Degradation of Lung Protective Angiotensin Converting Enzyme-2 by Meconium in Human Alveolar Epithelial Cells: A Potential Pathogenic Mechanism in Meconium Aspiration Syndrome.

BACKGROUND: Pancreatic digestive enzymes present in meconium might be responsible for meconium-induced lung injury. The local Renin Angiotensin System plays an important role in lung injury and inflammation. Particularly, angiotensin converting enzyme-2 (ACE-2) has been identified as a protective lung enzyme against the insult. ACE-2 converts pro-apoptotic Angiotensin II to anti-apoptotic Angiotensin 1-7. However, the effect of meconium on ACE-2 has never been studied before. OBJECTIVE: To study the effect of meconium on ACE-2, and whether inhibition of proteolytic enzymes present in the meconium reverses its effects on ACE-2. METHODS: Alveolar epithelial A549 cells were exposed to F-12 medium, 2.5% meconium, meconium + a protease inhibitor cocktail (PIc) and PIc alone for 16 h. At the end of incubation, apoptosis was measured with a nuclear fragmentation assay and cell lysates were collected for ACE-2 immunoblotting and enzyme activity. RESULTS: Meconium caused a fourfold increase in apoptotic nuclei (p < 0.001). The pro-apoptotic effect of meconium can be reversed by PIc. Meconium reduced ACE-2 enzyme activity by cleaving ACE-2 into a fragment detected at ~ 37 kDa by immunoblot. PIc prevented the degradation of ACE-2 and restored 50% of ACE-2 activity (p < 0.05). CONCLUSION: These data suggest that meconium causes degradation of lung protective ACE-2 by proteolytic enzymes present in meconium, since the effects of meconium can be reversed by PIc.

Correlations of Enzyme Levels at Birth in Stressed Neonates with Short-Term Outcomes.

INTRODUCTION: Multi-organ injury causes leakage of several intracellular enzymes into the circulation. We evaluated the correlation between the serum-leaked intracellular enzyme levels at the beginning of treatment and the outcome in perinatally stressed neonates. MATERIALS AND METHODS: We retrospectively studied neonates whose 1 minute Apgar score was < 7. We collected initial venous blood sample data, including aspartate transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH), and creatine kinase (CK) levels, and correlated these with patient short-term outcomes. RESULTS: Of 60 neonates, nine patients were treated with therapeutic hypothermia, and 32 needed mechanical ventilation. The therapeutic hypothermia group showed significantly larger base deficit, and higher lactate, AST, ALT, LDH, and CK (all p < 0.01). The duration of mechanical ventilation significantly correlated with AST, ALT, LDH, and CK levels (all p < 0.01). CONCLUSION: Initial enzyme levels are useful for predicting the duration of mechanical ventilation in stressed neonates.

Modified porcine surfactant enriched by recombinant human superoxide dismutase for experimental meconium aspiration syndrome.

AIMS: Combination of exogenous surfactant with antioxidant enzyme recombinant human superoxide dismutase (rhSOD) was tested in the treatment of experimental meconium aspiration syndrome as oxidative processes play key role in its pathogenesis. MATERIAL AND METHODS: Young New Zealand rabbits were instilled by saline (Sal group) or by meconium suspension (Mec group). Some of meconium-instilled animals were treated by surfactant alone (Surf group) or surfactant in combination with rhSOD (Surf + SOD group) and oxygen-ventilated for 5 h. PaO2/FiO2, oxygenation index, oxygen saturation, PaCO2, ventilation efficiency index and alveolar-arterial gradient were evaluated every hour; post mortem, cells in bronchoalveolar lavage were counted, inflammatory and oxidative markers were assessed using ELISA in lung tissue homogenates. KEY FINDINGS: Exogenous surfactant combined with rhSOD improved oxygenation during the first hour after the treatment more than surfactant alone (p = 0.039 to 0.0001 vs. Mec and Surf group). Amelioration was also seen in CO2 elimination (p = 0.049 to 0.0096 vs. Mec group), alveolar-arterial gradient diminution (p = 0.024 to 0.0019 vs. Mec and Surf group), prevention of oxidative damage and cytokine production (p = 0.049 to 0.002 vs. Mec group). SIGNIFICANCE: It seems that inhibition of oxidative signalization may be strong supporting factor in surfactant treatment of MAS.

High aspartate aminotransferase level predicts poor neurodevelopmental outcome in infants with meconium aspiration syndrome.

OBJECTIVE: The aim of our study is to clarify the perinatal predictive factors of meconium aspiration syndrome (MAS) with neurodevelopmental delay (ND) in infants. MATERIALS AND METHODS: In this retrospective study, data were collected from the infants born between 1990 and 2008. They all had primary diagnosis of MAS. Multivariable analyzed perinatal predictive factors of MAS with ND. The developmental status of these infants was followed at least 2 years with the Wechsler Intelligence Scale for Children. RESULTS: A total of 114 surviving babies met the criteria of MAS. Six babies were defined as ND group. Lower 5-minute Apgar score and diastolic blood pressure were significantly related to the ND group. Elevated asparatate aminotransferase (AST), nucleated red blood cells, and white blood cells at the time of admission were significantly high in ND group. Furthermore, AST had area under the receiver operating characteristic curve of 0.879, (95% confidence interval: 0.801, 0.934), p < 0.0001. At 96 mg/dL, it had 83.33% sensitivity, 80.81% specificity, and negative predictive value of 98.8. Multivariable logistic regression analysis revealed AST was the only significant predictive factor for MAS with ND. CONCLUSION: Early intervention should be recommended in infants having MAS with high AST level at birth for improving their neurodevelopmental outcomes.

Varespladib inhibits secretory phospholipase A2 in bronchoalveolar lavage of different types of neonatal lung injury.

Secretory phospholipase A2 (sPLA2), which links surfactant catabolism and lung inflammation, is associated with lung stiffness, surfactant dysfunction, and degree of respiratory support in acute respiratory distress syndrome and in some forms of neonatal lung injury. Varespladib potently inhibits sPLA2 in animal models. The authors investigate varespladib ex vivo efficacy in different forms of neonatal lung injury. Bronchoalveolar lavage fluid was obtained from 40 neonates affected by hyaline membrane disease, infections, or meconium aspiration and divided in 4 aliquots added with increasing varespladib or saline. sPLA2 activity, proteins, and albumin were measured. Dilution was corrected with the urea ratio. Varespladib was also tested in vitro against pancreatic sPLA2 mixed with different albumin concentration. Varespladib was able to inhibit sPLA2 in the types of neonatal lung injury investigated. sPLA2 activity was reduced in hyaline membrane disease (P < .0001), infections (P = .003), and meconium aspiration (P = .04) using 40 µM varespladib; 10 µM was able to lower enzyme activity (P = .001), with an IC(50) of 87 µM. An inverse relationship existed between protein level and activity reduction (r = 0.5; P = .029). The activity reduction/protein ratio tended to be higher in hyaline membrane disease. Varespladib efficacy was higher in vitro than in lavage fluids obtained from neonates (P < .001).

Role of distinct phospholipases A2 and their modulators in meconium aspiration syndrome in human neonates.

PURPOSE: Meconium aspiration syndrome (MAS) is a life-threatening neonatal lung injury, whose pathophysiology has been mainly studied in animal models. In such models, pancreatic secretory phospholipase A2 (sPLA2-IB) and proinflammatory cytokines present in meconium challenge the lungs, catabolising surfactant and harming the alveoli. Locally produced phospholipases might perpetuate the injury and influence clinical pictures and therapeutic approaches. Our aim is to verify whether pulmonary phospholipase A2 (sPLA2-IIA) is involved in the damage and to determine if phospholipases and their modulators are associated with MAS clinical pictures. METHODS: We studied distinct phospholipases A2 and their modulators in broncho-alveolar lavage (BAL) fluids and in meconium of five MAS neonates and in five control neonates ventilated for extrapulmonary reasons. RESULTS: MAS patients have higher amounts of pulmonary phospholipase (sPLA2-IIA; P = 0.016) and Clara cell secretory protein (CCSP; P = 0.032). The local production of such proteins by the lung is confirmed by their very low levels in meconium. sPLA2-IIA contributes to the higher total enzyme activity in MAS patients, as compared to controls (P = 0.008). Cytosolic phospholipase was not detected in meconium or alveolar fluid. sPLA2 activity and sPLA2-IIA concentrations are correlated with the TNFα and with the release of CCSP. sPLA2 total activity, sPLA2-IIA and TNFα concentrations in BAL fluids correlate with the oxygenation impairment and haemorrhagic lung oedema. CONCLUSIONS: Pulmonary sPLA2 is locally produced and contributes to the total sPLA2 activity during MAS. CCSP is also produced in trying to lower the inflammation. Both sPLA2 activity and sPLA2-IIA are significantly correlated with oxygenation impairment and haemorrhagic lung oedema.

Association of circulating angiotensin converting enzyme activity with respiratory muscle function in infants.

BACKGROUND: Angiotensin converting enzyme (ACE) gene contains a polymorphism, consisting of either the presence (I) or absence (D) of a 287 base pair fragment. Deletion (D) is associated with increased circulating ACE (cACE) activity. It has been suggested that the D-allele of ACE genotype is associated with power-oriented performance and that cACE activity is correlated with muscle strength. Respiratory muscle function may be similarly influenced. Respiratory muscle strength in infants can be assessed specifically by measurement of the maximum inspiratory pressure during crying (Pimax). Pressure-time index of the respiratory muscles (PTImus) is a non-invasive method, which assesses the load to capacity ratio of the respiratory muscles.The objective of this study was to determine whether increased cACE activity in infants could be related to greater respiratory muscle strength and to investigate the potential association of cACE with PTImus measurements as well as the association of ACE genotypes with cACE activity and respiratory muscle strength in this population. METHODS: Serum ACE activity was assayed by using a UV-kinetic method. ACE genotyping was performed by polymerase chain reaction amplification, using DNA from peripheral blood. PTImus was calculated as (Pimean/Pimax) x (Ti/Ttot), where Pimean was the mean inspiratory pressure estimated from airway pressure, generated 100 milliseconds after an occlusion (P0.1), Pimax was the maximum inspiratory pressure and Ti/Ttot was the ratio of the inspiratory time to the total respiratory cycle time. Pimax was the largest pressure generated during brief airway occlusions performed at the end of a spontaneous crying effort. RESULTS: A hundred and ten infants were studied. Infants with D/D genotype had significantly higher serum ACE activity than infants with I/I or I/D genotypes. cACE activity was significantly related to Pimax and inversely related to PTImus. No association between ACE genotypes and Pdimax measurements was found. CONCLUSIONS: These results suggest that a relation in cACE activity and respiratory muscle function may exist in infants. In addition, an association between ACE genotypes and cACE activity, but not respiratory muscle strength, was demonstrated.

Pancreatic phospholipase A2 contributes to lung injury in experimental meconium aspiration.

To investigate the role of pancreatic (group I) secretory PLA2 (sPLA2-I) in the pathogenesis of meconium aspiration syndrome, human particulate meconium or its supernatant either before or after extraction of PLA2-I was insufflated into rat lungs. In addition, the pulmonary effects of intra-tracheal human and bovine PLA2-I were studied. Lungs with saline instillation served as controls. Intrapulmonary particulate meconium (both before and after PLA2-I extraction), unlike meconium supernatant, resulted in markedly elevated lung tissue PLA2 catalytic activity and human PLA2-I concentrations when compared with controls. On the other hand, tissue concentrations of the group II PLA2 remained unchanged in all meconium lungs. Pulmonary PLA2-I concentrations further correlated positively with lung injury scores. Instillation of meconium-derived human PLA2-I, at a concentration of one-third of that in particulate meconium, did not raise PLA2 activity or concentrations of PLA2-I or PLA2-II in the lung tissue from the control level, but still resulted in significantly elevated lung wet/dry ratio and injury score. In contrast, insufflation of bovine pancreatic PLA2 increased the lung tissue enzyme activity and wet/dry ratio from the control level, but had no effect on the type II PLA2 concentration or lung injury score. Our data thus indicate that human pancreatic PLA2, introduced in high amounts within aspirated meconium especially in particulate form, is a potent inducer of lung tissue inflammatory injury.

Apoptosis of airway epithelial cells in response to meconium.

Meconium aspiration syndrome (MAS) is common among newborn children but its mechanism is unclear. The syndrome is known to produce a strong inflammatory reaction in the lungs resulting in massive cell death. In this work we studied lung cell death by apoptosis after meconium aspiration in forty two-week-old rabbit pups. Analyzing lung samples by ISEL-DNA end labeling demonstrated the specific spread of apoptotic bodies throughout the lungs. These bodies were shrunken and smaller in size compared to normal cells and many of them were lacking cell membranes. About 70% of all apoptotic bodies were found among the airway epithelium cell eight hours after meconium instillation. In comparison, among lung alveolar cells, only about 20% cells were apoptotic in the same animals. In meconium-treated lungs and A549 cells, a significant increase of angiotensinogen mRNA and Caspase-3 expression were observed. The pretreatment of cells with Caspase-3 inhibitor ZVAD-fmk significantly inhibited meconium-induced lung cell death by apoptosis. These findings demonstrate the apoptotic process in meconium-instilled lungs or A549 cells in culture. Our results show lung airway epithelial and A549 cell apoptosis after meconium instillation. We suggest that studies of lung airway epithelial cell death are essential to understanding the pathophysiology of MAS and may present a key point in future therapeutic applications.

Meconium aspiration syndrome: a role for phospholipase A2 in the pathogenesis?

UNLABELLED: The pathophysiology of neonatal meconium aspiration syndrome (MAS), often resulting in severe respiratory failure, is complex and still largely unclear. Factors involved in the propagation of acute lung injury after perinatal aspiration of meconium include obstruction of the airways, ventilation/perfusion mismatch, increase of the pulmonary vascular resistance and a rapidly developing parenchymal and alveolar inflammatory reaction with associated surfactant dysfunction. CONCLUSION: Although the early pulmonary inflammatory response is believed to play a central pathogenetic role in the meconium-induced acute lung damage, its initiating mechanisms are still poorly defined. However, increasing evidence indicates a direct toxic effect of meconium.