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Molecular Aspects of Medicine 31 (2010) 468–477

Contents lists available at ScienceDirect

Molecular Aspects of Medicine

journal homepage: www.elsevier.com/locate/mam

Review

Dietary isoflavones and vascular protection: Activation of cellular

antioxidant defenses by SERMs or hormesis?
Richard C.M. Siow, Giovanni E. Mann ⇑
Cardiovascular Division, British Heart Foundation Centre of Research Excellence, School of Medicine, King’s College London, 150 Stamford Street,
London SE1 9NH, UK

a r t i c l e i n f o a b s t r a c t

Keywords: During the past decade nutrigenomic studies in humans, animal models and cultured cells
Cardiovascular disease have provided important and novel insights into the mechanisms by which dietary isoflav-
Atherosclerosis
ones afford protection against vascular dysfunction through the amelioration of oxidative
Redox signaling
modifications and upregulation of endogenous antioxidant signaling pathways. In this
Antoxidant defenses
Nrf2/Keap1 review, we highlight that increased generation of nitric oxide (NO) and reactive oxygen
Hormesis species (ROS) in the vessel wall in response to dietary isoflavones enhance the activity of
antioxidant defense enzymes in endothelial and smooth muscle cells. The estrogenic prop-
erties of isoflavones are likely to contribute to the molecular mechanisms by which these
compounds activate signal transduction pathways involved in sustaining endothelial func-
tion and transcriptional activation of antioxidant defense genes in vascular cells. We eval-
uate the recent literature that estrogenic and hormetic properties of phytoestrogens are of
benefit for the maintenance of vascular function, and conclude that dietary isoflavones can
protect against cardiovascular diseases by virtue of their ability to activate signaling path-
ways leading to increased NO bioavailability and regulation of phase II and antioxidant
enzyme expression via the redox sensitive transcription factor Nrf2. In context of epigenet-
ics and the developmental origins of adult disease, it is noteworthy that exposure to dietary
soy during fetal development reduces the susceptibility to CVD and obesity in adulthood.
Thus, the Nrf2/Keap1 defense pathway provides a key mechanism by which isoflavones can
act as hormetic agents to modulate intracellular redox signaling in the vasculature to pro-
long healthspan and reduce the incidence of age-related cardiovascular diseases.
Ó 2010 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
2. Isoflavones as dietary modulators of cardiovascular function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
3. Cellular and molecular targets of isoflavones. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470
3.1. Regulation of vascular tone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470
3.2. Regulation of vascular redox status and antioxidant gene transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470
4. Hormetic properties of isoflavones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472
5. Epidemiological and clinical perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
5.1. Benefits of dietary isoflavones in atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
5.2. Benefit of dietary isoflavones in stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473

⇑ Corresponding author. Address: Cardiovascular Division, School of Medicine, Franklin-Wilkins Bld. (Rm. 3.01), King’s College London, 150 Stamford
Street, London SE1 9NH, UK. Tel.: +44 20 7848 4306; fax: +44 20 7848 4500.
E-mail address: giovanni.mann@kcl.ac.uk (G.E. Mann).

0098-2997/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.mam.2010.09.003
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R.C.M. Siow, G.E. Mann / Molecular Aspects of Medicine 31 (2010) 468–477 469

6. Concluding remarks – isoflavones for enhancing ‘healthspan’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474

1. Introduction

Cardiovascular diseases such as hypertension, coronary heart disease (CHD) and atherosclerosis are more common in men
than in pre-menopausal women of similar age, with the increased incidence of CHD after menopause attributed to estrogen
deficiency (Mendelsohn and Karas, 2005; Vitale et al., 2009) and increased vascular oxidative stress (Stocker and Keaney,
2004; Forstermann, 2008). Gender differences in the regulation of blood pressure and vascular function have been investi-
gated extensively over the past decade, and female sex hormones are implicated not only in the protective effects of gender
on the vasculature (Ballard and Edelberg, 2005; Mendelsohn and Karas, 2005; Vitale et al., 2009; Vina and Borras, 2010) but
also the expression of longevity-associated genes (Vina and Borras, 2010). Estrogens and structurally similar plant derived
phytoestrogens have direct antioxidant properties due to the presence of phenolic groups in their steroid structure, however
phytoestrogens, such as the soy isoflavones genistein and daidzein, can also activate intracellular signaling pathways in a
similar manner to estrogen,(Mahn et al., 2005; Joy et al., 2006; Mann et al., 2007; Borras et al., 2006) potentially accounting
for their beneficial effects on hypertension, thrombosis and cardiovascular diseases(Jackman et al., 2007; Nagarajan et al.,
2008; Nagarajan, 2010; Mann et al., 2009).
Nutrigenomic approaches over the past decade have yielded valuable mechanistic insights at genomic, proteomic and
metabolomic levels to evaluate the therapeutic potential of isoflavones in the prevention and treatment of cardiovascular
diseases (Muller and Kersten, 2003; Messina, 2010). Soy isoflavones are structurally similar to estrogen, but have a prefer-
ential affinity for estrogen receptor-b (ERb) (Kuiper et al., 1997, 1998; McCarty, 2006) which has enabled their vascular
actions to be investigated with negligible risk of stroke and breast cancer associated with prolonged hormone replacement
therapy (Hulley and Grady, 2004). Estrogen and isoflavones acutely activate intracellular kinases linked to gene transcrip-
tion(Mahn et al., 2005; Joy et al., 2006; Siow et al., 2007; Mann et al., 2007; Borras et al., 2006) and inhibition of NADPH
oxidase mediated superoxide production (Xu et al., 2004). In view of their non-genomic actions on kinase signaling pathways
(Joy et al., 2006; Mann et al., 2007; Martin et al., 2008) and the mitochondrial respiratory chain (Fuchs et al., 2007; Rasbach
and Schnellmann, 2008; Borras et al., 2010), it is likely that hormetic properties of isoflavones play a significant role in medi-
ating longer-term cardiovascular protection (Lindsay, 2005; Son et al., 2008).
As summarized in Fig. 1, aging is associated with impaired mitochondrial function (Madamanchi and Runge, 2007), eNOS
uncoupling (Yang et al., 2009), increased NADPH oxidase activity (Krause, 2007; Muller, 2009) and a decrease in endogenous
antioxidant enzyme defenses and the cysteine/cystine redox potential (Go et al., 2010), all of which exacerbate oxidative
stress and the risk of cardiovascular disease. In this review, we evaluate the evidence that estrogenic and/or hormetic actions
of isoflavones counteract oxidative stress by upregulating the activity and expression of endothelial NO synthase (eNOS) and
phase II and antioxidant defense enzymes (Mann et al., 2007, 2009), providing a clinically relevant therapeutic target to pro-
long healthspan and reduce the incidence of age-related cardiovascular diseases.

2. Isoflavones as dietary modulators of cardiovascular function

Interest in the health benefits of dietary isoflavones is based on epidemiological evidence that an increased intake of soya
products is associated with a lower incidence of cardiovascular disease (CVD) in oriental populations (Robertson et al., 1977;

Cardiovascular Diseases
Ageing Atherosclerosis
Hypertension
ROS generation Stroke
Mitochondria, eNOS
NADPH oxidase Oxidized status
Hormesis
Reduced status Antioxidant and Phase 2 defense
e.g. GSH, Nrf2 / ARE genes

Dietary Isoflavones

Fig. 1. Altered redox status in vascular disease and aging: hormetic actions of dietary isoflavones. Altered vascular redox homeostasis in cardiovascular
diseases associated with aging results from an imbalance between oxidative stress and endogenous antioxidant defenses. The process of aging shifts the
cellular redox balance from a reduced to a more oxidized status, thereby increasing the risk of atherosclerosis, hypertension and stroke. Exposure to dietary
isoflavones in utero or in later life may restore vascular redox balance via Nrf2/ARE mediated upregulation of antioxidant and phase II defense genes.
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Setchell and Cassidy, 1999). Soy products contain significant amounts of the isoflavones genistein and daidzein either in an
unconjugated aglycone form or in different glycoside conjugates (Setchell, 1998). Plasma concentrations of genistein may
only reach 40 nM in Europeans consuming Western diets, but up to 4 lM in Japanese consuming a traditional soy enriched
diet (Adlercreutz and Mazur, 1997; Adlercreutz et al., 1999). Although the Framingham Offspring Study highlighted a favor-
able cardiovascular risk profile in postmenopausal women receiving isoflavone supplements (De Kleijn et al., 2002), the
American Heart Association attributed the benefits of isoflavones to the high content of polyunsaturated fats, fiber, vitamins,
minerals, and low content of saturated fat in soy products (Sacks et al., 2006). Several of the trials included in the AHA pro-
spective analysis were based on small cohorts of healthy subjects, hypertensive patients or postmenopausal women, with
many of these trials failing to evaluate the bioavailability of isoflavone supplements (Cassidy et al., 2006; Hooper et al.,
2008).
Physiologically relevant concentrations of soy isoflavones elicit acute endothelium-dependent increases in forearm blood
flow (Walker et al., 2001; Chin-Dusting et al., 2004) and randomized trials have reported cardiovascular benefits of longer-
term (2–12 months) isoflavone supplementation (Kreijkamp-Kaspers et al., 2005; Nestel et al., 2007). In a small study of
healthy postmenopausal women fed an isoflavone enriched low fat meal, endothelium-dependent relaxation in vivo was
shown to be increased within 5–7 h (Hall et al., 2008). However, the lack of unequivocal evidence for the cardiovascular ben-
efits of isoflavones in larger scale clinical trials may in part reflect that 30–50% of individuals lack intestinal bacteria required
to metabolize daidzein to equol, suggesting that effects of dietary soy may be different in equol ‘producers’ and ‘non-produc-
ers’ (Setchell et al., 2002; Setchell and Clerici, 2010). Daidzein is metabolized via the intermediate dihydrodaidzein to
S-()equol, and notably rodents have high plasma concentrations of S-()equol due to the enriched soy protein content
of commercial rodent diets (Setchell and Clerici, 2010).
We have reported that feeding rats a soy isoflavone deficient diet from conception and throughout adult life results in
hypertension and impaired vascular reactivity mediated by NO and endothelium-derived hyperpolarizing factor in aged
male offspring (Mahn et al., 2005; Knock et al., 2006). Notably, feeding an isoflavone enriched diet to rats for only
4–6 months has marginal benefits for endothelial function (Douglas et al., 2006), suggesting that isoflavone supplements
may only affect endothelial function in aged animals with existing CVD (Trujillo et al., 2005, 2008). Consistent with the
reported vasodilatory actions of genistein and dehydroequol in humans (Walker et al., 2001; Chin-Dusting et al., 2004),
we have shown that isoflavones evoke rapid endothelium-dependent and -independent relaxation of preconstricted arterial
rings (Joy et al., 2006). In this context, a recent meta-analysis of double-blind, randomized, placebo-controlled trials of
isoflavone supplementation in postmenopausal women concluded that dietary isoflavones only significantly improve
endothelial function in women with a low (<5.2%) baseline FMD (Li et al., 2010), suggesting that dietary isoflavones may
be more effective in subjects with an increased risk of CVD (Mann et al., 2009).

3. Cellular and molecular targets of isoflavones

3.1. Regulation of vascular tone

Our studies with human fetal endothelial cells have shown that genistein, daidzein and equol (1–100 nM) rapidly (2 min)
activate endothelial nitric oxide synthase (eNOS) at basal cytosolic Ca2+ levels,(Joy et al., 2006) implicating non-genomic
mechanisms in NO production. In contrast, higher concentrations of genistein (10–50 lM) inhibit tyrosine kinase activity
(Chu et al., 2005), and thus vascular actions of higher concentrations of isoflavones most likely result from non-selective
kinase inhibition (Siow et al., 2007; Mann et al., 2007, 2009). Phosphorylation of eNOS Ser1177 in human endothelial cells
induced by the daidzein metabolite equol is dependent on the activation of ERK1/2 and PI3-kinase/Akt, yet unaffected by
estrogen receptor (ER) antagonists ICI 182 780 and tamoxifen or uncoupling of G-protein receptors (Joy et al., 2006). Similar
studies in bovine aortic endothelial cells have shown that genistein elicits eNOS phosphorylation after via activation of PKA
but independent of ERK1/2 or PI3-kinase activation (Liu et al., 2004). In addition to acutely stimulating endothelial NO
production (Mann et al., 2007), isoflavones also act like other selective estrogen receptor modulators (SERMs) in regulating
vascular smooth muscle tone by inhibiting L-type Ca2+ channels (Figtree et al., 2000; Ruehlmann et al., 1998) or activating
Maxi K+ channels (Valverde et al., 1999; Dick et al., 2001). Recent evidence in bovine aortic endothelial cells establishes that
high genistein concentrations (50 lM) potentiate the activity of a TRPC5 cation channel, independently of inhibition of
tyrosine kinases or phospholipase C (Wong et al., 2010), thereby potentially modulating release of vasodilators, vascular
permeability and response of the endothelium to inflammatory mediators.

3.2. Regulation of vascular redox status and antioxidant gene transcription

Few studies have directly addressed the molecular targets of dietary isoflavones in the vasculature; however, studies with
cultured endothelial and smooth muscle cells implicate ROS in activation of intracellular signaling pathways linked to anti-
oxidant gene expression (Siow et al., 2007; Mann et al., 2009; Rimbach et al., 2008). Accumulating evidence suggests that
ROS act as a double-edged sword (Cave et al., 2006; Gao and Mann, 2009; Cheng et al., 2010), with by-products of normal
oxygen metabolism modulating cellular redox signaling, and an overproduction of ROS leading to oxidative stress and
damage of lipids, proteins and mitochondrial DNA (Droge, 2002), all of which are associated with the progression of
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atherosclerosis (Stocker and Keaney, 2004). ROS originate from various sources, e.g. mitochondrial electron transport
enzymes, NADPH oxidases, xanthine oxidase, cyclooxygenase, lipoxygenase and uncoupled eNOS (Droge, 2002). Under phys-
iological conditions, vascular cells generate ROS principally via NADPH oxidase (Forstermann, 2008), with mitochondrial
sources providing further regulation of redox signaling (Davidson and Duchen, 2007; Go et al., 2010; Gao and Mann,
2009; Cheng et al., 2010). Studies by Brownlee and colleagues have shown that increased mitochondrial ROS generation
may be a common mediator linking increased glucose metabolism via the polyol pathway, accumulation of advanced
glycation end-products (AGEs), activation of PKC and the hexosamine pathway with endothelial dysfunction (Nishikawa
et al., 2000; Brownlee, 2005). As antioxidant trials have largely failed to prevent or delay the development of vascular
complications associated with vascular complications in diabetes (Sano and Fukuda, 2008; Ristow and Zarse, 2010) and
atherosclerosis (Levy et al., 2009; Yoshihara et al., 2010), targeting endogenous antioxidant defense pathways may provide
a more effective treatment strategy.
Cells have evolved endogenous defense mechanisms to counteract oxidative stress, and the redox sensitive transcription
factor nuclear factor erythroid 2-related factor 2 (Nrf2) serves as a key regulator of cell survival (Ishii et al., 2000, 2004, 2010;
McMahon et al., 2003; Tong et al., 2007; Niture et al., 2010). As illustrated in Fig. 2, the coordinated induction of phase II and
antioxidant enzymes by Nrf2 is controlled through a cis-acting element designated the antioxidant response element (ARE or
electrophile response element, EpRE) in the promoter of target genes. Nrf2 is normally targeted for proteasomal degradation
via the cytosolic regulatory protein Kelch-like ECH-associated protein (Keap1) (Itoh et al., 1999; McMahon et al., 2003). ROS
and electrophilic agents lead to spatial alterations in the Nrf2-Keap1 complex, resulting in nuclear accumulation of Nrf2 and
upregulation of ARE-linked gene expression (Itoh et al., 2010). Although modification of Keap1 cysteine residues is key for
the activation of Nrf2 by a different stress agents, phosphorylation of serine/threonine residues in Nrf2 enhances nuclear
accumulation of Nrf2 (Surh et al., 2008) and kinases also modulate nuclear import and export of Nrf2 (Jain and Jaiswal,
2007; Surh et al., 2008). Targeted disruption of the Keap1 leads to constitutive expression of Nrf2-linked genes (Itoh

Fig. 2. Regulation of the Nrf2/Keap1 defense pathway by dietary isoflavones. Under basal conditions the redox sensitive transcription factor Nrf2 is targeted
via Keap1 for Cullin3 (Cul3)-mediated polyubiquitination (Ub) and proteasomal degradation. Under basal conditions, antioxidant genes are largely
downregulated with antioxidant response elements (ARE) in their promoter regions bound by small Maf proteins or repressive factors like Bach1. Dietary
isoflavones, ROS, reactive nitrogen species (RNS), electrophiles, lipid peroxidation products, and other putative ‘hormetic’ agents can modify cysteine (SH)
residues on Keap1 to alter its association with Nrf2. Isoflavones can activate kinases such as JNK, p38, PI3K and Akt which may phosphorylate Nrf2 to
enhance nuclear accumulation, while phosphorylation of Bach1 facilitates its nuclear export and Nrf2 binding to ARE (Kaspar and Jaiswal, 2010). Activity of
Nrf2 is also modulated by DJ-1, proposed to inhibit the association of Nrf2 with Keap1 (Malhotra et al., 2008; Collins et al., 2009). Antioxidant genes, such as
the cystine/glutamate anionic amino acid transporter (xCT), glutamate cystine ligase (GCL), glutathione reductase (GR), heme oxygenase-1 (HO-1),
NAD(P)H-quinone oxidoreductase-1 (NQO1) and sequestosome-1 (SQSTM1), are transcriptionally activated through Nrf2-small Maf dimers bound to ARE
sequences and play an integral role in restoring redox balance and ameliorating oxidative damage (Ishii et al., 2000; Itoh et al., 2010). Chronic oxidative
stress, associated with aging and cardiovascular diseases, decreases the expression or function of critical Nrf2 pathway modulators and may also increase
the abundance of the negative regulators Keap1 and Bach1, resulting in compromised Nrf2-linked antioxidant enzyme activity and impaired cellular redox
balance.
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472 R.C.M. Siow, G.E. Mann / Molecular Aspects of Medicine 31 (2010) 468–477

et al., 2004). Notably, Nrf2 deficient mice have higher levels of endogenous ROS production, diminished cystine transport
activity (Ishii et al., 2000) and are more sensitive to diabetes induced DNA damage and renal injury compared to wild type
mice (Yoh et al., 2008; Jiang et al., 2010).
Exposure of vascular endothelial and smooth muscle cells to ROS, oxidized low density lipoproteins, advanced glycation
end products or lipid peroxidation products such as 4-hydroxynonenal (4-HNE) enhances Nrf2/ARE-linked activation of
phase II defense enzymes, e.g. NAD(P)H:quinone oxidoreductase 1 (NQO1) (He et al., 2010), antioxidant enzymes, e.g. heme
oxygenase-1 (HO-1) (Churchman et al., 2009; He et al., 2010), and the cystine-glutamate anionic amino acid transporter
(xCT) (Siow et al., 1998). Phase II defense enzymes protect cells against the toxicity of ROS and 4-hydroxynonal (4-HNE)
(Forman et al., 2003; Zhang and Forman, 2009) and, as enzymatic removal of 4-HNE is decreased in diabetes (Traverso
et al., 2002), these pathways may be compromised in cardiovascular disease. HO-1 metabolises microsomal heme to the
chain-breaking antioxidants biliverdin/bilirubin and carbon monoxide (CO), which like NO can inhibit platelet aggregation,
act as a vasodilator under conditions of limited NO bioavailability (Siow et al., 1999). As recently reviewed (Forman et al.,
2009), the cystine-glutamate transporter (xCT) (Sasaki et al., 2002; Sato et al., 2001) and intracellular glutathione play an
important role in protecting cells from ROS and electrophile mediated damage.

4. Hormetic properties of isoflavones

The concept of hormesis describes the phenomenon where specific chemicals are able to induce biologically opposite
effects at different doses; with beneficial effects most commonly detected at lower doses and toxic effects at high doses
(Gems and Partridge, 2008). Dietary soy isoflavones are natural compounds with ‘hormetic’ properties, since they exhibit
beneficial actions in the cardiovascular system at low concentrations, but at high concentrations can lead to detrimental
effects in other organ systems (Wuttke et al., 2007). As illustrated in Fig. 3, hormesis may contribute to mechanisms under-
lying the ‘healthspan’ enhancing actions of isoflavones in relation to activation of Nrf2/Keap1 defense pathway (Maher and
Yamamoto, 2010). As dietary isoflavones also exhibit SERM properties, they are likely to exert organ specific effects, however
the ‘estrogenicity’ of isoflavones must be compared with the effects of estradiol, and the action of isoflavones as ER agonists
or antagonists will depend on their target tissues. In addition to their estrogenic properties, several other mechanisms may
account for their cellular actions of isoflavones, including their ability to either inhibit or activate a number of different
kinases depending on concentrations (Siow et al., 2007). However, many studies have reported use of high micromolar
concentrations in both in vitro and ex vivo experimental models which exceed the serum concentrations that can be achieved
by isoflavone consumption (Setchell, 1998; Mann et al., 2009). The ability of isoflavones to alter the redox state of cells, lead-
ing to activation of intracellular kinases and Nrf2/ARE-linked antioxidant enzymes, may underlie the adaptation of vascular
and other cell types to environmental and dietary stresses (Lindsay, 2005). However, the clinical evidence for therapeutic
benefits of long-term isoflavone supplementation on vascular redox homeostasis remains to be established (Hooper et al.,
2008; Li et al., 2010; Reynolds et al., 2006).

Beneficial
‘Healthspan’

Hormesis
Toxicity

Detrimental

Nrf2 activity

Fig. 3. Hormesis and vascular protection afforded by isoflavone-mediated activation of Nrf2. Physiologically relevant plasma concentrations of isoflavones
(<1 lM) activate intracellular signaling pathways linked to the upregulation of phase II and antioxidant genes via the redox sensitive transcription factor
Nrf2. We hypothesize that generation of reactive oxygen species in vascular endothelial and smooth muscle cells in response to low concentrations of
isoflavones, similar to shear stress mediated redox signaling (Hojo et al., 2002; Ungvari et al., 2006) serve as important ‘hormetic’ agents in the modulation
of kinases pathways upstream of Nrf2. There may only be a narrow ‘physiological window’ in which hormetic agents are able to regulate the adaptive,
cytoprotective actions of Nrf2. Diminished Nrf2 activity associated with aging (Suh et al., 2004; Shih and Yen, 2007; Collins et al., 2009) or over-expression
of Nrf2 (Wakabayashi et al., 2003) may compromise the effectiveness of hormetic agents.
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5. Epidemiological and clinical perspectives

5.1. Benefits of dietary isoflavones in atherosclerosis

Consumption of a traditional oriental diet high in soy is likely to play a key role in prevention of chronic vascular diseases
such as atherosclerosis (Setchell and Cassidy, 1999). The atheroprotective effects of soy-based diets have been partly attrib-
uted to the associated reduction in cholesterol levels in human studies (Jenkins et al., 2002; Reynolds et al., 2006) and similar
findings have also been reported in nonhuman primates fed soy-based diets (Register et al., 2005; Anthony et al., 1997).
However, studies in apolipoprotein E knock-out mice showed that atherosclerotic lesions are reduced when fed a soy-con-
taining diet despite unchanged serum lipid levels (Adams et al., 2002). These studies suggest that dietary soy inhibits ath-
erosclerotic lesion development by mechanisms other than lowering serum cholesterol. Findings from a recent study in aged
lipoprotein receptor knock-out mice has underscored the importance of oxidative stress coupled with a failure to up-regu-
late Nrf2-mediated antioxidant gene transcription in atherogenesis associated with diminished expression of the Nrf2 reg-
ulator DJ-1 (Collins et al., 2009). This model is especially relevant since multiple risk factors converge, in particular aging,
insulin resistance and a high fat diet. In the context of potential mechanisms underlying atherogenesis, it is of interest to
note that expression of CD36, a macrophage LDL scavenger receptor, is under the regulation of Nrf2 (Ishii et al., 2004).
Nrf2-mediated induction of CD36 may be beneficial only when the metabolism and export of oxidized LDL are not exceeded
and despite the convincing evidence that Nrf2 affords protection against oxidative stress in the cardiovascular system and
other organs (Mann et al., 2009; Lewis et al., 2010), regulation of CD36 by Nrf2 may be a critical step in the progression of
atherosclerosis. Recent studies in mice deficient in both Nrf2 and ApoE demonstrated that Nrf2 paradoxically makes athero-
sclerotic lesion progression significantly worse, despite the fact that inflammation and oxidative stress levels were dimin-
ished, due to increased uptake and accumulation of lipids within macrophages via Nrf2-mediated CD36 regulation and
the formation of foam cells (Sussan et al., 2008). The observation that Nrf2 mediates induction of CD36 and enhanced sus-
ceptibility to atherosclerosis clearly requires further investigation to ascertain whether up-regulation of Nrf2 in the vascu-
lature by isoflavones has similar consequences. Nevertheless, there is now compelling evidence that isoflavone
supplementation can represent an effective therapeutic strategy to enhance Nrf2 activity to protect the aging vasculature
(Adams et al., 2002; Register et al., 2005; Nagarajan, 2010; Mulvihill and Huff, 2010; Mann et al., 2009).

5.2. Benefit of dietary isoflavones in stroke

In vivo studies in rodent models of cerebral ischemia–reperfusion injury have shown that a diet enriched in soy isoflav-
ones is neuroprotective (Schreihofer et al., 2005; Burguete et al., 2006; Lovekamp-Swan et al., 2007). Treatment of ovarec-
tomized rats with either estrogen or a high soy isoflavone diet for two weeks significantly reduces the cerebral infarct size
following permanent middle cerebral artery occlusion (Schreihofer et al., 2005). In this same study, neuroprotection in
different regions of the brain was greater in estrogen-treated versus soy isoflavone fed rats. Similar studies in rats subjected
to transient middle cerebral artery occlusion (MCAo) for 90 min and 72 h reperfusion confirmed that cerebral infarct size was
significantly reduced by in animals fed a soy isoflavone versus isoflavone-free diet for 5 weeks (Burguete et al., 2006). More
recent studies established that a high soy isoflavone diet protects ovariectomized rats against transient MCAo induced injury
by increasing mRNA and protein expression of the anti-apototic gene bclxL and thereby attenuating cell death pathways
(Lovekamp-Swan et al., 2007). The ability of isoflavones to cross the blood–brain barrier and their documented neuroprotec-
tive action following stroke in animal models in vivo highlights the importance of further characterizing their mechanism(s)
of action in the brain. Notably, daidzein has been reported to protect CNS neurons from glutamate excitotoxicity and pro-
mote outgrowth of axons from hippocampal neurons (Zhao et al., 2002; Schreihofer and Redmond, 2009). Feeding ovarec-
tomized rats a soy isoflavone enriched diet increases eNOS expression in cerebral microvascular cells without measurable
changes in eNOS-Ser1177 phosphorylation (Schreihofer et al., 2010). Although recent studies establish that genistein and
equol reduce cerebral infarct size in both male and female rats following transient MCAo, there were negligible differences
in cerebral blood flow in rats fed an isoflavone-rich or deficient diet (Ma et al., 2010). It remains to be established whether
dietary isoflavones can affect the vascular reactivity of small cerebral vessels in vivo prior to and after transient MCAo-reper-
fusion injury. In the context of cerebral ischemia, a randomized, double-blinded, placebo-controlled study in patients with
prior ischemic stroke demonstrated that isoflavone supplementation (80 mg/day) for 12 weeks reduces serum C-reactive
protein levels and improves endothelium-dependent flow-mediated dilation (Chan et al., 2008). These findings resonate
with earlier reports that the beneficial actions of isoflavone supplementation may only be evident in patients with either
existing cardiovascular disease (Cassidy et al., 2006; Mann et al., 2009) or a low baseline brachial artery flow-mediated
dilation (Li et al., 2010).

6. Concluding remarks – isoflavones for enhancing ‘healthspan’

Despite the fact that dietary isoflavones are used regularly by postmenopausal women as alternatives for estrogen/prog-
estrin hormone replacement therapy, their cardiovascular health benefits remain controversial. There is evidence that post-
menopausal women supplemented with genistein show marked improvement in glycemic control and vascular reactivity
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compared to a placebo group (Villa et al., 2009). However, these authors emphasized that the pre-existing metabolic status
of patients/subjects could affect endothelial dependent responses to isoflavones. Thus, reported discrepancies in the cardio-
vascular benefits of isoflavone supplementation in clinical trials may reflect (i) differences in intestinal bacterial flora and
hence bioavailability of soy isoflavone metabolites (Cassidy et al., 2006; Setchell and Clerici, 2010), (ii) differences in
dose–response effects (Hooper et al., 2008), (iii) length of isoflavone supplementation, (iv) limited number of subjects and
(iv) importantly the pre-existing metabolic status of subjects included in supplementation trials (Villa et al., 2009).
In view of the importance of the developmental origins of adult disease (Barker, 2002; McMillen and Robinson, 2005;
Hanson and Gluckman, 2005), it is worth highlighting that exposure to dietary soy during fetal development and early life
may reduce the susceptibility to CVD and obesity in adulthood (Todaka et al., 2005; Dolinoy et al., 2006). Thus, by influencing
developmental plasticity in utero and in early postnatal development, isoflavones may alter the expression of genes associ-
ated with ‘metabolic memory’ and endothelial function (Dolinoy and Jirtle, 2008; Villeneuve and Natarajan, 2010). In the
context of epigenetics, hypermethylation of CpG sites in the Nrf2 promoter suppresses Nrf2 expression in transgenic mice
with prostate tumors (Yu et al., 2010), and treatment human prostate cancer cell lines with the soy isoflavones genistein
and daidzein leads to demethylation of the phase II defense enzyme glutathione S-transferase P1 (GSTP1) and an upregua-
tion of protein expression (Vardi et al., 2010). The cytoprotective effects of isoflavones may reflect their ability to inhibit the
activity of histone acetyltransferase and DNA methyltransferase (Gilbert and Liu, 2010). It remains to be established whether
dietary isoflavones can affect or protect against disease induced chromatin histone and/or DNA methylation in vascular
endothelial and smooth muscle cells.

Acknowledgements

This work in this review was supported in part by the British Heart Foundation, Heart Research UK, Biotechnology & Bio-
logical Sciences Research Council, The Henry Smith Charity, Great Britain Sasakawa Foundation and EU COST ACTION B35.
We gratefully acknowledge our collaborators in the cited references.

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