Platelet neuropeptide Y is critical for ischemic revascularization in mice.

We previously reported that the sympathetic neurotransmitter neuropeptide Y (NPY) is potently angiogenic, primarily through its Y2 receptor, and that endogenous NPY is crucial for capillary angiogenesis in rodent hindlimb ischemia. Here we sought to identify the source of NPY responsible for revascularization and its mechanisms of action. At d 3, NPY(-/-) mice demonstrated delayed recovery of blood flow and limb function, consistent with impaired collateral conductance, while ischemic capillary angiogenesis was reduced (~70%) at d 14. This biphasic temporal response was confirmed by 2 peaks of NPY activation in rats: a transient early increase in neuronally derived plasma NPY and increase in platelet NPY during late-phase recovery. Compared to NPY-null platelets, collagen-activated NPY-rich platelets were more mitogenic (~2-fold vs. ~1.6-fold increase) for human microvascular endothelial cells, and Y2/Y5 receptor antagonists ablated this difference in proliferation. In NPY(+/+) mice, ischemic angiogenesis was prevented by platelet depletion and then restored by transfusion of platelets from NPY(+/+) mice, but not NPY(-/-) mice. In thrombocytopenic NPY(-/-) mice, transfusion of wild-type platelets fully restored ischemia-induced angiogenesis. These findings suggest that neuronally derived NPY accelerates the early response to femoral artery ligation by promoting collateral conductance, while platelet-derived NPY is critical for sustained capillary angiogenesis.

Dipeptidyl peptidases as survival factors in Ewing sarcoma family of tumors: implications for tumor biology and therapy.

Ewing sarcoma family of tumors (ESFT) is a group of aggressive pediatric malignancies driven by the EWS-FLI1 fusion protein, an aberrant transcription factor up-regulating specific target genes, such as neuropeptide Y (NPY) and its Y1 and Y5 receptors (Y5Rs). Previously, we have shown that both exogenous NPY and endogenous NPY stimulate ESFT cell death via its Y1 and Y5Rs. Here, we demonstrate that this effect is prevented by dipeptidyl peptidases (DPPs), which cleave NPY to its shorter form, NPY(3-36), not active at Y1Rs. We have shown that NPY-induced cell death can be abolished by overexpression of DPPs and enhanced by their down-regulation. Both NPY treatment and DPP blockade activated the same cell death pathway mediated by poly(ADP-ribose) polymerase (PARP-1) and apoptosis-inducing factor (AIF). Moreover, the decrease in cell survival induced by DPP inhibition was blocked by Y1 and Y5R antagonists, confirming its dependence on endogenous NPY. Interestingly, similar levels of NPY-driven cell death were achieved by blocking membrane DPPIV and cytosolic DPP8 and DPP9. Thus, this is the first evidence of these intracellular DPPs cleaving releasable peptides, such as NPY, in live cells. In contrast, another membrane DPP, fibroblast activation protein (FAP), did not affect NPY actions. In conclusion, DPPs act as survival factors for ESFT cells and protect them from cell death induced by endogenous NPY. This is the first demonstration that intracellular DPPs are involved in regulation of ESFT growth and may become potential therapeutic targets for these tumors.

Differential effects of neuropeptide Y on the growth and vascularization of neural crest-derived tumors.

Neuropeptide Y (NPY) is a sympathetic neurotransmitter recently found to be potently angiogenic and growth promoting for endothelial, vascular smooth muscle and neuronal cells. NPY and its cognate receptors, Y1, Y2 and Y5, are expressed in neural crest-derived tumors; however, their role in regulation of growth is unknown. The effect of NPY on the growth and vascularization of neuroendocrine tumors was tested using three types of cells: neuroblastoma, pheochromocytoma, and Ewing's sarcoma family of tumors (ESFT). The tumors varied in expression of NPY receptors, which was linked to differential functions of the peptide. NPY stimulated proliferation of neuroblastoma cells via Y2/Y5Rs and inhibited ESFT cell growth by Y1/Y5-mediated apoptosis. In both tumor types, NPY receptor antagonists altered basal growth levels, indicating a regulatory role of autocrine NPY. In addition, the peptide released from the tumor cells stimulated endothelial cell proliferation, which suggests its paracrine angiogenic effects. In nude mice xenografts, exogenous NPY stimulated growth of neuroblastoma tumors, whereas it increased apoptosis and reduced growth of ESFT. However, in both tumors, NPY treatment led to an increase in tumor vascularization. Taken together, this is the first report of NPY being a growth-regulatory factor for neuroendocrine tumors, acting both by autocrine activation of tumor cell proliferation or apoptosis and by angiogenesis. NPY and its receptors may become targets for novel approaches in the treatment of these diseases, directed against both tumor cell proliferation and angiogenesis.

Hypoxia shifts activity of neuropeptide Y in Ewing sarcoma from growth-inhibitory to growth-promoting effects.

Ewing sarcoma (ES) is an aggressive malignancy driven by an oncogenic fusion protein, EWS-FLI1. Neuropeptide Y (NPY), and two of its receptors, Y1R and Y5R are up-regulated by EWS-FLI1 and abundantly expressed in ES cells. Paradoxically, NPY acting via Y1R and Y5R stimulates ES cell death. Here, we demonstrate that these growth-inhibitory actions of NPY are counteracted by hypoxia, which converts the peptide to a growth-promoting factor. In ES cells, hypoxia induces another NPY receptor, Y2R, and increases expression of dipeptidyl peptidase IV (DPPIV), an enzyme that cleaves NPY to a shorter form, NPY3-36. This truncated peptide no longer binds to Y1R and, therefore, does not stimulate ES cell death. Instead, NPY3-36 acts as a selective Y2R/Y5R agonist. The hypoxia-induced increase in DPPIV activity is most evident in a population of ES cells with high aldehyde dehydrogenase (ALDH) activity, rich in cancer stem cells (CSCs). Consequently, NPY, acting via Y2R/Y5Rs, preferentially stimulates proliferation and migration of hypoxic ALDHhigh cells. Hypoxia also enhances the angiogenic potential of ES by inducing Y2Rs in endothelial cells and increasing the release of its ligand, NPY3-36, from ES cells. In summary, hypoxia acts as a molecular switch shifting NPY activity away from Y1R/Y5R-mediated cell death and activating the Y2R/Y5R/DPPIV/NPY3-36 axis, which stimulates ES CSCs and promotes angiogenesis. Hypoxia-driven actions of the peptide such as these may contribute to ES progression. Due to the receptor-specific and multifaceted nature of NPY actions, these findings may inform novel therapeutic approaches to ES.

Interactions of multiple signaling pathways in neuropeptide Y-mediated bimodal vascular smooth muscle cell growth.

Neuropeptide Y (NPY), a sympathetic cotransmitter, acts via G protein-coupled receptors to stimulate constriction and vascular smooth muscle cell (VSMC) proliferation through interactions with its Y1 receptors. However, VSMC proliferation appears bimodal, with high- and low-affinity peaks differentially blocked by antagonists of both Y1 and Y5 receptors. Here, we sought to determine the signaling mechanisms of NPY-mediated bimodal mitogenesis. In rat aortic VSMCs, NPY's mitogenic effect at all concentrations was blocked by pertussis toxin and was associated with decreased forskolin-stimulated cAMP levels. NPY also increased intracellular calcium levels; in contrast to mitogenesis, this effect was dose dependent. The rise in intracellular Ca2+ depended on extracellular Ca2+ and was mediated via activation of Y1 receptors, but not Y5 receptors. Despite differences in calcium, the signaling pathways activated at low and high NPY concentrations were similar. The mitogenic effect of the peptide at all doses was completely blocked by inhibitors of calcium/calmodulin-dependent kinase II (CaMKII), protein kinase C (PKC), and mitogen-activated protein kinase kinase, MEK1/2. Thus, in VSMCs, NPY-mediated mitogenesis signals primarily via Y1 receptors activating 2 Ca2+-dependent, growth-promoting pathways -- PKC and CaMKII. At the high-affinity peak, these 2 pathways are amplified by Y5 receptor-mediated, calcium-independent inhibition of the adenylyl cyclase - protein kinase A (PKA) pathway. All 3 mechanisms converge to the extracellular signal-regulated kinases (ERK1/2) signaling cascade and lead to VSMC proliferation.