Modulating Angiogenesis by Proteomimetics of Vascular Endothelial Growth Factor

Abstract

Angiogenesis, formation of new blood vessels from the existing vascular network, is a hallmark of cancer cells that leads to tumor vascular proliferation and metastasis. This process is mediated through the binding interaction of VEGF-A with VEGF receptors. However, the balance between pro-angiogenic and anti-angiogenic effect after ligand binding yet remains elusive and is therefore challenging to manipulate. To interrogate this interaction, herein we designed a few sulfono-γ-AA peptide based helical peptidomimetics that could effectively mimic a key binding interface on VEGF (helix-α1) for VEGFR recognition. Intriguingly, although both sulfono-γ-AA peptide sequences V2 and V3 bound to VEGF receptors tightly, in vitro angiogenesis assays demonstrated that V3 potently inhibited angiogenesis, whereas V2 activated angiogenesis effectively instead. Our findings suggested that this distinct modulation of angiogenesis might be due to the result of selective binding of V2 to VEGFR-1 and V3 to VEGFR-2, respectively. These molecules thus provide us a key to switch the angiogenic signaling, a biological process that balances the effects of pro-angiogenic and anti-angiogenic factors, where imbalances lead to several diseases including cancer. In addition, both V2 and V3 exhibited remarkable stability toward proteolytic hydrolysis, suggesting that V2 and V3 are promising therapeutic agents for the intervention of disease conditions arising due to angiogenic imbalances and could also be used as novel molecular switching probes to interrogate the mechanism of VEGFR signaling. The findings also further demonstrated the potential of sulfono-γ-AA peptides to mimic the α-helical domain for protein recognition and modulation of protein-protein interactions.

Figure 1.

Structure of VEGF-A/VEGFR-2 complex, Protein Data Bank (PDB): 3V2A. Homodimeric VEGF-A (green) binding to, and dimerization of, VEGFR-2 (yellow). Key binding residues on VEGF-A helix-α1 (Phe17, Met18, Tyr21, and Tyr25) are highlighted in red.

Figure 2.

(A) Structure of sulfono-γ-AA peptide building block. “a” and “b” denote chiral side chain and sulfono side chain, respectively. (B) Schematic representation of side chain distribution in a left-handed sulfono-γ-AA peptide helix scaffold. (C and D) Crystal structure of sulfono-γ-AA peptide side view and top view, respectively.

Figure 3.

(A) Binding interaction of key residues on helix-α1 of VEGF-A (green) with VEGFR-2 (yellow), PDB code: 3V2A. (B) Structures of helix-α1 of VEGF-A (green), sulfono-γ-AA peptide mimic V3 (magenta), and overlay of key binding residues.

Figure 4.

CD spectra of sulfono-γ- AA peptides V1-V5 and QK measured at 100 μM, room temperature in PBS buffer.

Figure 5.

Transwell migration assay of HUVECs following treatment with VEGF mimics in the presence of VEGF₁₆₅. (A) Increased migration of HUVECs treated with V2 and QK. Marked inhibition of migration is observed in cells treated with V1 and V3. (B) Percentage of HUVECs migrating, compared to control, following treatments with VEGF mimics in the presence VEGF₁₆₅. Concentration of treatments: VEGF₁₆₅ (50 ng/ mL), all other peptidic sequences (10 μM), control (no treatment). Results presented as mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.

Wound healing assay. (A) Migration of HUVECs following treatments with VEGF mimics in the presence of VEGF₁₆₅ observed at 0 and 24 h. (B) Percentage of wound area remaining high (inhibition of migration) after 24 h in HUVECs treated with V1 and V3. However, V2 and QK, as compared to cells treated with VEGF₁₆₅ only, did not activate migration. Concentration of treatments: VEGF₁₆₅ (50 ng/mL), VEGF mimic sequences (10 μM), control (no treatment). Results presented as mean ± SD (n = 3), ***P < 0.001.

Figure 7.

Formation of capillary tube structures of HUVECs following treatment with VEGF mimics in the presence of VEGF₁₆₅ observed after 24 h. (A) Increased capillary tube formation in cells treated with QK and V2. (B) Graphical representation of percentage count, compared to control, of capillary tube nodes of HUVECs for (A). (C) Decreased capillary tube formation in cells treated with V1 and V3. (D) Graphical representation for (C). VEGF₁₆₅ (50 ng/mL), VEGF mimic sequences (2 μM), negative control (no treatment). Results presented as mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8.

Western blot data on the levels on phosphorylation of VEGFR-2 and Akt in HUVEC lysates following treatment with (A) V1 (0.1, 0.5, 1, 10 μM), (B) V3 (0.1, 0.5, 1, 10 μM), (C) V2 (0.1, 0.5, 1, 10 μM), and (D) QK (0.1, 0.5, 1, 10 μM) and (E) QK, V1, V2, and V3 (20 μM) with or without VEGF₁₆₅.

Figure 9.

Characterization of binding profiles of V2 and V2 toward VEGFR-1 and VEGFR-2 with immunofluorescence. HUVECs treated with no drug (control), V2, and V3, stained with anti-VEGFR-1 (A) and anti-VEGFR-1 (B) antibody, and detected by FITC-labeled goat anti-rabbit IgG secondary antibody followed by DAPI counterstain. (C and D) Bar graph of relative fluorescence intensity of V2 and V3 compared to control in cells treated with anti-VEGFR-1 and anti-VEGFR-2 antibodies. Results presented as mean ± SD (n = 3), **P < 0.01, ***P < 0.001.

Figure 10.

(A) Superimposition of V3 (teal) with critical residues of helix-α1 (green) on the binding surface of VEGFR-2 (yellow). (B) Binding interaction of key residues on helix-α1 of VEGF-A (green) with VEGFR-1 (bronze), PDB: 1flt. (C) Structures of helix-α1 of VEGF-A (green), sulfono-γ-AA peptide mimic V2 (magenta), and overlay of key binding residues. (D) Superimposition of V2 (magenta) with critical residues of helix-α1 (green) on the binding surface of VEGFR-1 (bronze). (E) Modulation of the angiogenic switch with sulfono-γ-AA peptide mimics of VEGF-A.

Figure 11.

Stability studies of VEGF-A mimics. HPLC traces of indicated control sequences and sequences incubated in Pronase for 24 h.