A short synthetic peptide inhibits signal transduction, migration and angiogenesis mediated by Tie2 receptor

Abstract

Tie2, an endothelial cell-specific receptor kinase, has an important role in tumour angiogenesis. In an attempt to identify peptides that specifically interact with and block the Tie2 pathway, a phage-displayed peptide library was screened on a recombinant Tie2 receptor. One peptide, NLLMAAS, completely abolished the binding to Tie2 of both angiopoietin 2 and angiopoietin 1 (Ang1). We further show that NLLMAAS specifically suppresses both Ang1-induced ERK activity and migration in human umbilical endothelial cells. Moreover, in vivo, this peptide inhibits angiogenesis in the chick chorioallantoic membrane assay. NLLMAAS is the first peptide described to interact with Tie2. Our results demonstrate that it is an efficient and specific antagonist of the binding of Tie2 ligands, and suggest that this peptide or its derivates may have potential applications in the treatment of angiogenesis diseases. It also represents a potent tool to dissect the molecular mechanisms involved in the Tie2 pathway.

Figure 1

Selected phage-displayed peptides specifically bind to Tie2. The binding of clones was analysed by ELISA as described in the Materials and methods section, and was compared to that of wild-type M13 phage particles as control (Wt phage). Results are representative of three independent assays. ^*P<0.05 versus control.

Figure 2

A synthetic peptide competes with Ang2 for Tie2 binding in ELISA. (A) Peptides were tested in competition with Ang2 for binding to Tie2-Fc. As a positive control, Tie2s was tested under the same conditions. (B) Increasing concentrations of peptide T4 were tested in competition with Ang2 or Ang1 for binding to Tie2. Data represent the mean and standard deviation of triplicates. Similar results were obtained in three independent experiments. ^*P<0.05 versus control.

Figure 3

T4 inhibits the binding of Ang1 and Ang2 to Tie2 in a dose-dependent manner in Biacore experiments. Ang1 or Ang2 were injected onto a Tie2 surface in the presence of a range of concentrations of peptides T4 or T7 (0–1 mM). The association rate was measured for each peptide concentration and divided by that obtained in the absence of peptide: the ratios (in %) are plotted for Ang2 (Fig 3A) and Ang1 (Fig 3B) against the concentration of T4 (bold lines) or T7 (thin lines).

Figure 4

T4 specifically inhibits Ang1-induced MAPK activation in HUVECs. (A) Tie2s blocks p42/p44 MAPK activation induced by Ang1. HUVECs were stimulated with Ang1 with or without Tie2s. (B) T4 inhibits the function of Ang1 in a dose-dependent manner. HUVECs were stimulated with Ang1 in the absence (control) or presence of various concentrations of either peptide T4 (0.125, 0.25, 0.5 and 1 mM) or T7 (1 mM). (C) Specificity of T4 inhibition. HUVECs were stimulated with Ang1 or FGF2 in the absence (control) or presence of peptide T4 (1 mM). Activation of p42/p44 MAPK was monitored as described in the Materials and methods section. Similar results were obtained in two different experiments.

Figure 5

T4 specifically inhibits Ang1-induced HUVEC migration. HUVECs were allowed to migrate in the presence or absence of Ang1 or VEGF with or without peptide T4 (1 mM). (A) Representative picture of each field group. (B) Migration score means and standard errors were measured for three fields. Similar results were obtained in two different experiments. ^*P<0.05 versus control. NS, not significant. HUVEC migration was determined as described in the Materials and methods section.

Figure 6

T4 inhibits CAM angiogenesis in vivo. (A) A network of blood vessels formed in control PBS-treated embryos. (B) No alterations of vessel morphology were observed after treatment with control T7 peptide. (C,D) T4 application on the CAM of two different embryos. (C) Dramatic reduction of vessel density in the T4-treated region. (D) Note the complete loss of capillaries in some regions of the application zone. Scale bar, 1 mm.