CircCRIM1 Ameliorates Endothelial Cell Angiogenesis in Aging through the miR-455-3p/Twist1/VEGFR2 Signaling Axis

Abstract

Background. Aging leads to vascular endothelial cell senescence. Decreased expression of VEGFA and VEGFR2 plays a crucial role in impairing angiogenesis in senescent endothelial cells. Noncoding RNAs, including circular RNAs (circRNAs) and microRNAs (miRNAs), regulate endothelial cell proliferation, differentiation, apoptosis, and migration and participate in the occurrence and development of vascular diseases. However, the mechanism of noncoding RNAs in age-related vascular endothelial dysfunction remains unclear. Here, we aimed to identify the circRNA that is associated with VEGF/VEGFR2 signaling pathway activation in angiogenesis. Methods. Immunoblotting, quantitative reverse transcription-polymerase chain reaction (qRT–PCR), in vitro and in vivo experiments, luciferase assays, and chromatin immunoprecipitation followed by qRT–PCR (ChIP–qPCR) assays were performed to clarify the roles played by circCRIM1 in mouse aortic endothelial cell (MAEC) angiogenesis. Results. CircCRIM1 expression was downregulated in both an aging mouse model of lower limb ischemia in vivo and aging MAECs in vitro. Overexpressing circCRIM1 mediated through a plasmid or adeno-associated virus (AAV) reversed the downregulation of angiogenesis-related phenotype acquisition during aging. MiR-455-3p was confirmed to be a potential target of circCRIM1 through luciferase assays followed by RNA fluorescence in situ hybridization (FISH), which revealed the colocalization of circCRIM1 and miR-455-3p. CircCRIM1 was found to be a competitive endogenous RNA that sponged miR-455-3p and regulated angiogenesis-related phenotypes in MAECs. Furthermore, Twist1 was found to be downstream of miR-455-3p. A ChIP–qPCR assay showed that Twist1 promoted VEGFR2 expression by binding to the promoter region, playing a vital role in angiogenesis. Conclusions. Decreased expression of circCRIM1 impaired angiogenesis in aging via the miR-455-3p/Twist1/VEGFR2 axis. Our findings suggest that overexpression of circCRIM1 may be an effective therapeutic strategy for promoting ischemic lower limb blood flow recovery.