This study shows that decreased Notch1 signaling predisposes to increased aortic valve calcification in mice and that inhibition of Notch1 in cultured aortic valve cells also induces calcification. In vivo and in vitro, Notch1 signaling repressed valvular Bmp2 expression, and de-repression of Bmp2 was involved in calcification induced by Notch1 inhibition. Thus, Notch1 signaling appears to prevent aortic valve calcification in part by repressing Bmp2 expression within the valve (Figure 6).
Genetic studies have provided compelling evidence that NOTCH1
mutations contribute to valve disease in humans [9
], and rare variants may also contribute to disease in some settings [10
]. Our in vivo
and in vitro
results suggest that Notch signaling may repress a default pathway of osteoblast gene expression and calcification. In AVICs, aortic valve endocardial cells, and a macrophage cell line, decreased Notch signaling resulted in increased Bmp2
levels. Given the current knowledge and our findings in AVICs, these myofibroblast cells may be the cell type that contributes most to the calcification event, although endocardial cells may contribute as well. Further study is required to determine the relative contribution of Notch1
within specific aortic valve cell types in repressing aortic valve calcification. The murine and cell culture model systems described here may be useful for studying the pathogenesis of aortic valve calcification and for developing novel therapeutics.
Although the murine model of Notch1 deficiency displayed an increase in valve calcification, it was much milder than the human disease and none of the Notch1+/− mice had bicuspid aortic valves. These findings indicate that the human aortic valve is more dose-sensitive to alterations in NOTCH1 function and that genetic background may also affect phenotypic severity. The presence of calcification in tricuspid aortic valve leaflets suggests independent functions of Notch1 in leaflet morphology during development and in repressing calcification postnatally. This observation is consistent with the presence of aortic valve calcification in several human subjects heterozygous for NOTCH1 who had tricuspid aortic valve leaflets.
Our findings suggest that the interplay between Notch1
has a key role in calcification within aortic valve cells. We showed that Notch1
can repress Bmp2
, although it is unknown whether this effect is mediated directly through the Bmp2 enhancer or indirectly. Another pro-calcification pathway involves Runx2, a central regulator of osteoblast development [25
expression is increased by treatment with Bmp2 [29
]. Notch1 attenuates the ability of Runx2
to activate the Osteocalcin
enhancer as a result of a physical interaction between Runx2 and the Hrt repressors [9
]. Thus, Notch1 may prevent aortic valve calcification by repressing not only Bmp2
expression but also the activity of osteogenic genes downstream of Bmp2, such as Runx2
Our observations raise the interesting possibility that valve calcification in humans with NOTCH1
mutations is a result of a cell fate switch of valve mesenchymal cells into an osteoblast-like lineage. This possibility is supported by evidence that Notch1 represses osteoblast differentiation [16
] and that Bmp2 and Runx2 promote osteoblast commitment. Although Bmp2
was necessary for Notch1
-related calcification, additional pathways may be involved. For example, several Wnt family members [31
] and β-catenin [32
] are involved in cardiovascular calcification, and Notch1 represses β-catenin/Wnt activity [16
]. Since Notch1 [35
], Bmp2 [38
], and Wnts [40
] have key roles in cell proliferation and lineage choices, the interplay between these signals are likely involved in regulation of a wide range of cell fate decisions.
Our findings also have several intriguing clinical implications. In conjunction with chemical inhibition of Notch in AVICs, Notch1+/−
mice could provide insights into the early molecular pathogenesis of aortic valve calcification. Future studies of calcification in AVICs derived from induced pluripotent stem cells [41
] made from patients with NOTCH1
mutations may also reveal early mechanisms and allow formal testing of the cell fate switch hypothesis. As further clinical genotypic/phenotypic data are obtained, it may be possible to link specific mutations with distinct clinical prognoses. Finally, the finding that inhibiting Notch signaling with a γ-secretase inhibitor in vitro
increases aortic valve calcification raises the question of whether patients treated with β-secretase inhibitors for Alzheimer's disease [43
] should be monitored for aortic valve calcification.