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Am J Physiol Heart Circ Physiol 293: H959-H967, 2007. First published March 30, 2007; doi:10.1152/ajpheart.00886.2006
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Possible roles of neuropeptide Y Y3-receptor subtype in rat aortic endothelial cell proliferation under hypoxia, and its specific signal transduction

Zhi-Yong Chen,2 Guo-Gang Feng,1 Kimitoshi Nishiwaki,2 Yasuhiro Shimada,2 Yoshihiro Fujiwara,3 Toru Komatsu,3 and Naohisa Ishikawa1

1Department of Pharmacology, Aichi Medical University School of Medicine, Nagakute, Aichi; 2Department of Anesthesiology, Nagoya University School of Medicine, Showa-ku, Nagoya; and 3Department of Anesthesiology, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan

Submitted 17 August 2006 ; accepted in final form 23 March 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study was undertaken to determine whether neuropeptide Y (NPY) induces proliferation of rat aortic endothelial cells (RAECs). Since NPY increased the permeability of RAEC monolayers to large molecules via the NPY Y3 receptor, RAEC proliferation has been evaluated in terms of NPY-receptor subtypes and also intracellular mechanisms. RAECs were incubated with gases containing 20, 15, or 10% O2 and a certain amount of N2, depending on the O2 content in 5% CO2 incubators. NPY (10–9–10–6 M) increased the RAEC numbers under hypoxic conditions, such as 15 or 10% O2. Peptide YY elicited no proliferative effect on RAEC, and NPY-(18-36) inhibited the NPY-induced increase in cell number, suggesting that NPY increases the RAEC count through the NPY Y3 receptor. Pertussis toxin, U-73122, GF-109203X, myristorylated autocamtide-2-related inhibitory peptide, and wortmannin inhibited the NPY-induced proliferation of RAEC concentration dependently. DY9760e little affected the proliferation caused by NPY. ML-9 and imatinib actually enhanced the NPY-induced proliferation of cells. These results indicated that the NPY Y3 receptor is coupled with Gi protein, and that NPY-induced increases in RAEC proliferation are mediated by phospholipase C-protein kinase C and/or phosphatidylinositol 3-kinase pathways. In intracellular Ca2+-calmodulin-dependent pathways, calmodulin-dependent protein kinase II partly participates in the NPY-induced cell proliferation. Regarding the previously reported effect of NPY on the permeability of RAEC monolayers to large molecules, it is probable that protein kinase C and phosphatidylinositol 3-kinase pathways are activated for both permeability and cell proliferation induced by NPY under hypoxia, relevant to new insights into the roles of NPY in ischemia-hypoxia.

protein kinase C; phosphatidylinositol 3-kinase; phospholipase C; calmodulin-dependent protein kinase

NEUROPEPTIDE Y (NPY), A SYMPATHETIC neurotransmitter (9, 31), exerts various physiological functions via G protein-coupled receptors from the cardiovascular system (27), including the control of food intake (1), the integration of emotional behavior (12), and cerebrocortical excitability (33). In cases of strong stress, NPY is released with norepinephrine from the peripheral sympathetic nerve terminals, possibly enhancing the permeability of vascular walls to large molecules [resulting in neurogenic pulmonary edema (13, 25)], the proliferation of vascular smooth muscles (6), and the formation of aortic sprouts in ischemic tissues of skeletal muscles [relevant to muscle training physiology (18)].

Among six different NPY-receptor subtypes, only the NPY Y3 receptor has not been cloned (17, 19). The NPY Y3 receptor is characterized pharmacologically by its inability to be activated by peptide YY (PYY), and an ability to be inhibited by NPY-(18-36), a Y3-receptor antagonist and also a Y2-receptor agonist (19). Using such criteria, Nan et al. (22) demonstrated that the NPY Y3-receptor mediates the permeability of rat aortic endothelial cell (RAEC) monolayers under hypoxic (5% O2) but not normoxic (20% O2) conditions. Furthermore, NPY stimulates angiogenesis of the skeletal muscle vasculature via NPY Y2- and Y5-receptor subtypes, especially under ischemic conditions (18, 35). Recent studies have provided evidence of a role for NPY as a growth factor, which stimulates the proliferation of a variety of cells, including neuronal precursors and angiogenesis (6, 11, 18, 24). Endothelial permeability is mediated via the NPY Y3 receptor, and proliferation via NPY Y2 and Y5 receptors.

Since substances with good penetrative ability are often recognized as angiogenetic factors, e.g., vascular endothelial growth factor (VEGF) (21, 32, 34) and hepatocyte growth factor (4, 14), common intracellular pathways may be involved for both actions. The present study has been undertaken to evaluate roles of NPY in increasing cultured vascular endothelial cell proliferation and to determine whether NPY Y3-receptor or Y2-/Y5-receptor subtypes mediate such responses. RAECs were cultured under normoxic or hypoxic conditions, and, to clarify intracellular signaling transduction pathways, inhibitors of protein kinases, such as phospholipase C (PLC), protein kinase C (PKC), myosin light chain kinase (MLCK), calmodulin-dependent protein kinase, phosphatidylinositol 3-kinase (PI3K), and Abelson (Abl) tyrosine kinase, were used to evaluate for the responses of RAECs to NPY.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Throughout the experiments, all animals were handled in accordance with the guidelines for animal experimentation set by the Japanese Association for Laboratory Animal Science. The protocol used in the present study was approved by the Animal Care Committee of Aichi Medical University.

Isolation and culture of RAECs. RAECs were isolated from male Wistar rats (100–150 g) and cultured according to the methods of Suh et al. (30). Briefly, rats were anesthetized with ketamine (50 mg/kg im) followed by pentobarbital sodium (25 mg/kg ip), and their aortas were removed and placed in phosphate-buffered saline (PBS). The vessels were cleaned, opened longitudinally, cut into 0.2- to 0.3-cm sections, and placed with their intimal side down on Matrigel-coated plates in growth medium (GM). The GM contained 10% fetal bovine serum (FBS), 75 µg/ml of endothelial cell growth supplement (ECGS), 10 U/ml of heparin, and 100 U/ml of penicillin-streptomycin, in minimum essential medium (MEM). RAECs were incubated in 20% O2 + 75% N2 + 5% CO2, at a temperature of 37°C, and GM was added to prevent drying out every 2nd day. After 4–7 days, the pieces were removed, and the cells were harvested. When assessed with Trypan blue, the viability of the primary cultured cells was >90%.

Identification of cultured RAECs. To identify the nature of cultured RAECs, we used an immunohistochemical technique to detect platelet endothelial cell adhesion molecule-1 (CD31) and von Willebrand factor (vWF, factor VIII). After three to four passages, RAECs were transferred into an 8-well chamber slide (Lab-Tek, Nalge Nunc International, Naperville, IL) and cultured overnight at 37°C, in a 5% CO2 incubator. Cells were washed with Tris-buffered saline (Tris 50 mM, pH 7.5, and NaCl 150 mM), followed by a fixative solution comprising a 7:3 mixture of methanol-acetone for 20 min at –20°C. After being washed with Tris-buffered saline containing 0.25% Triton X-100, RAECs were blocked with 4% normal rabbit serum for 1 h at room temperature and incubated with mouse anti-rat CD31 antibody (1:50) or sheep anti-rat vWF antibody (1:300) at 4°C overnight. They were then incubated with either biotinylated rabbit anti-sheep IgG or biotinylated goat anti-mouse IgG followed by avidin and biotin-peroxidase complex. Thereafter, they were incubated for <5 min in a solution of diaminobenzidine tetrahydrochloride in the presence of hydrogen peroxide, to visualize binding.

RAEC proliferation assay. RAECs passaged three to four times were used for all experiments. The cells were transferred into a 96-well plate (3,000 cells/well) in an assay medium comprising 5% FBS, 10 U/ml of heparin, and 100 U/ml of penicillin-streptomycin in MEM, which had the same composition as GM, but with 5% FBS and without ECGS. To allow them to attach to the plates and adapt to the assay medium, RAECs were incubated in 20% O2 + 75% N2 + 5% CO2, at a temperature of 37°C, overnight.

Incubation of RAECs during hypoxia or normoxia. Ninety-six-well plates were then moved to three different CO2 incubators, which were kept at 37°C so that the RAECs could be incubated under three sets of conditions: 10% O2 + 85% N2 + 5% CO2 and 15% O2 + 80% N2 + 5% CO2 using two multigas incubators (APM-30D; Astec-Bio, Fukuoka, Japan), and 20% O2 + 75% N2 + 5% CO2 in a CO2 incubator (MCO-20AIC, Sanyo, Tokyo, Japan). Throughout the proliferation assays, RAECs were cultured in assay medium, which was renewed every 2nd day. One, two, or four days were allowed to elapse after the cells were treated with reagents.

Measurement of RAEC numbers. RAEC numbers were assessed using a colorimetric method for determining numbers of viable cells with a CellTiter AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI). Once the incubation had finished, the plates were taken out from the respective incubators, and the incubation medium was replaced with 100 µl of assay medium and 20 µl of CellTiter AQueous One Solution reagent. Then the plates were incubated under normoxic conditions (20% O2 + 75% N2 + 5% CO2) for 3 h. Finally, absorbance at 490 nm was measured with a microplate reader (Vmax, Maxline) (10). Standard curves of proliferation were obtained by measuring the absorbance at various known cell counts. Cell numbers counted visually were closely correlated to those obtained with the colorimetric methods with a correlation coefficient of 0.996 (n = 40). Proliferation rates of RAECs were calculated by dividing the final cell number by the number at the starting point. Concentration-dependent responses to NPY and the conditions most favorable for proliferation in terms of the oxygen concentration and incubation time were first examined at a range of 10–14 to 10–6 M, and at 10–9 and 10–8 M, respectively.

Estimation of NPY-receptor subtypes participating in RAEC proliferation. To estimate NPY-receptor subtypes involved in the NPY-induced proliferation of RAECs, effects of NPY-receptor-specific agonists were examined at a concentration of 10–8 M under hypoxic conditions: PYY was used as an NPY Y1-, Y2-, Y4-, and Y5-receptor agonist; NPY-(3–36) as an NPY Y2- and Y5-receptor agonist; and pancreatic polypeptide (1-17)-(Ala31, Aib32)-NPY-(18-36) (Ala-NPY) as an NPY Y5-receptor agonist. Furthermore, effects of NPY-receptor-specific antagonists at 10–7 M on 10–8 M NPY-induced proliferation were examined. The antagonists used were N-[(1S)-4-(aminoiminomethyl)amino]-1-({[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl) ethyl] amino}carbonyl)butyl-1-(2-{4-[6,11-dihydro-6-oxo-5H-dibenz(b,e)azepin-11-yl]-1-piperazinyl}-2-oxoethyl)-cyclopentaneacetamide (BIIE 0246) as an NPY Y2-receptor antagonist; N-[(trans-4-{[(4-amino-2-quinazolinyl)amino]methyl}cyclohexyl)methyl]-1-naphthalenesulfonamide hydrochloride (CGP 71683) as an NPY Y5-receptor antagonist; and NPY-(18-36) as an NPY Y3-receptor antagonist.

Evaluation of caspase-3 activity. Caspase-3 is a cysteine protease, which cleaves substrates with a DEVD (Asp-Glu-Val-Asp) motif at the terminal aspartate (i.e., cysteine aspartase) (5,26). Homogenates of RAECs were incubated with a peptide DEVD-7-amino-4-trifluoromethyl coumarin (AFC), and the amount of fluorochrome AFC released into the medium was measured, using a CPP32/caspase-3 fluorometric protease assay kit (MBL, Nagoya, Japan) (3).

After their incubation overnight under 20% O2, RAECs were incubated for a further 2 h in assay medium, with or without 5% FBS under 10% O2. Concurrently, a control culture was performed for 2 h at 37°C, under 20% O2. To measure caspase-3 activity, RAECs were transferred to 96-well plates (2 x 105 cells/well). The cells were lysed, a colorimetrically labeled DEVD-AFC substrate was administered at a final concentration of 50 µM, and the mixture was incubated at 37°C for a further 2 h. Fluorochrome AFC concentrations were then measured with an excitation wavelength of 405 nm and emission wavelength of 510 nm, utilizing a multiplate fluorescence spectrophotometer (Fluoroskan Ascent FL, Labsystems). Caspase-3 activity was calculated as a percentage of that obtained with the control culture.

Estimation of the effects of signal transduction inhibitors on NPY-induced proliferation of RAECs. Utilizing the proliferation rate obtained with different incubation times and oxygen levels to determine the best experimental conditions, we estimated the proliferative responses of RAECs cultured in assay medium containing 10–8 M NPY under hypoxic conditions (10% O2 + 85% N2 + 5% CO2) in 4 days, in the presence or absence of 10–9–10–5 M of each of the following reagents: Gi protein inhibitor, pertussis toxin; PLC-beta and PLC-{gamma} inhibitor, 1-(6-{[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U-73122); PKC inhibitor, bisindolylmaleimide I (GF-109203X); MLCK inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl (ML-9); CaM-dependent kinase II inhibitor, myristorylated autocamtide-2 related inhibitory peptide (myristorylated AIP); CaM-dependent protein kinase inhibitor 3-{2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl}-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e); PI3K inhibitor, wortmannin; Abl tyrosine kinase inhibitor, imatinib; nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME); and VEGF Flk-1 kinase inhibitor, (E)-3-(3,5-diisopropyl-4-hydroxyphenyl)-2-[(3-phenyl-n-propyl)amino-carbonyl]acrylonitrite (SU1498).

Materials. MEM, FBS, trypsin-EDTA, penicillin-streptomycin, and heparin were purchased from Gibco Life Technologies (Eggenstein, Germany); ECGS, heparin, and pertussis toxin, from Sigma (St. Louis, MO); anti-vWF antibody, from Cedarlane Laboratories (Ontario, Canada); anti-CD31 antibody, from Serotec (Oxford, UK); biotinylate rabbit anti-sheep IgG (H+L), from Zymed Laboratories (South San Francisco, CA); biotinylated goat anti-mouse IgG (H+L) and ABC reagent, from Vector Laboratories (Burlingame, CA); PBS(–), diaminobenzidine tetrahydrochloride, and normal rabbit serum, from Wako (Osaka, Japan); NPY, from Peptide Institute (Osaka, Japan); NPY-(18-36), PYY, NPY-(3-36), and Ala-NPY, from Bachem (Bubendort, Switzerland); U-73122, GF-109203X, ML-9, myristorylated AIP, wortmannin, and SU1498, from Calbiochem (Darmstadt, Germany); and L-NAME, from Dojin Chemical (Kumamoto, Japan). Imatinib and DY-9760e were kindly supplied by Novartis Pharmaceuticals and Daiichi Pharmaceutical (Tokyo, Japan), respectively. BIIE 0246 and CGP 71683 were purchased from Tocris Cookson (Ellisville, MO). ML-9 was dissolved in 50% EtOH; U-73122, SU1498, GF-109203X, wortmannin, BIIE 0246, and CGP 71683, in DMSO; and imatinib, in glycine and NaCl solution (Sorensen buffer solution; pH adjusted to 4.5 with 0.1 M HCl). All reagents were at first dissolved to make high-concentration stock solutions and then diluted to final concentrations with PBS just before use. A preliminary study showed that the diluted EtOH, DMSO, and sorensen buffer solution did not affect the proliferation of RAECs.

Statistical analysis. Differences between means of cell counts were examined for significance with ANOVA. Statistical significance was evaluated using Scheffé's method (28) at a level of 0.05, with values expressed as the means ± SE. And significances for differences in proliferation rates and caspase-3 activities were analyzed with nonparametric statistical methods (28), Kruskal-Wallis one-way analysis of variance compared with no drug/without antagonist, and Mann-Whitney U-test compared with normoxia/without NPY or FBS, at a level of 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of RAECs. RAECs passaged three to four times were identified with endothelial cell markers, anti-CD31 and anti-vWF antibodies. More than 98% of cells reacted positively to anti-CD31 antibody, or anti-vWF antibody (Fig. 1A), and almost the same ratio was obtained even 7–11 days after the incubation of frozen cells.


Figure 1
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  		Fig. 1. Responses of rat aortic endothelial cells (RAECs) to neuropeptide Y (NPY) under 10, 15, and 20% O2 and their relation to incubation time. A: RAECs passaged 3–4 times were stained with endothelial cell markers, anti-CD31 antibody, or anti-von Willebrand factor (vWF) antibodies. Cells that reacted positively for either CD31 or vWF were obtained at a rate of >98%. B: RAEC numbers were assessed on the 1st, 2nd, or 4th day, after being incubated under 10, 15, or 20% O2 in the absence or presence of 10–9 or 10–8 M NPY. C: ratios of caspase-3 protease activity obtained for RAECs cultured under 10% O2 to under 20% O2 were compared between with and without 5% fetal bovine serum (FBS). PYY, peptide YY. The number of experiments was 4. Statistical significance at a level of *0.05 and **0.01, respectively, compared with the values obtained after 4 days without NPY. ##Statistical significance at a level of 0.01, compared with the values obtained with 5% FBS.

 
Effects of NPY on RAEC proliferation under normoxic or hypoxic conditions. Without NPY, the cell counts of RAEC increased along with incubation time until the 4th day under either 20, 15, or 10% O2. There were no significant differences among the values obtained at the same time points under the three different conditions without drugs (Fig. 1B).

In the presence of 10–9 M or 10–8 M NPY, RAEC numbers obtained on the 1st day under either 20, 15, or 10% O2 were similar to those obtained without NPY. On the 2nd and 4th days, 10–9 M NPY significantly increased the RAEC counts to 9,286 ± 170 cells/well under 15% O2 (n = 4) (P < 0.05) and 9,718 ± 14 cells/well under 10% O2 (n = 4) (P < 0.05), compared with the values obtained without NPY, 8,588 ± 186 and 9,207 ± 207 cells/well, respectively. NPY at 10–8 M further increased the RAEC counts to 9,508 ± 217 cells/well under 15% O2 (n = 4) (P < 0.01) and 10,713 ± 257 cells/well under 10% O2 (n = 4) (P < 0.01).

Influences of NPY and PYY on caspase-3 protease activity. In the presence of 5% FBS under 10% O2, the caspase-3 activity relative to that obtained under 20% O2 was 176.8 ± 18.4% without drugs, and 146.8 ± 11.9 and 147.6 ± 7.7% with 10–8 M NPY and 10–8 M PYY (n = 4, each), respectively, and no statistically significant differences were obtained (Fig. 1C). In the absence of FBS, caspase-3 activity was 846.2 ± 34.6% without drugs (n = 4), and 811.9 ± 19.5 and 873.2 ± 23.8% with 10–8 M NPY and 10–8 M PYY (n = 4, each), respectively. The protease activity was significantly increased by eliminating FBS from the incubation medium (P < 0.01) and was not affected by either NPY or PYY. These data indicated that the NPY-induced increase in RAEC number might not be mediated via inhibition of the apoptotic pathway through caspase-3.

Effects of NPY or NPY-receptor agonists on proliferation rates of RAECs under normoxia or hypoxia. Concentration-response curves for NPY-induced RAEC proliferation (Fig. 2A) showed a bimodal increase in the proliferation rate, i.e., in the lower range of NPY concentrations (10–14–10–12 M) and in the higher range (10–9–10–6 M). In the middle range (10–10–10–9 M), the rates were almost identical to that obtained without NPY (no drug). Under 20% O2, the lower range concentrations of NPY increased the rate significantly (P < 0.01 or 0.001), whereas the higher range concentrations did not. Under 10% O2, 10–12 M NPY increased the rate significantly (P < 0.05), and higher range concentrations of NPY increased it even more (P < 0.05 or 0.01). In the higher range, the NPY-induced increases in proliferation rates obtained under 10% O2 were significantly greater than those under 20% O2 (P < 0.05 or 0.01).


Figure 2
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  		Fig. 2. Effects of NPY and NPY-receptor-specific agonists and antagonists on RAECs. Proliferation rates were calculated by dividing RAEC numbers obtained on the 4th day with those obtained at the starting point. Each value represents the mean and its SE. The number of experiments was 4. A: concentration-response relationships of NPY were obtained under either normoxia (20% O2) or hypoxia (10% O2). B: effects of NPY and NPY-receptor-specific agonists, PYY (Y1, Y2, Y4, and Y5 receptors), NPY-(3-36) (Y2 and Y5 receptors), Ala-NPY (Y5 receptor), and NPY-(18-36) (Y2 agonist) on proliferation rates of RAECs. C: effects of NPY-receptor antagonists, BIIE0246 (Y2), CGP71683 (Y5), a combination of BIIE0246 and CGP71683 (Y2 and Y5), and NPY-(18-36) on the NPY-induced proliferation rates of RAECs were examined under hypoxia. Concentrations of agonists and antagonists were 10–8 and 10–7 M, respectively. Horizontal dashed lines (C) indicate levels of proliferation obtained without any drugs (no drug). Statistical significance at a level of *0.05 and **0.01, respectively, compared with the values obtained without antagonists (–); #0.05, ##0.01, and ###0.001, respectively, compared with the values obtained with "no drug"; and $0.05 and $$0.01, respectively, compared with the values obtained under 20% O2.

 
The four NPY-receptor agonists (10–8 M) had no effect on the proliferation rate under 10% O2 compared with the culture without any agonists (no drug), as shown in Fig. 2B. Neither PYY (NPY Y1-, Y2-, Y4-, and Y5-receptor agonist), NPY-(3-36) (NPY Y2- and Y5-receptor agonist), Ala-NPY (NPY Y5-receptor agonist), nor NPY-(18-36) increased the proliferation rate, which is no different from the result obtained without any agonists (no drug).

Effects of NPY-receptor antagonists and pertussis toxin on NPY-induced proliferation of RAECs under hypoxia. The greatest increases in the rate of proliferation of RAECs induced by the administration of 10–8 M NPY were obtained after incubation for 4 days under 10% O2, and therefore the following studies with NPY-receptor antagonists and kinase inhibitors were carried out in such experimental conditions.

BIIE 0246, an NPY Y2-receptor antagonist, completely inhibited the NPY-induced increase in the rate of proliferation (P < 0.01), whereas CGP 71683, an NPY Y5-receptor antagonist, did not (Fig. 2C). Furthermore, a combination of BIIE 0246 and CGP 71683 also inhibited the NPY-induced proliferation (P < 0.01), suggesting participation of the NPY Y2-receptor in the NPY-induced proliferation of RAECs.

NPY-(18-36), an NPY Y3-receptor antagonist and Y2-receptor agonist, concentration dependently diminished the NPY-induced proliferation of RAECs (Fig. 2C). The highest concentration of NPY-(18-36), 10–7 M, completely inhibited the NPY-induced proliferation to the baseline level obtained without NPY (P < 0.01). Pertussis toxin, a Gi protein inhibitor, concentration dependently diminished the NPY-induced increase in the rate of proliferation of RAECs (P < 0.05 or 0.01) (Fig. 3A). The highest concentration of pertussis toxin, 10–7 g/ml, almost recovered the NPY-induced proliferation to the baseline level (P < 0.01). Administration of pertussis toxin alone did not affect the proliferation of RAECs.


Figure 3
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  		Fig. 3. Effects of pertussis toxin, U-73122, and GF-109203X on NPY-induced proliferation of RAECs under hypoxia. Proliferation rates were calculated by dividing RAEC numbers obtained on the 4th day with those obtained at the starting point. RAECs were incubated under 10% O2. Each value represents the mean and its SE, and the number of experiments was 4. RAECs were incubated under 10% O2, in the presence or absence of 10–8 M NPY. A: pertussis toxin was administered at concentrations from 0 to 100 ng/ml. U-73122 (B) and GF-109203X (C) were administered at concentrations from 0 M (–) to 10–5 M. Statistical significance at a level of *0.05 and **0.01, respectively, compared with the values obtained without inhibitors; statistical significance at a level of #0.05 and ##0.01, respectively, compared with the values obtained without NPY.

 
Effects of U-73122 and GF-109203X on NPY-induced proliferation of RAECs under hypoxia. U-73122, a PLC inhibitor, did not affect the rate of proliferation of RAEC without NPY after incubation for 4 days under 10% O2, whereas U-73122 significantly diminished the NPY-induced proliferation in a concentration-dependent manner (P < 0.05 or 0.01) (Fig. 3B).

GF-109203X, a PKC inhibitor, significantly restored the NPY-induced proliferation of RAECs back to the value obtained without NPY after incubation for 4 days under 10% O2 (P < 0.05 or 0.01) (Fig. 3C). Without NPY, the same concentrations of GF-109203X little affected the proliferation rate.

Effects of ML-9, myristorylated AIP, and DY9760e on NPY-induced proliferation of RAECs during hypoxia. ML-9, a MLCK inhibitor, concentration dependently increased the NPY-induced proliferation of RAECs after incubation for 4 days under 10% O2 (P < 0.05 or 0.01) (Fig. 4A). The higher concentrations of ML-9, above 10–8 M, increased the NPY-induced proliferation most (P < 0.01). Without NPY, the same concentrations of ML-9 little affected the rate of proliferation.


Figure 4
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  		Fig. 4. Effects of ML-9, myristorylated autocamtide-2-related inhibitory peptide (AIP), and DY9760e on NPY-induced proliferation of RAECs under hypoxia. Proliferation rates were calculated by dividing RAEC numbers obtained on the 4th day with those obtained at the starting point. RAECs were incubated under 10% O2, in the presence or absence of 10–8 M NPY. Each value represents the mean and its SE. The number of experiments was 4. ML-9 (A), myristorylated AIP (B), and DY9760e (C) were administered at concentrations from 0 M (–) to 10–5 M. Statistical significance at a level of *0.05, **0.01, and ***0.001, respectively, compared with the values obtained without inhibitors; statistical significance at a levels of #0.05 and ##0.01, respectively, compared with the values obtained without NPY.

 
Myristorylated AIP, a CaM kinase II inhibitor, significantly inhibited the NPY-induced proliferation of RAECs to the value obtained without NPY after incubation for 4 days under 10% O2, concentration dependently (P < 0.05, 0.01, or 0.001) (Fig. 4B). Without NPY, the same concentrations of myristorylated AIP little affected the rate of proliferation.

DY9760e, a CaM-dependent protein kinase inhibitor, little affected the NPY-induced proliferation of RAECs after incubation for 4 days under 10% O2 (Fig. 4C).

Effects of wortmannin on NPY-induced proliferation of RAECs under hypoxia. Wortmannin, a PI3K inhibitor, significantly diminished the NPY-induced proliferation of RAECs after incubation for 4 days under 10% O2, concentration dependently (P < 0.05 or 0.01) (Fig. 5A). The highest concentration of wortmannin (10–5 M) inhibited the NPY-induced proliferation to the baseline obtained without NPY (P < 0.01). The same concentrations of wortmannin alone did not affect the rate of proliferation.


Figure 5
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  		Fig. 5. Effects of wortmannin and imatinib on NPY-induced proliferation of RAECs under hypoxia. Proliferation rates were calculated by dividing RAEC numbers obtained on the 4th day with those obtained at the starting point. RAECs were incubated under 10% O2, in the presence or absence of 10–8 M NPY. Each value represents the mean and its SE. The number of experiments was 4. Wortmannin (A) and imatinib (B) were administered at concentrations from 0 M (–) to 10–5 M. Statistical significance at a level of *0.05, **0.01, and ***0.001, respectively, compared with the values obtained without inhibitors; statistical significances at a level of #0.05 and ##0.01, respectively, compared with the values obtained without NPY.

 
Effects of imatinib on NPY-induced proliferation of RAECs during hypoxia. Imatinib, an Abl tyrosine kinase inhibitor, at a low-concentration range, 10–9–10–7 M, concentration dependently increased the NPY-induced proliferation of RAECs (P < 0.05 or 0.01), whereas, at a higher concentration range, 10–6–10–5 M, imatinib diminished the rate of proliferation (P < 0.01 or 0.001) in either 10 or 20% O2 (Fig. 5B). Notably, the highest concentration of imatinib reduced the rate of proliferation to below the basal level.

Effects of L-NAME and SU1498 on NPY-induced proliferation of RAECs during hypoxia. L-NAME, a NOS inhibitor, or SU1498, a VEGF Flt-1 kinase inhibitor, little affected the NPY (10–8 M)-induced proliferation under hypoxia at a concentration range of 10–9–10–6 M, similar to the changes obtained without NPY.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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NPY exerted bimodal effects on RAEC proliferation, as reported for human umbilical vein endothelial cells (20). A lower concentration range increased the rate of proliferation under both normoxic and hypoxic conditions, whereas a higher range did so only under hypoxia. Since low oxygen levels around vascular endothelial cells strongly influenced the vascular permeability evoked by NPY (16, 22), the present study has been undertaken utilizing a higher concentration range of NPY. Furthermore, when the oxygen content in the cell incubator was first examined using 10, 15, and 20% O2, and 5% O2 (data not shown, little proliferation was observed) as well, the greatest rate of proliferation induced by NPY was obtained under 10% O2. Although higher concentration ranges of NPY promoted RAEC proliferation, especially in the hypoxic conditions, it was noteworthy that NPY elicited no inhibitory action against apoptosis through a caspase pathway, because NPY showed no influence on caspase-3 activity when RAECs were incubated under hypoxia, without FBS in the medium. Since other sympathetic neurotransmitters such as norepinephrine and ATP elicited no effect on the RAEC proliferation under 10 and 20% O2 (in a preliminary study), NPY may be a sympathetic nerve-derived mediator for vascular endothelial cell proliferation.

NPY-induced proliferation was inhibited by NPY-(18-36), an NPY Y3 antagonist and Y2 agonist, concentration dependently and completely at final concentrations, and, furthermore, three Y2-receptor agonists, PYY, NPY-(3-36), and NPY-(18-36), failed to increase RAEC numbers, suggesting that the NPY-induced increase in vascular endothelial cells may be mediated through the NPY Y3-receptor subtype, not the NPY Y2-receptor subtype. Nevertheless, BIIE 0246, an NPY Y2-receptor antagonist, and a combination of BIIE 0246 and CGP 71683, a Y5-receptor antagonist, completely blocked the NPY-induced proliferation of RAEC, whereas CGP 71683 alone did not. Thus it is conceivable that, since the NPY Y3 receptor has not been cloned yet, the NPY Y3 receptor may be composed of altered configurations or a dimer/trimer of the NPY Y2 receptor and other receptor subtypes (Fig. 6), being Y2-receptor antagonist sensitive and agonist insensitive. Additionally, (Leu31, Pro34)NPY, an NPY Y1-receptor agonist, caused no increase in the proliferation rate (data not shown). Also, pertussis toxin inhibited the NPY-induced increase in RAEC numbers, suggesting that the NPY Y3 receptor may be coupled with Gi protein in signal transduction in the same manner as the other NPY-receptor subtypes.


Figure 6
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  		Fig. 6. Schemes of intracellular signal transduction for endothelial cell proliferation and cell-layer permeability. Abl tyrosine kinase, Abelson tyrosine kinase; CaMKII, calmodulin kinase II; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; MLCK, myosin light chain kinase; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C. The signal transduction pathways for endothelial cell migration and cell layer permeability have been referred to Movafagh et al. (20) and Nan et al. (22), respectively.

 
Lee et al. (18) reported angiogenesis in the skeletal muscle vasculature under ischemic conditions, which was mediated by NPY Y2 and Y5 receptors. The NPY-receptor subtypes participating in this angiogenesis differed from that for endothelial cell proliferation obtained in the present study, although the reasons for the difference are still unclear. They showed that the angiogenesis is mediated by VEGF and at most nitric oxide, whereas the present study showed no clear involvement of such mediators in the NPY-induced RAEC proliferation, since SU1498 and L-NAME only in part reduced the proliferation rate at high concentrations. Differences in conditions around the endothelial cells between studies in vivo and in vitro may imply that smooth muscles and some other cells participate in releasing VEGF and nitric oxide under ischemia, with enhanced sympathetic nerve activities, resulting in angiogenesis.

NPY-induced proliferation of RAECs may still be ascribed to its inhibitory action on apoptosis via the activation of PI3K-Akt signaling pathways, since wortmannin, a PI3K inhibitor, inhibited the increase in the cell count concentration dependently, just like the angiogenetic effects of sonic hedgehog protein through G protein-coupled receptors (8). Although the caspase-3 activity induced by incubating cells under hypoxia without FBS was not influenced by NPY, possible pathways for apoptosis other than the caspase cascades, e.g., Bad, GSK3, etc., could not be excluded in the NPY-induced RAEC proliferation. Nevertheless, it is probable that, similar to the effects of NPY on the permeability of RAEC monolayers (16), the PI3K signaling pathway is involved in the NPY-induced proliferation of RAECs.

To examine the signal transduction pathways involved in the NPY-induced increase in RAECs, furthermore, several protein kinase inhibitors were utilized, before the administration of NPY. U-73122 and GF-109203X, PLC and PKC inhibitors, respectively, diminished the NPY-induced increase in RAEC numbers, in a concentration-dependent manner, suggesting that both PLC and PKC are activated through G protein-coupled NPY Y3 receptors (Fig. 6). Diacylglycerol and inositol triphosphate, PLC products, respectively activate PKC and CaM-dependent protein kinases. It has been shown that NPY causes a transient increase in the intracellular Ca2+ concentration (22), through increases in inositol triphosphate. However, ML-9, a MLCK inhibitor, actually enhanced the NPY-induced increase in cells, suggesting that Ca2+-dependent MLCK may only modify/depress its primary pathway for the NPY's action indirectly, e.g., by altering cell shape, and so on. The diacylglycerol-PKC signaling pathway is involved in the proliferation of RAEC induced by NPY, downstream of PLC's activation.

Myristorylated AIP, a CaM-dependent protein kinase II inhibitor, inhibited the NPY-induced increase in the cell count over a wide range, 10–9–10–6 M, and completely at a high concentration, 10–5 M, whereas DY9760e, a CaM-dependent protein kinase inhibitor, caused no clear modification. These results suggest that DY9760e may block the activities of both MLCK and CaM kinase II, i.e., enhancement from MLCK inhibition and inhibition from CaM kinase II prevention, resulting in no clear changes. Previously, NPY's action on the permeability of cultured monolayers of RAECs (16) has been shown to be mediated by either contractile or tethering forces, the former mediated by intracellular Ca2+ concentrations. The NPY-induced permeability was mediated through both CaM-dependent protein kinases different from the endothelial cell proliferation, which was not mediated in such a Ca2+-dependent manner. Thus it is possible that PLC-diacylglycerol-PKC and PLC-Ca2+-CaM kinase II signaling pathways were common to both vascular permeability and endothelial cell proliferation induced by NPY. Furthermore, imatinib, an Abl tyrosine kinase inhibitor, did not inhibit the increase of cells, but rather enhanced it at a lower range, suggesting that Abl tyrosine kinase seems to be a specific pathway for the function of NPY in increasing vascular permeability, which was clearly blocked with imatinib at lower concentrations (half-maximal effective dose: 2 x 10–8 M). High concentrations of imatinib (>10–6 M), even without NPY, reduced the rate of proliferation markedly. Since imatinib also inhibits platelet-derived growth factor receptor, VEGF receptor, c-kit receptor, or other receptors competitively at IC50 values more than 10 times greater than that for Abl inhibition, the results showed nonspecific inhibitory actions.

Neither L-NAME nor SU1498 inhibited both RAEC proliferation and permeability (16, 22), suggesting that the two NPY-induced responses were not mediated via the release of VEGF and nitric oxide through the NPY Y3 receptor. Lee et al. (18) reported that the VEGF released by NPY induces aortic sprouts to form (capillary density), but not cell migration/proliferation (cell density). Furthermore, they demonstrated that, in endothelial NOS (eNOS–/–) mice, the NPY-induced formation of aortic sprouts was inhibited, suggesting an involvement of nitric oxide in the angiogenesis in ischemia, which was mediated via NPY Y2- and Y5-receptor subtypes. But the localization of these receptors and the cell types from which VEGF is released were not specified. Since VEGF may activate eNOS through PLC-PKC and PI3K-extracellular signal-regulated kinase signal transduction pathways in the endothelial cells (7, 15), nitric oxide may participate in the neovascularization in the response to VEGF. In such a context, the NPY Y3 receptor may not participate in releasing VEGF or activating eNOS, although the PLC-PKC and PI3K signal transduction pathways were involved in NPY-induced RAEC proliferation.

Spyridopoulos et al. (29) reported that the angiogenesis and vascular permeability induced by VEGF are mediated by mechanisms that ultimately diverge. They demonstrated that the activation of PKC is a major signaling pathway required for VEGF-induced proliferation and angiogenesis, whereas vascular permeability was enhanced by blocking PKC. Surprisingly, according to them, inhibition of PKC with a specific PKC inhibitor induced vascular permeability in vivo via a nitric oxide-dependent mechanism through the induction of NOS activity in endothelial cells.

In conclusion, the present study provided strong evidence of a role for NPY, a sympathetic neurotransmitter, in vascular endothelial cell proliferation, but not inhibitory action against the apoptosis induced by hypoxia and elimination of FBS. However, it should be noted that such an action of NPY specifically appeared when oxygen levels in the culture were low, i.e., hypoxic conditions. The NPY Y3 receptor coupled with G protein participated in the endothelial cell proliferation, the same as the RAEC monolayer's permeability observed under hypoxic conditions. Taken together, although the NPY Y3 receptor has not been cloned yet, it is likely that the physiological roles of NPY via the NPY Y3 receptor are greatly associated with hypoxia, which may be relevant to pathophysiological analyses in ischemia-hypoxia. The intracellular signaling pathways involved in NPY's action differed between permeability and proliferation, reflecting the results obtained with MLCK and Abl tyrosine kinase inhibitors, whereas PKC, CaM kinase II, and PI3K were common. These common pathways indicate not only the cytoplasmic mechanism for vascular permeability, but also the transcriptional mechanism in the nucleus for vascular endothelial cell proliferation, in response to NPY. Therefore, the authors conclude that the vascular endothelial cell proliferation in response to NPY is associated with an increase in vascular permeability, especially under hypoxic-ischemic conditions, probably resulting in angiogenesis, although the interactions between NPY and angiogenetical factors, such as hypoxia-inducible factors (23) and matrix metalloproteinases (2) released or induced by hypoxia, are still obscure.


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This study was supported, in part, by Ministry of Education, Science, Sports, Culture, and Technology, Japan, Grants 16591532, 16390448, and 17791294.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. Ishikawa, Dept. of Pharmacology, Aichi Medical Univ. School of Medicine, Nagakute, Aichi 480-1195, Japan (e-mail: nao{at}aichi-med-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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