Hyaluronan Release Research Articles

The effect of mechanical strain on hyaluronan metabolism in embryonic fibrocartilage cells.

The development of the synovial joint cavity between the cartilage anlagen of the long bones is thought to be mediated by differential matrix synthesis at the developing articular surfaces. In addition, many studies have shown that removal of movement-induced mechanical stimuli from developing diarthrodial joints prevents cavity formation or produces a secondary fusion of previously cavitated joints. Herein, we describe an inductive influence of mechanical strain on hyaluronan metabolism and the expression of hyaluronan-binding proteins in cultured cells isolated from the articular surface of the distal tibial condyles of 18-day chick embryos. The effect of 10 min of mechanical strain on hyaluronan release into culture media, intracellular uridine diphospho-glucose dehydrogenase activity (an enzyme required for hyaluronan saccharide precursor production), cell surface hyaluronan-binding protein expression and HA synthase mRNA expression were analysed up to 24 h later. Six hours after the application of strain, there was a significant increase in the accumulation of hyaluronan released into tissue culture media by strained fibrocartilage cells compared with controls, an effect still detectable after 24 h. Strained cells also showed increased activity for uridine diphospho-glucose dehydrogenase and expressed higher levels of the hyaluronan-binding protein CD44 at 24 h. In addition, at 24 h mRNA for HA synthase 2 was expressed in all samples whereas mRNA for HA synthase 3 was only expressed in strained cells. These results further highlight the role for movement-induced stimuli in differential extracellular matrix metabolism during joint development and also show that strain may facilitate differential HA synthase gene expression.

Retention of hyaluronic acid in alginate beads: aspects for in vitro cartilage engineering.

Alginate has been used successfully for three-dimensional chondrocyte cultures and may be important for cartilage transplant formation. However, alginate is not a natural component of the cartilage matrix. The aim of this study was (a) to supplement alginate with the extracellular matrix component hyaluronic acid; and (b) to analyze the hyaluronic acid retention in different alginate gels. Hyaluronan is assumed to improve proteoglycan retention and may be important for in vitro matrix formation, tissue turgor, and biomechanical quality. Alginate and hyaluronan were mixed with chondrocytes and polymerized as were alginate, hyaluronan, and fibrinogen. [3H]hyaluronan was used to quantitate the leakage of hyaluronan from the gel beads. After 28 days in culture, 1.2% alginate beads supplemented with 0.26% hyaluronan contained only 9% of the initial amount of hyaluronan whereas 2.4% alginate beads still contained about 55% of the initial 0.22% hyaluronan. Release of hyaluronan from the beads was significantly lower if the beads additionally contained fibrin. Alginate beads supplemented with hyaluronan or fibrin showed increased chondrocyte proliferation compared to controls. Supplemented hyaluronan greatly diffuses out of alginate gels of lower densities. It must be assumed also that most of the hyaluronan newly synthesized by chondrocytes in these cells diffuses into the surrounding culture medium. The in vitro development of a sufficiently hygroscopic cartilage ground substance therefore may be very limited. Sufficient hyaluronic acid retention can be achieved in alginate gels with concentrations above 1.2% or by addition of fibrin.

Release of Hyaluronan and Laminin into Pancreatic Secretions

Background: Development of fibrosis characterizes chronic pancreatitis. As it results from deposition of extracellular matrix (ECM) proteins such as hyaluronan (HA) or laminin, the release of these ECM components into blood or pancreatic secretion may be enhanced during the course of chronic pancreatitis. Patients, Material and Methods: Using immunoassays for HA and laminin, the concentration for these ECM components was measured in pancreatic juice and serum samples from 20 patients with and 20 patients without chronic pancreatitis. Pancreatic calculi of varying size and weight obtained from 13 patients with chronic pancreatitis were also examined for the content of ECM components. Tissue samples from normal pancreas and those showing chronic pancreatitis were investigated immunocytochemically with an antibody to HA synthetase (HAS). Results: After stimulation with secretin high levels of ECM components were found in the initial washout period in chronic pancreatitis patients as well as in controls. HA levels, however, were seen seven times higher in patients with chronic pancreatitis (mean ± SEM; 734 ± 301 vs. 95 ± 15 µg/l; p < 0.01). Serum HA levels correlated with the duration of chronic pancreatitis and with the levels of HA in pancreatic juice. HA and laminin were detected in the supernatants of pancreatic calculi (laminin 0.70 ± 0.30 U/l; HA 275 ± 85 µg/l). Immunocytochemically, strong staining for HAS was found in the duct epithelium and in centroacinar cells of chronic pancreatitis specimens. Conclusion: Demonstration of increased amounts of HA in pancreatic juice of chronic pancreatitis patients stimulated with secretin suggests enhanced production of this ECM component in the chronically inflamed pancreas. The source of HA appears to be the pancreatic ductal epithelium.

An Ectoprotein Kinase of Group C Streptococci Binds Hyaluronan and Regulates Capsule Formation

A 56-kDa protein had been isolated and cloned from protoplast membranes of group C streptococci that had erroneously been identified as hyaluronan synthase. The function of this protein was reexamined. When streptococcal membranes were separated on an SDS-polyacrylamide gel and renatured, a 56-kDa protein was detected that had kinase activity for a casein substrate. When this recombinant protein was expressed in Escherichia coli and incubated in the presence of [32P]ATP, it was responsible for phosphorylation of two proteins with 30 and 56 kDa that were not present in the control lysate. The 56-kDa protein was specifically phosphorylated in an immunoprecipitate of a detergent extract of the recombinant E. coli lysate with antibodies against the 56-kDa protein, indicating that it was autophosphorylated. The E. coli lysate containing the recombinant protein could bind hyaluronan, and hyaluronan binding was abolished by the addition of ATP. Kinetic analysis of hyaluronan synthesis and release from isolated protoplast membranes indicated that phosphorylation by ATP stimulated hyaluronan release and synthesis. Incubation of membranes with antibodies to the 56-kDa protein increased hyaluronan release. The addition of [32P]ATP to intact streptococci led to rapid phosphorylation of two proteins, 56 and 75 kDa each at threonine residues. This phosphorylation was neither observed with [32P]phosphate nor in the presence of trypsin, indicating that the kinase was localized extracellularly. The addition of ATP to growing group C streptococci led to increased hyaluronan synthesis and release. However marked differences were found between group A and group C streptococci. Antibodies against the 56-kDa protein from group C streptococci did not recognize proteins from group A strains, and a homologous DNA sequence could not be detected by polymerase chain reaction or Southern blotting. In addition, Group A streptococci did not retain a large hyaluronan capsule like group C strains. These results indicated that the 56-kDa protein is an ectoprotein kinase specific for group C streptococci that regulates hyaluronan capsule shedding by phosphorylation.

Increased hyaluronan flux from skin following burn injury

Hyaluronan (formerly hyaluronic acid) is an important constituent of the interstitial matrix in skin. Following major burn injury in animal models, plasma hyaluronan can increase to levels 10-fold greater than normal. The present experiments were designed to determine whether this is a result of the increased lymph flow (Q̇L) accompanying the injury or of an increased release of hyaluronan from the burned skin and subcutaneous tissue. The lateral saphenous vein and a prenodal lymphatic were cannulated in the hindpaw of five anesthetized canines. Hindpaw venous pressure was elevated until the total protein concentration in lymph declined to steady-state levels, and Q̇L and hyaluronan flux (Q̇L × [hyaluronan]) was measured. A minor burn was inflicted by immersion of the paw into 100°C water for five sec, and measurements were repeated at regular intervals for a minimum of 4 hr. Burn injury resulted in significant and persistent increases in Q̇L (154 ± 61 μl/min versus 562 ± 105 μl/min 4 hr postburn) and lymph total protein concentration (1.34 ± 0.04 g/dl versus 4.08 ± 0.18 g/dl 4 hr postburn), while lymph hyaluronan concentration fell (3.01 ± 0.20 μg/ml versus 2.1 ± 0.16 μg/ml 4 hr postburn). The resultant increase in hyaluronan flux (0.42 ± 0.13 μg/min versus 1.17 ± 0.22 μg/min 4 hr postburn) appears to be a function of lymph flow rather than burn-induced release of skin hyaluronan. Hence, the increased plasma concentration of hyaluronan following major burns is likely a consequence of increased lymph flow from the site of injury.

Effect of Interleukin-1 and Insulin Like Growth Factor-1 on the Release of Proteoglycan Components and Hyaluronan from Pig Articular Cartilage in Explant Culture

The turnover of proteoglycans was investigated in articular cartilage in explant culture by analysing the components released into the culture medium. The effect of IL-1± on the release of fragments derived from different proteoglycan domains and of hyaluronan (HA) was determined over 4 days in culture. The effect of IGF-1 (100 ng/ml) on matrix degradation of proteoglycan and its ability to inhibit the effects of IL-1 (10 ng/ml) was also assessed. The rate of release of G1 and G2 globular domains of proteoglycans into the culture medium was determined by radioimmunoassay.In unstimulated control cartilage there was a greater release of proteoglycan G2 domain than of G1 domain suggesting that cleavage occured between them and that some G1 was preferentially retained bound in the matrix. Compared with control cartilage IL-1 stimulated the release of all proteoglycan components and hyaluronan. IL-1 had a greater effect on the release of G1 than on G2 domain, but also resulted in some net loss of these proteins (∼ 45% as detected in the immunoassays). In explants treated with both IL-1 and IGF-1 there was much less release of proteoglycan fragments and evidence for less extensive degradation. IGF-1 was particularly affective in preventing any increase in HA release and also preventing the apparent loss of G 1 and G2 domains. It also partially inhibited the release of G1 and G2 domains and the sulphated glycosaminoglycan fragments. IGF-1 was therefore an effective antagonist of IL-1 action on cartilage. It is not known at what level it blocks the chondrocyte response to IL-1, but it clearly results in the suppression of matrix degradative activity.

Hyaluronan-binding proteins on cultured J 774 macrophages

Cultivated macrophages of murine-line J 774 were found to bind-molecular-weight (molecular weight average approx. 5·10 6) [ 3H]hyaluronan (HA) by a saturable mechanism at 4°C. Half-maximal binding was observed at 7–8 μg/ml (1.4–1.6 nM) and the maximal binding was reached at 30–40 μg/ml. Scatchard plot analysis revealed that approx. 20 000 molecules could bind to each cell with a K d of 1.5 nM. The binding could be effectively inhibited by unlabeled HA. Also chondroitin sulphate inhibited the binding, but only to about 50%. At 37°C the J 774 cells took up and degraded the polysaccharide effectively. Affinity chromatography on HA coupled to agarose of solubilized surface-iodinated J 774 cells, revealed that a protein of approx. 60 kDa, when analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis and autoradiography, could be specifically eluted with HA-oligosaccharides. Our results suggest that J 774 macrophages can bind HA by a mechanism compatible with receptor-binding, and carry a 60 kDa HA-binding protein on their surface. This receptor-binding may mediate uptake and degradation of the polysaccharide and influence the levels and turnover of HA in interstitial fluid as well as the release of HA into the bloodstream.