Inhibition of vascular and epithelial differentiation in murine nitrofen-induced diaphragmatic hernia

C. Coleman1, J. Zhao3, M. Gupta1, S. Buckley1, J. D. Tefft3, C. W. Wuenschell3, P. Minoo2,3, K. D. Anderson1, and D. Warburton1,3

1 Division of Pediatric Surgery and Developmental Biology Program, Childrens Hospital Los Angeles Research Institute, Los Angeles 90027; and 2 Divisions of Basic Research and Neonatology, Department of Pediatrics, Women and Children's Hospital, and 3 Center for Craniofacial and Molecular Biology, University of Southern California Schools of Medicine and Dentistry, Los Angeles, California 90033

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Neonates with congenital diaphragmatic hernia (DH) die of pulmonary hypoplasia and persistent pulmonary hypertension. We used immunohistochemical localization of alpha -smooth muscle actin (alpha -SMA), platelet endothelial cell adhesion molecule (PECAM)-1, thyroid transcription factor (TTF)-1, surfactant protein (SP) A, SP-C, and competitive RT-PCR quantitation of TTF-1, SP-A, SP-C, and alpha -SMA mRNA expression to characterize the epithelial and vascular phenotype of lungs from ICR fetal mice with a nitrofen-induced DH. Nitrofen (25 mg) was gavage fed to pregnant mice on day 8 of gestation. Fetal mice were delivered on day 17. The diaphragm was examined for a defect, and the lungs were either fixed, sectioned, and immunostained or processed for mRNA isolation. In comparison with control lungs, DH lungs showed increased expression of alpha -SMA mRNA, fewer and more muscular arterioles (alpha -SMA), less well-developed capillary networks (PECAM-1), delayed epithelial development marked by a persistence of TTF-1 in the periphery, and decreased SP-A mRNA and SP-A expression. These data suggest that in the murine nitrofen-induced DH, as in human congenital DH, pulmonary insufficiency is due to an inhibition of peripheral pulmonary development including terminal airway and vascular morphogenesis.

pulmonary hypertension; pulmonary hypoplasia

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE MORTALITY for congenital diaphragmatic hernia (CDH) remains between 30 and 50% despite the advent of therapies such as artificial surfactant extracorporeal membrane oxygenation and high-frequency oscillatory ventilation (3, 4). The majority of severely affected infants die of pulmonary hypoplasia, and even if sufficient lung capacity to sustain life is present initially, persistent pulmonary hypertension (PPH) gives rise to further morbidity and mortality (25, 31). The cause of CDH remains unknown, and although it is associated with several genetic defects, it does not appear to be of simple genetic origin (27).

Advances in our understanding of normal pulmonary development have provided a framework for studying the developmental defects that lead to pulmonary hypoplasia in the lungs of infants with CDH. Normal lung development proceeds in discrete developmental stages that are seen in all animal species (6). Hypoplastic lungs of infants with CDH appear to be delayed in their advancement through these stages. An apparent consequence of this developmental delay is that the lungs of CDH infants have fewer bronchial branches (2, 31). During the later stages of development, these lungs also show a delay in the differentiation of epithelial cells, with a resultant surfactant deficiency (2, 18, 31). Pulmonary vascular branching follows in tandem with airway development and involves the processes of angiogenesis and vasculogenesis (6). Angiogenesis is defined as the development of arterioles by the extension and branching of existing vessels, whereas vasculogenesis is defined as the differentiation of mesenchymal cells into endothelial cells, which then form vessels (23). The lungs of CDH infants have been noted to have fewer pulmonary vascular branches than the lungs of unaffected infants (9, 14).

Herein, we used a murine nitrofen-induced diaphragmatic hernia (DH) model to investigate the pulmonary hypoplasia and vascular changes associated with CDH (8, 19). We used immunostaining techniques to determine 1) the distribution of smooth muscle in the muscular arterioles formed by angiogenesis and in the airways, with alpha -smooth muscle actin (alpha -SMA) as a smooth muscle marker; 2) the development of peripheral capillary networks by vasculogenesis, with platelet endothelial cell adhesion molecule-1 (PECAM-1); 3) epithelial pattern formation by thyroid transcription factor-1 (TTF-1) expression; and, finally, 4) epithelial differentiation, with surfactant protein (SP) A and SP-C as markers. We also performed competitive RT-PCR to quantitate the mRNA for alpha -SMA, TTF-1, SP-A, and SP-C in normal, nitrofen-exposed, and nitrofen-induced DH lungs. The results of these studies document a delay in angiogenesis, vasculogenesis, and epithelial differentiation in murine lungs with nitrofen-induced DH.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Nitrofen-induced DH. A total of 21 timed-pregnant ICR mice (Simonsen, Gilroy, CA) were gavage fed 25 mg of 2,4-dichloro-4'-nitrodiphenyl ether (nitrofen; Wako Chemicals, Osaka, Japan) dissolved in 0.5 ml of olive oil on day 8 of gestation (8, 19). Control timed-pregnant females (n = 8) were gavage fed olive oil alone. The finding of a vaginal plug was counted as day 0. The animals were transported to Childrens Hospital Los Angeles on gestational (embryonic) day (ED) 14 and were killed on ED17. The total length of gestation in mice is 19 days. The fetuses were removed by cesarean section, washed in ice-cold Hanks' balanced salt solution, blotted dry on sterile gauze, weighed individually, and returned to the iced Hanks' balanced salt solution. A dissecting microscope and microsurgical instruments were used to dissect and inspect the fetal diaphragm for the presence of a hernia. This was accomplished through a transverse abdominal incision; the falciform ligament was grasped with fine forceps, and the liver was gently pushed down off the diaphragm. The diaphragm was then examined, and the location and content of the hernia were noted. Nitrofen-treated fetuses were classified as either nitrofen exposed (no hernia) or having nitrofen-induced DH. Only those animals with a left-sided defect were utilized in this study. A median sternotomy was then made, and the lungs and heart were removed en bloc. The heart was removed from the lungs, and the lungs were processed for either immunohistochemistry or mRNA extraction.

Preparation of tissue sections. Lungs from control, nitrofen-exposed, and herniated mice were fixed for 3 h in either Carnoy's solution (n = 5/group) for alpha -SMA, TTF-1, and SP-C staining or 4% paraformaldehyde-PBS (n = 5/group) for PECAM-1 and SP-A staining. After fixation, tissues were dehydrated in ethanol and embedded in paraffin. Sections were cut at 5-µm thickness and mounted on Histostick (Accurate Chemical and Scientific Company, Westbury, NY)-coated slides.

Antibodies. alpha -SMA is a marker of smooth muscle and is also seen in other contractile cells including myofibroblasts and pericytes (15). A mouse monoclonal antibody to alpha -SMA was purchased from Sigma (St. Louis, MO) and diluted to 1:500 for use.

PECAM-1 is a 130-kDa member of the immunoglobulin superfamily and is a major constituent of the endothelial cell intercellular junction (20). PECAM-1 is found in endothelial cells, circulating platelets, monocytes, neutrophils, and selected T-cell subsets. However, it is not present on fibroblasts, epithelium, muscle, or other nonvascular cells, and because of this, it was used as a marker for vascular endothelial cells (20). PECAM-1 purified rat anti-mouse monoclonal antibody was puchased from PharMingen (San Diego, CA) and diluted 1:250 for use.

TTF-1 is a member of the Nkx2 family of homeodomain transcription factors. TTF-1 plays an important role in lung and thyroid epithelial cell gene expression. TTF-1 transactivates promoter activities of the SP-A, SP-B, SP-C, Clara cell secretory protein, thyroglobulin, and thyroperoxidase genes by binding in trans to DNA binding sites located within the promoters of each of these genes (10, 13). Furthermore, there is evidence that TTF-1 plays a critical role in lung branching morphogenesis (13, 17). A polyclonal anti-TTF-1 antibody developed by P. Minoo was raised in rabbits against four peptide fragments derived from human TTF-1 and used at a dilution of 1:150.

Rabbit polyclonal antiserum directed against a 15-amino acid peptide corresponding to amino acids 186-200 of human SP-A was a gift from Dr. Richard J. King and was used at a dilution of 1:1,000 (32).

SP-C rabbit polyclonal antiserum directed against a recombinant protein containing amino acids 1-20 from the NH2 terminus of the human SP-C proprotein was a gift from Dr. Jeffrey Whitsett (28). The human SP-C proprotein antibody was used at a dilution of 1:250. The TTF-1, SP-A, and SP-C antisera all cross-react with the homologous mouse proteins.

Immunohistochemistry. Sections were deparaffinized in xylene, rehydrated, and treated with 3% H2O2 in methanol to eliminate endogenous peroxidase activity. Zymed Histostain SP kits (South San Francisco, CA) with the appropriate biotinylated secondary antibody and strepavidin-peroxidase conjugate were used to detect bound antibody. The subsequent addition of the chromogen aminoethyl carbazole generated a red color around the areas of primary antibody binding. Immunostaining was performed as per the manufacturer's instructions, with the exception that the primary antibody was diluted in PBS-0.05% Tween 20 (Sigma) and incubated on the slides overnight at 4°C. Once staining was completed, the slides were analyzed and photographed by light microscopy with an Olympus BH2 microscope. Each immunostaining experiment had the following controls: 1) negative reagent control where no primary antibody was added, 2) a positive antibody control with either a monoclonal or polyclonal anti-actin antibody (Sigma) at a 1:10 dilution as the primary antibody, and 3) a negative antibody control with normal rabbit serum in place of the primary antibody.

Morphometry. The number of muscularized arterioles < 60 µm as identified by positive alpha -SMA immunostaining was counted with an Olympus BH2 microscope equipped with a Nikon 100-µm ocular micrometer (1 division = 2.8 µm) at a magnification of ×100. Two independent observers counted the number of muscularized arterioles in three random high-power fields (HPF) per lung on four different coronal sections (12 fields/lung). This analysis was performed for five different specimens per group (control, nitrofen exposed, and nitrofen-induced DH). The mean ± SE was calculated for each group.

Next, the thickness of the alpha -SMA-stained arterial walls from normal, nitrofen-exposed, and nitrofen-induced DH lungs was measured at a magnification of ×250. We used only arteries that were approximately round [i.e., maximal external diameter did not exceed minimal external diameter by >50%] and were 20-35 µm wide in the largest external diameter. The thickness of the alpha -SMA positively stained arteriole walls was calculated by subtracting the length of the internal diameter from the external diameter and dividing this value in half. Six different locations on the arteriolar circumference were measured for six individual arterioles taken from five different specimens. The mean ± SE was calculated for each group.

The PECAM-1-positive capillary branch points present per HPF at a magnification of ×250 were counted in 2 random HPF/lung from four different coronal sections in five different specimens/group.

Primers and templates for alpha -SMA, TTF-1, SP-A, and SP-C competitive RT-PCR. A set of primers was designed based on the murine alpha -SMA cDNA sequence: primer 1 (upstream) was 5'-CTGGAGAAGAGCTACGAACTGC-3' and primer 2 (downstream) was 5'-CTGATCCACATCTGCTGGAAGG-3'. alpha -SMA cDNA was thus obtained by PCR amplification with primers 1 and 2 in reversed-transcribed products from ED17 control, nitrofen-exposed, and nitrofen-induced DH mouse lungs; PCR product length was 368 bp (Fig. 1A). Subsequently, two composite primers were used for alpha -SMA competitor construction (Fig. 1A); each composite primer had the target alpha -SMA gene primer sequence attached to opposite strands of a heterogeneous DNA fragment. The desired primer sequences were thus incorporated into the heterogeneous fragment during the PCR amplification. This ensured that all alpha -SMA competitor molecules had the same gene-specific primer sequences as the alpha -SMA cDNA. The heterogeneous DNA was derived from a piece of v-erbB DNA (5). alpha -SMA competitor PCR products with primers 1 and 2 were 480 bp in length. The identities of alpha -SMA and its competitor were both confirmed by DNA sequencing (24).


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Fig. 1.   Competitive RT-PCR for alpha -smooth muscle actin (alpha -SMA) mRNA quantitation. A: competitive PCR products for alpha -SMA. Construction of alpha -SMA and its competitor is described in MATERIALS AND METHODS. B: electrophoretic pattern of alpha -SMA competitive PCR. PCR products separated in a 3% agarose gel were stained by ethidium bromide. Lanes 1-10, PCR products from 1:2 serial dilutions of mouse alpha -SMA cDNA (1, 0 fg; 2, 0.625 fg; 3, 1.25 fg; 4, 2.5 fg; 5, 5 fg; 6, 10 fg; 7, 20 fg; 8, 40 fg; 9, 80 fg; 10, 160 fg) and a constant 10 fg of alpha -SMA competitor. An inverse relationship between intensities of alpha -SMA and its competitor is evident, indicating competition between 2 templates. C: standard curve for alpha -SMA competitive PCR. Log(alpha SMA/competitor) from densitometric data from gel in B was plotted against logarithm of indicated amounts of added recombinant alpha -SMA cDNA template. Solid line, linear regression line with a high coefficient of correlation. Next, 5, 10, 20, 40, 80, and 160 ng of total RNA extracted from embryonic day 17 (ED17) normal lungs were reverse transcribed. Each reverse-transcribed mixture was also coamplified with 10 fg of alpha -SMA competitor template. Resultant PCR products were electrophoresed and densitometrically analyzed as above. Log(cDNA/competitor) was also plotted against logarithm of initial amount of total RNA being reverse transcribed. Slope of regression line from reverse-transcribed total RNA (dashed line) is almost identical to line derived from alpha -SMA cDNA. Both regression lines have R2 values > 0.95.

Similar methods were utilized for primer design and competitor template construction for TTF-1, SP-C, and SP-A as previously described (33). The identity of TTF-1, SP-C, and SP-A competitive PCR products was confirmed by DNA sequencing.

Competitive PCR. PCR amplification was carried out in a DNA Robocycler (Stratagene, La Jolla, CA) using a modification of a previously described assay for matrix Gla protein (34, 35). Thirty-five cycles of denaturation at 93°C for 2 min, annealing at 62°C for 2 min, and extension at 72°C for 2 min were routinely performed after an initial 3-min denaturation at 94°C. The final cycle included a 5-min extension step. The reaction mixture contained 10 mM Tris (pH 9.4), 50 mM KCl, 2 mM MgCl2 (optimized), 0.01% gelatin, 0.2% Triton X-1000, 20 pmol primer sets, 100 µM deoxynucleotide triphosphate, and 0.5 units Taq thermostable DNA polymerase (Promega, Madison, WI). A reaction mixture containing 1 pg/µl of alpha -SMA competitor was added to reverse-transcribed samples derived from 50 ng of total RNA or to dilutions of standard alpha -SMA templates in a volume of 50 µl. The concentration of cDNA standard solutions was determined spectrophotometrically by absorbance at 260 nm. The methodology of TTF-1, SP-C, and SP-A competitive PCR was similar to that of alpha -SMA. Primers and template construction for beta -actin competitive PCR was previously described (35).

Electrophoresis and densitometric analysis. Electrophoresis was performed in 3% agarose gels (3:1 mixture of NuSieve and SeaKem, FMC BioProducts, Rockland, ME), where target and competitor PCR products were separated by size. Gels were stained with 5 µg/ml of ethidium bromide and photographed with Polaroid 667 film. The intensity of each band was determined by densitometric analysis with ImageQuan band-analyzing software (Molecular Dynamics, Sunnyvale, CA).

RNA extraction and RT. Total RNA from normal control, nitrofen-exposed, and nitrofen-induced DH lungs (n = 6/group) was extracted by guanidinium thiocyanate after homogenization with an RNeasy total RNA purification kit (Qiagen, Northridge, CA). Extracted total RNA was reverse transcribed as described previously (1). Samples were incubated at 37°C for 1 h in 20 µl of 10 mM Tris (pH 8.4)-50 mM KCl-3 mM MgCl2-1 mM dithiothreitol-5 units of Moloney murine leukemia virus RT (USB Specialty Biochemicals, Cleveland, OH). The reaction was terminated by heating for 5 min at 95°C. Reverse-transcribed products were then used for competitive PCR assay.

Statistical analysis. All data are reported as means ± SD unless otherwise stated. Differences between the means were tested by ANOVA and Student's t-test. P values < 0.05 were considered to be statistically significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Fetal mice exposed to nitrofen weighed less than normal mice. The mice exposed to nitrofen in utero weighed less than their age-matched controls. The weight for the ED17 nitrofen-exposed mice (n = 242) was 0.66 ± 0.12 g (<OVL>x</OVL> ± SD) compared with the average weight of 0.76 ± 0.18 g in the ED17 control mice (n = 71; P < 0.001 by unpaired Student's t-test).

Nitrofen-induced DH was more prevalent on the left side. A total of 242 nitrofen-exposed fetal mice were examined. A DH was found in 63 mice (26%). Of these, 75% were left sided, 17% right sided, 6% central, and 3% bilateral. All of the left, right, and bilateral hernias were posterolateral in position. In the left-sided hernias, the abdominal contents were often seen in the left thoracic cavity. The stomach, liver, and small intestine were present in 55, 30, and 25%, respectively, of left-sided cases. All of the right-sided hernias contained the liver in the right thoracic cavity. Coronal sections through the chest and abdomen of an ED17 mouse fetus with a left-sided CDH are illustrated in Fig. 2, A (ventral) and B (dorsal). The stomach and small intestine are seen to lie within the left chest, causing apparent posterior displacement and compression of the left lung similar to that seen in human infants with a Bochdalek-type CDH (25). The liver was removed during the dissection so that only a small remnant of the liver and right diaphragm is seen in Fig. 2B.


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Fig. 2.   Ventral (A) and dorsal (B) coronal sections and corresponding line diagrams of chest and abdomen of an ED17 mouse fetus with a typical nitrofen-induced left-sided diaphragmatic hernia (DH). Histological sections through chest and abdomen of a typical ED17 mouse with a DH stained by routine hematoxylin and eosin show that stomach and small intestine lie within chest, causing apparent compression of left lung. star , Area of intestinal herniation into chest. Dashed lines, normal location of left hemidiaphragm. Liver was removed to visualize hernia during dissection. Intact right diaphragm is visualized at left bottom in B. Bar, 200 µm.

Both nitrofen-exposed and nitrofen-induced DH lungs had fewer alpha -SMA positive arterioles, muscular walls were thickened, and there was abundant alpha -SMA staining in lung mesenchyme. In control, nitrofen-exposed, and herniated lungs, immunostaining for alpha -SMA was localized to the mesenchyme surrounding the pulmonary bronchioles and arterioles. However, there were notably fewer bronchial and arterial branches in the nitrofen-induced hypoplastic lung with DH. The arterioles in the lungs of mice with a diaphragm defect had thickened muscular walls compared with those of control lungs (Fig. 3, A and B). ED17 nitrofen-exposed lungs showed a phenotype that was intermediate between the normal and nitrofen-induced DH lungs.


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Fig. 3.   Nitrofen-induced DH lungs have fewer, thickened arterioles and abundant diffuse alpha -SMA staining in peripheral lung mesenchyme. A and B: ED17 normal and nitrofen-induced DH lungs, respectively, immunostained with alpha -SMA (red) that localizes primarily around bronchioles and arterioles (star ). Note increased thickness of nitrofen-induced DH arteriole wall (B) compared with normal arterioles (A). Bar, 50 µm. C and D: ED17 normal and nitrofen-induced DH peripheral lung sections, respectively, of a distal bronchiole (b) and surrounding mesenchyme immunostained with alpha -SMA (red) and counterstained with hematoxylin (blue). alpha -SMA staining is found throughout peripheral mesenchyme of nitrofen-induced DH lung (D) compared with staining in normal lung (C), in which it is primarily located immediately adjacent to peripheral bronchiole. Bar, 10 µm. E: number of peripheral small and medium alpha -SMA-positive arterioles counted/high-power field (HPF) in lungs of normal control, nitrofen-exposed, and nitrofen-induced DH lungs. Right and left lungs were counted independently. Nitrofen-exposed and nitrofen-induced DH lungs had ~30% fewer arterioles than normal control lungs (* P < 0.001 by ANOVA). However, nitrofen-induced DH lung also had ~25% fewer arterioles in left compared with right lung (** P < 0.01 by Student's t-test).

The distribution of alpha -SMA in the mesenchymal cells that surround the distalmost epithelial branches in ED17 normal and nitrofen-induced DH mouse lungs is compared in Fig. 3, C and D. The alpha -SMA-positive mesenchymal cells were well localized immediately adjacent to the lung epithelium at airway branch points in ED17 normal murine lungs (Fig. 3C). However, in the ED17 nitrofen-induced DH lungs, a more abundant and diffusely distributed alpha -SMA staining pattern was evident in the peripheral lung mesenchyme. The staining was still most abundant immediately adjacent to the pulmonary epithelium, but it was not confined to this area as it was in the normal lung.

The number of peripheral small and medium alpha -SMA-positive arterioles was counted, and the results are shown graphically in Fig. 3E. The visual observation that ED17 normal lungs had a greater number of small to medium arterioles than either the ED17 nitrofen-exposed or nitrofen-induced DH lungs was confirmed. The number of alpha -SMA-positive arterioles per HPF was 3.6 ± 0.1 (right; <OVL>x</OVL> ± SE) and 3.6 ± 0.11 (left) for ED17 normal lungs, 2.4 ± 0.11 (right) and 2.4 ± 0.1 (left) for ED17 nitrofen-exposed lungs, and 2.3 ± 0.09 (right) and 1.6 ± 0.08 (left) for ED17 nitrofen-induced DH lungs. The nitrofen-induced DH and nitrofen-exposed lungs had statistically fewer arterioles than the control lungs (P < 0.001 by ANOVA). In addition, the DH lung had statistically fewer vascular branches in the left compared with the right side (P < 0.01). These data demonstrate that the lungs of fetal mice with a nitrofen-induced DH had bilateral vascular hypoplasia that was worse on the side with the hernia and that the nitrofen-exposed lungs had vascular hypoplasia that was equal on both sides.

The thickness of the alpha -SMA staining, reflecting the muscular component of the small muscular arterioles, revealed that the arterioles in the nitrofen-induced DH lungs had a thickened muscular wall. The average muscular thickness was 4.1 ± 0.17 µm for ED17 normal lung arterioles, 4.75 ± 0.18 µm for ED17 nitrofen-exposed lung arterioles, and 5.6 ± 0.2 µm for ED17 nitrofen-induced DH lung arterioles. This difference was statistically significant (P < 0.001 by ANOVA). Therefore, the thickness of the smooth muscle layer in the small arteriole walls of the nitrofen-induced DH was 1.4 times thicker than the smooth muscle layer in normal arterioles.

The lungs from mice with DH had fewer and thicker arterioles than both the normal control and nitrofen-exposed lungs.

Lungs with DH have fewer peripheral capillaries and demonstrate a thickened airway-capillary interface. The appearance of the peripheral vasculature in ED17 normal versus nitrofen-induced DH lungs, immunostained with a marker for vascular endothelial cells (PECAM-1), is compared in Fig. 4, A-F. PECAM-1 staining of the ED17 normal lung (Fig. 4, A, C, and E) showed a highly branched, dense peripheral vascular network. Essentially the entire area of the normal lung that was not occupied by respiratory epithelium (unstained) contained PECAM-1-positive vascular branches. At the highest magnification, PECAM-1-stained vascular endothelium directly abutted the unstained respiratory epithelium, with little or no space between these two structures. In contrast, the pattern of PECAM-1 staining in the ED17 nitrofen-induced DH lungs (Fig. 4, B, D, and F) showed a much less densely packed vasculature. The higher magnifications illustrate that there is less branching of the pulmonary capillaries and a considerable amount of unstained mesenchyme between the capillary endothelium and the neighboring respiratory epithelium in lungs with a DH. The increased mesenchymal tissue between the airway and its vasculature could represent a delay in the development of the air-vascular interface in the DH lungs. This could present a functional barrier to oxygen diffusion at birth.


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Fig. 4.   Lungs with nitrofen-induced DH have fewer peripheral capillaries and demonstrate a thickened air-capillary interface. A, C, and E: ED17 normal mouse lungs have a highly branched, dense peripheral vascular network as demonstrated by these lung sections stained with platelet endothelial cell adhesion molecule (PECAM)-1. In control lungs (A and C), vascular capillary endothelium directly abuts unstained respiratory epithelium, with little or no space between these 2 structures. B, D, and F: ED17 nitrofen-induced DH lungs show an underdeveloped vascular network marked by less PECAM-1 staining. D and F illustrate that there are few peripheral pulmonary capillary branches and that a substantial amount of unstained mesenchyme fills area between capillary endothelium and neighboring respiratory epithelium (bracket in F). Increase in mesenchymal tissue between airway and its vasculature in DH lungs may represent a developmental delay in formation of air-vascular interface and could present a functional barrier to oxygen diffusion at birth. Bars: 100 µm in A and B; 25 µm in C and D: 10 µm in E and F. G: number of PECAM-1-positive capillary branches counted/HPF in lungs of normal control, nitrofen-exposed, and nitrofen-induced DH lungs. ED17 nitrofen-induced DH lungs have 55% fewer capillary branches than either normal control or nitrofen-exposed lungs (* P < 0.001 by ANOVA). There were no differences in number of capillaries between right and left lungs.

The reduction in pulmonary capillary branching was quantified by branch counting, and the results are graphically depicted in Fig. 4G. The pulmonary capillary branching of ED17 normal and nitrofen-exposed mouse lungs was not significantly different: the ED17 normal lungs had 28.7 ± 1.21 branches/HPF on the right (<OVL>x</OVL> ± SE) and 27.5 ± 1.03 branches/HPF on the left. The ED17 nitrofen-exposed lungs had 25 ± 1.1 branches/HPF on the right and 26.0 ± 1.17 branches/HPF on the left. The ED17 nitrofen-induced DH lungs had 55% fewer capillary branches (11.8 ± 0.83 branches/HPF on the right and 11.9 ± 5.1 branches/HPF on the left) than either the normal control or the nitrofen-exposed lungs (P < 0.001). The difference in the number of capillary branches in the left versus the right lung was not significant in any of the groups. We conclude from these data that the lungs in the mice with nitrofen-induced DH show a delay in peripheral vasculogenesis that is due to the presence of a DH and independent of the effect of the administration of nitrofen.

Staining of the respiratory epithelium for TTF-1, SP-A, and SP-C revealed a delay in epithelial differentiation. The process of cellular differentiation requires the expression of a cell-specific array of genes that defines the cell phenotype (7). TTF-1 is a homeodomain transcriptional factor that is necessary for lung epithelial morphogenesis (13, 17). In the early embryo (ED10), TTF-1 nuclear protein is detectable within the ventral edge of the lung bud. As airway branching occurs, TTF-1 expression is found in the bronchial epithelium (ED12-16). By ED17, the TTF-1 expression is still present in the epithelial cells lining the conducting airways. However, in the distal lung parenchyma, the TTF-1 expression is restricted to type II epithelial cells (10).

The immunostaining for TTF-1 in ED17 normal lungs (Fig. 5, A and C) demonstrated a continuous nuclear TTF-1 staining of the bronchial epithelial cells, which became discontinuous in the lung periphery where TTF-1 staining became restricted to cells presumably destined to become type II pneumocytes. In contrast, the immunostaining of TTF-1 in the nitrofen-induced DH lungs was continuous in pattern throughout, with the TTF-1-positive staining extending all the way to the periphery (Fig. 5, B and D). Furthermore, because TTF-1 staining was restricted to the epithelium, the markedly thickened unstained mesenchyme occupying the space between the respiratory epithelial branches was made apparent.


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Fig. 5.   Staining of respiratory epithelium for thyroid transcription factor (TTF)-1 suggests a delay in epithelial differentiation in nitrofen-induced DH lungs. A and C: TTF-1 immunostaining in ED17 normal lungs demonstrates a continuous pattern of nuclear staining of bronchial epithelial cells, which becomes discontinuous in lung periphery where TTF-1 becomes restricted to cells presumably destined to become type II pneumocytes. Arrow, area of transition between bronchial and peripheral patterns of staining. B and D: TTF-1 staining of nitrofen-induced DH lungs demonstrates a continuous bronchiolar-type staining all the way to the periphery. Furthermore, because TTF-1 staining is restricted to epithelium, markedly thickened mesenchyme is apparent. Bars: 50 µm in A and B; 25 µm in C and D.

SP-C immunostaining is first detected in the mouse lung on ED11. It is present in early development in only a few distal bronchial epithelial cells and is thought to be a lung-specific marker for the progenitors of type II pneumocytes (29). Around ED17, SP-C staining becomes restricted to the type II epithelial cells and the secretion of SP-C increases with advancing gestation. Staining for SP-C was located in the peripheral lung of both the normal and nitrofen-induced DH lungs. The distribution was patchy in both groups of lungs, and there was no apparent difference in the staining pattern or quantity (data not shown).

SP-A is produced by two cell types in the lung epithelium, the type II cells and the distal small-airway cells. The expression of SP-A is first seen later in development (on ED14) than SP-C (26). In the small peripheral airways, the expression of SP-A rapidly increases in the distal epithelium until it reaches adult levels at birth (18). Comparison of the staining of SP-A in ED17 normal (Fig. 6A) and nitrofen-induced DH lungs (Fig. 6B) shows a greater amount of staining in the periphery of normal lungs, which appeared to be secondary to significantly more distal epithelial branches in normal than in DH lungs. However, the cellular location and pattern of SP-A staining was similar in both the normal and DH lungs.


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Fig. 6.   Immunostaining for surfactant protein (SP) A. A: this section of ED17 normal lung shows the numerous distal epithelial branches that stain positive for SP-A in cytoplasm. B: this section of nitrofen-induced DH lung shows that the distal epithelial cells do stain positive for SP-A; however, there is a markedly reduced number of distal epithelial branches. Bar, 50 µm.

Measurement of alpha -SMA, TTF-1, SP-A, and SP-C mRNA levels by competitive RT-PCR. A highly sensitive and quantitative competitive RT-PCR method was utilized to measure and compare the endogenous alpha -SMA, TTF-1, SP-A, and SP-C mRNA amounts in individual ED17 normal, nitrofen-exposed, and nitrofen-induced DH whole lungs. We have previously shown that competitive RT-PCR is ideal for low-abundance mRNA determination in small samples and enables both accurate and quantitative mRNA levels for specific genes to be determined in single lungs (33).

To determine the feasibility of using recombinant alpha -SMA as a standard, sequential 1:2 dilutions of a mouse alpha -SMA cDNA template were prepared, and a constant amount (10 fg) of an alpha -SMA competitor cDNA template was added to each tube. After PCR coamplification of the mouse alpha -SMA cDNA and alpha -SMA competitor cDNA, the PCR products were separated by 3% agarose gel electrophoresis (Fig. 1B). The resulting bands were then densitometrically analyzed, and the intensities were quantified. It was apparent that the alpha -SMA competitor band intensity decreased (from lanes 1 to 10), whereas the alpha -SMA band intensity increased in proportion to the amount of alpha -SMA cDNA included in each tube. When the logarithm of the ratio of alpha -SMA cDNA to its competitor PCR products was plotted against the logarithm of the initial amount of mouse alpha -SMA cDNA, a linear correlation was obtained (R2 > 95; Fig. 1C).

In the next step, sequential dilutions of total RNA extracted from ED17 mouse lung were reversed transcribed to form cDNA. Competitive PCR measurements of these reverse-transcribed cDNA products and a constant amount of alpha -SMA competitor are shown (Fig. 1C). The standard curve of reverse-transcribed products had a slope identical to that of the standard curve from the alpha -SMA cDNA (Fig. 1C). The identical slopes indicated that recombinant alpha -SMA behaves identically in competitive PCR to alpha -SMA cDNA made from RT. Therefore, the initial alpha -SMA mRNA in the RT reaction can be quantitated with recombinant alpha -SMA as a standard. The linear equation derived from the recombinant alpha -SMA cDNA standard curve can be used to determine the unknown amount of alpha -SMA as its cDNA equivalent. The detection limit for alpha -SMA was 2.5 fg of cDNA or 5 ng of total mRNA in competitive PCR (Fig. 1C).

The standard curves for TTF-1, SP-A, and SP-C competitive RT-PCR were all generated as described above for alpha -SMA. They were all linear and have been published previously (33).

Comparison of alpha -SMA, TTF-1, SP-A, and SP-C mRNA levels in ED17 normal, nitrofen-exposed, and nitrofen-induced DH mouse lungs. Six different whole lungs from ED17 control, nitrofen-exposed, and nitrofen-induced DH mice were individually extracted for total RNA, which was subsequently reverse transcribed to generate cDNA. The reverse-transcribed cDNA from each individual lung sample was then coamplified with either alpha -SMA, TTF-1, SP-A, or SP-C competitor cDNA in the appropriate competitive PCR assay. The equations drawn from the linear regressions for each of the standard curves were used to interpolate the alpha -SMA, TTF-1, and SP-A mRNA amount from its cDNA equivalent amount in each lung sample. To control for potential variations due to the efficiency of RNA extraction and RT, beta -actin mRNA was also quantitated in the same sample in which alpha -SMA, TTF-1, SP-A, and SP-C were measured. We designed the beta -actin competitive PCR using a methodology similar to that described for the measurement of alpha -SMA, TTF-1, SP-A, and SP-C mRNA levels by competitive RT-PCR. The typical electrophoretic pattern for beta -actin is also shown in Fig. 7A. The beta -actin mRNA level was the same under all the conditions tested. The alpha -SMA, TTF-1, SP-A, and SP-C mRNA levels were normalized to beta -actin.


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Fig. 7.   Comparison of alpha -SMA, TTF-1, SP-A, and SP-C mRNA levels in ED17 normal (C), nitrofen-exposed (N), and nitrofen-induced DH (H) mouse lungs. A: typical electrophoretic pattern for alpha -SMA, TTF-1, SP-A, SP-C, beta -actin, and their respective competitors. mRNA was extracted from lungs (n = 6) from each group, and mRNA quantity of alpha -SMA, TTF-1, SP-A, and SP-C was measured from its cDNA equivalent. beta -Actin mRNA levels were measured in an identical fashion in all groups and conditions tested as an internal control for RNA extraction and cDNA production. B: mRNA values from cDNA equivalents were calculated from densitometric data for each sample; this value was then normalized by dividing by value for beta -actin and taking an average of similar samples (n = 6 lungs/group). mRNA levels are presented as multiple of normal values for normal control, nitrofen-exposed, and nitrofen-induced DH lungs. Quantity of TTF-1 and SP-C mRNA was the same in all 3 groups. Quantity of alpha -SMA mRNA was significantly different in the 3 groups, with a 2.4-fold increase in alpha -SMA mRNA in nitrofen-exposed group and a 4.5-fold increase in alpha -SMA mRNA in nitrofen-induced DH group (P < 0.001). SP-A mRNA levels were also significantly different but showed a 50% decrease in SP-A mRNA in nitrofen-exposed group and an 84% decrease in SP-A mRNA in nitrofen-induced DH lungs (P < 0.001).

To compare the levels of alpha -SMA, TTF-1, SP-A, and SP-C on the same graphic scale, the values were all divided by the value for the ED17 normal lungs. Figure 7B shows the mRNA levels for alpha -SMA, TTF-1, SP-A, and SP-C in the ED17 normal control, nitrofen-exposed, and nitrofen-induced DH groups. There was a 2.4-fold increase in alpha -SMA mRNA in the nitrofen-exposed group and a 4.5-fold increase in alpha -SMA mRNA in the nitrofen-induced DH group. This increase was significant for both groups (P < 0.001). There was a 50% decrease in the SP-A mRNA in the nitrofen-exposed group and an 84% decrease in the SP-A mRNA in the nitrofen-induced DH lungs (P < 0.001). The levels of TTF-1 and SP-C mRNA were not significantly different from the normal group compared with either the nitrofen-exposed or nitrofen-induced DH group.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Human infants with CDH have fewer bronchiolar branches, fewer arterioles, and excessive muscularization of preacinar arteries (2, 14). The PPH in CDH infants is postulated to be caused by decreased vascular lumen size as well as by vasoconstriction of the abnormally muscularized vascular tree, leading to increased pulmonary vascular resistance (9). Herein, we utilized the nitrofen-induced mouse model of DH to further elucidate the vascular abnormalities such as fewer and more muscular small arterioles and delayed epithelial cell differentiation associated with CDH.

As in the human condition, the nitrofen-induced DH defect occurred most frequently on the left side (25). The lungs from nitrofen-exposed fetal mice showed markedly decreased numbers of medium to small pulmonary arterioles whether or not a hernia was present. However, the deficiency in muscular arterioles in the nitrofen-induced DH mice was greater than in the nitrofen-exposed mice as well as being even greater on the side with a hernia. These findings suggest that the mice with nitrofen-induced DH have bilaterally hypoplastic lungs but that the vascular defect is worse on the ispsilateral side with the hernia.

Further examination of alpha -SMA staining at a higher power revealed that in the periphery of the normal lungs, alpha -SMA was primarily limited to a narrow band immediately surrounding the bronchioles and arterioles. However, in the nitrofen-induced DH lungs, the alpha -SMA staining was not only confined to the area adjacent to the pulmonary bronchioles and arterioles but was also found throughout the distal parenchyma between the distalmost airway branches. Therefore, we speculate that there are more contractile cells in the mesenchyme of the nitrofen-induced DH lungs bilaterally and that alpha -SMA is expressed in a less well-organized distribution than in the normal lungs.

We quantitated the expression of alpha -smooth muscle mRNA by competitive RT-PCR and found that there was a 4.5-fold increase in this smooth muscle marker in the mice with a nitrofen-induced DH, as well as a 2.4-fold increase in nitrofen-exposed animals without a hernia, over levels observed in normal ED17 mouse lungs. The thickened arteriolar walls and the increase in contractile cells in the distal mesenchyme may explain the significant increase in alpha -SMA mRNA in both the nitrofen-exposed and nitrofen-induced DH mice.

Previously, Okazaki et al. (21) reported that in the rat model of nitrofen-induced DH, the smooth muscle phenotype expressed in the hernia lung was confined to differentiated smooth muscle localized around vessels and bronchioles. Although we also observed smooth muscle staining around the peripheral arterioles and bronchioles, the alpha -SMA-positive staining observed throughout the periphery did not appear to be limited to the previously described differentiated smooth muscle cells. The diffuse peripheral staining may therefore actually represent staining of another contractile cell type such as myofibroblasts, which also express alpha -SMA. These alpha -SMA-positive cells in the normal lung may play a role in the regulation of the matching of ventilation and perfusion (12, 15). Thus the increase in differentiated smooth muscle, along with the increased and abnormal distribution of contractile cells, may play a role in the pathogenesis of PPH.

The nitrofen-induced DH lungs showed a markedly underdeveloped peripheral capillary network by PECAM-1 staining compared with both the normal and nitrofen-exposed lungs, and this deficiency affected both the ipsilateral and contralateral sides equally. Thus the vascular hypoplasia seen in these fetal mice with a DH cannot be attributed solely to the effects of nitrofen alone nor can it be attributed solely to the presence of a hernia because the defect is seen bilaterally. Therefore, the diaphragmatic defect and delay in peripheral vascular development may be two parallel processes pertaining to mesenchymal abnormalities. This supports the hypothesis that the pulmonary hypoplasia seen in DH is a primary hypoplasia and that the diaphragmatic defect is a concurrent event or may even be a consequence of the lung hypoplasia resulting from abnormal developmental signals (11, 31).

The factors responsible for endothelial cell differentiation during lung development are not well characterized, but the spatial and temporal expression of peptide growth factors, extracellular matrix composition, and mesenchymal-epithelial interactions are all thought to be involved (23). One growth factor known to promote vasculogenesis is vascular endothelial growth factor (VEGF) (16). Nitrofen-induced DH rat lungs have been shown to have a developmental delay in the expression of VEGF, and the deficiency of a soluble peptide growth factor such as VEGF could contribute to both the decrease in peripheral capillary vasculogenesis and its bilateral distribution (21).

Apposition of alveolar air spaces and peripheral capillaries must occur in order to develop functional gas-exchanging units. When PECAM-1 staining was examined at high power, a marked increase in the distance between the capillary and the respiratory epithelium was noted in the DH lung compared with the normal lung capillaries. The nitrofen-induced DH lungs not only had fewer capillaries but also had a thickened air-capillary interface, thus creating a potential structural barrier to oxygen diffusion and uptake into the circulation. Therefore, we speculate that the hypoxia seen in CDH may be due to the combination of a decreased air-vascular surface area, increased diffusion barrier, and a defective diaphragm hampering breathing movements. Experiments by O'Toole et al. (22) and Wilcox et al. (30) in the surgical lamb model of DH have shown a marked improvement in gas exchange and a decreased peripheral vascular resistance by surfactant therapy and perfluorocarbon-associated gas exchange during mechanical ventilation due to improved alveolar recruitment, improved alveolar stability, and total lung capacity. In the face of a decreased air-vascular surface area, like that seen in the lamb model of DH, the improvement in recruitment of unused and/or dysfunctional alveolar segments allows for maximal utilization of the available surface area for gas exchange (22, 30). Furthermore, stabilization of the alveolar wall may improve gas exchange even across a thickened air-capillary interface due to improved capillary blood flow and decreased alveolar surface tension.

The process of differentiation requires the expression of a cell-specific array of genes that define the cell phenotype (7). Whereas relatively little is known about the genetic factors that lead to the differentiation of the lung vasculature, several factors such as TTF-1, SP-A to -D, and the Clara cell 10-kDa protein are known markers of pulmonary epithelial differentiation (10, 29). The distribution and expression of these proteins in the lung epithelium represent differentiation of the specific cell types of the peripheral pulmonary epithelium.

TTF-1 is expressed in the epithelial cells of the conducting airways. During distal airway acinar development, TTF-1 expression becomes restricted to cells of the type II pneumocyte lineage (13, 17). The level of expression of TTF-1 mRNA was the same in the ED17 normal, nitrofen-exposed, and nitrofen-induced DH lungs. However, immunostaining with TTF-1 revealed decreased expression in the peripheral pulmonary epithelium in the normal lungs but not in the nitrofen-induced hernia lungs. The spatial distribution of TTF-1 staining in the normal lung reflects advanced development of the peripheral acini, with TTF-1 expression isolated to the type II pneumocytes and loss of TTF-1 expression in all other peripheral epithelial cell types. In contrast, the age-matched lungs from fetuses with DH exhibited a delay in acinar development and continued to display a conducting-airway phenotype in which all respiratory epithelial cells expressed TTF-1 all the way out to the pleura. The apparently equal expression of TTF-1 mRNA in the normal and nitrofen-induced DH lungs may be due to the fact that the nitrofen-induced DH lungs had both less total epithelium and a higher fraction of TTF-1-expressing cells compared with the normal lungs. These two opposing effects may balance one another, leading to no significant change in total lung TTF-1 mRNA level in the nitrofen-induced DH lungs versus the normal lungs.

SP-C expression data paralleled those of TTF-1. We found no difference in the quantity of SP-C mRNA among the control, nitrofen-exposed, and nitrofen-induced DH lungs. Furthermore, we found no differences in the intensity or location of SP-C immunostaining among the different groups. We speculate that there is no difference in SP-C mRNA for the same reason that there is no change in the level of TTF-1 mRNA. There were no differences in SP-C staining among the groups, partly due to the fact that the immunostaining was patchy in distribution in all three lung groups, and thus subtle differences in the distribution patterns of the staining were not apparent.

SP-C expression begins early in development and increases gradually throughout development. SP-A expression, on the other hand, begins later in development via transcriptional amplification (29). In the human, SP-A synthesis parallels the synthesis of surfactant phospholipids. SP-A mRNA and SP-A are undetectable in human amniotic fluid until the 28th week of gestation and then rapidly increase until birth. Moya et al. (18) measured SP-A levels in amniotic fluid obtained prenatally in humans with CDH and found a marked decrease in SP-A production. Furthermore, those infants who survived had 3.4-fold higher SP-A levels than those infants who died. SP-A begins expression on day 14 in the mouse and is expressed in both distal epithelial cells and type II pneumocytes. Herein, the production of SP-A mRNA was reduced by 84% in the lungs with a nitrofen-induced DH compared with the normal lungs. Staining for SP-A revealed fewer distal bronchial branches in the DH than in the normal lungs. Delay of upregulation of SP-A mRNA may contribute to the low levels of SP-A mRNA seen in mouse DH and human CDH. The deficiency of SP-A mRNA and SP-A expression further supports the speculation that the epithelium in the nitrofen-induced DH animals is less differentiated compared with the normal animals and also supports the finding that surfactant therapy may be beneficial in the treatment of CDH (22).

In conclusion, the nitrofen-induced murine model of DH (8, 19) mimics the human disease with reference to a decrease in branching morphogenesis of the pulmonary epithelium and vasculature as shown in our present study. The vascular abnormalities include abnormal muscularization, fewer capillary networks, and a thickened, underdeveloped air-capillary interface. Differentiation of the pulmonary epithelium was delayed, with a decrease in the expression of SP-A but not of SP-C. We speculate that the panhypoplasia of both the pulmonary vasculature and the epithelium, which occurs bilaterally, may be caused by impaired autocrine/paracrine growth factor signaling. The characterization of abnormalities of growth factor signaling in animal models of DH may lead to a greater understanding of the molecular mechanisms that cause the abnormal morphogenesis in CDH and may lead to the development of new rational therapeutic approaches.

    ACKNOWLEDGEMENTS

We thank both Pablo Bringas and Valentino Santos at the Center for Craniofacial Molecular Biology (University of Southern California, Los Angeles) for excellent assistance and guidance in the completion of this work. We also thank Drs. Robert Cilley and Marla Chinoy at Pennsylvania State University (Hershey) for input on the correct dosage of nitrofen in the early stages of this work.

    FOOTNOTES

This study was supported by a Childrens Hospital Los Angeles Research Institute Career Development Award (to C. Coleman) and National Heart, Lung, and Blood Institute Grants HL-44060, HL-4977 (both to D. Warburton), and HL-56590 (to P. Minoo).

Address for reprint requests: D. Warburton, Developmental Biology Program, Childrens Hospital Los Angeles Research Institute, 4650 Sunset Blvd., Mailstop #35, Los Angeles, CA 90027.

Received 18 June 1997; accepted in final form 10 December 1997.

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Abstract
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Materials & Methods
Results
Discussion
References

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