A cystine-knot miniprotein from tomato fruit inhibits endothelial cell migration and angiogenesis by affecting vascular endothelial growth factor receptor (VEGFR) activation and nitric oxide production

Scope: Cystine-knot miniproteins are bioactive molecules with a broad range of potential therapeuticapplications. Recently, it was demonstrated that two tomato cystine-knot miniproteins(TCMPs) exhibit in vitro antiangiogenic activity on human umbilical vein cells. The aim of thepresent study was to investigate the effects of a fruit-specific cystine-knot miniprotein of tomatoon in vitro endothelial cell migration and in vivo angiogenesis using a zebrafish model.Methods and results: The cystine-knot protein purified from tomato fruits using gel filtration LCand RP-HPLC inhibited cell migration when tested at 200 nM using the wound healing assay,and reduced nitric oxide formation probed by 4-amino-5-methylamino-27-difluorofluoscescindiacetate. RT-PCR and Western blot analyses demonstrated that vascular endothelium growthfactor A dependent signaling was the target of TCMP bioactivity. Angiogenesis was inhibitedin vivo in zebrafish embryos treated with 500 nM TCMP.Conclusion: Our results demonstrate that cystine-knot miniproteins present inmature tomatofruits are endowed with antiangiogenic activity in vitro and in vivo. These moleculesmay conferbeneficial effects to tomato dietary intake, along with lycopene and other antioxidants. Furtherinvestigation is warranted to explore the potential of these compounds as model scaffolds forthe development of new drugs.