MECHANICAL STRAIN-INDUCED REACTIVE OXYGEN SPECIES AND THEIR ROLE IN VALVE INTERSTITIAL CELL CALCIFICATION

Zhong, Aileen 1, 2; Simmons, Craig 1, 2, 3

1. Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research; 
2. Institute of Biomaterials and Biomedical Engineering, University of Toronto; 
3. Department of Mechanical and Industrial Engineering, University of Toronto

Background and Purpose: Hypertension is thought to contribute to fibrosis and ectopic mineralization in calcific aortic valve disease (CAVD) by exposing the valve leaflets to pathophysiological levels of strain that promote valve interstitial cells (VICs) to differentiate to osteoblast-like cells. In vascular smooth muscle cells, mechanical strain promotes reactive oxygen species (ROS) production and oxidative stress-induced pathological changes. While oxidative stress is a hallmark of CAVD, the effect of mechanical strain-induced ROS production and its role on the regulation of VIC calcification have not been established. The Wnt/β-catenin signaling pathway is upregulated in CAVD and it can be perturbed by hydrogen peroxide, suggesting a mechanistic link between ROS signaling and VIC calcification. 
Hypothesis: We hypothesized that pathophysiological strain will elevate ROS production in VICs to stabilize β-catenin and activate downstream target genes to promote VIC calcification.
Methods: Porcine aortic VICs were subjected to normal (10%) or pathophysiological (15%) strain using a Flexcell bioreactor. VICs were transfected with HyPer, a fluorescent hydrogen peroxide sensor and ROS production was measured using two-photon imaging. To model oxidative stress, VICs were cultured in osteogenic media supplemented with hydrogen peroxide for up to 8 days. ROS-induced Wnt/β-catenin pathway activation was measured using TOPFlash luciferase assay and immunostaining of VICs for β-catenin to analyze nuclear translocation. VIC osteogenesis was characterized by alkaline phosphatase (ALP) staining. 
Results: Intracellular hydrogen peroxide levels, measured by HyPer staining, were elevated 1.6-fold with strain. Hydrogen peroxide induced β-catenin nuclear translocation and a 1.8-fold increase in TOPFlash activity (p<0.01), characteristic of activated Wnt/β-catenin signaling. In the presence of hydrogen peroxide, there was a 1.4-fold increase in ALP-positive calcified nodules than in osteogenic media alone (p<0.01). 
Conclusions: Mechanical strain induces hydrogen peroxide production in VICs and hydrogen peroxide activates Wnt/β-catenin signaling and promotes VIC calcification. On-going work will focus on identifying the mechanism that regulates mechanical strain-induced ROS production and characterizing the mechanistic link between ROS and β-catenin stabilization in VIC calcification.