− knockout mice fed normal diet

Atherosclerosis 243 (2015) 268e277

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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis

Chronic administration of methamphetamine promotes atherosclerosis formation in ApoE
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knockout mice fed normal diet Bo Gao, Lun Li, Pengfei Zhu, Mingjing Zhang, Lingbo Hou, Yufei Sun, Xiaoyan Liu, Xiaohong Peng**, Ye Gu* Department of Cardiology, Heart Center at Puai Hospital, Puai Hospital, Huazhong University of Science and Technology, Wuhan 430030, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 March 2015 Received in revised form 22 August 2015 Accepted 2 September 2015 Available online 8 September 2015

Objective: Chronic methamphetamine (METH) abuse could induce neurotoxicity due to reactive oxygen species generation and sympathetic activation. Both factors are associated with atherosclerosis, so we tested the hypothesis that chronic METH administration might also promote atherosclerosis formation in Apo E
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knockout mice fed normal diet. Methods and Results: Male ApoE
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mice (6 weeks-old) were treated with saline (NS) or METH [4 mg/ kg/day (M4) or 8 mg/kg/day (M8) through intraperitoneal injection] for 24 weeks. Atherosclerotic lesion area on oil red O stained en face aorta was dose-dependently increased in M4 and M8 groups compared to NS group. Percentage of atherosclerotic lesion area was significantly higher in M8 group compared to NS and M4 groups. Plasma CRP was increased and inflammatory cytokine (ICAM-1, VCAM-1, TNF-a, and INF-g) expression on aortic root was upregulated in METH groups compared to NS group. Neuropeptide Y (NPY) protein and mRNA expressions in aortic root and myocardial tissue were determined by Western blot and real time PCR, which were significantly upregulated in M4 and M8 groups. Moreover, mRNA expressions of NPY1R, NPY2R and NPY5R in aortic and myocardial tissue were also significantly upregulated in M4 and M8 groups. Raw264.7 cells were treated with NPY, NPY receptor antagonists, METH (10 mM or 100 mM) with or without lipopolysaccharide (LPS), and the expressions of TNF-a, CRP, MCP-1 and reactive oxygen species (ROS) production were significantly increased in METH and LPS þ METH groups compared to control and LPS groups. Co-treatment with NPY1R antagonist decreased the expressions of TNF-a, CRP and MCP-1 in NPY and METH treated cells. Conclusions: Chronic METH administration can promote inflammation and atherosclerotic plague formation in ApoE
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mice fed normal chow. NPY might be involved in the pathogenesis of METH-induced atherogenic effects through NPY Y1 receptor pathway. © 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Atherosclerosis Methamphetamine NPY NPYR

1. Introduction Abbreviations: NPY, neuropeptide Y; ApoE, Apolipoprotein E; a-SMA, a-smooth muscle actin; NE, norepinephrine; NPYR1, neuropeptide Y receptor 1; NPYR2, neuropeptide Y receptor 2; NPYR5, neuropeptide Y receptor 5; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule 1; TNF-a, tumor necrosis factor alpha; MCP-1, monocyte chemoattractant protein-1; IFN-g, interferon gamma; HDL, high density lipoprotein; LDL, low density lipoprotein; CRP, C reaction protein; ELISA, enzyme-linked immunosorbent assay; RT-PCR, real-time polymerase chain reaction; LPS, lipopolysaccharide; ROS, reactive oxygen species. * Corresponding author. Department of Cardiology, HeartCenter at PuaiHospital, HuazhongUniversity of Science and Technology, HanZheng Street 473#, QiaoKou District, Wuhan, 430033, China. ** Corresponding author. Department of Cardiology, HeartCenter at PuaiHospital, HuazhongUniversity of Science and Technology, HanZheng Street 473#, QiaoKou District, Wuhan, 430033, China. E-mail addresses: [email protected] (X. Peng), [email protected] (Y. Gu). http://dx.doi.org/10.1016/j.atherosclerosis.2015.09.001 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.

Methamphetamine (METH) abuse is a societal problem and could result in chronic relapsing disorders, joined by a wide range of neuropsychological deficits [1] and other neurotoxic effects [2,3]. It is known that the reactive nitrogen species, peroxynitrite, plays a major role in METH-induced dopaminergic neurotoxicity and selective antioxidants and peroxynitrite decomposition catalysts could protect against METH-induced neurotoxicity [4,5]. METH is chemically similar to dopamine and norepinephrine and readily crosses the blood/brain barrier, it produces its effects by causing dopamine and norepinephrine to be released into the synapse in several areas of the brain. Additionally, METH blocks the breakdown of excess dopamine and norepinephrine and could finally result in neurotoxicity through numerous interdependent mechanisms including excitotoxicity,

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mitochondrial damage and oxidative stress [6]. Besides the known neurotoxic effects post METH abuse, METHinduced effects on the cardiovascular system are drawing the attention of researchers nowadays. In a large cohort of METH users, Hawley et al. reported that myocardial ischemia evidence were found in 82 cases, accounting for 6.5% cases reviewed, suggesting that METH increases cardiovascular complications and there might be a potential link between METH use and acute coronary syndromes [7]. METH-induced oxidative stress and excess norepinephrine release are known risk factors for atherosclerosis [8,9]. Moreover, animal study showed that increased central superoxide levels could further enhance sympathoexcitation [10e12] while chronic subcutaneously norepinephrine treatment could also increase oxidative stress in circulating leukocytes [13]. Deo et al. demonstrated that norepinephrine increased reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived superoxide in human peripheral blood mononuclear cells via aadrenergic receptors [14]. These findings suggest a possible role of METH on the pathogenesis of atherosclerosis. In fact, in a retrospective review on the cardiovascular outcomes of METH exposures (n ¼ 2356) reported to both the California Poison Control System and two Level 1 trauma centers, 584 cases were coded as minor, 450 as moderate, 208 as major cardiovascular effects, and 28 as cardiovascular deaths [7] and Kaye et al. showed that coronary artery atherosclerosis was detected in 54% of decedents due to METH abuse [15]. Thus, there is a possible clinical association between METH exposures and coronary artery atherosclerosis and acute coronary syndrome. To date, the potential impacts of METH use on atherosclerosis and related mechanisms remain largely unknown. We therefore observed the chronic effects of METH on atherosclerosis in male ApoE
/
mice fed a normal chow diet.

company, Shanghai, and high density lipoprotein cholesterol (HDLC) and low-density lipoprotein (LDL-C) were measured by using commercial kits from SEKISUI company, Japan] and the rest two portions were stored at
80  C for ELISA analysis of NPY and Creactive protein (CRP). Mice were then humanely euthanized by deep anesthesia [intraperitoneal pentobarbital anesthesia (80 mg/ kg)] followed by cervical dislocation and then organ removal. The whole aorta or 6-mm-thick frozen sections of the aortic sinus were stained with Oil Red O [16e18]. Digital microphotographs of the aortic sinus were analyzed for lesion size in specific regions by measuring the stained surface area using Image J software (National Institutes of Health, Bethesda, MD). Atherosclerotic lesion percentage of luminal cross sectional area (LCSA) was evaluated.

2. Materials and methods

2.5. Real-time polymerase chain reaction measurements for mRNA expression of inflammatory cytokines in aorta and heart

2.3. Plasma NPY and CRP detection by ELISA Plasma NPY level was measured with the NPY enzyme immunoassay kit (RayBiotech, USA) following the manufacturer's instructions. The absorbance was recorded at 450 nm. Plasma CRP was measured with the ELISAs kit (Elabscience Biotechnology Co., Wuhan, Hubei Province, China) following manufacturer's instructions. 2.4. Immunohistochemistry Immunohistochemistry staining was made on frozen sections of aortic root. Macrophage (F4/80), a-smooth muscle actin(a-SMA) and NPY were identified with respective monoclonal antibodies (F4/80 1:600, a-SMA 1:200, NPY 1:3000). Anti-F4/80 (MCA497GA) was obtained from Bio-Rad (USA), anti-SMA (BM0002) from Boster (China), and anti-NPY (ab30914) from Abcam (U.K.). Macrophages, smooth muscle cells and NPY were evaluated by assessing the percent positive area of total plague for each marker.

2.1. Animals and study protocol Male ApoE
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mice on C57BL/6 background (6-week-old, Jackson Laboratory, Bar Harbor, ME, USA) were obtained from The Animal Center of Beijing University. Mice were fed with normal chow diet in a pathogen-free animal facility with 12 h light and dark cycles under conditions of controlled temperature (25  C). All experimental procedures were performed under the guidelines of the Care and Use of Laboratory Animals (Science and Technology Department of Hubei Province, China, 2005). Study protocol was approved by the Tongji Medical College Council on the Animal Care Committee of Huazhong University of Science and Technology (Wuhan, China). Mice were divided into three groups (saline control, NS, n ¼ 10, 4 mg/kg METH, M4, n ¼ 11 and 8 mg/kg METH, M8, n ¼ 11) and treated for 24 weeks. METH was dissolved in saline and the drug concentrations were 0.4 mg/ml and 0.8 mg/ml, respectively. Mice in control group received daily intraperitoneal saline injection, and mice in the two METH groups were treated with intraperitoneal 4 mg/kg/day or 8 mg/kg/day METH injection, respectively. The injection time was between 9 and 10 AM. 2.2. Weight monitoring, blood and tissue collection, atherosclerotic plague assessment Body weight was recorded at baseline and every two weeks thereafter. After 24 weeks treatments, blood was collected under intraperitoneal pentobarbital anesthesia (40 mg/kg) via abdominal vena cava.Plasma obtained was divided into 3 portions, one used to detect the lipid variables [plasma cholesterol (TC) and triglyceride (TG) were measured by using commercial kits from DIASYS

Total RNA was extracted from abdominal aorta connected with common iliac artery and left ventricular free wall using Total RNA kit (Takara, Japan) according to the manufacturer's instructions. Reverse transcription and cDNA synthesis were accomplished using RNA PCR kit (Takara, Japan). Real-time polymerase chain reaction was performed to detect the expression of various cytokines by Step One SYBR Green Mix Kit (Takara, Japan) and ABI Prism Sequence Detection System (Applied Biosystems, USA) according to the manufacturer's instructions. The conditions of amplification reaction were 95  C for 30 s, 95  C for 5 s, 60  C for 30sec, and PCR was done for 40 cycles. PCR primers are shown in Table 1. Relative gene expression was calculated using the 2
DDCT method [19]. 2.6. Western blot Total proteins were extracted from heart and abdominal aorta connected with common iliac artery. Protein concentrations were determined through bicinchoninic acid (BCA) method. After electrophoresis (SDS-PAGE), transmembrane, blocking (milk protein), incubation with primary anti-NPY monoclonal antibody ab30914 (Abcam, England) and secondary antibody (Goat-anti-rabbit 0741506, KPL, USA), and coloration, immunoreactive bands were obtained. Then the images were captured and semi-quantitatively analyzed by Quantity One (Bio-Rad, USA). 2.7. In vitro pro-inflammatory effects of METH on cultured macrophages The inflammatory profile was tested in macrophages post METH

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Table 1 RT-PCR Forward/Reverse (F/R) primers sequences. Forward/Reverse

Sequence (50 -30 )

NPY

F: 50 -AATGAAGGAAAGCACAGAAAACG-30 R: 50 -ATGAGATGAGATGAGGGTGGAAA-30 F: 50 -ATGTGTCTCCCGTTCACTTTTGT-30 R: 50 -CTATTGTTTGGTCTCCACCCTCT-30 F: 50 -CATATCTTTCTCCTACACCCGTATC-30 R: 50 -TTTGGTCATTTTGTGCCTTCG-30 F: 50 -GCTAAGCAGCAAGTATTTGTGTGTT-30 R: 50 -TCGAGTCTGTTTTCTTTGTGGGA-30 F: 50 -CCATAAAACTCAAGGGACAAGCC-30 R: 50 -TACCATTCTGTTCAAAAGCAGCA-30 F: 50 -ACTTTCTATTTCACTCACACCAGCC-30 R: 50 -ATCTTCACAGGCATTTCAAGTCTCT-30 F: 50 -CCTACCTTCAGACCTTTCCAGAT-30 R: 50 -GGCCTTCCAAATAAATACATTCA-30 F: 50 -AGGTCCCTGTCATGCTTCTGG-30 R: 50 -TGGTGATCCTCTTGTAGCTCTCC-30 F: 50 -GTCTCTTCTTGGATATCTGGAGGA-30 R: 50 -ATTCAATGACGCTTATGTTGTTGC-30 F:50 -TGAGAGGGAAATCGTGCGTGAC-30 R:50 -GCTCGTTGCCAATAGTGATGACC-30

NPY1R NPY2R NPY5R ICAM-1 VCAM-1 TNF-a MCP-1 IFN-g

b-Actin

NPY, neuropeptide Y; NPY1R, neuropeptide Y receptor 1; NPY2R, neuropeptide Y receptor 2; NPY5R, neuropeptide Y receptor 5; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule 1; TNF-a, tumor necrosis factor alpha; MCP-1, monocyte chemoattractant protein-1; IFN-g, interferon gamma.

and LPS in the presence or absence of NPY1R and NPY2R antagonist. Briefly, mouse macrophage RAW264.7 cell line was purchased from the Shanghai Institute of Cell Biology (Shanghai, China). They were cultured in Roswell Park Memorial Institute (RPMI) 1640 media (Sigma Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Gibico, USA), 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37  C. The cells were seeded at a density of 2.5  105 cells/ml in a 24-well plate. When the cells reached sub-confluence, they were incubated with 10 mM or 100 mM METH with or without LPS (10 ng/ ml) (Sigma, Aldrich) at the times indicated in the figure legends. In the experiments with NPY and NPY receptor antagonists, the macrophages were pretreatment for 30 min with Y1-specific peptide antagonist BIBO3304 at a concentration of 1 mM or/and Y2specific peptide antagonist BIIE0246 at a concentration of 1 mM (R&D, USA), then added 0.1 mM NPY (R&D, USA), 100 mM METH with or without LPS (10 ng/ml) at the times indicated in the figure legends. 2.8. TNF- a and CRP detection in culture supernatant by ELISA TNF-a and CRP in culture supernatant were measured with commercial ELISA kits (Elabscience Biotechnology Co., Wuhan, Hubei Province, China) following manufacturer's instructions. All of the samples were measured in triplicate. 2.9. Real-time polymerase chain reaction measurements for mRNA expression of inflammatory cytokines in macrophages

cell-permeable fluorescent-probe 20 -70 -dichlorofluorescein-diacetate (DCFDA) (Sigma, Aldrich). Briefly, RAW264.7 cells were seeded at a density of 5  105 cells/ml in a 6-well plate. When the cells reached sub-confluence, they were incubated with 10 mM or 100 mM METH with or without LPS (10 ng/ml) (Sigma, Aldrich) at the times indicated in the figure legends. Cells were washed twice with phosphate buffered saline (PBS) and then incubated with DCFDA probe (20 nM) at 37  C for 30 min in the dark. Then the cells were washed twice with PBS and fixed with 4% Poly formaldehyde for 30 min. After washing twice with PBS, fluorescence staining was visualized using a fluorescence microscope (Olympus, IX71), and fluorescence intensity was analyzed with IPP software as the means of ratio of total intensity and area of visual field. All experiments were performed with biological triplicates and datum were representative of at least three independent experiments. 3. Statistical analysis Data were shown as mean ± SD. One-way ANOVA and Tukey's post hoc test was used to test the difference between the means of various groups and P < 0.05 was considered as statistically significant. 4. Results 4.1. Body weight and plasma lipids measurements As shown in Table 2, body weight of ApoE
/
mice was similar at week 6 and significantly higher in M4 (4 mg/kg/day treated mice for 24 weeks) and M8 (8 mg/kg/day treated mice for 24 weeks) groups at week 30 compared to week 6 and NS (normal control) group. Plasma lipoprotein levels were similar among the three groups. 4.2. METH promoted atherosclerosis formation Results from oil Red O stained en face aorta demonstrated that atherosclerotic lesion area was dose-dependently increased in M4 and M8 groups compared to NS group (9.17 ± 0.99% vs. 7.15 ± 1.14%, P ¼ 0.126; 12.66 ± 1.89% vs. 7.15 ± 1.14%, P < 0.01; Fig. 1A and B). Fig. 2A showed the oil Red O stained aortic root of various groups, aortic atherosclerotic lesion area tended to be larger in M8 group compared to NS group (376.3 ± 77.31  103 mm2 vs. 304.08 ± 22.12  103 mm2, P ¼ 0.071; Fig. 2B) and percentage of atherosclerotic luminal cross sectional area (LCSA) was significantly higher in M8 group compared to NS and M4 groups (31.18 ± 5.89%, P < 0.01; and 25.18 ± 1.66% vs. 22.27 ± 3.48%, P < 0.05; Fig. 2C). There was no significant correlation between body weight and lesion area (r ¼ 0.287, P ¼ 0.3; Fig. 2D). Table 2 Body weight and plasma lipids. Body weight (g)

Total RNA was extracted from cultured macrophages using Total RNA kit (Takara, Japan) according to the manufacturer's instructions. Reverse transcription, cDNA synthesis, and real-time polymerase chain reaction were manipulated as previously described [19]. 2.10. Measurement of intracellular ROS production Evaluation of intracellular ROS production was performed using

NS Week 6 Week 30 Plasma lipids Cholesterol (mg/dl) Triglycerides (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl)

M4

22.11 ± 1.19 26.14 ± 1.89 384.18 188.9 38.46 166.02

± ± ± ±

71.73 41.88 7.66 38.88

M8

22.59 ± 1.15 29.40 ± 1.33*y 427.04 201.8 48.25 162.8

± ± ± ±

115.33 57.6 13.8 66.32

23.12 ± 2.34 29.70 ± 2.46*y 449.67 218.06 46.38 203.66

± ± ± ±

62.03 61.37 6.0 76.07

HDL, high density lipoprotein; LDL, low density lipoprotein. *P < 0.05 vs. Week 6. y P < 0.05 vs. NS.

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4.4. Cytokine mRNA expression in abdominal aorta and plasma CRP, NPY measurements

Fig. 1. Chronic administration of METH was associated with increased atherosclerotic lesion area. Images of the oil Red O staining of surface lesion area in the whole aorta are showed in A. Dot plots graphs represent quantitative value of oil Red Oepositive staining area as a percentage of the atheroma area are showed in B. *P < 0.05, **P < 0.01.

4.3. Immunohistochemistry results of aortic root Smooth muscle cells and mono-macrophage cell in the arterial wall may play a crucial role in the process of atherosclerosis formation. a-SMA immunohistochemistry staining results on aortic root showed that a-SMA positive area was significantly higher in M8 group compared to NS group (25.46 ± 4.58% vs. 20.04 ± 3.42%, P < 0.05; Fig. 3A and D), F4/80 immunohistochemistry staining demonstrated significantly increased F4/80 positive area in aortic root in M4 and M8 groups compared to NS group (17.61 ± 2.39% vs. 9.81 ± 3.4%, P < 0.05; 21.78 ± 3.09% vs. 9.81 ± 3.4%, P < 0.05; Fig. 3B and E). Fig. 3C and F showed that NPY expression was also significantly higher in M8 group compared to NS group (24.23 ± 4.17% vs. 16.71 ± 4.91, P < 0.05; Fig. 3C and F).

Enhanced inflammatory responses are known to contribute to the pathogenesis of atherosclerosis. As shown in Fig. 4 and Table 3, aortic ICAM-1 expression increased dose-dependently post 24 weeks METH treatment (P < 0.05; Fig. 4A), a trend of dosedependent increase of VCAM-1 and TNF-alpha expression in aorta was also observed and which was significantly higher in M8 group than in NS group (P < 0.01; Fig. 4A and B). Upregulation of MCP-1 mRNA expression was more significant in M4 group than in M8 group compared to NS group (P < 0.01; Fig. 4C). Plasma CRP value was significantly higher in M4 and M8 groups (P < 0.01; Fig. 4D). Plasma NPY level was similar between M4 and M8 groups and tended to be higher than in NS group. 4.5. NPY (receptor) protein and mRNA expression in aortic and myocardial tissue Sympathetic activation might be one of the mechanisms responsible for METH induced atherosclerosis formation. We thus detected the protein and mRNA expression of NPY in aorta and heart by Western blot and real time PCR as well as mRNA expressions of NPY1R, NPY2R and NPY5R in aorta and heart tissues. NPY protein expression in aortic (Fig. 5A and B) and heart (Fig. 5C and D) as well as mRNA expressions of NPY, NPY1R, NPY2R and NPY5R in aorta (Fig. 6A) and heart (Fig. 6B) were significantly upregulated in M8 group compared to NS group. 4.6. METH induced inflammation expression of macrophages in vitro As shown in Fig. 7, the expression of TNF-a, CRP and MCP-1 of RAW264.7 macrophages were obviously higher in METH and LPS þ METH groups compared to control and LPS groups

Fig. 2. METH promoted atherosclerosis development in area of aortic sinus. Sections of the aortic sinus stained with oil Red O are shown in A. Quantification of atherosclerotic lesion area and percentage of atherosclerotic lesion area are showed in B and C. The correlation of body weight and lesion area is shown in D. *P < 0.05, **P < 0.01. Black scale bar represents 125 mm.

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Fig. 3. METH up-regulated a-SMA, F4/80 and NPY expression in aorta root. Immunohistochemistry staining for a-SMA, F4/80 and NPY are shown in A, B and C. Quantification of areas with positive staining in the three groups are shown in the dot plots graphs D, E and F. *P < 0.05, **P < 0.01. Black scale bar represents 100 mm in A and 3B, 50 mm in C.

(Fig. 7AeD). At the same time, the expression of TNF-a, CRP and MCP-1 of RAW264.7 macrophages was dose-dependently increased post METH stimulation. 4.7. METH enhanced ROS production of macrophages in vitro It is known that METH might induce oxidative stress. We measured ROS production in RAW264.7 macrophages post METH and LPS stimulation. Our result showed that ROS production of RAW264.7 macrophages in METH and LPS þ METH groups were significantly higher compared to control and LPS groups. Meanwhile, a dose-dependent ROS production increase was evidenced in the RAW264.7 cells treated with 10 mM or 100 mM METH (Fig. 8A and B). 4.8. Potential impact of NPY in METH induced inflammation expression on macrophages in vitro Fig. 9 showed that NPY alone cannot induce expression of TNF-a,

CRP and MCP-1 of RAW264.7 macrophages (data not shown). But added NPY together with METH could clearly increase the expression of TNF-a, CRP and MCP-1 of RAW264.7 macrophages, these effects could be significantly attenuated by NPY1R antagonist, but not by NPY2R antagonist (Fig. 9AeD). 5. Discussion The major finding of the present study is as follows: 1) Chronic METH treatment promoted atherosclerosis formation without affecting plasma lipid levels in APOE
/
mice fed normal chow. 2) Immumochemistry results evidenced increased smooth muscle cells, mono-macrophage cell and NPY expression in aortic root post chronic METH treatment. 3) Plasma CRP was increased and aortic expression of inflammatory cytokines including ICAM-1, VCAM, TNF-a and MCP-1 were upregulated post chronic METH treatment. 4) These changes were accompanied by slightly increased plasma NPY level and significantly upregulated aortic and myocardial NPY and NPY receptors expression in APOE
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mice fed with normal

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Fig. 4. METH was associated with increased pro-inflammatory cytokine mRNA expression in aorta and increased CRP expression in plasma. The mRNA expression of ICAM-1, VCAM1, TNF-a, IFN-g and MCP-1 were detected by RT-PCR (A to C). Plasma CRP, NPY expressions were measured by ELISA (D and E). *P < 0.05, **P < 0.01.

Table 3 Cytokine mRNA expression in aorta and plasma CRP, NPY measurements. NS ICAM-1 VCAM-1 TNF-a IFN-g CRP (pg/ml) NPY (ng/ml)

0.79 1.15 0.93 1.21 427.3 3.26

M4 ± ± ± ± ± ±

0.15 0.21 0.32 0.29 34.17 0.72

8.15 1.73 2.06 2.04 1123.137 5.91

might also be involved in METH induced pro-inflammation and atherogenic effects through NPY Y1 receptor pathway.

M8 ± ± ± ± ± ±

y

3.61 0.95 1.07yy 1.15 58.93yy 1.6

9.77 2.27 3.63 1.05 616.77 6.32

± ± ± ± ± ±

3.4y 0.08yy 0.15yyzz 0.12y 104.54yz 1.24

y

P < 0.05 vs. NS. yyP < 0.01 vs. NS. zP < 0.05 vs. M4. zzP < 0.01 vs. M4.

chow diet. 5) In vitro, METH increased inflammation expression and ROS production of macrophages, which could be partly attenuated by NPY Y1 receptor antagonist. Taken together, chronic METH administration enhances inflammatory responses and promotes atherosclerosis formation without affecting serum lipids level. NPY

6. METH, weight gain, oxidative stress, inflammation and atherosclerosis Our results showed that METH treatment is related to increased body weights in APOE
/
mice fed normal diet. Increased NPY and epinephrine post chronic METH exposure might be responsible for the weight gain. Previous studies showed that both NPY [20] and epinephrine [21] could enhance adipogenesis. However, Pearson correlation analysis showed that there is no correlation between plaque area and body weight in this study. Till now, METH induced inflammatory responses were largely explored in terms of neurotoxic effects of METH [22e24]. In line with previous findings [8,9], we showed that ROS production was does-dependently increased in METH treated RAW264.7 macrophages suggesting a potential

Fig. 5. METH promoted NPY protein expression in aorta and heart. The NPY protein expression in aorta (A and B) and in heart (C and D) is detected by western-blot. *P < 0.05, **P < 0.01.

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Fig. 6. METH promoted NPY and NPY receptors mRNA expression in aorta and heart. The mRNA expression of NPY and NPYR in aorta (A) and in heart (B) is detected by RT-PCR. *P < 0.05, **P < 0.01.

role of oxidative stress on METH-induced atherogenic effects. Inflammation is a well-known stimulating factor of atherosclerosis and acute coronary artery disease [25,26]. In fact, previous clinical studies clearly demonstrated that prolonged exposure to METH is related to the acute coronary syndrome events [27]and coronary atherosclerotic changes might directly responsible for METHrelated deaths [15]. In a pathologic study, Karch et al. also showed that coronary artery disease was significantly more common in METH users (19.1%) than in controls (5.3%, p ¼ 0.0004) [28]. In the present study, we showed that chronic exposure to METH could increase plasma CRP and upregulate pro-inflammatory cytokine expression in aortic root, and these changes were joined with enhanced aortic tissue expression of smooth muscle cells, mono-macrophage cells. In vitro, treatment of RAW264.7 macrophages with METH and/or LPS induced an increased expression of TNF-a, CRP and MCP-1. These results are in line with previous report by Liu et al. in that METH increased LPS-mediated expression of pro-inflammatory cytokines in human macrophages [29], thus, the proinflammatory effects of METH might serve as one important lipid-independent mechanism for METH-induced atherosclerosis in this model. It is to note that plasma CRP level and aortic MCP-1 expression increased more in the M4 group than in M8 group, the underlying reason remains unclear now. On the other hand, there were also reports suggesting the immunosuppresion role of METH exposure, Harm et al. showed that METH administration led to a decrease in abundance of natural killer (NK) cells and the

remaining NK cells possessed a phenotype suggesting reduced responsiveness. Dendritic cells (DCs) and Gr-1(high) monocytes/ macrophages were also decreased in abundance while Gr-1(low) monocytes/macrophages appear to show signs of perturbation. CD4 and CD8 T cell subsets were affected by methamphetamine, both showing a reduction in antigen-experienced subsets in a murine model that simulates the process of METH consumption in a typical addict [30]. Talloczy et al. also reported a direct immunosuppressive effect on dendritic cells and macrophages of METH at pharmacological concentrations [31]. Future studies are warranted to explore the exact role of METH under various circumferences. 7. Potential role of NPY activation on METH-induced atherosclerosis Atherosclerosis is a multifactorial, pathological process and a leading cause of ischemic vascular diseases. Previous studies suggested potential involvement of NPY in METH-induced neurotoxic effects [32e35]. In the present study, we observed if NPY might also be involved in the pathogenesis of METH-induced atherogenic effects. As a sympathetic and central cotransmitter, NPY is known to mediate pleiotropic activities by activating multiple Gi/o-coupled Y1eY6 receptors. Besides the vasoconstriction via the Y1 receptor, NPY also mediates vascular smooth muscle cell growth, leading to neointima formation via both Y1 and Y1 receptors. Y1 receptors

Fig. 7. Treatment of RAW264.7 macrophages with METH and/or LPS induced an increased expression of TNF-a, CRP and MCP-1. Cells were treated with METH at various concentrations for 12 h, with 10 ng/ml LPS for 12 h and with both METH and LPS for 12 h, after which they were harvested. mRNA expression of TNF-a, CRP and MCP-1 is evaluated with RT-PCR (A and C). TNF-a, CRP and MCP-1 expression in culture supernatant is measured by ELISA (B and D). *P < 0.05; **P < 0.01.

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Fig. 8. Treatment of RAW264.7 macrophages with METH and/or LPS enhanced ROS production. Cells were treated with METH at different concentrations for 12 h or 10 ng/ml LPS at 12 h alone or in combination. Fluorescence microscopic pictures of ROS production are shown in A. The intensity of ROS production was analyzed by IPP software (B). *P < 0.05; **P < 0.01. Black scale bar represents 50 mm.

also mediate NPY's potent vascular growth-promoting activity leading in vivo neointima formation in rodents. This and the association of a polymorphism of the NPY signal peptide with carotid artery thickening in humans strongly suggest a crucial role of NPY activation in atherosclerosis [36,37]. Li et al. demonstrated that angioplasty injury significantly upregulated platelet and vascular NPY systems, which could then contribute to neointima formation via Y1 and Y5 receptor activation. In fact, increasing NPY to high stress levels triggered formation of a thrombotic atheroscleroticlike lesion and vessel occlusion in rat carotid artery, indicating NPY might be a risk factor for accelerated atherosclerosis [38]. In line with above hypothesis, we observed upregulated myocardial and aortic NPY and NPY receptors expression in APOE
/
mice fed with normal chow diet. In vitro, we found NPY participated in METH induced inflammation expression of macrophages through NPY Y1 receptor pathway. These results strongly suggest a potential role of NPY on METH-induced atherogenic effects. However, present study did not supply evidence demonstrating the mechanistic link between the accelerated atherosclerosis and NPY post METH €€ €inen et al. exposure in APOE
/
mice. On the contrary, Ja askela reported that systemic treatment with neuropeptide Y receptor Y1antagonist enhanced atherosclerosis and stimulates IL-12 expression in ApoE deficient mice suggesting NPY could have athero-

protective effects [39]. Subsequent studies using double NPY and ApoE mice or ApoE
/
mice treated simultaneously with METH and a NPY receptor antagonist are needed to explore the causal relationship between NPY and atherogenic effects of METH. It is possible that other mechanisms besides the postulated NPY-related mechanism might also play crucial roles on METH-induced proinflammatory and atherogenic effects and future studies are warranted to explore the related mechanisms. 8. Study limitation Present study has several limitations: 1) Body weights were significantly increased post METH treatments, although there was no correlation between body weight and lesion area and blood lipid was similar among groups, it is still reasonable to speculate that the pro-atherogenic effects of METH is at least partly due to the metabolic disorders induced by METH (obesity and diabetes), parameters like insulin sensitivity and blood glucose were not measured in the present study, future studies are therefore warranted to explore the impact of METH on metabolic issues. 2) to observe the lipid-independent impact of METH on atherosclerosis, APOE
/
mice were not fed with high fat diet in the present study, although we observed increased atherosclerotic burden in METH

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Fig. 9. Expression of TNF-a, CRP and MCP-1 on RAW264.7 macrophages post METH, NPY and/or LPS in the presence or absence of NPY1R or NPY2R antagonist. Cells were pretreatment for 30 min with 1 mM BIBO3304 or/and 1 mM BIIE0246, then treated with 0.1 mM NPY and/or 100 mM METH, or 10 ng/ml LPS, 100 mM METH plus 10 ng/ml LPS for 12 h. mRNA expression of TNF-a, CRP and MCP-1 is detected by RT-PCR (A and C). TNF-a, CRP and MCP-1 expression in culture supernatant is measured by ELISA (B and D). *P < 0.05; **P < 0.01.

treated APOE
/
mice fed regular diet, however, the classic mice atherosclerotic model used widely by researchers is the APOE
/
mice model fed high fat diet [40,41], moreover, present study did not quantify the plaque vulnerability issues, plaque vulnerability, apoptosis and neovessels should therefore be examined by future studies in APOE
/
mice treated with chronic METH and fed with high fat diet. In conclusion, chronic METH exposure is associated with promoted atherogenic pathology and this effect is linked with increased inflammatory responses in APOE
/
mice fed with normal chow diet. NPY might also be involved in the pathogenesis of METH-induced atherogenic effects through NPY Y1 receptor pathway. Sources of funding None. Disclosures None. Conflict of interest None. Acknowledgment None. References [1] J.C. Scott, S.P. Woods, G.E. Matt, R.A. Meyer, R.K. Heaton, J.H. Atkinson, I. Grant, Neurocognitive effects of methamphetamine: a critical review and metaanalysis, Neuropsychol. Rev. 17 (2007) 275e297.

[2] A.K. Cho, W.P. Melega, R. Kuczenski, D.S. Segal, Relevance of pharmacokinetic parameters in animal models of methamphetamine abuse, Synapse 39 (2001) 161e166. [3] D.E. Chapman, G.R. Hanson, R.P. Kesner, K.A. Keefe, Long-term changes in basal ganglia function after a neurotoxic regimen of methamphetamine, J. Pharmacol. Exp. Ther. 296 (2001) 520e527. [4] S.Z. Imam, J. el-Yazal, G.D. Newport, Y. Itzhak, J.L. Cadet, W. Slikker Jr., S.F. Ali, Methamphetamine-induced dopaminergic neurotoxicity: role of peroxynitrite and neuroprotective role of antioxidants and peroxynitrite decomposition catalysts, Ann. N. Y. Acad. Sci. 939 (2001) 366e380. [5] E.L. Riddle, A.E. Fleckenstein, G.R. Hanson, Mechanisms of methamphetamineinduced dopaminergic neurotoxicity, AAPS J. 8 (2006) E413eE418. [6] L.E. Halpin, S.A. Collins, B.K. Yamamoto, Neurotoxicity of methamphetamine and 3,4-methylenedioxy methamphetamine, Life Sci. 97 (2014) 37e44. [7] L.A. Hawley, J.D. Auten, M.J. Matteucci, L. Decker, N. Hurst, W. Beer, R.F. Clark, Cardiac complications of adult methamphetamine exposures, J. Emerg. Med. 45 (2013) 821e827. [8] A. Rezvan, C.W. Ni, N. Alberts-Grill, H. Jo, Animal, in vitro, and ex vivo models of flow-dependent atherosclerosis: role of oxidative stress, Antioxid. Redox Signal. 15 (2011) 1433e1448. [9] H.J. Bauch, J. Grunwald, P. Vischer, U. Gerlach, W.H. Hauss, A possible role of catecholamines in atherogenesis and subsequent complications of atherosclerosis, Exp. Pathol. 31 (1987) 193e204. [10] V.M. Campese, S. Ye, H. Zhong, V. Yanamadala, Z. Ye, J. Chiu, Reactive oxygen species stimulate central and peripheral sympathetic nervous system activity, Am. J. Physiol. Heart Circ. Physiol. 287 (2004) H695eH703. [11] M.C. Zimmerman, R.L. Davisson, Redox signaling in central neural regulation of cardiovascular function, Prog. Biophys. Mol. Biol. 84 (2004) 125e149. [12] M.C. Zimmerman, E. Lazartigues, J.A. Lang, P. Sinnayah, I.M. Ahmad, D.R. Spitz, R.L. Davisson, Superoxide mediates the actions of angiotensin ii in the central nervous system, Circ. Res. 91 (2002) 1038e1045. [13] E. Schraml, P. Quan, I. Stelzer, R. Fuchs, M. Skalicky, A. Viidik, K. Schauenstein, Norepinephrine treatment and aging lead to systemic and intracellular oxidative stress in rats, Exp. Gerontol. 42 (2007) 1072e1078. [14] S.H. Deo, N.T. Jenkins, J. Padilla, A.R. Parrish, P.J. Fadel, Norepinephrine increases nadph oxidase-derived superoxide in human peripheral blood mononuclear cells via alpha-adrenergic receptors, Am. J. Physiol. Regul. Integr. Comp. Physiol. 305 (2013) R1124eR1132. [15] S. Kaye, S. Darke, J. Duflou, R. McKetin, Methamphetamine-related fatalities in australia: demographics, circumstances, toxicology and major organ pathology, Addiction 103 (2008) 1353e1360. [16] N. Ishii, T. Matsumura, H. Kinoshita, H. Motoshima, K. Kojima, A. Tsutsumi, S. Kawasaki, M. Yano, T. Senokuchi, T. Asano, T. Nishikawa, E. Araki, Activation

B. Gao et al. / Atherosclerosis 243 (2015) 268e277

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25] [26] [27]

[28]

of amp-activated protein kinase suppresses oxidized low-density lipoproteininduced macrophage proliferation, J. Biol. Chem. 284 (2009) 34561e34569. T. Matsumura, H. Kinoshita, N. Ishii, K. Fukuda, H. Motoshima, T. Senokuchi, K. Taketa, S. Kawasaki, T. Nishimaki-Mogami, T. Kawada, T. Nishikawa, E. Araki, Telmisartan exerts antiatherosclerotic effects by activating peroxisome proliferator-activated receptor-gamma in macrophages, Arterioscler. Thromb. Vasc. Biol. 31 (2011) 1268e1275. K. Taketa, T. Matsumura, M. Yano, N. Ishii, T. Senokuchi, H. Motoshima, Y. Murata, S. Kim-Mitsuyama, T. Kawada, H. Itabe, M. Takeya, T. Nishikawa, K. Tsuruzoe, E. Araki, Oxidized low density lipoprotein activates peroxisome proliferator-activated receptor-alpha (pparalpha) and ppargamma through mapk-dependent cox-2 expression in macrophages, J. Biol. Chem. 283 (2008) 9852e9862. K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method, Methods 25 (2001) 402e408. L.E. Kuo, J.B. Kitlinska, J.U. Tilan, L. Li, S.B. Baker, M.D. Johnson, E.W. Lee, M.S. Burnett, S.T. Fricke, R. Kvetnansky, H. Herzog, Z. Zukowska, Neuropeptide y acts directly in the periphery on fat tissue and mediates stress-induced obesity and metabolic syndrome, Nat. Med. 13 (2007) 803e811. R. Han, J.B. Kitlinska, W.R. Munday, G.I. Gallicano, Z. Zukowska, Stress hormone epinephrine enhances adipogenesis in murine embryonic stem cells by up-regulating the neuropeptide y system, PloS One 7 (2012) e36609. A.K. Halladay, A. Kusnecov, L. Michna, T. Kita, C. Hara, G.C. Wagner, Relationship between methamphetamine-induced dopamine release, hyperthermia, self-injurious behaviour and long term dopamine depletion in balb/c and c57bl/6 mice, Pharmacol. Toxicol. 93 (2003) 33e41. A. Nakajima, K. Yamada, T. Nagai, T. Uchiyama, Y. Miyamoto, T. Mamiya, J. He, A. Nitta, M. Mizuno, M.H. Tran, A. Seto, M. Yoshimura, K. Kitaichi, T. Hasegawa, K. Saito, Y. Yamada, M. Seishima, K. Sekikawa, H.C. Kim, T. Nabeshima, Role of tumor necrosis factor-alpha in methamphetamine-induced drug dependence and neurotoxicity, J. Neurosci. 24 (2004) 2212e2225 the official journal of the Society for Neuroscience. J.M. Loftis, D. Choi, W. Hoffman, M.S. Huckans, Methamphetamine causes persistent immune dysregulation: a cross-species, translational report, Neurotox. Res. 20 (2011) 59e68. P. Libby, G.K. Hansson, Inflammation and immunity in diseases of the arterial tree: players and layers, Circ. Res. 116 (2015) 307e311. C.G. Santos-Gallego, B. Picatoste, J.J. Badimon, Pathophysiology of acute coronary syndrome, Curr. Atheroscler. Rep. 16 (2014) 401. S.D. Turnipseed, J.R. Richards, J.D. Kirk, D.B. Diercks, E.A. Amsterdam, Frequency of acute coronary syndrome in patients presenting to the emergency department with chest pain after methamphetamine use, J. Emerg. Med. 24 (2003) 369e373. S.B. Karch, B.G. Stephens, C.H. Ho, Methamphetamine-related deaths in san

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40] [41]

277

francisco: demographic, pathologic, and toxicologic profiles, J. forensic Sci. 44 (1999) 359e368. X. Liu, P.S. Silverstein, V. Singh, A. Shah, N. Qureshi, A. Kumar, Methamphetamine increases lps-mediated expression of il-8, tnf-alpha and il-1beta in human macrophages through common signaling pathways, PloS One 7 (2012) e33822. R. Harms, B. Morsey, C.W. Boyer, H.S. Fox, N. Sarvetnick, Methamphetamine administration targets multiple immune subsets and induces phenotypic alterations suggestive of immunosuppression, PloS One 7 (2012) e49897. Z. Talloczy, J. Martinez, D. Joset, Y. Ray, A. Gacser, S. Toussi, N. Mizushima, J.D. Nosanchuk, H. Goldstein, J. Loike, D. Sulzer, L. Santambrogio, Methamphetamine inhibits antigen processing, presentation, and phagocytosis, PLoS Pathog. 4 (2008) e28. K.A. Horner, S.C. Westwood, G.R. Hanson, K.A. Keefe, Multiple high doses of methamphetamine increase the number of preproneuropeptide y mrnaexpressing neurons in the striatum of rat via a dopamine d1 receptordependent mechanism, J. Pharmacol. Exp. Ther. 319 (2006) 414e421. F.H. Kobeissy, J.A. Jeung, M.W. Warren, J.E. Geier, M.S. Gold, Changes in leptin, ghrelin, growth hormone and neuropeptide-y after an acute model of mdma and methamphetamine exposure in rats, Addict. Biol. 13 (2008) 15e25. P.S. Frankel, M.E. Alburges, L. Bush, G.R. Hanson, S.J. Kish, Brain levels of neuropeptides in human chronic methamphetamine users, Neuropharmacology 53 (2007) 447e454. J. Goncalves, C.F. Ribeiro, J.O. Malva, A.P. Silva, Protective role of neuropeptide y y(2) receptors in cell death and microglial response following methamphetamine injury, Eur. J. Neurosci. 36 (2012) 3173e3183. J. Pons, E.W. Lee, L. Li, J. Kitlinska, Neuropeptide y: multiple receptors and multiple roles in cardiovascular diseases, Curr. Opin. Investig. Drugs 5 (2004) 957e962. Z. Zukowska, J. Pons, E.W. Lee, L. Li, Neuropeptide y: a new mediator linking sympathetic nerves, blood vessels and immune system? Can. J. Physiol. Pharmacol. 81 (2003) 89e94. L. Li, E.W. Lee, H. Ji, Z. Zukowska, Neuropeptide y-induced acceleration of postangioplasty occlusion of rat carotid artery, Arterioscler. Thromb. Vasc. Biol. 23 (2003) 1204e1210. A.E. Jaaskelainen, S. Seppala, T. Kakko, U. Jaakkola, J. Kallio, Systemic treatment with neuropeptide y receptor y1-antagonist enhances atherosclerosis and stimulates il-12 expression in apoe deficient mice, Neuropeptides 47 (2013) 67e73. n ~ ez, Modelos experimentales de C.G. Santos-Gallego, J.J. Badimon, B. Iba ~ ola Cardiol. Supl. 13 (2013) 3e12. aterosclerosis, Rev. Espan S. Zadelaar, R. Kleemann, L. Verschuren, J. de Vries-Van der Weij, J. van der Hoorn, H.M. Princen, T. Kooistra, Mouse models for atherosclerosis and pharmaceutical modifiers, Arterioscler. Thromb. Vasc. Biol. 27 (2007) 1706e1721.