Bicuspid aortic valve stenosis is characterized by increased angiogenesis, inflammation, and a higher valvular-to-systemic calcification ratio than tricuspid aortic valve stenosis

Abstract Calcific aortic valve stenosis is the most common valvular heart disease requiring treatment. Although both tricuspid (AVS) and bicuspid (bAVS) aortic valve stenoses become calcified and functionally impaired in advanced stages, the pathophysiology of these conditions remains unclear. We investigated this using a multitechnology approach on explanted AVS, bAVS, and aortic regurgitation (AR) control valves. Because of technical limitations in processing heavily calcified aortic valve tissue, we established Kawamoto’s film method for human aortic valve tissue, enabling the production of well-preserved cryosections and high-quality immunostainings. Both bulk RNA-seq analysis and immunostainings revealed that angiogenesis, inflammation, and calcification are key features distinguishing bAVS from AVS. In fact, we found that angiogenic genes and CD31 + cells, as well as inflammatory genes and CD45 + cells, are significantly elevated in bAVS. The most striking difference between bAVS and AVS was the prominent expression of specific genes involved in tissue calcification, such as matrix metallopeptidase 12 ( MMP12 ), dentin matrix acidic phosphoprotein 1 ( DMP1 ), and proteoglycan 4 ( PRG4 ), along with approximately 1.7-fold increased calcification as shown by micro-CT and von Kossa staining analysis in bAVS. These findings were corroborated in a retrospective analysis of 1108 AVS and bAVS patients who underwent transcatheter aortic valve implantation (TAVI). The bAVS patients exhibited significantly stronger aortic valve calcifications (1.6-fold) but a significantly lower vascular calcification burden. These data further suggest that AVS and bAVS are distinct disease entities, with bAVS exhibiting increased local inflammation, angiogenesis, and calcification, findings that may guide future therapeutic strategies.