Adenosine analogs and electromagnetic fields inhibit prostaglandin E2 release in bovine synovial fibroblasts M. De Mattei, Ph.D., K. Varani, Ph.D., F.F.

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Adenosine analogs and electromagnetic fields inhibit prostaglandin E2 release in bovine synovial fibroblasts M. De Mattei, Ph.D., K. Varani, Ph.D., F.F. Masieri, Sc.D., A. Pellati, Sc.D., A. Ongaro, Ph.D., M. Fini, M.D., R. Cadossi, M.D., F. Vincenzi, Sc.D., P.A. Borea, Ph.D., A. Caruso, Ph.D. Osteoarthritis and Cartilage Volume 17, Issue 2, Pages 252-262 (February 2009) DOI: 10.1016/j.joca.2008.06.002 Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Bovine SFs in culture. (A) Phase contrast. (B) Vimentin expression by immunofluorescence. Nuclei were counterstained in blue with DAPI. (C) CD14 mRNA expression in macrophages (MC) and in bovine SFs (C, upper panel). M is 100bp DNA ladder marker (Biolabs). One microgram of total RNA was loaded for lane and stained with ethidium bromide to confirm equal RNA quantity used for RT-PCR (C, lower panel). Osteoarthritis and Cartilage 2009 17, 252-262DOI: (10.1016/j.joca.2008.06.002) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Effects of increasing doses of TNF-α and LPS on PGE2 production in bovine SFs. TNF-α and LPS induced a dose response increase on PGE2 levels. PGE2 levels were measured after 24h treatment. Values are expressed as mean±S.E. (n=5). * Indicates statistical significance vs control (C). For each stimulus, ** indicates statistical significance vs the previous dose. Differences were considered significant at P<0.05. Osteoarthritis and Cartilage 2009 17, 252-262DOI: (10.1016/j.joca.2008.06.002) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Effects of adenosine receptor agonists and EMFs on basal and TNF-α- or LPS-induced PGE2 production in bovine SFs in the presence of endogenous adenosine. Adenosine agonists and EMFs did not modify basal PGE2 production (A) and inhibited PGE2 release in TNF-α-treated (B) or LPS-treated (C) SFs, SF treatments in the absence of EMFs; ▪ SF treatments in the presence of EMFs. Values are expressed as mean±S.E. (n=6). * Indicates statistical significance vs control (C). ▴ Indicates statistical significance vs the same treatment in the absence of EMFs. ○ Indicates statistical significance vs inflammatory stimuli (TNF-α (10ng/ml), B; LPS (1μg/ml), C). Differences were considered significant at P<0.05. Osteoarthritis and Cartilage 2009 17, 252-262DOI: (10.1016/j.joca.2008.06.002) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Effects of depletion of endogenous adenosine with increasing doses of ADA (0.5–4IU/ml) on PGE2 production (A) and cell proliferation/viability evaluated by MTT test (B). Values are expressed as mean±S.E. (n=5).* Indicates statistical significance vs control (C). Differences were considered significant at P<0.05. Osteoarthritis and Cartilage 2009 17, 252-262DOI: (10.1016/j.joca.2008.06.002) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Effects of adenosine receptor agonists and EMFs on TNF-α- or LPS-induced PGE2 production in bovine SFs in the presence of ADA (2IU/ml). Depletion of endogenous adenosine with ADA enhanced adenosine agonists effects but limited EMF-inhibitory effects on PGE2 release in TNF-α-treated (A) or LPS-treated (B) SFs, SF treatments in the absence of EMFs; ▪ SF treatments in the presence of EMFs. Values are expressed as mean±S.E. (n=6). * Indicates statistical significance vs control (C). ○ Indicates statistical significance vs inflammatory stimuli (TNF-α (10ng/ml), A; LPS (1μg/ml), B). Differences were considered significant at P<0.05. Osteoarthritis and Cartilage 2009 17, 252-262DOI: (10.1016/j.joca.2008.06.002) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Effects of adenosine receptor agonists and EMFs on control and TNF-α (upper panel) or LPS (lower panel) induced COX-2 expression evaluated by RT-PCR, at 24h treatment. GAPDH expression was used as control gene. M is DNA molecular weight marker VIII (Roche Diagnostics GmbH, Germany). Osteoarthritis and Cartilage 2009 17, 252-262DOI: (10.1016/j.joca.2008.06.002) Copyright © 2008 Osteoarthritis Research Society International Terms and Conditions