Stefan Kahlert

Estrogen Modulates AT1 Receptor Gene Expression In Vitro and In Vivo

BACKGROUND The AT1 receptor has been implicated in the pathogenesis of hypertension and atherosclerosis. Estrogen deficiency is also associated with cardiovascular diseases. Therefore, we examined the AT1 receptor gene expression in ovariectomized rats with and without estrogen replacement therapy and the influence of estrogen on AT1 receptor expression in cultured vascular smooth muscle cells. METHODS AND RESULTS Rat aortic tissue was examined 5 weeks after ovariectomy. In one group, estrogen (1.7 mg estradiol) was administered during the 5-week period. Functional experiments assessed angiotensin II-induced contraction of aortic rings. AT1 receptor mRNA levels were measured by quantitative polymerase chain reaction and Northern blotting. AT1 receptor density was assessed by radioligand binding assays. These techniques were also applied in cultured vascular smooth muscle cells. The efficacy of angiotensin II on vasoconstriction was significantly increased in aortas from ovariectomized rats. As assessed by radioligand binding assays, AT1 receptor density was increased to 160% without changes in receptor affinity during estrogen deficiency. AT1 receptor mRNA levels were consistently increased to 187% in ovariectomized rats compared with sham-operated animals. Estrogen substitution therapy in ovariectomized rats reversed this AT1 receptor overexpression. To explore the underlying mechanisms, the direct influence of estradiol on AT1 receptor expression was investigated in VSMCs. Estradiol (1 micromol/L) led to a time-dependent downregulation of AT1 receptor mRNA, with a maximum of 33.3% at 12 hours. There was a correlative decrease in AT1 receptor density. CONCLUSIONS This novel observation of estrogen-induced downregulation of AT1 receptor expression could explain the association of estrogen deficiency with hypertension and atherosclerosis, because activation of the AT1 receptor plays a key role in the regulation of blood pressure, fluid homeostasis, and vascular cell growth.

Cardiac myocytes and fibroblasts contain functional estrogen receptors

Gender‐based differences found in cardiovascular diseases raise the possibility that estrogen may have direct effects on cardiac tissue. Therefore we investigated whether cardiac myocytes and fibroblasts express functional estrogen receptors. Immunofluorescence demonstrated estrogen receptor protein expression in both female and male rat cardiac myocytes and fibroblasts. Nuclear translocation of the estrogen receptor protein was observed after stimulation of cardiomyocytes with 17β‐estradiol (E2). Cells transfected with an estrogen‐responsive reporter plasmid showed that treatment with E2 induced a significant increase in reporter activity. Furthermore, E2 induced a significant increase in expression of the estrogen receptors α and β, progesterone receptor and connexin 43 in cardiac myocytes. Cardiac myocytes and fibroblasts contain functional estrogen receptors and estrogen regulates expression of specific cardiac genes. These data suggest that gender‐based differences in cardiac diseases may in part be due to direct effects of estrogen on the heart.

17β-Estradiol stimulates expression of endothelial and inducible NO synthase in rat myocardium in-vitro and in-vivo

Objectives: NO production has been attributed to play a major role in cardiac diseases such as cardiac hypertrophy and cardiac remodeling after myocardial infarction which display significant gender-based differences. Therefore we assessed the effect of 17β-estradiol (E2) on estrogen receptor (ER) α and β and endothelial and inducible NO synthase in neonatal and adult rat cardiomyocytes. Methods: The presence of ERα and ERβ was demonstrated by immunofluorescence and western blot analysis as well as the expression pattern of inducible NO synthase (iNOS) and endothelial NOS (eNOS) in isolated cardiomyocytes from neonatal and adult rats. Furthermore, regulation of myocardial iNOS and eNOS expression by estrogen was evaluated in the myocardium from ovariectomized or sham-operated adult Wistar-Kyoto rats. Results: Incubation with E2 led to translocalization of the ER into the nucleus and increased receptor protein expression. E2 stimulated expression of iNOS and eNOS in both neonatal and adult cardiac myocytes. Coincubation with the pure anti-estrogen ICI 182.780 inhibited upregulation of ER and NOS expression. In ovariectomized rats myocardial iNOS and eNOS protein levels were significantly lower compared to sham-operated female animals. Conclusion: Taken together, these results show that E2 stimulates the expression of iNOS/eNOS in neonatal and adult cardiomyocytes in-vivo and in-vitro. These novel findings provide a potential mechanism of how estrogen may modulate NOS expression and NO formation in the myocardium.

Toxic effects of X-linked adrenoleukodystrophy-associated, very long chain fatty acids on glial cells and neurons from rat hippocampus in culture

Saturated very long chain fatty acids (VLCFAs; > or =C22:0) accumulate in X-linked adrenoleukodystrophy (X-ALD, OMIM 300100), a severe hereditary neurodegenerative disease, due to peroxisomal impairment. Previous studies analysed the development of X-ALD in humans and gene knockout animal models. However, the toxic effect of VLCFA leading to severe symptoms with progressive and multifocal demyelination, adrenal insufficiency and inflammation still remains unclear. To understand the toxic effects of VLCFA in the brain, here we exposed neural cells to VLCFA and analysed the cellular consequences. We found that oligodendrocytes and astrocytes challenged with docosanoic- (C22:0), tetracosanoic- (C24:0) and hexacosanoic acids (C24:0) die within 24 h. VLCFA-induced depolarization of mitochondria in situ and increased intracellular Ca2+ level in all three brain cell types provides indications about the mechanism of toxicity of VLCFA. Interestingly, VLCFAs affect to the largest degree the myelin-producing oligodendrocytes. In isolated mitochondria, VLCFAs exert a detrimental effect by affecting the inner mitochondrial membrane and promoting the permeability transition. In conclusion, we suggest that there is a potent toxic activity of VLCFA due to dramatic cell physiological effects with mitochondrial dysfunction and Ca2+ deregulation. This provides the first evidence for mitochondrial-based cell death mechanisms in neurodegenerative disease with peroxisomal defects and subsequent VLCFA accumulation.

Differential effects of 17β-estradiol on mitogen-activated protein kinase pathways in rat cardiomyocytes

Cardiac myocytes contain functional estrogen receptors, however, the effect of estrogen on growth-related signaling pathways such as mitogen-activated protein kinases (MAPK) in the pathogenesis of cardiac disease is unclear. MAPKs are critically involved in regulatory signaling pathways which ultimately lead to cardiac hypertrophy. Here we show that 17beta-estradiol (E2) activates extracellular signal-regulated kinase (ERK1/2), c-Jun-NH2-terminal protein kinase (JNK) and p38 in rat cardiomyocytes in a distinctive pattern. As shown by immunoblot analysis and phosphorylation assays, E2 (10(-9) M) induced a rapid and transient activation of ERK1/2 and a rapid but sustained increase of JNK phosphorylation. In contrast, E2 had only a marginal effect on p38 activation. Furthermore, MAPK phosphatase expression was induced by E2 and E2-stimulated expression of endothelial and inducible NO synthase was inhibited by PD 98059, an inhibitor of the ERK pathway. These novel observations may help to explain the role of estrogen in gender-based differences found in cardiac disease.

Modulation of hypertensive heart disease by estrogen

UNLABELLED Left ventricular hypertrophy is an independent risk factor for morbidity and mortality in patients with hypertensive heart disease. Cardiac hypertrophy, associated with increased cardiac fibrosis and myocardial relaxation impairment, shows gender-based differences with significantly higher mortality in men. The role of estrogen in the pathogenesis of this process is poorly understood. After our previous demonstration that cardiac myocytes and fibroblasts contain functional estrogen receptors, we therefore investigated: 1) the influence of different estrogen metabolites on cardiac fibroblast growth; 2) the influence of different estrogen metabolites on the expression of the immediate early gene c-Fos; 3) the influence of estrogen on the L-type calcium channel in cardiomyocytes. METHODS 1) Neonatal rat cardiac fibroblasts were incubated with 17 beta-estradiol, estrone, 2-hydroxyestrone, and 2-methoxyestradiol (all 10(-9) M). Bromodeoxyuridine incorporation was measured after 24 h. 2) c-Fos expression was demonstrated by immunoblotting. 3) L-type (Ca2+) current with and without 17 beta-estradiol was assessed in adult guinea pig cardiomyocytes by whole cell patch clamp. RESULTS Cardiac fibroblast growth was stimulated by estrogen metabolites with 2-hydroxyestrone as the most potent activator; in addition, 10(-5) M 17 beta-estradiol reduced the L-type Ca2+ current by about 20% in cardiomyocytes. CONCLUSIONS Estrogen induces both short term effects (non-genomic) and long term effects (genomic) on the heart and may therefore account for gender- and age-based differences in hypertensive heart disease.

Angiotensin converting enzyme inhibition modulates cardiac fibroblast growth

Background The progression of left ventricular hypertrophy and cardiac fibrosis in hypertensive heart disease is influenced by sex and age. Although angiotensin converting enzyme inhibition has been shown to prevent progression of the disease in postmenopausal women, the interaction of angiotensin II and estrogen in this process before and after the menopause is poorly understood. Objective To investigate the influence of the angiotensin converting enzyme inhibitor moexiprilat on serum, estrogen and angiotensin II-induced cardiac fibroblast growth. Methods Neonatal rat cardiac fibroblasts were incubated with 1 and 10% fetal calf serum, 10−7 mol/l angiotensin II, 10−9 mol/l estrone, 10–9 mol/l 17β-estradiol and 10−8 mol/l moexiprilat. Proliferation was measured in terms of incorporation of bromodeoxyuridine. Western blot analysis was performed using antibodies directed against the growth-related immediate early genes c-fos and Sp-1. All experiments were performed at least three times. Results Fetal calf serum stimulated cardiac fibroblast proliferation (1% fetal calf serum 2.0 ± 0.028-fold; 10% fetal calf serum 2.7 ± 0.028-fold). Angiotensin II and estrone stimulated proliferation of cardiac fibroblasts grown in the absence of fetal calf serum (angiotensin II 4.2 ± 0.075-fold; estrone 2.9 ± 0.034-fold) and further increased proliferation in the presence of 1% fetal calf serum (angiotensin II 4.3 ± 0.072-fold); (estrone 3.8 ± 0.045-fold) and 10% fetal calf serum (angiotensin II 4.8 ± 0.112-fold; estrone 4.1 ± 0.047-fold). Coincubation with moexiprilat specifically inhibited proliferation induced by angiotensin II and estrone but not by serum, and angiotensin II type 1 receptor blockade inhibited angiotensin II-induced but not estrone-induced cell growth. Western blot analysis showed that the expression of c-fos and Sp-1 was induced in a time-dependent fashion by angiotensin II (to maxima of 5.0-fold for c-fos and 3.0-fold for Sp-1) and estrone (15.2-fold for c-fos and 6.2-fold for Sp-1). This effect was completely inhibited by moexiprilat. Conclusions Angiotensin converting enzyme inhibition modulates cardiac fibroblast growth induced by angiotensin II and estrone. This mechanism might contribute to the beneficial effects of angiotensin converting enzyme inhibition in postmenopausal patients with hypertensive heart disease. J Hypertens 16:377–384

Effects of moexiprilat on oestrogen-stimulated cardiac fibroblast growth

The effects of 2‐2‐(1‐(ethoxycarbonyl)‐3‐phenylpropyl)‐[amino‐oxopropyl]−6,7‐dimethoxy‐1,2,3,4‐tetrahydroisoquinoline‐3 carboxylic acid (moexiprilat), 17β‐oestradiol (E2), oestrone (ES) and angiotensin II (AII) on growth and activation of oestrogen receptors and the immediate‐early gene egr‐1 were investigated in neonatal rat cardiac fibroblasts of female and male origin. In BrdU proliferation assays, oestrone (10−7–10−9 M) stimulated cardiac fibroblast growth in a concentration‐dependent fashion (maximum at 10−7 M, 4.0 fold±0.14 in female and 3.1 fold±0.06 in male cells, n=9, P<0.05), while E2 (10−7–10−9 M) had no effect. Moexiprilat (10−7 M) completely inhibited oestrone‐induced cardiac fibroblast growth. Angiotensin II (10−7 M) induced cardiac fibroblast growth (female 4.1 fold±0.1/male 3.9 fold±0.2; n=9, P<0.05). Angiotensin II induced oestrogen receptor (maximum 21.8 fold at 60 min) and egr‐1 (maximum 47.5 fold at 60 min) expression in a time‐dependent fashion. In immunoblot experiments, oestrogen activated oestrogen receptor (ES: 12.8 fold±2.0; E2: 14.7 fold±4.9; n=3, P<0.05) and egr‐1 (ES: 5.1 fold,±0.24; E2: 3.8 fold,±0.25; n=3, P<0.05) expression. The induction of oestrogen receptor and egr‐1 protein expression was time‐dependent and inhibited by moexiprilat. Our results show that oestrone and 17β‐oestradiol reveal a significant difference in their potential to activate cardiac fibroblast growth in female and male cells and that oestrone‐stimulated growth is inhibited by moexiprilat. The inhibition of oestrone‐stimulated cardiac fibroblast growth by moexiprilat may contribute to the beneficial effects seen in postmenopausal women with hypertensive heart disease treated with ACE inhibitors.