Valvular endothelial cell heterogeneity reflects different pathogenesis of tricuspid and bicuspid aortic valve stenosis in humans

Tricuspid aortic valve stenosis (tAVS) is a common disease in the elderly. It is characterized by inflammation, fibrosis, and calcification of the valve leaflets leading to impaired valve opening and inadequate blood flow. In contrast to tAVS, bicuspid aortic valve stenosis (bAVS) has a genetic basis, presents earlier in life, and progresses more rapidly, requiring earlier medical intervention; all these points suggest a different pathophysiology than in tAVS. Recent evidence suggests that altered endothelial cell biology contributes to disease development in calcified aortic disease in general and bAVS in particular. Given the prominent etiologic and clinical differences between tAVS and bAVS, we analyzed gene expression patterns of valvular endothelial cells (VECs) from fibrosa (aortic side of the leaflet) and ventricularis (ventricular side of the leaflet) in these two patient cohorts in humans. To address this, we isolated VECs from explanted human calcified valves of tAVS and bAVS taking advantage of the modified Haeutchen method and performed bulk RNAseq analysis of whole valves and VECs. We found alterations of VECs specifically in the fibrosa of tAVS but on both sides of bAVS regarding cardiac muscle growth, osteoblast differentiation and cell cycle activity. In tAVS we found calcium deposits in von Kossa staining, as well as deposition of the extracellular matrix molecules osteopontin and tenascin C preferentially in the fibrosa. In contrast, in bAVS, such changes were detected on both sides of the valve leaflets without the typical side specificity of tAVS. In summary, given the striking differences in etiology, clinical onset, and severity of tAVS vs bAVS, we explored potential molecular differences in VECs.