Neuropeptide Y: a new mediator linking sympathetic nerves,blood vessels and immune system?

Neuropeptide Y (NPY(1-36)), a sympathetic cotransmitter and neurohormone, has pleiotropic activities ranging from the control of obesity to anxiolysis and cardiovascular function. Its actions are mediated by multiple Gi/o-coupled receptors (Y1-Y5) and modulated by dipeptidyl peptidase IV (DPPIV/cd26), which inactivates NPY's Y1-agonistic activity but generates the Y2 and Y5-agonist, NPY(3-36). Released by sympathetic activity, NPY is a major mediator of stress, responsible for prolonged vasoconstriction via Y1 receptors. Y1 receptors also mediate NPY's potent vascular growth-promoting activity leading in vivo in rodents to neointima formation. This and the association of a polymorphism of the NPY signal peptide with increased lipidemia and carotid artery thickening in humans strongly suggest NPY's role in atherosclerosis. NPY and DPPIV/cd26 are also coexpressed in the endothelium, where the peptide activates angiogenesis. A similar system exists in immune cells, where NPY and DPPIV/cd26 are coactivated and involved in the modulation of cytokine release and immune cell functions. Thus, NPY, both a messenger and a modulator for all three systems, is poised to play an important regulatory role facilitating interactions among sympathetic, vascular and immune systems in diverse pathophysiological conditions such as hypertension, atherosclerosis and stress-related alterations of immunity.     

Neuropeptide Y-induced angiogenesis in aging

Age-related changes in NPY-driven angiogenesis were investigated using Matrigel and aortic sprouting assays in young (2 months.) and aged (18 months.) mice. In both assays, NPY-induced vessel growth decreased significantly with age. In parallel, aged mice showed reduced expression (RT-PCR) of Y2 receptors and the NPY converting enzyme, dipeptidyl peptidase IV (DPPIV), in spleens. Aging of human microvascular endothelial cells in vitro led to a loss of their mitogenic responses to NPY accompanied by a lack of NPY receptor mRNAs. Thus, NPY-dependent angiogenesis is impaired with age, which is associated with a decreased expression of endothelial NPY receptors (Y2) and DPPIV.     

Stress, NPY and vascular remodeling: Implications for stress-related diseases

Neuropeptide Y (NPY) has long been known to be involved in stress, centrally as an anxiolytic neuromodulator, and peripherally as a sympathetic nerve- and in some species, platelet-derived vasoconstrictor. The peptide is also a vascular mitogen, via Y1/Y5, and is angiogenic via Y2/Y5 receptors. Arterial injury activates platelet NPY and vascular Y1 receptors, inducing medial hypertrophy and neointima formation. Exogenous NPY, dipeptidyl peptidase IV (DPPIV, forming an Y2/Y5-selective agonist) and chronic stress augment these effects and occlude vessels with atherosclerotic-like lesions, containing thrombus and lipid-laden macrophages. Y1 antagonist blocks stress-induced vasoconstriction and post-angioplasty occlusions, and hence may be therapeutic in angina and atherosclerosis/restenosis. Conversely, tissue ischemia activates neuronal and platelet-derived NPY, Y2/Y5 and DPPIV, which stimulate angiogenesis/arteriogenesis. NPY-Y2-DPPIV agonists may be beneficial for ischemic revascularization and wound healing, whereas antagonists may be therapeutic in retinopathy, tumors, and obesity. Since stress is an underestimated risk factor in many of these conditions, NPY-based drugs may offer new treatment possibilities.     

Regulation of Dipeptidyl Peptidase IV in the Post-stroke Rat Brain and In Vitro Ischemia: Implications for Chemokine-Mediated Neural Progenitor Cell Migration and Angiogenesis

Cerebral ischemia evokes abnormal release of proteases in the brain microenvironment that spatiotemporally impact angio-neurogenesis. Dipeptidyl peptidase IV (DPPIV), a cell surface and secreted protease, has been implicated in extracellular matrix remodeling by regulating cell adhesion, migration, and angiogenesis through modifying the functions of the major chemokine stromal-derived factor, SDF1. To elucidate the possible association of DPPIV in ischemic brain, we examined the expression of DPPIV in the post-stroke rat brain and under in vitro ischemia by oxygen glucose deprivation (OGD). We further investigated the effects of DPPIV on SDF1 mediated in vitro chemotactic and angiogenic functions. DPPIV protein and mRNA levels were significantly upregulated during repair phase in the ischemic cortex of the rat brain, specifically in neurons, astrocytes, and endothelial cells. In vitro exposure of Neuro-2a neuronal cells and rat brain endothelial cells to OGD resulted in upregulation of DPPIV. In vitro functional analysis showed that DPPIV decreases the SDF1-mediated angiogenic potential of rat brain endothelial cells and inhibits the migration of Neuro-2a and neural progenitor cells. Western blot analyses revealed decreased levels of phosphorylated ERK1/2 and AKT in the presence of DPPIV. DPPIV inhibitor restored the effects of SDF1. Proteome profile array screening further revealed that DPPIV decreases matrix metalloproteinase-9, a key downstream effector of ERK-AKT signaling pathways. Overall, delayed induction of DPPIV in response to ischemia/reperfusion suggests that DPPIV may play an important role in endogenous brain tissue remodeling and repair processes. This may be mediated through modulation of SDF1-mediated cell migration and angiogenesis.     

Neuropeptide Y and its receptor analogs differentially modulate the immunoreactivity for neuronal or endothelial nitric oxide synthase in the rat brain following focal ischemia with reperfusion

Summary An intracerebroventricular (icv) injection of neuropeptide Y (NPY) or [Leu³¹, Pro³⁴]-NPY (non-Y2 receptor agonist) given during middle cerebral artery occlusion (MCAO) increases the infarct volume and nitric oxide (NO) overproduction in the rat brain. An icv injection of NPY3-36 (non-Y1 receptor agonist) has no effects, while BIBP3226 (selective Y1 receptor antagonist) reduces the infarct volume and NO overproduction. This study examined the effects of NPY or its receptor analog on the immunoreactivity (ir) for three isoforms of NO synthase (NOS) following 1h of MCAO and 3h of reperfusion. Focal ischemia/reperfusion led to increased ir for neuronal NOS (nNOS) within the ipsilateral caudate putamen and insular cortex. NPY or [Leu³¹, Pro³⁴]-NPY enhanced but BIBP3226 suppressed such increase in the nNOS-ir. Focal ischemia/reperfusion also led to an ipsilateral increase in extent and/or intensity of the ir for endothelial NOS (eNOS) in the caudate putamen and/or parietal cortex. NPY or [Leu³¹, Pro³⁴]-NPY suppressed but BIBP3226 enhanced such change in the eNOS-ir. NPY3-36 did not consistently influence the nNOS-ir or eNOS-ir following MCAO. Specific ir for inducible NOS was undetectable. These opposing effects of NPY-Y1 receptor activation or inhibition on nNOS and eNOS may lead to harmful or beneficial consequences following ischemia/reperfusion.     

Neuropeptide Y receptor alterations in the hypothalamus of lactating rats.

Hypothalamic neuropeptide Y (NPY) neurons are influenced by circulating levels of insulin and leptin and are thought to be involved in mediating hunger following underfeeding. We have investigated hypothalamic NPY receptor subtypes in lactating rats, which are markedly hyperphagic throughout the day and night. NPY receptors were measured by using [125I] peptide YY, a high-affinity ligand, and Y1 receptors were masked by using the highly specific antagonist BIBP 3226. Freely fed lactating rats showed no changes in the densities of Y1, or non-Y1, NPY binding sites in whole hypothalamic homogenates or in individual hypothalamic regions (measured by quantitative autoradiography) examined during the day or night (P > 0.05; n = 10/group, and n = 6/group, respectively). However, reducing food intake by 35% had a more profound effect on NPY receptor density in lactating than in control rats, producing down-regulation of non-Y1 receptors in the ventromedial, dorsomedial, and perifornical lateral areas (all P < 0.05; n = 7/group) and reduction of plasma insulin and leptin levels (both P < 0.01). Thus, although the NPY system may not have a major role in the hyperphagia of freely fed lactating rats, it appears to have an important function in the response to undernutrition in such animals.     

Neuropeptide Y Y5-receptor activation on breast cancer cells acts as a paracrine system that stimulates VEGF expression and secretion to promote angiogenesis

Accumulating data implicate a pathological role for sympathetic neurotransmitters like neuropeptide Y (NPY) in breast cancer progression. Our group and others reported that NPY promotes proliferation and migration in breast cancer cells, however the angiogenic potential of NPY in breast cancer is unknown. Herein we sought to determine if NPY promotes angiogenesis in vitro by increasing vascular endothelial growth factor (VEGF) expression and release from 4T1 breast cancer cells. Western blot analysis revealed that NPY treatment caused a 52 ± 14% increase in VEGF expression in the 4T1 cells compared to non-treated controls. Using selective NPY Y-receptor agonists (Y1R, Y2R and Y5R) we observed an increase in VEGF expression only when cells were treated with Y5R agonist. Congruently, using selective Y1R, Y2R, or Y5R antagonists, NPY-induced increases in VEGF expression in 4T1 cells were attenuated only under Y5R antagonism. Endothelial tube formation assays were conducted using conditioned media (CM) from NPY treated 4T1 cells. Concentration-dependent increases in number of branch points and complete endothelial networks were observed in HUVEC exposed to NPY CM. CM from Y5R agonist treated 4T1 cells caused similar increases in number of branch points and complete endothelial networks. VEGF concentration was quantified in CM (ELISA) from agonist experiments; we observed a 2-fold and 2.5-fold increase in VEGF release from NPY and Y5R agonist treated 4T1 cells respectively. Overall these data highlight a novel mechanism by which NPY may promote breast cancer progression, and further implicate a pathological role of the NPY Y5R.     

Modulation of intracellular calcium changes and glutamate release by neuropeptide Y1 and Y2 receptors in the rat hippocampus: differential effects in CA1, CA3 and dentate gyrus

In the present work, we investigated the role of pre- and post-synaptic neuropeptide Y1 (NPY1) and Y2 receptors on the calcium responses and on glutamate release in the rat hippocampus. In cultured hippocampal neurones, we observed that only NPY1 receptors are involved in the modulation of intracellular free calcium concentration ([Ca(2+)](i)). In 88% of the neurones analysed, the increase in the [Ca(2+)](i), in response to depolarization with 50 mM KCl, was inhibited by 1 microM [Leu31,Pro34]NPY, whereas 300 nM NPY13-36 was without effect. However, studies with hippocampal synaptosomes showed that both NPY1 and Y2 receptors can modulate the [Ca(2+)](i) and glutamate release. The pharmacological characterization of the NPY-induced inhibition of glutamate release indicated that Y2 receptors play a predominant role, both in the modulation of Ca(2+)-dependent and -independent glutamate release. However, we could distinguish between Y1 and Y2 receptors by using [Leu31,Pro34]NPY and NPY13-36. Active pre-synaptic Y1 receptors are present in the dentate gyrus (DG) as well as in the CA3 subregion, but its activity was not revealed by using the endogenous agonist, NPY. Concerning the Y2 receptors, they are present in the three subregions (CA1, CA3 and DG) and were activated by either NPY13-36 or NPY. The present data support a predominant role for NPY2 receptors in mediating NPY-induced inhibition of glutamate release in the hippocampus, but the physiological relevance of the presently described DG and CA3 pre-synaptic NPY1 receptors remains to be clarified.     

Neuropeptide Y-Y1 receptor agonist worsens while antagonist improves survival of cultured Y1-expressing neuronal cells following oxygen and glucose deprivation

In this in vitro study, we investigated the influence of neuropeptide Y (NPY) Y1 receptor activation or inhibition on the viability of cultured neuronal or glial cells following oxygen glucose deprivation (OGD). Viability of cultured cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. When compared to the vehicle-treated control group, treatment with NPY or [Leu³¹,Pro³⁴]-NPY (Y1 agonist) reduced viability of cultured SK-N-MC (Y1-expressing) human neuronal cells at 24 h after 1 h of OGD, while BIBP3226 (Y1 antagonist) improved viability. Except at the highest concentration of NPY used in the study, treatment with NPY or NPY3-36 (Y2 agonist) did not influence viability of cultured SH-SY5Y (Y2-expressing) human neuronal cells at 24 h after 1 h of OGD. In addition, treatment with NPY, [Leu³¹,Pro³⁴]-NPY, NPY3-36, or BIBP3226 did not affect viability of cultured primary astrocytes at 24 h after 4 h of OGD. The present results agree with those of a recent in vivo study. Activation of NPY-Y1 receptors may mediate ischemic pathophysiological processes, and inhibiting the Y1 receptors may be protective. The combination of OGD and cultured neuronal cells may be useful in future studies on the neuroprotective and harmful mechanisms of NPY-Y1 receptor inhibition and activation during ischemia, respectively.     

The cloned guinea pig neuropeptide Y receptor Y1 conforms to other mammalian Y1 receptors.

We have cloned the guinea pig neuropeptide Y (NPY) Y1 receptor and found it to be 92-93% identical to other cloned mammalian Y1 receptors. Porcine NPY and peptide YY (PYY) displayed affinities of 43 pM and 48 pM, respectively. NPY2-36 and NPY3-36 had 6- and 46-fold lower affinity, respectively, than intact NPY. Functional coupling was measured by using a microphysiometer. Human NPY and PYY were equipotent in causing extracellular acidification with EC50 values of 0.59 nM and 0.69 nM, respectively, whereas NPY2-36 and NPY3-36 were about 15-fold and 500-fold less potent, respectively, than NPY. The present study shows that the cloned guinea pig Y1 receptor is very similar to its orthologues in other mammals, both with respect to sequence and pharmacology. Thus, results from previous studies on guinea pig NPY receptors might imply the existence of an additional Y1-like receptor sensitive to B1BP3226.     

Dipeptidyl-peptidase-IV by cleaving neuropeptide Y induces lipid accumulation and PPAR-γ expression

We evaluated the effects of dipeptidyl peptidase-IV (DPPIV), and its inhibitor, vildagliptin, on adipogenesis and lipolysis in a pre-adipocyte murine cell line (3T3-L1). The exogenous rDPPIV increased lipid accumulation and PPAR-γ expression, whereas an inhibitor of DPPIV, the anti-diabetic drug vildagliptin, suppresses the stimulatory role of DPPIV on adipogenesis and lipid accumulation, but had no effect on lipolysis. NPY immunoneutralization or NPY Y(2) receptor blockage inhibited DPPIV stimulatory effects on lipid accumulation, collectively, indicating that DPPIV has an adipogenic effect through NPY cleavage and subsequent NPY Y(2) activation. Vildagliptin inhibits PPAR-γ expression and lipid accumulation without changing lipolysis, suggesting that this does not impair the ability of adipose tissue to store triglycerides inside lipid droplets. These data indicate that DPPIV and NPY interact on lipid metabolism to promote adipose tissue depot.     

NPY in rat retina is present in neurons, in endothelial cells and also in microglial and Müller cells

NPY is present in the retina of different species but its role is not elucidated yet. In this work, using different rat retina in vitro models (whole retina, retinal cells in culture, microglial cell cultures, rat Müller cell line and retina endothelial cell line), we demonstrated that NPY staining is present in the retina in different cell types: neurons, macroglial, microglial and endothelial cells. Retinal cells in culture express NPY Y(1), Y(2), Y(4) and Y(5) receptors. Retina endothelial cells express all NPY receptors except NPY Y(5) receptor. Moreover, NPY is released from retinal cells in culture upon depolarization. In this study we showed for the first time that NPY is present in rat retina microglial cells and also in rat Müller cells. These in vitro models may open new perspectives to study the physiology and the potential pathophysiological role of NPY in the retina.     

The role of PYY in feeding regulation

Peptide YY (PYY), a 36-amino-acid peptide, is secreted primarily from L-cells residing in the intestinal mucosa of the ileum and large intestine. PYY, which belongs to a family of peptides including neuropeptide Y (NPY) and pancreatic polypeptide, is released into the circulation as PYY(1-36) and PYY(3-36); the latter is the major form of PYY in gut mucosal endocrine cells and throughout the circulation. Plasma PYY levels begin to rise within 15 min after starting to eat and plateau within approximately 90 min, remaining elevated for up to 6 h. Exogenous administration of PYY(3-36) reduces energy intake and body weight in both humans and animals. Via Y2 receptors, the satiety signal mediated by PYY inhibits NPY neurons and activates pro-opiomelanocortin neurons within the hypothalamic arcuate nucleus. Peripheral PYY(3-36) binds Y2 receptors on vagal afferent terminals to transmit the satiety signal to the brain. PYY(3-36) may have therapeutic potential in human obesity.     

SNX9 determines the surface levels of integrin β1 in vascular endothelial cells: Implication in poor prognosis of human colorectal cancers overexpressing SNX9

Angiogenesis, the formation of new blood vessels, is involved in a variety of diseases including the tumor growth. In response to various angiogenic stimulations, a number of proteins on the surface of vascular endothelial cells are activated to coordinate cell proliferation, migration, and spreading processes to form new blood vessels. Plasma membrane localization of these angiogenic proteins, which include vascular endothelial growth factor receptors and integrins, are warranted by intracellular membrane trafficking. Here, by using a siRNA library, we screened for the sorting nexin family that regulates intracellular trafficking and identified sorting nexin 9 (SNX9) as a novel angiogenic factor in human umbilical vein endothelial cells (HUVECs). SNX9 was essential for cell spreading on the Matrigel, and tube formation that mimics in vivo angiogenesis in HUVECs. SNX9 depletion significantly delayed the recycling of integrin β1, an essential adhesion molecule for angiogenesis, and reduced the surface levels of integrin β1 in HUVECs. Clinically, we showed that SNX9 protein was highly expressed in tumor endothelial cells of human colorectal cancer tissues. High-level expression of SNX9 messenger RNA significantly correlated with poor prognosis of the patients with colorectal cancer. These results suggest that SNX9 is an angiogenic factor and provide a novel target for the development of new antiangiogenic drugs.     

Interaction of NPY compounds with the rat glucocorticoid-induced receptor (GIR) reveals similarity to the NPY-Y2 receptor

The rat glucocorticoid-induced receptor (rGIR) is an orphan G protein-coupled receptor awaiting pharmacological characterization. Among known receptors, rGIR exhibits highest sequence similarity to the neuropeptide Y (NPY)-Y(2) receptor (38-40%). The pharmacological profile of rGIR was investigated using (125)I-PYY(3-36), a Y(2)-preferring radioligand and several NPY analogs. rGIR displayed a similar displacement profile as reported for the Y(2) receptor, in that the Y(2)-selective C terminus fragments of NPY and PYY (NPY(3-36) and PYY(3-36)) showed high affinity binding and activation of rGIR (low nanomolar range). The rank order potency for displacement was NPY(3-36)>PYY(3-36)=NPY>NPY(13-36)>Ac, Leu NPY(24-36)>[D-Trp(32)]-NPY>Leu(31), Pro(34)-NPY=hPP. NPY and Y(2)-selective agonists NPY(3-36) and PYY(3-36) led to significant activation of (35)S-GTPgammaS binding to rGIR transfected cells. BIIE0246, a specific Y(2) antagonist, displaced (125)I-PYY(3-36) binding to rGIR with high affinity (95nM). Activation of (35)S-GTPgammaS binding by Y(2)-selective agonist in rGIR transfected cells was also completely abolished by BIIE0246. Our data report, for the first time, an interaction of NPY ligands with rGIR expressed in vitro, and indicate similarities between GIR and the NPY-Y(2) receptor.     

Neuropeptide Y induced modulation of dopamine synthesis in the striatum

The purpose of the present study was to determine whether the activation of NPY receptors alters catecholamines (CA) synthesis in the central nervous system and, if so, to identify the NPY receptor subtype(s) mediating this effect. Tyrosine hydroxylation, the rate-limiting step in CA synthesis, was assessed by measuring the accumulation of 3,4-dihydroxyphenyalanine (DOPA) by high pressure liquid chromatography coupled to electrochemical detection (HPLC-EC) in rat striatal dices following incubation of the tissue with the aromatic L-amino acid decarboxylase inhibitor m-hydroxybenzyl hydrazine (NSD 1015). Treatment with NSD 1015 resulted in an increase in DOPA accumulation that was increased even further following depolarization with a high potassium (KCl) buffer. PYY13-36 and NPY13-36 both produced a significant enhancement of the KCl-induced increase in DOPA accumulation. The effect of PYY13-36 was completely attenuated by the selective Y2 antagonist BIIE0246 suggesting that activation of Y2 receptors enhanced the synthesis of dopamine. In contrast to the effects of NPY13-36 and PYY13-36; NPY, PYY and PYY3-36 all produced a significant attenuation of the KCl-induced increase in DOPA accumulation. The Y1 antagonist BIBO3304 and the Y5-antagonist CGP71683A, both prevented the inhibitory effect of NPY converting it to a stimulatory effect. The enhancement of the NPY induced increase in DOPA accumulation observed by BIBO3304 was attenuated when examined in the presence of the Y2 antagonist BIIE0246. These results suggest that activation of NPY receptors can modulate the synthesis of CA in the rat striatum. The Y1 and Y5 receptor appear to be involved in attenuation, while Y2 receptors are involved in the stimulation of synthesis.     

Differential regulation of neuropeptide Y receptors in the brains of NPY knock-out mice

To study the effect of NPY deletion on the regulation of its receptors in the NPY knockout (NPY KO) mice, the expression and binding of NPY receptors were investigated by in situ hybridization and receptor autoradiography using (125)I-[Leu(31),Pro(34)]PYY and (125)I-PYY(3-36) as radioligands. A 6-fold increase in Y2 receptor mRNA was observed in the CA1 region of the hippocampus in NPY KO mice, but a significant change could not be detected for Y1, Y4, Y5 and y6 receptors. Receptor binding reveals a 60-400% increase of Y2 receptor binding in multiple brain areas. A similar increase in Y1 receptor binding was seen only in the hypothalamus. These results demonstrate the NPY receptor expression is altered in mice deficient for its natural ligand.