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

Abstract

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.