Stimulation of serum- and glucocorticoid-regulated kinase-1 gene expression by endothelin-1.

The serum- and glucocorticoid-regulated kinase-1 (SGK1) participates in the regulation of sodium homeostasis and blood pressure by mineralocorticoids. Aldosterone rapidly induces SGK1 transcription, which contributes to the activation of renal epithelial sodium channels. Another important regulator of blood pressure is the vasoactive hormone endothelin-1 (ET-1) that is systemically upregulated in chronic renal failure. In the present study, we investigated whether ET-1 modulates SGK1 expression, and thereby might explain some of its hypertensive effects. As assessed by real-time PCR analysis, ET-1 triggered the rapid increase of SGK1 mRNA levels in A-10 smooth muscle cells and also in intact aortas of adult rats. In A-10 cells transcriptional activation was associated with a more than 6-fold upregulation of SGK1 protein expression and in similar range as found after treatment with aldosterone. A stimulatory effect of ET-1 was not only observed in isolated cells, but also in an animal model. Upon subtotal nephrectomy (SNX) of rats, myocardial ET-1 levels strongly increased, which was followed by a more than 2-fold induction of SGK1 expression in the left ventricle. The myocardial upregulation of SGK1 was completely abrogated by a specific ET(A) receptor antagonist, thereby substantiating the in vivo role of ET-1 in SGK1 expression. Thus, these data demonstrate that ET-1 increases expression of SGK1 in vivo and in vitro, and therefore indicate that SGK1 upregulation might be involved in ET-1-dependent regulation of blood pressure and cardiac modelling during mild renal failure.

Modulation of the beta-adrenergic receptor system of vascular smooth muscle cells in vitro and in vivo by chronically elevated endothelin-1 levels.

Endothelin-1 (ET-1) levels are chronically elevated in several cardiovascular diseases and correlate with an increased mortality. However, in contrast to acute biological activities such as vasoconstriction, little is known about long-term effects of ET-1. In this study we determined the effects of ET-1 on the beta(2)-adrenergic receptor (AR) system. Incubation of smooth muscle cells with ET-1 for 72 hr led to increased beta(2)AR density as determined by radioligand binding. Experiments with inhibitors of protein and RNA synthesis as well as RT-PCR revealed that beta(2)AR upregulation required de novo synthesis. In addition, protein kinase C but neither NO nor prostaglandin metabolism were involved in this effect. The enhanced expression of beta(2)AR was associated with an increased expression of its stimulatory G-protein and the receptor's ability to stimulate adenylyl cyclase. To study chronic effects of ET-1 in vivo, rats were infused with ET-1 for 3 weeks. Similarly as in cultured cells, prolonged ET-1 exposure led to increased betaAR expression in vivo. As a consequence, beta(2)AR-induced vasodilatation was increased in aortic rings from ET-1-treated animals. Our results therefore suggest that chronically elevated ET-1 levels in vitro and in vivo induce counterregulatory mechanisms by increasing betaARs that attenuate the vasoconstrictive effects of ET-1.

Effect of nebivolol on left ventricular function in patients with chronic heart failure: a pilot study.

BACKGROUND: Sympathetic activity is a significant predictor of a poor prognosis in heart failure. Beta-blockers have been shown to improve the prognosis of patients with heart failure. AIM: This pilot study examined the tolerability and efficacy of the new beta-blocker nebivolol on left ventricular function in patients with chronic heart failure. METHODS AND RESULTS: Twelve patients with an ejection fraction of 13-39% were included in this double blind, placebo-controlled randomized trial of nebivolol administered in addition to standard therapy. Exercise time, heart rate, left ventricular function and tolerability were examined at baseline and after 3 months of orally administered nebivolol (2.5 and 5 mg, n = 6) or placebo (n = 6). Nebivolol was well tolerated and the NYHA class improved in four patients. Heart rate decreased while the maximal exercise duration and performance remained stable. Left ventricular function increased (ejection fraction 31.5 +/- 10.11 to 42.0 +/- 10.99%, P < or = 0.01) after 12 weeks of nebivolol. The left ventricular end-systolic diameter decreased in the nebivolol-group from 56.5 +/- 9.40 to 50.2 +/- 9.43 mm (P < or = 0.02). CONCLUSION: These data indicate that nebivolol might improve cardiac function in patients with reduced left ventricular function.

Endothelin-1 regulates protein kinase C isoforms differently in smooth muscle cells and in cardiomyocytes.

Endothelin-1 (ET-1) is known to increase the mitotic response of different growth factors but also to different vasoactive hormones already at threshold concentrations. The aim of this study was to investigate the influence of chronic elevated ET-1 concentrations on protein kinase C (PKC) isoforms in vitro and in vivo. Smooth muscle cells were incubated with ET-1 at a concentration of 10 mol/L. The incubation lasted for 1-96 hours. For in vivo studies, rats were chronically infused with ET-1 for 4 weeks using minipumps. Specific antibodies were used to determine the amount of PKC isoform in western blots. Incubation of smooth muscle cells with ET-1 revealed an increase in PKC-alpha (48 hours, 314 +/- 31.7%). In vivo PKC-alpha was augmented to 166 +/- 17.5%. In vitro PKC-epsilon showed an elevated concentration after 18 hours of 213 +/- 15.9% (maximum, 339 +/- 4.5% at 72 hours). In vivo PKC-epsilon was elevated to 162 +/- 4.2%. In the immunofluorescence microscopy after 48 hours of ET-1 incubation PKC-alpha was localized in the cytoplasm. Addition of angiotensin II resulted in a translocation of it into the nucleus. These data show that chronic elevated ET-1 concentrations modulate PKC isoforms. This might explain the increased response to different vasoactive hormones after ET-1 incubation.

Endothelin-1 increases serum-glucocorticoid-regulated kinase-1 expression in smooth muscle cells.

The mechanism of salt-sensitive hypertension is not fully understood. Several studies point to a possible role of endothelin (ET)-1 in this form of hypertension. Serum-regulated and glucocorticoid-regulated kinase-1 (SGK1) mediates trafficking of the renal epithelial sodium channel. The aim of the study was to find out whether ET-1 regulates SGK1. Rat smooth muscle cells were incubated with ET-1 (10(-7) M, 0-120 minutes). After 30 minutes a significant increase in SGK1 mRNA was found (122 +/- 4.2%), and a maximum was reached after 120 minutes (217 +/- 7.6%). Incubation of smooth muscle cells with ET-1 (10(-7) mol/L) in the presence of an ETA receptor antagonist inhibited SGK1 gene transcription (93 +/- 3.7%). Western blot analysis showed a time-dependent increase in SGK1 protein in smooth muscle cells. These data indicate that ET-1 increases SKG1 mRNA and protein concentration. Inhibition of ET-1 by ET antagonism prevented a SGK1 increase. Therefore, ET antagonism might influence blood pressure by regulating the sodium balance through reducing SGK1 gene expression.

[Influence of the beta-blocker nebivolol on left ventricular function in patients with chronic heart failure]. / Beeinflussung der linksventrikulären Funktion durch Nebivolol bei Patienten mit chronischer Herzinsuffizienz NYHA-Klasse II-III. Eine Pilotstudie.

BACKGROUND: Sympathetic activity is a significant predictor of a poor prognosis in heart failure. beta-blockers such as carvedilol, metoprolol or bisoprolol have been shown to improve the prognosis of patients with heart failure. AIM: This pilot study examined the tolerability and effect of the new beta-blocker nebivolol on left ventricular ejection fraction in patients with chronic heart failure. PATIENTS AND METHODS: Twelve patients with an ejection fraction of 13-39% were included into a double-blind, placebo-controlled, randomized trial with nebivolol on top of a standard therapy. Exercise time, heart rate, left ventricular function, and tolerability were examined at baseline and after 3 months of orally administered nebivolol (2.5 mg and 5 mg, n = 6) or placebo (n = 6). RESULTS: Nebivolol was well tolerated and NYHA stage improved in four patients. Heart rate decreased while the maximal exercise duration and performance remained stable. Left ventricular function improved (ejection fraction: increase from 29.8 +/- 10.66% to 41.2 +/- 10.53%; p = 0.007) after 12 weeks of nebivolol whereas placebo did not improve cardiac function statistically significant. Left ventricular endsystolic diameter decreased from 56.5 +/- 9.40 mm to 50.2 +/- 9.43 mm in the nebivolol group (p < or = 0.02). CONCLUSION: These data indicate that nebivolol might improve cardiac function in patients with chronic heart failure.

Major differences in gene expression in human coronary smooth muscle cells after nebivolol or metoprolol treatment.

OBJECTIVE: Vascular smooth muscle cells play a pivotal role in all stages of atherogenesis. Targeting their inflammatory and proliferative qualities might therefore inhibit the progression of atherosclerosis. This study aimed to characterize and compare the effects of the beta-receptor antagonists nebivolol and metoprolol on gene expression in human coronary artery smooth muscle cells (hcaSMC). METHODS AND RESULTS: hcaSMC were incubated with nebivolol or metoprolol (10(-5) mol/l) for 72 h. The downregulated genes are involved in inflammatory processes, oxidative stress and smooth muscle cell proliferation: i.e. downregulated were by nebivolol: interleukin-1alpha, cyclooxygenase-2, tumor-necrosis-factor (TNF)-alpha-induced protein 6, PDGF-A, growth-related oncogenes 2 and 3. Metoprolol increased the expression of interleukin-1alpha, cyclooxygenase-1, TNF-alpha-induced protein 3, heme oxygenase 1 and granulocyte/macrophage-colony-stimulating factor. In addition downregulated was monocyte chemoattractant protein 1 (MCP-1) mRNA by nebivolol. Nebivolol (10(-5) mol/l) reduced the amount of basal NF-kappaB after 48 and 52 h but not metoprolol. In the culture supernatants, MCP-1 concentrations were reduced by nebivolol. CONCLUSIONS: Nebivolol induced changes in the expression of inflammatory mediators in hcaSMC. These results add to data that suggest specific anti-inflammatory qualities of a beta-blocker of the third generation in comparison to metoprolol.

Influence of nebivolol and metoprolol on inflammatory mediators in human coronary endothelial or smooth muscle cells. Effects on neointima formation after balloon denudation in carotid arteries of rats treated with nebivolol.

OBJECTIVE AND BACKGROUND: Inflammation plays a critical role in all stages of atherogenesis. Proliferating vascular smooth muscle cells (SMC) and endothelial cells (EC) enhancing the inflammatory response, both contribute to the progression of atherosclerosis. Anti-proliferative, anti-inflammatory and anti-oxidative therapy seems to be a promising therapeutic strategy. The aim of this study was to assess the anti-proliferative and anti-inflammatory effect of the beta-blocker nebivolol in comparison to metoprolol in vitro and to find out whether nebivolol inhibits neointima formation in vivo. METHODS AND RESULTS: Real-time-RT-PCR revealed a decrease in VCAM-1, ICAM-1, PDGF-B, E-selectin and P-selectin mRNA expression in human coronary artery EC and SMC incubated with nebivolol for 72 hours while metoprolol did not have this effect. Nebivolol reduced MCP-1 and PDGF-BB protein in the culture supernatant of SMC and EC, respectively. Sprague-Dawley rats were treated with nebivolol for 0 or 35 days before and 28 days after carotid balloon injury. Immunohistological analyses showed that pre-treatment with nebivolol was associated with a decreased number of SMC layers and macrophages and an increased lumen area at the site of the arterial injury. The intima area was reduced by 43% after pre-treatment. CONCLUSION: We found that nebivolol reduced the expression of proinflammatory genes in endothelial cells and vascular smooth muscle cells in vitro whereas metoprolol did not. In vivo, nebivolol inhibited neointima formation by reducing SMC proliferation and macrophage accumulation.

Secondary syphilis after liver transplantation: case report and review of the literature.

Syphilitic disease is uncommon, but its incidence has increased worldwide in the last few years. An unusual manifestation of secondary syphilis after orthotopic liver transplantation is described which confirms that lues should be considered in patients with immune deficiency and abnormal liver function tests.

Effects of combined endothelin and angiotensin II antagonism on growth factor-induced proliferation of vascular smooth muscle cells isolated from uremic rats.

BACKGROUND: The activation of both the renin-angiotensin-aldosterone and endothelin (ET) system in uremia contributes to the development of cardiovascular disease. The combination of ET receptor antagonists and inhibitors of the angiotensin-converting enzyme (ACE) or angiotensin II type 1 (AT,) receptor could therefore inhibit atherogenesis. We studied the effects of different medications on growth-factor-induced proliferation of vascular smooth muscle cells (VSMC) isolated from uremic rats. METHODS: Subtotally nephrectomized rats (SNX) were treated with an ETA receptor antagonist, losartan, trandolapril, or the combinations of an ETA receptor antagonist and losartan or trandolapril for 12 weeks. Proliferation induced by different growth factors in isolated aortal SMC was measured using a BrdU-ELISA. RESULTS: Maximum proliferation in response to PDGF-BB, bFGF, and TNF-alpha which was higher in untreated SNX than in controls (PDGF-BB: 486.60 +/- 8.27% versus 346.74 +/- 4.60%, n=8), was reduced after monotherapy with losartan or the ETA receptor antagonist. VSMC from trandolapril-treated rats showed an increased response to all growth factors (PDGF-BB: 663.48 +/- 7.00%, n=8). After combined therapy with the ETA receptor antagonist and trandolapril, maximum proliferation was lower than in untreated SNX (PDGF-BB: 162.6 +/- 1.40%; n=8; p < or = 0.01). Combined treatment with losartan and the ETA receptor antagonist attenuated the maximum levels of VSMC proliferation induced by PDGF-BB, bFGF, and TNF. CONCLUSIONS: In contrast to an increased response after trandolapril monotherapy, combined treatment of SNX with an ETA receptor antagonist and trandolapril reduced growth-factor-induced VSMC proliferation in vitro. Further investigations in uremic patients have to clarify whether the combination of endothelin receptor antagonists and ACE inhibitors inhibits atherogenesis.

Influence of growth factors on the proliferation of vascular smooth muscle cells isolated from subtotally nephrectomized rats after endothelin or angiotensin II antagonism.

BACKGROUND: Cardiovascular disease is the most important cause of death in patients with end-stage renal disease. In uraemia, the renin-angiotensin-aldosterone and endothelin (ET) systems are activated. It is not known whether inhibition of these systems attenuates the proliferation of isolated smooth muscle cells of uraemic rats. METHODS: Subtotally nephrectomized (SNX) rats were treated with an ET(A) receptor antagonist, an ET(AB) receptor antagonist, the angiotensin type 1 (AT1) receptor antagonist losartan (all 10 mg/kg body weight/day) or the angiotensin-converting enzyme (ACE) inhibitor trandolapril (0.1 mg/kg body weight/day) or received no medication (SNX) for 12 weeks. Then, aortal smooth muscle cells (SMCs) were isolated and cultivated. After incubation of SMCs with different growth factors (5-7 days), proliferation was measured using a bromodeoxyuridine enzyme-linked immunosorbent assay (BrdU ELISA). RESULTS: Higher maximum levels of proliferation were found in SMCs from untreated SNX rats than in SMCs from control animals [platelet-derived growth factor-BB (PDGF-BB) 486.60+/-8.27 vs 346.74+/-4.60%, basic fibroblast growth factor (bFGF) 176.68+/-6.50 vs 123.71+/-1.49%, tumour necrosis factor-alpha (TNF-alpha) 153.38+/-10.16 vs 122.27+/-1.41%]. Treatment with ET receptor antagonists or losartan attenuated growth factor-stimulated proliferation (PDGF-BB: ET(A) receptor antagonist, 135.71+/-1.08%; ET(AB) receptor antagonist, 122.72+/-0.58%; losartan: 103.69+/-1.83%, n = 8). SMCs from trandolapril-treated rats showed an increased response (PDGF-BB 663.48+/-7.00%, n = 8). CONCLUSIONS: Treatment of SNX rats with ET receptor antagonists or losartan reduced growth factor-induced SMC proliferation in vitro. However, further investigations with uraemic patients have to clarify whether angiotensin or ET receptor antagonists inhibit the development of atherosclerosis.

Chronic elevated endothelin-1 concentrations regulate mitogen-activated protein kinases ERK 1 and ERK 2 in vascular smooth muscle cells.

Increased endothelin-1 (ET-1) levels are found in patients with atherosclerosis. ET-1 is known to increase the mitotic response of different growth factors already at threshold concentrations. The aim of this study was to investigate the influence of ET-1 on the mitogen-activated protein (MAP) kinases, extracellular signal-regulated protein kinase (ERK) 1 and ERK 2. Smooth muscle cells were incubated with ET-1 at a concentration of 10(-7) M for 1-120 h. ERK 1 and ERK 2 were determined in cell homogenates by electrophoresis. Specific antibodies were used to investigate the amount of ERK 1 or ERK 2 in the homogenate. The functional activity of ERK 1 and ERK 2 was determined. Immunofluorescence microscopy was performed to analyse the translocation of the MAP kinases into the nucleus. ET-1 incubation for 12 h decreased ERK 1 concentration by -51%. After 36 h of ET-1 application the concentration of ERK 1 increased to control levels again. When the cells were incubated for 120 h ERK 1 rose by +65% above control. The incubation with ET-1 in the presence of an ET(A) receptor antagonist inhibited the increase of ERK 1. ERK 2 showed a comparable time course with an initial decrease in the protein concentration followed by an increase after 120 h. Incubation with an ET(A) receptor antagonist inhibited the increase in protein concentration after 120 h. However, the functional activity of both MAP kinases remained unchanged between 1 and 120 h. Especially, after 120 h of ET-1 incubation no translocation into the nucleus was observed. However, an additional stimulus with angiotensin II resulted in translocation of ERK into the nucleus. These data show that ET-1 increases the protein concentration of MAP kinases ERK 1 and ERK 2 but not their basal activity. Only an additional stimulation with angiotensin II leads to the translocation of ERK into the cell nucleus.

Infectious risk factors for atherosclerotic vascular disease in hemodialysis patients--Chlamydia pneumoniae but not Helicobacter pylori or cytomegalovirus is associated with increased C-reactive protein.

BACKGROUND: Cardiovascular disease is a major problem in patients with chronic renal failure leading to increased mortality. Several infectious agents have been implicated to be associated with atherosclerosis. We were interested to evaluate whether there is a correlation between a past infection with Chlamydia pneumoniae (Cpn), Helicobacter pylori (Hp) or cytomegalovirus (CMV) and the manifestation of a symptomatic atherosclerotic disease in patients with endstage renal failure. METHODS AND RESULTS: Patients (n=267) on hemodialysis were investigated. In 101 patients with an apparent atherosclerotic disease (case group) increased IgA levels against Cpn were found (p < or = 0.0001 vs. controls; n=33). Nearly 45% of the case group had a history of myocardial infarction (MI) (p < or = 0.0001). Prior stroke was found in about 30% of patients in the case group (p < or = 0.002). Elevated CRP levels were identified as an independent risk factor for atherosclerosis in all groups. IgA seropositivity against Cpn correlated with elevated CRP values for all atherosclerotic patients (p < or = 0.001), especially in the group of patients with MI (p < or = 0.019) and peripheral vascular disease (p < or = 0.005). There was no correlation between CMV (IgG, IgM) or Hp (IgA, IgG) seropositivity and atherosclerotic disease. CONCLUSION: IgA seropositivity for Cpn and elevated CRP values but not Hp or CMV was associated with an increased rate of symptomatic atherosclerotic manifestations as MI, stroke, cerebral or peripheral atherosclerosis in patients with endstage renal disease on hemodialysis.

Influence of endothelin receptor antagonists on myocardial protein kinase C isoforms in uraemic cardiomyopathy.

Increased endothelin-1 (ET-1) levels were found in patients with chronic renal failure and these correlate with the severity of renal failure. Increased mortality due to cardiovascular problems is observed in patients with elevated ET-1 concentrations. The aim of this study was to find out the influence of ET-1 and ET receptor antagonists on myocardial protein kinase C (PKC) regulation in uraemic cardiomyopathy. Male rats were subtotally nephrectomized and treated with an ET(A)-receptor antagonist (30 mg x kg(-1) x day(-1), LU302146) or an ET(AB)-receptor antagonist (30 mg x kg(-1) x day(-1), LU302872) for 12 weeks. One group was left untreated (SNX) and one group was sham-operated (sham). Systolic blood pressure, myocardial weight and the changes of the protein kinase C isoforms in the heart were determined. PKC isoforms alpha and delta were investigated by Western blot analysis using specific antibodies. In the SNX group, systolic blood pressure rose to 154+/-5 mmHg after 12 weeks. The ET(A) receptor antagonist prevented this increase in blood pressure, but ET(AB) antagonism did not. Left ventricular weight increased in SNX; this increase was inhibited by the ET(A) receptor antagonist. In comparison with the sham group, PKC isoform alpha increased by 19% in SNX animals. When the SNX animals were treated with ET(A) or ET(AB) antagonists, PKC isoform alpha levels decreased by 31%. PKC isoform delta levels decreased by 35% in SNX animals. Treatment with both ET(A) or ET(AB) antagonists increased PKC isoform delta levels to normal. In the myocardium of uraemic rats PKC isoforms are differentially regulated with an increase in alpha isoform but a decrease in delta isoform. ET receptor blockers normalize these PKC isoforms.