Spatiotemporal dynamics of interleukin 6 in calcific aortic valve disease: a multi-omics and translational study

Background Valve interstitial cells (VICs) play a central role in calcific aortic valve disease (CAVD) and show a great heterogeneity. Herein, the study systematically characterizes the subpopulations of VICs and identifies interleukin 6 (IL6)’ spatiotemporal dynamics, therapeutic target, underlying mechanisms, and predictive value. Methods Public and in-house single-cell RNA-sequencing (scRNA-seq) data of 11 human aortic valves were integrated and the heterogeneity of major celltypes and VIC subpopulations was characterized. The spatiotemporal dynamics of cellular populations and IL6 was explored using scRNA-seq, bulk RNA-sequencing, and pathological staining. The in vitro and in vivo experiments were conducted to explore the calcification-promoting effect and molecular mechanism of IL6. Case-control plasma samples and UK Biobank were used to validate the predictive value of plasma concentration of IL6 in calcific AS. Results VICs were classified into 14 heterogeneous subclusters, but with silent or activated phenotypes. IL6 was identified as the key player in CAVD by single cell WGCNA analysis. IL6 presents an increasing trend as CAVD progresses from non-diseased (ND) to fibrotic to calcific stage, broadly distributed around three layers of aortic valve. Neutralizing antibody of IL6 effectively alleviates the calcific phenotype in two CAVD mouse models and human primary VICs. The pro-calcific effects of conditional medium on VICs are effectively abolished upon addition of an IL6-neutralizing antibody, indicating IL6 induces the calcific activation of VICs through a paracrine mechanism. Mechanic experiments on VICs indicate IL6 promotes calcification via IL6/JAK2/STAT3 pathway. Plasma IL6 levels are significantly elevated in patients with AS compared to non-AS subjects, with the area under the curve of 0.768 and 0.605 in our cohort and UK Biobank, respectively. After adjusting confounders, IL6 presents an independent predictor of the incidence of both AS (HR =1.24, 1.15-1.35) and AS-related adverse events (HR = 1.19, 1.04-1.35). Conclusions IL6 promotes aortic valve calcification via IL6/JAK2/STAT3 and acts in a paracrine way among VICs, being a promising therapeutic target and predictive biomarker of CAVD.