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DEVELOPMENTAL DYNAMICS 211:215–227 (1998)
VEGF Enhances Pulmonary Vasculogenesis
and Disrupts Lung Morphogenesis In Vivo
XIN ZENG,1 SUSAN E. WERT,1 ROBERT FEDERICI, 2 KEVIN G. PETERS,2 AND JEFFREY A. WHITSETT1*
1Children’s Hospital Medical Center, Division of Pulmonary Biology, Cincinnati, Ohio
2Duke University Medical Center, Department of Internal Medicine, Cardiology Division, Durham, North Carolina
ABSTRACT
Vascular endothelial growth factor (VEGF) was expressed in developing respiratory epithelial cells under control of the promoter from the human surfactant protein C (SP-C)
gene. SP-C-VEGF transgenic mice did not survive
after birth. When obtained by hysterectomy on
embryonic day 15 (E15) or 17 (E17), abnormalities
in the transgenic mice were confined to the lung
and were correlated with the expression of transgene mRNA as revealed by in situ hybridization.
On E15 and E17, marked abnormalities in lung
morphogenesis were observed in transgenic mice.
Lungs consisted of large dilated tubules with
increased peritubular vascularity. The mRNA levels of the VEGF receptor, Flk-1, and the endothelial cell specific receptor tyrosine kinase, Tie-1,
were increased in lung mesenchyme of the transgenic mice. The numbers of acinar tubules and
the abundance of mesenchyme were decreased.
Endogenous VEGF mRNA was expressed in the
respiratory epithelial cells of the developing
lungs, and the levels of VEGF mRNA were increased in the SP-C-VEGF transgenic mice. Although the normal pattern of immunostaining
for SP-C and Clara cell secretory protein (CCSP)
indicated that epithelial cell differentiation was
relatively unaltered by the transgene, electron
microscopic analysis revealed a lack of alveolar
Type I cell differentiation at E18. Expression of
VEGF in the developing respiratory epithelium
of transgenic mice increased growth of the pulmonary blood vessels, disrupted branching morphogenesis of the lung and inhibited Type I cell
differentiation. Dev. Dyn. 1998;211:215–227.
r 1998 Wiley-Liss, Inc.
Key words: transgenic mice; lung development;
alveolarization; vasculogenesis
bules have a relatively small lumen and the mesenchyme remains prominent. However, from E15 to birth
(canalicular and saccular periods), the terminal buds
dilate and the mesenchyme thins. The developing pulmonary vasculature comes into increasing proximity to
the epithelial cells in the periphery of the lung with
advancing gestation. The close apposition of the pulmonary vasculature to the respiratory epithelium is temporally associated with differentiation of the respiratory
epithelium during the canalicular and saccular stages
of lung development, ultimately forming the functional
gas exchange regions in the alveolus. In the mature
alveolus, thin sheets of capillary endothelial cells form
a network surrounding the alveolar saccules which are
lined primarily by Type I and Type II epithelial cells
(Burri, 1984; Adamson, 1991). Respiratory epithelial
cells undergo extensive cytodifferentiation during the
perinatal and postnatal period of development (Ten
Have-Opbroek, 1991; Hilfer, 1983, Weaver and Whitsett, 1991). Inductive interactions between the pulmonary epithelium and mesenchyme are required for the
process of branching morphogenesis, sacculation, and
epithelial cell differentiation. Autocrine/paracrine signaling, cell-cell and cell-extracellular matrix interactions have been implicated in the process of lung
morphogenesis and pulmonary epithelial cell differentiation (Guzowski et al., 1990; Minoo and King, 1994;
Peters et al., 1994; Hilfer, 1996).
The early stages of lung vascular development involve both angiogenesis and vasculogenesis (deMello et
al., 1997, Pardanaud et al., 1989). The signaling molecules that control this process are not known. Vascular
endothelial growth factor (VEGF) is a selective mitogen
for endothelial cells, influencing angiogenesis and vasculogenesis during normal development (Dvorak et al.,
1995). In the mouse, VEGF exists in at least three
distinct isoforms that are generated by alternative
splicing from a single gene (Breier et al., 1992). VEGF164
INTRODUCTION
Lung development in the mouse begins on embryonic
day 9.5 (E9.5) as the lung buds, derived from the
primitive foregut endoderm, invade the splanchnic
mesenchyme. These primitive respiratory tubules, lined
by undifferentiated columnar epithelial cells, undergo
dichotomous branching to form the conducting airways.
During this early developmental stage, the lung tu-
r 1998 WILEY-LISS, INC.
Grant sponsor: RDP Center for the Cystic Fibrosis Foundation
(X.Z., S.E.W., J.A.W.); Grant sponsor: Center for Gene Therapy for
CF and Other Lung Diseases (X.Z., S.E.W., J.A.W.); Grant number:
HL51832; Grant sponsor: James S. McDonnell Foundation
(R.F., K.G.P.).
*Correspondence to: Jeffrey A. Whitsett, M.D., Children’s Hospital
Medical Center, Divisions of Neonatology and Pulmonary Biology,
3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail:
WHITJO@CHMCC.ORG
Received 26 June 1997; Accepted 19 November 1997
216
ZENG ET AL.
is a secreted form of the protein that is expressed in
many tissues (Ferrara et al., 1996). VEGF is required
for early embryogenesis because genetic inactivation of
a single allele of VEGF in the mouse caused abnormal
vasculature resulting in early embryonic lethality (Carmeliet et al., 1996; Ferrara et al., 1996). VEGF is also
expressed at high levels in well-vascularized organs including the glomeruli in adult kidney, choroid plexus,
lung, liver, and corpus luteum (Monacci et al., 1993,
Berse et al., 1992). VEGF activates endothelial cells by
binding to high affinity VEGF receptors, flt-1 (VEGFR-1),
and Flk-1/KDR (VEGFR-2), both of which are expressed by endothelial cells (DeVries et al., 1992; Quinn
et al., 1993; Mustonen and Alitalo, 1995). In adult lung,
VEGF is synthesized primarily by alveolar epithelial
cells and macrophages (Monacci et al., 1993). VEGF
mRNA is enhanced in alveolar epithelial cells during
hypoxia (Tuder et al., 1995) and hyperoxia (Maniscalco
et al., 1995). In the embryonic lung, VEGF mRNA is
detected in the airway epithelium (Breier et al., 1992;
Millauer et al., 1993). In contrast, Flk-1 and flt-1
mRNAs are detected in lung mesenchyme (Millauer et
al., 1993; Breier et al., 1995), including the mesenchymal progenitors of pulmonary blood vessels, supporting
the concept that VEGF presents a paracrine signal
from the epithelium activating target receptors on
vessel precusor cells in the stroma.
To study the function of VEGF in the vasculogenesis
during mouse lung development and the influence of
the developing vascular system in the lung mesenchyme on the differentiation of respiratory epithelium,
we expressed murine VEGF in the respiratory epithelium of transgenic mice using the promoter from the
human surfactant protein-C gene (Glasser et al., 1991;
Wert et al., 1993). The altered vascularity caused by the
VEGF transgene in transgenic mice was associated
with marked disruption of acinar structure.
RESULTS
VEGF Disrupts Pulmonary Morphogenesis
in Transgenic Mice
The SP-C-VEGF DNA construct was microinjected
into oocytes harvested from FVB/N mice and transferred to surrogate dams. Of the 40 potential founder
mice born, 10 died at birth. When screened by PCR or
Southern blot analysis, two of the pups bore the VEGF
transgene. None of the surviving animals bore the
transgene. Analysis of tissues from the dead transgenic
animals revealed severe disruption of lung morphogenesis associated with the presence of the transgene (data
not shown). Thereafter, embryos were obtained by
hysterectomy on E15 and E17, representing the late
pseudoglandular and late canalicular stages of mouse
lung development, respectively. Of 31 embryos obtained, seven bore the SP-C-VEGF transgene. Weight
and appearance of SP-C-VEGF mice were similar to
their normal littermates. However, histological examination on E15 and E17 revealed a consistent pattern of
abnormalities that were confined to the lungs from all
transgenic embryos (Fig. 1). During this period of
embryonic development, the bronchial tubules of the
normal mouse lung undergo extensive branching, forming acinar buds in the periphery of the lung, which
begin to form dilated saccules by E17. In contrast, the
lungs from transgenic embryos on E15 and E17 consisted of large, abnormally dilated tubules, and the
number of acinar buds and saccules was markedly
reduced. The lumen of the remaining acinar buds and
saccules were dilated, and the amount of mesenchyme
was decreased in the transgenic mice. Except for the
marked disruption of lung morphogenesis, the morphology of other organs from the SP-C-VEGF transgenic
mice was indistinguishable from those of controls.
Consistent histological abnormalities were observed in
lungs from all nine transgenic founder animals (including the two dead transgenic pups).
Expression of SP-C-VEGF Transgene
In order to correlate the lung abnormalities from
SP-C-VEGF transgenic embryos with the expression of
the transgene, in situ hybridization using transgene
specific probes generated from the SV40 t-intron-polyA
sequence was performed to determine the presence and
the expression pattern of SP-C-VEGF transgenic mRNA
(Fig. 2A). Transgene mRNA was detected in the lung on
cross sections (E17) and sagittal sections (E15) from the
transgenic embryos. The hybridization signal was confined to pulmonary epithelial cells. Transgenic mRNA
was restricted to epithelial cells of dilated bronchioles,
acinar tubules and buds and was detected at low levels
in epithelial cells of larger airways, consistent with the
expression pattern noted for cDNAs expressed with the
human SP-C promoter in embryonic lungs of transgenic
mice reported previously (Korfhagen et al., 1990; Glasser
et al., 1991; Wert et al., 1993; Simonet et al., 1995; Zhou
et al., 1996a). Transgene mRNA was not detected in
other tissues or in lungs of nontransgenic embryos.
In situ hybridization using riboprobes generated
from VEGF cDNA was also performed to compare the
level and expression pattern of VEGF mRNA in lungs
from both transgenic and nontransgenic control embryos (Fig. 2B). The endogenous VEGF mRNA was
detected primarily in distal airway epithelial cells in
normal mouse lung at both E15 and E17. High intensity
of the hybridization signal, indicating the elevated level
of VEGF mRNA, was observed in the epithelial cells
lining the dilated tubules and acinar buds in lungs from
transgenic embryos. Consistent with results from in
situ hybridization, the VEGF protein, as identified by
immunohistochemical staining using the VEGF antibody, was also detected in the distal airway epithelial
cells in nontransgenic control embryos and in the
epithelial cells lining the dilated acinar tubules and
buds in lungs from transgenic embryos at E15 and E17
(Fig. 2C).
Fig. 1. Morphology of lungs from SP-C-VEGF transgenic embryos
and control littermates. Sagittal sections prepared from embryos on E15
(a,b) and cross sections from embryos on E17 (c,d) were stained with
hematoxylin and eosin and photographed under bright field microscopy.
The panel on the right is a higher magnification of the same field seen on
the left. In the normal lung on E15 and E17, extensive branching of the
airway epithelium is associated with the formation of numerous terminal
acinar buds (B) and acinar saccules (S) with small lumen (a,c). In the
transgenic embryos, respiratory tubules (T) and saccules (S) are markedly dilated and the numbers of terminal buds markedly decreased (b,d).
Epithelial cells of the dilated tubules at E15 are columnar in transgenic
embryos (f, arrow), and are primarily cuboidal in nontransgenic embryos
of the same age (e, arrow). On E17, epithelial cells of the dilated airways
in transgenic embryos are cuboidal and comparable to those lining the
acinar tubules of nontransgenic controls (h,g, arrows). Bars 5 100 µm.
218
ZENG ET AL.
Fig. 2. In situ hybridization for transgene specific and VEGF mRNA
and immunohistochemical staining for VEGF protein. Cross (E17) and
sagittal (E15) sections of embryos were prepared from both transgenic
and nontransgenic mice. Tissue sections were hybridized overnight with
35S-labeled anti-sense and sense (latter not shown) riboprobes generated
from transgene specific t-intron-polyA sequence (2A) or VEGF cDNA (2B)
or stained with VEGF antibody (2C). 2A: Transgene specific mRNA was
readily detectable in the epithelial cells lining the dilated tubules on E15
(c) and the malformed saccules on E17 (d) from the transgenic embryos.
The hybridization signal was not observed in bronchi or larger bronchioles
in the transgenic embryos. Signal was confined to the lung and was not
detected in nontransgenic embryos (a,b).
Expression of VEGF in Respiratory Epithelial
Cells Induced Ingrowth of Blood Vessels
were generally found in close contact with the respiratory epithelium, a situation that does not normally
occur until the saccular stage (E17-birth) of normal
lung development.
VEGF receptor Flk-1 and its related endothelial cell
specific receptor tyrosine kinase Tie-1 are expressed in
the mesenchyme of developing lung (Korhonen et al.,
1994; Breier et al., 1995). To determine whether the
increased VEGF level affected the VEGF receptor expression, in situ hybridization was performed on tissue
sections from transgenic and nontransgenic embryos at
E15. The higher intensity of the hybridization signal for
both Flk-1 and Tie-1 mRNA indicated increased vascularity and/or enhanced Flk-1 and Tie-1 mRNAs in the
lung mesenchyme (Fig. 4), but not in other tissues (data
not shown) in the SPC-VEGF transgenic embryos.
To detect other alterations in mesenchymal components, lung sections were stained for a-smooth muscle
actin (a-sma). Staining for a-sma, normally detected
surrounding the bronchiolar stalks and in the clefts at
branch points of bronchial tubules, appeared in a
Since VEGF receptors are expressed in the precursors of endothelial cells in mesenchyme, the pulmonary
blood vessels are most likely to be affected directly by
the expression of VEGF in the airway epithelium. To
determine the distribution of pulmonary blood vessels
in SP-C-VEGF transgenic mice, immunohistochemical
staining for PECAM-1 (CD31), a marker for endothelial
cells (Xie et al., 1993; Vecchi et al., 1994), was performed on tissue sections from the SP-C-VEGF transgenic embryos and nontransgenic control littermates.
On E15 of normal mouse lung development, microvessels were noted within the pulmonary mesenchyme and
were distributed around the distal acinar buds at high
density (Fig. 3a,c). Vessels within the mesenchyme
were not in close contact with the respiratory epithelium. In contrast, vasculogenesis was markedly advanced in lungs from the transgenic embryos on E15
(Fig. 3b,d). Lumenal diameter of the blood vessels was
increased in the transgenic mice. Furthermore, vessels
VEGF EXPRESSION
219
Fig. 2B. High levels of VEGF mRNA were detected in epithelial cell
lining dilated and malformed acinar tubules on E15 (c) and E17 (d) in the
developing lungs of transgenic embryos and colocalized with the transgene specific t-intron-polyA mRNA expression in (A). The endogenous
VEGF mRNA was detected at low levels in epithelial cells lining the distal
acinar tubules on E15 (a) and slightly increased on E17 (b) in the lungs
from nontransgenic littermates.
diffuse pattern surrounding the dilated tubules in
lungs from the transgenic embryos on E15 (Fig. 5).
lial cells forming the abnormal vessels seen in SP-CVEGF mice were joined by tight junctions. No fenestrations or gaps were observed along the endothelium.
Thus, overexpression of VEGF in pulmonary epithelium disrupted the normal interface between the pulmonary epithelial and capillary endothelial cells, inhibiting cytodifferentiation of Type I alveolar cells and
maturation of Type II alveolar cells. The expression of
VEGF in the pulmonary epithelium increased the
lumenal size of pulmonary blood vessels and caused
areas of interstitial edema consistent with the known
effects of VEGF on vascular permeability.
Electron Microscopy
During the saccular stage of normal mouse lung
development (E17.5–birth), the epithelial cells lining
the saccules differentiate, producing mature Type II
and Type I cells that line the distal airspaces (Fig. 6).
Vascularity increases and endothelial cells come into
close proximity to the developing squamous Type I cells.
In contrast, epithelial cells of the lungs from transgenic
mice remained primarily columnar and failed to form
the squamous epithelium characteristic of the normal
lung in late gestation (Fig. 6A). The respiratory epithelium was lined predominately by low columnar cells
containing extensive glycogen pools and, frequently,
lamellar bodies. The lumen of pulmonary blood vessels
was enlarged, and the organization of the surrounding
stroma was loosely arranged, containing areas of extracellular fluid accumulation, perhaps related to the
effect of VEGF on vascular permeability (Fig. 6B).
Platelets were frequently observed in dilated, tortuous
vessels adjacent to the respiratory epithelium. Endothe-
Expression of SP-C and CCSP Genes
in the SP-C-VEGF Mice
In normal mouse lung, immunostaining of the
proSP-C protein in distal airway epithelial cells increases markedly during the canalicular and saccular
stage of development in association with other aspects
of morphologic maturation and Type II cell cytodifferentiation (Zhou et al., 1996b). CCSP, a marker for Clara
cells, is first detected in the mouse conducting airway
epithelial cells at E16–17 (Wert et al., 1993; Zhou et al.,
220
ZENG ET AL.
Fig. 2C. Immunoperoxidase staining for VEGF protein was detected
in the epithelial cells of the malformed acinar tubules and saccules in the
lungs from SP-C-VEGF transgenic embryos on E15 (c) and E17 (d). The
endogenous VEGF protein was also expressed in the epithelial cells of
the acinar buds and saccules in the lungs from nontransgenic control
embryos on E15 (a) and E17 (b). Bars 5 50 µm.
1996b). These markers were therefore chosen to assess
proximal and distal airway epithelial cell differentiation in the SP-C-VEGF mice (Fig. 7). ProSP-C was
detected in epithelial cells of acinar tubules from nontransgenic embryos on E15 and E17 and also in epithelial cells of the abnormally dilated tubules seen in the
lungs from transgenic mice (Fig. 7A), supporting their
identity as terminal acinar tubules. CCSP was present
in the bronchiolar epithelium of lungs from both transgenic and nontransgenic embryos on E17 (Fig. 7B), but
was not detected in the abnormally dilated acinar
tubules in the transgenic mice. Thus, lung morphogenesis was markedly altered in SP-C-VEGF mice, while
cytodifferentiation of the respiratory epithelial cells, as
assessed by the distribution of CCSP and proSP-C, was
only modestly altered by the expression of the transgene.
transgene mRNA was restricted to the pulmonary
epithelium and was associated with consistent histological alterations in lung morphology that resulted in
perinatal death in transgenic offspring. Branching morphogenesis in the SP-C-VEGF mice on E15 in the
transgenic embryos was markedly disrupted. Acinar
tubules were dilated in association with decreased
numbers of terminal buds and decreased pulmonary
mesenchyme compared to age-matched controls. Blood
vessels were precociously developed and grew in close
proximity to the respiratory epithelium. While the
expression of SP-C and CCSP in pulmonary epithelial
cells were relatively unaltered in the SP-C-VEGF transgenic mice, the differentiation of Type II into Type I
epithelial cells was inhibited. Endogeneous VEGF
mRNA and protein were detected in the developing
respiratory epithelium, while the VEGF receptor, Flk-1,
was detected in lung mesenchyme. These findings
support a role of VEGF in vessel formation in the
developing lung that in turn influences acinar development.
The human SP-C promoter has been used to express
genes or cDNAs in the pulmonary epithelium in a
DISCUSSION
In the present study, transgenic mice were generated
in which the mouse VEGF164 was directed to the lungs
of fetal mice using the promoter/enhancer element
derived from the human SP-C gene. Expression of the
VEGF EXPRESSION
221
Fig. 3. PECAM-1 staining in the lungs from SP-C-VEGF transgenic
mice. The pattern of pulmonary blood vessel distribution was assessed in
sagittal sections prepared from SP-C-VEGF transgenic embryos (b,d)
and nontransgenic embryos (a,c) on E15 using PECAM-1 (CD31)
antibody. The panel on the right is a higher magnification of the field in the
window on the left. In lungs from nontransgenic embryos, blood vessels
were observed surrounding the distal airways and terminal acinar buds,
but remain embedded within the mesenchyme (a,c). In the transgenic
developing embryos, the blood vessels are seen in close approximation to
the respiratory epithelium (b,d). Note the unusually large lumen of the
vessels surrounding the distal epithelial tubules in (d, arrows) and the
columnar shape of the epithelial cells in close contact with the blood
vessels (d, arrowheads). Bars 5 50 µm. Staining is representative of 2
separate embryos from each group.
variety of transgenic mouse models. Transgenic mRNA
expression begins as early as E10 and was present only
in cells of the distal airway epithelium in transgenic
embryos (Wert et al., 1993). The cellular sites of expression of endogeneous VEGF mRNA and protein spatially
overlaps with that of the VEGF transgene expressed by
SP-C promoter. However, the endogeneous VEGF mRNA
was only detectable in respiratory epithelial cells after
E15, as determined by in situ hybridization. Therefore,
in the present study, the VEGF transgene was likely
expressed at higher levels and at an earlier developmental stages in distal airway epithelial cells in SP-CVEGF transgenic embryos. This suggests that the
extensive vascularization surrounding the dilated airways of the SP-C-VEGF transgenic mice is related to
increased activity or dysregulation of VEGF. This hypothesis is supported by previous studies that have also
shown alterations in vascularization in response to
VEGF. For example, increased expression of VEGF in
avian wing bud mesenchyme increased vascularization
(Flamme et al., 1995), while malformation of the vascular networks and neovascularization of normally avascular areas were induced by VEGF in developing quail
embryos (Drake et al., 1995). In the present study,
abnormalities of vascularity in pulmonary mesenchyme were observed in the SP-C-VEGF transgenic
mice, including the production of ectopic blood vessels
and the formation of vessels with increased lumenal
diameter. These findings demonstrated the paracrine
interactions between VEGF with its target cells, the
endothelial cells, during vasculogenesis. These findings
also support the hypothesis that precise regulation of
VEGF by the respiratory epithelium is critical for
pulmonary vasculogenesis. The finding that targeted
inactivation of a single allele of the VEGF gene in the
mouse led to abnormal blood vessel formation and early
embryonic lethality suggests that the precise control of
VEGF production is critical for normal embryonic vessel development (Carmeliet et al., 1996; Ferrara et al.,
1996).
222
ZENG ET AL.
Fig. 4. In situ hybridization for Flk-1 and Tie-1 mRNA. Tissue sections
from transgenic (b,d) and nontransgenic control (a,c) embryos at E15
were hybridized with 35S labeled antisense Flk-1 (a,b) and Tie-1 (c,d)
riboprobes. The higher intensity of the hybridization signal for Flk-1 and
Tie-1 was observed in lung mesenchyme of the SPC-VEGF transgenic
embryos. No hybridization signal was observed using the sense probe
(data not shown). Bars 5 50 µm.
The coordinate expression of VEGF and its high
affinity receptors during early embryogenesis (Breier et
al., 1992; Jakeman et al., 1993; Peters et al., 1993;
Breier et al., 1995) suggests that this signal transduction system may be involved in paracrine interactions
that result in increasing vascularization in developing
organs. In the present study, increased VEGF mRNA
expression in respiratory epithelium lead to the increased intensity of the in situ hybridization signals for
Flk-1 mRNA, demonstrating a paracrine induction of
the VEGF receptor in vivo. Although the endothelial
cell specific receptor tyrosine kinase Tie-1 is not a
cognate receptor for VEGF, the increased intensity of in
situ hybridization signals for Tie-1 mRNA detected in
SPC-VEGF transgenic lung may indicate that VEGF is
involved in the regulation of Tie-1.
Expression of VEGF in the respiratory epithelium of
transgenic mice disrupted branching morphogenesis of
the lung. These findings are distinct from previous
observations in which embryonic pattern formation
was relatively unaltered after exposure of avian wing
bud and developing quail embryo to VEGF, even though
VEGF caused marked malformation of the vasculature
in those experimental systems (Drake et al., 1995;
Flamme et al., 1995). The changes in lung morphology
seen in the SP-C-VEGF mice were highly distinct from
those produced by KGF and TGF-b. In contrast to
VEGF, KGF and TGF-b are mesenchymal signals that
directly affect epithelial differentiation. When expressed in pulmonary epithelium by SP-C promoter,
KGF induced cystadenomatoid malformation of the
lung (Simonet et al., 1995), while TGF-b markedly
arrested lung sacculation and inhibited branching morphogenesis (Zhou et al., 1996a).
The disruption of lung morphogenesis seen in the
SP-C-VEGF mice on E15 may be related to the precocious or dysregulated growth of pulmonary blood vessels, perhaps altering cell-cell or cell-extracellular matrix interactions that are involved in critical aspects of
acinar maturation and sacculation. Decreased a-smooth
muscle actin staining surrounding the bronchioles of
the transgenic embryos indicated that smooth muscle
and myofibroblast differentiation or organization within
the mesenchymal cell compartment has been altered.
VEGF EXPRESSION
223
Fig. 5. a-Smooth muscle actin staining. Sections of SP-C-VEGF
transgenic and nontransgenic control embryos on E15 were stained with
an FITC-conjugated a-sma antibody and photographed under fluorescence microscopy. a: Reactivity was observed surrounding the blood
vessels (V) and bronchioles (B) (bronchioles, stalks, clefts) of lungs from
nontransgenic embryos on E15. Note the lack of staining at the terminal
buds and leading edges of the branching epithelial tubules of the acinar
(a, arrow). b: In transgenic lung, staining was diffuse in regions surrounding the acinar tubules (B). Condensed staining in the vessels (V) was
maintained in lungs from transgenic embryos. Bar 5 100 µm.
Myofibroblasts may stabilize branch points of the developing bronchiolar tree and restrict expansion of the
proximal bronchioles (Mitchell et al., 1990), a process
critical to normal lung morphogenesis. The loss of the
stabilizing influence of the smooth muscle cell/myofibroblast cell may therefore contribute to the early dilation
and decreased development of acinar structures seen in
the lungs of the SP-C-VEGF mice. The observed changes
in lung structure may represent the indirect effect of
VEGF mediated changes in vascularity that in turn
influence branching morphogenesis. The precise temporal and spatial regulation of VEGF or VEGF receptor
signaling may therefore be critical to normal growth
and morphogenesis of acinar structures.
Fig. 6. Lung tissue from E18 transgenic and nontransgenic litter
mates was processed for ultrastructural analysis as described in Materials
and Methods. A: Alveolar septae from E18 nontransgenic control mice (a)
were composed of fibroblasts and capillaries containing red blood cells.
The overlying epithelium was composed of squamous Type I cells (solid
arrowheads) and cuboidal Type II cells (open arrowheads) that contained
a few lamellar bodies and little glycogen. Secreted pulmonary surfactant,
composed of lamellar bodies and tubular myelin, was found in the alveolar
lumen (arrows). Dilated pulmonary tubules found in the SP-C-VEGF
transgenic mice (b) were lined by immature, low-columnar to cuboidal
Type II cells containing abundant glycogen (asterisks) and lamellar bodies
(arrows), seen at higher magnification in panel c (arrows) along with
several multivesicular bodies (arrowhead). Scale bar for panel a 5 10 µm;
panel b 5 5 µm; and panel c 5 2 µm.
224
ZENG ET AL.
Fig. 6B. In the SP-C-VEGF transgenic mice (a), loosely organized,
edematous interstitial tissue is found adjacent to a large, dilated vessel
(asterisk). An irregular, tortuous blood vessel, containing red blood cells
and numerous platelets (arrows), is seen adjacent and closely apposed to
the overlying cuboidal epithelium in an oblique section through the basal
side of the epithelium (b). 1 5 endothelial cells; 2 5 epithelial cells. Scale
bar for panels a and b 5 5 µm.
Epithelial cell differentiation in the lungs from
SP-C-VEGF mice was not markedly altered as assessed
by immunostaining for proSP-C and CCSP. The increased growth of blood vessels did not enhance epithelial cytodifferentiation. The acinar tubules of the transgenic mice were lined primarily by cuboidal epithelial
cells with features of mature Type II cells such as
surfactant protein C and lamellar bodies. However,
during normal lung development from the canalicular
stage to the postnatal period, the increasingly close
apposition of capillaries to the respiratory epithelium is
associated with thinning of the respiratory epithelium
in the periphery of the lung (Burri, 1984), producing
a squamous epithelium lined by Type I cells characteristic of the normal postnatal lung. The distal respiratory epithelium remained primarily cuboidal in the
SP-C-VEGF mice in spite of the extensive vascularity of
the tissue. While the alterations in the pulmonary
vasculature induced by VEGF disrupted acinar development and branching morphogenesis, the changes in
vascularity did not enhance the respiratory epithelial
cell differentiation of Type II cells to form Type I cells.
In summary, the expression of VEGF in the respiratory epithelium caused an abnormal pattern of blood
vessel distribution in the lung mesenchyme which, in
turn, disrupted lung morphogenesis. Since VEGF is
expressed in alveolar epithelial cells in the adult lung
and is subject to regulation by external stimuli such as
hyperoxia (Tuder et al., 1995) and hypoxemia (Maniscalco et al., 1995), precise regulation of VEGF may also
be critical to repair following lung injury associated
with respiratory distress syndrome and bronchopulmonary dysplasia.
MATERIALS AND METHODS
Transgene Preparation
A cDNA (600 bp) encoding mouse VEGF164 (164
amino acid peptide) was isolated by RT-PCR from total
RNA purified from mouse kidney. The murine VEGF164
cDNA sequence was verified by analyzing two independent clones and by comparison with published sequence
(Breier et al., 1992). The full length (0.6 kb) cDNA of
mouse VEGF164 was then inserted into the EcoRI site of
the human SP-C promoter plasmid SP-C-3.7 SV40
t-intron-polyA (Glasser et al., 1991). The proper orientation of the construct was verified by restriction enzyme
digestion. The SP-C-VEGF chimeric gene was excised
with Ndel and Notl and purified from 0.8% agarose gel
by Qiaex Extraction Kit (Qiagen Inc, Chatsworth, CA).
Purified DNA was dialyzed extensively into 5 mM
Tris-HCl (pH 7.4), 0.1 mM EDTA, and microinjected
into fertilized eggs of FVB/N mice. The embryos from
VEGF EXPRESSION
225
Fig. 7. Immunohistochemical staining for proSP-C, CCSP. 7A: ProSP-C staining was detected in epithelial
cells of acinar buds on E15 (a, arrows) and differentiated Type II cells on E17 (b, arrow) in control embryos.
Comparable staining was also seen in the epithelium of the dilated acinar tubules in age matched transgenic
embryos (c,d, arrows).
pregnant mothers were obtained by hysterectomy at
embryonic day 15 (E15) and 17 (E17) for analysis.
Transgenic founders were identified by PCR analysis of
tail DNA using oligonucleotide primers that amplified a
800 bp fragment spanning the VEGF cDNA and the
SV40 derived t-intron-polyA sequence and further confirmed by Southern blot analysis on tail DNA probed
with the SV40 derived t-intron-polyA sequence.
Tissue Preparation
Hysterectomy was performed after lethal injection of
pentobarbital. The embryos were weighed and fixed in
neutral buffered 4% paraformaldehyde at 4°C for 16 hr.
Tissues were dehydrated through a graded series of
ethanol solutions and embedded in paraffin. Tissues
were sectioned transversely through the thorax (E17)
or sagittally (E15) at 5 µm thickness and stained with
hematoxylin and eosin.
Antibodies and Immunohistochemistry
Rabbit antiserum against rat CCSP was the kind gift
of Dr. Gurmukh Singh (Dept. of Veterans Affairs Medi-
cal Center, Pittsburgh, PA) and used at a dilution of
1:1,000. Rabbit antiserum against human proSP-C
(#68514) was used at a 1:1,000 dilution. Rabbit antiserum against human VEGF (A-20; Santa Cruz Biotechnology, Inc., San Diego, CA) was used at a dilution of
1:600. Immunostaining for CCSP, VEGF, and proSP-C
was performed as previously described (Vorbroker et
al., 1995; Zhou et al., 1996a,b). FITC-conjugated mouse
monoclonal antibody against a-smooth muscle actin
(Sigma Chemicals, St. Louis, MO) was used at a
dilution of 1:50 as previously described (Zhou et al.,
1996a). Immunostaining with monoclonal rat-antimouse PECAM-1 (CD31) antibody (Pharmingen, San
Diego, CA) was performed based on the procedure for
proSP-C immunostaining with minor modifications.
Briefly, 5 µm paraffin sections were deparaffinized and
rehydrated. After 15 min incubation at room temperature (RT) with 3% H2O2 to quench endogenous peroxidase, a brief incubation with 0.1% trypsin (Difco Laboratories, Detroit, MI), pH 7.6 at 37°C was performed to
unmask the antigen. Sections were blocked with 2%
normal rabbit serum in phosphate buffered saline with
226
ZENG ET AL.
plasmid with mouse VEGF cDNA in pCRScript SK (1)
was linearized and used to generate radiolabeled sense
and antisense transcripts by in vitro transcription in
the presence of 35S-labeled UTP (1310 Ci/mmole, Dupont NEN, Boston, MA) using T3 or T7 polymerase and
reagents contained in the Maxscript in vitro transcription kit (Ambion, Inc., Austin, TX). The mouse Flk-1
and Tie-1 partial cDNA clone containing the kinase
domain were generated by PCR from the full length
Flk-1 and Tie-1 cDNA respectively and used to generate
the radiolabeled antisense transcript. In situ hybridization was performed as previously described (Wert et al.,
1993) except that the overnight hybridization was
carried out at 55°C.
Electron Microscopy
Fetal lung tissue was minced into 1–2 mm3 pieces,
fixed overnight in the cold with 2.5% glutaraldehyde
and 2% paraformaldehyde in 0.1 M sodium cacodylate
buffer (SCB) at pH 7.3, and post-fixed in 1% osmium
tetraoxide in 0.1 M SCB for 2 hr at room temperature.
The tissue was then dehydrated through a graded
series of alcohol solutions, washed in propylene oxide,
and infiltrated and embedded in Embed-812 (Electron
Microscopy sciences, Ft. Washington, PA). Semithin
plastic sections were stained with toluidine blue and
photographed with a Nikon Microphot FXA photomicroscope. Ultrathin sections were stained with lead citrate
and uranyl acetate and examined with JEOL 100CX
electron microscope.
Fig. 7B. CCSP was detected in bronchial epithelial cells in lungs from
nontransgenic (a) and transgenic embryos on E17 (b).
2% Triton X-100 (PBST) for 2 hr at RT. After overnight
incubation with primary antibody (diluted to 1 µg/ml in
blocking serum) at 4°C, sections were washed in PBST
and incubated with biotinylated rabbit anti-rat antibody (Novocastra Laboratories Ltd, UK), diluted 1:200
in blocking solution, for 30 min at RT. Sections were
then washed, incubated with avidin-biotin-peroxidase
complex from a Vectastain Elite ABC standard kit
(Novocastra Laboratories, UK) diluted in PBST. Reaction products were visualized by incubation with nickelcontaining 3,38-diaminobenzidine, followed by a brief
incubation with Tris cobalt to achieve a black precipitate. Sections were then counter-stained with nuclear
fast red.
In Situ Hybridization
In situ hybridization using transgene specific riboprobes generated from SV40 derived t-intron-polyA
sequences was performed on tissue sections from transgenic and nontransgenic embryos as previously described (Zhou et al., 1996a).
In situ hybridization using VEGF, Flk-1, and Tie-1
riboprobes were performed on tissue sections from
transgenic and normal control mouse embryos. The
ACKNOWLEDGMENTS
The authors thank Dr. Lan Zhou for helpful discussions and Dr. Karen Yaeger, Sherri Proffit, Kevin Kirwin, and Chitta R. Dey for technical assistance during
the course of these studies.
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