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Adrenomedullin is up-regulated in nitrofen-induced fetal pulmonary hypoplasia
Journal of Pediatric Surgery (2005) 40, 1562 – 1567
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Adrenomedullin is up-regulated in nitrofen-induced fetal pulmonary hypoplasia Masafumi Kamiyama*, Noriaki Usui, Shinkichi Kamata, Masahiro Fukuzawa, Noritoshi Nagaya, Kenji Kangawa Department of Pediatric Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan Department of Biochemistry, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan Index words: Congenital diaphragmatic hernia; Adrenomedullin; Pulmonary hypoplasia; Hypoplastic lung; Nitrofen
Abstract Background/Purpose: Pulmonary hypoplasia associated with congenital diaphragmatic hernia remains a major therapeutic problem. Adrenomedullin (AM), a multifunctional regulatory peptide, has been suggested to play a role in the mechanisms of fetal lung differentiation and maturation. The aim of this study was to investigate the pulmonary expression of AM in nitrofen-induced fetal pulmonary hypoplasia. Materials and Methods: Pulmonary hypoplasia was induced in timed-pregnant Sprague-Dawley rats after administration of 100 mg nitrofen on day 9.5 of gestation. Fetal and neonatal lungs were excised on gestational days 16.5, 19, and 21 and 1 hour after birth and divided into the following 3 groups: nitrofen with diaphragmatic defect, nitrofen without diaphragmatic defect, and control (without nitrofen). Pulmonary levels of AM and AM messenger RNA expression were measured by radioimmunoassay and real time quantitative reverse transcriptase polymerase chain reaction, respectively. Localization of pulmonary AM was identified by immunohistochemical staining. Results: There was a significant increase in pulmonary level of AM in the nitrofen-treated groups on gestational days 19 and 21. Real time quantitative reverse transcriptase polymerase chain reaction on gestational day 19 confirmed an increase of AM gene expression in the nitrofen-treated groups. Adrenomedullin immunoreactivity was more strongly expressed in airway epithelial cells in the nitrofentreated groups than in the control. Conclusion: Nitrofen up-regulates expression of AM in the fetal lung, which suggests that AM has some pathophysiological role in the differentiation and/or maturation processes of pulmonary hypoplasia in congenital diaphragmatic hernia. D 2005 Published by Elsevier Inc.
The prognosis of infants with severe congenital diaphragmatic hernia (CDH) still remains unfavorable despite recent therapeutic progress [1]. Because persistent pulmonary hypertension resulting from pulmonary hypoplasia seems to be a major cause of the high mortality [2,3], numerous
T Corresponding author. Tel.: +81 6 6879 3753; fax: +81 6 6879 3759. E-mail address: [email protected] (M. Kamiyama). 0022-3468/$ – see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.jpedsurg.2005.06.005
efforts have been made to elucidate the pathophysiology of pulmonary hypoplasia in CDH. A nitrofen-induced murine pulmonary model of CDH has been extensively used to investigate the pathogenesis of CDH because of its phenotypic similarities to human CDH [4,5]. Recently, a number of regulatory factors such as fibroblast growth factor 10 and transforming growth factor b (TGF-b) and the molecular mechanisms underlying pulmonary hypoplasia in a nitrofeninduced model of CDH have been investigated [6,7].
Upregulation of adrenomedullin in fetal hypoplastic lung Table 1 Control DD ( ) DD (+) Left Right Bilateral
Numbers of experimental animals Gd 16.5
Gd 19
Gd 21
After birth
24 15 23 11 7 5
15 6 30 15 12 3
18 16 24 11 6 7
27 11 13 10 3 0
Gd indicates gestational day.
1563 Table 2 day 19
Lung Placenta Liver Heart Kidney
Organ-to-body weight ratios (%) on gestational Nitrofen with DD (n = 30)
Nitrofen without DD (n = 6)
Control (n = 15)
3.03 17.4 7.12 0.67 0.56
3.20 16.5 6.90 0.76 0.58
4.16 17.3 9.70 0.74 0.61
F F F F F
0.25T 2.6 0.74T 0.08 0.08
F F F F F
0.09T 1.3 0.65T 0.10 0.10
F F F F F
0.45 1.7 1.28 0.07 0.09
* P b .05, significant difference from control.
Adrenomedullin (AM) is a peptide originally isolated from extracts of human pheochromocytoma as a potent vasorelaxant [8], which consists of 52 amino acids in humans and 50 amino acids in the rat and structurally belongs to the calcitonin gene–related peptide superfamily. Although AM has been reported as a multifunctional regulatory peptide, many recent studies revealed that AM is especially involved in various types of cell proliferation [9,10]. The expression of AM during embryogenesis has been analyzed using mouse and rat embryos [11]. Adrenomedullin immunoreactivity was markedly increased during the late organogenetic and early fetal growth periods. These observations, taken together with data linking AM to growth control in various types of cells, suggested the possible involvement of AM in the control of embryonic cell proliferation and differentiation. Particularly in murine fetal lungs, AM expression was found in the bronchial epithelium, airway smooth muscle cells, mesenchymal cells, and vascular endothelial cells [11]. The expression of pulmonary AM in the late organogenic period and its increased staining during fetal lung development suggest its possible role in fetal lung differentiation and maturation [12]. It has been reported that plasma levels of AM are elevated in children with primary and secondary pulmonary hypertension [13]. We have also shown that plasma levels of AM in the umbilical artery and vein were elevated in patients with persistent pulmonary hypertension of the newborn [14]. Hypothesizing a pathophysiological role of AM in the regulation of lung differentiation and/or maturation in the hypoplastic lung, we investigated the pulmonary expression of AM in a nitrofen-induced rat model of pulmonary hypoplasia.
examined for diaphragmatic defects (DDs), both lungs were excised. The fetuses and neonates from nitrofen-treated dams (nitrofen-treated group) were divided into 2 groups according to the presence or absence of DD. In addition to the lungs, the placenta, liver, heart, and kidneys were excised at 19 days of gestation. Tissues were weighed, frozen, and kept at 808C until measurement of AM. Another series of fetal rat lungs at 19 days of gestation were obtained for real time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical staining. Fetal lungs were kept frozen at 808C for real time quantitative RT-PCR and fixed in 4% paraformaldehyde for immunohistochemical staining. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Osaka University Graduate School of Medicine.
1.2. Radioimmunoassay Each tissue was boiled in water to inactivate intrinsic proteases. After cooling, acetic acid was added and the mixture was homogenized. The supernatant of the extract, obtained after centrifugation, was lyophilized. For assay, the lyophilized material was dissolved in radioimmunoassay buffer, and the clear solution was subjected to radioimmunoassay [17]. The levels of AM were determined by
1. Materials and methods 1.1. Experimental animals Female Sprague-Dawley rats (Oriental Yeast Co, Osaka, Japan) were used for the experiment. After mating, the animals were given 100 mg nitrofen (Supelco, Bellefonte, Pa), dissolved in 1 mL olive oil, via a gastric tube on day 9.5 of gestation [15,16]. Control animals were given 1 mL olive oil only. Fetuses on days 16.5, 19, and 21 of gestation were killed before any breathing occurred, and neonates were killed 1 hour after birth. After the animals were weighed and
Fig. 1 Lung-to-body weight ratio on gestational days 16.5, 19, and 21 and 1 hour after birth. Numbers of experimental animals are shown in Table 1. Gd indicates gestational day. Asterisk indicates P b .05, significant difference from control at same age. Dagger indicates P b .05, significant difference from nitrofen without DD at same age.
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Table 3 Organ levels of AM (femtomol per milligram wet tissue) on gestational day 19
Lung Placenta Liver Heart Kidney
Nitrofen with DD (n = 30)
Nitrofen without DD (n = 6)
Control (n = 15)
4.51 2.27 0.063 0.93 0.48
4.37 2.31 0.056 1.36 0.54
1.36 2.56 0.072 1.00 0.36
F F F F F
0.78T 0.61 0.022 0.45 0.23
F F F F F
1.20T 0.60 0.021 0.60 0.19
F F F F F
0.31 0.36 0.033 0.40 0.11
* P b .05, significant difference from control.
immunoradiometric assays using specific kits (Shionogi Pharmaceutical Co, Ltd, Osaka, Japan) [18,19]. The radioactivity was measured by a gamma counter (ARC1000M, Aloka, Tokyo, Japan). All assay procedures were performed in duplicate.
1.3. Real time quantitative RT-PCR Total RNA was isolated using an RNA isolation system (Ambion Inc, Austin, Tex). Total RNA was then reverse transcribed using a first standard complementary DNA kit (Clontech Laboratories Inc, Palo Alto, Calif). We purchased Pre-Developed TaqMan Assay Reagents for AM and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Applied Biosystems, Foster City, Calif). Real time PCR amplification was performed using a sequence detector (ABI PRISM 7700, Applied Biosystems). The relative standard curve method was used to quantitative AM messenger RNA (mRNA) expression [20]. Data were shown as the ratio to GAPDH mRNA.
1.4. Immunohistochemistry Polyclonal antibody raised from rabbits against purified rat AM was purchased as primary antibody from Phoenix
Fig. 2 Levels of pulmonary AM on gestational days 16.5, 19, and 21 and 1 hour after birth. Numbers of experimental animals are shown in Table 1. Asterisk indicates P b .05, significant difference from control at same age.
Pharmaceuticals (Belmont, Calif). The fixed tissues were embedded in paraffin, then trimmed and mounted. After deparaffinization, tissues were autoclaved. Then tissues were incubated in diluted primary antibody for 1 hour at room temperature and then incubated in antirabbit immunoglobulin conjugated to peroxidase-labeled dextran polymer system as secondary antibody (Dako, Kyoto, Japan) for 1 hour at room temperature. Antibody labeling was revealed by a 3,3-diaminobenzidine tetrahydrochloride product. Lung immunohistochemical staining in each group was scored in a blinded manner, assigning a value of 3 as very strong staining; 2, strong staining; 1, weak staining; and 0, no staining [21,22]. Four areas (200) in each slide were selected blindly, and the intensity of staining was scored with respect to each histologic part.
1.5. Statistics All data were expressed as mean F SD. Statistical analysis was performed by 1-way analysis of variance, and significant differences were identified by Scheffe´ post hoc analysis. Scores for immunohistochemical staining were analyzed by nonparametric test using Mann-Whitney U test. All statistical analyses were performed using a commercially available statistics package (Stat View, SAS Institute Inc, Cary, NC). P b .05 was considered statistically significant.
2. Results 2.1. Incidence and side of DDs induced by nitrofen The number of fetuses or neonates subjected to measurement of organ-to-body weight ratios and AM on gestational days 16.5, 19, and 21 and after birth were 38, 36, 40, and 24, respectively (Table 1). The incidence of
Fig. 3 Pulmonary AM gene expression by real time quantitative RT-PCR on gestational day 19. Data are shown as the ratio to GAPDH mRNA. Asterisk indicates P b .05, significant difference from control.
Upregulation of adrenomedullin in fetal hypoplastic lung defects was around 60% to 80%, with a predominance of left-sided defects.
1565 Table 4 Scores of immunohistochemical intensity for AM in fetal lung on gestational day 19
2.2. Organ-to-body weight ratios In the nitrofen-treated groups, the organ-to-body weight ratio was decreased in the lung and liver on gestational day 19 as compared with the control (Table 2). There was no significant difference between nitrofen-treated groups with and without DD. A decrease of lung-to-body weight ratio in
Nitrofen with Nitrofen without Control DD (n = 16) DD (n = 8) (n = 8) Airway 1.75 F 0.68T 2.13 F 0.84T epithelial cells Vascular 1.50 F 0.52 1.75 F 0.89 endothelial cells Mesenchymal 1.06 F 0.25 1.13 F 0.35 cells
1.13 F 0.35 1.13 F 0.35
0.88 F 0.35
The intensity of immunohistochemical staining was scored, assigning a value 0 to 3 (3: very strong staining; 2: strong staining; 1: weak staining; 0: no staining). T P b .05, significant difference from control.
nitrofen-treated groups was also recognized on gestational days 16.5 and 21 and 1 hour after birth (Fig. 1). Lung-tobody weight ratio of nitrofen-treated rats with DD was lower than that of nitrofen-treated rats without DD on gestational day 21 and after birth.
2.3. Levels of AM in organs In the control, the placenta showed the highest concentration of AM on gestational day 19 (Table 3). A significant increase of pulmonary AM was observed in nitrofen-treated groups as compared with control, but no significant difference was observed between nitrofen-treated groups with and without DD. However, pulmonary level of AM in the control was increased as compared with that in the nitrofen-treated groups 1 hour after birth (Fig. 2).
2.4. Real time quantitative RT-PCR on gestational day 19 Pulmonary AM gene expression was up-regulated in the nitrofen-treated groups. No significant difference was observed in pulmonary AM mRNA level between nitrofen-treated groups with and without DD (Fig. 3).
2.5. Immunohistochemistry on gestational day 19 In the control, distinct immunoreactivity of AM was found in airway epithelial cells, vascular endothelial cells, and mesenchymal cells dominantly (Fig. 4A). The expression of AM immunoreactivity was more dominant in the nitrofen-treated groups with DD (Fig. 4B) and without DD (Fig. 4C) than in the control. However, a significant difference in scores of the intensity of AM immunohistochemical staining of airway epithelial cells was observed between the nitrofen-treated groups and the control (Table 4). Fig. 4 Immunohistochemical staining of AM in fetal lungs on gestational day 19. Adrenomedullin immunoreactivity is seen in airway epithelial cells (black arrows), vascular endothelial cells (white arrows), and mesenchymal cells (asterisks). Immunohistochemical staining is stronger in the nitrofen groups with (B) and without (C) DD than in control (A).
3. Discussion To investigate the pathophysiology of pulmonary hypoplasia in CDH, we used a nitrofen-induced rat model of
1566 CDH. In this model, the DD predominantly occurs on the left, and the incidence of CDH is compatible with that in previous reports [23,24]. It has been reported that embryos with homozygous deletion of the AM gene do not survive because of vascular fragility [25]. It is also reported that antagonism of AM function during rat pregnancy causes fetal growth restriction [26]. Montuenga et al [11] reported that the level of AM immunoreactivity was increased during the late organogenetic and early fetal growth periods during embryogenesis. These observations suggest the possible involvement of AM in promoting embryonic cell proliferation and differentiation. The present study showed up-regulation of pulmonary AM during late gestation in the nitrofen-treated groups. Gene expression data obtained by real time quantitative RT-PCR indicated increased production of AM in the lung. Immunohistochemical study showed that AM was preferentially expressed in airway epithelial cells in nitrofeninduced hypoplastic lungs. These results suggest that AM plays a role in fetal pulmonary growth, especially in promoting branching morphogenesis and airway cell proliferation or maturation. In the present study, the placenta was the organ with the highest AM level. It is reported that antagonism of AM function during rat pregnancy caused decreased placental size and gross necrosis of the placental margins and amniotic membrane [26], suggesting an important role of AM in vascular development of the placenta. It is also reported that the level of AM in the umbilical vein was higher than that in the umbilical artery [14], and the main clearance site of AM is the lung [27]. These observations suggest that AM produced in the placenta may also play a role in the regulation of fetal development, including lung differentiation and/or maturation. There was no difference in the pulmonary levels of protein and gene expression of AM between the nitrofentreated groups with and without DDs. This seems compatible with recent studies that showed that pulmonary hypoplasia in a nitrofen-induced model occurs early in the developmental period simultaneously with primary abnormalities independent of DDs [4,16,28,29]. In the present study, a decrease of lung-to-body weight ratio was recognized on gestational day 16.5, supporting early induction of pulmonary hypoplasia in nitrofen-induced models. Furthermore, there was a significant decrease of lung-to-body weight ratio in the nitrofen-treated group with DD compared with the nitrofen-treated group without DD on gestational day 21 and after birth, indicating that a defect of the diaphragm also affects the development of the lungs, which was described by Keijzer et al [29] as the bdual-hit hypothesis.Q The level of pulmonary AM in normal fetuses increased sharply after birth and was higher than that in the nitrofentreated groups. Adrenomedullin is a vasodilating peptide, and it has been reported that AM physiologically acts as a vasodilator immediately after birth to reduce pulmonary
M. Kamiyama et al. artery pressure in neonates [13,14] The increase of pulmonary AM observed after birth in the control group may be related to pulmonary vasodilatation and expression of AM in vascular endothelial cells. Therefore, the decreased level of pulmonary AM in the nitrofen-treated groups may suggest low ability of AM production for pulmonary vasodilation, although many factors may be involved in the level of AM in the lungs immediately after birth. Although many recent studies revealed that AM is involved in various types of cell proliferation [9,10], the function of AM in cell proliferation still remains unclear. It is reported that TGF-b plays a role in the control of differentiation and morphogenesis in the developing bronchi, respiratory epithelium, blood vessels, and surrounding mesenchyma [30]. It is also reported that TGF-b is an inhibitory factor for pulmonary morphogenesis, and administration of TGF-b resulted in pulmonary hypoplasia [31,32]. Interestingly, AM is expressed concomitant with the expression of TGF-b [30]. Moreover, it is reported that expression of AM is affected by the existence of TGF-b [33]. If AM acts complementarily with TGF-b, pulmonary hypoplasia may be induced by AM. Conversely, if AM acts contrary to TGF-b, AM production may be induced to promote fetal pulmonary growth. There may be two different interpretations for the results of our experiment. One possibility is that the pulmonary hypoplasia in nitrofen models was induced by overexpression of AM, which acts as inhibitory factor for pulmonary morphogenesis. The other is that AM expression was induced as a result and a negative feedback of pulmonary hypoplasia to promote the fetal lung growth. Although it is not clear whether up-regulation of AM promotes or suppresses fetal pulmonary growth, our data indicated that AM is involved in the pathophysiology, including the differentiation processes, of nitrofen-induced pulmonary hypoplasia. Further investigation such as direct administration of AM to fetal cultured lung is needed to clarify the effects of AM on pulmonary hypoplasia.
References [1] Lally KP, Jakstic T, Wilson JM, et al. The congenital diaphragmatic hernia study group: estimating disease severity of congenital diaphragmatic hernia in the first 5 minutes of life. J Pediatr Surg 2001;36:141 - 5. [2] Thebaud B, Mercier JC, Dinh-Xuan AT. Congenital diaphragmatic hernia. A cause of persistent pulmonary hypertension of the newborn, which lacks an effective therapy. Biol Neonate 1998;74:323 - 36. [3] Dillon PW, Cilley RE, Mauger D, et al. The relationship of pulmonary artery pressure and survival in congenital diaphragmatic hernia. J Pediatr Surg 2004;39:307 - 12. [4] Iritani I. Experimental study on embryogenesis of congenital diaphragmatic hernia. Anat Embryol 1984;169:133 - 9. [5] Tenbrinck R, Tibboel D, Gaillard JLJ, et al. Experimentally induced congenital diaphragmatic hernia in rats. J Pediatr Surg 1990;25:426 - 9. [6] Chinoy MR. Pulmonary hypoplasia and congenital diaphragmatic hernia: advances in the pathogenetics and regulation of lung development. J Surg Res 2002;106:209 - 23.
Upregulation of adrenomedullin in fetal hypoplastic lung [7] Schnitzer JJ. Control and regulation of pulmonary hypoplasia associated with congenital diaphragmatic hernia. Semin Pediatr Surg 2004;13:37 - 43. [8] Kitamura K, Kangawa K, Kawamoto M, et al. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 1993;192:553 - 60. [9] Beltowski J, Jamroz A. Adrenomedullin—what do we know 10 years since its discovery? Pol J Pharmacol 2004;56:5 - 27. [10] Hinson JP, Kapas S, Smith DM. Adrenomedullin, a multifunctional regulatory peptide. Endocr Rev 2000;21:138 - 67. [11] Montuenga LM, Martinez A, Miller MJ, et al. Expression of adrenomedullin and its receptor during embryogenesis suggests autocrine or paracrine modes of action. Endocrinology 1997;138: 440 - 51. [12] Marinoni E, Iorio RD, Alo P, et al. Immunohistochemical localization of adrenomedullin in fetal and neonatal lung. Pediatr Res 1999;45:282 - 5. [13] Yoshibayashi M, Kamiya T, Kitamura K, et al. Plasma levels of adrenomedullin in primary and secondary pulmonary hypertension in patients b20 years of age. Am J Cardiol 1997;79:1556 - 8. [14] Kamata S, Kamiyama M, Usui N, et al. Is adrenomedullin involved in the pathophysiology of persistent pulmonary hypertension of the newborn? Pediatr Surg Int 2004;20:24 - 6. [15] Kluth D, Kangah R, Reich P, et al. Nitrofen-induced diaphragmatic hernias in rats: an animal model. J Pediatr Surg 1990;25:850 - 4. [16] Jesudason EC, Connell MG, Fernig DG, et al. Early lung malformations in congenital diaphragmatic hernia. J Pediatr Surg 2000;35:124 - 8. [17] Makino I, Makino Y, Yoshihara F, et al. Decreased mature adrenomedullin levels in feto-material tissues of pregnant women with histologic chorioamnionitis. Biochem Biophys Res Commun 2003;301:437 - 42. [18] Ohta H, Tsuji T, Asai S, et al. One-step direct assay for maturetype adrenomedullin with monoclonal antibody. Clin Chem 1999; 45:244 - 251. [19] Oya H, Nagaya N, Furuichi S, et al. Comparison of intravenous adrenomedullin with atrial natriuretic peptide in patients with congestive heart failure. Am J Cardiol 2000;86:94 - 8. [20] Heid CA, Stevens J, Livak KJ, et al. Real time quantitative PCR. Genome Res 1996;6:986 - 94.
1567 [21] Belik J, Davidge ST, Zhang W, et al. Airway smooth muscle changes in the nitrofen-induced congenital diaphragmatic hernia rat model. Pediatr Res 2003;53:737 - 43. [22] Nikitenko LL, MacKenzie IZ, Rees MCP, et al. Adrenomedullin is an autocrine regulator of endothelial growth in human endometrium. Mol Hum Reprod 2000;6:811 - 9. [23] Migliazza L, Xia H, Alvarez JI, et al. Heart hypoplasia in experimental congenital diaphragmatic hernia. J Pediatr Surg 1999;34:706 - 11. [24] Yu J, Gonzalez S, Diez-Pardo JA, et al. Effect of early embryonal exposure to dexamethasone on malformations of neural-crest derivatives induced by nitrofen in rats. Pediatr Surg Int 2002;18: 606 - 10. [25] Shindo T, Kurihara Y, Nishimatu H, et al. Vascular abnormalities and elevated blood pressure in mice lacking adrenomedullin gene. Circulation 2001;104:1964 - 71. [26] Witlin AG, Li ZY, Wimalawansa SJ, et al. Placental and fetal growth in late rat gestation is dependent on adrenomedullin. Biol Reprod 2002;67:1025 - 31. [27] Nishikimi T, Matsuoka H, Shimada K, et al. Production and clearance sites of two molecular forms of adrenomedullin in human plasma. Am J Hypertens 2000;13:1032 - 4. [28] Guilbert TW, Gebb SA, Shannon JM. Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development. Am J Physiol Lung Cell Mol Physiol 2000;279:L1159 - 71. [29] Keijzer R, Liu J, Deimling J, et al. Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia. Am J Pathol 2000;156:1299 - 306. [30] Montuenga LM, Mariano JM, Prentice MA, et al. Coordinate expression of transforming growth factor-b1 and adrenomedullin in rodent embryogenesis. Endocrinology 1998;139:3946 - 57. [31] Zhao J, Bu D, Lee M, et al. Abrogation of transforming growth factor-b type II receptor stimulates embryonic mouse lung branching morphogenesis in culture. Dev Biol 1996;180:242 - 57. [32] Serra R, Pelton RW, Moses HL. TGF-b inhibits branching morphogenesis and N-myc expression in lung bud organ culture. Development 1994;120:2153 - 61. [33] Bodegas E, Martinez A, Ozbun LL, et al. Depressed adrenomedullin in the embryonic transforming growth factor-b1 null mouse become elevated postnatally. Int J Dev Biol 2004;48:67 - 77.
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Adrenomedullin is up-regulated in nitrofen-induced fetal pulmonary hypoplasia Masafumi Kamiyama*, Noriaki Usui, Shinkichi Kamata, Masahiro Fukuzawa, Noritoshi Nagaya, Kenji Kangawa Department of Pediatric Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan Department of Biochemistry, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan Index words: Congenital diaphragmatic hernia; Adrenomedullin; Pulmonary hypoplasia; Hypoplastic lung; Nitrofen
Abstract Background/Purpose: Pulmonary hypoplasia associated with congenital diaphragmatic hernia remains a major therapeutic problem. Adrenomedullin (AM), a multifunctional regulatory peptide, has been suggested to play a role in the mechanisms of fetal lung differentiation and maturation. The aim of this study was to investigate the pulmonary expression of AM in nitrofen-induced fetal pulmonary hypoplasia. Materials and Methods: Pulmonary hypoplasia was induced in timed-pregnant Sprague-Dawley rats after administration of 100 mg nitrofen on day 9.5 of gestation. Fetal and neonatal lungs were excised on gestational days 16.5, 19, and 21 and 1 hour after birth and divided into the following 3 groups: nitrofen with diaphragmatic defect, nitrofen without diaphragmatic defect, and control (without nitrofen). Pulmonary levels of AM and AM messenger RNA expression were measured by radioimmunoassay and real time quantitative reverse transcriptase polymerase chain reaction, respectively. Localization of pulmonary AM was identified by immunohistochemical staining. Results: There was a significant increase in pulmonary level of AM in the nitrofen-treated groups on gestational days 19 and 21. Real time quantitative reverse transcriptase polymerase chain reaction on gestational day 19 confirmed an increase of AM gene expression in the nitrofen-treated groups. Adrenomedullin immunoreactivity was more strongly expressed in airway epithelial cells in the nitrofentreated groups than in the control. Conclusion: Nitrofen up-regulates expression of AM in the fetal lung, which suggests that AM has some pathophysiological role in the differentiation and/or maturation processes of pulmonary hypoplasia in congenital diaphragmatic hernia. D 2005 Published by Elsevier Inc.
The prognosis of infants with severe congenital diaphragmatic hernia (CDH) still remains unfavorable despite recent therapeutic progress [1]. Because persistent pulmonary hypertension resulting from pulmonary hypoplasia seems to be a major cause of the high mortality [2,3], numerous
T Corresponding author. Tel.: +81 6 6879 3753; fax: +81 6 6879 3759. E-mail address: [email protected] (M. Kamiyama). 0022-3468/$ – see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.jpedsurg.2005.06.005
efforts have been made to elucidate the pathophysiology of pulmonary hypoplasia in CDH. A nitrofen-induced murine pulmonary model of CDH has been extensively used to investigate the pathogenesis of CDH because of its phenotypic similarities to human CDH [4,5]. Recently, a number of regulatory factors such as fibroblast growth factor 10 and transforming growth factor b (TGF-b) and the molecular mechanisms underlying pulmonary hypoplasia in a nitrofeninduced model of CDH have been investigated [6,7].
Upregulation of adrenomedullin in fetal hypoplastic lung Table 1 Control DD ( ) DD (+) Left Right Bilateral
Numbers of experimental animals Gd 16.5
Gd 19
Gd 21
After birth
24 15 23 11 7 5
15 6 30 15 12 3
18 16 24 11 6 7
27 11 13 10 3 0
Gd indicates gestational day.
1563 Table 2 day 19
Lung Placenta Liver Heart Kidney
Organ-to-body weight ratios (%) on gestational Nitrofen with DD (n = 30)
Nitrofen without DD (n = 6)
Control (n = 15)
3.03 17.4 7.12 0.67 0.56
3.20 16.5 6.90 0.76 0.58
4.16 17.3 9.70 0.74 0.61
F F F F F
0.25T 2.6 0.74T 0.08 0.08
F F F F F
0.09T 1.3 0.65T 0.10 0.10
F F F F F
0.45 1.7 1.28 0.07 0.09
* P b .05, significant difference from control.
Adrenomedullin (AM) is a peptide originally isolated from extracts of human pheochromocytoma as a potent vasorelaxant [8], which consists of 52 amino acids in humans and 50 amino acids in the rat and structurally belongs to the calcitonin gene–related peptide superfamily. Although AM has been reported as a multifunctional regulatory peptide, many recent studies revealed that AM is especially involved in various types of cell proliferation [9,10]. The expression of AM during embryogenesis has been analyzed using mouse and rat embryos [11]. Adrenomedullin immunoreactivity was markedly increased during the late organogenetic and early fetal growth periods. These observations, taken together with data linking AM to growth control in various types of cells, suggested the possible involvement of AM in the control of embryonic cell proliferation and differentiation. Particularly in murine fetal lungs, AM expression was found in the bronchial epithelium, airway smooth muscle cells, mesenchymal cells, and vascular endothelial cells [11]. The expression of pulmonary AM in the late organogenic period and its increased staining during fetal lung development suggest its possible role in fetal lung differentiation and maturation [12]. It has been reported that plasma levels of AM are elevated in children with primary and secondary pulmonary hypertension [13]. We have also shown that plasma levels of AM in the umbilical artery and vein were elevated in patients with persistent pulmonary hypertension of the newborn [14]. Hypothesizing a pathophysiological role of AM in the regulation of lung differentiation and/or maturation in the hypoplastic lung, we investigated the pulmonary expression of AM in a nitrofen-induced rat model of pulmonary hypoplasia.
examined for diaphragmatic defects (DDs), both lungs were excised. The fetuses and neonates from nitrofen-treated dams (nitrofen-treated group) were divided into 2 groups according to the presence or absence of DD. In addition to the lungs, the placenta, liver, heart, and kidneys were excised at 19 days of gestation. Tissues were weighed, frozen, and kept at 808C until measurement of AM. Another series of fetal rat lungs at 19 days of gestation were obtained for real time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical staining. Fetal lungs were kept frozen at 808C for real time quantitative RT-PCR and fixed in 4% paraformaldehyde for immunohistochemical staining. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Osaka University Graduate School of Medicine.
1.2. Radioimmunoassay Each tissue was boiled in water to inactivate intrinsic proteases. After cooling, acetic acid was added and the mixture was homogenized. The supernatant of the extract, obtained after centrifugation, was lyophilized. For assay, the lyophilized material was dissolved in radioimmunoassay buffer, and the clear solution was subjected to radioimmunoassay [17]. The levels of AM were determined by
1. Materials and methods 1.1. Experimental animals Female Sprague-Dawley rats (Oriental Yeast Co, Osaka, Japan) were used for the experiment. After mating, the animals were given 100 mg nitrofen (Supelco, Bellefonte, Pa), dissolved in 1 mL olive oil, via a gastric tube on day 9.5 of gestation [15,16]. Control animals were given 1 mL olive oil only. Fetuses on days 16.5, 19, and 21 of gestation were killed before any breathing occurred, and neonates were killed 1 hour after birth. After the animals were weighed and
Fig. 1 Lung-to-body weight ratio on gestational days 16.5, 19, and 21 and 1 hour after birth. Numbers of experimental animals are shown in Table 1. Gd indicates gestational day. Asterisk indicates P b .05, significant difference from control at same age. Dagger indicates P b .05, significant difference from nitrofen without DD at same age.
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Table 3 Organ levels of AM (femtomol per milligram wet tissue) on gestational day 19
Lung Placenta Liver Heart Kidney
Nitrofen with DD (n = 30)
Nitrofen without DD (n = 6)
Control (n = 15)
4.51 2.27 0.063 0.93 0.48
4.37 2.31 0.056 1.36 0.54
1.36 2.56 0.072 1.00 0.36
F F F F F
0.78T 0.61 0.022 0.45 0.23
F F F F F
1.20T 0.60 0.021 0.60 0.19
F F F F F
0.31 0.36 0.033 0.40 0.11
* P b .05, significant difference from control.
immunoradiometric assays using specific kits (Shionogi Pharmaceutical Co, Ltd, Osaka, Japan) [18,19]. The radioactivity was measured by a gamma counter (ARC1000M, Aloka, Tokyo, Japan). All assay procedures were performed in duplicate.
1.3. Real time quantitative RT-PCR Total RNA was isolated using an RNA isolation system (Ambion Inc, Austin, Tex). Total RNA was then reverse transcribed using a first standard complementary DNA kit (Clontech Laboratories Inc, Palo Alto, Calif). We purchased Pre-Developed TaqMan Assay Reagents for AM and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Applied Biosystems, Foster City, Calif). Real time PCR amplification was performed using a sequence detector (ABI PRISM 7700, Applied Biosystems). The relative standard curve method was used to quantitative AM messenger RNA (mRNA) expression [20]. Data were shown as the ratio to GAPDH mRNA.
1.4. Immunohistochemistry Polyclonal antibody raised from rabbits against purified rat AM was purchased as primary antibody from Phoenix
Fig. 2 Levels of pulmonary AM on gestational days 16.5, 19, and 21 and 1 hour after birth. Numbers of experimental animals are shown in Table 1. Asterisk indicates P b .05, significant difference from control at same age.
Pharmaceuticals (Belmont, Calif). The fixed tissues were embedded in paraffin, then trimmed and mounted. After deparaffinization, tissues were autoclaved. Then tissues were incubated in diluted primary antibody for 1 hour at room temperature and then incubated in antirabbit immunoglobulin conjugated to peroxidase-labeled dextran polymer system as secondary antibody (Dako, Kyoto, Japan) for 1 hour at room temperature. Antibody labeling was revealed by a 3,3-diaminobenzidine tetrahydrochloride product. Lung immunohistochemical staining in each group was scored in a blinded manner, assigning a value of 3 as very strong staining; 2, strong staining; 1, weak staining; and 0, no staining [21,22]. Four areas (200) in each slide were selected blindly, and the intensity of staining was scored with respect to each histologic part.
1.5. Statistics All data were expressed as mean F SD. Statistical analysis was performed by 1-way analysis of variance, and significant differences were identified by Scheffe´ post hoc analysis. Scores for immunohistochemical staining were analyzed by nonparametric test using Mann-Whitney U test. All statistical analyses were performed using a commercially available statistics package (Stat View, SAS Institute Inc, Cary, NC). P b .05 was considered statistically significant.
2. Results 2.1. Incidence and side of DDs induced by nitrofen The number of fetuses or neonates subjected to measurement of organ-to-body weight ratios and AM on gestational days 16.5, 19, and 21 and after birth were 38, 36, 40, and 24, respectively (Table 1). The incidence of
Fig. 3 Pulmonary AM gene expression by real time quantitative RT-PCR on gestational day 19. Data are shown as the ratio to GAPDH mRNA. Asterisk indicates P b .05, significant difference from control.
Upregulation of adrenomedullin in fetal hypoplastic lung defects was around 60% to 80%, with a predominance of left-sided defects.
1565 Table 4 Scores of immunohistochemical intensity for AM in fetal lung on gestational day 19
2.2. Organ-to-body weight ratios In the nitrofen-treated groups, the organ-to-body weight ratio was decreased in the lung and liver on gestational day 19 as compared with the control (Table 2). There was no significant difference between nitrofen-treated groups with and without DD. A decrease of lung-to-body weight ratio in
Nitrofen with Nitrofen without Control DD (n = 16) DD (n = 8) (n = 8) Airway 1.75 F 0.68T 2.13 F 0.84T epithelial cells Vascular 1.50 F 0.52 1.75 F 0.89 endothelial cells Mesenchymal 1.06 F 0.25 1.13 F 0.35 cells
1.13 F 0.35 1.13 F 0.35
0.88 F 0.35
The intensity of immunohistochemical staining was scored, assigning a value 0 to 3 (3: very strong staining; 2: strong staining; 1: weak staining; 0: no staining). T P b .05, significant difference from control.
nitrofen-treated groups was also recognized on gestational days 16.5 and 21 and 1 hour after birth (Fig. 1). Lung-tobody weight ratio of nitrofen-treated rats with DD was lower than that of nitrofen-treated rats without DD on gestational day 21 and after birth.
2.3. Levels of AM in organs In the control, the placenta showed the highest concentration of AM on gestational day 19 (Table 3). A significant increase of pulmonary AM was observed in nitrofen-treated groups as compared with control, but no significant difference was observed between nitrofen-treated groups with and without DD. However, pulmonary level of AM in the control was increased as compared with that in the nitrofen-treated groups 1 hour after birth (Fig. 2).
2.4. Real time quantitative RT-PCR on gestational day 19 Pulmonary AM gene expression was up-regulated in the nitrofen-treated groups. No significant difference was observed in pulmonary AM mRNA level between nitrofen-treated groups with and without DD (Fig. 3).
2.5. Immunohistochemistry on gestational day 19 In the control, distinct immunoreactivity of AM was found in airway epithelial cells, vascular endothelial cells, and mesenchymal cells dominantly (Fig. 4A). The expression of AM immunoreactivity was more dominant in the nitrofen-treated groups with DD (Fig. 4B) and without DD (Fig. 4C) than in the control. However, a significant difference in scores of the intensity of AM immunohistochemical staining of airway epithelial cells was observed between the nitrofen-treated groups and the control (Table 4). Fig. 4 Immunohistochemical staining of AM in fetal lungs on gestational day 19. Adrenomedullin immunoreactivity is seen in airway epithelial cells (black arrows), vascular endothelial cells (white arrows), and mesenchymal cells (asterisks). Immunohistochemical staining is stronger in the nitrofen groups with (B) and without (C) DD than in control (A).
3. Discussion To investigate the pathophysiology of pulmonary hypoplasia in CDH, we used a nitrofen-induced rat model of
1566 CDH. In this model, the DD predominantly occurs on the left, and the incidence of CDH is compatible with that in previous reports [23,24]. It has been reported that embryos with homozygous deletion of the AM gene do not survive because of vascular fragility [25]. It is also reported that antagonism of AM function during rat pregnancy causes fetal growth restriction [26]. Montuenga et al [11] reported that the level of AM immunoreactivity was increased during the late organogenetic and early fetal growth periods during embryogenesis. These observations suggest the possible involvement of AM in promoting embryonic cell proliferation and differentiation. The present study showed up-regulation of pulmonary AM during late gestation in the nitrofen-treated groups. Gene expression data obtained by real time quantitative RT-PCR indicated increased production of AM in the lung. Immunohistochemical study showed that AM was preferentially expressed in airway epithelial cells in nitrofeninduced hypoplastic lungs. These results suggest that AM plays a role in fetal pulmonary growth, especially in promoting branching morphogenesis and airway cell proliferation or maturation. In the present study, the placenta was the organ with the highest AM level. It is reported that antagonism of AM function during rat pregnancy caused decreased placental size and gross necrosis of the placental margins and amniotic membrane [26], suggesting an important role of AM in vascular development of the placenta. It is also reported that the level of AM in the umbilical vein was higher than that in the umbilical artery [14], and the main clearance site of AM is the lung [27]. These observations suggest that AM produced in the placenta may also play a role in the regulation of fetal development, including lung differentiation and/or maturation. There was no difference in the pulmonary levels of protein and gene expression of AM between the nitrofentreated groups with and without DDs. This seems compatible with recent studies that showed that pulmonary hypoplasia in a nitrofen-induced model occurs early in the developmental period simultaneously with primary abnormalities independent of DDs [4,16,28,29]. In the present study, a decrease of lung-to-body weight ratio was recognized on gestational day 16.5, supporting early induction of pulmonary hypoplasia in nitrofen-induced models. Furthermore, there was a significant decrease of lung-to-body weight ratio in the nitrofen-treated group with DD compared with the nitrofen-treated group without DD on gestational day 21 and after birth, indicating that a defect of the diaphragm also affects the development of the lungs, which was described by Keijzer et al [29] as the bdual-hit hypothesis.Q The level of pulmonary AM in normal fetuses increased sharply after birth and was higher than that in the nitrofentreated groups. Adrenomedullin is a vasodilating peptide, and it has been reported that AM physiologically acts as a vasodilator immediately after birth to reduce pulmonary
M. Kamiyama et al. artery pressure in neonates [13,14] The increase of pulmonary AM observed after birth in the control group may be related to pulmonary vasodilatation and expression of AM in vascular endothelial cells. Therefore, the decreased level of pulmonary AM in the nitrofen-treated groups may suggest low ability of AM production for pulmonary vasodilation, although many factors may be involved in the level of AM in the lungs immediately after birth. Although many recent studies revealed that AM is involved in various types of cell proliferation [9,10], the function of AM in cell proliferation still remains unclear. It is reported that TGF-b plays a role in the control of differentiation and morphogenesis in the developing bronchi, respiratory epithelium, blood vessels, and surrounding mesenchyma [30]. It is also reported that TGF-b is an inhibitory factor for pulmonary morphogenesis, and administration of TGF-b resulted in pulmonary hypoplasia [31,32]. Interestingly, AM is expressed concomitant with the expression of TGF-b [30]. Moreover, it is reported that expression of AM is affected by the existence of TGF-b [33]. If AM acts complementarily with TGF-b, pulmonary hypoplasia may be induced by AM. Conversely, if AM acts contrary to TGF-b, AM production may be induced to promote fetal pulmonary growth. There may be two different interpretations for the results of our experiment. One possibility is that the pulmonary hypoplasia in nitrofen models was induced by overexpression of AM, which acts as inhibitory factor for pulmonary morphogenesis. The other is that AM expression was induced as a result and a negative feedback of pulmonary hypoplasia to promote the fetal lung growth. Although it is not clear whether up-regulation of AM promotes or suppresses fetal pulmonary growth, our data indicated that AM is involved in the pathophysiology, including the differentiation processes, of nitrofen-induced pulmonary hypoplasia. Further investigation such as direct administration of AM to fetal cultured lung is needed to clarify the effects of AM on pulmonary hypoplasia.
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