Interactions of pentosan polysulfate with cartilage matrix proteins and synovial fibroblasts derived from patients with osteoarthritis.

Pentosan polysulfate (PPS) has been shown to improve symptoms of patients with osteoarthritis (OA) when studied under double-blinded conditions. Laboratory studies indicated that this drug exhibits multiple actions, including the preservation of articular cartilage (AC) proteoglycans in animal models of OA and the stimulation of hyaluronan synthesis by synovial fibroblasts in vitro and in vivo. As PPS is strongly anionic and has a molecular weight of approximately 5700 Da its ability to enter connective tissues rich in proteoglycans and interact with the resident cells has been questioned. In the present studies, experiments were undertaken to isolate and characterize proteins in human AC which have the potential to bind PPS. Thrombospondin was identified in 4.0 M GuHCl extracts of human AC as a PPS-binding protein. Furthermore, synovial fibroblasts derived from OA joints were shown to secrete thrombospondin and also bind PPS. Using bovine erythrocytes conjugated with PPS a rosetting of the synovial fibroblast could be demonstrated. The level of rosetting was not affected by pre-incubating cultures with thrombospondin antibody suggesting that PPS was interacting directly with the cells. Kinetic studies of 3H-PPS uptake by synovial fibroblasts showed saturation of binding sites within 30 min when cells were maintained at 4 degrees C but preservation of drug uptake for up to 120 min when cells were cultured at 37 degrees C. These data, together with the finding that cells labeled with drug at 37 degrees C showed higher incorporation, than at 4 degrees C after trypsin digestion suggests that PPS first binds to the cell membrane when at 37 degrees C is internalized, possibly by pinocytosis.

Interactions of hyaluronan (hyaluronic acid) with phospholipids as determined by gel permeation chromatography, multi-angle laser-light-scattering photometry and 1H-NMR spectroscopy.

The chain flexibility of solutions of hyaluronan (HA) of different molecular weights was determined by 1H-NMR spectroscopy in the absence and presence of the phospholipid dipalmitoyl-D,L-alpha-phosphatidylcholine (DPC). Sonication of high- or low-molecular-weight HA with DPC for periods of up to 120 min markedly increased HA chain flexibility as determined by observing the half-peak linewidths (delta V1/2) for the methyl protons of the acetamidodeoxyglucose residues of the HA molecules. Gel permeation chromatography of mixtures of purified high-molecular-weight HA (Healon) with 3H-DPC or 3H-platelet activating factor (PAF) showed exclusion of these radioactively labelled molecules from the gel in the presence of HA but not in its absence. Studies using multi-angle laser-light-scattering (MALLS) photometry of sonicates of DPC and Healon after Superose 6 chromatography revealed increases in HA Mw, Mn, Mz and their corresponding root mean square radii relative to control sonicates of HA without DPC. From these data, we have deduced that DPC binds to HA by competing for those hydrophobic centres along the HA chain which are normally responsible for the inter- and intra-chain interactions and which confer stiffness to the HA molecule. It is proposed that such interactions in arthritic joints could reduce synovial fluid viscoelasticity thereby diminishing the ability of this medium to protect articular cartilage from mechanical injury.

Pentosan polysulphate and glycosaminoglycan polysulphate stimulate the synthesis of hyaluronan in vivo.

Pentosan polysulphate (PPS) and glycosaminoglycan polysulphate (GAGPS) were examined for their ability to alter hyaluronan synthesis in vivo. The inflamed rat subcutaneous air pouch model was used for the study. PPS or GAGPS injected into the air pouch at a dose of 2.5 mg/kg daily for 7 days resulted in higher molecular weight hyaluronan in the pouch fluid compared with control non-drug-treated pouch fluid. The quantity of the hyaluronan was increased by PPS, but not by GAGPS. We concluded that both drugs could be beneficial in the treatment of inflammatory arthritides in which a decrease in normal synovial hyaluronan concentration and molecular weight occurs.

Effects of hydrogen peroxide on the metabolism of human rheumatoid and osteoarthritic synovial fibroblasts in vitro.

The effects of hydrogen peroxide (H2O2) on the metabolism of cultured human synovial fibroblasts derived from joints of four patients with rheumatoid arthritis and three with osteoarthritis have been investigated. The exposure of rheumatoid cell cultures to this oxygen derived species at sublethal concentrations (1-100 mumol/l) induced a dose related inhibition of both hyaluronic acid (HA) and DNA synthesis. In contrast, in osteoarthritic cell lines a biphasic response was shown. At low concentrations of H2O2 (less than 10 mumol/l) a stimulatory effect on HA synthesis was noted, whereas in the presence of higher concentrations (greater than 10 mumol/l) a significant inhibition of synthesis occurred. These deleterious effects of H2O2 were partially reduced by the addition of catalase to the culture media. The finding that both HA and DNA synthesis were inhibited at concentrations of H2O2 less than those which caused loss of cell integrity (greater than 200 mumol/l) suggests oxidation of intracellular components, such as glyceraldehyde-3-phosphate dehydrogenase, and subsequent depletion of ATP concentrations.

Binding of haptoglobin, inter-alpha-trypsin inhibitor, and alpha 1 proteinase inhibitor to synovial fluid hyaluronate and the influence of these proteins on its degradation by oxygen derived free radicals.

Synovial fluid from 201 normal and pathological knee joints was subjected to gel filtration by Sepharose CL-2B chromatography to separate hyaluronic acid (HA) from unbound proteins, which were retarded on this column. HA from all normal fluids was excluded from the gel and contained 1% or less bound protein. Synovial fluids taken from joints of patients with rheumatoid arthritis (RA) contained considerably more protein bound to HA. In 46% of RA samples the level of protein was greater than 4%, whereas only one fluid examined from osteoarthritic joints contained this amount. The proteins bound to HA from RA joints were identified by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) and immunodiffusion techniques as the acute phase proteins alpha 1 proteinase inhibitor, inter-alpha-trypsin inhibitor, and haptoglobin. The average relative percentages of these proteins bound to HA were 17.6%, 32.6%, and 29.2% respectively. These HA-protein complexes could be generated in vitro by mixing normal (low protein) HA with any one of the three acute phase proteins. The HA-protein complexes formed in vitro with inter-alpha-trypsin inhibitor or haptoglobin, and those isolated from RA synovial fluids, were more resistant to degradation by oxygen derived free radicals (ODFR) than HA from normal fluids. From these findings we conclude that certain acute phase proteins diffusing into synovial fluid during inflammatory episodes may play an important part in protecting HA from depolymerisation by activated phagocytes.

Liver procollagen prolyl hydroxylase in Opisthorchis viverrini infected hamsters after praziquantel administration.

Infection of hamsters by the human liver fluke Opisthorchis viverrini elevated liver procollagen prolyl hydroxylase activity, reflecting increased collagen biosynthesis. The increase was proportional to the intensity of infection. However, the infected liver procollagen prolyl hydroxylase activity decreased after administration of praziquantel 300 mg kg-1 body weight, and approached normal levels two weeks after treatment. In the infected hamsters, praziquantel, at a curative dose, caused a transient increase in serum aminotransferase levels and a small but persistent rise in serum alkaline phosphatase. The drug, however, did not cause changes in these enzyme activities in the uninfected hamsters.

Increased liver prolyl hydroxylase activity in hamsters infected with the human liver fluke Opisthorchis viverrini.

A parallel increase in liver collagen content and prolyl hydroxylase activity was observed in hamsters infected with the human liver fluke Opisthorchis viverrini. They were elevated at 2 weeks after infection, gradually increased to approximately 2-fold at 7-11 weeks of infection, and then declined as with duration of infection time increasing from 11 to 22 weeks.

Stimulation of Ca2+ uptake in the human liver fluke Opisthorchis viverrini by praziquantel.

45Ca2+ uptake by the human liver fluke Opisthorchis viverrini is enhanced by praziquantel. The drug-induced 45Ca2+ uptake was dependent on the presence of Ca2+ and was attenuated in the presence of 10 mM Mg2+. La3+ and vanadate at concentration of 1 mM partially reduced the amount of 45Ca2+ uptake into the liver fluke in response to praziquantel treatment. The stimulating effect of praziquantel was eliminated in the presence of 10 microM verapamil. These findings suggest that praziquantel increases the permeability of the liver fluke tegument to Ca2+ probably by interfering with the mechanism that regulates Ca2+ binding or transport across the tegumental membrane.

Liver collagen turnover in hamsters during infection by the human liver fluke, Opisthorchis viverrini.

Hamsters infected with Opisthorchis viverrini, a liver fluke of man, showed an increased deposition of collagen in their livers. However, increased collagen breakdown as well as its synthesis were observed in the infected livers. Thus, stimulated synthesis might be the main factor responsible for the net increase in collagen content. Synthesis and degradation increased to a greater extent in short-term infection than in long-term infection whereas the hepatic collagen content was equally elevated in both cases. The results, therefore, suggested a difference in collagen metabolism of short-term and long-term infected livers.