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Department of Obstetrics and Gynecology (R.K.D., B.I.), Clinic for Reproductive Endocrinology, University Hospitak Zurich, 8091 Zurich, Switzerland; and Departments of Medicine and Pharmacology (R.K.D., E.K.J.), Center for Clinical Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260
Address all correspondence and requests for reprints to: Raghvendra K. Dubey, Department of Obstetrics and Gynecology, Clinic for Reproductive Endocrinology (D217, NORD-1), University Hospital Zurich, Frauenklinikstrasse 10, 8091 Zurich, Switzerland. E-mail: raghvendra.dubey{at}usz.ch.
Evidence from several epidemiological studies suggests that endogenous human estrogens as well as estrogen replacement therapy protect women from progression of cardiovascular disease (1, 2, 3). Additional evidence for a cardioprotective action of estrogens comes from multiple animal studies and small clinical trials (1, 2, 3). In contrast, the outcomes of two large randomized clinical trials [Heart and Estrogen/progestin Replacement Study (HERS) and Women’s Health Initiative (WHI)] failed to demonstrate protection against cardiovascular disease by exogenous estrogens (4, 5).
Several explanations could account for these divergent findings (3). In this regard, some argue that the HERS and WHI studies were negative because they used conjugated equine estrogens, which is a mixture of horse estrogens containing negligible amounts of estradiol, the major endogenous human estrogen. Others speculate that the HERS and WHI studies were negative because the participants were older women with established cardiovascular pathology. Still others speculate that the oral route of administration of estrogens leads to inflammation and that the transdermal route would be cardioprotective (3).
Happily, the dark cloud cast off by the WHI and HERS has a silver lining, i.e. a renewed interest in the mechanisms by which estrogens influence the cardiovascular system, with potentially important spin-offs. For example, selective estrogen receptor modulators (SERMs; e.g. raloxifene) and xenoestrogens are classes of drugs that interact with estrogen receptors (ERs) in a tissue-selective manner. These compounds are being developed as potentially safer and more effective agents for estrogen therapy to induce beneficial effects on the cardiovascular system without increasing the risk for uterine and breast cancer or thromboembolic disorders. A limitation of this approach, however, is that the effects of SERMS and xenoestrogens are dependent on the expression of the ERs. Because the expression of ERs is a dynamic process (6), these drugs may not offer the same protective effects in all subjects at all times.
Naturally occurring metabolites of estradiol may offer a distinct safety advantage over conjugated equine estrogens, SERMs, or xenoestrogens. In particular, recent studies provide evidence that 2-methoxyestradiol (2-ME), a downstream metabolite of estradiol with little affinity for ERs, can mimic the antiproliferative actions of estradiol on vascular smooth muscle cells (SMCs) and injury-induced neointima formation (7, 8, 9). Moreover, in contrast to estradiol, 2-ME also inhibits growth of multiple cancer cells lines including ER-positive and -negative breast cancer cells, inhibits angiogenesis, and prevents tumor growth (10). Based on its anticarcinogenic effects, 2-ME is undergoing phase II clinical trials for potential use as an anticancer drug (11). Taken together these findings suggest that 2-ME may represent a novel drug for hormone therapy that may mimic the cardioprotective actions of estradiol but without the risk of inducing breast and uterine cancer.
Although 2-ME is protective against vascular remodeling associated with abnormal growth of vascular SMCs, whether 2-ME protects against atherosclerosis, a multifactorial disease that involves processes in addition to vascular SMC proliferation, was unclear until Bourghardt et al. (12) examined this aspect of 2-ME pharmacology in this issue of Endocrinology. Importantly, Bourghardt et al. (12) provide the first in vivo evidence that 2-ME reduces atherosclerotic lesion formation in female apolipoprotein E-deficient mice, a well-established mouse model of atherosclerosis. The authors demonstrate that treatment with 66.6 µg/d, but not 6.66 µg/d, of 2-ME for 90 d decreases the surface area of atherosclerotic lesions by a remarkable 52%. Moreover, at both doses, 2-ME lowers total cholesterol levels by similar extents (
20%), suggesting that the atheroprotective effects are not solely due to the cholesterol lowering actions of 2-ME.
The authors also report that doses of 2-ME that protect against atherosclerosis also decrease body weight gain and increase uterine weight. As shown in Fig. 1
, low concentrations 2-ME can be demethylated locally by CYP450s to 2-hydroxyestradiol (2-HE) (13), which has some, albeit modest, affinity for ERs (1, 2). Hence, it is feasible that the estrogenic effects of 2-ME on the uterus are due to the local conversion of 2-ME to 2-HE.
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Although 2-ME could be a novel drug to treat abnormal growth processes associated with cardiovascular disease and carcinogenesis and tumor progression, several key issues need to be investigated before 2-ME could be considered a substitute for estradiol for estrogen replacement therapy. In this regard, it would be important to investigate whether 2-ME is protective against hot flushes. Also, although one study demonstrated a positive effect of 2-ME on bone homeostasis (14), the effects of 2-ME in osteoporosis are unknown and in need of better clarification. Moreover, those of us interested in 2-ME should conduct a thorough investigation of the biochemical and molecular mechanisms by which 2-ME protects against atherosclerosis, plaque formation, and neointimal thickening. However, we should keep in mind that even if 2-ME does not fulfill all of the criteria for an ideal estrogen replacement therapy, 2-ME’s cardiovascular and anticarcinogenic benefits may in and of themselves be of therapeutic importance.
The receptor (or receptors) via which 2-ME induces its biological effects remains undefined. Clearly, 2-ME interacts with a colchicine binding site and disrupts tubulin polymerization (1, 2, 9). However, whether 2-ME’s antimitogenic, antiatherosclerotic, and cholesterol-lowering effects are mediated solely via this tubulin receptor remains unknown. Studies show that 2-ME reduces endothelin-1 production, induces antioxidant effects, stimulates cyclooxygenase-2 expression and prostacyclin synthesis, and blocks cell cycle regulatory pathways that contribute to the etiology of abnormal vascular SMC growth and vascular remodeling in cardiovascular disease (1, 2, 3, 9). Whether these effects are associated with a unique receptor or involve an interaction with tubulin remains undefined and needs to be investigated. Also, the biochemical mechanisms by which 2-ME lowers cholesterol levels and inhibits plaque formation remains undefined and warrants further investigation. In particular, a careful examination of the binding sites of 2-ME using state-of-the art proteomics to identify the molecular targets would be of considerable value.
Importantly, 2-ME also induces antiproliferative actions on several other cell types including glomerular mesangial cells and cardiac fibroblasts that contribute to the pathophysiology of glomerular remodeling in glomeruosclerosis and renal disease and cardiac remodeling in left ventricular hypertrophy (1, 2, 3). There is also increasing evidence that estradiol metabolites afford cardiorenal protection in vivo. For example, 2-ME inhibits balloon injury-induced neointima formation (9). Also, in genetically obese ZSF1 rats, 2-HE (a prodrug of 2-ME because 2-HE is rapidly and quantitatively converted to 2-ME in vivo) attenuates the development of obesity, the metabolic syndrome, hypercholesterolemia, and vascular and renal dysfunction (15). Interestingly, in ovariectomized rats, both 2-HE and 2-ME reduce circulating cholesterol levels (1, 2), as was also observed by Bourghardt et al. (12) with 2-ME in mice by. Moreover, 2-HE attenuates renal disease in chronic puromycin aminonucleoside nephropathy (16). The protective effects are associated with inhibitory actions on glomerular remodeling [decrease in proliferating cells, collagen IV synthesis, and infiltration of ectodysplasin-A (ED1) positive macrophages]. Similarly, both 2-HE and 2-ME attenuate renal and cardiovascular injury induced by chronic nitric oxide synthase inhibition, and 2-ME lowers blood pressure and increases endothelium-dependent relaxation (17). Finally, more recent studies provide evidence that both 2-HE and 2-ME abrogate the development and retard the progression of monocrotaline-induced pulmonary hypertension in rats (18). In this regard, the protective actions are associated with a significant decrease in wall thickness of small-sized pulmonary arteries, suggesting that the inhibitory actions of 2-HE and 2-ME on proliferating cells may be the key mechanism via which they attenuate pulmonary hypertension.
Based on the conventional mechanisms of steroid action, the received view is that the beneficial effects of endogenous human estrogens are mediated via ERs. However, this conclusion may be premature. Multiple studies using pharmacological and molecular inhibitors as well as knockout mouse have shown that the sequential metabolism of estradiol to 2-ME by CYP450 and catechol-O-methyltransferase is responsible for the antimitogenic actions of estradiol in vascular SMCs, glomerular mesangial cells, and cardiac fibroblasts (1, 2, 3, 19). This contention is further supported by the fact that the antiproliferative actions of estradiol are not lost in mice lacking both ER
and ERß (20). The above findings together with the fact that 2-ME protects against atherosclerosis and neointimal thickening suggests that estradiol may induce its cardiovascular protective effects via ER-independent mechanisms. An important corollary of this concept is that use of conjugated equine estrogens that contains little or no estradiol may result in lack of 2-ME formation and explain the lack of protective effects observed in the HERS and WHI studies.
As mentioned, low concentrations of 2-ME can be demethylated to 2-HE (13) and induce estrogenic effects on estrogen-sensitive tissues like the uterus. However, these effects are quite different from those observed with estrogenic molecules like estradiol and pose little or no risk of carcinogenicity (21). The possibility that 2-ME may form large amounts of 2-HE can also be ruled out because at higher concentrations 2-ME inhibits CYP450 enzymes via feedback inhibition thereby inhibiting or limiting its demethylation (13).
In conclusion, the findings of Bourghardt et al. (12) provide additional crucial evidence for an ER-independent protective effect of 2-ME in atherosclerosis, the major pathway to fatal coronary artery disease. Their findings, together with the fact that 2-ME inhibits injury-induced neointimal thickening as well as carcinogenesis within estrogen sensitive tissues, imply that 2-ME or its analogs may serve as a new therapeutic hormone that confers cardiovascular protection in both men and women. Importantly, these actions would not be influenced by the dynamic changes in the expression of ERs within the target tissues. The first long-acting formulation of 2-ME (PR Pharmaceuticals, Inc., Fort Collins, CO) is now in phase I clinical trials, so perhaps the cardiovascular protection hypothesis will ultimately be put to the test!
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Disclosure Statement: R.K.D. is an inventor on U.S. Patent 6998395. B.I. has nothing to declare. E.K.J. is an inventor on U.S. Patents 6998395 and 7192941.
Abbreviations: ER, Estrogen receptor; 2-HE, 2-hydroxyestradiol; 2-ME, 2-methoxyestradiol; SERM, selective ER modulator; SMC, smooth muscle cell.
Received April 20, 2007.
Accepted for publication June 4, 2007.
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and ß in human aortic smooth muscle cells by oligonucleotides and estradiol. J Clin Endocrinol Metab 89:2373–2381,ß (double knockout) mice. Circ Res 89:534–539
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, inhibition.