Leila Seres

Circulating and cardiac levels of apelin, the novel ligand of the orphan receptor APJ, in patients with heart failure.

The orphan receptor APJ and its recently identified endogenous ligand, apelin, are expressed in the heart. However, their importance in the human cardiovascular system is not known. This study shows that apelin-like immunoreactivity is abundantly present in healthy human heart and plasma. Gel filtration HPLC analysis revealed that atrial and plasma levels of high molecular weight apelin, possibly proapelin, were markedly higher than those of mature apelin-36 itself. As assessed by quantitative RT-PCR analysis, left ventricular apelin mRNA levels were increased 4.7-fold in chronic heart failure (CHF) due to coronary heart disease (p<0.01) and 3.3-fold due to idiopathic dilated cardiomyopathy (p<0.05), whereas atrial apelin mRNA levels were unchanged. Atrial and plasma apelin-like immunoreactivity as well as atrial and ventricular APJ receptor mRNA levels were significantly decreased in CHF. Our results suggest that a new cardiac regulatory peptide, apelin, and APJ receptor may contribute to the pathophysiology of human CHF.

The peroxynitrite decomposition catalyst FP15 improves ageing-associated cardiac and vascular dysfunction

Overproduction of oxidants and free radicals in ageing tissues induces nitro-oxidative stress, which has recently been implicated in the pathogenesis of cardiovascular dysfunction associated with ageing. Peroxynitrite, a strong cytotoxic oxidant damages proteins and DNA and activates several pathways causing tissue injury, including the peroxynitrite-poly(ADP-ribose) polymerase (PARP) pathway. In this study, we investigated the effectiveness of the peroxynitrite decomposition catalyst FP15 on ageing-associated cardiac and vascular dysfunction. Young and ageing rats were treated with vehicle or FP15 intraperitoneally. Using a microtip Millar pressure catheter we performed left ventricular blood pressure analysis to assess systolic and diastolic function. Endothelium-dependent and -independent vasorelaxation of isolated aortic rings were investigated by using acetylcholine and sodium nitroprusside. Ageing animals showed a marked reduction of systolic and diastolic cardiac function and loss of endothelium-dependent relaxant responsiveness of aortic rings. FP15-treatment significantly improved cardiac performance and endothelial function. Immunohistochemical staining confirmed that FP15 effectively reduced nitrosative stress and prevented the activation of PARP in the aortic wall of ageing rats. Our results demonstrate the importance of endogenous peroxynitrite-overproduction in the pathogenesis of ageing-associated cardiovascular dysfunction. Pharmacological decomposition of peroxynitrite by FP15 may represent a novel therapeutic utility to improve cardiac and vascular dysfunction associated with ageing.

Human blood plasma advanced oxidation protein products (AOPP) correlates with fibrinogen levels

In 1996 a novel oxidative stress biomarker, referred to as advanced oxidation protein products (AOPP) was detected in the plasma of chronic uremic patients. The aim of the present studies was to find out that which plasma fraction(s) is responsible for AOPP reactivity. Thermal treatment of pooled samples of human citrate-plasma or EDTA-plasma at 50°C resulted in a rapid and parallel loss of fibrinogen concentration and AOPP reactivity. On the basis of time course and t1/2 values following thermal treatment, AOPP was indistinguishable from fibrinogen. There was a statistically significant (p < 0.0001) correlation between levels of blood plasma fibrinogen and AOPP in patients (n = 61) with various peripheral vascular or cardiovascular diseases. There was also a significant (p < 0.0001) relationship between plasma levels of fibrinogen and molar AOPP/fibrinogen ratio indicating that higher fibrinogen concentrations were associated with more oxidatively transformed groups on the molecule. Results of the present studies suggest that post-translationally modified fibrinogen is a key molecule responsible for human plasma AOPP reactivity. It remains to be elucidated what is the pathophysiological significance of the post-translationally modified fibrinogen in the inflammation-associated events of atherosclerosis, in platelet aggregation, and as a cardiovascular risk biomarker.

Advanced oxidation protein products (AOPP) for monitoring oxidative stress in critically ill patients : a simple, fast and inexpensive automated technique

Abstract Oxidative stress is known to be involved in many human pathological processes. Although there are numerous methods available for the assessment of oxidative stress, most of them are still not easily applicable in a routine clinical laboratory due to the complex methodology and/or lack of automation. In research into human oxidative stress, the simplification and automation of techniques represent a key issue from a laboratory point of view at present. In 1996 a novel oxidative stress biomarker, referred to as advanced oxidation protein products (AOPP), was detected in the plasma of chronic uremic patients. Here we describe in detail an automated version of the originally published microplate-based technique that we adapted for a Cobas Mira Plus clinical chemistry analyzer. AOPP reference values were measured in plasma samples from 266 apparently healthy volunteers (university students; 81 male and 185 female subjects) with a mean age of 21.3years (range 18–33). Over a period of 18months we determined AOPP concentrations in more than 300 patients in our department. Our experiences appear to demonstrate that this technique is especially suitable for monitoring oxidative stress in critically ill patients (sepsis, reperfusion injury, heart failure) even at daily intervals, since AOPP exhibited rapid responses in both directions. We believe that the well-established relationship between AOPP response and induced damage makes this simple, fast and inexpensive automated technique applicable in daily routine laboratory practice for assessing and monitoring oxidative stress in critically ill or other patients.

Single dose treatment with PARP-inhibitor INO-1001 improves aging-associated cardiac and vascular dysfunction.

Overproduction of reactive oxygen species in aging tissues has been implicated in the pathogenesis of aging-associated cardiovascular dysfunction. Oxidant-induced DNA-damage activates the poly(ADP-ribose) polymerase (PARP) pathway, leading to tissue injury. In this study we investigated the acute effects of the PARP inhibitor INO-1001 on aging-associated cardiac and endothelial dysfunction. Using a pressure-volume conductance catheter, left ventricular pressure-volume analysis of young and aging rats was performed before and after a single injection of INO-1001. Endothelium-dependent and -independent vasorelaxation of isolated aortic rings were investigated by using acetylcholine and sodium nitroprusside. Aging animals showed a marked reduction of myocardial contractility and endothelium-dependent relaxant responsiveness of aortic rings. Single dose INO-1001-treatment resulted in acute improvement in their cardiac and endothelial function. Immunohistochemistry for nitrotyrosine and poly(ADP-ribose) confirmed enhanced nitro-oxidative stress and PARP-activation in aging animals. Acute treatment with INO-1001 decreased PARP-activation, but did not affect nitrotyrosine-immunoreactivity. Our results demonstrate that the aging-associated chronic cardiovascular dysfunction can be improved, at least, short term, by a single treatment course with a PARP-inhibitor, supporting the role of the nitro-oxidative stress -- PARP -- pathway in the age-related functional decline of the cardiovascular system. Pharmacological inhibition of PARP may represent a novel therapeutic utility to improve aging-associated cardiovascular dysfunction.

INO-1001 a novel poly(ADP-ribose) polymerase (PARP) inhibitor improves cardiac and pulmonary function after crystalloid cardioplegia and extracorporal circulation.

Poly(ADP-ribose) polymerase (PARP) activation plays a key role in free radical–induced injury in the context of systemic inflammation and ischemia/reperfusion. In the present preclinical study, we investigated the effects of INO-1001, a novel PARP inhibitor, on cardiac and pulmonary function during reperfusion in an experimental model of cardioplegic arrest and extracorporal circulation. Twelve anesthetized dogs underwent hypothermic cardiopulmonary bypass. After 60 min of hypothermic cardiac arrest, reperfusion was started after application of either saline vehicle (control, n = 6), or INO-1001 (1 mg/kg), a potent PARP inhibitor (n = 6). Biventricular hemodynamic variables were measured by combined pressure-volume-conductance catheters. Coronary and pulmonary blood flow and vasodilative responses to acetylcholine and sodium nitroprusside as well as pulmonary gas exchange were also determined. The administration of INO-1001 led to a significantly better recovery of left and right ventricular systolic function (P < 0.05) after 60 min of reperfusion. Coronary blood flow was also significantly higher in the INO-1001 group (P < 0.05). Although the vasodilative response to sodium nitroprusside was similar in both groups, acetylcholine resulted in a significantly greater increase in coronary and pulmonary blood flow in the INO-1001 group (P < 0.05). Pulmonary function in terms of alveolar arterial oxygen difference was better preserved in the INO-1001–treated group (P < 0.05). Thus, PARP inhibition improves the recovery of myocardial and endothelial function after hypothermic cardiac arrest and reduces pulmonary injury associated with extracorporal circulation.

Tetrahydrobiopterin improves cardiac and pulmonary function after cardiopulmonary bypass.

OBJECTIVE Tetrahydrobiopterin (BH4) is an important cofactor of endogenous nitric oxide synthesis. In the present preclinical study, we investigated the effects of BH4 on cardiac and pulmonary function during early reperfusion in an experimental model of cardioplegic arrest and extracorporal circulation. METHODS Twelve anesthetized dogs underwent hypothermic cardiopulmonary bypass. After 60 min of hypothermic cardiac arrest, reperfusion was started after application of either saline vehicle (control, n = 6), or BH4 (n = 6). Left-ventricular end-systolic pressure volume relationship (E(es)) was measured by a combined pressure-volume conductance catheter at baseline and after 60 min of reperfusion. Left anterior descending (LAD) coronary (CBF) and pulmonary blood flow (PBF), endothelium-dependent vasodilatation to acetylcholine (ACh), endothelium-independent vasodilatation to sodium nitroprusside (SNP) and alveolo-arterial O₂ gradient were determined. RESULTS The administration of BH4 led to a significantly better recovery of E(es) (given as percent of baseline: 85 ± 22 vs 46 ± 15%, p<0.05). CBF was also significantly higher in the BH4 group (38 ± 5 vs 22 ± 5 ml min⁻¹, p<0.05). While the vasodilatatory response to SNP was similar in both groups, injection of ACh resulted in a significantly higher increase in CBF (64 ± 12 vs 25 ± 12%, p < 0.05) and PBF (49 ± 15 vs 36 ± 14%, p<0.05) in the BH4-treated animals. Alveolo-arterial O₂ gradient was significantly lower after BH4 supplementation (80 ± 15 vs 49 ± 14 mm Hg, p < 0.05). CONCLUSIONS Application of BH4 improves myocardial, endothelial and pulmonary function after cardiopulmonary bypass with hypothermic cardiac arrest. The observed protective effects indicate that BH4 could be a novel therapeutic option in the treatment of ischemia/reperfusion injury.

Kinetic assay for the determination of the oxidative stress biomarker, advanced oxidation protein products (AOPP) in the human blood plasma

UNLABELLED A number of human diseases and pathological conditions were found to be associated with increased oxidative stress. In the literature several techniques are available for the assessment of oxidative stress, but most of them are not applicable for a routine medical laboratory due to the complex methodology and/or financial reasons. We report here on a simple, inexpensive, kinetic assay for the determination of the oxidative stress biomarker, advanced oxidation protein products (AOPP) in the human blood plasma. METHODS This study involved 70 patients (47M/23F; mean age: 64.6 y; range: 16-85) admitted to our Department with a wide range of cardiovascular and peripheral vascular diseases. Three critically ill patients were assigned for monitoring purposes. Plasma AOPP were simultaneously determined using an end-point assay as reference method and by a kinetic method developed in our laboratory. Plasma fibrinogen concentration was measured according to the Clauss method. RESULTS There was a highly significant correlation (r2 = 0.588; p < 0.0001) between AOPP concentration (reference method) and AOPP reactivity (kinetic method). Both AOPP concentration and AOPP reactivity also significantly correlated with plasma fibrinogen concentration (r2 = 0.780; p < 0.0001; r2 = 0.564; p < 0.0001). The three representative cases presented appear to support the relevance of our novel method in the monitoring of critically ill patients. CONCLUSIONS This simple and inexpensive kinetic assay can be widely used in any routine laboratory interested in oxidative stress research. It is especially recommended for monitoring critically ill or other patients.

Mesenteric injury after cardiopulmonary bypass: Role of poly(adenosine 5′-diphosphate-ribose) polymerase

Objectives:To investigate the effects of the ultrapotent poly(adenosine 5′-diphosphate-ribose) polymerase (PARP) inhibitor INO-1001 on cardiac and mesenteric function during reperfusion in an experimental model of cardiopulmonary bypass with cardioplegic arrest. Design:Prospective, randomized, and blinded experimental study. Setting:Research laboratory. Subjects:Twelve anesthetized dogs underwent cardiopulmonary bypass with hypothermic cardioplegic cardiac arrest. Interventions:After 60 mins of hypothermic cardiac arrest, either PARP inhibitor INO-1001 (1 mg/kg, n = 6) or vehicle (control, n = 6) was administered during reperfusion. Measurements and Main Results:Left ventricular hemodynamic variables were measured by combined pressure-volume-conductance catheters. Coronary and mesenteric blood flow and vasodilatory responses to acetylcholine and sodium nitroprusside as well as mesenteric lactate and creatinine phosphokinase release were also determined. The administration of INO-1001 led to a significantly improved recovery of left ventricular systolic function (p < .05) after 60 mins of reperfusion. Coronary and mesenteric blood flow were also significantly higher in the INO-1001 group (p < .05). Although the vasodilatory response to sodium nitroprusside was similar in both groups before and after cardiopulmonary bypass and similar in response to acetylcholine before cardiopulmonary bypass, PARP-inhibited dogs had lower mesenteric vascular resistance after cardiopulmonary bypass (p < .05). Mesenteric lactate and creatinine phosphokinase release was significantly lower in the PARP inhibitor treated group (p < .05). Conclusion:PARP inhibition with INO-1001 improves the recovery of myocardial function and prevents mesenteric vascular dysfunction and tissue injury after cardiopulmonary bypass with hypothermic cardiac arrest.

Endothelin-A-receptor antagonist LU 135.252 Inhibits the formation of ventricular arrhythmias caused by intrapericardial infusion of endothelin-1

We observed that heterozygous knockout (+/-, KO) of either endothelin-A- (ET(A)) or -B- (ET(B)) receptors significantly reduced the pressor responses to systemically administered endothelin-1 (ET-1) in ET(A) or ET(B) (+/-) KO mice when compared to wild-type (WT) mice (data not shown). Also, we observed that basal mean arterial pressure (MAP) is significantly higher in ET(B) (+/-) (92.7 +/- 1.2 mmHg) (n = 53, p < 0.05) but not ET(A) (+/-) KO mice (70.6 +/- 1.8 mmHg) (n = 23) when compared to their anaesthetized WT littermates (70.1 +/- 0.7 mmHg) (n = 118). A 90 min treatment with either BQ-123 (10 mg/kg), an ET(A)-selective antagonist, or BQ-928 (10 mg/kg), a mixed ET(A)/ET(B) antagonist, administered intraperitoneally, significantly reduced basal MAP of ET(B) (+/-) KO mice almost to the level of their WT treated counterparts (94.9 +/- 4.9 mmHg) (n = 6) vs (+ BQ-123: 59.7 +/- 0.3 mmHg, n = 8); (+ BQ-928: 72.4 +/- 2.6 mmHg, n = 5). It is worthy of note that BQ-123 significantly reduced basal MAP in WT mice but to a lesser extent than in ET(B) (+/-) KO mice (69.6 +/- 2.3 mmHg, n = 8) vs (+ BQ-123: 57.3 +/- 1.4 mmHg, n = 8). In contrast, the ET(B)-selective antagonist, BQ-788 (10 mg/kg i.p.), had no significant effect on MAP even after 90 min of treatment (ET(B) (+/-) KO: (92.3 +/- 2.3 mmHg, n = 6) vs (+ BQ-788: 89.7 +/- 3.1 mmHg, n = 6); WT: (70.5 +/- 3.7 mmHg, n = 7) vs (+ BQ-788: 71.2 +/- 2.0 mmHg, n = 6). Therefore heterozygous KO of either ET(A)- or ET(B)-receptors significantly alters the phenotypic pressor properties of ET-1. We also suggest that there is less ET clearance in ET(B) (+/-) KO mice than in WT mice, which can explain the ET(A)-dependent hypertensive state of the former strain.We evaluated the role of endothelin-B- (ET(B)) receptor-mediated action in the development and maintenance of deoxycorticosterone acetate (DOCA)-salt-induced hypertension, cardiovascular hypertrophy and renal damage, using the spotting lethal (sl) rat which carries a naturally occurring deletion in the ET(B)-receptor gene. Homozygous (sl/sl) rats exhibit abnormal development of the neural crest-derived epidermal melanocytes and the enteric nervous system (ENS), and do not live beyond 1 month because of intestinal aganglionosis and resulting intestinal obstruction. Therefore, the dopamine-beta-hydroxylase (D betaH) promoter was used to direct ET(B) transgene expression in sl/sl rats to support normal ENS development. D betaH-ET(B) sl/sl rats live into adulthood and are healthy, expressing ET(B)-receptor in adrenals and other adrenergic neurons. When homozygous (sl/sl) and wild-type (WT) (+/+) rats, all of which were transgenic, were treated with DOCA and salt for 4 weeks, the homozygous rats exhibited significantly earlier and higher increases in systolic blood pressure than WT rats. The daily oral administration of ABT-627, a selective ET(A)-receptor antagonist, almost completely suppressed the DOCA-salt-induced hypertension in both groups. Renal dysfunction and histological damage induced by DOCA-salt treatment were more severe in homozygous than in WT rats. Increased and marked vascular hypertrophy of the aorta was also observed in homozygous rats, compared with WT rats. Renal and vascular injuries induced by DOCA and salt were significantly improved by ABT-627 administration. We propose that ET(B)-receptor-mediated actions are protective factors in the pathogenesis of DOCA-salt-induced hypertension. ET(A)-mediated actions are at least partly responsible for the increased susceptibility to DOCA-salt-induced hypertension and related tissue injuries in ET(B)-receptor-deficient rats.