Peptides Derived from Vascular Endothelial Growth Factor B Show Potent Binding to Neuropilin-1.

Vascular endothelial growth factors (VEGFs) regulate significant pathways in angiogenesis, myocardial and neuronal protection, metabolism, and cancer progression. The VEGF-B growth factor is involved in cell survival, anti-apoptotic and antioxidant mechanisms, through binding to VEGF receptor 1 and neuropilin-1 (NRP1). We employed surface plasmon resonance technology and X-ray crystallography to analyse the molecular basis of the interaction between VEGF-B and the b1 domain of NRP1, and developed VEGF-B C-terminus derived peptides to be used as chemical tools for studying VEGF-B - NRP1 related pathways. Peptide lipidation was used as a means to stabilise the peptides. VEGF-B-derived peptides containing a C-terminal arginine show potent binding to NRP1-b1. Peptide lipidation increased binding residence time and improved plasma stability. A crystal structure of a peptide with NRP1 demonstrated that VEGF-B peptides bind at the canonical C-terminal arginine binding site. VEGF-B C-terminus imparts higher affinity for NRP1 than the corresponding VEGF-A165 region. This tight binding may impact on the activity and selectivity of the full-length protein. The VEGF-B167 derived peptides were more effective than VEGF-A165 peptides in blocking functional phosphorylation events. Blockers of VEGF-B function have potential applications in diabetes and non-alcoholic fatty liver disease.

A cyclic peptide reproducing the α1 helix of VEGF-B binds to VEGFR-1 and VEGFR-2 and inhibits angiogenesis and tumor growth.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are pivotal regulators of angiogenesis. The VEGF-VEGFR system is therefore an important target of anti-angiogenesis therapy. Based on the X-ray structure of VEGF-B/VEGFR-1 D2, we designed a cyclic peptide (known as VGB1) reproducing the α1 helix and its adjacent region to interfere with signaling through VEGFR-1. Unexpectedly, VGB1 bound VEGFR-2 in addition to VEGFR-1, leading to inhibition of VEGF-stimulated proliferation of human umbilical vein endothelial cells and 4T1 murine mammary carcinoma cells, which express VGEFR-1 and VEGFR-2, and U87 glioblastoma cells that mostly express VEGFR-2. VGB1 inhibited different aspects of angiogenesis, including proliferation, migration and tube formation of endothelial cells stimulated by VEGF-A through suppression of extracellular signal-regulated kinase 1/2 and AKT (Protein Kinase B) phosphorylation. In a murine 4T1 mammary carcinoma model, VGB1 caused regression of tumors without causing weight loss in association with impaired cell proliferation (decreased Ki67 expression) and angiogenesis (decreased CD31 and CD34 expression), and apoptosis induction (increased TUNEL staining and p53 expression, and decreased Bcl-2 expression). According to far-UV circular dichroism (CD) and molecular dynamic simulation data, VGB1 can adopt a helical structure. These results, for the first time, demonstrate that α1 helix region of VEGF-B recognizes both VEGFR-1 and VEGFR-2.

A peptide mimicking the binding sites of VEGF-A and VEGF-B inhibits VEGFR-1/-2 driven angiogenesis, tumor growth and metastasis.

Interfering with interactions of vascular endothelial growth factors (VEGFs) with their receptors (VEGFRs) effectively inhibits angiogenesis and tumor growth. We designed an antagonist peptide of VEGF-A and VEGF-B reproducing two discontinuous receptor binding regions of VEGF-B (loop 1 and loop3) covalently linked together by a receptor binding region of VEGF-A (loop3). The designed peptide (referred to as VGB4) was able to bind to both VEGFR1 and VEGFR2 on the Human Umbilical Vein Endothelial Cells (HUVECs) surface and inhibited VEGF-A driven proliferation, migration and tube formation in HUVECs through suppression of ERK1/2 and AKT phosphorylation. The whole-animal fluorescence imaging demonstrated that fluorescein isothiocyanate (FITC)-VGB4 accumulated in the mammary carcinoma tumors (MCTs). Administration of VGB4 led to the regression of 4T1 murine MCT growth through decreased expression of p-VEGFR1 and p-VEGFR2 and abrogation of ERK1/2 and AKT activation followed by considerable decrease of tumor cell proliferation (Ki67 expression) and angiogenesis (CD31 and CD34 expression), induction of apoptosis (increased p53 expression, TUNEL staining and decreased Bcl2 expression), and suppression of metastasis  (increased E-cadherin and decreased N-cadherin, NF-κB and MMP-9 expression). These findings indicate that VGB4 may be applicable for antiangiogenic and antitumor therapy.

Identification of Peptidic Antagonists of Vascular Endothelial Growth Factor Receptor 1 by Scanning the Binding Epitopes of Its Ligands.

Cancer angiogenesis is mainly initiated by vascular endothelial growth factors (VEGFs). On the basis of the reported crystal structures of three natural ligands (VEGF-A, -B, and PlGF) with the major receptors VEGFR-1 and VEGFR-2, we scanned receptor-binding epitopes of these ligands by designing linear and cyclic peptides with the aim to disrupt the VEGF-A/VEGFR-1 interaction, which is implicated in cancer development. The ability of peptides to inhibit this interaction was evaluated by an ELISA-based assay. Several peptides, especially those mimicking loop 1 (L1) of these ligands that binds primarily to domain D3 of VEGFRs, have demonstrated higher inhibition for VEGF-A/VEGFR-1 binding. They have also shown inhibitory effects on VEGF-induced tube formation in HUVECs (human umbilical vein endothelial cells). These results validate the domain D3 of VEGFRs as an efficient target for the design of VEGFR antagonists.

Vascular endothelial growth factor-B in physiology and disease.

Vascular endothelial growth factor-B (VEGF-B), discovered over 15 years ago, has long been seen as one of the more ambiguous members of the VEGF family. VEGF-B is produced as two isoforms: one that binds strongly to heparan sulfate in the pericellular matrix and a soluble form that can acquire binding via proteolytic processing. Both forms of VEGF-B bind to VEGF-receptor 1 (VEGFR-1) and the neuropilin-1 (NRP-1) coreceptor, which are expressed mainly in blood vascular endothelial cells. VEGF-B-deficient mice and rats are viable without any overt phenotype, and the ability of VEGF-B to induce angiogenesis in most tissues is weak. This has been a puzzle, as the related placenta growth factor (PlGF) binds to the same receptors and induces angiogenesis and arteriogenesis in a variety of tissues. However, it seems that VEGF-B is a vascular growth factor that is more tissue specific and can have trophic and metabolic effects, and its binding to VEGFR-1 shows subtle but important differences compared with that of PlGF. VEGF-B has the potential to induce coronary vessel growth and cardiac hypertrophy, which can protect the heart from ischemic damage as well as heart failure. In addition, VEGF-B is abundantly expressed in tissues with highly active energy metabolism, where it could support significant metabolic functions. VEGF-B also has a role in neuroprotection, but unlike other members of the VEGF family, it does not have a clear role in tumor progression. Here we review what is hitherto known about the functions of this growth factor in physiology and disease.

The basis for the distinct biological activities of vascular endothelial growth factor receptor-1 ligands.

Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development through VEGF receptors (VEGFRs). The VEGFR immunoglobulin homology domain 2 (D2) is critical for ligand binding, and D3 provides additional interaction sites. VEGF-B and placenta growth factor (PlGF) bind to VEGFR-1 with high affinity, but only PlGF is angiogenic in most tissues. We show that VEGF-B, unlike other VEGFs, did not require D3 interactions for high-affinity binding. VEGF-B with a PlGF-derived L1 loop (B-L1P) stimulated VEGFR-1 activity, whereas PlGF with a VEGF-B-derived L1 loop (P-L1B) did not. Unlike P-L1B and VEGF-B, B-L1P and PlGF were also angiogenic in mouse skeletal muscle. Furthermore, B-L1P also bound to VEGFR-2 and activated downstream signaling. These results establish a role for L1-mediated D3 interactions in VEGFR activation in endothelial cells and indicate that VEGF-B is a high-affinity VEGFR-1 ligand that, unlike PlGF, cannot efficiently induce signaling downstream of VEGFR-1.

Soluble vascular endothelial growth factor decoy receptor FP3 exerts potent antiangiogenic effects.

The binding of vascular endothelial growth factor (VEGF) to its receptors stimulates tumor growth; therefore, modulation of VEGF would be a viable approach for antiangiogenic therapy. We constructed a series of soluble decoy receptors containing different VEGF receptor 1 (FLT1) and VEGF receptor 2 (KDR) extracellular domains fused with the Fc region of human immunoglobulin (Ig) and evaluated their antiangiogenic effects and antitumor effects. Results of in vitro binding and cell proliferation assays revealed that decoy receptor FP3 had the highest affinity to VEGF-A and -B. Compared with bevacizumab, FP3 more effectively inhibited human umbilical vein endothelial cell (HUVEC) migration and vessel sprouting from rat aortic rings. FP3 significantly reduced phosphorylation of AKT and ERK1/2, critical proteins in the VEGF-mediated survival pathway in endothelial cells. Moreover, FP3 inhibited tumor growth in human hepatocellular carcinoma (HepG2), breast cancer (MCF-7), and colorectal cancer (LoVo) tumor models, and reduced microvessel density in tumor tissues. The FP3-mediated inhibition of tumor growth was significantly higher than that of bevacizumab at the same dose. FP3 also demonstrated synergistic antitumor effects when combined with 5-fluorouracil (5-FU). Taken together, FP3 shows a high affinity for VEGF and produced antiangiogenic effects, suggesting its potential for treating angiogenesis-related diseases such as cancer.

Vascular endothelial growth factor-B is neuroprotective in an in vivo rat model of Parkinson's disease.

Developing novel neuroprotective strategies for the treatment of Parkinson's disease (PD) is of great importance. We have previously shown that vascular endothelial growth factor-B (VEGF-B) is up-regulated in an in vitro model of PD using the neurotoxin rotenone. Addition of exogenous VEGF-B(167) was neuroprotective in this same model, suggesting that VEGF-B is a natural response to neurodegenerative challenges. Now we have extended this research using in vivo experiments. We tested a single intra-striatal injection of 3 µg VEGF-B(186), the more diffusible VEGF-B isoform, in a mild progressive unilateral 6-hydroxydopamine (6-OHDA) rat in vivo PD model. Treatment with VEGF-B(186) 6h prior to lesioning with 6-OHDA improved amphetamine-induced rotations and forepaw preference at 2, 4 and 6 weeks post-injection, indicating a neuroprotective effect. Immunohistochemical analysis showed that VEGF-B(186) treatment partially protected dopaminergic fibers in the striatum and demonstrated a partial rescue of the dopaminergic neurons in the caudal sub-region of the substantia nigra. Altogether our data suggest that VEGF-B(186) could be a new candidate trophic factor for the treatment of PD.

Structural insights into the binding of vascular endothelial growth factor-B by VEGFR-1(D2): recognition and specificity.

The formation of blood vessels (angiogenesis) is a highly orchestrated sequence of events involving crucial receptor-ligand interactions. Angiogenesis is critical for physiological processes such as development, wound healing, reproduction, tissue regeneration, and remodeling. It also plays a major role in sustaining tumor progression and chronic inflammation. Vascular endothelial growth factor (VEGF)-B, a member of the VEGF family of angiogenic growth factors, effects blood vessel formation by binding to a tyrosine kinase receptor, VEGFR-1. There is growing evidence of the important role played by VEGF-B in physiological and pathological vasculogenesis. Development of VEGF-B antagonists, which inhibit the interaction of this molecule with its cognate receptor, would be important for the treatment of pathologies associated specifically with this growth factor. In this study, we present the crystal structure of the complex of VEGF-B with domain 2 of VEGFR-1 at 2.7 A resolution. Our analysis reveals that each molecule of the ligand engages two receptor molecules using two symmetrical binding sites. Based on these interactions, we identify the receptor-binding determinants on VEGF-B and shed light on the differences in specificity towards VEGFR-1 among the different VEGF homologs.

Crystal structure of vascular endothelial growth factor-B in complex with a neutralising antibody Fab fragment.

Vascular endothelial growth factor (VEGF) B effects blood vessel formation by binding to VEGF receptor 1. To study the specifics of the biological profile of VEGF-B in both physiological and pathological angiogenesis, a neutralising anti-VEGF-B antibody (2H10) that functions by inhibiting the binding of VEGF-B to VEGF receptor 1 was developed. Here, we present the structural features of the 'highly ordered' interaction of the Fab fragment of this antibody (Fab-2H10) with VEGF-B. Two molecules of Fab-2H10 bind to symmetrical binding sites located at each pole of the VEGF-B homodimer, giving a unique U-shaped topology to the complex that has not been previously observed in the VEGF family. VEGF-B residues essential for binding to the antibody are contributed by both monomers of the cytokine. Our detailed analysis reveals that the neutralising effect of the antibody occurs by virtue of the steric hindrance of the receptor-binding interface. These findings suggest that functional complementarity between VEGF-B and 2H10 can be harnessed both in analysing the therapeutic potential of VEGF-B and as an antagonist of receptor activation.

Crystal structure of human vascular endothelial growth factor-B: identification of amino acids important for receptor binding.

The development of blood vessels (angiogenesis) is critical throughout embryogenesis and in some normal postnatal physiological processes. Pathological angiogenesis has a pivotal role in sustaining tumour growth and chronic inflammation. Vascular endothelial growth factor-B (VEGF-B) is a member of the VEGF family of growth factors that regulate blood vessel and lymphatic angiogenesis. VEGF-B is closely related to VEGF-A and placenta growth factor (PlGF), but unlike VEGF-A, which binds to two receptor tyrosine kinases VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), VEGF-B and PlGF bind to VEGFR-1 and not VEGFR-2. There is growing evidence of a role for VEGF-B in physiological and pathological blood vessel angiogenesis. VEGF-B may provide novel therapeutic strategies for the treatment of vascular disease and be a potential therapeutic target in aberrant vessel formation. To help understand at the molecular level the differential receptor binding profile of the VEGF family of growth factors we have determined the crystal structure of human VEGF-B(10-108) at 2.48 Angstroms resolution. The overall structure is very similar to that of the previously determined cysteine-knot motif growth factors: VEGF-A, PlGF and platelet-derived growth factor-B (PDGF-B). We also present a predicted model for the association of VEGF-B with the second domain of its receptor, VEGFR-1. Based on this interaction and the present structural data of the native protein, we have identified several putative residues that could play an important role in receptor recognition and specificity.