Cell-type Specific GTPase Regulation of Aortic Valve Homeostasis and CAVD Progression

Abstract

Calcific Aortic Valve Disease (CAVD) involves active remodeling of the thin fibrous leaflet matrix into large hypercellular and mineralized lesions, indicative of profound cellular interactions that have not been well explored. Herein, longitudinal live imaging via dual Optical Coherence Tomography (OCT) and confocal microscopy enabled elaboration of specific phases of 3D lesion progression and cell-type specific interactions between valvular endothelial (VECs) and interstitial cells (VICs) in biomechanically anchored co-culture. Homeostatic VECs maintained monolayer integrity with high Rac1-GTP activity, while healthy VICs exhibited low RhoA-GTP and Rac1-GTP. CAVD progression exhibited stages of endothelial disruption, VIC aggregation, 3D raised lesion formation, and matrix calcification, each engaging differential activation of small GTPases. Osteogenic conditions however, elevated RhoA-GTP >40% in VECs and 70% in VICs, while Rac1 activity remained unchanged. Human leaflets replicated the dynamics of cell-type GTPase activity in healthy and CAVD progression. Single cell RNA-sequencing revealed differential enrichment of RhoA, Rac1, and their regulatory GEFs and GAPs among quiescent, activated and differentiated cellular subpopulations. Pharmacological modulation of Rac1 or ROCK exhibited differential efficacy in preventing CAVD onset and/or progression. Further, valve cell type specific conditional overactivation of RhoA induced hemodynamic stenosis, leaflet calcification, VIC osteogenic differentiation, and left ventricular mass (p ≤ 0.05) in vivo. Cell-type specific regulation of valve cell kinematics via GTPase networks are key mediator of CAVD progression, targeting which could offer promising therapeutic avenues for CAVD.