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Nutritional Immunology

Dietary Soy Protein Isolate Ameliorates Atherosclerotic Lesions in Apolipoprotein E-Deficient Mice Potentially by Inhibiting Monocyte Chemoattractant Protein-1 Expression^1–3,

⁴ Arkansas Children's Nutrition Center, ⁵ Department of Microbiology and Immunology, and ⁶ Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205

^* To whom correspondence should be addressed. E-mail: nagarajanshanmugam{at}uams.edu.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Soy-based diets reportedly protect against the development of atherosclerosis; however, the underlying mechanism(s) for this protection remains unknown. In this report, the mechanism(s) contributing to the atheroprotective effects of a soy-based diet was addressed using the apolipoprotein E knockout (apoE–/–) mice fed soy protein isolate (SPI) associated with or without phytochemicals (SPI⁺ and SPI^–, respectively) or casein (CAS). Reduced atherosclerotic lesions were observed in aortic sinus and enface analyses of the descending aorta in SPI⁺- or SPI^–-fed apoE–/– mice compared with CAS-fed mice. SPI⁺-fed mice showed 20% fewer lesions compared with SPI^–-fed mice. Plasma lipid profiles did not differ among the 3 groups, suggesting alternative mechanism(s) could have contributed to the atheroprotective effect of soy-based diets. Real-time quantitative PCR analyses of proximal aorta showed reduced expression of monocyte chemoattractant protein-1 (MCP-1), a monocyte chemokine, in mice fed both soy-based diets compared with the CAS-fed mice. These findings paralleled the reduced number of macrophages observed in the lesion site in the aorta of SPI⁺- or SPI^–-fed mice compared with CAS-fed mice. In an in vitro LPS-induced inflammation model, soy isoflavones (genistein, daidzein, and equol alone or in combination) dose dependently inhibited LPS-induced MCP-1 secretion by macrophages, suggesting a role for soy isoflavones for the protective in vivo effects. Collectively, these findings suggest that the reduction in atherosclerotic lesions observed in mice fed the soy-based diet is mediated in part by inhibition of MCP-1 that could result in reduced monocyte migration, an early event during atherogenesis.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Atherosclerosis is a chronic vascular inflammatory response (5). The cascades of leukocyte-endothelial cell interactions and leukocyte infiltration are crucial processes in atherogenesis (6). Leukocyte recruitment to inflammatory sites is regulated by the expression of cytokines and chemokines (7,8) produced locally. Monocyte chemoattractant protein-1 (MCP-1), a member of the CC chemokines, is characterized by its chemokine activity for inflammatory cells, primarily monocytes (7,8). MCP-1 is secreted by inflamed endothelium and activated monocytes (9) and is involved in recruitment of monocytes into arterial walls (10). Studies using CC-chemokine receptor 2/apoE–/– mice have shown that despite having hypercholesterolemic conditions, these mice had reduced atherosclerotic lesions (7,8), suggesting that MCP-1 is necessary for the progression of the disease. Growing evidence suggests that MCP-1 plays a pivotal role in the inflammatory processes associated with the pathogenesis and progression of atherosclerosis (7,8).

The purpose of this study was to determine the underlying mechanism(s) of atheroprotective effects of soy-based diets. In light of the major role of MCP-1 in the initiation and/or progression of atherosclerosis, we hypothesize that the atheroprotective effect of soy-based diets could be mediated by inhibiting the expression of MCP-1. This investigation was carried out using atherosclerosis-prone apoE–/– mice fed AIN-93G diets made with casein (CAS), soy protein isolate (SPI⁺), or SPI processed to remove phytochemicals associated with the protein (SPI^–).

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Mice and diets. ApoE–/– mice (11) bred onto a C57BL/6 background purchased from the Jackson Laboratory were housed in micro-isolator cages with filter tops and maintained on a 12-h-light/-dark cycle in a temperature-controlled room. Sixty female mice (4 wk) were randomly assigned to 3 groups (n = 20) and fed the indicated diet for 12 wk. Although each group had 20 mice, samples from the indicated numbers of mice were used for some variables. Each group was fed 1 of 3 semipurified diets made according to the AIN-93G diet formula (12) (except that corn oil replaced soybean oil) and the protein source was either CAS (New Zealand Milk Products), SPI⁺, or SPI^– (Solae Company). The SPI⁺ diet had 430 mg total isoflavones/kg containing 276 mg/kg genistein and 132 mg/kg daidzein. The SPI^– diets had been commercially processed by successive ethanol washes to be essentially devoid of phytochemicals (i.e. <5% of the isoflavones found in SPI⁺) (13). Diet pellets were prepared by Harlan-Teklad and the dietary composition of each diet was confirmed by reanalysis (Eurofins). Mice consumed food and water ad libitum throughout the study period. These studies were conducted under the guidelines and protocols approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences.

    MRI analysis. Body weights of mice fed different diets were recorded weekly. MRI analysis was performed to measure lean body mass and body fat content. Whole-body scans were analyzed using the software provided by the manufacturer (Echo Medical Systems). Both parameters were normalized to the weight of the mice. Data are presented as percentage of body fat content and of lean body mass (Table 1).

    Analysis of serum lipids. We measured concentrations of plasma total cholesterol, HDL-cholesterol, and triglycerides by enzymatic methods using kits from Synermed as described earlier (15). LDL-cholesterol was calculated using Friedewald's formula [LDL-cholesterol = (total cholesterol – HDL-cholesterol- {triglycerides/5})] (16,17).

    Quantitative PCR analysis. The aorta were perfused with nuclease-free PBS and total RNA was isolated from a proximal portion of the descending aorta (aortic arch and aorta proximal to the subclavian artery) using Versagene RNA Purification kits (Gentra Systems) according to the manufacturer's instructions. Real-time PCR for tumor necrosis factor (TNF)- and MCP-1 expression was determined after RT of total RNA (0.5 µg) as described earlier (15). PCR primers for β-actin, TNF, and MCP-1 were generated using Beacon Designer software (Bio-Rad) (described in Supplemental Table 1). Expression of TNF and MCP-1 was calculated using CT method (15) using threshold cycles for β-actin as normalization reference. All real-time PCR were carried out at least twice from independent cDNA preparations. RNA without RT served as a negative control.

    Immunohistochemistry. Serial aortic sinus cryosections (10 µm) were stained with anti mouse monocyte/macrophage monoclonal antibody (MOMA-2, 1:25 dilution) followed by Vectastain ABC reagent (Vector Labs). The sections were developed with diaminobenzidine and counterstained with Mayer's hematoxylin. Images were captured using a Carl Zeiss inverted microscope. Four individuals unaware of the identity of the samples counted macrophages in the aortic sinus sections.

    Cell treatment. Human monocytic cell line THP-1 cells were cultured in phenol red-free RPMI 1640 medium supplemented with 10% charcoal/dextran-treated fetal bovine serum (Hyclone) as described earlier (18). Cells (1 x 10⁵ cells per well) in a 96-well plate were pretreated with various concentrations of individual soy isoflavones (genistein, daidzein, and equol) or combinations of isoflavones for 18 h. Cells were then stimulated with Escherichia coli K12 LPS (Invivogen) at 100 µg/L for indicated time periods. For mRNA analysis, cells were incubated for 3 h and the conditioned medium was collected after 6 h for protein analysis. Cells treated identically with vehicle (dimethyl sulfoxide, 0.1% final concentration) were used as a control.

    MCP-1 expression analysis. MCP-1 levels in the supernatant were determined by sandwich ELISA using mouse anti-human MCP-1 IgG (clone 10F7.2, 2 mg/L) and biotinylated murine anti-human MCP-1 IgG (clone 5D3-F7, 0.5 mg/L) as capture and detecting antibodies (BD-Biosciences), respectively. Human MCP-1 mRNA expression was carried out using RNA isolated from THP-1 cells treated with or without isoflavones. Real-time quantitative RT-PCR was performed as described earlier (15), using specific primers (Supplemental Table 1). Human glyceraldehyde 3-phosphate dehydrogenase mRNA levels were used as normalization reference.

    ELISA. MCP-1 and TNF in mouse plasma were determined by sandwich ELISA using kits specific for mouse MCP-1 and TNF (BD-Biosciences).

    Data analysis. Results are expressed as means ± SD. Serum lipid levels and lesion size data were analyzed by 1-way ANOVA followed by Tukey's multiple comparison post hoc test. Data of inhibition of MCP-1 expression by combination or individual soy isoflavones were analyzed by 1-way and 2-way ANOVA, respectively, and Tukey's multiple comparison for post hoc analysis. Differences were considered significant at P < 0.05. All analyses were carried out with SigmaStat 9 program (Systat Software).

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Atherosclerotic lesions in apoE–/– mice were inhibited by soy-based diets. The atherosclerotic lesion areas in the aortic sinus were extensive in the CAS-fed apoE–/– mice (Fig. 1A). The mean lesion areas for apoE–/– mice fed the SPI⁺ or SPI^– diet were lower (75% and 56%, respectively) compared with those in CAS-fed mice (P < 0.001; Fig. 1B). Progression of atherosclerotic lesions, assessed by fatty streak lesions, was 20% less in SPI⁺-fed mice compared with that in the SPI^–-fed mice (P < 0.01). Enface analysis of the descending aorta showed fewer lesions in SPI⁺- or SPI^–-fed groups than in CAS-fed mice (Fig. 1C). Microscopic analyses of the lesions in aortic arch, thoracic (Fig. 1C), and abdominal regions (data not shown) showed soy-based diets (SPI⁺ and SPI^–) inhibited atherosclerotic lesion development (P < 0.01). Atherosclerotic lesion area in the aortic tree was less in SPI⁺- and SPI^–-fed mice compared with their CAS-fed cohorts (P < 0.01; Fig. 1D).

View larger version (59K): [in this window] [in a new window]   FIGURE 1  Soy-based diets attenuate early atherosclerotic fatty streak lesions in apoE–/– mice. A, Light microscopy of aortic sinus stained with Oil Red O (10x magnification). Arrows indicate Oil Red O positive fatty streak lesions. B, Atherosclerotic plaque area in aortic sinus of mice fed CAS or SPI+ or SPI– diets. A total of 6 slides per mouse were analyzed. C, Photomicrographs showing the fatty streak lesions in the proximal portion (includes aortic arch and thoracic aorta) of the descending aorta from apoE–/– mice. Arrows indicate fatty streak lesions. D, Lesion areas were measured at the end of the curvature of the aortic arch to the iliac bifurcation. Total mean lesion area (n = 7 mice/diet group) was analyzed by quantitative image analysis software AxioVision. Values are means ± SD, n = 7. Bars without a common letter differ, P < 0.01.

    SPI feeding does not affect plasma lipid profiles. Plasma total, HDL-, and LDL-cholesterol and triglyceride concentrations in mice fed SPI⁺ or SPI^– diets did not differ from those of CAS-fed mice (Table 1).

    ApoE–/– mice fed soy-based diets had fewer macrophages and lower MCP-1 expression in lesions compared with CAS-fed mice. Immunohistochemical analyses of the aortic sinus showed SPI⁺- and SPI^–-fed apoE–/– mice had fewer (P < 0.01) macrophages in aortic lesions compared with CAS-fed mice (Fig. 2A,B). To determine the potential mechanism(s) contributing to the reduction in lesions and number of macrophages in a soy-based diet fed to apoE–/– mice, expression of MCP-1 and TNF was determined. Plasma TNF and MCP-1 protein levels did not differ among the groups (data not shown). However, real-time quantitative RT-PCR analyses showed mRNA expression of the monocyte chemokine MCP-1 in the lesion site of the proximal aorta (aortic arch to thoracic aorta) was decreased (P < 0.01) in SPI⁺- and SPI^–-fed mice compared with CAS-fed mice (Fig. 3A). Similarly, mRNA levels of pro-inflammatory cytokine TNF were reduced (P < 0.01) in aorta collected from SPI⁺- or SPI^–-fed apoE–/– mice (Fig. 3B). Immunohistochemical staining of the aortic arch and the aortic sinus did not yield a quantitative measure for the MCP-1 and TNF protein expression (data not shown).

View larger version (12K): [in this window] [in a new window]   FIGURE 2  Reduced number of macrophages in aortic lesions in mice fed soy-based diets. A, Immunohistochemical localization of macrophages in aortic sections from CAS-, SPI+-, or SPI–-fed apoE–/– mice (10 x magnification). Arrows indicate regions of macrophage positive staining. Under similar conditions, nonspecific staining of aortic sections with an isotype-matched rat IgG control was minimal (data not shown). B, Quantification of number of macrophages in the lesions was carried out in a blinded fashion as described. Mean positive macrophages from n = 5 mice per diet group and 3 slides per mouse is represented. Values are means ± SD. Bars without a common letter differ, P < 0.01.

View larger version (17K): [in this window] [in a new window]   FIGURE 3  Soy-based diets inhibit MCP-1 and TNF expression in mouse aortic samples. Relative MCP-1 (A) and TNF (B) mRNA expression levels in aortic samples from apoE–/– mice fed SPI+ or SPI– or CAS diets. Values are means ± SD, n = 6. Data represent percent reduction in expression by taking expression in CAS-fed group as 100%. Bars without a common letter differ, P < 0.01.

View larger version (11K): [in this window] [in a new window]   FIGURE 4  Soy isoflavones inhibit inflammation-induced MCP-1 expression. MCP-1 protein secretion by THP-1 cells was cocultured with soy isoflavones in combination (A). MCP-1 mRNA expression in THP-1 cells was cocultured soy isoflavones in combination or individually in the presence of LPS (B). Values are means ± SD, n = 2 (2 independent experiments performed in triplicate). Bars without a common letter differ, P < 0.001.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

There are 2 major components with potential bioactivity in soy foods or SPI: phytochemicals associated with soy protein and the soy proteins or peptides generated from the soy proteins. However, the component(s) of soy responsible for its atheroprotective effects are uncertain. Our findings show SPI⁺ (relative to CAS) inhibits atherosclerotic lesion formation in apoE–/– mice. The results showed that the atherosclerotic lesion areas were smaller (P < 0.05) in SPI⁺-fed mice compared with SPI^–-fed mice, suggesting a role for phytochemical components contributing to the atheroprotective effect (4,19). In vitro studies have shown that endothelial cells treated with genistein, one of the isoflavones, inhibited monocyte adhesion (20,21), suggesting that the atheroprotective effect of soy isoflavones could be mediated by regulating endothelial cell functions. Recently, we reported that sera from rats fed SPI⁺-containing diets blocked monocyte adhesion to CD54, whereas minimal effects were observed with sera from rats fed CAS diets (22). These studies suggest that the atheroprotective effect of SPI⁺-containing diets could be mediated by soy components or endogenous factors that are influenced by components of SPI.

Feeding the SPI^– (isoflavone-reduced) diet inhibited atherosclerotic lesion development in apoE–/– mice, suggesting a role for the protein component of SPI. We also showed reduced MCP-1 mRNA expression in aorta and number of macrophages in lesions in SPI^–-fed compared with CAS-fed apoE–/– mice. The molecular signature of peptides or protein components in the SPI^– diet that may contribute to the inhibition of inflammation-induced MCP-1 expression resulting in the atheroprotection is still unknown. Earlier, it was reported that isoflavone-poor soybean products markedly reduced plasma cholesterol levels in hypercholesterolemic patients (23). β-Conglycinin (or 7S globulins) and glycinin (or 11S globulins) are the major proteins present in soybeans. In vivo and in vitro studies have suggested that 7S globulin reduced plasma cholesterol levels in rats (24) and stimulated expression of LDL receptors in HepG2 cells (25). Studies by Adams et al. (26) have demonstrated that β-conglycinin had a pronounced inhibitory effect on the development of atherosclerosis in the LDL receptor knockout mouse model. These studies suggest that there are bioactive small peptide fractions produced by the digestion of soy protein that are absorbed from the intestinal tract that have favorable effects in preventing atherosclerosis. Therefore, it is most likely that the protein portion of intact SPI⁺ contributed to lowering the atherosclerotic lesion area.

Studies using MCP-1 receptor/apoE–/– mice have shown that the progression of atherosclerotic lesion development is reduced (7), suggesting that MCP-1 plays a pivotal role in the initiation of atherosclerosis (5). We showed that a soy-based diet inhibits MCP-1 mRNA expression in the aorta (Fig. 3). This observation is supported by a reduced number of macrophages in atherosclerotic lesions in mice fed soy-based diets compared with CAS-fed mice (Fig. 2). However, plasma MCP-1 levels did not differ among the 3 groups studied, suggesting soy-based diets inhibit pro-inflammatory cytokine and chemokine expression at the local inflamed site. The differences in atherosclerotic lesion area and aortic MCP-1 mRNA expression between the SPI⁺- and SPI^–-fed mice were minimal but significant (P < 0.05). These findings suggest that soy isoflavones may have an antiinflammatory function that could have contributed to the atheroprotective effect of the SPI⁺ diet. Immunohistochemical analyses to determine MCP-1 protein expression in aortic lesions did not give a quantitative measure to unequivocally demonstrate the antiinflammatory effect of SPI⁺ diet (our unpublished data). Hence, to address whether soy isoflavones inhibit inflammation-induced MCP-1 expression by macrophages, an in vitro LPS-induced macrophage inflammation model was used (27). Although >80% inhibition of inflammation-induced MCP-1 secretion was observed at high isoflavone concentrations (30 µmol/L), significant inhibition (25%) was also observed at a physiological concentration of 1 µmol/L isoflavone (a combination of 0.33 µmol/L each of genistein, daidzein, and equol). Notably, our findings also show that individual soy isoflavones (genistein, daidzein, and equol) inhibited inflammation-induced MCP-1 expression as low as 1 µmol/L. We recognize that the concentrations of soy isoflavones used in this study may be high. However, earlier studies have reported that plasma concentrations of soy isoflavones, particularly genistein and daidzein, can reach a total of 1–2 µmol/L following consumption of soy meal (28,29). The specific mechanisms by which soy isoflavones(s) inhibit MCP-1 expression are under investigation.

In conclusion, our data show that a soy-based diet effectively attenuates atherosclerotic fatty streak lesion formation, inhibits macrophage infiltration, and decreases MCP-1 mRNA expression in aortas of apoE–/– mice with hypercholesterolemia-induced systemic inflammation. We also demonstrated that soy isoflavones significantly inhibited MCP-1 mRNA and protein expression by human monocytic cell lines in vitro. Our findings provide novel insights into the pleiotropic effects of soy-based diets and the antiinflammatory effects of soy that may provide a promising approach of reducing the risk of atherosclerosis.

	ACKNOWLEDGMENTS

 
We thank Drs. Martin Ronis, Prajitha Thampi, and Uma Nagarajan for their critical review of the manuscript. We thank Mathew Ferguson for his help with the animal experiments, Mark Robinette for data analysis, and John Gregan and Phaedra Yount for their help with manuscript preparation.

	FOOTNOTES

 
¹ Supported by a grant from the USDA (CRIS 6251-51000-005-00D to S.N.).

² Author disclosures: S. Nagarajan, R. L. Burris, B. W. Stewart, J. E. Wilkerson, and T. M. Badger, no conflicts of interest.

³ Supplemental Table 1 is available with the online posting of this paper at jn.nutrition.org.

⁷ Abbreviations used: ApoE–/–, apolipoprotein E knockout; CAS, casein; MCP-1, monocyte chemoattractant protein-1; SPI, soy protein isolate; SPI⁺, soy protein isolate containing isoflavones; SPI^–, low isoflavone soy protein isolate; TNF, tumor necrosis factor.

Manuscript received 24 September 2007. Initial review completed 4 October 2007. Revision accepted 3 December 2007.

	LITERATURE CITED

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This Article Abstract Full Text (PDF) Online Supporting Material Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Nagarajan, S. Articles by Badger, T. M. Search for Related Content PubMed PubMed Citation Articles by Nagarajan, S. Articles by Badger, T. M.