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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1473-1480.)
© 1996 American Heart Association, Inc.

Articles

Esterified Estrogens With and Without Methyltestosterone Decrease Arterial LDL Metabolism in Cynomolgus Monkeys

Janice D. Wagner; Li Zhang; J. Koudy Williams; Thomas C. Register; Dennis M. Ackerman; Brinda Wiita; Thomas B. Clarkson; Michael R. Adams

the Comparative Medicine Clinical Research Center (J.D.W., L.Z., J.K.W., T.C.R., T.B.C., M.R.A.), Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC, and Solvay Pharmaceuticals, Inc (D.M.A., B.W.), Marietta, Ga.

Correspondence to Janice D. Wagner, DVM, PhD, Department of Comparative Medicine, Bowman Gray School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157-1040. E-mail j wagner@cpm.bgsm.edu.

	Abstract

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Although both epidemiological and experimental evidence suggests that estrogen replacement therapy reduces the risk of coronary heart disease, the mechanisms for this beneficial effect are largely unknown. Furthermore, the addition of progestins or androgens to estrogen replacement therapy is of concern. The objective of this study was to examine the effects of esterified estrogens alone or in combination with an androgen on arterial LDL metabolism and early atherogenesis in ovariectomized female cynomolgus monkeys. Arterial LDL metabolism was assessed by using dual-labeled LDL that was injected 24 hours before necropsy. Arterial LDL degradation was reduced by 64% to 84% and cholesteryl ester content was decreased by 50% in the thoracic aorta in both treatment groups compared with controls. In addition, aortic lipid peroxidation products, as assessed by thiobarbituric acid reaction, were significantly lower in animals treated with esterified estrogens, with a similar trend for combined estrogen-androgen treatment. Both treatments also reduced plasma concentrations of apoB-containing lipoproteins, reduced LDL particle size, and increased total-body LDL catabolism. The combination of decreased arterial LDL metabolism, decreased arterial lipid peroxidation, and improved plasma lipoprotein metabolism may explain some of the protective effects of estrogens on coronary heart disease and indicate that beneficial actions extend to a combination of estrogen and androgen.

Key Words: estrogen • androgen • women's health • cardiovascular disease • menopause

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Estrogen replacement therapy is associated with a 50% reduction in risk of CHD in postmenopausal women.^{1 2} The mechanisms for this beneficial effect on CHD risk remain largely unexplained, since effects on plasma lipids account for <50% of the benefit.² While there is evidence that estrogen monotherapy markedly reduces CHD risk in postmenopausal women, the cardiovascular effects of estrogen-progestin or estrogen-androgen regimens are less clear. The use of estrogen-androgen combinations has gained favor in recent years because they appear to be as effective as other modalities in ameliorating or preventing the somatic complaints associated with estrogen deficiency and also improve multiple psychological symptoms, libido, and sexual satisfaction.^{3 4} However, the fact that some estrogen-androgen combinations may lower plasma HDL-C concentrations^{5 6} has led to concerns that they may increase cardiovascular risk, despite evidence that other HDL-C–lowering compounds (ie, oral contraceptives) do not increase cardiovascular risk.^{7 8 9}

At menopause, there is a decrease in estrogen production and androgen levels. The effect of androgen deficiency on cardiovascular risk is not understood. However, endogenous androgens have not been implicated as risk factors in men or women. In fact, men with coronary artery disease (as determined by angiography) have similar to slightly lower androgen levels than healthy men.¹⁰ Oral and systemic androgen replacement with testosterone in men may result in beneficial changes in CHD risk,¹¹ but supraphysiological doses suppress HDL-C.¹² Whereas oral androgens decrease HDL-C in women,^{5 6} androgens administered parenterally do not alter HDL-C.¹³ Thus, androgens, like progestins, may modulate estrogen-dependent cardiovascular protection, but the mechanism and extent of this modulation are as yet unknown.

Because the initiation and progression of coronary artery atherosclerosis and related cardiovascular diseases are exceedingly difficult to study prospectively in human subjects, for the present study we used a nonhuman primate model of atherosclerosis. In this model, the physiological replacement of estrogen or estrogen-progesterone inhibits the progression of coronary artery atherosclerosis while having little effect on plasma lipid concentrations.¹⁴ One mechanism for this beneficial effect on atherosclerosis progression may be the direct effects of estrogens at the level of the arterial wall. In support of this hypothesis, we have found^{15 16} that parenteral estrogen-progesterone replacement therapy reduces the uptake and accumulation of LDL by the arterial wall. These effects are associated with decreases in the size of plasma LDL particles but not with changes in plasma lipid concentrations. The goals of the current study were to further define the effects of estrogen therapy on indices of early atherogenesis and lipoprotein metabolism and to determine if and to what extent the addition of an androgen affects these end points.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Animal Studies
Thirty-six adult female cynomolgus monkeys (Macaca fascicularis) were imported from Indonesia and quarantined for 3 months, during which time they ate monkey chow (High Protein Monkey Chow, Ralston Purina Co). After the quarantine the monkeys were fed a moderately atherogenic diet containing 0.28 mg cholesterol/kcal¹⁴ for 1 month to assess the response of TPC, HDL-C, and TG concentrations to this diet. Monkeys were then randomized to one of three treatment groups by using a stratified randomization scheme to achieve similar plasma TPC and HDL-C concentrations and similar variability in response to dietary cholesterol. Monkeys were again fed monkey chow and ovariectomized 2 months before the beginning of the treatment phase, which consisted of an atherogenic diet containing (as percent of calories) 44% fat, 38% carbohydrate, 18% protein, and 0.28 mg cholesterol/kcal with or without drug treatment. From earlier results,^{9 15 16} a 16-week treatment phase was chosen to avoid expected differences in plaque extent with longer treatment that may affect arterial LDL metabolism. The three groups were no drug treatment (control), EE, which is a mixture of sulfate esters containing primarily sodium estrone sulfate and to a lesser extent sodium equilin sulfate (Estratab, Solvay Pharmaceuticals), and EE+MT (Estratest, Solvay Pharmaceuticals).

Monkeys were housed in pairs and trained to come to the front of the cage to drink a dosing solution from a syringe held by a laboratory technician. The solution contained vehicle, the appropriate treatment (none, EE, or EE+MT) and, to improve flavor, a commercial beverage mix (Crystal Lite) and sugar. The solutions were prepared so each animal in the EE group received 42 µg EE·kg body wt^-1·d^-1, and each animal in the EE+MT group received the same EE dose plus 84 µg MT·kg body wt^-1·d^-1. Estrogen doses were calculated to be equivalent to a 60-kg woman receiving 2.5 mg EE and 5.0 mg MT and were selected on the basis of data from a pilot pharmacokinetic study in ovariectomized cynomolgus monkeys and comparisons of animal and human pharmacokinetic data. The ratio of EE to MT that was used (1:2) is the same as that used clinically.^{5 6}

Ovariectomies and femoral catheterizations for LDL studies were done while the animals were anesthetized with ketamine hydrochloride (10 mg/kg IM) and butorphanol tartrate (0.05 mg/kg IM), and blood sampling was done while the animals were sedated with ketamine hydrochloride (15 mg/kg IM). All procedures involving animals were conducted in compliance with state and federal laws, standards of the Department of Health and Human Services, and guidelines established by the Institutional Animal Care and Use Committee.

Clinical Chemistry Measurements
Blood samples for plasma lipid and lipoprotein measurements were collected in tubes containing EDTA (final concentration, 1 mg/mL) after the animals were fasted for 18 hours. TPC and HDL-C were determined at week 4 of the dietary challenge and TPC, HDL-C, combined VLDL and IDL cholesterol, and TG concentrations were determined at weeks 0, 4, 8, 12, and 15 of the experimental period. TPC,¹⁷ HDL-C,¹⁸ and TG¹⁹ measurements were in full standardization with the Centers for Disease Control and the National Heart, Lung, and Blood Institute Lipid Standardization Program. Lipoproteins were fractionated by using ultracentrifugation and high-performance liquid chromatography,²⁰ and cholesterol distributions were determined enzymatically.²¹ Determinations of plasma apoA-I²² and apoB²³ and assessments of LDL size (LDL molecular weight) by column chromotography^{24 25} were done at 0, 4, 8, 12, and 15 weeks.

Determinations of plasma estrone, equilin, and MT concentrations were done by Phoenix International Life Sciences at the time of anticipated peak levels, ie, 2 hours after dosing.

LDL Metabolic Studies
Lipoprotein Preparation
LDL particles to be used for labeling and reinjection were isolated from pooled blood obtained from a group of 33 intact female monkeys consuming an atherogenic diet with a fat and cholesterol content similar to those consumed by the study groups. Three blood pools of 325 mL each were collected in tubes containing aprotinin and D-phenyl-alanyl-L-prolyl-arginine chloromethyl ketone at final concentrations of 25 kallikrein inhibitory units/mL and 1 µmol/L, respectively, to limit degradation of apoB by proteolysis and 1 mg/mL Na₂EDTA to prevent oxidation.⁹ The serine protease inhibitor phenylmethylsulfonyl fluoride and the antioxidant butylated hydroxytoluene were added to isolated plasma at final concentrations of 0.5 mmol/L and 20 µmol/L, respectively. The average cholesterol and TG concentrations of the pooled plasma were 382±3 and 37±2 mg/dL, respectively. LDL was isolated (see below) from the three plasma pools; half was labeled immediately, and the other half was labeled 1 week later. Each labeled LDL preparation was injected into 6 monkeys (2 from each treatment group) within 1 week of labeling (all LDL was dialyzed and stored in the dark at 4°C under nitrogen to avoid oxidative damage).

LDL (d=1.020 to 1.063 g/mL) was isolated by using differential ultracentrifugation followed by exhaustive dialysis against buffer (0.9% NaCl and 2 mmol/L EDTA, pH 7.4).¹⁵ LDL protein was determined by using bovine serum albumin as a standard.²⁶ Each LDL preparation (50 mg) was labeled with ¹³¹I by using 1,3,4,6-tetrachloro-3,6-diphenylglycoluril (Iodogen) before being coupled to ¹²⁵I-TC.^{15 27 28} Specific activities for the dual-labeled LDL averaged 591±46 and 59±8 cpm/ng protein for ¹²⁵I-TC and ¹³¹I, respectively (mean±SEM) for six preparations. Trichloroacetic acid (final concentration, 10%)–soluble radioactivities averaged 1.1±0.3% for ¹²⁵I-TC and 0.4±0.04% for ¹³¹I; radioactivities extractable in chloroform-methanol²⁹ averaged 3.7±0.3% and 7.3±1.2%, respectively. LDL preparations were sterilized before injection by filtration with a 0.45-µm Millipore filter.

Study Protocol
After the monkeys had consumed the atherogenic diet for 16 weeks, dual-labeled LDL (2.89±0.30x10⁹ cpm ¹²⁵I and 2.92±0.45x10⁸ cpm ¹³¹I) was injected through an indwelling femoral venous catheter 24 hours before necropsy.¹⁵ Subsequent blood samples were collected from nonanesthetized animals in tubes containing EDTA (final concentration, 1 mg/mL) from the arterial catheter at 3, 10, 20, 40, and 60 minutes and 2, 4, 6, 20, and 24 hours after injection to determine the plasma decay of labeled LDL. The FCR of LDL by the whole body was calculated from coefficients and exponents determined by the biexponential equation fitted to data for the decline of protein-bound radioactivity in the plasma.^{15 30}

After the final (24-hour) blood sample was collected, the animals were deeply anesthetized with sodium pentobarbital (80 mg/kg body wt IV). The cardiovascular system was flushed via the left ventricle with lactated Ringer's solution (1 L) and perfused with a modified Karnovsky's solution at 100 mm Hg for an additional 20 minutes to provide adequate fixation for radiolabeled LDL.³¹ The thoracic and abdominal aortas, right common iliac artery, common carotid arteries and carotid bifurcations, and left anterior descending and left circumflex coronary arteries were removed (Fig 1).

View larger version (14K): [in this window] [in a new window]   Figure 1. Schematic diagram depicting arterial segments analyzed for radioactivity (C), cholesterol content (CHOL), lipid peroxidation (LPO), and histology (H). TA indicates thoracic aorta; AA, abdominal aorta; RCI, right common iliac artery; LCI, left common iliac artery; RC, right common carotid artery; LC, left common carotid artery; LAD, left anterior descending coronary artery; and LCX, left circumflex coronary artery.

Analysis of LDL Metabolism
Samples were fixed for an additional 24 hours in modified Karnovsky's solution, which preserves products of ¹²⁵I-TC–LDL degradation as well as undegraded ¹²⁵I-TC–LDL and ¹³¹I-LDL.³¹ Thus, accumulation of radioactivity from ¹²⁵I-TC represents both undegraded LDL and products of LDL degradation. The arterial ¹²⁵I-TC radioactivity (in counts per minute per gram) was normalized by the area under the curve of protein-bound ¹²⁵I-TC radioactivity in plasma during the metabolic experiment (expressed as [counts per minute per microliter]xhour) to express the arterial ¹²⁵I-TC radioactivity in a form (microliters per gram per hour) independent of the plasma LDL concentration and amount of labeled LDL injected.¹⁵

The rates of LDL degradation and the calculated concentration of undegraded LDL were determined.^{9 16} Since the [¹³¹I]iodotyrosine released during cellular degradation is leached from arteries during fixation in modified Karnovsky's solution, the remaining ¹³¹I radioactivity represents undegraded LDL. The ¹²⁵I-TC representing LDL degraded by the artery can then be determined by subtracting the arterial ¹³¹I radioactivity from the total arterial ¹²⁵I-TC radioactivity, taking into account the relative activities of these two isotopes in plasma LDL at the time of necropsy.

Arterial LDL degradation was first calculated in fractional terms and expressed as a fraction of the plasma LDL pool degraded per hour per gram of tissue (FCR_tissue). FCR_tissue was calculated as the product of the whole-body FCR determined from the plasma decay curve³⁰ and the ratio of LDL degradation products per gram of tissue to the LDL degraded by the whole body (dose injected multiplied by the fraction of the dose irreversibly degraded by the whole body).^{9 16 31} Degradation of LDL in absolute terms (ie, micrograms LDL-C per gram of tissue per hour) was calculated as the product of the fractional rate of LDL degradation by the tissue and the total amount of LDL-C in plasma, which was calculated as the product of the plasma LDL-C concentration and the plasma volume.^{9 16}

The arterial concentration of undegraded LDL (in micrograms LDL-C per gram) was calculated as the ratio of ¹³¹I radioactivity in the tissue (in counts per minute per gram) to that in plasma (in counts per minute per milliliter) at the time of necropsy multiplied by the plasma LDL-C concentration of individual animals.^{9 16}

Radioactivities in all samples were corrected for overlap of the energy spectra of the two isotopes, for background radioactivity, and for isotopic decay. Samples were counted for 60 minutes, giving a 2 counting error of <1.0% for ¹²⁵I and <3.0% for ¹³¹I. Background was counted until a minimum of 10 000 counts accumulated, resulting in a 2 counting error of <2%.

Atherosclerosis and Lipid Measurements
Atherosclerosis was assessed at two different sites in the left anterior descending and left circumflex coronary arteries to obtain an average over the length of the artery segment counted (Fig 1). Sections of coronary artery for histological study were dehydrated, embedded in paraffin, and stained with Verhoeff–van Gieson's stain. Sections were projected onto a screen, and the area occupied by intima was measured by using a digitizer. The extent of atherosclerosis was expressed as the mean intimal area in square millimeters.⁹

Arterial cholesterol content was determined in the thoracic and abdominal aortas, as these areas had the greatest amount of cholesterol in early studies (Fig 1). Lipid extracts of aortic tissue were prepared by using the method of Folch et al.³² TG and total and free cholesterol concentrations were determined enzymatically as described by Carr et al.³³ CE content was determined as the difference between measured total and free cholesterol.

Lipid peroxidation was assessed in serum and abdominal aorta (Fig 1) by using the TBARS method. Serum TBARS were determined as described by Yagi³⁴ with minor modifications. Briefly, serum was diluted twofold with 0.9% NaCl and incubated for 1 hour at 4°C after adding an equal volume of 20% trichloroacetic acid. The precipitate was redissolved in 4 mL distilled water and 1 mL of 0.67% 2-TBA and heated at 95°C for 1 hour. After cooling to room temperature, 5 mL of n-butanol was added, and the samples were shaken vigorously and then centrifuged at 3000 rpm for 15 minutes. The absorbance at 532 nm was measured in the n-butanol layer, as malondialdehyde, a secondary product of lipid peroxidation, changes color in the presence of TBA.

Arterial lipid peroxidation was determined as described by Ohkawa et al.³⁵ Tissues were homogenized by using a microhomogenizer and prepared in a ratio of 1 g wet tissue/9 mL ice-cold 0.9% NaCl. The homogenate (0.1 mL) was combined with 0.2 mL of 8.1% sodium dodecyl sulfate, 1.5 mL of 20% acetic acid (pH 3.5), and 1.5 mL of 0.8% TBA, diluted up to 4 mL with distilled water, and heated at 95°C for 1 hour. Water (1 mL) and 5 mL n-butanol–pyridine (15:1, vol/vol) were added. The absorbance at 532 nm was measured in the organic layer as described above. Values (TBARS) are reported as nanomoles of malondialdehyde equivalents per gram of wet weight or milliliter of serum.

Statistical Analysis
To reduce skewness and equalize group variances, data for LDL metabolism were analyzed after logarithmic transformation. Treatment groups were compared statistically by ANOVA for each arterial site separately as well as by repeated-measures ANOVA for all arterial sites. Post hoc analyses were done by using the method of Duncan when significant differences were found for individual treatment differences by ANOVA. Pearson product-moment correlations were used to assess the relationships among variables. In all cases, two-sided tests were used to assess statistical significance. Analyses were performed with the BMDP statistical package (Programs 2V, 7D, and 8D; BMDP Statistical Software). Means presented in the text are reported with the SEM.

Of 36 animals initially randomized, 32 completed this study protocol. Three animals died of cardiac arrhythmias during a separate study protocol involving coronary angiography. A fourth animal died of catheter complications in the LDL metabolism study. Data are presented only for animals completing all aspects of the study.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Clinical Chemistry
Temporal changes in plasma lipids and lipoproteins are shown in Fig 2. There were no differences among the treatment groups at baseline (time 0). After consuming the atherogenic diet for 1 month, TPC increased in all treatment groups. However, TPC was lower after 12 weeks in both treatment groups (P<.05) and after 15 weeks in the EE+MT group (P<.05) than in the control group. Plasma HDL-C did not differ significantly among treatment groups. Plasma TG concentrations were lowest in the control group. EE treatment increased TGs at 8, 12, and 15 weeks (P<.05), while EE+MT significantly increased TGs at 8 and 12 weeks compared with the control group (P<.05). In addition, plasma LDL-C and combined VLDL and IDL cholesterol were decreased after 15 weeks of treatment (P=.09, Table 1). LDL particle size was reduced significantly in both treatment groups (P<.01) compared with control and baseline levels (2.72±0.09 g/µmol). Plasma apoA-II concentrations were moderately decreased with treatment. No differences were found in apoA-I or apoB concentrations. Treatment with EE alone significantly increased the whole-body FCR for LDL (P<.05), with a similar trend for treatment with EE+MT (P=.27; Table 1). Plasma levels of estrone and equilin in this study were similar to those reported in a study in which postmenopausal women received 1.25 mg EE.³⁶

View larger version (30K): [in this window] [in a new window]   Figure 2. Line graphs show changes in plasma lipid and lipoprotein levels in millimoles per liter over time for ovariectomized control (solid line) and EE (cross-hatched line) and EE+MT (open line) treatment groups. TRIG indicates TG; HDLC, HDL-C. *P<.05 vs control.

View this table: [in this window] [in a new window]   Table 1. Effect of EEs With and Without MT on Clinical Chemistry Measurements After a 15-Week Treatment

Arterial LDL Metabolism
Treatment with EEs, alone or with MT, decreased all measures of arterial LDL metabolism (LDL degradation, amount of undegraded LDL, and ¹²⁵I-TC accumulation) (P.03 for all; Fig 3 and Table 2). Furthermore, there were no differences between the two treatment groups (P.22 for all). As in previous studies,^{9 16} hormone treatments had the greatest effect on the arterial LDL degradation rate, which was reduced by 64% to 84% in the different arterial sites (Fig 3).

View larger version (11K): [in this window] [in a new window]   Figure 3. Bar graph shows arterial LDL degradation rates for samples from coronary arteries (Coronary), common carotid arteries (Carotid), thoracic aorta (TAorta), abdominal aorta (AAorta), common carotid bifurcation (CBif), and iliac arteries (Iliac) for ovariectomized control (solid bar) and EE (cross-hatched bar) and EE+MT (open bar) treatment groups. Groupxtissue site ANOVA: effect of treatment, P=.015; effect of site, P<.0001; and treatment by site interaction, P=.77. Post hoc analysis: control vs EE, P=.03; control vs EE+MT, P=.006; and EE vs EE+MT, P=.50.

View this table: [in this window] [in a new window]   Table 2. Effect of EEs With and Without MT on Arterial LDL Metabolism

LDL metabolism differed among arterial sites (P<.0001 for all). In all cases, LDL metabolism was greatest at the carotid bifurcation, followed by the coronary arteries, common carotid arteries, and thoracic aorta, with lesser amounts in the abdominal aorta and iliac artery. Treatment and arterial site did not interact (P.10 for all), indicating that while arterial LDL metabolism was quantitatively different at different arterial sites, hormone treatment had a similar effect at all arterial sites.

Atherosclerosis
The extent of atherosclerosis was assessed in the left circumflex and the left anterior descending coronary arteries (Table 3). As expected, after 16 weeks of consuming an atherogenic diet, only minimal intimal involvement was found, and there were no treatment effects. Mean coronary intimal area did, however, correlate significantly with the coronary artery LDL degradation in the control group (r=.69, P<.02) and in all animals (r=.59, P<.001).

View this table: [in this window] [in a new window]   Table 3. Effect of EEs With and Without MT on Coronary Artery Intimal Area and Aortic and Hepatic Lipid Content

Treatment resulted in a moderate reduction of total cholesterol content of the thoracic aorta (P=.07; Table 3) that was accounted for by a significant reduction of esterified cholesterol (P=.04). Free cholesterol and TG concentrations in the thoracic aorta were not affected by treatment. In contrast, there were no differences in cholesterol content in the abdominal aorta, but a significant reduction of aortic TG content was found with EE+MT treatment (P=.02).

Lipid Peroxidation
There was >50% decrease in the accumulation of lipid peroxidation products (TBARS) in the abdominal aorta of animals treated with EE compared with controls (P=.01; Fig 4). A similar trend (23% reduction, P=.07) was seen with EE+MT. There were no apparent effects of treatment on TBARS in serum (4.21±0.67, 3.92±0.48, and 4.25±0.66 nmol/mL for control, EE, and EE+MT, respectively; P=.91). Aortic TBARS did not correlate significantly with the abdominal aortic cholesterol content (r=-.26, P>.05, all animals) or aortic LDL degradation (r=.24, P>.05, all animals).

View larger version (12K): [in this window] [in a new window]   Figure 4. Bar graph shows amount of lipid peroxidation products (ie, TBARS, expressed as nanomoles of malondialdehyde [MDA] per gram) in the abdominal aorta of ovariectomized control (CTL) and EE and EE+MT treatment groups. Effect of treatment: P<.05 by ANOVA. Post hoc analysis: control vs EE, P=.01; control vs EE+MT, P=.07.

Correlations
LDL molecular weight is highly associated with atherogenesis and coronary artery atherosclerosis.^{9 16 37} Correlational analyses for LDL size and other measures are shown in Table 4. LDL size was highly associated with plasma cholesterol concentrations in all groups (P<.01 for all). Among all animals and within the EE group, there was a significant correlation between LDL size and plasma TG concentration (P<.05). Among all animals, LDL size was highly correlated with a number of end points of atherogenesis, ie, coronary intimal area, coronary LDL degradation, and thoracic aortic CE content (P<.01 for all). These correlations were not significant within the EE group.

View this table: [in this window] [in a new window]   Table 4. Correlation Coefficients for LDL Molecular Weight and Other Measures

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Arterial LDL Metabolism
EEs, administered alone or in combination with MT, decreased all indices of arterial LDL metabolism (Fig 3 and Table 2). Varying decreases were found in coronary and all other arteries examined. Due to the relatively short duration (16 weeks) of the atherogenic stimulus and hormone treatment, there were no effects of treatment on coronary artery intimal area (Table 3). However, both treatments resulted in decreased CE content in the thoracic aorta. These findings concur with our past studies, which show parallel effects of combined estrogen-progestin treatment on arterial LDL metabolism^{15 16} and plaque development.¹⁴ This suggests that the estrogen-androgen combination of the current study might also inhibit atherosclerosis progression with a longer treatment duration. Thus, as we have suggested,¹⁵ the decrease in uptake and metabolism of LDL by the artery may, by inhibiting atherosclerosis progression, reduce the risk of CHD.

Arterial LDL metabolism (absolute and fractional degradation, amount of undegraded LDL, or total LDL accumulation) varied with arterial site, in agreement with our early studies.¹⁶ The greatest LDL metabolism occurred in the carotid bifurcation, with intermediate amounts in the coronary and common carotid arteries and thoracic aorta and the smallest amount in the abdominal aorta and iliac artery. The regional differences in LDL metabolism at these times (only 16 weeks of treatment) may explain why we found a difference in CE in the thoracic but not the abdominal aorta. However, the treatments resulted in similar effects on arterial LDL metabolism at all arterial sites. Furthermore, there were no significant differences between EE alone and EE+MT.

Of the various indices of arterial LDL metabolism, the greatest effect induced by hormone treatment was a decrease in the absolute degradation rate of LDL (Fig 3). EE and EE+MT treatments decreased the arterial LDL degradation rate, on average, by 73% and 75%, respectively. This is consistent with our previous study of the effects of combined estradiol and progesterone, in which treatment decreased the LDL degradation rate by 78% compared with ovariectomized controls.¹⁶ The greater percent decreases in absolute degradation (73% and 75%) compared with fractional degradation (32% and 44%) with EE and EE+MT, respectively, are due to the decreased plasma LDL-C concentrations (Table 1) in the treatment groups (absolute degradation is determined from the fractional degradation multiplied by total plasma LDL-C [see "Methods"]).

Lipid Peroxidation
Both LDL supplemented with estrogen in vitro³⁸ and LDL isolated from estrogen-treated women³⁹ have increased resistance to lipid peroxidation. Estrogen treatment of LDL also inhibits LDL accumulation in macrophages.⁴⁰ Thus, estrogens also may decrease the risk of atherosclerosis by acting as antioxidants. In the current study, no significant changes were found in serum TBARS levels, but a 50% reduction was found in the aorta with EE treatment compared with controls (Fig 4). An intermediate effect was seen with combined EE+MT. This suggests that estrogens may have greater antioxidant effects at the level of the arterial wall and that these effects are partially antagonized by the inclusion of androgens. In the abdominal aorta there was no significant difference in lipid content among the groups and no correlation was found between arterial TBARS and CE content, indicating that the decrease in lipid peroxidation with estrogen was not due to a decrease in lipid content. Perhaps the hormone effect on TBARS seen in the artery and not in serum was due to greater local concentrations of estrogen in the artery, to a greater concentration of oxidized particles in the arterial walls of untreated animals, or to a relative paucity of other antioxidants in the artery. If, as suggested by the decreased arterial TBARS, estrogens decrease the oxidative modification of LDL in the arterial wall, this could explain the decrease in arterial LDL metabolism seen presently and in our past studies.^{9 15 16} However, no correlation was found between arterial TBARS levels and LDL metabolism. This may suggest additional mechanisms for reduction in arterial LDL degradation by estrogens, such as reduced LDL-proteoglycan binding and reduced arterial retention of LDL particles, which were not specifically addressed in this study.

The lack of treatment effect on serum TBARS is not surprising, since there are a number of antioxidants present in the serum.⁴¹ However, since small LDL particles are more susceptible to oxidation,⁴² an antioxidant effect of estrogens may prevent the smaller LDL particles produced with estrogen treatment from being atherogenic in tissues such as arteries, where fewer endogenous antioxidants are present.

Plasma Lipoprotein Metabolism
Both treatments resulted in reductions of TPC, LDL-C, and combined VLDL and IDL cholesterol concentrations (Table 1 and Fig 2). This decrease in apoB-containing lipoproteins was most likely due to the increased whole-body LDL FCR (Table 1). Increased catabolism of LDL also occurs in women treated orally with conjugated equine estrogens⁴³ and in men treated with pharmacological doses of estrogens⁴⁴ and may be due to upregulation of LDL receptors by estrogens.^{45 46} Interestingly, parenteral estradiol, both at doses used for postmenopausal estrogen replacement in women⁴³ and combined with progesterone in monkeys,¹⁵ does not decrease plasma LDL-C concentrations or increase total-body LDL catabolism. The greater effect on plasma lipoproteins and LDL catabolism observed with oral medications in the current study and by Walsh et al⁴³ may be due either to the increased delivery of estrogen to the liver following absorption from the portal circulation or to varying doses and potencies of the various hormone treatments. However, beneficial effects on arterial LDL metabolism^{15 16} and atherosclerosis¹⁴ were also seen in monkeys treated with parenteral estrogen. This suggests that while oral estrogens may have beneficial effects on lipoproteins, which may mediate some of the beneficial effects in the artery, estrogens also directly affect arterial metabolism independent of plasma lipid concentrations.

In addition to decreased concentrations of apoB-containing lipoproteins, there was also a decrease in the size of LDL particles in both treatment groups (Table 1). This is consistent with results of studies of estrogen treatment in both nonhuman primates^{9 15 16 47} and women.^{48 49 50 51} The decrease in LDL size may be secondary to an increase in plasma TG concentrations and the enrichment of VLDL with TGs, which promotes the exchange with LDL CEs.^{52 53} The subsequent lipolysis of LDL TGs results in smaller LDL particles.⁵⁴ This is consistent with the negative correlation between LDL size and TG concentrations found in this study (Table 4). Alternatively, or in addition to this mechanism, larger lipoprotein particles may be selectively removed, leaving behind smaller particles. One mechanism for selective removal of large LDL particles is that large particles are more apoE enriched^{47 55} and have high affinity for the apoB/E receptor. They also may be removed by the chylomicron or lipoprotein-related protein receptor, for which apoE is the principal apoprotein ligand.⁵⁶ The FCR measured in this study does not differentiate between LDL receptor–mediated catabolism and other receptor- or nonreceptor-mediated catabolisms.

In conclusion, EEs with or without MT decreased LDL metabolism in all arterial sites studied and reduced CE accumulation in the thoracic aorta. Both treatments also reduced plasma cholesterol concentrations, reduced LDL size, and increased total-body LDL catabolism. Furthermore, LDL size was highly associated with indices of atherogenesis. Estrogen alone also significantly decreased the amount of lipid peroxidation in the aorta. Future studies are ongoing to determine the interaction among these events. In addition to these inhibitory effects on early atherogenesis, both treatments also improved coronary artery reactivity.⁵⁷ These effects may help explain the beneficial effects of estrogens on the risk of CHD and indicate that the addition of a small dose of androgen has no adverse influence.

	Selected Abbreviations and Acronyms

 

CE = cholesteryl ester CHD = coronary heart disease EE = esterified estrogen FCR = fractional catabolic rate HDL-C = HDL cholesterol LDL-C = LDL cholesterol MT = methyltestosterone TBA = thiobarbituric acid TBARS = thiobarbituric acid–reactive substance TC = tyramine cellobiose TG = triglyceride TPC = total plasma cholesterol

	Acknowledgments

 
This study was supported in part by a grant from Solvay Pharmaceuticals, Inc, and a grant from the National Center for Research Resources (KO1 RR00072) to Dr Wagner. The authors thank Dr Dawn Schwenke for valuable advice; Debbie Golden and Dr Michele Martino for technical assistance; and Karen Potvin Klein for editorial assistance.

	Footnotes

 
Presented in part at the 43rd Annual Clinical Meeting of the American College of Obstetrics and Gynecology, San Francisco, Calif, May 9, 1995.

Received March 4, 1996; revision received June 17, 1996;

	References

Top Abstract Introduction Methods Results Discussion References

 
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