High Hyaluronic Acid Research Articles

Hyaluronic acid modulates glioma cell proliferation through its interaction with CD44H in vitro

In a recent study we showed that hyaluronic acid (HA) induces cell detachment and promotes migration and invasion of glioma cells in vitro through its interaction with the cell surface molecule CD44H. In this study, the role of HA in proliferation in eight human glioma-derived cell Lines was investigated. We demonstrate that HA exerts a dose dependent proliferative and anti-proliferative effect on glioma cells. This effect was found to be partially counteracted by a CD44H monoclonal antibody, suggesting the involvement of its high affinity receptor, CD44H and other HA receptors in this process.

Biotinylated hyaluronic acid: a new tool for probing hyaluronate-receptor interactions.

Hyaluronic acid (HA) is a linear polysaccharide composed of repeating disaccharide units of D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc). Hyaluronate plays an important role in many biological processes as mediated by its interactions with a number of HA-binding proteins (the "hyaladherins") and with the cell surface HA-receptor, CD44. Studies of hyaluronate-hyaladherin interactions would be greatly facilitated by the availability of molecular probes derived from HA. We recently reported a convenient chemical modification of hyaluronate that introduces multiple pendant amine functionalities onto the HA carboxylate residues. We now report the preparation of biotinylated hyaluronic acid (molecular weight = 1.2 x 10(6) Da) as a probe for histochemical and immunochemical characterization of HA-binding proteins. Approximately one-third of the available HA glucuronate residues could be readily biotinylated in high molecular weight HA.

Effect of Hyaluronic Acid on Rabbit Profundus Flexor Tendon Healing in Vitro

We performed an in-depth biomechanical evaluation of the effect of hyaluronic acid (HA) on the healing of rabbit profundus tendons cultured in vitro. Seventy-eight flexor tendons from 13 rabbits were transected and reapproximated at their Zone II midpoints. Tendons were divided into left and right forepaw groups. Each tendon from the left forepaw group was incubated in one of four possible culture media: control (no HA), low (0.1 mg/ml), medium (0.5 mg/ml), or high (1.0 mg/ml) HA media. HA was added on the first day of incubation. Each tendon from the right forepaw group was cultured in low, medium, or high concentrations of HA, but HA was added after 1 week of incubation in control media. All tendons were cultured for 8 weeks, after which time tenorrhaphies were disrupted and the following biomechanical parameters were determined: apparent maximum stress, apparent strain at apparent maximum stress, normalized energy absorption, and tangent modulus before failure. Comparisons using these parameters showed no statistically significant differences among the various tendon groups. We believe this is the first study of its kind to show no effect of hyaluronic acid on the functional strength of tendon after healing in vitro.

Effects of hyaluronic acid on macrophage phagocytosis and active oxygen release.

The effects of hyaluronic acid (HA) on macrophage function, in terms of the phagocytosis of latex beads and superoxide anion and hydrogen peroxide release stimulated by phorbol myristate acetate (PMA), were studied in guinea-pig peritoneal macrophages. Phagocytosis was inhibited in a dose- and molecular-weight-dependent manner by HA. The addition of PMA to the culture, at a dose of more than 10 ng ml-1, caused an increase in the release of active oxygens. The release of active oxygens was inhibited by high molecular weight HA (MW 2.02 x 10(6), HA-202) in a dose-dependent manner. In cell-free systems, HA-202 had a negligible effect in scavenging these active oxygens. Of the three molecular sizes of HA (MW: 0.28 x 10(6), 0.98 x 10(6) and 2.02 x 10(6)), HA-202 most strongly inhibited the active oxygen release. These results indicate that high-molecular-weight HA acts directly on macrophages to inhibit phagocytosis and active oxygen formation, which, in turn, ameliorates the progression of chronic inflammation.

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

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

Evidence of hyaluronic acid and hyaluronic acid binding sites on human corneal endothelium

A highly specific hyaluronic acid (HA) recognizing protein (HABR) was used to study whether the human corneal endothelium is covered by HA and to quantify the amount. Tritiated high molecular weight HA was used to determine the capacity of the human endothelium to bind exogenous HA. Human corneas were obtained from keratoconus patients having corneal transplantation and from postmortem eyes. The corneas were immersed in a 4% formaldehyde solution containing 1% cetylpyridine chloride for histochemistry, frozen for biochemistry, or used for 3H-HA (Mr 3 × 106) binding. For the biochemical determinations, 125I-labeled HABR was used. Tritiated HA was used for the binding experiment. A specific layer of HA covering the endothelial cells of the corneal buttons was demonstrated. The biochemical analysis also revealed the presence of HA. Finally, the human endothelial cells had specific hyaluronic acid binding sites.

Characterization of a hyaluronic acid-binding protein from sheep brain comparison with human brain hyaluronectin

1. 1. A hyaluronic acid (HA)-binding glycoprotein from sheep brain was characterized. 2. 2. The specific affinity for HA was shown in vitro by high performance liquid chromatography, polyacrylamide gel electrophoresis and ELISA methods. 3. 3. The K D for high molecular weight HA was 5.4 10 −9 M at 37°C and lower than 10 −10 M at 4°C. 4. 4. No link protein was found and HA molecules could bind up to 10 times their weight of the glycoprotein. 5. 5. The specific site for interaction was the HA-derived decasaccharide Ha 10. 6. 6. The protein is composed of one polypeptidic chain. Tryptophan and lysine play a prominent role in the conformation of the binding site to HA. 7. 7. Enzyme analysis indicated that the protein different forms are due to differences in glycosylation and that N- and O-linkages coexist in the molecules. 8. 8. Immunohistochemistry localized the glycoprotein at the nodes of Ranvier and at the periphery of neurons. The perineuronal labeling was seen around all neurons studied in the cerebellum whereas it was almost undetectable in the cerebral hemispheres. 9. 9. HA is not saturated by hyaluronectin (HN) in the sheep nervous system. 10. 10. The glycoprotein is largely similar to human brain HN, and different from the hyaluronate-binding protein characterized in the cartilage.

Effect of hyaluronic acid on the emergence of neural crest cells from the neural tube of the quail, Coturnix coturnix japonica.

Hyaluronic acid (HA) added to the medium of quail neural tubes explanted in vitro influences the number of migratory neural crest cells that emerge, compared with controls. Neural crest cells were counted with an ocular grid after 20 h of migration into 0.1 mm wide areas or 'bins' lying parallel to the neural tube, and the results were analyzed by linear regression. A low concentration of HA (5 micrograms/ml) significantly decreased the total number of neural crest cells in all bins adjacent to the neural tube, whereas several high concentrations of HA (250, 500, and 1000 micrograms/ml) significantly increased the number of neural crest cells. Intermediate concentrations of HA (50 and 100 micrograms/ml) did not differ from that of controls. Linear regressions of number of cells versus distance from the tube showed no significant differences among the slopes of control, low HA, and high HA treatments, providing evidence that HA does not influence the rate of cell migration. Scanning electron microscopy showed that cells in neuroepithelia exposed to low HA (5 micrograms/ml) appeared in tighter contact, while cells of neuroepithelia in high HA (500 micrograms/ml) appeared more loosely organized, compared with controls. Cells in tight contact could be restrained from leaving the neuroepithelium, whereas cells in loose contact could more readily move out of the neural tube, thus explaining the differences in cell numbers in low HA and high HA, respectively. We conclude that HA can be a factor in the differential adhesivity among neuroepithelial cells and may be important in the initial separation of the neural crest from the neural tube.

Elevation of urinary hyaluronic acid in Werner's syndrome and progeria.

Werner's syndrome and Hutchinson-Gilford progeria syndrome (progeria) are human genetic diseases which may serve as models for the study of premature aging. The basic defects underlying these diseases are unknown. An abnormally high level of urinary hyaluronic acid (HA) excretion has been previously reported in several Werner's and one progeria subject, all from Japan. To determine if a high HA level is a reliable marker for these diseases, we quantitated the urinary excretion of HA in three progeria subjects, one subject with an atypical progeroid syndrome, and a Werner's syndrome subject. Compared to controls, the total urinary HA was found to be markedly increased in the three progeria samples and in the Werner's syndrome sample. These findings support the previous observations indicating elevated HA may be a specific marker for these diseases.

The effects of synovial fluid macrophages and interleukin-1 on hyaluronic acid synthesis by normal synovial fibroblasts.

The effects of peripheral blood monocyte and rheumatoid synovial fluid macrophage conditioned media were studied on hyaluronic acid (HA) metabolism of normal synovial fibroblasts. Both media stimulated HA synthesis about two-fold compared to controls (1% fetal calf serum). The activated mononuclear phagocyte conditioned media did not contain HA-degrading activity in these experiments. The effects of various concentrations of interleukin-1 (IL-1) on HA synthesis and proliferation of synovial fibroblasts were studied. Even at very low concentrations (0.1 IU IL-1/ml) HA synthesis was stimulated. With increasing concentrations HA synthesis did not increase but proliferation was stimulated. Stimulated fibroblasts synthesized mainly high molecular weight HA. Thus with IL-1-activation, normal synovial fibroblasts could not produce increased amounts of abnormal HA with decreased molecular weight.

Separate effects of exogenous hyaluronic acid on proteoglycan synthesis and deposition in pericellular matrix by cultured chick embryo limb chondrocytes

The effects of exogenous hyaluronic acid on cell cultures of chick embryo limb chondrocytes are reported in this paper. The evidence shows that exogenous hyaluronic acid (HA) can both depress the incorporation of 35SO 4 into glycosaminoglycans and cause a displacement of newly synthesized proteoglycan from the cell layer to the culture medium. The results demonstrate that these two effects are mediated by distinct mechanisms. The displacement effect has a rapid onset (by 2 hr) while the effect of exogenous HA on 35SO 4 incorporation has a long latency (12 hr). The displacement effect is produced by a lower concentration (5 μg/ml) of hyaluronate oligomers than the effect on 35SO 4 incorporation (50 μg/ml). In addition, displacement is produced only by hyaluronate oligomers that are decasaccharides or larger. The depression of 35SO 4 incorporation is produced by tetrasaccharides as well as high molecular weight HA. In fact tetrasaccharides can depress 35SO 4 incorporation without causing the displacement effect.

Hyaluronic acid synthesis and turnover by myotubes in culture

Analysis of glycosaminoglycan (GAG) synthesis by high density and clonal cultures of chick myoblasts and fibroblasts, and by cultures enriched for myotubes, indicates that all culture types produce hyaluronic acid (HA) and sulfated polysaccharides (S-GAG). Of these cell types the myotube synthesizes the most HA-rich complement of polysaccharides and has the most HA-rich cell surface. It appears that this is the result of decreased S-GAG synthesis rather than increased HA synthesis. Myotube cell surface HA is qualitatively different from HA released to the medium; cell surface HA is predominantly a single high molecular weight species, whereas HA isolated from the medium comprises a heterogeneous mixture of lower molecular weight HA. Since HA molecules of similar size classes can be generated by treating high molecular weight HA with hyaluronidase, an endogenous hyaluronidase could be involved. Alternatively, myotube-enriched cultures may synthesize and secrete HA polymers of several intermediate lengths.

The effect of oestrogenic treatment on the acid mucopolysaccharide pattern in skin of mice.

Acid mucopolysaccharides obtained from the skin of the back of mice were determined after precipitation with cetylpyridinium chloride and cellulose‐column fractionation. The results are given as μmol uronic acid per gram dry fat free tissue. Following treatment with oestradiol monobenzoate the hyaluronic acid (HA) increased from 2.7 to 14.6, while the chondroitin sulphates were unaffected (3.7 to 4.2). The increase (from 1.3 to 10.8) of a fraction probably consisting of low molecular HA was considered as reflecting an intensified metabolic activity of HA in the connective tissue. A very close linear correlation (r = 0.924) was demonstrated between the increase in the high molecular HA and the increase in tissue water.