The Pulmonary Mesenchymal Tissue Layer Is Defective in an in Vitro Recombinant Model of Nitrofen-Induced Lung Hypoplasia
2011; Elsevier BV; Volume: 180; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2011.09.032
ISSN1525-2191
AutoresRhiannon B. van Loenhout, Irene Tseu, Emily Fox, Zhen Huang, Dick Tibboel, Martin Post, Richard Keijzer,
Tópico(s)Pleural and Pulmonary Diseases
ResumoDespite modern treatments, congenital diaphragmatic hernia (CDH) remains associated with variable survival and significant morbidity. The associated pulmonary hypoplasia is a major determinant of outcome. To develop better treatments, improved comprehension of the pathogenesis of lung hypoplasia is warranted. We developed an in vitro cell recombinant model to mimic pulmonary hypoplasia and specifically to investigate epithelial-mesenchymal interactions and to decipher which tissue layer is primarily defective in nitrofen-induced CDH-associated lung hypoplasia. Epithelial cells (E) and fibroblasts (F) were isolated from E19 control (C) and nitrofen-induced hypoplastic rat lungs (N). Cells were recombined and cultured as either homotypic [(FC)(EC) and (FN)(EN)] or heterotypic [(FC)(EN) and (FN)(EC)] recombinants. Recombinants containing FN fibroblasts had a thickened fibroblast tissue layer and there were fewer organized alveolar-like epithelial structures compared with those in control (FC)(EC) recombinants. These FN recombinants exhibited a decrease in terminal deoxynucleotidyl transferase dUTP nick end labeling and cleaved caspase-3 positive cells. Cell proliferation was arrested in recombinants containing FN fibroblasts, which also exhibited increased p27Kip1 and p57Kip2 expression. In conclusion, fibroblasts, and not epithelial cells, appear to be the defective cell type in nitrofen-induced hypoplastic lungs due to a decreased ability to undergo apoptosis and maintain overall proliferation. This may explain the characteristic pulmonary interstitial thickening and hypoplasia observed in both nitrofen-induced hypoplastic lungs as well as human hypoplastic CDH lungs. Despite modern treatments, congenital diaphragmatic hernia (CDH) remains associated with variable survival and significant morbidity. The associated pulmonary hypoplasia is a major determinant of outcome. To develop better treatments, improved comprehension of the pathogenesis of lung hypoplasia is warranted. We developed an in vitro cell recombinant model to mimic pulmonary hypoplasia and specifically to investigate epithelial-mesenchymal interactions and to decipher which tissue layer is primarily defective in nitrofen-induced CDH-associated lung hypoplasia. Epithelial cells (E) and fibroblasts (F) were isolated from E19 control (C) and nitrofen-induced hypoplastic rat lungs (N). Cells were recombined and cultured as either homotypic [(FC)(EC) and (FN)(EN)] or heterotypic [(FC)(EN) and (FN)(EC)] recombinants. Recombinants containing FN fibroblasts had a thickened fibroblast tissue layer and there were fewer organized alveolar-like epithelial structures compared with those in control (FC)(EC) recombinants. These FN recombinants exhibited a decrease in terminal deoxynucleotidyl transferase dUTP nick end labeling and cleaved caspase-3 positive cells. Cell proliferation was arrested in recombinants containing FN fibroblasts, which also exhibited increased p27Kip1 and p57Kip2 expression. In conclusion, fibroblasts, and not epithelial cells, appear to be the defective cell type in nitrofen-induced hypoplastic lungs due to a decreased ability to undergo apoptosis and maintain overall proliferation. This may explain the characteristic pulmonary interstitial thickening and hypoplasia observed in both nitrofen-induced hypoplastic lungs as well as human hypoplastic CDH lungs. Congenital diaphragmatic hernia (CDH) is a developmental defect of the diaphragm that allows abdominal organs to herniate into the thoracic cavity during lung development. CDH occurs in 1 in 2500 live births.1Kays D.W. Congenital diaphragmatic hernia and neonatal lung lesions.Surg Clin North Am. 2006; 86 (ix): 329-352Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 2Rottier R. Tibboel D. Fetal lung and diaphragm development in congenital diaphragmatic hernia.Semin Perinatol. 2005; 29: 86-93Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar Despite modern treatments CDH remains associated with variable survival and significant morbidity, mainly due to the associated anomalies of the lung.3Logan J.W. Rice H.E. Goldberg R.N. Cotten C.M. Congenital diaphragmatic hernia: a systematic review and summary of best-evidence practice strategies.J Perinatol. 2007; 27: 535-549Crossref PubMed Scopus (169) Google Scholar, 4van den Hout L. Sluiter I. Gischler S. De Klein A. Rottier R. Ijsselstijn H. Reiss I. Tibboel D. Can we improve outcome of congenital diaphragmatic hernia?.Pediatr Surg Int. 2009; 25: 733-743Crossref PubMed Scopus (94) Google Scholar Although the exact pathogenesis is unknown, lung hypoplasia is a major associated anomaly. Characteristics of human lung hypoplasia in CDH are thickened alveolar walls, an increase in interstitial tissue, reduced alveolar air spaces and reduced gas-exchange surface area.2Rottier R. Tibboel D. Fetal lung and diaphragm development in congenital diaphragmatic hernia.Semin Perinatol. 2005; 29: 86-93Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 5Areechon W. Eid L. Hypoplasia of lung with congenital diaphragmatic hernia.Br Med J. 1963; 1: 230-233Crossref PubMed Scopus (214) Google Scholar, 6George D.K. Cooney T.P. Chiu B.K. Thurlbeck W.M. Hypoplasia and immaturity of the terminal lung unit (acinus) in congenital diaphragmatic hernia.Am Rev Respir Dis. 1987; 136: 947-950Crossref PubMed Scopus (132) Google Scholar, 7Kitagawa M. Hislop A. Boyden E.A. Reid L. Lung hypoplasia in congenital diaphragmatic hernia A quantitative study of airway, artery, and alveolar development.Br J Surg. 1971; 58: 342-346Crossref PubMed Scopus (325) Google Scholar Apart from the gas-exchange layer, well-documented changes are present in the vascular components consisting of medial hyperplasia, peripheral muscularization of pre-acinar vessels, and adventitial thickening.8Sluiter I. Reiss I. Kraemer U. Krijger R. Tibboel D. Rottier R.J. Vascular abnormalities in human newborns with pulmonary hypertension.Expert Rev Respir Med. 2011; 5: 245-256Crossref PubMed Scopus (23) Google Scholar It has been demonstrated that the lung, in itself, is defective, and that lung hypoplasia is not caused by compression of the lungs by herniated abdominal organs alone.9Keijzer R. Liu J. Deimling J. Tibboel D. Post M. Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia.Am J Pathol. 2000; 156: 1299-1306Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar However, as to which of the two major tissue layers (epithelial or fibroblast/mesenchyme) in the lung is defective has never been addressed in detail. Such knowledge is required to design tissue-specific treatment modalities that can exclusively target the defective tissue layer in lung hypoplasia, and thereby modulate or potentially prevent lung hypoplasia in CDH. Several animal models have been used to study CDH and/or the associated lung hypoplasia such as the nitrofen rodent model, a surgical lamb or rabbit model, and multiple genetic mouse models. The nitrofen model is the preferred model to study the pathogenetic aspects of CDH. For the past three decades, the nitrofen model has been used extensively to investigate the anomalies associated with CDH. Originally, nitrofen was used as an herbicide, and while toxicology screens on adult rats did not reveal any apparent toxicological effects, the administration to pregnant dams during midgestation caused developmental anomalies in the lungs, diaphragm, heart, thymus, parathyroid glands, and skeleton of the offspring.10Costlow R.D. Manson J.M. The heart and diaphragm: target organs in the neonatal death induced by nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether).Toxicology. 1981; 20: 209-227Crossref PubMed Scopus (114) Google Scholar, 11Ambrose A.M. Larson P.S. Borzelleca J.F. Smith Jr., R.B. Hennigar Jr., G.R. Toxicologic studies on 2,4-dichlorophenyl-p-nitrophenyl ether.Toxicol Appl Pharmacol. 1971; 19: 263-275Crossref PubMed Scopus (79) Google Scholar, 12Yu J. Gonzalez S. Rodriguez J.I. Diez-Pardo J.A. Tovar J.A. Neural crest-derived defects in experimental congenital diaphragmatic hernia.Pediatr Surg Int. 2001; 17: 294-298Crossref PubMed Scopus (35) Google Scholar Numerous groups including our own demonstrated that nitrofen-induced diaphragmatic hernias were strikingly similar to the human condition. The specific location and extent of the diaphragmatic defects were very comparable, but also the similarities in the CDH-associated anomalies, including pulmonary hypoplasia and persistent pulmonary hypertension, and cardiovascular and skeletal defects as well, were impressive.13Migliazza L. Otten C. Xia H. Rodriguez J.I. Diez-Pardo J.A. Tovar J.A. Cardiovascular malformations in congenital diaphragmatic hernia: human and experimental studies.J Pediatr Surg. 1999; 34: 1352-1358Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 14Migliazza L. Xia H. Alvarez J.I. Arnaiz A. Diez-Pardo J.A. Alfonso L.F. Tovar J.A. Heart hypoplasia in experimental congenital diaphragmatic hernia.J Pediatr Surg. 1999; 34 (discussion 710–701): 706-710Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 15Migliazza L. Xia H. Diez-Pardo J.A. Tovar J.A. Skeletal malformations associated with congenital diaphragmatic hernia: experimental and human studies.J Pediatr Surg. 1999; 34: 1624-1629Abstract Full Text PDF PubMed Scopus (40) Google Scholar, 16Tenbrinck R. Tibboel D. Gaillard J.L. Kluth D. Bos A.P. Lachmann B. Molenaar J.C. Experimentally induced congenital diaphragmatic hernia in rats.J Pediatr Surg. 1990; 25: 426-429Abstract Full Text PDF PubMed Scopus (142) Google Scholar This model was demonstrated to interfere with the retinoic acid (RA) signaling pathway (RA hypothesis), which was recently proven relevant in human CDH patients as well.17Beurskens N. Klaassens M. Rottier R. de Klein A. Tibboel D. Linking animal models to human congenital diaphragmatic hernia.Birth Defects Res A Clin Mol Teratol. 2007; 79: 565-572Crossref PubMed Scopus (58) Google Scholar, 18van Loenhout R.B. Tibboel D. Post M. Keijzer R. Congenital diaphragmatic hernia: comparison of animal models and relevance to the human situation.Neonatology. 2009; 96: 137-149Crossref PubMed Scopus (84) Google Scholar, 19Beurskens L.W. Tibboel D. Lindemans J. Duvekot J.J. Cohen-Overbeek T.E. Veenma D.C. de Klein A. Greer J.J. Steegers-Theunissen R.P. Retinol status of newborn infants is associated with congenital diaphragmatic hernia.Pediatrics. 2010; 126: 712-720Crossref PubMed Scopus (80) Google Scholar Previously we used this nitrofen model to demonstrate that CDH-associated lung hypoplasia is a result of two hits: an intrinsic problem in the hypoplastic lungs itself before development of the diaphragmatic defect, and interference with fetal breathing movements and competition for space of the lungs due to herniation of abdominal organs through the diaphragmatic defect.9Keijzer R. Liu J. Deimling J. Tibboel D. Post M. Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia.Am J Pathol. 2000; 156: 1299-1306Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar Thus, lung hypoplasia is already present before development of the diaphragmatic defect, but will worsen as a result of interference with fetal breathing movements and compression by the abdominal organs. In the present study, we were interested in the primary cause of the lung hypoplasia, and merely focused on "the first hit," the intrinsic lung defect of the nitrofen model. Previous studies by others and us have demonstrated abnormal patterns in proliferation, apoptosis, and cell differentiation in hypoplastic nitrofen-lungs, but the defective tissue layer has not been identified.9Keijzer R. Liu J. Deimling J. Tibboel D. Post M. Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia.Am J Pathol. 2000; 156: 1299-1306Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 20Jesudason E.C. Connell M.G. Fernig D.G. Lloyd D.A. Losty P.D. Cell proliferation and apoptosis in experimental lung hypoplasia.J Pediatr Surg. 2000; 35: 129-133Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Forty years ago it was reported that interactions between the different tissue layers are crucial for proper embryonic lung development.21Wessells N.K. Mammalian lung development: interactions in formation and morphogenesis of tracheal buds.J Exp Zool. 1970; 175: 455-466Crossref PubMed Scopus (194) Google Scholar Since then, many studies have contributed to the analysis of molecular determinants of lung growth.22Cardoso W.V. Lu J. Regulation of early lung morphogenesis: questions, facts and controversies.Development. 2006; 133: 1611-1624Crossref PubMed Scopus (463) Google Scholar, 23Maeda Y. Dave V. Whitsett J.A. Transcriptional control of lung morphogenesis.Physiol Rev. 2007; 87: 219-244Crossref PubMed Scopus (364) Google Scholar Previously we have demonstrated that fetal lung epithelial cells, recombined with fetal lung fibroblasts, reorganize in alveolar-like structures in vitro and that fibroblasts direct epithelial morphogenesis.24Deimling J. Thompson K. Tseu I. Wang J. Keijzer R. Tanswell A.K. Post M. Mesenchymal maintenance of distal epithelial cell phenotype during late fetal lung development.Am J Physiol Lung Cell Mol Physiol. 2007; 292: L725-L741Crossref PubMed Scopus (24) Google Scholar In addition, distal lung embryonic mesenchyme has been shown to induce expression of distal epithelial markers in proximal (tracheal) lung epithelial cells.25Shannon J.M. Induction of alveolar type II cell differentiation in fetal tracheal epithelium by grafted distal lung mesenchyme.Dev Biol. 1994; 166: 600-614Crossref PubMed Scopus (157) Google Scholar Thus, lung fibroblasts are essential for proper lung organogenesis. Consequently, a defective fibroblast layer could result in abnormal lung formation. Knowing the defective tissue layer in CDH-related lung hypoplasia is critical for designing improved treatment modalities specifically targeted at this defective tissue layer. To determine which lung tissue layer is defective in CDH, we used the above mentioned in vitro cell recombinant model.24Deimling J. Thompson K. Tseu I. Wang J. Keijzer R. Tanswell A.K. Post M. Mesenchymal maintenance of distal epithelial cell phenotype during late fetal lung development.Am J Physiol Lung Cell Mol Physiol. 2007; 292: L725-L741Crossref PubMed Scopus (24) Google Scholar Because access to human (hypoplastic) lungs to perform such experiments is at best very limited, we used the nitrofen rodent model to develop a novel in vitro model for pulmonary hypoplasia to address this question. Epithelial cells and fibroblasts isolated from control and nitrofen-treated lungs were recombined as either homotypic (control epithelial cells plus control fibroblasts or nitrofen epithelial cells plus nitrofen fibroblasts) or heterotypic (nitrofen epithelial cells or fibroblasts with healthy control fibroblasts or epithelial cells, respectively) recombinants (Figure 1). This approach enabled us to investigate the actual tissue interactions and the effects of a healthy opposing layer on a "diseased" tissue layer, thereby gaining new insights into the pathogenesis of lung hypoplasia in CDH and the potential role for epithelial–mesenchymal interactions. These recombination studies demonstrated that the fibroblast (mesenchymal) layer is the defective tissue layer in hypoplastic lungs due to a decreased ability to undergo apoptosis and maintain overall proliferation. This may explain the characteristic nitrofen-induced pulmonary interstitial thickening and hypoplasia as well as similar features noted in hypoplastic lungs in children with CDH. The animal care committee of the Hospital for Sick Children approved all experimental procedures. Timed-pregnant Sprague-Dawley rats (Rattus norvegicus) were ordered from Charles River (St. Constant, Quebec, Canada). Congenital diaphragmatic hernia and lung hypoplasia were induced in pregnant rats using 2,4-dichlorophenyl-p-nitrophenyl ether (nitrofen) (Cerilliant, Round Rock, TX) as described previously.9Keijzer R. Liu J. Deimling J. Tibboel D. Post M. Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia.Am J Pathol. 2000; 156: 1299-1306Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar E19 embryos (term = 22 days) were collected by Caesarian section under aseptic conditions. Thoracic contents were removed and collected per group in ice-cold Hanks' Balanced Salt Solution (HBSS; Invitrogen, Burlington, ON, Canada). Lungs were microscopically separated from all other tissues including removal of the major airways. In nitrofen-treated embryos, lungs from both hernia-positive and hernia-negative embryos were used, as lung hypoplasia is present in 100% of these embryos. In a control experiment, lungs from embryos with a hernia were separated from embryos without a hernia to investigate differences in the severity of hypoplasia in our recombinant model. We did not observe any obvious differences between recombinants of lung cells isolated from hernia-positive or hernia-negative embryos (see Supplemental Figure S1 at http://ajp.amjpathol.org). E19 whole lungs from nitrofen-treated and control rats served as in vivo controls for the recombinants. Fetal epithelial cells and fibroblasts were isolated by primary culture as described previously.26Caniggia I. Tseu I. Han R.N. Smith B.T. Tanswell K. Post M. Spatial and temporal differences in fibroblast behavior in fetal rat lung.Am J Physiol. 1991; 261: L424-L433PubMed Google Scholar After overnight culture, cells were washed, trypsinized and collected by centrifugation. Cells were counted and recombined (3.0 × 106 epithelial cells with 3.0 × 106 fibroblasts in solution) in four different combinations, as depicted in Figure 1. Subsequently, recombined cells were spun down, and supernatant was removed. Cells were lightly stirred and incubated for 1 hour, and transferred onto inserts (Millipore, Etobicoke, ON, Canada). The recombined cells were cultured according to Deimling et al.24Deimling J. Thompson K. Tseu I. Wang J. Keijzer R. Tanswell A.K. Post M. Mesenchymal maintenance of distal epithelial cell phenotype during late fetal lung development.Am J Physiol Lung Cell Mol Physiol. 2007; 292: L725-L741Crossref PubMed Scopus (24) Google Scholar After 5 days of culture, recombinants were fixed in 4% paraformaldehyde overnight at 4°C, dehydrated and embedded in paraplast, and 5-μm sections were cut. To ensure that isolated cells did not differ between the groups before recombination with respect to differentiation markers and number of apoptotic cells, freshly isolated cells were grown on coverslips for immunocytochemical analysis. Sections were rehydrated and stained with hematoxylin (Sigma, Oakville, ON, Canada). Slides were rinsed with warm tap water for 30 minutes and dehydrated to 95% ethanol. Subsequently, the sections were stained with 0.5% eosin (Sigma) in 95% ethanol, dehydrated, and mounted with 70% permount (Fisher, Pittsburgh, PA) in xylene. Immunofluorescence (IF) analysis was performed as described previously.24Deimling J. Thompson K. Tseu I. Wang J. Keijzer R. Tanswell A.K. Post M. Mesenchymal maintenance of distal epithelial cell phenotype during late fetal lung development.Am J Physiol Lung Cell Mol Physiol. 2007; 292: L725-L741Crossref PubMed Scopus (24) Google Scholar Briefly, tissue sections were rehydrated and antigen retrieval was performed in 10 mmol/L pH 6.0 sodium citrate using a pressure cooker in a microwave for 15 minutes at maximum wattage. Slides were incubated with blocking solution consisting of 10% (w/v) normal goat serum (NGS) and 1% (w/v) bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 1 hour. Subsequently, the primary antibody in blocking solution was added and sections were incubated overnight at 4°C. Following three washes with PBS containing 0.05% (v/v) Tween-20 (PBST), slides were incubated with a secondary antibody in blocking solution for 1 hour. The slides were then washed and the samples were mounted with DAPI hard mounting medium (Vector, Burlington, ON, Canada). Primary antibodies were: 1:500 rabbit anti-cytokeratin (Dako, Mississauga, ON, Canada), 1:50 mouse anti-vimentin (Dako), 1:400 mouse anti-α-smooth muscle actin (αSMA; NeoMarkers, Fremont, CA), 1:200 rabbit anti-pro-surfactant protein-C (pro-SFTPC; Abcam, Cambridge, MA), 1:200 rabbit anti-clara cell secretory protein (CCSP; Santa Cruz Biotechnology, Santa Cruz, CA), 1:1000 rabbit anti-platelet endothelial cell adhesion molecule (PECAM; Santa Cruz Biotechnology). Secondary antibodies (dilution of 1:200) were fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG (Calbiochem, San Diego, CA) for vimentin and αSMA, rhodamine-labeled anti-mouse IgG (Invitrogen, Eugene, OR) for vimentin, rhodamine-labeled anti-rabbit IgG (Invitrogen) for cytokeratin, pro-SFTPC, CCSP and PECAM. Whole lungs of E19 rat embryos were used as positive controls for immunofluorescence. Following antigen retrieval, slides were treated with 3% (v/v) hydrogenperoxide in methanol to block endogenous peroxidase activity. Blocking solution, containing avidin (Vector), was added for 1 hour according to the manufacturer's instructions. Primary antibody in blocking solution containing biotin (Vector) was added and slides were incubated overnight at 4°C. The following day a biotinylated secondary antibody was added for 1 hour in blocking solution. Subsequently, ABC-complex (Vector) was added for 30 minutes. Slides were developed using ImmPACT 3,3′-Diaminobenzidine (DAB; Vector), and counterstained with hematoxylin. Slides were mounted with 70% permount in xylene. Primary antibodies used for DAB staining were 1:100 rabbit anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA), 1:2000 rabbit anti-ki67 (Dako), 1:1000 mouse-anti-cyclin D3 (Abcam), 1:1000 rabbit anti-cyclin E (Abcam), 1:400 rabbit anti-phosphohistone H3 (pH3) (Upstate, Atlanta, GA), and 1:100 rabbit anti-thyroid transcription factor-1 (TITF-1) (NeoMarkers) in blocking solution. Biotinylated anti-mouse IgG (for cyclin D3; Vector) and anti-rabbit IgG (for cleaved caspase-3, ki67, cyclin E, pH3, and TITF-1; Calbiochem) were used as secondary antibodies in a dilution of 1:200. Whole lungs of E19 rat embryos were used as positive controls for immunohistochemistry. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-assay (Roche, Toronto, ON, Canada) was carried out according to the manufacturer's instructions. Rehydration and antigen retrieval were performed as described above. Slides were washed twice with PBS. TUNEL solution was added to the slides and the slides were incubated for 1 hour at 37°C. Slides were washed three times with PBS and counterstained with DAPI mounting medium. 5-Ethynyl-2′-deoxyuridine (EdU)-incorporation assay was carried out according to the manufacturer's instructions (Invitrogen). Briefly, recombinants were incubated for 2 hours with 10 μmol/L EdU component A on day 5 of culture before fixation. Sections were incubated with 0.5% (v/v) Triton X-100 for 20 minutes to make the sample permeable. EdU detection was performed by incubation with a freshly prepared reaction cocktail of fluorescein isothiocyanate–labeled anti-EdU antibody for 30 minutes. Slides were washed with 3% (w/v) BSA in PBS and counterstained with vimentin and DAPI. Cells on coverslips were made permeable with 0.2% Triton-X100 (Sigma Life Science, St Louis, MO) in 1% BSA for 5 minutes. Subsequently, IF analysis for cytokeratin, vimentin, pro-SFTPC, αSMA, and PECAM was performed and apoptosis measured using the TUNEL-assay as described above. Mitotic index was quantified by counting pH3-positive cells for each type of recombinant. Per recombinant five different randomly selected areas with alveolar-like structures were counted. Simultaneously, we determined cell origin (epithelial cell or fibroblast). Volocity4 software (Quorum Technologies Inc., Guelph, ON, Canada) was used to quantify the surface area of cytokeratin (epithelial cells) and vimentin (fibroblasts)-positive cells in all four types of recombinants. In each group, recombinants from at least four separate experiments were analyzed with an average of eight pictures per recombinant. Fibroblasts from nitrofen-treated and control lungs were isolated at E19 and cultured for 24 hours. Cells were collected in radioimmunoprecipitation assay buffer and sonicated. Western blot analysis was performed as previously described.27Cao L. Wang J. Tseu I. Luo D. Post M. Maternal exposure to endotoxin delays alveolarization during postnatal rat lung development.Am J Physiol Lung Cell Mol Physiol. 2009; 296: L726-L737Crossref PubMed Scopus (51) Google Scholar Primary antibodies (1:1000 dilution) were rabbit-anti-p27Kip1, rabbit-anti-p57Kip2, and mouse-anti-p21Waf/Cip1 (all from Cell signaling Technology). Anti-rabbit and anti-mouse secondary antibodies (Vector) were used in a concentration of 1:5000. All recombination experiments were repeated at least four times. Data are presented as mean ± SEM. For statistical analyses we used an analysis of variance and Bonferroni multiple comparisons test to compare fibroblast and epithelial cell area fractions and ratios, and the pH3-positive cells per field. Subsequently, a Student's t-test was used for pairwise comparison of the four recombinant groups and the P values were adjusted for multiple comparison error. Significance was defined as P <0.025. In all four types of recombinants (Figure 1), cells spontaneously organized in alveolar-like structures (Figure 2, A–D). IF staining for cytokeratin (epithelial cell marker) and vimentin (fibroblast marker) revealed fewer organized alveolar-like epithelial structures and a thickened fibroblast (mesenchymal) tissue layer in FN-containing recombinants versus FC-containing recombinants (Figure 2, F and H versus E and G). Area measurements of the four types of recombinants demonstrated significant differences between FC-containing recombinants and (FN)(EN) recombinants in the epithelial and fibroblast surface areas (Figure 3A). Fraction of fibroblasts were significantly different between (FC)(EC) and (FN)(EN) recombinants (Figure 3B).Figure 3Quantitative analysis of epithelial cells versus fibroblasts surface area in recombinants. To quantify the observed differences in Figure 2E–H, the surface area of epithelial cells and fibroblasts was measured using Volocity4 software (Quorum Technologies Inc). The epithelial-to-fibroblast (E/F) ratio (A) and fraction (B) of fibroblasts in the recombinants were determined. The E/F ratio was significantly decreased whereas the number of fibroblasts increased in (FN)(EN) recombinants compared with FC-containing recombinants. Both the E/F ratio and number of fibroblasts trended to either decrease or increase in (FN)(EC) recombinants, respectively. *P <0.025. The bars represent SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Freshly isolated epithelial cells and fibroblasts from nitrofen-treated and control lungs separately grown on coverslips did not differ in differentiation markers and number of apoptotic cells (see Supplemental Figure S2 at http://ajp.amjpathol.org). To determine whether the observed differences were due to apoptotic changes we performed TUNEL-assays and immunohistochemistry for cleaved caspase-3. Both analyses revealed less apoptosis, primarily in the fibroblast (mesenchymal) layer, in FN-containing recombinants when compared to FC-containing recombinants (Figure 4, B, D, F, and H versus A, C, E, and G). Freshly isolated FN and FC cultures were analyzed for TUNEL-positive cells, but no differences between the cultures were observed before recombination (see Supplemental Figure S2K and L at http://ajp.amjpathol.org). E19 nitrofen-treated rat lungs also demonstrated less apoptosis compared with E19 control rat lungs (see Supplemental Figure S3 at http://ajp.amjpathol.org). We then assessed cell proliferation by immunohistochemical analyses of ki67 (general proliferation marker), cyclin D3 ("first gap" G1-phase marker), cyclin E (G1/S-phase marker), and pH3 (mitosis marker). In addition, we measured the uptake of EdU into DNA (S-phase). Immunoreactivity for cyclin D3 (Figure 5, E–H) and cyclin E (results not shown) were similar in all four types of recombinants. However, EdU-incorporation assays demonstrated less cells in the S-phase of the cell cycle in (FN)(EN) recombinants compared with (FC)(EC) recombinants (Figure 5, J versus I). Ki-67 (Figure 5, A–D) and pH3 (Figure 5, K–N) immunohistochemistry corroborated the finding of less proliferating/dividing cells in FN-containing recombinants versus FC-containing recombinants (Figure 5, B, D, L, and N versus 5, A, C, K, and M; Figure 6). The ratio of proliferating cell types (epithelial versus fibroblast) remained the same in all four groups. No differences in total cell amount for each type of recombinant were observed. Western blot analysis demonstrated increased protein levels of cyclin-dependent kinase (Cdk) inhibitors p27Kip1 and p57Kip2 in nitrofen-treated fibroblasts compared to control fibroblasts (Figure 7). These inhibitors are known to inhibit the transition from the G1- to S-phase of the cell cycle. The other Cip/Kip family member of Cdk inhibitors, p21Waf1/Cip1, was not detectable in either fibroblast population (results not shown). E19 nitrofen-treated rat lungs had less ki67- and pH3-positive cells but equal numbers of cyclin D3-positive cells when compared to E19 contro
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