The Effect of Mechanotransduction and ECM Proteins on the Development of Calcific Nodules: A Model System for CAVD

Abstract

Test an in vitro model of calcific aortic valve nodule growth that modulates stiffness and substrate composition in polyacrylamide (PAAM) gels to understand progression of calcific aortic valve disease (CAVD). Isolated valve interstitial cells (VICs) were used to seed PAAM gels incorporated with either collagen I or hyaluronic acid (HA). To induce calcification, TFG-β1 was added to the culture medium. Additional murine leaflets were isolated and placed in culture media with TFG-β1. Nodule formation has been shown in both the rat leaflet model systems and the PAAM gel system using three separate visualization techniques: the calcium fluorescence indicator fluo-4 AM, Alizarin red staining, and two photon excited fluorescence (TPEF). This is also confirmed in human valves with CAVD. Nodule formation, as measured by fluo-4 imaging, shows an increase in average nodule size with an increase in Young's modulus, but also a dependence on the type of binding protein used. Preliminary studies looking at fiber organization in explanted rat leaflets shows an increase in nodule formation with a decreased in collagen organization – shown using second harmonic generation (SHG) signal. Using silk films to mimic the natural valve environment, we aim to study the effect of fiber alignment on nodule growth. Average nodule size is affected by both the stiffness of the PAAM gel and the binding protein indicating that changes in valve extracellular matrix (ECM) content during disease progression may lead to CAVD advancement. Additionally, preliminary studies indicate that fiber orientation may play a role in initial nodule formation that may be exacerbated by changes in ECM composition.