1 Center for Clinical & Basic Research, Ballerup Byvej 222, 2750 Ballerup, Denmark and 2 Department of Vascular Pharmacology, N.V.Organon, Molenstraat 110, 5340 BH Oss, The Netherlands
3 To whom correspondence should be addressed. e-mail: pa{at}ccbr.dk
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Abstract |
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Key words: atherosclerosis/estrogen/progestins/rabbits/vascular reactivity
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Introduction |
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The relative preponderance in venous events (e.g. deep venous thrombosis) as compared with arterial events (e.g. myocardial infarction) in pre-menopausal women is gradually equalized as the menopause is reached, so that the relative frequency of these events is close to 1:1 in peri-menopausal women. Since OC are prescribed for millions of pre-menopausal (and peri-menopausal) women who use these formulations for many years, it would be of the utmost public health importance to establish even a small increase in the relative risk. Therefore, the issue of OC in relation to arterial disease is highly relevant. It should be borne in mind, however, that it is possible that for both OC and HRT users, there may be prothrombotic mechanisms in relation to arterial as well as venous complications that are not necessarily based on atherosclerosis, but that are reflected in the population-based studies. Primary (Rossouw et al., 2002) and secondary (Grady et al., 2002
) prevention studies of HRT have failed to show cardioprotection in post-menopausal women.
We report here the results from an experimental study in rabbits of atherosclerosis designed to investigate the effect of estrogen (ethinyl estradiol, EE) in combination with levonorgestrel (LNG), desogestrel (DSG), or gestodene (GSD) on vascular reactivity, lipoprotein metabolism, and the aortic accumulation of cholesterol.
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Materials and methods |
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Key effect variables of the study comprised aortic atherosclerosis (i.e. fatty streaks and plaque formation), and vascular reactivity (primary key variables); and body weight, serum lipids and lipoproteins, uterus wet weight, hepatic cholesterol content, uterine estrogen receptor content, liver enzyme concentration, haemoglobin, and white cell count (secondary key variables).
The study was approved and overviewed by the Experimental Animals Committee under the Danish Ministry of Justice. All procedures 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 cholesterol-rich chow was prepared by first dissolving the hormone or the combination of hormones (all provided by N.V. Organon, The Netherlands) in ethanol (96%; 0.50 ml per animal per day), then mixing with maize oil (Unikem, Denmark). Another mixture was prepared by dissolving cholesterol (SIGC-8503; Bie & Berntsen A/S, Denmark) in maize oil by slow heating. The hormone solution and the cholesterol solution 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, Denmark), as previously described (Alexandersen et al., 1998). Food consumption was monitored weekly by weighing remaining chow. All animals had free access to water.
Blood samples
Blood samples were taken at baseline (week 0) and in weeks 6, 14 and 30. Blood samples were collected from a lateral ear vein on fasting animals (24 h) and analysed at the CCBR laboratory (Ballerup, Denmark) immediately after collection, except for the progestin concentrations that were assessed at Organon.
Safety variables
Haemoglobin, haematocrit, red blood cell count, leukocyte count (Sysmex K-1000; Toa Medical Electronics, Inc., USA) and alanine aminotransferase (ALAT) (Cobas Mira Plus; Roche Diagnostic Systems, Inc., F.HoffmannLa Roche, Switzerland) were determined in weeks 0, 6, 14 and 30.
Serum lipids and lipoproteins
Total serum cholesterol (TC) and triglycerides (TG) were measured enzymatically by kinetic colorimetric methods (Cobas Mira). Ultracentrifuged lipoproteins were determined regularly throughout the study as described in detail elsewhere (Haarbo et al., 1991; 1992; Alexandersen et al., 1998
).
Serum progestin concentrations
A kinetic study was performed after 16 weeks of treatment to determine the serum concentrations of the respective progestins. Blood samples were taken before dosing, and then again 1, 2, 3, 4, 6, 8 and 24 h after dosing, but taking only two samples per animal in each group (providing 40 samples per group), to give an impression of the pharmacokinetic profile of these compounds. These hormone concentrations were determined at Organons laboratories.
Desogestrel
DSG study samples were determined according to a validated assay. The limit of quantification for this study was 1.0200 ng DSG per ml plasma DSG and its internal standard (IS), an analogue of DSG, were isolated from 0.1 ml of rabbit plasma by solid-phase extraction (SPE) with C-18 cartridges. The plasma extracts were analysed using an API 365 LC-MS-MS system. The liquid chromatograph was equipped with an analytical Luna Phenyl Hexyl column. Ion spray was applied as ionization technique, monitoring m/z 325.4 (M + H) with fragment ion m/z 147.2 for DSG and m/z 339.20 (M + H) with fragment ion m/z 229.1 for its IS.
Gestodene
GSD study samples were determined according to a validated assay. The limit of quantification for this study was 1.0200 ng of GSD per ml plasma. GSD and its IS, an analogue of GSD, were isolated from 0.1 ml of rabbit plasma by SPE with C-18 cartridges. The plasma extracts were analysed using an API 365 LC-MS-MS system. The liquid chromatograph was equipped with an analytical Hypersil BDS C18 column. Atmospheric pressure chemical ionization was applied as ionization technique, monitoring m/z 311.0 (M + H) with fragment ion m/z 109.1 for GSD and m/z 339.10 (M + H) with fragment ion m/z 229.20 for its IS.
Levonorgestrel
LNG study samples were determined according to a validated assay. The limit of quantification for this study was 1.0200 ng of LNG per ml plasma. LNG and its IS, an analogue of LNG, were isolated from 0.1 ml of rabbit plasma by SPE with C-18 cartridges. The plasma extracts were analysed using an API 365 LC-MS-MS system. The liquid chromatograph was equipped with an analytical Luna Phenyl Hexyl column. Ion spray was applied as ionization technique, monitoring m/z 313.3 (M + H) with fragment ion m/z 109.2 for LNG and m/z 339.20 (M + H) with fragment ion m/z 229.10 for its IS.
Aortic accumulation of cholesterol
Necropsy (week 32) was done with an i.v. injection of 12 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 was rinsed with saline. The vessel was fixed at the corners with pins onto a piece of paper on a corkboard. 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 utilized 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 analysed. 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 h. The lipids were separated from the proteins (Haarbo et al., 1991). 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 (1951
). The weight of the heart was recorded.
Morphometric analysis of aortic plaque area
The aorta (comprising the ascendant part, the arch, and the descendant part, from the aortic valves and to the first intercostal artery) was opened longitudinally and rinsed in 50% ethanol and dyed in Sudan Red for 1 min. Each aortic tissue dyed was projected onto a horizontal surface with a projecting videocamera (JAI 2040 Protec, Japan) and videotaped under microscope (Zeiss Stemi 2000/C, Germany). The images obtained were then digitized (ImagePro Plus, USA) to determine the surface involvement of atherosclerotic lesions (fatty streaks) and the total area occupied by the atheroma plaque (see below). Surface involvement by atherosclerosis in an animal was assessed by tracing the contours of the lumen expressed as percentage of the total aortic area. Summing the degree of surface involvement per animal and dividing by the number of animals in the group, the mean degree of surface involvement by atherosclerosis in a treatment group was calculated. Sudan Red was found not to significantly interfere with chemical determination of aortic accumulation of cholesterol (data not shown).
Preparation of aortic rings and tension monitoring
Isolated vascular segments (34 mm transverse sections) from the thoracic aorta were prepared from the newly killed rabbit (Furchgott and Zawadzki 1980). Five to ten rabbits randomly selected from each group were used. The rings were immediately placed in ice-cold Krebs solution and cleaned under careful protection of the endothelium. The Krebs solution consisted of (mmol/l): NaCl 118.0, KCl 4.7, CaCl2 2.6, MgSO4 1.2, KH2PO4 1.2, NaCHO3 24.9, and glucose 11.1. The isolated rings were mounted in the organ bath on two parallel and horizontal stainless steel wires (40 µm in diameter) inserted into the lumen of the vessel. The bath contained Krebs solution at 37°C, carbonized with 95%/5% of O2/CO2. One hook was fixed, and the other connected to a force transducer measuring the isometric tension of the ring (Myograph 400; JP Trading A/S, Denmark). Initially, the rings were stretched to a basal tension of 2.0 g and allowed to equilibrate for
45 min. From other experiments, it was found that a basal tension of 2.0 g developed the maximal active tension in the rings (data not shown), and the basal tension was therefore increased to 2.0 g before each experiment and allowed to equilibrate for ≥30 min. The rings were then contracted twice with a 126 mmol/l K+ Krebs solution, which is identical to Krebs solution, except that Na+ in the Krebs was exchanged with K+ on a molar basis. The experiment began with repeated contraction with phenylephrine to
40% of their maximal contraction with high dose potassium (126 mmol/l). Cumulative doseresponse curves to acetylcholine were then obtained in the concentration range of 108 to 105 mol/l. The rings were washed and allowed to relax. The vessels were then stimulated with phenyleprine again to
50% of the maximal contractile response to 126 mmol/l of K+ , and doseresponse curves were subsequently obtained for sodium nitroprusside (4x108 to 1.3x105 mol/l).
Liver accumulation of cholesterol
The amount of cholesterol accumulated during the study was determined after homogenization of a liver biopsy taken at the time of necropsy. Hepatic cholesterol concentrations were assessed after homogenization and adjusted for hepatic protein similarly as described for aortic cholesterol determinations (Haarbo et al., 1991).
The uterus and endometrial tissue
The bicornuate uterus was cut at the level of the vagina and beginning of the salpinges, removed and the wet weight determined. A sample of endometrial tissue was excised and immediately frozen in liquid nitrogen, and stored at 85°C until analysis. For analysis, the endometrial tissue was homogenized and centrifuged at 800 g. The supernatant was then further centrifuged at 105 000 g, and the obtained supernatant (cytosol) was used for determination of cytosolic estrogen-binding capacity by steroid-binding assay with dextran-coated charcoal separation (Thorpe, 1987). The estrogen-binding capacity was adjusted for the protein concentration in the cytosol (Bradford, 1976
). The 800 g pellet was washed, the nuclear receptors extracted by 0.6 mol/l KCl (Thorpe et al., 1986
) and the nuclear estrogen receptor content determined by an enzyme immunoassay (Abott Laboratories). The inter-assay variation of the estrogen-binding capacity and the estrogen receptor (immunoassay) and protein determination were 7, 6 and 5% respectively. All analyses were done without knowledge of the treatment group.
Statistics
The mean levels of serum lipids and lipoproteins during the treatment period were calculated as the area under the curve (AUC). Analysis of variance (ANOVA) was performed for the primary and secondary key variables. If ANOVA indicated statistical significance, Students t-test was used to compare groups against the placebo group using Dunnetts correction for multiple comparisons. The relationship between aortic accumulation of cholesterol and the averaged serum total cholesterol (and lipoprotein) level was determined by correlation analysis. Doseresponse curves for acetylcholine were performed for each treatment group (n = 510), and ANOVA was used to test for statistical differences among groups at each concentration of acetylcholine. Linear correlation was performed between accumulation of cholesterol and vascular response to acetylcholine. Analysis of co-variance (ANCOVA) was used to investigate the significance of serum lipids and lipoproteins and of other non-lipid-mediated effects of the hormone treatments (independent variables) on the accumulation of cholesterol (dependent variable), and to study the degree of endothelial dysfunction (dependent variable) after correction for aortic accumulation of cholesterol and treatment (independent variables). All statistical analyses were performed with 5% as the level of significance.
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Results |
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Discussion |
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Data on the direct effect of OC formulations on the human arterial system are lacking (Kuhl, 1996). We found evidence that the OC formulations used in this study had a direct effect on the arteries of cholesterol-fed rabbits. Acetylcholine-mediated relaxation of precontracted aortic rings was increased in the EE plus progestin groups, although less than in the EE group alone as compared with placebo. EEs significant effect on restoring vasorelaxation was found to be independent of the accumulation of cholesterol in the aortic wall. However, we also found that the addition of the progestins influenced the estrogen-induced vasorelaxation (Figure 3), although by an unknown mechanism of action. Recently, in a study of precontracted rabbit jugular veins, EE, LNG, DSG and GSD were reported to induce relaxations in vessels with intact endothelium (Herkert et al., 2000
). However, this area warrants further investigation.
It is well known that cholesterol-fed rabbits show alterations in their lipoprotein metabolism that differ from the human situation (Haarbo et al., 1991; 1992). Combination of EE with a progestin in this study reflected the estrogenic effect. Furthermore, the three combined treatments lowered serum lipids and the atherogenic lipoprotein levels significantly and similarly to EE monotherapy. In contrast, treatment with a progestin alone did not affect these variables differently from the controls, in accordance with earlier findings (Haarbo et al., 1992
). In women, OC frequently increases serum triglycerides (Gevers Leuven et al., 1990
; Kuhl et al., 1990
; Leuven et al., 1990
; Lobo et al., 1996
; Cheung et al., 1999
).
The dose of EE was selected to reflect serum concentrations of EE in peri-menopausal women taking OC. However, the duration of the present study was longer than in many previous studies (20 weeks). Among the animals receiving EE alone, 40% died after only ≥21 weeks of treatment, whereas animals given combined treatment did not die prematurely. This suggests that the accumulated estrogen dose may have been too high and/or the study too long, as also indicated by the safety variables of the EE-treated animals at week 30 (Table IV), but also that adding a progestin was able to negate this toxic effect. Progestins were used in equipotent doses (i.e. in combination with EE they inhibit endometrial stimulation equally in humans) relative to each other. The selected dose of the progestins (µg per kg body weight) was chosen based on previous experience (van der Vies and de Visser, 1983
; Sulistiyani et al., 1995
; Zandberg et al., 2001
) and in-house studies (in Organon), but may be considered as high doses. All three OC formulations significantly decreased the concentration of the cytosolic ER concentration relative to controls, suggesting that these formulations affect the endometrium through a down-regulation of the cytosolic ER. Addition of a progestin in this study also down-regulated the ER although less than found for EE, and when combining EE with a progestin, the estrogen component dominated the ER regulation. It should, however, be emphasized that the lack of modifying effect of the progestins relative to the EE dose on the endometrium should not be taken as a lack of progestogenic effect, since the primary intention was to investigate the effect of these hormone combinations on atherosclerosis and arterial responsiveness.
A type II statistical error is not likely to have occurred in our study. However, the accumulation of cholesterol (and amount of fatty streaks) in the EE group was significantly lower than that of the placebo animals. For a type II error to occur, the null hypothesis (that there was no difference in aortic accumulation of cholesterol between the EE and the placebo group) would not be true, and despite this, we would obtain a non-significant result, i.e. a false negative result.
In conclusion, the present study demonstrates that in ovariectomized cholesterol-fed rabbits, the progestins investigated (LNG, DSG, or GSD) can be combined with EE without attenuating the anti-atherogenic effect of EE, partly by decreasing atherogenic lipoproteins, and partly by a direct effect on the endothelium, modulating the aortic vasomotor response in vitro.
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Submitted on December 13, 2002; resubmitted on February 20, 2003; accepted on March 25, 2003.