Targeting angiogenesis: structural characterization and biological properties of a de novo engineered VEGF mimicking peptide

Abstract

Modulating angiogenesis is an attractive goal because many pathological conditions depend on the growth of new vessels. Angiogenesis is mainly regulated by the VEGF, a mitogen specific for endothelial cells. In the last years, many efforts have been pursued to modulate the angiogenic response targeting VEGF and its receptors. Based on the x-ray structure of VEGF bound to the receptor, we designed a peptide, QK, reproducing a region of the VEGF binding interface: the helix region 17-25. NMR conformation analysis of QK revealed that it adopts a helical conformation in water, whereas the peptide corresponding to the alpha-helix region of VEGF, VEGF15, is unstructured. Biological assays in vitro and on bovine aorta endothelial cells suggested that QK binds to the VEGF receptors and competes with VEGF. VEGF15 did not bind to the receptors indicating that the helical structure is necessary for the biological activity. Furthermore, QK induced endothelial cells proliferation, activated cell signaling dependent on VEGF, and increased the VEGF biological response. QK promoted capillary formation and organization in an in vitro assay on matrigel. These results suggested that the helix region 17-25 of VEGF is involved in VEGF receptor activation. The peptide designed to resemble this region shares numerous biological properties of VEGF, thus suggesting that this region is of potential interest for biomedical applications, and molecules mimicking it could be attractive for therapeutic and diagnostic applications.

Fig. 1.

Peptide sequences and circular dichroism spectroscopy. (a) Amino acid sequences of the peptides VEGF15 and QK. The numbering is referred to the VEGF sequence. (b) CD spectra of QK (solid line) and VEGF15 (dashed line) in 10 mM phosphate (pH 7.1) at 20°C. CD spectra are converted and displayed in molar residue ellipticity [θ].

Fig. 2.

NMR structure of QK. (a) Superposition of the backbone of the best 20 cyana QK structures. (b) Backbone superposition of the QK representative structure (yellow) and VEGF helix (red) bound to Flt-1_D2. Side chain of the interacting residues and the Flt-1_D2 electrostatic surface are shown.

Fig. 3.

VEGF receptors binding and activation. (a) VEGF competitive binding on BAEC. One microgram of membrane protein was plated with QK and [¹²⁵I]-VEGF (500,000 cpm, 10^-10 M). (b) KDR activation. After stimulation, total KDR was immunoprecipitated from a whole-cell protein extracts and phospho-tyrosine was visualized by a specific antibody, anti-rabbit horseradish peroxidase-conjugated secondary antibody and standard chemiluminescence. (c) Flt-1 activation. After stimulation, total Flt-1 was immunoprecipitated from a whole-cell protein extract, and phospho-tyrosine was visualized by a specific antibody, anti-rabbit horseradish peroxidase-conjugated secondary antibody, and standard chemiluminescence.

Fig. 4.

Effect of QK and VEGF15 on ERK1/2 activation. Serum-deprived BAEC were treated with QK (a) or with VEGF15 (b) in the absence or presence of VEGF₁₆₅ (100 ng/ml) for 15 min at 37°C and then dissolved in radioimmunoprecipitation assay-SDS buffer. Total ERK1/2 and the phosphorylated form of ERK1/2 were visualized by specific antibodies.

Fig. 5.

Effect of QK on cell proliferation. (a) DNA synthesis. BAEC were incubated in DMEM with [³H]thymidine and QK in the absence or presence of VEGF₁₆₅ (100 ng/ml). After 24 h, cells were fixed and lysed. Scintillation liquid was added, and [³H]thymidine incorporation was evaluated. (b) Cell proliferation. BAEC were stimulated with the indicated amount of QK in the absence or presence of VEGF₁₆₅ (100 ng/ml). Cell number was determined at 24 h after stimulation. (c) RB phosphorylation. phospho-retinoblastoma protein (p-RB) was evaluated at 12 and 18 h after stimulation with QK (10^-6 M), VEGF₁₆₅ (100 ng/ml), and VEGF15 (10^-6 M).

Fig. 6.

In vitro angiogenic properties of QK. Human endothelial cells were cocultured with other human cells in a specially designed medium in a 24-well plate. Every 3 days, QK alone (c) or a combination of QK and VEGF₁₆₅ (100 ng/ml) (d) was added. On the 11th day, cells were fixed with ice cold 70% ethanol, and tubule formation was visualized by staining for anti-human CD31 (PECAM-1). (a–d) Sample images are reported. Suramine (20 μM) (a) and VEGF₁₆₅ (b) were used as negative and positive controls, respectively. (e) The number of cellular connections and the total tubule length were determined by using software that analyzes the images after digitalization.