Single-Cell RNA Sequencing Unveils Macrophage Involvement in Aortic Valve Stenosis Progression
Introduction: Calcific aortic stenosis (AS) is an age-related progressive disease with increasing global prevalence. Although chronic inflammation is a key factor in its pathogenesis, the detailed immune cell profiles and their interactions with non-immune cells remain poorly understood. This study aimed to characterize the immune cell landscape in calcific AS at a single-cell level and evaluate the potential of anti-inflammatory therapy. Methods: Human aortic valve samples were obtained from four patients with non-calcified aortic regurgitation (AR) and 11 patients with AS undergoing surgical aortic valve replacement. Single-cell 3' gene expression sequencing (scRNA-seq (3')) was performed on CD45+ immune cells, while single-cell RNA sequencing Flex (scRNA-seq (Flex)) and bulk RNA sequencing were conducted on unsorted cells. Additionally, an anti-TNFa antibody experiment was performed in a murine AS model induced by aortic valve wire injury. Results: In myeloid cells, LYVE1 resident macrophages were significantly decreased, whereas classical monocytes expressing pro-inflammatory genes and C1Q macrophages were increased in AS valves compared to AR valves. These macrophages exhibited strong interactions with valve interstitial cells, particularly with chondrogenic subtypes expressing COMP and SPARC. Anti-TNFa treatment in the murine model significantly reduced aortic valve calcification without hematopoietic complications. Conclusions: This study is the first to integrate scRNA-seq (3') and scRNA-seq (Flex) analyses of human aortic valves, revealing single cell-level characteristics of immune cells and their interactions with non-immune cells in calcific AS. These findings suggest that anti-inflammatory therapy could be a therapeutic target for calcific AS. Overall design: scRNA-seq of surgically extracted human aortic valve tissues (aortic regurgitation vs. aortic stenosis), which includes scRNA-seq (3') for CD45 sorted immune cells and scRNA-seq (Flex) for non-sorted cells. Bulk RNA-seq was also conducted for human aortic valve tissues. AS model mice induced by aortic valve wire injury were treated either TNFa antibody or IgG control, then murine aortic valve tissues were evaluted using spatial transcriptomics (visium HD).