Objective: Leaflet thickening, fibrosis, and hardening are early pathological features of calcific aortic valve disease (CAVD). An inadequate understanding of the resident aortic valve cells involved in the pathological process may compromise the development of therapeutic strategies. We aim to construct a pattern of the human aortic valve cell atlas in healthy and CAVD clinical specimens, providing insight into the cellular origins of CAVD and the complex cytopathological differentiation process. Approach and Results: We used unbiased single-cell RNA sequencing for the high-throughput evaluation of cell heterogeneity in 34 632 cells isolated from 6 different human aortic valve leaflets. Cellular experiments, in situ localization, and bulk sequencing were performed to verify the differences between normal, healthy valves and those with CAVD. By comparing healthy and CAVD specimens, we identified 14 cell subtypes, including 3 heterogeneous subpopulations of resident valve interstitial cells, 3 types of immune-derived cells, 2 types of valve endothelial cells, and 6 novel valve-derived stromal cells found particularly in CAVD leaflets. Combining additional verification experiments with single-cell transcriptome profiling provided evidence of endothelial to mesenchymal transition involved in lesion thickening of the aortic valve leaflet. Conclusions: Our findings deconstructed the aortic valve cell atlas and suggested novel functional interactions among resident cell subpopulations. Our findings may provide insight into future targeted therapies to prevent CAVD.

Lactate plays a critical role as an energy substrate, metabolite, and signaling molecule in hepatocellular carcinoma (HCC). Intracellular lactate‐derived protein lysine lactylation (Kla) is identified as a contributor to the progression of HCC. Liver cancer stem cells (LCSCs) are believed to be the root cause of phenotypic and functional heterogeneity in HCC. However, the impact of Kla on the biological processes of LCSCs remains poorly understood. Here enhanced glycolytic metabolism, lactate accumulation, and elevated levels of lactylation are observed in LCSCs compared to HCC cells. H3K56la was found to be closely associated with tumourigenesis and stemness of LCSCs. Notably, a comprehensive examination of the lactylome and proteome of LCSCs and HCC cells identified the ALDOA K230/322 lactylation, which plays a critical role in promoting the stemness of LCSCs. Furthermore, this study demonstrated the tight binding between aldolase A (ALDOA) and dead box deconjugate enzyme 17 (DDX17), which is attenuated by ALDOA lactylation, ultimately enhancing the regulatory function of DDX17 in maintaining the stemness of LCSCs. This investigation highlights the significance of Kla in modulating the stemness of LCSCs and its impact on the progression of HCC. Targeting lactylation in LCSCs may offer a promising therapeutic approach for treating HCC.

Valve interstitial cells (VICs) undergo a transition to intermediate state cells before ultimately transforming into the osteogenic cell population, which is a pivotal cellular process in calcific aortic valve disease (CAVD). Herein, this study successfully delineated the stages of VIC osteogenic transformation and elucidated a novel key regulatory role of lumican (LUM) in this process. Single-cell RNA-sequencing (scRNA-seq) from nine human aortic valves was used to characterize the pathological switch process and identify key regulatory factors. The in vitro, ex vivo, in vivo, and double knockout mice were constructed to further unravel the calcification-promoting effect of LUM. Moreover, the multi-omic approaches were employed to analyse the molecular mechanism of LUM in CAVD. ScRNA-seq successfully delineated the process of VIC pathological transformation and highlighted the significance of LUM as a novel molecule in this process. The pro-calcification role of LUM is confirmed on the in vitro, ex vivo, in vivo level, and ApoE^−/−//LUM^−/− double knockout mice. The LUM induces osteogenesis in VICs via activation of inflammatory pathways and augmentation of cellular glycolysis, resulting in the accumulation of lactate. Subsequent investigation has unveiled a novel LUM driving histone modification, lactylation, which plays a role in facilitating valve calcification. More importantly, this study has identified two specific sites of histone lactylation, namely, H3K14la and H3K9la, which have been found to facilitate the process of calcification. The confirmation of these modification sites’ association with the expression of calcific genes Runx2 and BMP2 has been achieved through ChIP-PCR analysis. The study presents novel findings, being the first to establish the involvement of lumican in mediating H3 histone lactylation, thus facilitating the development of aortic valve calcification. Consequently, lumican would be a promising therapeutic target for intervention in the treatment of CAVD. Single-cell RNA-sequencing (scRNA-seq) delineated the process of valve interstitial cell (VIC) pathological transformation and highlighted the significance of lumican (LUM) as a novel molecule in this process. The pro-calcification role of LUM was confirmed in the in vitro, ex vivo, and ApoE^−/−//LUM^−/− double knockout mice. The LUM induced osteogenesis in VICs via augmentation of cellular glycolysis, resulting in the accumulation of lactate. Subsequent multi-omic approaches and molecular biological verification unveiled a novel LUM driving H3 histone modification, lactylation, which plays a role in facilitating valve calcification