Endothelial KLF15/VASN Axis Inhibits Angiogenesis via Activation of Notch1 Signaling

BACKGROUND: Angiogenesis is a dynamic process fine-tuned by transcription factors in endothelial cells. The KLF15 (Krüppel-like factor 15)-mediated transcriptional regulation mechanism is critical for cardiovascular diseases. However, the role of KLF15 in governing angiogenesis remains unknown. METHODS: KLF15 and VASN (vasorin) were deleted from endothelial cells using tamoxifen-inducible Cdh5 promoter–driven Cre recombinase in EC-KLF15 knockout (KO) and EC-VASN KO mice, respectively. EC-KLF15 KO, EC-VASN KO, and control mice were subjected to retinal angiogenesis or tumor cell transplantation. The RNA sequencing, assay for transposase-accessible chromatin using sequencing, and chromatin immunoprecipitation sequencing were conducted to identify VASN as a downstream effector of KLF15. Cell proliferation, wound healing, tube formation, and sprouting assays were performed to delineate endothelial cell function. RESULTS: In EC-KLF15 KO mice and adenovirus-mediated KLF15 overexpression mice, we showed that KLF15 negatively regulated retinal angiogenesis, as confirmed in cultured endothelial cells. KLF15 opened chromatin, bound to the promoters of GC-rich sequences, and transactivated the expression of VASN. Subsequently, VASN suppressed endothelial angiogenic function, which was essential for Dll4 (delta-like ligand 4)-induced Notch1 signaling activation. Moreover, increased expression of VASN in EC-KLF15 KO mice suppressed retinal angiogenesis, which was attenuated by γ-secretase inhibitor. EC-VASN KO mice recapitulated the promotion of retinal angiogenesis in EC-KLF15 KO mice. Finally, the EGF (epidermal growth factor)-like domain of VASN was essential for its interaction with Notch1, and VASN EGF-like domain-derived peptides activated Notch1 signaling and suppressed angiogenesis. CONCLUSIONS: The KLF15/VASN axis negatively regulates angiogenesis by activating Notch1 signaling. KLF15 and VASN might represent novel therapeutic targets for the treatment of impaired angiogenesis-related diseases and tumors.