L-citrulline is protective in hyperoxic lung damage and improves matrix remodelling and alveolarization

Moderate hyperoxia alters alveolar and vascular lung morphogenesis. Nitric oxide (NO) and matrix metalloproteinases (MMP) have a crucial role in the homeostasis of the matrix and bronchoalveolar structure and may be regulated abnormally by exposure to hyperoxia. Disruption of vascular endothelial growth factor (VEGF)-NO signaling impairs vascular growth and contributes to hyperoxia-induced vascular disease in bronchopulmonary dysplasia (BPD). We hypothesize that L-citrulline, by raising the serum levels of L-arginine and enhancing endogenous NO synthesis, might attenuate hyperoxia-induced lung injury in an experimental model of BPD. Neonatal rats (1 day old) were exposed to 60% oxygen or room air for 14 days and administered L-citrulline or a vehicle (sham). Lung morphometry were performed; Serum was tested for arginine level; Matrix metalloproteinases2 (MMP2) gene expression, VEGF gene and protein expression and endothelial NO synthase (eNOS) protein expression were compared. Mean linear intercept was higher in the hyperoxia and sham groups when compared with the room air (RA) and L-citr+hyperoxia treated group (p<0.02). Secondary crests number was higher in L-citrulline treated and RA when compared to hyperoxia and sham group (p<0.02). L-Arginine level rose in the L-citrulline-treated group (p<0.05). L-citrulline did not affect MMP2 gene expression, but it regulated the MMP2 active protein, which rose in bronchoalveolar lavage fluid (p<0.05), presumably due to a post-transductional effect. Compared with RA controls, hyperoxia significantly decreased VEGF and eNOS protein expression. At the same time, an increased lung VEGF gene and protein expression (p<0.05) were also seen in the rats treated with L-citrulline. We conclude that: (i) hyperoxia decreases growth and disrupts VEGF-NO signaling of lung; (ii) the main effects of L-citrulline are an increased serum level of arginine, as a promoter and a substrate of the nitric oxide synthase; and (ii) a better alveolar growth and matrix control than in hyperoxia-induced lung damage seems promising

L-citrulline has a protective effect in hyperoxic lung damage and improves matrix remodeling and alveolarization

Even moderate hyperoxia alters alveolar and vascular lung morphogenesis. Nitric oxide (NO) and matrix metalloproteinases (MMP) have a crucial role in the homeostasis of the matrix and bronchoalveolar structure, and may be regulated abnormally by exposure to hyperoxia. We hypothesize that L-citrulline, by raising the serum levels of L-arginine and enhancing endogenous NO synthesis, might attenuate hyperoxia-induced lung injury in an experimental model of bronchopulmonary dysplasia (BPD). Neonatal rats (1 day old) were exposed to 60% oxygen or room air for 14 days and administered L-citrulline or a vehicle. Serum was tested for arginine level, which rose in the L-citrulline-treated group (p<0.05). Alveolar number decreased in the hyperoxia and vehicle-treated groups, when compared with the room air and L-citrulline treated group (p<0.05). L-citrulline did not affect matrix metalloproteinase2 (MMP2) gene expression, but MMP2 active protein resulted to be higher in bronchoalveolar lavage fluid of the citrulline-treated rats (p<0.05). At the same time, an increased lung VEGF gene expression (p<0.05) and protein immunostaining were also seen in the rats treated with L-citrulline. We conclude that: (i) the main effects of L-citrulline are an increased serum level of arginine, as a promoter and a substrate of the nitric oxide synthase; and (ii) a better alveolar growth and matrix control in hyperoxia-induced lung damage seems promising.Nonostante i progressi tecnologici e terapeutici degli ultimi anni, la displasia broncopolmonare (BPD) rimane ancora causa principale di morbilità respiratoria nei neonati prematuri di peso molto basso (VLBW). L’ossido nitrico (NO) e le metalloproteasi di matrice (MMP) hanno un ruolo cruciale nell’omeostasi della struttura e della matrice broncoalveolare. In particolare, sempre maggiore è l’evidenza scientifica che attribuisce all’ossido nitrico endogeno un ruolo vitale nello sviluppo alveolare e vascolare del polmone immaturo. Oltre ad essere un potente vasodilatatore, l’NO ha molti effetti biologici rilevanti: migliora lo scambio gassoso, ha azione antiinfiammatoria e antiossidante, stimola appunto l’angiogenesi e la crescita del polmone immaturo. Il potenziale ruolo di una terapia con NO inalatorio per la prevenzione della BPD è stato recente oggetto di vari studi, ma non è stato ancora chiaramente definito (Horst et al. Am J Physiol Lung Cell Mol Physiol, 2007; Lin et al, Pediatr Res, 2005; McCurnin et al, Am J Physiol Lung Cell Mol Physiol 2005). L’arginina è il substrato dell’ossido nitrico sintetasi per la produzione di NO. Ci sono evidenze che la supplementazione con L-arginina aumenti la vasodilatazione endotelio-dipendente, mediata dall’NO (Bode-Boger et al, Vasc Med 2003; Piatti et al, Diabetes Care, 2001). Dopo somministrazione orale però l’arginina è soggetta a un intenso metabolismo presistemico; è stato dimostrato che la supplementazione orale con L-citrullina aumenta significativamente le concentrazioni plasmatiche di arginina, in modo dose dipendente, più che la somministrazione di L-arginina stessa (Kuhn et al. Circulation 2002; Schwedhelm et al. Br J Clin Pharmacol, 2007). Infatti la citrullina viene convertita in arginina sia nelle cellule del tubulo renale prossimale, che in molte cellule in grado di produrre NO (cellule endoteliali e macrofagi), che utilizzano la citrullina come substrato per produrre arginina. Obiettivi del progetto di ricerca Scopo di questo progetto era valutare se la somministrazione di L-citrullina, aumentando i livelli plasmatici e/o tissutali di arginina e stimolando la produzione endogena di NO, potesse attenuare il danno polmonare indotto dall’iperossia in un modello sperimentale di BPD. Materiali e metodi Ratti neonati wild-type Sprague-Dawley (n=40) sono stati esposti a moderata iperossia moderata (FiO2 60%) per i primi 14 giorni di vita (Man Yi, Am J Respir Crit Care Med, 2004): 12 non hanno ricevuto alcun trattamento, 18 sono stati trattati con L-citrullina 1g/kg/die intraperitoneale, 10 sono stati trattati quotidianamente con placebo (soluzione salina intraperitoneale). Un gruppo di ulteriori 10 ratti sono stati mantenuti in aria ambiente per i primi 14 giorni di vita e utilizzati come controlli. Tutte le procedure sugli animali sono state condotte in maniera conforme al D. L. 116/1992 e secondo autorizzazione n. 173/2006-B del 4.12.2006. Per valutare l’effetto del trattamento è stata analizzata la morfometria su tessuto polmonare. Ulteriori marker di sviluppo polmonare sono stati analizzati, in particolare VEGF e metalloproteasi 2 (MMP2). Risultati Il numero di alveoli /mm2 risultava più basso nei ratti esposti a iperossia e in quelli trattati con placebo rispetto ai ratti cresciuti in aria ambiente e a quelli trattati con L-citrullina (p<0.05). I livelli sierici di arginina risultavano più elevati nel gruppo trattato con L-citrullina (p<0.05). Il trattamento con L-citrullina non modificava l’espressione del gene per la metalloproteasi2 (MMP2), ma la proteina attiva MMP2 risultava più elevata nel lavaggio broncoalveolare dei ratti trattati con L-citrullina (p<0.05). Nei ratti trattati con L-citrullina, inoltre, si evidenziava un incremento significativo dell’espressione del gene per il VEGF a livello polmonare (p<0.05). Conclusioni La somministrazione di L-citrullina sembra elevare i livelli plasmatici di arginina e potrebbe pertanto promuovere la produzione endogena di NO: il trattamento con citrullina sembra promettente per migliorare la crescita alveolare e il controllo della matrice nel danno polmonare indotto dall’iperossia

L-citrulline prevents alveolar and vascular derangement in a rat model of moderate hyperoxia-induced lung injury

BackgroundModerate normobaric hyperoxia causes alveolar and vascular lung derangement in the newborn rat. Endogenous nitric oxide (NO), which promotes lung growth, is produced from the metabolism of L-arginine to L-citrulline in endothelial cells. We investigated whether administering L-citrulline by raising the serum levels of L-arginine and enhancing NO endogenous synthesis attenuates moderate hyperoxia-induced lung injury.MethodsNewborn rats were exposed to FiO(2) = 0.6 or room air for 14 days to induce lung derangement and then were administered L-citrulline or a vehicle (sham). Lung histopathology was studied with morphometric features. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected for analysis. Lung vascular endothelial growth factor (VEGF), nitric oxide synthase (eNOS), and matrix metalloproteinase 2 (MMP2) gene and protein expressions were assessed.ResultsSerum L-arginine rose in the L-citr + hyperoxia group (p = 0.05), as well as the Von Willebrand factor stained vessels count (p = 0.0008). Lung VEGF immune staining, localized on endothelial cells, was weaker in the sections under hyperoxia than the L-citr + hyperoxia and room air groups. This pattern was comparable with the VEGF gene and protein expression profiles. Mean alveolar size increased in the untreated hyperoxia and sham-treated groups compared with the groups reared in room air or treated with L-citrulline under exposure to hyperoxia (p = 0.0001). Lung VEGF and eNOS increased in the L-citrulline-treated rats, though this treatment did not change MMP2 gene expression but regulated the MMP2 active protein, which rose in BALF (p = 0.003).ConclusionsWe conclude that administering L: -citrulline proved effective in improving alveolar and vascular growth in a model of oxygen-induced pulmonary damage, suggesting better lung growth and matrix regulation than in untreated groups

Association of maternal hypertension and chorioamnionitis with preterm outcomes

OBJECTIVES: We compared the relative effect of hypertensive disorders of pregnancy and chorioamnionitis on adverse neonatal outcomes in very preterm neonates, and studied whether gestational age (GA) modulates these effects. METHODS: A cohort of neonates 23 to 30 weeks' GA, born in 2008 to 2011 in 82 hospitals adhering to the Italian Neonatal Network, was analyzed. Infants born from mothers who had hypertensive disorders (N = 2096) were compared with those born after chorioamnionitis (N = 1510). Statistical analysis employed logistic models, adjusting for GA, hospital, and potential confounders. RESULTS: Overall mortality was higher after hypertension than after chorioamnionitis (odds ratio [OR], 1.39; 95% confidence interval [CI], 1.08-1.80), but this relationship changed across GA weeks; the OR for hypertension was highest at low GA, whereas from 28 weeks' GA onward, mortality was higher for chorioamnionitis. For other outcomes, the relative risks were constant across GA; infants born after hypertension had an increased risk for bronchopulmonary dysplasia (OR, 2.20; 95% CI, 1.68-2.88) and severe retinopathy of prematurity (OR, 1.48; 95% CI, 1.02-2.15), whereas there was a lower risk for early-onset sepsis (OR, 0.25; 95% CI, 0.19-0.34), severe intraventricular hemorrhage (OR, 0.65; 95% CI, 0.48-0.88), periventricular leukomalacia (OR, 0.70; 95% CI, 0.48-1.01), and surgical necrotizing enterocolitis or gastrointestinal perforation (OR, 0.47; 95% CI, 0.31-0.72). CONCLUSIONS: Mortality and other adverse outcomes in very preterm infants depend on antecedents of preterm birth. Hypertension and chorioamnionitis are associated with different patterns of outcomes; for mortality, the effect changes across GA weeks. Copyright \uc2\ua9 2014 by the American Academy of Pediatrics