Comparison of the antiatherosclerotic effect of tibolone with that of estradiol and ethinyl estradiol in cholesterol-fed, ovariectomized rabbits.

OBJECTIVE: Tibolone is a synthetic steroid with tissue-specific estrogenic, progestogenic, and androgenic properties. The drug relieves climacteric symptoms and prevents osteoporosis but does not stimulate the endometrium. We have previously shown that in laboratory animals tibolone inhibits the atherogenesis induced by a high-cholesterol diet. Therefore, we compared the antiatherosclerotic effect of oral tibolone at different dose levels with that of oral 17beta-estradiol (E2) and ethinyl estradiol (EE). DESIGN: Atherosclerotic lesion formation (increase in vessel wall cholesterol deposition and fatty streak formation) was measured in ovariectomized rabbits after 20 weeks on an atherogenic diet (fed daily 80 g of a rabbit chow containing 0.4% cholesterol, 3.75% peanut oil, and 3.75% coconut oil) in eight groups: group 1, placebo (n = 35); group 2, control (n = 34) received normal rabbit chow; group 3, E2 group (E2 4 mg, n = 12); group 4, EE group (EE 60 microg, n = 10); and groups 5-8, tibolone (6 mg, n = 12; 2 mg, n = 13; 0.6 mg, n = 25; and 0.15 mg, n = 11, respectively). During the study, blood samples were obtained for the evaluation of plasma triglycerides, cholesterol, lipoproteins, and glutamate pyruvate transaminase. After 20 weeks, the animals were killed, and cholesterol concentration and the formation of fatty streaks in the wall of the aortic arch were evaluated. RESULTS: In the placebo group, the atherogenic diet induced a mean increase in total plasma cholesterol concentration from 1.1+/-0.1 mmol/L (control group) to 34.1+/-1.8 mmol/L (mean +/- SE). This resulted in an accumulation of cholesterol in the aortic arch from 48+/-4 (control group) to 608+/-44 nmol/mg protein and in the formation of fatty streaks (41.8+/-3.2% of the surface of the aortic arch was covered with fatty streaks). Tibolone had strong dose-dependent antiatherosclerotic effects. It reduced the accumulation of cholesterol in the aortic arch at doses of 6 to 0.15 mg by 99, 97, 87, and 57% and the formation of fatty streaks by 98, 97, 81, and 38%, respectively. E2 had only a marginal antiatherosclerotic effect, whereas EE showed an effect comparable to that of tibolone at doses of 2 to 0.6 mg. With EE, the accumulation of cholesterol in the vessel wall was reduced by 93% and the formation of fatty streaks by 73%. Mean plasma cholesterol concentrations were also reduced by tibolone (64, 70, 61, and 47%) and EE (57%). This reduction was mainly mediated via a reduction in beta-very-low-density lipoprotein cholesterol. Analysis, however, indicated that the observed antiatherosclerotic effects of tibolone and EE, at least partly, are due to a direct effect on the vessel wall and independent of the changes in plasma cholesterol. At equipotent antiatherosclerotic doses, EE showed a stronger uterotropic effect (measured as the increase in uterine weight) than tibolone. EE increased uterine weight from 0.57 g/kg body weight (BW) (control group) to 3.5 g/kg BW; tibolone at doses of 6, 2, 0.6, and 0.15 mg increased uterine weight to 2.5, 2.8, 2.2, and 1.3 g/kg BW, respectively. CONCLUSION: Tibolone can protect the arterial vessel wall against atherosclerotic lesions induced by a hypercholesterolemic diet. However, it has much less estrogenic effects on the uterus compared with EE at equipotent doses, indicating tissue selectivity for tibolone. The clinical implications of these findings require investigation.

Tibolone prevents atherosclerotic lesion formation in cholesterol-fed, ovariectomized rabbits.

Tibolone (Org OD14), a synthetic steroid with estrogenic and progestogenic/androgenic properties, is clinically effective for the treatment of climacteric symptoms and the prevention and treatment of osteoporosis in postmenopausal women. The effect on atherogenesis, however, is not known. In the current study, we investigated the effect of tibolone in comparison with that of estradiol and norethisterone acetate on atherogenesis in 140 ovariectomized New Zealand White rabbits that had been induced by an atherogenic diet (0.4% cholesterol, 20 weeks). Tibolone at 18, 6, or 2 mg/d orally completely prevented cholesterol accumulation and fatty streak formation in the aorta; the impairment of endothelium-dependent smooth muscle relaxation of the aorta; and complex lesion formation after endothelial denudation in the carotid artery. Tibolone also reduced the increased postovariectomy plasma lipid concentrations. Analysis of the results, however, indicated that a substantial part of the strong, beneficial effects were plasma lipid independent. Compared with subcutaneous estradiol decanoate (150 microgram once weekly) and oral 17beta-estradiol (4 mg/d), the effects of tibolone were more pronounced at equipotent uterotropic activity. Norethisterone acetate (1 mg/d) did not affect atherosclerotic lesion formation. There are no indications that the progestogenic/androgenic properties of tibolone counteracted its atheroprotective effect on the vessel wall. Therefore, tibolone has the intrinsic potential to be a compound that protects the arterial vessel wall against atherosclerotic processes.

Formation of intimal cushions in the ductus arteriosus as a model for vascular intimal thickening. An immunohistochemical study of changes in extracellular matrix components.

There is a great resemblance in the sequence of events that take place in the pathological development of intimal thickening, so called arteriosclerosis and the formation of intimal cushions in both the normal ductus arteriosus (DA) and the persistent ductus arteriosus (PDA). The human DA was used as a model to study the changes in the extracellular matrix during this process with immunohistochemistry. The formation of intimal cushions was studied in 4 normal fetal DA, 4 normal mature DA and 3 persistent DA. The process of intimal thickening in the fetus starts in the second trimester of pregnancy with an accumulation of glycosaminoglycans in the subendothelial region (SER), accompanied by separation of endothelial cells from the internal elastic lamina and followed by migration of smooth muscle cells into the subendothelial region. This phenomenon was also observed in the mature DA in the neonate, indicating that cushion formation is a continuous process. Intimal cushions had also developed in the persistent DA, although they were morphologically different from the cushions found in the normal mature DA. It was remarkable that two elastic lamellae could be distinguished: one at the original site on the borderline of intimal cushion and media and the other in a subendothelial position. The endothelial cells were firmly attached to this subendothelial lamina, which was wrapped in the basal lamina components laminin and type IV collagen. The main morphological difference between the normal mature DA and the persistent DA is the close relation between endothelial cells and the subendothelial elastic lamina, suggesting an altered elastin metabolism in the PDA. PGI2 synthase was increased in the wall of both the normal and persistent DA as compared with the aorta. It may be related to a role of PGI2 in the formation of intimal cushions.

Changes in distribution of elastin and elastin receptor during intimal cushion formation in the ductus arteriosus.

Changes in distribution of elastin and elastin receptor were studied during maturation of the ductus arteriosus. In this vessel intimal thickening is a normal process. It starts with separation of endothelial cells from the internal elastic lamina by the formation of a wide subendothelial region, followed by an increase in number of radially orientated cells in the inner media and subendothelial region. For the first time the nature of these originally inner media cells could be definitely determined as being derived from smooth muscle cells. Areas rich in these modified smooth muscle cells contained less elastin as compared with regions rich in circularly orientated smooth muscle cells. The internal elastic lamina had disappeared underneath intimal cushions of the canine ductus arteriosus, whereas in the fetal human ductus arteriosus, splitting of the internal elastic lamina was observed. Elastin was not present underneath separated endothelial cells, which suggests that these cells do not synthesize elastin, at least not after separation. Both smooth muscle cells and endothelial cells demonstrated the presence of the elastin receptor. Changes in its distribution were not observed. The temporal and spatial association between the altering distribution of elastin and the absence of normal cushion formation in the persistent ductus arteriosus suggests a role of the elastin receptor in the process of intimal thickening.

Distribution of prostacyclin synthase, 6-keto-prostaglandin F1 alpha, and 15-hydroxy-prostaglandin dehydrogenase in the normal and persistent ductus arteriosus of the dog.

The presence of prostacyclin synthase (PGI2 synthase), 6-keto-prostaglandin F1 alpha (6k-PGF1 alpha), and the stable hydrolysis product of prostacyclin (PGI2), prostaglandin E2 (PGE2), as well as the activity of 15-hydroxy-prostaglandin dehydrogenase (PGDH) were studied in the aorta, pulmonary artery, the normal ductus arteriosus (DA), and persistent DA (PDA) of the dog using histochemical and immunohistochemical techniques. The normal DA is characterized by the development of intimal thickening, a process that does not occur in the persistent DA. Distribution of PGI2 synthase was identical in the aorta, pulmonary artery, and persistent DA. In these vessels endothelial cells contained higher levels of PGI2 synthase as compared with medial smooth muscle cells. In the normal DA, levels of PGI2 synthase were clearly higher in smooth muscle cells at the sites of intimal thickening than at other sites. Distribution of 6-keto-PGF1 alpha resembled the localization of PGI2 synthase. Presence of PGE2 and activity of PGDH could not be demonstrated. The results demonstrated existence of a clear relationship between ductal morphology and the presence of PGI2 synthase. This finding suggests a more important role for PGI2 in regulating ductal patency than has heretofore been appreciated. It was assumed that the role of PGI2 in regulating ductal patency is, at birth, at least overruled by the constrictive effect of the cytochrome P450 mono-oxygenase mechanism. It is still possible to attribute a role to PGI2 in the regulation of cushion formation. Once smooth muscle cell activity has been enhanced by the presence of a glycosaminoglycan rich environment, increase in PGI2 may produce a concurrent inhibition of smooth muscle cell growth.

Ultrastructural and immunohistochemical changes of the extracellular matrix during intimal cushion formation in the ductus arteriosus of the dog.

The changes of the intima during subendothelial edema formation were studied by ultrastructural and immunohistochemical methods in the ductus arteriosus (DA) of the dog. Subendothelial edema formation is the first stage in the development of intimal cushions in the DA. Development of intimal cushions is a physiological process preceding normal spontaneous closure after birth. The material consisted of normal canine DA and DA from a strain of dogs with hereditary persistence of the DA (PDA). In the normal DA intimal thickening starts with separation of the endothelial cells from the internal elastic lamina by a widened subendothelial region (SR). Initially this SR is, at the ultrastructural level, composed of granular and amorphous material. Collagen fibrils and elastin are not detected. During the formation of the SR a shedding of the basal lamina underneath the endothelial cells is observed. In the PDA the endothelial cells remain attached to the internal elastic lamina. The topography of the extracellular matrix components collagen type I, III, IV, fibronectin and laminin were studied immunohistochemically. These are important factors in the adherence of the endothelial cells to the underlying intimal layers. Laminin and collagen type I are diffusely present before but absent after detachment of the endothelial cells. Collagen type III, barely detectable before detachment, becomes visible underneath the detached cells. No changes are observed in the distribution of collagen type IV and fibronectin. Comparison of the normal DA with the various types of the PDA strain and controls allowed the conclusion that the observed changes in the extracellular matrix components were confined to those parts of the vessel wall that showed development of intimal thickening. The observed alterations both at the ultrastructural and immunohistochemical level do not explain the initiation of the process of endothelial cell detachment, which have been shown in a previous study to be related to an increase in hyaluronic acid.

Hyaluronic acid accumulation and endothelial cell detachment in intimal thickening of the vessel wall. The normal and genetically defective ductus arteriosus.

The closing ductus arteriosus (DA) was studied as a model for the development of intimal thickening of vessel walls using ultrastructural and immunohistochemical techniques. The material consisted of DA from neonatal dogs of three types: normal beagles, DA-defective pups from a line of mixed poodles with a genetic defect in the closure of the DA leading to persistent ductus arteriosus (PDA line), and normal litter-mates of DA-defective pups in the PDA line. The DA of the normal litter-mates of DA-defective pups did not differ from those of normal beagles. In the DA of normal beagles and normal PDA-line pups, closure is preceded by intimal thickening characterized by formation of a widened subendothelial region (SR), detachment of endothelial cells, invagination of endothelial cells, and migration of smooth muscle cells into the SR. It was observed that immediately before and after endothelial cell detachment, there was an increase in hyaluronic acid (HA) in the SR and inner media. In the DA-defective pups, the increase in hyaluronic acid failed to occur and there was no intimal thickening. The SR failed to expand, endothelium remained attached to the internal elastic membrane, and there was no invagination of endothelium or migration of smooth muscle cells. It is hypothesized that the increased synthesis of HA is an important early event leading to intimal thickening in the normal DA and perhaps to abnormal intimal thickening of other vessels. By its hygroscopic properties, HA may be directly involved in the formation of a wide SR, inducing endothelial cell detachment and favoring smooth muscle cell migration. In affected pups of the PDA line, there is a genetically-determined "block" in the normal process of intimal thickening at or before the initiation of increased HA synthesis.