Abstract 18010: Induced Pluripotent Stem Cell-Based Modeling of Human NOTCH1 Mutations Reveals Novel Pathways Regulating Aortic Valve Disease

Abstract

In humans, NOTCH1 mutations result in congenital heart defects including valve malformations and severe valve calcification in adults. Valve calcification typically occurs on the aortic side of the valve leaflets that is not exposed to laminar shear stress, suggesting a protective role of flow sensed by the endothelial lining. To understand the mechanisms by which NOTCH1 mutations in endothelial cells (ECs) cause disease, we generated induced pluripotent stem cell (iPSC) lines from fibroblasts of four individuals from two families affected with aortic valve disease due to heterozygous non-sense mutations in NOTCH1. We differentiated control and mutant iPSC lines into ECs and exposed the ECs to either static or fluid shear stress conditions. NOTCH1 mRNA levels were decreased by ~50% in the NOTCH1+/- ECs under static or shear stress conditions. RNA-seq revealed that 165 genes were differentially expressed in static conditions and 193 genes responded abnormally to shear stress in NOTCH1+/- ECs compared to NOTCH1+/+ ECs. Differentially expressed genes included canonical NOTCH1 targets HRT2 and EFNB2 as well as novel targets involved in vascular development, inflammation, and endochondral ossification. Anti-calcific genes uniquely upregulated in shear stress were dysregulated in NOTCH1+/- ECs, indicating that they were unable to mount the normal protective response induced by shear stress in the valve. Generating isogenic NOTCH1+/+ and NOTCH1+/- cell lines using TALEN genome editing identified genes specifically dysregulated due to NOTCH1 heterozygosity rather than differentially expressed due to genetic background and showed rescue of this dysregulation in TALEN-corrected NOTCH1+/+ ECs. We have mapped the gene networks dysregulated in NOTCH1+/- ECs as determined by NOTCH1 ChIP-seq, differentially methylated regions of DNA, and genome-wide differences in the progression of activating and repressive chromatin states that begin to explain the mechanisms by which heterozygosity of a transcription factor leads to disease-specific changes. Determining the consequence of NOTCH1 heterozygous mutations in human patient-specific ECs will reveal novel mechanisms underlying aortic valve disease, leading to potential targets for intervention.