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. 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 (≥25 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’s 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 ± 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.