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. 2018 Dec;36(12):3247-3255.
doi: 10.1002/jor.24126. Epub 2018 Sep 7.

High Molecular Weight Hyaluronan Protects Cartilage From Degradation by Inhibiting Aggrecanase Expression

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Free PMC article

High Molecular Weight Hyaluronan Protects Cartilage From Degradation by Inhibiting Aggrecanase Expression

Takashi Ohtsuki et al. J Orthop Res. .
Free PMC article

Abstract

Hyaluronan (HA) is an extracellular matrix (ECM) component of articular cartilage and has been used to treat patients with osteoarthritis (OA). A disintegrin and metalloproteinases with thrombospondin motifs (ADAMTSs) play an important role in cartilage degradation in OA. We have previously reported that ADAMTS4 and ADAMTS9 were induced by cytokine stimulation. However, the effect of HA on the cytokine-inducible ADAMTS9 has never been investigated. Moreover, it is unclear whether HA protects cartilage by suppressing aggrecan degradation. Here, we examined the effects of HA on ADAMTS expression in vitro and on cartilage degradation in vivo. ADAMTS9 expression was higher than that of the other aggrecanases (ADAMTS4 and 5) in human chondrocytes, chondrocytic cells, and rat cartilage. ADAMTS4 and 9 mRNA levels were upregulated in cytokine-stimulated chondrocytes and chondrocytic cells. Pre-incubation with HA significantly inhibited ADAMTS9 mRNA expression in cytokine-stimulated cells. In a rat OA model, Adamts5 and 9 mRNA levels were transiently increased after surgery; intra-articular HA injections attenuated the induction of Adamts5 and 9 mRNA. HA also blocked aggrecan cleavage by aggrecanase in OA rats in a molecular size-dependent manner. These results demonstrate that HA attenuates induced aggrecanases expression in OA and thereby protects articular cartilage degradation by this enzyme. Our findings provide insight into the molecular basis for the beneficial effects of HA in OA. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 36:3247-3255, 2018.

Keywords: aggrecan; chondrocyte; hyaluronic acid; metalloproteinase.

Figures

Figure 1
Figure 1
Cytokine‐induced ADAMTS expression in chondrocytes and chondrocytic cells. (A) NHAC‐kn and (B) OUMS‐27 cells were stimulated for 0–48 h with 10 ng/ml IL‐1β and TNFα. ADAMTS4, 5, and 9 transcript levels were evaluated relative to levels in unstimulated cells by qRT‐PCR. Basal expression of ADAMTS4, 5, and 9 mRNA determined by qRT‐PCT were represented as threshold cycles (Ct) on the top of graph. Values represent mean ± S.D. (n = 6 per group). Open bars, ADAMTS4; solid bars, ADAMTS5; hatched bars, ADAMTS9. *p < 0.05 versus control.
Figure 2
Figure 2
HA suppresses ADAMTS/MMP mRNA expression in cytokine‐stimulated chondrocytic cells. OUMS‐27 cells were pretreated with or without 1.0 mg/ml HA2700 for 5 h, then stimulated with 10 ng/ml IL‐1β and TNFα for 6 h. Open bars; nontreatment, dotted bars; cytokine stimulation for 6 h without HA pretreatment, horizontal bars; HA pretreatment; and cytokine stimulation for 6 h. HA pretreatment of OUMS‐27 significantly attenuated ADAMTS4, ADAMTS9, and MMP‐13 mRNA expression induced by cytokines. The decline ratio was 27%, 27%, 35%, and 34% of ADAMTS4 mRNA, ADAMTS9 mRNA, and MMP‐13 mRNA respectively.
Figure 3
Figure 3
OARSI score and ADAMTS mRNA expression in a rat OA model. (A) Histological analysis of medial femoral condyle cartilage of OA rats assigned an OARSI score after surgery (n = 10 per group). *p < 0.05 versus sham‐operated rats. (B) ADAMTS4, 5, and 9 mRNA expression was determined by qRT‐PCR (n = 10 per group). Basal expression of ADAMTS4, 5, and 9 mRNA determined by qRT‐PCR were represented as threshold cycles (Ct) on the top of graph. Total RNA was extracted 0, 1, 2, 3, and 4 week(s) after knee surgery. Open bars; ADAMTS4, closed bars; ADAMTS5, hatched bars; ADAMTS9. Levels of ADAMTS5 and 9 mRNA were upregulated in OA rats. * and # indicate p < 0.05 versus sham‐operated rats.
Figure 4
Figure 4
Intra‐articular HA injection attenuates ADAMTS5 and ADAMTS9 mRNA expressions in a rat OA model. HA was injected twice (days 5 and 7 post‐surgery) into the knee joint and mRNA levels were determined on day 10. ADAMTS4, 5, and 9 transcript levels were compared between rats with and without HA2700 injection (n = 10 per group). The expression level of mRNA in sham‐operated rat was indicated as 1.0 and relative mRNA expression level in each OA rats was indicated as—fold change. Open bars, sham operation; dot bars, OA operation with PBS‐injection; horizontal stripe bars, OA operation with HA‐injection. *p < 0.05 versus PBS‐injected rats. Please note that the levels of ADAMTS5 and 9 were suppressed by intra‐articular HA injection.
Figure 5
Figure 5
Intra‐articular HA injection protects against knee cartilage destruction in a rat OA model. (A) Influence of HA size. Rats were divided into five treatment groups (sham, PBS, HA300, HA800, and HA2700: n = 10 per group). The left panel shows OARSI scores 6 weeks post‐surgery. *p < 0.01 versus PBS‐injected rats; # and ## p < 0.05 versus other HA‐injected rats. OARSI scores were lower in rats receiving intra‐articular HA injection; the effect was HA size‐dependent. Effects of intra‐articular HA injection (twice weekly for 6 weeks) were examined by immunohistochemistry using an anti‐NITEGE antibody (a–e) and by Safranin O staining (f–j). (B) The left panel shows OARSI scores 6 weeks post‐surgery. **p < 0.01 versus PBS‐injected rats. OARSI scores with short‐term intra‐articular HA injection (from days 5 to 19 after surgery) were lower as compared with that of PBS‐injected cartilage (n = 10 per group). Please note that the impact of short‐term HA injection (five injections within 19 days post‐surgery) was comparable with that of long‐term HA injection (eleven injections within 6 weeks) in rat OA cartilages as measured by OARSI scoring system. The cartilage destruction by ADAMTS was examined by immunohistochemistry using an anti‐NITEGE antibody (a and b) and by Safranin O staining (c and d). Scale bar, 30 µm.

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