A Novel Role of Extracellular Nucleotides in Valve Calcification by Lana Osman, Adrian H. Chester, Mohamed Amrani, Magdi H. Yacoub FRS, and Ryszard T. Smolenski MD Circulation Volume 114(1 suppl):I-566-I-572 July 4, 2006 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Activity of ALP in human valve ICs treated for 21 days with osteogenic media (OST), ATP (adenosine 5′-triphosphate) (100 μmol/L), or treated with the 2 stable agonists to the P2Y receptor; ATP-γ-S (Adenosine 5′-(3-thiotriphosphate Tetralithium salt) (ATP-Y) (10 μmol/L) and 2-Methylthio-ADP (2-Methylthio-ADP Trisodium salt) (ADP-Y) (10 μmol/L). Figure 1. Activity of ALP in human valve ICs treated for 21 days with osteogenic media (OST), ATP (adenosine 5′-triphosphate) (100 μmol/L), or treated with the 2 stable agonists to the P2Y receptor; ATP-γ-S (Adenosine 5′-(3-thiotriphosphate Tetralithium salt) (ATP-Y) (10 μmol/L) and 2-Methylthio-ADP (2-Methylthio-ADP Trisodium salt) (ADP-Y) (10 μmol/L). Values are expressed as nmol/min per mg of protein±SEM (*P<0.001 vs control, n=3). Lana Osman et al. Circulation. 2006;114:I-566-I-572 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. ALP activity in human valve ICs treated with adenosine (30 μmol/L) in the presence and absence of osteogenic media (OST) for 21 days. Figure 2. ALP activity in human valve ICs treated with adenosine (30 μmol/L) in the presence and absence of osteogenic media (OST) for 21 days. Inhibitors of adenosine breakdown (deamination deoxycoformycin [dCf]) (5 μmol/L) and phosphorylation aminoadenosine (AA) (5 μmol/L) were added to cells treated with adenosine only. Values are expressed as nmol/min per mg of protein±SEM (*P<0.001 vs control and #P=<0.001 vs osteogenic treatment, n=3). Lana Osman et al. Circulation. 2006;114:I-566-I-572 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. Expression of ALP in human valve ICs (A) treated with osteogenic media (B), ATP (100 μmol/L) (C), ATP-γ-S (10 μmol/L) (D), 2-Methylthio-ADP (10 μmol/L) (E); adenosine (30 μmol/L) with dCF and AA (F) for 21 days (n =3). Figure 3. Expression of ALP in human valve ICs (A) treated with osteogenic media (B), ATP (100 μmol/L) (C), ATP-γ-S (10 μmol/L) (D), 2-Methylthio-ADP (10 μmol/L) (E); adenosine (30 μmol/L) with dCF and AA (F) for 21 days (n =3). Lana Osman et al. Circulation. 2006;114:I-566-I-572 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. Activity of ALP in human valve ICs treated for 21 days with atorvastatin (Atorva) (100 μmol/L) in the presence and absence of ATP (100 μmol/L). Figure 4. Activity of ALP in human valve ICs treated for 21 days with atorvastatin (Atorva) (100 μmol/L) in the presence and absence of ATP (100 μmol/L). Values are expressed as nmol/min per mg protein±SEM (*P<0.001 vs control and #P<0.001 versus ATP, n=3). Lana Osman et al. Circulation. 2006;114:I-566-I-572 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. Extracellular ATP breakdown into its metabolites by atorvastatin treatment in human valve ICs. Valve ICs treated with 10 μmol/L of ATP in the absence (black circle) or in the presence of 1 μmol/L atorvastatin (white circle), 10 μmol/L atorvastatin (filled triangle), or 100 μmol/L atorvastatin (white triangle) for 24 hours. Figure 5. Extracellular ATP breakdown into its metabolites by atorvastatin treatment in human valve ICs. Valve ICs treated with 10 μmol/L of ATP in the absence (black circle) or in the presence of 1 μmol/L atorvastatin (white circle), 10 μmol/L atorvastatin (filled triangle), or 100 μmol/L atorvastatin (white triangle) for 24 hours. Samples were analyzed for ATP breakdown (A) and formation of ADP (B), AMP (C), and adenosine (D). Values are expressed as the mean concentration in nmol/mg of protein±SEM and experiment was performed in quadruplicate. *P<0.001, ‡P<0.01 vs control (black circle). Lana Osman et al. Circulation. 2006;114:I-566-I-572 Copyright © American Heart Association, Inc. All rights reserved.

Figure 6. Activity of enzymes on extracellular nucleotide degradation in human valve ICs treated with atorvastatin for 24 hours. Figure 6. Activity of enzymes on extracellular nucleotide degradation in human valve ICs treated with atorvastatin for 24 hours. The activities of ATPase (A), ADPase (B), and eN (C) enzymes were determined. Lactate dehydrogenase (LDH) activity (E) was also measured in all samples. Values are expressed as nmol/min per mg of protein±SEM (n =8). *P<0.001, #P=0.004, §P=0.001 vs control (black circle). Lana Osman et al. Circulation. 2006;114:I-566-I-572 Copyright © American Heart Association, Inc. All rights reserved.