Substitution of the SEMA3A basic domain with the VEGF165 heparin-binding domain enhances anti-angiogenic potency in ovo

Angiogenesis plays a critical role in tumor development, with neuropilin receptors (NRPs) involved in this process through their interactions with vascular endothelial growth factor (VEGF) and semaphorins. SEMA3A, a member of class 3 semaphorins, demonstrates strong antiangiogenic potential. In this study, the C-terminal basic domain of SEMA3A was replaced with the heparin-binding domain (HBD) of VEGF165, hypothesizing that the resultant chimeric protein SEMA-PSI-IG-HBD would enhance antiangiogenic efficacy by: (1) competing with VEGF for NRPs (2) utilizing the HBD as a molecular anchor to increase the residence time and local concentration of the hybrid within the tumor microenvironment. To assess the effects of SEMA-PSI-IG-HBD on angiogenesis, a series of in vitro assays including microcapillary network formation, 3D cell spheroid sprouting, proliferation and interaction with NRP1 were carried out, whereas in ovo experiments were performed by using the chicken embryo chorioallantoic membrane (CAM) model. Pull-down assays confirmed that the SEMA-PSI-IG-HBD hybrid specifically interacted with secreted NRP1 in a manner similar to the wild-type SEMA3A, validating the structural integrity of the chimeric protein’s receptor-binding interface. SEMA-PSI-IG-HBD and SEMA3A showed comparable inhibition in 2D microcapillary formation and 3D spheroid sprouting assays. However, the hybrid demonstrated markedly superior anti-angiogenic activity in the complex 3D environment of the CAM model. Our findings demonstrate that by combining competitive occupancy of NRP1 with potent anti-angiogenic signaling, SEMA-PSI-IG-HBD represents a promising therapeutic candidate for the regulation of pathological vascularization in cancer.