Binding of ovarian cancer cells to immobilized hyaluronic acid

Ovarian cancer has the highest mortality rate of any gynaecological malignancy. This is caused by metastatic deposits obstructing the intestinal tract. Very little is known about the molecules involved in the initial attachment of the metastatic tumour cells to the peritoneal mesothelial lining. Previously, we showed that many ovarian tumour lines express the adhesion molecule, CD44, on their cell surface. The major ligand for CD44 is the extracellular matrix glycosaminoglycan, hyaluronic acid (HA). Because mesothelial cells have a pericellular cost that contains large amounts of HA, it was postulated that the CD44/HA interaction is an important stage in ovarian cancer spread. However, it was difficult to demonstrate this interaction in an in vitro adhesion assay with mesothelial cells as most of the HA, and presumably the bound tumour cells, were lost from the mesothelial cells during the washing steps of the assay. In order to try and clarify the situation, the adhesion of six ovarian tumour lines to immobilized HA was measured. Four lines expressed high levels of CD44 and two lines expressed negligible amounts. Preliminary experiments were carried out with one of the CD44-expressing lines. After coating a plate overnight with 3 mg ml−1 HA, the 5 min adhesion of this line varied between 2% and 73% according to the type of plate that was used. Falcon Micro Test III flexible plates gave the highest adhesion and was used for further experiments. Plates were coated with concentrations of HA between 0.001 mg ml−1 and 3 mg ml−1. All CD44 expressing lines adhered to HA, but the maximum adhesion and the adhesion strength varied with the line studied and was not closely related to the total CD44 expression. These results suggest that CD44 on ovarian tumour cells binds to HA on mesothelial cells. As much of the HA can be very easily lost from the mesothelial cell surface, additional factors such as the strength of the CD44/HA interaction, and the formation of bonds by the tumour cells with other membrane adhesion molecules, such as integrins, are also important in promoting tumour spread. This revised version was published online in November 2006 with corrections to the Cover Date.     

Binding of ovarian cancer cells to immobilized hyaluronic acid

Ovarian cancer has the highest mortality rate of any gynaecological malignancy. This is caused by metastatic deposits obstructing the intestinal tract. Very little is known about the molecules involved in the initial attachment of the metastatic tumour cells to the peritoneal mesothelial lining. Previously, we showed that many ovarian tumour lines express the adhesion molecule, CD44, on their cell surface. The major ligand for CD44 is the extracellular matrix glycosaminoglycan, hyaluronic acid (HA). Because mesothelial cells have a pericellular cost that contains large amounts of HA, it was postulated that the CD44/HA interaction is an important stage in ovarian cancer spread. However, it was difficult to demonstrate this interaction in an in vitro adhesion assay with mesothelial cells as most of the HA, and presumably the bound tumour cells, were lost from the mesothelial cells during the washing steps of the assay. In order to try and clarify the situation, the adhesion of six ovarian tumour lines to immobilized HA was measured. Four lines expressed high levels of CD44 and two lines expressed negligible amounts. Preliminary experiments were carried out with one of the CD44-expressing lines. After coating a plate overnight with 3 mg ml-1 HA, the 5 min adhesion of this line varied between 2% and 73% according to the type of plate that was used. Falcon Micro Test III flexible plates gave the highest adhesion and was used for further experiments. Plates were coated with concentrations of HA between 0.001 mg ml−1 and 3 mg ml−1. All CD44 expressing lines adhered to HA, but the maximum adhesion and the adhesion strength varied with the line studied and was not closely related to the total CD44 expression. These results suggest that CD44 on ovarian tumour cells binds to HA on mesothelial cells. As much of the HA can be very easily lost from the mesothelial cell surface, additional factors such as the strength of the CD44/HA interaction, and the formation of bonds by the tumour cells with other membrane adhesion molecules, such as integrins, are also important in promoting tumour spread. This revised version was published online in November 2006 with corrections to the Cover Date.     

A self-assembled monolayer-based micropatterned array for controlling cell adhesion and protein adsorption

We developed a surface micropatterning technique to control the cell adhesion and protein adsorption. This micropatterned array system was fabricated by a photolithography technique and self-assembled monolayer (SAM) deposition. It was hypothesized that the wettability and functional terminal group would regulate cell adhesion and protein adsorption. To demonstrate this hypothesis, glass-based micropatterned arrays with various functional terminal groups, such as amine (NH(2)) group (3-aminopropyl-triethoxysilane, APT), methyl (CH(3)) group (trichlorovinylsilane, TVS), and fluorocarbon (CF(3)) group (trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane, FOTS), were used. The contact angle was measured to determine the hydrophilic and hydrophobic properties of materials, demonstrating that TVS and FOTS were hydrophobic, whereas APTs were relatively hydrophilic. The cell adhesion was significantly affected by the wettability, showing that the cells were not adhered to hydrophobic surfaces, such as TVS and FOTS. Thus, the cells were selectively adhered to glass substrates within TVS- and FOTS-based micropatterned arrays. However, the cells were randomly adhered to APTs-based micropatterned arrays due to hydrophilic property of APTs. Furthermore, the protein adsorption of the SAM-based micropatterned array was analyzed, showing that the protein was more absorbed to the TVS surface. The surface functional terminal group enabled the control of protein adsorption. Therefore, this SAM-based micropatterned array system enabled the control of cell adhesion and protein adsorption and could be a potentially powerful tool for regulating the cell-cell interactions in a well-defined microenvironment.     

Facile synthesis and characterization of disulfide-cross-linked hyaluronic acid hydrogels for protein delivery and cell encapsulation

Injectable hyaluronic acid (HA) hydrogels cross-linked via disulfide bond are synthesized using a thiol-disulfide exchange reaction. The production of small-molecule reaction product, pyridine-2-thione, allows the hydrogel formation process to be monitored quantitatively in real-time by UV spectroscopy. Rheological tests show that the hydrogels formed within minutes at 37 °C. Mechanical properties and equilibrium swelling degree of the hydrogels can be controlled by varying the ratio of HA pyridyl disulfide and macro-cross-linker PEG-dithiol. Degradation of the hydrogels was achieved both enzymatically and chemically by disulfide reduction with distinctly different kinetics and profiles. In the presence of hyaluronidase, hydrogel mass loss over time was linear and the degradation was faster at higher enzyme concentrations, suggesting surface-limited degradation. The kinetics of hydrogel erosion by glutathione was not linear, nor did the erosion rate correlate linearly with glutathione concentration, suggesting a bulk erosion mechanism. A cysteine-containing chemokine, stromal cell-derived factor 1α, was successfully encapsulated in the hydrogel and released in vitro without chemical alteration. Several different cell types, including fibroblasts, endothelial cells, and mesenchymal stem cells, were successfully encapsulated in the hydrogels with high cell viability during and after the encapsulation process. Substantial cell viability in the hydrogels was maintained up to 7 days in culture despite the lack of adhesion between the HA matrix and the cells. The facile synthesis of disulfide-cross-linked, dual-responsive degradable HA hydrogels may enable further development of bioactive matrices potentially suitable for tissue engineering and drug delivery applications.     

Carbohydrate-mediated cell adhesion to glycoproteins immobilized on nitrocellulose

A method that allows the estimation of carbohydrate-mediated cell adhesion to glycoproteins and polysaccharides immobilized to a nitrocellulose matrix is described. Specificity of adhesion by indicator cells (Chang liver) has been verified using glycoconjugates with defined carbohydrate structure. Two independent receptor systems with beta-galactose or alpha-fucose specificity, respectively, have been demonstrated by this method to occur on Chang liver cells. The method is also applicable for other indicator cells like murine fibrosarcoma cells and has been used for the analysis of dot-blots and Western blots of glycoproteins.     

Carbohydrate-specific cell adhesion is mediated by immobilized glycolipids

We describe a technique for examining the ability of one important class of cell surface complex carbohydrates, glycosphingolipids, to mediate carbohydrate-specific cell recognition and adhesion. Analogs of natural glycosphingolipids were synthesized, consisting of 1-glycosyl derivatives of 3-deoxyceramide (N-palmitoyl-2-aminostearol) radiolabeled in the fatty acid portion. Methods were developed to efficiently adsorb both these synthetic glycolipids and natural glycosphingolipids (including gangliosides) from aqueous ethanol solution onto plastic wells. The glycolipids remained firmly attached to the surface in aqueous solutions, but could be recovered using detergents or organic solvents. The ability of the adsorbed glycolipids to elicit specific adhesion of intact hepatocytes was tested using specific adhesion of intact hepatocytes was tested using a cell adhesion assay based on that of McClay, D. R., Wessel, G. M., and Marchase, R. B. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4975-4979. When otherwise nonadhesive plastic surfaces were adsorbed with N-acetylglucosaminyl 3-deoxyceramide, they supported adhesion of 80-95% of the chicken hepatocytes added to the well. No adhesion above background levels (10-25%) was observed to surfaces adsorbed with other synthetic glycolipids including glucosyl, galactosyl, mannosyl, or lactosyl 3-deoxyceramide, 3-deoxyceramide, or to the naturally occurring glycosphingolipids, lactosyl ceramide or ganglioside GM1. Chicken hepatocyte adhesion to surfaces adsorbed with N-acetylglucosaminyl 3-deoxyceramide was inhibited by soluble N-acetylglucosamine (IC50 = 3 m M), but not by other soluble sugars. Rat hepatocytes adhered preferentially to surfaces adsorbed with lactosyl 3-deoxyceramide, but not to surfaces adsorbed with the N-acetylglucosaminyl derivative. These studies demonstrate the ability of adsorbed glucolipids to mediate carbohydrate- and cell-specific adhesion from intact cells. Using these techniques, the ability of naturally occurring complex glycosphingolipids to elicit specific cellular responses from a variety of cell types can be examined.     

Tailored poly(2-oxazoline) polymer brushes to control protein adsorption and cell adhesion

POx bottle-brush brushes (BBBs) are synthesized by SIPGP of 2-isopropenyl-2-oxazoline and consecutive LCROP of 2-oxazolines on 3-aminopropyltrimethoxysilane-modified silicon substrates. The side chain hydrophilicity and polarity are varied. The impact of the chemical composition and architecture of the BBB upon protein (fibronectin) adsorption and endothelial cell adhesion are investigated and prove extremely low protein adsorption and cell adhesion on BBBs with hydrophilic side chains such as poly(2-methyl-2-oxazoline) and poly(2-ethyl-2-oxazoline). The influence of the POx side chain terminal function upon adsorption and adhesion is minor but the side chain length has a significant effect on bioadsorption.     

The role of hyaluronic acid in intercellular adhesion of cultured mouse cells

Aggregation of cultured mouse cells was measured by the rate of disappearance of particles from a suspension of single cells. Treatment with several enzymes which degrade hyaluronic acid (testicular hyaluronidase, streptomyces hyaluronidase, streptococcal hyaluronidase and chondroitinase ABC) inhibited the aggregation of SV-3T3 and several other cell types. Since streptomyces and streptococcal hyaluronidases are specific for hyaluronic acid, it is suggested that hyaluronic acid is involved in the observed aggregation. Hyaluronidase-induced inhibition of aggregation was complete in the absence of divalent cations, but only partial in their presence. This finding is consistent with the hypothesis that two separate mechanisms are responsible for aggregation; one dependent upon and the other independent of calcium and magnesium. Aggregation was also inhibited by high levels of hyaluronic acid. A similar effect was obtained with fragments of hyaluronic acid consisting of six sugar residues or more. Chondroitin (desulfated chondroitin 6-sulfate) and to a lesser extent desulfated dermatan sulfate also inhibited aggregation. Other glycosaminoglycans (chondroitin 4-sulfate, chondroitin 6-sulfate, heparin and heparan sulfate) had little or no effect on aggregation. It is suggested that the hyaluronic acid inhibits aggregation by competing with endogenous hyaluronic acid for cell surface binding sites.     

Extracellular mycobacterial DNA-binding protein 1 participates in mycobacterium-lung epithelial cell interaction through hyaluronic acid

Mycobacterium tuberculosis infects not only host macrophages but also nonprofessional phagocytes, such as alveolar epithelial cells. Glycosaminoglycans (GAGs) are considered as the component of mycobacterial adherence to epithelial cells. Here we show that extracellularly occurring mycobacterial DNA-binding protein 1 (MDP1) promotes mycobacterial infection to A549 human lung epithelial cells through hyaluronic acid (HA). Both surface plasmon resonance analysis and enzyme-linked immunosorbent assay revealed that MDP1 bound to HA, heparin, and chondroitin sulfate. Utilizing synthetic peptides, we next defined heparin-binding site of 20 amino acids from 31 to 50 of MDP1, which is responsible for the specific DNA-binding site of MDP1. MDP1 bound to A549 cells, and exogenous DNA and HA interfered with the interaction. The binding was also abolished by treatment of A549 cells with hyaluronidase, suggesting that HA participates in the MDP1-A549 cell interaction. Adherence of bacillus Calmette-Guérin (BCG) and M. tuberculosis to A549 cells was inhibited by addition of HA, DNA, and anti-MDP1 antibody, showing that MDP1 participates in the interaction between mycobacteria-alveolar epithelial cells. Simultaneous treatment of intratracheal BCG-infected mice with HA reduced the growth of BCG in vivo. Taken together, theses results suggest that HA participates in Mycobacterium-lung epithelium interaction and has potential for therapeutic and prophylactic interventions in mycobacterial infection.     

In vivo real-time bioimaging of hyaluronic acid derivatives using quantum dots

The effect of chemical modification of hyaluronic acid (HA) on its distribution throughout the body was successfully visualized in nude mice through real-time bioimaging using quantum dots (QDots). Adipic acid dihydrazide modified HA (HA-ADH) was synthesized and conjugated with QDots having carboxyl terminal ligands activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide. The formation of HA-QDot conjugates could be confirmed by gel permeation chromatography, fluorometry, transmission electron microscopy, and zeta-size analysis. According to the real-time bioimaging of HA-QDot conjugates after subcutaneous injection to nude mice, the fluorescence of HA-QDot conjugates with a near infrared wavelength of 800 nm could be detected up to 2 months, whereas that with an emission wavelength of 655 nm disappeared almost completely within 5 days. The results can be ascribed to the fact that near-infrared light has a high penetration depth of about 5-6 cm in the body compared to that of about 7-10 mm for visible light. Thereby, using QDots with a near-infrared emission wavelength of 800 nm, the distribution of HA-QDot conjugates throughout the body was bioimaged in real-time after their tail-vein injection into nude mice. HA-QDot conjugates with 35 mol% ADH content maintaining enough binding sites for HA receptors were mainly accumulated in the liver, while those with 68 mol% ADH content losing much of HA characteristics were evenly distributed to the tissues in the body. The results are well matched with the fact that HA receptors are abundantly present in the liver with a high specificity to HA molecules.     

Affinity binding of the cartilage proteoglycan protein-keratan sulfate core to immobilized hyaluronic acid.

The protein-keratan sulfate core of bovine nasal cartilage proteoglycan was purified by affinity chromatography on a column of immobilized hyaluronic acid. The hyaluronic acid was immobilized by reaction with a hydrazido-alkyl derivative of Sepharose in the presence of borohydride. Proteoglycan was digested with chondroitinase ABC and the entire mixture was passed over a column of the Sepharose-hyaluronic acid maintained at 4°C. After the digested chondroitin sulfate chains were washed from the column, the bound protein-keratan sulfate core was eluted with 4m guanidinium chloride. The protein-keratan sulfate core interacts with the affinity matrix through its hyaluronic acid binding site as shown by the inhibition of binding by free hyaluronic acid and hyaluronic acid decasaccharide.     

RGD-grafted poly-L-lysine-graft-(polyethylene glycol) copolymers block non-specific protein adsorption while promoting cell adhesion

A novel class of surface-active copolymers is described, designed to protect surfaces from nonspecific protein adsorption while still inducing specific cell attachment and spreading. A graft copolymer was synthesized, containing poly-(L-lysine) (PLL) as the backbone and substrate binding and poly(ethylene glycol) (PEG) as protein adsorption-resistant pendant side chains. A fraction of the grafted PEG was pendantly functionalized by covalent conjugation to the peptide motif RGD to induce cell binding. The graft copolymer spontaneously adsorbs from dilute aqueous solution onto negatively charged surfaces. The performance of RGD-modified PLL-g-PEG copolymers was analyzed in protein adsorption and cell culture assays. These coatings efficiently blocked the adsorption of serum proteins to Nb(2)O(5) and tissue culture polystyrene while specifically supporting attachment and spreading of human dermal fibroblasts. This surface functionalization technology is expected to be valuable in both the biomaterial and biosensor fields, because different signals can easily be combined, and sterilization and application are straightforward and cost-effective.     

Fabrication of hyaluronic acid hydrogel beads for cell encapsulation

Hyaluronic acid (HA) hydrogel beads were prepared by photopolymerization of methacrylated HA and N-vinylpyrrolidone using alginate as a temporal spherical mold. Various fabrication conditions for preparing the hydrogel beads, such as the concentration of methacrylated HA and UV irradiation time, were optimized to control swelling properties and enzymatic degradability. A new concept for cell encapsulation is proposed in this paper. Viable cells were directly injected into the hydrogel beads using a microinjection technique. When bovine articular chondrocytes were injected into HA hydrogel beads and cultivated for 1 week, the cells could proliferate well within the HA beads. HA hydrogel beads could be potentially used as injectable cell delivery vehicles for regenerating tissue defects.     

Joint action of protein-chondroitin-4-keratan-sulfate and hyaluronic acid on erythrocyte aggregation and adhesion]

It was shown that the rate and the degree of erythrocytes aggregation brought about by a mixture of protein-chondroitin-4-keratan sulfate (PCKS) and hyaluronic acid (HA) was greater than the sum of the values of the corresponding indices observed during separate independent action of these proteoglycans on the aggregation of the mentioned cells concentrations as in the mixtures. It may be supposed that such phenomenon is connected with formation in the mixture of a hybrid PCKS-HA complex which is more active in respect to the erythrocyte aggregation than its components separately.     

Bioactivity studies of oxysporone and several derivatives

Recently oxysporone, a phytotoxic dihydrofuropyranone, was isolated along with two closely related compounds, afritoxinones A and B, from liquid cultures of Diplodia africana, an invasive fungal pathogen of Phoenicean juniper. In this study, eight derivatives were hemisynthesized and assayed for their phytotoxic and antifungal activities in comparison to the parent compound. Each compound was tested on non-host plants and on four destructive plant pathogens such as Athelia rolfsii, Diplodia corticola, Phytophthora cinnamomi and P. plurivora. The results on the phytotoxic activity showed that the dihydrofuropyranone carbon skeleton and both the double bond the hydroxy group of dihydropyran ring appeared to be structural features important in conferring activity. Although the data concerning the antifungal activity did not allow to extract any structure–activity relationships, it should be underlined that the conversion of oxysporone into the corresponding 4-O-benzoyl derivative led to a compound showing a good antifungal activity towards three out of the four organisms tested.