Multi-omics And Single Nucleus Transcriptomics Reveal Aberrant Adaptive Immune Responses In Human Bicuspid Aortic Valves

Introduction: Bicuspid aortic valve (BAV) is the most common cause of adult congenital heart disease. Patients with BAVs comprise <2% of the population but represent ~50% of valve replacement surgeries and the pathogenic mechanisms underlying this disproportionate incidence of calcific aortic valve disease (CAVD) are unknown. We aimed to identify molecular contributors to accelerated disease in BAV-CAVD. Methods: Bulk multi-omics (miRNA-seq, RNA-seq, proteomics) was performed on 70 human aortic valves (11 non-diseased; fibrotic and calcific portions of 32 tricuspid aortic valves [TAVs] and 27 BAVs with CAVD). Single nuclei RNA-seq (snRNA-seq) was completed on 54,608 nuclei from 13 additional human valves (4 non-diseased; 5 TAVs and 4 BAVs with CAVD). Results: Calcific burden did not differ between diseased TAVs and BAVs (p=0.77), but BAV mineralization was significantly accelerated by 11.5±1.7 years vs. TAVs (p<0.001). Latent factor-based integration of covariate-corrected multi-omics analyses (q<0.05), CT-derived valve calcification, and 29 clinical parameters uncovered extensive involvement of adaptive immune responses where T cell/B cell inflammatory activation associated with autoimmune signals was enhanced in the early stages of BAV disease. Integrated multi-omics also revealed significantly elevated platelet activation potentially as a result of lifelong exposure to the high-shear BAV microenvironment, and implicated altered SLIT-ROBO signaling in BAV-CAVD progression. snRNA-seq identified 5 major cell types (27 states) in human aortic valves: valvular interstitial cells (65% of cells; 7 states), endothelial cells (10%; 4 states), macrophages (13%, 7 states), T cells (8%; 5 states), and B cells (3%; 4 states). CAVD drove differentiation of valve interstitial cells along distinct myofibrogenic ( ACTA2/CARMN/MYH11 -high) vs. osteogenic ( RUNX2/CDH11/POSTN -high) lineages, induction of endothelial-to-mesenchymal transition in valve endothelium and significant accumulation of specific macrophage and T-cell states (p<0.05) in BAV-CAVD. Conclusions: Aberrant adaptive immune responses are critical drivers of BAV-CAVD, and our findings delineate a novel strategy for discovery of morphology-specific therapeutics.