Fas-Ligand-Induced Apoptosis of Respiratory Epithelial Cells Causes Disruption of Postcanalicular Alveolar Development
2008; Elsevier BV; Volume: 173; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2008.071123
ISSN1525-2191
AutoresMonique E. De Paepe, Sravanthi Gundavarapu, Umadevi Tantravahi, John R. Pepperell, Sheila A. Haley, François I. Luks, Quanfu Mao,
Tópico(s)Congenital Diaphragmatic Hernia Studies
ResumoPremature infants are at risk for bronchopulmonary dysplasia, a complex condition characterized by impaired alveolar development and increased alveolar epithelial apoptosis. The functional involvement of pulmonary apoptosis in bronchopulmonary dysplasia- associated alveolar disruption remains undetermined. The aims of this study were to generate conditional lung-specific Fas-ligand (FasL) transgenic mice and to determine the effects of FasL-induced respiratory epithelial apoptosis on alveolar remodeling in postcanalicular lungs. Transgenic (TetOp)7-FasL responder mice, generated by pronuclear microinjection, were bred with Clara cell secretory protein (CCSP)-rtTA activator mice. Doxycycline (Dox) was administered from embryonal day 14 to postnatal day 7, and lungs were studied between embryonal day 19 and postnatal day 21. Dox administration induced marked respiratory epithelium-specific FasL mRNA and protein up-regulation in double-transgenic CCSP-rtTA+/(TetOp)7-FasL+ mice compared with single-transgenic CCSP-rtTA+ littermates. The Dox-induced FasL up-regulation was associated with dramatically increased apoptosis of alveolar type II cells and Clara cells, disrupted alveolar development, decreased vascular density, and increased postnatal lethality. These data demonstrate that FasL-induced alveolar epithelial apoptosis during postcanalicular lung remodeling is sufficient to disrupt alveolar development after birth. The availability of inducible lung-specific FasL transgenic mice will facilitate studies of the role of apoptosis in normal and disrupted alveologenesis and may lead to novel therapeutic approaches for perinatal and adult pulmonary diseases characterized by dysregulated apoptosis. Premature infants are at risk for bronchopulmonary dysplasia, a complex condition characterized by impaired alveolar development and increased alveolar epithelial apoptosis. The functional involvement of pulmonary apoptosis in bronchopulmonary dysplasia- associated alveolar disruption remains undetermined. The aims of this study were to generate conditional lung-specific Fas-ligand (FasL) transgenic mice and to determine the effects of FasL-induced respiratory epithelial apoptosis on alveolar remodeling in postcanalicular lungs. Transgenic (TetOp)7-FasL responder mice, generated by pronuclear microinjection, were bred with Clara cell secretory protein (CCSP)-rtTA activator mice. Doxycycline (Dox) was administered from embryonal day 14 to postnatal day 7, and lungs were studied between embryonal day 19 and postnatal day 21. Dox administration induced marked respiratory epithelium-specific FasL mRNA and protein up-regulation in double-transgenic CCSP-rtTA+/(TetOp)7-FasL+ mice compared with single-transgenic CCSP-rtTA+ littermates. The Dox-induced FasL up-regulation was associated with dramatically increased apoptosis of alveolar type II cells and Clara cells, disrupted alveolar development, decreased vascular density, and increased postnatal lethality. These data demonstrate that FasL-induced alveolar epithelial apoptosis during postcanalicular lung remodeling is sufficient to disrupt alveolar development after birth. The availability of inducible lung-specific FasL transgenic mice will facilitate studies of the role of apoptosis in normal and disrupted alveologenesis and may lead to novel therapeutic approaches for perinatal and adult pulmonary diseases characterized by dysregulated apoptosis. Preterm infants who require assisted ventilation and supplemental oxygen are at risk for bronchopulmonary dysplasia (BPD), a chronic lung disease of newborn infants associated with significant mortality and long-term morbidity.1Jobe AH Bancalari E Bronchopulmonary dysplasia.Am J Respir Crit Care Med. 2001; 163: 1723-1729Crossref PubMed Scopus (3434) Google Scholar, 2Lemons JA Bauer CR Oh W Korones SB Papile LA Stoll BJ Verter J Temprosa M Wright LL Ehrenkranz RA Fanaroff AA Stark A Carlo W Tyson JE Donovan EF Shankaran S Stevenson DK Very low birth weight outcomes of the National Institute of Child health and human development neonatal research network, January 1995 through December 1996 NICHD Neonatal Research Network.Pediatrics. 2001; 107: E1Crossref PubMed Google Scholar The pathological hallmark of BPD in the postsurfactant era is an impairment of alveolar development, resulting in large and simplified airspaces that show little evidence of vascularized ridges (secondary crests) or alveolar septa.3Husain AN Siddiqui NH Stocker JT Pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia.Hum Pathol. 1998; 29: 710-717Abstract Full Text PDF PubMed Scopus (543) Google Scholar, 4Jobe AJ The new BPD: an arrest of lung development.Pediatr Res. 1999; 46: 641-643Crossref PubMed Scopus (790) Google Scholar In addition, the lungs of infants with BPD show structurally abnormal microvasculature and variable degrees of interstitial fibrosis.5Coalson JJ Pathology of chronic lung disease in early infancy.in: Bland RD Coalson JJ Chronic Lung Disease in Early Infancy. 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Among these, prematurity, oxygen toxicity, and barotrauma are considered central to a final common outcome.1Jobe AH Bancalari E Bronchopulmonary dysplasia.Am J Respir Crit Care Med. 2001; 163: 1723-1729Crossref PubMed Scopus (3434) Google Scholar, 8Palta M Gabbert D Weinstein MR Peters ME Multivariate assessment of traditional risk factors for chronic lung disease in very low birth weight neonates. The Newborn Lung Project.J Pediatr. 1991; 119: 285-292Abstract Full Text PDF PubMed Scopus (152) Google Scholar There are variable contributions of infection/inflammation, glucocorticoid exposure, chorioamnionitis, and genetic polymorphisms.1Jobe AH Bancalari E Bronchopulmonary dysplasia.Am J Respir Crit Care Med. 2001; 163: 1723-1729Crossref PubMed Scopus (3434) Google Scholar, 9Jobe AH Antenatal factors and the development of bronchopulmonary dysplasia.Semin Neonatol. 2003; 8: 9-17Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar The precise mechanisms whereby these predisposing conditions result in disrupted alveolar development remain primarily unknown. Our research efforts in recent years have focused on the role of alveolar epithelial apoptosis in postcanalicular alveolar development. We10De Paepe ME Mao Q Embree-Ku M Rubin LP Luks FI Fas/FasL-mediated apoptosis in perinatal murine lungs.Am J Physiol. 2004; 287: L730-L742Crossref Scopus (70) Google Scholar, 11De Paepe ME Rubin LP Jude C Lesieur-Brooks AM Mills DR Luks FI Fas ligand expression coincides with alveolar cell apoptosis in late-gestation fetal lung development.Am J Physiol. 2000; 279: L967-L976Google Scholar, 12De Paepe ME Sardesai MP Johnson BD Lesieur-Brooks AM Papadakis K Luks FI The role of apoptosis in normal and accelerated lung development in fetal rabbits.J Pediatr Surg. 1999; 34: 863-870Abstract Full Text PDF PubMed Scopus (38) Google Scholar and others13Kresch MJ Christian C Wu F Hussain N Ontogeny of apoptosis during lung development.Pediatr Res. 1998; 43: 426-431Crossref PubMed Scopus (94) Google Scholar, 14Schittny JC Djonov V Fine A Burri PH Programmed cell death contributes to postnatal lung development.Am J Respir Cell Mol Biol. 1998; 18: 786-793Crossref PubMed Scopus (157) Google Scholar have demonstrated that moderate and precisely timed alveolar epithelial type II cell apoptosis is an integral component of physiological postcanalicular lung remodeling in mice, rats, and rabbits. Although the exact biological role of apoptosis in alveologenesis remains uncertain, its choreographed occurrence across mammalian species strongly suggests apoptotic elimination of surplus type II cells during perinatal alveolar remodeling is a naturally occurring and developmentally relevant event. Although moderate and well timed apoptosis appears to represent a physiological phenomenon during postcanalicular lung development, we speculate that exaggerated and/or premature alveolar epithelial apoptosis may play a critical role in the pathogenesis of BPD-associated alveolar disruption. Several recent reports described increased levels of alveolar epithelial apoptosis in the lungs of ventilated preterm infants with respiratory distress syndrome or early BPD.15Hargitai B Szabo V Hajdu J Harmath A Pataki M Farid P Papp Z Szende B Apoptosis in various organs of preterm infants: histopathologic study of lung, kidney, liver, and brain of ventilated infants.Pediatr Res. 2001; 50: 110-114Crossref PubMed Scopus (63) Google Scholar, 16Lukkarinen HP Laine J Kaapa PO Lung epithelial cells undergo apoptosis in neonatal respiratory distress syndrome.Pediatr Res. 2003; 53: 254-259Crossref PubMed Scopus (38) Google Scholar, 17May M Strobel P Preisshofen T Seidenspinner S Marx A Speer CP Apoptosis and proliferation in lungs of ventilated and oxygen-treated preterm infants.Eur Respir J. 2004; 23: 113-121Crossref PubMed Scopus (58) Google Scholar The temporal patterns of apoptosis and alveolar disruption in ventilated preterm lungs are suggestive of a causative relationship; however, functional involvement of alveolar epithelial apoptosis in disrupted alveologenesis has not been demonstrated thus far. In this study, we used a gain-of-function approach to determine the functional role of alveolar epithelial apoptosis in alveolar remodeling. Enhanced respiratory epithelial apoptosis was achieved by means of a transgenic Fas-ligand (FasL) overexpression system. 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Subsequent activation of the effector caspases through mitochondria-dependent or -independent pathways results in activation of caspase-3, the key effector caspase. Activated caspase-3 cleaves a variety of substrates, including DNA repair enzymes, cellular and nuclear structural proteins, endonucleases, and many other cellular constituents, culminating in effective cell death.18Nagata S Fas ligand-induced apoptosis.Annu Rev Genet. 1999; 33: 29-55Crossref PubMed Scopus (670) Google Scholar, 19Sharma K Wang RX Zhang LY Yin DL Luo XY Solomon JC Jiang RF Markos K Davidson W Scott DW Shi YF Death the Fas way: regulation and pathophysiology of CD95 and its ligand.Pharmacol Ther. 2000; 88: 333-347Crossref PubMed Scopus (184) Google Scholar, 20Walczak H Krammer PH The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems.Exp Cell Res. 2000; 256: 58-66Crossref PubMed Scopus (546) Google Scholar, 21Wajant H The Fas signaling pathway: more than a paradigm.Science. 2002; 296: 1635-1636Crossref PubMed Scopus (716) Google Scholar Selection of the Fas/FasL system as inducer of alveolar epithelial apoptosis for this study was a logical choice. First, we previously demonstrated that perinatal murine respiratory epithelial cells are exquisitely sensitive to Fas-mediated apoptosis in vitro and in vivo.10De Paepe ME Mao Q Embree-Ku M Rubin LP Luks FI Fas/FasL-mediated apoptosis in perinatal murine lungs.Am J Physiol. 2004; 287: L730-L742Crossref Scopus (70) Google Scholar, 22De Paepe ME Mao Q Chao Y Powell JL Rubin LP Sharma S Hyperoxia-induced apoptosis and Fas/FasL expression in lung epithelial cells.Am J Physiol. 2005; 289: L647-L659Google Scholar Furthermore, the Fas/FasL signaling pathway lends itself better to experimental manipulation than other, in particular intrinsic (mitochondrial-dependent) pathways. 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We previously tested the in vivo effect of Fas-activation in perinatal lungs by systemic administration of a Fas-activating antibody to newborn mice.10De Paepe ME Mao Q Embree-Ku M Rubin LP Luks FI Fas/FasL-mediated apoptosis in perinatal murine lungs.Am J Physiol. 2004; 287: L730-L742Crossref Scopus (70) Google Scholar This approach allowed us to demonstrate that perinatal alveolar epithelial cells are susceptible to Fas-mediated apoptosis.10De Paepe ME Mao Q Embree-Ku M Rubin LP Luks FI Fas/FasL-mediated apoptosis in perinatal murine lungs.Am J Physiol. 2004; 287: L730-L742Crossref Scopus (70) Google Scholar However, systemic Fas activation resulted in rapid death from liver failure before the effects of exaggerated alveolar apoptosis on alveolar remodeling could be ascertained. To circumvent the deleterious effects of prolonged and systemic FasL exposure, we generated a tetracycline-inducible (tet-on) lung epithelial-specific FasL-overexpressing mouse, adapted from the Tet system of Gossen and colleagues,34Gossen M Freundlieb S Bender G Muller G Hillen W Bujard H Transcriptional activation by tetracyclines in mammalian cells.Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2026) Google Scholar to target apoptosis to respiratory epithelial cells during perinatal lung development. Our results demonstrate that increased apoptosis of respiratory epithelial cells during postcanalicular alveolar remodeling is sufficient to disrupt alveolar development and results in BPD-like alveolar simplification. These findings support our hypothesis that excessive or premature postcanalicular alveolar epithelial apoptosis is a pivotal event in the pathogenesis of BPD. Elucidation of the role and regulation of postcanalicular alveolar epithelial apoptosis may result in important insights into the regulation of alveologenesis and the pathogenesis of BPD. This, in turn, may open new therapeutic opportunities for the prevention and treatment of this disease, as well as other pulmonary conditions associated with dysregulated alveolar epithelial apoptosis, such as acute lung injury, emphysema, and neoplasia. The tetracycline-inducible system in vivo consists of two independent transgenic mouse lines, an activator line and a responder line. The activator line expresses the reverse tetracycline responsive transactivator (rtTA) in a tissue- or cell-specific manner, whereas the responder line carries a transgene of interest under control of the tet-operator (TetOp). We selected a tet-on tetracycline-dependent overexpression system to achieve conditional respiratory epithelium-specific FasL transgene expression. In the tet-on system, transgene expression is induced by binding of the tetracycline analogue, doxycycline (Dox) to rtTA, which in turn activates the (tetOp)7-CMV target promoter, activating transcription of the gene of interest.34Gossen M Freundlieb S Bender G Muller G Hillen W Bujard H Transcriptional activation by tetracyclines in mammalian cells.Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2026) Google Scholar, 35Kistner A Gossen M Zimmermann F Jerecic J Ullmer C Lubbert H Bujard H Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice.Proc Natl Acad Sci USA. 1996; 93: 10933-10938Crossref PubMed Scopus (661) Google Scholar Tetracycline-dependent transgene expression was targeted to respiratory epithelial cells by using transgenic CCSP-rtTA activator mice in which the rtTA is placed under control of Clara cell secretory protein (CCSP, CC-10) promoter elements.36Whitsett JA Clark JC Picard L Tichelaar JW Wert SE Itoh N Perl AK Stahlman MT Fibroblast growth factor 18 influences proximal programming during lung morphogenesis.J Biol Chem. 2002; 277: 22743-22749Crossref PubMed Scopus (67) Google Scholar, 37Perl AK Tichelaar JW Whitsett JA Conditional gene expression in the respiratory epithelium of the mouse.Transgenic Res. 2002; 11: 21-29Crossref PubMed Scopus (192) Google Scholar The specific CCSP-rtTA transactivator mice used for these studies have been shown previously to be robust activators that effectively drive transgene expression not only in nonciliated bronchial epithelial (Clara) cells, but also in alveolar type II cells.36Whitsett JA Clark JC Picard L Tichelaar JW Wert SE Itoh N Perl AK Stahlman MT Fibroblast growth factor 18 influences proximal programming during lung morphogenesis.J Biol Chem. 2002; 277: 22743-22749Crossref PubMed Scopus (67) Google Scholar, 37Perl AK Tichelaar JW Whitsett JA Conditional gene expression in the respiratory epithelium of the mouse.Transgenic Res. 2002; 11: 21-29Crossref PubMed Scopus (192) Google Scholar, 38Tichelaar JW Lu W Whitsett JA Conditional expression of fibroblast growth factor-7 in the developing and mature lung.J Biol Chem. 2000; 275: 11858-11864Crossref PubMed Scopus (274) Google Scholar, 39Clark JC Tichelaar JW Wert SE Itoh N Perl AK Stahlman MT Whitsett JA FGF-10 disrupts lung morphogenesis and causes pulmonary adenomas in vivo.Am J Physiol. 2001; 280: L705-L715Google Scholar, 40Zhu Z Ma B Homer RJ Zheng T Elias JA Use of the tetracycline-controlled transcriptional silencer (tTS) to eliminate transgene leak in inducible overexpression transgenic mice.J Biol Chem. 2001; 276: 25222-25229Crossref PubMed Scopus (110) Google Scholar, 41Zheng T Zhu Z Wang Z Homer RJ Ma B Riese Jr, RJ Chapman Jr, HA Shapiro SD Elias JA Inducible targeting of IL-13 to the adult lung causes matrix metalloproteinase- and cathepsin-dependent emphysema.J Clin Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (540) Google Scholar, 42Yang L Naltner A Yan C Overexpression of dominant negative retinoic acid receptor alpha causes alveolar abnormality in transgenic neonatal lungs.Endocrinology. 2003; 144: 3004-3011Crossref PubMed Scopus (36) Google Scholar The 2.3-kb rat CCSP promoter element used in these activator mice is thus expressed differently from the native murine CCSP gene, which is limited to Clara cells.43Stripp BR Sawaya PL Luse DS Wikenheiser KA Wert SE Huffman JA Lattier DL Singh G Katyal SL Whitsett JA cis-Acting elements that confer lung epithelial cell expression of the CC10 gene.J Biol Chem. 1992; 267: 14703-14712Abstract Full Text PDF PubMed Google Scholar Consistent with the endogenous expression pattern of CCSP, which is active from embryonal day 12.5 (E12.5) onward, CCSP-rtTA activator mice have been shown to be particularly suitable for studies of gene function in late gestation and postnatal lungs.37Perl AK Tichelaar JW Whitsett JA Conditional gene expression in the respiratory epithelium of the mouse.Transgenic Res. 2002; 11: 21-29Crossref PubMed Scopus (192) Google Scholar CCSP-rtTA activator mice, generated in a FVB/N background, were a generous gift from Dr. J. Whitsett37Perl AK Tichelaar JW Whitsett JA Conditional gene expression in the respiratory epithelium of the mouse.Transgenic Res. 2002; 11: 21-29Crossref PubMed Scopus (192) Google Scholar, 38Tichelaar JW Lu W Whitsett JA Conditional expression of fibroblast growth factor-7 in the developing and mature lung.J Biol Chem. 2000; 275: 11858-11864Crossref PubMed Scopus (274) Google Scholar (Cincinnati Children's Hospital Medical Center, Cincinnati, OH). To generate (tetOp)7-FasL responder mice, the 943-bp murine FasL cDNA containing the entire coding region of the protein, a kind gift from Dr. S. Nagata44Takahashi T Tanaka M Brannan CI Jenkins NA Copeland NG Suda T Nagata S Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand.Cell. 1994; 76: 969-976Abstract Full Text PDF PubMed Scopus (1468) Google Scholar (Osaka University Medical School, Osaka, Japan), was subcloned between a CMV minimal promoter and bovine growth hormone intronic and polyadenylation sequences in the pTRE2 vector (BD Biosciences, Franklin Lakes, NJ) (Figure 1). Restriction enzyme digestion and direct sequencing confirmed the orientation of the insert. The transgene was microinjected into fertilized FVB/N oocytes at the Cincinnati Children's Hospital Transgenic Core Facility. Founders were identified by transgene-specific polymerase chain reaction (PCR) using upstream primer 5′-CGCCTGGAGACGCCATC-3′ (pTRE2) or 5′-GTGCCATGCAGCAGCCCATGA-3′ (FasL) and downstream primer 5′-CCATTCTAAACAACACCCTG-3′ (pTRE2). Five (tetOp)7-FasL transgenic mouse lines were expanded in our laboratory. Genotype, transgene copy number, and number of integration sites were determined by Southern blot hybridization initially, and monitored by real-time PCR subsequently (FasL primers, PPM02926A; SuperArray Bioscience Corp., Frederick, MD). Transgenic (tetOp)7-FasL progeny derived from founders A through E were crossed with CCSP-rtTA mice to yield a mixed offspring of double-transgenic (CCSP-rtTA+/(tetOp)7-FasL+) and single-transgenic (CCSP-rtTA+/(tetOp)7-FasL−) littermates. For the sake of brevity, double-transgenic mice will be denoted in the text as CCSP+/FasL+ mice, whereas single-transgenic mice will be denoted as CCSP+/FasL− mice. Dox (1.0 mg/ml) was added to the drinking water of pregnant and nursing dams between E14 and postnatal day 7 (P7). The progeny (CCSP+/FasL+ and CCSP+/FasL−) were sacrificed at E19, P7 (early alveolarization stage45Amy RW Bowes D Burri PH Haines J Thurlbeck WM Postnatal growth of the mouse lung.J Anat. 1977; 124: 131-151PubMed Google Scholar), or P21 (late alveolarization stage) by pentobarbital overdose. The cages were inspected twice daily to record interval postnatal death. Body and wet lung weights were recorded. Pups were genotyped by PCR analysis of tail genomic DNA using the primers described above. For molecular analysis, lungs were snap-frozen in liquid nitrogen and stored at −80°C. For morphological studies, fetal lungs were immersion-fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline, pH 7.4. The lungs of newborn mice were fixed by tracheal instillation of paraformaldehyde at a constant pressure of 20 cm H2O. After overnight fixation, the lungs were dehydrated in graded ethanol solutions, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Controls consisted of Dox-treated single-transgenic CCSP+/FasL− littermates, and age-matched CCSP+/FasL+ and CCSP+/FasL− animals that were not treated with Dox. All animal experiments were conducted in accordance with institutional guidelines for the care and use of laboratory animals. Protocols were approved through the Institutional Animal Care and Use Committee. Alveolar type II cells were isolated from fetal mice (E19) by a modification of the methods described by Corti and colleagues46Corti M Brody AR Harrison JH Isolation and primary culture of murine alveolar type II cells.Am J Respir Cell Mol Biol. 1996; 14: 309-315Crossref PubMed Scopus (275) Google Scholar and Rice and colleagues,47Rice WR Conkright JJ Na CL Ikegami M Shannon JM Weaver TE Maintenance of the mouse type II cell phenotype in vitro.Am J Physiol. 2002; 283: L256-L264Google Scholar as described in detail elsewhere.10De Paepe ME Mao Q Embree-Ku M Rubin LP Luks FI Fas/FasL-mediated apoptosis in perinatal murine lungs.Am J Physiol. 2004; 287: L730-L742Crossref Scopus (70) Google Scholar Briefly, type II cells were isolated by protease digestion and differential adherence to CD45- and CD32-coated dishes. After isolation and purification, the cells were resuspended in culture medium (HEPES-buffered Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin). Purity was assessed by modified Papanicolaou stain48Dobbs LG Isolation and culture of alveolar type II cells.Am J Physiol. 1990; 258: L134-L147PubMed Google Scholar and anti-SP-C immunohistochemistry.10De Paepe ME Mao Q Embree-Ku M Rubin LP Luks FI Fas/FasL-mediated apoptosis in perinatal murine lungs.Am J Physiol. 2004; 287: L730-L742Crossref Scopus (70) Google Scholar Viability was assessed by trypan blue exclusion. After 24 hours, cells were assayed for FasL mRNA expression as detailed below. FasL mRNA levels were quantified by real-time PCR analysis. Total cellular RNA was extracted from whole lung or cell lysates using Trizol reagent (Invitrogen, Carlsbad, CA). Total RNA (2 μg) was DNase-treated (Turbo DNA-free kit; Ambion, Austin, TX) and reverse-transcribed using the reverse transcriptase2Lemons JA Bauer CR Oh W Korones SB Papile LA Stoll BJ Verter J Temprosa M Wright LL Ehrenkranz RA Fanaroff AA Stark A Carlo W Tyson JE Donovan EF Shankaran S Stevenson DK Very low birth weight outcomes of the National Institute of Child health and human development neonatal research network, January 1995 through December 1996 NICHD Neonatal Research Network.Pediatrics. 2001; 107: E1Crossref PubMed Google Scholar first strand kit (SuperArray BioScience) according to the manufacturer's protocols. The cDNA templates were amplified with mouse β-actin (Superarray catalog no. PPM02945A) and FasL (PPM02926A) primer pairs in independent sets of PCR using reverse transcriptase.2Lemons JA Bauer CR Oh W Korones SB Papile LA Stoll BJ Verter J Temprosa M Wright
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