Notch activation downregulates EZH2 to thereby attenuate endothelial cell proliferation and angiogenesis via MYC...

Angiogenesis, a tightly regulated process involving dynamic endothelial cell (EC) proliferation, is critical in both physiological and pathological contexts such as ocular neovascular disorders. While Notch signaling is known to regulate angiogenesis, its downstream molecular mechanisms remain incompletely understood.

Gene-modified mice with a Cdh5-Cre^ERT transgene were generated to block Notch signaling in ECs. Primary HUVECs were cultured in vitro. Gene expression were analyzed via qRT-PCR, western blotting, and immunofluorescence. Transcriptional regulation was investigated using reporter and ChIP assays. EC proliferation and migration were assessed through EdU incorporation, Transwell, and wound healing assays, respectively. Angiogenesis was evaluated in vivo using Matrigel plug, retinal angiogenesis, oxygen-induced retinopathy (OIR), and choroidal neovascularization (CNV) models.

Notch activation upregulated, whereas Notch blockade downregulated EZH2 expression in both mRNA and protein levels. Mechanistically, Notch signaling directly suppresses EZH2 promoter activity, thereby transcriptionally repressing EZH2 expression. Functionally, EZH2 inhibition impaired EC proliferation and sprouting angiogenesis, while EZH2 overexpression enhanced these processes. Furthermore, EZH2 inhibition reversed the pro-angiogenic effects of Notch blockade. At the molecular level, EZH2 stabilized MYC protein by modulating Thr58 phosphorylation and regulating MYC stability factors, thereby preventing its proteasomal degradation. MYC overexpression rescued the angiogenesis defects caused by EZH2 inhibition. Importantly, EZH2 was upregulated in OIR and CNV models, and pharmacological EZH2 inhibition (GSK126) effectively suppressed pathological angiogenesis.

Our findings not only elucidate the novel role of EZH2 in mediating Notch function in angiogenesis but also provide a promising therapeutic strategy for treating neovascularization-related diseases with EZH2 inhibitors.