Studio delle vie di segnale coinvolte nello sviluppo di ipertensione polmonare da sovraccarico cronico di volume del ventricolo destro e valutazione degli effetti di un nuovo donatore di ossido nitrico.

Background. Il sovraccarico di volume cronico del ventricolo destro determina a lungo termine la comparsa di un quadro patologico di scompenso ventricolare destro e ipertensione arteriosa polmonare, una condizione emodinamica e fisiopatologica progressiva e complessa caratterizzata da una pressione arteriosa polmonare media a riposo superiore, o uguale, a 25 mmHg, un rimodellamento sostanziale della struttura dei vasi polmonari e un elevato grado di ipertrofia ventricolare destra. Gli esatti meccanismi molecolari coinvolti nell\u2019eziopatogenesi dell\u2019ipertensione arteriosa polmonare, nonch\ue9 nel rimodellamento ventricolare e vascolare da sovraccarico di volume cronico del ventricolo destro, sono tuttora poco chiari, motivo per cui questa condizione clinica continua a rimanere devastante, con sintomi progressivamente debilitanti ed alta mortalit\ue0 nella popolazione affetta. Obiettivi. Nella prima fase dello studio (fase di valutazione, osservazionale e descrittiva) sono state eseguite analisi istologiche, biochimiche e molecolari allo scopo di fornire una valutazione approfondita del modello sperimentale di ipertensione polmonare da sovraccarico cronico di volume del ventricolo destro, essenzialmente un modello di ipertensione arteriosa polmonare associata a cardiopatie congenite (sottogruppo 1.4.4). Nella seconda fase dello studio (fase di trattamento) sono stati valutati gli effetti della somministrazione in acuto di albumina nitrosilata, un nuovo agente donatore di ossido nitrico (NO-donor), sull\u2019ipertensione polmonare e sul rimodellamento del ventricolo destro. Metodi. 35 ratti maschi adulti Sprague-Dawley (15 nella prima fase, gruppo SHUNT, 20 nella seconda fase, gruppi SHUNT+HSA e SHUNT+S-NO-HSA) del peso di 400 \ub1 50 grammi, sono stati sottoposti ad intervento di creazione di una fistola aorto-cavale mentre ratti sham-operated (n=15) sono stati considerati come controlli nella prima fase dello studio. A distanza di 20 settimane dall\u2019operazione, sezioni di ventricolo destro e arteriole polmonari degli animali della prima fase sperimentale (n=5 per gruppo) sono state analizzate con tecniche istologiche, mentre lo stato di fosforilazione di ERK1/2, Akt e cTnI e i livelli di espressione/attivazione di eNOS e iNOS sono stati valutati mediante analisi Western blot su biopsie di ventricolo destro e polmone di tutti i ratti considerati in entrambe le fasi dello studio. L\u2019analisi HPLC ha infine consentito di determinare sugli stessi campioni i rapporti tra glutatione ossidato e ridotto (GSSG/GSH) e il contenuto dei fosfati ad alta energia. Risultati. L\u2019analisi Western blot eseguita su campioni di miocardio ventricolare destro a distanza di 20 settimane dall\u2019esecuzione dello shunt aorto-cavale, ha evidenziato un significativo incremento dei livelli di fosforilazione di ERK1/2 (p<0.001) e di cTnI (p<0.01), una significativa riduzione dello stato di fosforilazione di Akt (p<0.05) e una significativa down-regolazione dei livelli di espressione di iNOS (p<0.05) nel gruppo SHUNT rispetto al gruppo SHAM. I ratti del gruppo SHUNT+S-NO-HSA hanno invece mostrato una significativa riduzione dei livelli di fosforilazione di ERK1/2 (p<0.01) e di cTnI (p<0.01) nel ventricolo destro rispetto al gruppo di controllo SHUNT+HSA, mentre 6 risultano caratterizzati da una significativa up-regolazione dei livelli di espressione di iNOS (p<0.05) dopo 20 settimane di esposizione allo shunt aorto-cavale. I campioni di tessuto polmonare degli animali del gruppo SHUNT, analizzati mediante Western blot a distanza di 20 settimane dalla creazione della fistola aorto-cavale, hanno mostrato un significativo aumento dei livelli di fosforilazione di ERK1/2 (p<0.01) e dell\u2019espressione di iNOS (p<0.05) rispetto ai ratti sham-operated (SHAM), mentre il trattamento in acuto con albumina S-nitrosilata (gruppo SHUNT+S-NO-HSA) ha ulteriormente contribuito ad un sostanziale incremento dei livelli di espressione di iNOS (p<0.05) rispetto ai ratti di controllo (SHUNT+HSA). La persistenza dello shunt aorto-cavale per 20 settimane \ue8 risultata indurre un significativo incremento dei livelli di glutatione ossidato (%GSSG, p<0.05) nel ventricolo destro dei ratti SHUNT rispetto ai ratti di controllo SHAM, mentre nessuna variazione statisticamente significativa \ue8 stata evidenziata negli animali trattati con S-NO-HSA (SHUNT+S-NO-HSA) rispetto al gruppo di controllo (SHUNT+HSA) dopo 20 settimane di esposizione allo shunt aorto-cavale. Nel tessuto polmonare i livelli di GSSG sono risultati sostanzialmente aumentati (p<0.01) nel gruppo SHUNT rispetto al gruppo SHAM ma significativamente ridotti (p<0.01) in seguito alla somministrazione acuta di S-NO-HSA (gruppo SHUNT+S-NO-HSA) dopo 20 settimane di esposizione allo shunt aorto-cavale. La funzionalit\ue0 mitocondriale dei cardiomiociti ventricolari destri \ue8 risultata complessivamente compromessa (diminuzione di ATP, p<0.05) negli animali del gruppo SHUNT rispetto agli animali sham-operated (SHAM), dopo 20 settimane dall\u2019operazione, con una generale tendenza alla preservazione in seguito al trattamento con S-NO-HSA (gruppo SHUNT+S-NO-HSA) rispetto al gruppo di controllo (SHUNT+HSA). Variazioni significative dei livelli di AMP e ATP (p<0.05, p<0.01 rispettivamente) sono state evidenziate nel tessuto polmonare dei ratti SHUNT rispetto al gruppo SHAM in seguito alla persistenza della fistola aorto-cavale; lo stato energetico polmonare \ue8 risultato significativamente preservato (diminuzione AMP: p<0.05; aumento ATP: p<0.01) nel gruppo SHUNT+S-NO-HSA rispetto agli animali non trattati (SHUNT+HSA). Conclusioni. Il modello studiato di sovraccarico cronico di volume del ventricolo destro con progressivo sviluppo di ipertensione polmonare dimostra il coinvolgimento di vie di segnale attivate nella patogenesi dell\u2019insufficienza ventricolare destra con ipertensione del circolo arterioso polmonare e in particolare: -ERK 1/2 nell\u2019ipertrofia cardiaca e della componente vascolare polmonare; -Akt nella sopravvivenza cellulare; -cTnI nella regolazione della funzione contrattile dei cardiomiociti; -eNOS e iNOS nell\u2019omeostasi endoteliale e nella generazione di ossido nitrico. In questo contesto sperimentale, che riproduce condizioni cliniche specifiche, l\u2019utilizzo di un nuovo NO-donor quale la S-NO-HSA per via sistemica in acuto, risulta essere vantaggioso non solo per il profilo emodinamico e biochimico ma poich\ue9 attenua le alterazioni di mediatori coinvolti in vie biomolecolari di fondamentale importanza per le possibili implicazioni cliniche.Background. Chronic volume overload of right ventricle induces right ventricular failure and pulmonary arterial hypertension, a progressive and multifaceted disease process characterized by high morbidity and mortality, high mean pulmonary artery pressure at rest ( 6525 mmHg), pulmonary vascular remodeling and right ventricular hypertrophy. The exact molecular mechanisms involved in the pathogenesis of pulmonary arterial hypertension remain still unclear as those underlying the development of chronic volume overload-induced right ventricular and pulmonary vascular remodeling. Objectives. In the first part of the study (evaluation phase) the animal model of right ventricular chronic volume overload-induced pulmonary hypertension was assessed by histological, biochemical and molecular analysis. This model was essentially designed to reproduce a particular clinical condition such as pulmonary arterial hypertension associated with congenital heart diseases (clinical subset 1.4.4). In the second part (treatment phase) the effects of a new NO-donor\u2019s acute administration, S-nitroso-albumin, were evaluated in respect of pulmonary hypertension and right ventricular remodeling. Methods. Aorto-caval shunt was surgically created in 35 adult male Sprague-Dawley rats weighting 400 \ub1 50 g (first part, SHUNT group, n=15; second part, SHUNT+HSA and SHUNT+S-NO-HSA groups, n=10 each one). Sham-operated rats (n=15) were considered as controls only for the first part of the study (SHAM group). 20 weeks after surgery histological sections were obtained from right ventricles and small pulmonary arteries of SHAM and SHUNT animals (n=5 each group), stained in hematoxylin-eosin and finally analyzed. The phosphorylation status of ERK1/2, Akt and cTnI and protein expression/activation levels of eNOS and iNOS were evaluated by Western blot analysis in right ventricles and lungs of all rats in both phases of the study after 20 weeks from surgery. The same samples were used even to determine the ratios between oxidized and reduced glutathione (GSSG/GSH) and high energy phosphates content by HPLC analysis. Results. Western blot analysis on right ventricular myocardium has shown a significant increase of ERK1/2 and cTnI phosphorylation levels (p<0.001 and p<0.01, respectively), a decrease of Akt phosphorylation (p<0.05) and a significant downregulation of iNOS expression (p<0.05) in SHUNT group compared to the SHAM group, 20 weeks after the aorto-caval shunt. ERK1/2 and cTnI phosphorylation levels were significantly reduced in right ventricle of SHUNT+S-NO-HSA rats compared to the control group (SHUNT+HSA) (p<0.01), while the treatment significantly upregulated the expression of iNOS (p<0.05) after 20 weeks from surgery. As shown by Western blot analysis performed on lung tissue biopsies 20 weeks after the aorto-caval shunt, there was a significant increase of ERK1/2 phosphorylation levels and iNOS expression in SHUNT group compared to sham-operated rats (p<0.01 and p<0.05 respectively), while S-NO-HSA acute administration induced a further increase of iNOS expression in SHUNT+S-NO-HSA group compared to the control (p<0.05). Twenty week-aorto-caval shunt induced a significant increase of oxidixed glutathione levels in right ventricle of SHUNT rats compared to the control group (p<0.05), while no 8 changes were shown after S-NO-HSA treatment compared to the SHUNT+HSA group 20 weeks after surgery. Lung tissue levels of GSSG were significantly increased in SHUNT group (p<0.01) but substantially reduced after acute administration of S-NO-HSA (p<0.01) at 20 weeks from aorto-caval shunt. As shown by the significant decrease of ATP levels in rigth ventricle of SHUNT animals compared to the control group (p<0.05), mitochondrial function of right ventricular cardiomyocytes was substantially reduced after 20 weeks from aorto-caval shunt while seemed to be preserved by the treatment with S-NO-HSA compared to the untreated animals. Significant changes of AMP and ATP content were shown in lung tissue of SHUNT rats compared to the control group (p<0.05, p<0.01 respectively) 20 weeks after the aorto-caval shunt while acute administration of S-NO-HSA significantly preserved lung energy status compared to the untreated group (p<0.05 for the decrease of AMP; p<0.01 for the increase of ATP). Conclusions.This model of right ventricle chronic volume overload with progressive development of pulmonary hypertension showed the involvement of signaling pathways in the pathogenesis of right ventricular failure with pulmonary arterial hypertension, in particular: -ERK 1/2 in cardiac hypertrophy and pulmonary vascular remodelling; -Akt in cell survival; -cTnI in regulation of cardiomyocyte contractile function; -eNOS and iNOS in endothelial homeostasis and nitric oxide generation. In this experimental setting acute administration of a new NO-donor, such as S-NO-HSA, has been shown overall haemodynamic and biochemical effects and a reduction of the altered mediators involved in some pathways of pivotal role in the clinical field

Role of calcium desensitization in the treatment of myocardial dysfunction after deep hypothermic circulatory arrest

Abstract Introduction Rewarming from deep hypothermic circulatory arrest (DHCA) produces calcium desensitization by troponin I (cTnI) phosphorylation which results in myocardial dysfunction. This study investigated the acute overall hemodynamic and metabolic effects of epinephrine and levosimendan, a calcium sensitizer, on myocardial function after rewarming from DHCA. Methods Forty male Wistar rats (400 to 500 g) underwent cardiopulmonary bypass (CPB) through central cannulation and were cooled to a core temperature of 13°C to 15°C within 30 minutes. After DHCA (20 minutes) and CPB-assisted rewarming (60 minutes) rats were randomly assigned to 60 minute intravenous infusion with levosimendan (0.2 μg/kg/min; n = 15), epinephrine (0.1 μg/kg/min; n = 15) or saline (control; n = 10). Systolic and diastolic functions were evaluated at different preloads with a conductance catheter. Results The slope of left ventricular end-systolic pressure volume relationship (Ees) and preload recruitable stroke work (PRSW) recovered significantly better with levosimendan compared to epinephrine (Ees: 85 ± 9% vs 51 ± 11%, P\u3c0.003 and PRSW: 78 ± 5% vs 48 ± 8%, P\u3c0.005; baseline: 100%). Levosimendan but not epinephrine reduced left ventricular stiffness shown by the end-diastolic pressure-volume relationship and improved ventricular relaxation (Tau). Levosimendan preserved ATP myocardial content as well as energy charge and reduced plasma lactate concentrations. In normothermia experiments epinephrine in contrast to Levosimendan increased cTnI phosphorylation 3.5-fold. After rewarming from DHCA, cTnI phosphorylation increased 4.5-fold in the saline and epinephrine group compared to normothermia but remained unchanged with levosimendan. Conclusions Levosimendan due to prevention of calcium desensitization by cTnI phosphorylation is more effective than epinephrine for treatment of myocardial dysfunction after rewarming from DHCA

Antiplatelet Agents Inhibit the Generation of Platelet-Derived Microparticles

Platelet microparticles (PMPs) contribute to thrombogenesis but the effects of antiplatelet drugs on PMPs generation is undefined. The present study investigated the cellular events regulating PMP shedding, testing in vitro platelet agonists and inhibitors. Platelet-rich plasma from healthy subjects was stimulated with arachidonic acid, U46619, collagen type-I (10 and 1.5 µg/mL), epinephrine, ADP or TRAP-6 and pre-incubated with acetylsalicylic acid (ASA, 100 and 10 µmol/L), SQ-29,548, apyrase, PSB-0739, or eptifibatide. PMPs were detected by flow-cytometry using CD61 and annexin-V as fluorescent markers. Platelet agonists induced annexin V-positive PMP shedding. The strongest response was to high concentration collagen. ADP-triggered PMP shedding was dose-independent. ASA reduced PMPs induced by arachidonic acid- (645, 347-2946 vs 3061, 446-4901 PMPs/µL; median ad range, n=9, P<0.001), collagen 10 µg/mL (5317, 2027-15935 vs 10252, 4187-46316 PMPs/µL; n=13, P<0.001), collagen 1.5 µg/mL (1078, 528-2820 vs 1465, 582-5948 PMPs/µL; n=21, P<0.001) and TRAP-6 (2008, 1621-2495 vs 2840, 2404-3031 PMPs/µL; n=3, P<0.01) but did not affect the response to epinephrine or ADP. The ADP scavenger apyrase reduced PMPs induced by U46619 (1256, 395-2908 vs 3045, 1119-5494 PMPs/µL, n=6, P<0.05), collagen 1.5 µg/mL (1006, 780-1309 vs 2422, 1839-3494 PMPs/µL, n=3, P<0.01) and TRAP-6 (904, 761-1224 vs 2840, 2404-3031 PMPs/µL, n=3, P<0.01). The TP receptor antagonist SQ-29,548 and the P2Y12 receptor antagonist PSB-0739 markedly inhibited PMPs induced by low doses of collagen. Except for high-dose collagen, eptifibatide abolished agonist-induced PMP release. Both TXA2 generation and ADP secretion are required as amplifiers of PMP shedding. The crucial role of the fibrinogen receptor and the collagen receptor in PMPs generation, independently of platelet aggregation, was identified

In reply to Sch\ue4fer et\ua0al: new evidence on the role of endothelin-1 axis as a potential therapeutic target in multiple myeloma

no abstract availabl

G\u3b115 in early onset of pancreatic ductal adenocarcinoma

The GNA15 gene is ectopically expressed in human pancreatic ductal adenocarcinoma cancer cells. The encoded G\u3b115 protein can promiscuously redirect GPCR signaling toward pathways with oncogenic potential. We sought to describe the distribution of GNA15 in adenocarcinoma from human pancreatic specimens and to analyze the mechanism driving abnormal expression and the consequences on signaling and clinical follow-up. We detected GNA15 expression in pre-neoplastic pancreatic lesions and throughout progression. The analysis of biological data sets, primary and xenografted human tumor samples, and clinical follow-up shows that elevated expression is associated with poor prognosis for GNA15, but not any other GNA gene. Demethylation of the 5' GNA15 promoter region was associated with ectopic expression of G\u3b115 in pancreatic neoplastic cells, but not in adjacent dysplastic or non-transformed tissue. Down-modulation of G\u3b115 by shRNA or CRISPR/Cas9 affected oncogenic signaling, and reduced adenocarcimoma cell motility and invasiveness. We conclude that de novo expression of wild-type GNA15 characterizes transformed pancreatic cells. The methylation pattern of GNA15 changes in preneoplastic lesions coincident with the release a transcriptional blockade that allows ectopic expression to persist throughout PDAC progression. Elevated GNA15 mRNA correlates with poor prognosis. In addition, ectopic G\u3b115 signaling provides an unprecedented mechanism in the early steps of pancreas carcinogenesis distinct from classical G protein oncogenic mutations described previously in GNAS and GNAQ/GNA11

Long-term survival and cure fraction estimates for childhood cancer in Europe (EUROCARE-6): results from a population-based study

Background: The EUROCARE-5 study revealed disparities in childhood cancer survival among European countries, giving rise to important initiatives across Europe to reduce the gap. Extending its representativeness through increased coverage of eastern European countries, the EUROCARE-6 study aimed to update survival progress across countries and years of diagnosis and provide new analytical perspectives on estimates of long-term survival and the cured fraction of patients with childhood cancer. Methods: In this population-based study, we analysed 135 847 children (aged 0–14 years) diagnosed during 2000–13 and followed up to the end of 2014, recruited from 80 population-based cancer registries in 31 European countries. We calculated age-adjusted 5-year survival differences by country and over time using period analysis, for all cancers combined and for major cancer types. We applied a variant of standard mixture cure models for survival data to estimate the cure fraction of patients by childhood cancer and to estimate projected 15-year survival. Findings: 5-year survival for all childhood cancer combined in Europe in 2010–14 was 81% (95% CI 81–82), showing an increase of three percentage points compared with 2004–06. Significant progress over time was observed for almost all cancers. Survival remained stable for osteosarcomas, Ewing sarcoma, Burkitt lymphoma, non-Hodgkin lymphomas, and rhabdomyoscarcomas. For all cancers combined, inequalities still persisted among European countries (with age-adjusted 5-year survival ranging from 71% [95% CI 60–79] to 87% [77–93]). The 15-year survival projection for all patients with childhood cancer diagnosed in 2010–13 was 78%. We estimated the yearly long-term mortality rate due to causes other than the diagnosed cancer to be around 2 per 1000 patients for all childhood cancer combined, but to approach zero for retinoblastoma. The cure fraction for patients with childhood cancer increased over time from 74% (95% CI 73–75) in 1998–2001 to 80% (79–81) in 2010–13. In the latter cohort, the cure fraction rate ranged from 99% (95% CI 74–100) for retinoblastoma to 60% (58–63) for CNS tumours and reached 90% (95% CI 87–93) for lymphoid leukaemia and 70% (67–73) for acute myeloid leukaemia. Interpretation: Childhood cancer survival is increasing over time in Europe but there are still some differences among countries. Regular monitoring of childhood cancer survival and estimation of the cure fraction through population-based registry data are crucial for evaluating advances in paediatric cancer care. Funding: European Commission