Angiotensin converting enzyme inhibitors block mitogenic signalling pathways in rat cardiac fibroblasts.

We studied the effects of angiotensin converting enzyme (ACE) inhibitors on angiotensin II (Ang II) induced growth related signalling pathways in neonatal rat cardiac fibroblasts. In BrdU proliferation assays, Ang II (10(-9)-10(-7) M) stimulated cardiac fibroblast growth in a dose-dependent fashion (maximum at 10(-7) M, 5.22 +/- 0.01-fold, n = 9). 2-2-(1-(ethoxycarbonyl)-3-phenylpropyl)[amino-oxopropyl]-6,7-dimethoxy- 1,2,3,4-tetrahydroisoquinoline-3 carboxylic acid (moexiprilat) led to a dose-dependent inhibition of the Ang II induced cardiac fibroblast growth. A less pronounced effect on cellular proliferation was seen with the ACE inhibitor enalaprilat. To elucidate the mechanisms involved in this direct antiproliferative effect of ACE inhibitors in cardiac fibroblasts, we studied the activation of mitogen-activated protein kinases [MAPKs: extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38-MAPK] and JAK/STAT janus kinases/signal transducer and activator of transcription) signal transduction pathways. Ang II (10(-7) M) caused an increase in MAPKs activity with an increased phosphorylation of ERK1/2 (1.7-fold) and p38-MAPK (3.6-fold). This effect was completely inhibited by moexiprilat (10(-7) M) and enalaprilat (10(-7) M). Stimulation with Ang II (10(-7) M) also led to an increased phosphorylation of STAT3, which is one of the key effector proteins in the JAK/STAT signalling pathway. This effect was also completely inhibited by moexiprilat (10(-7) M) and enalaprilat (10(-7) M). These data show that the ACE inhibitors moexiprilat and enalaprilat inhibit Ang II induced proliferation of cardiac fibroblasts according to their relative potency of ACE inhibition in vitro. This novel effect of ACE inhibitors is accompanied by blocking the Ang II induced activation of several intracellular signal transduction pathways (ERK1/2, p38-MAPK and STAT3).

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 17beta-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 11 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.

Expression of oestrogen receptor alpha and beta in rat heart: role of local oestrogen synthesis.

The role of cardiac oestrogen receptor expression and local oestrogen synthesis in the pathogenesis of cardiovascular disease is poorly understood. Therefore we studied the effects of the oestrogen precursors androstendione and testosterone on the expression of cyp450 aromatase, oestrogen receptor alpha and beta, and inducible NO synthase (iNOS) in neonatal rat cardiac myocytes. Here, we show that cyp450 aromatase is expressed in cardiac myocytes and incubation of cardiac myocytes with oestrogen precursors leads to sexual dimorphic transactivation of an oestrogen-responsive reporter plasmid. Furthermore, incubation with oestrogen precursors stimulated expression of oestrogen receptor alpha and beta, and iNOS in a gender-specific fashion. These data suggest that local oestrogen biosynthesis of the heart is effective to activate oestrogen receptor alpha and beta, and downstream target genes in a gender-based fashion and may therefore contribute to the beneficial effects of oestrogen in the pathogenesis of cardiovascular disease.

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 17beta-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 alpha and beta, 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.

Effects of moexiprilat on oestrogen-stimulated cardiac fibroblast growth.

1. The effects of 2-2-(1-(ethoxycarbonyl)-3-phenylpropyl)-[amino-oxopropyl]-6,7-dimethoxy- 1,2,3,4-tetrahydroisoquinoline-3 carboxylic acid (moexiprilat), 17beta-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. 2. 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. 3. 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. 4. 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. 5. Our results show that oestrone and 17beta-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.

Effects of estrogen on skeletal myoblast growth.

To determine the role of estrogen in skeletal muscle growth, we investigated estrogen receptor-mediated effects on proliferation in skeletal myoblasts. In L6, C2C12 and Sol8 myoblasts estrogen receptor was demonstrated by immunoblotting, immunofluorescence microscopy and transfection studies. Estrone induced a significant increase in myoblast growth whereas 17 beta-estradiol had no effect. Furthermore in L6-cells estrone (c-fos: 3.9-fold, egr-1: 4.6-fold) induced immediate-early gene induction significantly stronger than 17 beta-estradiol (c-fos: 1.7-fold, egr-1: 2.3-fold; p < 0.05). Skeletal myoblasts express functional estrogen receptors. Estrogens differ in the activation of skeletal myoblast growth and immediate-early gene induction.

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.