타이틀

Abstract

Aim

Atherosclerosis is the complex lesion that consists of endothelial inflammation, macrophage foam cell formation, vascular smooth muscle cell (VSMC) migration and proliferation, and extracellular matrix production. Human urocortin 1 (Ucn1), a 40-amino acid peptide member of the corticotrophin-releasing factor/urotensin I family, has potent cardiovascular protective effects. This peptide induces potent and long-lasting hypotension and coronary vasodilation. However, the relationship of Ucn1 with atherosclerosis remains unclear. The present study was performed to clarify the effects of Ucn1 on atherosclerosis.

Methods

We assessed the effects of Ucn1 on the inflammatory response and proliferation of human endothelial cells (ECs), human macrophage foam cell formation, migration and proliferation of human VSMCs, extracellular matrix expression in VSMCs, and the development of atherosclerosis in apolipoprotein E-deficient (Apoe−/−) mice.

Results

Ucn1 significantly suppressed cell proliferation without inducing apoptosis, and lipopolysaccharide-induced up-regulation of monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 in human ECs. Ucn1 significantly reduced oxidized low-density lipoprotein-induced foam cell formation with a significant down-regulation of CD36 and acyl-CoA:cholesterol acyltransferase 1 in human monocyte-derived macrophages. Ucn1 significantly suppressed the migration and proliferation of human VSMCs and increased the activities of matrix metalloproteinase-2 (MMP2) and MMP9 in human VSMCs. Intraperitoneal injection of Ucn1 into Apoe−/− mice for 4 weeks significantly retarded the development of aortic atherosclerotic lesions.

Conclusions

This study provided the first evidence that Ucn1 prevents the development of atherosclerosis by suppressing EC inflammatory response and proliferation, macrophage foam cell formation, and VSMC migration and proliferation. Thus, Ucn1 could serve as a novel therapeutic target for atherosclerotic cardiovascular diseases.

Figures

   

Citation: Hasegawa A, Sato K, Shirai R, Watanabe R, Yamamoto K, Watanabe K, et al. (2014) Vasoprotective Effects of Urocortin 1 against Atherosclerosis In Vitro and In Vivo. PLoS ONE 9(12): e110866. doi:10.1371/journal.pone.0110866

Editor: Gianfranco Pintus, University of Sassari, Italy

Received: June 4, 2014; Accepted: September 18, 2014; Published: December 2, 2014

Copyright: © 2014 Hasegawa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.

Funding: The authors received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Atherosclerosis is a chronic inflammatory response to the injury in the arterial wall [1]. Endothelial inflammation is characterized by increased production of pro-atherogenic molecules and inflammatory cytokines such as interleukin-6 (IL6), monocyte chemoattractant protein-1 (MCP1), intercellular adhesion molecule-1 (ICAM1), and E-selectin in endothelial cells (ECs), and monocyte adhesion and infiltration into the neointima lesion, followed by oxidized low-density lipoprotein (oxLDL)-induced transformation of macrophages into foam cells [2]. Accumulation of cholesterol ester (CE) in macrophages is a hallmark of foam cell formation [2]. This accumulation depends on the balance between the uptake of oxLDL via CD36 and the efflux of free cholesterol (FC) controlled by ATP-binding cassette transporter A1 (ABCA1) [2]. To protect the cells from the toxicity that would result from excessive FC accumulation, the FC is esterified to CE by acyl-CoA:cholesterol acyltransferase-1 (ACAT1) [2]. Apart from accumulation of macrophage foam cells, the migration and proliferation of vascular smooth muscle cells (VSMCs), EC proliferation, and the production of extracellular matrix (ECM) components, such as collagens, matrix metalloproteinases (MMPs), fibronectin, and elastin, contribute to the progression of atherosclerotic plaques [1], [3].

Urocortin 1 (Ucn1), a 40-amino-acid peptide related to the corticotrophin-releasing factor (CRF)/urotensin I family, was originally cloned from rat and thereafter the human brain [4]. In the cardiovascular system, Ucn1 and its receptors, CRF-R1 and CRF-R2, are expressed in cardiomyocytes, ECs, VSMCs, and macrophages [5]–[7]. Both animal and human studies have shown that Ucn1 is released when the heart is under stress, such as ischemia or heart failure[8], [9]. Secretion of Ucn1 is stimulated by reactive oxygen species (ROS), angiotensin II (AngII), lipopolysaccharide (LPS), and inflammatory cytokines, such as IL6, interferon-γ, and tumor necrosis factor-α (TNFα) [10], [11]. Thereby, Ucn1 exerts cardioprotective effects, such as causing coronary vasodilatation, positive inotropic effect, and an anti-apoptotic effect in the myocardium after ischemia-reperfusion injury [8], [12]. In clinical practice, plasma Ucn1 levels are elevated in patients with acute myocardial infarction or heart failure [13], [14]. A genomics array analysis highlighted Ucn1 as a favorable molecule for cardiovascular diseases [15]. However, the direct association between Ucn1 and atherogenesis has not yet been reported.

In the present study, we assessed the suppressive effects of Ucn1 on the inflammatory response and proliferation of human ECs, human macrophage foam cell formation, the migration, proliferation, and ECM production in human VSMCs in vitro, and the development of atherosclerotic lesions in apolipoprotein E-deficient (Apoe−/−) mice, an animal model of atherosclerosis, in vivo.

Materials and Methods

Human Cell Culture

This investigation was approved by the Ethics Committee of Tokyo University of Pharmacy and Life Sciences. Written informed consent was obtained from 15 healthy volunteers (7 men, 8 women; aged 19–22) who were free of hypertension, diabetes, dyslipidemia, and arteriosclerotic vascular diseases and were taking no medications. Human peripheral mononuclear cells were isolated from their blood. Monocytes purified using anti-CD14 antibody-conjugated magnetic microbeads (Miltenyi Biotec, Auburn, CA) were seeded onto 3.5-cm dishes (1×106 cells/1 ml/dish) for cholesterol esterification assay and immunoblotting analysis[16]–[19]. Cells were incubated at 37°C in 5% CO2 for 7 days in RPMI-1640 medium supplemented with 10% human serum, 0.05 mg/ml streptomycin, 50 U/ml penicillin, and the indicated concentrations of human Ucn1 (Abgent, San Diego, CA). The medium in each dish was replaced with fresh medium containing Ucn1 every 3 days.

Cholesterol Esterification Assay

Human macrophages differentiated by 7-day culture with the indicated concentrations of Ucn1 were incubated for 19 h with 50 µg/ml human oxLDL in the presence of 0.1 mmol/l [3H]oleate (PerkinElmer, Yokohama, Japan) conjugated with bovine serum albumin [16]. Cellular lipids were extracted and the radioactivity of cholesterol-[3H]oleate was determined by thin-layer chromatography.

Migration Assay

Human aortic smooth muscle cells (HASMCs; Lonza, Walkersville, MD) at passage 7 were seeded onto 3.5-cm dishes (5×104 cells/1 ml/dish). Cells were incubated at 37°C in 5% CO2 for 7 h in smooth muscle cell basal medium (SmBM; Lonza) supplemented with 0.5 ng/ml human epidermal growth factor, 5 µg/ml insulin, 2 ng/ml human fibroblast growth factor, 50 µg/ml gentamicin, 50 ng/ml amphotericin B, and 5% fetal bovine serum (FBS). Subsequently, while HASMCs were incubated in serum-free SmBM with or without the indicated concentrations of AngII (Sigma, St. Louis, MO) and/or Ucn1 for 5 h, photographs of cells were taken at 10-min intervals. The average migration distance of 10 cells randomly selected in each dish was measured using a BIOREVO BZ-9000 microscope (Keyence, Osaka, Japan) [16].

Proliferation Assay

HASMCs or human EA.hy926 ECs at passage 5–10 were seeded onto 96-well plates (1×104cells/100 µl/well) and incubated at 37°C in 5% CO2 for 24 h in SmBM supplemented with 5% FBS and above additives or Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% FBS, 4.5 mg/ml d-glucose, 0.584 mg/ml l-glutamine, 0.05 mg/ml streptomycin, and 50 U/ml penicillin G, respectively. Cells were incubated for 48 h with the indicated concentrations of Ucn1 with renewal of each medium. Subsequently, 10 µl of WST-8 solution (Cell Count Reagent SF; Nacalai Tesque, Kyoto, Japan) was added to each well [16]. After 1 h of incubation, the amount of formazan product was determined by measuring the absorbance at 450 nm using a Sunrise Remote R-micro plate reader (Tecan, Kawasaki, Japan).

Immunocytochemistry

HASMCs or human EA.hy926 ECs were seeded onto 12-well plates (1×105 cells/1 ml/well) and incubated at 37°C in 5% CO2 for 24 h in the same conditioning medium, followed by 48 h-incubation with the indicated concentrations of Ucn1. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and stained with rabbit polyclonal anti-Ki-67 antibody (Leica Biosystems, Newcastle upon Tyne, UK), followed by anti-rabbit Alexa Fluor 488 (Life Technologies, Carlsbad, CA). Terminal deoxynucleotidyl transferase- mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) staining was performed using an In Situ Apoptosis Detection Kit (Takara Bio, Otsu, Japan). Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining. All samples were mounted with Fluorescent Mounting Medium (Dako, Glostrup, Denmark). Fluorescence-stained cells were examined on confocal microscope (FV1000D, Olympus, Tokyo, Japan). Fluorescence was detected with wavelengths for excitation at 488 nm (Alexa Fluor 488) and 360 nm (DAPI).

Western Blotting

Aliquots of 20 µg of protein extracts from human macrophages and HASMCs were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblotting with the following antibodies: CD36 (R&D Systems, Minneapolis, MN), ACAT1 (Santa Cruz Biotechnology, Santa Cruz, CA), ABCA1, collagen-1 (Novus Biologicals, Littleton, CO), collagen-3, fibronectin, α-tubulin, MMP2 (GeneTex, Irvine, CA), MMP9 (EnoGene, Atlanta, GA), elastin (Bioss, Woburn, MA), or β-actin (Sigma) [16]–[19]. The densities of the bands were measured using a Densitograph System (Ez-Capture II and CS Analyzer 3.0, ATTO, Tokyo, Japan).

Zymography

The activities of MMP2 and MMP9 were determined using a gelatin-zymography kit (Cosmo Bio, Tokyo, Japan) [20]. The culture supernatants of HASMCs (15 µg) were mixed with 5 µl of sample buffer and fractionated by 10% SDS-PAGE using 0.1% gelatin. After electrophoresis, the gel was washed with renaturing buffer (2% Triton X-100) for 1 h. The gel was incubated for 20 h at 37°C in a reaction buffer (1% Triton X-100) and then stained with Coomassie brilliant blue. The densities of the bands were measured using an image analyzer (NIH ImageJ, Bethesda, MD).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Human umbilical vein endothelial cells (HUVECs; Kurabo, Osaka, Japan) were pre-treated with or without an indicated concentration of Ucn1 in HuMedia-EG2 medium (Kurabo) for 30 min, and further incubated with Ucn1+LPS (1 µg/ml) for 2 h. Total RNA was extracted using a High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. Complementary DNAs were synthesized from isolated RNA templates with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The mRNAs of IL6, MCP1, ICAM1, E-selectin, and glyceraldehyde-3-dehydrogenase (GAPDH) in 50 ng of each sample were detected by RT-PCR, using GoTaq Green Master Mix (Promega, Madison, WI). The sequence of the primers and product size are listed in Table 1. The PCR products were shown by 2% agarose gel electrophoresis, and densitometric analyses were performed as described above.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image (60KB)
• TIFF
  original image (266KB)

Table 1. Primer Sequences Used for RT-PCR.

 

doi:10.1371/journal.pone.0110866.t001

Animal Experiments

Animal experiments were performed in accordance with the NIH 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. A total of 19 male spontaneously hyperlipidemic Apoe−/− mice (C57BL/6. KOR/StmSlc-Apoeshl mice) at the age of 9 weeks were purchased from Japan SLC Inc. (Hamamatsu, Japan) and kept on a normal diet until the age of 13 weeks. Subsequently, a high cholesterol diet (Oriental Yeast, Tokyo, Japan) was started [16]. At 17 weeks of age, 3 mice were sacrificed as a control before injection. The remaining 16 mice were divided into 2 groups; 7 mice and 9 mice, were intraperitoneally injected once daily for 4 weeks with saline (vehicle) or Ucn1 (64 nmol/kg/day; Abgent), respectively. The doses of Ucn1 and its administration methods were decided on the basis of our preliminary examinations.

Animal Measurements

Body weight and food intake were measured during a protocol. Systolic and diastolic blood pressures were measured using the indirect tail-cuff method (Kent Scientific, Torrington, CT). Blood samples were collected after a 4-h fast. Plasma concentrations of glucose and total cholesterol were measured by enzymatic methods [16]. Plasma Ucn1 concentration was measured by enzyme-linked immunosorbent assay (ELISA Kit for Ucn1, Uscn Life Science, Houston, TX).

Atherosclerotic Lesion Assessment

After 4 weeks of injection, the Apoe−/− mice were anesthetized with diethyl ether. The whole aorta was washed by perfusion with PBS, and fixed with 4% formaldehyde. The aorta was excised from the aortic sinus to the abdominal area and the connective and adipose tissues were carefully removed. The entire aorta and cross-sections of the aortic sinus were stained with oil red O for assessment of atherosclerotic lesions [16], [17], [21]. Monocyte/macrophage and VSMC contens in the atherosclerotic lesions were visualized by staining with anti-MOMA-2 antibody (Millipore, Billerica, MA) or anti-α-smooth muscle actin (SMA) antibody (Sigma), respectively [16]. These areas of the aortic wall were traced by an investigator blind to the treatment and measured as described previously [16], [17], [21].

Statistical Analysis

All values are expressed as means ± SEM. The data were compared by unpaired Student's ttest between 2 groups and 1-way ANOVA followed by Bonferroni's post hoc test among ≥3 groups using Statview-J 5.0 (SAS Institute, Cary, NC). A value of P<0.05 was considered to be statistically significant.

Results

Effects of Ucn1 on Inflammatory Response and Cell Proliferation in Human ECs