1. The NPY System

92 J. B. Kitlinska & Z. Zukowska

always constitutive and depends on animal species, ethnicity and pathological conditions. For example, rats and some murine strains contain high platelet-derived NPY.6 On the other hand, platelet NPY is not detected in healthy humans, but has been found in chronically depressed patients.9

NPY is a pleiotropic factor (Fig. 1). In the brain, the peptide exerts a variety of functions, but is most known for its ability to stimulate food intake, inhibit anxiety and regulate energy balance and pituitary secretion. The best known peripheral actions of NPY include vasoconstriction, potentiation of other vasoconstrictors’ actions, inhibition of neurotransmitter release and regulation of immune responses.3 The peptide mediates stress-induced, slow-onset, prolonged vasoconstriction of small resistance vessels, and hence it has been suggested to be involved in coronary vasospasm and ischemic heart disease.10 Recently, however, new growth-regulatory activities of NPY have emerged, which occur at concentrations lower than the vasoconstrictive ones, indicating that NPY’s primary physiological role may not be as a vasoconstrictor but that of a neurogenic trophic factor. The peptide exerts proliferative effects in a variety of cells, including neuronal precursors, lymphocytes and tumor cells, such as neuroblastoma and prostate cancer cells.11−16 On the other hand, NPY can also inhibit cell growth, as seen in Ewing’s sarcoma and retinal glial cells.13,17 However, some of the most potent

Fig. 1. Structure and functions of NPY peptides, enzymatic cleavage and receptor subtypes.

activities of NPY involve its vascular growth promoting and angiogenic actions, and this chapter will focus on these effects.

Diverse actions of NPY are mediated by five Gi/o-protein-coupled receptors designated Y1–Y5. The Y1 receptor is the predominant vascular receptor mediating vasoconstriction and the major brain receptor involved in anxiety and, together with Y5, in regulation of food intake The Y2 receptor, on the other hand, is the primary receptor responsible for the presynaptic neuro-inhibitory actions of NPY in the central and peripheral nervous system.3,18 Receptor expression pattern is also an important factor determining effects of NPY on cell growth. For example, Y1 receptor mediates NPY-induced proliferation of neuronal precursors, while Y2 is the main receptor responsible for its mitogenic effect in neuroblastoma cells.12,13 On the other hand, activation of both Y1 and Y5 receptors in Ewing’s sarcoma cells leads to cell death.13 Hence, growth-regulatory effects of NPY are celland receptor-specific.

In addition, actions of NPY are modified by a serine protease, dipeptidyl peptidase IV (DPPIV), which functions in the NPY system as a “converting enzyme” and a “receptor switch.” The protease converts the full length NPY1−36 to a shorter form, NPY3−36, which is no longer able to bind to the Y1 receptor but retains affinity for all other receptors.19 Hence, DPPIV is an important regulatory molecule in the NPY system shifting the actions of the peptide from Y1to non-Y1 receptor mediated (Fig. 1).

2. NPY as a Growth Factor for Vascular Cells

Zukowska’s group was the first to discover that NPY, at sub-picomolar concentrations, exerts growth-promoting effects on both endothelial and vascular smooth muscle cells (VSMCs), and is potently angiogenic (Fig. 2).8,20,21 The angiogenic properties of the peptide have been established using a variety of models, such as aortic sprouting, rodent hindlimb ischemia, retinopathy and wound healing.20,22−25 The role of NPY in vascularization has been further supported by hypervascularization of skeletal muscles of NPY overexpressing rats20 and reduced basal angiogenic activity in NPY−/− mice, which exogenous peptide restores.

94 J. B. Kitlinska & Z. Zukowska

Fig. 2. NPY and its receptor-specific vascular growth-promoting and angiogenic actions: role in neovascularization in various pathological conditions.

Human and rodent endothelial cells constitutively express NPY and its Y1 receptors, whereas Y2 and Y5 receptors are not always expressed but are upregulated/induced during ischemia and after stimulation with NPY.20,21 The simultaneous expression of the ligand and its receptors

in the endothelium suggests possible autocrine effects of the peptide. Indeed, NPY has been found to be a potent growth factor for endothelial cells. The peptide stimulates not only their proliferation, but also other processes involved in angiogenesis, such as cell adhesion, migration and capillary tube formation.21

Despite the presence of Y1 receptors, NPY growth-promoting actions in endothelial cells seem to be mediated mainly by Y2, with some contribution of Y5 receptors. The Y2 receptors are abundantly expressed in newly formed microvessels in murine corneas implanted with NPY, as well as bFGF and VEGF pellets.22 The Y2 is also a predominant NPY receptor in growing vasculature of neuroblastoma tumors (unpublished data) and in neointimal atherosclerotic-like lesions induced by NPY administered at the time of arterial balloon angioplasty.26 The effect of NPY on endothelial cell proliferation and migration can be mimicked by Y2/Y5 receptor agonist, NPY3−36.21 Moreover, both Y2 and Y5 receptors are upregulated in ischemia, while age-dependent impairment of angiogenesis is associated with Y2 receptor downregulation.20,27 The angiogenic activities of NPY, as measured by its effect on aortic sprouting, revascularization of ischemic tissues and vascularization of mouse corneas, are either completely abolished or significantly reduced in Y2−/− mice.22,28 Moreover, deletion of Y2 receptor in mice results in severe impairment of angiogenesis-related processes, such as oxygen-induced retinopathy and wound healing but also reduced vascularization of non-ischemic limbs.22,24 Hence, Y2 seems to be the main angiogenic receptor in the NPY system, active during physiological developmental angiogenesis as well as in pathological conditions. The presence of the Y5 receptor, on the other hand, appears to provide an additional amplification of the angiogenic signal (see below: “mechanisms”).

In addition to its growth-promoting effects on endothelial cells, NPY is also a growth factor for VSMCs. However, in contrast to Y2/Y5 receptor-dependent proliferation of endothelial cells, the mitogenic effect of the peptide in VSMCs is mediated primarily by Y1, and again, amplified by Y5 receptors.8,29 Physiological consequences of NPY-induced VSMCs proliferation include its role in vascular remodeling and atherosclerosis.26 This process is also important in neovascularization. Even though the first steps of angiogenesis involve

96 J. B. Kitlinska & Z. Zukowska

mainly activation of endothelial cells and formation of capillaries, development of the mature blood vessels additionally requires cooption of VSMCs. Thus, by combining proliferative effects on both endothelial and VSMCs (Fig. 1), NPY is able to stimulate formation of functional, mature arteries. Such simultaneous activation of angioand arteriogenesis upon NPY treatment has been observed, e.g. in rat hindlimb ischemia model and in neuroblastoma xenografts (see below).13,20

Although mitogenic actions of NPY in endothelial and VSMCs are mediated by different receptors, in both types of cells the peptide exerts its proliferative effects at sub-picomolar and subnanomolar concentrations, significantly lower than those necessary for vasoconstriction.8,21,27,29 Moreover, in both endothelial and VSMCs, NPY stimulates proliferation in a bi-modal fashion, with two peaks of activity — a high affinity peak at picomolar concentrations and a low affinity peak at nanomolar concentrations of the peptide.8,21,27,29 Interestingly, this bi-modality seems to be universal for a variety of NPYinduced processes, such as neuroblastoma cell proliferation, endothelial cell migration and differentiation of adipocytes.21,27 The mechanism of this phenomenon has not been elucidated yet. However, some experimental data indicate that it may involve interaction between multiple NPY receptors, since the bi-modal effect occurs only in cells expressing more than one receptor subtype. In endothelial cells expressing only Y2 receptors, as well as in CHO-K1 cells stably transfected with single Y1 or Y2 receptors, NPY stimulates proliferation only at high concentrations, corresponding to the low affinity peak.30 Interestingly, the high affinity peak occurs at concentrations below Kd values for any known single NPY receptors. Thus, this first peak of activity seems to be dependent on the presence of multiple NPY receptors and their interactions leading to amplification of NPY’s growth-promoting actions.

Such effects have previously been reported to occur as a result of receptor oligomerization, described for other G protein-coupled receptors, and recently also found to be the case for NPY receptors.31,32 NPY receptor homodimerization has been shown for Y1, Y2 and Y4 receptors, whereas heterodimerization so far only for Y1 and Y5 receptors.33−35 These interactions are believed to change receptor affinity, trafficking and signaling pathways. Further studies are necessary