Lipids (2015) 50:241–251 DOI 10.1007/s11745-015-3991-2

ORIGINAL ARTICLE

Octacosanol Enhances the Proliferation and Migration of Human Umbilical Vein Endothelial Cells via Activation of the PI3K/Akt and MAPK/Erk Pathways Yu‑Wei Liu · Pei‑Yuan Zuo · Xiang‑Nan Zha · Xing‑Lin Chen · Rong Zhang · Xiao‑Xiao He · Cheng‑Yun Liu 

Received: 28 October 2014 / Accepted: 16 January 2015 / Published online: 1 February 2015 © AOCS 2015

Abstract  Atherosclerosis is characterized by endothelial dysfunction, lipid deposition, fibro-proliferative reactions and inflammation. Octacosanol is a high-molecularweight primary aliphatic alcohol. As the main component of a cholesterol-lowering drug, octacosanol could inhibit lipids accumulation and cholesterol metabolism. To explore the indication of octacosanol on endothelial protection, we evaluated its effects on the proliferation and migration of human umbilical vein endothelial cells (HUVEC). Cell viability assay using methyl thiazolyl tetrazolium and 5-ethynyl-2′-deoxyuridine revealed that 3.125 μg/ ml octacosanol promoted the proliferation of HUVEC. A cell migration assay indicated that 0.781 and 3.125 μg/ml octacosanol increased the migration of HUVEC. Moreover, the phosphorylation levels of Akt and Erk1/2 were significantly elevated under exposure to octacosanol. Blocking the activation of Akt and Erk with their potent inhibitors LY294002 and PD98059, respectively, markedly attenuated the octacosanol-induced proliferation and migration of HUVEC. These findings demonstrated for the first time that octacosanol enhanced the proliferation and migration of HUVEC and mediated these effects through activation of the PI3K/Akt and MAPK/Erk1/2 signaling pathways. Keywords  Octacosanol · Migration · Proliferation · Human umbilical vein endothelial cells · PI3K/Akt signaling pathway · MAPK/Erk1/2 signaling pathway · p-Akt · p-Erk1/2

Y.‑W. Liu · P.‑Y. Zuo · X.‑N. Zha · X.‑L. Chen · R. Zhang · X.‑X. He · C.‑Y. Liu (*)  Department of Geriatrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022, People’s Republic of China e-mail: [email protected]

Abbreviations HUVEC Human umbilical vein endothelial cells PI3K/Akt Phosphatidylinositol-3-kinase/Akt MAPK/Erk1/2 Mitogen-activated protein kinases/Erk1/2 HMGR Hydroxymethylglutaryl-coenzyme A reductase VEGF Vascular endothelial growth factor CHD Coronary heart disease MTT 3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide EdU 5-Ethynyl-2′-deoxyuridine FBS Fetal bovine serum

Introduction Octacosanol (CH3(CH2)26CH2OH), a high-molecularweight primary aliphatic alcohol, is the main component of a natural wax product extracted from plants. Dietary incorporation of octacosanol into a high-fat diet decreased the serum triacylglycerol concentration and enhanced the concentration of serum fatty acids, which overall suppressed lipids accumulation in the perirenal adipose tissue of rats [1]. Octacosanol administration to human decreased in the concentration of fecal cholesterol end products implying the effects of octacosanol on cholesterol metabolism [2]. Policosanol is a mixture of higher primary aliphatic alcohols isolated from sugar cane wax, whose main component is octacosanol. The mixture has been shown to lower cholesterol in animal models, healthy volunteers, and patients with type II hypercholesterolemia [3]. The effects of octacosanol and policosanol on the regulation of Hydroxymethylglutaryl-coenzyme A reductase (HMGR) in HUVEC and HepG2 human hepatoma cells were compared, which demonstrated that octacosanol was as effective as

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policosanol in inhibiting the upregulation of HMGR [4], indicating that octacosanol could efficiently inhibit cholesterol biosynthesis. The causal relationship between blood cholesterol and atherosclerosis is no longer in doubt [5]. The notably premature coronary heart disease (CHD) in patients with familial hypercholesterolemia strongly indicated that a very high cholesterol level, especially a high LDL-cholesterol level, was in itself enough to account for severe atherosclerotic diseases [6]. Oxidized LDL induced monocyte binding to endothelial cells, caused endothelial dysfunction, triggered inflammation and the aggregation of lipid-rich macrophages and T lymphocytes within the intima of the artery wall, and ultimately resulted in the formation of atherosclerotic plaques [7]. Some researchers believed that the endothelial dysfunction resulting from exposure to oxLDL and its products was the critical initiators of atherosclerosis [6]. By lowering the cholesterol and LDL levels, octacosanol helped to slow the formation of obstructive atherosclerotic plaques and therefore reduced the occurrences of potentially life-threatening events in CHD patients [8]. It was recently demonstrated that policosanol exhibited positive effects on vascular endothelial protection [9]. However, the specific components and the underlying mechanisms involved in the vascular endothelial protection remain to be further elucidated. Previously, we demonstrated that the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway plays an important role in the proliferation and migration of endothelial progenitor cells [10]. The mitogen-activated protein kinases/Erk (MAPK/Erk) pathway also participated in the transduction of endothelial cell proliferation signals [11]. The aim of this study was to investigate the effects of octacosanol on the proliferation and migration of human umbilical vein endothelial cells (HUVEC) and to further address the underlying mechanisms. To our knowledge, there were few reports focusing on the proliferation and migration of HUVEC induced by octacosanol and the pathways involved in these effects. With DNA labeling, Transwell migration assays and Western blotting to unveil the signaling pathways involved, we hoped that this study was able to broaden our understanding of the effects of octacosanol and further enlighten the clinical management of CHD.

Materials and Methods Experimental Protocols EA.Hy926 HUVEC were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). Octacosanol (2 mg; Formula weight: 410.76 g/mol;

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purity (GC): ≥99 %; Product number: #O3379, Sigma, St. Louis, Missouri, USA) was dissolved in 50 μl absolute ethanol and diluted in 10 ml medium. The concentrations ultimately chosen for the following experiments were 0.195 μg/ml (0.48 μM), 0.781 μg/ml (1.90 μM), 3.125 μg/ ml (7.61 μM), and 12.5 μg/ml (30.43 μM); these were chosen based on the former studies [4, 12]. The concentration of absolute ethanol in the subsequent experiments was lower than 0.1 % and had no effect on cell viability. After being exposed to different levels of octacosanol for 48 h, cell viability and proliferation were measured by methyl thiazolyl tetrazolium (MTT) and DNA labeling assays respectively via the well documented methods [13, 14]. To evaluate the effects of octacosanol on HUVEC migration, a Transwell chamber was employed after HUVEC were incubated with octacosanol for 6 h according to the method described in the previous study [15]. Vascular endothelial growth factor (VEGF, 10 ng/ml), which has been well documented to stimulate the proliferation and survival of endothelial cells and promote angiogenesis [16], served as the positive control. The serum-free medium was taken as the negative control. Western blotting was used to detect the activation of signaling pathways involved in the impact of octacosanol on HUVEC based on the well documented protocol [17]. To confirm the participation of the identified signaling pathways in octacosanol-induced HUVEC proliferation and migration, potent inhibitors of the PI3K/Akt and MAPK/Erk1/2 signaling pathways, LY294002 (Cell Signaling Technology, Danvers, Massachusetts, USA) and PD98059 (Cell Signaling Technology, Danvers, Massachusetts, USA), respectively, were used. According to previous research and the product instructions, 1 h pretreatment with 50 μM LY294002 and 50 μM PD98059 could effectively block the PI3K/Akt and MAPK/Erk1/2 pathways, respectively [18]. The specific procedures and materials for each assay are described below. Cell Culture HUVEC were cultured in 25-cm2 cell culture flasks (Corning, Oneonta, New York, USA). Cultures were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone, South Logan, Utah, USA) supplemented with 10 % fetal bovine serum (FBS) (Tianhang BioTech, Zhejiang, China) in an incubator at 37 °C with 5 % CO2. The cells were subcultured at a split ratio of 1:3 when the flasks reached 80–90 % confluence. To do this, the medium was removed, the cell surface was rinsed with phosphate buffered saline (PBS) twice, and a 0.05 % (w/v) solution of trypsin/EDTA (HyClone, South Logan, Utah, USA) was added to detach the cells from the flask. The detached cells were resuspended in standard culture medium and seeded into fresh culture flasks.

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Cell Viability Assay The 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay (Dingguo Changsheng Biotech, Beijing, China) was used to measure the viability of HUVEC. HUVEC in the logarithmic growth phase were digested, centrifuged, and inoculated in 96-well flat-bottomed plates. Each well contained 1 × 103 HUVEC in suspension. After being initially cultured in DMEM with 10 % FBS for 12 h, the cells were placed in serum-free medium for another 24 h to achieve cell cycle synchronization at G0/G1. The cells were cultured with the different concentrations of octacosanol described above for 48 h. MTT solution (5 mg/ ml) was added to each well. Following incubation at 37 °C for 4 h, the cell culture medium was removed and 100 μl of dimethylsulfoxide (DMSO) (Amresco, Solon, Ohio, USA) was added to each well. The absorbance of each well was measured with an ELx800 Universal Microplate Reader (Bio-Tek, Winooski, Vermont, USA) at a detection wavelength of 570 nm. The viability of cells in the experimental groups was expressed as a percentage of the viability of control cells (which was taken to be 100 %). The results were calculated from four experiments, with five replicates in each experiment. Cell Proliferation Assay The proliferation of HUVEC was measured using a DNA labeling assay (RiboBio, Guangzhou, China). The procedure was as follows. HUVEC were seeded in 96-well flatbottomed plates. Each well contained 1 × 103 HUVEC in suspension. First, the cells were placed in serum-free media for another 24 h to achieve cell cycle synchronization at G0/G1. Then, the cells were treated with various concentrations of octacosanol for 48 h. Third, cells were incubated with 50 μM 5-ethynyl-2′-deoxyuridine (EdU) for 2 h at 37 °C. To show the proliferative cells, HUVEC were incubated with Apollo® fluorescent dye for 30 min at room temperature and washed twice in phosphate-buffered saline (PBS) for 5 min. As the analogue of thymidylic acid, EdU participated in DNA replication and provided a red fluorescent signal after being conjugated with Apollo® fluorescent dye. To further display all of the cells on the plates, Hoechst 33,258 staining was used to provide a blue fluorescent signal. After being washed twice in PBS for 5 min, the nuclear morphology of the proliferative cells and the total cells was visualized, respectively, by fluorescence microscopy (Nikon, Tokyo, Japan) (magnification, 200×) and evaluated with Image Pro-Plus 6.0 (Media Cybernetics, Rockville Maryland, USA). The proliferation rate of the cells in each experimental group was expressed as a percentage of the proliferative cells versus the total cells. All experiments were performed in triplicate. To investigate the involvement

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of the PI3K/Akt and MAPK/Erk pathways in octacosanolinduced HUVEC proliferation, HUVEC were pretreated with the indicated concentrations of 50 μM LY294002 or 50 μM PD98059 for 1 h, and then, the cells were cultured in the absence or presence of 3.125 μg/ml octacosanol. The steps that followed were the same as those described above. Cell Migration Assay The migratory ability of the HUVEC was evaluated using a Transwell migration assay (Costar, St Lowell, Massachusetts, USA) with 6.5-mm-diameter polycarbonate filters (8 μm pore size). As the presence of serum may affect the judgment of the influence of octacosanol on cell migration, octacosanol and VEGF were dissolved in the endothelial medium containing only 0.5 % FBS, which maintained the normal growth of HUVEC but had little influence on cell migration. Various concentrations of octacosanol plus endothelial medium with 0.5 % fetal bovine serum (FBS) were placed in the lower wells. HUVEC (1 × 104 cells/ well) were seeded into the upper chamber and supplemented with serum-free medium. After 6 h of incubation, the upper chamber was removed and wiped clean with a cotton swab; the lower side of the filter was washed with PBS and fixed with 4 % paraformaldehyde for 30 min. For quantification, the cell nuclei were stained with crystal violet for 30 min. Cell migration into the lower chamber and attachment to the lower side of the filter were visualized in 8 randomly selected fixed microscopic fields (magnification, 400×) (Nikon, Tokyo, Japan) and counted manually with the NIH Image J 3.0 software (National Institutes of Health, Bethesda, Maryland, USA) by independent investigators blinded to the treatment regimen. Each test was performed in duplicate, and assays were repeated three times independently. To investigate the involvement of the PI3K/ Akt and MAPK/Erk pathways on the migration ability of the cells, we raised the number of cells in each well to 10 × 104, considering that the inhibitors may exert strong effects on the migration of HUVEC. After the HUVEC were pretreated with 50 μM LY294002 or 50 μM PD98059 for 1 h, the cells were cultured in the absence or presence of 3.125 μg/ml octacosanol. The steps that followed were the same as those described above. Western Blotting Analysis HUVEC pre-treated with 3.125 μg/ml octacosanol were lysed with RIPA buffer containing protease inhibitors (Beyotime Biotech, Shanghai, China). The total protein concentration was determined by the BCA protein assay (Beyotime Biotech, Shanghai, China). The proteins were electrophoresed on 10 % SDS-PAGE gels at 120 V for 1.5 h and then electroblotted onto a PVDF membrane at 300 mA

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for 1 h. The membrane was incubated with 5 % fat-free milk in distilled water or 3 % bovine serum albumin (BSA) in distilled water for 1 h at room temperature. The membrane was later incubated with anti-Akt, anti-p44/42 MAPK (Erk1/2) (1:1,000; Cell Signaling Technology, Danvers, Massachusetts, USA), anti-phospho-Akt (Ser473) and anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:1,000; Cell Signaling Technology, Danvers, Massachusetts, USA) mouse monoclonal antibodies, followed by the addition of a goat anti-mouse antibody (LI-COR, Lincoln, Nebraska, USA). The immunoreactive bands were then visualized with Odyssey (LI-COR, Lincoln, Nebraska, USA). The density of each band was quantified using Photoshop CS5 (Adobe, San Jose, California, USA). Protein quantifications were normalized to the corresponding levels of GAPDH, which were not altered dramatically between the different treatment conditions. All of the assays were performed at least three times independently. Statistical Analysis Results were obtained from at least three independent experiments. Data are presented as the mean ± SD. Student’s t test was performed for statistical comparisons between two groups, and ANOVA was used for comparisons between more than two groups using SPSS version 20.0 (IBM, Armonk, New York, USA). A p value