Shay Soker

Neuropilin-1 Is Expressed by Endothelial and Tumor Cells as an Isoform-Specific Receptor for Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF), a major regulator of angiogenesis, binds to two receptor tyrosine kinases, KDR/Flk-1 and Flt-1. We now describe the purification and the expression cloning from tumor cells of a third VEGF receptor, one that binds VEGF165 but not VEGF121. This isoform-specific VEGF receptor (VEGF165R) is identical to human neuropilin-1, a receptor for the collapsin/semaphorin family that mediates neuronal cell guidance. When coexpressed in cells with KDR, neuropilin-1 enhances the binding of VEGF165 to KDR and VEGF165-mediated chemotaxis. Conversely, inhibition of VEGF165 binding to neuropilin-1 inhibits its binding to KDR and its mitogenic activity for endothelial cells. We propose that neuropilin-1 is a novel VEGF receptor that modulates VEGF binding to KDR and subsequent bioactivity and therefore may regulate VEGF-induced angiogenesis.

Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo

Arterial conduits are increasingly preferred for surgical bypass because of inherent functional properties conferred by arterial endothelial cells, especially nitric oxide production in response to physiologic stimuli. Here we tested whether endothelial progenitor cells (EPCs) can replace arterial endothelial cells and promote patency in tissue-engineered small-diameter blood vessels (4 mm). We isolated EPCs from peripheral blood of sheep, expanded them ex vivo and then seeded them on decellularized porcine iliac vessels. EPC-seeded grafts remained patent for 130 days as a carotid interposition graft in sheep, whereas non-seeded grafts occluded within 15 days. The EPC-explanted grafts exhibited contractile activity and nitric-oxide–mediated vascular relaxation that were similar to native carotid arteries. These results indicate that EPCs can function similarly to arterial endothelial cells and thereby confer longer vascular-graft survival. Due to their unique properties, EPCs might have other general applications for tissue-engineered structures and in treating vascular diseases.

REGULATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR-DEPENDENT RETINAL NEOVASCULARIZATION BY INSULIN-LIKE GROWTH FACTOR-1 RECEPTOR

Although insulin-like growth factor 1 (IGF-1) has been associated with retinopathy, proof of a direct relationship has been lacking. Here we show that an IGF-1 receptor antagonist suppresses retinal neovascularization in vivo, and infer that interactions between IGF-1 and the IGF-1 receptor are necessary for induction of maximal neovascularization by vascular endothelial growth factor (VEGF). IGF-1 receptor regulation of VEGF action is mediated at least in part through control of VEGF activation of p44/42 mitogen-activated protein kinase, establishing a hierarchical relationship between IGF-1 and VEGF receptors. These findings establish an essential role for IGF-1 in angiogenesis and demonstrate a new target for control of retinopathy. They also explain why diabetic retinopathy initially increases with the onset of insulin treatment. IGF-1 levels, low in untreated diabetes, rise with insulin therapy, permitting VEGF-induced retinopathy.

VEGF165 mediates formation of complexes containing VEGFR‐2 and neuropilin‐1 that enhance VEGF165‐receptor binding

Co‐expression of NRP1 and (VEGFR‐2) KDR on the surface of endothelial cells (EC) enhances VEGF165 binding to KDR and EC chemotaxis in response to VEGF165. Overexpression of NRP1 by prostate tumor cells in vivo results in increased tumor angiogenesis and growth. We investigated the molecular mechanisms underlying NRP1‐mediated angiogenesis by analyzing the association of NRP1 and KDR. An intracellular complex containing NRP1 and KDR was immunoprecipitated from EC by anti‐NRP1 antibodies only in the presence of VEGF165. In contrast, VEGF121, which does not bind to NRP1, did not support complex formation. Complexes containing VEGF165, NRP1, and KDR were also formed in an intercellular fashion by co‐culture of EC expressing KDR only, with cells expressing NRP1 only, for example, breast carcinoma cells. VEGF165 also mediated the binding of a soluble NRP1 dimer to cells expressing KDR only, confirming the formation of such complexes. Furthermore, the formation of complexes containing KDR and NRP1 markedly increased 125I‐VEGF165 binding to KDR. Our results suggest that formation of a ternary complex of VEGF165, KDR, and NRP1 potentiates VEGF165 binding to KDR. These complexes are formed on the surface of EC and in a juxtacrine manner via association of tumor cell NRP1 and EC KDR. J. Cell. Biochem. 85: 357–368, 2002.

Characterization of Novel Vascular Endothelial Growth Factor (VEGF) Receptors on Tumor Cells That Bind VEGF via Its Exon 7-encoded Domain

Vascular endothelial growth factor (VEGF), a potent angiogenic factor, uses two receptor tyrosine kinases, FLK/KDR and FLT, to mediate its activities. We have cross-linked I-VEGF to the cell surface of various tumor cell lines and of human umbilical vein endothelial cells. High molecular mass (220 and 240 kDa) and/or lower molecular mass (165 and 175 kDa) labeled complexes were detected depending on the cell type. The 220- and 240-kDa labeled complexes were shown to contain FLT and FLK/KDR receptors, respectively. On the other hand, the 165- and 175-kDa complexes did not seem to contain FLK/KDR or FLT but instead appeared to contain novel VEGF receptors with relatively low molecular masses of approximately 120 and 130 kDa. These receptors were further characterized in breast cancer MDA MB 231 cells (231), which did not form the high molecular mass complexes and which did not express detectable amounts of flk/kdr or flt mRNA. The 231 cells displayed one VEGF binding site, with a K of 2.8 × 10M and 0.95-1.1 × 105 binding sites per cell. By comparison, human umbilical vein endothelial cells had two binding sites, one with a K of 7.5 × 10M, presumably FLK/KDR, and the other with a K of 2 × 10M, a value similar to the VEGF binding sites on 231 cells. These lower affinity/molecular mass receptors on 231 cells cross-linked I-VEGF but not I-VEGF. Accordingly, exon 7 of VEGF, which encodes the 44 amino acids present in VEGF that are absent in VEGF, was fused to glutathione S-transferase (GST). The GST-VEGF-exon 7 fusion protein bound to heparin-Sepharose with a similar affinity as VEGF and inhibited the binding of I-VEGF to 231 cells. Cross-linking of I-GST-VEGF-exon 7 to 231 cells resulted in the formation of 150- and 160-kDa labeled complexes that presumably contained the 120- and 130-kDa lower affinity/molecular mass VEGF receptors. It was concluded that certain tumor-derived cell lines express novel surface-associated receptors that selectively bind VEGF via the exon 7-encoded domain, which is absent in VEGF.

Principals of neovascularization for tissue engineering.

The goals in tissue engineering include the replacement of damaged, injured or missing body tissues with biological compatible substitutes such as bioengineered tissues. However, due to an initial mass loss after implantation, improved vascularization of the regenerated tissue is essential. Recent advances in understanding the process of blood vessel growth has offered significant tools for therapeutic neovascularization. Several angiogenic growth factors including vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) were used for vascularization of ischemic tissues. Three approaches have been used for vascularization of bioengineered tissue: incorporation of angiogenic factors in the bioengineered tissue, seeding endothelial cells with other cell types and prevascularization of matrices prior to cell seeding. This paper reviews the process of blood vessel growth and tissue vascularization, and discuss strategies for efficient vascularization of engineered tissues.

Differential Binding of Vascular Endothelial Growth Factor B Splice and Proteolytic Isoforms to Neuropilin-1

Vascular endothelial growth factor B (VEGF-B) is expressed in various tissues, especially strongly in the heart, and binds selectively to one of the VEGF receptors, VEGFR-1. The two splice isoforms, VEGF-B167 and VEGF-B186, have identical NH2-terminal cystine knot growth factor domains but differ in their COOH-terminal domains which give these forms their distinct biochemical properties. In this study, we show that both splice isoforms of VEGF-B bind specifically to Neuropilin-1 (NRP1), a receptor for collapsins/semaphorins and for the VEGF165isoform. The NRP1 binding of VEGF-B could be competed by an excess of VEGF165. The binding of VEGF-B167 was mediated by the heparin binding domain, whereas the binding of VEGF-B186 to NRP1 was regulated by exposure of a short COOH-terminal proline-rich peptide upon its proteolytic processing. In immunohistochemistry, NRP1 distribution was found to be overlapping or adjacent to known sites of VEGF-B expression in several tissues, in particular in the developing heart, suggesting the involvement of VEGF-B in NRP1-mediated signaling.

Tissue specific regulation of VEGF expression during bone development requires Cbfa1/Runx2

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis during development, but little is known about the factors that control its expression. We provide the first example of tissue specific loss of VEGF expression as a result of targeting a single gene, Cbfa1/Runx2. During endochondral bone formation, invasion of blood vessels into cartilage is associated with upregulation of VEGF in hypertrophic chondrocytes and increased expression of VEGF receptors in the perichondrium. This upregulation is lacking in Cbfa1 deficient mice, and cartilage angiogenesis does not occur. Finally, over-expression of Cbfa1 in fibroblasts induces an increase in their VEGF mRNA level and protein production by stimulating VEGF transcription. The results demonstrate that Cbfa1 is a necessary component of a tissue specific genetic program that regulates VEGF during endochondral bone formation.

Tumor necrosis factor-α regulates expression of vascular endothelial growth factor receptor-2 and of its co-receptor neuropilin-1 in human vascular endothelial cells

Tumor necrosis factor-α (TNF-α) modulates gene expression in endothelial cells and is angiogenic in vivo. TNF-α does not activate in vitro migration and proliferation of endothelium, and its angiogenic activity is elicited by synthesis of direct angiogenic inducers or of proteases. Here, we show that TNF-α up-regulates in a dose- and time-dependent manner the expression and the function of vascular endothelial growth factor receptor-2 (VEGFR-2) as well as the expression of its co-receptor neuropilin-1 in human endothelium. As inferred by nuclear run-on assay and transient expression of VEGFR-2 promoter-based reporter gene construct, the cytokine increased the transcription of the VEGFR-2 gene. Mithramycin, an inhibitor of binding of nuclear transcription factor Sp1 to the promoter consensus sequence, blocked activation of VEGFR-2, suggesting that the up-regulation of the receptor required Sp1 binding sites. TNF-α increased the cellular amounts of VEGFR-2 protein and tripled the high affinity125I-VEGF-A165 capacity without affecting theK d of ligand-receptor interaction. As a consequence, TNF-α enhanced the migration and the wound healing triggered by VEGF-A165. Since VEGFR-2 mediates angiogenic signals in endothelium, our data indicate that its up-regulation is another mechanism by which TNF-α is angiogenic and may provide insight into the mechanism of neovascularization as occurs in TNF-α-mediated pathological settings.

Inhibition of Vascular Endothelial Growth Factor (VEGF)-induced Endothelial Cell Proliferation by a Peptide Corresponding to the Exon 7-Encoded Domain of VEGF165

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells (EC) in vitro and a major regulator of angiogenesis in vivo. VEGF121 and VEGF165 are the most abundant of the five known VEGF isoforms. The structural difference between these two is the presence in VEGF165 of 44 amino acids encoded by exon 7 lacking in VEGF121. It was previously shown that VEGF165 and VEGF121 both bind to KDR/Flk-1 and Flt-1 but that VEGF165 binds in addition to a novel receptor (Soker, S., Fidder, H., Neufeld, G., and Klagsbrun, M. (1996)J. Biol. Chem. 271, 5761–5767). The binding of VEGF165 to this VEGF165-specific receptor (VEGF165R) is mediated by the exon 7-encoded domain. To investigate the biological role of this domain further, a glutathioneS-transferase fusion protein corresponding to the VEGF165 exon 7-encoded domain was prepared. The fusion protein inhibited binding of125I-VEGF165 to VEGF165R on human umbilical vein-derived EC (HUVEC) and MDA-MB-231 tumor cells. The fusion protein also inhibited significantly125I-VEGF165 binding to KDR/Flk-1 on HUVEC but not on porcine EC which express KDR/Flk-1 alone. VEGF165had a 2-fold higher mitogenic activity for HUVEC than did VEGF121. The exon 7 fusion protein inhibited VEGF165-induced HUVEC proliferation by 60% to about the level stimulated by VEGF121. Unexpectedly, the fusion protein also inhibited HUVEC proliferation in response to VEGF121. Deletion analysis revealed that a core inhibitory domain exists within the C-terminal 23-amino acid portion of the exon 7-encoded domain and that a cysteine residue at position 22 in exon 7 is critical for inhibition. It was concluded that the exon 7-encoded domain of VEGF165 enhances its mitogenic activity for HUVEC by interacting with VEGF165R and modulating KDR/Flk-1-mediated mitogenicity indirectly and that exon 7-derived peptides may be useful VEGF antagonists in angiogenesis-associated diseases.