Inhibition of experimental metastasis and cell adhesion of murine melanoma cells by chondroitin sulfate-derivatized lipid, a neoproteoglycan with anti-cell adhesion activity.

Chondroitin sulfate dipalmitoylphosphatidylethanolamine (CS-PE), when immobilized onto substratum, inhibited the adhesion of B16F10 mouse melanoma cells to fibronectin-coated dishes (anti-adhesion activity). CS-PE showed the most potent anti-adhesion activity for the melanoma cells among various GAG-PEs. CS-PE also inhibited the adhesion of B16F10 cells to Matrigel and the invasion of the cells into Matrigel. In the in vivo system of experimental metastasis, administration of B16F10 cells with CS-PE into C57BL/6 mice significantly inhibited lung metastasis. The inhibition degree of CS or hyaluronic acid-PE was lower than CS-PE. CS-PE administered intravenously into mice before the injection of B16F10 cells also inhibited metastasis. Pretreatment of B16F10 cells with CS-PE caused some but a lower degree of inhibition. When CS-PE was injected intravenously into mice, more binding in the lung was found than when CS was injected. CS-PE but not CS inhibited the retention in the lung of fluorochrome-labeled B16F10 cells when injected intravenously into mice. Since there was no significant effect of CS-PE on the viability and growth of B16F10 cells, the results suggest that CS-PE immobilized onto the subendothelial matrix may prevent melanoma cells from adhering to the subendothelial substrata of lung capillaries and inhibit subsequent invasion processes of metastasis.

Antithrombotic activity of avian crown dermatan sulfate.

The antithrombotic activity of dermatan sulfate from avian crown with the mean molecular weight of 38000 was compared to those from bovine intestine with the mean molecular weight of 16000 in vivo and in vitro. In an in vitro test, bovine intestine dermatan sulfate exhibited stronger effects on stimulation of heparin cofactor II and activation of Glu-plasminogen by tissue plasminogen activator. In vivo, avian crown dermatan sulfate more effectively prevented the development of thrombus in a rat deep vein thrombosis model. The measurement of plasma levels of these two kinds of dermatan sulfate revealed that avian crown dermatan sulfate circulated in higher concentration and longer duration than bovine intestine dermatan sulfate after intravenous administration to rats.

Contradictory functions of sulfatide in the blood coagulation system as coagulant and anticoagulant.

Sulfatide (galactosylceramide I3 -sulfate) has been reported to activate blood coagulation factor XII (Hageman factor), which suggests that it exhibits coagulant activity (Fujikama et al., 1980 Biochemistry 19, 1322-1330) However, sulfatide administered into animals as a bolus shot without subsequent thrombus formation, prolonged conventional clotting times and bleeding time (Hara et al., 1996 Glycoconjugate J. 13, 187-194). These findings suggest that it may exhibit anticoagulant rather than coagulant activity. Following this suggestion we found in vitro that binding of sulfatide to fibrinogen resulted in disturbance of fibrin formation. To examine a possible pharmacological effect of sulfatide on blood coagulation in vivo we continuously infused sulfatide into rats through plastic cannulae and found formation of giant thrombi around the tips of the cannulae. These data suggest that sulfatide may exhibit contradictory functions in the blood coagulation system.

Roles of galactose and sulfate residues in sulfatides for their antagonistic functions in the blood coagulation system.

We previously reported that the sulfatide (galactosylceramide I3-sulfate) may have contradictory functions, namely both coagulant and anticoagulant roles in vivo: sulfatide induced giant thrombi formation when injected into rats with vein ligation, whereas no thrombi were formed when sulfatide was injected into rats without vein ligation. Rather it prolonged bleeding time. To investigate the structural features of sulfatide for both functions, a synthetic sulfatide (galactosylceramide I6-sulfate) which does not occur naturally, cholesterol 3-sulfate and ganglioside GM4 were examined together with naturally occurring sulfatide. Both sulfatides and cholesterol 3-sulfate induced giant thrombi in the rats with vein ligation within ten minutes of injection, although cholesterol 3-sulfate exhibited weaker coagulant activity than the sulfatides. On the contrary, both sulfatides significantly prolonged bleeding time but cholesterol 3-sulfate barely prolonged it when injected without vein ligation. GM4 exhibited neither coagulant nor anticoagulant activity. These results suggested that sulfate moiety in the sulfatides is essential for coagulant activity and that galactose residue enhances the activity, whereas both galactose and sulfate residues seem to be important for anticoagulant activity. This is because the sulfatides possess both residues but GM4 possesses galactose without sulfate and cholesterol 3-sulfate possesses sulfate without galactose. We previously reported that the possible mechanism of anticoagulation by sulfatide was due to its binding to fibrinogen, thereby inhibiting the conversion to fibrin. In this paper we reveal that both sulfatides inhibited thrombin activity independent of heparin cofactor II, thus providing evidence of another anticoagulation mechanism for the sulfatides.

Sulfatide can markedly enhance thrombogenesis in rat deep vein thrombosis model.

Although sulfatide (galactosylceramide I3-sulfate) has been reported to activate blood coagulation factor XII (Hageman factor), it has been administered to animals without subsequent thrombus formation. We recently found that sulfatide binds to fibrinogen and thus disturbs fibrin formation in vitro, suggesting its possible role as an anticoagulant rather than as a coagulant. We therefore examined the in vivo effects of sulfatide on thrombogenesis by using a rat deep vein thrombosis model in which thrombus is induced by ligating the inferior vena cava. Sulfatide and gangliosides were each separately administered to rats 1 min before the vein ligation, and after 3 h, sulfatide but not gangliosides markedly (P < .001) enhanced the thrombogenesis. A kinetic turbidmetric assay of plasma coagulation initiated by CaCl2 in the wells of a microtiter plate revealed that coagulation was also markedly accelerated in the presence of sulfatide but not gangliosides, the results of which seemed to be very consistent with those of the in vivo experiments. Because sulfatide could not induce thrombosis without vein ligation in rats, the enhancement of thrombogenesis by sulfatide in the in vivo model might require endothelial damage and/or venous congestion, both of which could be induced by vein ligation.

[Experimental knee pain model in rats and analgesic effect of sodium hyaluronate (SPH)].

Using the model of knee pain reaction induced by intra-articular injection of endogenous pain substances, especially bradykinin (BK) in rats, the mechanism of the analgesic effect of sodium hyaluronate (SPH) was investigated. The simultaneous administration of prostaglandin E2 with BK or hyaluronidase digestion of endogenous hyaluronic acid (HA) in our experiments brought remarkable hyperalgesia on BK-induced knee pain. These results suggest that higher sensitivity to the pain reaction is induced in a diseased joint (higher prostaglandin content, lower concentration and molecular size of HA in synovial fluid) than in a normal one. SPH definitely decreased BK-induced pain, and its analgesic effect was observed for a longer period, depending on its dose in pre-treatment and the degree of its distribution in synovial tissues. As the analgesic effect of SPH was observed in the hyaluronidase-treated joint as well, it is suggested that the increasing viscosity of synovial fluid caused by increasing HA concentration can decrease the pain even without normalizing molecular size of HA in the joint. HA oligomer and other compounds with similar viscosity or with similar polyanionic character as SPH showed no analgesic effect. From these results, it seems that the characteristic steric configurations of higher molecular HA are needed for the manifestation of the analgesic effect. SPH seems to show its analgesic effect by covering pain receptors in synovial tissues and holding endogenous pain substances in its molecule.

Effects of the molecular weight of hyaluronic acid and its action mechanisms on experimental joint pain in rats.

OBJECTIVES: It has been shown previously that hyaluronic acid (HA) has an analgesic action on bradykinin induced pain in the knee joints of rats. This study further clarifies the effects of the molecular weight of HA and its mechanism of action in the same model using HA of molecular weight 800 to 2.3 x 10(6) daltons and a bradykinin antagonist. METHODS: Bradykinin and the test HA preparations were given to rats by intra-articular injection, and the severity of pain was evaluated by a change in the walking behaviour. RESULTS: HA with a molecular weight greater than 40 kilodaltons produces analgesic effects with a simultaneous or earlier injection. The ID50 values of HA with molecular weight 40, 310, 860, and 2300 kilodaltons were greater than 2.5, 0.6, 0.07, and 0.06 mg/joint respectively. The duration of the analgesic effect of 860 and 2300 kilodalton HA was 72 hours at 10 mg/ml, whereas that of 310 kilodalton HA was short, being undetectable after 24 hours. The analgesic action of HA of 860 kilodaltons was not changed by pretreatment with four saccharide HA and inhibited by pretreatment with HA larger than six to eight saccharides, capable of binding to HA receptors. Further, HA did not interfere with the analgesic action of the bradykinin antagonist, indicating that HA does not directly bind with bradykinin receptors. CONCLUSIONS: HA with a molecular weight of greater than 40 kilodaltons produced an analgesic effect, and HA of 860 and 2300 kilodaltons produced high and long-lasting analgesia. These effects of HA appear to be caused by the interaction between HA and HA receptors.

Alterations of proteoglycan synthesis in rabbit articular cartilage induced by intra-articular injection of papain.

In order to investigate the biochemical alteration of proteoglycan (PG) synthesis during cartilage repair, reversible destruction was induced by injecting papain into the knee joint cavity of rabbits. The PG synthesis in the cartilage was examined using Na2 35SO4 and high performance liquid chromatography (HPLC). PGs labeled with 35SO4(2-) (35S-PGs) were extracted from normal and papain-treated cartilage, and the amount of synthesis, ability to aggregate with hyaluronan (HA), and the composition of glycosaminoglycan and chondroitin sulfate isomer labeled with 35SO4(2-) (35S-GAG and 35S-CS isomer) were analyzed. Synthesis of 35S-PGs, especially those that were unable to aggregate with HA (nonaggregating 35S-PGs), increased in papain-treated cartilage compared with that in normal cartilage. The acceleration and qualitative change in PG synthesis in the papain-treated cartilage are considered to be responses to the supplementation of the loss of cartilage PGs induced by papain. The compositions of 35S-GAG and 35S-CS isomer of the nonaggregating 35S-PGs differed from those of 35S-PGs which were able to aggregate with HA (aggregating 35S-PGs) in the papain-treated cartilage as well as in the normal cartilage. However, the compositions of both nonaggregating and aggregating 35S-PGs in the papain-treated and normal cartilage were similar. These results indicate that most of the nonaggregating 35S-PGs in papain-treated cartilage have properties similar to those in normal cartilage and are not simple degradation products of aggregating 35S-PGs; they also suggest that the supplementary reaction for PG content in the cartilage during its repair process is not simple acceleration in PG turn-over but the enhancement of PG synthesis accompanied by alterations in aggregating ability and the compositions of GAG and CS isomer.

Effects of dermatan sulfate, a heparin cofactor II mediated thrombin inhibitor, on the endotoxin-induced disseminated intravascular coagulation model in the rat: comparison with low-molecular weight heparin, nafamostat mesilate and argathroban.

Effects of dermatan sulfate (DS) on the endotoxin-induced disseminated intravascular coagulation (DIC) rat model were compared with those of low-molecular weight heparin (LMWH), nafamostat mesilate (NM) and argathroban (AR). At doses of 5, 10 or 20 mg/kg/4 hr, DS significantly ameliorated the decrease of fibrinogen (Fbg), the increase of fibrin-fibrinogen degradation products (FDP) and except at the highest dose (20 mg/kg/4 hr), the prolongation of thrombin clotting time (TCT). It also decreased the glomerular fibrin deposits (%GFD) at doses of 10 or 20 mg/kg/4 hr. LMWH suppressed the decrease of Fbg and the increase of FDP at doses of 1.4 or 2.8 mg/kg/4 hr. Only the highest dose of LMWH suppressed the decrease of the platelet count (PL), the prolongation of prothrombin time, and improved the %GFD. AR suppressed the decrease of PL and improved the %GFD. At the dose required to improve the %GFD, DS (5, 10 mg/kg/4 hr) significantly suppressed the prolongation of TCT, which is related to the bleeding frequency, while LMWH and AR further increased the prolongation of the TCT. These results suggest that DS has potential as a therapeutic drug with a lower hemorrhagic risk as compared with LMWH and AR in the treatment of DIC.