Angiogenesis contributes to plaque instability in atherosclerosis and further increases cardio-cerebrovascular risk. Circular RNAs (circRNAs) are promising biomarkers and potential therapeutic targets for atherosclerosis. Previous studies have demonstrated that tetramethylpyrazine (TMP) and paeoniflorin (PF) combination treatment (TMP-PF) inhibited oxidized low-density lipoprotein (ox-LDL)-induced angiogenesis in vitro. However, whether circRNAs regulate angiogenesis in atherosclerosis and whether TMP-PF can regulate angiogenesis-related target circRNAs in atherosclerosis are unknown. In this study, human RNA sequencing (RNA-seq) data were analysed to identify differentially expressed (DE) circRNAs in atherosclerosis and to obtain angiogenesis-associated circRNA-microRNA (miRNA)-messenger RNA (mRNA) networks. Target circRNA-related mechanisms in angiogenesis in atherosclerosis and the regulatory effects of TMP-PF on target circRNA signalling were studied in ox-LDL-induced human umbilical vein endothelial cells (HUVECs) by cell proliferation, migration, tube formation, and luciferase reporter assays, real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. A novel circRNA (circular stimulator of chondrogenesis 1, circSCRG1) was initially identified associated with angiogenesis in atherosclerosis, and circSCRG1 silencing up-regulated miR-1268b expression, increased nuclear receptor subfamily 4 group A member 1 (NR4A1) expression and then promoted ox-LDL-induced angiogenesis. TMP-PF (1 μmol/L TMP combined with 10 μmol/L PF) up-regulated circSCRG1 expression, mediated miR-1268b to suppress NR4A1 expression and then inhibited ox-LDL-induced angiogenesis. However, circSCRG1 silencing abolished the inhibitory effects of TMP-PF on ox-LDL-induced angiogenesis, which were rescued by the miR-1268b inhibitor. In conclusion, circSCRG1 might serve as a new target regulating angiogenesis in atherosclerosis via the circSCRG1/miR-1268b/NR4A1 axis and TMP-PF could regulate the circSCRG1/miR-1268b/NR4A1 axis to inhibit angiogenesis in atherosclerosis in vitro, indicating a novel angiogenesis signalling circSCRG1/miR-1268b/NR4A1 pathway in atherosclerosis and the regulatory effects of TMP-PF, which might provide a new pharmaceutical strategy to combat atherosclerotic plaque instability.
Keywords: Chinese medicine; atherosclerosis; cardio-cerebrovascular disease; circRNA; neovascularization; paeoniflorin; plaque stability; tetramethylpyrazine.