Diet-Induced Aortic Valve Disease in Mice Haploinsufficient for the Notch Pathway Effector RBPJK/CSL

Abstract

Objective— Calcific aortic valve disease is similar to atherosclerosis in that both diseases result from chronic inflammation and endothelial dysfunction. Heterozygous NOTCH1 mutations have been associated to calcific aortic disease and a bicuspid aortic valve. We investigated whether mice with genetic inactivation of the Notch signaling pathway are prone to develop valve disease when exposed to a predisposing diet. Methods and Results— Using Doppler echocardiography, histology, immunohistochemistry, quantitative gene expression analysis, and cell culture assays, we examined the effect of a hypercholesterolemic diet supplemented with vitamin D on mice heterozygous for null mutations in the Notch1 receptor or the effector transcription factor gene RBPJk . After 16 weeks on the hyperlipidemic diet, calcific aortic disease was detected in heterozygous RBPJk mice. Analysis of valve leaflets revealed macrophage infiltration, enhanced collagen deposition, proosteogenic protein expression, and calcification. Heterozygous null Notch1 mice displayed milder histopathologic changes and did not develop any significant hemodynamic disturbance. Valvular disease correlated with reduced expression of the Notch target gene Hey1 in valves of RBPJk heterozygous mice fed the hyperlipidemic diet. Consistent with the in vivo data, Notch signaling inhibition in porcine valve interstitial cells led to downregulation of HEY1 transcription, activation of osteogenic markers, and increased calcified nodule formation. Conclusion— We show that Notch signaling disruption via RBPJk heterozygous inactivation results in aortic valve disease. Notch1 heterozygous mice do not show functional impairment, suggesting that additional Notch receptors may be involved in aortic valve homeostasis and disease. Our data establish a genetic mouse model of calcific aortic valve disease and may help to identify a patient population with reduced valvular NOTCH signaling at risk for developing this disease.

The intracellular domains of Notch1 and 2 are functionally equivalent during development and carcinogenesis

“…To confirm that the N12 allele is hypomorphic, we investigated whether haploinsufficient cardiac phenotypes associated with Notch1 (de la Pompa, 2009;de la Pompa and Epstein, 2012;Garg et al, 2005;Nus et al, 2011) +/12 and 39 were N1 +/+ ). By contrast, N1 12/12 embryos constituted ∼25% of the litters examined at E9.5, E14.5 and E18.5.…”

The pathology and pathobiology of bicuspid aortic valve: State of the art and novel research perspectives

“…Transfection of Sox‐9 into VICs rescued the hypermineralising phenotype during Notch inhibition, suggesting that Notch signalling prevents mineralisation of the aortic valve in a Sox‐9‐dependent manner 59. Mice haploinsufficient for the Rbpjκ transcription factor and supplemented with a cholesterol‐rich diet and vitamin D develop CAVD but do not have BAV 60. Intriguingly, GATA5 ‐/‐ mice develop BAV (∼25% of littermates) and have lower expression of Jag1 and higher levels of mRNA encoding for Rbpjκ in embryonic tissues, suggesting dysregulation of the Notch pathway in these mice 61.…”

Notch Signaling in Aortic Valve Development and Disease

“…These studies demonstrated that loss of Notch signaling contributes to aortic valve calcification via a Sox9-dependent mechanism. Additional work by other investigators has supported these conclusions and have also demonstrated a role for Bmp2 as a downstream target of Notch1 signaling in this process and found that Notch1 haploinsufficient mice develop aortic valve calcification with aging [14,15].…”