Susceptibility to Early Atherosclerosis in Male Mice Is Mediated by Estrogen Receptor
http://www.100md.com
《动脉硬化血栓血管生物学》
From the Department of Internal Medicine (A.V., L.S.), University of California, Davis; the Department of Biochemistry (D.L.), University of Missouri, Columbia; and the Center for Comparative Medicine (K.L., S.B.), University of California, Davis.
ABSTRACT
Objective— Vascular tissues express 2 types of estrogen receptors (ERs): ER and ER?. Their role in early atherosclerosis remains poorly understood, particularly in males. We developed and characterized an atherosclerosis model in ER knockout male mice to investigate directly its role in atheroma.
Methods and Results— Cholesterol-fed ER knockout and wild-type mice developed early atheroma characterized by fatty streaks and foam cells. ER wild-type mice developed 3.8-fold greater lesion area, more advanced lesions, more extensive lesion distribution, twice the number of lesions, and at a 2.2-fold faster rate than ER knockout mice. Lesion development and atheroma susceptibility in ER wild-type and knockout mice were independent of serum cholesterol, triglycerides, high-density lipoproteins, 17?-estradiol, and testosterone levels. In contrast, castration eliminated the predilection of ER wild-type mice for atheroma, suggesting that testosterone mediates ER-dependent atheroma formation in males.
Conclusions— This study is the first to report that the ER mediates susceptibility to early atherosclerosis in male mice by a testosterone-dependent pathway, suggesting that local production of estrogen from testosterone in the vessel wall may promote atheroma formation in ER males. Our findings may have implications for selective targeting of ER in atherosclerotic disease.
The role of ERs ER and ER? in early atherosclerosis remains poorly understood in males. We developed and characterized an atherosclerosis model in ER knockout male mice to investigate directly its role in atheroma. In males, the ER mediates susceptibility to early atherosclerosis by a testosterone-dependent pathway.
Key Words: vascular ? hormone ? lipids ? gender ? atheroma ? aromatase ? estrogen ? testosterone
Introduction
Atherosclerosis is a complex progressive disease characterized by alterations in endothelial function, smooth muscle proliferation, coagulation, and inflammation.1 Atherogenesis is largely dependent on hyperlipidemia and is also clearly modulated by hormonal influences.2 Important gender differences in atherosclerotic cardiovascular disease have been demonstrated, although they are mechanistically not yet fully understood. In females, estrogen reduces smooth muscle cell proliferation, attenuates coagulation, reduces vasoconstriction, and lowers atherogenic lipoproteins.3 However, the role of sex steroid hormones and their cognate receptors in atherosclerosis in males is not well defined.
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The long-term effects of estrogens are generally ascribed to transcriptional modulation of target genes through estrogen receptors (ERs).4 Estrogens influence gene expression and a number of physiological functions in target tissues (eg, cell growth and cellular differentiation) by activation of these receptors. Two ERs, ER (ESR1) and ER? (ESR2), have been characterized.5,6 They are encoded on 2 separate genes (Esr1, Esr2), are distinct structurally and functionally, and have overlapping although not identical tissue expression patterns.6–8 Vascular endothelial cells, 9 smooth muscle cells, 10,11 and macrophages12 express ERs.
The availability of ER and ER? null mice and their characterization13 has elucidated the role of ERs in vascular function. In mice, both ERs are necessary and sufficient for estrogen-mediated protection against vascular injury. ERs also regulate a number of vasodilatory and vasoconstrictor proteins, including several components of the renin-angiotensin and nitric oxide systems. However, the actions of vascular ERs in atherosclerosis have not been directly studied experimentally in males. An association between disruption of ER and possible premature atherosclerosis in a single man14 and the epidemiological association between ER gene variation and cardiovascular disease, particularly in males,15 are the only data to implicate ER as a potential modulator of atherosclerosis in men. Therefore, determining the relative contribution of ER to atherogenesis is of considerable interest, as well as in relation to the use of pharmaceutical agents specific to this receptor type.
We therefore sought to develop a model of atherosclerosis using the mouse lacking the ER gene (ER–/–) to investigate the specific physiological role of ER in atherogenesis in male mice. Here, we report that ER is a significant mediator of susceptibility to early atherosclerosis in male mice because susceptibility is substantially less in male mice that lack ER.
Materials and Methods
Experiments were performed in compliance with National Institutes of Health Guidelines and in accordance with protocols approved by the University of California, Davis Animal Care and Use Committee.
Generation and Use of Mice
Mice heterozygous for the ER gene were obtained from Dr. Dennis Lubahn (University of Missouri) and mated to yield progeny ER–/– and wild-type littermate controls having intact ER (ER+/+). Mice were housed in a temperature-, light-, and humidity-controlled environment in a dedicated pathogen-free barrier facility at the University of California, Davis. ER–/– mice have a mixed genetic background from strains 129/J and C57BL/6J, an atherosclerosis-susceptible strain.
ER gene-deletion mice are infertile but otherwise phenotypically normal.16 They do not produce a fully functional ER receptor and have no demonstrable full-length wild-type ER protein.16 However, the disrupting neo sequence previously used to generate the knockout results in a variant ER protein (not the full-length ER wild-type protein). This alternative ER protein has residual low-level, high-affinity, estradiol-binding activity17,18 but lacks the specificity to be from either ER or ER? when tested for estrogenic activity in uteri of ER–/– mice (data not shown). Thus, the insensitivity of ER–/– mice to estrogen is well established.17
Atherogenesis Model and Experimental Design
To induce atherogenic lesions, 6- to 7-week-old ER–/– (n=22) and ER+/+ littermate control (n=24) male mice were fed a high-fat, high-cholesterol Paigen diet19 containing 15% butter fat and 1.25% cholesterol (wt/wt, Purina). The diet contained 0.5% cholic acid, which is required for cholesterol absorption in the mouse.20 Aortas (5 to 7 from each genotype) were sampled for lesion formation at baseline (age 6 to 7 weeks, immediately before cholesterol feeding) and after 2, 4, and 6 months on the atherogenic diet. In a parallel set of experiments, ER–/– (n=15) and ER+/+ littermate control (n=15) male mice were surgically castrated by bilateral orchiectomy at baseline before initiation of the atherogenic diet. Aortas (5) were sampled at the experimental time intervals noted above.
Evaluation of Aortic Atherosclerosis
To determine extent of atheromatous lesions in aortas and regional differences in atherosclerosis susceptibility, we adapted and optimized previously published and reproducible methods for atheroma quantification.21 (For details please see Figure IA through ID, available online at http://atvb.ahajournals.org). At the specified experimental time intervals, aortas were excised intact, fixed, and cut into 3 segments (proximal segment, aortic arch; midsegment, suprarenal aorta; distal segment, infrarenal aorta proximal to the iliac bifurcation). Cryosections (10-μm thick) simultaneously sampled all 3 segments of the aorta, permitting extensive sampling of each (108 sections per aorta, 36 sections per segment). A total of 1000 μm from each aorta and 360 μm from each segment were analyzed.
Oil red O-stained (Sigma) cryosections defined and characterized neutral lipid in the vessel wall, permitting histological assessment and quantitative evaluation of lesions in artery segments. Sections were analyzed for lesion location, quantity, and area by direct imaging (Olympus BX-40 microscope, Olympus DP11 camera). Mean lesion size (μm2±SEM) for each atheroma was determined by morphometric analysis with a computer-assisted imaging system (Image Pro Plus, version 4.1). Qualitative morphological assessment of lesion complexity was performed by light microscopy. Parameters were: absence or presence of foam cells, wall thickness, disruption of vessel wall architecture, nuclear proliferation, and lumenal extension.
Quantification of Plasma Lipids
Plasma lipid levels were measured in study mice at baseline and at 2, 4, and 6 months on the atherogenic diet to determine whether lesion formation was related to changes in lipids. Lipids were assayed as follows: total cholesterol (TC) using a GM7 Analox analyzer (Analox), triglyceride (TG) with triglyceride reagent (Sigma), and high-density lipoprotein (HDL)-cholesterol (HDL-C) with a colororimetric reagent kit (Wako Biochemicals) after precipitating the apolipoprotein B (apoB)-containing particles. All assays were performed in duplicate on nonpooled plasma samples.
Plasma Hormone Analysis
The impact of circulating estrogen and testosterone on lesion development was determined by assessment of free testosterone and 17?-estradiol plasma levels in study mice at baseline and at 2, 4, and 6 months on the atherogenic diet. Assays were performed in duplicate by enzyme immunoassay (Cayman Chemical) on nonpooled plasma samples.
Statistical Analysis
Values are reported as mean±SEM. Comparisons between ER–/– and ER+/+ mice were made using Student t test for independent samples (2-tailed) and ANOVA using Microsoft Office 2000 Excel and Sigma Stat version 2.03 (SPSS). Correlations between parameters were analyzed using simple linear regression. Probability values <0.05 identified all statistically significant comparisons.
Results
Atheroma Development Was Greater and Faster in ER +/+ Than in ER –/– Male Mice
The atherogenic diet resulted in aortic lesions after 10 to 16 weeks, consistent with previously published reports for atherosclerosis susceptible mouse strains.19 Representative lesions of male ER–/– and ER+/+ mice at each of the time points are shown in Figure 1. Early atherosclerotic lesions were present in the intimal layer of aortas of both groups after 2 months of cholesterol feeding, with more robust lesions developing over time. Atheromatous lesions were distinguished by lipid infiltration into the vessel wall, the presence of fatty streaks, and occasional raised intimal lesions. With time, lesions were more advanced than simple fatty streaks, having deposits of intracellular fat (lipid lakes; eg, ER+/+ at 4 months), and contained large numbers of foam cells (eg, ER+/+ at 6 months) but were not complicated lesions. Compared with ER–/– mice, atherosclerotic lesions in ER+/+ mice were more advanced, larger, had greater cellular architecture disruption and cellular disarray, and greater proliferation of nuclei in the intima. Over the entire 6-month study period, lesion area in male ER+/+ mice averaged 3.8-fold larger than ER–/– mice (P<0.05).
Figure 1. Representative images (magnification x20) of oil red O-stained cross-sections (10 μm) of proximal segments of aortic lesions from cholesterol-fed ER–/– (left) and ER+/+ (right) male mice demonstrating atheroma formation over time (2, 4, and 6 months). A normal vessel before the initiation of cholesterol feeding and baseline is shown for comparison. Cross-sections are oriented with the lumen to the right and the adventitial surface to the left of each image. Size markers=100 μm.
Atheroma Number and Distribution Is Greater in ER+/+ Than in ER–/– Male Mice
We characterized atheroma by quantity, rate of progression, and segment location. Over the entire period of cholesterol feeding, aortas of ER+/+ mice averaged twice the number of atheromatous lesions (24±8) than aortas of male ER–/– mice (12±6). Because of variability in lesion number, this did not reach statistical significance. Atheroma quantity was initially low but increased steadily over time in both groups of mice. However, ER+/+ mice developed lesions at nearly twice the rate of ER–/– mice (slope for mean lesion number/time=11.5 for ER+/+ and 5.1 for ER–/– mice, respectively; P<0.05; Figure 2A).
Figure 2. A, Rate of lesion development over time in aortas of ER–/– and ER+/+ mice. The total number of lesions in the aortas of ER–/– (n=22) and ER+/+ male mice (n=24) were measured at baseline, 2, 4, and 6 months of cholesterol feeding (n=5 to 7 mice/genotype/time point). Rate of lesion development=slope of number of lesions/time. B, Atheroma distribution in aortic segments. Total mean lesion area (μm2) for atheroma in proximal, mid, and distal aortic segments of male ER–/– (n=60 segments) and ER+/+ (n=72 segments) mice (n=5 to 7 mice/genotype/time point).
Distribution of atheroma in aortic segments of ER+/+ (n=72 segments) and ER–/–mice (n=60 segments) is shown in Figure 2B. When averaged over the period of atherogenic diet, there were very few or no atheroma in the distal aortas of ER+/+ and ER–/– mice (1% and 3%, respectively). The proximal aorta was the primary lesion predilection site for atheroma formation in ER–/– mice (80% of atheroma), with only 17% in the midsegment. The proximal aorta displayed greater lipid deposition, larger lesions, and more lesions than other segments. However, in ER+/+ mice, atheroma were distributed more extensively throughout the aorta and more evenly between proximal and midsegments (49% and 50% of atheroma, respectively). Lesion area in the proximal and midsegments of ER+/+ mice was 2.3- and 11.3-fold larger, respectively (P<0.05), than corresponding segments in ER–/– mice.
ER-Mediated Atheroma Susceptibility in Male Mice Is Independent of Lipoprotein Serum Levels
The Table summarizes plasma lipids (TC, TG, and HDL) in ER–/– and ER+/+ mice over the atherogenic diet time period. After 6 months on the diet, fasting TC levels increased nearly 50% in ER mice compared with baseline values (P<0.05). However, the level of hypercholesterolemia attained by ER–/– and ER+/+ mice on the atherogenic diet correlated poorly with observed differences in lesion formation in the 2 mouse groups (r2=0.16 and 0.5, respectively; data not shown). There were no significant differences in baseline TG levels between ER–/– and ER+/+ mice. While on the atherogenic diet, fasting levels of plasma TG dropped by 59% in both groups of mice compared with baseline values (P<0.05). However, changes in TG levels did not correlate with lesion formation in either group (r0.01; data not shown). Fasting levels of HDL-C were similar at baseline in both groups, with no significant change in HDL-C levels on the atherogenic diet in either group compared with baseline.
Plasma Lipids in ER–/– and ER+/+ Male Mice
ER-Mediated Atheroma Susceptibility in Male Mice Is Independent of Serum Hormone Levels
No significant differences were observed between ER–/– and ER+/+ mice in mean levels of serum 17?-estradiol (18±2 pg/mL and 19±2 pg/mL , respectively) or serum testosterone (2689±231 pg/mL and 2978±227 pg/mL , respectively) during the study period. We found no correlation between lesion area and plasma levels of either hormone (r0.01; data not shown).
Effect of Castration on Lesion Development
To understand the mechanism underlying the differences in atheroma susceptibility between ER+/+ and ER–/– mice, we studied atheroma in castrated ER+/+ and ER–/– mice. Castration eliminated differences in atheroma susceptibility previously observed between ER+/+ and ER–/– mice. Castration resulted in a dramatic reduction (P<0.01) in atheroma in both ER+/+ and ER–/– mice (91% and 96%, respectively; Figure 3A). In addition, compared with intact mice of the same genotype, castration resulted in a reduction in the number of lesions in ER+/+ mice and ER–/– mice (by 33% and 83% , respectively). There were no differences in the atheroma distribution pattern of intact and castrated ER+/+ mice and ER–/– mice (data not shown).
Figure 3. Atheroma and hormone levels in castrated and intact ER–/– (KO) and ER+/+ (WT) male mice. A, Time-averaged aortic lesion area (μm2) in intact ER–/– (n=22) and ER+/+ (n=24) and castrated ER–/– (n=15) and ER+/+ (n=15) male mice at baseline, 2, and 4 to 6 months of cholesterol feeding (n=5 to 7 aortas/genotype/time point for intact mice; n=5 aortas/genotype/time point for castrated mice). *The bars in A denote statistically significant (P<0.05) differences between groups. B, Time average mean plasma testosterone levels. C, Time average mean plasma estradiol levels. See text for details of B and C.
As expected, after castration, mean time-averaged plasma testosterone levels declined significantly (P<0.001) in both ER–/– and ER+/+ mice to 144±18 pg/mL (n=5) and 218±40 pg/mL (n=5), respectively (Figure 3B). Concomitantly, mean time-averaged plasma 17?-estradiol levels also declined significantly (P<0.001) in both ER–/– and ER+/+ mice to 3±1 pg/mL (n=5) and 7±2 pg/mL (n=5), respectively (Figure 3C). As with intact mice, serum testosterone and 17?-estradiol did not significantly differ between castrated ER–/– and ER+/+ mice.
Demonstration of P450 Aromatase Expression in Aortas of ER–/– and ER+/+ mice
We considered the possibility of local estrogen synthesis from testosterone in vessels of ER+/+ and ER–/– male mice. P450 aromatase is the key enzyme regulating conversion of testosterone to physiologically active estrogens in males.22,23 To determine whether P450 aromatase was present in aortas of ER males, we performed immunocytochemical studies to evaluate P450 antigen expression in normal aortas of ER+/+ and ER–/– male mice using standard immunohistochemical techniques and a well-studied polyclonal antibody to P450 aromatase (kindly provided by Dr. N. Harada, Fujita Health University, Japan). (Please see www. ahajournals.org for details.) Figure 4 demonstrates P450 aromatase immunostaining in all layers of the vessel wall in aortas of ER males.
Figure 4. Immunostaining of P450 aromatase in aortas of ER–/– and ER+/+ male mice. A, Positive control tissue demonstrating P450 aromatase in pig testicular stroma (brown immunostaining, arrow). B, Comparison of negative control (deletion of primary antibody) in ER mouse aorta. C and D, Positive immunostaining for P450 aromatase in normal male ER –/– and ER +/+ aorta, respectively.
Discussion
The ER is implicated in a diverse array of physiological functions and plays a pivotal role in vascular biology. To test the role of ER gene expression on atheroma in males, we developed a model of early atherosclerosis in male mice lacking ER. Our data provide previously undocumented direct in vivo evidence of ER-mediated regulation of susceptibility to early atherogenesis in males.
The atheroprotective effect of estradiol in mice with targeted inactivation of the low-density lipoprotein (LDL) receptor24 and apoE25,26 has been demonstrated. In these models, under certain conditions, mice spontaneously develop advanced atherosclerosis. In contrast, we have developed a model of early atherosclerosis in cholesterol-fed ER male mice, permitting the study of fatty streaks, foam cell formation, and early lesion progression. The development of fatty streaks and foam cells, the earliest stage of atherosclerosis, is biologically important because it can lead to formation of more advanced lesions that characterize clinical events in humans.
The pathology of vessel wall architecture and lesion morphology in our system was robust and comparable with that reported previously for atherosclerosis-susceptible mouse strains.21 The gross morphology of lesions reported here resembles early lesions in fat-fed C57BL/6 mice, other model species, and humans. Studying lesions in the proximal, mid, and distal aorta of ER–/– and ER+/+ mice provides a better estimate of relative changes in regional susceptibility to lesion development than previous estimates recording lesions in the aortic free wall or at the base of the aortic sinus.21,27 Thus, our results agree overall with previous reports in analogous systems of diet-induced atherosclerosis19,28 and are consistent with immature lesions observed in other animal species fed an atherogenic diet.29,30
Changes in age, body weight, and brown/white fat weight distribution did not appear to explain observed differences in atherosclerosis susceptibility between ER–/– and ER+/+ mice. Without cholesterol feeding, C57BL/6 mice do not develop atheroma as they age.19 In addition, because the ER–/– and ER+/+ mice were age matched, aging did not influence observed atheroma susceptibility differences. ER–/– and ER+/+ mice also maintained similar total body weights throughout the study, so weight does not explain observed differences. Brown adipose tissue weight is similar in ER–/– and ER+/+ males at all ages.31 Although white adipose weight differs between ER–/– and ER+/+ male mice, 31 these changes are observed between 270 and 360 days and thus occur in older mice than those studied here.
A relationship has been established previously between high cholesterol, low HDL-C, and susceptibility to aortic lesions in humans and mice. Therefore, we investigated a possible differentiating role for serum lipids in the atheroma differences observed in our study. In ER–/– and ER+/+ mice, lipid levels at baseline and after the atherogenic diet were consistent with those published previously for atherosclerosis susceptible mouse strains.19 However, the greater susceptibility to atherosclerosis we observed in male ER+/+ mice compared with ER–/– mice was independent of lipid changes, and therefore, presumably occurring at the artery wall level. There are numerous examples of vessel wall pathophysiological states that are independent of lipid effects, including alterations in vascular reactivity, vascular responses to injury, and vascular inflammation.32–34 Indeed, previous animal models of atherosclerosis have also demonstrated that atheroma may not be linked to degree of hyperlipidemia.19,21,35
We then considered the possible role of sex steroid hormones in observed differences for atheroma development between ER–/– and ER+/+ male mice. Although testosterone is the predominant sex steroid hormone in male mice, estrogen is also present, albeit at low levels. In males, peripheral production of estrogen occurs in a number of tissues36,37 and in vascular cells38 via enzymatic activity of P450 aromatase. We specifically assayed the levels of serum-free testosterone and 17?-estradiol in our study mice and found them to be in the expected range for male mice and to not differ significantly between ER–/– and ER+/+ mice. This suggested that the effects of ER on atherosclerosis susceptibility were independent of circulating levels of these 2 hormones. After castration, we observed the expected decline in serum testosterone in ER–/– and ER+/+ animals and a concomitant decline in serum 17?-estradiol, again without significant differences observed in plasma testosterone and 17?-estradiol levels between castrated ER–/– and ER+/+ animals. However, because castration eliminated the predilection for atheroma observed previously in ER+/+ animals, our data support the postulate that in early atherosclerosis, the actions of ER are mediated by testosterone at the level of the artery wall. Our findings differ from those in the LDL receptor-deficient mouse, in which testosterone appears to attenuate atherogenesis.39
Because ER binds estrogens but not androgens, we postulate that the greater susceptibility of ER+/+ mice for atheroma formation demonstrated in our studies may be mediated by in situ local conversion of testosterone to estrogen in vessel walls of mouse aortas. This would implicate a role for vascular aromatase in mice. Recent reports of aromatase activity in the vessel wall of humans support the feasibility of this mechanism.39,40 Indeed, we were able to immunolocalize P450 aromatase in aortas of ER+/+ male mice. We interpret these data to support the tenet that vascular P450 aromatase in aortas of ER–/– and ER +/+ male mice can participate in the local conversion of testosterone to estrogen in vessel walls. Thus, locally produced estrogens, serving as substrate for activation of the ER, could predispose ER+/+ males but not ER–/– males to atheroma. Future studies will need to investigate this potentially new mechanism of atheroma formation in ER males. Nonetheless, our discovery highlights a previously unidentified role for ER in male mice well beyond the reproductive axis.
Differences in susceptibility to atherosclerosis observed in ER–/– and ER+/+ mice could also result from receptor-mediated changes in regulatory processes associated with atherosclerosis, including ER modulation of biological responsiveness, activation of second messenger signaling pathways, and immuno-inflammatory components of atherogenesis.41 Further, ER-mediated atherosclerosis susceptibility in males may result from interactions of the ER with functional or genetic targets in vessel walls other than aromatase. The possibility that alternatively spliced variants of ER or ER? may play a role in ER-mediated atherosclerosis susceptibility in male mice also needs to be considered.
Regarding the biological relevance of our findings to human disease, 3 recent clinical association studies implicate ER deletion and ER variants in cardiovascular disease in men: (1) a single published case report of premature coronary heart disease and estrogen insensitivity in a young male lacking ER;42,43 (2) a common ER gene variant (c.454.397T>C) manifesting as higher myocardial infarction risk in a predominantly male cohort of the Framingham study;15 and (3) more complex atherosclerotic plaque pathology in men with an ESR1 variant.44 These reports provide the first clues that ER may be clinically significant in atherosclerosis in males, although the nature and direction of the association need to be better defined.
In summary, this is the first report to establish a direct mechanistic link between ER and susceptibility to early atherosclerosis in male mice, defining ER as an important gene mediating atheroma formation in males. The results of this study also discover a key role for ER in manifesting a previously unrecognized function in initiation and progression of early atherosclerotic lesions in male mice. These studies carry potentially significant implications for the selective targeting of ER in prevention and treatment of atherosclerotic disease, and for understanding molecular and cellular mechanisms underlying its role in atherogenesis.
Acknowledgments
This work was supported by National Institutes of Health K01-HL04142 award (A.V.). The authors acknowledge personnel at the Murine Targeted Genomics Laboratory of the University of California, Davis, Mouse Biology Program for assistance with rederivation and breeding of mice, especially Carla French, Renee Araiza, and Pollyanna Mraz; Michael Lee and Shannon Smith for technical assistance; Christina Lannen for administrative assistance with manuscript preparation, and Dr. Beverly Paigen for advice regarding atheroma quantification methodology.
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Lehtimaki T, Kunnas TA, Mattila KM, Perola M, Penttila A, Koivula T, Karhunen PJ. Coronary artery wall atherosclerosis in relation to the estrogen receptor 1 gene polymorphism: an autopsy study. J Mol Med. 2002; 80: 176–180.(Amparo Villablanca; Denni)
ABSTRACT
Objective— Vascular tissues express 2 types of estrogen receptors (ERs): ER and ER?. Their role in early atherosclerosis remains poorly understood, particularly in males. We developed and characterized an atherosclerosis model in ER knockout male mice to investigate directly its role in atheroma.
Methods and Results— Cholesterol-fed ER knockout and wild-type mice developed early atheroma characterized by fatty streaks and foam cells. ER wild-type mice developed 3.8-fold greater lesion area, more advanced lesions, more extensive lesion distribution, twice the number of lesions, and at a 2.2-fold faster rate than ER knockout mice. Lesion development and atheroma susceptibility in ER wild-type and knockout mice were independent of serum cholesterol, triglycerides, high-density lipoproteins, 17?-estradiol, and testosterone levels. In contrast, castration eliminated the predilection of ER wild-type mice for atheroma, suggesting that testosterone mediates ER-dependent atheroma formation in males.
Conclusions— This study is the first to report that the ER mediates susceptibility to early atherosclerosis in male mice by a testosterone-dependent pathway, suggesting that local production of estrogen from testosterone in the vessel wall may promote atheroma formation in ER males. Our findings may have implications for selective targeting of ER in atherosclerotic disease.
The role of ERs ER and ER? in early atherosclerosis remains poorly understood in males. We developed and characterized an atherosclerosis model in ER knockout male mice to investigate directly its role in atheroma. In males, the ER mediates susceptibility to early atherosclerosis by a testosterone-dependent pathway.
Key Words: vascular ? hormone ? lipids ? gender ? atheroma ? aromatase ? estrogen ? testosterone
Introduction
Atherosclerosis is a complex progressive disease characterized by alterations in endothelial function, smooth muscle proliferation, coagulation, and inflammation.1 Atherogenesis is largely dependent on hyperlipidemia and is also clearly modulated by hormonal influences.2 Important gender differences in atherosclerotic cardiovascular disease have been demonstrated, although they are mechanistically not yet fully understood. In females, estrogen reduces smooth muscle cell proliferation, attenuates coagulation, reduces vasoconstriction, and lowers atherogenic lipoproteins.3 However, the role of sex steroid hormones and their cognate receptors in atherosclerosis in males is not well defined.
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The long-term effects of estrogens are generally ascribed to transcriptional modulation of target genes through estrogen receptors (ERs).4 Estrogens influence gene expression and a number of physiological functions in target tissues (eg, cell growth and cellular differentiation) by activation of these receptors. Two ERs, ER (ESR1) and ER? (ESR2), have been characterized.5,6 They are encoded on 2 separate genes (Esr1, Esr2), are distinct structurally and functionally, and have overlapping although not identical tissue expression patterns.6–8 Vascular endothelial cells, 9 smooth muscle cells, 10,11 and macrophages12 express ERs.
The availability of ER and ER? null mice and their characterization13 has elucidated the role of ERs in vascular function. In mice, both ERs are necessary and sufficient for estrogen-mediated protection against vascular injury. ERs also regulate a number of vasodilatory and vasoconstrictor proteins, including several components of the renin-angiotensin and nitric oxide systems. However, the actions of vascular ERs in atherosclerosis have not been directly studied experimentally in males. An association between disruption of ER and possible premature atherosclerosis in a single man14 and the epidemiological association between ER gene variation and cardiovascular disease, particularly in males,15 are the only data to implicate ER as a potential modulator of atherosclerosis in men. Therefore, determining the relative contribution of ER to atherogenesis is of considerable interest, as well as in relation to the use of pharmaceutical agents specific to this receptor type.
We therefore sought to develop a model of atherosclerosis using the mouse lacking the ER gene (ER–/–) to investigate the specific physiological role of ER in atherogenesis in male mice. Here, we report that ER is a significant mediator of susceptibility to early atherosclerosis in male mice because susceptibility is substantially less in male mice that lack ER.
Materials and Methods
Experiments were performed in compliance with National Institutes of Health Guidelines and in accordance with protocols approved by the University of California, Davis Animal Care and Use Committee.
Generation and Use of Mice
Mice heterozygous for the ER gene were obtained from Dr. Dennis Lubahn (University of Missouri) and mated to yield progeny ER–/– and wild-type littermate controls having intact ER (ER+/+). Mice were housed in a temperature-, light-, and humidity-controlled environment in a dedicated pathogen-free barrier facility at the University of California, Davis. ER–/– mice have a mixed genetic background from strains 129/J and C57BL/6J, an atherosclerosis-susceptible strain.
ER gene-deletion mice are infertile but otherwise phenotypically normal.16 They do not produce a fully functional ER receptor and have no demonstrable full-length wild-type ER protein.16 However, the disrupting neo sequence previously used to generate the knockout results in a variant ER protein (not the full-length ER wild-type protein). This alternative ER protein has residual low-level, high-affinity, estradiol-binding activity17,18 but lacks the specificity to be from either ER or ER? when tested for estrogenic activity in uteri of ER–/– mice (data not shown). Thus, the insensitivity of ER–/– mice to estrogen is well established.17
Atherogenesis Model and Experimental Design
To induce atherogenic lesions, 6- to 7-week-old ER–/– (n=22) and ER+/+ littermate control (n=24) male mice were fed a high-fat, high-cholesterol Paigen diet19 containing 15% butter fat and 1.25% cholesterol (wt/wt, Purina). The diet contained 0.5% cholic acid, which is required for cholesterol absorption in the mouse.20 Aortas (5 to 7 from each genotype) were sampled for lesion formation at baseline (age 6 to 7 weeks, immediately before cholesterol feeding) and after 2, 4, and 6 months on the atherogenic diet. In a parallel set of experiments, ER–/– (n=15) and ER+/+ littermate control (n=15) male mice were surgically castrated by bilateral orchiectomy at baseline before initiation of the atherogenic diet. Aortas (5) were sampled at the experimental time intervals noted above.
Evaluation of Aortic Atherosclerosis
To determine extent of atheromatous lesions in aortas and regional differences in atherosclerosis susceptibility, we adapted and optimized previously published and reproducible methods for atheroma quantification.21 (For details please see Figure IA through ID, available online at http://atvb.ahajournals.org). At the specified experimental time intervals, aortas were excised intact, fixed, and cut into 3 segments (proximal segment, aortic arch; midsegment, suprarenal aorta; distal segment, infrarenal aorta proximal to the iliac bifurcation). Cryosections (10-μm thick) simultaneously sampled all 3 segments of the aorta, permitting extensive sampling of each (108 sections per aorta, 36 sections per segment). A total of 1000 μm from each aorta and 360 μm from each segment were analyzed.
Oil red O-stained (Sigma) cryosections defined and characterized neutral lipid in the vessel wall, permitting histological assessment and quantitative evaluation of lesions in artery segments. Sections were analyzed for lesion location, quantity, and area by direct imaging (Olympus BX-40 microscope, Olympus DP11 camera). Mean lesion size (μm2±SEM) for each atheroma was determined by morphometric analysis with a computer-assisted imaging system (Image Pro Plus, version 4.1). Qualitative morphological assessment of lesion complexity was performed by light microscopy. Parameters were: absence or presence of foam cells, wall thickness, disruption of vessel wall architecture, nuclear proliferation, and lumenal extension.
Quantification of Plasma Lipids
Plasma lipid levels were measured in study mice at baseline and at 2, 4, and 6 months on the atherogenic diet to determine whether lesion formation was related to changes in lipids. Lipids were assayed as follows: total cholesterol (TC) using a GM7 Analox analyzer (Analox), triglyceride (TG) with triglyceride reagent (Sigma), and high-density lipoprotein (HDL)-cholesterol (HDL-C) with a colororimetric reagent kit (Wako Biochemicals) after precipitating the apolipoprotein B (apoB)-containing particles. All assays were performed in duplicate on nonpooled plasma samples.
Plasma Hormone Analysis
The impact of circulating estrogen and testosterone on lesion development was determined by assessment of free testosterone and 17?-estradiol plasma levels in study mice at baseline and at 2, 4, and 6 months on the atherogenic diet. Assays were performed in duplicate by enzyme immunoassay (Cayman Chemical) on nonpooled plasma samples.
Statistical Analysis
Values are reported as mean±SEM. Comparisons between ER–/– and ER+/+ mice were made using Student t test for independent samples (2-tailed) and ANOVA using Microsoft Office 2000 Excel and Sigma Stat version 2.03 (SPSS). Correlations between parameters were analyzed using simple linear regression. Probability values <0.05 identified all statistically significant comparisons.
Results
Atheroma Development Was Greater and Faster in ER +/+ Than in ER –/– Male Mice
The atherogenic diet resulted in aortic lesions after 10 to 16 weeks, consistent with previously published reports for atherosclerosis susceptible mouse strains.19 Representative lesions of male ER–/– and ER+/+ mice at each of the time points are shown in Figure 1. Early atherosclerotic lesions were present in the intimal layer of aortas of both groups after 2 months of cholesterol feeding, with more robust lesions developing over time. Atheromatous lesions were distinguished by lipid infiltration into the vessel wall, the presence of fatty streaks, and occasional raised intimal lesions. With time, lesions were more advanced than simple fatty streaks, having deposits of intracellular fat (lipid lakes; eg, ER+/+ at 4 months), and contained large numbers of foam cells (eg, ER+/+ at 6 months) but were not complicated lesions. Compared with ER–/– mice, atherosclerotic lesions in ER+/+ mice were more advanced, larger, had greater cellular architecture disruption and cellular disarray, and greater proliferation of nuclei in the intima. Over the entire 6-month study period, lesion area in male ER+/+ mice averaged 3.8-fold larger than ER–/– mice (P<0.05).
Figure 1. Representative images (magnification x20) of oil red O-stained cross-sections (10 μm) of proximal segments of aortic lesions from cholesterol-fed ER–/– (left) and ER+/+ (right) male mice demonstrating atheroma formation over time (2, 4, and 6 months). A normal vessel before the initiation of cholesterol feeding and baseline is shown for comparison. Cross-sections are oriented with the lumen to the right and the adventitial surface to the left of each image. Size markers=100 μm.
Atheroma Number and Distribution Is Greater in ER+/+ Than in ER–/– Male Mice
We characterized atheroma by quantity, rate of progression, and segment location. Over the entire period of cholesterol feeding, aortas of ER+/+ mice averaged twice the number of atheromatous lesions (24±8) than aortas of male ER–/– mice (12±6). Because of variability in lesion number, this did not reach statistical significance. Atheroma quantity was initially low but increased steadily over time in both groups of mice. However, ER+/+ mice developed lesions at nearly twice the rate of ER–/– mice (slope for mean lesion number/time=11.5 for ER+/+ and 5.1 for ER–/– mice, respectively; P<0.05; Figure 2A).
Figure 2. A, Rate of lesion development over time in aortas of ER–/– and ER+/+ mice. The total number of lesions in the aortas of ER–/– (n=22) and ER+/+ male mice (n=24) were measured at baseline, 2, 4, and 6 months of cholesterol feeding (n=5 to 7 mice/genotype/time point). Rate of lesion development=slope of number of lesions/time. B, Atheroma distribution in aortic segments. Total mean lesion area (μm2) for atheroma in proximal, mid, and distal aortic segments of male ER–/– (n=60 segments) and ER+/+ (n=72 segments) mice (n=5 to 7 mice/genotype/time point).
Distribution of atheroma in aortic segments of ER+/+ (n=72 segments) and ER–/–mice (n=60 segments) is shown in Figure 2B. When averaged over the period of atherogenic diet, there were very few or no atheroma in the distal aortas of ER+/+ and ER–/– mice (1% and 3%, respectively). The proximal aorta was the primary lesion predilection site for atheroma formation in ER–/– mice (80% of atheroma), with only 17% in the midsegment. The proximal aorta displayed greater lipid deposition, larger lesions, and more lesions than other segments. However, in ER+/+ mice, atheroma were distributed more extensively throughout the aorta and more evenly between proximal and midsegments (49% and 50% of atheroma, respectively). Lesion area in the proximal and midsegments of ER+/+ mice was 2.3- and 11.3-fold larger, respectively (P<0.05), than corresponding segments in ER–/– mice.
ER-Mediated Atheroma Susceptibility in Male Mice Is Independent of Lipoprotein Serum Levels
The Table summarizes plasma lipids (TC, TG, and HDL) in ER–/– and ER+/+ mice over the atherogenic diet time period. After 6 months on the diet, fasting TC levels increased nearly 50% in ER mice compared with baseline values (P<0.05). However, the level of hypercholesterolemia attained by ER–/– and ER+/+ mice on the atherogenic diet correlated poorly with observed differences in lesion formation in the 2 mouse groups (r2=0.16 and 0.5, respectively; data not shown). There were no significant differences in baseline TG levels between ER–/– and ER+/+ mice. While on the atherogenic diet, fasting levels of plasma TG dropped by 59% in both groups of mice compared with baseline values (P<0.05). However, changes in TG levels did not correlate with lesion formation in either group (r0.01; data not shown). Fasting levels of HDL-C were similar at baseline in both groups, with no significant change in HDL-C levels on the atherogenic diet in either group compared with baseline.
Plasma Lipids in ER–/– and ER+/+ Male Mice
ER-Mediated Atheroma Susceptibility in Male Mice Is Independent of Serum Hormone Levels
No significant differences were observed between ER–/– and ER+/+ mice in mean levels of serum 17?-estradiol (18±2 pg/mL and 19±2 pg/mL , respectively) or serum testosterone (2689±231 pg/mL and 2978±227 pg/mL , respectively) during the study period. We found no correlation between lesion area and plasma levels of either hormone (r0.01; data not shown).
Effect of Castration on Lesion Development
To understand the mechanism underlying the differences in atheroma susceptibility between ER+/+ and ER–/– mice, we studied atheroma in castrated ER+/+ and ER–/– mice. Castration eliminated differences in atheroma susceptibility previously observed between ER+/+ and ER–/– mice. Castration resulted in a dramatic reduction (P<0.01) in atheroma in both ER+/+ and ER–/– mice (91% and 96%, respectively; Figure 3A). In addition, compared with intact mice of the same genotype, castration resulted in a reduction in the number of lesions in ER+/+ mice and ER–/– mice (by 33% and 83% , respectively). There were no differences in the atheroma distribution pattern of intact and castrated ER+/+ mice and ER–/– mice (data not shown).
Figure 3. Atheroma and hormone levels in castrated and intact ER–/– (KO) and ER+/+ (WT) male mice. A, Time-averaged aortic lesion area (μm2) in intact ER–/– (n=22) and ER+/+ (n=24) and castrated ER–/– (n=15) and ER+/+ (n=15) male mice at baseline, 2, and 4 to 6 months of cholesterol feeding (n=5 to 7 aortas/genotype/time point for intact mice; n=5 aortas/genotype/time point for castrated mice). *The bars in A denote statistically significant (P<0.05) differences between groups. B, Time average mean plasma testosterone levels. C, Time average mean plasma estradiol levels. See text for details of B and C.
As expected, after castration, mean time-averaged plasma testosterone levels declined significantly (P<0.001) in both ER–/– and ER+/+ mice to 144±18 pg/mL (n=5) and 218±40 pg/mL (n=5), respectively (Figure 3B). Concomitantly, mean time-averaged plasma 17?-estradiol levels also declined significantly (P<0.001) in both ER–/– and ER+/+ mice to 3±1 pg/mL (n=5) and 7±2 pg/mL (n=5), respectively (Figure 3C). As with intact mice, serum testosterone and 17?-estradiol did not significantly differ between castrated ER–/– and ER+/+ mice.
Demonstration of P450 Aromatase Expression in Aortas of ER–/– and ER+/+ mice
We considered the possibility of local estrogen synthesis from testosterone in vessels of ER+/+ and ER–/– male mice. P450 aromatase is the key enzyme regulating conversion of testosterone to physiologically active estrogens in males.22,23 To determine whether P450 aromatase was present in aortas of ER males, we performed immunocytochemical studies to evaluate P450 antigen expression in normal aortas of ER+/+ and ER–/– male mice using standard immunohistochemical techniques and a well-studied polyclonal antibody to P450 aromatase (kindly provided by Dr. N. Harada, Fujita Health University, Japan). (Please see www. ahajournals.org for details.) Figure 4 demonstrates P450 aromatase immunostaining in all layers of the vessel wall in aortas of ER males.
Figure 4. Immunostaining of P450 aromatase in aortas of ER–/– and ER+/+ male mice. A, Positive control tissue demonstrating P450 aromatase in pig testicular stroma (brown immunostaining, arrow). B, Comparison of negative control (deletion of primary antibody) in ER mouse aorta. C and D, Positive immunostaining for P450 aromatase in normal male ER –/– and ER +/+ aorta, respectively.
Discussion
The ER is implicated in a diverse array of physiological functions and plays a pivotal role in vascular biology. To test the role of ER gene expression on atheroma in males, we developed a model of early atherosclerosis in male mice lacking ER. Our data provide previously undocumented direct in vivo evidence of ER-mediated regulation of susceptibility to early atherogenesis in males.
The atheroprotective effect of estradiol in mice with targeted inactivation of the low-density lipoprotein (LDL) receptor24 and apoE25,26 has been demonstrated. In these models, under certain conditions, mice spontaneously develop advanced atherosclerosis. In contrast, we have developed a model of early atherosclerosis in cholesterol-fed ER male mice, permitting the study of fatty streaks, foam cell formation, and early lesion progression. The development of fatty streaks and foam cells, the earliest stage of atherosclerosis, is biologically important because it can lead to formation of more advanced lesions that characterize clinical events in humans.
The pathology of vessel wall architecture and lesion morphology in our system was robust and comparable with that reported previously for atherosclerosis-susceptible mouse strains.21 The gross morphology of lesions reported here resembles early lesions in fat-fed C57BL/6 mice, other model species, and humans. Studying lesions in the proximal, mid, and distal aorta of ER–/– and ER+/+ mice provides a better estimate of relative changes in regional susceptibility to lesion development than previous estimates recording lesions in the aortic free wall or at the base of the aortic sinus.21,27 Thus, our results agree overall with previous reports in analogous systems of diet-induced atherosclerosis19,28 and are consistent with immature lesions observed in other animal species fed an atherogenic diet.29,30
Changes in age, body weight, and brown/white fat weight distribution did not appear to explain observed differences in atherosclerosis susceptibility between ER–/– and ER+/+ mice. Without cholesterol feeding, C57BL/6 mice do not develop atheroma as they age.19 In addition, because the ER–/– and ER+/+ mice were age matched, aging did not influence observed atheroma susceptibility differences. ER–/– and ER+/+ mice also maintained similar total body weights throughout the study, so weight does not explain observed differences. Brown adipose tissue weight is similar in ER–/– and ER+/+ males at all ages.31 Although white adipose weight differs between ER–/– and ER+/+ male mice, 31 these changes are observed between 270 and 360 days and thus occur in older mice than those studied here.
A relationship has been established previously between high cholesterol, low HDL-C, and susceptibility to aortic lesions in humans and mice. Therefore, we investigated a possible differentiating role for serum lipids in the atheroma differences observed in our study. In ER–/– and ER+/+ mice, lipid levels at baseline and after the atherogenic diet were consistent with those published previously for atherosclerosis susceptible mouse strains.19 However, the greater susceptibility to atherosclerosis we observed in male ER+/+ mice compared with ER–/– mice was independent of lipid changes, and therefore, presumably occurring at the artery wall level. There are numerous examples of vessel wall pathophysiological states that are independent of lipid effects, including alterations in vascular reactivity, vascular responses to injury, and vascular inflammation.32–34 Indeed, previous animal models of atherosclerosis have also demonstrated that atheroma may not be linked to degree of hyperlipidemia.19,21,35
We then considered the possible role of sex steroid hormones in observed differences for atheroma development between ER–/– and ER+/+ male mice. Although testosterone is the predominant sex steroid hormone in male mice, estrogen is also present, albeit at low levels. In males, peripheral production of estrogen occurs in a number of tissues36,37 and in vascular cells38 via enzymatic activity of P450 aromatase. We specifically assayed the levels of serum-free testosterone and 17?-estradiol in our study mice and found them to be in the expected range for male mice and to not differ significantly between ER–/– and ER+/+ mice. This suggested that the effects of ER on atherosclerosis susceptibility were independent of circulating levels of these 2 hormones. After castration, we observed the expected decline in serum testosterone in ER–/– and ER+/+ animals and a concomitant decline in serum 17?-estradiol, again without significant differences observed in plasma testosterone and 17?-estradiol levels between castrated ER–/– and ER+/+ animals. However, because castration eliminated the predilection for atheroma observed previously in ER+/+ animals, our data support the postulate that in early atherosclerosis, the actions of ER are mediated by testosterone at the level of the artery wall. Our findings differ from those in the LDL receptor-deficient mouse, in which testosterone appears to attenuate atherogenesis.39
Because ER binds estrogens but not androgens, we postulate that the greater susceptibility of ER+/+ mice for atheroma formation demonstrated in our studies may be mediated by in situ local conversion of testosterone to estrogen in vessel walls of mouse aortas. This would implicate a role for vascular aromatase in mice. Recent reports of aromatase activity in the vessel wall of humans support the feasibility of this mechanism.39,40 Indeed, we were able to immunolocalize P450 aromatase in aortas of ER+/+ male mice. We interpret these data to support the tenet that vascular P450 aromatase in aortas of ER–/– and ER +/+ male mice can participate in the local conversion of testosterone to estrogen in vessel walls. Thus, locally produced estrogens, serving as substrate for activation of the ER, could predispose ER+/+ males but not ER–/– males to atheroma. Future studies will need to investigate this potentially new mechanism of atheroma formation in ER males. Nonetheless, our discovery highlights a previously unidentified role for ER in male mice well beyond the reproductive axis.
Differences in susceptibility to atherosclerosis observed in ER–/– and ER+/+ mice could also result from receptor-mediated changes in regulatory processes associated with atherosclerosis, including ER modulation of biological responsiveness, activation of second messenger signaling pathways, and immuno-inflammatory components of atherogenesis.41 Further, ER-mediated atherosclerosis susceptibility in males may result from interactions of the ER with functional or genetic targets in vessel walls other than aromatase. The possibility that alternatively spliced variants of ER or ER? may play a role in ER-mediated atherosclerosis susceptibility in male mice also needs to be considered.
Regarding the biological relevance of our findings to human disease, 3 recent clinical association studies implicate ER deletion and ER variants in cardiovascular disease in men: (1) a single published case report of premature coronary heart disease and estrogen insensitivity in a young male lacking ER;42,43 (2) a common ER gene variant (c.454.397T>C) manifesting as higher myocardial infarction risk in a predominantly male cohort of the Framingham study;15 and (3) more complex atherosclerotic plaque pathology in men with an ESR1 variant.44 These reports provide the first clues that ER may be clinically significant in atherosclerosis in males, although the nature and direction of the association need to be better defined.
In summary, this is the first report to establish a direct mechanistic link between ER and susceptibility to early atherosclerosis in male mice, defining ER as an important gene mediating atheroma formation in males. The results of this study also discover a key role for ER in manifesting a previously unrecognized function in initiation and progression of early atherosclerotic lesions in male mice. These studies carry potentially significant implications for the selective targeting of ER in prevention and treatment of atherosclerotic disease, and for understanding molecular and cellular mechanisms underlying its role in atherogenesis.
Acknowledgments
This work was supported by National Institutes of Health K01-HL04142 award (A.V.). The authors acknowledge personnel at the Murine Targeted Genomics Laboratory of the University of California, Davis, Mouse Biology Program for assistance with rederivation and breeding of mice, especially Carla French, Renee Araiza, and Pollyanna Mraz; Michael Lee and Shannon Smith for technical assistance; Christina Lannen for administrative assistance with manuscript preparation, and Dr. Beverly Paigen for advice regarding atheroma quantification methodology.
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