Author + information
- Received April 13, 2006
- Revision received June 6, 2006
- Accepted July 3, 2006
- Published online November 7, 2006.
- Shu-Lin Liu, MS⁎,†,
- Yi-Heng Li, MD, PhD‡,§,
- Guey-Yueh Shi, PhD⁎,‡,
- Yung-Huan Chen, MS⁎,
- Chia-Wei Huang, MS⁎,†,
- Jau-Shyong Hong, PhD∥ and
- Hua-Lin Wu, PhD⁎,‡,⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Hua-Lin Wu, Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan.
Objectives We investigated whether naloxone could reduce macrophage activation and influence atherosclerotic lesion formation in mice.
Background Macrophages play an important role in the inflammatory process in atherosclerosis. Naloxone could inhibit activation of microglia, the resident macrophage in the nervous system.
Methods The anti-inflammatory effect of naloxone was evaluated by stimulating the macrophage cell culture and FVB mice with lipopolysaccharide or oxidized low-density lipoprotein with and without naloxone pretreatment. Apolipoprotein-E (apoE)-deficient mice received naloxone injection for 10 weeks, and the severity of aortic atherosclerosis was measured. The left common carotid arteries of C57BL/6 mice were ligated near the carotid bifurcation. The mice then received naloxone injection for 4 weeks after ligation, and the severity of neointima formation was evaluated.
Results Naloxone pretreatment significantly suppressed the production of tumor necrosis factor-α (TNF-α), interleukin-6, monocyte chemoattractant protein-1, and superoxide in macrophages after stimulation. In FVB mice, naloxone reduced the TNF-α level in circulation, inflammatory cell infiltration in lungs, and superoxide production in aorta. Naloxone injection significantly decreased the severity of aortic atherosclerosis in the apoE-deficient mice and carotid neointima formation in the C57BL/6 mice after ligation.
Conclusions Naloxone, with its novel anti-inflammatory effect, significantly reduces atherosclerosis and neointima formation in mice.
Atherosclerosis remains a leading cause of morbidity and mortality worldwide. Central to the pathogenesis of atherosclerosis is the infiltration of monocytes/macrophages in the arterial wall and the involvement of inflammation (1,2). Macrophages play a diverse array of roles in atherogenesis. They function as scavenger cells that takes up oxidized low-density lipoprotein (oxLDL) and become foam cells in the initial lesion of atherosclerosis (3,4). After activation, macrophages are capable of producing free radicals and pro-inflammatory factors, all of which are critical for the promotion of cellular proliferation and inflammation in atherosclerosis (3,4). Evidence indicates that atherosclerotic lesions could be decreased if monocyte/macrophage extravasation and activation is inhibited (5,6). Naloxone is a non-selective antagonist of the opioid receptors that are widely expressed not only in the central nervous system but also on the endothelium and monocytes (7,8). Previous animal studies found that naloxone can significantly decrease the inflammatory response in septic shock. Naloxone improves hypotension, leukocytosis, and prolonged survival time in endotoxin-induced shock rats (9,10). It inhibits the production of tumor necrosis factor-α (TNF-α) induced by lipopolysaccharide (LPS) in mice. Recent studies also show that naloxone reduces the pro-inflammatory factors and superoxide generation from LPS-induced microglia, the resident macrophages within the nervous system (11,12). Because of the importance of macrophage-related inflammation in the pathogenesis of atherosclerosis, we sought to investigate the influence of naloxone on macrophage activation and determine the treatment effect of naloxone on atherosclerosis. In this study, we report that naloxone treatment can reduce pro-inflammatory factors and superoxide production in macrophage cell cultures and mice after stimulation. Naloxone injection might decrease the severity of atherosclerotic lesion development in apolipoprotein-E (apoE)-deficient mice and the neointima formation in mouse vascular remodeling model.
RPMI 1640 medium, phorbol 12-myristate-13-acetate, LPS (Escherichia coli0111:B4), and naloxone were purchased from Sigma-Aldrich (St. Louis, Missouri). The human acute monocytic leukemia cell line (THP-1) was purchased from the Food Industry Research and Development Institute (Hsin Chu, Taiwan). Levels of TNF-α, interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) in the medium or plasma were determined with monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) kits purchased from the R&D Systems (Minneapolis, Minnesota). ApoE-deficient mice were obtained from the Jackson Laboratories (Bar Harbor, Maine). All animal experiments were approved by the Institutional Animal Care and Use Committee, National Cheng Kung University.
Pro-inflammatory factor and superoxide in THP-1 cell culture
The THP-1 cells were grown in the RPMI-1640 medium containing 10% fetal bovine serum at 37°C in 5% carbon dioxide. The cells were differentiated to macrophages after treatment of the culture with 100 nmol/l phorbol 12-myristate-13-acetate for 24 h. The cell suspension (5 × 105) was added, 0.5 ml into each well of the tissue culture plates. For each experiment, naloxone was prepared immediately before use. The LPS was dissolved in sterile water and stored at −70°C in aliquots. The LDL was isolated from human endotoxin-free heparin plasma and was oxidized with copper(II) sulphate (CuSO4) as described previously (13). In brief, LDL (1 ml, 1 mg/ml) was dialyzed in 500 ml phosphate-buffered saline (PBS) (pH 7.4) overnight. Copper sulfate was added to a final concentration of 5 μmol/l, and the LDL was allowed to oxidize at room temperature for 24 h.
First, we examined the effective naloxone concentration that inhibited TNF-α production from macrophages. Because a previous study (11) demonstrated that 1 μmol/l naloxone could protect neurons through inhibition of microglia activation, our initial experiment began with this concentration. The THP-1 cell culture was pretreated for 1 h with various concentrations of naloxone (1 to 10−6μmol/l) before treatment with 100 ng/ml LPS for 24 h. The TNF-α level in the supernatant was determined by ELISA. The most effective naloxone concentrations (1, 0.1, and 0.01 μmol/l) were chosen for all the following experiments.
For study of pro-inflammatory factors, the THP-1 cell culture was pretreated for 1 h with naloxone (1, 0.1, or 0.01 μmol/l) before treatment with 100 ng/ml LPS or 10 μg/ml oxLDL for up to 24 h. The supernatants were harvested, and the TNF-α, IL-6, and MCP-1 levels were determined by ELISA. Superoxide production in the THP-1 cell culture was measured by lucigenin-enhanced chemiluminescence as described previously (14). The LPS-treated THP-1 cells were treated with PBS containing 1.25 mmol/l lucigenin, and counts were obtained for a 10-min period in a luminometer (Berthold Technologies, Germany) as relative light units (RLU) emitted. Background counts determined in cell-free preparations were subtracted. Superoxide levels were reported as RLU/10 min and were normalized to the volume (ml) of cell suspension added (i.e., RUL/10 min/ml).
Cell viability was determined with the 3 (4,5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT) assay in each treatment group previously described. Briefly, fresh medium was added to cells together with 10% MTT (5 mg/ml). Each plate was maintained at 37°C for 2 h, and subsequently formazan crystals were dissolved in dimethylsulfoxide (DMSO). Absorbance was read at a wavelength of 550 nm with a SPECTRAmax PLUS384spectrophotometer (Molecular Devices, Sunnyvale, California).
Pro-inflammatory factor and superoxide production in mice
The anti-inflammatory effect of naloxone in adult male FVB mice (8 to 12 weeks old) was examined. The mice received pretreatment with intraperitoneal (IP) naloxone (10, 20, or 25 mg/kg) or PBS 30 min before the experiment. All mice then received 20 mg/kg LPS by IP injection. Blood samples were obtained at 6 h after LPS injection from a catheter placed in the left carotid artery. Blood samples were centrifuged, and the plasma TNF-α levels were measured by ELISA. Lungs were harvested from the animals 6 h after LPS injection in each group as previously described. The tissues were fixed in 4% paraformaldehyde and embedded in paraffin for histopathological examination. The presence of inflammatory cells in lungs was determined by immunofluorescence. The sections were incubated with rat monoclonal antibody against mouse CD45, a leukocyte common antigen (1:50 dilution; Pharmingen, San Diego, California). After washing, fluorescein isothiocyanate-conjugated sheep anti-mouse immunoglobulin (Amersham Pharmacia Biotech, Piscataway, New Jersey) was applied as a secondary antibody. A laser scanning confocal microscope (Leica Microsystems, Wetzler, Germany) was used to examine the samples, and the CD45-positive cells were demonstrated by green immunofluorescence labeling. The total CD45-positive cell number was counted in 5 randomly selected sections under high-power field magnification (400×) for each mouse.
Oxidative fluorescent microtopography with the oxidative fluorescent dye dihydroethidium (DHE) was used to evaluate the in situ production of superoxide in aorta as described previously (15). Aortas were harvested from the animals at 6 h after LPS injection in each group as previously described. Unfixed frozen ring segments were cut into 30-μm-thick sections and placed on a glass slide. The DHE (10 μmol/l) was topically applied to each tissue section, and a cover slip was applied. Slides were incubated in a light-protected humidified chamber at 37°C for 30 min. Fluorescence was detected with a laser scanning confocal microscope (Leica Microsystems) with excitation at 488 nm and detection at 585 nm with a long-pass filter.
Naloxone treatment in apoE-deficient mice
ApoE-deficient mice were fed with a high cholesterol diet containing 21% fat and 0.15% cholesterol (PMI LabDiet 40097, Richmond, Indiana) from 8 to 17 weeks of age. The apoE-deficient mice received IP naloxone (10, 20, or 25 mg/kg/day) or PBS injection for 10 weeks during this period. After completing the treatment, blood samples were collected by direct heart puncture when killing the animals. The serum levels of total cholesterol, LDL, high-density lipoprotein (HDL), and triglycerides were measured by enzymatic methods with an automatic analyzer (Model 747, Hitachi Ltd. Co., Tokyo, Japan). The aorta was dissected from the aortic valve to the iliac bifurcation. To identify lipid-rich atherosclerotic lesions, the aorta was rinsed in 50% isopropanol for 2 min, incubated in 0.67% Oil-Red-O for 15 min, and washed by 10% isopropanol for 2 min. With a dissection microscope, the area of each atherosclerotic lesion was measured with Image Pro Plus software (Version 3.0.1; Media Cybernetics, Inc., Silver Spring, Maryland) and expressed as percentage of atherosclerotic area / total area of the aorta.
Effect of naloxone treatment in a mouse vascular remodeling model
Adult C57BL/6 mice (8 to 12 weeks) were anesthetized by IP injection of sodium pentobarbital. The left common carotid artery was isolated and ligated completely with a 6-0 silk suture near the carotid bifurcation as described previously (16). The mice received IP naloxone (10, 20, or 25 mg/kg/day) or PBS injection for 4 weeks immediately after surgery. After completing the treatment, the animals were killed and the segment of the left common carotid artery just proximal to the ligation was excised, fixed in 4% paraformaldehyde, and embedded in paraffin. Five transverse sections/animal were cut at 100-μm intervals and stained with hematoxylin-eosin. The borders of the internal lumen, internal elastic lamina (IEL), and external elastic lamina (EEL) were traced on a digitizing board with Meta Imaging Series 5.0 software (Adobe Inc., San Jose, California). The luminal, IEL, and EEL areas were measured. The neointima area was calculated by subtracting the luminal area from the IEL area, and the media area was calculated by subtracting the IEL area from the EEL area. The total vascular area was represented by EEL area. The ratio of neointima to media area (N/M ratio) was calculated. Average values were obtained from morphometric analysis of each section of the animal.
Data were given as mean values ± SD. The Mann-Whitney Utest was used to compare continuous variables between the 2 groups. Because there was a tremendous variability of vascular remodeling along the ligated carotid artery and the distance to the ligation site might influence the thickness of neointima formation, multiple regression analysis was performed to simultaneously analyze the contribution of the distance to ligation site and naloxone treatment on the response of carotid ligation (17). All statistical analyses were performed with SPSS 12.0 (SPSS Inc., Chicago, Illinois). The statistical significance level was set at p < 0.05, 2-tailed.
Effect of naloxone on macrophage activation
The initial experiment demonstrated that the most effective naloxone concentration range was 0.01 to 1 μmol/l. These concentrations could reduce TNF-α production by 50% in the THP1 cell culture after LPS stimulation (Table 1).Subsequent experiments were performed with these effective naloxone concentrations. The LPS treatment induced a dramatic increase of TNF-α (8.02 ± 0.42 ng/ml), IL-6 (5.02 ± 0.14 ng/ml), and MCP-1 (9.22 ± 1.17 ng/ml) in the medium of the THP-1 cell culture when compared with the PBS treatment (TNF-α 0.02 ± 0.02 ng/ml, IL-6 0.03 ± 0.02 ng/ml, MCP-1 0.03 ± 0.04 ng/ml). Naloxone pretreatment (0.01, 0.1, and 1 μmol/l) significantly reduced the macrophage production of TNF-α (0.01 μmol/l, 4.28 ± 0.39 ng/ml; 0.1 μmol/l, 4.10 ± 0.37 ng/ml; 1 μmol/l, 4.41 ± 0.36 ng/ml vs. 8.02 ± 0.42 ng/ml, all p < 0.05), IL-6 (0.01 μmol/l, 3.74 ± 0.31 ng/ml; 0.1 μmol/l, 3.28 ± 0.14 ng/ml; 1 μmol/l, 3.44 ± 0.48 ng/ml vs. 5.02 ± 0.14 ng/ml, all p < 0.05), and MCP-1 (0.01 μmol/l, 7.08 ± 0.16 ng/ml; 0.1 μmol/l, 7.32 ± 0.33 ng/ml; 1 μmol/l, 6.16 ± 0.16 ng/ml vs. 9.22 ± 1.17 ng/ml, all p < 0.05) in the medium of THP-1 cells after LPS stimulation (Fig. 1).
After oxLDL stimulation, there was also a dramatic increase of TNF-α (1.70 ± 0.06 ng/ml), IL-6 (1.51 ± 0.08 ng/ml), and MCP-1 (2.23 ± 0.34 ng/ml) in the medium of the THP-1 cell culture when compared with the PBS treatment (TNF-α 0.02 ± 0.01 ng/ml, IL-6 0.01 ± 0.01 ng/ml, MCP-1 0.16 ± 0.03 ng/ml). Naloxone pretreatment (0.01, 0.1, and 1 μmol/l) significantly reduced the macrophage production of TNF-α (0.01 μmol/l, 0.85 ± 0.12 ng/ml; 0.1 μmol/l, 0.62 ± 0.04 ng/ml; 1 μmol/l, 0.60 ± 0.05 ng/ml; vs. 1.70 ± 0.06 ng/ml, all p < 0.05), IL-6 (0.01 μmol/l, 1.05 ± 0.03 ng/ml; 0.1 μmol/l, 1.01 ± 0.01 ng/ml; 1 μmol/l, 1.05 ± 0.07 ng/ml vs. 1.51 ± 0.08 ng/ml, all p < 0.05), and MCP-1 (0.01 μmol/l, 0.95 ± 0.28 ng/ml; 0.1 μmol/l, 1.18 ± 0.19 ng/ml; 1 μmol/l, 1.09 ± 0.24 ng/ml vs. 2.23 ± 0.34 ng/ml, all p < 0.05) in the medium of THP-1 cells after oxLDL stimulation (Fig. 2).
Figure 3shows the production of superoxide in the THP-1 cell culture in the different treatment groups. Superoxide production was significantly increased after LPS stimulation (817,000 ± 90,000 RLU/10 min/ml) when compared with the control group treated with only PBS (183,000 ± 39,000 RLU/10 min/ml). Naloxone pretreatment (0.01, 0.1, and 1 μmol/l) significantly suppressed the elevation of macrophage superoxide production (0.01 μmol/l, 434,000 ± 124,000 RLU/10 min/ml; 0.1 μmol/l, 364,000 ± 202,000 RLU/10 min/ml; 1 μmol/l, 362,000 ± 24,000 RLU/10 min/ml vs. 817,000 ± 90,000 RLU/10 min/ml, all p < 0.05).
The MTT assays demonstrated that there were no significant changes of the cell viability between the THP-1 cell cultures receiving PBS, LPS, 0.01, 0.1, or 1 μmol/l naloxone or LPS plus 0.01, 0.1, or 1 μmol/l naloxone pretreatment.
Effect of naloxone on TNF-α and superoxide production in mice
Figure 4shows the effect of naloxone on the TNF-α and superoxide production in the mice after stimulation. The plasma level of TNF-α was significantly elevated in the LPS-treated (n = 9) compared with the PBS-treated (n = 7) mice (576.1 ± 43.6 pg/ml vs. 27.8 ± 13.1 pg/ml, p < 0.001). Pretreatment of the mice with naloxone (n = 8 in 10 mg/kg, n = 7 in 20 mg/kg, and n = 5 in 25 mg/kg naloxone) effectively reduced the elevation of the plasma TNF-α levels (10 mg/kg, 452.9 ± 55.0 pg/ml, p < 0.05; 20 mg/kg, 251.9 ± 59.6 pg/ml, p < 0.01; 25 mg/kg, 288.0 ± 33.9 pg/ml, p < 0.01 vs. 576.1 ± 43.6 pg/ml). There was more pulmonary inflammatory cell infiltration in the mice after LPS stimulation (n = 7) compared with PBS injection only (n = 9; 103 ± 16 vs. 45 ± 10, p < 0.01). In the mice pretreated with naloxone (n = 5 in 10 mg/kg, n = 6 in 20 mg/kg, and n = 5 in 25 mg/kg naloxone), the infiltration of inflammatory cells in the lungs was significantly decreased (10 mg/kg, 74 ± 10; 20 mg/kg, 58 ± 15; 25 mg/kg, 62 ± 12, all p < 0.01 vs. 103 ± 16). Aortic sections from the mice were stained with DHE and then imaged with a laser scanning confocal microscope. The LPS-treated mice showed a marked increase in fluorescence, reflecting an increase in superoxide production in aorta (Fig. 5).Naloxone pretreatment (10, 20, and 25 mg/kg) significantly reduced the increase of fluorescence intensity in the aortic sections from the LPS-treated mice.
Effect of naloxone on atherosclerosis and neointima formation
The apoE-deficient mice received IP naloxone (n = 7 in 10 mg/kg, n = 9 in 20 mg/kg, and n = 8 in 25 mg/kg naloxone) or PBS (n = 10) injection for 10 weeks. There were no significant differences of the total cholesterol, triglyceride, LDL, and HDL levels between the PBS-treatment and naloxone-treatment groups (Fig. 6).Naloxone treatment for 10 weeks significantly reduced the severity of aortic atherosclerotic lesions (10 mg/kg/day, 5.2 ± 3.8%, p < 0.01; 20 mg/kg/day, 3.4 ± 1.4%, p < 0.005; 25 mg/kg/day, 1.8 ± 1.2%, p < 0.005 vs. PBS 12.9 ± 6.1%) in the apoE-deficient mice (Fig. 7).
There was a progressively decreased lumen area and increased neointima formation in C57BL/6 mice after carotid artery ligation (Fig. 8).The lumen area was significantly larger (10 mg/kg/day, 24,475 ± 5,261 μm2; 20 mg/kg/day, 26,452 ± 9,024 μm2; 25 mg/kg/day, 29,650 ± 5,643 μm2, all p < 0.05 vs. PBS 12,859 ± 7,258 μm2) at 28 days in mice receiving naloxone treatment (n = 5 in 10, n = 7 in 20, and n = 5 in 25 mg/kg/day) compared with the control subjects receiving PBS only (n = 7). The neointima area was significantly reduced (10 mg/kg/day, 20,052 ± 4,372 μm2, p < 0.01; 20 mg/kg/day, 14,233 ± 4,040 μm2, p < 0.005; 25 mg/kg/day, 13,865 ± 3,494 μm2, p < 0.005 vs. PBS 33,623 ± 9,377 μm2) after naloxone treatment. The N/M ratio also decreased in the naloxone-treatment group (10 mg/kg/day, 0.66 ± 0.15; 20 mg/kg/day, 0.41 ± 0.18; 25 mg/kg/day, 0.39 ± 0.07, all p < 0.005 vs. PBS 1.25 ± 0.42) at 28 days after surgery (Fig. 8). In the regression model (Table 2),naloxone treatment had a statistically significant influence on the lumen, neointima, and total vascular areas in carotid remodeling after ligation, and the effect was independent of the distance to ligation.
The present study is the first demonstration that naloxone treatment can inhibit LPS-induced and oxLDL-induced macrophage activation and decrease proinflammatory factors and superoxide production. We show that treatment with naloxone decreases the vascular lesion in apoE-deficient mice and the neointima formation in C57BL/6 mice receiving carotid artery ligation.
Anti-inflammatory effect of naloxone
It has been reported that the opioid receptor antagonists naloxone and naltrexone display a novel anti-inflammatory effect. Recent studies demonstrated that naltrexone protects mice from septic shock induced by LPS (18,19). The compound suppresses plasma TNF-α elevation and reduces superoxide production in the aorta (18,19). Naloxone treatment inhibits the activation of microglia, the resident macrophage in brain, and its production of TNF-α, IL-1beta, and superoxide (11,12,20). Naloxone also can decrease cerebral ischemic injury by reducing neutrophil accumulation and chemokine expression (21). However, the mechanisms of naloxone’s anti-inflammatory effect have not been fully elucidated. The protective effect might not be directly related to opioid receptor binding, because the naloxone stereoisomer (+)-naloxone, an ineffective opioid receptor antagonist, also shows a similar anti-inflammatory effect (22). Recent evidence indicates that microglial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase might be a target of naloxone action. Microglial NADPH oxidase has been shown to be responsible for the production of superoxide that is toxic to neurons and amplified microglial proinflammatory gene expression (23). The naloxone-mediated anti-inflammatory effect disappeared in NADPH oxidase-deficient mice, indicating that inhibition of this enzyme is critical to the protective mechanism of naloxone and its related compound (23–25). Our study results further extend the previous observation of naloxone in microglia. We found that naloxone could inhibit the macrophage activation after LPS and oxLDL stimulation. Naloxone reduced the TNF-α production up to 50% (Figs. 1 and 2) in macrophage after LPS and oxLDL stimulation. It also decreased the superoxide production about 40% in macrophage stimulated by LPS (Fig 3). These results are in accordance with the previous report that microglia from NADPH oxidase-deficient mice display a 50% reduction in TNF-α messenger ribonucleic acid expression in response to LPS stimulation and are unable to produce superoxide (23). The superoxide derived from NADPH oxidase enhances the pro-inflammatory gene expression (26). It is likely that naloxone blocks the NADPH oxidase-dependent superoxide production, accounting for the 50% reduction of macrophage TNF-α production after stimulation. Further studies with opioid receptor knockout mice are necessary to elucidate the naloxone anti-inflammatory mechanism more clearly.
Naloxone and atherosclerosis
We used the apoE-deficient mice and vascular remodeling murine models to test the hypothesis that naloxone could decrease atherosclerosis through its inhibitory effect on macrophage activation. We started from the naloxone dose (10 mg/kg) that rescues rats or mice from septic shock (18,19). The 10 to 25 mg/kg naloxone doses were approximately equivalent to about 10−3to 10−4μmol/l blood concentrations in mice. These concentrations were effective in reducing TNF-α production from macrophage (Table 1). The apoE-deficient mice develop lesions that have many of the histological features of human atherosclerosis, including lipid deposition and macrophage infiltration (27,28). Monocytes attachment to the endothelium could be observed at 6 weeks, and foam cell lesions could be developed as early as 8 weeks of age. In our study, naloxone treatment that was started from 8 weeks of age in the apoE-deficient mice significantly attenuated the severity of atherosclerotic lesion formation. Our results demonstrate that naloxone inhibits macrophage activation in an early stage and the severity of atherosclerosis lesion decreases. In the vascular remodeling model, because the arterial pulsation is still present, there is only minimal thrombus formation near the ligation site (16,29). The altered flow condition after ligation not only increases the expression of endothelial-leukocyte adhesion molecules but also leads to the monocytes activation, with a subsequent influx of monocytes into the vessel wall (16,30,31). Although there is no lipid component in this model, flow cessation produces significant vessel wall inflammation and causes a smooth muscle cell-rich neointima. A previous study supported an important role of macrophage infiltration in the up-regulation of matrix metalloproteinase and vascular smooth muscle cell proliferation in the arterial remodeling process and neointima formation (32). In our study, naloxone treatment for 4 weeks after carotid ligation could reduce the severity of neointima formation and alter the remodeling process. This effect might have important clinical implications, because the major source of oxidative stress in the artery wall is the monocyte/macrophage NADPH oxidase (33). Naloxone blockage of macrophage activation with reduced oxidative stress and inflammation in the vasculature is likely to prevent the deterioration of atherosclerosis or neointima formation. Further studies are required to determine the effect of naloxone on vascular disease and restenosis in humans. For statistical analyses in the study, although there were many individual comparisons within studies, the statistical tests were performed according to the hypotheses that were determined before the experiment. So the multiple comparisons should not be a major concern.
On the basis of these observations, we propose that naloxone can inhibit macrophage activation after LPS and oxLDL stimulation with reduced oxidative stress. It can serve as a protective anti-oxidative and anti-inflammatory agent in the prevention of atherosclerotic plaque and neointima formation in the vascular system.
The authors would like to thank How-Ran Guo, MD, PhD, for his kind help in statistical analysis of the data.
This work was supported by the Ministry of Education Program for Promoting Academic Excellence of Universities under the grant number 91-B-FA09-2-4 and the National Science Council, Grants NSC 94-2320-B-006-030, NSC 95-2752-B-006-003-PAE, and NSC 95-2752-B-006-005-PAE, Taipei, Taiwan. This study was presented in part at the Annual Scientific Session of the American Heart Association, Dallas, Texas, November 13–16, 2005.
- Abbreviations and Acronyms
- external elastic lamina
- enzyme-linked immunosorbent assay
- high-density lipoprotein
- internal elastic lamina
- monocyte chemoattractant protein-1
- 3 (4,5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide
- nicotinamide adenine dinucleotide phosphate
- neointima/media area ratio
- oxidized low-density lipoprotein
- phosphate-buffered saline
- relative light units
- human acute monocytic leukemia cell line
- tumor necrosis factor-α
- Received April 13, 2006.
- Revision received June 6, 2006.
- Accepted July 3, 2006.
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