Seiji Takashima

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.

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.

REACTIVE OXYGEN INTERMEDIATES INCREASE VASCULAR ENDOTHELIAL GROWTH FACTOR EXPRESSION IN VITRO AND IN VIVO

Elevated vascular endothelial growth factor (VEGF) levels are required for ocular and tumor angiogenesis in animal models. Ischemic hypoxia is strongly correlated with increased VEGF expression in these systems and is considered a physiologically relevant stimulus. Because ischemic hypoxia is often followed by reperfusion and reactive oxygen intermediate (ROI) generation, we examined the potential role of ROI in the control of VEGF gene expression. Human retinal pigment epithelial cells exposed to superoxide or hydrogen peroxide rapidly increased VEGF mRNA levels. Superoxide-associated mRNA increases were dose dependent, blocked by antioxidants, and associated with elevated VEGF protein levels in conditioned media. Increases in VEGF mRNA levels were also observed in cultured human melanoma and rat glioblastoma cells with superoxide or hydrogen peroxide. Cycloheximide prevented the ROI-associated increases in VEGF mRNA. Transcriptional inhibition with actinomycin D revealed an inducible increase in VEGF mRNA half-life, but nuclear run-on experiments showed no increase in VEGF transcriptional rate. Reoxygenation of human retinal pigment epithelial cells in vitro and ocular reperfusion in vivo increased retinal VEGF mRNA levels. Antioxidants prevented the reperfusion-associated VEGF mRNA increases in retina. We conclude that ROIs increase VEGF gene expression in vitro and during the reperfusion of ischemic retina in vivo. The ROI-associated increases are mediated largely through increases in VEGF mRNA stability.

Inhibition of Endothelial Cell Growth by Macrophage-like U-937 Cell-derived Oncostatin M, Leukemia Inhibitory Factor, and Transforming Growth Factor β1

Conditioned media were collected from phorbol ester-treated human macrophage-like U-937 cells and analyzed for the presence of inhibitors of endothelial cell (EC) proliferation. By a combination of ion exchange and reverse-phase liquid chromatography, three inhibitors were purified to homogeneity as ascertained by microsequencing of 14-17 N-terminal amino acids. These inhibitors were identified as oncostatin M (OSM), leukemia inhibitory factor (LIF), and transforming growth factor β1 (TGF-β1). The identities of the three EC growth inhibitors were confirmed by demonstrating that recombinant human OSM, LIF, and TGF-β1 were inhibitory in the same concentration range. Inhibition of EC proliferation by OSM was a newly described property of this cytokine. OSM was the most potent inhibitor with a half-maximal inhibition by recombinant material of 0.15-.2 ng/ml compared with 0.6-0.9 and 0.9-1.0 ng/ml for LIF and TGF-β1, respectively. The three factors inhibited basal, vascular endothelial cell growth factor-stimulated, and fibroblast growth factor 2-stimulated EC proliferation. Interleukin-6 and ciliary neurotrophic factor, two cytokines related structurally to OSM and LIF, were not active as EC growth inhibitors. It was concluded that macrophage-like cells secrete a variety of potent EC growth inhibitors and that one of these, OSM, is among the most potent EC growth inhibitors yet reported.

Neuropilin 1 expression correlates with differentiation status of epidermal cells and cutaneous squamous cell carcinomas

Neuropilins (NRPs) are cell surface receptors for vascular endothelial growth factor (VEGF) and SEMA3 (class 3 semaphorin) family members. The role of NRPs in neurons and endothelial cells has been investigated, but the expression and role of NRPs in epithelial cells is much less clear. Herein, the expression and localization of NRP1 was investigated in human and mouse skin and squamous cell carcinomas (SCCs). Results indicated that NRP1 mRNA and protein was expressed in the suprabasal epithelial layers of the skin sections. NRP1 staining did not overlap with that of keratin 14 (K14) or proliferating cell nuclear antigen, but did co-localize with staining for keratin 1, indicating that differentiated keratinocytes express NRP1. Similar to the expression of NRP1, VEGF-A was expressed in suprabasal epithelial cells, whereas Nrp2 and VEGFR2 were not detectable in the epidermis. The expression of NRP1 correlated with a high degree of differentiation in human SCC specimens, human SCC xenografts, and mouse K14-HPV16 transgenic SCC. UVB irradiation of mouse skin induced Nrp1 upregulation. In vitro, Nrp1 was upregulated in primary keratinocytes in response to differentiating media or epidermal growth factor-family growth factors. In conclusion, the expression of NRP1 is regulated in the skin and is selectively produced in differentiated epithelial cells. NRP1 may function as a reservoir to sequester VEGF ligand within the epithelial compartment, thereby modulating its bioactivity.