Of Mice, Men, and Hormones
http://www.100md.com
《动脉硬化血栓血管生物学》
From the Departments of Surgery (V.M.M.), Physiology and Bioengineering (V.M.M.), Urology (D.J.T.), and Internal Medicine, Division of Endocrinology (P.Y.L.), Mayo Clinic College of Medicine, Rochester, MN.
Incidence of cardiovascular disease is consistently greater in men than in women during the first five decades of life.1 Because this gender disparity begins early in life, it is likely that a combination of genetic, environmental (possibly intrauterine) factors, and actions of sex-specific hormones contribute to the disease process (see Nagel and vom Saal2). However, most research has focused on understanding how sex steroid hormones, estrogens and androgens, affect vascular function in men and women.
See page 1055
Historically, an "estrogen protection" hypothesis, where estrogens limit development of atherosclerosis, has been tested, although an "androgen detrimental" hypothesis is also plausible, but less studied. Although these hypotheses provide a starting point for design of experiments to study effects of hormones on vascular function, review of available data suggests a more complex interaction among the sex steroids than this simple dichotomy.3 Therefore, new approaches are needed to dissect mechanisms of how sex steroids interact in development of vascular disease in both sexes.
The study by Villablanca et al, reported in this issue, uses an approach of examining the contribution of estrogen receptor alpha (ESR1) in development of early atheroma in male mice.4 The design is based on two published lines of research: one which forms the basis of the estrogen "protection" hypothesis, and the other which suggests that disruption and/or polymorphisms in ESR1 are associated with accelerated atherosclerosis and cardiovascular events in men.
The estrogen protection hypothesis arose predominately from large cohort studies, where perimenopausal women treated with estrogen or estrogen-progestin for relief of menopausal symptoms had reduced cardiovascular disease compared with untreated women.5,6 From experimental studies, estrogen treatment of ovariectomized animals affected several processes that would promote repair of damaged vascular endothelium, reduce adhesion of aggregating platelets and leukocytes, and cause vasodilatation and thus limit development of occlusive vascular lesions (see Mendelsohn and Karas7 for review). In addition, estrogen affects lipid metabolism such that estrogen treatment increases serum concentrations of high-density lipoproteins while decreasing low-density lipoproteins. Estrogen also reduces lipid uptake by macrophages and their adhesion to endothelial cells, which should limit formation of fatty streaks in the arterial wall.
In males with normal testicular function, endogenous estrogen is produced enzymatically from testosterone by aromatase in many tissues, including the arterial wall. Theoretically, then, some vascular effects of testosterone could be mediated indirectly through conversion to 17?-estradiol.3 Indeed, inhibition of aromatase in men decreases circulating levels of 17?-estradiol.8
Estrogen effects are mediated through two receptors, estrogen receptor (ESR1) and ? (ESR2). These receptors, located on separate somatic chromosomes 6 and 14, respectively, are present in men and women. Little is known about how expression of these receptors is regulated in vascular tissue of men and women. However, ESR2 as measured by mRNA is the predominant subtype found in human blood vessels collected as surgical waste, and there is a suggestion that the ratio of ESR1:ESR2 may be greater in blood vessels from males compared with females.9
The second line of evidence supporting the design of the studies from Villablanca et al comes from observations that loss of ESR1 in men is associated with loss of flow-mediated dilation of the brachial artery.10 The fact that this flow-mediated dilatation is diminished in men treated with aromatase inhibitors8 suggests a link between the physiological response and testosterone-derived estrogen activation of ESR1. Polymorphisms in ESR1 in men are also associated with accelerated atherosclerosis and increased risk for myocardial infarction.11–13 Therefore, it is attractive to propose that in men, estrogen produced through local aromatization of testosterone would also provide "protection" against development of cardiovascular disease through mechanisms involving ESR1.
To test this hypothesis requires reliable and characterized animal models. To that end, Villablanca and colleagues4 sought to characterize atheroma in male mice lacking ESR1 receptors. In their experiments, disruption of ESR1 was performed in mice of a mixed genetic background (129/J and C57BL/J6). Animals were fed a high fat diet. At time points up to six months, aortae were removed for histological quantification of number and extent of atheromatic lesions. Lesions were characterized by deposits of intracellular fat and large numbers of foam cells but were not considered complex lesions nor did they contain fibrotic caps or calcification. The rate of development and number of fatty lesions were greater in wild-type compared with ESR1 knockout animals. This was unexpected given the association in humans between the disruption and polymorphisms of ESR1 and extent of cardiovascular disease in men.
Discrepancies between observations in experimental animals as models for disease and disease in humans must be reconciled. One consideration is that the genetic manipulation of ESR1 results in a variant gene product that might have some biological activity.14–16 There are over 300 single nucleotide polymorphisms in the human estrogen receptor public database.17 Relative prevalence of a particular genetic variant while showing association to disease progression in humans may not exist in isolation of other genetic variants.
Alternatively, the hypothesis that effects on the vasculature are "protective" and mediated solely through conversion of testosterone to estrogen and ESR1-mediated mechanisms may be na?ve and should be revised. Aromatase was observed by immunohistochemistry in the endothelium, media, and adventia of the aortic wall suggesting that enzymatic conversion of testosterone to 17?-estradiol could occur within the aorta. However, androgen receptors are present in vascular tissue and nonaromatizable androgens initiate vascular effects.3,18 Therefore, given the potential for stoichiometric interactions of testosterone with aromatase and androgen receptors, it is unlikely that all physiologically relevant testosterone would be completely converted to estradiol independent of activation of androgen receptors (Figure). Indeed, sex-specific differences in vascular function are still observed in male pigs where estrogen levels exceed those of females by 10-fold.19–22 In contrast to estrogen receptors, the gene for the androgen receptor is on the X chromosome. It is unknown at this time whether genetic variation in this receptor show sex-related associations with cardiovascular disease.
Schematic representation of possible mechanisms by which testosterone could affect vascular function. Testosterone is synthesized from DHEA in extravascular tissue. Whether DHEA activates a membrane receptor is controversial. Testosterone can be aromatized to 17?-estradiol in extravascular or vascular tissue. Both androgen and estrogen receptors are present in vascular tissue. However, little is known about their regulation in cells of the vascular wall or how they might interact to regulate nongenomic or genomic pathways that participate in development of atherosclerosis including regulation of vascular tone, adhesion of macrophages, aggregation of platelets, cellular apoptosis, differentiation, or proliferation. Therefore, expression of an atherosclerotic phenotype associated with a particular polymorphism in, for example, an estrogen receptor may depend on the type of initiating stimulus or combination of stimuli (endothelial denudation, lipid peroxidation, infection), coregulators needed for receptor activation, duration of stimulus, and/or hormone exposure, genetic sex, and hormonal status. Although this schematic depicts actions of steroids in endothelial cells, similar mechanisms are likely to be present in smooth muscle cells, macrophages, and platelet-precursors, megakaryocytes.36,37 Abbreviations: Akt, a serine-threonine kinase important for regulation of several cellular processes; AR, androgen receptor; ARom, aromatase; DHEA, dihydroepiandrosterone; DHEAR, dihydroepiandrosterone receptor; E2, 17?-estradiol; ER, estrogen receptor alpha () or beta (?); eNOS, endothelial nitric oxide synthase; Gi, guanine nucleotide regulator protein subunit which inhibits guanylate cyclase; NO, nitric oxide; Rm, membrane receptor; ?, denotes unknown interactions or pathways needing additional research.
In the study by Villablanca et al, castration (which reduced both systemic testosterone and estradiol exposure) reduced the average area of the atheroma lesion by over 90%, but a difference in atheroma between wild-type and ESR1 knockout mice was still present although not statistically significant.4 Whether all of testosterone’s effects are mediated by aromatization cannot be answered by this study because the effect of castration was not evaluated with concomitant inhibition of aromatase. Furthermore, whether the effect of castration could be reversed by selective replacement of testosterone was not confirmed, leaving open the possibility that another testicular secretion could contribute to vascular effects. Therefore, it is possible that vascular actions of testosterone are mediated directly through stimulation of androgen receptors as well as indirectly through conversion to 17?-estradiol and stimulation of ESR2.
It should be recognized that cross talk exists between the steroid receptors. For example, estrogen modulates androgen receptor expression as well as transcriptional activity.23,24 In addition, nonaromatizable androgens downregulate expression of estrogen receptors.25 Thus, ESR1 knockout mice may have compensatory changes in androgenic pathways that could affect the phenotype.
Factors affecting quantity and distribution of vascular androgen receptors are unknown. Although activation of androgen receptors can initiate both nongenomic and genomic effects in vascular tissue,3 it is not known how the androgen receptor regulates the synthesis or activity of endothelial nitric oxide synthase (eNOS).26 In contrast, regulation and activation of eNOS by estrogen receptors is well established.27
The animal model studied by the Villablanca group may be useful in understanding hormonal interactions in early atheroma as defined by adhesion of macrophages and subendothelial accumulation of fat and foam cells. These responses are consistent with effects of nonaromatizable testosterone and androgen receptor blockade on increased expression of vascular cell adhesion molecule (VCAM)-1 in cultured endothelial cells and enhanced apoptosis of endothelial cells.28,29 Effects of androgens on lipogenesis are beginning to be defined, and there is evidence that sterol regulatory element–binding proteins are key mediators of lipogenic effects of both androgens and estrogens in a variety of tissues.30–32 Studies taking advantage of androgen receptor antagonists, aromatase inhibitors, castration, and/or replacement of testosterone or estrogen to castrated animals could be designed to investigate the stages of atheroma development.
Finally, vascular effects of ESR2 in ESR1 knockout mice should be considered. ESR2 inhibits proliferation of vascular smooth muscle.33 Responses regulated by ESR1 and 2 may be opposite and differentially regulated in adipose and endothelial cells.34,35 Therefore, experiments are also needed to understand the contribution of ESR2 in atheroma development in both male and female animals.
Atherosclerosis in humans is a multifactorial disease process which includes lipid accumulation, remodeling of the vascular wall, infection, and calcification. In the era of sex-based medicine and genomics it is important to define similarities and differences in these processes as regulated by sex steroids in males and females. Therefore, studies in experimental animals that relate genetic sex, hormonal status, and receptor polymorphisms to vascular healing after mechanical injury16 or lipid accumulation, as in the article by Villablanca et al, provide important tools to understand effects of hormones on various stages of the disease process. However, each method provides an incomplete picture of disease progression in humans, and discovery of novel therapeutics will be hampered by generalization of hormonal functions as being "beneficial," "protective," or "detrimental" when sex- and cell-specific actions of hormones remain incompletely understood. Further investigation is needed to understand actions of testosterone on the vascular wall, contributions of androgen and estrogen receptor polymorphisms to vascular function, and better definition of responses mediated by ESR2 in various stages of the atherogenic process.
References
Vargas CM, Burt VL, Gillum RF. Cardiovascular disease in the NHANES III. Ann Epidemil. 1997; 7: 523–525.
Nagel SC, vom Saal FS. Endocrine control of sexual differentiation: effects of the maternal-fetal environment and endocrine disrupting chemicals. In: Miller VM and Hay, M eds. Principles of Sex-Based Differences in Physiology. Volume 34 of: Bittar EG, Series Ed. Advances in Molecular and Cell Biology. The Netherlands: Elsevier Science; 2004: 15–37.
Liu PY, Death AK, Handelsman DJ. Androgens and cardiovascular disease. Endocrine Reviews. 2003; 24: 313–340.
Villablanca A, Lubahn D, Shelby L, Lloyd K, Barthold S. ZSusceptibility to early atherosclerosis in male mice is mediated by estrogen receptor . Arterioscler Thromb Vasc Biol. 2004: 24: 1055–1061.
Barrett-Connor E, Bush TL. Estrogen and coronary heart disease in women. JAMA. 1991; 265: 1861–1867.
Kannel WB, Hjortland MC, McNamara PM, Gordon T. Menopause and risk of cardiovascular disease: the Framingham Study. Ann Int Med. 1976; 85: 447–452.
Mendelsohn ME, Karas RH. The protective effects of estrogen on the cardiovascular system. N Engl J Med. 1999; 340: 1801–1811.
Lew R, Komesaroff PA, Williams M, Dawood T, Sudhir K. Endogenous estrogens influence endothelial function in young men. Circ Res. 2003; 93: 1127–1133.
Hodges YK, Tung L, Yan X-D, Graham D, Horwitz KB, Horwitz LD. Estrogen receptors and ?. Prevalence of estrogen receptor ? mRNA in human vascular smooth muscle and transcriptional effects. Circulation. 2000; 101: 1792–1798.
Sudhir K, Chou TM, Messina LM, Hutchison SJ, Korach KS, Chatterjee K, Rubanyi GM. Endothelial dysfunction in a man with disruptive mutation in oestrogen-receptor gene. Lancet. 1997; 349: 1146–1147.
Shearman AM, Cupples LA, Demissie S, Peter I, Schmid CH, Karas RH, Mendelsohn ME, Housman DE, Levy D. Association between estrogen receptor gene variation and cardiovascular disease. JAMA. 2003; 290: 2263–2270.
Sudhir K, Chou TM, Chatterjee K, Smith EP, Williams TC, Kane JP, Malloy MJ, Korach KS, Rubanyi GM. Premature coronary artery disease associated with a disruptive mutation in the estrogen receptor gene in a man. Circulation. 1997; 96: 3774–3777.
Kunnas TA, Laippala P, Penttila A, Lehtimaki T, Karhunen PJ. Association of polymorphism of human oestrogen receptor gene with coronary artery disease in men: A necropsy study. BMJ. 2000; 321: 273–274.
Couse JF, Curtis SW, Washburn TF, Lindzey J, Golding TS, Lubahn DB, Smithies O, Korach KS. Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene. Mol Endocrinol. 1995; 9: 1441–1454.
Toran-Allerand CD, Guan X, MacLusky NJ, Horvath TL, Diano S, Singh M, Connolly ESJ, Nethrapalli IS, Tinnikov AA. ER-X: a novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury. J Neurosci. 2002; 22: 8391–8401.
Pare G, Krust A, Karas RH, Dupont S, Aronovitz M, Chambon P, Mendelsohn ME. Estrogen receptor- mediates the protective effects of estrogen against vascular injury. Circ Res. 2002; 90: 1087–1092.
Shearman AM. Hormone receptor polymorphisms. In: Miller VM and Hay M, eds. Principles of Sex-Based Differences in Physiology Volume 34 of: Bittar EG, Series Ed. Advances in Molecular and Cell Biology. The Netherlands: Elsevier Science; 2004: 59–69.
Death AK, McGrath KC, Sader MA, Nakhla S, Jessup W, Handelsman DJ, Celermajer DS. Dihydrotestosterone promotes vascular cell adhesion molecule-1 expression in male human endothelial cells via a nuclear factor{kappa}B-dependent pathway. Endocrinology. 2004; 145: 1889–1897.
Wang X, Barber DA, Lewis DA, McGregor CGA, Sieck GC, Fitzpatrick LA, Miller VM. Gender and transcriptional regulation of endothelial nitric oxide synthase and endothelin-1 in porcine aortic endothelial cells. Am J Physiol. 1998; 273: H1962–H1967.
Barber DA, Miller VM. Gender differences in endothelium-dependent relaxations do not involve NO in porcine coronary arteries. Am J Physiol. 1997; 273: H2325–H2332.
Barber DA, Burnett JC Jr., Fitzpatrick LA, Sieck GC, Miller VM. Gender and relaxation to C-Type natriuretic peptide in porcine coronary arteries. J Cardiovasc Pharmacol. 1998; 32: 5–11.
Miller VM, Lewis DA, Barber DA. Gender differences and endothelium- and platelet-derived factors in the coronary circulation. Clin Exp Pharmacol Physiol. 1999; 26: 132–136.
Woodham C, Birch L, Prins GS. Neonatal estrogen down-regulates prostatic androgen receptor through a proteosome-mediated protein degradation pathway. Endocrinology. 2003; 144: 4841–4850.
Kumar MV, Leo ME, Tindall DJ. Modulation of androgen receptor transcriptional activity by the estrogen receptor. J Androl. 1994; 15: 534–542.
Cardenas H, Pope WF. Attenuation of estrogenic effects by dihydrotestosterone in the pig uterus is associated with downregulation of the estrogen receptors. Biol Reprod. 2004; 70: 297–302.
Chatrath R, Ronningen KL, Severson SR, LaBreche P, Jayachandran M, Bracamonte MP, Miller VM. Endothelium-dependent responses in coronary arteries are changed with puberty in male pigs. Am J Physiol. 2003; 285: H1168–H1176.
Chambliss KL, Shaul PW. Estrogen modulation of endothelial nitric oxide synthase. Endocrine Reviews. 2002; 23: 665–686.
Hatakeyama H, Nishizawa M, Nakagawa A, Nakano S, Kigoshi T, Uchida K. Testosterone inhibits tumor necrosis factor--induced vascular cell adhesion molecule-1expression in human aortic endothelial cells. FEBS Lett. 2002; 530: 129–132.
Ling S, Dai A, Williams MRI, Myles K, Dilley RJ, Komesaroff PA, Sudhir K. Testosterone (T) enhances apoptosis-related damage in human vascular endothelial cells. Endocrinology. 2002; 143: 1119–1125.
Swinnen JV, Heemers H, Heyns W. Androgen regulation of lipogenesis. Adv Exp Med Biol. 2002; 506 (Pt. A): 379–387.
Lopez D, Sanchez MD, Shea-Eaton W, McLean MP. Estrogen activates the high-density lipoprotein receptor gene via binding to estrogen response elements and interaction with sterol regulatory element binding protein-1A. Endocrinology. 2002; 143: 2155–2168.
Machinal-Quelin F, Dieudonne MN, Pecquery R, Leneveu MC, Giudicelli Y. Direct in vitro effects of androgens and estrogens on ob gene expression and leptin secretion in human adipose tissue. Endocrine. 2002; 18: 179–184.
Watanabe T, Akishita M, Nakaoka T, Kozaki K, Miyahara Y, He H, Ohike Y, Ogita T, Inoue S, Muramatsu M, Yamashita N, Ouchi Y. Estrogen receptor ? mediates the inhibitory effect of estradiol on vascular smooth muscle cell proliferation. Cardiovasc Res. 2003; 59: 734–744.
Tschugguel W, Dietrich W, Zhegu Z, Stonek F, Kolbus A, Huber JC. Differential regulation of proteasome-dependent estrogen receptor and ? turnover in cultured human uterine artery endothelial cells. J Clin Endocrinol Metab. 2003; 88: 2281–2287.
Naaz A, Zakroczymski M, Heine P, Taylor J, Saunders P, Lubahn DB, Cooke PS. Effect of ovariectomy on adipose tissue of mice in the absence of estrogen receptor (ER): A potential role for estrogen receptor ? (ER?). Hormone and Metabolic Research. 2002; 34: 758–763.
Jayachandran M, Miller VM. Human platelets contain estrogen receptor , caveolin-1 and estrogen receptor associated proteins. Platelets. 2003; 14: 75–81.
Jayachandran M, Owen WG, Miller VM. Effects of ovariectomy on aggregation, secretion, and metalloproteinases in porcine platelets. Am J Physiol. 2003; 284: H1679–H1685.(Virginia M. Miller; Donal)
Incidence of cardiovascular disease is consistently greater in men than in women during the first five decades of life.1 Because this gender disparity begins early in life, it is likely that a combination of genetic, environmental (possibly intrauterine) factors, and actions of sex-specific hormones contribute to the disease process (see Nagel and vom Saal2). However, most research has focused on understanding how sex steroid hormones, estrogens and androgens, affect vascular function in men and women.
See page 1055
Historically, an "estrogen protection" hypothesis, where estrogens limit development of atherosclerosis, has been tested, although an "androgen detrimental" hypothesis is also plausible, but less studied. Although these hypotheses provide a starting point for design of experiments to study effects of hormones on vascular function, review of available data suggests a more complex interaction among the sex steroids than this simple dichotomy.3 Therefore, new approaches are needed to dissect mechanisms of how sex steroids interact in development of vascular disease in both sexes.
The study by Villablanca et al, reported in this issue, uses an approach of examining the contribution of estrogen receptor alpha (ESR1) in development of early atheroma in male mice.4 The design is based on two published lines of research: one which forms the basis of the estrogen "protection" hypothesis, and the other which suggests that disruption and/or polymorphisms in ESR1 are associated with accelerated atherosclerosis and cardiovascular events in men.
The estrogen protection hypothesis arose predominately from large cohort studies, where perimenopausal women treated with estrogen or estrogen-progestin for relief of menopausal symptoms had reduced cardiovascular disease compared with untreated women.5,6 From experimental studies, estrogen treatment of ovariectomized animals affected several processes that would promote repair of damaged vascular endothelium, reduce adhesion of aggregating platelets and leukocytes, and cause vasodilatation and thus limit development of occlusive vascular lesions (see Mendelsohn and Karas7 for review). In addition, estrogen affects lipid metabolism such that estrogen treatment increases serum concentrations of high-density lipoproteins while decreasing low-density lipoproteins. Estrogen also reduces lipid uptake by macrophages and their adhesion to endothelial cells, which should limit formation of fatty streaks in the arterial wall.
In males with normal testicular function, endogenous estrogen is produced enzymatically from testosterone by aromatase in many tissues, including the arterial wall. Theoretically, then, some vascular effects of testosterone could be mediated indirectly through conversion to 17?-estradiol.3 Indeed, inhibition of aromatase in men decreases circulating levels of 17?-estradiol.8
Estrogen effects are mediated through two receptors, estrogen receptor (ESR1) and ? (ESR2). These receptors, located on separate somatic chromosomes 6 and 14, respectively, are present in men and women. Little is known about how expression of these receptors is regulated in vascular tissue of men and women. However, ESR2 as measured by mRNA is the predominant subtype found in human blood vessels collected as surgical waste, and there is a suggestion that the ratio of ESR1:ESR2 may be greater in blood vessels from males compared with females.9
The second line of evidence supporting the design of the studies from Villablanca et al comes from observations that loss of ESR1 in men is associated with loss of flow-mediated dilation of the brachial artery.10 The fact that this flow-mediated dilatation is diminished in men treated with aromatase inhibitors8 suggests a link between the physiological response and testosterone-derived estrogen activation of ESR1. Polymorphisms in ESR1 in men are also associated with accelerated atherosclerosis and increased risk for myocardial infarction.11–13 Therefore, it is attractive to propose that in men, estrogen produced through local aromatization of testosterone would also provide "protection" against development of cardiovascular disease through mechanisms involving ESR1.
To test this hypothesis requires reliable and characterized animal models. To that end, Villablanca and colleagues4 sought to characterize atheroma in male mice lacking ESR1 receptors. In their experiments, disruption of ESR1 was performed in mice of a mixed genetic background (129/J and C57BL/J6). Animals were fed a high fat diet. At time points up to six months, aortae were removed for histological quantification of number and extent of atheromatic lesions. Lesions were characterized by deposits of intracellular fat and large numbers of foam cells but were not considered complex lesions nor did they contain fibrotic caps or calcification. The rate of development and number of fatty lesions were greater in wild-type compared with ESR1 knockout animals. This was unexpected given the association in humans between the disruption and polymorphisms of ESR1 and extent of cardiovascular disease in men.
Discrepancies between observations in experimental animals as models for disease and disease in humans must be reconciled. One consideration is that the genetic manipulation of ESR1 results in a variant gene product that might have some biological activity.14–16 There are over 300 single nucleotide polymorphisms in the human estrogen receptor public database.17 Relative prevalence of a particular genetic variant while showing association to disease progression in humans may not exist in isolation of other genetic variants.
Alternatively, the hypothesis that effects on the vasculature are "protective" and mediated solely through conversion of testosterone to estrogen and ESR1-mediated mechanisms may be na?ve and should be revised. Aromatase was observed by immunohistochemistry in the endothelium, media, and adventia of the aortic wall suggesting that enzymatic conversion of testosterone to 17?-estradiol could occur within the aorta. However, androgen receptors are present in vascular tissue and nonaromatizable androgens initiate vascular effects.3,18 Therefore, given the potential for stoichiometric interactions of testosterone with aromatase and androgen receptors, it is unlikely that all physiologically relevant testosterone would be completely converted to estradiol independent of activation of androgen receptors (Figure). Indeed, sex-specific differences in vascular function are still observed in male pigs where estrogen levels exceed those of females by 10-fold.19–22 In contrast to estrogen receptors, the gene for the androgen receptor is on the X chromosome. It is unknown at this time whether genetic variation in this receptor show sex-related associations with cardiovascular disease.
Schematic representation of possible mechanisms by which testosterone could affect vascular function. Testosterone is synthesized from DHEA in extravascular tissue. Whether DHEA activates a membrane receptor is controversial. Testosterone can be aromatized to 17?-estradiol in extravascular or vascular tissue. Both androgen and estrogen receptors are present in vascular tissue. However, little is known about their regulation in cells of the vascular wall or how they might interact to regulate nongenomic or genomic pathways that participate in development of atherosclerosis including regulation of vascular tone, adhesion of macrophages, aggregation of platelets, cellular apoptosis, differentiation, or proliferation. Therefore, expression of an atherosclerotic phenotype associated with a particular polymorphism in, for example, an estrogen receptor may depend on the type of initiating stimulus or combination of stimuli (endothelial denudation, lipid peroxidation, infection), coregulators needed for receptor activation, duration of stimulus, and/or hormone exposure, genetic sex, and hormonal status. Although this schematic depicts actions of steroids in endothelial cells, similar mechanisms are likely to be present in smooth muscle cells, macrophages, and platelet-precursors, megakaryocytes.36,37 Abbreviations: Akt, a serine-threonine kinase important for regulation of several cellular processes; AR, androgen receptor; ARom, aromatase; DHEA, dihydroepiandrosterone; DHEAR, dihydroepiandrosterone receptor; E2, 17?-estradiol; ER, estrogen receptor alpha () or beta (?); eNOS, endothelial nitric oxide synthase; Gi, guanine nucleotide regulator protein subunit which inhibits guanylate cyclase; NO, nitric oxide; Rm, membrane receptor; ?, denotes unknown interactions or pathways needing additional research.
In the study by Villablanca et al, castration (which reduced both systemic testosterone and estradiol exposure) reduced the average area of the atheroma lesion by over 90%, but a difference in atheroma between wild-type and ESR1 knockout mice was still present although not statistically significant.4 Whether all of testosterone’s effects are mediated by aromatization cannot be answered by this study because the effect of castration was not evaluated with concomitant inhibition of aromatase. Furthermore, whether the effect of castration could be reversed by selective replacement of testosterone was not confirmed, leaving open the possibility that another testicular secretion could contribute to vascular effects. Therefore, it is possible that vascular actions of testosterone are mediated directly through stimulation of androgen receptors as well as indirectly through conversion to 17?-estradiol and stimulation of ESR2.
It should be recognized that cross talk exists between the steroid receptors. For example, estrogen modulates androgen receptor expression as well as transcriptional activity.23,24 In addition, nonaromatizable androgens downregulate expression of estrogen receptors.25 Thus, ESR1 knockout mice may have compensatory changes in androgenic pathways that could affect the phenotype.
Factors affecting quantity and distribution of vascular androgen receptors are unknown. Although activation of androgen receptors can initiate both nongenomic and genomic effects in vascular tissue,3 it is not known how the androgen receptor regulates the synthesis or activity of endothelial nitric oxide synthase (eNOS).26 In contrast, regulation and activation of eNOS by estrogen receptors is well established.27
The animal model studied by the Villablanca group may be useful in understanding hormonal interactions in early atheroma as defined by adhesion of macrophages and subendothelial accumulation of fat and foam cells. These responses are consistent with effects of nonaromatizable testosterone and androgen receptor blockade on increased expression of vascular cell adhesion molecule (VCAM)-1 in cultured endothelial cells and enhanced apoptosis of endothelial cells.28,29 Effects of androgens on lipogenesis are beginning to be defined, and there is evidence that sterol regulatory element–binding proteins are key mediators of lipogenic effects of both androgens and estrogens in a variety of tissues.30–32 Studies taking advantage of androgen receptor antagonists, aromatase inhibitors, castration, and/or replacement of testosterone or estrogen to castrated animals could be designed to investigate the stages of atheroma development.
Finally, vascular effects of ESR2 in ESR1 knockout mice should be considered. ESR2 inhibits proliferation of vascular smooth muscle.33 Responses regulated by ESR1 and 2 may be opposite and differentially regulated in adipose and endothelial cells.34,35 Therefore, experiments are also needed to understand the contribution of ESR2 in atheroma development in both male and female animals.
Atherosclerosis in humans is a multifactorial disease process which includes lipid accumulation, remodeling of the vascular wall, infection, and calcification. In the era of sex-based medicine and genomics it is important to define similarities and differences in these processes as regulated by sex steroids in males and females. Therefore, studies in experimental animals that relate genetic sex, hormonal status, and receptor polymorphisms to vascular healing after mechanical injury16 or lipid accumulation, as in the article by Villablanca et al, provide important tools to understand effects of hormones on various stages of the disease process. However, each method provides an incomplete picture of disease progression in humans, and discovery of novel therapeutics will be hampered by generalization of hormonal functions as being "beneficial," "protective," or "detrimental" when sex- and cell-specific actions of hormones remain incompletely understood. Further investigation is needed to understand actions of testosterone on the vascular wall, contributions of androgen and estrogen receptor polymorphisms to vascular function, and better definition of responses mediated by ESR2 in various stages of the atherogenic process.
References
Vargas CM, Burt VL, Gillum RF. Cardiovascular disease in the NHANES III. Ann Epidemil. 1997; 7: 523–525.
Nagel SC, vom Saal FS. Endocrine control of sexual differentiation: effects of the maternal-fetal environment and endocrine disrupting chemicals. In: Miller VM and Hay, M eds. Principles of Sex-Based Differences in Physiology. Volume 34 of: Bittar EG, Series Ed. Advances in Molecular and Cell Biology. The Netherlands: Elsevier Science; 2004: 15–37.
Liu PY, Death AK, Handelsman DJ. Androgens and cardiovascular disease. Endocrine Reviews. 2003; 24: 313–340.
Villablanca A, Lubahn D, Shelby L, Lloyd K, Barthold S. ZSusceptibility to early atherosclerosis in male mice is mediated by estrogen receptor . Arterioscler Thromb Vasc Biol. 2004: 24: 1055–1061.
Barrett-Connor E, Bush TL. Estrogen and coronary heart disease in women. JAMA. 1991; 265: 1861–1867.
Kannel WB, Hjortland MC, McNamara PM, Gordon T. Menopause and risk of cardiovascular disease: the Framingham Study. Ann Int Med. 1976; 85: 447–452.
Mendelsohn ME, Karas RH. The protective effects of estrogen on the cardiovascular system. N Engl J Med. 1999; 340: 1801–1811.
Lew R, Komesaroff PA, Williams M, Dawood T, Sudhir K. Endogenous estrogens influence endothelial function in young men. Circ Res. 2003; 93: 1127–1133.
Hodges YK, Tung L, Yan X-D, Graham D, Horwitz KB, Horwitz LD. Estrogen receptors and ?. Prevalence of estrogen receptor ? mRNA in human vascular smooth muscle and transcriptional effects. Circulation. 2000; 101: 1792–1798.
Sudhir K, Chou TM, Messina LM, Hutchison SJ, Korach KS, Chatterjee K, Rubanyi GM. Endothelial dysfunction in a man with disruptive mutation in oestrogen-receptor gene. Lancet. 1997; 349: 1146–1147.
Shearman AM, Cupples LA, Demissie S, Peter I, Schmid CH, Karas RH, Mendelsohn ME, Housman DE, Levy D. Association between estrogen receptor gene variation and cardiovascular disease. JAMA. 2003; 290: 2263–2270.
Sudhir K, Chou TM, Chatterjee K, Smith EP, Williams TC, Kane JP, Malloy MJ, Korach KS, Rubanyi GM. Premature coronary artery disease associated with a disruptive mutation in the estrogen receptor gene in a man. Circulation. 1997; 96: 3774–3777.
Kunnas TA, Laippala P, Penttila A, Lehtimaki T, Karhunen PJ. Association of polymorphism of human oestrogen receptor gene with coronary artery disease in men: A necropsy study. BMJ. 2000; 321: 273–274.
Couse JF, Curtis SW, Washburn TF, Lindzey J, Golding TS, Lubahn DB, Smithies O, Korach KS. Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene. Mol Endocrinol. 1995; 9: 1441–1454.
Toran-Allerand CD, Guan X, MacLusky NJ, Horvath TL, Diano S, Singh M, Connolly ESJ, Nethrapalli IS, Tinnikov AA. ER-X: a novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury. J Neurosci. 2002; 22: 8391–8401.
Pare G, Krust A, Karas RH, Dupont S, Aronovitz M, Chambon P, Mendelsohn ME. Estrogen receptor- mediates the protective effects of estrogen against vascular injury. Circ Res. 2002; 90: 1087–1092.
Shearman AM. Hormone receptor polymorphisms. In: Miller VM and Hay M, eds. Principles of Sex-Based Differences in Physiology Volume 34 of: Bittar EG, Series Ed. Advances in Molecular and Cell Biology. The Netherlands: Elsevier Science; 2004: 59–69.
Death AK, McGrath KC, Sader MA, Nakhla S, Jessup W, Handelsman DJ, Celermajer DS. Dihydrotestosterone promotes vascular cell adhesion molecule-1 expression in male human endothelial cells via a nuclear factor{kappa}B-dependent pathway. Endocrinology. 2004; 145: 1889–1897.
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