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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 17, 2005 as doi:10.1096/fj.05-4008fje. |
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* Cardiovascular Division,
Reproductive Health, Endocrinology and Development Division,
Asthma, Allergy and Lung Biology Division, School of Biomedical and Health Sciences and School of Medicine, King’s College London, London, UK;
Departamento de Fisiologia, Facultad de Medicina, Universidad de Valencia, Valencia, Spain; and
|| Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, Edinburgh, Scotland, UK
1 Correspondence: Cardiovascular Division, School of Biomedical and Health Sciences, King’s College London, Guy’s Campus, London SE1 1UL, UK. E-mail: giovanni.mann{at}kcl.ac.uk
SPECIFIC AIMS
The cardiovascular risks associated with hormone replacement therapy in postmenopausal women have precipitated a search for alternative estrogen receptor modulators. Genistein, a major isoflavone in soy protein, binds to estrogen receptor ß (ERß) with much higher affinity than ER
, and plasma concentrations of genistein and daidzein range from 50 to 800 ng mL–1 in adults consuming soy-rich foods. The aim of the present study was to compare the effects of a soy protein (SP) versus a soy-deficient (SD) diet on indices of oxidative stress, endothelial function in vitro and arterial blood pressure in vivo in adult male rats. Because imbalanced maternal nutrition during pregnancy can lead to the development of cardiovascular disease in adulthood, dams were maintained on SP or SD diets for 4 months before gestation and weaning, and male offspring were then fed the same diets for up to 16 months. To assess whether impaired vascular reactivity in SD animals could be reversed, SD males were switched after 10 months to the SP diet for 2 or 6 months.
PRINCIPAL FINDINGS
We found that soy protein deprivation leads to reduced eNOS and antioxidant gene expression, impaired vascular reactivity in vitro and elevated blood pressure in vivo in aged male rats. Refeeding soy-deficient animals for 6 months with a soy protein diet increased mRNA levels for eNOS and antioxidant enzymes 2- to 3-fold, improved endothelium-dependent relaxation and lowered blood pressure. The "cardioprotective" actions of a soy protein diet may in part be mediated by increased NO synthesis and/or bioavailability due to transcriptional activation of antioxidant enzymes.
1. Vascular reactivity is impaired in male offspring fed a soy protein deficient diet
Plasma concentrations of the soy isoflavone genistein were respectively 2.4 ± 2 ng mL–1, 163 ± 53 ng mL–1 and 126 ± 33 ng mL–1 in rats fed SD, SP, or SD + 6 month SP diets, confirming previous measurements in soy-deficient rats. Vasoconstrictor responses to the -adrenoceptor agonist phenylephrine were not significantly different in rats fed either SP or SD diets (Fig. 1
A). ACh-mediated, endothelium-dependent relaxation was significantly enhanced in SP compared to SD fed rats (Fig. 1B, C
) and refeeding SD rats with the SP diet for 6 months, but not 2 months (Fig. 1B
), restored endothelial function to levels in SP animals (Fig. 1C
). In concentration response experiments, ACh-mediated relaxation was abolished by the NO synthase inhibitor L-NAME (Fig. 1D
), implicating endothelium-derived NO in the dilator response. Refeeding SD animals for 6 months with the SP diet restored impaired endothelial function (Fig. 1D
). In these same animals, relaxation elicited by SNP was unaffected by dietary manipulations (Fig. 1E
), suggesting that the NO-cGMP signal transduction pathway in rat aortic vascular smooth muscle was insensitive to soy protein deprivation. We obtained similar findings in isolated intrapulmonary artery ring preparations (not shown).
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2. Soy protein diet increases glutathione levels and expression of eNOS and antioxidant genes
Mitochondrial levels of glutathione (GSH) were significantly lower in SD (6.72±0.64 nmol mg protein–1) compared to SP (10.29±1.03 nmol mg protein–1, mean±SE, n=5, P<0.01) fed rats, suggesting that production of reactive oxygen species (ROS) was elevated in SD rats. Malondialdehyde levels were elevated in SD compared to SP rats (4.8±1.6 vs 1.5±0.3 nmol/mg protein, P<0.05), confirming an increased generation of lipid peroxides. Real-time PCR provided the first direct evidence that an SP diet significantly increases mRNA levels for eNOS in aortic tissue (Fig. 2
A), potentially accounting for the improved endothelial function in aortic and intrapulmonary artery ring preparations in vitro. mRNA levels for the antioxidant genes cytochrome c oxidase and 
-glutamylcysteine synthetase (catalytic unit) were also 2- to 3-fold higher in aortic tissue from rats fed the SP compared to SD diet (Fig. 2A
). However, refeeding SD rats with the SP diet for 2 months did not significantly increase mRNA levels for eNOS or antioxidant enzymes in aortic tissue.
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eNOS, cytochrome c oxidase, and Mn-SOD mRNA levels were 
3-fold higher in liver homogenates from SP compared to SD fed rats (Fig. 2B
). We hypothesized that if increased eNOS and antioxidant gene expression are responsible for improved endothelial function in SP rats, refeeding SD animals for 6 months with the SP diet should increase the expression of cytoprotective genes. As shown in Fig. 2B
, gene expression was increased to levels in age-matched rats fed the SP diet throughout life.
3. Soy protein diet lowers blood pressure in vivo
The foregoing results establish that an SP diet improves vascular reactivity in vitro. To determine whether an SP diet causes generalized vascular improvements in vivo, we obtained direct hemodynamic measurements of blood pressure, heart rate, and motor activity in conscious rats implanted with telemetric transducers. As impaired vascular reactivity and eNOS and antioxidant gene expression in SD animals was only improved after refeeding the SP diet for 6 months, systolic and diastolic blood pressures were recorded at 12 h intervals over 4 days in 16-month-old male rats. Systolic (Fig. 2C
) and diastolic (Fig. 2D
) blood pressure were significantly decreased in SP compared to SD rats. Refeeding 10-month-old SD male rats with the SP diet for 6 months significantly lowered blood pressure.
CONCLUSIONS AND SIGNIFICANCE
We have identified a physiological role for dietary soy protein in enhancing gene expression of eNOS and antioxidant defense enzymes, resulting in improved endothelial function and lower blood pressure in vivo. Soy-deficient animals had significantly elevated systolic and diastolic blood pressure, which was lowered after refeeding a soy protein diet for 6 months, suggesting that potential ’fetal programming’ of vascular function could be overridden by long-term refeeding of a soy isoflavone-rich diet. ERß deficient rodents also have elevated blood pressure, and since genistein binds to ERß with high affinity, it seems likely that ERß plays an important role in mediating the cardiovascular protection afforded by soy isoflavones.
Classical actions of estrogens and phytoestrogens are mediated via transcriptional activation of estrogen responsive genes involving intracellular estrogen receptors. The receptor-hormone complex binds to a specific estrogen response element (ERE) in the promoter region of target genes, leading to transcriptional activation (Fig. 3
). However, activation of target genes by estrogens may also be mediated by other transcription factors, including AP-1 and NF-
B independent of the ERE. Recent evidence implicates cell surface receptors in the rapid responses to estrogen and phytoestrogens in vascular endothelial and smooth cells. We and others have shown that binding of estrogen and the anti-estrogen tamoxifen to the ß-subunit of the Maxi K+ (hSlo) channel in vascular smooth muscle cells leads to changes in channel activity. Infusion of genistein at µM concentrations detected in Asian populations causes a rapid increase in forearm blood flow in humans, mediated by endothelium-derived NO and inhibition of L-type Ca2+ channels in vascular smooth muscle.
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Mitochondrial GSH levels were significantly lower in soy-deficient animals, suggesting that like estrogen soy isoflavones can reduce ROS production in vivo. Although high concentrations of estrogens are thought to act as free radical scavengers, recent studies implicate estrogen in the induction of endogenous antioxidant enzymes such as Mn-SOD, heme oxygenases, thioredoxin, GPx, and phase II detoxification enzymes such as glutathione S-transferases and NADPH quinone reductase. The absence of EREs in the promoter region of these genes suggests that phosphorylation of mitogen-activated protein kinases such as ERK1/2 could lead to transcriptional activation.
Our findings also have important implications for the design of alternative estrogen receptor modulators in the treatment of postmenopausal women. We propose that improved antioxidant and eNOS gene expression in adult male rodents fed a soy protein diet leads to decreased ROS production, improved endothelial function and lower blood pressure in vivo. Since the beneficial effects on vascular function were observed when soy-deficient animals were switched to a soy protein diet for 6 months, this suggests that patients and postmenopausal women at risk of coronary heart disease may benefit from an increased intake of dietary soy isoflavones.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4008fje;
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