Chondroitin sulfate increases hyaluronan production by human synoviocytes through differential regulation of hyaluronan synthases: Role of p38 and Akt.

OBJECTIVE: To uncover the mechanism by which chondroitin sulfate (CS) enhances hyaluronan (HA) production by human osteoarthritic (OA) fibroblast-like synoviocytes (FLS). METHODS: The production of HA was investigated by exposing human OA FLS to CS in the presence or absence of interleukin-1beta (IL-1beta). HA levels were determined by enzyme-linked immunosorbent assay, and levels of messenger RNA (mRNA) for HA synthase 1 (HAS-1), HAS-2, and HAS-3 were determined by real-time polymerase chain reaction analysis. The effect of CS and IL-1beta on signaling pathways was assessed by Western blotting. Specific inhibitors were used to determine their effects on both HA production and HAS expression. The molecular size of HA was analyzed by high-pressure liquid chromatography. RESULTS: CS increased HA production by FLS through up-regulation of the expression of HAS1 and HAS2. This was associated with activation of ERK-1/2, p38, and Akt, although to a lesser extent. Both p38 and Akt were involved in CS-induced HA accumulation. IL-1beta increased HA production and levels of mRNA for HAS1, HAS2, and HAS3. CS enhanced the IL-1beta-induced level of HAS2 mRNA and reduced the level of HAS3 mRNA. IL-1beta-induced activation of p38 and JNK was slightly decreased by CS, whereas that of ERK-1/2 and Akt was enhanced. More high molecular weight HA was found in CS plus IL-1beta-treated FLS than in FLS treated with IL-1beta alone. CONCLUSION: CS stimulates the synthesis of high molecular weight HA in OA FLS through up-regulation of HAS1 and HAS2. It reduces the IL-1beta-enhanced transcription of HAS3 and increases the production of HA of large molecular sizes. These effects may be beneficial for maintaining viscosity and antiinflammatory properties in the joint.

Differential effects of hyaluronan and its fragments on fibroblasts: relation to wound healing.

Hyaluronan (HA) is involved in wound healing and its biological properties depend on its molecular size. The effects of native HA and HA-12 and HA-880 saccharide fragments on human fibroblast proliferation and expression of matrix-related genes were studied. The three HA forms promoted cell adhesion and proliferation. Matrix metalloproteinase-1 and -3 mRNA were increased by all HA forms, whereas only HA-12 stimulated the expression of the tissue inhibitor of metalloproteinase 1. HA-12 enhanced type I collagen and transforming growth factor-beta (TGF-beta) 1 expression. Interestingly, HA-12 and native HA stimulated type III collagen and TGF-beta3. HA and its fragments activated Akt and extracellular-regulated kinases 1/2 and p38. Inhibition of these signaling pathways suggested their implication in most of the effects. Only native HA activated nuclear factor-kappaB and activating protein 1. Use of CD44 siRNA suggests that this HA receptor is partly implicated in the effects, although it does not rule out the involvement of other receptors. Depending on its size, HA may exert differential regulation on the wound-healing process. Furthermore, the HA up-regulation of type III collagen and TGF-beta3 expression suggests that it may promote a fetal-like cell environment that favors scarless healing.

Comparative effects of 2 antioxidants, selenomethionine and epigallocatechin-gallate, on catabolic and anabolic gene expression of articular chondrocytes.

OBJECTIVE: . To determine the effects of selenomethionine (Se-met) and epigallocatechin-gallate (EGCg) on gene expression, activation of mitogen-activating kinases, and DNA binding of nuclear factor-kappaB (NF-kappaB) and apolipoprotein-1 (AP-1) in articular chondrocytes. METHODS: Chondrocytes, cultured in low-oxygen tension, were pretreated with L-selenomethionine or EGCg for 24 h, followed by interleukin 1 (IL-1beta) for 1 h (nuclear and cytoplasmic extracts) or 24 h (RNA extraction). Reverse transcription-polymerase chain reaction was performed to determine mRNA levels of matrix metalloproteinases (MMP-1, -3, -13), aggrecanases (-1, -2), IL-1beta, inducible nitric oxide synthase, cyclooxygenases (-1, -2), type II collagen and aggrecan, and transforming growth factor-beta (TGF-beta1, -2, -3) and their receptors I and II. Activity of mitogen-activating protein kinases (MAPK) was assayed by Western blot and AP-1/NF-kB DNA binding by electrophoretic mobility shift assay. RESULTS: Pretreatment with 0.5 microM Se-met prevented IL-1beta-induced MMP-1 and aggrecanase-1 expression, and reduced the cytokine inhibitory effect on type II collagen, aggrecan core protein, and TGF-beta receptor II (TGF-betaRII) mRNA levels. EGCg was more efficient in modulating the effects of IL-1beta on the genes studied. Whereas EGCg inhibited the IL-1beta-activated MAPK, NF-kappaB, and AP-1, Se-met stimulated that signaling pathway. This could account for the differential effects exerted by these antioxidants on chondrocytes. CONCLUSION: Our data provide insights into the mechanisms whereby ECGg and selenium modulate chondrocyte metabolism. Despite their differential mechanisms of action, the 2 compounds may exert global beneficial effects on articular cartilage.

Inhibition of arterial cells proliferation in vivo in injured arteries by hyaluronan fragments.

It has been demonstrated previously that administration of high levels of high molecular mass hyaluronan (hyaluronic acid, HA) to rats was able to reduce in a significant way neointima formation in the injured arteries. In the present study, our aim was to verify whether small forms of HA (4-16 saccharides) are still able to reduce the proliferative response of ASMC to aortic injury. Treated rats received a total of 8 injections of a fixed dose of HA fragments (27 mg/kg rat contained in a volume of 550 microl). Two injections were given on the day of balloon catheter injury (BCI): one, intravenous, 10 min before BCI and one, subcutaneous, immediately after the BCI. The others injections (subcutaneous) were at 2, 4, 6, 8, 10 and 12 days after BCI. Control rats received an equivalent volume of the dissolving buffer containing only hyaluronidase, which has been destroyed before injection to rats. Neointima formation was analysed 14 days after the BCI. Intima-media wet weight and DNA content were significantly reduced in rats receiving HA fragments in comparison to controls (2P=0.01 for wet weight and 0.03 for DNA). This finding was confirmed by the histomorphometric study which showed that both neointima area and the ratio neointima/neointima+media were significantly decreased in treated rats (2P=0.03 for intima area and 0.049 for the ratio). Our data showed thus and for the first time that administration of HA fragments with a very low molecular mass (4-16 saccharides) reduces the proliferative reaction of aorta to injury in vivo. In conclusion, HA fragments, which are components with an excellent safety profile, may offer hope for the prevention of restenosis after angioplasty.