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Regular Articles |
From the Section of Pulmonary Diseases, Critical Care and
Environmental Medicine of the Department of Medicine,*
and
the Department of Pathology and Laboratory
Medicine,
Tulane University Medical Center,
New Orleans, Louisiana
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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granules of platelets. PDGF was subsequently found to be produced
by a variety of cell types, including macrophages and epithelial,
endothelial, mesenchymal, and neuronal cells.2
PDGF has
been demonstrated to be essential for normal
development3,4
and has been implicated in disease
processes such as atherosclerosis and lung fibrosis.5,6
Active PDGF consists of two homologous subunits, A and B, which can
form three dimeric PDGF isoforms (AA, AB, and BB).7-9
PDGF dimers bind to specific receptors that themselves are dimers
formed from two types of subunits, and ß, that differentially
bind PDGF isoforms.10,11
The ßß receptor dimer binds
only PDGF-BB, the ß receptor dimer binds PDGF-BB and PDGF-AB, and
the receptor dimer binds all three types of PDGF dimers. PDGF
receptors are found primarily on mesenchymal cells.12
Normally, PDGF is produced at low levels in adult tissues in
vivo, but its expression increases after injury or when cells are
removed for culture.12 PDGF is expressed at low levels in normal adult lung but is up-regulated in human interstitial lung disease and in animal models of lung fibrosis.13 Macrophages from patients with idiopathic pulmonary fibrosis expressed higher levels of PDGF message and secreted increased amounts of PDGF compared to macrophages from normal lung.14,15 Elevated levels of PDGF-B message and immunoreactivity have been found in epithelial cells and macrophages of patients with idiopathic pulmonary fibrosis.5 In another study, PDGF-B chain mRNA was detected in epithelium and macrophages in lung sections from patients with idiopathic pulmonary fibrosis, but PDGF-B immunoreactivity was detected only in macrophages.16 Elevated PDGF-related peptides have been detected in lavage fluid from patients with Hermansky-Pudlak syndrome, a condition characterized by severe pulmonary fibrosis.17 In an animal model of pulmonary fibrosis, asbestos inhalation rapidly induced up-regulation of PDGF-A and -B genes in epithelial cells and macrophages at sites of asbestos fiber deposition.18
Because of its documented effects on mesenchymal cell proliferation and extracellular matrix production and its expression in human interstitial lung disease and rodent models of lung fibrosis, PDGF is likely to play an important role in fibrogenic lung injury. Numerous cytokines and growth factors are elaborated during the development of fibroproliferative lung disease. To determine whether fibrotic lung disease can be initiated by PDGF alone, we generated transgenic mice that express the PDGF-B gene from the lung-specific surfactant protein C (SPC) promoter. Here we report that SPC-PDGFB transgenic mice develop lung disease characterized by enlarged airspaces, which in some animals were distributed throughout the lung, as well as inflammation and fibrosis, which were generally more focal in nature. Our results indicate that PDGF has potent effects on lung growth and development and that its expression can cause lung pathology characterized by features of two distinct diseases, emphysema and lung fibrosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The SPC-PDGFB DNA construct contained promoter sequences from the
human SPC gene, 5' untranslated sequences from the rat insulin gene,
and coding sequences from the human PDGF-B (c-sis) cDNA. To
obtain human SPC promoter sequences, oligonucleotides were synthesized
based on the published human SPC sequence19
and used to
amplify a 550-bp fragment from the first exon of the SPC gene by
polymerase chain reaction. The 550-bp SPC fragment was used as a probe
to screen a human DNA library in the vector 
EMBL3, and clones that
hybridized to the SPC probe were isolated. The 3.7-kb
HindIII-PstI fragment that confers lung-specific
expression in transgenic mice20,21
was isolated and cloned
into a plasmid vector for further manipulation. The identity of this
fragment was confirmed by restriction mapping and partial DNA
sequencing. An intron-containing fragment from the rat insulin II gene
was included in the SPC-PDGFB construct upstream from the PDGF-B cDNA.
This fragment contains bases +6 to +177 relative to the transcription
initiation site and encodes the transcription start site, the first
exon and intron, and a portion of the second exon.22
The
PDGF-B (c-sis) cDNA (clone pSM-1) was obtained from Dr. Lee
Ratner (Washington University, St. Louis, MO). The PDGF-B cDNA was
excised from pSM-1 with PstI and XhoI and cloned
downstream of the SPC and insulin fragments. This 2.5-kb PDGF-B
fragment contains from base +890 (relative to the transcription start
site) to the polyadenylation sequence and poly A tail and includes the
entire PDGF-B open reading frame.23
Transgenic mice were generated by microinjection of the linear 6.9-kb SPC-PDGFB construct into fertilized B6SJLF2 one-cell mouse embryos as described.24 SPC-PDGFB transgenic mice were identified by hybridization of tail biopsy DNA with a probe derived from human SPC promoter sequences. Transgenic mice were generated on a mixed genetic background of the C57BL/6 and SJL inbred strains, and transgenic lineages were maintained by breeding transgenic individuals to nontransgenic B6SJLF1 hybrid mice. Mice were housed under specific pathogen-free conditions in accordance with National Institutes of Health guidelines for the care and use of laboratory animals. No evidence of viruses or respiratory pathogens has been found in SPC-PDGFB mice or other mice from the facility in which the transgenic mice were housed. We have observed no inflammation in the lungs of nontransgenic mice or in mice carrying transgenes unrelated to PDGF.
RNA Analysis
RNA was prepared by ultracentrifugation of tissue homogenates through cesium chloride.25 RNA was subjected to Northern blot analysis26 with a probe from the human PDGF-B gene that hybridizes with both human and mouse PDGF-B RNA. Blots were subsequently hybridized with 36B4, a human cDNA encoding the ribosomal protein P0, as a loading control.27,28
PDGF Enzyme Immunoassay
PDGF was measured in lung homogenates using a previously described enzyme immunoassay that detects isoforms containing the PDGF-B chain.29,30 Lungs were perfused with PBS (10 mmol/L sodium phosphate, pH 7.4, containing 140 mmol/L NaCl and 3 mmol/L KCl) via the right ventricle and then homogenized in 1 ml of PBS containing 1 mmol/L phenylmethylsulfonyl fluoride, 4 mmol/L EDTA, and 7 µg/ml aprotinin. Homogenates were rapidly frozen and thawed and then centrifuged at 16,000 x g for 10 minutes at 4°C. PDGF was measured in aliquots of supernatants by enzyme immunoassay. PDGF levels in lung homogenates were normalized to protein content as determined by Bradford assay (BioRad, Hercules, CA) with bovine serum albumin as a standard.
Histological and Immunohistochemical Analyses
For histological analysis, lungs were fixed by intratracheal instillation of 10% neutral buffered formalin for 10–30 minutes at room temperature, followed by fixation by immersion overnight at 4°C. Lungs were embedded in paraffin and sectioned at 5 µm. Elastin staining was performed by the method of Miller, but Van Gieson counterstaining was omitted.31,32 Eosinophils were detected in tissue sections by the method of Luna.33 Immunohistochemistry was performed by an immunoperoxidase technique similar to that recently described.34 Briefly, lung sections were stained with PGF-007 (Mochida Pharmaceutical Co., Tokyo), a mouse monoclonal antibody directed against a peptide from the PDGF-B chain,6,35 at a concentration of 5 µg/ml. Bound antibody was detected by sequential incubation of the sections with biotinylated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:4000, streptavidin-conjugated horseradish peroxidase (Jackson ImmunoResearch) diluted 1:2000, and diaminobenzidine chromagen.
Morphometric Analysis
Measurements were performed by an investigator who was unaware of the identity of the samples. For each mouse, one section from each of four lobes was analyzed to determine the percentage of each lung section that exhibited abnormal histology. Areas were measured using V150 software36 (Oncor Imaging, Gaithersburg, MD) by tracing the perimeter of each section (total area) or by tracing histologically abnormal regions (abnormal area). Total area measurements were performed on images captured from an Olympus SZH dissecting microscope, whereas abnormal area measurements were performed on images captured from an Olympus BH-2 microscope using a 4X objective. For each mouse, the abnormal and total areas respectively from the four lobes were summed, and the percentage of the section areas that was abnormal was calculated.
Two parameters were measured in sections from neonatal transgenic and nontransgenic mice. Airspace area was measured to compare the size of the airspaces between the two groups of mice. A second parameter, which we have called septal chord length, was measured as an indication of the thickness of the septa. This is identical to the parameter called "airspace wall thickness" by previous investigators37 and is derived by overlaying a grid of parallel horizontal lines over the image, measuring the lengths of all intersections between the grid lines and airspace walls, and repeating these steps with a vertical grid. Septal chord length increases with increasing septal thickness. For measurement of airspace area and septal chord length, neonatal lung sections were viewed with a 20X objective, and images from the lungs were digitized, converted to tagged image format file, and analyzed using public domain NIH Image software (downloaded from http://rsb.info.nih.gov/nih- image/Default.html). For each lung, five fields lacking large airways or blood vessels were analyzed. For measurement of airspace area, images were edited to remove airways and blood vessels, thresholded manually, made binary, and inverted. A procedure was performed to retain the airspaces while eliminating the remainder of the image (Onion, written by Robert Homer and available from ftp://codon.nih.gov/pub/nih-image/contrib/ChordLength.SurfaceArea). Airspace areas were then measured using the "Analyze Particles" function of NIH Image. Areas from five fields were pooled and averaged to generate a mean airspace area for each mouse. Values for five transgenic and five nontransgenic mice were then averaged to evaluate the influence of the transgene on airspace area. Septal chord length was used as an indication of the thickness of the primary septa. Septal chord length was measured similarly to alveolar chord length as described previously,38 but the images were not inverted before analysis, so that the operations were performed on the airspace walls instead of the airspaces. Lengths from five fields were pooled and averaged to generate a mean septal chord length for each mouse. Values for five transgenic and five nontransgenic mice were averaged to evaluate the influence of the transgene on septal chord length.
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Results |
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Expression of the PDGF-B gene was directed to the lung by placing
PDGF-B coding sequences downstream from a 3.7-kb DNA fragment from the
5' region of the human SPC gene (Figure 1)
.19
This fragment has been
previously demonstrated to direct expression of transgenes to distal
lung epithelium, including type II alveolar epithelial cells and Clara
cells of the terminal bronchioles.20,21
The PDGF-B cDNA
fragment contains the entire PDGF-B open reading frame, 3' untranslated
sequences, and polyadenylation site. An intron-containing fragment from
the rat insulin II gene was placed upstream of the PDGF-B coding
sequences because this fragment has been shown to increase the
efficiency of expression of cDNA fragments in transgenic
mice.22,39
Five founder mice that carried the SPC-PDGFB
transgene shown in Figure 1
were generated. Three of the five founder
mice transmitted the transgene to progeny; one founder mouse sired
offspring but did not transmit the transgene to progeny; and one female
founder, designated 49-6, became sick and was euthanized before it bore
offspring.
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. The probe
hybridizes with the endogenous mouse PDGF-B RNA by virtue of the close
homology between the human and mouse PDGF-B genes. The size of the
mouse PDGF-B mRNA is 3.5 kb, whereas the SPC-PDGFB transgene message is
predicted to be approximately 2.7 kb because much of the 5'
untranslated region of the human PDGF-B cDNA is not present in the
transgene. Two of the lineages examined did not express detectable
transgene message (lanes 1 and 3); they yielded results identical to
those for nontransgenic mice (lanes 5 and 6). One founder mouse that
did not produce any transgenic offspring exhibited hybridizing RNA
species of higher molecular weight but no hybridizing band at the
predicted size (lane 4). These larger RNAs may be produced by improper
RNA processing or by integration of a truncated transgene that can form
a hybrid RNA with sequences at the integration site. Because this
founder mouse did not sire transgenic offspring, it was not possible to
determine whether PDGF protein was produced from these aberrant RNA
species. In one lineage, designated 47-5, Northern blot analysis
revealed a band of the predicted size that hybridized with the human
PDGF-B probe (lane 2, arrowhead). Mice of this lineage also exhibited
bands that were larger than the endogenous PDGF-B RNA; the identities
of these RNA species are not known. Analysis of RNA from nine other
organs in the 47-5 lineage indicated that lung was the only organ in
which transgene message could be detected (not shown). In summary,
Northern blot analysis revealed that one of four SPC-PDGFB lineages
produced a PDGF-B RNA of the predicted size specifically in the lung.
RNA from the 49-6 founder mouse, which became sick and was euthanized,
was not subjected to Northern blot analysis.
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. The concentration of PDGF in
plasma was not different between SPC-PDGFB and nontransgenic mice,
indicating that systemic PDGF levels were not increased in the
transgenic mice (not shown).
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One of the SPC-PDGFB founder mice, 49-6, became cachectic,
lethargic, and cyanotic starting at approximately 7 weeks of age. This
mouse was euthanized at 8 weeks of age and its lungs were fixed for
histological analysis. On gross examination, the lungs appeared larger
than normal and had airspaces that were obviously enlarged. Compared to
normal lungs (Figure 4A)
, histological
sections of the transgenic mouse revealed a severe disruption in the
architecture of the lung characterized by enlarged airspaces,
inflammation, and focal areas of fibrosis (Figure 4, B–D)
. Normal
alveoli were not present; instead, large emphysematous air spaces were
observed throughout the lung. These large airspaces were bounded by
septa that appeared thickened compared to normal alveolar septa. The
lungs showed a prominent inflammatory process that was dominated by
macrophages and eosinophils (Figure 4C
; also shown in greater detail in
Figure 5
). Many airspaces were filled
with macrophages containing phagocytosed red blood cells and cell
debris (Figure 4C)
. Focal areas of fibrosis were also observed (Figure 4D)
.
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. Some mice exhibited pathology throughout the lung that was
characterized by enlarged airspaces, inflammation, and fibrosis.
(Figure 5A)
. Other mice developed fibrotic lesions with less pronounced
airspace enlargement but more inflammation and fibrosis (Figure 5B)
.
Inflammation of the lungs by macrophages and eosinophils was observed
(Figure 5C and 5D)
. In many mice a less severe pathology was observed
that was characterized by focal airspace enlargement surrounded by
normal lung tissue (Figure 5E)
. These lesions were never observed in
nontransgenic littermates. Lung sections of some transgenic mice
exhibited no sign of the PDGF-induced pathology and appeared normal by
light microscopy (Figure 5F)
. The most common phenotype observed in the
47-5 lineage was enlarged airspaces in a variable proportion of the
lung. Inflammation and fibrotic lesions were less frequently visible.
Although some mice exhibited severely diseased lungs histologically,
all of the animals appeared outwardly healthy. We have observed no
excess mortality in the 47-5 lineage through 12 months of age. These
results indicated that constitutive expression of PDGF could induce
lung pathology, but there was incomplete penetration of the phenotype
induced by the SPC-PDGFB transgene in the 47-5 lineage.
A histological analysis of lungs from mice of different ages was
performed to determine whether the transgene-induced lung disease
progressed with age. Figure 6
shows the
extent of disease in lung sections from mice of different ages.
Significant lung pathology was found in a fraction of transgenic mice
of most ages but not in nontransgenic mice. Transgenic mice 
3
months of age appeared to fall into two groups: one in which the
majority of the lung was affected by PDGF-induced pathology, and one in
which the lung was normal or mildly affected. In transgenic mice
6 months of age, all mice exhibited moderate to severe pathology,
and no animals with normal lung histology were observed. These
observations are consistent with the existence of two populations of
mice that vary in the degree of PDGF-induced lung disease that
develops. One population is susceptible to the effects of the transgene
at an early age. The second population is resistant to
transgene-induced disease at young ages, but the disease eventually
develops in older mice.
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or in transgenic lungs if the
anti-PDGF-B monoclonal antibody was replaced with the same
concentration of normal mouse IgG (Figure 7B)
. In areas of transgenic
lung that appeared histologically normal, PDGF-B immunostaining was
detected in type II alveolar epithelial cells and more weakly
in macrophages (Figure 7C)
. In areas of airspace enlargement, PDGF-B
staining was observed in epithelial cells in thickened septa (Figure 7D)
. In fibrotic areas of diseased lung, PDGF-B staining was observed
in epithelial cells, macrophages, and in cells within the thickened
interstitium (Figure 7E)
. The staining observed in macrophages could
represent endogenous PDGF-B expressed by activated macrophages or
uptake of transgene-derived PDGF released from type II cells.
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In our initial histological analysis, we observed a transgenic
mouse that developed lung pathology by 2 weeks of age (Figure 6)
. This
observation suggested that mice with early-onset PDGF-induced lung
disease would exhibit abnormalities in lung development. We therefore
analyzed lung tissue from 1-week-old and neonatal mice. Lungs from four
of seven 1-week-old transgenic mice were abnormal and exhibited a
pathology similar to that observed in adult mice, including enlarged
airspaces, inflammation, and fibrosis (Figure 8B)
. Lungs from five of six neonatal
SPC-PDGFB mice were abnormal, but the pathology differed from that in
older mice. Lungs of neonatal mice are in the saccular stage of
development, with the air sacs divided by primary septa. During
postnatal days 4–14, secondary septal growth occurs to divide the
saccules into adult-type alveoli. SPC-PDGFB mice appeared to have
enlarged saccules and thickened primary septa but no inflammation was
observed (Figure 8D)
. In contrast to older mice, in which airspace
enlargement was heterogeneously distributed in the lung, neonatal mice
appeared to have uniformly enlarged airspaces. To examine these changes
quantitatively, morphometric measurements were made to compare abnormal
lungs from neonatal SPC-PDGFB mice with lungs from nontransgenic
littermates. Airspace area and septal chord length, a parameter that
increases with septal thickness, were significantly increased in the
transgenic mouse lungs (Figure 9)
. These
results indicated that in transgenic mice that were susceptible to
PDGF-induced disease, transgene expression affected lung development so
that an abnormal lung structure was present at the time of birth.
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and transgenic
(Figure 8F)
mice. There did not appear to be a generalized disruption
of alveolarization as has been described in PDGF-A knockout
mice3
and in transgenic mice expressing transforming
growth factor-.40
These results indicated that the
process of alveolarization can occur in SPC-PDGFB mice and that
inhibition of this process is not likely to explain the emphysematous
lesions observed in the older transgenic mice. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to fibrosis, SPC-PDGFB mice developed enlarged airspaces. This phenotype was observed in both neonatal and adult mice but was different in appearance at these two stages. At birth, the lungs of some SPC-PDGFB mice exhibited enlarged saccules and thickened primary septa. Airspace area in these mice was increased 62% over that in nontransgenic mice, and the increase appeared to be uniformly distributed through the lung. In adult SPC-PDGFB mice, emphysematous areas containing extremely enlarged airspaces were observed, but in most mice these were heterogeneously distributed and were interspersed with areas of normal lung architecture. The mechanisms by which PDGF-B expression produced this emphysema-like phenotype are not known. PDGF-B expression did not appear to affect the process of alveolarization, in that septal crests with elastin fibers concentrated at their tips were readily observed in lungs from 1-week-old SPC-PDGFB mice. Emphysema in human lungs appears to arise from disturbances in the balance between proteases and protease inhibitors. An imbalance leading to the proteolysis of extracellular matrix, particularly elastin fibers, causes a destruction of alveolar tissue and results in emphysema. These observations raise the possibility that proteolysis of elastin fibers may contribute to the emphysematous phenotype in SPC-PDGFB mice. This possibility is consistent with studies in vitro that have demonstrated the up-regulation of protease expression by PDGF-BB in some cell types.43-45
Airspace enlargement has been a commonly observed phenotype in
transgenic mice with pulmonary manipulation of cytokine and growth
factor expression. Mice deficient in PDGF-A chain,3
mice
overexpressing TGF- from the SPC promoter (SPC-TGF-
mice),40,46
and mice overexpressing interleukin 11 from
the Clara cell secretory protein promoter (CCSP-IL-11
mice)38,47
all developed enlarged airspaces as a result of
these genetic manipulations. In all of these cases, the enlarged
airspaces have been documented to occur as a result of abnormalities in
lung development. The lungs of PDGF-A knockout mice were normal at
birth but developed enlarged airspaces over the first 2 postnatal weeks
as a result of failed secondary septation.3
Neonatal lungs
from SPC-TGF- transgenic mice appeared to have slightly enlarged
saccules and developed dramatically enlarged airspaces because the
growth of secondary septa did not occur.40
CCSP-IL-11 mice
in which the transgene was expressed throughout lung development
exhibited airspace enlargement, but mice in which IL-11 expression was
not induced until adulthood did not develop this
phenotype.38
These observations suggest that there is a
delicate balance of factors throughout development that controls
alveolarization and airspace size. Disruption of this balance in any of
a number of ways, including altered production of a growth
factor that controls mesenchymal cell proliferation, may result in the
histopathological manifestation of enlarged airspaces.
Our results indicate, in accordance with previous studies, that PDGF is
an important growth factor in lung development. Expression of PDGF-A
and -B genes and PDGF- and -ß receptors has been documented in the
developing lung. In fetal lung, PDGF-A expression has been detected in
epithelium48-50
and PDGF-B expression has been detected
in epithelium and endothelium.48,49
PDGF- receptor is
expressed in mesenchymal cells underlying airway and distal
epithelium49,50
and PDGF-ß receptor is expressed in
mesenchymal cells surrounding blood vessels.49
These
expression patterns are consistent with PDGF isoforms functioning as
signaling molecules that control lung mesenchymal cell proliferation
and development. PDGF-A has been shown to be required for normal lung
development; disruption of the PDGF-A gene by homologous recombination
caused an emphysema-like lung disease.3
The lungs of
PDGF-A knockout mice were normal at birth, but developed enlarged
airspaces within the first 2 postnatal weeks. The defect in these mice
was found to be the lack of elastin-producing mesenchymal cells that
are required for secondary septal growth and the formation of
alveoli.49
In contrast, our SPC-PDGFB mice exhibited an
abnormal lung phenotype on the day of birth. Based on the distribution
of PDGF receptors, this phenotype is likely to arise from the action of
PDGF-B that is released by type II alveolar epithelial cells and binds
to PDGF receptors on mesenchymal cells in the lung interstitium. We
speculate that the thickened airspace walls in neonatal SPC-PDGFB mice
arise from excess mesenchymal cell proliferation mediated by PDGFB, and
that the enlarged airspaces develop as a result of an improper ratio
between epithelial and mesenchymal cell populations.
An inflammatory influx containing macrophages and eosinophils was observed in the lungs of SPC-PDGFB transgenic mice. Inflammation was generally not distributed throughout the lung but was confined to focal areas. These focal areas of inflammation were usually within or adjacent to areas of fibrosis or severe emphysema. This observation indicated an association between inflammation and other manifestations of the PDGF-induced phenotype, but whether these events were causally related was not determined. Within the areas of inflammation, macrophages were predominantly confined to the air spaces. Eosinophils were observed both within the interstitium and in the airspaces. It is not clear whether the cells are actively migrating into the airspaces or whether they appear there as a consequence of the tissue destruction that leads to the emphysematous lesions. The presence of eosinophils was of interest, because this cell type has recently been implicated as having a role in the pathogenesis of lung fibrosis.51 The expression of interleukin 5 and the influx of eosinophils correlated with fibrosis in a bleomycin model of lung fibrosis.51 Similarly, we observed a correlation between eosinophilic inflammation and fibrosis in SPC-PDGFB mice. Further studies will be required to establish a causal relationship between eosinophil influx and the pathogenesis of fibrosis in SPC-PDGFB mice.
Multiple potential mechanisms exist by which PDGF expression might induce an inflammatory response. The most straightforward mechanism would be a direct effect of PDGF on inflammatory cells. Expression of PDGF receptors on macrophages or eosinophils has not been demonstrated directly, but there is evidence that PDGF can act as a chemotactic factor for eosinophils.52 A second mechanism by which PDGF may promote inflammation is the induction of chemokines in mesenchymal cells that can respond to PDGF. Evidence that this process can occur comes from studies in cultured fibroblasts, in which treatment with PDGF caused up-regulation of expression of chemokines including the macrophage chemoattractant MCP-1 (JE).53 Finally, if PDGF can promote proteolysis of lung tissue as discussed above, the process of tissue destruction may induce a pulmonary inflammatory response.
The extent of lung disease that was observed in SPC-PDGFB mice varied significantly among individual mice within the same transgenic lineage. At birth, some mice exhibited enlarged saccules and thickened septa, but the lungs of other transgenic mice appeared normal. Among young adults (up to 3 months of age) some mice displayed normal lung histology or were mildly affected by airspace enlargement, whereas other mice were severely affected by airspace enlargement and also had focal areas of inflammation and fibrosis. All SPC-PDGFB mice aged 6 months and older had some degree of histological abnormality in the lung, but still varied in the extent of the lung that was affected. These results are consistent with the existence of two populations of SPC-PDGFB mice. One population is susceptible to PDGF-B overexpression during lung development. These mice have abnormal lungs at birth and develop severe lung disease as young adults. The second population is resistant to the developmental effects of PDGF-B overexpression. These mice have normal lungs at birth, have normal or mildly affected lungs as young adults, and do not develop significant lung disease until they are 6 months of age. One potential mechanism that would explain this variation in phenotype is a possible difference in transgene expression among individual animals or regional variation in expression within a single lung. Immunostaining of transgenic lungs for PDGF-B indicated that, in both normal and abnormal areas of the lung, the transgene was expressed at similar levels in type II alveolar epithelial cells. In areas of inflammation, strong PDGF-B immunostaining was observed in macrophages, but it was not clear whether this represented endogenous PDGF expressed by macrophages or transgene-derived PDGF that had been taken up by macrophages. Thus, the immunostaining results did not exclude variation in PDGF expression as a causative factor for differences in PDGF-induced lung disease. However, this explanation does not account for the existence of two discrete populations of mice with differing disease susceptibilities. Variation in PDGF expression would be most likely to result in a continuum of the disease phenotype rather than the observed results with two distinct populations. Another possibility is that genetic variation among mice within the 47-5 lineage is responsible for the variation in phenotype. The SPC-PDGFB transgenic mice were generated and maintained on a genetic background that is a mixture of two inbred strains, C57BL/6 and SJL. If these strains vary in the response of the lung to PDGF, then mice with a hybrid B6/SJL background could exhibit disease-sensitive and disease-resistant phenotypes. These phenotypes could result from the differential expression of secondary genes that control the response to PDGF, such as PDGF receptors, signaling molecules, or downstream genes induced by PDGF. Strain differences in susceptibility to lung injury and disease produced by environmental agents such as bleomycin54,55 and asbestos56 have been described. In these models, the production of and response to cytokines and growth factors in the lung appear to be important determinants in controlling the susceptibility to fibrotic agents.57 Thus, there is precedence for postulating that the C57BL/6 and SJL inbred strains could respond differently to PDGF overexpression.
In summary, PDGF overexpression in distal lung epithelium resulted in enlarged airspaces, inflammation, and fibrosis. In susceptible mice, the airspace enlargement appeared to result from developmental abnormalities that occurred both before and after birth. Variation in the severity of PDGF-induced lung disease suggested that the genetic background of the mice may be important in determining the response of the lung to PDGF expression. These results highlight the importance of PDGF in lung development and disease pathogenesis. Future studies may determine whether PDGF overexpression renders mice more susceptible to lung fibrosis induced by exogenous agents such as asbestos or bleomycin.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health HL58610 (to GWH), NIH ES06766 (to ARB), NIH ES/HL09242 (to ARB), and the Tulane/Xavier Center for Bioenvironmental Research.
Accepted for publication February 25, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  C. L. Fattman, F. Gambelli, G. Hoyle, B. R. Pitt, and L. A. Ortiz Epithelial expression of TIMP-1 does not alter sensitivity to bleomycin-induced lung injury in C57BL/6 mice Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L572 - L581. [Abstract] [Full Text] [PDF]
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 B. R. Gochuico, N. A. Avila, C. K. Chow, L. J. Novero, H.-P. Wu, P. Ren, S. D. MacDonald, W. D. Travis, M. P. Stylianou, and I. O. Rosas Progressive Preclinical Interstitial Lung Disease in Rheumatoid Arthritis Arch Intern Med, January 28, 2008; 168(2): 159 - 166. [Abstract] [Full Text] [PDF]
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 H.-W. Chen, S.-F. Su, C.-T. Chien, W.-H. Lin, S.-L. Yu, C.-C. Chou, J. J. W. Chen, and P.-C. Yang Titanium dioxide nanoparticles induce emphysema-like lung injury in mice FASEB J, November 1, 2006; 20(13): 2393 - 2395. [Abstract] [Full Text] [PDF]
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R. H. Alvarez, H. M. Kantarjian, and J. E. Cortes Biology of Platelet-Derived Growth Factor and Its Involvement in Disease Mayo Clin. Proc., September 1, 2006; 81(9): 1241 - 1257. [Abstract] [Full Text] [PDF]
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 R. Fernandez-Valdivia, Y. Zhang, S. Pai, M. L. Metzker, and A. Schumacher l7Rn6 Encodes a Novel Protein Required for Clara Cell Function in Mouse Lung Development Genetics, January 1, 2006; 172(1): 389 - 399. [Abstract] [Full Text] [PDF]
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 F. Chua, J. Gauldie, and G. J. Laurent Pulmonary Fibrosis: Searching for Model Answers Am. J. Respir. Cell Mol. Biol., July 1, 2005; 33(1): 9 - 13. [Abstract] [Full Text] [PDF]
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 C. Reinhard, B. Meyer, H. Fuchs, T. Stoeger, G. Eder, F. Ruschendorf, J. Heyder, P. Nurnberg, M. H. de Angelis, and H. Schulz Genomewide Linkage Analysis Identifies Novel Genetic Loci for Lung Function in Mice Am. J. Respir. Crit. Care Med., April 15, 2005; 171(8): 880 - 888. [Abstract] [Full Text] [PDF]
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 A. Abdollahi, M. Li, G. Ping, C. Plathow, S. Domhan, F. Kiessling, L. B. Lee, G. McMahon, H.-J. Grone, K. E. Lipson, et al. Inhibition of platelet-derived growth factor signaling attenuates pulmonary fibrosis J. Exp. Med., March 21, 2005; 201(6): 925 - 935. [Abstract] [Full Text] [PDF]
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 M. D. Wewers {alpha}1-Antitrypsin Deficiency: More Than a Protease Imbalance? Chest, May 1, 2004; 125(5): 1607 - 1609. [Full Text] [PDF]
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B. R. Vuillemenot, J. F. Rodriguez, and G. W. Hoyle Lymphoid Tissue and Emphysema in the Lungs of Transgenic Mice Inducibly Expressing Tumor Necrosis Factor-{alpha} Am. J. Respir. Cell Mol. Biol., April 1, 2004; 30(4): 438 - 448. [Abstract] [Full Text] [PDF]
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P.-N. Tsao, Y.-N. Su, H. Li, P.-H. Huang, C.-T. Chien, Y.-L. Lai, C.-N. Lee, C.-A. Chen, W.-F. Cheng, S.-C. Wei, et al. Overexpression of Placenta Growth Factor Contributes to the Pathogenesis of Pulmonary Emphysema Am. J. Respir. Crit. Care Med., February 15, 2004; 169(4): 505 - 511. [Abstract] [Full Text] [PDF]
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 S. C. Pitchford, Y. Riffo-Vasquez, A. Sousa, S. Momi, P. Gresele, D. Spina, and C. P. Page Platelets are necessary for airway wall remodeling in a murine model of chronic allergic inflammation Blood, January 15, 2004; 103(2): 639 - 647. [Abstract] [Full Text] [PDF]
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 F. Huaux, T. Liu, B. McGarry, M. Ullenbruch, Z. Xing, and S. H. Phan Eosinophils and T Lymphocytes Possess Distinct Roles in Bleomycin-Induced Lung Injury and Fibrosis J. Immunol., November 15, 2003; 171(10): 5470 - 5481. [Abstract] [Full Text] [PDF]
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 R. V. Hoch and P. Soriano Roles of PDGF in animal development Development, October 15, 2003; 130(20): 4769 - 4784. [Abstract] [Full Text] [PDF]
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 A. Ponten, X. Li, P. Thoren, K. Aase, T. Sjoblom, A. Ostman, and U. Eriksson Transgenic Overexpression of Platelet-Derived Growth Factor-C in the Mouse Heart Induces Cardiac Fibrosis, Hypertrophy, and Dilated Cardiomyopathy Am. J. Pathol., August 1, 2003; 163(2): 673 - 682. [Abstract] [Full Text] [PDF]
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Workshop on Lung Disease and the Environment: Where Do We Go from Here? Am. J. Respir. Crit. Care Med., July 15, 2003; 168(2): 250 - 254. [Full Text] [PDF]
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S. Shimizu, E. C. Gabazza, O. Taguchi, H. Yasui, Y. Taguchi, T. Hayashi, M. Ido, T. Shimizu, T. Nakagaki, H. Kobayashi, et al. Activated Protein C Inhibits the Expression of Platelet-derived Growth Factor in the Lung Am. J. Respir. Crit. Care Med., May 15, 2003; 167(10): 1416 - 1426. [Abstract] [Full Text] [PDF]
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R. F. Foronjy, Y. Okada, R. Cole, and J. D'Armiento Progressive adult-onset emphysema in transgenic mice expressing human MMP-1 in the lung Am J Physiol Lung Cell Mol Physiol, May 1, 2003; 284(5): L727 - L737. [Abstract] [Full Text] [PDF]
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 R Mahadeva and S D Shapiro Chronic obstructive pulmonary disease * 3: Experimental animal models of pulmonary emphysema Thorax, October 1, 2002; 57(10): 908 - 914. [Abstract] [Full Text] [PDF]
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P A Dawkins and R A Stockley Animal models of chronic obstructive pulmonary disease Thorax, December 1, 2001; 56(12): 972 - 977. [Full Text] [PDF]
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S. D. Shapiro Animal Models for Chronic Obstructive Pulmonary Disease . Age of Klotho and Marlboro Mice Am. J. Respir. Cell Mol. Biol., January 1, 2000; 22(1): 4 - 7. [Full Text]
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