M ⁶ A-modified circEZH2 Protects Endothelial Cells from Senescence and Suppresses Atherosclerosis by Stabilizing ZNF326

BACKGROUND

Endothelial cell senescence induces endothelial dysfunction, thereby contributing to atherosclerosis progression. Circular RNAs (circRNAs) play diverse roles in multiple physiological and pathological processes. N ⁶ -methyladenosine (m ⁶ A) is the most abundant internal RNA modification in eukaryotic RNAs and dynamically regulates RNA fate and function. However, the functions and therapeutic potential of m ⁶ A-modified circRNAs in endothelial cell senescence remain unknown.

METHODS

m6A-modified circRNAs associated with endothelial cell senescence were screened by circRNA expression and m6A-circRNA microarray profiling of endothelial cells and mouse aortic intima. circEZH2 expression was validated in endothelial cells, vascular tissues, and human atherosclerotic plaques by RT-qPCR, and RNA fluorescence in situ hybridization. The role of circEZH2 in endothelial senescence and atherosclerosis was assessed in vitro and in vivo . RNA pull-down, mass spectrometry, RNA immunoprecipitation, co-immunoprecipitation, ubiquitination assays, and rescue experiments were used to define the underlying mechanism.

RESULTS

We identified A novel m ⁶ A-modified circRNA, circEZH2, that was downregulated in the aged aortic intima and advanced plaques. CircEZH2 was stabilized by m6A reader IGF2BP2. Endothelial cell-specific overexpression of circEZH2 delayed senescence and suppressed atherosclerosis progression. At the cellular level, circEZH2 overexpression delayed senescence, decreased p53/p21 levels and increased angiogenic activity of endothelial cells, while circEZH2 knockdown exhibited the opposite effect. Mechanistically, circEZH2 functions as a scaffold to promote USP37-mediated deubiquitination, thereby stabilizing ZNF326. Moreover, endothelial cell-specific knockdown of ZNF326 counteracts the anti-senescent and anti-atherosclerotic effects mediated by circEZH2 overexpression.

CONCLUSIONS

In summary, the present study identifies circEZH2 as a novel suppressor of endothelial cell senescence, highlighting its potential as a therapeutic target for age-related atherosclerosis.

GRAPHIC ABSTRACT

A graphic abstract is available for this article.

Schematic model of m ⁶ A-modified circEZH2 regulation in endothelial cell senescence and atherosclerosis. In young endothelial cells, IGF2BP2 is highly expressed and recognizes m ⁶ A-modified circEZH2, thereby maintaining its RNA stability. CircEZH2 stabilizes ZNF326 protein through USP37-mediated deubiquitination, which leads to suppression of p21 and p53, delays endothelial cell senescence, ameliorates endothelial dysfunction, and ultimately suppresses the progression of atherosclerosis.

What Are the Clinical Implications?

This study identifies circEZH2 as a novel m6A-modified circular RNA that is reduced in the aged aortic intima and in endothelial cells within advanced atherosclerotic plaques. Endothelial circEZH2 overexpression delays endothelial cell senescence, preserves endothelial function, and suppresses atherosclerotic lesion formation, supporting an important role for circEZH2 in vascular aging-associated atherosclerosis. Mechanistically, circEZH2 acts as a scaffold to enhance USP37-mediated deubiquitination and stabilization of ZNF326, while endothelial ZNF326 knockdown counteracts the anti-senescent and anti-atherosclerotic effects of circEZH2. These findings reveal the circEZH2-ZNF326 axis as a previously unrecognized mechanism regulating endothelial senescence and atherosclerosis progression. Our work supports the potential of circEZH2-based gene therapy as a novel therapeutic approach for atherosclerosis.