Fibroblast activation protein expressing valvular interstitial cells define calcific remodelling in aortic stenosis

Introduction Aortic stenosis (AS) is the most prevalent valvular heart disease in ageing populations and lacks effective medical therapy. Improved understanding of the cellular mechanisms driving calcific remodelling is needed to enable targeted intervention. Purpose To identify transcriptomic cell states associated with aortic stenosis severity and determine their relationship to in vivo molecular imaging and calcification. Methods We performed single-nuclei RNA-sequencing (snRNA-seq) on cryopreserved human aortic valve tissue spanning the spectrum of aortic stenosis severity. In a separate cohort, individuals with AS and control subjects underwent [68Ga]FAPI-46 positron emission tomography (PET) and computed tomography (CT) to quantify fibroblast activation protein (FAP) expressing valvular interstitial cells (VICs) in vivo. For patients who underwent valve replacement, excised leaflets were co-registered to pre-operative imaging and subjected to Xenium spatial transcriptomics. Results snRNA-seq (control n=6, mild/moderate n=8, severe n=11) generated a comprehensive cellular atlas of the aortic valve and revealed expansion of a FAP+ VIC population with increasing disease severity (p=0.026; Figure 1). Among VIC subsets, FAP+ cells demonstrated the strongest extracellular matrix transcriptional programme and were enriched for chondrogenic and osteogenic genes, including POSTN, COL1A2, COMP, CRTAC1, and ENPP1. In vivo, [68Ga]FAPI-46 uptake was significantly elevated in AS (n=85) compared to controls (n=10; p<0.001). Tracer signal correlated with peak aortic jet velocity (r=0.514, p<0.001) and CT-derived calcific burden (r=0.561, p<0.001). Spatial transcriptomics with unbiased niche analysis (scNiche) identified a peri-nodular cellular microenvironment immediately adjacent to calcified nodules. This region was expanded in high-uptake areas and enriched for FAP+ VICs and macrophages (Figure 2). In contrast, segments with lower tracer uptake showed depletion of this niche and predominance of non-osteogenic VIC states. Conclusion Expansion of a FAP+ VIC population is linked to increasing AS severity and characterised by extracellular matrix remodelling and osteogenic gene expression. [68Ga]FAPI-46 PET detects this biology in vivo, with signal intensity reflecting CT-defined calcification and localising to peri-calcific cellular niches on matched spatial transcriptomics. FAP+ VICs represent both a marker of disease activity and a potential target for therapeutic modulation in aortic stenosis.