Elucidating the mechanosensitive pathways of physiological and pathological strain on valve cells in a novel human valve-on-chip system

Abstract

Calcific aortic valve disease (CAVD) is a progressive pathology driven by cellular-level processes in which the valve mechanical environment plays a significant role. Our engineered microphysiological system, a human cell valve-on-chip (VOC), mimics the aortic endothelium and fibrosa interface. This system can hold VICs (Valve Interstitial Cells) in a 3D hydrogel culture adjacent to a VEC (Valve Endothelial Cells) monolayer, integrated to a mechanical actuator to apply uniaxial cyclic stretch. Piezo1 is present in VICs, and stimulation of this mechanotransductor with Yoda1, a soluble agonist, was able to induce osteogenic differentiation in vitro, mediated by YAP activation downstream and GLS1 signaling. However, stimulation of this mechanosensitive pathway by mechanical stretch hasn’t been attempted. Our VOC is a PDMS construct containing a co-culture of VECs and VICs, which contains two chambers separated by a 10μm-thick PDMS membrane with 5μm pores. Human VICs and VECs were harvested from replacement surgery valves. We magnetically sorted for CD31 and obtained a stable endothelial phenotype, with VECs expressing CD31, VWF, and no αSMA. VICs were seeded in a 6mg/mL collagen hydrogel in the interstitial chamber. Within the endothelial chamber, VECs are seeded in a monolayer. We will apply static and cyclic stretch regimes (1Hz) of 10% (healthy) and 20% (pathological) and observe changes in cell alignment and morphology, VIC activation and VEC endothelial-to- mesenchymal transition and the correlation Piezo1 and GLS1 expression and YAP nuclear translocation using immunocytochemistry and qPCR. VECs from explanted valves exhibited expression of inflammatory markers and an activated phenotype.