Alternative splicing of vascular endothelial growth factor (VEGF) mRNA results in three distinct molecular forms of 121 or 165 (V165) amino acids that are released in the conditioned medium of cultured cells and one longer isoform of 189 amino acids (V189) that remains cell-associated. V189 has been expressed in wild type CHO-K1 cells and in glycosaminoglycan-deficient pgsA-745 Chinese hamster ovary (CHO) mutant cells. It could be released from CHO-K1 cell membranes by heparin or a synthetic peptide designed on the sequence encoded by exon 6 but was freely released from CHO mutant cells. In both cases, the immunoreactive V189 was mainly released as a 40-kDa cleaved form, provided that the serine protease urokinase, but not plasmin, was active. Recombinant V189 was purified from insect cells infected with a recombinant baculovirus as a nonmitogenic 50-kDa precursor that binds to the receptor Flt-1 but not to Flk-1. It could be matured by urokinase as a 38-kDa fragment able to bind to Flk-1 and to trigger cell proliferation. V165 and V189, however, could be cleaved by plasmin as 34-kDa fragments that exhibit a decreased mitogenic activity. These findings indicate that the carboxyl-terminal domain of V189 masks its binding domain to Flk-1.

An endothelial cell growth factor with unique specificity for vascular endothelial cells has been purified from the conditioned medium of the AtT‐20 pituitary cell line. This growth factor, which has been characterized as a homodimer composed of two subunits with mol. wts of 23 kd is a potent mitogen for vascular endothelial cells in vitro with activity detectable at 50 pg/ml and saturation at 1 ng/ml. It was also angiogenic in vivo. In contrast with other endothelial mitogens of the fibroblast growth factor family, it has a unique target cell specificity. It did not stimulate the growth of other cell types of the vascular system such as vascular smooth muscle cells or that of mesoderm and neuroectoderm derived cells. Microsequencing revealed an amino‐terminal sequence with no homology to any known protein. The release of this novel endothelial cell growth factor by pituitary derived cells and its unique target cell specificity suggest that it could play an important role in the angiogenic process.

Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR‐2) mediates vascularization and tumor‐induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF–KDR interaction. A phage epitope library was screened by affinity for membrane‐expressed KDR or for an anti‐VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell‐displayed KDR. In vitro, this effect led to the inhibition of the VEGF‐mediated proliferation of human vascular endothelial cells, in a dose‐dependent and endothelial cell type‐specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF‐induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis.

Vascular endothelial growth factor (VEGF), a major factor mediating endothelial cell survival, migration, and proliferation during angiogenesis, is expressed as five splice variants (121, 145, 165, 189, and 206 aminoacids) encoded by a single gene. Although the three shorter isoforms are mainly diffusible, the two longer ones are sequestered in cell membranes after secretion. However, their potential role as true components of the extracellular matrix has not been investigated. We determined that endothelial cells could adhere and spread on VEGF189 and VEGF165, but not on VEGF121. Adhesion was mediated by the alpha3beta1 and alpha(v)beta3 integrins and other alpha(v) integrins but not by the cognate VEGF receptors. Cells migrated on VEGF165 and VEGF189 and displayed a stellate morphology with numerous lamellopodia and FAK staining but no actin stress fibers. Tumstatin, an antiangiogenic peptide that interacts with the alpha(v)beta3 integrin, could inhibit adhesion on VEGF, and this effect was potentiated by anti-alpha(v)beta3 blocking antibody. Immobilized VEGF almost totally abolished endothelial cell apoptosis through interactions with integrins. The inhibition of alpha(v)beta3 engagement with immobilized VEGF by tumstatin inhibited most of its survival activity. We have thus determined a new VEGF receptor-independent role for immobilized VEGF in supporting cell adhesion and survival through interactions with integrins.

Netrins are secreted molecules with roles in axon guidance and angiogenesis. We identified Netrin-4 as a gene specifically overexpressed in VEGF-stimulated endothelial cells (EC) in vitro as well as in vivo. Knockdown of Netrin-4 expression in EC increased their ability to form tubular structures on Matrigel. To identify which receptor is involved, we showed by quantitative RT-PCR that EC express three of the six Netrin-1 cognate receptors: neogenin, Unc5B, and Unc5C. In contrast to Netrin-1, Netrin-4 bound only to neogenin but not to Unc5B or Unc5C receptors. Neutralization of Netrin-4 binding to neogenin by blocking antibodies abolished the chemotactic effect of Netrin-4. Furthermore, the silencing of either neogenin or Unc5B abolished Netrin-4 inhibitory effect on EC migration, suggesting that both receptors are essential for its function in vitro. Coimmunoprecipitation experiments demonstrated that Netrin-4 increased the association between Unc5B and neogenin on VEGF-or FGF-2-stimulated EC. Finally, we showed that Netrin-4 significantly reduced pathological angiogenesis in Matrigel and laser-induced choroidal neovascularization models. Interestingly, Netrin-4, neogenin, and Unc5B receptor expression was up-regulated in choroidal neovessel EC after laser injury. Moreover, Netrin-4 overexpression delayed tumor angiogenesis in a model of s.c. xenograft. We propose that Netrin-4 acts as an antiangiogenic factor through binding to neogenin and recruitment of Unc5B.

Vascular endothelial growth factor (VEGF) and Delta-like 4 ligand (DLL4) are the only genes whose haploinsufficiency results in vascular abnormalities. Although many common pathways are up-regulated in both vascular development and tumor angiogenesis and in vascular remodeling, the role of the Delta/Notch pathway has not been clearly defined in tumor angiogenesis. In this study, we assessed the expression of DLL4, Notch4, and ephrin B2 in transgenic mice developing hepatocarcinoma characterized by a strong remodeling of the tumor sinusoids. We also investigated the role of VEGF in the expression and biological functions of these molecules on human venous endothelial cells. In transgenic livers, we showed that DLL4, active Notch4, and ephrin B2 were gradually up-regulated within the hepatocarcinoma progression and expressed on tumor sinusoidal endothelial cells. In venous endothelial cells, we showed that VEGF up-regulates DLL4 and presenilin, and increased the activation of Notch4, leading to an up-regulation of ephrin B2 with a downregulation of Eph B4. We also showed that the activation of Notch4 is required for VEGF-induced up-regulation of ephrin B2 and the differentiation of human venous endothelial cells in vitro. Accordingly, the disruption of Notch4 signaling by pharmacologic inhibition of presenilin or addition of soluble DLL4 inhibited the effect of VEGF on human venous endothelial cell migration and differentiation. Our study strongly suggests that a coordinated activation of DDL4/ Notch4 and ephrin B2 pathways downstream of VEGF plays a key role in the abnormal remodeling of tumor vessels. (Cancer Res 2006; 66(17): 8501-10)

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HLA-G is a major histocompatibility complex class Ib molecule whose constitutive tissue distribution is restricted mainly to trophoblast cells at the maternal-fetal interface during pregnancy. In this study, we demonstrated the ability of the soluble HLA-G1 (sHLA-G1) isoform to inhibit fibroblast growth factor-2 (FGF2)-induced capillary-like tubule formation. Using a rabbit corneal neovascularization model, we further showed that sHLA-G1 inhibits FGF2-induced angiogenesis in vivo. We also demonstrated that sHLA-G1 induces endothelial cell apoptosis through binding to BY55/ CD160, a glycosylphosphatidylinositolanchored receptor expressed by endothelial cells. Furthermore, we showed that the specific CL1-R2 anti-CD160 monoclonal antibody mimics sHLA-G1-mediated inhibition of endothelial cell tube formation and induction of apoptosis. Thus, the engagement of CD160 in endothelial cells may be essential for the inhibition of angiogenesis. sHLA-G1/CD160-mediated antiangiogenic property may participate in the vascular remodeling of maternal spiral arteries during pregnancy, and, given that we found that CD160 is strongly expressed in the vasculature of a murine tumor, it offers an attractive therapeutic target for preventing pathologic neovascularization. ( IntroductionHLA-G is a human major histocompatibility complex (MHC) class Ib gene characterized by a unique promoter region, limited polymorphism, restricted constitutive tissue distribution, and several spliced transcripts encoding either membrane-bound or soluble proteins. 1 The soluble HLA-G1 (sHLA-G1) isoform derives from mRNA retaining intron 4, 2 which contains a stop codon that precludes translation of the transmembrane domain. Such intron 4 retention is unique among all HLA class I molecules described to date. This 37-kDa, intron 4-retaining sHLA-G1 isoform associates noncovalently with ␤2-microglobulin (␤2m). 2 Soluble HLA-G can also be generated by metalloproteinase-mediated release of surface HLA-G containing only extracellular domains. 3 The predominant expression of sHLA-G1 in the placenta, at a time when polymorphic HLA-A and HLA-B class Ia molecules are repressed, is consistent with important immunologic functions during pregnancy. 4 sHLA-G1 induces apoptosis of activated CD8 ϩ T and natural killer (NK) cells 5,6 and down-regulates the CD4 ϩ T-cell alloproliferation response. 7 The observation that some anti-HLA-G monoclonal antibodies bound to HLA-G-negative placental endothelial cells 8,9 led to our hypothesis that sHLA-G1 might bind to these cells and be involved in the modulation of placental angiogenesis or uterine vessel remodeling. 8 Several further observations are in line with such a novel function of HLA-G. Among them is that a defect of HLA-G expression in extravillous cytotrophoblast is associated with preeclampsia, 10,11 a common complication of pregnancy in which HLA-G ϩ endovascular trophoblast invasion of maternal spiral arteries is abrogated, compromising blood flow to the maternal interface. 12 In addition, it has been shown that HL...