PGC1α/ß Expression Predicts Therapeutic Response to Oxidative Phosphorylation Inhibition in Ovarian Cancer.

Ovarian cancer is the deadliest gynecologic cancer, and novel therapeutic options are crucial to improve overall survival. Here we provide evidence that impairment of oxidative phosphorylation (OXPHOS) can help control ovarian cancer progression, and this benefit correlates with expression of the two mitochondrial master regulators PGC1α and PGC1ß. In orthotopic patient-derived ovarian cancer xenografts (OC-PDX), concomitant high expression of PGC1α and PGC1ß (PGC1α/ß) fostered a unique transcriptional signature, leading to increased mitochondrial abundance, enhanced tricarboxylic acid cycling, and elevated cellular respiration that ultimately conferred vulnerability to OXPHOS inhibition. Treatment with the respiratory chain complex I inhibitor IACS-010759 caused mitochondrial swelling and ATP depletion that consequently delayed malignant progression and prolonged the lifespan of high PGC1α/ß-expressing OC-PDX-bearing mice. Conversely, low PGC1α/ß OC-PDXs were not affected by IACS-010759, thus pinpointing a selective antitumor effect of OXPHOS inhibition. The clinical relevance of these findings was substantiated by analysis of ovarian cancer patient datasets, which showed that 25% of all cases displayed high PGC1α/ß expression along with an activated mitochondrial gene program. This study endorses the use of OXPHOS inhibitors to manage ovarian cancer and identifies the high expression of both PGC1α and ß as biomarkers to refine the selection of patients likely to benefit most from this therapy. SIGNIFICANCE: OXPHOS inhibition in ovarian cancer can exploit the metabolic vulnerabilities conferred by high PGC1α/ß expression and offers an effective approach to manage patients on the basis of PGC1α/ß expression.

Ventilation With Argon Improves Survival With Good Neurological Recovery After Prolonged Untreated Cardiac Arrest in Pigs.

Background Ventilation with the noble gas argon (Ar) has shown neuroprotective and cardioprotective properties in different in vitro and in vivo models. Hence, the neuroprotective effects of Ar were investigated in a severe, preclinically relevant porcine model of cardiac arrest. Methods and Results Cardiac arrest was ischemically induced in 36 pigs and left untreated for 12 minutes before starting cardiopulmonary resuscitation. Animals were randomized to 4-hour post-resuscitation ventilation with: 70% nitrogen-30% oxygen (control); 50% Ar-20% nitrogen-30% oxygen (Ar 50%); and 70% Ar-30% oxygen (Ar 70%). Hemodynamic parameters and myocardial function were monitored and serial blood samples taken. Pigs were observed up to 96 hours for survival and neurological recovery. Heart and brain were harvested for histopathology. Ten animals in each group were successfully resuscitated. Ninety-six-hour survival was 60%, 70%, and 90%, for the control, Ar 50%, and Ar 70% groups, respectively. In the Ar 50% and Ar 70% groups, 60% and 80%, respectively, achieved good neurological recovery, in contrast to only 30% in the control group (P<0.0001). Histology showed less neuronal degeneration in the cortex (P<0.05) but not in the hippocampus, and less reactive microglia activation in the hippocampus (P=0.007), after Ar compared with control treatment. A lower increase in circulating biomarkers of brain injury, together with less kynurenine pathway activation (P<0.05), were present in Ar-treated animals compared with controls. Ar 70% pigs also had complete left ventricular function recovery and smaller infarct and cardiac troponin release (P<0.01). Conclusions Post-resuscitation ventilation with Ar significantly improves neurologic recovery and ameliorates brain injury after cardiac arrest with long no-flow duration. Benefits are greater after Ar 70% than Ar 50%.

Primary pulmonary arterial hypertension: Protocol to assess comprehensively in the rat the response to pharmacologic treatments.

The identification of new treatments for primary pulmonary arterial hypertension (PAH) is a critical unmet need since there is no a definitive cure for this disease yet. Due to the complexity of PAH, a wide set of methods are necessary to assess the response to a pharmacological intervention. Thus, a rigorous protocol is crucial when experimental studies are designed. In the present experimental protocol, a stepwise approach was followed in a monocrotaline-induced PAH model in the rat, moving from the dose finding study of treatment compounds to the recognition of the onset of disease manifestation, in order to identify when to start a curative treatment. A complete multidimensional evaluation of treatment effects represented the last step. The primary study endpoint was the change in right ventricular systolic pressure after 14 days of treatment; echocardiographic and biohumoral markers together with heart and pulmonary arterial morphometric parameters were considered as secondary efficacy and/or safety endpoints and for the evaluation of the biologic coherence in the different results.

Monocrotaline-induced pulmonary arterial hypertension: Time-course of injury and comparative evaluation of macitentan and Y-27632, a Rho kinase inhibitor.

Novel pharmacological approaches are needed to improve outcomes of patients with idiopathic pulmonary hypertension. Rho-associated protein kinase (ROCK) inhibitors have shown beneficial effects in preclinical models of pulmonary arterial hypertension (PAH), because of their role in the regulation of pulmonary artery vasoconstrictor tone and remodeling. We compared a ROCK inhibitor, Y-27632, for the first time with the dual endothelin receptor antagonist, macitentan, in a monocrotaline-induced rat pulmonary hypertension model. Different methods (echocardiography, hemodynamics, histology of right ventricle and pulmonary vessels, and circulating biomarkers) showed consistently that 100 mg/kg daily of Y-27632 and 10 mg/kg daily of macitentan slowed the progression of PAH both at the functional and structural levels. Treatments started on day 14 after monocrotaline injection and lasted 14 days. The findings of all experimental methods show that the selective ROCK inhibitor Y-27632 has more pronounced effects than macitentan, but a major limitation to its use is its marked peripheral vasodilating action.

Realistic Evaluation of Titanium Dioxide Nanoparticle Exposure in Chewing Gum.

There is growing concern about the presence of nanoparticles (NPs) in titanium dioxide (TiO2) as food additive (E171). To realistically estimate the number and the amount of TiO2 NPs ingested with food, we applied a transmission electron microscopy method combined with inductively coupled plasma optical emission spectrometry. Different percentages of TiO2 NPs (6-18%) were detected in E171 from various suppliers. In the eight chewing gums analyzed as food prototypes, TiO2 NPs were absent in one sample and ranged 0.01-0.66 mg/gum, corresponding to 7-568 billion NPs/gum, in the other seven. We estimated that the mass-based TiO2 NPs ingested with chewing gums by the European population ranged from 0.28 to 112.40 µg/kg b.w./day, and children ingested more nanosized titanium than adolescents and adults. Although this level may appear negligible it corresponds to 0.1-84 billion TiO2 NPs/kg b.w/day, raising important questions regarding their potential accumulation in the body, possibly causing long-term effects on consumers' health.

Duration of Untreated Cardiac Arrest and Clinical Relevance of Animal Experiments: The Relationship Between the "No-Flow" Duration and the Severity of Post-Cardiac Arrest Syndrome in a Porcine Model.

INTRODUCTION: The study investigated the effect of untreated cardiac arrest (CA), that is, "no-flow" time, on postresuscitation myocardial and neurological injury, and survival in a pig model to identify an optimal duration that adequately reflects the most frequent clinical scenario. METHODS: An established model of myocardial infarction followed by CA and cardiopulmonary resuscitation was used. Twenty-two pigs were subjected to three no-flow durations: short (8-10 min), intermediate (12-13 min), and long (14-15 min). Left ventricular ejection fraction (LVEF) was assessed together with thermodilution cardiac output (CO) and high sensitivity cardiac troponin T (hs-cTnT). Neurological impairment was evaluated by neurological scores, serum neuron specific enolase (NSE), and histopathology. RESULTS: More than 60% of animals survived when the duration of CA was ≤13 min, compared to only 20% for a duration ≥14 min. Neuronal degeneration and neurological scores showed a trend toward a worse recovery for longer no-flow durations. No animals achieved a good neurological recovery for a no-flow ≥14 min, in comparison to a 56% for a duration ≤13 min (P = 0.043). Serum NSE levels significantly correlated with the no-flow duration (r = 0.892). Longer durations of CA were characterized by lower LVEF and CO compared to shorter durations (P < 0.05). The longer was the no-flow time, the higher was the number of defibrillations delivered (P = 0.043). The defibrillations delivered significantly correlated with LVEF and plasma hs-cTnT. CONCLUSIONS: Longer no-flow durations caused greater postresuscitation myocardial and neurological dysfunction and reduced survival. An untreated CA of 12-13 min may be an optimal choice for a clinically relevant model.

Expression of A2V-mutated Aß in Caenorhabditis elegans results in oligomer formation and toxicity.

Although Alzheimer's disease (AD) is usually sporadic, in a small proportion of cases it is familial and can be linked to mutations in ß-amyloid precursor protein (APP). Unlike the other genetic defects, the mutation [alanine-673→valine-673] (A673V) causes the disease only in the homozygous condition with enhanced amyloid ß (Aß) production and aggregation; heterozygous carriers remain unaffected. It is not clear how misfolding and aggregation of Aß is affected in vivo by this mutation and whether this correlates with its toxic effects. No animal models over-expressing the A673V-APP gene or alanine-2-valine (A2V) mutated human Aß protein are currently available. Using the invertebrate Caenorhabditis elegans, we generated the first transgenic animal model to express the human Aß1-40 wild-type (WT) in neurons or possess the A2V mutation (Aß1-40A2V). Insertion of an Aß-mutated gene into this nematode reproduced the homozygous state of the human pathology. Functional and biochemical characteristics found in the A2V strain were compared to those of transgenic C. elegans expressing Aß1-40WT. The expression of both WT and A2V Aß1-40 specifically reduced the nematode's lifespan, causing behavioral defects and neurotransmission impairment which were worse in A2V worms. Mutant animals were more resistant than WT to paralysis induced by the cholinergic agonist levamisole, indicating that the locomotor defect was specifically linked to postsynaptic dysfunctions. The toxicity caused by the mutated protein was associated with a high propensity to form oligomeric assemblies which accumulate in the neurons, suggesting this to be the central event involved in the postsynaptic damage and early onset of the disease in homozygous human A673V carriers.

Ibuprofen plus isosorbide dinitrate treatment in the mdx mice ameliorates dystrophic heart structure.

BACKGROUND: Co-administration of ibuprofen (IBU) and isosorbide dinitrate (ISDN) provides synergistic beneficial effects on dystrophic skeletal muscle. Whether this treatment has also cardioprotective effects in this disease was still unknown. AIMS: To evaluate the effects of co-administration of IBU and ISDN (a) on left ventricular (LV) structure and function, and (b) on cardiac inflammatory response and fibrosis in mdx mice. METHODS: Three groups of mice were studied: mdx mice treated with IBU (50 mg kg⁻¹)+ISDN (30 mg kg⁻¹) administered daily in the diet, mdx mice that received standard diet without drugs and wild type aged-matched mice. Animals were analysed after 10-11 months of treatment. Structural and functional parameters were evaluated by echocardiography while histological analyses were performed to evaluate inflammatory response, collagen deposition, cardiomyocyte number and area. RESULTS: Treatment for 10-11 months with IBU+ISDN preserved LV wall thickness and LV mass. Drug treatment also preserved the total number of cardiomyocytes in the LV and attenuated the increase in cardiomyocyte size, when compared to untreated mdx mice. Moreover, a trend towards a decreased number of inflammatory cells, a reduced LV myocardial interstitial fibrosis and an enhanced global LV function response to stress was observed in treated mdx mice. CONCLUSIONS: Treatment for 10-11 months with IBU+ISDN is effective in preventing the alterations in LV morphology of mdx mice while not reaching statistical significance on LV function and cardiac inflammation.

Lipofuscin accumulation and gene expression in different tissues of mnd mice.

Neuronal ceroid lipofuscinoses (NCLs) are a group of lysosomal storage diseases characterized by neurological impairment and blindness. NCLs are almost always due to single mutations in different genes (CLN1-CLN8). Ubiquitous accumulation of undigested material and of a hydrophobic inner mitochondrial membrane protein, the subunit c of mitochondrial ATP synthase, has been described. Although protein mutation(s) in the endoplasmic reticulum-lysosomes axis can modify the trafficking and the recycling of different molecules, one of the upstream targets in these diseases may be represented by the balance of gene expression. To understand if and how neurons modify the levels of important genes during the first phases of the disease, it is important to characterize the mechanisms of neurodegeneration. Due to the impossibility of performing this analysis in humans, alternative models of investigation are required. In this study, a mouse model of human NCL8, the mnd mouse has been employed. The mnd mice recapitulate many clinical and histopathological features described in NCL8 patients. In this study, we found an altered expression of different genes in both central and peripheral organs associated with lipopigment accumulation. This is a preliminary approach, which could also be of interest in providing new diagnostic tools for NCLs.

Distinct cardiac and renal effects of ETA receptor antagonist and ACE inhibitor in experimental type 2 diabetes.

Diabetic nephropathy is associated with cardiovascular morbidity. Angiotensin-converting enzyme (ACE) inhibitors provide imperfect renoprotection in advanced type 2 diabetes, and cardiovascular risk remains elevated. Endothelin (ET)-1 has a role in renal and cardiac dysfunction in diabetes. Here, we assessed whether combination therapy with an ACE inhibitor and ET(A) receptor antagonist provided reno- and cardioprotection in rats with overt type 2 diabetes. Four groups of Zucker diabetic fatty (ZDF) rats were treated orally from 4 (when proteinuric) to 8 mo with vehicle, ramipril (1 mg/kg), sitaxsentan (60 mg/kg), and ramipril plus sitaxsentan. Lean rats served as controls. Combined therapy ameliorated proteinuria and glomerulosclerosis mostly as a result of the action of ramipril. Simultaneous blockade of ANG II and ET-1 pathways normalized renal monocyte chemoattractant protein-1 and interstitial inflammation. Cardiomyocyte loss, volume enlargement, and capillary rarefaction were prominent abnormalities of ZDF myocardium. Myocyte volume was reduced by ramipril and sitaxsentan, which also ameliorated heart capillary density. Drug combination restored myocardial structure and reestablished an adequate capillary network in the presence of increased cardiac expression of VEGF/VEGFR-1, and significant reduction of oxidative stress. In conclusion, in type 2 diabetes concomitant blockade of ANG II synthesis and ET-1 biological activity through an ET(A) receptor antagonist led to substantial albeit not complete renoprotection, almost due to the ACE inhibitor. The drug combination also showed cardioprotective properties, which however, were mainly dependent on the contribution of the ET(A) receptor antagonist through the action of VEGF.

Misplaced NMDA receptors in epileptogenesis contribute to excitotoxicity.

Pharmacological blockade of NR2B-containing N-methyl-d-aspartate receptors (NMDARs) during epileptogenesis reduces neurodegeneration provoked in the rodent hippocampus by status epilepticus. The functional consequences of NMDAR activation are crucially influenced by their synaptic vs extrasynaptic localization, and both NMDAR function and localization are dependent on the presence of the NR2B subunit and its phosphorylation state. We investigated whether changes in NR2B subunit phosphorylation, and alterations in its neuronal membrane localization and cellular expression occur during epileptogenesis, and if these changes are involved in neuronal cell loss. We also explored NR2B subunit changes both in the acute phase of status epilepticus and in the chronic phase of spontaneous seizures which encompass the epileptogenesis phase. Levels of Tyr1472 phosphorylated NR2B subunit decreased in the post-synaptic membranes from rat hippocampus during epileptogenesis induced by electrical status epilepticus. This effect was concomitant with a reduced interaction between NR2B and post-synaptic density (PSD)-95 protein, and was associated with decreased CREB phosphorylation. This evidence suggests an extra-synaptic localization of NR2B subunit in epileptogenesis. Accordingly, electron microscopy showed increased NR2B both in extra-synaptic and pre-synaptic neuronal compartments, and a concomitant decrease of this subunit in PSD, thus indicating a shift in NR2B membrane localization. De novo expression of NR2B in activated astrocytes was also found in epileptogenesis indicating ectopic receptor expression in glia. The NR2B phosphorylation changes detected at completion of status epilepticus, and interictally in the chronic phase of spontaneous seizures, are predictive of receptor translocation from synaptic to extrasynaptic sites. Pharmacological blockade of NR2B-containing NMDARs by ifenprodil administration during epileptogenesis significantly reduced pyramidal cell loss in the hippocampus, showing that the observed post-translational and cellular changes of NR2B subunit contribute to excitotoxicity. Therefore, pharmacological targeting of misplaced NR2B-containing NMDARs, or prevention of these NMDAR changes, should be considered to block excitotoxicity which develops after various pro-epileptogenic brain injuries.

The differentiation of cardiomyocytes from mouse embryonic stem cells is altered by dioxin.

2,3,7,8-Tetrachlorodibenzo-para-dioxin (TCDD) causes abnormalities during heart development. Cardiomyocytes derived from embryonic stem (ES) cells are a robust model for the study of early cardiomyogenesis. Here, we evaluated the effects of TCDD at key stages during the differentiation of mouse ES cells into cardiomyocytes analysing: (i) the transcription of lineage differentiation (Brachyury, Nkx-2.5, Actc-1), cardiac-specific (Alpk3, cTnT, cTnI, cTnC) and detoxification phase I (Cyp1A1, Cyp1A2 and Cyp1B1) and phase II (Nqo1, Gsta1 and Ugt1a6) genes; (ii) the global gene expression; (iii) the ultrastructure of ES-derived cardiomyocytes; (iv) level of ATP production and (v) the immunolocalisation of sarcomeric α-actinin, ß-myosin heavy chain and cTnT proteins. We show that TCDD affects the differentiation of ES cells into cardiomyocytes at several levels: (1) induces the expression of phase I genes; (2) down-regulates a group of heart-specific genes, some involved in the oxidative phosphorylation pathway; (3) reduces the efficiency of differentiation; (4) alters the arrangement of mitochondria, that show twisted and disrupted cristae, and of some sarcomeres, with misalignement or disarrangement of the myofibrillar organisation and (5) reduces ATP production. This study provides novel evidences that TCDD impairs cardiomyocyte differentiation. Sarcomeres and mitochondria could be a target for dioxin toxicity, their disruption representing a possible mechanism developing cardiac injury.

Tetracycline and its analogues protect Caenorhabditis elegans from ß amyloid-induced toxicity by targeting oligomers.

The accumulation and deposition of amyloid beta (Aß) peptide in extracellular dense plaques in the brain is a key phase in Alzheimer's disease (AD). Small oligomeric forms of Aß are responsible for the toxicity and the early cognitive impairment observed in patients before the amyloid plaque deposits appear. It is essential for the development of an efficient cure for AD to identify compounds that interfere with Aß aggregation, counteracting the molecular mechanisms involved in conversion of the monomeric amyloid protein into oligomeric and fibrillar forms. Tetracyclines have been proposed for AD therapy, although their effects on the aggregation of Aß protein, particularly their ability to interact in vivo with the Aß oligomers and/or aggregates, remain to be understood. Using transgenic Caenorhabditis elegans as a simplified invertebrate model of AD, we evaluated the ability of tetracyclines to interfere with the sequence of events leading to Aß proteotoxicity. The drugs directly interact with the Aß assemblies in vivo and reduce Aß oligomer deposition, protecting C. elegans from oxidative stress and the onset of the paralysis phenotype. These effects were specific, dose-related and not linked to any antibiotic activity, suggesting that the drugs might offer an effective therapeutic strategy to target soluble Aß aggregates.

Cognitive deficits associated with alteration of synaptic metaplasticity precede plaque deposition in AßPP23 transgenic mice.

Synaptic dysfunction is an early event in the development of Alzheimer's disease (AD) and relates closely to the cognitive impairment characterizing this neurodegenerative process. A causative association has been proposed, largely on the basis of in vitro studies, between memory decline, soluble amyloid-ß (Aß) oligomers and alterations of glutamatergic neurotransmission. We aimed here to characterize in vivo N-methyl-D-aspartate receptor (NMDAR)-mediated signaling, at an early stage of AD, before extracellular amyloid plaques are deposited. We assessed the functional link between cognitive abilities and NMDAR-mediated pharmacological responses of six-month-old AßPP23 transgenic mice (AßPP23tg), overexpressing the human amyloid-ß protein precursor carrying the Swedish double mutation. We found evidence of cognitive impairments in these mice, indicated by deficits in the delayed-non-matching-to-place task. Alterations of NMDAR-mediated signaling in this mouse model were confirmed by the reduced sensitivity of motor-activation and working memory to pharmacological inhibition of NMDAR activity. At the molecular level, AßPP23tg mice show hippocampal alterations in the trafficking of synaptic NMDAR subunits NR2A and NR2B and at an ultrastructural analysis show Aß oligomers intracellularly localized in the synaptic compartments. Importantly, the behavioral and biochemical alterations of NMDAR signaling are associated with the inhibition of long-term synaptic potentiation and inversion of metaplasticity at CA1 synapses in hippocampal slices from AßPP23tg mice. These results indicate a general impairment of synaptic function and learning and memory in young AßPP23tg mice with Aß oligomers but no amyloid plaques.

Disruption of the Ang II type 1 receptor promotes longevity in mice.

The renin-angiotensin system plays a role in the etiology of hypertension and the pathophysiology of cardiac and renal diseases in humans. Ang II is the central product of this system and is involved in regulating immune responses, inflammation, cell growth, and proliferation by acting through Ang II type 1 receptors (AT1 and AT2). Here, we show that targeted disruption of the Agtr1a gene that encodes AT1A results in marked prolongation of life span in mice. Agtr1a-/- mice developed less cardiac and vascular injury, and multiple organs from these mice displayed less oxidative damage than wild-type mice. The longevity phenotype was associated with an increased number of mitochondria and upregulation of the prosurvival genes nicotinamide phosphoribosyltransferase (Nampt) and sirtuin 3 (Sirt3) in the kidney. In cultured tubular epithelial cells, Ang II downregulated Sirt3 mRNA, and this effect was inhibited by an AT1 antagonist. These results demonstrate that disruption of AT1 promotes longevity in mice, possibly through the attenuation of oxidative stress and overexpression of prosurvival genes, and suggests that the Ang II/AT1 pathway may be targeted to influence life span in mammals.

Mutant prion protein expression causes motor and memory deficits and abnormal sleep patterns in a transgenic mouse model.

A familial form of Creutzfeldt-Jakob disease (CJD) is linked to the D178N/V129 prion protein (PrP) mutation. Tg(CJD) mice expressing the mouse homolog of this mutant PrP synthesize a misfolded form of the mutant protein, which is aggregated and protease resistant. These mice develop clinical and pathological features reminiscent of CJD, including motor dysfunction, memory impairment, cerebral PrP deposition, and gliosis. Tg(CJD) mice also display electroencephalographic abnormalities and severe alterations of sleep-wake patterns strikingly similar to those seen in a human patient carrying the D178N/V129 mutation. Neurons in these mice show swelling of the endoplasmic reticulum (ER) with intracellular retention of mutant PrP, suggesting that ER dysfunction could contribute to the pathology. These results establish a transgenic animal model of a genetic prion disease recapitulating cognitive, motor, and neurophysiological abnormalities of the human disorder. Tg(CJD) mice have the potential for giving greater insight into the spectrum of neuronal dysfunction in prion diseases.

Cardioprotective function of the long pentraxin PTX3 in acute myocardial infarction.

BACKGROUND: Despite widespread clinical use as a prognostic marker in ischemic heart disorders, the actual pathogenetic role of the short pentraxin, C-reactive protein, has not undergone stringent genetic testing because of evolutionary divergence between mouse and humans. The long pentraxin PTX3 is conserved in evolution, is expressed in the heart under inflammatory conditions, and is a candidate prognostic marker in acute myocardial infarction. It was therefore important to assess whether PTX3 plays a pathogenetic role in acute myocardial infarction. METHODS AND RESULTS: In a model of acute myocardial infarction caused by coronary artery ligation and reperfusion, tissue mRNA expression and circulating levels of PTX3 increased. The interleukin-1R-MyD88 pathway plays a pivotal role in the induction of PTX3 transcript after ischemia. ptx3-deficient mice showed exacerbated heart damage (33% larger infarcts in null mice; P=0.0047). Increased myocardial damage in ptx3-deficient mice was associated with a greater no-reflow area, increased neutrophil infiltration, decreased number of capillaries, and increased number of apoptotic cardiomyocytes. In addition, ptx3-deficient mice with acute myocardial infarction showed higher circulating levels of interleukin-6 and increased C3 deposition in lesional tissue. The phenotype was reversed by exogenous PTX3. CONCLUSIONS: Thus, PTX3 plays a nonredundant, regulatory, cardioprotective role in acute myocardial infarction in mice. Our results suggest that modulation of the complement cascade contributes to the cardioprotective function of PTX3.

Effect of beta-adrenergic and renin-angiotensin system blockade on myocyte apoptosis and oxidative stress in diabetic hypertensive rats.

Diabetes aggravates the clinical severity and represents an additional independent risk factor of hypertension. Since both diseases separately concur to cardiomyocyte apoptosis, a mechanism at least partly involving unbalanced oxidative stress, we investigated whether the combination of diabetes and hypertension potentiated cardiac cell death in experimental models, compared to either disease alone. We also evaluated the short-term effects of different drugs in these models. Streptozotocin-induced diabetic normotensive (WKY) or hypertensive (SHR) rats were treated for one week with a DA(2)/alpha(2) agonist (CHF-1024), a selective beta1 adrenergic blocker (metoprolol), an angiotensin II-receptor blocker (valsartan) or a radical scavenger (tempol). In separate experiments, isolated cardiomyocytes were cultured in high glucose medium (25 mM) containing the same drugs. Although the number of apoptotic cardiomyocytes and the myocardial density of oxygen radicals were higher in non diabetic hypertensive than in normotensive controls, diabetes raised these variables to comparable absolute levels in both strains. All drugs except metoprolol significantly reduced apoptosis and oxidative stress in the diabetic animals of both strains and in the isolated myocytes cultured with high glucose. In conclusion, hypertensive rat is no more susceptible than its normotensive control to acute apoptosis induced by diabetes. Oxidative stress might be considered the common trigger for cardiac myocyte apoptosis in both conditions.

The prognostic value of big endothelin-1 in more than 2,300 patients with heart failure enrolled in the Valsartan Heart Failure Trial (Val-HeFT).

BACKGROUND: Endothelin is elevated in heart failure and contributes to neurohormonal activation, hemodynamic deterioration, and cardiovascular remodeling. Here, we examined its prognostic value in a large population of patients with chronic heart failure. METHODS AND RESULTS: Big endothelin-1 (Big ET-1) and 4 other neurohormones were measured at study entry in 2359 patients enrolled in the Valsartan Heart Failure Trial (Val-HeFT) and their concentrations related to outcome over a median follow-up of 23 months. Baseline concentration of Big ET-1 (median 0.80 pmol/L) was proportional to severity of disease (New York Heart Association class, left ventricular structure and function). High circulating concentrations of brain natriuretic peptide (BNP), creatinine and bilirubin, advanced New York Heart Association class, elevated body mass index, and the presence of atrial fibrillation were independently associated to higher concentrations of Big ET-1. Big ET-1 (ranking second just behind BNP among neurohormonal factors) was an independent predictor of outcome defined as all-cause mortality (hazard ratio 1.49, 95% CI 1.20-1.84, P = .0003) or the combined endpoint of mortality and morbidity (hazard ratio 1.43, 95% CI 1.20-1.69, P < .0001) and provided incremental prognostic value compared with BNP. CONCLUSIONS: In a large population of patients with symptomatic heart failure, the circulating concentration of Big ET-1, a precursor of the paracrine and bioactive peptide ET-1, was an independent marker of mortality and morbidity. In this setting, BNP remained the strongest neurohormonal prognostic factor.

Cardiovascular oxidative stress is reduced by an ACE inhibitor in a rat model of streptozotocin-induced diabetes.

Blockade of the renin-angiotensin system (RAS) reduces cardiovascular morbidity and mortality in diabetic patients. Ang II-mediated generation of reactive oxygen species (ROS) has been suggested to be involved in several diabetic complications. We investigated whether the inhibition of Ang II production with an ACE inhibitor (ACEi) reduces oxidative stress and limits structural cardiovascular remodeling in a rat model of streptozotocin (STZ)-induced diabetes. Diabetic rats were treated for 7 weeks with an ACEi (lisinopril, 5 mg/kg/d), an antioxidant (N-acetyl-l-cysteine (NAC), 0.5 g/kg/d) and their combination. At sacrifice, ROS in the myocardium and thoracic aorta, LV myocyte number and size and aorta morphology were determined by quantitative histological methods. Superoxide and hydroxyl radical content, detected by dihydroethidium (DHE) and 8-hydroxydeoxyguanosine (8-OHdG), were 6.7 and 4.5-fold, respectively, higher in diabetic myocardium than in non-diabetic controls (p<0.001). The amount of superoxide was 5-fold higher in the thoracic aorta of diabetic rats compared to controls (p<0.001). Diabetes caused a modest increase in myocyte volume (+13%, p<0.01), a reduction of LV myocyte number (-43%, p<0.001), an accumulation of collagen around coronary arterioles (1.9-fold increase, p<0.01) and a decrease in arterial elastin/collagen ratio (-63%, p<0.001) compared to controls. Treatment with the ACEi attenuated ROS formation and prevented phenotypic changes in the heart (cardiomyocyte hypertrophy, perivascular fibrosis) and in the aorta of diabetic rats to the same extent as NAC. The absence of an additive effect, suggests a common mechanism of action, through the reduction of oxidative stress.