Several reports have demonstrated that SPP inhibits cell motility. SPP inhibited chemotactic motility of mouse melanoma B16, mouse fibroblast BALB/3T3 clone A31, and several tumor cell lines at nanomolar concentrations (17-19). Moreover, SPP immobilized on glass beads markedly inhibited melanoma cell motility. However, pertussis toxin treatment did not block the effect of SPP, suggesting that in these cells SPP acts through a cell surface receptor, independently of pertussis toxin-sensitive G-proteins (20). In contrast, SPP inhibits chemotaxis of human breast cancer cells only at high (micromolar) concentrations, acting independently of EDG-1 (21).We have recently identified SPP as a ligand for the G-protein-coupled receptor, endothelial differentiation gene-1 (EDG-1) (22). EDG-1 binds SPP with remarkable specificity and high affinity (K D ϭ 8 nM) (9,22). Binding of SPP to EDG-1 resulted in inhibition of adenylate cyclase and activation of mitogen-activated protein kinase (both G i -mediated), but did not mobilize calcium from internal stores (9, 23). In contrast, Okamoto et al. (12) found that in HEL cells overexpressing EDG-1, binding of SPP induced calcium mobilization (12).

Previously we discovered that NPY induces ischemic angiogenesis by activating Y2 and Y5 receptors. The receptors that mediate specific steps of the complex process of angiogenesis are unknown. Here, we studied in vitro NPY receptors subtypes involved in migration, proliferation, and differentiation of human endothelial cells. In cells that expressed Y1, Y2, and Y5 receptors, NPY bimodally stimulated migration and proliferation with a 2-fold increase at 10(-12) M and 10(-8) M (high- and low-affinity peaks, respectively). Preincubation of cells with NPY up-regulated the Y5 receptor and markedly enhanced endothelial cell migration and proliferation. NPY-induced endothelial cell migration was mimicked by agonists and fully blocked by antagonists for any specific NPY receptors (Y1, Y2, or Y5), while proliferation was blocked by any two antagonists (Y1+Y2, Y1+Y5, or Y2+Y5), and capillary tube formation on Matrigel was blocked by all three (Y1+Y2+Y5). Thus, NPY-induced angiogenesis requires participation of Y1, Y2, and Y5 receptor subtypes, with the Y5 receptor acting as an enhancer. We propose that these receptors form heteromeric complexes, and the Y1/Y2/Y5 receptor oligomer may be the uncloned Y3 receptor.

Sympathetic nerves have long been suspected of trophic activity, but the nature of their angiogenic factor has not been determined. Neuropeptide Y (NPY), a sympathetic cotransmitter, is the most abundant peptide in the heart and the brain. It is released during nerve activation and ischemia and causes vasoconstriction and smooth muscle cell proliferation. Here we report the first evidence that NPY is angiogenic. At low physiological concentrations, in vitro, it promotes vessel sprouting and adhesion, migration, proliferation, and capillary tube formation by human endothelial cells. In vivo, in a murine angiogenic assay, NPY is angiogenic and is as potent as a basic fibroblast growth factor. The NPY action is specific and is mediated by Y1 and Y2 receptors. The expression of both receptors is upregulated during cell growth; however, Y2 appears to be the main NPY angiogenic receptor. Its upregulation parallels the NPY-induced capillary tube formation on reconstituted basement membrane (Matrigel); the Y2 agonist mimics the tube-forming activity of NPY, whereas the Y2 antagonist blocks it. Endothelium contains not only NPY receptors but also peptide itself, its mRNA, and the "NPY-converting enzyme" dipeptidyl peptidase IV (both protein and mRNA), which terminates the Y1 activity of NPY and cleaves the Tyr1-Pro2 from NPY to form an angiogenic Y2 agonist, NPY3-36. Endothelium is thus not only the site of action of NPY but also the origin of the autocrine NPY system, which, together with the sympathetic nerves, may be important in angiogenesis during tissue development and repair.

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