Stefan Schäfer

Publications of Stefan Schäfer

• A role for coagulation factor Xa in experimental pulmonary arterial hypertension.

  Authors: Martina Delbeck, Katrin F Nickel, Elisabeth Perzborn, Peter Ellinghaus, Julia Strassburger, Raimund Kast, Volker Laux, Stefan Schäfer, Ralph T Schermuly, Georges von Degenfeld

  Cardiovascular research. 06/2011; 92(1):159-68.

  Anticoagulation with warfarin is recommended for the treatment of patients with pulmonary arterial hypertension (PAH). However, the therapeutic benefit of anticoagulation has not yet beenAnticoagulation with warfarin is recommended for the treatment of patients with pulmonary arterial hypertension (PAH). However, the therapeutic benefit of anticoagulation has not yet been demonstrated experimentally or clinically. Here, rivaroxaban, an oral, direct factor Xa (FXa) inhibitor, was compared with warfarin and enoxaparin in the prevention of right ventricular (RV) dysfunction and hypertrophy in the monocrotaline (MCT) model of pulmonary hypertension. Sprague-Dawley rats (n = 10 per group) were randomized to receive rivaroxaban, warfarin, enoxaparin, or placebo before receiving a subcutaneous injection of MCT 60 mg/kg or saline. Rivaroxaban and enoxaparin were administered for 28 days starting 4 h before MCT injection; warfarin was given for 35 days initiated 7 days before MCT injection. RV haemodynamics and hypertrophy were assessed 28 days after MCT administration. Rivaroxaban dose-dependently reduced systolic and end-diastolic RV pressure increase and RV hypertrophy. Warfarin reduced RV pressure increase only. Enoxaparin had no effect on either parameter. Severe bleeding occurred in four and five rats treated with warfarin and enoxaparin, respectively, whereas no overt bleeding was observed in rats treated with rivaroxaban. Selective, direct inhibition of FXa by rivaroxaban effectively prevented RV dysfunction and hypertrophy in MCT-injected rats, indicating a role for coagulation factors in experimental pulmonary hypertension. Clinical investigation of the impact of early and continued administration of a specific FXa inhibitor such as rivaroxaban on the course of PAH should be considered.

• Impaired left-ventricular cardiac function in male GPR30-deficient mice.

  Authors: Martina Delbeck, Stefan Golz, Richardus Vonk, Wiebke Janssen, Tim Hucho, Jörg Isensee, Stefan Schäfer, Christiane Otto

  Molecular medicine reports. 01/2011; 4(1):37-40.

  G-protein-coupled receptor 30 (GPR30) has been reported to act as a membrane-bound estrogen receptor that is involved in the mediation of non-genomic estradiol signalling. In this study, weG-protein-coupled receptor 30 (GPR30) has been reported to act as a membrane-bound estrogen receptor that is involved in the mediation of non-genomic estradiol signalling. In this study, we demonstrated that male, but not female, GPR30-deficient mice suffer from impaired left‑ventricular cardiac function. Left ventricles from male mutant mice were enlarged. There were no malformations in the valves or outflow tract of the heart. Both the contractility and relaxation capacity of the left ventricle were reduced, leading to increased left‑ventricular end-diastolic pressure in GPR30-deficient mice. In conclusion, our data support a role for GPR30 in the gender-specific aspects of heart failure.

• Mineralocorticoid receptor-mediated DNA damage in kidneys of DOCA-salt hypertensive rats.

  Authors: Nicole Schupp, Peter Kolkhof, Nina Queisser, Sabine Gärtner, Ursula Schmid, Axel Kretschmer, Elke Hartmann, Rajaraman G Oli, Stefan Schäfer, Helga Stopper

  FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 12/2010; 25(3):968-78.

  Epidemiological studies exploring the connection between hypertension and cancer demonstrate a higher cancer incidence, especially of kidney cancer, and a higher cancer mortality in hypertensiveEpidemiological studies exploring the connection between hypertension and cancer demonstrate a higher cancer incidence, especially of kidney cancer, and a higher cancer mortality in hypertensive patients. Hormones elevated in hypertension, i.e., aldosterone and angiotensin II, which exert genotoxic effects in vitro, could contribute to carcinogenesis in hypertension. The present study was conducted to investigate the possible DNA-damaging effect of aldosterone receptor activation in vivo. Crl:CD (Sprague-Dawley) rats were treated for 6 wk with desoxycorticosterone acetate (DOCA) and salt to induce a mineralocorticoid-dependent hypertension. DOCA-salt treatment caused increased blood pressure (+26 mmHg) compared to untreated rats, elevated markers of kidney failure (up to 62-fold for Kim-1), and the induction of several proinflammatory genes and proteins (up to 2.6-fold for tissue MCP-1). The mineralocorticoid receptor (MR) antagonist spironolactone (MR IC(50) 24 nM) and the novel nonsteroidal antagonist BR-4628 (MR IC(50) 28 nM) decreased these damage markers. DOCA-salt treatment also caused 8.8-fold increased structural DNA damage, determined with the comet assay, double-strand breaks (3.5-fold), detected immunohistochemically, and oxidative stress. Furthermore, the oxidatively modified mutagenic DNA base 7,8-dihydro-8-oxo-guanine (8-oxodG), quantified by LC-MS/MS, was almost 2-fold higher in DOCA-salt-treated kidneys. Our results suggest a mutagenic potential of high mineralocorticoid levels, frequent in hypertensive individuals.

• A new mode of mineralocorticoid receptor antagonism by a potent and selective nonsteroidal molecule.

  Authors: Jérôme Fagart, Alexander Hillisch, Jessica Huyet, Lars Bärfacker, Michel Fay, Ulrich Pleiss, Elisabeth Pook, Stefan Schäfer, Marie-Edith Rafestin-Oblin, Peter Kolkhof

  The Journal of biological chemistry. 09/2010; 285(39):29932-40.

  Limitations of current steroidal mineralocorticoid receptor (MR) antagonists have stimulated the search for a new generation of molecules. We screened for novel nonsteroidal compounds and identifiedLimitations of current steroidal mineralocorticoid receptor (MR) antagonists have stimulated the search for a new generation of molecules. We screened for novel nonsteroidal compounds and identified MR antagonists derived from the chemical class of dihydropyridines. Chemical optimization resulted in BR-4628, which displays high in vitro and in vivo MR potency as well as selectivity with respect to the other steroid hormone receptors and the L-type calcium channel. Biochemical studies demonstrated that BR-4628 forms complexes with MR that do not promote the recruitment of transcriptional co-regulators. Docking experiments, using the crystal structure of the MR ligand-binding domain in an agonist conformation, revealed that BR-4628 accommodates in the MR ligand-binding cavity differently in comparison with the classical steroidal MR antagonists. An alanine scanning mutagenesis approach, based on BR-4628 docking, allowed identifying its anchoring mode within the ligand-binding cavity. Altogether, we propose that BR-4628 is a bulky antagonist that inactivates MR through a passive mechanism. It represents the prototype of a new class of MR antagonists.

• Cardiac glycosides potently inhibit C-reactive protein synthesis in human hepatocytes.

  Authors: Peter Kolkhof, Andreas Geerts, Stefan Schäfer, Jan Torzewski

  Biochemical and biophysical research communications. 03/2010; 394(1):233-9.

  Elevated plasma levels of C-reactive protein (CRP), the prototype acute-phase protein (APP), are predictive for future cardiovascular events. Controversial evidence suggests that CRP may play aElevated plasma levels of C-reactive protein (CRP), the prototype acute-phase protein (APP), are predictive for future cardiovascular events. Controversial evidence suggests that CRP may play a causal role in cardiovascular disease. CRP synthesis inhibition is a potential approach for reducing cardiovascular mortality. We show here that endogenous and plant-derived inhibitors of the Na(+)/K(+)-ATPase, i.e. the cardiac glycosides ouabain and digitoxin, inhibit IL-1beta- and IL-6-induced APP expression in human hepatoma cells and primary human hepatocytes (PHH) at nanomolar concentrations. Inhibition is demonstrated on transcriptional and on protein level. The molecular target of cardiac glycosides, i.e. the alpha1 subunit of the Na(+)/K(+)-ATPase, is strongly expressed in human hepatocytes. Inhibition of APP synthesis correlates with the potency of cardiac glycosides at the Na(+)/K(+)-ATPase. The trigger for APP expression inhibition is an increase in intracellular calcium since the calcium ionophore calcimycin is also active. Qualified specificity of oubain for hepatocellular APP synthesis inhibition is demonstrated by lack of effectivity on IL-1beta-induced IL-6 release from primary human coronary artery smooth muscle cells. The inhibitory activity of cardiac glycosides on CRP expression may have important implications for the treatment of cardiovascular disease. Cardiac glycosides may be used for CRP synthesis inhibition in the future.

• Chronic inhibition of phosphodiesterase 5 does not prevent pressure overload induced right ventricular remodelling.

  Authors: Stefan Schäfer, Peter Ellinghaus, Wiebke Janssen, Frank Kramer, Klemens Lustig, Hendrik Milting, Raimund Kast, Martina Klein

  Cardiovascular research. 02/2009;

  AIMS: Inhibition of phosphodiesterase 5 (PDE5) decreases pulmonary pressure and improves symptoms in patients with pulmonary arterial hypertension. It is unclear however, whether inhibition of PDE5AIMS: Inhibition of phosphodiesterase 5 (PDE5) decreases pulmonary pressure and improves symptoms in patients with pulmonary arterial hypertension. It is unclear however, whether inhibition of PDE5 can prevent myocardial remodelling during right ventricular pressure overload. METHODS: Right ventricular pressure overload was produced in male rats in a pulmonary hypertension model (monocrotaline 60 mg/kg s.c.) or by surgical pulmonary artery banding. PDE5 inhibition using oral sildenafil (50 mg/kg/d in drinking water) or placebo was initiated 14 days after monocrotaline treatment and continued for 14 days until final examination. In the pulmonary artery banding groups, rats were treated with sildenafil (50 mg/kg/d) or placebo for 21 days following surgical pulmonary artery banding. At the final experiments, right ventricular haemodynamics were measured and remodelling was analyzed using histological, biochemical and gene expression markers. RESULTS: Both monocrotaline and pulmonary artery banding increased right ventricular systolic pressure to approximately 80 mmHg. In parallel, both interventions induced markers of hypertrophy (upregulation of natriuretic peptides, increase in myocyte diameter) and fibrosis (upregulation of collagen types 1A2 and 3A1) as well as mRNA expression of the tissue inhibitor of matrix metalloproteases 1 and osteopontin in the right ventricle. In monocrotaline model, sildenafil decreased pulmonary pressure, reduced right ventricular hypertrophy and prevented fibrosis marker gene upregulation. After pulmonary artery banding, in contrast, sildenafil increased markers of myocardial remodeling and right ventricular myocyte diameter. CONCLUSIONS: Sildenafil prevents myocardial remodelling in pulmonary hypertension through an indirect action via right ventricular unloading.

• Failure is an option: learning from unsuccessful proof-of-concept trials.

  Authors: Stefan Schäfer, Peter Kolkhof

  Drug discovery today. 12/2008; 13(21-22):913-6.

  Recent statistics indicate that the attrition rates during drug development remain high. Lack of clinical efficacy has meanwhile become the most frequent cause for discontinuation of a drugRecent statistics indicate that the attrition rates during drug development remain high. Lack of clinical efficacy has meanwhile become the most frequent cause for discontinuation of a drug development program. Consequently, attrition rates are highest in clinical Phase II, which usually includes the first evidence for pharmacodynamic action of the compound or, proof of concept. Interestingly, attrition is approximately 60-70% across a variety of therapeutic areas, including the central nervous system (where predictivity of animal models is usually low) and cardiovascular medicine (where animal models are considered to be more predictive). Obviously, the translation of animal data into clinical benefit remains suboptimal.

• Combined tyrosine and serine/threonine kinase inhibition by sorafenib prevents progression of experimental pulmonary hypertension and myocardial remodeling.

  Authors: Martina Klein, Ralph T Schermuly, Peter Ellinghaus, Hendrik Milting, Bernd Riedl, Sevdalina Nikolova, Soni S Pullamsetti, Norbert Weissmann, Eva Dony, Rajkumar Savai, Hossein A Ghofrani, Friedrich Grimminger, Andreas E Busch, Stefan Schäfer

  Circulation. 12/2008; 118(20):2081-90.

  BACKGROUND: Inhibition of tyrosine kinases, including platelet-derived growth factor receptor, can reduce pulmonary arterial pressure in experimental and clinical pulmonary hypertension. WeBACKGROUND: Inhibition of tyrosine kinases, including platelet-derived growth factor receptor, can reduce pulmonary arterial pressure in experimental and clinical pulmonary hypertension. We hypothesized that inhibition of the serine/threonine kinases Raf-1 (also termed c-Raf) and b-Raf in addition to inhibition of tyrosine kinases effectively controls pulmonary vascular and right heart remodeling in pulmonary hypertension. METHODS AND RESULTS: We investigated the effects of the novel multikinase inhibitor sorafenib, which inhibits tyrosine kinases as well as serine/threonine kinases, in comparison to imatinib, a tyrosine kinase inhibitor, on hemodynamics, pulmonary and right ventricular (RV) remodeling, and downstream signaling in experimental pulmonary hypertension. Fourteen days after monocrotaline injection, male rats were treated orally for another 14 days with sorafenib (10 mg/kg per day), imatinib (50 mg/kg per day), or vehicle (n=12 to 16 per group). RV systolic pressure was decreased to 35.0+/-1.5 mm Hg by sorafenib and to 54.0+/-4.4 mm Hg by imatinib compared with placebo (82.9+/-6.0 mm Hg). In parallel, both sorafenib and imatinib reduced RV hypertrophy and pulmonary arterial muscularization. The effects of sorafenib on RV systolic pressure and RV mass were significantly greater than those of imatinib. Sorafenib prevented phosphorylation of Raf-1 and suppressed activation of the downstream ERK1/2 signaling pathway in RV myocardium and the lungs. In addition, sorafenib but not imatinib antagonized vasopressin-induced hypertrophy of the cardiomyoblast cell line H9c2. CONCLUSIONS: The multikinase inhibitor sorafenib prevents pulmonary remodeling and improves cardiac and pulmonary function in experimental pulmonary hypertension. Sorafenib exerts direct myocardial antihypertrophic effects, which appear to be mediated via inhibition of the Raf kinase pathway. The combined inhibition of tyrosine and serine/threonine kinases may provide an option to treat pulmonary arterial hypertension and associated right heart remodeling.

• Inhibition of renin angiotensin system decreases renal protein oxidative damage in diabetic rats.

  Authors: Manuel Portero-Otín, Reinald Pamplona, Jordi Boada, Mariona Jové, Hugo Gonzalo, Marie Buleon, Wolfgang Linz, Stefan Schäfer, Ivan Tack, Jean-Pierre Girolami

  Biochemical and biophysical research communications. 05/2008; 368(3):528-35.

  Renin angiotensin system (RAS) worsens diabetic nephropathy (DN) by increasing oxidative stress. We compared the effect of three different RAS inhibitors: the angiotensin converting enzyme inhibitorRenin angiotensin system (RAS) worsens diabetic nephropathy (DN) by increasing oxidative stress. We compared the effect of three different RAS inhibitors: the angiotensin converting enzyme inhibitor Ramipril, the vasopeptidase inhibitor AVE7688 and the angiotensin receptor (AT1) antagonist Losartan on the formation of oxidative and carbonyl stress derived protein modifications in kidney from Zucker obese hyperglycemic rats (ZDFn Gm-fa/fa). Gas chromatography-mass spectrometry was used to measure representative markers of several protein oxidative pathways: direct oxidation [dinitrophenylhydrazine reactive carbonyls (DNP), glutamic (GSA), and aminoadipic (AASA) semialdehydes], mixed glyco- and lipoxidation [N(epsilon)-carboxyethyl-lysine (CEL) and N(epsilon)-(carboxymethyl)-lysine (CML)] and lipoxidation-[N(epsilon)-(malondialdehyde)-lysine-(MDAL)], as well as renal fatty acid composition. Urinary albumin (a marker of DN), DNP, GSA, and MDAL levels, were increased in all obese rats and were dose dependently decreased by AVE7688 whereas Ramipril and Losartan were less efficient. These results show that RAS inhibition improves DN at several levels, independently of its effects on blood pressure and glycemic control, via mechanisms depending of renal oxidative stress.

• One target-multiple indications: a call for an integrated common mechanisms strategy.

  Authors: Ulrich Nielsch, Stefan Schäfer, Hanno Wild, Andreas Busch

  Drug discovery today. 01/2008; 12(23-24):1025-31.

  Ever-increasing research and development costs are putting constant pressure on the pharmaceutical industry to improve their efficiency. Efforts to increase the output of the research pipeline haveEver-increasing research and development costs are putting constant pressure on the pharmaceutical industry to improve their efficiency. Efforts to increase the output of the research pipeline have yielded limited success. Traditionally, maximization of the value of a drug is attempted through life-cycle management, which is initiated late in development, or when the drug is already on the market. Validated targets can be exploited further through development of a follow-up drug, which may offer advantages regarding safety or convenience. In this article, we propose to systematically evaluate the full therapeutic potential of a drug target, proprietary chemical lead structure, or drug candidate as broad and as early as possible and we call this the 'common mechanism' approach.

• Vasopeptidase inhibition prevents target organ damage and improves survival in spontaneously hypertensive rats.

  Authors: Wolfgang Linz, Stefan Schäfer, Freni Afkham, Martin Gerl, Hans-Ludwig Schmidts, Hartmut Rütten

  Journal of the renin-angiotensin-aldosterone system : JRAAS. 10/2006; 7(3):155-61.

  BACKGROUND: Vasopeptidase inhibition has been shown to be an effective antihypertensive principle but its long-term effects on hypertensive target organ damage are not known. We investigated theBACKGROUND: Vasopeptidase inhibition has been shown to be an effective antihypertensive principle but its long-term effects on hypertensive target organ damage are not known. We investigated the myocardial, vascular and renal effects of chronic vasopeptidase inhibition in arterial hypertension. METHODS AND RESULTS: One hundred and thirty-nine male spontaneously hypertensive rats aged 15 months were treated chronically with either the pure angiotensin-converting enzyme (ACE) inhibitor, ramipril (1 mg/kg/d in drinking water, n=46), or the vasopeptidase inhibitor AVE7688 (30 mg/kg/d in chow, n=46), or placebo (n=47) and followed up until they died. After six months, both ramipril and AVE7688 had markedly reduced plasma ACE activity, normalised blood pressure (BP), reduced left ventricular mass and improved systolic function to similar extents. Acetylcholine mediated relaxation of aortic rings was improved by both ramipril and AVE7688. There was substantial albuminuria in the placebo group (albumin-to-creatinine ratio 107+/-54 microg/mg), which was significantly reduced by ramipril to 57+/-34 microg/mg, and practically abolished in the AVE7688 group (22+/-12 microg/mg, p<0.05 vs. placebo and ramipril). Tubulo-interstitial damage (semi-quantitative score) was significantly reduced by AVE7688 and ramipril. Overall mortality was markedly reduced in the ramipril and AVE7688 groups (13% and 16% at six months, respectively), both p<0.05 vs. placebo (71%). CONCLUSIONS: Vasopeptidase inhibition effectively controls BP and reduces myocardial, vascular and renal target organ damage, resulting in a markedly prolonged survival. At similar degrees of plasma ACE inhibition, AVE7688 compared to ramipril offers superior protection against hypertensive kidney damage.

• Decreased wheel-running activity in hamsters post myocardial infarction.

  Authors: Stefan Schäfer, Wolfgang Linz, Katja Hürland

  Journal of translational medicine. 01/2006; 4:51.

  Reduced exercise capacity is a key symptom and an independent determinant of mortality in patients with heart failure. We analyzed the running activity of hamsters with cardiac dysfunction afterReduced exercise capacity is a key symptom and an independent determinant of mortality in patients with heart failure. We analyzed the running activity of hamsters with cardiac dysfunction after myocardial infarction. In 39 male Syrian hamsters aged 10 to 12 weeks, a myocardial infarction (MI) was produced by permanent ligation of the left coronary artery. Spontaneous running activity in a wheel was monitored daily. After four weeks, left ventricular (LV) hemodynamics (catheter tip manometry) were measured at baseline and during inotropic stimulation (isoprenaline 0.03, 0.1 and 0.3 microg/kg/min i.v.). LV infarct size was quantified using planimetry. Four weeks post MI, daily running distance was reduced stepwise in animals with small (4-15% of LV: 9.8 +/- 3.4 km/d) and large (> 15% of LV: 7.5 +/- 3.5 km/d) MI, compared to sham-operated hamsters (11.5 +/- 1.5 km/d). Similar reductions were observed in maximum speed and distance of longest running period. MI size influenced daily running distance, maximum speed, and longest running period (linear correlations, all p < 0.05). MI size also impaired LV systolic and diastolic function under isoprenaline stimulation. The results suggest that myocardial infarction reduces running capacity and isoprenaline stimulated LV function in hamsters, mimicking impaired exercise performance in patients with heart failure. Analysis of running activity in hamsters with myocardial infarction offers a unique opportunity for non-invasive and serial functional assessment of heart failure in the experimental setting.

• Impaired left ventricular relaxation in type 2 diabetic rats is related to myocardial accumulation of N(epsilon)-(carboxymethyl) lysine.

  Authors: Stefan Schäfer, Jochen Huber, Cornelia Wihler, Hartmut Rütten, Andreas E Busch, Wolfgang Linz

  European journal of heart failure : journal of the Working Group on Heart Failure of the European Society of Cardiology. 01/2006; 8(1):2-6.

  Myocardial dysfunction in the absence of myocardial ischemia is frequent in patients with diabetes mellitus but the underlying pathomechanism is unclear. We investigated whether accumulation ofMyocardial dysfunction in the absence of myocardial ischemia is frequent in patients with diabetes mellitus but the underlying pathomechanism is unclear. We investigated whether accumulation of advanced glycation end products (AGEs) in the diabetic myocardium is related to its functional abnormalities. In 11 male homozygous Zucker diabetic fatty rats (ZDF/Gmi-fa/fa) aged 37 weeks (OBESE) and 11 non-obese, non-diabetic littermates (LEAN), we measured left ventricular function (pressure-volume catheter) and levels of N(epsilon)-(carboxymethyl) lysine (CML), a prototypical AGE, in serum and the left ventricle (competitive enzyme linked immuno-assay). Overt diabetes mellitus (HbA1c > 9%) was present in all OBESE animals but not in LEAN. Systolic left ventricular function was not different between the groups, but the markers of left ventricular relaxation, dP/dt(min) and the relaxation constant tau, were impaired in OBESE. In parallel, CML levels were increased in serum (273 +/- 15 vs. 197 +/- 10 ng/ml, p<0.05) and in the left ventricle (18.4 +/- 1.1 vs. 12.5 +/- 2.0 ng/mg protein, p < 0.05) in OBESE compared to LEAN. There was a linear correlation between tau and the left ventricular CML levels (r = 0.65; p < 0.05). We conclude that type 2 diabetes is associated with predominant left ventricular diastolic dysfunction. Myocardial accumulation of advanced glycation end products may contribute to relaxation abnormalities in type 2 diabetes.

• Down-regulation of calpain 9 is linked to hypertensive heart and kidney disease.

  Authors: André Markmann, Stefan Schäfer, Wolfgang Linz, Matthias Löhn, Andreas E Busch, Paulus Wohlfart

  Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology. 02/2005; 15(1-4):109-16.

  Calpains are a family of 14 intracellular calcium-dependent proteases, which have been implicated in cardiovascular diseases. We aimed to analyze specifically the expressional regulation of theCalpains are a family of 14 intracellular calcium-dependent proteases, which have been implicated in cardiovascular diseases. We aimed to analyze specifically the expressional regulation of the different calpain isoforms in hypertensive target organ damage. Using real-time PCR, we found calpain 6 and 9 down-regulated by more than 50% and the endogenous calpain inhibitor calpastatin up-regulated by 225%, respectively, in the hearts of Dahl salt-sensitive rats on a high salt (4% NaCl) compared to normal salt diet. On the protein level, calpain 9 but not calpastatin was regulated in the hypertensive target organs heart and kidney. Moreover, the myocardial expression of calpain 9 protein was inversely linked to left ventricular mass (r= -0.93, p<0.01), and renal expression of calpain 9 protein correlated inversely with albuminuria (r= -0.82, p<0.05). In the aorta, there was no regulation of calpain 9 on the protein level. We conclude that differential regulation of calpain 9 may play a role in hypertensive target organ damage.

• Role of activators of ferric sGC in cardiovascular disease

  Authors: Ursula Schindler, Martina Klein, Wolfgang Linz, Hartmut Rütten, Stefan Schäfer, Hartmut Strobel, Andreas Schäfer, Johann Bauersachs, Sven Wassmann, van Eickels Martin

  BMC Pharmacology. 01/2005;

• Protein kinase C pathway is involved in transcriptional regulation of C-reactive protein synthesis in human hepatocytes.

  Authors: Yuri Ivashchenko, Frank Kramer, Stefan Schäfer, Andrea Bucher, Kerstin Veit, Vinzenz Hombach, Andreas Busch, Olaf Ritzeler, Jürgen Dedio, Jan Torzewski

  Arteriosclerosis, thrombosis, and vascular biology. 01/2005; 25(1):186-92.

  C-reactive protein (CRP) is the prototype acute phase protein and a cardiovascular risk factor. Interleukin-1beta (IL-1beta) and IL-6 stimulate CRP synthesis in hepatocytes. We searched forC-reactive protein (CRP) is the prototype acute phase protein and a cardiovascular risk factor. Interleukin-1beta (IL-1beta) and IL-6 stimulate CRP synthesis in hepatocytes. We searched for additional pathways regulating CRP expression. Primary human hepatocytes (PHHs) were treated with IL-1beta, IL-6, and protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu). CRP was analyzed by quantitative RT-PCR and ELISA. PDBu significantly induced CRP transcription by 21.0+/-9.24-fold and protein release by 2.9+/-0.5-fold. Transcriptional regulation was studied in detail in hepatoma G2 (HepG2) cells stably transfected with the 1-kb CRP promoter (HepG2-ABEK14 cells). In these cells, PDBu significantly induced CRP transcription by 5.39+/-0.66-fold. Competitive inhibition with bisindolylmaleimide derivative LY333531 abolished PDBu-mediated promoter activation. Competitive inhibition with IkappaB kinase inhibitor I229 also inhibited PDBu effects. Importantly, IL-8 significantly induced CRP release in PHHs by 58.675+/-19.1-fold, which was blockable by LY333531. This study describes a novel PKC-dependent transcriptional regulation of CRP gene expression, which, in analogy to the classical IL-1beta and IL-6 pathways, is operational in hepatocytes only. It also identifies IL-8 as a potential physiological PKC activator. HepG2-ABEK14 cells may be useful for high throughput screening to identify inhibitors of CRP synthesis for the prevention of cardiovascular disease.

• Nephroprotection in Zucker diabetic fatty rats by vasopeptidase inhibition is partly bradykinin B2 receptor dependent.

  Authors: Stefan Schäfer, Hans-Ludwig Schmidts, Markus Bleich, Andreas E Busch, Wolfgang Linz

  British journal of pharmacology. 10/2004; 143(1):27-32.

  1. Vasopeptidase inhibition (i.e., the simultaneous inhibition of both angiotensin-converting enzyme (ACE) and neutral endopeptidase) can ameliorate diabetic nephropathy. We investigated whether this1. Vasopeptidase inhibition (i.e., the simultaneous inhibition of both angiotensin-converting enzyme (ACE) and neutral endopeptidase) can ameliorate diabetic nephropathy. We investigated whether this nephroprotection is mediated by the bradykinin B2 receptor. 2. In all, 43 obese Zucker diabetic fatty (ZDF/Gmi-fa/fa) rats aged 21 weeks were separated into four groups and treated for 26 weeks with either placebo, the bradykinin B2 receptor antagonist icatibant (500 microg kg(-1) day(-1) s.c. infusion), the vasopeptidase inhibitor AVE7688 (45 mg kg(-1) day(-1) in chow), or AVE7688 plus icatibant. Nephropathy was assessed as albuminuria at age 31 and 39 weeks, and by histopathologic scoring at the end of the treatment period. 3. All animals had established diabetes mellitus (blood glucose >20 mmol l(-1)) and marked albuminuria at baseline. Blood glucose was not influenced by any treatment. Icatibant alone did not influence albuminuria (8.6+/-1.6 vs placebo 9.5+/-1.3 mg kg(-1) h(-1)). AVE7688 reduced albuminuria at week 31 markedly to 1.1+/-0.1 mg kg(-1) h(-1) and reduced glomerular and tubulo-interstitial kidney damage at week 47. In the AVE7688 plus icatibant group, proteinuria was significantly higher than in the AVE7688 only group (2.0+/-0.6 mg kg(-1) h(-1)), but still reduced compared to placebo. In addition, icatibant partly antagonized the tubulo-interstitial protection mediated by AVE7688. 4. We conclude that vasopeptidase inhibition provides nephroprotection in rats with type II diabetic nephropathy, which is partly mediated by bradykinin B2 receptor activation.

• Inhibition of Na+-H+ exchange by cariporide reduces inflammation and heart failure in rabbits with myocardial infarction.

  Authors: Katrin Rungwerth, Ursula Schindler, Martin Gerl, Stefan Schäfer, Thomas Licher, Andreas E Busch, Hartmut Ruetten

  British journal of pharmacology. 08/2004; 142(7):1147-54.

  The aim of this study was to assess the effects of the Na+-H+ exchange inhibitor cariporide on left ventricular (LV) morphology and function as well as inflammation in rabbits with heart failure.The aim of this study was to assess the effects of the Na+-H+ exchange inhibitor cariporide on left ventricular (LV) morphology and function as well as inflammation in rabbits with heart failure. Rabbits with myocardial infarction (MI) and sham controls were randomized to receive either standard chow or chow supplemented with cariporide for 9 weeks. LV morphology was determined by echocardiography. LV systolic and diastolic function was assessed under load-dependent and -independent conditions by analysis of LV pressure-volume loops using piezo-electric crystals. Plasma concentrations of C-reactive protein and aldosterone were measured. Rabbits with MI developed LV dilatation that was reduced by cariporide. Systolic and diastolic LV function was impaired in rabbits with MI when compared to sham, as indicated by a decreased dP/dtmax (MI: 3537 +/- 718 mmHg s(-1), sham: 5839 +/- 247 mmHg s(-1), P < 0.05), the load-independent preload recruitable stroke work (PRSW)(MI: 22 +/-7 mmHg, sham: 81 +/- 23 mmHg, P < 0.05) and a reduction in the time constant of relaxation tau (tau) (MI: 27+/-1 ms, sham: 17+/-1 ms, P < 0.05), and significantly improved by cariporide (dP/dtmax: 4586 +/- 374 mmHg s(-1), PRSW: 67 +/- 18 mmHg, tau: 20 +/- 2 ms; P < 0.05 vs MI/control). Induction of MI was associated with an increase in aldosterone and CRP, indicating activation of the neurohormonal and the inflammatory system that were largely reduced by cariporide. Cariporide improves LV morphology and function post MI and suppresses inflammation and neurohormonal activation in congestive heart failure (CHF). Na+-H+ exchange inhibition may represent a new pharmaceutical approach for the treatment of CHF.

• Chronic vasopeptidase inhibition normalizes diabetic endothelial dysfunction.

  Authors: Stefan Schäfer, Kerstin Steioff, Wolfgang Linz, Markus Bleich, Andreas E Busch, Matthias Löhn

  European journal of pharmacology. 02/2004; 484(2-3):361-2.

  Type 2 diabetes mellitus is a major cause of vascular morbidity but animal models for this disease have not been adequately characterized. We demonstrate that endothelial dysfunction is present inType 2 diabetes mellitus is a major cause of vascular morbidity but animal models for this disease have not been adequately characterized. We demonstrate that endothelial dysfunction is present in the Zucker diabetic fatty (ZDF) rat. Vasopeptidase inhibition with AVE7688 (7-[[(2S)-2-(acetylthio)-1-oxo-3-methylpropyl]amino]-1,2,3,4,6,7,8,12b-octahydro-6-oxo-(4S,7S,12bR)-pyrido[2,1-a][2]benzazepine-4-carboxic acid), 45 mg/kg/day in chow for 6 weeks, normalized acetylcholine mediated relaxation of mesenteric artery rings. Thus, chronic vasopeptidase inhibition may prevent vascular complications related to type 2 diabetes mellitus.

• Vasopeptidase inhibition prevents nephropathy in Zucker diabetic fatty rats.

  Authors: Stefan Schäfer, Wolfgang Linz, Axel Bube, Martin Gerl, Jochen Huber, Gert Ulrich Kürzel, Markus Bleich, Hans-Ludwig Schmidts, Andreas E Busch, Hartmut Rütten

  Cardiovascular research. 12/2003; 60(2):447-54.

  BACKGROUND: Blocking the renin-angiotensin system is an established therapeutic principle in diabetic nephropathy. We investigated whether inhibition of both neutral endopeptidase and ACEBACKGROUND: Blocking the renin-angiotensin system is an established therapeutic principle in diabetic nephropathy. We investigated whether inhibition of both neutral endopeptidase and ACE (vasopeptidase inhibition) can prevent functional and morphological features of nephropathy in the Zucker diabetic fatty (ZDF) rat, an animal model of type II diabetes. METHODS: Homozygous (fa/fa) ZDF rats (each n=15) aged 10 weeks were treated with placebo, ramipril (1 mg/kg/day in drinking water), or the vasopeptidase inhibitor AVE7688 (45 mg/kg/day in chow). Metabolic parameters and renal function (metabolic cages) were assessed at baseline (age 10 weeks), and at age 17, 27, and 37 weeks. Twenty heterozygous animals (fa/-) served as lean, nondiabetic controls. At age 37 weeks, the animals were sacrificed and the kidneys analyzed histopathologically. RESULTS: Overt diabetes mellitus (blood glucose >20 mmol/l) was established at age 17 weeks in all homozygous ZDF rats. In the placebo group, urinary protein excretion increased progressively from 8+/-1 (baseline) to 342+/-56 mg/kg/day (week 37) whereas diabetes and proteinuria were absent in the lean control group. Ramipril tended to reduce albuminuria and morphological damage (p=ns) but AVE7688 virtually prevented albuminuria (33+/-12 mg/kg/day, p<0.05 vs. ZDF placebo) and drastically reduced the incidence and severity of glomerulosclerosis and tubulointerstitial damage. CONCLUSIONS: In ZDF rats, development of diabetes mellitus is accompanied by functional and morphological kidney damage that resembles human diabetic nephropathy. Diabetic nephropathy can be prevented by chronic vasopeptidase inhibition.