A Urokinase Receptor–Derived Peptide Inhibiting VEGF-Dependent Directional Migration and Vascular Sprouting

Abstract

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration, and uPAR [88][89][90][91][92] is the minimal sequence required to induce cell motility. We previously showed that soluble forms of uPAR elicit angiogenic responses through their uPAR 88-92 chemotactic sequence and that the synthetic peptide SRSRY exerts similar effects. By a drug design approach, based on the conformational analysis of the uPAR 88-92 sequence, we developed peptides (pERERY, RERY, and RERF) that potently inhibit signaling triggered by uPAR [88][89][90][91][92] . In this study, we present evidence that these peptides are endowed also with a clear-cut antiangiogenic activity, although to different extents. The most active, RERF, prevents tube formation by human endothelial cells exposed to SRSRY. RERF also inhibits VEGF-triggered endothelial cell migration and cord-like formation in a dose-dependent manner, starting in the femtomolar range. RERF prevents F-actin polymerization, recruitment of avb3 integrin at focal adhesions, and avb3/VEGFR2 complex formation in endothelial cells exposed to VEGF. At molecular level, the inhibitory effect of RERF on VEGF signaling is shown by the decreased amount of phospho-FAK and phospho-Akt in VEGF-treated cells. In vivo, RERF prevents VEGF-dependent capillary sprouts originating from the host vessels that invaded angioreactors implanted in mice and neovascularization induced by subcorneal implantation of pellets containing VEGF in rabbits. Consistently, RERF reduced the growth and vascularization rate of tumors formed by HT1080 cells injected subcutaneously in the flanks of nude mice, indicating that RERF is a promising therapeutic agent for the control of diseases fuelled by excessive angiogenesis such as cancer.

UPARANT: A Urokinase Receptor–Derived Peptide Inhibitor of VEGF-Driven Angiogenesis with Enhanced Stability and In Vitro and In Vivo Potency

“…Cell lysate protein content was measured by a colorimetric assay (Bio-Rad). Western blot analysis was performed as previously described (17,20). Briefly, 50 mg proteins were separated on 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane.…”

“…RERF has been shown to inhibit endothelial cell migration driven by VEGF (20). We compared the effects of RERF and Aib containing peptides in inhibiting endothelial cell migration directed toward 40 ng/mL VEGF 165 .…”

“…To test whether similar to RERF (20), UPARANT competes with fMLF for binding to FPR, HUVEC were preincubated at 4 C (to avoid receptor internalization) with diluents, 100 nmol/L concentration of fMLF, RERF, or UPARANT for 60 minutes at 4 C, and then exposed to 10 nmol/L N-formyl-NleLeu-Phe-Nle-Tyr-Lys-fluorescein (FITC-fMLF) for additional 60 minutes at 4 C. Quantification of cell-associated fluorescence was assessed by reading endothelial cell lysates with a fluorescence plate reader. An aliquot of each sample was subjected to Western blot analysis with anti-a-tubulin to control that equal amount of proteins/ sample has been read (inset to Fig.…”

“…UPARANT inhibits cytoskeletal organization, avb3 recruitment at focal adhesions, and adhesion onto vitronectin of endothelial cells stimulated with VEGF VEGF-triggered endothelial cell motility is the result of marked cytoskeletal reorganization and accumulation of stress fibers associated with new actin polymerization, and rapid formation of focal adhesions, which do not occur in the presence of RERF (20). To analyze the effects of UPARANT on cytoskeletal organization induced by VEGF, HUVEC were exposed to 40 ng/mL VEGF 165 , in the presence/absence of 10 nmol/L UPARANT, and then double stained for vinculin and F-actin.…”

“…It inhibits in vitro and in vivo responses promoted either by uPAR [88][89][90][91][92] or by VEGF. RERF also prevents cytoskeletal organization and the recruitment of avb3 integrin at the focal adhesions in endothelial cells exposed to VEGF, by forcing avb3 in an inactive state either directly or indirectly, through FPR (20).…”