Modulation of angiogenesis by a tetrameric tripeptide that antagonizes vascular endothelial growth factor receptor 1

Abstract

Vascular endothelial growth factor receptor-1 (VEGFR-1, also known as Flt-1) is involved in complex biological processes often associated to severe pathological conditions like cancer, inflammation, and metastasis formation. Consequently, the search for antagonists of Flt-1 has recently gained a growing interest. Here we report the identification of a tetrameric tripeptide from a combinatorial peptide library built using non-natural amino acids, which binds Flt-1 and inhibits in vitro its interaction with placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) A and B (IC(50) approximately 10 microm). The peptide is stable in serum for 7 days and prevents both Flt-1 phosphorylation and the capillary-like tube formation of human primary endothelial cells stimulated by PlGF or VEGF-A. Conversely, the identified peptide does not interfere in VEGF-induced VEGFR-2 activation. In vivo, this peptide inhibits VEGF-A- and PlGF-induced neoangiogenesis in the chicken embryo chorioallantoic membrane assay. In contrast, in the cornea, where avascularity is maintained by high levels of expression of the soluble form of Flt-1 receptor (sFlt-1) that prevents the VEGF-A activity, the peptide is able to stimulate corneal mouse neovascularization in physiological condition, as reported previously for others neutralizing anti-Flt-1 molecules. This tetrameric tripeptide represents a new, promising compound for therapeutic approaches in pathologies where Flt-1 activation plays a crucial role.

FIGURE 1.

Structure of 4-23-5 peptide. A, schematic representation of the 4-23-5 tetrameric tripeptide. l-Cys(Bzl), l-cysteine(S-benzyl); l-Cha, l-cyclohexylalanine. B, chemical structure of the 4-23-5 peptide that has a calculated MW of 2362.02 atomic mass units.

FIGURE 2.

Tetrameric peptide 4-23-5 inhibits the binding of VEGF family members with Flt-1 receptor. A-C, dose-dependent inhibition of PlGF (A), VEGF-A (B), and VEGF-B (C) interaction with Flt-1 exerted by 4-23-5 peptide in competitive ELISA-based assays. Tetrameric 4-23-5 (○), 4-23-A (▴), and 21-1-5 (▪) peptides were assayed at concentrations ranging between 1.56 and 50.0 μm. The results represent the average of three independent experiments. The error bars represent the S.D.

FIGURE 3.

Tetrameric peptide 4-23-5 binds specifically to Flt-1 receptor. The binding properties of selected peptide were assessed by ELISA-based assays. A, binding of human (black bar) or mouse (gray bar) Flt-1 receptors (250 pm) to 4-23-5 peptide coated on a microtiter plate at 20 μm. The two receptors failed to bind to three control peptides: 4-23-5 dimer, 4-23-A, and 21-1-5 coated at the same concentration. B, dose-dependent inhibition of Flt-1 (125 pm) interaction with coated 4-23-5 peptide (20 μm) exerted by PlGF (○) and VEGF-A (▴). Soluble growth factors were assayed at concentrations ranging from 0.05 to 6.25 nm. C, dose-dependent interaction of human and mouse Flt-1 (hFlt-1 and mFlt-1) assayed at 125 pm (black bar) and 250 pm,(gray bar) with 4-23-5 peptide coated at 20 μm. VEGFRs-2 (human KDR (hKDR) and mouse Flk-1 (mFlk-1)) failed to bind 4-23-5 peptide in the same conditions. The results represent the average of three independent experiments. The error bars represent the S.D.

FIGURE 4.

Stability and neutralizing properties of peptide 4-23-5. A, the resistance to enzyme degradation of the selected peptide was assessed by incubation in 10% fetal calf serum for 168 h. At time 0, after every hour within the first 12 h, and at 24, 72, 120, and 168 h, three aliquots were removed, centrifuged to remove proteins, and analyzed by HPLC (10 μl, 1 μg). Residual peptide quantity, expressed as the percentage of the initial amount versus time, was plotted. The results represent the average of three independent experiments. The error bars represent the S.D. The ability of selected peptide to inhibit the VEGFRs activation exerted by PlGF and VEGF-A was evaluated in receptor phosphorylation assays (B-D). Starved 293-hFlt1 cells were stimulated with 20 ng/ml PlGF (B) or 50 ng/ml of VEGF-A (C) for 10 min, in the presence or absence of active and control peptides. As controls, neutralizing anti-PlGF or anti-VEGF antibodies were used. Similarly, starved HUVECs were stimulated with VEGF-A (D). 100 μg of cell lysate were used for Western blot analyses. Anti-phospho-Flt-1 (pFlt-1) or anti-phospho-KDR (pKDR) antibodies were used to detect the level of receptor phosphorylation, whereas anti-Flt-1 (Flt-1) or anti-KDR (KDR) antibodies were used for normalization. The values of densitometry analyses, performed using ImageQuant 5.2 software (GE Healthcare) are shown. Values (in percentages) were calculated as the ratio of degree of receptor phosphorylation with respect to the total receptor amounts. The value of 100 has been arbitrarily assigned to PlGF- or VEGF-induced samples.

FIGURE 5.

Tetrameric tripeptide 4-23-5 inhibits capillary-like tube formation induced by PlGF or VEGF-A. A-N, representative pictures of capillary-like tube formation stimulated with 100 ng/ml PlGF (A-G) or VEGF-A (H-N) in the presence or absence of active and control peptides. PlGF (A) or VEGF-A (H) in endothelial basal medium (EBM-2) was able to stimulate the formation of structure similar to capillaries. 4-23-5 peptide at 20 μm completely prevented CTF induced by PlGF (B) or VEGF-A (I), whereas control peptides 4-23-A (C and J) and 21-1-5 (D and K) at 50 μm failed to block CTF. Tetrameric peptide 4-23-5 was still able to fully block CTF stimulated by both PlGF and VEGF-A at 4.0 μm (E and L). At 0.80 μm (F and M), a partial inhibitory effect was still present, whereas at 0.16 μm (G and N), peptide lost the capability to inhibit CTF (original magnification ×10).

FIGURE 6.

Tetrameric tripeptide 4-23-5 inhibits in vivo neovessels formation stimulated by VEGF-A or PlGF in CAM assay. A and B, alginate beads containing 150 ng/embryo of VEGF-A (A) or 250 ng/embryo of PlGF (B), with or without peptides, vehicle, or peptides alone, were placed on top of the CAM of fertilized White Leghorn chicken eggs at day 11 of incubation (6-8 eggs per experimental group). After 72 h, new blood vessels converging toward the implant were counted by two observers in a double-blind fashion under a stereomicroscope. Black bars, VEGF-A or PlGF; light gray bars, factors plus 0.25 nmol of peptides or peptides alone; dark gray bars, factors plus 0.025 nmol of peptide; white bars, vehicle. 4-23-5 peptide significantly inhibited either PlGF-induced or VEGF-A-induced angiogenesis in a dose-dependent manner in all the embryos tested. No inhibition was detected with the control peptide 21-1-5, except for a partial inhibition observed only in 2 out of the 6 embryos stimulated with VEGF-A and treated with 0.25 nmol of the control peptide. The error bars represent the S.D. ^*, p < 0.005 versus VEGF-A; ^#, p < 0.0001 versus VEGF-A; ^¶, p < 0.05 versus VEGF-A or PlGF; ^§, p < 0.005 versus PlGF.

FIGURE 7.

Tetrameric tripeptide 4-23-5 stimulates corneal neovascularization. A total of 0.4 (A and E), 4.0 (B and F), or 20.0 (C and G) nmol of 4-23-5 peptide and 20.0 nmol of control peptide 21-1-5 (D and H), in the same volume of DMSO, were injected in the corneas of BALB/c mice. After 7 days, corneas were photographed (A-D) and then harvested and flat-mounted. In A, new vessels are indicated with arrows. New vessels were immunostained (E-H) using anti-mouse CD31 antibodies (green) and anti-mouse-LYVE-1 antibodies (red). The areas corresponding to cornea have been indicated with a white circle. Blood vessels were defined as CD31-positive and LYVE-1-negative (original magnification ×4).