Angiotensin-Converting Enzyme 2 Improves Arterial Hypoxemia in Meconium-Induced Acute Lung Injury in Piglets

Objective. Meconium aspiration induces acute lung injury (ALI) in neonates born through meconium-stained amniotic fluid. As yet, there is no specific therapy for improving the outcome. Recently, angiotensin-converting enzyme 2 (ACE2), which inactivates angiotensin II (Ang II), has been shown to ameliorate murine ALI. Design. To evaluate the therapeutic potential of this substance, we studied ACE2 in a piglet model of ALI induced by meconium aspiration. Subjects. Twelve anesthetized piglets were subjected in an animal research laboratory. ALI was induced by tracheal meconium instillation.Thereafter, six animals were randomly assigned to the ACE2 group, while another 6 served as control.Measurements. Systemic, pulmonary hemodynamic, and blood gas exchange parameters and Ang II levels were examined before ALI induction and at various time points after administering ACE2 or saline. In addition, ventilation-perfusion distribution of the lung was assessed by the multiple inert gas elimination technique (MIGET). Main Results. Animals treated with ACE2 maintained significantly higher arterial partial pressures of oxygen (Pao 2 ) and lower arterial partial pressures of carbon dioxide (Paco 2 ), respectively. Furthermore, Ang II, which was substantially increased, returned to basal values. Conclusion. In summary, ACE2 improves blood gas exchange in meconium-induced ALI in piglets.


Introduction
In neonates, aspiration of meconium causes acute lung injury (ALI) and may lead to severe acute respiratory distress syndrome (ARDS), often associated with persistent fetal circulation [1,2].Death occurs in about 10% of the cases [3].Similar to the situation in adults, neonates with ARDS require supportive intensive care including aggressive mechanical ventilation, circulatory support by catecholamines, and, in most severe cases, extracorporeal membrane oxygenation.
Recently, angiotensin-converting enzyme 2 (ACE2) has been described as a new principal enzyme of the renin-angiotensin-aldosterone system [4,5].Angiotensin II (Ang II), a product of ACE, is cleaved by ACE2 generating vasodilator mediators [4][5][6].Experimental data indicate an important role of ACE2 in the development of ARDS.For instance, ACE2 protects wild type mice from severe lung injury induced by acid aspiration, colon ligation, or endotoxin shock, and ACE2-knockout mice are more susceptible to these forms of lung disease as compared to wild type animals [7].Furthermore, we recently showed that ACE2 ameliorates failure of oxygenation in a piglet model of lung injury induced by lipopolysaccharide (LPS) [8].
In our study, we investigated the potential influence of ACE2 on respiratory and hemodynamic parameters in a piglet model following meconium aspiration.This model is particularly suitable, because it allows the assessment of exactly the same parameters that are required for the monitoring of the disease in human intensive care medicine.Furthermore, this model allows the application of the multiple inert gas elimination technique (MIGET) [9][10][11] to assess ventilation/perfusion distribution in the lung tissue.

Subjects.
All experiments were approved by the Austrian Federal Animal Investigational Committee.Twelve healthy, eight-week-old crossbreed piglets (German landrace x Pietrain) of either sex were selected from a local stock regularly used for experimental research.Prior to induction of anesthesia the animals received ketamine (50 mg/kg i.m.) and atropine (0.01 mg/kg i.v.).Following endotracheal intubation, anaesthesia was maintained with i.v.propofol (10-15 mg/kg/h; Perfusor Secura FT, B. Braun Melsungen AG, Melsungen, Germany) and boluses of 15 mg piritramide.All pigs were mechanically ventilated in volume-controlled mode (Evita 2, Drägerwerk AG & Co. KGaA, Lübeck, Germany) at an inspiratory fraction of oxygen (FiO 2 ) of 0.21 and a tidal volume (VT) of 10 mL/kg at 15 breaths/min, positive end expiratory pressure set at 5 mmHg.Throughout the procedure, Ringer's solution (6 mL/kg/h) and a 3% gelatin solution (4 mL/kg/h) were infused.A standard lead II ECG (Datex Ohmeda AS/3, Datex-Ohmeda Inc., Wisconsin, USA) was used to monitor cardiac rhythm.Body temperature was maintained between 38 ∘ C and 39 ∘ C by using an electric heating blanket (Warm Air Model 134, Cincinnati Sub-Zero Inc., Ohio, USA).A 8.5 F pulmonary artery catheter (131HF7, Edwards Lifesciences, California, USA) was advanced from the internal jugular vein into a branch of the pulmonary artery to measure mean pulmonary arterial pressure (MPAP), pulmonary capillary wedge pressure (PCWP), central venous pressure (CVP), and cardiac output using the thermodilution technique (10 mL saline in triplicate) and to obtain mixed venous blood.A 6 F arterial catheter (SI-09600, Arrow International Inc., Pennsylvania, USA) introduced into the femoral artery was used to monitor systemic blood pressure and to take blood samples for blood gas and MIGET analyses.All catheters were filled with saline and connected to pressure transducers zeroed to ambient pressure at the level of the right atrium.Pulmonary vascular resistance (PVR) was calculated using standard formulas.

Experimental ARDS Protocol.
After surgical preparations, all animals were moved into prone position and left for stabilization for 30 min.Thereafter, baseline measurements (i.e., "0 min") including hemodynamics, blood gases, ventilation, and MIGET were taken.As a next step, acute lung injury was induced using deep intratracheal instillation of a 20% solution of pooled human meconium.First, all piglets received 0.5 mL/kg of meconium solution over a period of 3 min, and FiO 2 was set to 1.0 to counteract effects of acute hypoxia like pulmonary hypertension and acute right heart failure.After 3 min, boluses of 0.25 mL/kg meconium were titrated up to a maximum of 2.0 mL/kg.Meconium instillation was stopped after MPAP increased to values 50 percent above baseline and FiO 2 was set to 0.21.Both groups received approximately the same amount of meconium (data not shown).Thereafter, respirator settings were not changed for the remaining course of the experiment.
Every 30 min, measurements were obtained including hemodynamics, ventilation, and blood gas parameters.MIGET was performed at 0, 30, 60, 120, 180, and 240 min.Ang II and ACE2 levels in plasma and serum, respectively, were measured at 0, 30, 60, 120, 180, and 240 min.Plasma and serum samples were supplemented with a protease inhibitor cocktail (phenanthroline, pepstatin A, p-hydroxymercuribenzoic acid, and EDTA) and were stored frozen at −20 ∘ C until analysis.After 270 min, all piglets were euthanized by i.v.injection of 20 mL 1 M potassium chloride.The heart was removed and inspected to rule out any anatomical abnormalities.MIGET was performed as previously described [9,10].Six inert gases, prepared in sterile 0.9% sodium chloride solution, were continuously i.v.infused at a rate of 3 mL/min.Samples of expired gas and arterial and mixed venous blood were obtained in gas-tight glass syringes.The concentrations of the six inert gases in expired air and arterial and mixed venous blood were measured using a gas chromatograph (Hewlett-Packard 5890, series II).  / distribution reflects regional lung ventilation/perfusion, including lung units ranging from either shunt (i.e., unventilated lung units) or dead space ventilation (i.e., lung units without blood flow).  / was calculated using least squares best fit regression analysis.In the MIGET, adequacy of fit of the data to the model is assessed by the remaining sum of squares (RSS).RSS was ≥10.6 in 92.6% of all MIGET analysis, thereby indicating good data quality.

Histopathology.
For histological analyses, lung tissue samples were fixed in 10% formalin.After embedding in paraffin, sections were obtained and stained with haematoxylin and eosin (H&E) according to standard methods.Lung tissue sections were scored by two pathologists blinded to treatment.

Preparation of Human Meconium.
Pooled human meconium was suspended in physiological saline at a concentration of 20% and stored at −20 ∘ C until use.

ACE2 Preparation and Measurement of ACE2 and
Ang II in Plasma.The preparation of recombinant ACE2 and the measurements of ACE2 activity and Ang II levels in plasma samples were performed as previously described [8].

Statistical Analysis.
A two-way ANOVA was used to determine inter-and intragroup differences.Significant results were post hoc analyzed using the Newman-Keuls and Fisher's exact tests and the unpaired -test.Results are given as mean ± SD (standard deviation).Values of  ≤ 0.05 were considered significant.

ACE2 Treatment Reduces Systemic Ang II Levels in Acute
Lung Injury.Prior to meconium instillation, no (intrinsic) ACE2 activity could be found in the sera of piglets (Figure 1).
Basal Ang II levels were similar in both groups.Meconium instillation induced a nearly 35 percent increase of Ang II after 30 min in both groups (Figure 2).In controls, Ang II further increased to a 3-fold level at the end of the observation period.ACE2, however, decreased Ang II below baseline values immediately after application, remaining at this level until the end of the experiment.

Changes in Blood Gases and Hemodynamics.
Immediately after meconium instillation, Pao 2 substantially decreased (Figure 3(a)).In parallel, the alveoloarterial partial pressure difference of oxygen (AaDO 2 ) increased (Table 1).Furthermore, Paco 2 increased (Figure 3(b)), as did the mixed venous partial pressure of carbon dioxide (Pvco 2 ) (Table 1).Mixed venous partial pressure of oxygen (Pvo 2 ) decreased.Also, mean arterial pressure (MAP) decreased shortly after meconium instillation, and MPAP increased by approximately one-third.PVR nearly doubled 30 min after meconium and further increased until the end of the experiment (Table 1).
In controls, Pao 2 remained decreased but increased in the ACE2 group immediately after treatment and remained at this level (Figure 3(a)).Paco 2 constantly increased in controls but decreased in the ACE2 group (Figure 3(b)).Pvco 2 behaved in a similar manner (Table 1).
MAP in controls remained nearly unchanged, but ACE2 animals showed higher MAP levels after 180 and 240 min (Table 1).
Heart rate increased in both groups during the course of the experiment.All other hemodynamic and blood gas parameters, that is, PCWP, CVD, and the arterial pH (pHa), remained unchanged at all time points tested, regardless of whether the animals received ACE2 or saline (Table 1).

Changes in Ventilation-Perfusion Distribution.
Meconium led to an impairment of pulmonary blood flow as expressed by an increased LogSDQ (Table 2).This expression, the logarithmic standard deviation of perfusion distribution, describes the homogeneity of pulmonary blood flow.Furthermore, blood flow to unventilated lung units (i.e., shunt) increased, while blood flow to normally ventilated units (normal   / of ) decreased (Table 2).ACE2 administration did not change any of these MIGET parameters in a significant manner.Differences in shunt fraction and pH, immediately before and subsequent to ACE2-infusion, might have resulted from uneven response to meconium between individual animals.Therefore, the lack of significance might in part be due to the small sample size.

Changes in Lung
Histology.The histologic picture of meconium-induced ALI was characterized by extensive collapse of the alveolar system due to bronchiolar obstruction by meconium (Figure 4(a)).ACE2 did not alter the histopathologic pattern (not shown).

Discussion
The present study demonstrates that ACE2 ameliorates meconium-induced ALI in piglets by attenuating pulmonary gas exchange, that is, by increasing Pao 2 and decreasing Paco 2 .
Meconium induces expression of inflammatory cytokines and Ang II-mediated apoptotic processes in human and animal alveolar epithelial cells [12].In our model, meconium led to a substantial increase of Ang II, most probably by hyperactivity of ACE [13,14].Ang II binds to the Ang II subtype 1a (AT1a) receptor of the lung microvascular endothelium, resulting in edema and inflammation [7].Rosenfeld et al. showed that meconium leads to a 3-fold increase of total lung AT1 receptor protein and induces AT1 receptor-mediated apoptotic cell death in the newborn rabbit lung [15].Thus, Ang II seems to be an important mediator of acute lung injury induced by meconium aspiration.In agreement with our previous work in LPS-induced ARDS, ACE2 inactivates Ang II by cleavage to angiotensin 1-7 (AT 1-7) in vivo [8].Furthermore, AT 1-7 acts as an endogenous antagonist of Ang II and has recently been shown to prevent murine ventilator-or acid aspiration-induced ALI [16].AT 1-7 binds to the MAS receptor, and, in turn, administration of a MAS-inhibitor aggravates lung fibrosis [17].Altogether, these observations might provide the pharmacological basis for the mechanism by which ACE2 attenuates meconium-induced ALI.
Meconium instillation increased MPAP to 135% of baseline, and this value remained unchanged after ACE2 treatment.This is in contrast to the previously described LPS-ARDS model, where MPAP increased 2-fold and slightly dropped after ACE2.A lesser increase of Ang II levels after meconium instillation compared to LPS (35% versus 8-fold in the LPS model) could explain this difference.However, administration of ACE2 was followed by a return to basal Ang II values in both models.
One probable but unwanted side effect of ACE2 administration could be a drop in blood pressure.However, after 180 min, MAP even increased in the ACE2 group despite decreased Ang II levels.This might be important for clinical testing, as meconium aspiration often is associated with severe hypotension requiring catecholamines.
Apart from our experimental setting, well-established substances like the ACE inhibitor captopril and the angiotensin II receptor antagonist telmisartan also have been tested in experimental ALI.For example, captopril has been shown to attenuate ALI in rats [18] and Liu and Guo observed decreased TNF- levels and an increased PaO2 after captopril in rats subjected to hydrochloric acid-induced ALI [19].In meconium-or LPS-treated piglets, lisinopril or telmisartan led to severe hemodynamic instability requiring high doses of catecholamines.This rescue treatment, however, significantly impaired the accuracy of MIGET (unpublished observations).

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