Katharina Relle

Nitric oxide-independent stimulation of soluble guanylate cyclase reduces organ damage in experimental low- renin and high-renin models

Objectives The nitric oxide–soluble guanylate cyclase (sGC)–cGMP signal transduction pathway is impaired in different cardiovascular diseases, including pulmonary hypertension, heart failure and arterial hypertension. Riociguat is a novel stimulator of soluble guanylate cyclase (sGC). However, little is known about the effects of sGC stimulators in experimental models of hypertension. We thus investigated the cardio-renal protective effects of riociguat in low-renin and high-renin rat models of hypertension. Methods The vasorelaxant effect of riociguat was tested in vitro on isolated saphenous artery rings of normal and nitrate tolerant rabbits. The cardiovascular in-vivo effects of sGC stimulation were evaluated in hypertensive renin-transgenic rats treated with the nitric oxide-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) (high-renin model) and in rats with 5/6 nephrectomy (low-renin model). Results In both animal models, riociguat treatment improved survival and normalized blood pressure. Moreover, in the L-NAME study part, riociguat reduced cardiac target organ damage as indicated by lower plasma ANP, lower relative left ventricular weight and lower cardiac interstitial fibrosis, and reduced renal target organ damage as indicated by lower plasma creatinine and urea, less glomerulosclerosis and less renal interstitial fibrosis. In the 5/6 nephrectomy study part, riociguat reduced cardiac target organ damage as indicated by lower plasma ANP, lower relative left ventricular weight, lower myocyte diameter and lower arterial media/lumen ratio, and reduced renal target organ damage as indicated by improved creatinine clearance and less renal interstitial fibrosis. Conclusion We demonstrate for the first time that the novel sGC stimulator riociguat shows in two independent models of hypertension a potent protection against cardiac and renal target organ damage.

NO-independent activation of soluble guanylate cyclase prevents disease progression in rats with 5/6 nephrectomy

1 Chronic renal disease is associated with oxidative stress, reduced nitric oxide (NO) availability and soluble guanylate cyclase (sGC) dysfunction. Recently, we discovered BAY 58‐2667, a compound activating heme‐deficient or oxidized sGC in a NO‐independent manner. 2 We assessed potential of BAY 58‐2667 in preventing cardiac and renal target organ damage in rats with 5/6 nephrectomy. 3 Male Wistar rats were allocated to three groups: 5/6 nephrectomy, 5/6 nephrectomy treated with BAY 58‐2667 and sham operation. Study period was 18 weeks: blood pressure and creatinine clearance were assessed repeatedly. At study end blood samples were taken and hearts and kidneys harvested for histological studies. 4 BAY 58‐2667 markedly lowered blood pressure in animals with 5/6 nephrectomy (untreated versus treated animals: 189±14 versus 146±11 mmHg, P<0.001). Left ventricular weight, cardiac myocyte diameter as well as cardiac arterial wall thickness significantly decreased in comparison to untreated animals with 5/6 nephrectomy. Natriuretic peptide plasma levels were also improved by BAY 58‐2667. Kidney function and morphology as assessed by creatinine clearance, glomerulosclerosis, interstitial and perivascular fibrosis of intrarenal arteries were likewise significantly improved by BAY 58‐2667. 5 This is the first study showing that BAY 58‐2667 effectively lowers blood pressure, reduces left ventricular hypertrophy and slows renal disease progression in rats with 5/6 nephrectomy by targeting mainly oxidized sGC. Therefore, BAY 58‐2667 represents a novel pharmacological principle with potential clinical value in treatment of chronic renal disease.

Impact of maternal body mass index on neonatal outcome

IntroductionMaternal body mass index has an impact on maternal and fetal pregnancy outcome. An increased maternal BMI is known to be associated with admission of the newborn to a neonatal care unit. The reasons and impact of this admission on fetal outcome, however, are unknown so far.ObjectiveThe aim of our study was to investigate the impact of maternal BMI on maternal and fetal pregnancy outcome with special focus on the children admitted to a neonatal care unit.MethodsA cohort of 2049 non-diabetic mothers giving birth in the Charite university hospital was prospectively studied. The impact of maternal BMI on maternal and fetal outcome parameters was tested using multivariate regression analysis. Outcome of children admitted to a neonatal ward (n = 505) was analysed.ResultsIncreased maternal BMI was associated with an increased risk for hypertensive complications, peripheral edema, caesarean section, fetal macrosomia and admission of the newborn to a neonatal care unit, whereas decreased BMI was associated with preterm birth and lower birthweight. In the neonatal ward children from obese mothers are characterized by hypoglycaemia. They need less oxygen, and exhibit a shorter stay on the neonatal ward compared to children from normal weight mothers, whereas children from underweight mothers are characterized by lower umbilical blood pH and increased incidence of death corresponding to increased prevalence of preterm birth.ConclusionPregnancy outcome is worst in babies from mothers with low body mass index as compared to healthy weight mothers with respect to increased incidence of preterm birth, lower birth weight and increased neonate mortality on the neonatal ward. We demonstrate that the increased risk for neonatal admission in children from obese mothers does not necessarily indicate severe fetal impairment.

The adenosine A1 receptor antagonist SLV320 reduces myocardial fibrosis in rats with 5/6 nephrectomy without affecting blood pressure.

Myocardial fibrosis is an unwanted effect associated with chronic renal failure. The adenosine system is involved in cardiac and renal function. Therefore, we investigated the novel selective adenosine A1 receptor antagonist SLV320 focusing on its potential in preventing cardiomyopathy in rats with 5/6 nephrectomy.

Lack of endothelial nitric oxide synthase promotes endothelin-induced hypertension: lessons from endothelin-1 transgenic/endothelial nitric oxide synthase knockout mice.

Endothelin-1 (ET-1) is one of the most potent biologic vasoconstrictors. Nevertheless, transgenic mice that overexpress ET-1 exhibit normal BP. It was hypothesized that vascular effects of ET-1 may be antagonized by an increase of the endothelial counterpart of ET-1, nitric oxide (NO), which is produced by the endothelial NO synthase (eNOS). Therefore, cross-bred animals of ET transgenic mice (ET+/+) and eNOS knockout (eNOS-/-) mice and were generated, and BP and endothelial function were evaluated in these animals. Endothelium-dependent and -independent vascular function was assessed as relaxation/contraction of isolated preconstricted aortic rings. The tissue ET and NO system was determined in aortic rings by quantitative real-time PCR and Western blotting. Systolic BP was similar in ET+/+ and wild-type (WT) mice but was significantly elevated in eNOS-/- mice (117 +/- 4 mmHg versus 94 +/- 6 mmHg in WT mice; P < 0.001) and even more elevated in ET+/+ eNOS-/- cross-bred mice (130 +/- 4 mmHg; P < 0.05 versus eNOS-/-). Maximum endothelium-dependent relaxation was enhanced in ET+/+ mice (103 +/- 6 versus 87 +/- 4% of preconstriction in WT littermates; P < 0.05) and was completely blunted in eNOS-/- (-3 +/- 4%) and ET+/+ eNOS-/- mice (-4 +/- 4%), respectively. Endothelium-independent relaxation was comparable among all groups. Quantitative real-time PCR as well as Western blotting revealed an upregulation of the aortic ET(A) and ET(B) receptors in ET+/+ eNOS-/-, whereas eNOS was absent in aortic rings of eNOS-/- and ET+/+ eNOS-/- mice. ET-1 aortic tissue concentrations were similar in WT mice and ET+/+ eNOS-/- mice most probably as a result of an enhanced clearance of ET-1 by the upregulated ET(B) receptor. These data show for the first time that in transgenic mice that overexpress human ET-1, additional knockout of eNOS results in a further enhancement of BP as compared with eNOS-/- mice. The human ET+/+ eNOS-/- mice therefore represent a novel model of hypertension as a result of an imbalance between the vascular ET-1 and NO systems.

Endothelin-Converting Enzyme/Neutral Endopeptidase Inhibitor SLV338 Prevents Hypertensive Cardiac Remodeling in a Blood Pressure–Independent Manner

Hypertensive heart disease is a major contributor to cardiovascular mortality. Endothelin is a potent vasoconstrictive and profibrotic mediator produced by the endothelin-converting enzyme (ECE), whereas natriuretic peptides, degraded by the neutral endopeptidase (NEP), have diuretic, vasodilatory, and antifibrotic properties. Thus, combined ECE/NEP inhibition may halt hypertensive cardiac remodeling. This study examined effects of SLV338, a novel ECE/NEP inhibitor, on cardiac protection in experimental renovascular hypertension (2-kidney, 1-clip [2K1C]). Male rats were allocated to 5 groups: sham-operated rats, untreated animals with 2K1C, 2K1C animals treated with oral SLV338 (30 and 100 mg/kg per day), and 2K1C animals treated with oral losartan (20 mg/kg per day). Treatment duration was 12 weeks. Blood pressure was assessed every 4 weeks. At study end, hearts were taken for histology/computer-aided histomorphometry/immunohistochemistry. Pharmacological properties of SLV338 are described. SLV338 is a dual ECE/NEP inhibitor, as demonstrated both in vitro and in vivo. In the 2K1C study, losartan lowered blood pressure by ⩽46 mm Hg, whereas both dosages of SLV338 had no effect. However, SLV338 (both dosages) completely normalized cardiac interstitial fibrosis, perivascular fibrosis, myocyte diameter, and media:lumen ratio of cardiac arteries, as did losartan. Cardiac transforming growth factor-&bgr;1 expression was significantly enhanced in untreated 2K1C rats versus controls, whereas treatment with SLV338 and losartan prevented this effect. Taken together, dual ECE/NEP inhibitor SLV338 prevents cardiac remodeling to the same extent as losartan, but in a blood pressure–independent manner, in a rat model of renovascular hypertension. This effect is at least partially mediated via suppression of cardiac transforming growth factor-&bgr;1 expression.

Endothelin-1 overexpression restores diastolic function in eNOS knockout mice.

Background The cardiac nitric oxide and endothelin-1 (ET-1) systems are closely linked and play a critical role in cardiac physiology. The balance between both systems is often disturbed in cardiovascular diseases. To define the cardiac effect of excessive ET-1 in a status of nitric oxide deficiency, we compared left ventricular function and morphology in wild-type mice, ET-1 transgenic (ET+/+) mice, endothelial nitric oxide synthase knockout (eNOS−/−) mice, and ET+/+eNOS−/− mice. Methods and results eNOS−/− and ET+/+eNOS−/− mice developed high blood pressure compared with wild-type and ET+/+ mice. Left ventricular catheterization showed that eNOS−/− mice, but not ET+/+eNOS−/−, developed diastolic dysfunction characterized by increased end-diastolic pressure and relaxation constant tau. To elucidate the causal molecular mechanisms driving the rescue of diastolic function in ET+/+eNOS−/− mice, the cardiac proteome was analyzed. Two-dimensional gel electrophoresis coupled to mass spectrometry offers an appropriate hypothesis-free approach. ET-1 overexpression on an eNOS−/− background led to an elevated abundance and change in posttranslational state of antioxidant enzymes (e.g., peroxiredoxin-6, glutathione S-transferase mu 2, and heat shock protein beta 7). In contrast to ET+/+eNOS−/− mice, eNOS−/− mice showed an elevated abundance of proteins responsible for sarcomere disassembly (e.g., cofilin-1 and cofilin-2). In ET+/+eNOS−/− mice, glycolysis was favored at the expense of fatty acid oxidation. Conclusion eNOS−/− mice developed diastolic dysfunction; this was rescued by ET-1 transgenic overexpression. This study furthermore suggests that cardiac ET-1 overexpression in case of eNOS deficiency causes specifically the regulation of proteins playing a role in oxidative stress, myocytes contractility, and energy metabolism.

Soluble CD154 Is a Unique Predictor of Nonfatal and Fatal Atherothrombotic Events in Patients Who Have End-Stage Renal Disease and Are on Hemodialysis

Cardiovascular mortality is remarkably high in patients who are on hemodialysis. Soluble CD154 (sCD154), a protein that belongs to the TNF receptor superfamily, has been implicated in the pathogenesis of atheromatous plaque destabilization and thrombotic events. The predictive value of sCD154 as a marker for clinical outcome in patients with ESRD was investigated. A total of 232 patients were prospectively followed for 52 mo. At study entry, clinical characteristics were documented and plasma concentrations of sCD154 and those of conventional risk predictors were analyzed. The time and cause of any hospitalization and death were documented during the entire follow-up. Survival rates were compared by Kaplan-Meier and Cox regression analyses. A total of 122 patients died, 64 of cardiovascular disease, including 20 cases of fatal atherothrombotic diseases (myocardial infarction, stroke, mesenteric ischemia). All 20 cases of fatal atherothrombotic events had high sCD154 plasma levels (cutoff >6.42 ng/ml) at study entry. The total number of fatal and nonfatal atherothrombotic events was 66. Only five atherothrombotic nonfatal events occurred in patients with sCD154 <6.42 ng/ml, whereas 61 fatal and nonfatal events were seen in patients with sCD154 > or =6.42 ng/ml (P < 0.005). This was confirmed by Kaplan-Meier curves for fatal atherothrombotic events (P = 0.0214) and the combined end point fatal and nonfatal atherothrombotic events (P = 0.0039). Cox regression analysis revealed that high sCD154 is an independent predictor (relative risk 6.80; 95% confidence interval 1.64 to 28.26; P = 0.008) for the combined end point death or hospitalization as a result of atherothrombotic events. Death or hospitalizations as a result of any other reason (arrhythmia, heart failure, infectious diseases, and cancer) were not linked to sCD154 plasma concentrations. In conclusion, sCD154 predicts nonfatal and fatal atherothrombotic events (myocardial infarction, stroke, mesenteric ischemia) but not death and hospitalization as a result of any other reason in stable patients who have ESRD and are on hemodialysis.

Cell‐type specific interaction of endothelin and the nitric oxide system: pattern of prepro‐ET‐1 expression in kidneys of l‐NAME treated prepro‐ET‐1 promoter‐lacZ‐transgenic mice

Nitric oxide (NO) and endothelin‐1 (ET‐1) are known to play a major role in renal and vascular pathophysiology and exhibit a close interaction with ET‐1, stimulating NO production; NO in turn inhibits ET‐1 expression. Our objectives were (1) to establish a novel transgenic mouse model facilitating ET‐1 expression assessment in vivo, (2) to validate this model by assessing prepro‐ET‐1 promoter activity in mice embryos by means of our novel model and comparing expression sites to well‐established data on ET‐1 in fetal development and (3) to investigate renal ET–NO interaction by assessing prepro‐ET‐1 promoter activity in different structures of the renal cortex in the setting of blocked NO synthases via l‐NAME administration. We established transgenic mice carrying a lacZ reporter gene under control of the human prepro‐ET‐1 gene promoter sequence (8 kb of 5′ sequences). Bluo‐Gal staining of tissue sections revealed intracellular blue particles as indicators of prepro‐ET‐1 promoter activity. In mouse embryos, we detected high prepro‐ET‐1 promoter activity in the craniofacial region, as well as in bone and cartilage consistent with the literature. In order to investigate the interaction of ET‐1 and NO in the kidney in vivo, transgenic mice at the age of 3–4 months were treated with a single dose of the NO synthase inhibitor l‐NAME (25 mg (kg bw)−1i.p.) 12 h before kidney removal. Bluo‐Gal staining of kidney sections revealed intracellular blue particles as indicators of prepro‐ET‐1 promoter activity in tubular and vascular endothelium and glomerular cells. Particle count was closely correlated to kidney tissue ET‐1 content (R= 0.918, P < 0.001). Comparison of counts revealed an increase by 135 ± 53% in l‐NAME treated (n= 12) compared to non‐treated mice (n= 10, P= 0.001). Cell‐type specific evaluation revealed an increase of 136 ± 51% in tubular (P= 0.001) and 105 ± 41% in glomerular cells (P= 0.046), but no significant increase in vascular endothelium. In conclusion, our study revealed a close interaction of renal endothelin and the NO system in a cell‐type specific manner. Our new transgenic model provides a unique opportunity to analyse regulation of the ET system on a cellular level in vivo.

Endothelin-1 overexpression and endothelial nitric oxide synthase knock-out induce different pathological responses in the heart of male and female mice

AIMS The nitric oxide and endothelin systems are key components of a local paracrine hormone network in the heart. We previously reported that diastolic dysfunction observed in mice lacking the endothelial nitric oxide synthase (eNOS-/-) can be prevented by a genetic overexpression of ET-1. Sexual dimorphisms have been reported in both ET-1 and NO systems. Particularly, eNOS-/- mice present sex related phenotypic differences. MAIN METHODS We used the ET-1 transgenic (ET+/+), eNOS-/-, and crossbred ET+/+eNOS-/- mice, and wild type controls. We measured cardiac function by heart catheterization. Cardiac ventricles were collected for histological and molecular profiling. KEY FINDINGS We report here that (i) the level of ET-1 expression in eNOS-/- mice was elevated in males but not in females. (ii) Left ventricular end-diastolic blood pressure was higher in male eNOS-/- mice than in females. (ii) eNOS-/- males but not females developed cardiomyocyte hypertrophy. (iv) Perivascular fibrosis of intracardiac arteries developed in female ET+/+ and eNOS-/- mice but not in males. Additionally, (v) the cardiac expression of metalloprotease-9 was higher in eNOS-/- males compared to females. Finally, (vi) cardiac proteome analysis revealed that the protein abundance of the oxidative stress related enzyme superoxide dismutase presented with sexual dimorphism in eNOS-/- and ET+/+ mice. SIGNIFICANCE These results indicate that the cardiac phenotypes of ET-1 transgenic mice and eNOS knockout mice are sex specific. Since both systems are key players in the pathogenesis of cardiovascular diseases, our findings might be important in the context of gender differences in patients with such diseases.