High molecular weight hyaluronic acid down-regulates the gene expression of osteoarthritis-associated cytokines and enzymes in fibroblast-like synoviocytes.

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High molecular weight hyaluronic acid down-regulates the gene expression of osteoarthritis-associated cytokines and enzymes in fibroblast-like synoviocytes from patients with early osteoarthritis C.-T. Wang, M.D., Y.-T. Lin, M.D., Ph.D., B.-L. Chiang, M.D., Ph.D., Y.-H. Lin, M.Sc., S.-M. Hou, M.D., Ph.D., M.P.H. Osteoarthritis and Cartilage Volume 14, Issue 12, Pages 1237-1247 (December 2006) DOI: 10.1016/j.joca.2006.05.009 Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 1 Phenotypic features of synovial fluid-derived FLS (passages 4 through 6). (A) Light microscopy showed that FLS were homogeneous and spindle-shaped in morphology. (B) Flow cytometric analysis showed that more than 90% of FLS were positively stained with anti-fibroblast surface molecule mAb D7-FIB. (C) Immunocytochemistry revealed that the majority of FLS were positively stained with anti-prolyl-4-hydroxylase mAb 5B5. (D) Immunocytochemistry of negative control staining using isotype-matched control IgG did not display a positive reaction. Osteoarthritis and Cartilage 2006 14, 1237-1247DOI: (10.1016/j.joca.2006.05.009) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 2 Melting curve analysis and agarose gel electrophoresis of real-time Q-PCR assays. (A) Melting curve analysis showed a single melting peak in each real-time Q-PCR assay. (B) Agarose gel electrophoresis also showed a single PCR product in each real-time Q-PCR assay. Osteoarthritis and Cartilage 2006 14, 1237-1247DOI: (10.1016/j.joca.2006.05.009) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 3 Standard curves and amplification efficiencies of real-time Q-PCR assays. The standard curves of all real-time Q-PCR assays showed high linearity over a wide range of starting copy numbers of cDNA templates. The equation represents the relationship of the threshold cycle value (CT) and the log value of starting copy number of the sample from the dilution series. R2=correlation coefficient; Eff=calculated amplification efficiency. Osteoarthritis and Cartilage 2006 14, 1237-1247DOI: (10.1016/j.joca.2006.05.009) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 4 Effect of IL-1 (1ng/ml) stimulation on gene expression in FLS. Values are expressed in fold in comparison with control group after normalization to housekeeping gene. Data are expressed as mean±s.e.m. *P<0.05. Osteoarthritis and Cartilage 2006 14, 1237-1247DOI: (10.1016/j.joca.2006.05.009) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 5 Dose-response study of the effect of HMW-HA (10μg/ml, 100μg/ml, and 1mg/ml) on gene expression in FLS. (A) Effect of HMW-HA on gene expression in FLS without IL-1 stimulation. Values are expressed in fold in comparison with control group after normalization to housekeeping gene. (B) Effect of HMW-HA on gene expression in FLS with IL-1 (1ng/ml) stimulation. Values are expressed in fold in comparison with IL-1 group after normalization to housekeeping gene. Data are expressed as mean±s.e.m. Osteoarthritis and Cartilage 2006 14, 1237-1247DOI: (10.1016/j.joca.2006.05.009) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 6 Effect of HMW-HA (100μg/ml) on gene expression in FLS. (A) Effect of HMW-HA on gene expression in FLS without IL-1 stimulation. Values are expressed in fold in comparison with control group after normalization to housekeeping gene. (B) Effect of HMW-HA on gene expression in FLS with IL-1 (1ng/ml) stimulation. Values are expressed in fold in comparison with IL-1 group after normalization to housekeeping gene. Data are expressed as mean±s.e.m. *P<0.05. **There was significant difference between IL-1 group and control group (Fig. 4), but there was no significant difference between “IL-1+HMW-HA” group and control group. Osteoarthritis and Cartilage 2006 14, 1237-1247DOI: (10.1016/j.joca.2006.05.009) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 7 Effect of CD44 blocking on gene expression in FLS. FLS were stimulated with IL-1 (1ng/ml) and treated with HMW-HA (100μg/ml) with or without pretreatment with anti-CD44 blocking mAb 5F12 (20μg/ml) or isotype-matched control IgG (20μg/ml). Values are expressed in fold in comparison with IL-1 group after normalization to housekeeping gene. Osteoarthritis and Cartilage 2006 14, 1237-1247DOI: (10.1016/j.joca.2006.05.009) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions