1 Division of Pediatric Surgery
and Developmental Biology Program, Neonates with
congenital diaphragmatic hernia (DH) die of pulmonary hypoplasia and
persistent pulmonary hypertension. We used immunohistochemical
localization of
pulmonary hypertension; pulmonary hypoplasia
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 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 Antibodies. 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 Next, the thickness of the 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
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-smooth muscle actin (
-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
-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
-SMA mRNA,
fewer and more muscular arterioles (
-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.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-smooth muscle actin (
-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
-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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-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.
-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
-SMA was purchased from Sigma (St. Louis, MO) and diluted to 1:500
for use.
-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.
-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
-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.
-SMA, TTF-1, SP-A, and
SP-C competitive RT-PCR. A set of primers was designed
based on the murine
-SMA cDNA sequence: primer
1 (upstream) was
5'-CTGGAGAAGAGCTACGAACTGC-3' and primer
2 (downstream) was
5'-CTGATCCACATCTGCTGGAAGG-3'.
-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
-SMA competitor
construction (Fig. 1A); each
composite primer had the target
-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
-SMA competitor molecules had the same gene-specific primer sequences as the
-SMA cDNA. The heterogeneous DNA was derived from a piece of
v-erbB DNA (5).
-SMA competitor PCR
products with primers 1 and
2 were 480 bp in length. The
identities of
-SMA and its competitor were both confirmed by DNA
sequencing (24).
View larger version (9K):
[in a new window]
View larger version (18K):
[in a new window]
View larger version (21K):
[in a new window]
Fig. 1.
Competitive RT-PCR for -smooth muscle actin (
-SMA) mRNA
quantitation. A: competitive PCR
products for
-SMA. Construction of
-SMA and its competitor is
described in MATERIALS AND METHODS.
B: electrophoretic pattern of
-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
-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
-SMA competitor. An inverse relationship between intensities of
-SMA and its competitor is evident, indicating competition between 2 templates. C: standard curve for
-SMA competitive PCR. Log(
SMA/competitor) from densitometric data
from gel in B was plotted against
logarithm of indicated amounts of added recombinant
-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
-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
-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 -SMA competitor was added to
reverse-transcribed samples derived from 50 ng of total RNA or to
dilutions of standard
-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
-SMA.
Primers and template construction for
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 ( ± 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.
|
Both nitrofen-exposed and nitrofen-induced DH lungs
had fewer -SMA positive arterioles, muscular walls were thickened,
and there was abundant
-SMA staining in lung
mesenchyme. In control, nitrofen-exposed, and herniated
lungs, immunostaining for
-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.
|
The distribution of -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
-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
-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 -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
-SMA-positive arterioles per HPF was 3.6 ± 0.1 (right;
± 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 -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.
|
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 ( ± 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.
|
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.
|
Measurement of -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
-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 -SMA as a
standard, sequential 1:2 dilutions of a mouse
-SMA cDNA template were prepared, and a constant amount (10 fg) of an
-SMA competitor cDNA template was added to each tube. After PCR coamplification of the
mouse
-SMA cDNA and
-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
-SMA competitor band intensity decreased (from
lanes 1 to
10), whereas the
-SMA band
intensity increased in proportion to the amount of
-SMA cDNA
included in each tube. When the logarithm of the ratio of
-SMA cDNA
to its competitor PCR products was plotted against the logarithm of the
initial amount of mouse
-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 -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
-SMA cDNA (Fig.
1C). The identical slopes indicated
that recombinant
-SMA behaves identically in competitive PCR to
-SMA cDNA made from RT. Therefore, the initial
-SMA mRNA in the
RT reaction can be quantitated with recombinant
-SMA as a standard.
The linear equation derived from the recombinant
-SMA cDNA standard
curve can be used to determine the unknown amount of
-SMA as its
cDNA equivalent. The detection limit for
-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 -SMA. They were all linear and
have been published previously (33).
Comparison of -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
-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
-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,
-actin
mRNA was also quantitated in the same sample in which
-SMA, TTF-1,
SP-A, and SP-C were measured. We designed the
-actin competitive PCR
using a methodology similar to that described for the measurement of
-SMA, TTF-1, SP-A, and SP-C mRNA levels by competitive RT-PCR.
The typical electrophoretic pattern for
-actin is also shown in Fig.
7A.
The
-actin mRNA level was the same under all the conditions tested.
The
-SMA, TTF-1, SP-A, and SP-C mRNA levels were normalized to
-actin.
|
To compare the levels of -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
-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
-SMA mRNA in the nitrofen-exposed group and a 4.5-fold
increase in
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -SMA staining at a higher power revealed that
in the periphery of the normal lungs,
-SMA was primarily limited to
a narrow band immediately surrounding the bronchioles and arterioles.
However, in the nitrofen-induced DH lungs, the
-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
-SMA is expressed in a less
well-organized distribution than in the normal lungs.
We quantitated the expression of -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
-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
-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
-SMA. These
-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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Araki, N.,
F. D. Robinson,
and
S. K. Nishimoto.
Rapid and sensitive method for quantitation of bone Gla protein mRNA using competitive polymerase chain reaction.
J. Bone Miner. Res.
8:
131-122,
1993.
2.
Areechon, W.,
and
L. Reid.
Hypoplasia of lung with congenital diaphragmatic hernia.
Br. Med. J.
1:
230-233,
1963.
3.
Atkinson, J. B.,
E. G. Ford,
B. Humphries,
H. Kitagawa,
C. Lew,
M. Garg,
and
K. Bui.
The impact of extracorporeal membrane support in the treatment of congenital diaphragmatic hernia.
J. Pediatr. Surg.
26:
791-793,
1991[Medline].
4.
Azarow, K.,
A. Messineo,
R. Pearl,
R. Filler,
G. Barker,
and
B. Desmond.
Congenital diaphragmatic herniaa tale of two cities: the Toronto experience.
J. Pediatr. Surg.
32:
395-399,
1997[Medline].
5.
Bruskin, A.,
J. Jackson,
J. M. Bishop,
D. J. McCarley,
and
R. C. Schatzman.
Six amino acids from the retroviral gene gag greatly enhance the transforming potential of the oncogene verb-B.
Oncogene
5:
15-24,
1990[Medline].
6.
Burri, P. H.
Structural aspects of prenatal and postnatal development and growth of the lung.
In: Lung Growth and Development, edited by John McDonald. New York: Dekker, 1997, vol. 100, p. 1-35. (Lung Biol. Health Dis. Ser.)
7.
Cardoso, W. V.
Transcription factors and pattern formation in the developing lung.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L429-L442,
1995
8.
Cilley, R. E.,
S. E. Zgleszewski,
T. M. Krummel,
and
M. R. Chinoy.
Nitrofen dose-dependent gestational day-specific murine lung hypoplasia and left-sided diaphragmatic hernia.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L362-L371,
1997
9.
Geggel, R. L.,
J. D. Murphy,
D. Langleben,
R. K. Crone,
J. P. Vacanti,
and
L. M. Reid.
Congenital diaphragmatic hernia: arterial structural changes and persistent pulmonary hypertension after surgical repair.
J. Pediatr.
107:
457-464,
1985[Medline].
10.
Ikeda, K.,
J. C. Clark,
J. R. Shaw-White,
M. T. Stahlman,
C. J. Boutell,
and
J. A. Whitsett.
Gene structure and expression of human thyroid transcription factor-1 in respiratory epithelial cells.
J. Biol. Chem.
270:
8108-8114,
1995
11.
Iritani, I.
Experimental study on embryogenesis of congenital diaphragmatic hernia.
Anat. Embryol. (Berl.)
169:
133-139,
1984[Medline].
12.
Kapanchi, Y.,
A. Assimacopoulos,
C. Irie,
A. Zwahlen,
and
G. Gabbiani.
Contractile interstitial cells in pulmonary alveolar septa: a possible regulator of ventilation/perfusion ratio?
J. Cell Biol.
60:
375-392,
1974
13.
Kimura, S.
Role of thyroid-specific enhancer-binding protein in transcription, development and differentiation.
Eur. J. Endocrinol.
136:
128-136,
1997[Medline].
14.
Kitagawa, M.,
A. Hislop,
E. Boyden,
and
L. Reid.
Lung hypoplasia in congenital diaphragmatic hernia. A quantitative study of airway artery and alveolar development.
Br. J. Surg.
58:
342-346,
1971[Medline].
15.
Leslie, K.,
J. J. Mitchell,
J. L. Woodcock-Mitchell,
and
R. B. Low.
-Smooth muscle actin expression in developing and adult human lung.
Differentiation
44:
142-149,
1990.
16.
Leung, D.,
G. Cachianes,
W. J. Kuang,
D. V. Goeddel,
and
N. Ferrara.
Vascular endothelial growth factor is a secreted angiogenic mitogen.
Science
246:
1306-1309,
1989[Medline].
17.
Minoo, P.,
H. Hamdan,
D. Bu,
D. Warburton,
P. Stepanik,
and
R. deLemos.
TTF-1 regulates lung epithelial morphogenesis.
Dev. Biol.
172:
694-698,
1995[Medline].
18.
Moya, F. R.,
V. L. Thomas,
J. Romaguera,
M. R. Mysore,
M. Mayberry,
A. Bernard,
and
M. Freund.
Fetal lung maturation in congenital diaphragmatic hernia.
Am. J. Obstet. Gynecol.
173:
1401-1405,
1995[Medline].
19.
Nakao, Y.,
and
R. Ueki.
Congenital diaphragmatic hernia induced by nitrofen in mice and rats: characteristics as animal model and pathogenetic relationship between diaphragmatic hernia and lung hypoplasia.
Congenital Anom.
27:
397-417,
1987.
20.
Newman, P. J.
Cell adhesion in vascular biology: the biology of PECAM-1.
J. Clin. Invest.
99:
3-8,
1997
21.
Okazaki, T.,
H. S. Sharma,
M. Aikawa,
A. Yamataka,
R. Nagai,
T. Miyano,
and
D. Tibboel.
Pulmonary expression of vascular endothelial growth factor and myosin isoforms in rats with congenital diaphragmatic hernia.
J. Pediatr. Surg.
20:
391-394,
1997.
22.
O'Toole, S. J.,
H. L. Karamanoukian,
F. C. Morin III,
B. A. Holm,
E. A. Egan,
R. G. Azizikhan,
and
P. L. Glick.
Surfactant decreases pulmonary vascular resistance and increases pulmonary blood flow in the fetal lamb model of congenital diaphragmatic hernia.
J. Pediatr. Surg.
31:
507-511,
1996[Medline].
23.
Roman, J.
Cell-cell and cell-matrix interactions in development of the lung vasculature.
In: Lung Growth and Development, edited by John McDonald. New York: Dekker, 1997, vol. 100, p. 365-399. (Lung Biol. Health Dis. Ser.)
24.
Sanger, R.,
S. Nickren,
and
R. R. Coulson.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA
74:
5463-5467,
1977[Abstract].
25.
Skandalakis, J.,
and
S. W. Gray.
Embryology for Surgeons: The Embryologic Basis for the Treatment of Congenital Anomalies. Baltimore, MD: Williams and Wilkins, 1994, p. 491-539.
26.
Ten Have-Opbroek, A. A.
Immunological study of lung development in the mouse embryo.
Dev. Biol.
69:
408-423,
1979[Medline].
27.
Tibboel, D.,
and
A. V. Gaag.
Etiologic and genetic factors in congenital diaphragmatic hernia.
Clin. Perinatol.
23:
689-699,
1996[Medline].
28.
Vorbroker, D. K.,
C. Dey,
T. E. Weaver,
and
J. A. Whitsett.
Surfactant protein C precursor is palmitoylated and associates with subcellular membrane.
Biochim. Biophys. Acta
1105:
161-169,
1992[Medline].
29.
Weaver, T. E.,
and
J. A. Whitsett.
Function and regulation of expression of pulmonary surfactant-associated proteins.
Biochem. J.
273:
249-264,
1991[Medline].
30.
Wilcox, D. T.,
P. L. Glick,
H. L. Karamanoukian,
C. Leach,
F. C. Morin III,
and
B. P. Fuhrman.
Perfluorocarbon-associated gas exchange improves pulmonary mechanics, oxygenation, ventilation, and allows nitric oxide delivery in the hypoplastic lung congenital diaphragmatic hernia lamb model.
Crit. Care Med.
23:
1858-1863,
1995[Medline].
31.
Wilcox, D. T.,
M. S. Irish,
B. A. Holm,
and
P. L. Glick.
Pulmonary parenchymal abnormalities in congenital diaphragmatic hernia.
Clin. Perinatol.
23:
771-779,
1996[Medline].
32.
Wuenschell, C. W.,
M. E. Sunday,
G. Singh,
P. Minoo,
H. C. Slavkin,
and
D. Warburton.
Embryonic mouse lung epithelial progenitor cells co-express immunohistochemical markers of diverse mature cell lineages.
J. Histochem. Cytochem.
44:
113-123,
1996
33.
Wuenschell, C. W.,
J. Zhao,
J. D. Tefft,
and
D. Warburton.
Nicotine stimulates branching and expression of SP-A and SP-C mRNAs in embryonic mouse lung culture.
Am. J. Physiol.
274 (Lung Cell. Mol. Physiol. 18):
L165-L170,
1998
34.
Zhao, J.,
N. Araki,
and
S. K. Nishimoto.
Quantitation of matrix Gla protein mRNA by competitive polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase as an internal control.
Gene
155:
159-165,
1995[Medline].
35.
Zhao, J.,
and
S. K. Nishimoto.
An RNA-competitive polymerase chain reaction method for human matrix -carboxyglutamic acid protein mRNA measurement.
Anal. Biochem.
228:
162-164,
1995[Medline].