Fig. 7.

Modulation of adenosine metabolism by deoxycoformycin substantially decreases aortic valve thickness and reduces markers of calcification in a mouse model of CAVD. Representative images of 6-month old male wild type (WT) and ApoE−/−LDLR−/− mice aortic valve stained with Oil Red O (ORO) and Orcein Mertius Scarlet Blue (OMSB), AV aortic valve (a). The ratio of average leaflet thickness measured in 3 different places of aortic valve leaflet to leaflet area of WT and ApoE−/−LDLR−/− mice stained with ORO (b). Representative images of WT and ApoE−/−LDLR−/− mice aortic valve stained with immunofluorescence (red signal) for CD39, CD73, eNPP1, ALP, ADA (c). Results are shown as mean ± SEM; n = 3; *p < 0.05 vs. non-stenotic valve by Mann–Whitney test. The rates of ATP hydrolysis (d, g), AMP hydrolysis (e, h) and adenosine deamination (f, i) on the surface of male 3-month-old (d–f) and 10-month-old (g–i) WT and ApoE−/−LDLR−/− mice aortic roots and in the presence of ecto-enzyme inhibitors. Serum ALP activity (j), calcium (k), magnesium (l) and phosphate concentration (m). Results are shown as mean ± SEM; n = 4–6; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. WT by one-way Anova followed by Holm-Sidak post hoc test. Representative images of aortic valve of 6-month old male ApoE−/−LDLR−/− mice treated intraperitoneally with saline or 0.2 mg/kg deoxycoformycin (dCF) twice weekly for 2 months stained with Oil Red O (ORO), AV aortic valve (n). The ratio of average leaflet thickness measured in three different places of aortic valve leaflet to leaflet area of ApoE−/−LDLR−/− mice treated with saline or dCF stained with ORO (o). Aortic root ALP activity (p), serum ALP activity (q), calcium (r), magnesium (s) and phosphate (t) concentration. Results are shown as mean ± SEM; n = 5, *p < 0.05, ***p < 0.001 vs. saline-treated ApoE−/−LDLR−/− mice (−dCF) by Student’s t test. Wild type mice aortic root ALP activity after 96 h treatment with osteogenic medium with adenosine (50 μm) and adenosine receptor antagonists (50 μm) in the presence of 150 μm AOPCP, 5 μm dCF and 5 μm NBTI. Results are shown as mean ± SEM; n = 5, *p < 0.05, ***p < 0.001, ****p < 0.0001 by one-way Anova followed by Holm–Sidak post hoc test