Characterization of anti-angiogenic properties of miniproteins isolated from tomato

Angiogenesis, the formation of new blood vessels, is critical to tumour growth and inflammation. The discovery of angiogenesis inhibitors disclosed effective therapeutic strategies. Dietary food compounds have received increasingly attention in disease prevention. The fruit of tomato is rich in compounds with potential anti-angiogenic activity such as lycopene and cystine-knot miniproteins (TCMPs). TCMPs belong to a large family of cysteine-rich proteins present in many organisms and characterized by three intramolecular disulfide bonds forming a structural motif called cystine-knot. These proteins have been characterised for a broad spectrum of therapeutical activities. In a previous study Cavallini et al. (2011) identified two tomato cystine-knot miniproteins (TCMP-1 and -2) and described their antiangiogenic properties in vitro. The present thesis aims to further characterize and give a mechanistic insight into the anti-angiogenic activity of the TCMP-2 which is most abundant in mature tomato fruit and in tomato products. We first set up a method for the extraction and purification of TMCPs from fruits and processed tomato products. The purified TCMP-2 proved to be resistant to gastric peptidases in simulated in vitro digestion. Fruits from several tomato varieties were analyzed for their TCMPs content, observing variations in TCMPs levels particularly in fresh-market varieties. Regarding the characterization of the biological activity of TCMPs, we performed a qRT-PCR expression analysis of genes involved in angiogenesis comparing TCMP-2-treated and untreated human umbilical vein endothelial cells (HUVECs). We found that one of the main effects was the up-regulation of vascular endothelial growth factor (VEGF-A) gene, whose expression was doubled in TCMP-2-treated compared to untreated cells. VEGF is a potent angiogenic agent that regulates all the key steps of the angiogenic process, including endothelial cell proliferation and migration. In order to evaluate the potential effect of TCMPs on the phosphorylation state of VEGFR-2, we performed a western blot analysis on porcine artery endothelial cell (PAEC). This analysis provided evidence that 200 nM TCMP-2 partially inhibits the VEGFA-induced phosphorylation of VEGFR-2. Based on this evidence, we investigated the effects of TCMP-2 on cell migration and nitric oxide (NO) production in VEGF-induced endothelial cells. We found that TCMP-2 inhibits by approximately 50% the increase in cell migration induced by VEGF-A and significantly decreases the formation of NO. On the contrary, lycopene increased NO production in VEGF-A-treated HUVECs. Finally we explored the in vivo antiangiogenic potential of TCMPs using zebrafish embryos as an animal model of angiogenesis. Considering that during zebrafish development, at the stage 72-76 hour post-fertilization (hpf), a functioning vasculature, including subintestinal vessels (SIVs) has already formed, this time-frame was used to examine the TCMPs effects on vessels formation. We observed a significant inhibition of SIVs at 500 nM TCMPs after 76 hpf. These preliminary results suggest that TCMPs might exert antiangiogenic activity in vivo. The results of this thesis contribute to improving the knowledge of the beneficial effect of the tomato, a key component of the Mediterranean diet, in the prevention of diseases. Further investigations are required to evaluate the potential therapeutic implication of our findings.