Endorepellin Affects Angiogenesis by Antagonizing Diverse Vascular Endothelial Growth Factor Receptor 2 (VEGFR2)-evoked Signaling Pathways TRANSCRIPTIONAL REPRESSION OF HYPOXIA-INDUCIBLE FACTOR 1α AND VEGFA AND CONCURRENT INHIBITION OF NUCLEAR FACTOR OF ACTIVATED T CELL 1 (NFAT1) ACTIVATION

Endorepellin, the angiostatic C-terminal domain of the heparan sulfate proteoglycan perlecan, inhibits angiogenesis by simultaneously binding to the α2β1 integrin and the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) on endothelial cells. This interaction triggers the down-regulation of both receptors and the concurrent activation of the tyrosine phosphatase SHP-1, which leads to a signaling cascade resulting in angiostasis. Here, we provide evidence that endorepellin is capable of attenuating both the PI3K/PDK1/Akt/mTOR and the PKC/JNK/AP1 pathways. We show that hypoxia-inducible factor 1α (HIF-1α) transcriptional activity induced by VEGFA was inhibited by endorepellin independent of oxygen concentration and that only a combination of both PI3K and calcineurin inhibitors completely blocked the suppressive activity evoked by endorepellin on HIF1A and VEGFA promoter activity. Moreover, endorepellin inhibited the PKC/JNK/AP1 axis induced by the recruitment of phospholipase γ and attenuated the VEGFA-induced activation of NFAT1, a process dependent on calcineurin activity. Finally, endorepellin inhibited VEGFA-evoked nuclear translocation of NFAT1 and promoted NFAT1 stability. Thus, we provide evidence for a novel downstream signaling axis for an angiostatic fragment and for the key components involved in the dual antagonistic activity of endorepellin, highlighting its potential use as a therapeutic agent.