Stefan Schäfer

Combined Tyrosine and Serine/Threonine Kinase Inhibition by Sorafenib Prevents Progression of Experimental Pulmonary Hypertension and Myocardial Remodeling

Background— 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.

Adenosine A2-receptor activation at reperfusion reduces infarct size and improves myocardial wall function in dog heart

Reestablishment of blood supply to ischemic myocardium leads to biochemical and cellular changes which are believed to reduce the amount of potentially salvageable myocardium (reperfusion injury). In this situation, adenosine is known to have myocardial protective properties. Activation of adenosine A2-receptors may account for most of the beneficial effects of adenosine in reperfusion injury because A2-receptor activation mediates vasodilation, inhibits neutrophil adhesion to vascular endothelium and diminishes generation of free radicals by neutrophils, thus acting on some of the key mechanisms of reperfusion injury such as postischemic vascular dysfunction and neutrophil-mediated damage. Therefore, we investigated the effect of an intracoronary A2-agonist, CGS 21680, on regional postischemic myocardial function (measured as wall thickening) and infarct size [determined by triphenyltetrazolium chloride (TTC) staining]. Fourteen anesthetized open-chest dogs underwent 1-h left anterior descending artery (LAD) occlusion and 6-h reperfusion and were randomly assigned to receive intracoronary CGS 21680 or to serve as control. The drug was infused for 60 min starting 5 min before reperfusion with a concentration of 10 -7 M at a rate of 10 ml/min under anoxic conditions. The infusion was then continued for the first 55 min of reperfusion with 10 -6 M at a rate of 1 ml/min. Intracoronary infusion of CGS 21680 led to significant improvement in regional wall function in postischemic myocardium (p < 0.05 vs. control). Thickening fraction (percentage of baseline) increased from −13.1 ± 13.7% (mean ± SD) during occlusion to 15.3 ± 29.8% at 30 min of reperfusion in the CGS 21680 treatment group and remained at this level throughout the reperfusion period. In the control group, thickening fraction was - 23.7 ± 16.2% during occlusion and did not recover during reperfusion. A significant reduction in infarct size was noted in the treatment group (11.5 ± 7.9% of area at risk) as compared with the control group (28.6 ± 11.4%, p < 0.05). These findings support the hypothesis that the protective effect of adenosine on reperfusion injury is not due to replenishment of the nucleoside pool but rather is induced by stimulation of the adenosine A2-receptor.

Finerenone, a novel selective nonsteroidal mineralocorticoid receptor antagonist protects from rat cardiorenal injury.

Abstract: Pharmacological blockade of the mineralocorticoid receptor (MR) ameliorates end-organ damage in chronic heart failure. However, the clinical use of available steroidal MR antagonists is restricted because of concomitant hyperkalemia especially in patients with diminished kidney function. We have recently identified a novel nonsteroidal MR antagonist, finerenone, which uniquely combines potency and selectivity toward MR. Here, we investigated the tissue distribution and chronic cardiorenal end-organ protection of finerenone in comparison to the steroidal MR antagonist, eplerenone, in 2 different preclinical rat disease models. Quantitative whole-body autoradiography revealed that [14C]-labeled finerenone equally distributes into rat cardiac and renal tissues. Finerenone treatment prevented deoxycorticosterone acetate-/salt-challenged rats from functional as well as structural heart and kidney damage at dosages not reducing systemic blood pressure. Finerenone reduced cardiac hypertrophy, plasma prohormone of brain natriuretic peptide, and proteinuria more efficiently than eplerenone when comparing equinatriuretic doses. In rats that developed chronic heart failure after coronary artery ligation, finerenone (1 mg·kg−1·d−1), but not eplerenone (100 mg·kg−1·d−1) improved systolic and diastolic left ventricular function and reduced plasma prohormone of brain natriuretic peptide levels. We conclude that finerenone may offer end-organ protection with a reduced risk of electrolyte disturbances.

Vasopeptidase inhibition prevents nephropathy in Zucker diabetic fatty rats

BACKGROUND 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.

Regional contractile blockade at the onset of reperfusion reduces infarct size in the dog heart

An important mechanism of lethal myocardial reperfusion injury is the development of cellular hypercontracture at the onset of reperfusion. Hypercontracture can lead to cytolysis by mutual mechanical disruption of myocardial cells. 2,3-Butanedione monoxime (BDM) inhibits myofibrillar cross-bridge cycling and may therefore reduce infarct size in ischaemic reperfused myocardium. This study investigated whether a temporary presence of BDM protects against myocardial reperfusion injury in an intact-animal preparation. Anaesthetized open-chest dogs (n=10) underwent 1 h of left anterior descendent artery (LAD) occlusion and received intracoronary BDM (25 mM, n=5) or vehicle (n=5) for 65 min starting with an anoxic local infusion 5 min before reperfusion. Infarct size was assessed by triphenyltetrazolium staining after 6 h reperfusion. The infusion of BDM was accompanied by a transient reduction of left ventricular systolic pressure from 84.3±11.2 mm Hg during occlusion to 66.4±9.9 mm Hg at 30 min reperfusion (mean±SD, P<0.01 vs. control). LAD-flow and regional wall motion in the area at risk showed no difference between groups. Infarct size (% of area at risk) was reduced from 24.4±8.7 (control) to 6.6±2.0% (BDM) (P<0.01). The results demonstrate that development of necrosis in reperfused myocardium can be greatly reduced by temporary presence of the contractile inhibitor BDM at the onset of reperfusion.

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

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 of advanced glycation end products (AGEs) in the diabetic myocardium is related to its functional abnormalities.

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

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. 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 μg 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. 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. We conclude that vasopeptidase inhibition provides nephroprotection in rats with type II diabetic nephropathy, which is partly mediated by bradykinin B2 receptor activation.

Protein Kinase C Pathway Is Involved in Transcriptional Regulation of C-Reactive Protein Synthesis in Human Hepatocytes

Objective— C-Reactive protein (CRP) is the prototype acute phase protein and a cardiovascular risk factor. Interleukin-1&bgr; (IL-1&bgr;) and IL-6 stimulate CRP synthesis in hepatocytes. We searched for additional pathways regulating CRP expression. Methods and Results— Primary human hepatocytes (PHHs) were treated with IL-1&bgr;, 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. Competetive inhibition with bisindolylmaleimide derivative LY333531 abolished PDBu-mediated promoter activation. Competetive inhibition with I&kgr;B 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. Conclusions— This study describes a novel PKC-dependent transcriptional regulation of CRP gene expression, which, in analogy to the classical IL-1&bgr; 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.

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

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 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.

Vasopeptidase inhibition improves insulin sensitivity and endothelial function in the JCR:LA-cp rat.

The insulin-resistant, hyperinsulinemic, normoglycemic, and obese JCR:LA-cp rat was used to study the effects of ramipril (an ACE inhibitor) and AVE7688 (a dual inhibitor of ACE and neutral endopeptidases) on insulin sensitivity and vascular function. Both compounds reduced the surge of plasma insulin in a meal tolerance test by approximately 50%. Ramipril had no effect on acetylcholine-induced relaxation but increased the sensitivity to sodium nitroprus-side at low concentrations. AVE7688 significantly reduced the EC50 for acetylcholine to relax phenylephrine-contracted aortic rings. None of the compounds affected the baseline coronary flow and reactive hyperemia. Coronary flow response to bradykinin in AVE7688- and ramipril-treated rat hearts showed a significantly lower EC50 than in control rats. Maximum flow rate was not different between groups. In summary, both ramipril and AVE7688 had significant hypoinsulinemic and insulin-sensitizing effects. Whereas ramipril had limited vascular effects, AVE7688 had more marked beneficial vascular effects, probably of endothelial origin and possibly related to lowered insulin levels.