β-Endorphin Mediates the Development and Instability of Atherosclerotic Plaques

β-Endorphin, an endogenous opioid peptide, and its μ-opioid receptor are expressed in brain, liver, and peripheral tissues. β-Endorphin induces endothelial dysfunction and is related to insulin resistance. We clarified the effects of β-endorphin on atherosclerosis. We assessed the effects of β-endorphin on the inflammatory response and monocyte adhesion in human umbilical vein endothelial cells (HUVECs), foam cell formation, and the inflammatory phenotype in THP-1 monocyte-derived macrophages, and migration and proliferation of human aortic smooth muscle cells (HASMCs) in vitro. We also assessed the effects of β-endorphin on aortic lesions in Apoe−/− mice in vivo. The μ-opioid receptor (OPRM1) was expressed in THP-1 monocytes, macrophages, HASMCs, HUVECs, and human aortic endothelial cells. β-Endorphin significantly increased THP-1 monocyte adhesion to HUVECs and induced upregulation of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin via nuclear factor-κB (NF-κB) and p38 phosphorylation in HUVECs. β-Endorphin significantly increased HUVEC proliferation and enhanced oxidized low-density lipoprotein-induced foam cell formation in macrophages. β-Endorphin also significantly shifted the macrophage phenotype to proinflammatory M1 rather than anti-inflammatory M2 via NF-κB phosphorylation during monocyte-macrophage differentiation and increased migration and apoptosis in association with c-jun-N-terminal kinase, p38, and NF-κB phosphorylation in HASMCs. Chronic β-endorphin infusion into Apoe−/− mice significantly aggravated the development of aortic atherosclerotic lesions, with an increase in vascular inflammation and the intraplaque macrophage/smooth muscle cell ratio, an index of plaque instability. Our study provides the first evidence that β-endorphin contributes to the acceleration of the progression and instability of atheromatous plaques. Thus, μ-opioid receptor antagonists may be useful for the prevention and treatment of atherosclerosis.


Introduction
Atherosclerosis is a chronic inflammatory response to injury of the arterial wall [1,2]. Vascular inflammation stimulates the expression of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, in endothelial cells (ECs). ese effectors encourage monocyte adhesion and infiltration into the neointima lesion, followed by atheroma formation and subendothelial accumulation of lipid-laden macrophage foam cells [1,3]. Foam cell formation is characterized by intracytoplasmic accumulation of cholesterol ester and depends on the balance among the uptake of oxidized low-density lipoprotein (oxLDL) via CD36, cholesterol esterification by acyl-CoA:cholesterol acyltransferase-1 (ACAT-1), and the efflux of free cholesterol controlled by the ATP-binding cassette transporter A1 (ABCA1) [2,3]. Categorization of the macrophage phenotype as either pro-or anti-inflammatory (M1 and M2 phenotypes, respectively) has recently focused on atherosclerosis [2,4]. In addition, the migration and proliferation of vascular smooth muscle cells (VSMCs) also contribute to the development of atheromatous plaques [1,2].
In the present study, we aimed at clarifying the effects of β-endorphin in vitro on the inflammatory response and adhesion of human THP-1 monocytes to human umbilical vein ECs (HUVECs). We also assessed the inflammatory phenotype and foam cell formation in THP-1 monocytederived macrophages, as well as migration and proliferation of human aortic smooth muscle cells (HASMCs). Our in vivo studies focused on the development of atherosclerotic lesions in Apoe −/− mice.

Quantitative
Real-Time RT-PCR. HUVECs at passage 3-7 were seeded in 3.5 cm dishes and incubated at 37°C in a 5% CO 2 incubator for 24 h in EGM-2. Near-confluent HUVECs were incubated at 37°C in 5% CO 2 for 4 h with or without the indicated concentration of β-endorphin in EGM-2 [34,35,38]. Total RNA and cDNA were obtained as described above [34,35,38]. qPCR was performed with Power SYBR® Green PCR Master Mix (Applied Biosystems) to quantify mRNA for ICAM1, VCAM1, SELE (E-selectin gene), and GAPDH. All reactions were carried out on a StepOnePlus system (Applied Biosystems). Each sample was analyzed in triplicate at least three times for each PCR measurement. Melting curves were checked to ensure specificity. e relative quantification of mRNA expression was calculated using the standard curve method with normalization to GAPDH.

Migration Assay (Scratch Assay).
HASMCs at passage 6-8 were seeded in 24-well plates (1 × 10 5 cells/500 μL/well). Cells were grown to near confluence at 37°C in 5% CO 2 in SmGM-2. HASMCs were then serum-starved overnight in serum-free SmGM-2. To induce a migrating zone in a transverse scratch wound, each monolayer of HASMCs was scratched with a sterilized Cell Scratcher (Iwaki, Tokyo, Japan). HASMCs were gently rinsed to discard debris, initial photomicrographs were taken (0 h), and then cells were incubated for 24 h in serum-free SmGM-2 containing the indicated concentration of β-endorphin or PDGF-BB [32]. After overnight incubation (24 h), a second set of images was obtained to measure HASMC migration over the scratched area [32]. e migrating zone was examined and analyzed between 0 h and 24 h using ImageJ software.

Animal Experiments.
Animal experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Tokyo University of Pharmacy and Life Sciences (no. L19-16). A total of 21 male spontaneously hyperlipidemic Apoe −/− mice (KOR/StmSlc-Apoe shl mice) were purchased from Japan SLC (Hamamatsu, Japan). Mice were fed a high cholesterol diet containing 1.25% cholesterol, 3.0% lard, and 1.625% glucose (F2HFD1, Oriental Yeast, Tokyo, Japan), starting at 13 weeks of age [2, 29-37, 39, 40]. At 13 weeks of age, five mice were sacrificed as preinfusion controls. e remaining 16 were divided into two groups of eight each and were infused with saline (vehicle) or β-endorphin (5 μg/kg/h) using osmotic minipumps (Alzet Model 1002; Durect, Cupertino, CA, USA) for 4 weeks. Doses of β-endorphin were selected based on others' previous data and our preliminary data [32][33][34][35]. Once every 2 weeks, the minipumps were implanted subcutaneously into the dorsum under medetomidine-midazolam-butorphanol anesthesia (0.3 mg/kg, 4.0 mg/kg, and 5.0 mg/kg, respectively, intraperitoneal injection).

Statistical Analyses.
Data are expressed as the means ± standard error of the mean. e data were compared using the unpaired Student's t-test between two groups. Comparison of several groups was performed with one-way analysis of variance followed by Bonferroni's post hoc test. A value of p < 0.05 was considered to be statistically significant.

Expression of μ-Opioid Receptor in Human Vascular Cells.
First, the gene expression of the μ-opioid receptor (OPRM1) was investigated in the human vascular cells used in this study. OPRM1 was expressed at high levels in THP-1 monocyte-derived macrophages and HAECs, and at low levels in THP-1 monocytes, HASMCs, and HUVECs (Figure 1(a)).
Exposure of HUVECs to 10 and 100 pM β-endorphin for 24 h resulted in 2.7-and 2.9-fold increases in THP-1 monocyte adhesion compared with the untreated control, respectively (Figure 1(j)).
β-Endorphin significantly increased the proliferation of HUVECs in a concentration-dependent manner, with a maximal increase of 11% with 100 pM (Figure 1(k)).

Effects of β-Endorphin on the Inflammatory Phenotype in
Human Macrophages. After 6 days of THP-1 monocyte culture, the differentiation of human monocytes into macrophages was confirmed by increased protein expression of CD68, a macrophage differentiation marker (Figure 2(e)). β-Endorphin (10 pM) did not affect the differentiation of monocytes into macrophages. However, β-endorphin significantly increased the protein expression of MARCO, an M1 marker, and significantly decreased that of arginase-1, an M2 marker (Figure 2(e)). Likewise, β-endorphin significantly increased NF-κB phosphorylation, but not PPAR-c protein expression (Figure 2(e)). ese observations indicate that β-endorphin shifted the macrophage phenotype to the M1 rather than M2 phenotype, which was associated with NF-κB phosphorylation during monocyte-macrophage differentiation.

Effects of β-Endorphin on Atherosclerotic Lesion Development in Apoe
In Apoe −/− mice, the aortic atherosclerotic lesion area and atheromatous plaque size accompanied by the intraplaque pentraxin-3-positive area and monocyte/macrophage and VSMC contents as well as plasma total cholesterol concentration were significantly increased at 17 weeks of age compared with 13 weeks of age by 8.8-fold, 7.2-fold, 15.2-fold, 7.9-fold, and 8.1-fold, respectively (Figure 4(a) (A, B, D, G, H, J, K, M, and N) and 4(b)-4(f ) and Table 1). Infusion of β-endorphin (5 μg/ kg/h) significantly enhanced the aortic atherosclerotic lesion area and atheromatous plaque size by 1.9-fold and 1.5-fold, respectively (Figure 4(a) (B, C, E, and F) and 4(b) and 4(c)), with significant increases in the pentraxin-3positive area and monocyte/macrophage infiltration by 1.7-fold and 2.1-fold, respectively, and a significant decrease in the VSMC content (Figure 4(a) (H, I, K, L, N, and O) and 4(d)-4(f )). In addition, the ratio of monocyte/ macrophage contents : VSMC contents within atheromatous plaques was significantly increased by β-endorphin by 2.1-fold (Figure 4(g)). We found no significant differences in body weight; food intake; systolic and diastolic blood pressures; plasma levels of total cholesterol, International Journal of Endocrinology triglycerides, free fatty acids, glucose, and insulin; or HOMA-IR between the two groups of 17-week-old Apoe −/− mice (Table 1).

Discussion
e present study provides the first evidence that β-endorphin stimulates atherosclerosis by increasing the inflammation in ECs and macrophages, mediating oxLDLinduced foam cell formation in association with CD36 and ACAT-1 upregulation, and inducing migration and apoptosis in VSMCs. Moreover, β-endorphin accelerates vascular inflammation and the development and instability of atherosclerotic plaques in Apoe −/− mice.
We discuss the reason why β-endorphin increases HUVEC proliferation but decreases HASMC proliferation. A previous study has shown that β-endorphin stimulates the migration and proliferation of HUVECs, leading to angiogenesis [41]. is report is consistent with our results. We also showed that β-endorphin increased the migration HASMCs via stimulation of JNK phosphorylation. However, in these cells, the peptide decreased proliferation and increased apoptosis via suppression of ERK1/2 and Akt phosphorylation and Bcl-2 expression, and via stimulation of p38, Bax, and caspase-3 expression. Parra et al. have reported that μ-opioid receptor agonists show biphasic action on vasocontraction, which is suppressed at low concentrations but stimulated at high concentrations [42]. In the present study, β-endorphin reduced the proliferation of VSMCs in vitro and in vivo and thus contributed to the instability of atheromatous plaques.  Several studies have shown that plasma β-endorphin levels increase with psychological stress and exercise stress in patients with CAD [43,44]. Plasma β-endorphin levels at onset are increased in patients with acute myocardial infarction with chest pain compared to those without chest pain [45]. In patients with symptomatic myocardial ischemia, plasma β-endorphin levels decrease after percutaneous transluminal coronary angioplasty [46]. Plasma β-endorphin levels are increased in patients with essential hypertension and heart failure [28,47]. Cozzolino et al. have shown that a short-term infusion of β-endorphin improves cardiac function and systemic vascular resistance in patients with heart failure [48]. ese findings suggest that β-endorphin may play a key role in cardioprotection against pain, Statistical comparisons of the atherosclerotic lesion area, atheromatous plaque size, pentraxin-3-positive area, monocyte/macrophage infiltration, and VSMC contents, and the ratio of monocyte-macrophage contents : VSMC contents within atheromatous plaques among three groups. Data are presented as the means ± standard error of the mean. * p < 0.0001, † p < 0.0005, ‡ p < 0.005, I p < 0.05. 8 International Journal of Endocrinology stress, and pressure overload. However, the present study reveals that long-term infusion of β-endorphin accelerates the progression and instability of atheromatous plaques. Further studies are needed to demonstrate the stimulatory effect of β-endorphin on plaque rupture in aged Apoe −/− mice. e physiological relevance of β-endorphin concentrations used in the present in vitro and in vivo experiments warrants further discussion. First, the concentrations of β-endorphin (10-100 pM) that were needed to influence multiple responses by HUVECs, THP-1 monocyte-derived macrophages, and HASMCs were almost equivalent to the average plasma concentration of β-endorphin in patients with CAD (10-75 pM) [27,49]. β-Endorphin affects HASMCs at a maximum concentration of 1000 pM, which is ∼100-fold higher than the plasma concentration. In the vascular wall, ECs and macrophages generate high amounts of β-endorphin in an autocrine/paracrine manner [15]. In a previous study, local levels of angiotensin II were increased by ∼100-fold [50]. erefore, the observation that local levels of β-endorphin were increased in the microenvironment of the cells that secrete the peptide to a similar degree as other vasoactive agents is not surprising. e present study has some limitations. Future experiments with overexpression of β-endorphin, knockout of the μ-opioid receptor, and administration of a selective μ-opioid receptor antagonist, naloxonazine dihydrochloride, in Apoe −/− mice may strengthen our observation of the stimulatory effects of β-endorphin on atherosclerosis. Likewise, experiments using β-endorphin with naloxonazine dihydrochloride may be required in all in vitro experiments in future studies.

Conclusions
e results of the present study indicated that β-endorphin accelerates atherosclerosis by enhancing inflammatory responses and monocyte adhesion in ECs, the inflammatory phenotype and foam cell formation in macrophages, and migration of VSMCs. In addition, β-endorphin may contribute to plaque instability by increasing vascular inflammation and the intraplaque macrophage : VSMC ratio. erefore, μ-opioid receptor antagonists may serve as novel potential therapeutic agents for atherosclerosis and related diseases.

Data Availability
All data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest with respect to this manuscript.