Akt phosphorylation induced by CD44/osteopontin (OPN) is required to protect human sclerotic valve interstitial cells (VICs) from calcium deposition. A, Western blotting of pAkt, Akt 1/2, pSMAD 1/5/8, and GAPDH at 0 hours, 15 minutes, 4 hours, and 24 hours after bone morphogenetic protein 4 (BMP4; 100 ng/mL) in combination with neutralizing antibody (NAb) CD44 (5 μg/mL) or NAb OPN (5 μg/mL). B, Bar graph represents calcium deposition on aortic valve sclerotic VICs after 12 days treatment with BMP4 (100 ng/mL) in the absence or presence of Wortmannin (0.2 mmol/L). *P=0.05. C, Alizarin red staining representing calcium deposition (red) on VICs after 12 days treatment with BMP4 (100 ng/mL) in the presence or absence of Wortmannin (0.2 mmol/L).