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Phytoestrogens and cardiovascular health
Journal of the American College of Cardiology © 2000 by the American College of Cardiology Published by Elsevier Science Inc.
Vol. 35, No. 6, 2000 ISSN 0735-1097/00/$20.00 PII S0735-1097(00)00590-8
REVIEW ARTICLE
Phytoestrogens and Cardiovascular Health Lynette Wroblewski Lissin, MD, John P. Cooke, MD, PHD, FACC Stanford, California Coronary artery disease is the leading overall cause of mortality for women and increases dramatically after menopause. Estrogen has many beneficial cardiovascular actions although concerns have been raised about its effects on the progression of breast and uterine neoplasms and its tendency to increase coagulability. Selective estrogen agonists may be superior to conventional estrogens. A dietary source of a partial estrogen agonist is the plant-based group of phytoestrogens, which include isoflavones, lignans and coumestans. Phytoestrogens have a similar structure to estradiol and have weak affinity for the estrogen receptor. Epidemiologic data indicate that women ingesting high amounts of phytoestrogens, particularly as isoflavones in soy products, have less cardiovascular disease, breast and uterine cancer and menopausal symptoms than those eating Western diets. Preclinical and clinical studies have found that isoflavones have lipid-lowering effects as well as the ability to inhibit low-density lipoprotein oxidation. They have been shown to normalize vascular reactivity in estrogendeprived primates. Furthermore, phytoestrogens have antineoplastic effects with inhibition of cellular proliferation as well as angiogenesis, properties that could be protective against cancer development. Finally, menopausal symptoms and bone density may be favorably influenced by phytoestrogens. In summary, phytoestrogens, in the form of dietary isoflavones, represent a new area to explore in pursuit of nutritional approaches to cardiovascular protection. (J Am Coll Cardiol 2000;35:1403–10) © 2000 by the American College of Cardiology
ESTROGEN AND CARDIOVASCULAR DISEASE IN WOMEN Coronary artery disease (CAD) is the leading cause of death among women in developed nations. It is well recognized that its incidence increases substantially after menopause, purportedly due to the loss of estrogen’s protection. The increased incidence of cardiovascular events in younger, surgically postmenopausal women supported this hypothesis. The role of estrogen deficiency in cardiovascular disease (CVD) was further indicated by preclinical studies demonstrating that estrogen could inhibit atherosclerosis. Population-based, observational studies revealed that the risk of cardiovascular events in women taking hormones was cut in half (i.e., a relative risk of 0.5), providing support for a benefit of estrogen (1–3). Subsequently, a number of clinical trials have demonstrated that, in postmenopausal women, estrogen has favorable effects upon lipid profiles, endothelial cell function, vascular reactivity and hemostatic factors. The Postmenopausal Estrogen/Progestin Interventions Trial (4) convincingly showed that women taking estrogen replacement therapy experienced a significant decline in levels of low-density lipoprotein (LDL) and li-
From the Stanford University Medical Center, Stanford, California. This work was supported, in part, by a grant from the National Heart, Lung and Blood Institute (HL58638 and HLO7708). Dr. Cooke is an established investigator of the American Heart Association. Dr. Lissin is an American College of Cardiology/ Merck Fellowship Awardee. Manuscript received March 8, 1999; revised manuscript received November 24, 1999; accepted January 13, 2000.
poprotein(a) while the high-density lipoprotein (HDL) favorably increased. Estrogen influences vascular reactivity through effects on smooth muscle cells as well as the endothelium. The multiple effects of estrogen on vasomotor responses were recently summarized by Farhat et al. (5) and include relaxation of precontracted vessels, inhibition of calcium entry, enhanced expression of nitric oxide synthase, stimulation of prostacyclin production and prevention of myointimal hyperplasia. Estrogen has also been shown to potentiate endothelium-dependent vasodilation of the coronary (6,7) and brachial (8,9) arteries in both postmenopausal women and primates (10 –12). Because of the extent of this supportive data, in the last decade, postmenopausal hormone replacement therapy (HRT) has been used in an effort to reduce CVD in women. Today, the evidence of estrogen’s efficacy for prevention of CVD has been called into question due to the negative results of the Heart and Estrogen/Progestin Interventions Trial (13). In this first randomized clinical trial performed in women with known CAD, cardiovascular events were not prevented by combination therapy of oral estrogen and progesterone, and, surprisingly, early events were actually increased in treated patients. This study, as well as others, underscored the known risks of estrogen therapy, which include hypertriglyceridemia, endometrial hyperplasia, tumorigenesis and hypercoagulable states. Unopposed estrogen therapy causes symptomatic and premalignant endometrial hyperplasia, which can be ameliorated with the addition of progesterone although at a cost of attenuating the lipid-lowering effect of estrogen (4). The
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Abbreviations CAD CVD HDL HRT ICAM-1 LDL VCAM-1 VLDL
and Acronyms ⫽ coronary artery disease ⫽ cardiovascular disease ⫽ high density lipoprotein ⫽ hormone replacement therapy ⫽ intercellular adhesion molecule-1 ⫽ low-density lipoprotein ⫽ vascular cell adhesion molecule-1 ⫽ very low density lipoprotein
modest, but nevertheless concerning, increased incidence of breast cancer in women taking estrogens is a major limitation of this prophylactic therapy (14). Angiogenesis, a fundamental process necessary for tumorigenesis as well as atherosclerotic progression, can be enhanced by estrogen (15), which induces expression of cellular adhesion molecules and organization of endothelial cells into primitive blood vessels, upon which tumors depend. The effect of estrogen upon the coagulation system is variable. Platelets incubated with estradiol manifest decreased adherence (16) and aggregation (17) although studies in women on HRT have not shown similar antiplatelet effects (18,19). Other reports have noted decreased fibrinogen (4) and decreased plasminogen activator inhibitor (20 –22) levels in women taking HRT, which may be protective, although thrombotic events are actually increased in this population (13).
PHYTOESTROGENS: AN ALTERNATIVE TO ESTROGEN? Given the demonstrated risks to conventional HRT, many women and their practitioners have been in search of alternatives. Phytoestrogens are naturally occurring estrogens that may have beneficial effects on the cardiovascular system and may also alleviate common illnesses afflicting women, such as menopausal symptoms, osteoporosis and breast cancer (Table 1). Phytoestrogens may have advantages over conventional estrogens in that they may lower LDL cholesterol without inducing hypertriglyceridemia (23); they may relieve menopausal symptoms without increasing the risk of uterine or breast neoplasia (24); they may enhance vascular function without accelerating pathological angiogenesis (25); and there are no reports of increased thrombotic events. There is not yet enough evidence from large randomized clinical trials to make an unqualified recommendation about the use of phytoestrogens, but accumulating data indicate that phytoestrogens may be an alternative therapy for postmenopausal women at risk for CVD. The evidence for cardiovascular benefit of phytoestrogens is the subject of this review.
PHARMACOLOGY AND PHYSIOLOGY OF PHYTOESTROGENS Phytoestrogens are naturally occurring, plant based diphenolic compounds that are similar in structure and function
JACC Vol. 35, No. 6, 2000 May 2000:1403–10 Table 1. Effects of Phytoestrogens in Basic, Animal and Human Studies* Preclinical Studies 2 Cholesterol (44,45,50) 2 LDL oxidation (46) 1 Antioxidant enzymes (48) 2 Atherosclerotic lesions (45) 1 Vascular reactivity (54,55) 2 Platelet aggregation (51,52) 2 Expression of ICAM-1 and VCAM-1 (49) 2 Angiogenesis (25,61) 2 Neoplastic proliferation (62–66,68) 2 Bone loss (88,89) Clinical Studies Beneficial 2 Cholesterol (23,41,42) 2 LDL oxidation (47) 2 Perimenopausal hot flashes (56) 2 Cancer incidence (69,73–84) 1 Bone mineral density (90) ?Detrimental 1 breast secretions (85) 1 proliferation of breast epithelium (86) *See text for details. ICAM-1 ⫽ intercellular adhesion molecule; VCAM-1 ⫽ vascular cell adhesion molecule.
to estradiol (Fig. 1). There are many types of phytoestrogens, but the major categories include isoflavones, lignans and coumestans. Common and significant sources of phytoestrogens are soybeans (isoflavones), cereals and oilseeds such as flaxseed (lignans) and alfalfa sprouts (coumestans). These estrogen-like compounds were identified in the 1940s, when an epidemic of infertility among sheep was investigated. The animals were grazing on clover (Trifolium sp)—a plant with a high content of formononetin, which is converted by ruminal bacteria to isoflavones, the predominant type of phytoestrogens.
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The most common and best studied phytoestrogen is the class of isoflavones. The most abundant active components of isoflavones are genistein and daidzein. These agents appear to have selective estrogenic actions, i.e., in some tissues they display proestrogenic responses, whereas in others, they inhibit estrogenic effects. The recent identification of a second subtype of estrogen receptor lends support to the theory of selective estrogenic action (26,27). Estrogenic activity is dependent on the affinity of binding to the estrogen receptors, which is determined by the presence of the aromatic ring as well as hydroxyl groups at specific sites (28). Compared with estradiol, genistein and daidzein bind estrogen receptors with 100 and 1,000 times less affinity, respectively (29). Nevertheless, in the quantities that can be consumed in the diet, isoflavones can have biological effects. Antiestrogenic effects may be due to competitive inhibition at the estrogen receptor, interference with gonadotrophins, inhibition of estrogen synthesis or increased synthesis of estrogen-binding protein. Phytoestrogens do not have feminizing effects in male primates as reflected by unchanged weights of the reproductive organs in these animals, although prenatal exposure of rats resulted in diminished weights of ovaries and uteri in females (30). Isoflavones are widely consumed by Asian populations, predominantly in the form of soy. The typical concentration of genistein in soy foods is 1 to 2 mg per g of protein, and Asians consume 20 to 80 mg of genistein per day in the usual diet. By contrast, the average American ingests only 1 to 3 mg per day (31). Dietary studies can be inconsistent, however, as there is significant variation of isoflavone content between varieties of soy beans, the particular crop, as well as the processessing methods. Isoflavones are inactive molecules when in the form of glycosides (genistin, daidzin), but as aglycones (genistein, daidzein) intestinal absorption is possible. Intestinal absorption varies greatly between individuals, which may be related to the content of dietary fiber and the state of intestinal microflora. After absorption, isoflavones are reconjugated to glucuronides and excreted unchanged in the urine (32). Plasma levels increase 6 1/2 h after ingestion in a dose dependent fashion (33), and urinary levels increase dramatically after supplemented diets have begun. For example, urinary levels of genistein increased from 0.8 umol/day to 26 umol/day and daidzein from 0.3 umol/day to 23 umol/day after a nine day ingestion of 100 g tofu and 45 g soy protein isolate (34). Concentrations of the phytoestrogens and their metabolites can be measured in urine, plasma and other bodily fluids and vary widely, even during controlled nutrition studies. As dietary phytoestrogen metabolism is profoundly influenced by gastrointestinal flora, antibiotic use or bowel disease will modify metabolism and therefore, bioavailability. Equol is a metabolite of daidzein, which shows enormous variation in urinary excretion. Some studies have distinguished individuals as either equol excreters or nonexcreters because the excretion rates vary so substantially (35). However, this appears to be independent of the
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dose-dependent excretion of genistein and daidzein. Chronic ingestion of isoflavones also affects their metabolism. For example, after one month of a high soy diet (36 oz of soymilk containing 100 mg daidzein and 115 mg genistein), young, healthy women exhibit increased absorption rates, as indicated by a higher peak serum level, but also accelerated excretion based on the percentage of ingested isoflavone recovered in the urine (36). There are many other natural agents classified as phytoestrogens, based on their structure or function. Resveratrol is another potentially important phytoestrogen with a structure similar to diethylstilbesterol. Resveratrol is present in grapes and, therefore, wine and has been proposed to be the agent responsible for the “French-paradox” (37). The French paradox derives from the epidemiological observation that the French and Italian populations suffer from less CVD than other Europeans and Americans, despite a relatively high intake of dietary fat. Resveratrol has been shown to bind to human estrogen receptors, initiate transcriptional activity dependent on the estrogen-response element, activate estrogen-regulated genes, cause proliferation of estrogen-dependent breast cancer cell lines and inhibit the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in human endothelial cells (38). Its action can also be inhibited by estrogen antagonists such as tamoxifen (39).
BENEFICIAL CARDIOVASCULAR EFFECTS Favorable effects of phytoestrogens on lipid profiles, vascular reactivity, thrombosis and cellular proliferation have been reported. It is plausible that the lower incidence of CAD in populations ingesting diets high in phytoestrogen is due to an improved lipid profile. In a study of 24 healthy normocholesterolemic men assigned to either a low fat soy diet or low fat animal protein diet, no difference was observed in total cholesterol levels (40), but when patients with type II hyperlipoproteinemia (mean TC 409 mg/dL) were placed on high soy diets for four weeks, the total cholesterol and LDL decreased by 16%. These changes were attributed to increased LDL degradation (41). A later study designed specifically to examine diets differing only in their protein source randomly assigned healthy men to diets either: 1) high in fat, 2) low in fat with soy protein, or 3) low in fat with animal protein. Both of the low fat diets decreased total cholesterol levels and blood pressure compared with the high fat diet, but the soy protein had a more potent hypocholesterolemic effect (10% vs. 5% decline in total cholesterol) (42). Although some published studies have failed to observe a fall in cholesterol values on a soy-based diet, a meta-analysis of 38 trials of soy protein consumption in humans revealed an improvement in total cholesterol by 9% and LDL by 13%, as well as a decrease in triglyceride levels of 10% (23). In these trials, the average intake of soy was 47 g/day. The
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extent of reduction was dependent upon the baseline level of cholesterol; for example, in subjects with moderate hypercholesterolemia (259 –333 mg/dL), a decrease in total cholesterol of 7.4% was observed, whereas subjects with severe hypercholesterolemia (⬎335 mg/dL) achieved a decline of 19.6%. Three theories of possible mechanisms for the hypocholesterolemic effects of phytoestrogens have been proposed. One explanation is that phytoestrogens cause an increase in the excretion of bile acids and, therefore, enhance removal of LDL. Others have proposed that phytoestrogens initiate a hyperthyroid state, supported by the finding in some studies of increased free thyroxine levels after feeding soy proteins to animals (43). The third and best supported mechanism of phytoestrogen lipid-lowering is that of altered hepatic metabolism with augmented LDL and VLDL removal by hepatocytes. Sirtori et al. (44) demonstrated substantial reductions in cholesterol levels with enhanced LDL degradation and increased binding of radiolabeled LDL to receptors. A study using the LDL-receptor deficient mouse has provided further support for an effect of isoflavones on LDL receptor activity (45). When normocholesterolemic C57BL/6J mice were fed a high cholesterol, isoflavone-poor diet, they developed hyperlipidemia and intimal lesions in the proximal aorta. However, those animals fed an isoflavone-rich diet exhibited a decrease in total cholesterol and VLDL cholesterol. By contrast, this benefit of an isoflavone-rich diet was not observed in LDL-r null mice, suggesting that isoflavones may reduce lipid levels by increasing the activity of the LDL receptor. Furthermore, there was a 50% reduction in intimal lesion area in isoflavone treated C57BL/6J but not LDLr-null mice, again implicating an effect of isoflavones on LDL receptor activity in conferring cardiovascular protection. Isoflavones may also inhibit oxidation of LDL. Kapiotis and colleagues (46) observed that LDL oxidation products, assayed as Thiobarbituric Acid–Reactive Substances in either cell free or endothelial cell systems, were strongly inhibited by genistein, somewhat inhibited by daidzein but not affected by genistin (the glycosylated form of genistein) or control. Furthermore, both genistein and daidzein were found to protect against cytotoxic effects of oxidized LDL as assessed by cellular morphologic features and lactate dehydrogenase release by cultured endothelial cells. A trial in humans enrolling healthy volunteers consuming three soy bars per day for two weeks (total 36 mg genistein and 21 mg daidzein per day) demonstrated a significant amount of genistein and daidzein within the LDL fractions, as well as a significant prolongation of the lag time to LDL oxidation, implying an antioxidant effect (47). Genistein has also been shown to inhibit hydrogen peroxide production and increase the activity of antioxidant enzymes, such as catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase. Furthermore, genistein, and to a lesser degree daidzein, can inhibit superoxide anion generation by xanthine/xanthine oxidase (48).
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Atherosclerosis is initiated by monocytes binding to the endothelium and migrating into the intimal layer to develop into foam cells. The adhesiveness of endothelial cells is due to lipid-induced, oxidant-sensitive transcription of adhesion molecules and chemokines, which promote monocyte binding. This binding is reliant upon intercellular signalling and oxidant-sensitive transcription of adhesion molecules. Phytoestrogens may further protect against atherosclerosis by interfering with these initial processes. Although scant information has been published thus far, genistein is capable of inhibiting the expression of ICAM-1 and VCAM-1 on human endothelial cells co-cultured with monocytes (49). Furthermore, by reducing oxidative stress, it is possible that phytoestrogens deactivate the transcriptional pathways leading to the expression of genes involved in monocyte adhesion and infiltration. The response to isoflavones is influenced by gender. Monkeys that were fed an atherogenic diet followed by a soy protein phytoestrogen-rich diet in a six-month crossover trial had a lowering of LDL and VLDL levels compared with control values. However, female animals additionally manifested an increase in HDL, decrease of lipoprotein (a), lower LDL molecular weight, decrease in apolipoprotein B, AI, A2 and no hypertriglyceridemia (50). Phytoestrogens may have less prothrombotic tendencies than the currently used formulations of estrogen. In vitro studies of genistein and daidzein reveal decreased platelet aggregation by collagen and thromboxane (51,52), which may be due to an inhibition of binding of the thromboxane receptor or diminished tyrosine phosphorylation. However, in a small study of 20 healthy, normocholesterolemic men fed a soy diet for 28 days, a significant increase in plasma levels of genistein and daidzein occurred but without a change in platelet aggregation (53). It was suggested that on such supplements, serum levels of the active compound do not reach levels comparable with those required to affect platelet aggregation in the in vitro studies. Preclinical studies suggest that vascular reactivity may be favorably influenced by phytoestrogens. Primates manifest an improvement in endothelium mediated vasodilation when treated with phytoestrogens. Specifically, postmenopausal monkeys on a phytoestrogen-rich diet for six months exhibited normal coronary artery vasodilation in response to locally-administered acetylcholine, whereas a vasoconstrictive response was seen in male animals as well as female monkeys with a low intake of phytoestrogens (54). In vitro studies of isolated vessels have examined the mechanisms of phytoestrogen-induced vasodilation (55). Estradiol-17B, genistein and daidzein were all found to relax mesenteric arterial rings of rats in a dose dependent manner. Consistent with receptor affinity assays, estradiol was the most potent vasodilator, followed by genistein, then daidzein. In these isolated arterial rings, response was independent of gender. The vasorelaxation was endothelium-independent and was not blocked by antagonists of nitric oxide or prostacyclin production.
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Estrogen withdrawal results in symptomatic hot flashes in perimenopausal women and may represent an alteration in vasomotor response to endocrinologic stimuli. As a population, Asian women experience less perimenopausal hot flashes, a benefit attributed to phytoestrogens. Also, in a randomized study of 58 postmenopausal non-Asian women, those consuming soy protein supplementation in the form of 45 g of soy flour per day suffered less from hot flashes than control women (56), invoking another potential use for phytoestrogen supplementation. Phytoestrogens may have electrophysiological effects as well. The sensitivity of ventricular myocytes to betaadrenergic stimulation can be enhanced by genistein and daidzein, implicating a role of tyrosine kinase regulation of cardiac ion channels (57). However, there is, as yet, no known clinical effect of phytoestrogens on arrhythmias.
BENEFICIAL NONCARDIOVASCULAR EFFECTS The most powerful argument against the routine use of HRT for postmenopausal women is the increased risk of cancer. Phytoestrogens do not appear to increase the risk of breast and uterine cancer; in fact, observations from in vitro and animal studies indicate that phytoestrogens may have antineoplastic effects. These antineoplastic effects may be related to suppression of angiogenesis, inhibition of tyrosine kinase activity, or partial antagonism of estrogen receptors. Tumor growth is dependent upon angiogenesis, which is a tightly regulated, complex process (58). Endothelial cells play a pivotal role in angiogenesis, which requires endothelial cell attachment, migration, proliferation, protease production and subsequent organization into vascular structures. Whereas estradiol has been demonstrated to augment various steps in the angiogenic process (59), components of phytoestrogens may actually inhibit angiogenesis. Genistein reduces basic fibroblast growth factor induced endothelial cell migration (60) and capillary-like tube formation, perhaps by inhibiting tyrosine kinase or by decreasing levels of plasminogen activator and plasminogen-inhibitor and, thus, interrupting the tightly regulated balance of proteolytic degradation (25). In the setting of atherosclerosis, the enhancement of angiogenesis may hold therapeutic potential to relieve myocardial ischemia by increasing blood flow. Therapeutic angiogenesis has become a very active field of research with ongoing clinical studies designed to explore the safety and efficacy of various growth factors, delivered as protein or gene therapy. However, the neovascularization of atherosclerotic plaques could lead to plaque instability and resultant negative consequences. Indeed, Folkman and colleagues (61) have recently demonstrated that endostatin, an inhibitor of angiogenesis, can slow the development of aortic lesions in the hypercholesterolemic apoE-deficient mouse. Phytoestrogens, particularly genistein, also have antiproliferative effects. This has been demonstrated in multiple cell lines including prostate cancer cells (62), breast cancer
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cells (both estrogen receptor positive as well as negative) (63) and other tumor cells (neuroblastoma, sarcoma, retinoblastoma), as well as vascular endothelial cells and fibroblasts (64). The in vitro effect of genistein, a known tyrosine kinase inhibitor, on breast cancer cell growth was evaluated by quantifying DNA and total cellular protein levels compared with the effect of estrogen (65). Over a range of 0.1 to 1 uM, genistein had a stimulatory, estrogen-like effect on estrogen receptor positive cell proliferation, but at ⬎10 uM of genistein, growth was totally inhibited. However, estrogen receptor negative cells showed no response to, or possibly inhibition by, genistein even at low concentrations (10 nM–1 uM). Another group of investigators demonstrated that the effect of phytoestrogens in inhibiting proliferation is a reversible, cytostatic effect (60). Even at concentrations of up to 200 umol/L, genistein was not cytotoxic in quiescent cells. The mechanism of this inhibitory effect of genistein is speculated to be its antagonism of tyrosine kinase. However, other investigators have observed a genistein-induced inhibition of proliferation despite adequate levels of tyrosine kinase activity (66). This antiestrogenic effect of phytoestrogens may provide protection analogous to the estrogen partial-agonist, tamoxifen, in the treatment or prevention of breast cancer. Animal models support an antineoplastic role for phytoestrogens; for example, rats given soy-derived phytoestrogens develop fewer mammary tumors after exposure to carcinogens (67). The evidence that phytoestrogens may protect against cancer development derives largely from in vitro studies of human cell lines, animal models of neoplasia and epidemiological data. Overall, most epidemiological studies have indicated a protective effect of soy ingestion (68). Epidemiologic studies reveal a decreased incidence of breast, prostate and endometrial cell cancer in populations ingesting high amounts of soy. Asian women develop breast cancer four to six times less often than their American counterparts, but this advantage wanes after living in the U.S. (69 –71), implicating a protective influence of traditional diets. Furthermore, breast cancer patients have lower urinary isoflavone levels, suggesting that they have decreased ingestion, absorption, excretion or overall bioavailability of isoflavones compared with women without breast cancer (72,73). The benefits of dietary phytoestrogens may extend to other neoplasms, and to men as well as women. Hawaiians of Japanese descent who maintain a traditional diet with high soy content have less risk of prostate cancer than nonJapanese Hawaiians (74), and consumption of a diet enriched with lignans (legumes and fruit) is associated with decreased prostate cancer risk in Seventh Day Adventists (75). If these beneficial effects of diet are due to phytoestrogen content, the benefit may be linked to the observation that certain phytoestrogens (i.e., genistein and biochanin A) have been observed to inhibit the growth of androgendependent and independent human prostate cancer cell lines (62). High soy consumption (as tofu, miso or soybean sprouts) is associated with reduced risks of lung, gastric and
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rectal cancer in Chinese and Japanese men and women (76 – 83). Rates of breast, ovarian, prostate and colon cancer are negatively correlated with cereal and phytoestrogen intake when comparing cancer mortality rates and food availability data among countries (84). Despite the plethora of evidence that phytoestrogens have antineoplastic effects, two concerning clinical reports have been published. One study reported the effect upon monthly nipple aspirations in pre- and postmenopausal women ingesting soy protein containing 38 mg genistein/day (85). An increased total fluid volume was recovered in premenopausal women and postmenopausal women during and after phytoestrogen treatment. Additionally, there was an increased frequency of cellular hyperplasia in the aspirate. However, there were no controls in this study, and the aspirations themselves could account for the abnormalities. Nevertheless, the study does raise some concern for high risk patients. Another report of 29 women taking 60 g of soybean supplement daily found increased proliferation of breast lobular epithelium assessed by 3H-thymidine labeling (86). However, there are no human studies that have linked dietary phytoestrogens to the development of breast cancer. Similar to other selective estrogen antagonists, phytoestrogens may also protect against osteoporosis. Studies of aging or ovariectomized rats have shown that a soy protein diet protects against bone loss (87,88) although this effect is not mimicked by supplemental genistein (89), suggesting that some other component of soy protein may be responsible for the beneficial effect. However, a trial in postmenopausal women revealed an increase in bone mineral density in the lumbar spine after isoflavone supplementation (90).
SUMMARY To conclude, there are accumulating data from epidemiologic studies, human trials, animal models and in vitro analysis to support the increased use of phytoestrogens, particularly in individuals at high risk of CVD. Favorable effects upon lipid profiles, vascular reactivity, angiogenesis and tumorigenesis have been attributed to the isoflavones genistein and, to a lesser extent, daidzein. The increasing use by the general public of alternative therapies including these agents necessitates a greater understanding of their effects, as well as a need for clinical investigations to guide practitioners in their recommendations. The current state of knowledge indicates that the world of plant based estrogens has much to offer, but significant questions remain. Do phytoestrogen-enriched diets alleviate symptoms of atherosclerosis or reduce the risk of vascular events? If so, can this effect be attributed to a specific phytoestrogen, and can the benefit be mimicked by supplementation with that specific compound? How do the benefits of phytoestrogen therapy on the lipoprotein profile or vascular reactivity compare with other therapies? Which subgroups of the population might benefit most from phytoestrogen use? What are the side effects of such supplementation? Is it safe for women with a
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personal or family history of breast or uterine cancer to use phytoestrogens? Answers to these questions, which could be obtained by large, randomized clinical trials, may lead to a greater understanding of the hormonal effects upon the cardiovascular system and provide women with more options for preventative treatment. In the interim, it seems reasonable to recommend that women at high risk of cardiovascular morbidity and mortality, particularly those without a personal or family history of breast or uterine cancer, increase their consumption of foods rich in phytoestrogens such as soy protein. Reprint requests and correspondence: John P. Cooke, Division of Cardiovascular Medicine, 300 Pasteur Drive, Stanford, California 94305-5406. E-mail: [email protected].
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Vol. 35, No. 6, 2000 ISSN 0735-1097/00/$20.00 PII S0735-1097(00)00590-8
REVIEW ARTICLE
Phytoestrogens and Cardiovascular Health Lynette Wroblewski Lissin, MD, John P. Cooke, MD, PHD, FACC Stanford, California Coronary artery disease is the leading overall cause of mortality for women and increases dramatically after menopause. Estrogen has many beneficial cardiovascular actions although concerns have been raised about its effects on the progression of breast and uterine neoplasms and its tendency to increase coagulability. Selective estrogen agonists may be superior to conventional estrogens. A dietary source of a partial estrogen agonist is the plant-based group of phytoestrogens, which include isoflavones, lignans and coumestans. Phytoestrogens have a similar structure to estradiol and have weak affinity for the estrogen receptor. Epidemiologic data indicate that women ingesting high amounts of phytoestrogens, particularly as isoflavones in soy products, have less cardiovascular disease, breast and uterine cancer and menopausal symptoms than those eating Western diets. Preclinical and clinical studies have found that isoflavones have lipid-lowering effects as well as the ability to inhibit low-density lipoprotein oxidation. They have been shown to normalize vascular reactivity in estrogendeprived primates. Furthermore, phytoestrogens have antineoplastic effects with inhibition of cellular proliferation as well as angiogenesis, properties that could be protective against cancer development. Finally, menopausal symptoms and bone density may be favorably influenced by phytoestrogens. In summary, phytoestrogens, in the form of dietary isoflavones, represent a new area to explore in pursuit of nutritional approaches to cardiovascular protection. (J Am Coll Cardiol 2000;35:1403–10) © 2000 by the American College of Cardiology
ESTROGEN AND CARDIOVASCULAR DISEASE IN WOMEN Coronary artery disease (CAD) is the leading cause of death among women in developed nations. It is well recognized that its incidence increases substantially after menopause, purportedly due to the loss of estrogen’s protection. The increased incidence of cardiovascular events in younger, surgically postmenopausal women supported this hypothesis. The role of estrogen deficiency in cardiovascular disease (CVD) was further indicated by preclinical studies demonstrating that estrogen could inhibit atherosclerosis. Population-based, observational studies revealed that the risk of cardiovascular events in women taking hormones was cut in half (i.e., a relative risk of 0.5), providing support for a benefit of estrogen (1–3). Subsequently, a number of clinical trials have demonstrated that, in postmenopausal women, estrogen has favorable effects upon lipid profiles, endothelial cell function, vascular reactivity and hemostatic factors. The Postmenopausal Estrogen/Progestin Interventions Trial (4) convincingly showed that women taking estrogen replacement therapy experienced a significant decline in levels of low-density lipoprotein (LDL) and li-
From the Stanford University Medical Center, Stanford, California. This work was supported, in part, by a grant from the National Heart, Lung and Blood Institute (HL58638 and HLO7708). Dr. Cooke is an established investigator of the American Heart Association. Dr. Lissin is an American College of Cardiology/ Merck Fellowship Awardee. Manuscript received March 8, 1999; revised manuscript received November 24, 1999; accepted January 13, 2000.
poprotein(a) while the high-density lipoprotein (HDL) favorably increased. Estrogen influences vascular reactivity through effects on smooth muscle cells as well as the endothelium. The multiple effects of estrogen on vasomotor responses were recently summarized by Farhat et al. (5) and include relaxation of precontracted vessels, inhibition of calcium entry, enhanced expression of nitric oxide synthase, stimulation of prostacyclin production and prevention of myointimal hyperplasia. Estrogen has also been shown to potentiate endothelium-dependent vasodilation of the coronary (6,7) and brachial (8,9) arteries in both postmenopausal women and primates (10 –12). Because of the extent of this supportive data, in the last decade, postmenopausal hormone replacement therapy (HRT) has been used in an effort to reduce CVD in women. Today, the evidence of estrogen’s efficacy for prevention of CVD has been called into question due to the negative results of the Heart and Estrogen/Progestin Interventions Trial (13). In this first randomized clinical trial performed in women with known CAD, cardiovascular events were not prevented by combination therapy of oral estrogen and progesterone, and, surprisingly, early events were actually increased in treated patients. This study, as well as others, underscored the known risks of estrogen therapy, which include hypertriglyceridemia, endometrial hyperplasia, tumorigenesis and hypercoagulable states. Unopposed estrogen therapy causes symptomatic and premalignant endometrial hyperplasia, which can be ameliorated with the addition of progesterone although at a cost of attenuating the lipid-lowering effect of estrogen (4). The
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Abbreviations CAD CVD HDL HRT ICAM-1 LDL VCAM-1 VLDL
and Acronyms ⫽ coronary artery disease ⫽ cardiovascular disease ⫽ high density lipoprotein ⫽ hormone replacement therapy ⫽ intercellular adhesion molecule-1 ⫽ low-density lipoprotein ⫽ vascular cell adhesion molecule-1 ⫽ very low density lipoprotein
modest, but nevertheless concerning, increased incidence of breast cancer in women taking estrogens is a major limitation of this prophylactic therapy (14). Angiogenesis, a fundamental process necessary for tumorigenesis as well as atherosclerotic progression, can be enhanced by estrogen (15), which induces expression of cellular adhesion molecules and organization of endothelial cells into primitive blood vessels, upon which tumors depend. The effect of estrogen upon the coagulation system is variable. Platelets incubated with estradiol manifest decreased adherence (16) and aggregation (17) although studies in women on HRT have not shown similar antiplatelet effects (18,19). Other reports have noted decreased fibrinogen (4) and decreased plasminogen activator inhibitor (20 –22) levels in women taking HRT, which may be protective, although thrombotic events are actually increased in this population (13).
PHYTOESTROGENS: AN ALTERNATIVE TO ESTROGEN? Given the demonstrated risks to conventional HRT, many women and their practitioners have been in search of alternatives. Phytoestrogens are naturally occurring estrogens that may have beneficial effects on the cardiovascular system and may also alleviate common illnesses afflicting women, such as menopausal symptoms, osteoporosis and breast cancer (Table 1). Phytoestrogens may have advantages over conventional estrogens in that they may lower LDL cholesterol without inducing hypertriglyceridemia (23); they may relieve menopausal symptoms without increasing the risk of uterine or breast neoplasia (24); they may enhance vascular function without accelerating pathological angiogenesis (25); and there are no reports of increased thrombotic events. There is not yet enough evidence from large randomized clinical trials to make an unqualified recommendation about the use of phytoestrogens, but accumulating data indicate that phytoestrogens may be an alternative therapy for postmenopausal women at risk for CVD. The evidence for cardiovascular benefit of phytoestrogens is the subject of this review.
PHARMACOLOGY AND PHYSIOLOGY OF PHYTOESTROGENS Phytoestrogens are naturally occurring, plant based diphenolic compounds that are similar in structure and function
JACC Vol. 35, No. 6, 2000 May 2000:1403–10 Table 1. Effects of Phytoestrogens in Basic, Animal and Human Studies* Preclinical Studies 2 Cholesterol (44,45,50) 2 LDL oxidation (46) 1 Antioxidant enzymes (48) 2 Atherosclerotic lesions (45) 1 Vascular reactivity (54,55) 2 Platelet aggregation (51,52) 2 Expression of ICAM-1 and VCAM-1 (49) 2 Angiogenesis (25,61) 2 Neoplastic proliferation (62–66,68) 2 Bone loss (88,89) Clinical Studies Beneficial 2 Cholesterol (23,41,42) 2 LDL oxidation (47) 2 Perimenopausal hot flashes (56) 2 Cancer incidence (69,73–84) 1 Bone mineral density (90) ?Detrimental 1 breast secretions (85) 1 proliferation of breast epithelium (86) *See text for details. ICAM-1 ⫽ intercellular adhesion molecule; VCAM-1 ⫽ vascular cell adhesion molecule.
to estradiol (Fig. 1). There are many types of phytoestrogens, but the major categories include isoflavones, lignans and coumestans. Common and significant sources of phytoestrogens are soybeans (isoflavones), cereals and oilseeds such as flaxseed (lignans) and alfalfa sprouts (coumestans). These estrogen-like compounds were identified in the 1940s, when an epidemic of infertility among sheep was investigated. The animals were grazing on clover (Trifolium sp)—a plant with a high content of formononetin, which is converted by ruminal bacteria to isoflavones, the predominant type of phytoestrogens.
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The most common and best studied phytoestrogen is the class of isoflavones. The most abundant active components of isoflavones are genistein and daidzein. These agents appear to have selective estrogenic actions, i.e., in some tissues they display proestrogenic responses, whereas in others, they inhibit estrogenic effects. The recent identification of a second subtype of estrogen receptor lends support to the theory of selective estrogenic action (26,27). Estrogenic activity is dependent on the affinity of binding to the estrogen receptors, which is determined by the presence of the aromatic ring as well as hydroxyl groups at specific sites (28). Compared with estradiol, genistein and daidzein bind estrogen receptors with 100 and 1,000 times less affinity, respectively (29). Nevertheless, in the quantities that can be consumed in the diet, isoflavones can have biological effects. Antiestrogenic effects may be due to competitive inhibition at the estrogen receptor, interference with gonadotrophins, inhibition of estrogen synthesis or increased synthesis of estrogen-binding protein. Phytoestrogens do not have feminizing effects in male primates as reflected by unchanged weights of the reproductive organs in these animals, although prenatal exposure of rats resulted in diminished weights of ovaries and uteri in females (30). Isoflavones are widely consumed by Asian populations, predominantly in the form of soy. The typical concentration of genistein in soy foods is 1 to 2 mg per g of protein, and Asians consume 20 to 80 mg of genistein per day in the usual diet. By contrast, the average American ingests only 1 to 3 mg per day (31). Dietary studies can be inconsistent, however, as there is significant variation of isoflavone content between varieties of soy beans, the particular crop, as well as the processessing methods. Isoflavones are inactive molecules when in the form of glycosides (genistin, daidzin), but as aglycones (genistein, daidzein) intestinal absorption is possible. Intestinal absorption varies greatly between individuals, which may be related to the content of dietary fiber and the state of intestinal microflora. After absorption, isoflavones are reconjugated to glucuronides and excreted unchanged in the urine (32). Plasma levels increase 6 1/2 h after ingestion in a dose dependent fashion (33), and urinary levels increase dramatically after supplemented diets have begun. For example, urinary levels of genistein increased from 0.8 umol/day to 26 umol/day and daidzein from 0.3 umol/day to 23 umol/day after a nine day ingestion of 100 g tofu and 45 g soy protein isolate (34). Concentrations of the phytoestrogens and their metabolites can be measured in urine, plasma and other bodily fluids and vary widely, even during controlled nutrition studies. As dietary phytoestrogen metabolism is profoundly influenced by gastrointestinal flora, antibiotic use or bowel disease will modify metabolism and therefore, bioavailability. Equol is a metabolite of daidzein, which shows enormous variation in urinary excretion. Some studies have distinguished individuals as either equol excreters or nonexcreters because the excretion rates vary so substantially (35). However, this appears to be independent of the
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dose-dependent excretion of genistein and daidzein. Chronic ingestion of isoflavones also affects their metabolism. For example, after one month of a high soy diet (36 oz of soymilk containing 100 mg daidzein and 115 mg genistein), young, healthy women exhibit increased absorption rates, as indicated by a higher peak serum level, but also accelerated excretion based on the percentage of ingested isoflavone recovered in the urine (36). There are many other natural agents classified as phytoestrogens, based on their structure or function. Resveratrol is another potentially important phytoestrogen with a structure similar to diethylstilbesterol. Resveratrol is present in grapes and, therefore, wine and has been proposed to be the agent responsible for the “French-paradox” (37). The French paradox derives from the epidemiological observation that the French and Italian populations suffer from less CVD than other Europeans and Americans, despite a relatively high intake of dietary fat. Resveratrol has been shown to bind to human estrogen receptors, initiate transcriptional activity dependent on the estrogen-response element, activate estrogen-regulated genes, cause proliferation of estrogen-dependent breast cancer cell lines and inhibit the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in human endothelial cells (38). Its action can also be inhibited by estrogen antagonists such as tamoxifen (39).
BENEFICIAL CARDIOVASCULAR EFFECTS Favorable effects of phytoestrogens on lipid profiles, vascular reactivity, thrombosis and cellular proliferation have been reported. It is plausible that the lower incidence of CAD in populations ingesting diets high in phytoestrogen is due to an improved lipid profile. In a study of 24 healthy normocholesterolemic men assigned to either a low fat soy diet or low fat animal protein diet, no difference was observed in total cholesterol levels (40), but when patients with type II hyperlipoproteinemia (mean TC 409 mg/dL) were placed on high soy diets for four weeks, the total cholesterol and LDL decreased by 16%. These changes were attributed to increased LDL degradation (41). A later study designed specifically to examine diets differing only in their protein source randomly assigned healthy men to diets either: 1) high in fat, 2) low in fat with soy protein, or 3) low in fat with animal protein. Both of the low fat diets decreased total cholesterol levels and blood pressure compared with the high fat diet, but the soy protein had a more potent hypocholesterolemic effect (10% vs. 5% decline in total cholesterol) (42). Although some published studies have failed to observe a fall in cholesterol values on a soy-based diet, a meta-analysis of 38 trials of soy protein consumption in humans revealed an improvement in total cholesterol by 9% and LDL by 13%, as well as a decrease in triglyceride levels of 10% (23). In these trials, the average intake of soy was 47 g/day. The
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extent of reduction was dependent upon the baseline level of cholesterol; for example, in subjects with moderate hypercholesterolemia (259 –333 mg/dL), a decrease in total cholesterol of 7.4% was observed, whereas subjects with severe hypercholesterolemia (⬎335 mg/dL) achieved a decline of 19.6%. Three theories of possible mechanisms for the hypocholesterolemic effects of phytoestrogens have been proposed. One explanation is that phytoestrogens cause an increase in the excretion of bile acids and, therefore, enhance removal of LDL. Others have proposed that phytoestrogens initiate a hyperthyroid state, supported by the finding in some studies of increased free thyroxine levels after feeding soy proteins to animals (43). The third and best supported mechanism of phytoestrogen lipid-lowering is that of altered hepatic metabolism with augmented LDL and VLDL removal by hepatocytes. Sirtori et al. (44) demonstrated substantial reductions in cholesterol levels with enhanced LDL degradation and increased binding of radiolabeled LDL to receptors. A study using the LDL-receptor deficient mouse has provided further support for an effect of isoflavones on LDL receptor activity (45). When normocholesterolemic C57BL/6J mice were fed a high cholesterol, isoflavone-poor diet, they developed hyperlipidemia and intimal lesions in the proximal aorta. However, those animals fed an isoflavone-rich diet exhibited a decrease in total cholesterol and VLDL cholesterol. By contrast, this benefit of an isoflavone-rich diet was not observed in LDL-r null mice, suggesting that isoflavones may reduce lipid levels by increasing the activity of the LDL receptor. Furthermore, there was a 50% reduction in intimal lesion area in isoflavone treated C57BL/6J but not LDLr-null mice, again implicating an effect of isoflavones on LDL receptor activity in conferring cardiovascular protection. Isoflavones may also inhibit oxidation of LDL. Kapiotis and colleagues (46) observed that LDL oxidation products, assayed as Thiobarbituric Acid–Reactive Substances in either cell free or endothelial cell systems, were strongly inhibited by genistein, somewhat inhibited by daidzein but not affected by genistin (the glycosylated form of genistein) or control. Furthermore, both genistein and daidzein were found to protect against cytotoxic effects of oxidized LDL as assessed by cellular morphologic features and lactate dehydrogenase release by cultured endothelial cells. A trial in humans enrolling healthy volunteers consuming three soy bars per day for two weeks (total 36 mg genistein and 21 mg daidzein per day) demonstrated a significant amount of genistein and daidzein within the LDL fractions, as well as a significant prolongation of the lag time to LDL oxidation, implying an antioxidant effect (47). Genistein has also been shown to inhibit hydrogen peroxide production and increase the activity of antioxidant enzymes, such as catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase. Furthermore, genistein, and to a lesser degree daidzein, can inhibit superoxide anion generation by xanthine/xanthine oxidase (48).
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Atherosclerosis is initiated by monocytes binding to the endothelium and migrating into the intimal layer to develop into foam cells. The adhesiveness of endothelial cells is due to lipid-induced, oxidant-sensitive transcription of adhesion molecules and chemokines, which promote monocyte binding. This binding is reliant upon intercellular signalling and oxidant-sensitive transcription of adhesion molecules. Phytoestrogens may further protect against atherosclerosis by interfering with these initial processes. Although scant information has been published thus far, genistein is capable of inhibiting the expression of ICAM-1 and VCAM-1 on human endothelial cells co-cultured with monocytes (49). Furthermore, by reducing oxidative stress, it is possible that phytoestrogens deactivate the transcriptional pathways leading to the expression of genes involved in monocyte adhesion and infiltration. The response to isoflavones is influenced by gender. Monkeys that were fed an atherogenic diet followed by a soy protein phytoestrogen-rich diet in a six-month crossover trial had a lowering of LDL and VLDL levels compared with control values. However, female animals additionally manifested an increase in HDL, decrease of lipoprotein (a), lower LDL molecular weight, decrease in apolipoprotein B, AI, A2 and no hypertriglyceridemia (50). Phytoestrogens may have less prothrombotic tendencies than the currently used formulations of estrogen. In vitro studies of genistein and daidzein reveal decreased platelet aggregation by collagen and thromboxane (51,52), which may be due to an inhibition of binding of the thromboxane receptor or diminished tyrosine phosphorylation. However, in a small study of 20 healthy, normocholesterolemic men fed a soy diet for 28 days, a significant increase in plasma levels of genistein and daidzein occurred but without a change in platelet aggregation (53). It was suggested that on such supplements, serum levels of the active compound do not reach levels comparable with those required to affect platelet aggregation in the in vitro studies. Preclinical studies suggest that vascular reactivity may be favorably influenced by phytoestrogens. Primates manifest an improvement in endothelium mediated vasodilation when treated with phytoestrogens. Specifically, postmenopausal monkeys on a phytoestrogen-rich diet for six months exhibited normal coronary artery vasodilation in response to locally-administered acetylcholine, whereas a vasoconstrictive response was seen in male animals as well as female monkeys with a low intake of phytoestrogens (54). In vitro studies of isolated vessels have examined the mechanisms of phytoestrogen-induced vasodilation (55). Estradiol-17B, genistein and daidzein were all found to relax mesenteric arterial rings of rats in a dose dependent manner. Consistent with receptor affinity assays, estradiol was the most potent vasodilator, followed by genistein, then daidzein. In these isolated arterial rings, response was independent of gender. The vasorelaxation was endothelium-independent and was not blocked by antagonists of nitric oxide or prostacyclin production.
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Estrogen withdrawal results in symptomatic hot flashes in perimenopausal women and may represent an alteration in vasomotor response to endocrinologic stimuli. As a population, Asian women experience less perimenopausal hot flashes, a benefit attributed to phytoestrogens. Also, in a randomized study of 58 postmenopausal non-Asian women, those consuming soy protein supplementation in the form of 45 g of soy flour per day suffered less from hot flashes than control women (56), invoking another potential use for phytoestrogen supplementation. Phytoestrogens may have electrophysiological effects as well. The sensitivity of ventricular myocytes to betaadrenergic stimulation can be enhanced by genistein and daidzein, implicating a role of tyrosine kinase regulation of cardiac ion channels (57). However, there is, as yet, no known clinical effect of phytoestrogens on arrhythmias.
BENEFICIAL NONCARDIOVASCULAR EFFECTS The most powerful argument against the routine use of HRT for postmenopausal women is the increased risk of cancer. Phytoestrogens do not appear to increase the risk of breast and uterine cancer; in fact, observations from in vitro and animal studies indicate that phytoestrogens may have antineoplastic effects. These antineoplastic effects may be related to suppression of angiogenesis, inhibition of tyrosine kinase activity, or partial antagonism of estrogen receptors. Tumor growth is dependent upon angiogenesis, which is a tightly regulated, complex process (58). Endothelial cells play a pivotal role in angiogenesis, which requires endothelial cell attachment, migration, proliferation, protease production and subsequent organization into vascular structures. Whereas estradiol has been demonstrated to augment various steps in the angiogenic process (59), components of phytoestrogens may actually inhibit angiogenesis. Genistein reduces basic fibroblast growth factor induced endothelial cell migration (60) and capillary-like tube formation, perhaps by inhibiting tyrosine kinase or by decreasing levels of plasminogen activator and plasminogen-inhibitor and, thus, interrupting the tightly regulated balance of proteolytic degradation (25). In the setting of atherosclerosis, the enhancement of angiogenesis may hold therapeutic potential to relieve myocardial ischemia by increasing blood flow. Therapeutic angiogenesis has become a very active field of research with ongoing clinical studies designed to explore the safety and efficacy of various growth factors, delivered as protein or gene therapy. However, the neovascularization of atherosclerotic plaques could lead to plaque instability and resultant negative consequences. Indeed, Folkman and colleagues (61) have recently demonstrated that endostatin, an inhibitor of angiogenesis, can slow the development of aortic lesions in the hypercholesterolemic apoE-deficient mouse. Phytoestrogens, particularly genistein, also have antiproliferative effects. This has been demonstrated in multiple cell lines including prostate cancer cells (62), breast cancer
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cells (both estrogen receptor positive as well as negative) (63) and other tumor cells (neuroblastoma, sarcoma, retinoblastoma), as well as vascular endothelial cells and fibroblasts (64). The in vitro effect of genistein, a known tyrosine kinase inhibitor, on breast cancer cell growth was evaluated by quantifying DNA and total cellular protein levels compared with the effect of estrogen (65). Over a range of 0.1 to 1 uM, genistein had a stimulatory, estrogen-like effect on estrogen receptor positive cell proliferation, but at ⬎10 uM of genistein, growth was totally inhibited. However, estrogen receptor negative cells showed no response to, or possibly inhibition by, genistein even at low concentrations (10 nM–1 uM). Another group of investigators demonstrated that the effect of phytoestrogens in inhibiting proliferation is a reversible, cytostatic effect (60). Even at concentrations of up to 200 umol/L, genistein was not cytotoxic in quiescent cells. The mechanism of this inhibitory effect of genistein is speculated to be its antagonism of tyrosine kinase. However, other investigators have observed a genistein-induced inhibition of proliferation despite adequate levels of tyrosine kinase activity (66). This antiestrogenic effect of phytoestrogens may provide protection analogous to the estrogen partial-agonist, tamoxifen, in the treatment or prevention of breast cancer. Animal models support an antineoplastic role for phytoestrogens; for example, rats given soy-derived phytoestrogens develop fewer mammary tumors after exposure to carcinogens (67). The evidence that phytoestrogens may protect against cancer development derives largely from in vitro studies of human cell lines, animal models of neoplasia and epidemiological data. Overall, most epidemiological studies have indicated a protective effect of soy ingestion (68). Epidemiologic studies reveal a decreased incidence of breast, prostate and endometrial cell cancer in populations ingesting high amounts of soy. Asian women develop breast cancer four to six times less often than their American counterparts, but this advantage wanes after living in the U.S. (69 –71), implicating a protective influence of traditional diets. Furthermore, breast cancer patients have lower urinary isoflavone levels, suggesting that they have decreased ingestion, absorption, excretion or overall bioavailability of isoflavones compared with women without breast cancer (72,73). The benefits of dietary phytoestrogens may extend to other neoplasms, and to men as well as women. Hawaiians of Japanese descent who maintain a traditional diet with high soy content have less risk of prostate cancer than nonJapanese Hawaiians (74), and consumption of a diet enriched with lignans (legumes and fruit) is associated with decreased prostate cancer risk in Seventh Day Adventists (75). If these beneficial effects of diet are due to phytoestrogen content, the benefit may be linked to the observation that certain phytoestrogens (i.e., genistein and biochanin A) have been observed to inhibit the growth of androgendependent and independent human prostate cancer cell lines (62). High soy consumption (as tofu, miso or soybean sprouts) is associated with reduced risks of lung, gastric and
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rectal cancer in Chinese and Japanese men and women (76 – 83). Rates of breast, ovarian, prostate and colon cancer are negatively correlated with cereal and phytoestrogen intake when comparing cancer mortality rates and food availability data among countries (84). Despite the plethora of evidence that phytoestrogens have antineoplastic effects, two concerning clinical reports have been published. One study reported the effect upon monthly nipple aspirations in pre- and postmenopausal women ingesting soy protein containing 38 mg genistein/day (85). An increased total fluid volume was recovered in premenopausal women and postmenopausal women during and after phytoestrogen treatment. Additionally, there was an increased frequency of cellular hyperplasia in the aspirate. However, there were no controls in this study, and the aspirations themselves could account for the abnormalities. Nevertheless, the study does raise some concern for high risk patients. Another report of 29 women taking 60 g of soybean supplement daily found increased proliferation of breast lobular epithelium assessed by 3H-thymidine labeling (86). However, there are no human studies that have linked dietary phytoestrogens to the development of breast cancer. Similar to other selective estrogen antagonists, phytoestrogens may also protect against osteoporosis. Studies of aging or ovariectomized rats have shown that a soy protein diet protects against bone loss (87,88) although this effect is not mimicked by supplemental genistein (89), suggesting that some other component of soy protein may be responsible for the beneficial effect. However, a trial in postmenopausal women revealed an increase in bone mineral density in the lumbar spine after isoflavone supplementation (90).
SUMMARY To conclude, there are accumulating data from epidemiologic studies, human trials, animal models and in vitro analysis to support the increased use of phytoestrogens, particularly in individuals at high risk of CVD. Favorable effects upon lipid profiles, vascular reactivity, angiogenesis and tumorigenesis have been attributed to the isoflavones genistein and, to a lesser extent, daidzein. The increasing use by the general public of alternative therapies including these agents necessitates a greater understanding of their effects, as well as a need for clinical investigations to guide practitioners in their recommendations. The current state of knowledge indicates that the world of plant based estrogens has much to offer, but significant questions remain. Do phytoestrogen-enriched diets alleviate symptoms of atherosclerosis or reduce the risk of vascular events? If so, can this effect be attributed to a specific phytoestrogen, and can the benefit be mimicked by supplementation with that specific compound? How do the benefits of phytoestrogen therapy on the lipoprotein profile or vascular reactivity compare with other therapies? Which subgroups of the population might benefit most from phytoestrogen use? What are the side effects of such supplementation? Is it safe for women with a
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personal or family history of breast or uterine cancer to use phytoestrogens? Answers to these questions, which could be obtained by large, randomized clinical trials, may lead to a greater understanding of the hormonal effects upon the cardiovascular system and provide women with more options for preventative treatment. In the interim, it seems reasonable to recommend that women at high risk of cardiovascular morbidity and mortality, particularly those without a personal or family history of breast or uterine cancer, increase their consumption of foods rich in phytoestrogens such as soy protein. Reprint requests and correspondence: John P. Cooke, Division of Cardiovascular Medicine, 300 Pasteur Drive, Stanford, California 94305-5406. E-mail: [email protected].
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