Impaired angiogenesis in neuropeptide Y (NPY)-Y2 receptor knockout mice
Introduction
Angiogenesis, generally defined as the formation of new vessels from pre-existing vessels [35], is a complex and highly regulated process particularly active during embryogenesis [5]. In adulthood, angiogenesis slows down and is limited to certain organs and conditions, such as wound healing and reproduction [26]. It can be re-activated in pathophysiological conditions, as it occurs in tumors and metastasis [16], [46] or retinopathies [3]. On the other hand, stimulation of angiogenesis using growth factors has become a new therapeutic target for treatment of ischemic heart and vascular diseases [7], [41]. A rich source of growth factors is provided by endothelial and vascular smooth muscle cells, the most known of which are basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) [22], [38].
Recently, our laboratory has shown that sympathetic nerves also release angiogenic factors and contribute to angiogenesis [21]. We found that neuropeptide Y (NPY), a sympathetic co-transmitter and previously known as a vasoconstrictor, is potently mitogenic for vascular smooth muscle cells [48] and angiogenic in vivo and in vitro [49]. In vivo, endogenous NPY appears to contribute to ischemic tissue revascularization in rats and mice and is also involved in angiogenesis in aging [18]. Exogenous NPY administration, at physiological concentrations, was found to have therapeutic potential since the peptide can fully restore not only skeletal muscle vascularity and blood flow, but also its function [21]. However, it has not been elucidated which of NPY’s multiple receptor(s) mediate(s) NPY’s angiogenic activity.
Six NPY receptors have been cloned and/or identified pharmacologically: Y1–Y6 [15], [24], [36], [42]. Among them, Y3 is the only one, which has not yet been cloned [39] and Y6 is a non-functioning receptor in species other than mouse [4]. All of NPY’s receptors belong to a family of Gi/o-heptahelical receptors [2]. The Y1 receptor is the predominant vascular receptor, mediating vasoconstriction [47] and vascular smooth muscle proliferation [50]. In the brain, it is also involved in anxiolytic action of NPY [30] and body weight regulation [9]. The Y2 receptor plays a major role in the inhibition of transmitter release in both central and peripheral nervous systems [11], [14]. The Y5 receptor appears to be NPY’s main hypothalamic receptor for feeding and body weight regulation [10].
There has been no evidence that Y5 receptors are also expressed in the cardiovascular system and evidence for Y2-mediated actions in blood vessels is also sparse [13], [33]. In some vessels, Y2-mediated vasoconstriction [32] and in others, nitric oxide-mediated vasodilation [45] has been described. Recently, however, we have found that Y2, in addition to Y1 receptors, are constitutively expressed on human endothelial cells [49] and in some vascular smooth muscle cells [50]. We have also showed that the Y5 receptor, although not expressed in intact vessels or non-growing cells, can be induced in both endothelial and vascular smooth muscle cells following their ischemia or injury [23], [50]. In vascular smooth muscle cells, the Y1 and Y5 receptors, but not Y2 receptors, appear to mediate NPY’s mitogenic effects [31], [50]. In contrast, our previous in vitro data suggested that Y2 might be the primary angiogenic receptor [49], although the role of Y1 and Y5 receptors has not been sufficiently addressed. Hence, the goal of this study was to determine the contribution of each of the receptor subtypes to NPY-mediated angiogenesis using transgenic Y2 knockout mice and receptor-specific antagonists in a variety of in vitro and in vivo models of angiogenesis.
Section snippets
Animals
We used 3–6-month-old male wild-type (Y2+/+) 129 SV mice and NPY Y2 receptor knockout mice (Y2−/−), devoid of Y2 receptor expression [28] developed from 129 SV embryonic stem cells (gift from AstraZeneca®, Sweden). The study was approved by the Animal Care and Use Committee of Georgetown University and all experimental procedures were carried out in accordance with the National Institutes of Health (NIH, USA) guidelines.
Materials
NPY was purchased from Peninsula Laboratory (Belmont, CA), VEGF (121, 165,
NPY-induced aortic sprouting in vitro
To determine if NPY-mediated angiogenesis is affected by a deficiency of the Y2 receptor, an ex vivo aortic sprouting assay was performed on aortas from Y2+/+ and Y2−/− mice (Fig. 1). NPY, at 1×10−8 M, was administrated to the rings in the presence or absence of NPY receptor antagonists, all at 1×10−7 M concentration, to block NPY’s effect. In previous dose response studies, these doses were tested in vascular smooth muscle and endothelial cells as maximally effective in blocking NPY’s mitogenic
Discussion
Based on our previous in vitro data, we have speculated that non-Y1 receptor(s), Y2 and/or Y5, is (are) responsible for NPY-mediated angiogenesis [27], [49]. Here we investigated in a variety of in vivo and ex vivo models, the role of each of NPY receptors, using transgenic Y2−/− mice and receptor-specific antagonists. We now report that NPY-mediated angiogenesis is markedly reduced in the Y2−/− mice in all angiogenic assays.
No changes were found, however, in spontaneous capillary sprouting of
References (50)
- et al.
Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia: dose-response effect of basic fibroblast growth factor
J. Vasc. Surg.
(1992) - et al.
Distribution of a novel hypothalamic neuropeptide Y receptor gene and it’s absence in rat
Brain Res. Mol. Brain Res.
(1998) - et al.
BIIE0246: a selective and high affinity neuropeptide Y Y(2) receptor antagonist
Eur. J. Pharmacol.
(1999) - et al.
Characterization of a selective antagonist of neuropeptide Y at the Y2 receptor. Synthesis and pharmacological evaluation of a Y2 antagonist
J. Biol. Chem.
(1997) - et al.
Neuropeptide Y-induced angiogenesis in aging
Peptides
(2002) - et al.
Cloning of a human receptor of the NPY receptor family with high affinity for pancreatic polypeptide and peptide YY
J. Biol. Chem.
(1995) - et al.
Chronic modulation of the GABA(A) receptor complex regulates Y1 receptor gene expression in the medial amygdala of transgenic mice
Neuropharmacology
(2000) Prolonged non-adrenergic inhibition of cardiac vagal action following sympathetic stimulation: neuromodulation by neuropeptide Y?
Neurosci. Lett.
(1985)- et al.
A novel neuropeptide Y analog, N-acetyl [Leu28, Leu31]neuropeptide Y-(24–36), with functional specificity for the presynaptic (Y2) receptor
Eur. J. Pharmacol.
(1994) - et al.
Neuropeptide Y and epilepsy: varying effects according to seizure type and receptor activation
Peptides
(2001)
Cloning and functional expression of a cDNA encoding a human type 2 neuropeptide Y receptor [published erratum appears in J Biol Chem 1995 Dec 1;270(48):29038]
J. Biol. Chem.
Design, synthesis and SAR of a series of 2-substituted 4-amino-quinazoline neuropeptide Y Y5 receptor antagonists
Bioorg. Med. Chem. Lett.
Endothelial-pericyte interdigitations in rat subcutaneous disc implanted angiogenesis
Microvasc. Res.
Cloning and expression of a novel neuropeptide Y receptor
J. Biol. Chem.
Insulin stimulates the growth and tube formation of human microvascular endothelial cells through autocrine vascular endothelial growth factor
Microvasc. Res.
Both platelet-derived growth factor receptor (PDGFR)-alpha and PDGFR-beta promote murine fibroblast cell migration
Biochem. Biophys. Res. Commun.
Mechanisms of vascular growth-promoting effects of neuropeptide Y: role of its inducible receptors
Regul. Pept.
Mitogenic effect of neuropeptide Y in rat vascular smooth muscle cells
Peptides
Y-receptor subtypes—how many more?
Trends Neurosci.
Angiogenesis in embryos and ischemic diseases
Thromb. Haemost.
Molecular analysis of blood vessel formation and disease
Am. J. Physiol.
Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions
Nat. Med.
Angiogenesis for the treatment of vascular diseases [editorial]
Int. Angiol.
Food intake regulation in rodents: Y5 or Y1 NPY receptors or both?
Can. J. Physiol. Pharmacol.
Species differences in the expression and distribution of the neuropeptide Y, Y1, Y2, Y4, and Y5 receptors in rodents, guinea pig, and primates brains
J. Comp. Neurol.
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