask are prepared to expose selectively the film to UV light (in a physiological buffer), without any direct contact between the photomask and the soft film. We show that these micropatterns are chemically homogeneous and flat, without any preferential adsorption of adhesive proteins. Three groups of pattern geometries differing by their shape (circles or lines), size (form 2 to 100 μm) or interspacing distance between the motifs are used to study the adhesion and spatial organization of myoblast cells. On large circular micropatterns, the cells form large assemblies that are confined to the stiffest parts. Conversely, when the size of the rigidity patterns is subcellular, the cells respond by forming protrusions. Finally, on linear micropatterns of rigidity, myoblasts align and their nuclei drastically elongate in specific conditions. These results pave the way for the study of the different steps of myoblast fusion in response to matrix rigidity in well-defined geometrical conditions. PMID:25100929

Alteration of pancreatic carcinoma and promyeloblastic cell adhesion in liver microvasculature by co-culture of hepatocytes, hepatic stellate cells and endothelial cells in a physiologically-relevant model.

PubMed

Danoy, Mathieu; Shinohara, Marie; Rizki-Safitri, Astia; Collard, Dominique; Senez, Vincent; Sakai, Yasuyuki

2017-04-18

In vitro models of the liver microvasculature, especially with respect to cancer cell extravasation, should include not only endothelial and cancer cells but also surrounding cells to mimic the physiological situation. To this end, in the present study, we established a physiologically-relevant hierarchical co-culture model by stacking layers of primary rat hepatocytes (Hep), hepatic stellate cells embedded in collagen gel (LX-2) and endothelial cells (HUVECs) on a specially designed oxygen-permeable polydimethylsiloxane PDMS bottom plate. The model was used to investigate the role and contribution of each of the three cell types in pancreatic cancer and promyeloblast cell adhesion. In particular, we showed an increase in albumin production by the primary hepatocytes and in the consumption of the produced vascular endothelial growth factors (VEGFs). Furthermore, in co-culture, the HUVECs exhibited a mature vascular endothelial and non-inflamed phenotype, as evidenced by Stabilin-1, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), intercellular adhesion molecule (ICAM-1), and vascular adhesion protein-1 (VAP-1) expression. The HUVECs were also successfully activated with an inflammatory cytokine and their ICAM-1 response was found to be higher in monoculture compared to co-culture. Additionally, the adhesion of MiaPaCa-2 pancreatic cancer cells and HL60 promyeloblasts was tested in both cases (i.e.: activation or not by an inflammatory cytokine). It has been found that their adhesion was always reduced in the co-culture model. These results highlight the importance of integrating hepatic stellate cells in the design of biomimetic models of the hepatic endothelial barrier.

Response of human dental pulp cells to a silver-containing PLGA/TCP-nanofabric as a potential antibacterial regenerative pulp-capping material.

PubMed

Cvikl, Barbara; Hess, Samuel C; Miron, Richard J; Agis, Hermann; Bosshardt, Dieter; Attin, Thomas; Schmidlin, Patrick R; Lussi, Adrian

2017-02-27

Damage or exposure of the dental pulp requires immediate therapeutic intervention. This study assessed the biocompatibility of a silver-containing PLGA/TCP-nanofabric scaffold (PLGA/Ag-TCP) in two in vitro models, i.e. the material adapted on pre-cultured cells and cells directly cultured on the material, respectively. Collagen saffolds with and without hyaluronan acid (Coll-HA; Coll) using both cell culturing methods and cells growing on culture plates served as reference. Cell viability and proliferation were assessed after 24, 48, and 72 h based on formazan formation and BrdU incorporation. Scaffolds were harvested. Gene expression of interleukin(IL)-6, tumor necrosis factor (TNF)-alpha, and alkaline phosphatase (AP) was assessed 24 h after stimulation. In both models formazan formation and BrdU incorporation was reduced by PLGA/Ag-TCP on dental pulp cells, while no significant reduction was found in cells with Coll and Coll-HA. Cells with PLGA/Ag-TCP for 72 h showed similar relative BrdU incorporation than cells stimulated with Coll and Coll-HA. A prominent increase in the pro-inflammatory genes IL-6 and TNF-α was observed when cells were cultured with PLGA/Ag-TCP compared to the other groups. This increase was parallel with a slight increase in AP expression. Overall, no differences between the two culture methods were observed. PLGA/Ag-TCP decreased viability and proliferation rate of human dental pulp cells and increased the pro-inflammatory capacity and alkaline phosphatase expression. Whether these cellular responses observed in vitro translate into pulp regeneration in vivo will be assessed in further studies.

Modular Extracellular Matrices: Solutions for the Puzzle

PubMed Central

Serban, Monica A.; Prestwich, Glenn D.

2008-01-01

The common technique of growing cells in two-dimensions (2-D) is gradually being replaced by culturing cells on matrices with more appropriate composition and stiffness, or by encapsulation of cells in three-dimensions (3-D). The universal acceptance of the new 3-D paradigm has been constrained by the absence of a commercially available, biocompatible material that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. The challenge – the puzzle that needs a solution – is to replicate the complexity of the native extracellular matrix (ECM) environment with the minimum number of components necessary to allow cells to rebuild and replicate a given tissue. For use in drug discovery, toxicology, cell banking, and ultimately in reparative medicine, the ideal matrix would therefore need to be highly reproducible, manufacturable, approvable, and affordable. Herein we describe the development of a set of modular components that can be assembled into biomimetic materials that meet these requirements. These semi-synthetic ECMs, or sECMs, are based on hyaluronan derivatives that form covalently crosslinked, biodegradable hydrogels suitable for 3-D culture of primary and stem cells in vitro, and for tissue formation in vivo. The sECMs can be engineered to provide appropriate biological cues needed to recapitulate the complexity of a given ECM environment. Specific applications for different sECM compositions include stem cell expansion with control of differentiation, scar-free wound healing, growth factor delivery, cell delivery for osteochondral defect and liver repair, and development of vascularized tumor xenografts for personalized chemotherapy. PMID:18442709

Synthesis and characterization of a lubricin mimic (mLub) to reduce friction and adhesion on the articular cartilage surface.

PubMed

Lawrence, Alexandra; Xu, Xin; Bible, Melissa D; Calve, Sarah; Neu, Corey P; Panitch, Alyssa

2015-12-01

The lubricating proteoglycan, lubricin, facilitates the remarkable low friction and wear properties of articular cartilage in the synovial joints of the body. Lubricin lines the joint surfaces and plays a protective role as a boundary lubricant in sliding contact; decreased expression of lubricin is associated with cartilage degradation and the pathogenesis of osteoarthritis. An unmet need for early osteoarthritis treatment is the development of therapeutic molecules that mimic lubricin function and yet are also resistant to enzymatic degradation common in the damaged joint. Here, we engineered a lubricin mimic (mLub) that is less susceptible to enzymatic degradation and binds to the articular surface to reduce friction. mLub was synthesized using a chondroitin sulfate backbone with type II collagen and hyaluronic acid (HA) binding peptides to promote interaction with the articular surface and synovial fluid constituents. In vitro and in vivo characterization confirmed the binding ability of mLub to isolated type II collagen and HA, and to the cartilage surface. Following trypsin treatment to the cartilage surface, application of mLub, in combination with purified or commercially available hyaluronan, reduced the coefficient of friction, and adhesion, to control levels as assessed over macro-to micro-scales by rheometry and atomic force microscopy. In vivo studies demonstrate an mLub residency time of less than 1 week. Enhanced lubrication by mLub reduces surface friction and adhesion, which may suppress the progression of degradation and cartilage loss in the joint. mLub therefore shows potential for treatment in early osteoarthritis following injury. Copyright © 2015 Elsevier Ltd. All rights reserved.

Synergistic effect of defined artificial extracellular matrices and pulsed electric fields on osteogenic differentiation of human MSCs.

PubMed

Hess, Ricarda; Jaeschke, Anna; Neubert, Holger; Hintze, Vera; Moeller, Stephanie; Schnabelrauch, Matthias; Wiesmann, Hans-Peter; Hart, David A; Scharnweber, Dieter

2012-12-01

In vivo, bone formation is a complex, tightly regulated process, influenced by multiple biochemical and physical factors. To develop a vital bone tissue engineering construct, all of these individual components have to be considered and integrated to gain an in vivo-like stimulation of target cells. The purpose of the present studies was to investigate the synergistic role of defined biochemical and physical microenvironments with respect to osteogenic differentiation of human mesenchymal stem cells (MSCs). Biochemical microenvironments have been designed using artificial extracellular matrices (aECMs), containing collagen I (coll) and glycosaminoglycans (GAGs) like chondroitin sulfate (CS), or a high-sulfated hyaluronan derivative (sHya), formulated as coatings on three-dimensional poly(caprolactone-co-lactide) (PCL) scaffolds. As part of the physical microenvironment, cells were exposed to pulsed electric fields via transformer-like coupling (TC). Results showed that aECM containing sHya enhanced osteogenic differentiation represented by increases in ALP activity and gene-expression (RT-qPCR) of several bone-related proteins (RUNX-2, ALP, OPN). Electric field stimulation alone did not influence cell proliferation, but osteogenic differentiation was enhanced if osteogenic supplements were provided, showing synergistic effects by the combination of sHya and electric fields. These results will improve the understanding of bone regeneration processes and support the development of effective tissue engineered bone constructs. Copyright © 2012 Elsevier Ltd. All rights reserved.

HABP1/p32/gC1qR induces aberrant growth and morphology in Schizosaccharomyces pombe through its N-terminal {alpha} helix

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mallick, Jaideep; Datta, Kasturi

2005-10-01

Hyaluronan binding protein (HABP1), located on human chromosome 17p13.3, was identified and characterized as being involved in cellular signaling from our laboratory. Here, we demonstrate that HABP1 expression in Schizosaccharomyces pombe induces growth inhibition, morphological abnormalities like elongation, multinucleation and aberrant cell septum formation in several strains of S. pombe, implicating its role in cell cycle progression and cytokinesis. This argument is further strengthened by an observed delay in the maximal expression of cell cycle regulatory proteins like CDC 2 and CDC 25 coupled to the direct interaction of HABP1 with CDC 25. In order to pinpoint the interacting domainmore » of HABP1, its N- and C-terminal truncated variants ({delta}N.HABP1 and {delta}C.HABP1, respectively) were utilized which revealed that while expression of the former did not alter the phenotype, the latter generated morphological changes similar to those imparted upon HABP1 expression. It was also noted that along with HABP1, {delta}C.HABP1 too directly interacts with CDC 25 while {delta}N.HABP1 does not. Taken together, these data suggest that HABP1 induces morphological changes and modulates the cell cycle by interacting with proteins like CDC 25 through its N-terminal {alpha}-helix.« less

Stem cells from human exfoliated deciduous teeth (SHED) enhance wound healing and the possibility of novel cell therapy.

PubMed

Nishino, Yudai; Yamada, Yoichi; Ebisawa, Katsumi; Nakamura, Sayaka; Okabe, Kazuto; Umemura, Eri; Hara, Kenji; Ueda, Minoru

2011-05-01

In recent years, stem cells from human exfoliated deciduous teeth (SHED) have received attention as a novel stem cell source with multipotent potential. We examined the effect on wound-healing promotion with unique stem cells from deciduous teeth as a medical waste. An excisional wound-splinting mouse model was used and the effect of wound healing among SHED, human mesenchymal stromal cells (hMSCs), human fibroblasts (hFibro) and a control (phosphate-buffered saline; PBS) was evaluated by macroscopy, histology and enzyme-linked immunosorbent assay (ELISA), and the expression of hyaluronan (HA), which is related to wound healing, investigated. SHED and hMSCs accelerated wound healing compared with hFibro and the control. There was a statistically significant difference in wound healing area among hFibro, hMSCs and SHED compared with the control after day 5. At days 7 and 14 after cell transplantation, the histologic observation showed that transplanted PKH26-positive cells were surrounded by human HA binding protein, especially in hMSCs and SHED. HA expression volume values were 1558.41 ± 60.33 (control), 2092.75 ± 42.56 (hFibro), 2342.07 ± 188.10 (hMSCs) and 2314.85 ± 164.91 (SHED) ng/mg, respectively, and significantly higher in hMSCs and SHED compared with hFibro and control at days 7 and 14 (P < 0.05). Our results show that SHED hMSCs have similar effects of wound-healing promotion as hFibro and controls. This implies that SHED might offer a unique stem cell resource and the possibility of novel cell therapies for wound healing in the future.

Group B streptococcus alters properties of vaginal epithelial cells in pregnant women.

PubMed

Scholl, Jessica; Nasioudis, Dimitrios; Boester, Allison; Speleotes, Maureen; Grunebaum, Amos; Witkin, Steven S

2016-03-01

Group B streptococcus (GBS) infection in pregnancy is a major cause of maternal and neonatal morbidity. An understanding of the mechanisms responsible for GBS persistence in the genital tract, as well as recognition of host defenses employed to combat its presence, are crucial to our efforts to reduce maternal GBS colonization and prevent the acquisition of neonatal infections. However, alterations in vaginal immunity in response to GBS colonization in pregnant women remain incompletely defined. Whether GBS modulates autophagy, a major host defense mechanism and contributor to the control of intracellular microbial infections, also remains unclear. We sought to identify differences in the extent of autophagy as well as in the concentration of biomarkers previously shown to be involved in vaginal innate immunity between GBS-positive and GBS-negative pregnant women. We performed a prospective cohort study of healthy pregnant women, who had vaginal secretions obtained at 35-37 weeks of gestation, just prior to the standard GBS rectovaginal sample collection. The contents of the swabs were released into tubes containing 1 mL of sterile phosphate-buffered saline. Samples were centrifuged, and supernatant and cell pellet fractions were collected and stored separately at -80°C until used for analysis. Epithelial cells were then lysed, and the extent of autophagy was determined by measuring the residual level of p62 remaining in the cytoplasm. p62 is a protein that is consumed during autophagy, and so its concentration detectable in the cytoplasm is inversely related to the extent of autophagy induction. The intracellular level of the inducible 70-kDa heat shock protein (hsp70), an inhibitor of autophagy, was also measured. The cell-free fraction was assayed for D- and L-lactic acid, neutrophil gelatinase-associated lipocalin, extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinase (MMP)-8, alpha amylase, hyaluronan, and total protein. Laboratory personnel were blinded to all clinical data. There were 145 women included in the study, of which 45 (31%) were culture-positive for GBS. Vaginal cells from GBS-positive women had elevated intracellular levels of p62 (2.1 vs 0.7 pg/mL, P < .01) and hsp70 (16.9 vs 9.6 ng/mL, P = .03) as compared to GBS-negative women. The p62 and hsp70 levels were highly correlated in both groups of subjects (P < .01). In vaginal fluid, concentrations of neutrophil gelatinase-associated lipocalin (1.1 vs 0.7 ng/μg total protein, P = .01), MMP-8 (21.9 vs 11.1 pg/μg total protein, P = .01), and extracellular MMP inducer (8.8 vs 7.2 pg/μg total protein, P = .03) were highest in GBS-positive women. There were no differences in the concentrations of D- and L-lactic acid, alpha amylase, or hyaluronan between the 2 groups of women. The inhibition of autophagy in vaginal epithelial cells by GBS-induced hsp70 production is associated with its persistence. Concurrently, alterations in components known to influence vaginal bacterial colonization or facilitate microbial passage to the upper genital tract also occur in relation to GBS carriage. Copyright © 2016 Elsevier Inc. All rights reserved.

Encapsulated Stem Cells Loaded With Hyaluronidase-expressing Oncolytic Virus for Brain Tumor Therapy

PubMed Central

Martinez-Quintanilla, Jordi; He, Derek; Wakimoto, Hiroaki; Alemany, Ramon; Shah, Khalid

2015-01-01

Despite the proven safety of oncolytic viruses (OV) in clinical trials for glioblastoma (GBM), their efficacy has been hindered by suboptimal spreading within the tumor. We show that hyaluronan or hyaluronic acid (HA), an important component of extracellular matrix (ECM), is highly expressed in a majority of tumor xenografts established from patient-derived GBM lines that present both invasive and nodular phenotypes. Intratumoral injection of a conditionally replicating adenovirus expressing soluble hyaluronidase (ICOVIR17) into nodular GBM, mediated HA degradation and enhanced viral spread, resulting in a significant antitumor effect and mice survival. In an effort to translate OV-based therapeutics into clinical settings, we encapsulated human adipose-derived mesenchymal stem cells (MSC) loaded with ICOVIR17 in biocompatible synthetic extracellular matrix (sECM) and tested their efficacy in a clinically relevant mouse model of GBM resection. Compared with direct injection of ICOVIR17, sECM-MSC loaded with ICOVIR17 resulted in a significant decrease in tumor regrowth and increased mice survival. This is the first report of its kind revealing the expression of HA in GBM and the role of OV-mediated HA targeting in clinically relevant mouse model of GBM resection and thus has clinical implications. PMID:25352242

Extracellular matrix components in breast cancer progression and metastasis.

PubMed

Oskarsson, Thordur

2013-08-01

The extracellular matrix (ECM) is composed of highly variable and dynamic components that regulate cell behavior. The protein composition and physical properties of the ECM govern cell fate through biochemical and biomechanical mechanisms. This requires a carefully orchestrated and thorough regulation considering that a disturbed ECM can have serious consequences and lead to pathological conditions like cancer. In breast cancer, many ECM proteins are significantly deregulated and specific matrix components promote tumor progression and metastatic spread. Intriguingly, several ECM proteins that are associated with breast cancer development, overlap substantially with a group of ECM proteins induced during the state of tissue remodeling such as mammary gland involution. Fibrillar collagens, fibronectin, hyaluronan and matricellular proteins are matrix components that are common to both involution and cancer. Moreover, some of these proteins have in recent years been identified as important constituents of metastatic niches in breast cancer. In addition, specific ECM molecules, their receptors or enzymatic modifiers are significantly involved in resistance to therapeutic intervention. Further analysis of these ECM proteins and the downstream ECM mediated signaling pathways may provide a range of possibilities to identify druggable targets against advanced breast cancer. Copyright © 2013 Elsevier Ltd. All rights reserved.

Inductions of granulosa cell luteinization and cumulus expansion are dependent on the fibronectin-integrin pathway during ovulation process in mice.

PubMed

Kitasaka, Hiroya; Kawai, Tomoko; Hoque, S A Masudul; Umehara, Takashi; Fujita, Youko; Shimada, Masayuki

2018-01-01

It has been known that EGF-like factor secreted from LH-stimulated granuloma cells acts on granulosa cells and cumulus cells to induce ovulation process. Granulosa cells are changed the morphology with differentiating cell functions to produce progesterone. Cumulus cells are detached to make a space between the cells to accumulate hyaluronan rich matrix. LH also changes extracellular matrix (ECM) components including fibronectin in the follicular walls and granulosa cell layers. EGF like factor and fibronectin synergistically play important roles in numerous cell functions, especially cancer cell migration, estimating that fibronectin would impact on granulosa cells and cumulus cells. To clear this hypothesis, the localizations of fibronectin and its receptor integrin were observed by immunofluorescence technique. The functions were monitored by the detection of downstream signaling pathway, focal adhesion kinase (FAK). The pharmacological approach in both in vivo and in vitro were used for analyzing the physiological roles of FAK during ovulation process. The immunofluorescence staining revealed that fibronectin and integrin were observed in granulosa cells, cumulus cells and the space between cumulus cells and oocyte at 4 and 8 h after hCG injection. Concomitantly with the changes of fibronectin-integrin localization, FAK was phosphorylated in periovulatory follicles. The injection of FAK inhibitor suppressed not only ovulation but also luteinization of granulosa cells and cumulus expansion. In cultured-granulosa cells, fibronectin-integrin synergistically activated FAK with amphiregulin (AREG). Such cooperative stimulations induced a morphological change in granulosa cells, which resulted in the maximum level of progesterone production via the induction of Hsd3b. When cumulus-oocyte complexes (COCs) were cultured with AREG in the presence of serum, the maximum level of cumulus expansion was observed. The AREG-induced cumulus expansion was also suppressed by FAK inhibitor. Thus, it is concluded that fibronectin and AREG synergistically activate FAK not only in granulosa cells and cumulus cells to induce successful ovulation process.

Phycocyanin-encapsulating hyalurosomes as carrier for skin delivery and protection from oxidative stress damage.

PubMed

Castangia, Ines; Manca, Maria Letizia; Catalán-Latorre, Ana; Maccioni, Anna Maria; Fadda, Anna Maria; Manconi, Maria

2016-04-01

The phycobiliprotein phycocyanin, extracted from Klamath algae, possesses important biological properties but it is characterized by a low bioavailability due to its high molecular weight. To overcome the bioavailability problems, phycocyanin was successfully encapsulated, using an environmentally-friendly method, into hyalurosomes, a new kind of phospholipid vesicles immobilised with hyaluronan sodium salt by the simple addition of drug/sodium hyaluronate water dispersion to phospholipids. Liposomes were used as a comparison. Vesicles were small in size and homogeneously dispersed, being the mean size always smaller than 150 nm and PI never higher than 0.31. Liposomes were unilamellar and spherical, the addition of the polymer slightly modify the vesicular shape which remain spherical, while the addition of PEG improve the lamellarity of vesicles being multilamellar vesicles. In all cases phycocyanin was encapsulated in good amount especially using hyalurosomes and PEG hyalurosomes (65 and 61% respectively). In vitro penetration studies suggested that hyalurosomes favoured the phycocyanin deposition in the deeper skin layers probably thanks to their peculiar hyaluronan-phospholipid structure. Moreover, hyalurosomes were highly biocompatible and improved phycocyanin antioxidant activity on stressed human keratinocytes respect to the drug solution.

Effect of growth factors on hyaluronan production by canine vocal fold fibroblasts.

PubMed

Hirano, Shigeru; Bless, Diane M; Heisey, Dennis; Ford, Charles N

2003-07-01

Hyaluronan (HYA) is considered to be a crucial factor in scarless wound healing and in maintaining tissue viscosity of the vocal fold lamina propria. In this study focusing on the effects of growth factors, we examined how HYA is produced and controlled in canine cultured vocal fold fibroblasts. Fibroblasts were taken from the lamina propria of the vocal folds of 8 dogs and cultured with and without growth factors. The production of HYA in the supernatant culture was quantitatively examined by enzyme-linked immunosorbent assay. Hepatocyte growth factor, epidermal growth factor, basic fibroblast growth factor, and transforming growth factor beta1 all stimulated HYA synthesis from vocal fold fibroblasts. These effects differed with the concentration of growth factors and the incubation period. We also examined how frequently the growth factors had to be administered in order to maintain appropriate levels of HYA. A single administration was sufficient to maintain appropriate HYA levels for at least 7 days. The present studies have demonstrated positive effects of growth factors in stimulating HYA production. Further in vivo study is needed to clarify the usefulness of these growth factors in the management of vocal fold scarring.

Dual pH/redox responsive and CD44 receptor targeting hybrid nano-chrysalis based on new oligosaccharides of hyaluronan conjugates.

PubMed

Chen, Daquan; Dong, Xue; Qi, Mengjiao; Song, Xiaoyan; Sun, Jingfang

2017-02-10

A smart hybrid microenvironment-mediated dual pH/redox-responsive polymeric nanoparticles combined with inorganic calcium phosphate (CaP) was fabricated, which we term as armored nano-chrysalis inspired by butterfly pupa. The nano-chrysalis has an inner core composed of specially designed oligosaccharides of hyaluronan (oHA) targeting CD44 receptor. The inner core has two functions, i.e., the dual pH/redox responsive polymeric conjugate and the fluorescent curcumin-prodrug function. The prepared nano-chrysalis possessed a smaller size (102.5±4.6nm) than the unarmored nano-chrysalis (122.5±6.6nm). Interestingly, while the nano-chrysalis were stable under pH 7.4, when incubated under the tumor acidic conditions (pH 6.5) the outer CaP armor would dissolve in a pH-dependent, sustained manner. Moreover, nano-chrysalis was demonstrated to present the most effective antitumor efficacy than other formulations. This study provides a promising smart nano-carrier platform to enhance the stability, decrease the side effects, and improve the therapeutic efficacy of anticancer drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Oral hyaluronan relieves wrinkles: a double-blinded, placebo-controlled study over a 12-week period.

PubMed

Oe, Mariko; Sakai, Seigo; Yoshida, Hideto; Okado, Nao; Kaneda, Haruna; Masuda, Yasunobu; Urushibata, Osamu

2017-01-01

Hyaluronan (HA) has critical moisturizing property and high water retention capacity especially for human skin. This study aimed to evaluate the effect of oral intake of HA. The mean molecular weight (MW) of HA is 2 k and 300 k. Sixty Japanese male and female subjects aged 22-59 years who presented with crow's feet wrinkles were randomly assigned to the HA 2 k or HA 300 k at 120 mg/day or the placebo group. The subjects were administered HA at a rate of 120 mg/day or a placebo for 12 weeks. The skin wrinkles were evaluated by image analysis of skin wrinkle replicas, and their skin condition was evaluated using a questionnaire survey. During the study period, the HA groups showed better level of the whole sulcus volume ratio, wrinkle area ratio, and wrinkle volume ratio than the placebo group. After 8 weeks of ingestion, the HA 300 k group showed significantly diminished wrinkles compared with the placebo group. Skin luster and suppleness significantly improved after 12 weeks in all groups compared with the baseline. The results suggest that oral HA (both HA 2 k and HA 300 k) inhibits skin wrinkles and improves skin condition.

Mutations in HYAL2, Encoding Hyaluronidase 2, Cause a Syndrome of Orofacial Clefting and Cor Triatriatum Sinister in Humans and Mice

PubMed Central

Hasan, S. Naimul; Mark, Brian; Harlalka, Gaurav V.; Patton, Michael A.; Ishida, Miho; Sharma, Sanjay; Faqeih, Eissa; Blakley, Brian; Jackson, Mike; Lees, Melissa; Dolinsky, Vernon; Cross, Leroy; Stanier, Philip; Salter, Claire; Baple, Emma L.; Crosby, Andrew H.

2017-01-01

Orofacial clefting is amongst the most common of birth defects, with both genetic and environmental components. Although numerous studies have been undertaken to investigate the complexities of the genetic etiology of this heterogeneous condition, this factor remains incompletely understood. Here, we describe mutations in the HYAL2 gene as a cause of syndromic orofacial clefting. HYAL2, encoding hyaluronidase 2, degrades extracellular hyaluronan, a critical component of the developing heart and palatal shelf matrix. Transfection assays demonstrated that the gene mutations destabilize the molecule, dramatically reducing HYAL2 protein levels. Consistent with the clinical presentation in affected individuals, investigations of Hyal2-/- mice revealed craniofacial abnormalities, including submucosal cleft palate. In addition, cor triatriatum sinister and hearing loss, identified in a proportion of Hyal2-/- mice, were also found as incompletely penetrant features in affected humans. Taken together our findings identify a new genetic cause of orofacial clefting in humans and mice, and define the first molecular cause of human cor triatriatum sinister, illustrating the fundamental importance of HYAL2 and hyaluronan turnover for normal human and mouse development. PMID:28081210

Mesenchymal stem cells in renal function recovery after acute kidney injury: use of a differentiating agent in a rat model.

PubMed

La Manna, Gaetano; Bianchi, Francesca; Cappuccilli, Maria; Cenacchi, Giovanna; Tarantino, Lucia; Pasquinelli, Gianandrea; Valente, Sabrina; Della Bella, Elena; Cantoni, Silvia; Claudia, Cavallini; Neri, Flavia; Tsivian, Matvey; Nardo, Bruno; Ventura, Carlo; Stefoni, Sergio

2011-01-01

Acute kidney injury (AKI) is a major health care condition with limited current treatment options. Within this context, stem cells may provide a clinical approach for AKI. Moreover, a synthetic compound previously developed, hyaluronan monoesters with butyric acid (HB), able to induce metanephric differentiation, formation of capillary-like structures, and secretion of angiogenic cytokines, was tested in vitro. Thereafter, we investigated the effects of human mesenchymal stem cells from fetal membranes (FMhMSCs), both treated and untreated with HB, after induction of ischemic AKI in a rat model. At reperfusion following 45-min clamping of renal pedicles, each rat was randomly assigned to one of four groups: CTR, PBS, MSC, and MSC-HB. Renal function at 1, 3, 5, and 7 days was assessed. Histological samples were analyzed by light and electron microscopy and renal injury was graded. Cytokine analysis on serum samples was performed. FMhMSCs induced an accelerated renal functional recovery, demonstrated by biochemical parameters and confirmed by histology showing that histopathological alterations associated with ischemic injury were less severe in cell-treated kidneys. HB-treated rats showed a minor degree of inflammation, both at cytokine and TEM analyses. Better functional and morphological recovery were not associated to stem cells' regenerative processes, but possibly suggest paracrine effects on microenvironment that induce retrieval of renal damaged tissues. These results suggest that FMhMSCs could be useful in the treatment of AKI and the utilization of synthetic compounds could enhance the recovery induction ability of cells.

LYVE1 and PROX1 in the reconstruction of hepatic sinusoids after partial hepatectomy in mice.

PubMed

Meng, F

2017-01-01

Revascularisation is crucial to liver regeneration after liver injury, but the process remains unclear. This study investigated changes in the levels and distribution of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) and prospero homeobox protein 1 (PROX1) in liver tissue sections after partial hepatectomy in mice. Mice were subjected to partial hepatectomy. Control animals were sham-operated. From days 1 through 8, the remaining liver tissues were collected from 8 animals each day. Histology showed that after partial hepatectomy, the remaining liver tissue samples underwent initial degeneration and then hepatocyte proliferation and regeneration. Using immunohistochemical analysis, relative to the control a significantly higher number of vascular endothelial growth factor A (VEGFA)-positive hepatocytes was observed on days 4 and 5 after partial hepatectomy. LYVE1 was mainly present in the liver sinusoidal endothelial cells and the number of LYVE1-positive cells gradually increased with time. PROX1 was detected in some of the hepatocytes, but liver sinusoidal endothelial cells, artery, and vein were negative for PROX1 staining in the early stage after liver injury. The presence of PROX1 could be observed in some central veins as well as liver sinusoidal endothelial cells. Seven days after partial hepatectomy, colocalisation of PROX1 and LYVE1 was observed in liver sinusoidal endothelial cells and veins. This study revealed the dynamic process of revascularisation and hepatic sinusoid reconstruction during liver regeneration in response to liver injury in mice. PROX1 and LYVE1 may participate in this process and serve as biomarkers for identification of newly formed liver sinusoidal endothelial cells.

HC-HA/PTX3 Purified From Amniotic Membrane as Novel Regenerative Matrix: Insight Into Relationship Between Inflammation and Regeneration.

PubMed

Tseng, Scheffer C G

2016-04-01

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. Review of published data to determine the molecular action mechanism explaining how AM exerts the aforementioned therapeutic actions. 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. 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.

HYAL-2–WWOX–SMAD4 Signaling in Cell Death and Anticancer Response

PubMed Central

Hsu, Li-Jin; Chiang, Ming-Fu; Sze, Chun-I; Su, Wan-Pei; Yap, Ye Vone; Lee, I-Ting; Kuo, Hsiang-Ling; Chang, Nan-Shan

2016-01-01

Hyaluronidase HYAL-2 is a membrane-anchored protein and also localizes, in part, in the lysosome. Recent study from animal models revealed that both HYAL-1 and HYAL-2 are essential for the metabolism of hyaluronan (HA). Hyal-2 deficiency is associated with chronic thrombotic microangiopathy with hemolytic anemia in mice due to over accumulation of high molecular size HA. HYAL-2 is essential for platelet generation. Membrane HYAL-2 degrades HA bound by co-receptor CD44. Also, in a non-canonical signal pathway, HYAL-2 serves as a receptor for transforming growth factor beta (TGF-β) to signal with downstream tumor suppressors WWOX and SMAD4 to control gene transcription. When SMAD4 responsive element is overly driven by the HYAL-2–WWOX–SMAD4 signaling complex, cell death occurs. When rats are subjected to traumatic brain injury, over accumulation of a HYAL-2–WWOX complex occurs in the nucleus to cause neuronal death. HA induces the signaling of HYAL-2–WWOX–SMAD4 and relocation of the signaling complex to the nucleus. If the signaling complex is overexpressed, bubbling cell death occurs in WWOX-expressing cells. In addition, a small synthetic peptide Zfra (zinc finger-like protein that regulates apoptosis) binds membrane HYAL-2 of non-T/non-B spleen HYAL-2+ CD3− CD19− Z lymphocytes and activates the cells to generate memory anticancer response against many types of cancer cells in vivo. Whether the HYAL-2–WWOX–SMAD4 signaling complex is involved is discussed. In this review and opinion article, we have updated the current knowledge of HA, HYAL-2 and WWOX, HYAL-2–WWOX–SMAD4 signaling, bubbling cell death, and Z cell activation for memory anticancer response. PMID:27999774

Size selectivity of hyaluronan molecular sieving by extracellular matrix in rabbit synovial joints

PubMed Central

Sabaratnam, S; Arunan, V; Coleman, PJ; Mason, RM; Levick, JR

2005-01-01

In joint fluid the polymer hyaluronan (HA) confers viscous lubrication and greatly attenuates trans-synovial fluid loss (outflow buffering). Outflow buffering arises from the molecular sieving (reflection) and concentration polarization of HA at the synovial membrane surface. Outflow buffering declines if HA chain length is reduced, as in arthritis, and this has been attributed to reduced HA reflection. This was tested directly in the present study. Infused solutions of HA of ∼2200 kDa (HA2000, 0.2 mg ml−1) or ∼500 kDa (HA500, 0.2 mg ml−1) or ∼140 kDa (HA140, 0.2–4.0 mg ml−1) were filtered across the synovial lining of the knee joint cavity of anaesthetized rabbits at a constant rate, along with a freely permeating reference solute, 20 kDa fluorescein–dextran (FD20). After a priming period the femoral lymph was sampled over 3 h. Mixed intra-articular (i.a.) fluid and subsynovial fluid were sampled at the end. Fluids were analysed by gel exclusion chromatography. The trans-synovial concentration profile was found to depend on polymer size. The i.a. concentration of HA2000 increased substantially relative to infusate and the subsynovial and lymph concentrations fell substantially. For HA500 and HA140 the trans-synovial concentration gradients were less pronounced, and absent for FD. The reflected fractions for HA2000, HA500 and HA140 across the cavity-to-lymph barrier were 0.65 ± 0.05 (n = 10), 0.43 ± 0.09 (n = 3) and 0.19 ± 0.05 (n = 7), respectively, at matched filtration rates (P < 0.0001, analysis of variance). Reflected fractions calculated from HA i.a. accumulation or subsynovial dilution showed the same trend. The results demonstrate size-selective molecular sieving by the synovial extracellular matrix, equivalent to steric exclusion from cylindrical pores of radius 33–59 nm. The findings underpin the concentration polarization-outflow buffering theory and indicate that reduced HA chain length in arthritis exacerbates lubricant loss from a joint. PMID:15961430

Development of Novel Faster-Dissolving Microneedle Patches for Transcutaneous Vaccine Delivery

PubMed Central

Ono, Akihiko; Ito, Sayami; Sakagami, Shun; Saito, Mio; Quan, Ying-Shu; Kamiyama, Fumio; Hirobe, Sachiko; Okada, Naoki

2017-01-01

Microneedle (MN) patches are promising for transcutaneous vaccination because they enable vaccine antigens to physically penetrate the stratum corneum via low-invasive skin puncturing, and to be effectively delivered to antigen-presenting cells in the skin. In second-generation MN patches, the dissolving MNs release the loaded vaccine antigen into the skin. To shorten skin application time for clinical practice, this study aims to develop novel faster-dissolving MNs. We designed two types of MNs made from a single thickening agent, carboxymethylcellulose (CMC) or hyaluronan (HN). Both CMC-MN and HN-MN completely dissolved in rat skin after a 5-min application. In pre-clinical studies, both MNs could demonstrably increase antigen-specific IgG levels after vaccination and prolong antigen deposition compared with conventional injections, and deliver antigens into resected human dermal tissue. In clinical research, we demonstrated that both MNs could reliably and safely puncture human skin without any significant skin irritation from transepidermal water loss measurements and ICDRG (International Contact Dermatitis Research Group) evaluation results. PMID:28771172

Cisplatin Cross-Linked Multifunctional Nanodrugplexes for Combination Therapy.

PubMed

Zhang, Weiqi; Tung, Ching-Hsuan

2017-03-15

Combination therapy efficiently tackles cancer by hitting multiple action mechanisms. However, drugs administered, simultaneously or sequentially, may not reach the targeted sites with the desired dose and ratio. The outcomes of combination therapy could be improved with a polymeric nanoparticle, which can simultaneously transport an optimal combination of drugs. We have demonstrated a simple one-pot strategy to formulate nanomedicines based on platinum coordination and the noncovalent interactions of the drugs. A naturally occurring polymer, hyaluronan (HA), was chosen as the building scaffold to form a nanodrugplex with cisplatin and aromatic-cationic drugs. The platinum coordination between cisplatin and HA induces the formation of a nanocomplex. The aromatic-cationic drugs are tightly packed by an electrostatic interaction and π-π stacking. The nanodrugplex bears excellent flexibility in drug combination and size control. It is stable in storage and has favorable release kinetics and targeting capabilities toward CD44, a receptor for HA that is highly expressed on many types of cancer cells.

Regulation of hyaluronan secretion into rabbit synovial joints in vivo by protein kinase C

PubMed Central

Anggiansah, C L; Scott, D; Poli, A; Coleman, P J; Badrick, E; Mason, R M; Levick, J R

2003-01-01

Hyaluronan (HA) is important for joint cavitation, lubrication, volume regulation and synovial fluid drainage but little is known about the regulation of joint HA synthesis/secretion in vivo. We investigated whether HA secretion into joints in vivo can be regulated by protein kinase C (PKC). Secretion into the knee joint cavity of anaesthetised rabbits was measured over 6 h by washout and chromatography. Joints received intra-articular injections of Ringer vehicle (control) or an activator of classical PKC isoforms, phorbol-12-myristate-13-acetate (PMA), at 20–2000 ng ml−1. The effects of PKC inhibition by bisindolylmaleimide (BIM) and protein synthesis inhibition by cycloheximide (CX) on basal and stimulated HA secretion were also studied. The endogenous HA mass, 181 ± 8 μg (n = 26, mean ± s.e.m.), and basal secretion rate, 4.4 ± 0.4 μg h−1, indicated a turnover time of 41 h. Secretion rate showed a dose-dependent response to PMA (n = 30), rising 5-fold to 21.7 ± 5.0 μg h−1 (n = 5) at 2000 ng ml−1 PMA (P < 0.0001, one-way ANOVA). PMA-induced stimulation was partially suppressed by CX (HA secretion: 5.8 ± 1.7 μg h−1, n = 8, P < 0.01) and totally blocked by BIM (HA secretion: 3.2 ± 0.6 μg h−1, n = 9, P < 0.001). Basal HA secretion was unaffected by CX over 6 h (4.2 ± 0.7 μg h−1, n = 8) but was reduced by 29 % by BIM (3.1 ± 0.6 μg h−1, n = 10, P = 0.03). It is concluded that: (1) PKC can stimulate HA secretion into joints in vivo through mechanisms involving protein synthesis de novo as well as phosphorylation; (2) basal HA secretion is only partially PKC dependent; and (3) hyaluronan synthase turnover time is > 6 h in vivo, which is slower than in vitro (< 2–3 h). PMID:12766248

Harnessing cellular differentiation to improve ALA-based photodynamic therapy in an artificial skin model

NASA Astrophysics Data System (ADS)

Maytin, Edward; Anand, Sanjay; Sato, Nobuyuki; Mack, Judith; Ortel, Bernhard

2005-04-01

During ALA-based photodynamic therapy (PDT), a pro-drug (aminolevulinic acid; ALA) is taken up by tumor cells and metabolically converted to a photosensitizing intermediate (protoporphyrin IX; PpIX). ALA-based PDT, while an emerging treatment modality, remains suboptimal for most cancers (e.g. squamous cell carcinoma of the skin). Many treatment failures may be largely due to insufficient conversion of ALA to PpIX within cells. We discovered a novel way to increase the conversion of ALA to PpIX, by administering agents that can drive terminal differentiation (i.e., accelerate cellular maturation). Terminally-differentiated epithelial cells show higher levels of intracellular PpIX, apparently via increased levels of a rate-limiting enzyme, coproporphyrinogen oxidase (CPO). To study these mechanisms in a three-dimensional tissue, we developed an organotypic model that mimics true epidermal physiology in a majority of respects. A line of rat epidermal keratinocytes (REKs), when grown in raft cultures, displays all the features of a fully-differentiated epidermis. Addition of ALA to the culture medium results in ALA uptake and PpIX synthesis, with subsequent death of keratinocytes upon exposure to blue light. Using this model, we can manipulate cellular differentiation via three different approaches. (1) Vitamin D, a hormone that enhances keratinocyte differentiation; (2) Hoxb13, a nuclear transcription factor that affects the genetically-controlled differentiation program of stratifying cells (3) Hyaluronan, an abundant extracellular matrix molecule that regulates epidermal differentiation. Because the raft cultures contain only a single cell type (no blood, fibroblasts, etc.) the effects of terminal differentiation upon CPO, PpIX, and keratinocyte cell death can be specifically defined.

VEGF-C and TGF-β reciprocally regulate mesenchymal stem cell commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes.

PubMed

Igarashi, Yasuyuki; Chosa, Naoyuki; Sawada, Shunsuke; Kondo, Hisatomo; Yaegashi, Takashi; Ishisaki, Akira

2016-04-01

The direction of mesenchymal stem cell (MSC) differentiation is regulated by stimulation with various growth factors and cytokines. We recently established MSC lines, [transforming growth factor-β (TGF-β)-responsive SG‑2 cells, bone morphogenetic protein (BMP)-responsive SG‑3 cells, and TGF-β/BMP-non-responsive SG‑5 cells], derived from the bone marrow of green fluorescent protein-transgenic mice. In this study, to compare gene expression profiles in these MSC lines, we used DNA microarray analysis to characterize the specific gene expression profiles observed in the TGF-β-responsive SG‑2 cells. Among the genes that were highly expressed in the SG‑2 cells, we focused on vascular endothelial growth factor (VEGF) receptor 3 (VEGFR3), the gene product of FMS-like tyrosine kinase 4 (Flt4). We found that VEGF-C, a specific ligand of VEGFR3, significantly induced the cell proliferative activity, migratory ability (as shown by Transwell migration assay), as well as the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the SG‑2 cells. Additionally, VEGF-C significantly increased the expression of prospero homeobox 1 (Prox1) and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), which are lymphatic endothelial cell markers, and decreased the expression of osteogenic differentiation marker genes in these cells. By contrast, TGF-β significantly increased the expression of early-phase osteogenic differentiation marker genes in the SG‑2 cells and markedly decreased the expression of lymphatic endothelial cell markers. The findings of our study strongly suggest the following: i) that VEGF-C promotes the proliferative activity and migratory ability of MSCs; and ii) VEGF-C and TGF-β reciprocally regulate MSC commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes, respectively. Our findings provide new insight into the molecular mechanisms underlying the regenerative ability of MSCs.

Hyaluronidase 2 Deficiency Causes Increased Mesenchymal Cells, Congenital Heart Defects, and Heart Failure.

PubMed

Chowdhury, Biswajit; Xiang, Bo; Liu, Michelle; Hemming, Richard; Dolinsky, Vernon W; Triggs-Raine, Barbara

2017-01-01

Hyaluronan (HA) is required for endothelial-to-mesenchymal transition and normal heart development in the mouse. Heart abnormalities in hyaluronidase 2 (HYAL2)-deficient ( Hyal2 - /- ) mice and humans suggested removal of HA is also important for normal heart development. We have performed longitudinal studies of heart structure and function in Hyal2 -/- mice to determine when, and how, HYAL2 deficiency leads to these abnormalities. Echocardiography revealed atrial enlargement, atrial tissue masses, and valvular thickening at 4 weeks of age, as well as diastolic dysfunction that progressed with age, in Hyal2 -/- mice. These abnormalities were associated with increased HA, vimentin-positive cells, and fibrosis in Hyal2 -/- compared with control mice. Based on the severity of heart dysfunction, acute and chronic groups of Hyal2 -/- mice that died at an average of 12 and 25 weeks respectively, were defined. Increased HA levels and mesenchymal cells, but not vascular endothelial growth factor in Hyal2 -/- embryonic hearts, suggest that HYAL2 is important to inhibit endothelial-to-mesenchymal transition. Consistent with this, in wild-type embryos, HYAL2 and HA were readily detected, and HA levels decreased with age. These data demonstrate that disruption of normal HA catabolism in Hyal2 -/- mice causes increased HA, which may promote endothelial-to-mesenchymal transition and proliferation of mesenchymal cells. Excess endothelial-to-mesenchymal transition, resulting in increased mesenchymal cells, is the likely cause of morphological heart abnormalities in both humans and mice. In mice, these abnormalities result in progressive and severe diastolic dysfunction, culminating in heart failure. © 2016 The Authors.

Pirfenidone reduces subchondral bone loss and fibrosis after murine knee cartilage injury.

PubMed

Chan, Deva D; Li, Jun; Luo, Wei; Predescu, Dan N; Cole, Brian J; Plaas, Anna

2018-01-01

Pirfenidone is an anti-inflammatory and anti-fibrotic drug that has shown efficacy in lung and kidney fibrosis. Because inflammation and fibrosis have been linked to the progression of osteoarthritis, we investigated the effects of oral Pirfenidone in a mouse model of cartilage injury, which results in chronic inflammation and joint-wide fibrosis in mice that lack hyaluronan synthase 1 (Has1 -/- ) in comparison to wild-type. Femoral cartilage was surgically injured in wild-type and Has1 -/- mice, and Pirfenidone was administered in food starting after 3 days. At 4 weeks, Pirfenidone reduced the appearance, on micro-computed tomography, of pitting in subchondral bone at, and cortical bone surrounding, the site of cartilage injury. This corresponded with a reduction in fibrotic tissue deposits as observed with gross joint surface photography. Pirfenidone resulted in significant recovery of trabecular bone parameters affected by joint injury in Has1 -/- mice, although the effect in wild-type was less pronounced. Pirfenidone also increased Safranin-O staining of growth plate cartilage after cartilage injury and sham operation in both genotypes. Taken together with the expression of selected extracellular matrix, inflammation, and fibrosis genes, these results indicate that Pirfenidone may confer chondrogenic and bone-protective effects, although the well-known anti-fibrotic effects of Pirfenidone may occur earlier in the wound-healing response than the time point examined in this study. Further investigations to identify the specific cell populations in the joint and signaling pathways that are responsive to Pirfenidone are warranted, as Pirfenidone and other anti-fibrotic drugs may encourage tissue repair and prevent progression of post-traumatic osteoarthritis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:365-376, 2018. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

The role of the conformational profile of polysaccharides on skin penetration: the case of hyaluronan and its sulfates.

PubMed

Cilurzo, Francesco; Vistoli, Giulio; Gennari, Chiara G M; Selmin, Francesca; Gardoni, Fabrizio; Franzè, Silvia; Campisi, Monica; Minghetti, Paola

2014-04-01

The literature data suggest the capacity of biomacromolecules to permeate the human skin, even though such a transdermal permeation appears to be governed by physicochemical parameters which are significantly different compared to those ruling the skin permeation of small molecules. On these grounds, the present study was undertaken to investigate the in vitro diffusion properties through the human epidermis of hyaluronic acid and their sulfates. Low- and medium-molecular-weight hyaluronic acids and the corresponding derivatives at two degrees of sulfation were then tested. In vitro studies evidenced that the sulfated polymers permeate better than the corresponding hyaluronic acid, despite their vastly greater polarity, while the observed permeation markedly decreases when increasing the polymer's molecular weight regardless of the sulfation degree. Using a fluorescent-labeled polysaccharide, it was also evidenced that hyaluronans have a great affinity for corneocytes and likely cross the stratum corneum mainly through a transcellular route. The molecular-dynamics study revealed how the observed permeations for the investigated polysaccharides can be rationalized by monitoring their conformational profiles, since the permeation was found to be directly related to their capacity to assume extended and flexible conformations. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

Peripheral Nerve Repair in Rats Using Composite Hydrogel-Filled Aligned Nanofiber Conduits with Incorporated Nerve Growth Factor

PubMed Central

Jin, Jenny; Limburg, Sonja; Joshi, Sunil K.; Landman, Rebeccah; Park, Michelle; Zhang, Qia; Kim, Hubert T.

2013-01-01

Repair of peripheral nerve defects with current synthetic, tubular nerve conduits generally shows inferior recovery when compared with using nerve autografts, the current gold standard. We tested the ability of composite collagen and hyaluronan hydrogels, with and without the nerve growth factor (NGF), to stimulate neurite extension on a promising aligned, nanofiber poly-L-lactide-co-caprolactone (PLCL) scaffold. In vitro, the hydrogels significantly increased neurite extension from dorsal root ganglia explants. Consistent with these results, the addition of hydrogels as luminal fillers within aligned, nanofiber tubular PLCL conduits led to improved sensory function compared to autograft repair in a critical-size defect in the sciatic nerve in a rat model. Sensory recovery was assessed 3 and 12 weeks after repair using a withdrawal assay from thermal stimulation. The addition of hydrogel did not enhance recovery of motor function in the rat model. The NGF led to dose-dependent improvements in neurite out-growth in vitro, but did not have a significant effect in vivo. In summary, composite collagen/hyaluronan hydrogels enhanced sensory neurite outgrowth in vitro and sensory recovery in vivo. The use of such hydrogels as luminal fillers for tubular nerve conduits may therefore be useful in assisting restoration of protective sensation following peripheral nerve injury. PMID:23659607

Chronic UVB-irradiation actuates perpetuated dermal matrix remodeling in female mice: Protective role of estrogen

PubMed Central

Röck, Katharina; Joosse, Simon Andreas; Müller, Julia; Heinisch, Nina; Fuchs, Nicola; Meusch, Michael; Zipper, Petra; Reifenberger, Julia; Pantel, Klaus; Fischer, Jens Walter

2016-01-01

Chronic UVB-exposure and declined estradiol production after menopause represent important factors leading to extrinsic and intrinsic aging, respectively. Remodeling of the extracellular matrix (ECM) plays a crucial role in both responses. Whether the dermal ECM is able to recover after cessation of UVB-irradiation in dependence of estradiol is not known, however of relevance when regarding possible treatment options. Therefore, the endogenous sex hormone production was depleted by ovariectomy in female mice. Half of the mice received estradiol substitution. Mice were UVB-irradiated for 20 weeks and afterwards kept for 10 weeks without irradiation. The collagen-, hyaluronan- and proteoglycan- (versican, biglycan, lumican) matrix, collagen cleavage products and functional skin parameters were analyzed. The intrinsic aging process was characterized by increased collagen fragmentation and accumulation of biglycan. Chronic UVB-irradiation additionally augmented the lumican, versican and hyaluronan content of the dermis. In the absence of further UVB-irradiation the degradation of collagen and accumulation of biglycan in the extrinsically aged group was perpetuated in an excessive matter. Whereas estradiol increased the proteoglycan content, it reversed the effects of the perpetuated extrinsic response on collagen degradation. Suspension of the intrinsic pathway might therefore be sufficient to antagonize UVB-evoked long-term damage to the dermal ECM. PMID:27460287

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.

Reparative Effects of Astaxanthin-Hyaluronan Nanoaggregates against Retrorsine-CCl₄-Induced Liver Fibrosis and Necrosis.

PubMed

Wu, Yi Jhen; Wu, Yu Chiuan; Chen, I-Fen; Wu, Yi-Lung; Chuang, Chin Wen; Huang, Han Hsiang; Kuo, Shyh Ming

2018-03-22

Astaxanthin (Asta), a xanthophyll carotenoid, has been reported to be a strong antioxidative agent and has anti-inflammatory, antitumor and free radical-scavenging activities. However, inadequate stability and water solubility results in its low bioavailability. This study incorporated Asta into hydrophilic hyaluronan nanoparticles (HAn) to produce Asta-HAn aggregates (AHAna) using an electrostatic field system and investigated the restorative effects of AHAna on retrorsine-CCl₄-induced liver fibrosis in rats in vivo. Transmission electron microscopy (TEM) revealed that the prepared HAn were approximately 15 ± 2.1 nm in diameter and after the incorporation of Asta into HAn, the size increased to 210-500 nm. The incorporation efficiency of Asta was approximately 93% and approximately 54% of Asta was released after incubation for 18 h. Significant reductions in alanine aminotransferase and aspartate aminotransferase levels were observed after the rats were intraperitoneally injected with AHAna. Histopathological findings revealed the greatest reduction in hepatic fibrosis and hepatocyte necrosis in the rats after 2 weeks of intraperitoneal injection with AHAna, which is consistent with the data acquired from serum biochemical analysis. The restorative effects on liver damage displayed by AHAna in vivo demonstrated that Asta aggregated through HAn incorporation exerts therapeutic effects on liver fibrosis and necrosis.

Multi-functional polyelectrolyte multilayer films: combining mechanical resistance, biodegradability and bioactivity

PubMed Central

Schneider, Aurore; Vodouhê, Constant; Richert, Ludovic; Francius, Gregory; Le Guen, Erell; Schaaf, Pierre; Voegel, Jean-Claude; Frisch, Benoît; Picart, Catherine

2008-01-01

Cross-linked polyelectrolyte multilayer films (CL PEM) have an increased rigidity and are mechanically more resistant than native (e.g. uncrosslinked) films. However, they are still biodegradable, which make them interesting candidates for biomedical applications. In this study, CL PEM films have been explored for their multifunctional properties as i) mechanically resistant ii) biodegradable and iii) bioactive films. Toward this end, we investigated drug loading into CL chitosan/hyaluronan (CHI/HA) and poly(L-lysine)/hyaluronan (PLL/HA) films by simple diffusion of the drugs. Sodium diclofenac and paclitaxel were chosen as model drugs and were successfully loaded into the films. The effect of varying the number of layers in the (CHI/HA) films as well as the cross-linker concentration on diclofenac loading were studied. Diclofenac was released from the film in about ten hours. Paclitaxel was also found to diffuse within CL films. Its activity was maintained after loading in the CL films and cellular viability could be reduced by about 55% over three days. Such simple approach may be applied to other types of cross-linked films and to other drugs. These results prove that it is possible to design multifunctional multilayer films that combine mechanical resistance, biodegradability and bioactivity properties into a single PEM architecture. PMID:17206799

Oral hyaluronan relieves wrinkles: a double-blinded, placebo-controlled study over a 12-week period

PubMed Central

Oe, Mariko; Sakai, Seigo; Yoshida, Hideto; Okado, Nao; Kaneda, Haruna; Masuda, Yasunobu; Urushibata, Osamu

2017-01-01

Background Hyaluronan (HA) has critical moisturizing property and high water retention capacity especially for human skin. This study aimed to evaluate the effect of oral intake of HA. Methods The mean molecular weight (MW) of HA is 2 k and 300 k. Sixty Japanese male and female subjects aged 22–59 years who presented with crow’s feet wrinkles were randomly assigned to the HA 2 k or HA 300 k at 120 mg/day or the placebo group. The subjects were administered HA at a rate of 120 mg/day or a placebo for 12 weeks. The skin wrinkles were evaluated by image analysis of skin wrinkle replicas, and their skin condition was evaluated using a questionnaire survey. Results During the study period, the HA groups showed better level of the whole sulcus volume ratio, wrinkle area ratio, and wrinkle volume ratio than the placebo group. After 8 weeks of ingestion, the HA 300 k group showed significantly diminished wrinkles compared with the placebo group. Skin luster and suppleness significantly improved after 12 weeks in all groups compared with the baseline. Conclusion The results suggest that oral HA (both HA 2 k and HA 300 k) inhibits skin wrinkles and improves skin condition. PMID:28761365

Ingestion of hyaluronans (molecular weights 800 k and 300 k) improves dry skin conditions: a randomized, double blind, controlled study.

PubMed

Kawada, Chinatsu; Yoshida, Takushi; Yoshida, Hideto; Sakamoto, Wakako; Odanaka, Wataru; Sato, Toshihide; Yamasaki, Takeshi; Kanemitsu, Tomoyuki; Masuda, Yasunobu; Urushibata, Osamu

2015-01-01

Hyaluronan (HA) has been increasingly used as a dietary supplement to improve the skin. However, the effect of ingested HA may depend on its molecular weight (MW) because its physiological activities in the body vary with its MW. In this study, we examined the effects of ingested HA with varying MW on the skin. In this randomized, double blind, placebo controlled study, 61 subjects with dry skin received oral HA (120 mg/day), of MWs 800 k and 300 k or placebo, for 6 weeks. The skin moisture contents of the first two groups increased more than those of the placebo group during the ingestion period. In addition, group HA 300 k exhibited significant improvements in skin moisture content 2 weeks after ingestion ended compared with the placebo group. A questionnaire survey about subjective facial aging symptoms showed that the HA treated groups exhibited significantly improved the skin condition compared with the placebo treated group. Furthermore, dermatologists objectively evaluated the clinical symptoms of the facial and whole body skin, showing that no adverse events were related to daily ingestion of HA. This study shows that both of ingesting HAs (MWs 800 k and 300 k) improved the skin condition by increasing the moisture content.

Impact of salt exposure on N-acetylgalactosamine-4-sulfatase (arylsulfatase B) activity, glycosaminoglycans, kininogen, and bradykinin

PubMed Central

Kotlo, Kumar; Bhattacharyya, Sumit; Yang, Bo; Feferman, Leonid; Tejaskumar, Shah; Linhardt, Robert; Danziger, Robert

2013-01-01

N -acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) is the enzyme that removes sulfate groups from the N-acetylgalactosamine-4-sulfate residue at the non-reducing end of chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Previous studies demonstrated reduction in cell-bound high molecular weight kininogen in normal rat kidney (NRK) epithelial cells when chondroitin-4-sulfate content was reduced following overexpression of ARSB activity, and chondroitinase ABC produced similar decline in cell-bound kininogen. Reduction in the cell-bound kininogen was associated with increase in secreted bradykinin. In this report, we extend the in vitro findings to in vivo models, and present findings in Dahl salt-sensitive (SS) rats exposed to high (SSH) and low salt (SSL) diets. In the renal tissue of the SSH rats, ARSB activity was significantly less than in the SSL rats, and chondroitin-4-sulfate and total sulfated glycosaminoglycan content were significantly greater. Disaccharide analysis confirmed marked increase in C4S disaccharides in the renal tissue of the SSH rats. In contrast, unsulfated, hyaluronan-derived disaccharides were increased in the rats on the low salt diet. In the SSH rats, with lower ARSB activity and higher C4S levels, cell-bound, high-molecular weight kininogen was greater and urinary bradykinin was lower. ARSB activity in renal tissue and NRK cells declined when exogenous chloride concentration was increased in vitro. The impact of high chloride exposure in vivo on ARSB, chondroitin-4-sulfation, and C4S-kininogen binding provides a mechanism that links dietary salt intake with bradykinin secretion and may be a factor in blood pressure regulation. PMID:23385884

Impact of salt exposure on N-acetylgalactosamine-4-sulfatase (arylsulfatase B) activity, glycosaminoglycans, kininogen, and bradykinin.

PubMed

Kotlo, Kumar; Bhattacharyya, Sumit; Yang, Bo; Feferman, Leonid; Tejaskumar, Shah; Linhardt, Robert; Danziger, Robert; Tobacman, Joanne K

2013-10-01

N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) is the enzyme that removes sulfate groups from the N-acetylgalactosamine-4-sulfate residue at the non-reducing end of chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Previous studies demonstrated reduction in cell-bound high molecular weight kininogen in normal rat kidney (NRK) epithelial cells when chondroitin-4-sulfate content was reduced following overexpression of ARSB activity, and chondroitinase ABC produced similar decline in cell-bound kininogen. Reduction in the cell-bound kininogen was associated with increase in secreted bradykinin. In this report, we extend the in vitro findings to in vivo models, and present findings in Dahl salt-sensitive (SS) rats exposed to high (SSH) and low salt (SSL) diets. In the renal tissue of the SSH rats, ARSB activity was significantly less than in the SSL rats, and chondroitin-4-sulfate and total sulfated glycosaminoglycan content were significantly greater. Disaccharide analysis confirmed marked increase in C4S disaccharides in the renal tissue of the SSH rats. In contrast, unsulfated, hyaluronan-derived disaccharides were increased in the rats on the low salt diet. In the SSH rats, with lower ARSB activity and higher C4S levels, cell-bound, high-molecular weight kininogen was greater and urinary bradykinin was lower. ARSB activity in renal tissue and NRK cells declined when exogenous chloride concentration was increased in vitro. The impact of high chloride exposure in vivo on ARSB, chondroitin-4-sulfation, and C4S-kininogen binding provides a mechanism that links dietary salt intake with bradykinin secretion and may be a factor in blood pressure regulation.

Mechanisms of skin aging induced by EGFR inhibitors.

PubMed

Gerber, Peter Arne; Buhren, Bettina Alexandra; Schrumpf, Holger; Hevezi, Peter; Bölke, Edwin; Sohn, Dennis; Jänicke, Reiner U; Belum, Viswanath Reddy; Robert, Caroline; Lacouture, Mario E; Homey, Bernhard

2016-10-01

The mechanisms of skin aging have not been completely elucidated. Anecdotal data suggests that EGFR inhibition accelerates aging-like skin changes. The objective of the study was to evaluate the clinical characteristics and investigate the cellular and molecular mechanisms underlying skin changes associated with the use of EFGRIs. Patients during prolonged treatment with EGFRIs (>3 months) were analyzed for aging-like skin changes. Baseline EGFR expression was compared in young (<25 years old) vs. old (> 65 years old) skin. In addition, the regulation of extracellular matrix, senescence-associated genes, and cell cycle status was measured in primary human keratinocytes treated with erlotinib in vitro. There were progressive signs of skin aging, including xerosis cutis, atrophy, rhytide formation, and/or actinic purpura in 12 patients. Keratinocytes treated with erlotinib in vitro showed a significant down-modulation of hyaluronan synthases (HAS2 and HAS3), whereas senescence-associated genes (p21, p53, IL-6, maspin) were upregulated, along with a G1 cell cycle arrest and stronger SA β-Gal activity. There was significantly decreased baseline expression in EGFR density in aged skin, when compared to young controls. EGFR inhibition results in molecular alterations in keratinocytes that may contribute to the observed skin aging of patients treated with respective targeted agents.

Mechanisms of skin aging induced by EGFR inhibitors

PubMed Central

Gerber, Peter Arne; Buhren, Bettina Alexandra; Schrumpf, Holger; Hevezi, Peter; Bölke, Edwin; Sohn, Dennis; Jänicke, Reiner; Belum, Viswanath Reddy; Robert, Caroline; Lacouture, Mario E.; Homey, Bernhard

2017-01-01

BACKGROUND The mechanisms of skin aging have not been completely elucidated. Anecdotal data suggests that EGFR inhibition accelerates aging-like skin changes. OBJECTIVE To evaluate the clinical characteristics and investigate the cellular and molecular mechanisms underlying skin changes associated with the use of EFGRIs. PATIENTS AND METHODS Patients during prolonged treatment with EGFRIs (>3 months) were analyzed for aging-like skin changes. Baseline EGFR expression was compared in young (< 25 years old) vs. old (> 65 years old) skin. In addition, the regulation of extracellular matrix, senescence-associated genes, and cell cycle status was measured in primary human keratinocytes treated with erlotinib in vitro. RESULTS Progressive signs of skin aging, including xerosis cutis, atrophy, rhytide formation and/or actinic purpura in 12 patients. Keratinocytes treated with erlotinib in vitro showed a significant down-modulation of hyaluronan synthases (HAS2 and HAS3), whereas senescence-associated genes (p21, p53, IL-6, maspin) were upregulated, along with a G1 cell cycle arrest and stronger SA β-Gal activity. There was significantly decreased baseline expression in EGFR-density in aged skin, when compared to young controls. CONCLUSIONS EGFR inhibition results in molecular alterations in keratinocytes that may contribute to the observed skin aging of patients treated with respective targeted agents. PMID:27165055

Implantation of preadipocyte-loaded hyaluronic acid-based scaffolds into nude mice to evaluate potential for soft tissue engineering.

PubMed

Hemmrich, Karsten; von Heimburg, Dennis; Rendchen, Raoul; Di Bartolo, Chiara; Milella, Eva; Pallua, Norbert

2005-12-01

The reconstruction of soft tissue defects following extensive deep burns or tumor resections remains an unresolved problem in plastic and reconstructive surgery since adequate implant materials are still not available. Preadipocytes, immature precursor cells found between mature adipocytes in adipose tissue, are a potential material for soft tissue engineering since they can proliferate and differentiate into adipose tissue after transplantation. In previous studies, we identified hyaluronan benzyl ester (HYAFF 11) sponges to be promising carrier matrices. This study now evaluates, in vitro and in vivo, a new sponge architecture with pores of 400 microm either made of plain HYAFF 11 or HYAFF 11 coated with the extracellular matrix glycosaminoglycan hyaluronic acid. Human preadipocytes were isolated, seeded onto carriers and implanted into nude athymic mice. Explants harvested after 3, 8, and 12 weeks were examined for macroscopical appearance, thickness, weight, pore structure, histology, and immunohistochemistry. Compared to previous studies, we found better penetration of cells into both types of scaffolds, with more extensive formation of new vessels throughout the construct but with only minor adipose tissue. Our encouraging results contribute towards a better seeded and vascularised scaffold but also show that the enhancement of adipogenic conversion of preadipocytes remains a major task for further in vivo experiments.

Sporadic Premature Aging in a Japanese Monkey: A Primate Model for Progeria

PubMed Central

Oishi, Takao; Imai, Hiroo; Go, Yasuhiro; Imamura, Masanori; Hirai, Hirohisa; Takada, Masahiko

2014-01-01

In our institute, we have recently found a child Japanese monkey who is characterized by deep wrinkles of the skin and cataract of bilateral eyes. Numbers of analyses were performed to identify symptoms representing different aspects of aging. In this monkey, the cell cycle of fibroblasts at early passage was significantly extended as compared to a normal control. Moreover, both the appearance of senescent cells and the deficiency in DNA repair were observed. Also, pathological examination showed that this monkey has poikiloderma with superficial telangiectasia, and biochemical assay confirmed that levels of HbA1c and urinary hyaluronan were higher than those of other (child, adult, and aged) monkey groups. Of particular interest was that our MRI analysis revealed expansion of the cerebral sulci and lateral ventricles probably due to shrinkage of the cerebral cortex and the hippocampus. In addition, the conduction velocity of a peripheral sensory but not motor nerve was lower than in adult and child monkeys, and as low as in aged monkeys. However, we could not detect any individual-unique mutations of known genes responsible for major progeroid syndromes. The present results indicate that the monkey suffers from a kind of progeria that is not necessarily typical to human progeroid syndromes. PMID:25365557

Tissue engineering approaches for studying the effect of biochemical and physiological stimuli on cell behavior

NASA Astrophysics Data System (ADS)

Jimenez Vergara, Andrea Carolina

Tissue engineering (TE) approaches have emerged as an alternative to traditional tissue and organ replacements. The aim of this work was to contribute to the understanding of the effects of cell-material and endothelial cell (EC) paracrine signaling on cell responses using poly(ethylene glycol) diacrylate (PEGDA) hydrogels as a material platform. Three TE applications were explored. First, the effect of glycosaminoglycan (GAG) identity was evaluated for vocal fold restoration. Second, the influence of GAG identity was explored and a novel approach for stable endothelialization was developed for vascular graft applications. Finally, EC paracrine signaling in the presence of cyclic stretch, and hydrophobicity and inorganic content were studied for osteogenic applications. In terms of vocal fold restoration, it was found that vocal fold fibroblast (VFF) phenotype and extracellular matrix (ECM) production were impacted by GAG identity. VFF phenotype was preserved in long-term cultured hydrogels containing high molecular weight hyaluronan (HAHMW). Furthermore, collagen I deposition, fibronectin production and smooth muscle α-actin (SM-α-actin) expression in PEG-HA, PEG-chondroitin sulfate C and PEG-heparan sulfate (HS) gels suggest that CSC and HS may be undesirable for vocal fold implants. Regarding vascular graft applications, the impact of GAG identity on smooth muscle cell (SMC) foam cell formation was explored. Results support the increasing body of literature that suggests a critical role for dermatan sulfate (DS)-bearing proteoglycans in early atherosclerosis. In addition, an approach for fabricating bi-layered tissue engineering vascular grafts (TEVGs) with stable endothelialization was validated using PEGDA as an intercellular “cementing” agent between adjacent endothelial cells (ECs). Finally, mesenchymal stem cell (MSC) differentiation toward osteogenic like cells was evaluated. ECM and cell phenotypic data showed that elevated scaffold inorganic content and hydrophobicity were indeed correlated with increased osteogenic differentiation. Moreover, the present results suggest that EC paracrine signaling enhances MSC osteogenesis in the presence of cyclic stretch.

Hyaluronic Acid as a Target for Intervention in Prostate Cancer Metastases

DTIC Science & Technology

2012-06-01

hyaluronan synthase, commonly available in herbal supplements and, up to now, used mainly for digestion complaints. We proposed that it may be efficacious... herbal supplements and, up to now, has been utilized mainly for digestion complaints. We proposed that it may be efficacious in the prevention and...HAS. It is commonly available in herbal supplements and, up to now, has been utilized mainly in Europe for digestive complaints. We hypothesize that

TSH/IGF-1 Receptor Cross-Talk Rapidly Activates Extracellular Signal-Regulated Kinases in Multiple Cell Types.

PubMed

Krieger, Christine C; Perry, Joseph D; Morgan, Sarah J; Kahaly, George J; Gershengorn, Marvin C

2017-10-01

We previously showed that thyrotropin (TSH)/insulinlike growth factor (IGF)-1 receptor cross-talk appears to be involved in Graves' orbitopathy (GO) pathogenesis and upregulation of thyroid-specific genes in human thyrocytes. In orbital fibroblasts from GO patients, coadministration of TSH and IGF-1 induces synergistic increases in hyaluronan secretion. In human thyrocytes, TSH plus IGF-1 synergistically increased expression of the sodium-iodide symporter that appeared to involve ERK1/2 activation. However, the details of ERK1/2 activation were not known, nor was whether ERK1/2 was involved in this synergism in other cell types. Using primary cultures of GO fibroblasts (GOFs) and human thyrocytes, as well as human embryonic kidney (HEK) 293 cells overexpressing TSH receptors (HEK-TSHRs), we show that simultaneous activation of TSHRs and IGF-1 receptors (IGF-1Rs) causes rapid, synergistic phosphorylation/activation of ERK1 and ERK2 in all three cell types. This effect is partially inhibited by pertussis toxin, an inhibitor of TSHR coupling to Gi/Go proteins. In support of a role for Gi/Go proteins in ERK1/2 phosphorylation, we found that knockdown of Gi(1-3) and Go in HEK-TSHRs inhibited ERK1/2 phosphorylation stimulated by TSH and TSH plus IGF-1. These data demonstrate that the synergistic effects of TSH plus IGF-1 occur early in the TSHR signaling cascade and further support the idea that TSHR/IGF-1R cross-talk is an important mechanism for regulation of human GOFs and thyrocytes.

Chondrocytes, synoviocytes and dermal fibroblasts all express PH-20, a hyaluronidase active at neutral pH

PubMed Central

El Hajjaji, Hafida; Cole, Ada Asbury; Manicourt, Daniel-Henri

2005-01-01

Hyaluronan (HA), an important component of connective tissues, is highly metabolically active, but the mechanisms involved in its catabolism are still largely unknown. We hypothesized that a protein similar to sperm PH-20, the only mammalian hyaluronidase known to be active at neutral pH, could be expressed in connective tissue cells. An mRNA transcript similar to that of PH-20 was found in chondrocytes, synoviocytes, and dermal fibroblasts, and its levels were enhanced upon stimulation with IL-1. In cell layers extracted with Triton X-100 – but not with octylglucoside – and in culture media, a polyclonal antipeptide anti-PH-20 antibody identified protein bands with a molecular weight similar to that of sperm PH-20 (60 to 65 kDa) and exhibiting a hyaluronidase activity at neutral pH. Further, upon stimulation with IL-1, the amounts of the neutral-active hyaluronidase increased in both cell layers and culture media. These findings contribute potential important new insights into the biology of connective tissues. It is likely that PH-20 facilitates cell-receptor-mediated uptake of HA, while overexpression or uncontrolled expression of the enzyme can cause great havoc to connective tissues: not only does HA fragmentation compromise the structural integrity of tissues, but also the HA fragments generated are highly angiogenic and are potent inducers of proinflammatory cytokines. On the other hand, the enzyme activity may account for the progressive depletion of HA seen in osteoarthritis cartilage, a depletion that is believed to play an important role in the apparent irreversibility of this disease process. PMID:15987477

hmmr mediates anterior neural tube closure and morphogenesis in the frog Xenopus.

PubMed

Prager, Angela; Hagenlocher, Cathrin; Ott, Tim; Schambony, Alexandra; Feistel, Kerstin

2017-10-01

Development of the central nervous system requires orchestration of morphogenetic processes which drive elevation and apposition of the neural folds and their fusion into a neural tube. The newly formed tube gives rise to the brain in anterior regions and continues to develop into the spinal cord posteriorly. Conspicuous differences between the anterior and posterior neural tube become visible already during neural tube closure (NTC). Planar cell polarity (PCP)-mediated convergent extension (CE) movements are restricted to the posterior neural plate, i.e. hindbrain and spinal cord, where they propagate neural fold apposition. The lack of CE in the anterior neural plate correlates with a much slower mode of neural fold apposition anteriorly. The morphogenetic processes driving anterior NTC have not been addressed in detail. Here, we report a novel role for the breast cancer susceptibility gene and microtubule (MT) binding protein Hmmr (Hyaluronan-mediated motility receptor, RHAMM) in anterior neurulation and forebrain development in Xenopus laevis. Loss of hmmr function resulted in a lack of telencephalic hemisphere separation, arising from defective roof plate formation, which in turn was caused by impaired neural tissue narrowing. hmmr regulated polarization of neural cells, a function which was dependent on the MT binding domains. hmmr cooperated with the core PCP component vangl2 in regulating cell polarity and neural morphogenesis. Disrupted cell polarization and elongation in hmmr and vangl2 morphants prevented radial intercalation (RI), a cell behavior essential for neural morphogenesis. Our results pinpoint a novel role of hmmr in anterior neural development and support the notion that RI is a major driving force for anterior neurulation and forebrain morphogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Injectable gentamicin-loaded thermo-responsive hyaluronic acid derivative prevents infection in a rabbit model.

PubMed

Ter Boo, Gert-Jan A; Arens, Daniel; Metsemakers, Willem-Jan; Zeiter, Stephan; Richards, R Geoff; Grijpma, Dirk W; Eglin, David; Moriarty, T Fintan

2016-10-01

Despite the use of systemic antibiotic prophylaxis, the surgical fixation of open fractures with osteosynthesis implants is associated with high infection rates. Antibiotic-loaded biomaterials (ALBs) are increasingly used in implant surgeries across medical specialties to deliver high concentrations of antibiotics to the surgical site and reduce the risk of implant-associated infection. ALBs which are either less or not restricted in terms of spatial distribution and which may be applied throughout complex wounds could offer improved protection against infection in open fracture care. A thermo-responsive hyaluronic acid derivative (hyaluronic acid-poly(N-isopropylacrylamide) (HApN)) was prepared by a direct amidation reaction between the tetrabutyl ammonium (TBA) salt of hyaluronic acid and amine-terminated poly(N-isopropylacrylamide) (pN). The degree of grafting, and gelation properties of this gel were characterized, and the composition was loaded with gentamicin. The rheological- and release properties of this gentamicin-loaded HApN composition were tested in vitro and its efficacy in preventing infection was tested in a rabbit model of osteosynthesis contaminated with Staphylococcus aureus. The gentamicin-loaded HApN composition was able to prevent bacterial colonization of the implant site as shown by quantitative bacteriology. This finding was supported by histopathological evaluation of the humeri samples where no bacteria were found in the stained sections. In conclusion, this gentamicin-loaded HApN hydrogel effectively prevents infection in a complex wound, simulating a contaminated fracture treated with plating osteosynthesis. Fracture fixation after trauma is associated with high infection rates. Antibiotic loaded biomaterials (ALBs) can provide high local concentrations without systemic side effects. However, the currently available ALBs have limited accessibility to contaminated tissues in open fractures because of predetermined shape. Thus, a novel thermo-responsive hyaluronan based hydrogel with control over gelation temperature is reported. The efficacy of this gentamicin loaded hyaluronan derivative is demonstrated in an in vivo fracture model in the presence of fracture fixation hardware. The bacterial burden is cleared in all of the inoculated rabbits in the presence of the ALB. Thus, the proposed injectable thermo-responsive hyaluronan presents an effective ALB for the prevention of infection. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Warburg effect increases steady-state ROS condition in cancer cells through decreasing their antioxidant capacities (anticancer effects of 3-bromopyruvate through antagonizing Warburg effect).

PubMed

El Sayed, Salah Mohamed; Mahmoud, Ahmed Alamir; El Sawy, Samer Ahmed; Abdelaal, Esam Abdelrahim; Fouad, Amira Murad; Yousif, Reda Salah; Hashim, Marwa Shaban; Hemdan, Shima Badawy; Kadry, Zainab Mahmoud; Abdelmoaty, Mohamed Ahmed; Gabr, Adel Gomaa; Omran, Faten M; Nabo, Manal Mohamed Helmy; Ahmed, Nagwa Sayed

2013-11-01

Cancer cells undergo an increased steady-state ROS condition compared to normal cells. Among the major metabolic differences between cancer cells and normal cells is the dependence of cancer cells on glycolysis as a major source of energy even in the presence of oxygen (Warburg effect). In Warburg effect, glucose is catabolized to lactate that is extruded through monocarboxylate transporters to the microenvironment of cancer cells, while in normal cells, glucose is metabolized into pyruvate that is not extruded. Pyruvate is a potent antioxidant, while lactate has no antioxidant effect. Pyruvate in normal cells may be further metabolized to acetyl CoA and then through Krebs cycle with production of antioxidant intermediates e.g. citrate, malate and oxaloacetate together with the reducing equivalents (NADH.H+). Through activity of mitochondrial transhydrogenase, NADH.H+ replenishes NADPH.H+, coenzyme of glutathione reductase which replenishes reduced form of glutathione (potent antioxidant). This enhances antioxidant capacities of normal cells, while cancer cells exhibiting Warburg effect may be deprived of all that antioxidant capabilities due to loss of extruded lactate (substrate for Krebs cycle). Although intrinsic oxidative stress in cancer cells is high, it may be prevented from reaching progressively increasing levels that are cytotoxic to cancer cells. This may be due to some antioxidant effects exerted by hexokinase II (HK II) and NADPH.H+ produced through HMP shunt. Glycolytic phenotype in cancer cells maintains a high non-toxic oxidative stress in cancer cells and may be responsible for their malignant behavior. Through HK II, glycolysis fuels the energetic arm of malignancy, the mitotic arm of malignancy (DNA synthesis through HMP shunt pathway) and the metastatic arm of malignancy (hyaluronan synthesis through uronic acid pathway) in addition to the role of phosphohexose isomerase (autocrine motility factor). All those critical three arms start with the substrate G6P that is a direct product of HK II. 3-bromopyruvate (3BP, inhibitor of HK II) may prove as a promising anticancer and antimetastatic agent based on antagonizing the Warburg effect and disturbing the malignant behavior in cancer cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in Mice – Implications for Superficial Erosion

PubMed Central

Franck, Grégory; Mawson, Thomas; Sausen, Grasiele; Salinas, Manuel; Masson, Gustavo Santos; Cole, Andrew; Beltrami-Moreira, Marina; Chatzizisis, Yiannis; Quillard, Thibault; Tesmenitsky, Yevgenia; Shvartz, Eugenia; Sukhova, Galina K.; Swirski, Filip K.; Nahrendorf, Matthias; Aikawa, Elena; Croce, Kevin J.; Libby, Peter

2017-01-01

Rationale Superficial erosion currently causes up to a third of acute coronary syndromes (ACS), yet we lack understanding of its mechanisms. Thrombi due to superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. Objective This study tested in vivo the involvement of disturbed flow, and of neutrophils, hyaluronan, and TLR2 ligation in superficial intimal injury, a process implicated in superficial erosion. Methods and Results : In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell (EC) activation, neutrophil accumulation, EC death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. TLR2 agonism activated luminal ECs, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing EC injury and local thrombosis (p<0.05). Conclusions These results implicate flow disturbance, neutrophils, and TLR2 signaling as mechanisms that contribute to superficial erosion, a cause of ACS of likely growing importance in the statin era. PMID:28428204

Water-based polyurethane 3D printed scaffolds with controlled release function for customized cartilage tissue engineering.

PubMed

Hung, Kun-Che; Tseng, Ching-Shiow; Dai, Lien-Guo; Hsu, Shan-hui

2016-03-01

Conventional 3D printing may not readily incorporate bioactive ingredients for controlled release because the process often involves the use of heat, organic solvent, or crosslinkers that reduce the bioactivity of the ingredients. Water-based 3D printing materials with controlled bioactivity for customized cartilage tissue engineering is developed in this study. The printing ink contains the water dispersion of synthetic biodegradable polyurethane (PU) elastic nanoparticles, hyaluronan, and bioactive ingredients TGFβ3 or a small molecule drug Y27632 to replace TGFβ3. Compliant scaffolds are printed from the ink at low temperature. These scaffolds promote the self-aggregation of mesenchymal stem cells (MSCs) and, with timely release of the bioactive ingredients, induce the chondrogenic differentiation of MSCs and produce matrix for cartilage repair. Moreover, the growth factor-free controlled release design may prevent cartilage hypertrophy. Rabbit knee implantation supports the potential of the novel 3D printing scaffolds in cartilage regeneration. We consider that the 3D printing composite scaffolds with controlled release bioactivity may have potential in customized tissue engineering. Copyright © 2016 Elsevier Ltd. All rights reserved.

Imaging of Homeostatic, Neoplastic, and Injured Tissues by HA-Based Probes

PubMed Central

Veiseh, Mandana; Breadner, Daniel; Ma, Jenny; Akentieva, Natalia; Savani, Rashmin C; Harrison, Rene; Mikilus, David; Collis, Lisa; Gustafson, Stefan; Lee, Ting-Yim; Koropatnick, James; Luyt, Leonard G.; Bissell, Mina J.; Turley, Eva A.

2013-01-01

An increase in hyaluronan (HA) synthesis, cellular uptake, and metabolism occurs during the remodeling of tissue microenvironments following injury and during disease processes such as cancer. We hypothesized that multimodality HA-based probes selectively target and detectably accumulate at sites of high HA metabolism, thus providing a flexible imaging strategy for monitoring disease and repair processes. Kinetic analyses confirmed favorable available serum levels of the probe following intravenous (i.v.) or subcutaneous (s.c.) injection. Nuclear (technetium-HA, 99mTc-HA, and iodine-HA, 125I-HA), optical (fluorescent Texas Red-HA, TR-HA), and magnetic resonance (gadolinium-HA, Gd-HA) probes imaged liver (99mTc-HA), breast cancer cells/xenografts (TR-HA, Gd-HA), and vascular injury (125I-HA, TR-HA). Targeting of HA probes to these sites appeared to result from selective HA receptor-dependent localization. Our results suggest that HA-based probes, which do not require polysaccharide backbone modification to achieve favorable half-life and distribution, can detect elevated HA metabolism in homeostatic, injured, and diseased tissues. PMID:22066590

Transcription factor TBX4 regulates myofibroblast accumulation and lung fibrosis

PubMed Central

Xie, Ting; Liang, Jiurong; Liu, Ningshan; Huan, Caijuan; Zhang, Yanli; Liu, Weijia; Kumar, Maya; Xiao, Rui; D’Armiento, Jeanine; Metzger, Daniel; Chambon, Pierre; Papaioannou, Virginia E.; Stripp, Barry R.; Jiang, Dianhua

2016-01-01

Progressive tissue fibrosis is a major cause of the morbidity and mortality associated with repeated epithelial injuries and accumulation of myofibroblasts. Successful treatment options are limited by an incomplete understanding of the molecular mechanisms that regulate myofibroblast accumulation. Here, we employed in vivo lineage tracing and real-time gene expression transgenic reporting methods to analyze the early embryonic transcription factor T-box gene 4 (TBX4), and determined that TBX4-lineage mesenchymal progenitors are the predominant source of myofibroblasts in injured adult lung. In a murine model, ablation of TBX4-expressing cells or disruption of TBX4 signaling attenuated lung fibrosis after bleomycin-induced injury. Furthermore, TBX4 regulated hyaluronan synthase 2 production to enable fibroblast invasion of matrix both in murine models and in fibroblasts from patients with severe pulmonary fibrosis. These data identify TBX4 as a mesenchymal transcription factor that drives accumulation of myofibroblasts and the development of lung fibrosis. Targeting TBX4 and downstream factors that regulate fibroblast invasiveness could lead to therapeutic approaches in lung fibrosis. PMID:27400124

Molecular MR Imaging of CD44 in Breast Cancer with Hyaluronan-Based Contrast Agents

DTIC Science & Technology

2009-09-01

linear polysaccharide composed of alternating (β-1,4)-linked d- glucuronic acid and (β-1,3) N-acetyl-d-glucosamine residues with molecular weights as...enzymatic reactions in-vivo that generate polysaccharides of decreasing sizes, which in principle may facilitate the timely excretion of HA based...14CO2) or in urine (as low molecular weight HA or monosaccharide fragments). The same authors also reported that the total amount of excretion into

Vitreous floaters: Etiology, diagnostics, and management.

PubMed

Milston, Rebecca; Madigan, Michele C; Sebag, J

2016-01-01

Vitreous is a hydrated extracellular matrix comprised primarily of water, collagens, and hyaluronan organized into a homogeneously transparent gel. Gel liquefaction results from molecular alterations with dissociation of collagen from hyaluronan and aggregation of collagen fibrils forming fibers that cause light scattering and hence symptomatic floaters, especially in myopia. With aging, gel liquefaction and weakened vitreoretinal adhesion result in posterior vitreous detachment, the most common cause of primary symptomatic floaters arising from the dense collagen matrix of the posterior vitreous cortex. Recent studies indicate that symptomatic floaters are not only more prevalent, but also have a negative impact on the quality of life that is greater than previously appreciated. We review the literature concerning management of symptomatic vitreous floaters, currently either with observation, vitrectomy, or Nd:YAG laser. Published evidence is consistent with a low-risk profile and excellent success rate for floater vitrectomy, particularly with sutureless small gauge instruments and a limited core vitrectomy without PVD induction. Nd:YAG laser treatment of floaters, reported less commonly, claims resolution of floaters ranging between 0% and 100%; however, both peer-reviewed literature and assertions on web-based nonpeer-reviewed laser vitreolysis sites remain to be substantiated, and at present only vitrectomy has proven value. Prospective studies using objective, quantitative outcome measures are required to assess the relative efficacy and safety of these two procedures as well as new therapies such as pharmacologic vitreolysis. Copyright © 2016 Elsevier Inc. All rights reserved.

Hyaluronan 35kDa treatment protects mice from Citrobacter rodentium infection and induces epithelial tight junction protein ZO-1 in vivo.

PubMed

Kim, Yeojung; Kessler, Sean P; Obery, Dana R; Homer, Craig R; McDonald, Christine; de la Motte, Carol A

2017-10-01

Maintaining a healthy intestinal barrier, the primary physical barrier between intestinal microbiota and the underlying lamina propria, is critical for optimal health. Epithelial integrity is essential for the prevention of the entrance of luminal contents, such as bacteria and their products, through the large intestinal barrier. In this study, we investigated the protective functions of biosynthetic, specific sized, hyaluronan around 35kDa (HA35) on intestinal epithelium in healthy mice, as well as mice infected Citrobacter rodentium, an established model that mimics infection with a serious human pathogen, enteropathogenic E. coli (EPEC). Our results reveal that treatment with HA35 protects mice from Citrobacter infection and enhances the epithelial barrier function. In particular, we have found that HA35 induces the expression of tight junction protein zonula occludens (ZO)-1 in both healthy and Citrobacter infected mice, as demonstrated by immunoflurorescence and Western blot analyses. Furthermore, we determined that HA35 treatment enhances ZO-1 expression and reduces intestinal permeability at the early stages of dextran sulfate sodium (DSS)-induced colitis in mice. Together, our data demonstrate that the expression and functionality of tight junctions, are increased by HA35 treatment, suggesting a novel mechanism for the protection from Citrobacter infection. Copyright © 2016 Elsevier B.V. All rights reserved.

Interstitial matrix proteins determine hyaluronan reflection and fluid retention in rabbit joints: effect of protease

PubMed Central

Sabaratnam, S; Coleman, P J; Mason, R M; Levick, J R

2007-01-01

Hyaluronan (HA) retention inside the synovial cavity of joints serves diverse protective roles. We tested the hypothesis that HA retention is mediated by the network of extracellular matrix proteins in the synovial lining. Cannulated rabbit knee joints were infused with HA solution with or without pretreatment by chymopapain, a collagen-sparing protease. Trans-synovial fluid escape rate was measured and, after a period of trans-synovial filtration, samples of intra-articular fluid and subsynovial fluid were analysed for HA to assess its trans-synovial ultrafiltration. In control joints, HA ultrafiltration was confirmed by postfiltration increases in intra-articular HA concentration (259 ± 17% of infused concentration) and reduced subsynovial concentration (30 ± 8%; n = 11). The proportion of HA molecules reflected by the synovium was 57–75%. Chymopapain treatment increased the hydraulic permeability of the synovial lining ∼13-fold, almost abolished the trans-synovial difference in HA concentration and reduced the HA reflected fraction to 3–7% (n = 6; P < 0.001, ANOVA). Structural studies confirmed that chymopapain treatment depleted the matrix of proteoglycans but preserved its collagen. The findings thus demonstrate that HA ultrafiltration and synovial hydraulic permeability are determined by the network of non-collagen, extracellular matrix proteins. This may be important clinically, since protease activity is raised in rheumatoid arthritis, as are HA and fluid escape. PMID:17008373

Effects of transcutaneous topical injection of oxygen on vascular endothelial growth factor gene into the healing ligament in rats.

PubMed

Ishii, Yoshimasa; Ushida, Takashi; Tateishi, Tetsuya; Miyanaga, Yutaka

2003-11-01

The effects of intermittent exposure to oxygen injection on an experimentally induced ligament tear were studied in the right hind limb of 17 male Sprague-Dawley rats. Two rats were used for monitoring the partial oxygen pressure (pO(2)) of subcutaneous tissue and 15 rats were divided into the following three groups of 5 after an experimentally induced ligament tear: Group A, control group; Group B, injection of 0.5 ml hyaluronan to the wound transcutaneously; Group C, injection of 0.5 ml hyaluronan mixed with haemoglobin and oxygen (n=5). At 7 days post-ligament injury, we compared the ligaments of the three treatment groups for gross appearance, histology and expression of vascular endothelial growth factor (VEGF) mRNA by RT-PCR. Our results indicate that the pO(2) was immediately elevated to 334.6 mmHg by topical oxygen injection and this method was effective in promoting vessel formation in comparison to the control group (p<0.01). However, the expression of VEGF mRNA in the topical oxygen injection group (Group C) was lower than that in control group (p<0.05). Our results suggest that oxygen is able to accelerate vessel formation in spite of its effect of decreasing VEGF mRNA. Our method of using topical injection proved to be useful in healing the ligament and the wound.

Structure of a group A streptococcal phage-encoded virulence factor reveals a catalytically active triple-stranded beta-helix.

PubMed

Smith, Nicola L; Taylor, Edward J; Lindsay, Anna-Marie; Charnock, Simon J; Turkenburg, Johan P; Dodson, Eleanor J; Davies, Gideon J; Black, Gary W

2005-12-06

Streptococcus pyogenes (group A Streptococcus) causes severe invasive infections including scarlet fever, pharyngitis (streptococcal sore throat), skin infections, necrotizing fasciitis (flesh-eating disease), septicemia, erysipelas, cellulitis, acute rheumatic fever, and toxic shock. The conversion from nonpathogenic to toxigenic strains of S. pyogenes is frequently mediated by bacteriophage infection. One of the key bacteriophage-encoded virulence factors is a putative "hyaluronidase," HylP1, a phage tail-fiber protein responsible for the digestion of the S. pyogenes hyaluronan capsule during phage infection. Here we demonstrate that HylP1 is a hyaluronate lyase. The 3D structure, at 1.8-angstroms resolution, reveals an unusual triple-stranded beta-helical structure and provides insight into the structural basis for phage tail assembly and the role of phage tail proteins in virulence. Unlike the triple-stranded beta-helix assemblies of the bacteriophage T4 injection machinery and the tailspike endosialidase of the Escherichia coli K1 bacteriophage K1F, HylP1 possesses three copies of the active center on the triple-helical fiber itself without the need for an accessory catalytic domain. The triple-stranded beta-helix is not simply a structural scaffold, as previously envisaged; it is harnessed to provide a 200-angstroms-long substrate-binding groove for the optimal reduction in hyaluronan viscosity to aid phage penetration of the capsule.

Hyaluronidase and hyaluronan in insect venom allergy.

PubMed

King, Te Piao; Wittkowski, Knut M

2011-01-01

Insect venoms contain an allergen hyaluronidase that catalyzes the hydrolysis of hyaluronan (HA), a polymer of disaccharide GlcUA-GlcNAc in skin. HAs depending on their size have variable function in inflammation and immunity. This paper reports on whether hyaluronidase, HA polymers and oligomers can promote antibody response in mice. HA oligomers (8- to 50-mer; 3-20 kDa) were obtained by bee venom hyaluronidase digestion of HA polymers (750- to 5,000-mer; 300-2,000 kDa). Antibody responses in mice were compared following 3 biweekly subcutaneous injection of ovalbumin (OVA) with or without test adjuvant. OVA-specific IgG1 levels were approximately 2 times higher in BALB/c and C3H/HeJ mice receiving OVA and HA oligomer or polymer than those treated with OVA alone, and no increase in total IgE level was observed. In C57Bl/6 mice, observed increases in IgG1 and IgE were 3.5- and 1.7-fold, respectively, for the oligomer and 16- and 5-fold (p < 0.05), respectively, for the polymer. Hyaluronidase by its action on HA in skin can function indirectly as adjuvant to promote IgE and IgG1 response in mice. Insect venoms also have cytolytic peptides and phospholipases with inflammatory roles. These activities found in mice may contribute to venom allergenicity in susceptible people. Copyright © 2011 S. Karger AG, Basel.

Efficacy and safety of far infrared radiation in lymphedema treatment: clinical evaluation and laboratory analysis.

PubMed

Li, Ke; Zhang, Zheng; Liu, Ning Fei; Feng, Shao Qing; Tong, Yun; Zhang, Ju Fang; Constantinides, Joannis; Lazzeri, Davide; Grassetti, Luca; Nicoli, Fabio; Zhang, Yi Xin

2017-04-01

Swelling is the most common symptom of extremities lymphedema. Clinical evaluation and laboratory analysis were conducted after far infrared radiation (FIR) treatment on the main four components of lymphedema: fluid, fat, protein, and hyaluronan. Far infrared radiation is a kind of hyperthermia therapy with several and additional benefits as well as promoting microcirculation flow and improving collateral lymph circumfluence. Although FIR therapy has been applied for several years on thousands of lymphedema patients, there are still few studies that have reported the biological effects of FIR on lymphatic tissue. In this research, we investigate the effects of far infrared rays on the major components of lymphatic tissue. Then, we explore the effectiveness and safety of FIR as a promising treatment modality of lymphedema. A total of 32 patients affected by lymphedema in stage II and III were treated between January 2015 and January 2016 at our department. After therapy, a significant decrease of limb circumference measurements was noted and improving of quality of life was registered. Laboratory examination showed the treatment can also decrease the deposition of fluid, fat, hyaluronan, and protein, improving the swelling condition. We believe FIR treatment could be considered as both an alternative monotherapy and a useful adjunctive to the conservative or surgical lymphedema procedures. Furthermore, the real and significant biological effects of FIR represent possible future applications in wide range of the medical field.

Ingestion of hyaluronans (molecular weights 800 k and 300 k) improves dry skin conditions: a randomized, double blind, controlled study

PubMed Central

Kawada, Chinatsu; Yoshida, Takushi; Yoshida, Hideto; Sakamoto, Wakako; Odanaka, Wataru; Sato, Toshihide; Yamasaki, Takeshi; Kanemitsu, Tomoyuki; Masuda, Yasunobu; Urushibata, Osamu

2015-01-01

Hyaluronan (HA) has been increasingly used as a dietary supplement to improve the skin. However, the effect of ingested HA may depend on its molecular weight (MW) because its physiological activities in the body vary with its MW. In this study, we examined the effects of ingested HA with varying MW on the skin. In this randomized, double blind, placebo controlled study, 61 subjects with dry skin received oral HA (120 mg/day), of MWs 800 k and 300 k or placebo, for 6 weeks. The skin moisture contents of the first two groups increased more than those of the placebo group during the ingestion period. In addition, group HA 300 k exhibited significant improvements in skin moisture content 2 weeks after ingestion ended compared with the placebo group. A questionnaire survey about subjective facial aging symptoms showed that the HA treated groups exhibited significantly improved the skin condition compared with the placebo treated group. Furthermore, dermatologists objectively evaluated the clinical symptoms of the facial and whole body skin, showing that no adverse events were related to daily ingestion of HA. This study shows that both of ingesting HAs (MWs 800 k and 300 k) improved the skin condition by increasing the moisture content. PMID:25834304

Serum inter-alpha-trypsin inhibitor and matrix hyaluronan promote angiogenesis in fibrotic lung injury.

PubMed

Garantziotis, Stavros; Zudaire, Enrique; Trempus, Carol S; Hollingsworth, John W; Jiang, Dianhua; Lancaster, Lisa H; Richardson, Elizabeth; Zhuo, Lisheng; Cuttitta, Frank; Brown, Kevin K; Noble, Paul W; Kimata, Koji; Schwartz, David A

2008-11-01

The etiology and pathogenesis of angiogenesis in idiopathic pulmonary fibrosis (IPF) is poorly understood. Inter-alpha-trypsin inhibitor (IaI) is a serum protein that can bind to hyaluronan (HA) and may contribute to the angiogenic response to tissue injury. To determine whether IaI promotes HA-mediated angiogenesis in tissue injury. An examination was undertaken of angiogenesis in IaI-sufficient and -deficient mice in the bleomycin model of pulmonary fibrosis and in angiogenesis assays in vivo and in vitro. IaI and HA in patients with IPF were examined. IaI significantly enhances the angiogenic response to short-fragment HA in vivo and in vitro. lal deficiency Ieads to decreased angiogenesis in the matrigel model, and decreases lung angiogenesis after bleomycin exposure in mice. IaI is found in fibroblastic foci in IPF, where it colocalizes with HA. The colocalization is particularly strong in vascular areas around fibroblastic foci. Serum levels of IaI and HA are significantly elevated in patients with IPF compared with control subjects. High serum IaI and HA levels are associated with decreased lung diffusing capacity, but not FVC. Our findings indicate that serum IaI interacts with HA, and promotes angiogenesis in lung injury. IaI appears to contribute to the vascular response to lung injury and may lead to aberrant angiogenesis. Clinical trial registered with www.clinicaltrials.gov (NCT00016627).

Agarose and Polyacrylamide Gel Electrophoresis Methods for Molecular Mass Analysis of 5–500 kDa Hyaluronan

PubMed Central

Bhilocha, Shardul; Amin, Ripal; Pandya, Monika; Yuan, Han; Tank, Mihir; LoBello, Jaclyn; Shytuhina, Anastasia; Wang, Wenlan; Wisniewski, Hans-Georg; de la Motte, Carol; Cowman, Mary K.

2011-01-01

Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5–500 kDa have been investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffers systems were determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition, over which the relationship between HA mobility and logarithm of the molecular mass was linear. Excellent linear calibration was obtained for HA molecular mass as low as approximately 9 kDa in agarose gels. For higher resolution separation, and for extension to molecular masses as low as approximately 5 kDa, gradient polyacrylamide gels were superior. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in a sample, and calculation of weight-average and number-average values. The methods were validated for polydisperse HA samples with viscosity-average molecular masses of 112, 59, 37, and 22 kDa, at sample loads of 0.5 µg (for polyacrylamide) to 2.5 µg (for agarose). Use of the methods for electrophoretic mobility shift assays was demonstrated for binding of the HA-binding region of aggrecan (recombinant human aggrecan G1-IGD-G2 domains) to a 150 kDa HA standard. PMID:21684248

Biomaterial-enabled delivery of SDF-1α at the ventral side of breast cancer cells reveals a crosstalk between cell receptors to promote the invasive phenotype.

PubMed

Liu, Xi Qiu; Fourel, Laure; Dalonneau, Fabien; Sadir, Rabia; Leal, Salome; Lortat-Jacob, Hugues; Weidenhaupt, Marianne; Albiges-Rizo, Corinne; Picart, Catherine

2017-05-01

The SDF-1α chemokine (CXCL12) is a potent bioactive chemoattractant known to be involved in hematopoietic stem cell homing and cancer progression. The associated SDF-1α/CXCR4 receptor signaling is a hallmark of aggressive tumors, which can metastasize to distant sites such as lymph nodes, lung and bone. Here, we engineered a biomimetic tumoral niche made of a thin and soft polyelectrolyte film that can retain SDF-1α to present it, in a spatially-controlled manner, at the ventral side of the breast cancer cells. Matrix-bound SDF-1α but not soluble SDF-1α induced a striking increase in cell spreading and migration in a serum-containing medium, which was associated with the formation of lamellipodia and filopodia in MDA-MB231 cells and specifically mediated by CXCR4. Other Knockdown and inhibition experiments revealed that CD44, the major hyaluronan receptor, acted in concert, via a spatial coincidence, to drive a specific matrix-bound SDFα-induced cell response associated with ERK signaling. In contrast, the β1 integrin adhesion receptor played only a minor role on cell polarity. The CXCR4/CD44 mediated cellular response to matrix-bound SDF-1α involved the Rac1 RhoGTPase and was sustained solely in the presence of matrix-bound SDFα, in contrast with the transient signaling observed in response to soluble SDF-1α. Our results highlight that a biomimetic tumoral niche enables to reveal potent cellular effects and so far hidden molecular mechanisms underlying the breast cancer response to chemokines. These results open new insights for the design of future innovative therapies in metastatic cancers, by inhibiting CXCR4-mediated signaling in the tumoral niche via dual targeting of receptors (CXCR4 and CD44) or of associated signaling molecules (CXCR4 and Rac1). Copyright © 2017 Elsevier Ltd. All rights reserved.

Hyaluronan-Based Therapy for Metastatic Prostate Cancer

DTIC Science & Technology

2016-09-01

forward 5′-TGT GAC TTT GGT TGT TCC GTT GC-3′ and reverse 5′-ACC TGA GCC GAT GCA ACA ACA G-3′; DR5, forward 5′- AAG ACC CTT GTG CTC GTT GT-3′ and...reverse 5′-AGG TGG ACA CAA TCC CTC TG-3′; actin, forward 5′- TCC CTG GAG AAG AGC TAC GA-3′ and reverse 5′-AGC ACT GTG TTG GCG TAC AG-3′. 2.2.5. Death

A novel CD44-binding peptide from the pro-matrix metalloproteinase-9 hemopexin domain impairs adhesion and migration of chronic lymphocytic leukemia (CLL) cells.

PubMed

Ugarte-Berzal, Estefanía; Bailón, Elvira; Amigo-Jiménez, Irene; Albar, Juan Pablo; García-Marco, José A; García-Pardo, Angeles

2014-05-30

(pro)MMP-9 binds to CLL cells through the PEX9 domain and contributes to CLL progression. To biochemically characterize this interaction and identify potential therapeutic targets, we prepared GST-PEX9 forms containing structural blades B1B2 or B3B4. We recently described a sequence in blade B4 (P3 sequence) that bound α4β1 integrin and partially impaired cell adhesion and migration. We have now studied the possible contribution of the B1B2 region to cell interaction with PEX9. CLL cells bound to GST-B1B2 and CD44 was the primary receptor. GST-B1B2 inhibited CLL cell migration as effectively as GST-B3B4. Overlapping synthetic peptides spanning the B1B2 region identified the sequence FDAIAEIGNQLYLFKDGKYW, present in B1 and contained in peptide P6, as the most effective site. P6 inhibited cell adhesion to PEX9 in a dose-dependent manner and with an IC50 value of 90 μM. P6 also inhibited cell adhesion to hyaluronan but had no effect on adhesion to VCAM-1 (α4β1 integrin ligand), confirming its specific interaction with CD44. Spatial localization analyses mapped P6 to the central cavity of PEX9, in close proximity to the previously identified P3 sequence. Both P6 and P3 equally impaired cell adhesion to (pro)MMP-9. Moreover, P6 synergistically cooperated with P3, resulting in complete inhibition of CLL cell binding to PEX9, chemotaxis, and transendothelial migration. Thus, P6 is a novel sequence in PEX9 involved in cell-PEX9/(pro)MMP-9 binding by interacting with CD44. Targeting both sites, P6 and P3, should efficiently prevent (pro)MMP-9 binding to CLL cells and its pathological consequences. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Carbohydrate Polymers for Nonviral Nucleic Acid Delivery

PubMed Central

Sizovs, Antons; McLendon, Patrick M.; Srinivasachari, Sathya

2014-01-01

Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and these results are discussed with the focus on facile synthetic routes and favorable performance in biological systems. Many of these carbohydrates have been used to develop alternative types of biomaterials for nucleic acid delivery to typical polyplexes, and these novel materials are discussed. Also presented are polymeric vehicles that incorporate copolymerized carbohydrates into polymer backbones based on polyethylenimine and polylysine and their effect on transfection and biocompatibility. Unique scaffolds, such as clusters and polymers based on cyclodextrin (CD), are also discussed, with the focus on recent successes in vivo and in the clinic. These results are presented with the emphasis on the role of carbohydrate and charge on transfection. Use of carbohydrates as molecular recognition ligands for cell-type specific delivery is also briefly reviewed. We contend that carbohydrates have contributed significantly to progress in the field of non-viral DNA delivery, and these new discoveries are impactful for developing new vehicles and materials for treatment of human disease. PMID:21504102

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 HA injection was abrogated by Cd44 ablation, suggesting that interaction of the injected HA with CD44 is central to its protective effects on joint tissue remodeling and degeneration in OA progression. PMID:22721434

Biodegradable membrane-covered stent from chitosan-based polymers.

PubMed

Thierry, Benjamin; Merhi, Yahye; Silver, Jim; Tabrizian, Maryam

2005-12-01

Membrane-covered devices could help treat disease of the vasculature such as aneurysm, rupture, and fistulas. They are also investigated to reduce embolic complication associated with revascularization of saphenous vein graft. The aim of this study is to design a clinically applicable biodegradable membrane-covered stent based on the natural polysaccharide chitosan, which has been developed. The mechanical properties of the membrane is optimized through blending with polyethylene oxide (70:30% Wt CH:PEO). The membrane was able to sustain the mechanical deformation of the supporting self-expandable metallic stents during its deployment. The membrane was demonstrated to resist physiological transmural pressure (burst pressure resistance >500 mm Hg) and presented a high-water permeation resistance (1 mL/cm(2) min(-1) at 120 mmHg). The CH-PEO membrane showed a good hemocompatibility in an ex vivo assay. Heparin and hyaluronan surface complexation with the membrane further reduced platelet adhesion by 50.1 and 63% (p = 0.05). The ability of the membrane-covered devices to be used as a drug reservoir was investigated using the nitric oxide donor sodium nitroprusside (SNP). SNP-loaded membranes displayed significantly reduced platelet adhesion. (c) 2005 Wiley Periodicals, Inc.

Hyaluronan-decorated liposomes as drug delivery systems for cutaneous administration.

PubMed

Franzé, Silvia; Marengo, Alessandro; Stella, Barbara; Minghetti, Paola; Arpicco, Silvia; Cilurzo, Francesco

2018-01-15

The work aimed to evaluate the feasibility to design hyaluronic acid (HA) decorated flexible liposomes to enhance the skin penetration of nifedipine. Egg phosphatidylcholine (e-PC) based transfersomes (Tween 80) and transethosomes (ethanol) were prepared. HA was reacted with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (HA-DPPE) and two molar ratios (0.5 and 3%) of conjugate with respect to e-PC were tested. The presence of HA significantly increased the packing order of the bilayer (as verified by differential scanning calorimetry), reducing both the encapsulation efficiency and the flexibility of the decorated liposomes in a dose-dependent manner. In fact, at the highest HA content the constant of deformability (K, N/mm) increased and the carriers remained on the skin surface after topical application. The stiffening effect of HA was counterbalanced by the addition of ethanol as fluidizing agent that allowed to maintain the highest HA concentration, meanwhile reducing the K value of the vesicles. HA-transethosomes allowed a suitable nifedipine permeation (J ∼ 30 ng/cm 2 /h) and significantly improved the drug penetration, favouring the formation of a drug depot in the epidermis. These data suggest the potentialities of HA-transethosomes as drug delivery systems intended for the treatment of cutaneous pathologies and underline the importance of studying the effect of surface functionalization on carrier deformability to rationalize the design of such systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Decreasing the metastatic potential in cancers--targeting the heparan sulfate proteoglycans.

PubMed

Fjeldstad, K; Kolset, S O

2005-09-01

The heterogeneity of proteoglycans (PG)s contributes to their functional diversity. Many functions depend on their ability to bind and modulate the activity of components of the extracellular matrix (ECM). The ability of PGs to interact with other molecules, such as growth factors, is largely determined by the fine structure of the glycosaminoglycan (GAG) chains. Tumorigenesis is associated with changes in the PG synthesis. Heparan sulfate (HS) PGs are involved in several aspects of cancer biology including tumor progression, angiogenesis, and metastasis. PGs can have both tumor promoting and tumor suppressing activities depending on the protein core, the GAG attached, molecules they associate with, localization, the tumor subtype, stages, and degree of tumor differentiation. Perlecan is an angiogenic factor involved in tumor invasiveness. The C-terminal domain V of perlecan, named endorepellin, has however been shown to inhibit angiogenesis. Another angiogenic factor is endostatin, the COOH-terminal domain of the part-time PG collagen XVIII. Glypicans and syndecans may promote local cancer cell growth in some cancer tissues, but inhibit tissue invasion and metastasis in others. The GAG hyaluronan (HA) promotes cancer growth by providing a loose matrix for migrating tumor cells and mediates adhesion of cancer cells. HSPG degrading enzymes like heparanase, heparitinase, and other enzymes such as hyaluronidase and MMP are also important in tumor metastasis. Several different treatment strategies that target PGs have been developed. They have the potential to be effective in reducing tumor growth and inhibit the formation of metastases. PGs are also valuable tumor markers in several cancers.

Enzyme-responsive nanocomposites for wound infection prophylaxis in burn management: in vitro evaluation of their compatibility with healing processes

PubMed Central

Grützner, Verena; Unger, Ronald E; Baier, Grit; Choritz, Lars; Freese, Christian; Böse, Thomas; Landfester, Katharina; Kirkpatrick, C James

2015-01-01

Responsive, theranostic nanosystems, capable of both signaling and treating wound infections, is a sophisticated approach to reduce the most common and potentially traumatizing side effects of burn wound treatment: slowed wound healing due to prophylactic anti-infective drug exposure as well as frequent painful dressing changes. Antimicrobials as well as dye molecules have been incorporated into biodegradable nanosystems that release their content only in the presence of pathogens. Following nanocarrier degradation by bacterial enzymes, any infection will thus emit a visible signal and be effectively treated at its source. In this study, we investigated the effect of fluorescent-labeled hyaluronan nanocapsules containing polyhexanide biguanide and poly-L-lactic acid nanoparticles loaded with octenidine on primary human dermal microvascular endothelial cells, which play a major role in cutaneous wound healing. Microscopic and flow cytometric analysis indicated a time-dependent uptake of both the nanocapsules and the nanoparticles. However, enzyme immunoassays showed no significant influence on the expression of pro-inflammatory cell adhesion molecules and cytokines by the endothelial cells. Under angiogenic-stimulating conditions, the potential to form capillary-like structures in co-culture with dermal fibroblasts was not inhibited. Furthermore, cytotoxicity studies (the MTS and crystal violet assay) after short- and long-term exposure to the materials demonstrated that both systems exhibited less toxicity than solutions of the antiseptic agents alone in comparable concentrations. The results indicate that responsive antimicrobial nanocomposites could be used as an advanced drug delivery system and a promising addition to current best practice wound infection prophylaxis with few side effects. PMID:26150717

Impaired response of mature adipocytes of diabetic mice to hypoxia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Seok Jong, E-mail: seok-hong@northwestern.edu; Jin, Da P.; Buck, Donald W.

2011-10-01

Adipose tissue contains various cells such as infiltrated monocytes/macrophages, endothelial cells, preadipocytes, and adipocytes. Adipocytes have an endocrine function by secreting adipokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-{alpha}, leptin, and adiponectin. Dysregulation of adipokines in adipose tissues leads to a chronic low-grade inflammation which could result in atherosclerosis, hypertension, and type 2 diabetes. A sustained inflammatory state, which is characterized by prolonged persistence of macrophages and neutrophils, is found in diabetic wounds. In addition, subcutaneous adipocytes are enormously increased in amount clinically in type 2 diabetes. However, the function of subcutaneous adipocytes, which play an important role inmore » injured tissue subjected to hypoxia, has not been well characterized in vitro due to the difficulty of maintaining mature adipocytes in culture using conventional methods because of their buoyancy. In this study, we established a novel in vitro culture method of mature adipocytes by enclosing them in a hyaluronan (HA) based hydrogel to study their role in response to stress such as hypoxia. BrdU labeling and Ki67 immunostaining experiments showed that hydrogel enclosed mature adipocytes proliferate in vitro. Both mRNA and protein expression analyses for hypoxia regulated genes, such as vascular endothelial growth factor (VEGF) and heme oxygenase 1 (HO1), showed that mature adipocytes of wild type mice respond to hypoxia. In contrast, mature adipocytes of diabetic db/db and TallyHo mice did not efficiently respond to hypoxia. Our studies suggest that mature adipocytes are functionally active cells, and their abnormal function to hypoxia can be one of underlining mechanisms in type 2 diabetes.« less

Single-Molecule Unbinding Forces between the Polysaccharide Hyaluronan and Its Binding Proteins.

PubMed

Bano, Fouzia; Tammi, Markku I; Kang, David W; Harris, Edward N; Richter, Ralf P

2018-06-19

The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, the HA receptor for endocytosis (stabilin-2/HARE), and the HA receptor CD44. We find that the resistance to tensile stress for these hyaladherins correlates with the size of the HA-binding domain. The lowest mean rupture forces are observed for members of the type A subgroup (i.e., with the shortest HA-binding domains; TSG-6 and HARE). In contrast, the mechanical stability of the bond formed by aggrecan in complex with cartilage link protein (two members of the type C subgroup, i.e., with the longest HA-binding domains) and HA is equal or even superior to the high affinity streptavidin⋅biotin bond. Implications for the molecular mechanism of unbinding of HA⋅hyaladherin bonds under force are discussed, which underpin the mechanical properties of HA⋅hyaladherin complexes and HA-rich extracellular matrices. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Elastic fiber-mediated enthesis in the human middle ear

PubMed Central

Kawase, Tetsuaki; Shibata, Shunichi; Katori, Yukio; Ohtsuka, Aiji; Murakami, Gen; Fujimiya, Mineko

2012-01-01

Adaptation to constant vibration (acoustic oscillation) is likely to confer a specific morphology at the bone–tendon and bone–ligament interfaces at the ear ossicles, which therefore represent an exciting target of enthesis research. We histologically examined (i) the bone attachments of the tensor tympani and stapedius muscles and (ii) the annular ligament of the incudostapedial joint obtained from seven elderly donated cadavers. Notably, both aldehyde-fuchsin and elastic-Masson staining demonstrated that the major fibrous component of the entheses was not collagen fibers but mature elastic fibers. The positive controls for elastic fiber staining were the arterial wall elastic laminae included in the temporal bone materials. The elastic fibers were inserted deeply into the type II collagen-poor fibrocartilage covering the ear ossicles. The muscle tendons were composed of an outer thin layer of collagen fibers and an inner thick core of elastic fibers near the malleus or stapes. In the unique elastic fiber-mediated entheses, hyaluronan, versican and fibronectin were expressed strongly along the elastic fibers. The hyaluronan seemed to act as a friction-reducing lubricant for the elastic fibers. Aggrecan was labeled strongly in a disk- or plica-like fibrous mass on the inner side of the elastic fiber-rich ligament, possibly due to compression stress from the ligament. Tenascin-c was not evident in the entheses. The elastic fiber-mediated entheses appeared resistant to tissue destruction in an environment exposed to constant vibration. The morphology was unlikely to be the result of age-related degeneration. PMID:22803514

PRELIMINARY HIGH PERFORMANCE CAPILLARY ELECTROPHORESIS (HPCE) STUDIES OF ENZYMATIC DEGRADATION OF HYALURONIC ACID BY HYALURONIDASE IN THE PRESENCE OF POLYVALENT METAL IONS.

PubMed

Urbaniak, Bartosz; Plewa, Szymon; Kokot, Zenon Jozef

2017-01-01

The aim of this study was, at first, to examine the influence of metal ions on digestion process of hyaluronic acid by hyaluronidase (HAse) using high performance capillary electrophoresis (HPCE) method. The influence of copper(H), zinc(Il), manganese(II) ions on enzymatic degradation of HA by hyaluronidase enzyme (HA-se) were investigated. Secondly, the kinetic parameters, V(max), K(m), k(cat), and k (cat),/K(m) were determined to estimate the impact of these metal ions (Me) on digestion process of hyaluronic acid (HA). The two different HA-Me mole ratios were analyzed. The examined data were always compared to the digestion process of pure HA solution by hyaluronidase, to exhibit the differences in the digestion process of pure hyaluronan as well as the hyaluronan in the presence of metal ions. It was observed that all of the investigated metal ions have influenced the hyaluronic acid degradation process. The most important conclusion was a decrease of the kinetic parameters both K,, and V,. In the result, it can be assumed that in all of the studied samples with metal ions addition, the uncompetitive mechanism of enzyme inhibition occurred. The results of this study may give new insight into foregoing knowledge about hyaluronic acid behavior. Due to the fact that our study was carried out only for three different metal ions in two concentrations, it is necessary to continue further research comprising wider range of metal ions and their concentrations.

Effects of temperature and solvent condition on phase separation induced molecular fractionation of gum arabic/hyaluronan aqueous mixtures.

PubMed

Hu, Bing; Han, Lingyu; Gao, Zhiming; Zhang, Ke; Al-Assaf, Saphwan; Nishinari, Katsuyoshi; Phillips, Glyn O; Yang, Jixin; Fang, Yapeng

2018-05-14

Effects of temperature and solvent condition on phase separation-induced molecular fractionation of gum arabic/hyaluronan (GA/HA) mixed solutions were investigated. Two gum arabic samples (EM10 and STD) with different molecular weights and polydispersity indices were used. Phase diagrams, including cloud and binodal curves, were established by visual observation and GPC-RI methods. The molecular parameters of control and fractionated GA, from upper and bottom phases, were measured by GPC-MALLS. Fractionation of GA increased the content of arabinogalactan-protein complex (AGP) from ca. 11% to 18% in STD/HA system and 28% to 55% in EM10/HA system. The phase separation-induced molecular fractionation was further studied as a function of temperature and solvent condition (varying ionic strength and ethanol content). Increasing salt concentration (from 0.5 to 5 mol/L) greatly reduced the extent of phase separation-induced fractionation. This effect may be ascribed to changes in the degree of ionization and shielding of the acid groups. Increasing temperature (from 4 °C to 80 °C) also exerted a significant influence on phase separation-induced fractionation. The best temperature for GA/HA mixture system was 40 °C while higher temperature negatively affected the fractionation due to denaturation and possibly degradation in mixed solutions. Increasing the ethanol content up to 30% showed almost no effect on the phase separation induced fractionation. Copyright © 2018 Elsevier B.V. All rights reserved.

Chitosan Enriched Three-Dimensional Matrix Reduces Inflammatory and Catabolic Mediators Production by Human Chondrocytes

PubMed Central

Oprenyeszk, Frederic; Sanchez, Christelle; Dubuc, Jean-Emile; Maquet, Véronique; Henrist, Catherine; Compère, Philippe; Henrotin, Yves

2015-01-01

This in vitro study investigated the metabolism of human osteoarthritic (OA) chondrocytes encapsulated in a spherical matrix enriched of chitosan. Human OA chondrocytes were encapsulated and cultured for 28 days either in chitosan-alginate beads or in alginate beads. The beads were formed by slowly passing dropwise either the chitosan 0.6%–alginate 1.2% or the alginate 1.2% solution through a syringe into a 102 mM CaCl2 solution. Beads were analyzed histologically after 28 days. Interleukin (IL)-6 and -8, prostaglandin (PG) E2, matrix metalloproteinases (MMPs), hyaluronan and aggrecan were quantified directly in the culture supernatant by specific ELISA and nitric oxide (NO) by using a colorimetric method based on the Griess reaction. Hematoxylin and eosin staining showed that chitosan was homogeneously distributed through the matrix and was in direct contact with chondrocytes. The production of IL-6, IL-8 and MMP-3 by chondrocytes significantly decreased in chitosan-alginate beads compared to alginate beads. PGE2 and NO decreased also significantly but only during the first three days of culture. Hyaluronan and aggrecan production tended to increase in chitosan-alginate beads after 28 days of culture. Chitosan-alginate beads reduced the production of inflammatory and catabolic mediators by OA chondrocytes and tended to stimulate the synthesis of cartilage matrix components. These particular effects indicate that chitosan-alginate beads are an interesting scaffold for chondrocytes encapsulation before transplantation to repair cartilage defects. PMID:26020773

Serum Inter–α-Trypsin Inhibitor and Matrix Hyaluronan Promote Angiogenesis in Fibrotic Lung Injury

PubMed Central

Garantziotis, Stavros; Zudaire, Enrique; Trempus, Carol S.; Hollingsworth, John W.; Jiang, Dianhua; Lancaster, Lisa H.; Richardson, Elizabeth; Zhuo, Lisheng; Cuttitta, Frank; Brown, Kevin K.; Noble, Paul W.; Kimata, Koji; Schwartz, David A.

2008-01-01

Rationale: The etiology and pathogenesis of angiogenesis in idiopathic pulmonary fibrosis (IPF) is poorly understood. Inter-α-trypsin inhibitor (IaI) is a serum protein that can bind to hyaluronan (HA) and may contribute to the angiogenic response to tissue injury. Objectives: To determine whether IaI promotes HA-mediated angiogenesis in tissue injury. Methods: An examination was undertaken of angiogenesis in IaI-sufficient and -deficient mice in the bleomycin model of pulmonary fibrosis and in angiogenesis assays in vivo and in vitro. IaI and HA in patients with IPF were examined. Measurements and Main Results: IaI significantly enhances the angiogenic response to short-fragment HA in vivo and in vitro. lal deficiency Ieads to decreased angiogenesis in the matrigel model, and decreases lung angiogenesis after bleomycin exposure in mice. IaI is found in fibroblastic foci in IPF, where it colocalizes with HA. The colocalization is particularly strong in vascular areas around fibroblastic foci. Serum levels of IaI and HA are significantly elevated in patients with IPF compared with control subjects. High serum IaI and HA levels are associated with decreased lung diffusing capacity, but not FVC. Conclusions: Our findings indicate that serum IaI interacts with HA, and promotes angiogenesis in lung injury. IaI appears to contribute to the vascular response to lung injury and may lead to aberrant angiogenesis. Clinical trial registered with www.clinicaltrials.gov (NCT00016627). PMID:18703791

An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution.

PubMed

Lee, H G; Cowman, M K

1994-06-01

An electrophoretic method is described for determining the molecular weight distribution of hyaluronan (HA). The method involves separation of HA by electrophoresis on a 0.5% agarose gel, followed by detection of HA using the cationic dye Stains-All (3,3'-dimethyl-9-methyl-4,5,4'5'-dibenzothiacarbocyanine). The recommended sample load is 7 micrograms. Calibration of the method with HA standards of known molecular weight has established a linear relationship between electrophoretic mobility and the logarithm of the weight-average molecular weight over the range of approximately 0.2-6 x 10(6). The separated HA pattern may also be visualized after electrotransfer of HA from the agarose gel to a nylon membrane. The membrane may be stained with the dye alcian blue. Alternatively, specific detection of HA from impure samples can be achieved by probing the nylon membrane with biotin-labeled HA-binding protein and subsequent interaction with a streptavidin-linked gold reagent and silver staining for amplification. The electrophoretic method was used to analyze HA in two different liquid connective tissues. Normal human knee joint synovial fluid showed a narrow HA molecular weight distribution, with a peak at 6-7 x 10(6). Owl monkey vitreous HA also showed a narrow molecular weight distribution, with a peak at 5-6 x 10(6). These results agree well with available published data and indicate the applicability of the method to the analysis of impure HA samples which may be available in limited amounts.

Sonic-Hedgehog pathway inhibition normalizes desmoplastic tumor microenvironment to improve chemo- and nanotherapy

PubMed Central

Voutouri, Chrysovalantis; Kalli, Maria; Pirentis, Athanassios P.; Stylianopoulos, Triantafyllos

2017-01-01

Targeting the rich extracellular matrix of desmoplastic tumors has been successfully shown to normalize collagen and hyaluronan levels and re-engineer intratumoral mechanical forces, improving tumor perfusion and chemotherapy. As far as targeting the abundant cancer-associated fibroblasts (CAFs) in desmoplastic tumors is concerned, while both pharmacologic inhibition of the sonic-hedgehog pathway and genetic depletion of fibroblasts have been employed in pancreatic cancers, the results between the two methods have been contradictory. In this study, we employed vismodegib to inhibit the sonic-hedgehog pathway with the aim to i) elucidate the mechanism of how CAFs depletion improves drug delivery, ii) extent and evaluate the potential use of sonic-hedgehog inhibitors to breast cancers, and iii) investigate whether sonic-hedgehog inhibition improves not only chemotherapy, but also the efficacy of the most commonly used breast cancer nanomedicines, namely Abraxane® and Doxil®. We found that treatment with vismodegib normalizes the tumor microenvironment by reducing the proliferative CAFs and in cases the levels of collagen and hyaluronan. These modulations re-engineered the solid and fluid stresses in the tumors, improving blood vessel functionality. As a result, the delivery and efficacy of chemotherapy was improved in two models of pancreatic cancer. Additionally, vismodegib treatment significantly improved the efficacy of both Abraxane and Doxil in xenograft breast tumors. Our results suggest the use of vismodegib, and sonic hedgehog inhibitors in general, to enhance cancer chemo- and nanotherapy. PMID:28662901

Co-culture of chondrons and mesenchymal stromal cells reduces the loss of collagen VI and improves extracellular matrix production.

PubMed

Owida, H A; De Las Heras Ruiz, T; Dhillon, A; Yang, Y; Kuiper, N J

2017-12-01

Adult articular chondrocytes are surrounded by a pericellular matrix (PCM) to form a chondron. The PCM is rich in hyaluronan, proteoglycans, and collagen II, and it is the exclusive location of collagen VI in articular cartilage. Collagen VI anchors the chondrocyte to the PCM. It has been suggested that co-culture of chondrons with mesenchymal stromal cells (MSCs) might enhance extracellular matrix (ECM) production. This co-culture study investigates whether MSCs help to preserve the PCM and increase ECM production. Primary bovine chondrons or chondrocytes or rat MSCs were cultured alone to establish a baseline level for ECM production. A xenogeneic co-culture monolayer model using rat MSCs (20, 50, and 80%) was established. PCM maintenance and ECM production were assessed by biochemical assays, immunofluorescence, and histological staining. Co-culture of MSCs with chondrons enhanced ECM matrix production, as compared to chondrocyte or chondron only cultures. The ratio 50:50 co-culture of MSCs and chondrons resulted in the highest increase in GAG production (18.5 ± 0.54 pg/cell at day 1 and 11 ± 0.38 pg/cell at day 7 in 50:50 co-culture versus 16.8 ± 0.61 pg/cell at day 1 and 10 ± 0.45 pg/cell at day 7 in chondron monoculture). The co-culture of MSCs with chondrons appeared to decelerate the loss of the PCM as determined by collagen VI expression, whilst the expression of high-temperature requirement serine protease A1 (HtrA1) demonstrated an inverse relationship to that of the collagen VI. Together, this implies that MSCs directly or indirectly inhibited HtrA1 activity and the co-culture of MSCs with chondrons enhanced ECM synthesis and the preservation of the PCM.

Adult human mesenchymal stem cells delivered via intra-articular injection to the knee following partial medial meniscectomy: a randomized, double-blind, controlled study.

PubMed

Vangsness, C Thomas; Farr, Jack; Boyd, Joel; Dellaero, David T; Mills, C Randal; LeRoux-Williams, Michelle

2014-01-15

There are limited treatment options for tissue restoration and the prevention of degenerative changes in the knee. Stem cells have been a focus of intense preclinical research into tissue regeneration but limited clinical investigation. In a randomized, double-blind, controlled study, the safety of the intra-articular injection of human mesenchymal stem cells into the knee, the ability of mesenchymal stem cells to promote meniscus regeneration following partial meniscectomy, and the effects of mesenchymal stem cells on osteoarthritic changes in the knee were investigated. A total of fifty-five patients at seven institutions underwent a partial medial meniscectomy. A single superolateral knee injection was given within seven to ten days after the meniscectomy. Patients were randomized to one of three treatment groups: Group A, in which patients received an injection of 50 × 10⁶ allogeneic mesenchymal stem cells; Group B, 150 × 10⁶ allogeneic mesenchymal stem cells; and the control group, a sodium hyaluronate (hyaluronic acid/hyaluronan) vehicle control. Patients were followed to evaluate safety, meniscus regeneration, the overall condition of the knee joint, and clinical outcomes at intervals through two years. Evaluations included sequential magnetic resonance imaging (MRI). No ectopic tissue formation or clinically important safety issues were identified. There was significantly increased meniscal volume (defined a priori as a 15% threshold) determined by quantitative MRI in 24% of patients in Group A and 6% in Group B at twelve months post meniscectomy (p = 0.022). No patients in the control group met the 15% threshold for increased meniscal volume. Patients with osteoarthritic changes who received mesenchymal stem cells experienced a significant reduction in pain compared with those who received the control, on the basis of visual analog scale assessments. There was evidence of meniscus regeneration and improvement in knee pain following treatment with allogeneic human mesenchymal stem cells. These results support the study of human mesenchymal stem cells for the apparent knee-tissue regeneration and protective effects.

Autologous mesenchymal stem cells or meniscal cells: what is the best cell source for regenerative meniscus treatment in an early osteoarthritis situation?

PubMed

Zellner, Johannes; Pattappa, Girish; Koch, Matthias; Lang, Siegmund; Weber, Johannes; Pfeifer, Christian G; Mueller, Michael B; Kujat, Richard; Nerlich, Michael; Angele, Peter

2017-10-10

Treatment of meniscus tears within the avascular region represents a significant challenge, particularly in a situation of early osteoarthritis. Cell-based tissue engineering approaches have shown promising results. However, studies have not found a consensus on the appropriate autologous cell source in a clinical situation, specifically in a challenging degenerative environment. The present study sought to evaluate the appropriate cell source for autologous meniscal repair in a demanding setting of early osteoarthritis. A rabbit model was used to test autologous meniscal repair. Bone marrow and medial menisci were harvested 4 weeks prior to surgery. Bone marrow-derived mesenchymal stem cells (MSCs) and meniscal cells were isolated, expanded, and seeded onto collagen-hyaluronan scaffolds before implantation. A punch defect model was performed on the lateral meniscus and then a cell-seeded scaffold was press-fit into the defect. Following 6 or 12 weeks, gross joint morphology and OARSI grade were assessed, and menisci were harvested for macroscopic, histological, and immunohistochemical evaluation using a validated meniscus scoring system. In conjunction, human meniscal cells isolated from non-repairable bucket handle tears and human MSCs were expanded and, using the pellet culture model, assessed for their meniscus-like potential in a translational setting through collagen type I and II immunostaining, collagen type II enzyme-linked immunosorbent assay (ELISA), and gene expression analysis. After resections of the medial menisci, all knees showed early osteoarthritic changes (average OARSI grade 3.1). However, successful repair of meniscus punch defects was performed using either meniscal cells or MSCs. Gross joint assessment demonstrated donor site morbidity for meniscal cell treatment. Furthermore, human MSCs had significantly increased collagen type II gene expression and production compared to meniscal cells (p < 0.05). The regenerative potential of the meniscus by an autologous cell-based tissue engineering approach was shown even in a challenging setting of early osteoarthritis. Autologous MSCs and meniscal cells were found to have improved meniscal healing in an animal model, thus demonstrating their feasibility in a clinical setting. However, donor site morbidity, reduced availability, and reduced chondrogenic differentiation of human meniscal cells from debris of meniscal tears favors autologous MSCs for clinical use for cell-based meniscus regeneration.

Simultaneous determination of intracellular UDP-sugars in hyaluronic acid-producing Streptococcus zooepidemicus.

PubMed

Franke, Lukáš; Čožíková, Dagmar; Smirnou, Dzianis; Hermannová, Martina; Hanová, Tereza; Růžičková, Andrea; Velebný, Vladimír

2015-08-01

Two chromatographic methods for the quantitative analysis of uridine diphosphate (UDP) sugars involved in hyaluronan pathway of Streptococcus zooepidemicus (SEZ) were developed and compared. The sample preparation protocol using centrifugation and extraction in hot ethanol was employed prior to the analyses. Separation was achieved using an anion exchange Spherisorb SAX column or a Shodex QA-825 column connected with a photodiode array (PDA) detector. To increase the throughput of the chromatography method employing the Spherisorb SAX column, the solid phase extraction (SPE) procedure was introduced. Method validation results displayed that limits of detection (LODs) of UDP-glucose (UDP-Glc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) calculated according to QC Expert software were in the low micromolar range and the coefficient of correlation (R(2)) was above 0.997. However, the analytical technique using the Spherisorb SAX column resulted in 80-90% recoveries and low LODs (≤6.19μM), the Shodex QA-825 column showed better long-term stability and reproducible chromatographic properties (RSD≤5.60%). The Shodex QA-825 column was successfully used to monitor UDP-sugar levels during the growth rate of SEZ cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Unusual glycosaminoglycans from a deep sea hydrothermal bacterium improve fibrillar collagen structuring and fibroblast activities in engineered connective tissues.

PubMed

Senni, Karim; Gueniche, Farida; Changotade, Sylvie; Septier, Dominique; Sinquin, Corinne; Ratiskol, Jacqueline; Lutomski, Didier; Godeau, Gaston; Guezennec, Jean; Colliec-Jouault, Sylvia

2013-04-23

Biopolymers produced by marine organisms can offer useful tools for regenerative medicine. Particularly, HE800 exopolysaccharide (HE800 EPS) secreted by a deep-sea hydrothermal bacterium displays an interesting glycosaminoglycan-like feature resembling hyaluronan. Previous studies demonstrated its effectiveness to enhance in vivo bone regeneration and to support osteoblastic cell metabolism in culture. Thus, in order to assess the usefulness of this high-molecular weight polymer in tissue engineering and tissue repair, in vitro reconstructed connective tissues containing HE800 EPS were performed. We showed that this polysaccharide promotes both collagen structuring and extracellular matrix settle by dermal fibroblasts. Furthermore, from the native HE800 EPS, a low-molecular weight sulfated derivative (HE800 DROS) displaying chemical analogy with heparan-sulfate, was designed. Thus, it was demonstrated that HE800 DROS mimics some properties of heparan-sulfate, such as promotion of fibroblast proliferation and inhibition of matrix metalloproteinase (MMP) secretion. Therefore, we suggest that the HE800EPS family can be considered as an innovative biotechnological source of glycosaminoglycan-like compounds useful to design biomaterials and drugs for tissue engineering and repair.

A fermented barley and soybean formula enhances skin hydration.

PubMed

Lee, Sein; Kim, Jong-Eun; Suk, Sujin; Kwon, Oh Wook; Park, Gaeun; Lim, Tae-Gyu; Seo, Sang Gwon; Kim, Jong Rhan; Kim, Dae Eung; Lee, Miyeong; Chung, Dae Kyun; Jeon, Jong Eun; Cho, Dong Woon; Hurh, Byung Serk; Kim, Sun Yeou; Lee, Ki Won

2015-09-01

Skin hydration is one of the primary aims of beauty and anti-aging treatments. Barley (Hordeum vulgare) and soybean (Glycine max) are major food crops, but can also be used as ingredients for the maintenance of skin health. We developed a natural product-based skin treatment using a barley and soybean formula (BS) incorporating yeast fermentation, and evaluated its skin hydration effects as a dietary supplement in a clinical study. Participants ingested a placebo- (n = 33) or BS- (3 g/day) containing drink (n = 32) for 8 weeks. A significant increase in hydration in the BS group as compared to the placebo group was observed on the faces of subjects after 4 and 8 weeks, and on the forearm after 4 weeks. Decreases in stratum corneum (SC) thickness were also observed on the face and forearm. BS enhanced hyaluronan (HA) and skin barrier function in vitro and reduced Hyal2 expression in human dermal fibroblasts (HDF). BS also recovered ultraviolet (UV) B-induced downregulation of HA in HaCaT cells. These results suggest that BS has promising potential for development as a health functional food to enhance skin health.

A fermented barley and soybean formula enhances skin hydration

PubMed Central

Lee, Sein; Kim, Jong-Eun; Suk, Sujin; Kwon, Oh Wook; Park, Gaeun; Lim, Tae-gyu; Seo, Sang Gwon; Kim, Jong Rhan; Kim, Dae Eung; Lee, Miyeong; Chung, Dae Kyun; Jeon, Jong Eun; Cho, Dong Woon; Hurh, Byung Serk; Kim, Sun Yeou; Lee, Ki Won

2015-01-01

Skin hydration is one of the primary aims of beauty and anti-aging treatments. Barley (Hordeum vulgare) and soybean (Glycine max) are major food crops, but can also be used as ingredients for the maintenance of skin health. We developed a natural product-based skin treatment using a barley and soybean formula (BS) incorporating yeast fermentation, and evaluated its skin hydration effects as a dietary supplement in a clinical study. Participants ingested a placebo- (n = 33) or BS- (3 g/day) containing drink (n = 32) for 8 weeks. A significant increase in hydration in the BS group as compared to the placebo group was observed on the faces of subjects after 4 and 8 weeks, and on the forearm after 4 weeks. Decreases in stratum corneum (SC) thickness were also observed on the face and forearm. BS enhanced hyaluronan (HA) and skin barrier function in vitro and reduced Hyal2 expression in human dermal fibroblasts (HDF). BS also recovered ultraviolet (UV) B-induced downregulation of HA in HaCaT cells. These results suggest that BS has promising potential for development as a health functional food to enhance skin health. PMID:26388675

Unusual Glycosaminoglycans from a Deep Sea Hydrothermal Bacterium Improve Fibrillar Collagen Structuring and Fibroblast Activities in Engineered Connective Tissues

PubMed Central

Senni, Karim; Gueniche, Farida; Changotade, Sylvie; Septier, Dominique; Sinquin, Corinne; Ratiskol, Jacqueline; Lutomski, Didier; Godeau, Gaston; Guezennec, Jean; Colliec-Jouault, Sylvia

2013-01-01

Biopolymers produced by marine organisms can offer useful tools for regenerative medicine. Particularly, HE800 exopolysaccharide (HE800 EPS) secreted by a deep-sea hydrothermal bacterium displays an interesting glycosaminoglycan-like feature resembling hyaluronan. Previous studies demonstrated its effectiveness to enhance in vivo bone regeneration and to support osteoblastic cell metabolism in culture. Thus, in order to assess the usefulness of this high-molecular weight polymer in tissue engineering and tissue repair, in vitro reconstructed connective tissues containing HE800 EPS were performed. We showed that this polysaccharide promotes both collagen structuring and extracellular matrix settle by dermal fibroblasts. Furthermore, from the native HE800 EPS, a low-molecular weight sulfated derivative (HE800 DROS) displaying chemical analogy with heparan-sulfate, was designed. Thus, it was demonstrated that HE800 DROS mimics some properties of heparan-sulfate, such as promotion of fibroblast proliferation and inhibition of matrix metalloproteinase (MMP) secretion. Therefore, we suggest that the HE800EPS family can be considered as an innovative biotechnological source of glycosaminoglycan-like compounds useful to design biomaterials and drugs for tissue engineering and repair. PMID:23612369

Severe invasive streptococcal infection by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis.

PubMed

Watanabe, Shinya; Takemoto, Norihiko; Ogura, Kohei; Miyoshi-Akiyama, Tohru

2016-01-01

Streptococcus pyogenes, a group A Streptococcus (GAS), has been recognized as the causative pathogen in patients with severe invasive streptococcal infection with or without necrotizing fasciitis. In recent epidemiological studies, Streptococcus dysgalactiae subsp. equisimilis (SDSE) has been isolated from severe invasive streptococcal infection. Complete genome sequence showed that SDSE is the closest bacterial species to GAS, with approximately 70% of genome coverage. SDSE, however, lacks several key virulence factors present in GAS, such as SPE-B, the hyaluronan synthesis operon and active superantigen against human immune cells. A key event in the ability of GAS to cause severe invasive streptococcal infection was shown to be the acquisition of novel genetic traits such as phages. Strikingly, however, during severe invasive infection, GAS destroys its own covRS two-component system, which negatively regulates many virulence factor genes, resulting in a hyper-virulent phenotype. In contrast, this phenomenon has not been observed in SDSE. The present review describes the epidemiology of severe invasive streptococcal infection and the detailed pathogenic mechanisms of GAS and SDSE, emphasizing findings from their genome sequences and analyses of gene expression. © 2015 The Societies and John Wiley & Sons Australia, Ltd.

Dual-Functional Hydrazide-Reactive and Anhydride-Containing Oligomeric Hydrogel Building Blocks.

PubMed

Kascholke, Christian; Loth, Tina; Kohn-Polster, Caroline; Möller, Stephanie; Bellstedt, Peter; Schulz-Siegmund, Michaela; Schnabelrauch, Matthias; Hacker, Michael C

2017-03-13

Biomimetic hydrogels are advanced biomaterials that have been developed following different synthetic routes. Covalent postfabrication functionalization is a promising strategy to achieve efficient matrix modification decoupled of general material properties. To this end, dual-functional macromers were synthesized by free radical polymerization of maleic anhydride with diacetone acrylamide (N-(1,1-dimethyl-3-oxobutyl)acrylamide) and pentaerythritol diacrylate monostearate. Amphiphilic oligomers (M n < 7.5 kDa) with anhydride contents of 7-20% offered cross-linking reactivity to yield rigid hydrogels with gelatinous peptides (E = 4-13 kPa) and good cell adhesion properties. Mildly reactive methyl ketones as second functionality remained intact during hydrogel formation and potential of covalent matrix modification was shown using hydrazide and hydrazine model compounds. Successful secondary dihydrazide cross-linking was demonstrated by an increase of hydrogel stiffness (>40%). Efficient hydrazide/hydrazine immobilization depending on solution pH, hydrogel ketone content as well as ligand concentration for bioconjugation was shown and reversibility of hydrazone formation was indicated by physiologically relevant hydrazide release over 7 days. Proof-of-concept experiments with hydrazido-functionalized hyaluronan demonstrated potential for covalent aECM immobilization. The presented dual-functional macromers have perspective as reactive hydrogel building blocks for various biomedical applications.

Expression of N-acetyl-D-galactosamine associated epitope in synovium: a potential marker of glycoprotein production.

PubMed

El-Gabalawy, H; King, R; Bernstein, C; Ma, G; Mou, Y; Alguacil-Garcia, A; Fritzler, M; Wilkins, J

1997-07-01

To investigate synovial glycoprotein production in situ, a novel monoclonal antibody (Mab), A13D8, was used to evaluate the expression of an epitope containing N-acetyl-D-galactosamine (GalNAc) in normal and pathological synovium. Immunohistological and cytochemical analysis of synovial tissue samples was undertaken with single and double staining techniques using the A13D8 Mab, anti-CD68, vascular cell adhesion molecule-1 (VCAM-1), the hyaluronan associated enzyme uridine diphosphoglucose dehydrogenase (UDPGD), and the anti-Golgi Mab SSN/HR-1992. The specificity of the A13D8 Mab was established through blocking studies using carbohydrate residues, including GalNAc and N-acetylglucosamine (GlcNAc). A13D8 is expressed intensely in the cytoplasm of normal type B lining cells, which coexpress VCAM-1 and UDPGD, and is not expressed by CD68+ type A lining cells. In the lining layer of RA synovium, there is a negative correlation between A13D8 expression and the level of lymphocytic infiltration in the sublining areas (r = -0.43, p < 0.001). The endothelium of a subset of venules, typically in lymphocyte-rich aggregates, also stains intensely for A13D8. Pretreatment of the Mab with GalNAc completely eliminates the tissue staining, as well as the 110 kDa band seen on immunoblot, whereas pretreatment of A13D8 with GlcNAc and lactose has no effect. Double staining of HEp-2 cells with A13D8 and the anti-Golgi Mab SSN/HR-1992 reveals co-localization of the A13D8 epitope to the Golgi apparatus. Type B synovial lining cells and selected synovial endothelium express GalNAc containing epitope identified by Mab A13D8. Marked reduction in the expression of this epitope in the lining layer of inflamed RA synovium suggests that the synovial production of GalNAc containing glycoproteins, such as mucins, may be altered in this disorder.

Insights Into SMAD4 Loss in Pancreatic Cancer From Inducible Restoration of TGF-β Signaling

PubMed Central

Fullerton, Paul T.; Creighton, Chad J.

2015-01-01

Pancreatic ductal adenocarcinoma (PDAC) is the fourth-leading cause of cancer death in the United States. The TGF-β signaling protein SMAD family member 4 is lost in 60% of PDAC, and this has been associated with poorer prognosis. However, the mechanisms by which SMAD4 loss promotes PDAC development are not fully understood. We expressed SMAD4 in human PDAC cell lines BxPC3 and CFPAC1 by selection of stable clones containing an inducible SMAD4 tetracycline inducible expression system construct. After 24 hours of SMAD4 expression, TGF-β signaling-dependent G1 arrest was observed in BxPC3 cells with an increase in the G1 phase fraction from 48.9% to 71.5%. Inhibition of cyclin-dependent kinase inhibitor 1A by small interfering RNA eliminated the antiproliferative effect, indicating that up-regulation of cyclin-dependent kinase inhibitor 1A/p21 by TGF-β signaling is necessary for the phenotype. SMAD4 expression had no impact on invasion in BxPC3 cells, but reduced migration. Microarray analysis of gene expression at 8, 24, and 48 hours after SMAD4 expression characterized the regulatory impact of SMAD4 expression in a SMAD4-null PDAC cell line and identified novel targets of TGF-β signaling. Among the novel TGF-β targets identified are anthrax toxin receptor 2 (3.58× at 8 h), tubulin, β-3 class III (7.35× at 8 h), cell migration inducing protein, hyaluronan binding (8.07× at 8 h), IL-1 receptor-like 1 (0.403× at 8 h), regulator of G protein signaling 4 (0.293× at 8 h), and THAP domain containing 11 (0.262× at 8 h). The gene expression changes we observed upon restoration of TGF-β signaling provide numerous new targets for future investigations into PDAC biology and progression. PMID:26284758

Anti-aging effects of Piper cambodianum P. Fourn. extract on normal human dermal fibroblast cells and a wound-healing model in mice.

PubMed

Lee, Hyunji; Hong, Youngeun; Kwon, So Hee; Park, Jongsun; Park, Jisoo

2016-01-01

Aging of skin is associated with environmental factors such as ultraviolet rays, air pollution, gravity, and genetic factors, all of which can lead to wrinkling of skin. Previous reports suggest that the wound repair is impaired by the aging process and strategies to manipulate the age-related wound healing are necessary in order to stimulate repair. Several traditional plant extracts are well-known for their properties of skin protection and care. Piper cambodianum P. Fourn. (PPF), a member of Piperacecae, is a plant found in Vietnam that might have therapeutic properties. Therefore, the effects of PPF stem and leaf extract on aging process were investigated in vitro and in vivo. PPF extract dissolved in methanol was investigated using Western blotting, real-time quantitative reverse transcription-polymerase chain reaction, flow cytometry, and cell wound-healing assays. We assessed the anti-aging effect of PPF in mouse using the wound-healing assay. The results were analyzed by Student's unpaired t-test; *P<0.05 and **P<0.01 were considered to indicate significant and highly significant values, respectively, compared with corresponding controls. PPF treatment demonstrated in vitro and in vivo anti-aging activity. Western blot analysis of PPF-treated normal human dermal fibroblast cells showed a dose-dependent increase in the expression of extracellular matrix genes such as collagen and elastin, but decreased expression of the aging gene matrix metalloproteinase-3. Quantitative polymerase chain reaction showed that PPF-treated cells displayed dose-dependent increase in messenger RNA expression levels of collagen, elastin, and hyaluronan synthase-2 and decreased expression levels of matrix metalloproteinase-1 aging gene. PPF treatment led to decreased production of reactive oxygen species in cells subjected to ultraviolet irradiation. Furthermore, PPF extract showed positive wound-healing effects in mice. This study demonstrated the anti-aging and wound-healing effects of PPF extract. Therefore, PPF extract represents a promising new therapeutic agent for anti-aging and wound-healing treatments.

The Pathophysiology of Thyroid Eye Disease (TED): Implications for Immunotherapy

PubMed Central

Gupta, Shivani; Douglas, Raymond

2012-01-01

Purpose of Review Thyroid eye disease (TED) is a poorly understood autoimmune manifestation most commonly associated with Graves’ disease. Current nonspecific treatment paradigms offer symptomatic improvement but fail to target the underlying pathogenic mechanisms and thus, do not significantly alter the long-term disease outcome. The purpose of this review is to provide an update of the current understanding of the immunopathogenesis of TED and explore these implications for targeted immunotherapy. Recent Findings Orbital fibroblasts are integral to the pathogenesis of TED and may modulate immune responses by production of cytokines and hyaluronan in response to activation of shared autoantigens including thyrotropin receptor (TSHR) and insulin-like growth factor-1 receptor (IGF-R1). Fibrocytes share many of these phenotypic and functional features, suggesting a link between systemic and site-specific disease. Use of targeted immunotherapies in TED is limited, though data from the use Rituximab (RTX), a B cell depleting agent, are encouraging. Sustained clinical response has been seen with RTX in several reports, despite return of peripheral B cell levels to pretreatment levels. Additionally, this response appears to be independent to cytokine and antibody production, suggesting possible modulation of antigen presentation as a mechanism of its effect. Summary Progressive advances in the understanding of the immunopathogenesis of TED continue to spur clinical trials utilizing targeted immune therapies. Continued understanding of the molecular mechanisms of disease will expand potential treatments for TED patients and obviate the need for reconstructive surgical therapies. PMID:21730841

Repair of retropatellar cartilage defects in the knee with microfracture and a cell-free polymer-based implant.

PubMed

Becher, Christoph; Ettinger, Max; Ezechieli, Marco; Kaps, Christian; Ewig, Marc; Smith, Tomas

2015-07-01

To analyze magnetic resonance imaging (MRI) at 3T and the clinical outcome in a short-term pilot study after treatment of retropatellar cartilage defects with microfracturing and subsequent covering with the cell-free chondrotissue(®) polyglycolic acid-hyaluronan implant. Five consecutive patients after microfracturing and defect coverage with the chondrotissue(®) implant immersed with autologous serum were included. After a mean follow-up of 21 months (range 11-31 months), defect fill and repair tissue quality was assessed by 3-T MRI followed by applying established MRI scoring systems. The patients' situation was assessed using the Knee injury and Osteoarthritis Outcome Score (KOOS) and a patients' satisfaction questionnaire. Magnetic resonance imaging showed good to excellent defect fill with complete integration. The mean MOCART score was 61 (range 50-75) points. The mean Henderson score was 7 (range 6-9) points. All patients showed subchondral bone alterations. The KOOS showed good values in all sub-categories in 4 out of 5 patients and a mean overall score of 73 (range 40-90) points. Two patients rated the outcome as excellent, two as good and one as fair. All patients would have the procedure again and recommend it. In this small case series, the coverage of symptomatic retropatellar cartilage defects with the chondrotissue(®) implant after microfracturing was safe and feasible with improvement of the patients' situation at short-term follow-up. IV, case series.

Effect of an alpha-blocker (Nicergoline) and of a beta-blocker (Acebutolol) on the in vitro biosynthesis of vascular extracellular matrix.

PubMed

Moczar, M; Robert, A M; Jacotot, B; Robert, L

2001-05-01

The effect of an alpha-blocking agent and of a beta-blocking agent on the biosynthesis of extracellular matrix macromolecules of the arterial wall was investigated. Rabbit aorta explants were cultured up to 48 hours with radioactive proline, lysine or glucosamine. In presence of these drugs, at concentration shown to be effective for the inhibition of platelet-endothelial cell interactions (10(-7) M), the incorporation of 14C proline in total macromolecular proline was higher than in macromolecular hydroxyproline suggesting a relatively higher rate of biosynthesis of non-collagenous proteins as compared to collagens. The alpha-blocking increased the incorporation of 14C proline in collagenous and non-collagenous proteins after 18 hours of incubation. beta-blocking also increased the incorporation of proline in macromolecular proline and hydroxyproline as compared to control cultures. Both increased the incorporation of 3H glucosamine in newly synthesised glycosaminoglycans. beta-blocking increased mainly the neosynthesis of heparan sulphate, alpha-blocking that of hyaluronan. The incorporation of 14C-lysine in crosslinked, insoluble elastin was not modified. These experiments confirm that alpha and beta-blocking agents can influence not only the tonus of aortic smooth muscle cells but also the relative rates of biosynthesis of extracellular matrix macromolecules. This effect should be taken in consideration for the evaluation of the long range effect of alpha and beta-blocking drugs on the vascular wall.

Sericin Accelerates the Production of Hyaluronan and Decreases the Incidence of Polyspermy Fertilization in Bovine Oocytes During In Vitro Maturation

PubMed Central

HOSOE, Misa; YOSHIDA, Nao; HASHIYADA, Yutaka; TERAMOTO, Hidetoshi; TAKAHASHI, Toru; NIIMURA, Sueo

2014-01-01

Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%, 0.1% or 0.15% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine oocytes. PMID:24748396

Sericin accelerates the production of hyaluronan and decreases the incidence of polyspermy fertilization in bovine oocytes during in vitro maturation.

PubMed

Hosoe, Misa; Yoshida, Nao; Hashiyada, Yutaka; Teramoto, Hidetoshi; Takahashi, Toru; Niimura, Sueo

2014-01-01

Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%, 0.1% or 0.15% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine oocytes.

In vitro effects of platelet-rich gel supernatants on histology and chondrocyte apoptosis scores, hyaluronan release and gene expression of equine cartilage explants challenged with lipopolysaccharide.

PubMed

Carmona, Jorge U; Ríos, Diana L; López, Catalina; Álvarez, María E; Pérez, Jorge E; Bohórquez, Mabel E

2016-07-01

Platelet-rich plasma (PRP) preparations are a common treatment in equine osteoarthritis (OA). However, there are controversies regarding the ideal concentration of platelets and leukocytes in these biological substances necessary to induce an adequate anti-inflammatory and anabolic response in articular cartilage. The aims were to study the influence of leukocyte- and platelet-rich gel (L-PRG) and pure platelet-rich gel (P-PRG) supernatants on the histological changes of cartilage, the degree of chondrocyte apoptosis, the production of hyaluronan (HA) and the gene expression of nuclear factor kappa beta (NFkβ), matrix metalloproteinase 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), collagen type I alpha 1 (COL1A1), collagen type II alpha 1 (COL2A1) and cartilage oligomeric matrix protein (COMP) in normal cartilage explants (CEs) challenged with lipopolysaccharide (LPS). Overall, 25 % L-PRG supernatant (followed in order of importance by, 50 % P-PRG, 25 % P-PRG and 50 % L-PRG) represented the substance with the most important anti-inflammatory and anabolic effect. 25 % P-PRG supernatant presented important anabolic effects, but it induced a more severe chondrocyte apoptosis than the other evaluated substances. 25 % L-PRG supernatant presented the best therapeutic profile. Our results demonstrate that the biological variability of PRP preparations makes their application rather challenging. Additional in vivo research is necessary to know the effect of PRP preparations at different concentrations.

Cyclic movement stimulates hyaluronan secretion into the synovial cavity of rabbit joints

PubMed Central

Ingram, K R; Wann, A K T; Angel, C K; Coleman, P J; Levick, J R

2008-01-01

The novel hypothesis that the secretion of the joint lubricant hyaluronan (HA) is coupled to movement has implications for normal function and osteoarthritis, and was tested in the knee joints of anaesthetized rabbits. After washing out the endogenous synovial fluid HA (miscibility coefficient 0.4), secretion into the joint cavity was measured over 5 h in static joints and in passively cycled joints. The net static secretion rate (11.2 ± 0.7 μg h−1, mean ± s.e.m., n = 90) correlated with the variable endogenous HA mass (mean 367 ± 8 μg), with a normalized value of 3.4 ± 0.2 μg h−1 (100 μg)−1 . Cyclic joint movement approximately doubled the net HA secretion rate to 22.6 ± 1.2 μg h−1 (n = 77) and raised the normalized percentage to 5.9 ± 0.3 μg h−1 (100 μg)−1. Secretion was inhibited by 2-deoxyglucose and iodoacetate, confirming active secretion. The net accumulation rate underestimated true secretion rate due to some trans-synovial loss. HA turnover time (endogenous mass/secretion rate) was 17–30 h (static) to 8–15 h (moved) The results demonstrate for the first time that the active secretion of HA is coupled to joint usage. Movement–secretion coupling may protect joints against the damaging effects of repetitive joint use, replace HA lost during periods of immobility (overnight), and contribute to the clinical benefit of exercise therapy in moderate osteoarthritis. PMID:18202097

Elastic fiber-mediated enthesis in the human middle ear.

PubMed

Kawase, Tetsuaki; Shibata, Shunichi; Katori, Yukio; Ohtsuka, Aiji; Murakami, Gen; Fujimiya, Mineko

2012-10-01

Adaptation to constant vibration (acoustic oscillation) is likely to confer a specific morphology at the bone-tendon and bone-ligament interfaces at the ear ossicles, which therefore represent an exciting target of enthesis research. We histologically examined (i) the bone attachments of the tensor tympani and stapedius muscles and (ii) the annular ligament of the incudostapedial joint obtained from seven elderly donated cadavers. Notably, both aldehyde-fuchsin and elastic-Masson staining demonstrated that the major fibrous component of the entheses was not collagen fibers but mature elastic fibers. The positive controls for elastic fiber staining were the arterial wall elastic laminae included in the temporal bone materials. The elastic fibers were inserted deeply into the type II collagen-poor fibrocartilage covering the ear ossicles. The muscle tendons were composed of an outer thin layer of collagen fibers and an inner thick core of elastic fibers near the malleus or stapes. In the unique elastic fiber-mediated entheses, hyaluronan, versican and fibronectin were expressed strongly along the elastic fibers. The hyaluronan seemed to act as a friction-reducing lubricant for the elastic fibers. Aggrecan was labeled strongly in a disk- or plica-like fibrous mass on the inner side of the elastic fiber-rich ligament, possibly due to compression stress from the ligament. Tenascin-c was not evident in the entheses. The elastic fiber-mediated entheses appeared resistant to tissue destruction in an environment exposed to constant vibration. The morphology was unlikely to be the result of age-related degeneration. © 2012 The Authors Journal of Anatomy © 2012 Anatomical Society.

Viscoelasticity of rabbit vocal folds after injection augmentation.

PubMed

Dahlqvist, Ake; Gärskog, Ola; Laurent, Claude; Hertegård, Stellan; Ambrosio, Luigi; Borzacchiello, Assunta

2004-01-01

Vocal fold function is related to the viscoelasticity of the vocal fold tissue. Augmentation substances used for injection treatment of voice insufficiency may alter the viscoelastic properties of vocal folds and their vibratory capacity. The objective was to compare the mechanical properties (viscoelasticity) of various injectable substances and the viscoelasticity of rabbit vocal folds, 6 months after injection with one of these substances. Animal model. Cross-linked collagen (Zyplast), double cross-linked hyaluronan (hylan B gel), dextranomers in hyaluronan (DHIA), and polytetrafluoroethylene (Teflon) were injected into rabbit vocal folds. Six months after the injection, the animals were killed and the right- and left-side vocal folds were removed. Dynamic viscosity of the injected substances and the vocal folds was measured with a Bohlin parallel-plate rheometer during small-amplitude oscillation. All injected vocal folds showed a decreasing dynamic viscosity with increasing frequency. Hylan B gel and DiHA showed the lowest dynamic viscosity values, and vocal folds injected with these substances also showed the lowest dynamic viscosity (similar to noninjected control samples). Teflon (and vocal folds injected with Teflon) showed the highest dynamic viscosity values, followed by the collagen samples. Substances with low viscoelasticity alter the mechanical properties of the vocal fold to a lesser degree than substances with a high viscoelasticity. The data indicated that hylan B gel and DiHA render the most natural viscoelastic properties to the vocal folds. These substances seem to be appropriate for preserving or restoring the vibratory capacity of the vocal folds when glottal insufficiency is treated with augmentative injections.

pH-Amplified multilayer films based on hyaluronan: influence of HA molecular weight and concentration on film growth and stability.

PubMed

Shen, Liyan; Chaudouet, Patrick; Ji, Jian; Picart, Catherine

2011-04-11

In this study, we investigate the growth and internal properties of polyelectrolyte multilayer films made of poly(l-lysine) and hyaluronan (PLL/HA) under pH-amplified conditions, that is, by alternate deposition of PLL at high pH and HA at low pH. We focus especially on the influence of the molecular weight of HA in this process as well as on its concentration in solution. Film growth was followed by quartz crystal microbalance and by infrared spectroscopy to quantify the deposited mass and to characterize the internal properties of the films, including the presence of hydrogen bonds and the ionization degree of HA in the films. Film growth was significantly faster for HA of high molecular weight (1300 kDa) as compared with 400 and 200 kDa. PLL was found to exhibit a random structure once deposited in the films. Furthermore, we found that PLL-ending films are more stable when they are placed in PBS than their HA counterparts. This was explained on the basis of more cohesive interactions in the films for PLL-ending films. Finally, we quantified PLL(FITC) diffusion into the films and observed that PLL diffusion is enhanced when PLL is paired with the HA of high MW. All together, these results suggest that besides purely physicochemical parameters such as variation in pH, the molecular weight of HA, its concentration in solution, and the possibility to form intermolecular HA association play important roles in film growth, internal cohesion, and stability.

Modifications in Bone Matrix of Estrogen-Deficient Rats Treated with Intermittent PTH

PubMed Central

Campos, Jenifer Freitas; Katchburian, Eduardo; de Medeiros, Valquíria Pereira; Nader, Helena Bonciani; Nonaka, Keico Okino; Plotkin, Lilian Irene; Reginato, Rejane Daniele

2015-01-01

Bone matrix dictates strength, elasticity, and stiffness to the bone. Intermittent parathyroid hormone (iPTH), a bone-forming treatment, is widely used as a therapy for osteoporosis. We investigate whether low doses of intermittent PTH (1-34) change the profile of organic components in the bone matrix after 30 days of treatment. Forty 6-month-old female Wistar rats underwent ovariectomy and after 3 months received low doses of iPTH administered for 30 days: daily at 0.3 µg/kg/day (PTH03) or 5 µg/kg/day (PTH5); or 3 times per week at 0.25 µg/kg/day (PTH025). After euthanasia, distal femora were processed for bone histomorphometry, histochemistry for collagen and glycosaminoglycans, biochemical quantification of sulfated glycosaminoglycans, and hyaluronan by ELISA and TUNEL staining. Whole tibiae were used to estimate the bone mineral density (BMD). Histomorphometric analysis showed that PTH5 increased cancellous bone volume by 6% over vehicle-treated rats. In addition, PTH5 and PTH03 increased cortical thickness by 21% and 20%, respectively. Tibial BMD increased in PTH5-treated rats and this group exhibited lower levels of chondroitin sulfate; on the other hand, hyaluronan expression was increased. Hormonal administration in the PTH5 group led to decreased collagen maturity. Further, TUNEL-positive osteocytes were decreased in the cortical compartment of PTH5 whereas administration of PTH025 increased the osteocyte death. Our findings suggest that daily injections of PTH at low doses alter the pattern of organic components from the bone matrix, favoring the increase of bone mass. PMID:25695082

Expression of tumour necrosis factor alpha and accumulation of fibronectin in coronary artery restenotic lesions retrieved by atherectomy.

PubMed Central

Clausell, N.; de Lima, V. C.; Molossi, S.; Liu, P.; Turley, E.; Gotlieb, A. I.; Adelman, A. G.; Rabinovitch, M.

1995-01-01

BACKGROUND--The formation of coronary artery neointima experimentally induced in piglets after cardiac transplantation is related to an immune-inflammatory reaction associated with increased expression of T cells and inflammatory mediators (tumour necrosis factor alpha and interleukin 1 beta) and upregulation of fibronectin. In vivo blockade of tumour necrosis factor alpha in rabbits after cardiac transplantation results in reduced neointimal formation. The objective of this study was to investigate the hypothesis that coronary restenosis after atherectomy or percutaneous balloon angioplasty is associated with a similar inflammatory cascade initiated by mechanical injury. METHODS--Specimens taken at coronary atherectomy were analysed from 16 patients. Nine had had the procedure performed twice, firstly, to remove a primary lesion, and secondly, to remove a restenotic lesion. Seven had percutaneous balloon angioplasty after removal of restenotic tissue. Coronary atherectomy specimens were analysed by immunohistochemistry for the presence of T cells, macrophages, major histocompatibility complex II, interleukin 1 beta, tumour necrosis factor alpha, fibronectin, and the receptor for hyaluronan mediated motility. RESULTS--The groups were clinically and angiographically similar with equivalent lumens before and after atherectomy. Restenotic lesions had increased expression of tumour necrosis factor alpha and fibronectin compared with the primary lesions (P < 0.05 for both). There was also a trend towards a greater number of T cells and increased expression of interleukin 1 beta. CONCLUSIONS--Restenosis is associated with increased expression of tumour necrosis factor alpha and fibronectin, suggesting that an immune-inflammatory reaction probably contributes to neointimal formation and may represent a form of wound healing and repair secondary to mechanical injury. Images PMID:7626352

Proteomics-based network analysis characterizes biological processes and pathways activated by preconditioned mesenchymal stem cells in cardiac repair mechanisms.

PubMed

Di Silvestre, Dario; Brambilla, Francesca; Scardoni, Giovanni; Brunetti, Pietro; Motta, Sara; Matteucci, Marco; Laudanna, Carlo; Recchia, Fabio A; Lionetti, Vincenzo; Mauri, Pierluigi

2017-05-01

We have demonstrated that intramyocardial delivery of human mesenchymal stem cells preconditioned with a hyaluronan mixed ester of butyric and retinoic acid (MSCp + ) is more effective in preventing the decay of regional myocardial contractility in a swine model of myocardial infarction (MI). However, the understanding of the role of MSCp + in proteomic remodeling of cardiac infarcted tissue is not complete. We therefore sought to perform a comprehensive analysis of the proteome of infarct remote (RZ) and border zone (BZ) of pigs treated with MSCp + or unconditioned stem cells. Heart tissues were analyzed by MudPIT and differentially expressed proteins were selected by a label-free approach based on spectral counting. Protein profiles were evaluated by using PPI networks and their topological analysis. The proteomic remodeling was largely prevented in MSCp + group. Extracellular proteins involved in fibrosis were down-regulated, while energetic pathways were globally up-regulated. Cardioprotectant pathways involved in the production of keto acid metabolites were also activated. Additionally, we found that new hub proteins support the cardioprotective phenotype characterizing the left ventricular BZ treated with MSCp + . In fact, the up-regulation of angiogenic proteins NCL and RAC1 can be explained by the increase of capillary density induced by MSCp + . Our results show that angiogenic pathways appear to be uniquely positioned to integrate signaling with energetic pathways involving cardiac repair. Our findings prompt the use of proteomics-based network analysis to optimize new approaches preventing the post-ischemic proteomic remodeling that may underlie the limited self-repair ability of adult heart. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluating The Effect of Melatonin on HAS2, and PGR expression, as well as Cumulus Expansion, and Fertility Potential in Mice.

PubMed

Ezzati, Maryam; Roshangar, Leila; Soleimani Rad, Jafar; Karimian, Nahid

2018-04-01

Infertility is a worldwide health problem which affects approximately 15% of sexually active couples. One of the factors influencing the fertility is melatonin. Also, protection of oocytes and embryos from oxidative stress inducing chemicals in the culture medium is important. The aim of the present study was to investigate if melatonin could regulate hyaluronan synthase-2 (HAS2) and Progesterone receptor (PGR) expressions in the cumulus cells of mice oocytes and provide an in vitro fertilization (IVF) approach. In this experimental study, for this purpose, 30 adult female mice and 15 adult male mice were used. The female mice were superovulated using 10 U of pregnant mare serum gonadotropin (PMSG) and 24 hours later, 10 U of human chorionic gonadotropin (hCG) were injected. Next, cumulus oocyte complexes (COCs) were collected from the oviducts of the female mice by using a matrix-flushing method. The cumulus cells were cultured with melatonin 10 μM for 6 hours and for real-time reverse transcription-polymerase chain reaction (RT-PCR) was used for evaluation of HAS2 and PGR expression levels. The fertilization rate was evaluated through IVF. All the data were analyzed using a t test. The results of this study showed that HAS2 and PGR expressions in the cumulus cells of the mice receiving melatonin increased in comparison to the control groups. Also, IVF results revealed an enhancement in fertilization rate in the experimental groups compared to the control groups. To improve the oocyte quality and provide new approaches for infertility treatment, administration of melatonin as an antioxidant, showed promising results. Thus, it is concluded that fertility outcomes can be improved by melatonin it enhances PGR. Copyright© by Royan Institute. All rights reserved.

Doxycycline Inhibits Inflammation-Induced Lymphangiogenesis in Mouse Cornea by Multiple Mechanisms

PubMed Central

Huang, Jingwen; Zhou, Jingwen; Qiu, Sujuan; Liang, Dan

2014-01-01

Lymphangiogenesis is significantly involved in the pathogenesis of diseases, including graft rejection, cancer metastasis and various inflammatory conditions. The inhibition of lymphangiogenesis has become a new therapeutic target for the treatment of these diseases. Here, we explored the anti-lymphangiogenic effects of doxycycline in inflammation-induced lymphangiogenesis (ILA) in the cornea and the underlying mechanisms. In the present study, mice with ILA of the cornea were treated with topical doxycycline (0.1%) or vehicle control. Lymphangiogenesis was quantified using corneal immunostaining of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). Human dermal lymphatic endothelial cells (HDLECs) and a murine macrophage cell line (RAW264.7) were used to further explore the underlying mechanisms of doxycycline-mediated anti-lymphangiogenesis in vitro. Our results showed that doxycycline treatment dramatically inhibited ILA in the mouse cornea (p<0.001), with a significant decrease in vascular endothelial growth factor (VEGF)-C/VEGF receptor 3 signalling, macrophage infiltration and inflammatory cytokine expression. Doxycycline also significantly inhibited VEGF-C-induced HDLEC proliferation in vitro by modulating the PI3K/Akt/endothelial nitric oxide (NO) synthase (eNOS) pathway and significantly suppressed interleukin-1β (IL-1β), TNF-α and VEGF-C production in the RAW264.7 cell line by modulating the PI3K/Akt/nuclear factor-kappaB (NF-κB) pathway. Additionally, doxycycline treatment dramatically reduced the phosphorylation of NF-κBp65, Akt and eNOS in ILA and significantly inhibited matrix metalloproteinases (MMPs) activity in vitro and in ILA. In conclusion, doxycycline inhibited ILA, possibly through suppression of VEGF-C signalling, macrophage function and MMPs activity. This observation suggests that doxycycline is a potential therapeutic agent for lymphangiogenesis-related diseases. PMID:25268699

Evaluation of the UVB-screening capacity and restorative effects exerted by farnesol gel on UVB-caused sunburn.

PubMed

Wu, Guan Xuan; Huang, Han Hsiang; Chang, Huoy Rou; Kuo, Shyh Ming

2018-04-01

Farnesol, a natural 15-carbon organic compound, has various microbiological and cellular activities. It has been found to exert apoptosis-inducing effects against carcinoma cells as well as antiallergic and anti-inflammatory effects in vivo. In the current study, a series of formulations composed of various concentrations of hydroxypropyl methylcellulose (HPMC) with the addition of hyaluronan (HA) and xanthan gum (XG) was designed to evaluate the UVB-screening and H 2 O 2 -eliminating effects of farnesol in normal fibroblasts. Farnesol at 0.005, 0.0075, and 0.01% exhibited significant capacity for H 2 O 2 scavenging; at 0.0025%, it showed insignificant effects. Under 120-min UVB exposure, screening with plural gel composed of 0.0025% farnesol, 0.5% HA, and 0.5% XG containing 1.5% or 2% HPMC retained normal fibroblast viability. After 60-min exposure to UVB, screening with plural gel composed of farnesol, HA, XG, and 0.5%, 1.0%, 1.5%, or 2% HPMC decreased the ratio of the G1 phase and increased ratio of the S phase in comparison with the accumulated cell cycle of the normal fibroblasts without screening. The gel with 2% HPMC displayed the strongest cell cycle-reversal ability. In vivo histopathological results showed that the prepared plural gels with 0.5% or 2% HPMC and farnesol, HA, and XG had greater antiphotoaging and reparative effects against UVB-induced changes and damage in the skin. In conclusion, the current in vitro and in vivo results demonstrated that the prepared plural composed of 0.0025% farnesol, 0.5% HA, 0.5% XG, and 2% HPMC possessed the greatest UVB-screening capacity and the strongest restorative effects on UVB-induced sunburned skin. © 2018 Wiley Periodicals, Inc.

Modulation of Bleomycin-Induced Lung Fibrosis by Pegylated Hyaluronidase and Dopamine Receptor Antagonist in Mice

PubMed Central

Pershina, Olga Victorovna; Reztsova, Alena Mikhaylovna; Ermakova, Natalia Nikolaevna; Khmelevskaya, Ekaterina Sergeevna; Krupin, Vycheslav Andreevich; Stepanova, Inna Ernestovna; Artamonov, Andrew Vladimirovich; Bekarev, Andrew Alexandrovich; Madonov, Pavel Gennadjevich

2015-01-01

Hyaluronidases are groups of enzymes that degrade hyaluronic acid (HA). To stop enzymatic hydrolysis we modified testicular hyaluronidase (HYAL) by activated polyethylene oxide with the help of electron-beam synthesis. As a result we received pegylated hyaluronidase (pegHYAL). Spiperone is a selective D2 dopamine receptor antagonist. It was demonstrated on the model of a single bleomycin damage of alveolar epithelium that during the inflammatory phase monotherapy by pegHYAL or spiperone reduced the populations of hematopoietic stem /progenitor cells in the lung parenchyma. PegHYAL also reduced the levels of transforming growth factor (TGF)-β, interleukin (IL)-1β, tumor necrosis factor (TNF)-α in the serum and lungs, while spiperone reduced the level of the serum IL-1β. Polytherapy by spiperone and pegHYAL caused the increase of the quantity of hematopoietic stem/ progenitor cells in the lungs. Such an influx of blood cell precursors was observed on the background of considerable fall level of TGF-β and the increase level of TNF-α in the serum and lungs. These results show pegHYAL reduced the bleomycin-induced fibrosis reaction (production and accumulation of collagen) in the lung parenchyma. This effect was observed at a single and repetitive bleomycin damage of alveolar epithelium, the antifibrotic activity of pegHYAL surpassing the activity of testicular HYAL. The antifibrotic effect of pegHYAL is enhanced by an additional instillation of spiperone. Therapy by pegHYAL causes the flow of CD31‒CD34‒CD45‒CD44+CD73+CD90+CD106+-cells into the fibrous lungs. These cells are incapable of differentiating into fibroblast cells. Spiperone instillation separately or together with pegHYAL reduced the MSC-like cells considerably. These data enable us to assume, that pegHYAL is a new and promising instrument both for preventive and therapy of toxic pneumofibrosis. The blockage of D2 dopamine receptors with the following change of hyaluronan matrix can be considered as a new strategy in treatment of pneumofibrosis. PMID:25927611

Polysaccharide-Modified Synthetic Polymeric Biomaterials

PubMed Central

Baldwin, Aaron D.; Kiick, Kristi L.

2010-01-01

This review presents an overview of polysaccharide-conjugated synthetic polymers and their use in tissue-engineered scaffolds and drug-delivery applications. This topic will be divided into four categories: (1) polymeric materials modified with non-mammalian polysaccharides such as alginate, chitin, and dextran; (2) polymers modified with mammalian polysaccharides such as hyaluronan, chondroitin sulfate, and heparin; (3) multi-polysaccharide-derivatized polymer conjugate systems; and (4) polymers containing polysaccharide-mimetic molecules. Each section will discuss relevant conjugation techniques, analysis, and the impact of these materials as micelles, particles, or hydrogels used in in-vitro and in-vivo biomaterial applications. PMID:20091875

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 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. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr02787b

Comparison of Single Intra-Articular Injection of Novel Hyaluronan (HYA-JOINT Plus) with Synvisc-One for Knee Osteoarthritis: A Randomized, Controlled, Double-Blind Trial of Efficacy and Safety.

PubMed

Sun, Shu-Fen; Hsu, Chien-Wei; Lin, Huey-Shyan; Liou, I-Hsiu; Chen, Yin-Han; Hung, Chia-Ling

2017-03-15

Viscosupplementation has been widely used for the treatment of knee osteoarthritis. Because we found no well-controlled trial comparing single-injection regimens of hyaluronan for knee osteoarthritis, we compared the efficacy and safety of a single intra-articular injection of a novel cross-linked hyaluronan (HYA-JOINT Plus) with a single injection of Synvisc-One in patients with knee osteoarthritis. In a prospective, randomized, controlled, double-blind trial with a 6-month follow-up, 132 patients with knee osteoarthritis (Kellgren-Lawrence grade 2 or 3) were randomized to receive 1 intra-articular injection of 3 mL of HYA-JOINT Plus (20 mg/mL) (n = 66) or 6 mL of Synvisc-One (8 mg/mL) (n = 66). The primary outcome was the change from baseline in the visual analog scale (VAS) (0 to 100 mm) pain score at 6 months. Secondary outcome measures included the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC, Likert scale), Lequesne index, timed "Up & Go" (TUG) test, single-limb stance (SLS) test, use of rescue analgesics, and patient satisfaction. A total of 121 patients were available for the intention-to-treat analysis at 6 months. Both groups had a significant improvement in the VAS, WOMAC, and Lequesne index scores at each follow-up visit (p < 0.001). Patients who received HYA-JOINT Plus experienced a significantly greater improvement in the VAS pain score at 1, 3, and 6 months compared with those treated with Synvisc-One (adjusted mean difference: -12.0, -8.5, and -6.6; p = 0.001, 0.033, and 0.045, respectively). There were no significant between-group differences in any of the secondary outcomes except the WOMAC stiffness scores at 6 months, which favored HYA-JOINT Plus treatment (p = 0.043). The TUG time did not change significantly in either group during the study (p > 0.05), but the SLS time improved significantly in both the HYA-JOINT Plus and the Synvisc-One group (p = 0.004 and p = 0.022, respectively). No significant between-group differences were observed with respect to patient satisfaction or consumption of analgesics. No serious adverse events occurred following the injections. A single injection of either HYA-JOINT Plus or Synvisc-One is safe and effective for 6 months in patients with knee osteoarthritis. HYA-JOINT Plus is superior to Synvisc-One in terms of reducing the VAS pain score at 1, 3, and 6 months and the WOMAC stiffness score at 6 months, with similar safety. Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

Integrated genomic and immunophenotypic classification of pancreatic cancer reveals three distinct subtypes with prognostic/predictive significance.

PubMed

Wartenberg, Martin; Cibin, Silvia; Zlobec, Inti; Vassella, Erik; Eppenberger-Castori, Serenella M M; Terracciano, Luigi; Eichmann, Micha; Worni, Mathias; Gloor, Beat; Perren, Aurel; Karamitopoulou, Eva

2018-04-16

Current clinical classification of pancreatic ductal adenocarcinoma (PDAC) is unable to predict prognosis or response to chemo- or immunotherapy and does not take into account the host reaction to PDAC-cells. Our aim is to classify PDAC according to host- and tumor-related factors into clinically/biologically relevant subtypes by integrating molecular and microenvironmental findings. A well-characterized PDAC-cohort (n=110) underwent next-generation sequencing with a hotspot cancer panel, while Next-generation Tissue-Microarrays were immunostained for CD3, CD4, CD8, CD20, PD-L1, p63, hyaluronan-mediated motility receptor (RHAMM) and DNA mismatch-repair proteins. Previous data on FOXP3 were integrated. Immune-cell counts and protein expression were correlated with tumor-derived driver mutations, clinicopathologic features (TNM 8. 2017), survival and epithelial-mesenchymal-transition (EMT)-like tumor budding.  Results: Three PDAC-subtypes were identified: the "immune-escape" (54%), poor in T- and B-cells and enriched in FOXP3+Tregs, with high-grade budding, frequent CDKN2A- , SMAD4- and PIK3CA-mutations and poor outcome; the "immune-rich" (35%), rich in T- and B-cells and poorer in FOXP3+Tregs, with infrequent budding, lower CDKN2A- and PIK3CA-mutation rate and better outcome and a subpopulation with tertiary lymphoid tissue (TLT), mutations in DNA damage response genes (STK11, ATM) and the best outcome; and the "immune-exhausted" (11%) with immunogenic microenvironment and two subpopulations: one with PD-L1-expression and high PIK3CA-mutation rate and a microsatellite-unstable subpopulation with high prevalence of JAK3-mutations. The combination of low budding, low stromal FOXP3-counts, presence of TLTs and absence of CDKN2A-mutations confers significant survival advantage in PDAC-patients. Immune host responses correlate with tumor characteristics leading to morphologically recognizable PDAC-subtypes with prognostic/predictive significance. Copyright ©2018, American Association for Cancer Research.

Stimulation of the Superficial Zone Protein and Lubrication in the Articular Cartilage by Human Platelet-Rich Plasma

PubMed Central

Sakata, Ryosuke; McNary, Sean M.; Miyatake, Kazumasa; Lee, Cassandra A.; Van den Bogaerde, James M.; Marder, Richard A.; Reddi, A. Hari

2016-01-01

Background Platelet-rich plasma (PRP) contains high concentrations of autologous growth factors that originate from platelets. Intra-articular injections of PRP have the potential to ameliorate the symptoms of osteoarthritis in the knee. Superficial zone protein (SZP) is a boundary lubricant in articular cartilage and plays an important role in reducing friction and wear and therefore is critical in cartilage homeostasis. Purpose To determine if PRP influences the production of SZP from human joint-derived cells and to evaluate the lubricating properties of PRP on normal bovine articular cartilage. Study Design Controlled laboratory study. Methods Cells were isolated from articular cartilage, synovium, and the anterior cruciate ligament (ACL) from 12 patients undergoing ACL reconstruction. The concentrations of SZP in PRP and culture media were measured by enzyme-linked immunosorbent assay. Cellular proliferation was quantified by determination of cell numbers. The lubrication properties of PRP from healthy volunteers on bovine articular cartilage were investigated using a pin-on-disk tribometer. Results In general, PRP stimulated proliferation in cells derived from articular cartilage, synovium, and ACL. It also significantly enhanced SZP secretion from synovium- and cartilage-derived cells. An unexpected finding was the presence of SZP in PRP (2.89 ± 1.23 µg/mL before activation and 3.02 ± 1.32 µg/mL after activation). In addition, under boundary mode conditions consisting of high loads and low sliding speeds, nonactivated and thrombin-activated PRP decreased the friction coefficient (μ = 0.012 and μ = 0.015, respectively) compared with saline (μ = 0.047, P < 0.004) and high molecular weight hyaluronan (μ = 0.080, P < 0.006). The friction coefficient of the cartilage with PRP was on par with that of synovial fluid. Conclusion PRP significantly stimulates cell proliferation and SZP secretion by articular cartilage and synovium of the human knee joint. Furthermore, PRP contains endogenous SZP and, in a functional bioassay, lubricates bovine articular cartilage explants. Clinical Relevance These findings provide evidence to explain the biochemical and biomechanical mechanisms underlying the efficacy of PRP treatment for osteoarthritis or damage in the knee joint. PMID:25813869

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. Copyright © 2016 Elsevier B.V. All rights reserved.

A test of the hypothesis that oxalate secretion produces proximal tubule crystallization in primary hyperoxaluria type I

PubMed Central

Evan, Andrew P.; Coe, Fredric L.; Lingeman, James E.; Krambeck, Amy; Sommers, Andre; Phillips, Carrie L.; Milliner, Dawn

2013-01-01

The sequence of events by which primary hyperoxaluria type 1 (PH1) causes renal failure is unclear. We hypothesize that proximal tubule (PT) is vulnerable because oxalate secretion raises calcium oxalate (CaOx) supersaturation (SS) there, leading to crystal formation and cellular injury. We studied cortical and papillary biopsies from two PH1 patients with preserved renal function, and seven native kidneys removed from four patients at the time of transplant, after short-term (2) or longer term (2) dialysis. In these patients, and another five PH1 patients without renal failure, we calculated oxalate secretion, and estimated PT CaOx SS. Plasma oxalate was elevated in all PH1 patients and inverse to creatinine clearance. Renal secretion of oxalate was present in all PH1 but rare in controls. PT CaOx SS was >1 in all nonpyridoxine-responsive PH1 before transplant and most marked in patients who developed end stage renal disease (ESRD). PT from PH1 with preserved renal function had birefringent crystals, confirming the presence of CaOx SS, but had no evidence of cortical inflammation or scarring by histopathology or hyaluronan staining. PH1 with short ESRD showed CaOx deposition and hyaluronan staining particularly at the corticomedullary junction in distal PT while cortical collecting ducts were spared. Longer ESRD showed widespread cortical CaOx, and in both groups papillary tissue had marked intratubular CaOx deposits and fibrosis. CaOx SS in PT causes CaOx crystal formation, and CaOx deposition in distal PT appears to be associated with ESRD. Minimizing PT CaOx SS may be important for preserving renal function in PH1. PMID:24089413