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 α-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.
Get alerts for new articles, or get an alert when an article is cited.
Angiogenesis is a phenomenon intimately associated with endothelial cell (EC) migration and proliferation. During embryonic development, ECs rapidly proliferate, thereby forming new blood vessels. In adult life, however, EC turnover is very low but under a variety of pathological conditions such as chronic ischemia, cancer, proliferative retinopathy, and rheumatoid arthritis, these cells detach from their neighbors, migrate, proliferate, and subsequently form a new branch vessel with a lumen. The mediators of this proliferation have been identified in a series of vascular growth factors that can be released in response to many intermediates such as cytokines.
VEGF is a potent angiogenic factor, a mitogen specific for vascular ECs and plays a major role in angiogenesis. VEGF and its receptors are overexpressed in pathological angiogenesis, making this system a potential target for therapeutic and diagnostic applications.
VEGF is a homodimeric protein belonging to the cystine knot growth factor family. It is encoded by a single gene that is expressed in four different isoforms because of different splicing events. VEGF 165, the most abundant isoform, is a glycoprotein that binds to heparin with high affinity. The biological function of VEGF is mediated through binding to two tyrosine kinase receptors, the kinase domain receptor (KDR) and the Fms-like tyrosine kinase (Flt-1). VEGF induces receptor dimerization that stimulates EC mitogenesis. KDR and Flt-1 are localized on the cell surface of various EC types. Increased expression of these receptors occurs in response to several stimuli and results in priming of EC toward proliferation, migration, and angiogenesis. Oxygen tension plays a major role in the regulation of VEGF. Its mRNA expression is rapidly and reversibly induced by hypoxia in a variety of normal and transformed cultured cell types.
Several VEGF structures have been reported included the VEGF bound to the extracellular domain 2 of Flt-1. Two VEGF monomers, linked by disulfide bonds, bind to two receptor molecules that are localized at the poles of the VEGF antiparallel homodimer. The analysis of structural and mutagenesis data allowed to identify the residues involved in the binding to the receptors. They are distributed over a discontinuous surface that include residues from the N-terminal helix 17–25. KDR and Flt-1 share the VEGF binding region, in fact five of the seven most important VEGF binding residues are present in both interfaces.
Many approaches have been pursued to modulate the VEGF–receptors interaction, and new molecular entities as peptides and antibodies have been reported to bind to the extracellular region of the VEGF receptors. A large number of them show an antagonist activity and only few behave as agonists. Remarkably, the peptides modulating the VEGF–receptors interaction are mainly derived by phage display libraries screening, and only few examples of rational design approaches have been reported so far.
The aim of this work was to develop small peptides able to recognize VEGF receptors to modulate both endothelial cell proliferation and propensity toward angiogenesis. We designed, by a structure-based approach, a peptide, QK, reproducing the VEGF 17–25 helix region. In this paper, we report the design, the conformational analysis, and the biological properties of QK revealing its ability to assume a helical conformation in pure water and to modulate the angiogenic response mediated by VEGF.
Peptides were synthesized on solid phase by using Rink Amide MBHA resin (Novabiochem) by standard Fmoc (N-(9-Fluorenyl)methoxycarbonyl) chemistry. The side chain of the N-terminal lysine was protected with the methyltrytil group to allow selective deprotection and peptide labeling. Cleavage from the resin were achieved by treatment with trifluoracetic acid, triisopropyl silane, and water (95:2.5:2.5) at room temperature for 3 h. Purity and identity of the peptides were assessed by HPLC and MALDI-TOF mass spectrometry.
CD spectra were collected on a Jasco 715 instrument by using a 1 mm path-length quartz cuvette (Hellma) at 20°C in 10 mM phosphate buffer, pH 7.1. Peptide concentrations were 21.8 μM (QK) and 43.4 μM (VEGF15). The aggregation state of QK was checked, over a concentration range of 1 μM to 1.5 mM, by UV spectroscopy (absorbance at 280 nm) and NMR (line widths and chemical shift variations). All of the experimental data (data not shown) indicate the QK peptide does not aggregate up to 1.5 mM.
The NMR samples were prepared dissolving the QK peptide at a concentration of 1.0 × 10-3 M either in a H 2 O/2 H 2 O 90:10 mixture or in pure 2 H 2 O at pH 5.5. The NMR experiments were recorded on a Varian Inova 600 spectrometer at a temperature of 298 K. All of the spectra were processed with the software prosa and analyzed with the program xeasy.
Experimental distance restraints for structure calculations were derived from the cross-peak intensities in NOESY spectra recorded in H 2 O and 2 H 2 O. Structure calculations, which used the torsion angle dynamics protocol of cyana, were started from 100 randomized conformers. The 20 conformers with the lowest cyana target function were further refined by means of restrained energy minimization with the gromos 96 force field with the program spdb viewer. The color figures and the structure analysis have been performed with the program molmol.
ECs from bovine aorta, immortalized with SV40, were cultured in DMEM (Sigma) and supplemented with 10% FBS (Invitrogen) at 37°C in 95% air/5% CO 2. In all of the experiments, VEGF 165 (Alexis) was used at 100 ng/ml.
Cells were homogenized in lysis buffer (12.5 mM Tris, pH 6.8/5 mM EDTA/5 mM EGTA), and membranes were separated from the cytosol fraction by centrifugation. Membranes were suspended in binding buffer (75 mM Tris/12.5 mM MgCl 2/2 mM EDTA), and an equal amount of membrane protein (1 μg) was plated in 96-well plates with QK (10-13 to 10-8 M) and [125 I]-VEGF (Amersham Biosciences). VEGF binding was evaluated with a γ-counter.
Cells were plated on six-well dishes and serum starved overnight. On the next day, cells were treated with a different amount of peptide in the absence or in presence of VEGF 165 for 15 min at 37°C and then dissolved in radioimmunoprecipitation assay-SDS buffer (50 mM Tris·HCl, pH 7.5/150 mM NaCl/1% Nonidet P-40/0.25% deoxycholate/9.4 mg/50 ml sodium orthovanadate/20% SDS). In some experiments, total KDR and Flt-1 were immunoprecipitated from an equal amount of whole-cell protein extracts by using protein A/G agarose beads conjugated with antibodies raised against total KDR or Flt-1 (R & D Systems). Proteins from whole-cell extracts or immunocomplexes were resolved by PAGE and transferred to nitrocellulose. Total extracellular signal-regulated kinase 1 and 2 (ERK1/2), serine-tyrosin phosphorylated ERK1/2, phosphotyrosine (Cell Signaling Technology) and phospho-retinoblastoma protein (p-RB) (Santa Cruz Biotechnology) were visualized by specific antibodies, anti-rabbit horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) and standard chemiluminescence (Pierce).
Cells were serum starved for 24 h and then incubated in DMEM with [3 H]thymidine (Amersham Pharmacia) and QK alone (10-12 to 10-6 M) or with a combination of QK and VEGF 165. After 24 h, cells were fixed with trichloracetic acid (0.05%) and dissolved in 1M NaOH. Scintillation liquid was added and thymidine incorporation was evaluated with a beta counter.
Cells were seeded at a density of 10,000 per well in six-well plates, serum starved overnight, and then stimulated with QK (10-12 to 10-6 M) in the absence or presence of VEGF 165. Cell number was determined at 24 h after stimulation. The phospho-retinoblastoma cyclin was evaluated by Western blot 12 and 18 h after stimulation with QK (10-6 M), VEGF 165, and VEGF15 (10-6 M).
Human endothelial cells were cocultured with other human cells in a specially designed medium (Angiokit, TCS CellWorks, Buckingham, U.K.), in 24-well plates. Every 3 days, QK in the absence or presence of VEGF 165 was added to the cultures. VEGF and suramine (20 μM) were used as positive and negative controls, respectively. Cells subsequently begin to proliferate and then enter a migratory phase, during which they move through the matrix to form thread-like tubule structures. 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). Results were scored with the image analysis software, angiosys (TCS CellWorks).
Based on the x-ray structure of the VEGF/Flt-1 domain 2 (Flt-1 D2) complex, we designed and synthesized a peptide reproducing the VEGF binding region spanning the amino acid sequence Phe-17-Tyr-25. This region contains 5 (Phe-17, Met-18, Tyr-21, Gln-22, and Tyr-25) of 21 residues situated at <4.5 Å from the receptor and it assumes, in the natural protein, an α-helix conformation. The design strategy we adopted was to keep fixed the three-dimensional arrangement of the residues interacting with the receptor and stabilize the secondary structural motif. Mutagenesis data indicate that when Phe-17 is mutated to Ala, the affinity toward KDR is reduced by 90-fold, whereas mutations of the other four residues only slightly affect the binding. All of the five interacting residues occupy a face of the helix, and they make hydrophobic interaction with the receptor. Residue
