Increased aortic valve calcification in 10-month-old male Notch1^+/− mice fed a Western diet. A and B, von Kossa staining shows increased calcification of the aortic valve in Notch1^+/− mice. C and D, Higher magnification views of the boxed areas in A and B. The arrowhead indicates representative area of staining. E, Quantification of calcification in Notch1^+/− (n=6) or wildtype (WT) (n=4) valves (2.5% ± 0.64% vs. 0.47% ± 0.16%; *P=0.03).

Inhibition of Notch signaling in cultured sheep AVICs with DAPT increases calcification and activates osteoblast–specific genes. A, Calcification of AVICs treated with DMSO (negative control) or DAPT demonstrated by Alizarin Red staining (red-brown). AVICs treated with β-glycerol phosphate and asorbic acid served as positive controls. Arrowheads indicate representative areas of calcification. B, Quantification of Alizarin Red staining from seven independent samples for each condition. The percent area of calcification was significantly greater in DAPT-treated cells (75 μM, 27.3 ± 8%; 100 μM, 28.1 ± 9.3%) than in DMSO-treated controls (4.0 ± 2%; *P<0.03 vs control). C, von Kossa staining of DAPT-treated AVICs showed more calcification than in DMSO-treated cells. D, Runx2 mRNA levels assayed by qPCR were threefold higher in sheep AVICs treated with DAPT than in DMSO-treated controls (*P<0.002 vs control; n=3). E, Osteopontin (Opn) protein levels were higher by Western blot in sheep AVICs treated with DAPT. GAPDH was used as a loading control. F, Alkaline phosphatase protein levels were higher in DAPT-treated cells than in DMSO-treated controls by Western blot. G, DAPT-treated AVICs had increased alkaline phosphatase activity (red areas, arrowhead). Arrowheads indicate representative areas of alkaline phosphatase activity. H, AVICs treated with Notch1 siRNA for 2 weeks had increased osteopontin protein levels by Western blot. I, Western blot analysis shows increased alkaline phosphatase levels in Notch1-treated AVICs. J, Runx2 mRNA levels increased >40% in AVICs treated with Notch1 siRNA (P<0.06; n=3).

Decreased Notch1 signaling increases Bmp2 expression in vivo and in vitro. A, Bmp2 expression in aortic valve leaflets was more than threefold higher in Notch1^+/− mice than controls (*P<0.02; n=3;). B, AVICs treated with Notch1 siRNA had 73% more Bmp2 mRNA than cells treated with scrambled siRNA (*P<0.01; n=3). C, Bmp2 protein levels were higher in sheep AVICs treated with Notch1 siRNA than in control cells treated with scrambled siRNA. GAPDH was used as a loading control for Western blots. D, Bmp2 protein levels were higher in DAPT-treated than control DMSO-treated sheep AVICs. E, Cultured aortic valve endocardial cells treated with 50 μM DAPT had 45% more Bmp2 mRNA than DMSO-treated cells (*P<0.03; n=6). F, RAW264.7 cells treated with 50 μM DAPT had 130% more Bmp2 mRNA than DMSO-treated cells (*P<0.02; n=3).

Bmp2 expression is necessary for calcification induced by Notch inhibition. A, Western blot analysis showed that Bmp2 siRNA specifically knocked down Bmp2 but not Bmp4. B, Sheep AVICs treated with Bmp2 siRNA and DAPT for 3 weeks had >80% less calcification than cells treated with control siRNA and DAPT (3.4 ± 1.5% vs 20.1 ± 5.7%, *P=0.02 vs. control siRNA; n=5).

A potential model for repression of aortic valve calcification by NOTCH1. A, Notch is activated by its ligands, Delta or Jagged. The Notch intracellular domain (NICD) is cleaved from the cell surface and translocates to the nucleus, where it binds to its co-activator, recombination signaling protein-J (RBP-J). This complex binds to the promoter of transcriptional repressors such as HES or HRT, whose protein products may in turn reduce BMP2 expression, thereby repressing BMP2-induced aortic valve calcification. NOTCH1 can also repress the transcriptional activity of RUNX2, a central transcriptional regulator of osteoblast-specific genes [9]. B, With decreased NOTCH1 signaling, BMP2 and RUNX2 expression is increased resulting in increased aortic valve calcification. Adapted from Yoon et al. [13].