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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-0939
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 3 1734-1740
Copyright © 2005 by The Endocrine Society

Effects of Soy Isoflavones and Conjugated Equine Estrogens on Inflammatory Markers in Atherosclerotic, Ovariectomized Monkeys

Thomas C. Register, Jennifer A. Cann, Jay R. Kaplan, J. Koudy Williams, Michael R. Adams, Timothy M. Morgan, Mary S. Anthony, Robert M. Blair, Janice D. Wagner and Thomas B. Clarkson

Comparative Medicine Clinical Research Center (T.C.R., J.A.C., J.R.K., J.K.W., M.R.A., M.S.A., R.M.B., J.D.W., T.B.C.) and the Department of Public Health Sciences (T.M.M.), Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1040

Address all correspondence and requests for reprints to: Thomas C. Register, Ph.D., Comparative Medicine Clinical Research Center, Wake Forest University School of Medicine, Medical Center Boulevard, Winston Salem, North Carolina 27157-1040. E-mail: register{at}wfubmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of dietary soy isoflavones (IF) and conjugated equine estrogens (CEE) on circulating inflammatory markers were determined at the end of a 3-yr study of ovariectomized monkeys consuming a moderately atherogenic diet. Treatments were: 1) control, receiving alcohol-extracted soy-protein-based diet with low IF content (comparable to ~5 mg/d); 2) CEE, added to the control diet at a dose comparable to 0.625 mg/d; and 3) IF, consumed as a part of unextracted soy protein isolate at a dose comparable to 129 mg/d. Serum soluble vascular cell adhesion molecule-1 (sVCAM-1) was reduced by both IF (P < 0.006) and CEE (P < 0.0001) relative to controls. Serum monocyte chemoattractant protein (MCP)-1 was reduced by CEE (P < 0.0001) but not by IF (P = 1.00). Treatments did not affect serum IL-6 (P = 0.40), soluble E-selectin (P = 0.17), or C-reactive protein (P = 0.15). Serum MCP-1 and, to a lesser extent, IL-6 significantly correlated with atherosclerosis (plaque area) in the iliac and carotid arteries (all P < 0.05). Serum MCP-1 was also strongly associated with coronary artery atherosclerosis and with indices of plaque inflammation and matrix remodeling (matrix metalloproteinase-9) in the coronary artery intima (all P < 0.01). We conclude that, in this well-established nonhuman primate model of atherosclerosis, this dose of soy IF provided an antiinflammatory effect specific for sVCAM-1, whereas the effects of CEE extended to both sVCAM-1 and MCP1. It is possible that the atheroprotective effects of IF and CEE are mediated, at least in part, by effects on VCAM-1. The sites of IF inhibitory effects on sVCAM-1 production are not known, but likely candidates include the liver and/or the cardiovascular system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOY PROTEIN AND isoflavones (IF) comprise the most commonly consumed botanical supplements by peri- and postmenopausal women, in part because they are thought to be cardioprotective and to provide a substitute for traditional hormone replacement therapy (1, 2, 3, 4, 5). The IF found in soy, including genistein and daidzein, are of particular interest in this context because they are ligands for estrogen receptors (ERs) and thereby could have vascular effects similar to those reported for estrogens. Among the effects suggested for estrogens is the suppression of inflammatory activity, an outcome thought to comprise part of the mechanism by which estrogens inhibit atherosclerosis in experimental models and, perhaps, in pre- and early postmenopausal women.

Only a handful of randomized, prospective, double-blind, placebo-controlled clinical trials of the effects of soy phytoestrogens on inflammatory markers have been performed, and the results from these trials have been equivocal (6, 7, 8, 9). Trials to date have been of limited duration (all ≤ 6 wk diet duration), with a relatively small number of subjects. The difficulties of performing long-term, randomized trials of dietary treatments in humans and the inability to characterize effects of these treatments on tissue endpoints, such as arterial lesion cross-sectional areas and characteristics, can be circumvented through the use of animal models. In the study described here, we evaluated circulating markers of inflammation in ovariectomized cynomolgus monkeys that were atherosclerotic and that had been treated with soy IF or conjugated equine estrogens (CEE). The three objectives were: 1) to determine whether IF have antiinflammatory effects; 2) to establish how such effects compared with those observed in relation to CEE; and 3) to determine the relationship between these circulating biomarkers and both histologic indices of atherosclerosis (e.g. plaque size, necrosis) and an index of inflammation and atherosclerotic arterial matrix remodeling [immunoreactive matrix metalloproteinase-9 (MMP-9)].


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental design

Female cynomolgus macaques (Macaca fascicularis, n = 181) were imported as adults from Indonesia and used in a premenopausal investigation of atherosclerosis, during which animals were fed an atherogenic diet that, for half of individuals, also contained an oral contraceptive (10, 11). At the end of the premenopausal study, the monkeys underwent bilateral ovariectomies to create a menopausal condition. At the same time, a segment of the common iliac artery (0.5 cm in length) was removed and perfusion fixed using 10% neutral buffered formalin for measurement of atherosclerosis extent (cross-sectional lesion area). The animals then were randomly assigned to one of three dietary treatment conditions: 1) consumption of an atherogenic diet deriving its protein from alcohol-washed (IF-depleted) soy protein isolate (control, n = 57); 2) consumption of an atherogenic diet deriving its protein from IF-depleted soy protein and to which CEE had been added in an amount equivalent to a human dose of 0.625 mg CEE/d (n = 64); and 3) consumption of an atherogenic diet deriving its protein from a soy isolate containing 0.04 mg genistein, 0.01 mg daidzein, and 0.01 mg glycitein per gram, equivalent to a woman’s dose of 129 mg IF/d (n = 59). The diets were isocaloric for macronutrients [protein (19% of calories), carbohydrate (37%), and fat (44%)] and comparable for cholesterol (0.28 mg/cal), calcium (830 mg/1800 cal), and phosphorus (820 mg/1800 cal). Specific details of the overall design of this study, as well as outcomes with respect to plasma lipoproteins and coronary artery atherosclerosis and bone density, have been previously described (12, 13). All procedures were conducted in compliance with state and federal laws, standards of the United States Department of Health and Human Services, and regulations and guidelines established by the Wake Forest University Animal Care and Use Committee.

Necropsy procedures

After 36 months of treatment in the surgically postmenopausal period, the monkeys were killed using sodium pentobarbital (100 mg/kg, iv). The remaining common iliac artery (0.5 cm) was removed, perfusion-fixed with 10% neutral buffered formalin, and prepared for morphometric evaluation. The heart was removed, and the coronary arteries were perfusion-fixed for 1 h at 100 mm Hg pressure using 10% neutral buffered formalin. The common carotid arteries, carotid bifurcations, and internal carotid arteries were removed and fixed flat on cardboard in 10% neutral buffered formalin.

Assessment of atherosclerosis

Atherosclerosis was evaluated as previously described (12). Briefly, vascular tissues were embedded in paraffin, and 5-µm sections were made and stained with Verhoeff-van Gieson’s stain. Sections were projected onto a digitizer plate and quantified using a handheld stylus with a computer-assisted digitizer. The extent of atherosclerosis was measured as the cross-sectional area of intimal lesions in each of the sections of the artery segments as described previously (12). Coronary artery atherosclerosis was determined from 15 blocks [each 3 mm in length, cut perpendicular to the long axis of the arteries; five serial blocks each from the left circumflex (LCX), the left anterior descending, and the right coronary arteries]. For the iliac arteries, at the beginning of the postmenopausal phase, a biopsy of one common iliac artery was obtained for measurement of baseline atherosclerosis and the contralateral iliac artery taken at necropsy for terminal measures. Five 5-µm sections were used to assess atherosclerosis in each of the iliac arteries. The common iliac arterial site has been shown to have essentially the same plaque sizes in the left and right arteries (r = 0.97) and to be highly associated with coronary artery plaque extent (r = 0.86) (12). Carotid artery atherosclerosis was assessed in both the left and right common carotid arteries (three blocks each), carotid bifurcations (one block each), and left and right internal carotid arteries (two blocks each).

Evaluations of plaque complications

Ten histologic sections representative of the length of the LCX coronary artery were taken. Five of the sections were stained with hematoxylin and eosin, and five were stained with Verhoeff-van Gieson’s. The degree of inflammation associated with the monkey’s atherosclerotic plaque was based on the density of leukocytes accumulated both within the adventitia and within the intimal plaque. A subjective score of 0–4 was used for the most extensively involved of those cases within the data set. The intermediate grades 1, 2, and 3 were used relative to reference slides available in the laboratory. The amount of atheronecrosis was estimated as a percentage of the cross-sectional area of the plaque. The fibromuscular caps overlying the plaques were measured histomorphometrically at points of maximum and minimum thickness. These measurements were made blind to treatment group by a technician with more than 20 yr of experience and were randomly reevaluated by one of us (T.B.C.). Data were then averaged across all of the sections for an individual animal before statistical analysis.

Immunohistochemistry

For MMP-9 immunostaining, 5-µm sections were cut from the first serial block from each LCX artery. Sections were mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA), deparaffinized in xylenes, and rehydrated in graded alcohols. Antigen retrieval was achieved with 10 mM citrate (pH 6.0) under pressure for 30 min. Slides were preincubated with 5% normal goat serum for 30 min at room temperature and then incubated with a polyclonal rabbit antibody against gelatinase-B (RP3-MMP-9; Triple Point Biologics, Inc., Portland, OR) or nonimmune serum for 16 h at 4 C. Primary antibodies were localized with biotinylated antirabbit secondary antibody and tertiary avidin-biotin-complex stain (Vector Laboratories, Burlingame, CA). Sections were counterstained with Harris’ hematoxylin and examined by light microscopy. Immunostaining was quantified using Image Pro Plus Version 4.5 for Windows. Images were projected onto a computer monitor at x2 magnification and overlaid with a 52-pixel grid. Contrast and color settings were kept constant for all slides. Each point of intersection was then evaluated according to staining character and location. Location was defined as tunica intima, tunica media, or tunica adventitia, and staining character was classified as positive if immunostained or negative if not immunostained.

Serum biomarkers

Serum markers were assessed en bloc at the end of the study on serum samples obtained from fasted animals on the day of necropsy. Serum soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble E-selectin (sE-selectin), monocyte chemoattractant protein (MCP)-1, and IL-6 were assessed by ELISA using human-based assays and standards obtained from R&D Systems (Minneapolis, MN). Serum C-reactive protein (CRP) was determined by ELISA using a human-based assay from Diagnostic Systems Laboratories (Webster, TX). Intraassay coefficients of variation were less than 10%.

Statistical analyses

Statistical analyses were performed using SAS (Cary, NC) statistical software. Most of the variables had highly skewed distributions. Results are presented as means and SE of original data; however, all tests of hypotheses were based on nonparametric tests, Wilcoxon tests for group comparisons, and Spearman’s rank correlations for associations. Partial correlation coefficients were computed to adjust for effects of treatment. Bonferonni corrected t tests were used for post hoc between-group comparisons. A level of 0.05 or less was used to determine statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum MCP-1 was reduced by CEE (P < 0.0001) but not by IF (P = 1.00) (Fig. 1Go). In contrast, serum sVCAM-1 levels were reduced by IF (P < 0.006) and by CEE (P < 0.0001) relative to controls (Fig. 2Go), whereas IF and CEE did not differ from one another (P = 0.37). No effects of treatment were observed on serum sE-selectin (P = 0.17), IL-6 (P = 0.40), or CRP (P = 0.15) (Table 1Go).



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  		FIG. 1. Effects of treatments on serum MCP-1. Animals were ovariectomized and received a diet containing alcohol-extracted soy protein with low phytoestrogen content (Control), unextracted soy protein isolate containing soy phytoestrogens (IF), or CEE (admixed into the control diet) for 36 months. Data were analyzed by ANOVA. CEE treatment resulted in significantly lower circulating levels of MCP-1 compared with Control (P = 0.0001) and IF groups (P = 0.0002). No significant effect of IF consumption was observed (P = 1.00). MCP-1 value of 100% is defined as 478 pg/ml, based upon the use of human MCP-1 standards.

 


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  		FIG. 2. Effects of treatments on serum sVCAM-1. Serum sVCAM-1 levels were significantly reduced in CEE (P < 0.0001) and IF (P < 0.006)-treated animals compared with Controls. CEE and IF were not different from one another (P = 0.37). sVCAM-1 value of 100% is defined as 288 ng/ml, based upon the use of human sVCAM-1 standards.

 

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  		TABLE 1. Effects of treatment on serum markers associated with inflammatory processes

 
Arterial immunoreactivity for MMP-9 was assessed in the intima, media, and adventitia of the LCX, as an index of matrix remodeling (Table 2Go). The majority of MMP-9 immunoreactivity was observed in the intima, with approximately two thirds of the intimal area positive for MMP-9 as opposed to less than 5% immunoreactivity in the media and adventitial compartments. No effects of treatment were observed on MMP-9 immunoreactivity in any compartment (all P > 0.7).


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  		TABLE 2. Effects of treatment on MMP-9 immunoreactivity in the left circumflex coronary artery

 
Partial correlations (corrected for treatment effects) between serum markers and indices of atherosclerosis, inflammation, and matrix remodeling are shown in Tables 3Go and 4Go. For the baseline iliac artery biopsy sample taken at ovariectomy, atherosclerotic plaque size did not correlate with any of the serum markers assessed at the end of the study (Table 3Go). In contrast, significant correlations were observed between serum markers and plaque size in the contralateral iliac artery and in other arteries obtained at necropsy, which coincided with serum sampling. Serum MCP-1 levels were significantly correlated with numerous indices of atherosclerosis, including plaque area in the contralateral iliac artery obtained at necropsy, and in the coronary and carotid (common and internal) arteries. Serum MCP-1 was also found to correlate with estimates of plaque necrosis, inflammation, and matrix remodeling, the latter indicated by immunohistochemical analyses of MMP-9 (Table 4Go). Serum IL-6 was correlated with inflammation index and with atherosclerosis extent in the iliac and carotid artery but not in the coronary arteries. Serum CRP and sE-selectin showed no association with atherosclerosis parameters in this study.


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  		TABLE 3. Partial correlations and P values of circulating markers with plaque area in different vascular sites across all animals in the study

 

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  		TABLE 4. Partial correlations and P values of circulating markers with plaque characteristics determined in the left circumflex coronary artery across all animals in the study

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major findings of this study were that soy IF reduced circulating levels of sVCAM-1, an important mediator of the initiation and progression of atherosclerosis, whereas CEE reduced both sVCAM-1 and MCP-1. Of the soluble markers, MCP-1 reliably associated with atherosclerosis in this study, whereas IL-6 was also associated but to a lesser extent. Other soluble markers measured were not significantly associated with these parameters.

The results suggest that soy IF may provide an anti-inflammatory effect that appears to be specific for sVCAM-1 as opposed to other markers. The source of sVCAM-1 and the site of the inhibitory effects of dietary IF (and/or CEE) on its expression are not known, but likely candidates include the cardiovascular system and/or the liver. The mechanism by which the IF preferentially reduced circulating sVCAM-1, but not MCP-1, and CEE reduced both is also not known. It is possible that the sVCAM-1-specific effect of IF could be mediated through dosage-dependent and/or ligand-specific effects on ER{alpha} and ERß action. In addition to the well-known classical activation pathways involving estrogen response elements in the promoter regions of estrogen responsive genes, ER have been shown to influence MAPK pathways and to interact with other transcription factor pathways such as AP-1, nuclear factor {kappa}B (NF-{kappa}B), and peroxisome proliferator activated receptor-{gamma} (PPAR-{gamma}) (14, 15, 16, 17), any of which might be differentially influenced by IF and CEE. In most instances, ER-mediated suppression of gene expression has been associated with transcription factor cross-talk. The transcription factor NF-{kappa}B, a key mediator of inflammatory pathways that appears to play a central role in initiation and progression of atherosclerosis, is one likely target for antiinflammatory/atheroprotective effects of IF and CEE. Inhibition of NF-{kappa}B activation and/or activity by IF and CEE could be mediated through ligand-dependent ER-mediated processes, although antioxidant activity or other primary effects of these compounds might also result in reduced activation of NF-{kappa}B. However, global reduction of NF-{kappa}B activity would not appear to be the sole mechanism involved in the sVCAM-1-specific effect of IF, because the promoter regions of MCP-1, VCAM-1, E-selectin, and IL-6 genes all contain NF-{kappa}B response elements. It is possible that the different effects of CEE and IF seen here may relate to differences in the promoter regions of the VCAM-1 and MCP-1 genes. Although both contain NF-{kappa}B response elements, VCAM-1 is unique in that it contains two tandem GATA motifs (18) that are not present in the MCP-1 promoter. These GATA motifs have been shown to be targets for inhibition by antioxidants, estrogen, and genistein (19, 20, 21), providing a potential mechanism for VCAM-1-specific effects of IF in this study. Genistein, the major phytoestrogen in soy, also has activity as a tyrosine kinase inhibitor and many other properties that might differentially influence gene expression. Interestingly, genistein has been shown to inhibit the production/secretion of MCP-1 by cultured human umbilical vein endothelial cells (22). Many factors may be responsible for this apparent discrepancy between this in vitro study and our studies. First, the IF received by the monkeys as a part of unextracted soy protein are complex mixtures, containing genistein, daidzein, glycitein, and other components that may have biological activity. IF metabolites may also contribute to the biological activity. Current evidence suggests that monkeys may, on average, convert daidzein into equol more readily than 30–50% of humans studied to date (12, 23). Second, the peak plasma levels attained in the monkeys are generally lower than the concentrations commonly used in vitro. In the present study, the IF doses that the monkeys received were equivalent to approximately 129 mg/d for a woman. Plasma concentrations of total IF reached approximately 750 nM 4 h after receiving one third of the daily proportion of diet (12), which is the same order of magnitude as observed in humans consuming high levels of IF (24). Serum genistein levels attained 4 h after dosing were found to be approximately 0.1 µM, well below the relatively high concentrations (5 µM) used in many in vitro studies (22). Finally, MCP-1 is derived from a variety of cell types, and there are certain to be cell-type-specific effects of these complex IF mixtures. We are currently evaluating tissues from these animals for evidence of tissue-specific effects of these treatments.

Serum MCP-1 and, to a lesser extent, IL-6 were significantly correlated with the amount of atherosclerosis at necropsy in these animals. These proteins are produced by a variety of tissues and cell types, including endothelium, smooth muscle, macrophages, hepatocytes, adipocytes, and others. The correlations observed here with atherosclerosis suggest that these circulating proteins may originate, in large part, in the blood vessels, although they may be derived from liver, spleen, and/or other sites. It is possible that the circulating marker levels are also a global index of the overall inflammatory status of an individual animal. The strong association of serum MCP-1 levels with atherosclerosis variables suggests that it may be an excellent marker for overall atherosclerosis load. MCP-1 was strongly associated with coronary artery atherosclerosis, whereas IL-6 was not. Interestingly, CRP was not significantly correlated with atherosclerosis variables in this study. CRP has been traditionally assumed to be derived from the liver, although recent studies suggest that expression also occurs in atherosclerotic plaques in coronary arteries and other vessels (25, 26, 27). Circulating CRP has also been found to be an independent risk factor for clinical sequelae associated with coronary heart disease (28, 29). The relatively poor correlation between serum CRP and atherosclerosis-related variables in the present study illustrates that the plaque vulnerability to rupture (the key event leading to clinical events that are predicted by CRP levels in people) is not well represented by measures of plaque size or extent. Nevertheless, CRP was also unrelated to plaque characteristics in this study, whereas circulating MCP-1 was positively associated with several plaque characteristics assumed to be important in plaque vulnerability, including the degree of inflammation (by subjective assessment), thinness of the fibrous cap, and plaque expression of MMP-9. It will be interesting to determine whether circulating MCP-1 can serve as a predictor of clinical outcomes in the human population. Additional studies are underway to determine the effects of treatment on hepatic and vascular expression of these inflammatory mediators, as well as potential relationships between tissue expression and circulating levels.

Genistein and daidzein have received much attention because they have shown diverse biological activities in vitro, including the ability to bind to ER{alpha} and ERß (30) and induce estrogen response element-mediated transcription (31). Although ER{alpha} has approximately twice the affinity toward 17ß-estradiol as ERß, ERß has up to 3- to 5-fold higher affinity than ER{alpha} for some phytoestrogens (30). Thus, tissue-specific expression of ER{alpha} and ERß, combined with different affinities and actions of ligands, suggests that differential targeting of the receptors could lead to improved therapies for postmenopausal women. The effects of IF on sVCAM-1 in the present study could be mediated by either ER{alpha} or ERß, which are both expressed in monkey arteries at the mRNA (32) and protein levels (data not shown). It is conceivable that there are ethnic and species differences in ER expression in target organs, and that certain populations of women might benefit from IF more than others. Adaptations and genetic selection resulting from generations of people consuming high levels of phytoestrogens are perhaps even likely in some populations (i.e. Asian) but as yet unproven. Nevertheless, phytoestrogen action is not restricted to ERß in vivo, and several studies in transgenic mouse models suggest that their mechanisms of action often depend upon the presence of ER{alpha} and not necessarily ERß (33, 34, 35, 36).

Previous work from many laboratories has demonstrated that estrogens inhibit diet-induced atherosclerosis in a variety of animal models (37, 38, 39). Only a portion of this benefit has been explainable by effects on known risk factors (40, 41), suggesting direct influences of estrogen on arterial wall biology, presumably mediated through ERs (32, 42, 43). Atheroprotective effects of estrogens may be dependent, in part, on antiinflammatory effects that have been demonstrated in a variety of in vivo (44, 45, 46, 47, 48, 49) and in vitro (50) studies. Observational studies support the idea that estrogen is atheroprotective, and a recent longitudinal trial demonstrated that healthy women, without cardiovascular disease, who received estrogen replacement therapy initiated soon after menopause, had reduced progression of atherosclerosis, as defined by longitudinal evaluation of carotid artery intimal-medial thickness (51). Indeed, in the present study, we found atheroprotective effects in the coronary, iliac, and carotid arteries when CEE was administered soon after ovariectomy, even in the presence of some preexisting atherosclerosis that developed during 2 yr of atherogenic diet consumption while premenopausal (12). This lack of an estrogen-deficient peri-/postmenopausal interval before treatment started may have improved atheroprotective efficacy of the treatment. CEE treatment was also associated with a reduction in circulating MCP-1 levels, which were widely associated with several atherosclerosis endpoints and plaque characteristics. The atheroprotective effects of IF were weaker than CEE and not significantly different from control, except in the common and internal carotid arteries (12).

The current study evaluated CEE only, and not estrogen-medroxyprogesterone acetate combination therapies for which cardiovascular benefits have not been found either in women with preexisting coronary heart disease (52) or in older postmenopausal women in the Heart and Estrogen/Progestin Replacement Study trial (53) or in the Women’s Health Initiative (54). These therapies have had mixed effects on circulating markers associated with inflammation in postmenopausal women; CRP often increased in response to estrogen (48, 55, 56), whereas other markers such as circulating adhesion molecules were reduced by treatment (48, 57, 58, 59, 60). The impact of these divergent inflammatory marker responses on the development of atherosclerosis and/or clinical outcomes is not clear. Although we found inhibitory effects on circulating adhesion molecules, we found no effect of CEE (or IF) on serum CRP levels in the current study, perhaps due to the fact that the CEE was admixed into the diet and the dose was received in two daily feedings. Such a spreading of dose could result in reduced first-pass effects on the liver, the major source of CRP.

Strengths of the current study include the use of a well-established primate model of ovariectomy and atherosclerosis susceptibility that provides for detailed characterization of atherosclerosis in multiple arterial beds. In addition, compliance with long-term dietary regimens is ensured, and the experiments are well controlled in terms of environment and other activities that are difficult or impossible to control in clinical trials. The current study is limited in a number of ways. First, the markers were obtained near the end of a 5-yr study (3 yr post menopause), and we do not have any data on potential short-term changes in markers that may have occurred due to treatment. Likewise, our studies are not powered for or capable of detecting effects of treatments on susceptibility to clinically relevant outcomes such as plaque rupture and myocardial infarction, which are not readily detectable and which occur too infrequently to be an endpoint. Because the study was designed to determine the effects of IF vs. CEE on outcome measures, with all animals receiving soy protein, we have no information on the potential effects of the soy protein component itself on these parameters. In this regard, emerging evidence suggests that the soy protein and subfractions of soy protein (i.e. 7S globulins) may have beneficial effects on atherosclerosis that are independent of the IF (61). Conversely, several studies have provided evidence for independent atheroprotective or antiinflammatory effects of IF in animal models. For example, dietary IF aglycones were found to reduce aortic cholesterol content in an ovariectomized rabbit model of atherosclerosis progression (62). Atheroprotective effects of intact dietary soy protein relative to alcohol-extracted, IF-depleted soy protein have also been observed in two different atherosclerosis prone transgenic mouse models (63), and this benefit appeared to be dependent on the presence of ER{alpha} (36). Purified genistein has also been shown to alter inflammatory gene expression in the livers of ovariectomized female mice (44, 45).

In conclusion, we found divergent effects of dietary IF and estrogens on circulating serum markers associated with inflammation and atherosclerosis. Suppression of VCAM-1 expression may represent one mechanism by which IF inhibit the progression of atherosclerosis. Ongoing studies are being directed toward examination of the expression of VCAM-1 and MCP-1 in hepatic and vascular tissues from this study in an effort to determine whether there is tissue specificity to these effects. Elucidation of the mechanisms underlying the differential effects on sVCAM-1 and MCP-1 will require further study. Overall, studies from this experiment have demonstrated that consumption of IF as components of soy provided benefits against atherosclerosis (12), caused no apparent harmful effects to the reproductive system (64, 65), and had little or no benefit to the skeleton (13) in these animals. These findings have important implications for postmenopausal women who are seeking alternatives to traditional pharmaceutical therapies to counteract the adverse effects of estrogen loss.


   Acknowledgments
 
The authors gratefully acknowledge Solae LLC (St. Louis, MO; formerly Protein Technologies International) for their contribution of the soy products; and Tim Vest, Matt Dwyer, and Maryanne Post for their technical contributions.


   Footnotes
 
This work was supported, in part, by National Institutes of Health Grants HL45666 (to T.B.C. and J.R.K.) and AG18170 (to T.C.R.).

First Published Online December 7, 2004

Abbreviations: CEE, Conjugated equine estrogens; CRP, C-reactive protein; ER, estrogen receptor; IF, isoflavone(s); LCX, left circumflex; MCP-1, monocyte chemoattractant protein-1; MMP-9, matrix metalloproteinase-9; NF-{kappa}B, nuclear factor {kappa}B; sE-selectin, soluble E-selectin; sVCAM-1, serum soluble vascular cell adhesion molecule-1.

Received May 18, 2004.

Accepted November 24, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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