Nikolaj Nikolajewitsch Anitschkow (1885-1964) established the cholesterol-fed rabbit as a model for atherosclerosis research.

The cholesterol-fed rabbit is a widely used model for experimental atherosclerosis research. In regard to this, one name is periodically mentioned: Nikolaj Nikolajewitsch Anitschkow. Those infrequent reminders of an important name in modern medical history do not pay an adequate tribute to basic findings concerning the pathology and pathogenesis of atherosclerosis. In contrast to research groups at that time conducting experiments with protein enriched diets, Anitschkow demonstrated, in 1913 in St. Petersburg, that it was cholesterol only that caused these atherosclerotic changes in the rabbit arterial intima, which was very similar to human atherosclerosis. By analysing the plaque's development and histology, Anitschkow was able to identify the cell types, on which modern atherosclerosis research is now focussing with a new set of immunohistochemical methods: smooth muscle cells, macrophages and lymphocytes. He noted early (fatty streaks) and advanced (atheromatous plaques) lesions and, by standardizing cholesterol feeding, he discovered that the amount of cholesterol uptake was directly proportional to the degree of atherosclerosis formation. His explanation for this observation was what modern terminology calls 'response-to-injury'. With modern immunohistochemical and molecular-biological methods, the cholesterol-fed rabbit can be used to investigate the pathophysiological aspects which also contribute to human atherosclerosis, such as lipoproteins, diabetes, mitogens, growth-factors, adhesion molecules, endothelial-function, receptor-pathways or platelets. This model can be combined with a number of other methods causing endothelial dysfunction and injury, such as balloon denudation, electric stimulation, cuff implantation, artificial hypertension, diabetes or infection. Bred strains of hereditary hypercholesterolemic rabbits or those resistant to a cholesterol-diet provide further possibilities to expand experimental designs.

Inhibition of the protective effect of estrogen by progesterone in experimental atherosclerosis.

The aim of the present study was to determine the effect of progesterone on the action of estrogen in the development of atherosclerosis. A total of 48 female New Zealand white (NZW) rabbits were ovariectomized. The animals were separated into 6 groups of 8 animals each and received subsequently a 0.5% cholesterol diet for 12 weeks. During this cholesterol feeding period, either estradiol (1 mg/kg body weight (BW)/week), progesterone (25 mg/kg BW/week), or combined estradiol/progesterone (in above dosages) was administered intramuscularly in each group (n = 8 each) of ovariectomized rabbits. One additional group of 8 animals received a combined estrogen/ progesterone regimen, but with progesterone at one third of the above mentioned dosage. In another 8 rabbits, progesterone was reduced to one ninth of the maximum dosage above, whereas estrogen was kept the same, at 1 mg/kg BW/week. Eight ovariectomized animals served as the control group and received no hormone treatment. After 12 weeks, the animals were sacrificed and the proximal aortic arch was removed for further histological examination. An inhibitory effect of estrogen of intimal thickening was found, in comparison to the control group (intimal area: 0.7 +/- 0.5 mm2 vs. 3.7 +/- 2.5 mm2, P < 0.01), whereas progesterone alone did not show a significant effect on intimal plaque size (intimal area: 4.0 +/- 2.3 mm2). In combination with progesterone (high dose), estrogen was not able to reduce intimal atherosclerosis (intimal area: 3.4 +/- 2.4 mm2). However, the beneficial effect of estrogen was not affected by progesterone, when this was reduced respectively to one third (intimal area: 0.8 +/- 0.7 mm2), or to one ninth of the highest dosage (intimal area: 0.6 +/- 0.4 mm2). Interestingly, these differences in atherosclerotic plaque development were observed without significant changes in plasma cholesterol concentrations by the administered hormones. In conclusion, progesterone was dose-dependently able to completely inhibit the beneficial effect of estrogen in experimental atherosclerosis, suggesting that progesterone exerts a direct inhibitory effect on the athero-protective action of estrogen. In the context of recently published data, the present work confirms the importance of the 'non-lipid-mediated', anti-atherosclerotic effect of estrogen, probably due to an interaction with six hormone receptors in vascular smooth muscle cells (VSMC).

17beta-estradiol, gender independently, reduces atheroma development but not neointimal proliferation after balloon injury in the rabbit aorta.

The aim of the present study was to investigate anti-proliferative and anti-atherogenic properties of 17beta-estradiol in balloon injured female and male rabbit aortae. Thirty-two female and 32 male New Zealand White rabbits where gonadectomised. Vascular injury was performed with a balloon catheter in the lower abdominal aorta. Male and female rabbits were randomised into four groups of eight animals each. Only two of four groups received a 0.5% cholesterol-enriched diet. One cholesterol-diet group and one normal-diet group received intramuscular injections of estradiol valerate (1 mg/kg body weight/week). After 28 days, the denuded part of the abdominal aorta was excised and analysed by morphometry and immunohistochemistry. Estrogen treatment did not show an inhibitory effect on neointimal proliferation in normo-cholesterolemic male or female rabbits. A gender independent inhibitory effect of 17beta-estradiol was seen on atheroma development in cholesterol-fed female and male rabbits, while plasma total cholesterol levels were significantly reduced in male rabbits only. The 17beta-estradiol treatment was associated with a significantly decreased number of luminal endothelial cells in normo and hyper-cholesterolemic female rabbits, as evaluated by immunohistochemical staining for 'von Willebrand factor'. Staining for Ki-67-positive proliferating cells after 28 days showed a statistically significant increased proliferative activity in the neointima of hyper-cholesterolemic female rabbits. The neointimal content of macrophages increased significantly in all hyper-cholesterolemic rabbits. Under 17beta-estradiol treatment, the number of macrophages was increased in female and decreased in male rabbits by tendency. Additionally, the 'classical' vascular estrogen receptor was present in both female and male rabbit aortae without statistically significant differences. In conclusion, 17beta-estradiol did not reduce post-injury neointima formation in normo-cholesterolemic rabbits. However, in hyper-cholesterolemic rabbits, 17beta-estradiol reduced atheroma development gender independently. This effect cannot be explained by lowering of plasma cholesterol levels or endothelium-mediated pathways, and requires further investigation on, for example, antioxidative, antiproliferative or estrogen receptor mediated effects.

Effect of 17-beta estradiol on pre-existing atherosclerotic lesions: role of the endothelium.

The atheroprotective effects of estrogen during the process of atherogenesis is well documented, whereas limited information is available about the effect of estrogen on pre-existing atherosclerotic lesions. After bilateral ovariectomy, 24 New Zealand White rabbits were randomized into three groups of eight animals each and subsequently fed a 0.5% cholesterol diet. In group I, the vessels were excised at day 84, whereas in group II, the cholesterol diet was continued for a total of 168 days. In group III, the animals were first fed with a cholesterol diet for 84 days; in the second phase of the experiment, the cholesterol diet was continued for a further 84 days with a combined estrogen treatment (1 mg estradiol valerate per kg body weight per week intramuscularly). At the end of the experiment, the proximal aortic arch, right carotid artery, thoracical aorta and abdominal aorta of each animal were excised and prepared for histological and immunohistological examination. By day 168, morphometrical analysis displayed a significantly lower plaque development under estrogen therapy in the carotid artery (0.08+/-0.18 mm(2) vs. 0.60+/-0.39 mm(2)), the thoracic aorta (0.56+/-0.94 mm(2) vs. 3.63+/-2.06 mm(2)), and in the abdominal aorta (0.55+/-0.70 mm(2) vs. 1.71+/-1.05 mm(2)) in comparison with the corresponding 168 day control group. However, estrogen treatment has failed to reduce further atherosclerotic plaque development in the aortic arch (9.42+/-1.79 mm(2) vs. 11. 64+/-3.29 mm(2)). Immunohistological detection of the 'anti-human factor VIII related antigen', i.e. the 'von Willebrand factor' (vWF), showed a significantly lower number of luminal cells positive for vWF in the aortic arch in the 84-day cholesterol group, compared with the corresponding controls of normocholesterolemic rabbits (65. 9+/-12.4% vs. 83.1+/-6.2%; P<0.05). Estradiol was able to inhibit the further progression of atherosclerosis when moderate vessel wall alterations were present, whereas pre-existing severe atherosclerosis was associated with a failure of the anti-atherosclerotic estrogen action. As suggested by the in situ detection of vWF as a morphological marker for endothelial cells, an intact endothelial layer might play an important role in mediating the beneficial effect of estrogen in the process of atherosclerosis.

The phytoestrogens Genistein and Daidzein, and 17 beta-estradiol inhibit development of neointima in aortas from male and female rabbits in vitro after injury.

BACKGROUND: 17 beta-Estradiol and phytoestrogens are known to have beneficial effects on the cardiovascular systems of women. The exact mechanisms for how estrogens and phytoestrogens influence the cardiovascular system are not yet understood in detail. OBJECTIVE: The objective of this study was to investigate whether 17 beta-estradiol and the phytoestrogens Genistein and Daidzein have an effect on post-injury processes in vessel walls. METHODS: In this in-vitro experiment, the sex-specific effects of 50 micrograms/ml 17 beta-estradiol (equivalent to 180 mumol/l), and of the isoflavones Genistein (5 and 50 micrograms/ml, equivalent to 18.5 and 185 mumol/l), and Daidzein (5 and 50 micrograms/ml, equivalent to 19.7 and 197 mumol/l) on endothelium-denuded aortas from female and male rabbits after vascular injury were studied. Morphometry and immunohistochemistry were performed for quantitative and qualitative analysis. RESULTS: Neointimal cells were in part positive for alpha-actin staining of smooth muscle cells. Staining with 5'-bromo-2'deoxyuridine plus 2'-deoxycytidine showed that proliferative activity in the neointima had significantly decreased after 28 days for groups that had been treated with 50 micrograms/ml Genistein. Immunofluorescence staining for the expression of nuclear estrogen receptor protein in the arterial wall for aortic rings from female and male rabbits was positive. 17 beta-Estradiol, Genistein, and its analog Daidzein (with no protein tyrosine kinase activity) inhibited formation of neointima sex-independently at equivalent concentrations of 50 micrograms/ml. However, a concentration of 5 micrograms/ml Genistein decreased formation of neointima significantly for aortic rings from male rabbits only, whereas 5 micrograms/ml Genistein increased formation of neointima in rings from female rabbits, which corresponded to the increase in proliferative activity detected after 28 days. CONCLUSION: Genistein and Daidzein both inhibited proliferation at certain concentrations, so this effect is supposed to be independent from Genistein's protein tyrosine kinase activity. The antiproliferative properties of all three estrogens were observed in the absence of endothelium and therefore are independent from endothelium-mediated effects.

The discovery of the pathophysiological aspects of atherosclerosis--a review.

Considering the morphological findings in egyptian mummies at the beginning of the 20th century, atherosclerotic lesions were also apparent in pharaoh mummies more than 3500 years ago. Hippokrates (469-377 b.c.) described the sudden (cardiac) death, whereas Erasistratos had documented the typical claudication intermittens symptoms of peripheral arterial disease approximately 300 b.c. Later on in 1575, Fallopius observed severe pathological findings in arteries which he has characterized as a 'degeneration to bones', suggesting the presence of calcified atherosclerotic lesions. The relation between coronary lesions and the symptoms of angina pectoris was postulated in 1799 by Parry, however, only more than 80 years later angina pectoris was interpreted as a result of myocardial ischemia by Potain. During that time, the term 'arteriosclerosis' was firstly created by Lobstein in his 'Lehrbuch der pathologischen Anatomie', published in 1835. With the beginning of the last century, the pathophysiological aspects of plaque development were investigated in more detail by a number of researchers. In this context, people such as Saltykow, Chalatow and Anitschkow are important to notice. In 1914, Anitschkow firstly described the role of cholesterol accumulation in the vessel wall for the development of atherosclerosis. He used a cholesterol-fed rabbit model, which is the most important model of experimental atherosclerosis up to now. He also firstly described the 'Cholesterinesterphagozyten', which today commonly are known as foam cells, derived from macrophages. Using the cholesterol-fed rabbit model as well, already in 1942, Ludden et al. could demonstrate the atheroprotective effect of estrogen experimentally, a finding, which got later confirmed in the primate model and epidemiological studies. In the last three decades our knowledge has expanded by a large number of findings, based on morphological, immunohistological and molecular methods. In this context, one major contribution was the discovery of the LDL-receptor and its importance for the development of atherosclerosis by Brown and Goldstein, and the setting up of the 'response to injury hypothesis' by Ross and Glomset. At the present, we understand atherosclerosis as a complex (and at least in part as a physiological) phenomenon, beginning in the early childhood. The pathological aspect, making it to a disease, is depending on individual growth dynamics and plaque localization. The following key processes during the development of atherosclerosis are identified: 1) Endothelial injury, 2) intimal cholesterol accumulation and monocyte invasion with subsequent foam cell formation, 3) migration and proliferation of smooth muscle cells with expression of extracellular matrix 4) local thrombus formation with secondary organization 5) calcification and/or plaque rupture 6) final occlusion due to plaque rupture/thrombus formation. The classical concept of cardiovascular risk factors does only partially explain the origin of atherosclerosis. For the future, further mechanism(s) need to be identified and studied (genomic pathways, hormonal aspects, infective components, etc.) probably opening an effective therapeutical strategy to prevent and treat atherosclerotic diseases.

The value of phytoestrogens as a possible therapeutic option in postmenopausal women with coronary heart disease.

Large epidemiological studies have proved that the risk of coronary heart disease in postmenopausal women can be decreased by oestrogen replacement therapy. The effect is triggered by metabolic processes in the liver (decrease of LDL-cholesterol, increase of HDL-cholesterol) as well as by direct impact on the arterial wall (anti-oxidation, relaxation, anti-proliferation). The therapeutical usage of oestrogens is limited by an increased incidence of breast and endometrial cancer. Cyclic application of progestogens virtually eliminates the risk. Unfortunately, progestogens may antagonise the atheroprotective effect of oestrogens. Structurally modified oestrogens as well as selective oestrogen receptor modulators were investigated in clinical trials. They might provide the desired atheroprotective effects of oestrogen without negative side effects on the mammary gland or the endometrium. In this respect isoflavones also known as phytoestrogens, were analysed. They are widespread and occur naturally in many plants, especially in soy products. Cell culture and animal experiments as well as clinical studies revealed that phytoestrogens such as genistein and daidzein act atheroprotectively in the same way as oestrogen. Effects on the mammary gland or the endometrium could not be detected, but positive side effects on the bone metabolism and the decrease of certain types of cancer could be observed. In total, the therapeutical application of phytoestrogens in postmenopausal women seems to be of real and great benefit. We conclude that in women the risk of death from coronary heart disease increases after the onset of menopause. Recently discovered properties of phyto-oestrogens seem to be of great benefit as they do not seem to have any side effects on the mammary gland and the endometrium which are limiting factors for oestrogen replacement therapy.

[In vitro model to investigate the effect of estrogens on neointima development after endothelial injury in the rabbit aorta]. / In vitro Modell zur Untersuchung der Wirkung von östrogenen auf die Neointimabildung nach Endothelverletzung an der Kaninchenaorta.

Animal experiments are widely accepted in arterosclerosis research. Estrogens have lipid lowering properties and beneficial effects on the vasomotion. They act antiproliferative on those cells of the vascular wall which play a major role in lumen narrowing after vascular injury and in atherogenesis. The aim of the present study was to establish an organ culture model (rabbit aorta) to investigate post injury estrogen effects in the vessel wall. We chose the rabbit abdominal aorta which is the target organ in various animal experiments on this matter. The endothelial mono-layer was manipulated in a way that caused a measurable and reproducible post injury reaction (neointima formation). Then the effect of different estrogens (17beta-Estradiol, the phytoestrogens Genistein and Daidzein) on neointima development was investigated in male and female rabbit aortae. In equivalent dosages of 50 microg/ml all three estrogens inhibited the neointima formation significantly in male and female vessels. By the use of this organ culture model it was possible to show post injury effects of different estrogens in the vasculature while the consumption of animals was significantly reduced. Because 10 aortic segments could be taken from one aortic vessel, the number of animals that would have been necessary for an in vivo experiment could be reduced by the factor 10.

Post-injury ex vivo model to investigate effects and toxicity of pharmacological treatment in rings of rabbit aortic vessels.

Animal experiments are widely accepted in arteriosclerosis research. The aim of the present study was to establish an organ culture model (rings of rabbit aortic vessels) to investigate inhibitory estrogen effects on post injury neointima formation in the vessel wall and to examine whether these effects are cytotoxic. Estrogens are used for secondary prevention of atherosclerosis in postmenopausal women (estrogen replacement therapy/ERT). Phytoestrogens as well as the ovarian 17 beta-estradiol have been demonstrated to inhibit proliferation and migration of vascular smooth muscle cells which are key events in atherogenesis and restenosis after coronary angioplasty. In situ endothelial denudation of the thoracic and abdominal aorta was performed in female rabbits by a 3F Fogarty catheter. Segments of 5 mm were randomized in groups of n = 12 and held in culture. 17 beta-estradiol, Genistein and Daidzein were applied in concentrations of 20 microM, 30 microM, and 40 microM. Groups without estrogen treatment served as controls. The segments were investigated after 21 days. Afterwards, 3 further groups (n = 12) were held with the lowest concentrations of 17 beta-estradiol or the two phytoestrogens having been evaluated to inhibit the neointima formation significantly. After 21 days of treatment these sections were held in medium only for another 7 days to proof whether these segments were still able to proliferate. A denuded control group was held in medium only over 28 days. Compared to controls, 30 microM 17 beta-estradiol, 20 microM Genistein, and 40 microM Daidzein inhibited neointima formation significantly over 21 days. After another 7 days of cultivation in medium only the amount of neointima formation was comparable to that of non-estrogen-treated controls after 21 days. We therefore suggest that the demonstrated inhibitory effect is not explained by toxicity. In conclusion, by the use of this organ culture model it was possible to demonstrate non-toxic post injury effects of different estrogens in the vasculature. Because 24 aortic segments could be taken from one aortic vessel, the number of animals that would have been necessary for an experiment (8 to 10 per group for statistical reasons) could be markedly reduced. The results are of clinical interest because phytoestrogens and 17 beta-estradiol may offer therapeutic options for patients after coronary angioplasty regarding the process of restenosis. Because phytoestrogens do not affect the reproductive system they can also be used in men.

[The effects of estrogen in the cardiovascular system]. / Die Wirkungen von Ostrogen im kardiovaskulären System.

Estrogen replacement therapy (ERT) has been established for the treatment of perimenopausal symptoms and postmenopausal prevention of osteoporosis. Clinical (not randomized) cohort studies have shown an association of a significantly reduced cardiovascular mortality with ERT. However, a first randomized, double-blind and placebo-controlled study (HERS) could not support these findings. However, this study is limited by the use of (heterogeneous) conjugated estrogens and combined progestin treatment because animal experiments demonstrated an inhibition of protective estrogen effects by progesterone. On the other hand, experimental and clinical findings demonstrated beneficial estrogen effects on lipid metabolism, lipid-peroxidation, smooth-muscle-cell proliferation, hemostasis, and vasomotion. Actually, several authors are discussing the mediation of estrogen's effects by vascular estrogen receptors. Recent findings on different subtypes of estrogen receptors (ER-alpha and ER-beta) may explain some antagonistic effects as proliferation in the one (endometrium) and anti-proliferation in the other (vascular wall) tissue. In this context, the exact detection of the mechanism(s) of estrogen action may probably lead to new approaches in the treatment of coronary artery disease.

Does anti-atherogenic estradiol valerate treatment cause adverse effects on liver and uterus in NZW rabbits?

The rabbit has been a widely accepted animal model for atherosclerosis research since Anitschkow first used this animal in 1913 in identifying dietary-induced hypercholesterolemia as a major risk factor for atherogenesis. Experiments with cholesterol-fed rabbits have demonstrated the beneficial effects of estrogen treatment on the development of atheroma for more than 50 y. Clinical trials have found a reduction in cardiovascular events of up to 50% in postmenopausal women receiving estrogen replacement therapy. However, metabolic conditions in rabbits, as well as physiological estrogen serum levels, differ in some aspects from those in humans. In rabbits, experimentally-induced hormone levels are about 5- to 10-fold higher than those found in untreated animals. Normal physiological estrogen levels in rabbits are not cardioprotective under dietary-induced hypercholesterolemia. We investigated whether replacement induced "hyperestrogenemia" causes adverse effects on organs other than the cardiovascular system. Twenty-nine female rabbits were divided into 4 different groups, 2 without and 2 with estrogen treatment (1 mg estradiol valerate/kg body weight/w over 12 w). Organ weights, transaminases and uterine histology were examined. In rabbits treated with estrogen, we did not see relevant adverse effects on heart, kidney and liver weights, or on liver enzymes. But there was a significant increase in spleen weights, as well as notable changes in the endometrium with moderate inflammation. These findings indicate that the dosage of estrogen commonly used for atherosclerosis research does not cause serious disorders in the major organs of cholesterol-fed rabbits.

Aortic plaque size and endometrial response in cholesterol-fed rabbits treated with estrogen plus continuous or sequential progestin.

ERT is associated with a reduced incidence of coronary risk and cardiac events in postmenopausal women, but increases the risk of endometrial hyperplasia and carcinoma. Combined estrogen and progestin therapy protects the endometrium; however, its effects on heart disease risk factors are not completely known. In our study, 56 ovariectomized New Zealand White rabbits in 7 groups received a 0.5% cholesterol diet for 12 weeks. Controls were not treated with hormones. All other animals received (per kilogram body weight per week) intramuscular injections of either 0.3 mg estrogen (estradiol valerate) alone, 8.3 mg progestin (hydroxyprogesterone caproate) alone, estrogen and progestin continuously in 3 different dosages (0.3 and 8.3 mg; 1 and 8.3 mg; or 1 and 2.8 mg; estrogen and progestin, respectively), or 1 mg estrogen with 25 mg progestin sequentially in 2-week cycles. Eight non-ovariectomized animals served as further controls for endometrial analysis. Morphometric analysis of plaque size in the aortic arch showed that estrogen monotherapy, and the 3 combined therapies with 1 mg estrogen, significantly reduced intimal thickening (P<0.05). The application of progestin alone had no effect on plaque size. The endometrium was enlarged by 3-fold after estrogen treatment, and was decreased by half after progestin treatment, compared with control uteri (P<0.05). In all groups with combined hormone regimens, endometrial size was not significantly different from control uteri. However, these uteri showed more inflammatory reactions, especially when higher doses of hormones were given. In this animal model, doses of progestin that are able to successfully reduce the proliferative effect of estrogen on endometrium do not diminish the desirable antiatherosclerotic properties of estrogen.

Different effects of estrogen and progesterone on experimental atherosclerosis in female versus male rabbits. Quantification of cellular proliferation by bromodeoxyuridine.

BACKGROUND: The aim of the present study was to compare the effect of estrogen and progesterone on the development of experimental atherosclerosis in female versus male rabbits to assess possible sex-specific differences. METHODS AND RESULTS: A total of 32 female and 32 male New Zealand White rabbits were ovariectomized or castrated. In addition to a 0.5% cholesterol diet, the rabbits received estradiol alone (1 mg/kg body wt [BW] per week), progesterone alone (25 mg/kg BW per week), or combined estradiol-progesterone in these dosages during 12 weeks. Ovariectomized female and castrated male rabbits served as control groups without hormone treatment. Before excision of the vessels, bromodeoxyuridine labeling was performed to determine the extent of cellular proliferation in the atherosclerotic lesions. The aortic arch was analyzed immunohistologically and morphometrically. An inhibitory effect of estrogen on intimal plaque size was found in female rabbits compared with the ovariectomized control group (0.7 +/- 0.5 versus 3.7 +/- 2.5 mm2, P < .002; proliferating cells, 3.1 +/- 1.8% versus 8.5 +/- 2.6%, P < .002). In combination with progesterone, however, estrogen was not able to reduce intimal plaque size or cellular proliferation. In contrast, estradiol in castrated male rabbits was not associated with an inhibitory effect on cellular proliferation or intimal thickening compared with controls (estrogen treatment, 7.6 +/- 2.1% proliferating cells and 2.8 +/- 1.0 mm2 neointima; control group, 7.2 +/- 2.1% cellular proliferation and 2.9 +/- 1.2 mm2 intimal thickening). CONCLUSIONS: Our data suggest that the atheroprotective effect of estrogen is probably due to a mechanism that is present in female rabbits only.

Gender-specific differences in the effects of testosterone and estrogen on the development of atherosclerosis in rabbits.

The aim of the present study was to investigate whether there are gender-specific differences in the effects of testosterone and estrogen on the process of atherogenesis. Thirty-two castrated male and 32 ovariectomized female rabbits were separated into 4 study groups of 8 males and 8 females each and received postoperatively a 0.5% cholesterol diet for 12 weeks. During this period either no hormones, estradiol (1 mg/kg body wt/week), testosterone (25 mg/kg body wt/week IMM), or estrogen combined with testosterone in above dosages were administered. Computerized morphometric analysis of the intimal thickening in the proximal aortic arch showed a significant inhibitory effect of estrogen in female and of testosterone in male animals (P < .05). In the group with combined treatment, the plaque size in both sexes was smaller than in the animals of the control group (P < .05). These differences were independent of changes in plasma lipid parameters. The incorporation of 5'-bromo-2'-deoxyuridine, associated with cell proliferation, into cells of the neointima was not significantly affected by the different hormone application regimens in males. In females, the incorporation rate was significantly lowered in the estrogen treated group compared with the control group (P < .05). Due to the observed differences in the sex specific atheroprotective effects of testosterone and estrogen, these data suggest that complex hormone interactions, which are independent of changes in plasma lipids, may play an important role in the process of atherogenesis.