Spatial and Single-Cell Mapping Reveals Valvular Interstitial Cell and Macrophage Sex Differences in Calcific Aortic Valve Disease

BACKGROUND: Sex differences in calcific aortic valve disease (CAVD) progressing to aortic valve stenosis have been documented clinically, but the underlying cellular mechanisms that drive sex-dependent fibrocalcification in aortic valve tissue remain poorly understood. METHODS: Human aortic valve tissues were analyzed via histological techniques and scanning electron microscopy to visualize and quantify sites of calcification. Single-cell and spatial transcriptomics were used to investigate mechanisms that drive sex-dependent spatial organization of valvular interstitial cell (VIC) and macrophage gene expression near calcification sites in human male and female aortic valve tissues. RNA in situ hybridization was used to validate sex-dependent gene expression near sites of calcification, and cell communication analyses were performed using bioinformatics tools to determine sex-dependent pathological cell-cell interactions. RESULTS: Histological analyses of aortic valve tissues from healthy patients and patients with CAVD reveal increased valve calcification area in CAVD male aortic valves relative to female valves. Single-cell sequencing analysis of heterogeneous VIC populations reveals male-biased gene expression of the AP-1 (activator protein 1) transcription factor complex and female-biased ECM (extracellular matrix) remodeling genes. Spatial transcriptomics analyses of VIC populations near histologically defined calcification sites revealed male-dependent localization of COMP or cartilage oligomeric matrix protein, as opposed to nonlocalized COMP expression in female VICs. Female CAVD tissues with increased fibrotic density had elevated expression of SERPINE1 , encoding PAI-1 (plasminogen activator inhibitor-1), relative to male tissues. Female CAVD tissues also exhibited increased macrophage count heterogeneity relative to male tissues. Candidate ligand-receptor interactions, including osteopontin ( SPP1 ) interacting with CD44 , which encodes a cell-surface glycoprotein, were identified as contributors to inflammation-mediated CAVD. CONCLUSIONS: Our results reveal sex differences in VIC and macrophage heterogeneity and functions near sites of calcification in aortic valve tissue. Our results highlight the importance of sex-based transcriptomics analyses to understand the cellular phenotypes responsible for causing sex differences in aortic valve fibrocalcification.