Valve endothelial monolayer fissuring via RhoA activity induces 3D calcific aortic valve lesion emergence as revealed by a longitudinal live-imaging platform

Abstract

Calcific aortic valve disease (CAVD) is a degenerative disease with wide prevalence in the aging population and a low survival rate after onset of symptoms, yet there are no effective pharmacological treatments. Many patients present to the clinic with symptoms at end-stages of CAVD, when the disease may be irreversible. The ability to identify and live-trace calcific lesion emergence in-vivo would allow for the identification of disease biomarkers and discovery of therapeutic targets at earlier, more treatable stages. In this work, we establish a new multimodal in-vitro CAVD platform consisting of lineage traced-VEC and VIC cells in a 3D model combined with live optical coherence and fluorescence microscopy to unravel VEC, VIC, and matrix transitional events during calcific lesion formation. We discover that fissuring of the endothelial monolayer combined with the formation of dense aggregates in adjacent regions is a key biomarker of the onset of lesion formation. This coincides with the formation of dense VIC and ECM conglomerates under endothelial aggregates, an additional biomarker of pathogenesis. Further, we discover that fibrotic tissue compaction is correlated with but not necessary for lesion formation. Additionally, we identify RhoA activation in disease-treated samples. We demonstrate that RhoA inhibition through ROCK, but not Rac1 inhibition, prevents delamination of the endothelial monolayer, fibrotic remodeling, and emergence of calcific lesions. Together, this work establishes a new longitudinal live-imaging platform that identifies emergent cell and matrix biological signatures of CAVD onset and enables the evaluation of therapeutic interventions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/660489v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@dc378org.highwire.dtl.DTLVardef@19c37d5org.highwire.dtl.DTLVardef@11581c6org.highwire.dtl.DTLVardef@fb0a6a_HPS_FORMAT_FIGEXP M_FIG C_FIG