Michael Klagsbrun

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.

Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor

The role of low affinity, heparin-like binding sites for basic fibroblast growth factor (bFGF) was investigated in CHO cells mutant in their metabolism of glycosaminoglycans. Heparan sulfate-deficient mutants transfected to express a cloned mouse FGF receptor cDNA are not able to bind bFGF. It is demonstrated that free heparin and heparan sulfate can reconstitute a low affinity receptor that is, in turn, required for the high affinity binding of bFGF. These studies suggest that the low affinity receptor is an accessory molecule required for binding of bFGF to the high affinity site. Such an obligatory interaction of low and high affinity FGF receptors suggests a physiological role for heparin-like, low affinity receptors and constitutes a novel mechanism for the regulation of growth factor-receptor interactions.

A HEPARIN-BINDING GROWTH FACTOR SECRETED BY MACROPHAGE-LIKE CELLS THAT IS RELATED TO EGF

Macrophage-like U-937 cells secrete a 22-kilodalton heparin-binding growth factor that is mitogenic for BALB-3T3 fibroblasts and smooth muscle cells, but not endothelial cells. The amino acid sequence predicted from complementary DNA clones indicates that the mitogen is a new member of the epidermal growth factor (EGF) family. This heparin-binding EGF-like growth factor (HB-EGF) binds to EGF receptors on A-431 epidermoid carcinoma cells and smooth muscle cells, but is a far more potent mitogen for smooth muscle cells than is EGF. HB-EGF is also expressed in cultured human macrophages and may be involved in macrophage-mediated cellular proliferation.

Vascular endothelial growth factor and its receptors

Vascular endothelial growth factor (VEGF) is a prime regulator of endothelial cell proliferation, angiogenesis, vasculogenesis and vascular permeability. Its activity is mediated by the high affinity tyrosine kinase receptors, KDR/Fik-1 and Fit-1. In this article, recently discovered structural, molecular and biological properties of VEGF are described. Among the topics discussed are VEGF and VEGF receptor structure and bioactivity, the regulation of VEGF expression, the role of VEGF and its receptors in vascular development, and the involvement of VEGF and its receptors in normal and pathological (ocular and tumor) angiogenesis.

Neovascularization is associated with a switch to the export of bFGF in the multistep development of fibrosarcoma.

In a transgenic mouse model, dermal fibrosarcomas develop in a pathway comprised of at least three stages: mild fibromatosis, aggressive fibromatosis, and fibrosarcoma. The latter two stages are highly vascularized when compared with both the normal dermis and the initial mild lesion. Analysis of cell cultures derived from biopsies of these lesions has revealed that basic fibroblast growth factor (bFGF) is synthesized in all three stages and in normal dermal fibroblasts derived from the same mice. Unexpectedly, there is a change in the localization of bFGF from its normal cell-associated state to extracellular release in the latter two stages, which is concomitant both with the neovascularization seen in vivo and with the tumorigenicity of these cell lines. Thus, in this multistep tumorigenesis pathway there appears to be a discrete switch to the angiogenic phenotype that correlates with the export of bFGF, a known angiogenic factor.

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.

Vasculogenesis, Angiogenesis, and Growth Factors: Ephrins Enter the Fray at the Border

of Angiopoietin-2, the natural antagonist for Tie2 (Mai-sonpierre et al., 1997), all lead to embryonic lethality resulting from defects in angiogenesis. and eight ephrin ligands for these receptors have been Boston, Massachusetts 02115 described to date. The Eph receptors and their ligands can broadly be divided into two subclasses, A and B, In an important paper in this issue of Cell, David Ander-based on structural homologies and binding specifici-son and his associates provide insights into the early ties, with a great deal of redundancy within a subclass molecular determinants that distinguish arteries and in terms of receptor/ligand binding specificities (Gale veins (Wang et al., 1998). The key players include the et al., 1996; Flanagan and Vanderhaeghen, 1998). The Eph-B4 receptor from the Eph family—the largest known ephrin-B ligands are transmembrane proteins and pref-family of receptor tyrosine kinases—and its obligate erentially bind to receptors of the Eph-B subclass. Unlike membrane-bound ligand, ephrin-B2. Wang et al. also ligands for other receptor tyrosine kinases, the ephrins link the actions of ephrin-B2 and Eph-B4 to those of a can not act as soluble mediators, but rather must be recently defined family of angiogenic factors, the angio-membrane-bound in order to activate their receptors poietins, and their endothelial-specific receptor tyrosine (membrane linkage seemingly serves to cluster the li-kinases, the Ties. gands, and this clustering is required for their activity) Vasculogenesis Versus Angiogenesis, (Davis et al., 1994). Remarkably, there appears to be and Their Molecular Regulators reciprocal signaling between ligands and receptors in The adult vasculature consists of large arteries, inter-the B subclass, in that ephrin-B ligands not only activate nally lined by endothelial cells and well-ensheathed by their respective receptors, but are in turn activated upon smooth muscle cells, that progressively branch into engaging their receptors, as judged by tyrosine phos-phorylation of the ephrin-B cytoplasmic domains (Bruck-smaller and smaller vessels, terminating in precapillary ner et al., 1997; Holland et al., 1997). These findings arterioles that then give rise to capillaries. Capillaries suggested early on that the ephrins might provide key are comprised almost entirely of endothelial cells that bidirectional cues in an obligate cell-to-cell contact-are only occasionally coated by a smooth muscle cell-dependent fashion. like pericyte. Capillaries then feed into postcapillary ven-The ephrins and their Eph receptors have been most ules that progressively associate into larger and larger studied for their roles in the nervous system. Most nota-venous structures. The earliest stages of vascular devel-bly, they …

Heparin-binding EGF-like growth factor.

HB-EGF is a heparin-binding member of the EGF family that was initially identified in the conditioned medium of human macrophages. Soluble mature HB-EGF is proteolytically processed from a larger membrane-anchored precursor and is a potent mitogen and chemotactic factor for fibroblasts, smooth muscle cells but not endothelial cells. HB-EGF activates two EGF receptor subtypes, HER1 and HER4 and binds to cell surface HSPG. The transmembrane form of HB-EGF is a juxtacrine growth and adhesion factor and is uniquely the receptor for diphtheria toxin. HB-EGF gene expression is highly regulated, for example by cytokines, growth factors, and transcription factors such as MyoD. HB-EGF has been implicated as a participant in a variety of normal physiological processes such as blastocyst implantation and wound healing, and in pathological processes such as tumor growth, SMC hyperplasia and atherosclerosis.

Tumor-Associated Endothelial Cells with Cytogenetic Abnormalities

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Tumor blood vessels have been shown to differ from normal counterparts, for example, by changes in morphology. An important concept in tumor angiogenesis is that tumor endothelial cells are assumed to be genetically normal, although these endothelial cells are structurally and functionally abnormal. However, we hypothesized that given the phenotypic differences between tumor and normal blood vessels, there may be genotypic alterations as well. Mouse endothelial cells were isolated from two different human tumor xenografts, melanoma and liposarcoma, and from two normal endothelial cell counterparts, skin and adipose. Tumor-associated endothelial cells expressed typical endothelial cell markers, such as CD31. They had relatively large, heterogeneous nuclei. Unexpectedly, tumor endothelial cells were cytogenetically abnormal. Fluorescence in situ hybridization (FISH) analysis showed that freshly isolated uncultured tumor endothelial cells were aneuploid and had abnormal multiple centrosomes. The degree of aneuploidy was exacerbated by passage in culture. Multicolor FISH indicated that the structural chromosomal aberrations in tumor endothelial cells were heterogeneous, indicating that the cytogenetic alterations were not clonal. There was no evidence of human tumor-derived chromosomal material in the mouse tumor endothelial cells. In marked contrast, freshly isolated normal skin and adipose endothelial cells were diploid, had normal centrosomes, and remained cytogenetically stable in culture even up to 20 passages. FISH analysis of tumor sections also showed endothelial cell aneuploidy. We conclude that tumor endothelial cells can acquire cytogenetic abnormalities while in the tumor microenvironment.

Activation of HER4 by heparin‐binding EGF‐like growth factor stimulates chemotaxis but not proliferation

Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) is a potent mitogen and chemotactic factor for fibroblasts, smooth muscle cells and keratinocytes. It is demonstrated that HB‐EGF is not only a ligand for HER1, as previously reported, but for HER4 as well. HB‐EGF binds to NIH 3T3 cells over‐expressing either HER1 or HER4 alone, but not HER2 or HER3 alone. Binding to HER4 is independent of HER1. The ability of HB‐EGF to bind to two different receptors is in contrast to EGF which binds to HER1, but not to HER4, and heregulin‐β1 which binds to HER4, but not to HER1. Besides binding, HB‐EGF activates HER4. For example (i) it induces tyrosine phosphorylation of HER4 in cells overexpressing this receptor and of endogenous HER4 in MDA‐MB‐453 cells and astrocytes; (ii) it induces association of phosphatidylinositol 3‐kinase (PI3‐K) activity with HER4; and (iii) it is a potent chemotactic factor for cells overexpressing HER4. Chemotaxis is inhibited by wortmannin, a PI3‐K inhibitor, suggesting a possible role for PI3‐K in mediating HB‐EGF‐stimulated chemotaxis. On the other hand, HB‐EGF is not a mitogen for cells expressing HER4, in contrast to its ability to stimulate both chemotaxis and proliferation in cells expressing HER1. It was concluded that HER4 is a newly described receptor for HB‐EGF and that HB‐EGF can activate two EGF receptor subtypes, HER1 and HER4, but with different biological responses.