Screening for candidate genes and cell populations associated with atherosclerotic calcification using single-cell RNA sequencing.

Abstract

Calcification often occurs as a characteristic pathological manifestation in the progression of atherosclerosis (AS) plaques, but its mechanism is not fully understood yet. The purpose of this research was to supplement the exploration of key candidate genes and key cells involved in the calcification process of AS, building on existing insights into its underlying mechanisms. Through the examination of our internally generated single‑cell RNA sequencing (scRNA-seq) dataset derived from human carotid plaque samples, pivotal cellular populations associated with AS calcification were successfully identified. Following this identification, a comprehensive analytical approach was employed, incorporating differential gene expression profiling alongside the establishment of protein-protein interaction (PPI) networks, thereby enabling the extraction of critical genetic markers within these cellular subsets. Furthermore, a molecular regulatory framework was assembled, aiming to elucidate the mechanistic pathways through which these genetic determinants contribute to the calcification phenomena in AS pathology. Moreover, analysis of cell communication was applied to explore the interactions among cells. Pseudo-time analysis was employed to explore the expression of key candidate genes during the differentiation of key cells. Finally, monocytes were identified as key cells. WARS1, IFITM1, ANXA1, ADGRE2, and S100P were identified as key candidate genes. Moreover, 115 transcription factors such as THRB and 118 miRNAs such as hsa-miR-196a-5p were predicted to be associated with the key candidate genes. Across both calcified and non-calcified control specimens, the cellular communication between endothelial cells and natural killer (NK) T cell populations was consistently orchestrated via the PPBP-CXCR2 signaling axis. During monocytic differentiation trajectories, ADGRE2 expression exhibited a biphasic pattern characterized by initial gradual elevation followed by subsequent decline. Conversely, both ANXA1 and S100P demonstrated progressive upregulation throughout the differentiation process. The expression of IFITM1 and WARS1 first decreased, then increased, and finally decreased again. The present investigation successfully pinpointed five critical genes alongside one key cellular population, collectively providing potential molecular insights and candidate targets for further investigation into AS calcification.