Norethindrone Acetate Enhances the Antiatherogenic Effect of 17ß-Estradiol

A Secondary Prevention Study of Aortic Atherosclerosis in Ovariectomized Cholesterol-Fed Rabbits

From the Center for Clinical & Basic Research, Ballerup (P.A., J.H., I.S., H.L., C.C.); and Novo Nordisk A/S, Clinical Department, Gynecological Pharmaceuticals, Bagsvaerd (M.S.), Denmark.

Correspondence to Peter Alexandersen, MD, Center for Clinical & Basic Research, Ballerup Byvej 222, DK-2750 Ballerup, Denmark.

	Abstract

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Abstract—The influence of progestogens in combination with 17ß-estradiol (E2) on cardiovascular disease remains controversial. This study investigated the effect of norethindrone acetate (NETA) combined with E2 on aortic atherosclerosis. Eighty mature female rabbits were ovariectomized, then fed a cholesterol-rich diet (240 mg/d) for 14 weeks to induce aortic atherosclerosis. They were randomized to four equally large groups for the following 38-week intervention period. One group received placebo, another group oral E2 4 mg daily (E2), and the last two groups oral E2 4 mg daily combined with either NETA 1 mg (E2NETA1) or NETA 3 mg (E2NETA3). The cholesterol intake was reduced to a "maintenance" level of 80 mg/d during the intervention period. Total serum cholesterol and ultracentrifuged lipoproteins were analyzed enzymatically throughout the study. The cholesterol content in the aortic wall was 2.76±0.44 µmol/cm² (mean±SEM) in the E2NETA1 group, 1.77±0.37 µmol/cm² in the E2NETA3 group, 5.46±0.77 µmol/cm² in the E2 group, and 7.20±0.94 µmol/cm² in the placebo group (ANOVA P<0.0001). The difference (in the aortic cholesterol accumulation) between the E2 and each of the combined E2/NETA groups was statistically significant (P<0.01) but could only partly be explained by the differences in serum lipids and lipoproteins. In conclusion, NETA enhances the antiatherogenic effect of E2 in cholesterol-fed rabbits. This effect is only partially mediated through changes in serum lipids and lipoproteins.

Key Words: atherosclerosis • prevention • rabbits • estradiol • NETA

	Introduction

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Cardiovascular disease continues to be the primary cause of morbidity and mortality in postmenopausal women in the Western World.¹ Several epidemiological and clinical studies indicate that menopause is associated with a considerable increase in CVD but that postmenopausal estrogen replacement therapy reduces the incidence of CVD by at least 50%.^{1 2 3} However, estrogen monotherapy increases the risk of endometrial cancer,⁴ and therefore progestogens are usually added to reduce this risk.⁵ The influence of progestogens in combination with estrogen on CVD is highly controversial: some authors believe that progestogens negate the beneficial antiatherogenic effect of estrogens.⁶ This belief primarily rests on the negative influence exerted by progestogens on serum lipids, especially HDL cholesterol.^{6 7} This parameter is, however, known to be only one of several antiatherogenic mediators of estrogens. Some clinical data indicate, on the other hand, that NETA, a 19-nortestosterone derivative, may itself possess a beneficial effect on serum total cholesterol and LDL cholesterol in postmenopausal women.⁸ Accordingly, NETA may also enhance some of the beneficial effects of estrogens in terms of CVD. To investigate further the role of NETA, continuously combined with 17ß-estradiol (E2), on established atherosclerosis, we used the rabbit model, which has previously been found useful in atherosclerosis research.⁹

	Methods

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Study Design
Eighty sexually mature female rabbits of the Danish Country strain (SSC:CPH) were obtained from Statens Serum Institute, Copenhagen, Denmark. They were individually housed at room temperature (20±2°C), 55±5% relative humidity, with a 12-hour light cycle. The study was conducted in new facilities (approved by the Animal Experiments' Inspectorate, Denmark) at the Center for Clinical & Basic Research, Ledoeje, Denmark. After a 4-week period of acclimatization, the animals underwent bilateral ovariectomy (weeks 5 to 6). The rabbits were anesthetized with an intramuscular injection of Hypnorm 0.3 mL/kg (0.2 mg/mL fentanyl+10 mg/mL fluanizon, Janssen-Cilag Ltd).¹⁰ Before arrival at the Center and up to the time of surgery, the rabbits were fed a standard commercial rabbit chow. Postoperatively, all animals were fed a diet containing 240 mg of cholesterol per day for the next 14 weeks (weeks 7 through 20) to induce aortic atherosclerosis. The rabbits were then randomly assigned to one of the following four oral treatment groups: placebo (n=20), 4 mg of E2 daily (E2, n=20), or two groups receiving 4 mg of E2 continuously combined with either NETA 1 mg (E2NETA1, n=19) or NETA 3 mg (E2NETA3, n=20). During this 38-week intervention period (weeks 21 to 59), the cholesterol content in the chow was reduced to a "maintenance level" of 80 mg per day. The study was approved by the Animal Experiments' Inspectorate, Denmark. All procedues complied with the Danish guidelines for experimental animal studies.

Rabbit Chow
Each rabbit was fed 100 g of chow per day throughout the entire study. The chow was prepared by first dissolving the E2, E2+low-dose NETA, or E2+high-dose NETA (NovoNordisk A/S), in ethanol (96%; 0.50 mL per animal per day), then mixing with maize oil (Unikem). Another mixture was prepared by dissolving cholesterol (SIGC-8503, Bie & Berntsen A/S) in maize oil by slow heating. The two solutions containing maize oil (total daily intake of maize oil was 8 mL per animal) were then mixed manually together with the pellets (Altromin 2123, Brogaarden). For the placebo group, the chow was prepared as described; however, no hormone was added. For logistic reasons, the chow for a period of 5 weeks was produced, labeled, and stored at -20°C in daily portions. Food consumption was monitored by weighing any remaining chow each week. All animals had free access to water.

Blood Samples
Blood samples were taken in weeks 4, 13, 19, 25, 32, 40, 48, and 55, and always after a 24-hour fast.

Serum Lipids and Lipoproteins
Total serum cholesterol and triglycerides were measured enzymatically by means of kinetic colorimetric methods (Cobas Mira). To determine serum lipoproteins (HDL, density 1.063 g/mL; LDL, 1.019 g/mLdensity <1.063 g/mL; IDL, 1.006 g/mLdensity <1.019 g/mL; VLDL, density <1.006 g/mL), three aliquots from the serum samples were adjusted to specific densities, using solutions of NaCl and NaBr. The aliquots were then sealed and ultracentrifuged at 4°C at 1.58x10⁸ g/min for 14.5 hours in a Beckman 50.4 Ti rotor (Beckman Instruments, Inc). The top and bottom fractions of each aliquot were divided by tube slicing, and the cholesterol levels of the fractions were then measured enzymatically. The lipoproteins were calculated from the fractions after correction for recovery and dilution.

Serum E2
Serum levels of E2 were measured in weeks 4 and 55 by radioimmunoassay, with an intra-assay imprecision of 8.5%, an interassay imprecision of 12.3%, and a detection limit of 0.010 nmol/L.¹¹

Aortic Cholesterol Content
At the end of the study (week 59), the rabbits were killed with an intravenous injection of 1 to 2 mL of mebumal (pentobarbital) 20% solution. The thoracic aorta (just above the aortic valves to the level of the diaphragm) was dissected free, and the connective tissue adhering to the adventitia was then carefully removed under running saline. The aorta was cut longitudinally and the luminal surface rinsed with saline. The vessel was fixed at the corners with pins onto a piece of paper on a cork board. The aortic surface area was determined and the tissue was separated in two parts (a proximal and a distal part) at the level of the first intercostal arteries. The proximal part was used to strip the luminal layer containing the intima and part of the media from the underlying media/adventitia. The proximal part was weighed and stored at -20°C until analyzed.

For analysis, the luminal layer of the aortic tissue was minced and the lipids were extracted chemically with chloroform and methanol (2:1, vol/vol) over 24 hours. The lipids were separated from the proteins.¹² The total aortic cholesterol content in the tissue specimens was measured enzymatically after the fraction containing cholesterol had been taken to dryness by heating and then dissolved in 1.0 mL of 2-propanol. The amount of protein in the aorta was measured as described by Lowry et al.¹³ The weight of the heart was recorded.

The Uterus
The bicorn uterus was cut at the level of the vagina and beginning of the salpinges, respectively, removed, and the weight was determined.

Body Weight
Body weight was determined every 4 weeks throughout the study on the same scales.

Statistical Analysis
The average levels of serum cholesterol and lipoproteins during the treatment period were calculated as the area under the curve divided by the length of the intervention period (38 weeks). ANOVA was performed for the baseline values (Table 1), the average serum lipids and lipoprotein levels, aortic cholesterol content, and uterine weight. If ANOVA indicated statistical significance, a t test was used to compare groups two by two using the Bonferroni correction for multiple comparisons. The relation between aortic accumulation of cholesterol and the averaged serum total cholesterol (and lipoprotein) level was determined by correlation analysis. The influence of baseline total serum cholesterol and triglyceride levels, average serum cholesterol, triglycerides, and lipoprotein levels, and treatment (the independent variables) on aortic cholesterol accumulation (the dependent variable) was adjusted by ANCOVA. All statistical analyses were performed with the Statistical Analysis System (SAS) with 5% as the level of significance.¹⁴

	Results

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The baseline values for the four treatment groups are given in Table 1. The baseline body weight of the E2NETA3 group was lower than that of the other groups, despite randomization. Body weights were largely unchanged in all the groups throughout the study, although the E2NETA3 group tended to increase in weight, thereby eliminating the small initial difference. Five rabbits died prematurely: one in the placebo group (week 50) for unknown reasons; one in the estrogen group (week 42) because of intestinal obstruction; two in the E2NETA1 group: one (week 12) probably because of pericholangitis and one (week 48) after a limb trauma; and one in the E2NETA3 group (week 34) because of postoperative hernia. Thus, 93.8% completed the study.

Figure 1 shows the changes in total serum cholesterol during the study. Serum values peaked in week 19, ie, at the end of the atherosclerosis-induction period. Table 2 shows the average serum total cholesterol and lipoprotein levels during the treatment period. For the two estradiol/NETA groups, the averaged total serum cholesterol, triglycerides, IDL cholesterol, and VLDL cholesterol values were lower than those for the placebo and estradiol groups (ANOVA P<0.05). There was no statistically significant difference in these parameters between the two estradiol/NETA groups or between the placebo and the estradiol group. Serum estradiol was low and remained unchanged in the placebo group (0.17±0.04 versus 0.12±0.03 nmol/L, baseline versus week 55; mean±SEM) but increased significantly in the E2 group (from 0.10±0.01 to 0.21±0.04 nmol/L, P=0.02) and the estradiol/NETA groups (E2NETA1, from 0.11±0.01 to 0.22±0.03 nmol/L, and E2NETA3, from 0.08±0.01 to 0.16±0.03 nmol/L, P<0.01 for both). The uterine weight in the placebo group was 6.2±0.7 g (mean±SEM) and was significantly and equally higher in both estradiol/NETA groups (17.5±1.4 g for E2NETA1 and 19.6±2.1 g for E2NETA3), although lower than in the E2 group (28.3±1.8 g) (ANOVA P<0.0001). Thus, an increase in the NETA dose does not apparently add further to the reduction in uterine weight.

View larger version (20K): [in this window] [in a new window]   Figure 1. Changes in the total serum levels of cholesterol during the study for the four treatment groups. The levels were reduced more in the estradiol/NETA groups than in the placebo and estradiol groups (ANOVA P<0.05). Values are mean±SEM.

Figure 2 visualizes the accumulation of cholesterol in the luminal proximal layer of the thoracic aorta for the four groups, unadjusted (A), adjusted for the corresponding aortic protein content (B), adjusted for aortic wet weight (C), or adjusted for the aortic area (D). The differences in aortic cholesterol accumulation were highly statistically significant (ANOVA P<0.0001). When adjusted for the aortic area, the estrogen group had accumulated 75.7% of that found in the placebo group, and this was significantly more (P<0.01) than in the E2NETA1 and E2NETA3 groups, which had accumulated respectively only 38.2% and 24.6% of the aortic cholesterol in the placebo group. Aortic accumulation of cholesterol was significantly related to the averaged serum total cholesterol, VLDL cholesterol, IDL cholesterol, and LDL cholesterol (.54r0.71, P<0.0001 for these correlations) but not to HDL cholesterol (r=-0.05). ANCOVA was performed, with the aortic cholesterol content as the dependent variable and baseline and average total serum cholesterol, triglyceride, and lipoprotein levels, and treatment as covariates. This analysis showed that only treatment, LDL cholesterol, and VLDL cholesterol were significant (respectively, P=0.006, P=0.045, and P=0.011) independent predictors of aortic atherosclerosis. Compared with placebo, the estimates (mean±SEM) were as follows: with E2NETA1:-3.02±0.95 µmol/cm^-2 (P<0.01); with E2NETA3: -2.90±0.97 µmol/cm^-2 (P<0.01); with E2: -0.56±0.97 µmol/cm^-2 (NS); and for LDL cholesterol: 0.46±0.22 µmol/cm^-2 (P<0.05) and VLDL cholesterol: 0.24±0.09 µmol/cm^-2 (P<0.05). The amount of aortic cholesterol accumulation after correction for LDL cholesterol and VLDL cholesterol is depicted in Figure 3. In addition, each of the two E2/NETA groups had statistically significantly less aortic cholesterol accumulation than the E2 group (P<0.01 for both comparisons). Comparison of the two E2/NETA groups showed that the aortic cholesterol accumulation (when adjusted for aortic protein or surface area) tended to be lower in the high-dose NETA group than in the low-dose NETA group (P<0.1) (Figure 2). However, after correction for lipoproteins, the cholesterol content in the two groups was similar, which indicates a lipid-mediated, dose-response effect (Figure 3).

View larger version (46K): [in this window] [in a new window]   Figure 2. Accumulation of cholesterol in the proximal thoracic aorta, unadjusted (µmol; A), adjusted for aortic protein (nmol/mg; B), adjusted for tissue wet weight (nmol/mg; C), and adjusted for the area surface (µmol/cm2; D). Values are mean±SEM. *P<0.05; **P<0.01; ***P<0.001.

View larger version (35K): [in this window] [in a new window]   Figure 3. Accumulation of cholesterol in the proximal aorta after adjustment for LDL cholesterol and VLDL cholesterol (set at 4 mmol/L and 8 mmol/L, respectively) according to the different treatment groups. Values are mean±SEM.

	Discussion

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The present study demonstrated that in rabbits, NETA 1 and 3 mg per day induce different effects according to the target tissue studied. Thus, 1 mg of NETA and 3 mg of NETA daily were equally efficacious in counteracting the uterotrophic effects of estradiol 4 mg per day. In this study, the continuous treatment with NETA combined with E2 had a significant additional preventive antiatherogenic effect compared with E2 alone. This additive or synergistic action of NETA in reducing atherosclerosis was only partly explained by lower levels of serum lipids and lipoproteins. Hence, a direct hormonal effect on the arterial wall is indicated. Our results suggest that at least some combined estrogen-progestogen replacement therapies may have a nonlipid inhibitory effect on established atherosclerosis, greater than that of E2 monotherapy. The beneficial progestogenic action may perhaps seem somewhat surprising. However, with respect to serum lipids and lipoproteins, previous human data indicate that when NETA is added to estradiol treatment, it enhances the beneficial estrogenic changes in serum levels of total and LDL cholesterol and lipoprotein(a), which suggests that NETA has an additive antiatherogenic influence.^{8 15} This does not seem to be the case for other commonly used progestogens.¹⁶ Furthermore, clinical studies investigating other end points, such as postmenopausal bone mass, have indicated that NETA possesses an intrinsic estrogenic action.¹⁷ Whether this also applies to other progestogens is not yet known. Data from a previous rabbit study have suggested that aortic accumulation of cholesterol seems to be inhibited significantly more by NETA than by MPA.¹⁸ Such differences in the antiatherogenic properties of different progestogens are supported by other investigations. A recent study thus suggested that progesterone injected in high doses completely eliminates the beneficial estrogenic effect on aortic atherosclerosis in ovariectomized cholesterol-fed rabbits.¹⁹ Studies of ovariectomized cynomolgus monkeys have demonstrated that progesterone appears to have a neutral impact on the antiatherogenic effect of E2^{20 21} and that in combination with CEE, MPA may even negate the favorable estrogenic effect as measured by the endothelium-mediated dilatation of atherosclerotic coronary arteries.²² Data from a recently published study of ovariectomized monkeys fed an atherogenic diet and treated with either CEE, MPA, CEE+MPA, or no hormones showed that animals treated with MPA or CEE+MPA had similar extent of atherosclerosis compared with the controls and that animals in the CEE group had significantly less atherosclerosis than any of these groups.²³ Whether these discrepancies are due to differences between 19-nortestosterone and 17-hydroxyprogesterone derivatives is at present unknown. Methodological differences, ie, in the species/strain, study design, doses, or route of administration, may also be important. It might, however, be suggested that there are important fundamental differences in the ability of 17-hydroxyprogesterone and 19-nortestosterone derivatives to prevent the atherosclerotic process.

The possible mechanism by which NETA could enhance one or more of the beneficial estrogenic effects is, however, largely speculative. It has been suggested that accumulation and/or metabolism of LDL cholesterol in the arterial wall may be reduced during E2 therapy,²⁴ which, theoretically, could be explained by a potent estrogenic antioxidant capacity.²⁵ Recent data suggest that metabolites of NETA (3ß,5-NETA and 3,5-NETA, respectively) may exert an estrogenic (and perhaps an antioxidant) effect through binding to the estrogen receptor.²⁶ Indeed, some data indicate that NETA in vivo is also metabolized to ethinyl estradiol, which in turn exerts the estrogenic effect via the estrogen receptor.²⁷ In addition, the progestogenic effect of NETA is mediated via interaction of NETA with the progesterone receptor,²⁷ whereas other studies in the rabbit uterus have shown that MPA does not bind to the estrogen receptor.²⁸ In previous studies we found that NETA alone²⁹ and in combination with E2⁹ had a neutral effect on early atherogenic processes. It may therefore be speculated that NETA primarily has a synergistic beneficial effect together with estradiol in established and more advanced atherogenic stages.

Two recent epidemiological studies investigated the role of estradiol combined with progestogens on hard cardiovascular end points. One study³⁰ showed that postmenopausal women treated with an estrogen/progestogen combination (predominantly a 19-nortestosterone derivative other than NETA, namely levonorgestrel) tended to have fewer cardiovascular events (ie, myocardial infarctions) than women receiving E2 monotherapy, who in turn had fewer events than women not taking hormone replacement therapy. Very recently, data from the Nurses' Health Study³¹ indicated that the current use of MPA combined with oral conjugated estrogens was associated with markedly less risk of a major coronary heart disease condition than when hormone replacement therapy was never used. The relative risk was even lower than that of current estrogen use. These risk estimates were, however, based on a very small number of subjects.

If the estrogen-enhancing effect of NETA reported here is confirmed in future studies, it will undoubtedly have a great impact on future considerations regarding estrogen/progestogen therapy in the (secondary) prevention of CVD in postmenopausal women.

	Selected Abbreviations and Acronyms

 

CEE = conjugated equine estrogens CVD = cardiovascular disease E2 = 17ß-estradiol MPA = medroxyprogesterone acetate NETA = norethindrone acetate

Received September 26, 1997; accepted December 8, 1997.

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