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【摘要】
Objective— We recently identified esculeoside A, a new spirosolane-type glycoside, with a content in tomatoes that is 4-fold higher than that of lycopene. In the present study, we examined the effects of esculeoside A and esculeogenin A, a new aglycon of esculeoside A, on foam cell formation in vitro and atherogenesis in apoE-deficient mice.
Methods and Results— Esculeogenin A significantly inhibited the accumulation of cholesterol ester (CE) induced by acetylated low density lipoprotein (acetyl-LDL) in human monocyte-derived macrophages (HMDM) in a dose-dependent manner without inhibiting triglyceride accumulation, however, it did not inhibit the association of acetyl-LDL to the cells. Esculeogenin A also inhibited CE formation in Chinese hamster ovary cells overexpressing acyl-coenzymeA (CoA): cholesterol acyl-transferase (ACAT)-1 or ACAT-2, suggesting that esculeogenin A suppresses the activity of both ACAT-1 and ACAT-2. Furthermore, esculeogenin A prevented the expression of ACAT-1 protein, whereas that of SR-A and SR-BI was not suppressed. Oral administration of esculeoside A to apoE-deficient mice significantly reduced the levels of serum cholesterol, triglycerides, LDL-cholesterol, and the areas of atherosclerotic lesions without any detectable side effects.
Conclusions— Our study provides the first evidence that purified esculeogenin A significantly suppresses the activity of ACAT protein and leads to reduction of atherogenesis.
Esculeogenin A, a new aglycon of esculeoside A isolated from tomato, significantly inhibited the accumulation of cholesterol ester in macrophages by inhibiting acyl-CoA: cholesterol acyl-transferase (ACAT). Oral administration of esculeoside A to apoE-deficient mice significantly reduced the levels of serum cholesterol and the areas of atherosclerotic lesions.
【关键词】 esculeogenin A atherosclerosis ACAT human monocytederived macrophages foam cells
Introduction
The presence of a large cluster of macrophage-derived foam cells in situ in the subendothelial spaces is one of the characteristic features of early stage atherosclerotic lesions. 1 Macrophages take up chemically-modified low-density lipoproteins (LDL), such as oxidized LDL (Ox-LDL) and acetylated LDL (acetyl-LDL) through the scavenger receptors 2 such as class A scavenger receptor (SR-A), 3 class B scavenger receptor (CD36), 4 and class B scavenger receptor type-I (SR-BI). 5 Because free cholesterol, which is incorporated into the cells with modified LDL through the scavenger receptors, is toxic to the cells, it is esterified to the cholesterol ester (CE) by acyl CoA: cholesterol acyl-transferase (ACAT), an intracellular enzyme located in the rough endoplasmic reticulum. 6 These reactions change the macrophages to foam cells that are characterized by intracellular accumulation of CE. To date, 2 human ACAT isozymes (ACAT-1 and ACAT-2) are known. 7,8 ACAT-1 is highly expressed by macrophage-derived foam cells in atherosclerotic lesions and upregulated during monocytic differentiation into macrophages. 9 In addition, ACAT-1 is located in the Kuppfer cells of the liver, kidney, and adrenal cortical cells, whereas ACAT-2 is mainly located in hepatocytes and intestinal mucosal cells. 9 These findings are consistent with the notion that ACAT-1 plays a critical role in foam cell formation in macrophages; whereas ACAT-2 is responsible for the cholesterol absorption process in intestinal mucosal cells. 9 Because foam cell formation by these mechanisms is believed to play an essential role in the progression of early atherosclerotic lesions in vivo, prevention of foam cell formation is considered as one of the major targets for the treatment of atherosclerosis. From this point of view, many investigators have examined the usefulness of a number of antiatherosclerotic agents using various strategies such as prevention of LDL oxidation, 10 inhibition of scavenger receptor expression, 11 and ACAT activity. 12 We recently isolated esculeoside A, 13 a new spirosolane type glycoside, from the fruit of tomatoes ( Lycopersicon esculentum ) such as the cherry tomato and the Momotaro tomato. In addition, we identified a new compound named esculeogenin A 14 as aglycon of esculeoside A by hydrolysis of esculeoside A. Because the sugar chains of glycosides in natural products are degraded by the action of intestinal bacteria after oral administration, the aglycons act as physiologically active substances. 15,16 Therefore, esculeoside A also changes into esculeogenin A in the intestine, followed by production of esculeogenin A in vivo.
Because lifestyle related diseases such as atherosclerosis and diabetes progress gradually because of unfavorable dietary habits, improvement of daily nutritional intake is thought to prevent the pathogenesis of these diseases. For this reason, we have prepared 50 crude extracts and 80 purified compounds from natural products, and measured their inhibitory effect on foam cell formation in human monocyte-derived macrophages (HMDM). The results showed that purified esculeogenin A significantly suppresses the activity of ACAT protein and leads to reduction of atherogenesis.
Methods
Preparation of Esculeoside A and Esculeogenin A
Esculeoside A and esculeogenin A were prepared as previously described 13,14 (Please see supplemental Figure I, available online at http://atvb.ahajournals.org).
Isolation of Monocyte and Endocytic Uptake of Acetyl-LDL
Human peripheral mononuclear cells were isolated from the blood of healthy volunteers by Ficoll density gradient centrifugation (Ficoll-Paque from GE Healthcare Bio-Sciences). Acetyl-LDL was prepared by chemical modification of LDL with acetic anhydride as previously described. 17 Acetyl-LDL was labeled with 125 I as described by McFarlane. 18 The differentiated human monocyte-derived macrophages (HMDM) were incubated at 37°C for 5 hour with 50 µg/mL 125 I-acetyl-LDL in the presence of the indicated concentration of esculeogenin A, then the cell associated radioactivity and cell degradated radioactivity were measured as described supplementary information I.
Assay for Foam Cell Formation (CE-Accumulation)
HMDM monolayers were incubated with 50 µg/mL acetyl-LDL for 24 hours in the presence of 0.1 mmol/L [ 3 H]oleate conjugated with BSA, and cellular lipids were extracted for determination of radioactivity of cholesteryl-[ 3 H]oleate as described previously. 19
Assay for ACAT Activity and ACAT Expression
Microsomes prepared from HMDM were used as an enzyme source. Microsomes were incubated for 15 minutes with 250 µmol/L [ 14 C]oleoyl-CoA in the presence or absence of esculeogenin A, and the formation of cholesteryl [ 14 C]oleate was measured.
Expression level of ACAT-1 and scavenger receptors were measured by Western blotting analysis (please see supplementary information I).
In Vivo Antiatherosclerotic Activity
Apolipoprotein E (apoE)-deficient mice (C57BL/6.KOR- Apoe shl ) were fed a normal rodent chow diet (Clea) and esculeoside A (50 and 100 mg/kg of body weight) containing diets and administered orally every day for 90 days. Total cholesterol, LDL cholesterol and triglyceride levels in serum were determined. Whole aorta were collected and stained with Sudan IV, and cross sections of proximal aorta were prepared and stained with oil red as described 20 (please see supplementary information I).
For enhanced Materials and Methods of in vitro experiments used on this article, please see supplementary information I.
Results
Inhibitory Effect of Esculeogenin A on Acetyl-LDL–Induced CE Accumulation and Endocytic Uptake of 125 I-acetyl-LDL by HMDM
Incubation of HMDM for 24 hours with 50 µg/mL acetyl-LDL increased CE accumulation. Under the assay conditions used, esculeoside A and lycopene significantly inhibited CE accumulation. However, esculeogenin A, esculeoside A, and lycopene caused no morphological changes or cytotoxic effects on HMDM, even at 100 µmol/L (data not shown). Esculeogenin A showed the highest inhibitory effect among tested natural compounds in a dose-dependent manner ( Figure 1 A). Based on the result, we then focused on the inhibitory effect of esculeogenin A on CE accumulation. Esculeogenin A did not show any inhibitory effect on triglyceride (TG) synthesis even at the highest dose ( Figure 1 B), suggesting that esculeogenin A inhibits CE synthesis selectively in the HMDM. When 50 µg/mL of 125 I-acetyl-LDL was incubated at 37°C for 5 hours with the cells, significant amounts of 125 I-acetyl-LDL were associated with the cells ( Figure 1 C) and subjected to lysosomal degradation by the same cells ( Figure 1 C), whereas these cellular responses were not inhibited by esculeogenin A.
Figure 1. Inhibitory effect of esculeogenin A on CE accumulation by acetyl-LDL uptake in HMDM. HMDM were incubated with acetyl-LDL and [ 3 H]oleate conjugated with BSA in the absence or presence of escuoleogenin A ( ), esculeoside A () and lycopene ( ). [ 3 H]CE (A) and [ 3 H]TG (B) were measured. C, HMDM were incubated with esculeogenin A and 125 I-acetyl-LDL, followed by determination of cell-association () and cell-degradation ( ) of 125 I-acetyl-LDL. Data are mean±SD. * P <0.05; ** P <0.01; *** P <0.001 (esculeogenin A, esculeoside A. or lycopene vs without compounds).
Inhibitory Effect of Esculeogenin A on Acetyl-LDL–Induced CE Accumulation in Human ACAT Overexpressing CHO Cells
We subsequently measured the involvement of ACAT in the esculeogenin A–induced reduction of CE accumulation in the HMDM. Incubation of CHO cells overexpressing human ACAT-1 (hACAT-1 CHO) and human ACAT-2 (hACAT-2 CHO) for 24 hours with medium containing 10% fetal calf serum in the presence of [ 3 H]oleate increased CE accumulation. Under these conditions, esculeogenin A inhibited CE accumulation in both hACAT-1 and hACAT-2 CHO cells in a dose-dependent manner ( Figure 2A and 2 B). Esculeogenin A caused no morphological changes or cytotoxic effects on both cells even at 100 µmol/L esculeogenin A (data not shown). Because esculeogenin A showed a significant inhibitory effect on CE accumulation in both HMDM and hACAT CHO cells in a similar fashion, esculeogenin A may serve as an inhibitor of cholesterol esterification, possibly by inhibiting ACAT activity and/or ACAT expression.
Figure 2. Inhibitory effect of esculeogenin A on CE accumulation and ACAT activity in hACAT CHO cells and HMDM. (A) hACAT-1 CHO cells (; A) and hACAT-2 CHO cells (; B) were incubated with [ 3 H]oleate conjugated BSA and esculeogenin A. The ACAT activity of HMDM (C), hACAT-1 CHO cells (D), or hACAT-2 CHO cells (E) were measured. Data are mean±SD. * P <0.005; ** P <0.001; *** P <0.0001 (esculeogenin A vs without compounds).
Inhibitory Effects of Esculeogenin A on ACAT Activity in HMDM and hACAT CHO Cells
As shown in Figure 2 C, esculeogenin A inhibited ACAT activity in a dose-dependent manner. Similar results were observed by hACAT-1 and hACAT-2 CHO cells. Thus, formation of cholesteryl [ 14 C]oleate by microsomes prepared from hACAT-1 or hACAT-2 CHO cells was inhibited in the presence of esculeogenin A in a dose-dependent manner ( Figure 2D and 2 E). These data suggest that esculeogenin A has a significant inhibitory effect on foam cell formation from HMDM by inhibiting ACAT activity.
Inhibitory Effects of Esculeogenin A on SR-A, SR-BI, and ACAT-1 Expression in HMDM
Incubation of HMDM for 24 hours in the presence of indicated concentration of esculeogenin A resulted in inhibition of ACAT-1 expression in a dose-dependent manner, whereas expression of SR-A and SR-BI remained unchanged compared with the control (supplementary Figure II in supplementary information I). These results suggest that esculeogenin A has a significant inhibitory effect on foam cell formation from HMDM through the inhibition of both the expression and activity of ACAT.
Changes in Body Weight and Biochemical Data of Plasma Samples in ApoE-Deficient Mice
We next administrated esculeoside A to apoE-deficient mice to examine effect on atherogenesis. As shown in Figure 3 A, mean body weight gain was unchanged by administration of esculeogenin A for 90 days. Total cholesterol levels showed a trend toward reductions after the oral administration of esculeoside A (50 mg/kg/d), and were significantly reduced by approximately 25% after administration of 100 mg/kg/d ( Figure 3 B). Furthermore, administration of esculeoside A (100 mg/kg/d) significantly reduced serum levels of LDL cholesterol ( Figure 3 C) and triglycerides ( Figure 3 D) by approximately 25% ( Figure 3 C) and 45% ( Figure 3 D), respectively, without changing TC/HDL ratio (data not shown) compared with the control group.
Figure 3. Change in body weight and biochemical data of plasma samples in apoE-deficient mice. ApoE-deficient mice were fed diets with or without esculeoside A (EsA) (n=10, each group) and measured body weight (A), plasma total cholesterol (B), LDL cholesterol (C), TG (D). Data are mean±SD. * P <0.05; ** P <0.01.
Inhibition of ACAT Activity in ApoE-Deficient Mice by Esculeoside A Treatment
Administration of esculeoside A (100 mg/kg/d) to apoE-deficient mice for 21 days significantly reduced the ACAT activity in the liver (supplemental Figure IIIA in supplementary information I) and peritoneal macrophages (supplemental Figure IIIB in supplementary information I), whereas intestinal ACAT activity (supplemental Figure IIIC in supplementary information I) did not change compared with the control group.
Inhibition of Atherogenesis in ApoE-Deficient Mice by Esculeoside A Treatment
Although surface atherosclerotic lesions were observed in both groups mainly in the arch region, total surface lesions in the esculeoside A (100 mg/kg/d)-treated group were significantly reduced compared with the control group (8.2% versus 13.8%, P <0.01; Figure 4A, 4B, and 4 E). Cross sections of the aortic sinus showed a marked thickening of the intima filled with oil red O–positive foamy cells in the control mice (C), whereas such lesions were greatly reduced in esculeoside A-treated mice (D). The cross-sectional lesion area in the esculeoside A (100 mg/kg/d)-treated mice was significantly smaller (by 52%) than that of control mice (214,508 versus 444,555 µm 2, P <0.005; Figure 4C, 4D, and 4 F). LC-MS/MS analyses demonstrated that esculeogenin A was detected from the aorta of esculeoside A-treated apoE-deficent mice (Supplementary Figure IV in supplementary information I), demonstrating that orally administrated esculeoside A is converted into esculeogenin A by intestinal bacteria.
Figure 4. Inhibition of atherogenesis in apoE-deficient mice by esculeoside A. Pinned-out aortas showing Sudan-IV-positive lesions of apoE-deficient mice without esculeoside A treatment (A) and with esculeoside A (100 mg/kg/d) treatment (B), magnification x 3. Representative sections of aortic sinus atherosclerosis stained with oil red O in apoE-deficient mice without esculeoside A treatment (C) and with esculeoside A (100 mg/kg/d) treatment (D), magnification x 200. Quantitative evaluations of surface atherosclerotic lesions in whole aortae (E) and cross sectional lesions of aortic sinus (F). Data are mean±SD. * P <0.05; ** P <0.001; *** P <0.005.
Discussion
Because both of the ACAT isozymes are expressed in the liver and provide cholesteryl esters for VLDL, 9 an ACAT-1 inhibitor, such as K-604, significantly decreases the serum cholesterol level. 20 Furthermore, it is known that ACAT inhibitors block dietary cholesterol absorption in the intestines when animals are fed with high fat/high cholesterol diet. 21 Because we conducted animal experiments using apoE-deficient mice administered a normal diet, it is reasonable that the administration of esculeoside A inhibited the ACAT activity in the liver and macrophages rather than in the intestine, and decrease endogenous cholesterol production. However, the mechanism by which esculeoside A ameliorates the level of triglycerides remains unclear. A similar question has arisen regarding some reported ACAT inhibitors. Thus, oral administration of ACAT inhibitors such as R-755, 21 U-73482, 22 and CI-976 21 to rat reduce the level of serum triglycerides, whereas its mechanism still remains poorly understood. Furthermore, although 3-hydroxy-3-methylglutaryl (HMG) Co-A reductase inhibitor, such as atorvastatin, decreases not only the serum level of cholesterol but also the triglycerides, 23 little is known about its mechanism. Taken together, it is therefore likely that the administration of esculeoside A decreases endogenous cholesterol production by inhibiting the liver ACAT activity, followed by decreases in the serum triglycerides concentration like some reported ACAT inhibitors and atorvastatin. Field FJ et al 24 demonstrated that although the inhibitory effect of β-sitosterol on ACAT activity is weak, it inhibits cholesterol absorption in the intestine because cholesterol in the mixed micelles is replaced by β-sitosterol because of its high hydrophobicity. Because esculeogenin A possesses 3 hydroxyl groups and shows higher hydrophilicity than bete-sitosterol, the replacement of esculeogenin A with cholesterol in the mixed micelles is negligible or unlikely to occur. Thus, the physicochemical property of esculeogenin A differs from β-sitosterol. Taken together, the present study indicates that the administration of esculeoside A may inhibit the development of atherosclerosis by decreasing the serum cholesterol level through the inhibition of ACAT in the liver and the suppression of foam cell formation by inhibiting ACAT activity in the macrophage.
Many researchers have examined the usefulness of a number of antiatherosclerotic agents using as a strategy inhibition of ACAT activity. Mice lacking ACAT-2 exhibited a restricted capacity to absorb cholesterol and protection against diet-induced hypercholesterolemia and gallstone formation. 25 Nonselective ACAT inhibition is known to reduce atherosclerosis in apoprotein E–deficient mice. 26 NTE-122 and F-1394, a nonselective ACAT inhibitors, prevents the progression of atherosclerosis in cholesterol-fed rabbits. 27,28 Moreover, the ACAT inhibitor avasimibe reduces macrophage and matrix metalloproteinase (MMP) expression in atherosclerotic lesions of hypercholesterolemic rabbits, 29 and reduces atherosclerosis in addition to its cholesterol-lowering effect in apoE*3-Leiden mice. 30 Sakashita et al 31 demonstrated that ACAT-2 is also expressed in macrophage-derived foam cells in vitro and in vivo. Therefore, it is likely that esculeogenin A may have inhibited the activity of both ACAT-1 and ACAT-2 in the HMDM and showed a remarkable inhibitory effect of foam cell formation. However, Tardif et al 32 reported that avasimibe, administered for 2 years to patients with atherosclerosis, did not reduce plaque volume. Furthermore, unfavorable aspects of ACAT-1–deficient mice is reported (please see Supplementary information II). However, Sahi et al 33 reported that avasimibe increases CYP3A4 (Cytochrome P450, family 3, subfamily A, polypeptide 4) and multiple drug resistance protein 1 gene expression in vitro through activation of pregnane X receptor. Consequently, avasimibe causes clinically significant changes in the pharmacokinetics of the CYP3A4 substrate and P-glycoprotein substrate. The induction of CYP3A4 of P-glycoprotein may be the basis for the observed autoinduction of clearance for avasimibe itself. Therefore, it is speculated that avasimibe is degraded easily in the human body and does not function as an atheroprotective agent. 33 Generally, reported ACAT inhibitors, such as CI976 34 and avasimibe, 35 show high hydrophobicity attributable to their ring-shaped structure and the alkyl chain. However, because esculeogenin A has the hydroxyl and the amino group, but not the alkyl chain (please see supplementary figure I in supplementary information I), the hydrophobicity of escuelogenin A is considerably lower than that of cholesterol. Furthermore, although the reported ACAT inhibitors are divided roughly into urea compounds and amide compounds that contain an urea group and amide group, respectively, esculeogenin A does not belong to either of them.
We compared CI976, 36 a synthetic ACAT inhibitor. CI976 inhibited cholesterol ester (CE) accumulation by 90% when human monocyte-derived macrophages were incubated with 5 µmol/L CI976, whereas 50 µmol/L esculeogenin A were required to show same activity. Moreover, although 5 µmol/L CI976 inhibited ACAT activity by 90%, 50 µmol/L esculeogenin A inhibited by 40% (data not shown), thus indicating that the inhibitory effect of esculeogenin A on foam cell formation and ACAT activity is lower than that of known ACAT inhibitor. However, it is known that preventive medicine is the most important approach to prevent the lifestyle related diseases such as atherosclerosis and type II diabetes, and improvement of daily nutritional intake is thought to prevent the pathogenesis of these diseases. Therefore, ACAT inhibitors which can be taken from normal daily meals are preferred to strong synthetic ACAT inhibitors. Because esculeoside A is the natural compound isolated from tomatoes, we can intake esculeoside A from tomato products at daily life and it would thus be a promising strategy for preventive medicine. Therefore, in spite of lower inhibiting activity compared with synthetic ACAT inhibitor, we believe that daily intake of esculeoside A can thus play a beneficial role in preventing the pathogenesis of atherosclerosis. In the present study, we administered 50 mg/kg BW and 100 mg/kg BW of esculeoside A. Based on the esculeoside A content of a tomato, this dosage corresponds to about 10 kg when the human body weight is estimated as 60 kg. As described in the Methods, this dosage was decided based on a previous study by Kong W et al. 37 However, because human atherosclerosis progresses more mildly and takes over the years, we speculate that atherosclerosis can be ameliorated even when taking only 1/20 or 1/50 of the mice dosage if tomatoes are eaten on a daily basis. We will need to conduct additional study to evaluate the inhibitory effect of esculeoside A on the pathogenesis of atherosclerosis in human. Furthermore, because the tomato contains not only esculeoside A but also lycopene which inhibits oxidation of circulating LDL, 38 daily intake of tomatoes and tomato products may decrease the risk of cardiovascular diseases. Further studies will be required to elucidate the mechanism of esculeogenin A to inhibit activity and expression of ACAT.
Acknowledgments
We are grateful to Dr Chang TY of Department of Biochemistry, Dartmouth Medical School for supplying CHO cells stably overexpressing human ACAT-1 or ACAT-2, and valuable and fruitful discussion throughout this study. We are also grateful to Yu-ichiro Sakamoto, Masateru Ono, Ayumi Takaki, Yukie Uehara, Miho Yoshizaki, Keita Motomura, Katsumi Mera, Jyunichi Yoshida, and Mime Nagai for their collaborative endeavors.
Sources of Funding
This work was supported in part by Grants-in-Aid for scientific Research (No. 18790619 to Ryoji Nagai) from the Ministry of Education, Science, Sports, and Cultures of Japan.
Disclosures
None.
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作者单位：Departments of Medical Biochemistry (Y.F., N.K., M.H., R.N.) and Cellular Pathology (M.T.), Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan the Department of Natural Medicine (Y.F., N.K., S.M., T.I., T.N.), Graduate School of Medical and Pharmaceutical Sciences,