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From the Departments of Clinical Renal Regeneration and Internal Medicine (K.H., T.F.), Division of Nephrology and Endocrinology, University of Tokyo, Japan; and the Department of Pharmacology (T.N.), Teikyo University School of Medicine, Japan.
Correspondence to Keiichi Hishikawa, Department of Internal Medicine, Division of Nephrology and Endocrinology, University of Tokyo, Hongo, 7-3-1, Bunkyo-ku, Tokyo, 113-8655, Japan. E-mail hishikawa-tky@umin.ac.jp
Abstract
Objective— Caffeic acid phenethyl ester (CAPE), a natural flavonoid, specifically blocks activation of nuclear factor-B (NF-B). We examined the effects of oral CAPE supplementation on atherogenesis in apolipoprotein E–deficient (apoE–/–) mice.
Methods and Results— Ten-week-old male apoE–/– mice were supplemented orally with CAPE (30 mg/kg body weight) for 12 weeks. At the end of administration, atherosclerosis progression, NF-B activity, gene expression profiling by microarray analysis, and oxidative stress were studied. Treatment of apoE–/– mice with CAPE significantly reduced aortic atherosclerosis, NF-B activity, and expression of NF-B–related genes in the aorta. Moreover, expression of other gene clusters such as basic transcription factors, growth factors, cytokines, cell adhesion proteins, and extracellular matrix were also significantly reduced by treatment with CAPE. Plasma isoprostane level in apoE–/– mice was also significantly reduced by CAPE.
Conclusion— In apoE–/– mice, oral CAPE supplementation attenuates the atherosclerotic process. This may be attributable to direct inhibition of NF-B in the lesion and reduction of systemic oxidative stress.
In apoE–/– mice, oral caffeic acid phenethyl ester (CAPE) supplementation attenuates the atherosclerotic process and reduces NF-B activity and expression of NF-B–related genes in the aorta. This may be attributable to direct inhibition of NF-B in the lesion and reduction of systemic oxidative stress.
Key Words: atherosclerosis ? NF-B ? microarray ? oxidative stress ? flavonoid
Introduction
Nuclear factor-B (NF-B) is a good therapeutic target for cardiovascular disease,1,2 and numerous efforts are being made to develop safe NF-B inhibitors. Among the candidates are natural flavonoids, which constitute a promising class of dietary antioxidants that are found ubiquitously in fruits, vegetables, and tea. For example, resveratol, which is found in red wine, can inhibit NF-B activity, the abrogation of which may contribute to the ability of red wine to reduce mortality from coronary heart disease and cancer.3,4 Curcumin, another NF-B inhibitor, is an effective inhibitor of tumor initiation and promotion in different carcinogen-induced models.5 Among these natural flavonoids, the most promising is caffeic acid phenethyl ester (CAPE), which is structurally related to 3,4-dihydroxycinnamic acid and can be obtained from propolis, a honey constituent.6 CAPE is the only compound that has been shown to inhibit the HIV integrase enzyme needed for integration of HIV DNA into the host genome7 and is a potent and specific NF-B inhibitor.8 We clarified recently that CAPE induced apoptosis in human breast cancer cells, but not in normal cells, by inhibiting NF-B, leading to fas aggregation.9 The present studies were designed to examine whether CAPE could prevent atherosclerosis by inhibiting NF-B activity in vivo.
Methods
Animals and Compound
All experimental procedures were in accordance with institutional guidelines for animal research. Apolipoprotein E–deficient (apoE–/–) mice were purchased from Taconic. CAPE was obtained from Bachem AG. ApoE–/– mice received CAPE-supplemented chow for 12 weeks. Compliance with CAPE supplementation was confirmed by measuring the consumption of chow supplemented with CAPE every day. The average chow intake was 5 to 6 g per day in control and CAPE-supplemented groups.
Quantitation of Aortic Atherosclerosis
The extent of atherosclerosis in the mouse aorta was determined using an en-face method.10 Aortas were fixed in paraformaldehyde and stained with oil red O. Morphometric image analysis of the captured digitalized image of aorta was done using ImagePro Plus image software, and the lesion areas covering the aortic surface were quantitated.
NF-B Assay
Specimens from mice were fixed at –80°C. The tissue was diced into small pieces with a cooled razor blade and placed in lysis buffer. Nuclear extracts were prepared using a nuclear extract kit (Active Motif). Total protein (20 μg) was loaded in each well, and NF-B activity was measured using a TransAM NF-B p65 kit (Active Motif) according to manufacturer directions.
Plasma Lipid and F2-Isoprostane Measurement
Blood samples were collected from animals fasted overnight, and plasma lipid levels were measured using an automated analyzer (Fuji Dri-Chem 3500V). Plasma F2-isoprostane level was measured using a StressXpress 8-Iso PGF2a ELISA kit (StressGen).
Microarray Analysis
DNA microarray hybridization experiments were performed using Mouse 3.8 (Clontech) according to the protocol of the manufacturer. The protocol and the complete list of genes on Mouse 3.8 are available on the web. DNA arrays were scanned with a Gene Pix 4000.11,12
Statistics
Results are shown as mean±SEM. Data were analyzed by ANOVA and subsequently by Student unpaired 2-tailed test. P values <0.05 were considered significant.
Results
Body Weight and Plasma Lipids
At 10 weeks of age, apoE–/– mice were assigned randomly to 2 groups to receive normal chow or normal chow supplemented with CAPE for 12 weeks. We preliminarily examined the doses of 3 mg/kg body weight and 10 mg/kg body weight, but the effect was not significant (data not shown). It is reported that CAPE at a dose of 30 mg/kg body weight reduced neointimal proliferation by balloon injury,13 polysaccharide-induced colitis,14 and irradiation-induced inflammation.15 Therefore, we used 30 mg/kg in the following experiments. As shown in Figure 1A, there was no significant change in body weight through the treatment period. Compared with 10 weeks, plasma levels of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein–cholesterol (LDL-C) were significantly higher at 22 weeks in both groups, but there was no significant difference between values in the 2 groups (Figure 1B).
Figure 1. Effects of oral CAPE supplementation on body weight and lipid levels. A, Body weight was measured every week. B, Blood was collected at 10 weeks (black bars) and 22 weeks (white bars normal chow; gray bars chow supplemented with CAPE) for measurement of TC, TG, high-density lipoprotein–cholesterol (HDL-C), and LDL-C. Values are mean±SEM (n=12). *P<0.05 compared with 10 weeks; cont indicates control.
Effect of CAPE on Extent of Atherosclerosis
Mice were killed at 22 weeks of age and their aortas were harvested. Morphometric quantitation showed that the lesion area in the thoracic aorta was significantly less in CAPE-treated apoE–/– mice than in untreated mice (Figure 2A). When the total aortic surface area was measured, aortic lesion area was also significantly less in the CAPE-treated group than in the untreated group (Figure 2B). We also performed histopathologic analysis, but there was no obvious regression such as unstable plaque lesion in the lesion of CAPE-treated group. These results suggest that CAPE is effective to prevent initiation and progression of new lesion, but its effect is minimal for regression of it.
Figure 2. Effect of dietary CAPE supplementation on extent of atherosclerosis in apoE–/– mice. Areas of lesions in the thoracic aorta (A) and total aortic lesions (B) were determined at 10 weeks (black bars) and 22 weeks (white bars normal chow; gray bars chow supplemented with CAPE) in en-face photographs with oil red O staining. Values are mean±SEM (n=12). *P<0.05 compared with 10 weeks; **P<0.05 compared with 22 weeks with normal chow.
Effect of CAPE on NF-B Activation
Compared with control mice (C57/B6 22 weeks), NF-B activity (p65) was significantly higher in the aorta of apoE–/– mice but not in the left ventricle (LV), right ventricle (RV), and kidney (Kid). Activation of NF-B in the aorta of apoE–/– mice was significantly inhibited by treatment with CAPE, but CAPE showed no effect in the LV, RV, and Kid (Figure 3).
Figure 3. CAPE inhibits NF-B activity in apoE–/– mice. NF-B activity in the LV, RV, aorta (Ao), and kid was measured at 22 weeks (open bars C57/B6 with normal chow; black bars apoE–/– mice with normal chow; gray bars apoE–/– mice supplemented with CAPE). Values are mean±SEM (n=12). *P<0.05 compared with C57/B6 mice with normal chow; **P<0.05 compared with apoE–/– mice with normal chow.
Gene Expression in Aorta
To evaluate the effect of CAPE, comprehensive gene expression in the aorta was assessed by microarray analysis. Compared with control mice (C57/B6 22 weeks), 88.9% of genes (3343/3758) were highly expressed in the aorta of apoE–/– mice (Figure 4A). On the other hand, 63.4% of genes (2384 of 3758) were highly expressed in apoE–/– mice treated with CAPE (Figure 4B). Among 3758 genes, expression of NF-B related genes are shown in Figure 4C. Except for hemeoxygenase 1, these genes were significantly activated in apoE–/– compared with control (C57/B6) mice. Moreover, expression of these genes (tumor necrosis factor- [TNF-], interleukin-2 [IL-2], platelet-derived growth factor -BB, and E-selectin) in apoE–/– mice was significantly reduced by treatment with CAPE (Figure 4C). Gene expression of other NF-B–related genes such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 (ICAM-1) was evaluated by quantitative polymerase chain reaction, but CAPE had no effect on these 2 genes (data not shown). We further examined the effect of CAPE on expression of several gene clusters such as basic transcription factors (34 genes), growth factor cytokines (53 genes), cell adhesion proteins (29 genes), and extracellular matrix (50 genes; Figure 5). Average expression of all gene clusters was significantly higher compared with control mice (C57/B6 at 22 weeks). Although these gene clusters are not related to NF-B, treatment with CAPE significantly reduced the expression of all these gene clusters.
Figure 4. Microarray analysis of aorta of apoE–/– and C57/B6 mice. Scatter-plot analyses of gene expression (3758 genes) in aorta of apoE–/– vs that in C57/B6 mice (A), and apoE–/– mice supplemented with CAPE vs that of C57/B6 mice (B). Theoretically, genes that were equally expressed in apoE–/– and C57/B6 mice should be on the y=x line. The majority of points (3343 of 3758) are located in the top left area in apoE–/– mice (A). On the other hand, points are seen near the y=x line in apoE–/– mice supplemented with CAPE (B). C, Gene expression of NF-B–related genes in the aorta of C57/B6 mice (open bars; n=4), apoE–/– mice (black bars; n=6), and apoE–/– mice supplemented with CAPE (gray bars; n=6). HO-1 indicates hemeoxygenase 1. Values are mean±SEM. *P<0.05 compared with C57/B6 mice; **P<0.05 compared with apoE–/– mice.
Figure 5. Expression of gene clusters related to atherosclerosis. Average gene expression of basic transcription factors (34 genes), growth factors and cytokines (53 genes), cell adhesion proteins (29 genes), and extracellular matrix (50 genes) were examined in the aorta of C57/B6 mice (open bars; n=4), apoE–/– mice (black bars; n=6), and apoE–/– mice supplemented with CAPE (gray bars; n=6). Values are mean±SEM. *P<0.05 compared with C57/B6; **P<0.05 compared with apoE–/– mice.
Effect of CAPE on Systemic Oxidative Stress
Because CAPE not only inhibits gene expression of NF-B–related genes but also other gene clusters, we evaluated its effect on systemic oxidative stress. Plasma F2-isoprostane level was significantly higher in apoE–/– compared with control mice, but this was significantly reduced by treatment with CAPE (Figure 6). We also measured lipid peroxidation marker malondialdehyde-modified LDL by ELISA kit (LPO assay kit; BIOXYTEC LPO, Oxis), but CAPE had no effect on it.
Figure 6. CAPE reduced systemic oxidative stress. Blood was collected at 22 weeks for measurement of plasma F2-isoprospane level. Open bars, C57/B6 mice; black bars, apoE–/– mice; gray bars, apoE–/– mice supplemented with CAPE. Values are mean±SEM (n=12). *P<0.05 compared with C57/B6 mice; **P<0.05 compared with apoE–/– mice.
Discussion
This study demonstrated that a natural NF-B inhibitor, CAPE, prevents atherosclerosis even in the presence of a high level of plasma cholesterol without serious adverse effects. We confirmed that CAPE inhibits NF-B activity in the aorta, the target organ of hypercholesterolemia, and have also shown that CAPE reduced the expression of NF-B–related genes such as TNF-, IL-2, PDGF-BB, and E-selectin in the aorta, demonstrating that treatment with CAPE actually inhibited molecular events downstream of NF-B activation in vivo. NF-B is a good target for treating atherosclerosis, but there is no specific and potent inhibitor that can be applied to clinical therapy. CAPE, a structural derivative of flavonoids, has been shown to be a safe pharmacological compound with known anti-inflammatory, immunomodulatory, anticarcinogenic, and antioxidant properties. CAPE completely blocked activation of NF-B induced by a wide variety of inflammatory agents, including TNF-, phorbol ester, ceramide, okadaic acid, and H2O2 in vitro.8
NF-B inhibition by CAPE has been examined extensively in many in vitro studies. In human coronary artery endothelial cells, CAPE inhibited oxidized LDL–mediated degradation of IB and NF-B activation and inhibited oxidized LDL–induced upregulation of angiotensin II type 1 receptor expression.16 CAPE inhibited oxidized LDL–induced apoptosis in human aortic endothelial cells mediated by the action of the lectin-like endothelial receptor for oxidized LDL.17 CAPE concentration-dependently inhibited chlamydophila pneumoniae–induced ICAM-1 upregulation in human aortic endothelial cells by inhibiting NF-B.18 Moreover, CAPE specifically inhibited IL-2 gene transcription and IL-2 synthesis in stimulated T-cells and inhibited NF-B–dependent transcriptional activity without affecting degradation of the cytoplasmic NF-B inhibitory protein IB.19 Compounds that have potent NF-B inhibition frequently show a limited effect in vivo. On the other hand, CAPE shows a potent effect in vivo.20–25 Concerning vascular disease, CAPE also inhibited NF-B activation in rat carotid arteries induced by balloon injury in vivo.13 As Ceschel et al reported that CAPE has good permeability across mucosa in vitro,26 CAPE may also have good bioavailability pharmacologically in vivo.
Compared with control mice (C57/B6), microarray analysis of the aorta clarified that many genes were activated in apoE–/– mice, and CAPE normalized 40% of them. The precise mechanism is unclear, but several pathways could be involved, except NF-B inhibition. In smooth muscle cells from porcine coronary artery, CAPE arrested angiotensin II–dependent DNA synthesis and migration. CAPE prevented phosphorylation of cyclin-dependent kinase 2 and retinoblastoma protein.27 CAPE also modulates ion channels. CAPE increased the Ca2+-activated K+ current and slightly suppressed the voltage-dependent L-type Ca2+ current. CAPE-stimulated channel activity was dependent on membrane potential.28 CAPE inhibited the increase in cytosolic Ca2+ concentration triggered by stimulation of aortic smooth muscle cells with phenylephrine or KCl.29 Considering the structure of CAPE, we are not able to explain these effects, but these pathways could contribute to the protective effect of CAPE on atherosclerosis.
CAPE is well known to have a free radical–scavenging effect and also inhibits xanthine oxidase activity.30 Recently, the structure–activity relationships of synthetic caffeic acid amide and ester analogs as potential antioxidants and free radical scavengers have been investigated. The 2,2-diphenyl-1-picrylhydrazyl radical–scavenging activity of the test compounds was N-trans-caffeoyl-L-cysteine methyl ester > N-trans-caffeoyldopamine > N-trans-caffeoyltyramine > N-trans-caffeoyl-?-phenethylamine > Trolox C > CAPE > caffeic acid > ferulic acid. On the other hand, antioxidative activity order was CAPE > N-trans-caffeoyl-?-phenethylamine > N-trans-caffeoyldopamine > N-trans-caffeoyltyramine > N-trans-caffeoyl-L-cysteine methyl ester > caffeic acid > Trolox C > ferulic acid. These results suggested that the antioxidative activity of CAPE depends not only on the hydroxyl groups or catechol rings but also on the partition coefficient or hydrophobicity of the compounds.31 In rat bleomycin-induced pulmonary fibrosis models, an increase in catalase and superoxide dismutase activities and a decrease in myeloperoxidase activity were seen after CAPE application.32 CAPE was more effective in decreasing the tissue levels of NO, hydroxyproline, and malondialdehyde than vitamin E.32 Pratico et al reported that oxidative stress is increased in the apoE–/– mouse, is of functional importance in the evolution of atherosclerosis, and can be suppressed by oral administration of the antioxidant vitamin E.33 In our studies, treatment with CAPE reduced the plasma F2-isoprostane level and inhibited not only NF-B– related genes but also several gene clusters such as basic transcription factors, growth factor cytokines, cell adhesion proteins, and extracellular matrix. Because oxidative stress is well known to activate these genes, the antioxidant property of CAPE may play a key role to reduce atherosclerosis in apoE–/– mice. In conclusion, our results suggest that CAPE is a promising compound for treatment of atherosclerosis by inhibiting NF-B and by reducing oxidative stress.
Acknowledgments
This work was supported in part by Mochida Pharmaceutical Co, Ltd.
Received August 5, 2004; accepted October 5, 2004.
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