Endothelial Cell Differentiation-Related CircRNAs Drive the Differentiation of Vascular Endothelial Cells

The emergence and progression of a range of cardiovascular and cerebrovascular conditions are linked to endothelial dysfunction. Nevertheless, the specific pathways leading to damage in vascular endothelium remain to be elucidated. This study aimed to investigate the function and mechanisms of endothelial cell differentiation-related circular RNAs (circRNAs) in the differentiation of vascular endothelial cells. First, we screened for differentially expressed circRNAs (DECs) and genes (DEGs) during the differentiation of induced pluripotent stem cells (iPSCs) into endothelial cells, using human umbilical vein endothelial cells (HUVECs) as a reference model. Next, we predicted miRNAs associated with DECs and identified core genes related to the differentiation of stem cells into endothelial cells. Then, we verified the expression of endothelial cell differentiation-related circRNAs and the competing endogenous RNA (ceRNA) axis at the cellular and organoid level. Finally, we explored the ceRNA regulatory axis. Six core genes were initially identified, but we focused on a disintegrin and metalloproteinase with thrombospondin motifs 9 (ADAMTS9). At the cellular level, hsa_circ_0000010, hsa_circ_0005875, hsa_circ_0090122, and ADAMTS9 mRNA and protein levels were significantly increased in the HUVEC group, while hsa_circ_0000161, hsa_circ_0002824, hsa_circ_0074944, TXNIP mRNA, hsa-miR-548az-5p, and hsa-miR-548t-5p levels were significantly decreased compared to the iPSC group. At the organoid level, during the differentiation of stem cells into endothelial cells, hsa_circ_0090122 and ADAMTS9 mRNA and protein levels were increased, while hsa-miR-548az-5p and hsa-miR-548t-5p levels were decreased. Among the candidates, hsa_circ_0090122 and its related miRNAs (hsa-miR-548az-5p/hsa-miR-548t-5p) were prioritized because their expression changes were the most pronounced and directionally consistent with ADAMTS9, both in bioinformatic screening and validation experiments. Hsa_circ_0090122 may play a significant role in driving endothelial differentiation by regulating ADAMTS9 expression through competitive interaction with hsa-miR-548az-5p and hsa-miR-548t-5p.