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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1476-1483

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1476-1483.)
© 1999 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Estrogen-Mediated Increases in LDL Cholesterol and Foam Cell–Containing Lesions in Human ApoB100xCETP Transgenic Mice

Steven H. Zuckerman; Glenn F. Evans; Judi A. Schelm; Patrick I. Eacho; George Sandusky

From the Divisions of Cardiovascular Research (S.H.Z., G.F.E., J.A.S., P.I.E.) and Research Technology & Proteins (G.S.), Lilly Research Labs, DC0434, Indianapolis, Ind 46285.

Correspondence to Steven H. Zuckerman, Division of Cardiovascular Research, Lilly Research Labs, DC0434, Indianapolis, IN 46285. E-mail Zuckerman_Steven{at}Lilly.com


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—The murine double transgenic mouse expressing both human apoB100 and cholesteryl ester transfer protein (CETP), has been used as a model to understand the effects mediated by various therapeutic modalities on serum lipoproteins and on atherosclerotic lesion progression. In the present study the effects of estrogen therapy on serum lipoproteins were investigated after mice were placed on an atherosclerotic diet. The daily oral administration of 20 or 100 µg/kg of 17 {alpha}-ethinyl estradiol resulted in a significant, dose-dependent increase in LDL cholesterol over a 20-week regimen. These differences were apparent by 6 weeks and further increases were observed through the 20-week period. Although CETP did result in a reduction in total HDL, estrogen did not have any impact on the amount of CETP activity associated with the HDL particles. The significant increase in LDL cholesterol was associated with increases in the amount of apoB100 and B48 and apoE–containing particles. Hepatic apoB message levels, however, were not different between the experimental groups. Although the extent of atherosclerotic lesions was modest, <0.5% of the aortic surface area in the vehicle group, the high-dose estrogen group, showed an increase in lesion area consistent with the elevation in LDL cholesterol. These lesions, primarily restricted to the aortic root and aortic semilunar valves, were more intensely stained with Oil Red O in the high-dose estrogen group when compared with the vehicle controls.


Key Words: estrogen • apoproteins • cholesterol • lipoproteins • transgenic


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Estrogen replacement therapy has proven to have beneficial effects in reducing the incidence of cardiovascular disease and is associated with a more favorable lipoprotein profile in postmenopausal women.1 2 3 4 Estrogen therapy has been demonstrated to lower serum LDL cholesterol, lipoprotein (a), to increase HDL cholesterol, and to have cardioprotective properties that were independent of serum lipoprotein modulation including effects on endothelial cell function, lipoprotein oxidation, cytokine levels and on hemostasis.5 6 7 8 9 10 These cellular responses to exogenous estrogen are consistent with the efficacy of estrogens in preclinical models of atherosclerosis, including the cholesterol-fed rabbit11 and the apoE knockout mouse.12

In contrast to the beneficial effects of exogenous estrogens in murine apoE KO and apoE2 transgenic animals,13 high-dose estrogen increases apoB and VLDL synthesis in rats14 15 and increases total cholesterol in some mouse strains,16 17 while decreasing cholesterol in others.18 In the latter 2 studies, chronic estrogen treatment resulted in a shift in cholesterol from HDL to a significant increase in LDL cholesterol. The shift in LDL cholesterol observed in some normal mouse strains is associated with an increase in apoB and occurs at a posttranscriptional level.19 Clearly, a greater understanding of the role of selective estrogen agonists in modulating the lipid and vascular events associated with atherosclerosis is necessary as these classes of compounds are increasingly used in the clinic.

Murine transgenic models expressing human genes involved in lipoprotein metabolism have increasingly served as small mammalian models where the spectra of both normal and pathologic human serum lipid profiles can be simulated, and in several instances have demonstrated the formation of atherosclerotic lesions.20 The double transgenic model incorporating hemizygous expression of human apoB100 and cholesteryl ester transfer protein (CETP) represents 1 such model where decreased HDL and increased VLDL+LDL cholesterol reflects a murine counterpart of familial combined hyperlipidemia.21 22 In an attempt to characterize the effects of estrogen therapy on the lipoprotein and vascular changes in this model, mice were shifted to an atherogenic diet and were dosed orally with 17 {alpha}-ethinyl estradiol at 20 or 100 µg/kg daily for 20 weeks. Chronic estrogen administration resulted in a significant dose-dependent increase in total cholesterol with the increase primarily in LDL cholesterol. Concomitant with these changes were increases in both apoB and apoE levels by Western blot analysis.

However, although further increases in serum cholesterol were obtained with estrogen administration, atherosclerotic lesions after 20 weeks were small, focal, and of limited distribution within the aortic root. An increase in Oil Red O staining was detected in the high-dose estrogen group and was observed in lesions within the aortic root and the aortic semilunar valves. This model offers a different pharmacologic and vascular profile than that demonstrated with the apoE knockout for the evaluation of novel therapeutics targeted toward the management of atherosclerosis.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Mouse Model
Double transgenic female mice expressing both human CETP and apoB100 were obtained from Taconic Labs (Germantown, NY). These mice are hemizygous for both transgenes and are on a mixed (C57BL/6xSJL) background. At 6 weeks of age, 18 to 20 g weight, mice were shifted to an atherogenic diet consisting of 0.5% cholic acid, 1.0% cholesterol, and 18% fat (Harlan Teklad).23 Concomitant with the diet shift from normal laboratory chow to the atherogenic one, mice were randomly divided into 3 experimental groups and were dosed orally on a 7-day-per-week protocol throughout the duration of the study with 17 {alpha}-ethinyl estradiol at 100 or 20 µg/kg or the vehicle alone, 1% carboxymethyl cellulose with 0.25% Tween 80. Retro-orbital bleeds were performed after 6, 8, and 12 weeks on diet and mice were euthanized at 20 weeks. In comparative studies involving fast protein liquid chromatogram (FPLC), apoB100 transgenic mice were used to compare the FPLC profile with B100 transgenic mice expressing CETP. All animal experiments were performed in accordance with institutional guidelines.

Serum Lipoprotein Analysis
Sera from each group at the designated time points were pooled and a 200-ul aliquot was resolved by FPLC (Pharmacia) using tandem-linked Superose 6 columns as previously described.24 Total cholesterol was quantitated enzymatically (Wako Chemicals USA) from the individual sera as well as from 100-ul aliquots of the FPLC fractions. The relative amount of cholesterol within each peak was determined by area quantitation under the curve using the appropriate baseline modifications from the FPLC cholesterol tracings. Serum triglyceride levels were measured by enzymatic assay (Sigma).

Electrophoresis and Immunoblot Analysis of FPLC Fractions
Sequential fractions from the VLDL, LDL, and HDL peaks were subjected to denaturing 8% gradient gels (apoB and apoE analysis), 8% to 16% gradient gels (apoA1) or 4% to 12% gradient gels to distinguish B100 from B48. After electrophoresis, gels were stained with Coomassie blue and parallel gels were electroblotted at 2.5 mA/cm2 for 40 minutes onto nitrocellulose using semi-wet blotting conditions (ABN Polyblot, American Bionetics, Inc.). Nitrocellulose blots were probed with antisera against mouse apoA1 (rabbit polyclonal, Biodesign), human apoB, (mouse monoclonal, Calbiochem) and apoE (goat polyclonal, Chemicon). These antisera were determined in preliminary experiments to cross-react with the mouse apoproteins. Detection of immunoreactive bands was by chemiluminescence using Pierce Supersignal Ultra (Pierce), with the appropriate peroxidase-conjugated secondary antibody (Sigma) and the procedure described by the suppliers. Film (Hyperfilm-ECL, Amersham) was exposed to the chemiluminescent signal from 3 seconds to 5 minutes depending on the primary antibody used and the resulting band intensity.

Cholesteryl Ester Transfer Protein Assay
CETP activity was quantitated on sera as well as on the FPLC fractions from the corresponding HDL regions as described previously,24 using [3H]cholesteryl ester-labeled HDL (Amersham), and was based on minor modifications of the transfer assay described by Tall et al.25 The percentage transfer of [3H]cholesteryl ester to the LDL acceptor was determined by heparin-manganese chloride precipitation of the LDL fraction and quantitation of counts remaining with the HDL supernatant.

Lesion Analysis
At sacrifice, animals were prepared for en face aorta evaluation by opening the body and cutting the aorta below the bifurcation in the lower abdomen. The aorta was perfused with PBS from the heart down through the bifurcation. The heart was then removed, cutting the aorta just above the heart, flushed and frozen immediately in OCT Compound embedding medium (Miles Laboratories) using liquid nitrogen. Hearts were sectioned from the aortic leaflets through the aortic root with serial sections being microtomed every 10 µm. Three to 4 serial sections were placed on a single glass slide and all slides were stained with either an Elastic Van Gieson's stain or with Oil Red O. Aortas in parallel were cleaned of external fat, opened, flattened out on a microscope slide and covered with a coverglass. The tissue was fixed in 4% formalin and kept at 4°C in humidified chambers until imaging. Samples were visualized using a Nikon SMZ-10 stereo microscope fitted with a COHU high performance CCD camera26 connected to a Power MacIntosh 7500/100 computer. Images of the aortas were captured using NIH Image v1.62b7 and analyzed using a custom-written macro (Mike Esterman, Lilly Research Labs). The macro uses the threshold tool in NIH Image to select the lesion sites based on their image intensity, which is in contrast to the normal translucent arterial wall. Lesion area and total aortic area were expressed in mm2. Approximately 45 mm2 of total aortic surface were evaluated per aorta for lesion quantitation.

SemiQuantitative Reverse Transcriptase PCR
RNA was isolated from frozen liver using a Clontech Micro-Scale Total RNA separator kit (Clontech Laboratories). Frozen livers were pooled from >=3 animals per group and homogenized directly in the guanidinium lysing solution using a Virtis homogenizer (Virtis Co) at a speed setting of 60 for 1 minute. RNA was isolated by the alcohol precipitation and phenol chloroform extraction procedures as detailed in the Clontech kit. cDNA was synthesized from the total RNA using a Superscript Preamplification System kit (GIBCO/BRL, Life Technologies, Inc). PCR was performed using a thermocycler (MJ Research) and Advantage cDNA Polymerase Mix (which contains KlenTaq-1 polymerase, an additional proofreading DNA polymerase, and TaqStart antibody (Clontech). Semiquantitative PCR comparisons were made by first determining the number of cycles where a product could be visualized that was in the linear phase of the amplification reaction. Amplimers for both the apolipoprotein and control G3PDH were added together to enable apolipoprotein normalization with G3PDH. Due to the abundance of G3PDH message, tubes were run with the apolipoprotein amplimers for 2 to 5 cycles, and then the G3PDH amplimers were added and the reaction run for an additional 16 cycles. To minimize the amount of normalization needed, all cDNAs were adjusted to the same OD260 concentration before PCR analysis. PCR products were separated on 1.75% agarose gels stained with ethidium bromide. Gels were imaged with a Gel Doc 1000 (Bio-Rad) that captured the gel image to the computer using a CCD camera. Bands were quantitated using the Multi-analyst/PC image analysis software (Bio-Rad). Amplimers used to detect apolipoprotein sequences included mouse apoA127 and both mouse and human apoB100 (see below). The G3PDH amplimers were from Clontech.

Mouse ApoB100
5'-AAGCTCAATTCCTGGAGTTAAATCC (sense)

5'-GTCATTTCTGCCTTTGCGTCCTTG (antisense)

Human ApoB100
5'-ATGCACAACTCTCAAACCCTAAGAT (sense)

5'-GTAAACTCTGCCTTCCCTTCTCCA (antisense)

PCR products of 623 bp for the apoB amplimers, 680 for the apoA1, and 983 for murine G3PDH were predicted. Preliminary experiments with the human apoB amplimers demonstrated that they did not react with mouse apoB. Amplification of cDNA from livers of apoE knockout mice with these amplimers failed to detect any PCR products. Likewise, the mouse apoB amplimers gave no PCR product from human liver cDNA.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
The wild type, inbred strains of mice exhibit a well-characterized serum lipoprotein profile in which cholesterol is transported almost exclusively on HDL particles with {approx}85% of the total serum cholesterol within the HDL peak (Figure 1ADown). In agreement with a previous report,22 transgenic mice expressing the human apoB transgene displayed a significant increase in LDL cholesterol with {approx}50% of the cholesterol now in the LDL peak (Figure 1BDown). Transgenic mice that were hemizygous for both human apoB100 and CETP when compared with the B100 single transgenic demonstrated a further shift in the relative distribution of cholesterol across the HDL and LDL peaks with a reduction of the HDL cholesterol peak to 31% as compared with the single B100 transgenic of 48% in the HDL fraction (Figure 1CDown). The ratio of cholesterol transported on LDL particles when compared with HDL had now shifted toward LDL being the major carrier of serum cholesterol in the B100xCETP transgenic mice. These data, in agreement with earlier reports,22 suggest that expression of the human CETP transgene in these mice did result in a reduction in HDL cholesterol with a concomitant increase primarily in LDL cholesterol.



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  		Figure 1. FPLC serum lipoprotein profile on control mice. Sera from 3 to 6 mice per group on laboratory chow were pooled and 200 ul separated on FPLC. Fractions of 0.5 mL were collected and 100-ul aliquots were assayed for total cholesterol. C57BL/6 mice (A) were used as a wild type control and compared with the mice transgenic for human apoB100 (B) or double transgenic for human apoB100 and CETP (C). Note reduction in the HDL peak in panel C.

In view of the protective effects of estrogen in nonmurine atherosclerotic models, B100xCETP transgenic mice were shifted to an atherogenic diet containing cholic acid and were dosed with vehicle or 17 {alpha}-ethinyl estradiol at 20 or 100 µg/kg. These doses have been demonstrated to reduce serum cholesterol in rats within 5 to 10 days.28 A preliminary assessment of the sera from the control and experimental groups was performed 6 weeks into the dosing regimen (Table 1Down). These results, when compared with litter mates that had not been exposed to the atherogenic diet (T=0), demonstrated increases in serum cholesterol with the most significant elevation in the high-dose estrogen group. These increases in serum cholesterol occurred without any significant differences in weight between the groups. Furthermore, most of the increase in serum cholesterol was due to the cholesterol associated with the LDL fraction. CETP activity between the 3 groups was not significantly different, although lower than the nonatherogenic diet controls. Finally, serum triglycerides were not significantly different between the 3 groups.


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  		Table 1. Analysis of B100-CETP Transgenic Mice at 0 and 6 Weeks Into Study

In an attempt to further characterize the dose-dependent increases in LDL cholesterol, mice were bled after 8, 12, and 20 weeks on diet and pooled sera were again analyzed by FPLC. As demonstrated (Figure 2ADown), estradiol treatment resulted in a dose-dependent increase in serum cholesterol with the increase occurring primarily in the LDL peak. The effects of chronic estrogen administration seem to exacerbate this increase in LDL cholesterol, as the differences in LDL cholesterol relative to the vehicle control were further increased by 12 weeks (Figure 2BDown) and were still increasing at 20 weeks (Figure 2CDown). The percentage of total cholesterol in the LDL peak in the high-dose estrogen group was 87% at 8 weeks and 96% at euthanasia. In contrast, the vehicle group had 66% of its cholesterol in the LDL peak at 8 weeks and 71% by 20 weeks on diet. In addition, the 100-µg/kg dose of estradiol resulted in a modest shift in the HDL region to larger particles, which was most apparent at the 8- and 12-week time points.



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  		Figure 2. Effects of chronic estrogen administration on serum lipoprotein profiles. B100xCETP double transgenic mice were shifted to the modified Paigen diet and dosed orally with vehicle, 17{alpha}-ethinyl estradiol at 20 (Est20) or 100 (Est100) µg/kg and at 8 (A), 12 (B), or 20 (C) weeks mice were bled, sera pooled and 200 ul analyzed by FPLC as described in the legend to Figure 1Up. Note the progressive and dose-dependent increase in LDL cholesterol with estrogen treatment.

The increases in LDL cholesterol were associated with similar changes in the apolipoproteins. The most significant changes were observed at the 20-week time point when comparing the vehicle with the high-dose estrogen group. There was a significant increase in apoB within the LDL fraction (Figure 3ADown). This effect was less apparent at the lower estrogen dose or at the earlier time points. A similar increase was also observed in apoE, although these increases extended beyond the LDL fractions (Figure 3BDown). These apolipoprotein increases were not apparent, however, within the HDL particles where a decrease in A1 between the high-dose estrogen and vehicle groups was observed (Figure 3CDown). Finally, despite the reduction in total serum CETP activity at the high-dose estrogen group (Table 2Down), quantitation of CETP activity across the HDL fractions at both the 12- and 20-week time points revealed no significant differences in the amount of CETP within any of the 3 groups (data not shown).



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  		Figure 3. Detection of apoproteins on FPLC fractions. Fractions from the 20-week time point for the estrogen 100 µg/kg and vehicle groups were run on 8% gels (apoB and apoE) or 8% to 16% gradient gels (apoA1) and electroblotted. Apoproteins were detected by Western blot analysis and film exposures were within the linear range for band intensity. Note the increase in apoB in the LDL fractions with estrogen treatment compared with the vehicle (A). A similar result is seen with apoE (B) although the apoE was distributed over the LDL and VLDL fractions. In contrast, apoA1 was present in the HDL fractions in a reduced amount with estrogen treatment (C).


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  		Table 2. Analysis of B100xCETP Mice at 20 Weeks

Analysis of the sera from these animals at sacrifice revealed that while serum cholesterol had not changed significantly beyond the elevation observed at 6 weeks in the vehicle group, the estrogen groups demonstrated further increases in total serum cholesterol (Table 2Up). Chronic estrogen treatment did result in a dose-dependent reduction in body weight, which first became apparent by 10 weeks, and a decrease in serum CETP activity in the high-dose estrogen group. Consistent with the data at the 6-week time point, at the time of sacrifice, the major portion of the increase in serum cholesterol was associated with the LDL fraction. These changes in serum cholesterol occurred without a comparable change in serum triglyceride levels. There was only a modest increase observed at the highest estrogen group, which was statistically significant.

The increases in apoB with estrogen treatment were apparent for both B100 and B48 species when the peak LDL fractions were analyzed by denaturing 4% to 12% gradient gels (Figure 4Down). However, though increases in B100 and B48 were detected within the LDL fraction and were consistent with the increase in LDL cholesterol, RT-PCR did not reveal any significant changes in apoB message (Figure 5Down). There were no significant increases in either murine apoB (lanes 4 to 6) or human apoB (lanes 7 to 9) with estrogen treatment. Although the vehicle group had an increase in apoA1 PCR amplified product compared with the estrogen groups (lanes 1 to 3), when normalized to the upper G3PDH band this increase was not specific for A1. Therefore, the changes observed on Western blots for the apoproteins in the absence of RT-PCR changes would suggest posttranscriptional regulation.



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  		Figure 4. Estrogen treatment results in increased expression of apoB100 and B48. Peak FPLC fractions from the LDL region at the 20-week time point were run on denaturing 4% to 12% gradient gels and Coomassie blue stained. Note the progressive increase in both B48 and B100 when comparing the vehicle (lane 1) with the 20- (lane 2) and 100-µg/kg (lane 3) doses of estrogen.



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  		Figure 5. Reverse transcriptase PCR analysis of apoA1 and apoB hepatic mRNA levels with estrogen treatment. Total RNA was prepared from livers at the 20-week time point, with 3 to 4 livers per group pooled. Primers for human and mouse apoB and mouse A1 were chosen to permit amplification of a PCR product that could be resolved from the G3PDH internal control and coamplified in the same tubes. PCR products were analyzed on 1.75% agarose gels. Vehicle: lanes 1, 4, 7; Est20: lanes 2, 5, 8; Est100: lanes 3, 6, 9. The larger band is the mouse G3PDH, and the smaller bands are the apoproteins.

Histologic analysis of the hearts was then performed to determine whether the changes in serum lipoproteins were associated with any significant differences in the pathology of the atheromatous lesions detected within the aortic root. The hearts of all 3 groups were within normal limits and no evidence of chronic inflammation was seen. There were no major differences in the atherosclerotic plaque lesions between the groups, and these lesions were confined to the aortic root and the aortic semilunar valves. These lesions were not seen in the main branches of the coronaries, nor were lesions seen in the small branches in the coronary arteries in the myocardium. The main lesions around the aortic root and semilunar valves ranged from minimal to intermediate lesions, a mixture of a few to several layers of spindle-shaped cells and foam cells in the control mice, to minimal to intermediate plaques with a few small necrotic cores and a few foam cells covered with a fibrous cap in the estrogen-treated mice. Cholesterol clefts were seen in some of these lesions. Partial destruction of underlying medial smooth muscle cells was observed in a few of the intermediate lesions in the aortic wall. Chronic inflammation was not seen around these lesions; nor was evidence of hemorrhage or plaque rupture observed (Figure 6Down). The Oil Red O staining in the control groups was minimal. The lesions in the estrogen-treated groups were more intensely stained with Oil Red O when compared with similar lesions in the control animals.



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  		Figure 6. Proximal aortic root sections. Hearts and the aortic root were flash-frozen and 10-µm sections prepared. A, An EVG stain on vehicle-treated animals after 20 weeks on diet. B, The Oil Red O stain on an adjacent section. C represents the EVG stain after 20 weeks for the 100-µg/kg estradiol group, and D, the Oil Red O staining. Note the increased Oil Red O staining seen in the 100-µg/kg estradiol group compared with the control group.

En face analysis of the total aortas was consistent with the lesion analysis at the aortic root. Lesions were small and focal in the vehicle group and occupied <0.5% of the total surface area (Figure 7Down). There was a dose-dependent trend toward an increase in surface area occupied with lesions with estrogen treatment, although, due to the small number of animals available (n=4 to 6 in the 2 estrogen groups), this difference was only statistically significant in the high-dose estrogen group.



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  		Figure 7. Quantitation of aortic lesions by en face analysis. Lesion areas at 20 weeks were quantitated and expressed as mm2. Lesions were detected visually for 5/8 animals from the vehicle group, 3/6 from the 20-µg/kg estrogen dose, and from 3/4 of the 100-µg/kg estrogen group. Lesion area from the lesion-free animals was calculated as 0 and all animals were included in quantitating the amount of lesion involvement. The average total aortic area scanned was 48 mm2. The percentage of the aorta in which lesions could be detected was 0.2%, 0.5%, and 2.0% for the vehicle, low- and high-dose estrogen groups, respectively. The total lesion area in the high-dose estrogen group was significantly increased compared with the vehicle control (P<0.01) by a 2-tailed Student's t test. Brackets indicate standard deviation of the mean.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The development of murine transgenic models that simulate various aspects of atherosclerotic disease has provided significant information as to both genetic and nongenetic modifiers of the disease process.20 Transgenics expressing human genes involved in lipoprotein metabolism as well as mice in which targeted deletions have created functional knockouts of select genes have been established and are well characterized. The murine atherosclerosis model involving targeted disruption of the apoE gene has served as the most extensively studied of these transgenics with significant atherosclerotic lesion formation and exaggerated elevations in VLDL+LDL cholesterol observed.29 30 These animals have been crossed with other mutant strains including osteopetrotic mice31 to evaluate the role of the second mutation in modulating the atherogenic phenotype. Additional constructs, including the LDL receptor knockout32 and transgenics expressing human apoB100 alone33 or as the double hemizygous transgenic with apo(a)34 35 or CETP,22 have also been investigated, as the lipoprotein profiles in these animals, when the animals are placed on an atherosclerotic diet, display a more significant increase in LDL cholesterol similar to that observed in familial combined hyperlipidemia.

The use of these well-defined transgenic models permits a systematic evaluation of the changes at the cellular and molecular level that occur within the lesion, and offers a small animal model in which pharmacologic interventions can be evaluated during the preclinical assessment of compounds that proceed to clinical trials. Using this approach, for example, the effects of estrogen, probucol and the angiotensin II receptor antagonist Losartan12 32 36 have been evaluated in the apoE knockout, and with probucol, in the LDL receptor knockout.32 Whereas both Losartan and pharmacologic doses of 17ß-estradiol reduced lesion size in the apoE knockout, probucol actually increased lesion size in both of these models.12 32 36 37 Clearly, the mechanisms involved in the atheroprotective effects of these agents, including effects on vascular function as well as on lipoprotein oxidation and catabolism, remain to be defined. Estrogen, for example, was reported to decrease total serum cholesterol in both the apoE knockout12 19 and in nontransgenic mouse strains, yet in contrast to the rat did not result in an upregulation of hepatic LDL receptors.18 In a different model, downregulation of the hepatic LDL receptor at the transcriptional level was observed in B100xCETP transgenic mice when they were maintained on a Western style diet.38 This downregulation was reversed in vivo with the cytokine Oncostatin M.38

In the present study the effects of estrogen treatment were investigated in the B100xCETP transgenic mouse, as the lipoprotein profile was more similar to the human when compared with the apoE knockout. In contrast with this latter model, chronic estrogen administration actually induced an increase in total serum cholesterol resulting primarily from an elevation in LDL cholesterol. These effects were associated with increases in apoB100, B48, and apoE without any significant changes in total CETP activity or any transcriptional increase in apoB message. Furthermore, though lesions detected after 20 weeks on the modified Paigen diet were small and focal, Oil Red O staining revealed increased lipid accumulation within the lesions of the higher-dose estrogen group. Although these results appear contradictory to the previous study in apoE knockout mice,12 there are several fundamental differences in the experimental design, beyond the obvious genotypic and phenotypic ones when comparing these 2 models. These include differences in estrogen formulation (17ß-estradiol versus 17{alpha}-ethinyl estradiol), delivery (pellet implant versus oral gavage), diet (Western or laboratory chow versus a modified Paigen diet with cholic acid), and ovariectomy. Finally, Callow et al35 reported significant lesion formation in apoB100 transgenic mice characterized by high levels of transgene expression, whereas mice with lower levels of transgene expression had more modest lesions, which occupied <10% of the area of the high-expression lines. Whether the smaller lesions observed in the present study are due to lower levels of transgene expression remains to be determined.

As estrogen replacement therapy has been demonstrated to have cardioprotective effects in postmenopausal women,1 2 3 4 a greater understanding of its role in modulating the cardiovascular phenotype in both normal and transgenic mice will enable the development of more specific estrogen receptor modulators. Estrogen treatment over a 6-day period, for example, resulted in an increase in hepatic apoA4 mRNA levels concomitant with decreased hepatic apoA2 mRNA in responsive mouse strains.39 Increases in apoE and apoB have also been reported and are likely to occur at the hepatic level through posttranscriptional regulation.19 40 The shift in cholesterol distribution to the LDL fraction was apparent in certain strains of mice, ie, C3H/HeJ18 and in the MRL/lpr mouse, a model of systemic lupus erythematosus.17 Whereas the same dosing protocol also induced an increase in LDL cholesterol in both BALB/c and the wild type MRL/++ mice, the extent of increase was more modest. A similar increase in LDL cholesterol was also observed in the hyperlipidemic Zucker diabetic rat.15 These results suggest that underlying pathologies can significantly impact the beneficial effects of estrogen on serum lipoprotein profiles and presumably on plaque regression.

In summary, this study represents the first in which increases in LDL cholesterol were observed in a mouse transgenic line designed to simulate an atherosclerotic lipid profile. Furthermore, this also represents, to the best of our knowledge, the first study in which pharmacologic interventions in modulating serum lipoprotein levels and on lesion morphology have been attempted with this murine double transgenic model. The present results demonstrate that the modulating effects of estrogen therapy in well-characterized murine transgenic models of atherosclerosis are complex and not necessarly associated with reductions in serum cholesterol or lesion regression. The efficacy of estrogen treatment in these models can vary significantly, depending on the nature of the transgenes expressed.

It is of interest that although estrogen resulted in an increase in serum cholesterol of over 1000 mg/dL, at the time of sacrifice, atherosclerotic lesions were minimal and occupied only 2% of the total surface area of the aorta. This is in contrast to the apoE KO where similar serum cholesterol levels resulted in significant lesion accumulation throughout the aorta. The possibility exists that estrogen in this model had both negative (increased LDL cholesterol) and positive vascular effects, in reducing the amount of lesion one would have anticipated with this form of hypercholesterolemia. The extent of similarity in the pharmacologic profile of serum lipoproteins and lesion characteristics of more specific estrogen agonists, as well as that of the differences observed in the response of ovariectomized mice to these same agonists, represent questions that require further investigation.


*    Acknowledgments
 
The authors wish to acknowledge Drs Ray Kauffman and Jacques Mizrahi for their critical review of our manuscript and Mike Esterman for developing the software used for lesion quantitation.

Received August 13, 1998; accepted December 2, 1998.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Walsh BW, Schiff I, Rosner B, Greenberg L, Ravnikar V, Sacks FM. Effects of postmenopausal estrogen replacement on the concentrations and metabolism of plasma lipoproteins. N Engl J Med. 1991;325:1196–1204.[Abstract]
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