Notch1 Mutation Leads to Valvular Calcification Through Enhanced Myofibroblast Mechanotransduction

Abstract

Objective Calcific aortic valve disease (CAVD) is a significant cardiovascular disorder, and controversy exists as to whether it is primarily a dystrophic or osteogenic process in vivo. In this study, we sought to clarify the mechanism of CAVD by assessing a genetic mutation, Notch1 heterozygosity, which leads to CAVD with 100% penetrance in humans. Approach and Results Murine immortalized Notch1+/− aortic valve interstitial cells (AVICs) were isolated and expanded in vitro. Molecular signaling of wild type (WT) and Notch1+/− AVICs were compared to identify changes in pathways that have been linked to CAVD – TGFβ/BMP, MAPK and PI3K/Akt – and assessed for calcification potential. Additionally, AVIC mechanobiology was studied in a physiologically relevant, dynamic mechanical environment (10% cyclic strain) to investigate differences in responses between the cell types. We found that Notch1+/− AVICs resembled a myofibroblast-like phenotype expressing higher amounts of cadherin-11, a known mediator of dystrophic calcification, and decreased Runx2, a known osteogenic marker. We determined that cadherin-11 expression is regulated by Akt activity, and inhibition of Akt phosphorylation significantly reduced cadherin-11 expression. Moreover, in the presence of cyclic strain, Notch1+/− AVICs exhibited significantly upregulated phosphorylation of Akt at Ser473 and smooth muscle α-actin (αSMA) expression, indicative of a fully activated myofibroblast. Finally, these Notch1 mediated alterations led to enhanced dystrophic calcific nodule formation. Conclusions This study presents novel insights in our understanding of Notch1-mediated CAVD by demonstrating that the mutation leads to AVICs that are fully activated myofibroblasts, resulting in dystrophic, but not osteogenic, calcification.

The Role of High Mobility Group Box 1 Protein in Interleukin-18-Induced Myofibroblastic Transition of Valvular Interstitial Cells

“…Myofibroblastic phenotype might be the intermediate state of osteoblastic phenotype [21]. Recent study also supported this point in which reduced Notch1 signaling promoted VIC myofibroblastic differentiation and then dystrophic calcification [22]. In the present study, we demonstrated that IL-18 could induce the myofibroblastic transition of VICs, as evidenced by upregulation of α-SMA and enhanced fiber formation.…”

Genetic basis of aortic valvular disease

“…In order to define the role of Notch1 in AVICs, immortalized aortic VICs were isolated from Notch1 +/− Immortomice, a compound mutant mouse which is both heterozygous for Notch1 and expresses the immortalizing oncogene simian virus 40 (SV40) large tumor (T) antigen (Ag), and were found to not only have molecular differences at baseline but also had an exaggerated response to cyclic mechanical strain, which mimics in vivo diastolic loading, becoming fully activated and resulting in calcification [41]. Further mechanistic insight was found by studying calcified human aortic valves, where the long non-coding RNA, lncRNA H19 , was identified as a NOTCH1 repressor with a direct role in calcification of human aortic VICs.…”

Recent Advances in the Genetics of Atherothrombotic Disease and Its Determinants

“…98 Aortic valve interstitial cells from Notch1 heterozygous deficient mice become fully activated myofibroblasts leading to enhanced dystrophic calcification. 99 Genetic ablation of CaV3.2 channels enhances the arterial myogenic response through modulation of the RyR-BKCa (ryodine receptors-large conductance Ca 2+ -activated K + channels) axis. 100 Conditional knockout of the E-twenty six factor Ets variant 2 in endothelial cells resulted in impaired neovascularization in response to ischemic tissue injury.…”