Hartmut Ruetten

Fas receptor signaling inhibits glycogen synthase kinase 3β and induces cardiac hypertrophy following pressure overload

Congestive heart failure is a leading cause of mortality in developed countries. Myocardial hypertrophy resulting from hypertension often precedes heart failure. Understanding the signaling underlying cardiac hypertrophy and failure is of major interest. Here, we identified Fas receptor activation, a classical death signal causing apoptosis via activation of the caspase cascade in many cell types, as a novel pathway mediating cardiomyocyte hypertrophy in vitro and in vivo. Fas activation by Fas ligand induced a hypertrophic response in cultured cardiomyocytes, which was dependent on the inactivation of glycogen synthase kinase 3 beta (GSK3 beta) by phosphorylation. In vivo, lpr (lymphoproliferative disease) mice lacking a functional Fas receptor demonstrated rapid-onset left ventricular dilatation and failure, absence of compensatory hypertrophy, and significantly increased mortality in response to pressure overload induction that was accompanied by a failure to inhibit GSK3 beta activity. In contrast, Fas ligand was dispensable for the development of pressure overload hypertrophy in vivo. In vitro, neonatal cardiomyocytes from lpr mice showed a completely abrogated or significantly blunted hypertrophic response after stimulation with Fas ligand or angiotensin II, respectively. These findings indicate that Fas receptor signaling inhibits GSK3 beta activity in cardiomyocytes and is required for compensation of pressure overload in vivo.

Cardiovascular abnormalities with normal blood pressure in tissue kallikrein-deficient mice

Tissue kallikrein is a serine protease thought to be involved in the generation of bioactive peptide kinins in many organs like the kidneys, colon, salivary glands, pancreas, and blood vessels. Low renal synthesis and urinary excretion of tissue kallikrein have been repeatedly linked to hypertension in animals and humans, but the exact role of the protease in cardiovascular function has not been established largely because of the lack of specific inhibitors. This study demonstrates that mice lacking tissue kallikrein are unable to generate significant levels of kinins in most tissues and develop cardiovascular abnormalities early in adulthood despite normal blood pressure. The heart exhibits septum and posterior wall thinning and a tendency to dilatation resulting in reduced left ventricular mass. Cardiac function estimated in vivo and in vitro is decreased both under basal conditions and in response to βadrenergic stimulation. Furthermore, flow-induced vasodilatation is impaired in isolated perfused carotid arteries, which express, like the heart, low levels of the protease. These data show that tissue kallikrein is the main kinin-generating enzyme in vivo and that a functional kallikrein–kinin system is necessary for normal cardiac and arterial function in the mouse. They suggest that the kallikrein–kinin system could be involved in the development or progression of cardiovascular diseases.

Effect of calpain inhibitor I, an inhibitor of the proteolysis of IκB, on the circulatory failure and multiple organ dysfunction caused by endotoxin in the rat

We compared the effects of calpain inhibitor I (inhibitor of the proteolysis of IκB and, hence, of the activation of nuclear factor κB (NFκB)) and dexamethasone on (i) the circulatory failure, (ii) multiple organ dysfunction and (iii) induction of the inducible isoforms of nitric oxide (NO) synthase (iNOS) and cyclo‐oxygenase (COX‐2) in anaesthetized rats with endotoxic shock. Injection of lipopolysaccharide (LPS, E. coli, 10 mg kg−1, i.v.) resulted in hypotension and a reduction of the pressor responses elicited by noradrenaline. This circulatory dysfunction was attenuated by pretreatment of LPS‐rats with calpain inhibitor I (10 mg kg−1, i.v., 2 h before LPS) or dexamethasone (1 mg kg−1, i.v.). Endotoxaemia also caused rises in the serum levels of (i) urea and creatinine (renal dysfunction), (ii) alanine aminotransferase (ALT), aspartate aminotransferase (AST) (hepatocellular injury), bilirubin and γ‐glutamyl transferase (γGT) (liver dysfunction), (iii) lipase (pancreatic injury) and (iv) lactate. Calpain inhibitor I and dexamethasone attenuated the liver injury, the pancreatic injury, the lactic acidosis as well as the hypoglycaemia caused by LPS. Dexamethasone, but not calpain inhibitor I, reduced the renal dysfunction caused by LPS. Endotoxaemia for 6 h resulted in a substantial increase in iNOS and COX‐2 protein and activity in lung and liver, which was attenuated in LPS‐rats pretreated with calpain inhibitor I or dexamethasone. Thus, calpain inhibitor I and dexamethasone attenuate (i) the circulatory failure, (ii) the multiple organ dysfunction (liver and pancreatic dysfunction/injury, lactic acidosis, hypoglycaemia), as well as (iii) the induction of iNOS and COX‐2 protein and activity in rats with endotoxic shock. We propose that prevention of the activation of NF‐κB in vivo may be useful in the therapy of circulatory shock or of disorders associated with local or systemic inflammation.

Effects of tyrphostins and genistein on the circulatory failure and organ dysfunction caused by endotoxin in the rat: a possible role for protein tyrosine kinase

1 Here we compared the effects of various inhibitors of the activity of protein tyrosine kinase on (i) the expression of the activity of the inducible isoform of nitric oxide (NO) synthase (iNOS) caused by endotoxin (lipopolysaccharide, LPS) in cultured macrophages, (ii) the induction of iNOS and cyclo‐oxygenase 2 (COX‐2) protein and activity in rats with endotoxaemia, and (iii) the circulatory failure and organ dysfunction caused by LPS in the anaesthetized rat. 2 Activation of murine cultured macrophages with LPS (1 μg ml−1) resulted, within 24 h, in a significant increase in nitrite (an indicator of the formation of NO) in the cell supernatant. This increase in nitrite was attenuated by the tyrphostins AG126, AG556, AG490 or AG1641 or by genistein in a dose‐dependent fashion (IC50: ∼15 μM). In contrast, tyrphostin A1 (an analogue of tyrphostin AG126) or daidzein (an analogue of genistein) had no effect on the rise in nitrite caused by LPS. 3 Administration of LPS (E. coli, 10 mg kg−1, i.v.) caused hypotension and a reduction of the pressor responses elicited by noradrenaline (NA, 1 μg kg−1, i.v.). Pretreatment of rats with the tyrphostins AG126, AG490, AG556, AG1641 or A1 attenuated the circulatory failure caused by LPS. Although genistein attenuated the vascular hyporeactivity to NA, it did not affect the hypotension caused by LPS. Daidzein did not affect the circulatory failure caused by LPS. 4 Endotoxaemia for 360 min resulted in rises in the serum levels of (i) urea and creatinine (indicators of renal failure), (ii) alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin and γ‐glutamyl transferase (γGT) (indicators of liver injury/dysfunction), lipase (an indicator of pancreatic injury) as well as lactate (an indicator of tissue hypoxia). None of the tyrosine kinase inhibitors tested had a significant effect on the rise in the serum levels of urea, but the tyrphostins AG126, AG556 or A1 significantly attenuated the rises in the serum levels of creatinine caused by LPS. In addition, all tyrphostins and genistein attenuated the liver injury/failure, the pancreatic injury, the hypoglycaemia and the lactic acidosis caused by LPS. In contrast, daidzein did not reduce the organ injury/dysfunction or the lactic acidosis caused by LPS. 5 Injection of LPS resulted (within 90 min) in a substantial increase in the serum level of tumour necrosis factor α (TNFα), which was attenuated by pretreatment of LPS‐rats with any of the tyrphostins used. Genistein, but not daidzein, also reduced the rise in the serum levels of TNFα caused by LPS. Endotoxaemia for 6 h also resulted in a substantial increase in the expression of iNOS and COX‐2 protein and activity in the lung, which was attenuated by pretreatment of LPS‐rats with the tyrphostins AG126, AG556 or genistein, but not by daidzein. 6 Thus, tyrphostins (AG126, AG490, AG556, AG1641 or A1) and genistein, but not daidzein (inactive analogue of genistein), prevent the (i) circulatory failure, (ii) the multiple organ dysfunction (liver and pancreatic dysfunction/injury, lactacidosis, hypoglycaemia), as well as (iii) the induction of iNOS and COX‐2 protein and activity in rats with endotoxic shock

Improvement in Left Ventricular Remodeling by the Endothelial Nitric Oxide Synthase Enhancer AVE9488 After Experimental Myocardial Infarction

Background— Reduced endothelial nitric oxide (NO) bioavailability contributes to the progression of heart failure. In this study, we investigated whether the transcription enhancer of endothelial NO synthase (eNOS) AVE9488 improves cardiac remodeling and heart failure after experimental myocardial infarction (MI). Methods and Results— Starting 7 days after coronary artery ligation, rats with MI were treated with placebo or AVE9488 (25 ppm) as a dietary supplement for 9 weeks. AVE9488 therapy versus placebo substantially improved left ventricular (LV) function, reduced LV filling pressure, and prevented the rightward shift of the pressure-volume curve. AVE9488 also attenuated the extent of pulmonary edema, reduced LV fibrosis and myocyte cross-sectional area, and prevented the increases in LV gene expression of atrial natriuretic factor, brain natriuretic peptide, and endothelin-1. eNOS protein levels and calcium-dependent NOS activity were decreased in the surviving LV myocardium from placebo MI rats and normalized by AVE9488. The beneficial effects of AVE9488 on LV dysfunction and remodeling after MI were abrogated in eNOS-deficient mice. Aortic eNOS protein expression and endothelium-dependent NO-mediated vasorelaxation were significantly enhanced by AVE9488 treatment after infarction, whereas increased vascular superoxide anion formation was reduced. Moreover, AVE9488 prevented the marked depression of circulating endothelial progenitor cell levels in rats with heart failure after MI. Conclusions— Long-term treatment with the eNOS enhancer AVE9488 improved LV remodeling and contractile dysfunction after MI. Molecular alterations, circulating endothelial progenitor cell levels, and endothelial vasomotor dysfunction were improved by AVE9488. Pharmacological interventions designed to increase eNOS-derived NO constitute a promising therapeutic approach for the amelioration of postinfarction ventricular remodeling and heart failure.

Inhibition of endogenous nitric oxide synthase potentiates ischemia–reperfusion-induced myocardial apoptosis via a caspase-3 dependent pathway

OBJECTIVE Apoptosis of cardiomyocytes may contribute to ischemia-reperfusion injury. The role of nitric oxide (NO) in apoptosis is controversial. Therefore, we investigated the effect of NO synthase inhibition on apoptosis of cardiomyocytes during ischemia and reperfusion and elucidated the underlying mechanisms. METHODS AND RESULTS Isolated perfused rat hearts (n = 6/group) were subjected to ischemia (30 min) and reperfusion (30 min) in the presence or absence of the NO synthase inhibitor NG-mono-methyl-L-arginine. Reperfusion induced cardiomyocyte apoptosis as assessed by immunohistochemistry (TUNEL-staining) and the demonstration of the typical DNA laddering. Apoptosis during reperfusion was associated with the cleavage of caspase-3, the final down-stream executioner caspase, whereas the protein levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were unchanged. Inhibition of the NO synthase drastically increased ischemia and reperfusion-induced apoptosis of cardiomyocytes. Moreover, the NO synthase inhibitor enhanced the activation of caspase-3, suggesting that NO interferes with the activation of caspases in ischemia-reperfusion. CONCLUSION The results of the present study demonstrate that inhibition of endogenous NO synthesis during ischemia and reperfusion leads to an enhanced induction of apoptosis, suggesting that the endogenous NO synthesis protects against apoptotic cell death. Inhibition of NO synthesis thereby activates the caspase cascade, whereas the Bcl-2/Bax protein levels remained unchanged.

Antiatherosclerotic Effects of Small-Molecular-Weight Compounds Enhancing Endothelial Nitric-Oxide Synthase (eNOS) Expression and Preventing eNOS Uncoupling

Many cardiovascular diseases are associated with reduced levels of bioactive nitric oxide (NO) and an uncoupling of oxygen reduction from NO synthesis in endothelial NO synthase (eNOS uncoupling). In human endothelial EA.hy 926 cells, two small-molecular-weight compounds with related structures, 4-fluoro-N-indan-2-yl-benzamide (CAS no. 291756-32-6; empirical formula C16H14FNO; AVE9488) and 2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid indan-2-ylamide (CAS no. 450348-85-3; empirical formula C17H13F2NO3; AVE3085), enhanced eNOS promoter activity in a concentration-dependent manner; with the responsible cis-element localized within the proximal 263 base pairs of the promoter region. RNA interference-mediated knockdown of the transcription factor Sp1 significantly reduced the basal activity of eNOS promoter, but it did not prevent the transcription activation by the compounds. Enhanced transcription of eNOS by AVE9488 in primary human umbilical vein endothelial cells was associated with increased levels of eNOS mRNA and protein expression, as well as increased bradykinin-stimulated NO production. In both wild-type C57BL/6J mice and apolipoprotein E-knockout (apoE-KO) mice, treatment with AVE9488 resulted in enhanced vascular eNOS expression. In apoE-KO mice, but not in eNOS-knockout mice, treatment with AVE9488 reduced cuff-induced neointima formation. A 12-week treatment with AVE9488 or AVE3085 reduced atherosclerotic plaque formation in apoE-KO mice, but not in apoE/eNOS-double knockout mice. Aortas from apoE-KO mice showed a significant generation of reactive oxygen species. This was partly prevented by nitric-oxide inhibitor Nω-nitro-l-arginine methyl ester, indicating eNOS uncoupling. Treatment of mice with AVE9488 enhanced vascular content of the essential eNOS cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin and reversed eNOS uncoupling. The combination of an up-regulated eNOS expression and a reversal of eNOS uncoupling is probably responsible for the observed vasoprotective properties of this new type of compounds.

Inhibition of caspase-3 improves contractile recovery of stunned myocardium, independent of apoptosis-inhibitory effects

OBJECTIVES The aim of this study was to investigate whether the caspase-3 inhibitor Ac-DEVD-CHO functionally improves stunned myocardium. BACKGROUND Degradation of troponin I contributes to the pathogenesis of myocardial stunning, whereas the role of apoptosis is unknown. Caspase-3 is an essential apoptotic protease that is specifically inhibited by Ac-DEVD-CHO. METHODS Isolated working hearts of rats were exposed to 30 min of low-flow ischemia, followed by 30 min of reperfusion. Ac-DEVD-CHO (0.1 to 1 micromol/l) was added 15 min before ischemia/reperfusion or 5 min before reperfusion. Cardiac output, external heart power, left ventricular (LV) developing pressure and contractility (dp/dt(max)) were measured. Apoptosis was assessed by TUNEL staining and internucleosomal deoxyribonucleic acid fragmentation. Caspase-3 processing and troponin I cleavage were determined by immunoblotting. Caspase-3 activity was measured using a fluorogenic substrate. RESULTS The addition of Ac-DEVD-CHO before ischemia/reperfusion or before reperfusion dose-dependently and significantly (p < 0.05) improved post-ischemic recovery of cardiac output, external heart power, LV developing pressure and dp/dt(max), compared with the vehicle (0.01% dimethyl sulfoxide). Ac-DEVD-CHO was similarly effective when given before reperfusion. Ac-DEVD-CHO blocked ischemia/reperfusion-induced caspase-3 activation, but cardiomyocyte apoptosis was unaffected. Troponin I cleavage was not inhibited by Ac-DEVD-CHO. CONCLUSIONS Caspase-3 is activated in stunned myocardium. Inhibition of caspase-3 by Ac-DEVD-CHO significantly improves post-ischemic contractile recovery of stunned myocardium, even when given after the onset of ischemia. The mechanism(s) of protection by Ac-DEVD-CHO appear to be independent of apoptosis. Inhibition of caspase-3 is a novel therapeutic strategy to improve functional recovery of stunned myocardium.

Pharmacological Characterization of SAR407899, a Novel Rho-Kinase Inhibitor

Abstract—Recent advances in basic and clinical research have identified Rho kinase as an important target potentially implicated in a variety of cardiovascular diseases. Rho kinase is a downstream mediator of RhoA that leads to stress fiber formation, membrane ruffling, smooth muscle contraction, and cell motility. Increased Rho-kinase activity is associated with vasoconstriction and elevated blood pressure. We identified a novel inhibitor of Rho kinase (SAR407899) and characterized its effects in biochemical, cellular, tissue-based, and in vivo assays. SAR407899 is an ATP-competitive Rho-kinase inhibitor, equipotent against human and rat-derived Rho-kinase 2 with inhibition constant values of 36 nM and 41 nM, respectively. It is highly selective in panel of 117 receptor and enzyme targets. SAR407899 is ≈8-fold more active than fasudil. In vitro, SAR407899 demonstrated concentration-dependent inhibition of Rho-kinase-mediated phosphorylation of myosin phosphatase, thrombin-induced stress fiber formation, platelet-derived growth factor-induced proliferation, and monocyte chemotactic protein-1-stimulated chemotaxis. SAR407899 potently (mean IC50 values: 122 to 280 nM) and species-independently relaxed precontracted isolated arteries of different species and different vascular beds. In vivo, over the dose range 3 to 30 mg/kg PO, SAR407899 lowered blood pressure in a variety of rodent models of arterial hypertension. The antihypertensive effect of SAR407899 was superior to that of fasudil and Y-27632. In conclusion, SAR407899 is a novel and potent selective Rho-kinase inhibitor with promising antihypertensive activity.

Release of nitric oxide from endothelial cells stimulated by YC‐1, an activator of soluble guanylyl cyclase

In this study we examined the endothelium‐dependent effect of YC‐1–a benzyl indazole derivative which directly activates soluble guanylyl cyclase (sGC)–on vascular relaxation and nitric oxide (NO) and guanosine‐3′,5′‐cyclic monophosphate (cyclic GMP) in endothelial cells. In preconstricted rat aortic rings with intact endothelium, YC‐1 produced a concentration‐dependent relaxation. However, the concentration response curve was shifted rightward to higher concentrations of YC‐1, when (i) the aortas were pre‐treated with L‐NG‐nitroarginine methylester (L‐NAME) or (ii) the endothelium was removed. Incubation of bovine aortic endothelial cells (BAEC) with YC‐1 produced a concentration‐dependent NO synthesis and release as assessed using a porphyrinic microsensor. Pre‐incubating cells with L‐NAME or with 8‐bromo‐cyclic GMP decreased this effect indicating that the YC‐1 stimulation of NO synthesis is due to an activation of nitric oxide synthase, but not to an elevation of cyclic GMP. No direct effect of YC‐1 on recombinant endothelial constitutive NO synthase activity was observed. The YC‐1 stimulated NO release was reduced by 90%, when extracellular free calcium was diminished. In human umbilical vein endothelial cells (HUVEC), YC‐1 stimulated intracellular cyclic GMP production in a concentration‐ and time‐dependent manner. Stimulation of cyclic GMP was greater with a maximum concentration of YC‐1 compared to calcium ionophore A23187. Similar effects were observed in BAEC and rat microvascular coronary endothelial cells (RMCEC). When HUVEC and RMCEC were pre‐treated with L‐NG‐nitroarginine (L‐NOARG), the maximum YC‐1 stimulated cyclic GMP increase was reduced by 50%. These results indicate, that beside being a direct activator of sGC, YC‐1 stimulates a NO‐synthesis and release in endothelial cells which is independent of elevation of cyclic GMP but strictly dependent on extracellular calcium. The underlying mechanism needs to be determined further.