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a Neonatology Research Laboratories, Department of Pediatrics, Michael Reese Hospital, Chicago, Illinois
b Division of Neonatology, Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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DESIGN. Four groups of 2-week-old rabbit pups were used for the study: group 1, saline instilled rabbits; group 2, captopril-pretreated rabbits; group 3, meconium-instilled rabbits; and group 4, captopril-pretreated and then meconium-instilled rabbits. Each group was studied at different time points: 0, 2, 4, 8, and 24 hours after instillation of meconium. Experiments were done at the University of Illinois and Michael Reese Hospital at Chicago. After treatment and instillation of meconium, the right lung was fixed with formalin, and 2-µm slices were obtained for immunohistochemistry. The left lung was used for obtaining of lung lavage and measurement of total proteins (for enzyme-linked immunosorbent assay) and mRNA (for reverse transcription-polymerase chain reaction) purification.
RESULTS. We found that meconium induces inflammatory cytokine expression and apoptotic lung cell death. In situ end labeling revealed a dramatic DNA fragmentation in the meconium group, which supports the presence of apoptosis. Using enzyme-linked immunosorbent assay, we demonstrated increase of interleukin 6 and interleukin 8 cytokines in meconium-instilled lungs, which were significantly decreased in captopril-pretreated lungs. Captopril pretreatment also decreased meconium-induced cell death and angiotensinogen expression. We believe this effect is explained by the ability of captopril to decrease processing of ANGEN to angiotensinogen (ANG) I and finally to ANG II. It suggests that captopril inhibits ANG II-induced lung cell apoptosis.
CONCLUSION. Our results demonstrate that captopril pretreatment significantly inhibits meconium-induced lung cell death, cytokine, and ANGEN expression in newborn lungs.
Key Words: meconium aspiration syndrome • captopril • apoptosis • newborn lungs • cytokines
Abbreviations: MAS—meconium aspiration syndrome • ACE—angiotensin-converting enzyme • RT—reverse transcription • PCR—polymerase chain reaction • ELISA—enzyme-linked immunosorbent assay • ISEL—in situ end labeling • EtBr—ethidium bromide • TNF—tumor necrosis factor • IL—interleukin
Although meconium aspiration syndrome (MAS) is a major cause of newborn mortality and morbidity, there is a lack of a clear understanding of the disease process. Previous experimental models have shown that MAS includes accumulation of inflammatory cells, release of inflammatory cytokines, and massive cell death in experimental models.1, 2 Previous studies from our laboratory demonstrated that intratracheal instillation of meconium induces apoptosis of lung lavage cells.1 Maximal apoptosis was observed 8 hours after meconium instillation. Apoptosis is known to be induced by angiotensin II. It is strongly believed that conversion of angiotensin I to angiotensin II by angiotensin-converting enzyme (ACE) within the cell is the basis of apoptosis in the cells.3, 4 Some investigators have used captopril or other ACE inhibitors to suppress the apoptotic process,4 but this has not been studied in MAS.
We hypothesize that meconium aspiration causes global angiotensin II-induced apoptotic cell death in the lung, a process that can be significantly inhibited by captopril pretreatment. We also studied the effect of captopril on the expression of inflammatory cytokines in the newborn lungs.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Four groups of 2-week-old New Zealand white rabbit pups (LSP Industries, Union Grove, WI) were studied at different times (0, 2, 4, 8, and 24 hours) after saline or meconium instillation. Twenty rabbits per group were used: group 1, saline-instilled rabbits; group 2, captopril-pretreated rabbits; group 3, meconium-instilled rabbits; and group 4, captopril-pretreated and then meconium-instilled rabbits. The captopril (Sigma Co, St Louis, MO) groups (groups 2 and 4) received captopril (concentration: 500 mg/L for 48 hours) mixed in drinking water for a 48-hour period before the study.4 Other groups (groups 1 and 3) received plain water. Meconium-instilled rabbit pups were anesthetized with IP 10 mg/kg of ketamine and 1 mg/kg of xylazine. A small midline incision was made on the ventral aspect of the neck to expose the trachea, an endotracheal tube was placed, and 1.2 mL/kg of 10% sterile meconium supernatant was instilled into lungs followed by 5 mL of air injection. The skin incision was closed with 4–0 nylon suture, and rabbits were then allowed to breathe room air spontaneously for 2, 4, 8, and 24 hours. They were then killed using nembutal (100 mg/kg, IP). Immediately after sacrifice, the chest was opened by a midline incision, lungs were isolated, and lung lavage was performed as described below.
Meconium Preparation
Meconium was prepared according to a procedure published previously.5 Fifteen first-pass human meconium samples were obtained from term, healthy neonates. One gram of fresh newborn infant meconium was homogenized on ice in a blender with 9 mL of 0.9% NaCl to a 10% (weight/volume) final concentration and was spun down at 5000 rpm for 20 minutes (4°C) to separate the supernatant and pellet. The supernatant was filtered via a glass filter followed by sterilization via a 0.2-µm filter (both filters were from Millipore Co, Bedford, MA) and was used for instillation in the lungs of 2-week-old newborn rabbit pups.
Reagents
Captopril, ethidium bromide (EtBr), acridine orange, Tris-HCL, sodium chloride, and most of chemicals were obtained from Sigma Chemical Co (St Louis, MO). Primers for reverse transcription- (RT) polymerase chain reaction (PCR) were synthesized by Genemed Synthesis (San Francisco, CA). ELISA kits were obtained from R&D Systems Co (Minneapolis, MI) and Peninsula Laboratories (San Francisco, CA). All of the other materials were from different sources or were of reagent grade.
In Vivo Animal Model Experiments
Bronchial lavage was performed according to a procedure published previously.6 After the sacrifice of each rabbit, the trachea was cannulated, and lungs were isolated. To recover lung cells, bronchoalveolar lavage (total: 10 mL) was performed after mainstream bronchial cannulation of the left lobe of the lung (the right lobe was used for histologic studies as described below). The lobe was lavaged through the main stem bronchus using 5-mL aliquots of 37°C preheated phosphate-buffered saline complemented with 20 mM N-2-hydroxyethyl-piperazine-N'-2-ethane-sulphonate, HEPES (pH 7.3) to wash out alveolar cells. From the 10 mL of instilled normal saline, we recovered 
9 mL of lung lavage fluid. We used the lung lavage cells for studies without separation for their different subtypes.
In Vitro Cellular Model Experiments
We also conducted in vitro studies to see the effect of meconium on a lung epithelial cell line (A549 cells). Cultured cells were exposed to meconium and saline. Half of each group was pretreated with captopril, and half were not. Cell death was identified using the DNA fragmentation assay. We counted 
10 fields per slide under magnification of x100. For the DNA fragmentation assay, we used a staining method for formalin-fixed, paraffin-embedded tissue sections that involved an in situ end-labeling (ISEL) procedure performed according to Wijsman et al.7 This assay is able to differentiate fragmented DNA and is important in studies of apoptosis, necrosis, or high DNA repair process in cells.
Cell Culture
The human lung epithelial cell line A549 was obtained from American Type Culture Collection and cultured in Ham's F12 medium supplemented with 10% fetal bovine serum. We used A549 human lung epithelial cells, because many important reagents and PCR primers are available for human cells. Cells collected were >90% of purity assessed by acridine orange staining as discussed previously.8 All of the cells were seed in 24-well or 6-well chambers, and all of the experiments were conducted at densities of 80–90% in serum-free Ham's F12 medium.
Detection of apoptotic cells with propidium iodide or EtBr was studied as described earlier3, 4 after digestion of ethanol-fixed cells with DNase-free Rnase in phosphate-buffered saline containing 5 µg/mL propidium iodide (or EtBr). In all of the assays, detached cells were retained by centrifugation of the culture vessels during fixation with 70% ethanol or by retention of culture medium and recovery of cells by centrifugation before assay. As we described earlier, induction of apoptosis was verified by activation of caspase 3/CPP32/YAMA.3 Caspase 3 activity was assessed in "viable" A549 cells in suspension cultures, which were assayed fluorimetrically over the first hour after the addition of the caspase 3 substrate N-Acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Upstate Biotechnology, Saranac Lake, NY) at 200 µM in final concentration.
Studies of Cytokine Proteins Using ELISA
The lung lavage cells (10 mL) were centrifuged at 5000 rpm and disrupted by freeze/thaw 4 times. The cell debris was discarded by centrifugation in the same conditions, and supernatants were used for the ELISA assays.9 Broken cells were centrifuged at –4°C, total protein concentration in the supernatants were determined using bovine serum albumin protein assay reagents (Pierce Co, Rockford, IL), and the same amounts of protein from each sample (2 mg/mL) were used in ELISA experiments. We measured the tumor necrosis factor (TNF)-
, interleukin (IL) 1ß, IL-6, IL-8, and IL-10 cytokines expression and prostaglandin E2 levels using commercially available ELISA kits as described by the manufacturer. Cytokine quantitation was expressed in picograms per milliliter of lavage fluid.
Studies of Cytokine mRNA Expression by RT-PCR
PCR and RT-PCR analyses of angiotensinogen expression in human cells were conducted in our laboratory earlier. These techniques provide adequate and satisfactory results. RNA was purified from A549 lung cells using commercially available RNeasy Mini Protocol (Qiagen Co, Santa Clarita, CA). Complementary DNAs were obtained using RT. Then, complementary DNA was used in the PCR reaction to amplify the cytokine primer.3 Steps of the PCR reactions were: denaturation at 95°C for 30 seconds, annealing of primers at 45°C (40–60°) for 30 seconds, and elongation of the chain at 72°C for 1 minute using Taq DNA polymerase (Promega Co, Madison, WI). After that step, reaction samples were run on 1% agarose gel in Tris-borate buffer (pH 8.0). ß-Actin housekeeping gene was used in our study for a comparison purposes. Statistical significance was determined using analysis of variance (P < .05).
Data Analysis
Each experiment was repeated 4 times. Comparison of mRNA expression was done using a densitometry analysis. All of the measurements were compared using the analysis of variance. Results are mean ± SD of 4 experiments. In morphologic experiments, 500 cells per sample were counted. Unpaired evaluations were made for experimental and control conditions within each experiment, and significance was determined by Student t test.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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45%) level of cell death in the lungs. Pretreatment of lungs with captopril significantly decreased lung cell death to from 45% to 21%. Control rabbits, pretreated with captopril, without meconium treatment, show only 14% of lung cell death. Captopril was able to inhibit meconium-induced lung apoptosis by 
70%. A similar effect was demonstrated in our previous work where we used caspase 3 inhibitor N-benzyfoxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD-fmk).10 The massive cell loss because of apoptosis in meconium-instilled lungs was almost completely abrogated by pretreatment with caspase 3 inhibitor ZVAD-fmk, which suggests that observed meconium-induced cell loss is attributable primarily to apoptosis. Figure 2 shows the ISEL nuclear DNA fragmentation. Saline-instilled lungs do not show a positive ISEL (Fig 2A), as well as captopril-pretreated lungs (Fig 2B). Captopril-pretreated and then meconium-instilled lungs also show no statistically significant changes (Fig 2C). Figure 2D shown that meconium-instilled lungs without captopril pretreatment are strongly ISEL labeled. As mentioned before,11 positive ISEL demonstrates dramatic DNA fragmentation, DNA repair, apoptosis, and even necrosis. In the presence of the ACE inhibitor captopril, meconium-induced strong positive ISEL is seen as dark blue staining under a fluorescent microscope (Fig 2C).
So far, we used 2 independent techniques to evaluate apoptosis, cell death count and ISEL. The third technique used in our study to determine apoptosis is the evaluation of angiotensinogen expression. It was shown previously that expression of angiotensinogen is a powerful method indicating apoptosis.4 Identification of angiotensinogen expression was done using human lung epithelial cells A549. Obtained results demonstrate that saline-treated lung A549 cells do not express angiotensinogen (Fig 3A). Similar results were observed also in captopril-pretreated cells (Fig 3B). In contrast, meconium-treated cells dramatically express angiotensinogen (Fig 3C), which can also point to the apoptotic process in addition to the data mentioned above. The important finding here is the demonstration that expression of angiotensinogen can be completely inhibited by captopril pretreatment (Fig 3B). It shows the antiapoptotic effect of captopril.4 The ß-actin gene was expressed equally in captopril-pretreated and -untreated cells (Fig 3, bottom panel).
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, IL-6, IL-8, and IL-10 by their mRNA expression (Fig 4). We found that IL-8 and IL-10 cytokine mRNAs were weakly expressed in saline-treated A549 cells, but TNF- and IL-6 cytokines were not expressed (Fig 4). At 8 hours after meconium treatment expression of mRNA for TNF-, IL-6 and IL-8 were statistically significant (analysis of variance: P < .05). Pretreatment of rabbits with captopril in vivo significantly inhibited all of the above-mentioned cytokine mRNA expression except for antiinflammatory cytokine IL-10, which was not changed in all of the study groups (Fig 4).
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and IL-6 cytokines. In the middle column of the table, the expression of above-mentioned cytokines in meconium-instilled lungs in vivo are shown. As seen from the table, TNF-, IL-6, and IL-8 cytokines are dramatically expressed in meconium-instilled lungs. Expression of the above-mentioned cytokines were significantly different between the groups (P < .05). It shows that expression of inflammatory cytokines is significantly inhibited by pretreatment of rabbits by captopril (Table 1, right column).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Captopril, an angiotensin-converting enzyme inhibitor,4 has been shown to ameliorate radiation-induced fibrosis of the lung.15 We demonstrated here the effect of captopril pretreatment on cell death, DNA fragmentation, angiotensinogen, and cytokine expression. Our results show that pretreatment with captopril significantly inhibits meconium-induced lung cell death, angiotensinogen, and cytokines expression. These are the pivotal findings of our present studies.
Meconium-induced apoptosis is activated by a strong vasoconstrictor, angiotensin II, which is a final step of angiotensinogen degradation in the cell. The mentioned events usually lead to cell apoptosis via activation of surface AT1 receptors and stimulation of Ca2+-dependent DNAase I.16 This effect is modulated by 2 classes of angiotensin II receptors AT1 and AT2. Angiotensin II also increases production of TGF-1-ß1, platelet-activating factor, endothelin, TNF-, and platelet-derived growth factor.17 Apoptosis can be induced by a ligand binding to AT2 receptors through the involvement of mitogen-activated protein kinase.18 Previously in our laboratory we demonstrated a dose-dependent increase in apoptosis by purified angiotensin II in human lung epithelial cell line A549.3 The use of ACE inhibitors or selective antagonists of AT1 or AT2 receptors, which decrease the levels of angiotensin II or limit its action, may significantly inhibit apoptotic cell death. As discussed earlier,19 the scoring of cells on the basis of nuclear fragmentation provides an assay less ambiguous than the popular terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay, which does not discriminate differences among apoptotic, necrotic, and autolytic cells.11
The present study demonstrated that lung airway epithelial cells are heavily ISEL labeled for DNA fragmentation after meconium exposure. In contrast, saline exposure does not show a positive ISEL laddering for DNA fragmentation. Pretreatment with captopril significantly inhibited apoptosis and DNA fragmentation and also increases the survivability of rabbit pups from meconium. In vivo studies demonstrate that inhibition of apoptosis by captopril occurs in a concentration-dependent manner in different cells.20 The mechanism of its inhibitory effect is based on the ability of captopril to block the conversion of angiotensin I to the apoptotic-inducer angiotensin II. If AT2 is strongly expressed, it binds to angiotensin II, limiting its binding to its apoptotic AT1 receptors. In another words, there is a very limited binding of angiotensin II to its apoptotic AT1 receptors. Therefore, the total levels of apoptosis are very small. This suggests a new hypothesis: namely, AT1 receptors are important in meconium-induced lung apoptosis. We are going to address this issue in our next studies. The present results suggest that the ACE inhibitor captopril, and possibly other thiol components, may effectively inhibit meconium-induced cell death and lung damage and can be effectively used for treatment of MAS patients.
The present work also demonstrated that meconium induces expression of inflammatory cytokines TNF-, IL-6, and IL-8, but not IL-10, and expression of angiotensinogen gene and demonstrated a massive lung cell death by apoptosis. Pretreatment with captopril inhibits these findings. These observations suggest that captopril may play an important role in reducing meconium-induced inflammatory lung injury and further support its potential use as a new therapeutic intervention for MAS.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address correspondence to D. Vidyasagar, MD, Division of Neonatology, Department of Pediatrics, University of Illinois at Chicago, 804 S Wood St, Chicago, IL 60612. E-mail: dsagar{at}uic.edu
The authors have indicated they have no financial relationships relevant to this article to disclose.
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REFERENCES |
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and IFN-
: the indusibility of specific cell ICAM-1 in vivo.
Am J Respir Cell Mol Biol. 1996;15
:540
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B. Salvesen, M. Fung, O. D. Saugstad, and T. E. Mollnes Role of Complement and CD14 in Meconium-Induced Cytokine Formation Pediatrics, March 1, 2008; 121(3): e496 - e505. [Abstract] [Full Text] [PDF]
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