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Upregulation of keratinocyte growth factor in the tracheal ligation lamb model of congenital diaphragmatic hernia
Upregulation of Keratinocyte Growth Factor in the Tracheal Ligation Lamb Model of Congenital Diaphragmatic Hernia By Amanda J. MeCabe, Ulrike Carlino, Bruce A. Holm, and Philip L. Glick Buffalo, New York
Purpose: Congenital diaphragmatic hernia (CDH) carries a high mortality rate of 60% because of associated anomalies, pulmonary hypoplasia, pulmonary hypertension, and type II cell dysfunction. Prenatal tracheal ligation has been shown to improve lung growth in experimental models. This could be caused by a direct effect of increased endothoracic pressure in utero, secondary to the induction of specific growth factors, or both. Keratinocyte growth factor (KGF) is involved in normal lung organogenesis and is a potent mitogen of alveolar type II cells. The authors have therefore investigated the protein and mRNA levels of keratinocyte growth factor in the lung tissue of control, CDH, and CDH tracheal ligation lambs. Methods: Eight lambs had left-sided diaphragmatic hernias surgically created at 80 days' gestation. Tracheal ligation was performed at 110 days in 4 lambs, and they were delivered by cesarean section at 140 to 145 days. Twin littermates served as controls. The lungs were dissected en bloc and snap frozen. KGF protein levels were determined by ELISA. Total RNA was isolated, and a RNase protection assay was performed using an ovine cDNA probe for KGF, and a human cDNA probe for GAPDH (house keeping control). Densito-
NFANTS born with congenital diaphragmatic hernia (CDH) have a high perinatal mortality rate, which remains around 60%, despite prenatal diagnosis and advances in neonatal care.~ Death has been attributed to associated anomalies, pulmonary hypoplasia, pulmonary hypertension, and type II cell dysfunction. The degree of pulmonary hypoplasia varies between infants born with CDH.2. 3 It is thought, in part, that intraabdominal viscera occupy space in the chest during critical periods of lung growth and development, and that the severity of pulmonary hypoplasia is related to both the timing of herniation and the volume of herniated viscera. This view is supported by the fact that smaller hernias and those that develop later in gestation are
I
From the Buffalo Institute of Fetal Therapy (B1FT), The ChiMren's Hospital of Buffale (Kaleida Health), Departments of Surgery, Pediatrics, OBGYN, and Microbiology, The State University of New York at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, NY. Presented at the 31st Annual Meeting of the American Pediatric Surgical Association, Orlando, Florida, May 25-29, 2000. Address reprint requests to Philip L. Glick, MD, FACS, FAAP, FSCCM, FACPE, 219 Bryant St, Buffalo, NY 14222. Copyright © 2001 by W.B. Saunders Company 0022-3468/01/3601-0022503.00/0 doi: l O.1053/jpsu.2001.20029 128
metric analysis was used to quantify the relative amounts of mRNA in each sample. Results: There was a significant decrease in the KGF protein levels of the CDH samples (110 v 73.2 pg/mg protein, P = .02). This decrease was mirrored by a significant fall in the level of mRNA expression for KGF (0.694 v 0.235, P = .02). Tracheal ligation normalized the KGF protein levels (96.1 pg/mg protein). This elevation of KGF protein was accompanied by an upregulation of KGF gene expression to control levels (0.56). Conclusions: Tracheal ligation clearly is accompanied by an upregulation of keratinocyte growth factor protein and gene expression, It is not yet clear whether keratinocyte growth factor is solely responsible for the growth observed in these tracheal ligation preparations. Further growth factor blocking experiments are required. J Pediatr Surg 36:128-132. Copyright © 2001 by W.B. Saunders Company. INDEX WORDS: Congenital diaphragmatic hernia, keratinocyte growth factor, tracheal ligation, fetal lung growth.
associated with lower mortality rate, 4 and by the observation in animal experiments, that intrathoracic masses result in pulmonary hypoplasia. 5 In utero tracheal ligation is a potent stimulus for fetal lung growth. Animal studies have found that tracheal occlusion accelerates lung growth in normal lungs 6 and in models of lung hypoplasia such as oligohydramnios,7 bilateral nephrectomy, 8 and congenital diaphragmatic hernia. 9 The latter observation supports the rationale of selective in utero tracheal ligation of those CDH fetuses with a very poor predicted prognosis. 10 Currently, the precise mechanism by which tracheal ligation augments fetal lung growth is not known. A pressure phenomenon has been postulated whereby the ligation acts as an endobronchial stent, stretching the developing lung, and possibly stimulating the release of specific growth factors. 1H3 However, no specific stretch receptors have yet been identified. The complex process of branching morphogenesis in the lung is mediated by transcription factors, peptide growth factors, and extracellular matrix signals that interact in a tightly coordinated temporospatial pattern of epithelial-mesenchymal interaction. ~4 Keratinocyte growth factor (KGF), which is produced in mesenchyme but acts via receptors positioned in epithelium, has been Journal of Pediatric Surgery, Vol 36, No 1 (January), 2001: pp 128-132
UPREGULATION OF KGF IN CDH
i m p l i c a t e d in t h e r e g u l a t i o n o f e a r l y l u n g b r a n c h i n g 15 a n d a l s o h a s b e e n s h o w n to b e a p o t e n t s t i m u l a t o r o f e p i t h e lial t y p e II p n e u m o c y t e s in v i v o a n d i n vitro. 16,17 T h e c u r r e n t s t u d y w a s t h e r e f o r e d e s i g n e d to i n v e s t i g a t e t h e p o s s i b l e i n v o l v e m e n t o f K G F in t h e i n d u c t i o n o f l u n g g r o w t h in t h e C D H t r a c h e a l l i g a t i o n l a m b m o d e l . MATERIALS AND METHODS
Experimental Design and Fetal Surgical Procedures These studies were approved by the Animal Care Committee of the State University of New York at Buffalo, NY. Eight fetal lambs underwent the surgical creation of a left-sided diaphragmatic hernia via an open hysterotomy at 80 days' gestation, as previously described. TM This stage of gestation in the sheep corresponds with the pseudoglandular phase of lung development. The pathophysiologic and morphometric changes seen in this model mirror those seen in human babies with CDH29,2° At 110 days' gestation, 4 animals underwent a second hysterotomy. The fetal head and neck were delivered, and the trachea was exposed through a transverse incision. The trachea was isolated and ligated with umbilical tape. The fetus was once more returned to the uterus and the hysterotomy closed. Nonoperated littermates served as controls. At 140 days' gestation, all the fetuses were delivered by cesarean section. Each animal was killed immediately, the chest opened, and the lungs perfused in situ with normal saline via the pulmonary artery. The heart and lungs were removed en bloc and the lungs perfused once again. The lung tissue was then snap frozen in liquid nitrogen and stored at -80°C.
Determination of KGF Protein Levels Lung tissue samples were weighed and suspended in cell lysis buffer as modified by Bui et al. zl The mixture was homogenized and centrifuged (500 g, for 6 minutes). The supernatant was lyophilized overnight. Samples of cell lysis buffer and KGF standard (R&D Systems, Minneapolis, MN) were treated similarly. Alter reconstitution with more cell lysis buffer, the supernatant from each sample was treated according to a KGF enzyme-linked immunosorbent assay (ELISA; R&D Systems). The optical density of each well was read by Microplate Manager 4.0 (Bio-Rad Laboratories, Inc, Hercules, CA) at 450 nm. The protein content of the supernatants was assayed according to the Micro-Lowry method. 22 Results were expressed as picograms per milligram of protein.
RNA Isolation Total cellular RNA was isolated from homogenized lung tissue by the acid guanidinium thiocyanate, phenol/chloroform method (denaturing solution: 4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 4), 0.5% N-lauroylsarcosine, and 0.1 M 2-mercaptoethanol) and resuspended in DEPC-treated water. Each RNA sample was quantified by spectrophotometry (Az6o/A2s0 ratio). The quality of the RNA was further assessed by electrophoresis on a 1% agarose gel containing × formaldehyde and staining with ethidium bromide. Only intact samples were used for RPA experiments. RNA samples were stored at -80°C.
KGF Probe Synthesis cDNA was obtained by reverse transcription (Superscript II reverse transcriptase, Gibco BRL, Carlsbad, CA) of total RNA purified from the lung tissue of one of the control samples using the manufacturer's instructions. The previously published sheep KGF sequence was used to design forward and reverse primers for a polymerase chain reaction
129 (PCR). 23 The forward primer was: 5'-TATCTTGCAATGAACAAGGAA-3', the reverse primer was: 5'-TTAAGTTATTGCCATAGG-3'. These oligonucleotides were synthesized at the Center for Advanced Molecular Biology and Immunology Facility (SUNY at Buffalo, Buffalo, NY). To amplify the KGF gene from the lung cDNA, a PCR was performed in a Perkin Elmer GeneAmp PCR System 2400 thermal cycler. The conditions for the PCR were: 94°C for 5 minutes (1 cycle); 94°C for 1 minute, 60°C for 2 minutes, 72°C for 3 minutes (30 cycles); 72°C for 10 minutes (1 cycle). The PCR products were fractionated on 1.5% agarose gels stained with 0.5 /xg/mL ethidium bromide and visualized by ultraviolet illumination. The PCR product was blunted ended,24 ligated into the phagemid vector pBSCII SK-, and used to transform DH5a-competent Escherichia coli cells (Gibco BRL) according to the manufacturer's instructions. Successful ligations were confirmed by colony PCR. 25 Phagemid DNA was prepared using the "alkaline lysis method. ''26 Phagemids were sequenced at the Center for Advanced Molecular Biology and Immunology DNA Sequencing Facility (SUNY at Buffalo, Buffalo, NY), using automated DNA sequencing technology. The sequences obtained were compared with the published sheep KGF sequence. A single base mismatch was found in a total of 234 bases. Sequence analysis of the KGF gene in the vector allowed determination of its orientation with respect to the T3 and T7 promoters encoded in the vector. The KGF-containing plasmid (pKGF) was linearized with EcoRI and used as a template for RNA synthesis.
RPA Assay A RiboQuant Multi-Probe RNase Protection Assay System (Pharmingen, San Diego, CA) was used to detect and quantitate the mRNA in the lung tissue samples. The KGF template was used in place of the recommended kit template set, and a human GAPDH template was used as a housekeeper. Each template was transcribed with T7 RNA polymerase and then both were hybridized overnight with the RNA samples, according to RiboQuant directions. The samples were then treated with RNase A, and the RNase digests were extracted and purified according to kit directions. The resultant protected digests were electrophoresed on a 5% denaturing polyacrylamide gel at 50 watts. The dried gel image was intensified and developed using a BioRad GS-525 Molecular Imager System. Quantification of the radioactivity of each sample was achieved using molecular Analyst v.l.5 software (BioRad Laboratories), and each sample value was compared with that of the housekeeper (hGAPDH).
Statistical Analysis Data are expressed as mean +_ SEM. The groups were compared using unpaired t tests. P values of less than .05 were considered statistically significant. RESULTS
W h o l e a n i m a l w e i g h t s f o r all g r o u p s w e r e c o m p a r a b l e . T o t a l w e t l u n g w e i g h t s w e r e s i g n i f i c a n t l y d e c r e a s e d in t h e C D H p r e p a r a t i o n s (53 + 8 v 124 - 14 g), P = 0.02, a n d s i g n i f i c a n t l y i n c r e a s e d i n t h e l i g a t i o n m o d e l s ( 1 7 0 -21 v 124 -+ 14 g), P = .03, w h e n c o m p a r e d w i t h c o n t r o l s .
KGF Protein Levels T h e r e w e r e n o s i g n i f i c a n t d i f f e r e n c e s in K G F p r o t e i n l e v e l b e t w e e n f i g h t a n d l e f t l u n g s in t h e 3 e x p e r i m e n t a l g r o u p s . T h e r e w a s a s i g n i f i c a n t d e c r e a s e in t h e K G F p r o t e i n l e v e l in t h e C D H s a m p l e s ( 7 3 . 2 -+ 19 p g / m g p r o t e i n ) c o m p a r e d w i t h c o n t r o l s ( 1 1 0 . 4 _+ 35.8 p g / m g p r o t e i n ) , P = .02. T r a c h e a l l i g a t i o n r e s t o r e d t h e K G F
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the gene expression of KGF have been restored back to control levels in the lung tissue of those CDH fetuses that were ligated. Although ligation has been shown to induce KGF production, it is still not clear if the subsequent lung growth is solely a response to the presence of KGF. There may be many other factors induced in response to ligation. For example, the saline replacement of lung fluid from tracheaMigated lambs inhibited the lung hypertrophy usually seen in the tracheal ligation model. 13 It was concluded that the tracheal fluid composition rather than intratracheal pressure was critical in promoting pulmonary growth. More specifically, we have previously identified that platelet-derived growth factor-BB (PDGF-BB) also is induced in the tracheal ligation lamb model of CDH. 27 In this context it is interesting to note the elegant in vitro experiments of Liu et al. 2s Using an in vitro organotypic fetal rat lung cell culture they showed that an intermittent mechanical strain regimen, which simulated fetal breathing movements, enhanced DNA synthesis, stimulated cell division, and increased the mRNA levels of PDGF-BB. Antisense PDGF-BB oligonucleotides abolished the strain-enhanced DNA synthesis and cell growth5 8 The upregulation of PDGF gene expression in response to strain suggests a mechanism for the transduction of a physical force causing fetal lung growth. In the current study, similar blocking experiments would help to clarify whether the growth effect caused by tracheal ligation was solely, or in part, a response to the presence of KGF. Despite the apparent positive effects of tracheal ligation, the intervention must be viewed with caution. Animal experiments have shown that the resultant lungs
120
/
lOO
~ so ~ ao
2O •
CONTROL
r
CDH
-
-
LIGATION
Fig 1. Lung KGF protein content, expressed as picograms per milligram protein. There is a significant decrease in protein content in the CDH samples when compared with both controls and ligated samples (*P = .02).
protein level back to control levels (96.1 _+ 27.6 pg/mg protein), P = .2 (Fig 1).
KGF mRNA Expression The pattern of mRNA expression for KGF mirrored the pattern already seen with the levels of KGF protein in the 3 experimental groups. There was a significant decrease of mRNA KGF expression in the CDH group (0.235 -+ 0.09) when compared with controls, (0.694 -4- 0.22), P = .02. Tracheal ligation restored the level of expression back to control levels (0.56 _+ 0.24), P = .04 (Fig 2). DISCUSSION The precise mechanism by which tracheal ligation augments lung growth in the developing fetus is not known. The current study investigated whether keratinocyte growth factor was involved in the growth process induced by tracheal ligation of the CDH lamb model. It has been shown clearly that both KGF protein levels, and
KGFhGAPDHA
Z" J
0.8 0.7 0.6
0.5 0.4
.< z 0.3 E 0.2
0 0.1 0 B
CONTROL
l/ CDH
r
L1GATION
Fig 2. Expression of mRNA for KGF in lung tissue from control, CDH, and ligation samples. (A) RPA bands for KGF and hGAPDH. (B) Desitometric analysis of RPA bands. There is a significant fall in mRNA expression in the CDH samples (*P = .02); ligation has restored the mRNA expression back to control levels (**P = .04).
UPREGULATION OF KGF IN CDH
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are p h y s i o l o g i c a l l y i n s u f f i c i e n t a n d s u r f a c t a n t depleted. 29 T h e s u r f a c t a n t d e f i c i e n c y is b o t h q u a n t i t a t i v e , b e c a u s e o f a d r a m a t i c d e c r e a s e i n t h e d e n s i t y o f t y p e II p n e u m o c y t e s in l i g a t e d lungs, 3° a n d qualitative, as h i g h l i g h t e d b y t h e functional measure of poor choline incorporation of type II p n e u m o c y t e s i s o l a t e d f r o m l i g a t e d lungs. 31 M o r e recently, t h e t i m i n g o f l i g a t i o n h a s b e e n s h o w n to b e o f critical i m p o r t a n c e i n t h e b a l a n c e b e t w e e n a c h i e v i n g sufficient t i m e for a d e q u a t e l u n g g r o w t h , w h i l e a v o i d i n g d e t r i m e n t a l effects o n the t y p e II cells. 32 K n o w l e d g e r e g a r d i n g K G F i n d u c t i o n in t h e d i f f e r e n t t i m e f r a m e s o f
t h e s e e x p e r i m e n t s m i g h t o f f e r f u r t h e r i n s i g h t into the m e c h a n i s t i c effects o f K G F a n d u l t i m a t e l y a s s e s s i n g its potential use therapeutically. Fetuses with C D H w h o h a v e a p o o r prognosis n o w can b e offered in utero tracheal occlusion b y fetoscopic surgery in 1 center. 1° A prospective r a n d o m i z e d clinical trial o f the treamaent has n o t b e e n carried out. It is therefore o f p r i m e i m p o r t a n c e that the u n d e r l y i n g m e c h a n i s m s operating to produce lung g r o w t h in the p r e s e n c e o f tracheal ligation are further investigated and u n d e r s t o o d before a m o r e widespread clinical application o f the practice.
R E FE R E N C E S
1. Harrison MR, Adzick NS, Estes JM: A prospective study of the outcome for fetuses with diaphragmatic hernia. JAMA 271:382-384, 1994 2. Berdon WE, Baker DH, Amoury R: The role of pulmonary hypoplasia in the prognosis of newborn infants with diaphragmatic hernia and eventration. Am J Roentgenol 103:413-421, 1968 3. Nguyen L, Guttman FM, de Chadarevian JP: The mortality of congenital diaphragmatic hernia: Is total pulmonary mass inadequate, no matter what? Ann Surg 198:766-770, 1983 4. Adzick NS, Harrison MR, Glick PL: Diaphragmatic hernia in the fetus: Prenatal diagnosis and outcome in 94 cases. J Pediatr Surg 20:357-361, 1985 5. Harrison MR, Jester JA, Ross NA: Correction of congenital diaphragmatic hernia in utero I. The Model: Lntrathoracic balloon produces fatal pulmonary hypoplasia. Surgery 88:174-182, 1980 6. Alcorn D, Adamson TM, Lambert TF: Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J Anat 123:649-660, 1977 7. Adzick NS, Harrison MR, Glick PL: Experimental pulmonary hypoplasia and oligohydramnios: Relative contributions of lung fluid and fetal breathing movements. J Pediatr Surg 19:658-665, 1984 8. Wilson JM, DiFore JW, Peters CA: Experimental fetal tracheal ligafion prevents the pulmonary hypoplasia associated with fetal nephrectomy: Possible application for congenital diaphragmatic hernia. J Pediatr Surg 28:1433-1439, 1993 9. Hedrick MH, Estes JM, Sullivan KM: Plug the lung until it grows (PLUG): A new method to treat congenital diaphragmatic hernia in utero. J Pediatr Surg 29:612-617, 1994 10. Harrison MR, Mychaliska GB, Albanese CT, et al: Correction of congenital diaphragmatic hernia in utero IX; Fetuses with poor prognosis (Liver herniation and low lung-to-head ratio) can be saved by fetoscopic temporary tracheal occlusion. J Pediatr Surg 33:1017-1023, 1998 11. DiFiore JW, Fauza DO, Slavin R: Experimental fetal tracheal ligation reverses the structural and physiological effects of pulmonary hypoplasia in congenital diaphragmatic hernia. J Pediatr Surg 29:248255, 1994 12. Liu M, Skinner SJ, Xu J: Stimulation of fetal rat lung cell proliferation in vitro by mechanical stretch. Am J Physiol 263:376-383, 1992 13. Papadakis K, Luks FI, De Paepe ME, et al: Fetal lung growth after tracheal ligation is not solely a pressure phenomenon. J Pediatr Snrg 32:347-351, 1997 14. Warburton D, Lee MK: Current concepts on lung development. Curt Opin Pediatr 11:188-192, 1999 15. Post M, Souza P, Liu J, et al: Keratinocyte growth factor and its receptor are involved in regulating early lung branching. Development 122:3107-3115, 1996 16. Panos RJ, Rubin JS, Csaky KG, et al: Keratinocyte growth factor and hepatocyte growth factor are heparin-binding factors for
alveolar type II cells in fibroblast-conditioned medium. J Clin Invest 92:969-977, 1993 17. Ulich TR, Yi ES, Longmuir K, et al: Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo. J Clin Invest 93:1298-1306, 1994 18. Glick PL, Stannard VA, Leach CL: Pathophysiology of congenital diaphragmatic hernia II. The fetal lamb model is surfactant deficient. J Pediatr Surg 27:382-388, 1992 19. de Lorimier AA, Tierney DF, Parker HR: Hypoplastic lungs in fetal lambs with surgically produced diaphragmatic hernia. Surgery 62:12-17, 1967 20. Adzick NS, Outwater KM, Harrison MR: Correction of congenital diaphragmatic hernia in utero IV. An early gestational fetal lamb model for pulmonary vascular morphometric analysis. J Pediatr Snrg 20:673-680, 1985 21. Bui KC, Buckley S, Wu F: Induction of A and D type cyclins and cdc2 kinase activity during recovery from short-term hyperoxic lung injury. Am J Physiol 12:L625-636, 1995 22. Lowry DH, Rosebrough NJ, Fan" AL, et al: Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275, 1951 23. Doyle MP, Morris CF, Biltz RE: Keratinocyte growth factor and keratinocyte growth factor receptor orthologs [monkey, pig, rabbit]. In Vitro Cell Dev Biol 32:318-320, 1996 24. Bhat GJ, Lodes MJ, Myler PJ, et al: A simple method for cloning blunt ended DNA fragments. Nucl Acids Res 19:398, 1991 25. Chomczynsld P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analyt Biochem 162:156-159, 1987 26. Ausbel FA, Brent R, Kingston RE, et al: Alkaline lysis DNA preparation, in Current Protocols in Molecular Biology. New York, NY, John Wiley and Sons, 1999 27. McCabe AJ, Patel A, Kapur P, et al: Keratinocyte and platelet derived growth factors in the tracheal ligation lamb model of congenital diaphragmatic hernia. Am J Respir Crit Care Med 159:A665, 1999 28. Liu M, Liu J, Buch S, et al: Antisense oligonucleotides for PDGF-BB and its receptor inhibit mechanical strain-induced fetal lung cell growth. Am J Physiol 269:L178-L184, 1995 29. O'Toole SJ, Sharma A, Karamanoukian HL, et al: Tracheal ligation does not correct the surfactant deficiency associated with congenital diaphragmatic hernia. J Pediatr Surg 31:546-550, 1996 30. Piedboeuf B, Laberge J, Ghitulescu G, et al: Deleterious effect of tracheal obstruction on type lI pneumocytes in fetal sheep. Pediatr Res 41:473-479, 1997 31. Wilcox DT, Glick PL, Karamanoukian HL, et al: Contributions by individual lungs to the surfactant status in congenital diaphragmatic hernia. Pediatr Res 41:686-691, 1997 32. Paepe ME, Johnson BD, Papadakis K, et al: Lung growth response after tracheal occlusion in fetal rabbits is gestational agedependent. Am J Respir Cell Biol 21:65-76, 1999
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Discussion A. Haller (Baltimore, MD): I would like to ask you the question I asked earlier but now directed to your experimental model. If the occlusion is begun earlier than the time that you have, do you have data to suggest that there is a difference in the level of the growth factor? The second question is, if it is released, is there a diminution in this concentration suggesting that it is truly the obstruction that is the reason or are there other factors associated with the hypoplasia? A.J. McCabe (response): We have not done any earlier timing experiments, so I cannot comment about that.
Again, with the release experiments we have not looked at the growth factor in those tissues just yet. D. Teitelbaum (Ann Arbor, MI): We do similar KGF work in intestine--have you looked at whether a major source of KGF is lymphocytic? It is intraepithelial lymphocytes that are also found in the lung. Have you looked at that as a potential etiology? ff not, perhaps a useful way of approaching that would be to eliminate your CD8-positive population because the Gamma delta CD8-positive population is the source of KGF in those 2 sources. A.J. McCabe (response): We have only looked at total lung tissue, but, yes, that is a good thought.
Purpose: Congenital diaphragmatic hernia (CDH) carries a high mortality rate of 60% because of associated anomalies, pulmonary hypoplasia, pulmonary hypertension, and type II cell dysfunction. Prenatal tracheal ligation has been shown to improve lung growth in experimental models. This could be caused by a direct effect of increased endothoracic pressure in utero, secondary to the induction of specific growth factors, or both. Keratinocyte growth factor (KGF) is involved in normal lung organogenesis and is a potent mitogen of alveolar type II cells. The authors have therefore investigated the protein and mRNA levels of keratinocyte growth factor in the lung tissue of control, CDH, and CDH tracheal ligation lambs. Methods: Eight lambs had left-sided diaphragmatic hernias surgically created at 80 days' gestation. Tracheal ligation was performed at 110 days in 4 lambs, and they were delivered by cesarean section at 140 to 145 days. Twin littermates served as controls. The lungs were dissected en bloc and snap frozen. KGF protein levels were determined by ELISA. Total RNA was isolated, and a RNase protection assay was performed using an ovine cDNA probe for KGF, and a human cDNA probe for GAPDH (house keeping control). Densito-
NFANTS born with congenital diaphragmatic hernia (CDH) have a high perinatal mortality rate, which remains around 60%, despite prenatal diagnosis and advances in neonatal care.~ Death has been attributed to associated anomalies, pulmonary hypoplasia, pulmonary hypertension, and type II cell dysfunction. The degree of pulmonary hypoplasia varies between infants born with CDH.2. 3 It is thought, in part, that intraabdominal viscera occupy space in the chest during critical periods of lung growth and development, and that the severity of pulmonary hypoplasia is related to both the timing of herniation and the volume of herniated viscera. This view is supported by the fact that smaller hernias and those that develop later in gestation are
I
From the Buffalo Institute of Fetal Therapy (B1FT), The ChiMren's Hospital of Buffale (Kaleida Health), Departments of Surgery, Pediatrics, OBGYN, and Microbiology, The State University of New York at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, NY. Presented at the 31st Annual Meeting of the American Pediatric Surgical Association, Orlando, Florida, May 25-29, 2000. Address reprint requests to Philip L. Glick, MD, FACS, FAAP, FSCCM, FACPE, 219 Bryant St, Buffalo, NY 14222. Copyright © 2001 by W.B. Saunders Company 0022-3468/01/3601-0022503.00/0 doi: l O.1053/jpsu.2001.20029 128
metric analysis was used to quantify the relative amounts of mRNA in each sample. Results: There was a significant decrease in the KGF protein levels of the CDH samples (110 v 73.2 pg/mg protein, P = .02). This decrease was mirrored by a significant fall in the level of mRNA expression for KGF (0.694 v 0.235, P = .02). Tracheal ligation normalized the KGF protein levels (96.1 pg/mg protein). This elevation of KGF protein was accompanied by an upregulation of KGF gene expression to control levels (0.56). Conclusions: Tracheal ligation clearly is accompanied by an upregulation of keratinocyte growth factor protein and gene expression, It is not yet clear whether keratinocyte growth factor is solely responsible for the growth observed in these tracheal ligation preparations. Further growth factor blocking experiments are required. J Pediatr Surg 36:128-132. Copyright © 2001 by W.B. Saunders Company. INDEX WORDS: Congenital diaphragmatic hernia, keratinocyte growth factor, tracheal ligation, fetal lung growth.
associated with lower mortality rate, 4 and by the observation in animal experiments, that intrathoracic masses result in pulmonary hypoplasia. 5 In utero tracheal ligation is a potent stimulus for fetal lung growth. Animal studies have found that tracheal occlusion accelerates lung growth in normal lungs 6 and in models of lung hypoplasia such as oligohydramnios,7 bilateral nephrectomy, 8 and congenital diaphragmatic hernia. 9 The latter observation supports the rationale of selective in utero tracheal ligation of those CDH fetuses with a very poor predicted prognosis. 10 Currently, the precise mechanism by which tracheal ligation augments fetal lung growth is not known. A pressure phenomenon has been postulated whereby the ligation acts as an endobronchial stent, stretching the developing lung, and possibly stimulating the release of specific growth factors. 1H3 However, no specific stretch receptors have yet been identified. The complex process of branching morphogenesis in the lung is mediated by transcription factors, peptide growth factors, and extracellular matrix signals that interact in a tightly coordinated temporospatial pattern of epithelial-mesenchymal interaction. ~4 Keratinocyte growth factor (KGF), which is produced in mesenchyme but acts via receptors positioned in epithelium, has been Journal of Pediatric Surgery, Vol 36, No 1 (January), 2001: pp 128-132
UPREGULATION OF KGF IN CDH
i m p l i c a t e d in t h e r e g u l a t i o n o f e a r l y l u n g b r a n c h i n g 15 a n d a l s o h a s b e e n s h o w n to b e a p o t e n t s t i m u l a t o r o f e p i t h e lial t y p e II p n e u m o c y t e s in v i v o a n d i n vitro. 16,17 T h e c u r r e n t s t u d y w a s t h e r e f o r e d e s i g n e d to i n v e s t i g a t e t h e p o s s i b l e i n v o l v e m e n t o f K G F in t h e i n d u c t i o n o f l u n g g r o w t h in t h e C D H t r a c h e a l l i g a t i o n l a m b m o d e l . MATERIALS AND METHODS
Experimental Design and Fetal Surgical Procedures These studies were approved by the Animal Care Committee of the State University of New York at Buffalo, NY. Eight fetal lambs underwent the surgical creation of a left-sided diaphragmatic hernia via an open hysterotomy at 80 days' gestation, as previously described. TM This stage of gestation in the sheep corresponds with the pseudoglandular phase of lung development. The pathophysiologic and morphometric changes seen in this model mirror those seen in human babies with CDH29,2° At 110 days' gestation, 4 animals underwent a second hysterotomy. The fetal head and neck were delivered, and the trachea was exposed through a transverse incision. The trachea was isolated and ligated with umbilical tape. The fetus was once more returned to the uterus and the hysterotomy closed. Nonoperated littermates served as controls. At 140 days' gestation, all the fetuses were delivered by cesarean section. Each animal was killed immediately, the chest opened, and the lungs perfused in situ with normal saline via the pulmonary artery. The heart and lungs were removed en bloc and the lungs perfused once again. The lung tissue was then snap frozen in liquid nitrogen and stored at -80°C.
Determination of KGF Protein Levels Lung tissue samples were weighed and suspended in cell lysis buffer as modified by Bui et al. zl The mixture was homogenized and centrifuged (500 g, for 6 minutes). The supernatant was lyophilized overnight. Samples of cell lysis buffer and KGF standard (R&D Systems, Minneapolis, MN) were treated similarly. Alter reconstitution with more cell lysis buffer, the supernatant from each sample was treated according to a KGF enzyme-linked immunosorbent assay (ELISA; R&D Systems). The optical density of each well was read by Microplate Manager 4.0 (Bio-Rad Laboratories, Inc, Hercules, CA) at 450 nm. The protein content of the supernatants was assayed according to the Micro-Lowry method. 22 Results were expressed as picograms per milligram of protein.
RNA Isolation Total cellular RNA was isolated from homogenized lung tissue by the acid guanidinium thiocyanate, phenol/chloroform method (denaturing solution: 4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 4), 0.5% N-lauroylsarcosine, and 0.1 M 2-mercaptoethanol) and resuspended in DEPC-treated water. Each RNA sample was quantified by spectrophotometry (Az6o/A2s0 ratio). The quality of the RNA was further assessed by electrophoresis on a 1% agarose gel containing × formaldehyde and staining with ethidium bromide. Only intact samples were used for RPA experiments. RNA samples were stored at -80°C.
KGF Probe Synthesis cDNA was obtained by reverse transcription (Superscript II reverse transcriptase, Gibco BRL, Carlsbad, CA) of total RNA purified from the lung tissue of one of the control samples using the manufacturer's instructions. The previously published sheep KGF sequence was used to design forward and reverse primers for a polymerase chain reaction
129 (PCR). 23 The forward primer was: 5'-TATCTTGCAATGAACAAGGAA-3', the reverse primer was: 5'-TTAAGTTATTGCCATAGG-3'. These oligonucleotides were synthesized at the Center for Advanced Molecular Biology and Immunology Facility (SUNY at Buffalo, Buffalo, NY). To amplify the KGF gene from the lung cDNA, a PCR was performed in a Perkin Elmer GeneAmp PCR System 2400 thermal cycler. The conditions for the PCR were: 94°C for 5 minutes (1 cycle); 94°C for 1 minute, 60°C for 2 minutes, 72°C for 3 minutes (30 cycles); 72°C for 10 minutes (1 cycle). The PCR products were fractionated on 1.5% agarose gels stained with 0.5 /xg/mL ethidium bromide and visualized by ultraviolet illumination. The PCR product was blunted ended,24 ligated into the phagemid vector pBSCII SK-, and used to transform DH5a-competent Escherichia coli cells (Gibco BRL) according to the manufacturer's instructions. Successful ligations were confirmed by colony PCR. 25 Phagemid DNA was prepared using the "alkaline lysis method. ''26 Phagemids were sequenced at the Center for Advanced Molecular Biology and Immunology DNA Sequencing Facility (SUNY at Buffalo, Buffalo, NY), using automated DNA sequencing technology. The sequences obtained were compared with the published sheep KGF sequence. A single base mismatch was found in a total of 234 bases. Sequence analysis of the KGF gene in the vector allowed determination of its orientation with respect to the T3 and T7 promoters encoded in the vector. The KGF-containing plasmid (pKGF) was linearized with EcoRI and used as a template for RNA synthesis.
RPA Assay A RiboQuant Multi-Probe RNase Protection Assay System (Pharmingen, San Diego, CA) was used to detect and quantitate the mRNA in the lung tissue samples. The KGF template was used in place of the recommended kit template set, and a human GAPDH template was used as a housekeeper. Each template was transcribed with T7 RNA polymerase and then both were hybridized overnight with the RNA samples, according to RiboQuant directions. The samples were then treated with RNase A, and the RNase digests were extracted and purified according to kit directions. The resultant protected digests were electrophoresed on a 5% denaturing polyacrylamide gel at 50 watts. The dried gel image was intensified and developed using a BioRad GS-525 Molecular Imager System. Quantification of the radioactivity of each sample was achieved using molecular Analyst v.l.5 software (BioRad Laboratories), and each sample value was compared with that of the housekeeper (hGAPDH).
Statistical Analysis Data are expressed as mean +_ SEM. The groups were compared using unpaired t tests. P values of less than .05 were considered statistically significant. RESULTS
W h o l e a n i m a l w e i g h t s f o r all g r o u p s w e r e c o m p a r a b l e . T o t a l w e t l u n g w e i g h t s w e r e s i g n i f i c a n t l y d e c r e a s e d in t h e C D H p r e p a r a t i o n s (53 + 8 v 124 - 14 g), P = 0.02, a n d s i g n i f i c a n t l y i n c r e a s e d i n t h e l i g a t i o n m o d e l s ( 1 7 0 -21 v 124 -+ 14 g), P = .03, w h e n c o m p a r e d w i t h c o n t r o l s .
KGF Protein Levels T h e r e w e r e n o s i g n i f i c a n t d i f f e r e n c e s in K G F p r o t e i n l e v e l b e t w e e n f i g h t a n d l e f t l u n g s in t h e 3 e x p e r i m e n t a l g r o u p s . T h e r e w a s a s i g n i f i c a n t d e c r e a s e in t h e K G F p r o t e i n l e v e l in t h e C D H s a m p l e s ( 7 3 . 2 -+ 19 p g / m g p r o t e i n ) c o m p a r e d w i t h c o n t r o l s ( 1 1 0 . 4 _+ 35.8 p g / m g p r o t e i n ) , P = .02. T r a c h e a l l i g a t i o n r e s t o r e d t h e K G F
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the gene expression of KGF have been restored back to control levels in the lung tissue of those CDH fetuses that were ligated. Although ligation has been shown to induce KGF production, it is still not clear if the subsequent lung growth is solely a response to the presence of KGF. There may be many other factors induced in response to ligation. For example, the saline replacement of lung fluid from tracheaMigated lambs inhibited the lung hypertrophy usually seen in the tracheal ligation model. 13 It was concluded that the tracheal fluid composition rather than intratracheal pressure was critical in promoting pulmonary growth. More specifically, we have previously identified that platelet-derived growth factor-BB (PDGF-BB) also is induced in the tracheal ligation lamb model of CDH. 27 In this context it is interesting to note the elegant in vitro experiments of Liu et al. 2s Using an in vitro organotypic fetal rat lung cell culture they showed that an intermittent mechanical strain regimen, which simulated fetal breathing movements, enhanced DNA synthesis, stimulated cell division, and increased the mRNA levels of PDGF-BB. Antisense PDGF-BB oligonucleotides abolished the strain-enhanced DNA synthesis and cell growth5 8 The upregulation of PDGF gene expression in response to strain suggests a mechanism for the transduction of a physical force causing fetal lung growth. In the current study, similar blocking experiments would help to clarify whether the growth effect caused by tracheal ligation was solely, or in part, a response to the presence of KGF. Despite the apparent positive effects of tracheal ligation, the intervention must be viewed with caution. Animal experiments have shown that the resultant lungs
120
/
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~ so ~ ao
2O •
CONTROL
r
CDH
-
-
LIGATION
Fig 1. Lung KGF protein content, expressed as picograms per milligram protein. There is a significant decrease in protein content in the CDH samples when compared with both controls and ligated samples (*P = .02).
protein level back to control levels (96.1 _+ 27.6 pg/mg protein), P = .2 (Fig 1).
KGF mRNA Expression The pattern of mRNA expression for KGF mirrored the pattern already seen with the levels of KGF protein in the 3 experimental groups. There was a significant decrease of mRNA KGF expression in the CDH group (0.235 -+ 0.09) when compared with controls, (0.694 -4- 0.22), P = .02. Tracheal ligation restored the level of expression back to control levels (0.56 _+ 0.24), P = .04 (Fig 2). DISCUSSION The precise mechanism by which tracheal ligation augments lung growth in the developing fetus is not known. The current study investigated whether keratinocyte growth factor was involved in the growth process induced by tracheal ligation of the CDH lamb model. It has been shown clearly that both KGF protein levels, and
KGFhGAPDHA
Z" J
0.8 0.7 0.6
0.5 0.4
.< z 0.3 E 0.2
0 0.1 0 B
CONTROL
l/ CDH
r
L1GATION
Fig 2. Expression of mRNA for KGF in lung tissue from control, CDH, and ligation samples. (A) RPA bands for KGF and hGAPDH. (B) Desitometric analysis of RPA bands. There is a significant fall in mRNA expression in the CDH samples (*P = .02); ligation has restored the mRNA expression back to control levels (**P = .04).
UPREGULATION OF KGF IN CDH
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are p h y s i o l o g i c a l l y i n s u f f i c i e n t a n d s u r f a c t a n t depleted. 29 T h e s u r f a c t a n t d e f i c i e n c y is b o t h q u a n t i t a t i v e , b e c a u s e o f a d r a m a t i c d e c r e a s e i n t h e d e n s i t y o f t y p e II p n e u m o c y t e s in l i g a t e d lungs, 3° a n d qualitative, as h i g h l i g h t e d b y t h e functional measure of poor choline incorporation of type II p n e u m o c y t e s i s o l a t e d f r o m l i g a t e d lungs. 31 M o r e recently, t h e t i m i n g o f l i g a t i o n h a s b e e n s h o w n to b e o f critical i m p o r t a n c e i n t h e b a l a n c e b e t w e e n a c h i e v i n g sufficient t i m e for a d e q u a t e l u n g g r o w t h , w h i l e a v o i d i n g d e t r i m e n t a l effects o n the t y p e II cells. 32 K n o w l e d g e r e g a r d i n g K G F i n d u c t i o n in t h e d i f f e r e n t t i m e f r a m e s o f
t h e s e e x p e r i m e n t s m i g h t o f f e r f u r t h e r i n s i g h t into the m e c h a n i s t i c effects o f K G F a n d u l t i m a t e l y a s s e s s i n g its potential use therapeutically. Fetuses with C D H w h o h a v e a p o o r prognosis n o w can b e offered in utero tracheal occlusion b y fetoscopic surgery in 1 center. 1° A prospective r a n d o m i z e d clinical trial o f the treamaent has n o t b e e n carried out. It is therefore o f p r i m e i m p o r t a n c e that the u n d e r l y i n g m e c h a n i s m s operating to produce lung g r o w t h in the p r e s e n c e o f tracheal ligation are further investigated and u n d e r s t o o d before a m o r e widespread clinical application o f the practice.
R E FE R E N C E S
1. Harrison MR, Adzick NS, Estes JM: A prospective study of the outcome for fetuses with diaphragmatic hernia. JAMA 271:382-384, 1994 2. Berdon WE, Baker DH, Amoury R: The role of pulmonary hypoplasia in the prognosis of newborn infants with diaphragmatic hernia and eventration. Am J Roentgenol 103:413-421, 1968 3. Nguyen L, Guttman FM, de Chadarevian JP: The mortality of congenital diaphragmatic hernia: Is total pulmonary mass inadequate, no matter what? Ann Surg 198:766-770, 1983 4. Adzick NS, Harrison MR, Glick PL: Diaphragmatic hernia in the fetus: Prenatal diagnosis and outcome in 94 cases. J Pediatr Surg 20:357-361, 1985 5. Harrison MR, Jester JA, Ross NA: Correction of congenital diaphragmatic hernia in utero I. The Model: Lntrathoracic balloon produces fatal pulmonary hypoplasia. Surgery 88:174-182, 1980 6. Alcorn D, Adamson TM, Lambert TF: Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J Anat 123:649-660, 1977 7. Adzick NS, Harrison MR, Glick PL: Experimental pulmonary hypoplasia and oligohydramnios: Relative contributions of lung fluid and fetal breathing movements. J Pediatr Surg 19:658-665, 1984 8. Wilson JM, DiFore JW, Peters CA: Experimental fetal tracheal ligafion prevents the pulmonary hypoplasia associated with fetal nephrectomy: Possible application for congenital diaphragmatic hernia. J Pediatr Surg 28:1433-1439, 1993 9. Hedrick MH, Estes JM, Sullivan KM: Plug the lung until it grows (PLUG): A new method to treat congenital diaphragmatic hernia in utero. J Pediatr Surg 29:612-617, 1994 10. Harrison MR, Mychaliska GB, Albanese CT, et al: Correction of congenital diaphragmatic hernia in utero IX; Fetuses with poor prognosis (Liver herniation and low lung-to-head ratio) can be saved by fetoscopic temporary tracheal occlusion. J Pediatr Surg 33:1017-1023, 1998 11. DiFiore JW, Fauza DO, Slavin R: Experimental fetal tracheal ligation reverses the structural and physiological effects of pulmonary hypoplasia in congenital diaphragmatic hernia. J Pediatr Surg 29:248255, 1994 12. Liu M, Skinner SJ, Xu J: Stimulation of fetal rat lung cell proliferation in vitro by mechanical stretch. Am J Physiol 263:376-383, 1992 13. Papadakis K, Luks FI, De Paepe ME, et al: Fetal lung growth after tracheal ligation is not solely a pressure phenomenon. J Pediatr Snrg 32:347-351, 1997 14. Warburton D, Lee MK: Current concepts on lung development. Curt Opin Pediatr 11:188-192, 1999 15. Post M, Souza P, Liu J, et al: Keratinocyte growth factor and its receptor are involved in regulating early lung branching. Development 122:3107-3115, 1996 16. Panos RJ, Rubin JS, Csaky KG, et al: Keratinocyte growth factor and hepatocyte growth factor are heparin-binding factors for
alveolar type II cells in fibroblast-conditioned medium. J Clin Invest 92:969-977, 1993 17. Ulich TR, Yi ES, Longmuir K, et al: Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo. J Clin Invest 93:1298-1306, 1994 18. Glick PL, Stannard VA, Leach CL: Pathophysiology of congenital diaphragmatic hernia II. The fetal lamb model is surfactant deficient. J Pediatr Surg 27:382-388, 1992 19. de Lorimier AA, Tierney DF, Parker HR: Hypoplastic lungs in fetal lambs with surgically produced diaphragmatic hernia. Surgery 62:12-17, 1967 20. Adzick NS, Outwater KM, Harrison MR: Correction of congenital diaphragmatic hernia in utero IV. An early gestational fetal lamb model for pulmonary vascular morphometric analysis. J Pediatr Snrg 20:673-680, 1985 21. Bui KC, Buckley S, Wu F: Induction of A and D type cyclins and cdc2 kinase activity during recovery from short-term hyperoxic lung injury. Am J Physiol 12:L625-636, 1995 22. Lowry DH, Rosebrough NJ, Fan" AL, et al: Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275, 1951 23. Doyle MP, Morris CF, Biltz RE: Keratinocyte growth factor and keratinocyte growth factor receptor orthologs [monkey, pig, rabbit]. In Vitro Cell Dev Biol 32:318-320, 1996 24. Bhat GJ, Lodes MJ, Myler PJ, et al: A simple method for cloning blunt ended DNA fragments. Nucl Acids Res 19:398, 1991 25. Chomczynsld P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analyt Biochem 162:156-159, 1987 26. Ausbel FA, Brent R, Kingston RE, et al: Alkaline lysis DNA preparation, in Current Protocols in Molecular Biology. New York, NY, John Wiley and Sons, 1999 27. McCabe AJ, Patel A, Kapur P, et al: Keratinocyte and platelet derived growth factors in the tracheal ligation lamb model of congenital diaphragmatic hernia. Am J Respir Crit Care Med 159:A665, 1999 28. Liu M, Liu J, Buch S, et al: Antisense oligonucleotides for PDGF-BB and its receptor inhibit mechanical strain-induced fetal lung cell growth. Am J Physiol 269:L178-L184, 1995 29. O'Toole SJ, Sharma A, Karamanoukian HL, et al: Tracheal ligation does not correct the surfactant deficiency associated with congenital diaphragmatic hernia. J Pediatr Surg 31:546-550, 1996 30. Piedboeuf B, Laberge J, Ghitulescu G, et al: Deleterious effect of tracheal obstruction on type lI pneumocytes in fetal sheep. Pediatr Res 41:473-479, 1997 31. Wilcox DT, Glick PL, Karamanoukian HL, et al: Contributions by individual lungs to the surfactant status in congenital diaphragmatic hernia. Pediatr Res 41:686-691, 1997 32. Paepe ME, Johnson BD, Papadakis K, et al: Lung growth response after tracheal occlusion in fetal rabbits is gestational agedependent. Am J Respir Cell Biol 21:65-76, 1999
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Discussion A. Haller (Baltimore, MD): I would like to ask you the question I asked earlier but now directed to your experimental model. If the occlusion is begun earlier than the time that you have, do you have data to suggest that there is a difference in the level of the growth factor? The second question is, if it is released, is there a diminution in this concentration suggesting that it is truly the obstruction that is the reason or are there other factors associated with the hypoplasia? A.J. McCabe (response): We have not done any earlier timing experiments, so I cannot comment about that.
Again, with the release experiments we have not looked at the growth factor in those tissues just yet. D. Teitelbaum (Ann Arbor, MI): We do similar KGF work in intestine--have you looked at whether a major source of KGF is lymphocytic? It is intraepithelial lymphocytes that are also found in the lung. Have you looked at that as a potential etiology? ff not, perhaps a useful way of approaching that would be to eliminate your CD8-positive population because the Gamma delta CD8-positive population is the source of KGF in those 2 sources. A.J. McCabe (response): We have only looked at total lung tissue, but, yes, that is a good thought.