Christina Lenz

Effect of Testosterone on Plaque Development and Androgen Receptor Expression in the Arterial Vessel Wall

Background —Recent studies have suggested that testosterone has a protective effect in the arterial vascular system. However, little is known about the molecular aspects of the mechanism(s) involved in these processes. The aim of the present study was to investigate the effect of testosterone on neointimal plaque development and on the expression of the vascular androgen receptor. Methods and Results —Neointimal plaque formation was induced by endothelial denudation in the aortas of male New Zealand White rabbits. Aortic ring segments were cultured for 21 days after endothelial denudation. Testosterone was applied to the culture medium in different doses. Compared with the non–hormone-treated control group, a significant inhibition of neointimal plaque development (expressed as the intima/media ratio) was found at testosterone concentrations of 10 ng/mL (P =0.037) and 100 ng/mL (P =0.012; intima/media ratios: median of controls, 0.25; median of 10 ng/mL testosterone group, 0.15; median of 100 ng/mL testosterone group, 0.16). Associated with this inhibitory effect on plaque size was a 50% increase of the amount of androgen receptor mRNA in the arterial segments treated with testosterone. Conclusion —The beneficial effects of testosterone on postinjury plaque development underlines, at least in males, the important role of androgens in the vascular system. As our data suggest, the vascular androgen receptor is probably involved in these processes. Further studies are required to characterize the androgen receptor–dependent pathways in the vascular system.

Incidence of intimal proliferation and apoptosis following balloon angioplasty in an atherosclerotic rabbit model

OBJECTIVE The aim of this study was to determine the occurrence of apoptosis in relation to the proliferative response in the intimal layer after experimental balloon angioplasty of a pre-existing plaque. METHODS After induction of an intimal plaque in the right carotid artery by electrical stimulation, 26 rabbits underwent balloon angioplasty. Twelve animals served as a control group without performance of angioplasty after plaque induction. To study the time course of intimal apoptosis and cell proliferation the vessels were excised on day 7, 14 and 28 after balloon angioplasty. For in situ detection of apoptosis, the TUNEL-technique (TdT-mediated d-UTP fluorescein nick end labeling) was used. In addition, bromodeoxyuridine labeling in all animals allowed the determination of the percentage of cells undergoing DNA synthesis in the neointimal area. Additionally, smooth muscle cells were detected by immunostaining of alpha-actin and macrophages by a specific antibody (RAM 11). RESULTS Within 28 days of balloon angioplasty, the number of cells undergoing apoptosis remained at a very low level and was not significantly different to the control group without interventional treatment (controls: 0.1 +/- 0.15%; 7 days: 0.44 +/- 0.68%; 14 days: 0.13 +/- 0.11%; 28 days: 0.1 +/- 0.1%). In contrast, the number of cells undergoing DNA synthesis was significantly increased at day 7 after angioplasty (3.72 +/- 2.0% vs. 0.51 +/- 0.29% in controls), resulting in an increase of the total intimal area from 0.088 +/- 0.037 mm2 in the control animals up to 0.256 +/- 0.172 mm2 at day 28 following balloon dilatation. CONCLUSIONS Our data showed that significant changes in the occurrence of apoptosis are not involved in the regulation of cellular turnover during the examined time period after vessel wall injury. The lacking up-regulation of apoptosis in comparison to the increased cell proliferation in order to maintain the tissue balance is perhaps an important regulatory mechanism leading to intimal hyperplasia after vascular injury in this animal model. Overall, we suggest that there may be a delicate balance between cell proliferation and apoptosis in smooth muscle cells of the vessel wall, and only small shifts in this balance could account for both cellular accumulation in restenotic lesions as well as cell death in mature atheroma.

Reduction of post injury neointima formation due to 17β-estradiol and phytoestrogen treatment is not influenced by the pure synthetic estrogen receptor antagonist ICI 182,780 in vitro

BackgroundAnimal and organ culture experiments have shown beneficial inhibitory estrogen effects on post injury neointima development. The purpose of this study was to investigate whether such estrogen effects are influenced by the estrogen receptor antagonist ICI 182,780. Different concentrations of 17β-estradiol and the phytoestrogens genistein and daidzein were tested.MethodsF emale New Zealand White rabbits were benumbed. In situ vascular injury of the thoracic and abdominal aorta was performed by a 3F Fogarty catheter. Segments of 5 mm were randomised and held in culture for 21 days. Three test series were performed: 1) control group – 20 μM ICI – 30 μM ICI – 40 μM ICI. 2) control group – 20 μM ICI – 40 μM 17β-estradiol – 40 μM 17β-estradiol + 20 μM ICI. 3) control group – 20 μM ICI – 40 μM daidzein – 40 μM daidzein + 20 μM ICI – 20 μM genistein – 20 μM genistein + 20 μM ICI. After 21 days the neointima-media-ratio was evaluated.Results1) Treatment with ICI 182,780 did not reduce neointima formation significantly (p = 0.05). 2) 40 μM 17β-estradiol alone (p < 0.0001) and in combination with 20 μM ICI (p < 0.0001) reduced neointima formation significantly. 3) 20 μM genistein alone (p = 0.0083) and combined with 20 μM ICI (p = 0.0053) reduced neointima formation significantly. 40 μM daidzein did not have a significant (p = 0.0637) effect.ConclusionsThe estrogen receptor antagonist ICI 182,780 did not modulate the inhibitory estrogen effects on post injury neointima formation. These results do not support the idea that such effects are mediated by vascular estrogen receptors.

Die Wirkungen von Östrogen 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-α and ER-β) 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. Die Östrogen- substitution ist ein etabliertes Therapieverfahren in der Behandlung perimenopausaler Beschwerden und der postmenopausalen Osteoporose-Prophylaxe. Darüber hinaus war die Östrogensubstitution in klinischen (nicht randomisierten) Kohortenstudien mit einer signifikanten Reduktion der Koronarmortalität assoziiert. Allerdings konnte die erste randomisierte, Plazebo-kontrollierte Doppelblindstudie unter Verwendung einer Kombination aus konjugierten Pferdeöstrogenen und Progesteron diese Ergebnisse nicht stützen. Limitiert wird diese Studie jedoch durch die Auswahl der (heterogenen) Östrogenkomponente sowie die kontinuierliche kombinierte Progesterongabe, da tierexperimentell eine Hemmung protektiver Östrogeneffekte durch bestimmte Gestagene demonstriert wurde. Andererseits wurden experimentell und klinisch günstige Wirkungen von Östrogen auf den Lipidmetabolismus, die Lipidoxidation, Proliferation glatter Gefäßmuskelzellen, die Hämostase und die Vasomotion gezeigt. Verschiedene Autoren diskutieren derzeit eine mögliche Wirkungsvermittlung von Östrogen über gefäßwandständige Östrogenrezeptoren. Jüngste Entdeckungen von zwei Subtypen des Östrogenrezeptors (Östrogenrezeptor α und β) könnten solch gegensätzliche Effekte wie die proliferative Wirkung in dem einen (Endometrium) und die antiproliferative Wirkung in einem anderen (Gefäßwand) Gewebe erklären. Möglicherweise kann die exakte Aufklärung des Wirkprinzips von Östrogen auf Gefäßwandebene in der Zukunft neue Ansatzpunkte in der Behandlung der koronaren Herzkrankheit ergeben.

Cardiovascular oestrogen effects in women

Introduction Oestrogen replacement therapy (ERT) is an established treatment for perimenopausal symptoms and for prevention of postmenopausal osteoporosis. Clinical (not randomised) cohort studies have shown a strong association of reduced cardiovascular mortality with ERT. However, a first randomised, double-blinded and placebo controlled study (HERS) could not support these findings. This study was limited by the use of (heterogeneous) conjugated oestrogens together with combined progesterone treatment because animal experiments have demonstrated an inhibition of the protective oestrogen effects by progesterone. In addition, the observation period was just 4·1 years compared to 18 years in the cohort Nurses’ Health Study. Experimental and clinical findings demonstrated beneficial oestrogen effects on lipid metabolism, lipidperoxidation, smooth-muscle-cell proliferation, haemostasis and vasomotion. Several authors are correctly debating the mediation of oestrogen effects by vascular oestrogen receptors. Recent findings on different subtypes of oestrogen receptors (ER-a and ER-b) may explain some antagonistic effects as proliferation in the one (endometrium) and antiproliferation in the other (vascular wall) tissue. In this context, the exact detection of the mechanism(s) of oestrogen action may probably lead to new approaches in the treatment of coronary artery disease.

EFFECT OF THE ANTIOXIDANT NICANARTINE ON THE PROLIFERATIVE AND INFLAMMATORY RESPONSE AFTER EXPERIMENTAL BALLOON ANGIOPLASTY

BackgroundAntioxidant treatment seems to reduce the development of restenosis after percutaneous transluminal angioplasty. In this study, the effect of Nicanartine, a new antioxidant drug with both antiproliferative and lipid-lowering properties, on the proliferative and inflammatory response after balloon angioplasty was investigated in a rabbit model of restenosis. MethodsTo induce pre-interventional plaques in the common carotid artery of 48 New Zealand White rabbits, electrostimulation was carried out for 28 days. After a break of 7 days, balloon angioplasty was performed in 36 animals, of which 18 received Nicanartine at a dose of 120 mg/kg body weight; the other 18 served as a control group. The vessels were excised by day 7 and 28 after balloon angioplasty and examined for intimal plaque size, macrophage content and proliferative activity. Bromodeoxyuridine labeling was used to determine proliferating cells in the dilated segment; macrophages were detected using the RAM-11 antibody. ResultsIn the Nicanartine-treated group, immunohistological quantification 7 days after intervention showed a statistically significant (P < 0.05) reduction of both cells undergoing DNA synthesis (1.6 ± 1.4% versus 3.7 ± 2.2%) and intimal macrophages (0.7 ± 1.2% versus 1.3 ± 0.6%). Twenty-eight days after balloon angioplasty, proliferative activity in both groups was decreased to a level comparable to the non-dilated control groups. A clear trend towards smaller plaques could be seen in the Nicanartine group (0.146 ± 0.077 mm2 versus 0.255 ± 0.174 mm2). Total cholesterol levels did not differ significantly between the groups. ConclusionUnder treatment with Nicanartine a clear reduction in the proliferative and inflammatory response after balloon angioplasty was observed. Antioxidant treatment, especially with compounds having antiproliferative and lipid-lowering properties, appears to be an effective secondary preventive strategy after interventional treatment in patients with coronary artery disease.