Hyperlipidemia is one of the major independent risk factors for the development of atherosclerosis. More than 50% of heart and coronary artery diseases are attributable to blood lipid abnormalities (1). Atherogenesis is initiated by accumulation of lipid components in the vascular vessel walls. Treatment of hyperlipidemia may be an important component for the management of atherogenesis in its early stages (2, 3). Several lines of evidence support the notion that atherosclerosis is a chronic inflammatory disease in nature (4–6). The chemokine interleukin-1β (IL-1β)/chemokine (C-X-C motif) ligand 8 (CXCL8/IL-8) has been known to play an important role in monocyte migration into the subendothelial space in the early phase of atherosclerosis (7, 8). Elevated levels of IL-8 are associated with increased risks of future coronary artery diseases (9–11). It has also been reported that IL-8 is highly inducible in human vascular smooth muscle cells (VSMCs) when cells are activated by IL-1β or tumor necrosis factor (TNF) (12). IL-8 is suggested be an important naturally occurring mitogen and chemoattractant for VSMCs and play a role in arterial intimal thickening (13).
IL-8 interacts with the VSMCs through two seven domain-transmembrane G protein-coupled receptors, CXCR1 and CXCR2, and the ELR-CXC chemokine receptors. CXCR1 binds to CXCL6 granulocyte chemoattractant protein-2 (GCP-2) and IL-8 with high affinity while CXCR2 binds to all of the ELR-CXC chemokines with lower affinity (14, 15). It is reported that CXCR2 is involved in a number of inflammatory and neoplastic diseases, and inflammatory response was diminished and the tumor growth was inhibited in CXCR2 knockout mice (16–19). G31P is a recombinant compound produced from human IL-8 using gene site-directed mutagenesis by introducing a Lys11 to Arg substitution and a Gly31 to Pro substitution to generate a broad-spectrum ELR-CXC chemokine antagonist, human CXCL8(3-72) K11R/G31P (G31P) (20). It has been verified that G31P with no discernible neutrophil agonist activity apparently binds to neutrophils (via both the CXCR1 and CXCR2) with higher affinity than IL-8 and can block neutrophil responses to IL-8 better than other analogues (21, 22).
In this study, we investigated the therapeutic efficacy of G31P with a mouse model of hyperlipidemia and the potential mechanisms of G31P through the VSMC proliferation and migration in a cell line.
Materials and Methods
The study followed the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Male BALB/c mice (6-week-old) were obtained from Dalian Medical University Laboratory Animal Center. Mice were randomly divided into the control group (n = 6, standard chow fed, treated with normal saline), the high-fat diet fed group (n = 12, treated with normal saline) and the G31p-treated high-fat diet fed group (n = 12). Those in the high-fat diet group were fed with high-fat diet (common feeds 78.8%, lard oil 10%, yolk powder 10%, cholesterol 1%, bile salt from pig 0.2%) and treated with normal saline. Those of G31P-treated group were fed with high-fat diet and treated with 500 μg/kg G31P (presented by Prof. Fang Li, Department of immunology of Dalian Medical University) injection, once per week. The other two groups were injected with normal saline, hypodermic. Eight months afterward, the blood was collected through eyeballs and kept in water bath at 37 °C for 10 min, then centrifuged at 2,700g for 10 min. Serum was collected and stored at −20 °C for keratinocyte chemoattractant (KC) and lipid profile analysis. Aortas were excised, snap frozen in liquid nitrogen, and stored at −80 °C for reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Samples of aortic root were fixed in 4% paraformaldehyde and embedded in paraffin using standard procedures for subsequent immunohistochemistry analyses. The animal protocol was approved by the local research ethics review board of the Animal Ethics Committee of Dalian Medical University.
Total RNA was initially extracted using RNAiso Plus (TaKaRa, Japan) according to the manufacture's protocol. RNA from each sample (300 ng) was reverse transcribed to cDNA with random 9-mers and amplified in a total volume of 50 μl using the TaKaRa RNA PCR Kit (TAKARA, Japan). Primers used in RT-PCR were as follows: GAPDH: 5′-CCATCACTGCCAC TCAGAAGAC-3′ (sense) and 5′-TACTCCTTGGAGGCCATGTA GG' (antisense), KC: 5′-GATTCACTCAAGAACATCCAGA -3′ (sense) and 5′-GGACACCTTTTAGCACTTTTGG-3′ (antisense), MIP-2 (macrophage inflammatory protein-2): 5′-AACATCCAGAGCTTGAGTGTGAC-3′ (sense) and 5′-GCCTTGCCTTTGTT CAGTATCTT-3′ (antisense); CXCR2: 5′-CATCCCGTTTGAGGG TCGTA-3′ (sense) and 5′-GCCAGCAGAGCAGGAAGACA-3′ (antisense), TNF-α: 5′-CAGCCGATTTGCTATCTCATACC-3′ (sense) and 5′-GTACTTGGGCAGATTGACCTCAG-3′ (antisense), interferon (IFN)-γ,: 5′–TCAAGTGGCATAGATGTGGAAGA-3′ (sense) and 5′-TCAGGTGTGATTCAATGACGCT-3′ (antisense). PCR cycling parameters (35 cycles) were set as denaturation (94 °C, 30 sec), annealing (56 °C, 30 sec), and extension (72 °C, 1 min). Equal amounts of PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining. The specific bands of PCR products were analyzed by Image-Pro Plus 6.0 system with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control for normalization. RT-PCR was performed in triplicates independently.
Aortas were processed in serials sections at 3 μm and stained with hematoxylin/eosin. Additional aortic sections were used for immunohistochemistry staining to detect matrix metalloproteinases MMP-2, MMP-9 (Bioss, China) and PCNA (proliferating cell nuclear antigen, Santa Cruz Biotechnology, Santa Cruz, CA). Briefly, endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 min. The samples were rinsed three times with PBS, incubated for 15 min at room temperature with a protein-blocking solution of 5% normal horse serum in PBS (pH 7.5), washed three times with PBS, then incubated with the primary antibodies (MMP-2 1:100, MMP-9 1:100, PCNA 1:100) at 4 °C overnight. The samples were then rinsed three times with PBS and incubated for 40 min at 37 °C with the biotinylated secondary antibody for 30 min. Finally, the specimens were incubated in diaminobenzidine for 10 min and then counterstained with hematoxylin. The primary antibody was omitted for staining as negative controls. Results were calculated using Image-Pro 6.0 Microsoft (MediaCybernetics, Bethesda, MD).
KC and Lipid Profile Assays
Serum levels of high density lipoprotein (HDL)-C, low density lipoprotein (LDL)-C, triglyceride (TG), and total cholesterol were determined by selective precipitation or enzymatic method (Beijing BHKT Clinical Reagent, China) according to the manufacturer's protocol. KC levels in the serum were measured by enzyme-linked Immunosorbnent Assay (ELISA) (Assay Biotechnology Company, California's San Francisco Bay Area). Serum samples were placed in assay plate and incubated at 37 °C for 30 min. After five washes, samples were added horseradish peroxidase (HRP)-Conjugate Reagent and incubated at 37 °C for 30 min. Finally after another five washes, samples were incubated with Chromogen Solution A and B at 37 °C for 10 min and then the reactions were read at 490 nm in a microplate reader.
The A7r5 cell line, originally derived from embryonic rat aorta (23), was purchased from the American Type Culture Collection (ATCC, Manassas, VA). The cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Langley, OK) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a CO2 incubator (in a humidified atmosphere of 5% CO2 and 95% air). Prior to stimulation, 80–90% confluent VSMCs were serum-starved overnight by incubating in DMEM without FBS.
Cell Migration Assay
Boyden Chamber Assay
Cell migration was monitored using a Boyden chamber (Neuroprobe, Gaithersburg, MD) as previously described (24). In all experiments, collagen-coated polycarbonate filters (pore size, 8 μm) were used. A7r5 cells (2 × 105 cells/well) suspended in DMEM containing 0.5% bovine serum albumin (DMEM-BSA) were loaded into the upper wells of the chamber. The lower wells were filled with DMEM-BSA containing 1–100 ng/l IL-8 (Sigma-Aldrich, St Louis, MO) or vehicle.
After 5 h of incubation at 37 °C, the cells that had migrated to the lower surface of the filter were fixed in methanol, stained with Giemsa solution, and observed under microscope. Each condition was tested in quadruplicate, and the number of cells from a randomly chosen high-power field (400× magnification) was counted in each well. In some experiments, cells were pretreated with G31P for 1 h. During the assay, G31P was added to the upper and lower wells. The migration assay was conducted in at least three independent replicates.
Wound Healing Assay
The wound healing assay was performed as previously described (25). Briefly, cells were cultured in six-well plates under standard culture conditions and allowed to reach 100% confluence. A “wound” was initiated in the confluent monolayers with the tip of a 200 μl pipette tip, and the wells were washed three times with phosphate buffer saline (PBS). Cells were then incubated in serum-free medium at 37 °C (5% CO2) for 24 h in the presence of 20 ng/ml of IL-8. The migration of the A7r5 cells into the “wounded” area was evaluated using an inverted microscope. The cell's wound-healing rates were quantified as the distance across the injury line during the culture, as determined at three different sites in each well.
For the study of cell viability, 1–5 × 103 cells were plated in each well of a flat-bottom 96-well culture plate and incubated in 200 μl DMEM medium for 24 h. After treatment with IL-8 with/without G31P, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well to a final concentration of 0.5 mg/ml, and they were incubated for 4 h at 37 °C in a humidified incubator containing 5% CO2. To dissolve the cells, we used 150 μl of dimethyl sulphoxide (DMSO) and measured the absorbance of the resulting solutions at 570 nm using a microplate reader (Multiskan Ascent, Thermo Scientific, Barrington, IL). Results were obtained as percentage cell viability (optical density [OD] of the experiment samples/OD of the control).
A7r5 cells stimulated with IL-8 or pretreated with G31P were grown on sterile glass coverslips, which were washed in ice-cold PBS and fixed for 20 min at room temperature with 4% paraformaldehyde. After fixation, cells were permeabilized with 0.1% Triton X-100 for 10 min and blocked with 5% BSA in PBS for 20 min. Cells were then incubated with rabbit anti-F-actin-conjugated tetramethyl rhodamine iso-thiocyanate (1: 2,000, Sigma-Aldrich) for 40 min. Cells were then washed thrice with PBS and mounted onto slides for photography using a fluorescence microscope (BX-51, TR32000, Olympus, Tokyo, Japan) with a charge-coupled device system.
Flow Cytometry Assay
Flow cytometry was used to determine the suppressive effects of the dose response of G31P on A7R5 stimulated by IL-8. Cells (2 × 105 cells/tube) in different groups were washed with PBS twice then collected with trypsinization. After centrifugation (380g, 5 min), the cell pellet was suspended in 500 μl PBS/0.02% ethylenediaminetetraacetic acid (EDTA) (pH 7.4) and 500 μl 100% methanol, then incubated 10 min at room temperature. Cells were centrifuged again (670g, 5 min) at room temperature and after discarding supernatant, cells were incubated with 500 μl PBS and 5 μl RNase A (10 mg/ml) for 30 min at 37 °C, then in a solution containing 50 μl (1 mg/ml) propidium iodide (Sigma) in 450 ml PBS for 30 min in the dark and analyzed by flow cytometry on a FACScalibur (BD Biosciences, San Jose, CA) using a 560 nm dichromatic mirror and a 600 nm band pass filter.
Data were expressed as mean ± S.E.M. All statistical analyses were done by SPSS17.0. Comparisons between two groups were conducted by using the unpaired t-test. For multiple groups, a Tukey multiple comparison test was used. P < 0.05 was considered as statistically significant.
Therapeutic Effects of CXCL8(3-73)K11R/G31P on Hyperlipidemia
High-fat diet increased body weight from 1 to 6 months of feeding, G31P remarkably reduced high-fat diet-induced weight gain (P < 0.05) (Fig. 1A). G31P treatment also showed significant suppression on the high-fat diet-induced hyperlipidemia, that is, increase of serum TG, cholesterol, HDL-C, and LDL-C (all P < 0.05). Consistent with its effects on body weight and lipid profile, G31P displayed significant suppression to the control level on the high-fat diet-induced increase of serum KC (P < 0.01, Fig. 1C).
G31P Suppressed High-Fat Diet-Induced Inflammatory Factors' mRNA Expressions in Aorta
We investigated the effect of G31P on the high-fat diet-induced expression of chemokine mRNAs including MIP-2, KC, CXCR2, TNF-α, IFN-γ in the mouse root of aorta. As expected, high-fat diet induced significant increases of the mRNA expression of the inflammatory factors. G31P demonstrated robust suppression to almost the control level on the high-fat diet-induced mRNA levels of the inflammatory factors (Fig. 2).
G31P Suppressed Cell Proliferation In Vitro and In Vivo
To study the biological roles of G31P on VSMCs proliferation in vitro, we performed an MTT assay. IL-8 induced VSMCs proliferation in a dose-dependent manner when its concentrations were above 10 ng/ml (P < 0.05, Fig. 3A), G31P (100 ng/ml) significantly suppressed IL-8 (20 ng/ml)-induced VSMCs proliferation (P < 0.05, Fig. 3B). In addition, G31P (100 ng/ml) also suppressed the IL-8 (100 ng/ml)-induced increase of S phase cell on the growth of A7R5 cell lines (Fig. 4). Furthermore, G31P significantly suppressed high-fat diet-induced PCNA immunostaining of mouse aorta (P < 0.01, Fig. 5).
G31P Inhibited Cell Migration In Vitro and In Vivo
To investigate the role of G31P in VSMC migration, we first assessed the effects of IL-8 on VSMC wound healing. A7r5 cells treated with IL-8 remarkably accelerated the wound closure. However, G31P prevented these effects and significantly decreased the rate of wound healing (P < 0.01, Fig. 6A). As wound healing analysis involves multiple cellular processes, we then measured the migratory properties of VSMCs by using Boyden Chambers. Similar inhibitory effects of G31P were observed (P < 0.05, Fig. 6B). G31P also reduced IL-8-induced F-actin stress fibers in VSMCs (Fig. 6C).
After 6 months of high-fat diet feeding, MMP2 and MMP9 were highly induced in the mouse aorta (Figs. 7A and 7C). G31P significantly suppressed about 45% of MMP2 and 1/3 of MMP9 expression (both P < 0.01) compared with the high-fat diet fed group (Figs. 7B and 7D).
In line with the inflammation hypothesis of atherosclerosis (26, 27), some inflammation factors have been found to participate in the development of atherosclerosis (28, 29). In this study, through an animal model with hyperlipidemia, we found that the expression of γ-IFN, TNF-α, CXCR2, MIP-2, and KC on the blood vessels were induced to higher levels than the controls, as well as the serum KC expression was elevated; together these data suggest that the immunoinflammatory factors and chemotactic factors were upregulated before the formation of foam cells on the blood vessel wall. It is conclusive the inflammation is the pathophysiological base of the initiation and development of atherosclerosis. IL-8 as a vital regulatory factor in the immunoinflammation response is a marker for coronary arterial lesion and inflammation in angina patients, and it is also a signal for the instability of coronary arterial lesions (30). It has been shown IL-8 expression is elevated in the atherosclerosis patients and the animal model (31–36), which is consistent with our current study. Based on these results, we speculate that there is a positive correlation between IL-8 and other inflammatory factors, such as γ-IFN, TNF-α, CXCR2, MIP-2, and KC. In addition, these inflammatory factors that act as an acute inflammatory reactant have been shown to be highly associated with the acute damage of artery and the development of atherosclerosis, reflecting the intensity of the inflammatory response and the instability of arterial lesion (37). Herein, these inflammatory factors were considered as the potential dangerous risk factors of the initiation and development of the cardiovascular diseases.
Given that hyperlipidemia causes the release of IL-8, we speculate the pivotal role of IL-8 in atherosclerosis. As a very special chemotactic factor of neutrophile granulocytes, IL-8 promotes the aggregation of neutrophile granulocytes and the release of cytotoxin from them (38), which would worsen the inflammatory response by enhancing the expression levels of γ-IFN, TNF-α, CXCR2, MIP-2, and KC. The upregulated inflammatory factors could induce the attachment of monocytes on the blood vessel endothelium, the proliferation of smooth muscle cells, and finally the initiation and development of atherosclerosis. IL-8 initiates the inflammatory response, induces the elevation of the inflammatory response of vessel walls, and hence plays a vital role in the regulation of hyperlipidemia and atherosclerosis. When we treated the mice with G31P, the antagonist of IL-8 receptor, the KC (KC is IL-8 homologs in mice) expression level in mice was obviously decreased to the level similar with the controls, while the inflammatory factors were notably decreased. These results demonstrate that, by blocking the IL-8 receptors and inhibiting the biologic activity of IL-8, G31P can downregulate the inflammatory factors and ameliorate the lesions of the blood vessel. We suspect that through competing with the IL-8 receptor, the biologic activity of IL-8 and the inflammatory response can be suppressed. The suppressed inflammatory response will in turn reversibly reduce the release of inflammatory factors including the expression of IL-8 and the receptor of CXCR2.
During the early stage of atherosclerosis, under the stimulation of inflammatory factors, the VSMCs migrate from the media to the intima then proliferate and secrete excessive cell matrix proteins. These processes are thought to play a pivotal role in the development of chronic atherosclerosis and in restenosis after angioplasty of human coronary arteries (39). PCNA plays important roles in DNA replication, promotion of cell proliferation, and itself is a good marker for the cell proliferation. Compared with the control, PCNA had higher expression in the hyperlipidemia model mice. These results demonstrated that, during hyperlipidemia period, although there is no notable inner membrane increment, proliferation and migration of vessel smooth muscle cells are induced. G31P significantly suppressed cell proliferation as shown with reduced PCNA in aorta and decreased MTT activity and S phase cells in vitro through the stimulation of A7r5 cells with IL-8. G31P can effectively inhibit cell proliferation in vitro. We speculate G31P inhibits cell proliferation through interaction with CXCR1/2 and consequent blockage of IL-8.
The prerequisite of the movement of VSMCs migrating through tunica media to tunica intima is the degradation of extracellular matrix (ECM). MMPs are a family of Zn-dependent endopeptidases, which are produced by VSMCs, macrophage, and so on. MMPs can degrade most of the ECM, contributing to the migration of VSMCs (40–42). As members of MMPs family, MMP-2,9 are the most important candidates to degrade ECM and promote the movement of VSMCs. Currently, it is conceivable that the imbalance of synthesizing and the degradation of ECM is a vital process in the initiation of atherosclerosis. By degrading ECM, MMPs impair the barrier role of the blood vessel endothelium, promote the migration and proliferation of VSMCs on the tunica media vasorum, and furthermore, MMPs can boost the invasion of inflammatory cells, worsening the change of blood vessel structure (43). We found that both MMP2 and MMP9 were highly expressed during the hyperlipidemia period. Interestingly, G31P significantly downregulated the expression of MMP-2 and MMP-9. In the in vitro experiments, IL-8 stimulated the migration of the A7r5 cells and the cells appeared with more numbers of filiformed pseudopodium. After treatment with G31P, the cell migration was notably decreased, the numbers of filiformed pseudopodium were reduced, and cell surfaces were much smoother than the controls. Consequently, through antagonizing IL-8 by blocking its interaction with CXCR1/2, G31P inhibits the biological activity of IL-8, inhibits the proliferation and migration of VSMCs, and furthermore ameliorates the development of hyperlipidemia and atherosclerosis.
In conclusion, our results indicate G31P as a potent CXCR1/2 receptor antagonist that markedly inhibits the proliferation and migration capacity of VSMCs in hyperlipidemia. These findings might provide a new approach to prevent and treat hyperlipidemia and atherosclerosis.