# Combined Application of 17β-Estradiol and Progesterone Enhance Vascular Endothelial Growth Factor and Surfactant Protein Expression in Cultured Embryonic Lung Cells of Mice

Authors: Andreas Trotter; Markus Kipp; Roland Matthias Schrader; Cordian Beyer
Journal: International Journal of Pediatrics
 (2009)

---

## Abstract

Preterm delivery is associated with disruption of the placental supply with 17β-estradiol (E2) and progesterone (P). The aim is to evaluate the role of E2 and P on the regulation of key proteins in lung development in embryonic lung cells. Alveolar cell type II (AT-II) and central lung fibroblast cultures were established from mouse embryos. Cells were exposed for 24 hours to E2 and/or P, the estrogen receptor antagonist ICI 182.780 (ICI) and the progesterone receptor antagonist mifepristone (RU 486). The mRNA expression of vascular endothelial growth factor (VEGF) and surfactant protein B and C (SB-B, SB-C) was determined, and protein levels of VEGF were measured. Only the combined treatment with E2 and P increased mRNA expression and VEGF protein in AT-II cells and lung fibroblasts. Combined treatment also promoted SP-B and SP-C expression in AT-II cells. Pretreatment with ICI and RU 486 completely abolished the E2 and P induced effects. E2 and P enhanced expression of VEGF and surfactant proteins in primary embryonic lung cells and may be involved in regulating expression of key molecules for the prenatal lung development and postnatal lung function.


---

## Body

## 1. Introduction

Respiratory
distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) remain major
factors for morbidity and mortality in extremely preterm infants.
Histopathological studies in preterm infants dying from BPD demonstrate an
arrest of lung development with reduced alveologenesis [1]. The concept
has evolved that postnatal lung development in extremely preterm infants is
arrested by lack of factors that regulate lung differentiation and maturation
in utero [2]. During mid
and late gestation, the human fetus is exposed to high amounts of 17β-estradiol
(E2) and progesterone (P) [3] produced by the placenta from precursors originating
from the mother and the fetus [4]. Delivery disrupts the
placental supply of both hormones. Within one day, the levels of E2 and P drop
100-fold [5]. This is a physiologic
condition for the term infant. The extremely preterm infant is disrupted from
the supply of these hormones at a much earlier developmental stage. The uterus
as a known estrogen responsive target grows in utero until the end of gestation
but stops growing in preterm infants [6].  It is conceivable that the withdrawal of E2
and P at this early developmental stage also affects lung development. Replacement
of E2 and P in extremely preterm infants tailored to maintain in utero plasma
levels of E2 and P was associated with a trend toward a reduced incidence of
BPD [5, 7].In mice lung mRNA
expression of estrogen and progesterone receptors suggests that E2 and P are
likely to be involved in mammalian fetal lung development [8]. The number of
alveolar crests and alveolar type II cells [9] as well as lamellar bodies in
type II cells [10] increased in rat fetuses
after maternal E2 administration and E2 stimulates fetal lung
surfactant production in rabbits [11, 12]. In newborn
piglets antagonism of E2 and P during mid to late pregnancy decreased alveolarization
[13].Vascular
endothelial growth factor (VEGF) is a major mitogen for vasculogenesis and
angiogenesis [14] and is essential for
embryonic development. Loss of a single VEGF allele results in embryonic
lethality [15]. Absence of
isoforms of VEGF impairs lung microvascular development and delays airspace
maturation in mice fetuses reflecting the essential role of VEGF for normal
lung development [16]. Pneumotrophic
effects may be mediated through modulation of VEGF expression with the potential
to accelerate lung maturation in preterm infants [17]. Intra-amniotic
injection of VEGF in preterm rats resulted in increased surfactant protein B (SP-B)
mRNA expression [18]. Type 2
pneumocytes respond to VEGF by increasing their expression of SP-B and SP-C [17].Both sex
steroids E2 and P and VEGF are described to induce surfactant proteins in the
developing lung. Whereas it is established that E2 and P induce VEGF gene
transcription in breast tumor cell lines [19] and the endometrium [20–22] no data from
the literature is available about the effects of E2 and P on VEGF gene
transcription in lung tissue. The aim of this study was to investigate if E2 and
P are involved in the developmental regulation of VEGF and surfactant protein B
(SB-B) and C (SB-C) mRNA expression using an in vitro model of cultured embryonic
lung cells.


## 2. Material and Methods

All experiments
followed the local guidelines according to the Federation of
European Laboratory Animal Science Associations recommendations and were
approved by local executives.


### 2.1. Cell Culturing and Treatment

Highly
enriched alveolar cell type II (AT-II) and central lung fibroblast cultures
were established from embryonic day (ED) 18 BALB/c mice as followed. Since
E18 mouse lungs can be dissected from the surrounding vessels in a reliable
manner we did choose this developmental stage to isolate purified AT-II cells
from the mouse lung. Briefly, the fetuses were removed on ED 18 by caesarian
section and the lungs aseptically explanted from the thorax. Lungs were then
washed three times in 10 mL ice cold HBSS, mechanically dissected from
surrounding vessels, and then incubated in a 2.5% trypsin solution containing
200μL of DNAse 1 (2 mg/mL, Worthington) for 10 minutes at 37°C in a shaking
water bath (60 rpm). Finally, the cell suspension was filtered through a nylon
mesh with 100-μm pore size. The cell suspension was centrifuged twice at 420
and then 120 g for 4 minutes, and the pellet was resuspended in minimal
essential medium (MEM). The resulting cell suspension contained the AT-II cells
and attaching fibroblasts. After adding 1.5 ML collagenase (1250 U/mL,
Worthington) and 150 μL DNAse 1 (2 mg/mL, Worthington) the suspension was
incubated for 15 minutes at 37°C. After stopping the collagenase activity by incubating in ice-cold MEM (supplemented with
10% FCS) the cell suspension was centrifuged as
above for 4 minutes and the pellet was resuspended in MEM + 10% FCS. Cells were
seeded and cultivated in a fibronectin-coated culture flask for 1 hour at 37°C
under 5% CO2/21%  O2. During this time, fibroblasts were attached
to the flask, while AT-II cells did not due to their differential adherence
characteristics. This procedure was repeated twice. Finally, approximately 1 ×
105 AT-II cells/cm2 were seeded at 24-well culture
plates, cultured for one day in MEM supplemented with 10% FCS. Medium was
changed after 24 hours to Cellgro complete medium (Mediatech, Virginia, USA,
without phenol red). Attached fibroblasts were resuspended by trypsination and
replated. When cells reached a confluence of approximately 80% the medium was
changed and the final treatment was started. Cells were exposed for 24 hours to
E2 and/or P and dexamethasone (all from Sigma-Aldrich, Germany) in
concentrations ranging from 10–10 M to 10–6 M. Pretreatment with the estrogen
receptor antagonist ICI 182,780 (TOCRIS bioscience, UK, 0.1 μM) and the progesterone
receptor antagonist mifepristone (RU 486 from Biomol, Germany, 0.1 μM) was
performed 1 hour before hormone exposure. Concentrations
of ICI and RU 468 were derived by preparing a 10–2 M stock solution in ethanol
100% p.a. quality and by further diluting in medium. Appropriate ethanol
concentrations served as controls.


### 2.2. Gene Expression Analysis

The mRNA expression
of VEGF, SP-B, and SP-C was quantified using the rtPCR technology (BioRad,
Germany), QTM SYBR Green Supermix (BioRad, Germany), and a standardized
protocol as described previously [23, 24]. Isolation
of total RNA was performed with peq Gold (PeqLab,
Germany). RNA
concentration and purity were assessed using OD260 and OD260/OD280 ratio,
respectively, and reverse transcribed using an Invitrogen M-MLV RT-kit and
random hexanucleotide primers. The rtPCR reactions were carried out in a
reaction mixture consisting of 2 μL cDNA, 6 μL RNAse-free water, 10 μL hot StartTaq
DNA-polymerase, and 1 μL of primer (10 pmol). Reactions were conducted in
standard tubes using the MyIQ rtPCR Detection System (BioRad, Germany) under
following conditions: 10-minute
enzyme activation at 95°C, 45 cycles of 15-second denaturation at 95°C, 30-second annealing at
individual temperatures, 30-second
amplification at 72°C, and 5-second fluorescence measurement at 80°C. Primer sequences to
detect VEGF, SP-B, and SP-C are shown in Table 1. Relative quantification was
performed using the ΔCt method, which results in ratios between target genes
and a housekeeping reference gene (HPRT). As the validity of this method
critically depends on the constant expression of the housekeeping gene,
constant expression of HPRT was tested against other housekeeping genes (not
shown). In each run, external standard curves were generated by several fold
dilutions of target genes. The concentration of the target genes was calculated
by comparing Ct values in each sample with Ct values of the internal standard
curve. Finally, data were expressed as the ratio of the amount of each
transcript versus the concentration of HPRT. Melting curves and gel
electrophoresis of the PCR products were routinely performed to determine the
specificity of the PCR reaction.Table 1
Primer
sequences for mRNA detection of the different gene products.
GenForwardReversebpATVEGF*cca cgt cag aga gca aca tcatca ttc tct cta tgt gct ggc ttt7160SP-Bcca cct cct cac aaa gat gacttg ggg tta atc tgg ctc tgg17460SP-Catg gac atg agt
agc aaa gag gtcac gat gag aag
gcg ttt gag11960ER-acgt gtg caa tga cta tgc ctcttt
cat cat gcc cacttcgtaa19962ER-bctg
tga tga act acagtg ttc ccgca
gtg ggt ggc taa agg a12462PRcca
act tca caa aac ttc tcg acaggc
agc aat aac ttc aga cat ca12764HPRTgct
ggt gaa aaggac ctc tcac
agg act aga aca cct gc24862*recognizes transcript variants VEGF 120, 144, and 164.


### 2.3. Protein Analysis

An ELISA for
VEGF was conducted according to the manufacturer’s instructions (RayBiotech
ELISA Kit, specific for VEGF-A). For each sample, blank values (i.e., those for
serum-free media) were subtracted, and mean results were normalized per 105 cells, counted after trypsination in a Neubauer's counting chamber. This
assay was performed in triplicate experiments.


### 2.4. Immunostaining

Purity of cell
cultures was determined by immunohistochemistry for vimentin (fibroblasts,
abcam, Germany, 1:100) and
cytokeratin (AT-II cells, abcam, Germany, 1:75). Cells were grown on
top of autoclaved 13 mm circular glass cover slips placed in
each well of a 24-well plate. After incubation, the media were aspirated, and cells
were washed twice in PBS. Cells were fixed in methanol and incubated with 1%
BSA and 2% FCS in PBS for 20 minutes at 23°C prior to exposure to primary antibodies
for 18 hours at 4°C. Appropriate secondary antibodies and the AEC Kit (Zymed Laboratories,
Calif, USA) were used for staining.


### 2.5. Statistical Analysis

Differences between groups were tested by one-way analysis of variance
(ANOVA) followed by Tukey’s
post hoc multiple range test using SPSS software (SPSS Inc., Chicago, IL, USA). AP-value of less than .05 was considered
significant. Results in figures are presented as means with standard error of
the mean.


## 2.1. Cell Culturing and Treatment

Highly
enriched alveolar cell type II (AT-II) and central lung fibroblast cultures
were established from embryonic day (ED) 18 BALB/c mice as followed. Since
E18 mouse lungs can be dissected from the surrounding vessels in a reliable
manner we did choose this developmental stage to isolate purified AT-II cells
from the mouse lung. Briefly, the fetuses were removed on ED 18 by caesarian
section and the lungs aseptically explanted from the thorax. Lungs were then
washed three times in 10 mL ice cold HBSS, mechanically dissected from
surrounding vessels, and then incubated in a 2.5% trypsin solution containing
200μL of DNAse 1 (2 mg/mL, Worthington) for 10 minutes at 37°C in a shaking
water bath (60 rpm). Finally, the cell suspension was filtered through a nylon
mesh with 100-μm pore size. The cell suspension was centrifuged twice at 420
and then 120 g for 4 minutes, and the pellet was resuspended in minimal
essential medium (MEM). The resulting cell suspension contained the AT-II cells
and attaching fibroblasts. After adding 1.5 ML collagenase (1250 U/mL,
Worthington) and 150 μL DNAse 1 (2 mg/mL, Worthington) the suspension was
incubated for 15 minutes at 37°C. After stopping the collagenase activity by incubating in ice-cold MEM (supplemented with
10% FCS) the cell suspension was centrifuged as
above for 4 minutes and the pellet was resuspended in MEM + 10% FCS. Cells were
seeded and cultivated in a fibronectin-coated culture flask for 1 hour at 37°C
under 5% CO2/21%  O2. During this time, fibroblasts were attached
to the flask, while AT-II cells did not due to their differential adherence
characteristics. This procedure was repeated twice. Finally, approximately 1 ×
105 AT-II cells/cm2 were seeded at 24-well culture
plates, cultured for one day in MEM supplemented with 10% FCS. Medium was
changed after 24 hours to Cellgro complete medium (Mediatech, Virginia, USA,
without phenol red). Attached fibroblasts were resuspended by trypsination and
replated. When cells reached a confluence of approximately 80% the medium was
changed and the final treatment was started. Cells were exposed for 24 hours to
E2 and/or P and dexamethasone (all from Sigma-Aldrich, Germany) in
concentrations ranging from 10–10 M to 10–6 M. Pretreatment with the estrogen
receptor antagonist ICI 182,780 (TOCRIS bioscience, UK, 0.1 μM) and the progesterone
receptor antagonist mifepristone (RU 486 from Biomol, Germany, 0.1 μM) was
performed 1 hour before hormone exposure. Concentrations
of ICI and RU 468 were derived by preparing a 10–2 M stock solution in ethanol
100% p.a. quality and by further diluting in medium. Appropriate ethanol
concentrations served as controls.


## 2.2. Gene Expression Analysis

The mRNA expression
of VEGF, SP-B, and SP-C was quantified using the rtPCR technology (BioRad,
Germany), QTM SYBR Green Supermix (BioRad, Germany), and a standardized
protocol as described previously [23, 24]. Isolation
of total RNA was performed with peq Gold (PeqLab,
Germany). RNA
concentration and purity were assessed using OD260 and OD260/OD280 ratio,
respectively, and reverse transcribed using an Invitrogen M-MLV RT-kit and
random hexanucleotide primers. The rtPCR reactions were carried out in a
reaction mixture consisting of 2 μL cDNA, 6 μL RNAse-free water, 10 μL hot StartTaq
DNA-polymerase, and 1 μL of primer (10 pmol). Reactions were conducted in
standard tubes using the MyIQ rtPCR Detection System (BioRad, Germany) under
following conditions: 10-minute
enzyme activation at 95°C, 45 cycles of 15-second denaturation at 95°C, 30-second annealing at
individual temperatures, 30-second
amplification at 72°C, and 5-second fluorescence measurement at 80°C. Primer sequences to
detect VEGF, SP-B, and SP-C are shown in Table 1. Relative quantification was
performed using the ΔCt method, which results in ratios between target genes
and a housekeeping reference gene (HPRT). As the validity of this method
critically depends on the constant expression of the housekeeping gene,
constant expression of HPRT was tested against other housekeeping genes (not
shown). In each run, external standard curves were generated by several fold
dilutions of target genes. The concentration of the target genes was calculated
by comparing Ct values in each sample with Ct values of the internal standard
curve. Finally, data were expressed as the ratio of the amount of each
transcript versus the concentration of HPRT. Melting curves and gel
electrophoresis of the PCR products were routinely performed to determine the
specificity of the PCR reaction.Table 1
Primer
sequences for mRNA detection of the different gene products.
GenForwardReversebpATVEGF*cca cgt cag aga gca aca tcatca ttc tct cta tgt gct ggc ttt7160SP-Bcca cct cct cac aaa gat gacttg ggg tta atc tgg ctc tgg17460SP-Catg gac atg agt
agc aaa gag gtcac gat gag aag
gcg ttt gag11960ER-acgt gtg caa tga cta tgc ctcttt
cat cat gcc cacttcgtaa19962ER-bctg
tga tga act acagtg ttc ccgca
gtg ggt ggc taa agg a12462PRcca
act tca caa aac ttc tcg acaggc
agc aat aac ttc aga cat ca12764HPRTgct
ggt gaa aaggac ctc tcac
agg act aga aca cct gc24862*recognizes transcript variants VEGF 120, 144, and 164.


## 2.3. Protein Analysis

An ELISA for
VEGF was conducted according to the manufacturer’s instructions (RayBiotech
ELISA Kit, specific for VEGF-A). For each sample, blank values (i.e., those for
serum-free media) were subtracted, and mean results were normalized per 105 cells, counted after trypsination in a Neubauer's counting chamber. This
assay was performed in triplicate experiments.


## 2.4. Immunostaining

Purity of cell
cultures was determined by immunohistochemistry for vimentin (fibroblasts,
abcam, Germany, 1:100) and
cytokeratin (AT-II cells, abcam, Germany, 1:75). Cells were grown on
top of autoclaved 13 mm circular glass cover slips placed in
each well of a 24-well plate. After incubation, the media were aspirated, and cells
were washed twice in PBS. Cells were fixed in methanol and incubated with 1%
BSA and 2% FCS in PBS for 20 minutes at 23°C prior to exposure to primary antibodies
for 18 hours at 4°C. Appropriate secondary antibodies and the AEC Kit (Zymed Laboratories,
Calif, USA) were used for staining.


## 2.5. Statistical Analysis

Differences between groups were tested by one-way analysis of variance
(ANOVA) followed by Tukey’s
post hoc multiple range test using SPSS software (SPSS Inc., Chicago, IL, USA). AP-value of less than .05 was considered
significant. Results in figures are presented as means with standard error of
the mean.


## 3. Results

### 3.1. Cell Culture Purity and Receptor Expression

The culturing protocol for
lung fibroblast yielded a greater than 98% homogeneity of vimentin-positive fibroblasts.
The culturing protocol for AT-II cells yielded a greater than 95% homogeneity
of cytokeratin-positive cells. Cross contamination of purified fibroblasts cell
cultures was excluded by immunohistochemical staining of fibroblast cell
cultures with anti-cytokeratin antibody (Figure1). No expression of SP-B and
SP-C was detected in lung fibroblasts further proving purity of cell cultures. Semiquantitative
PCR revealed that all three hormone receptors, namely, estrogen receptor alpha
(ER-α), estrogen receptor beta (ER-β), and the progesterone receptor (PR) are
expressed in purified central lung fibroblast and AT-II cell cultures (Figure
2).Alevolar
cells type II (AT-II) and central lung fibroblasts stained with cytokeratin
antibody. Note that AT-II stain positive for cytokeratin whereas fibroblasts do
not. Therefore, contamination of AT-II cells in fibroblast cell cultures can be
excluded.
(a)(b)Semiquantitative
analysis of estrogen receptor alpha (ER-α), estrogen receptor beta (ER-β), and the
progesterone receptor (PR) expression in central lung fibroblast and AT-II cell
cultures. Note that
all three hormone receptors
are expressed in both cell cultures.
(a)(b)


### 3.2. Hormonal Effects on Central Lung Fibroblasts

The
treatment of cultured fibroblast with either E2-8 M or P-8 M alone for
48 hours had no significant influence on VEGF mRNA expression (Figure3). However,
a combined application with both steroids resulted in a significant increase by
approximately 100% compared to controls in VEGF mRNA levels (P < .01, Figure 3). Dexamethasone was applied as positive control (P < .01, Figure 3). The
combined application at concentrations lower than 10–8 M did not significantly affect
VEGF mRNA expression (Figure 4), and higher concentrations (10–6 M) did not
further promote VEGF mRNA expression compared to 10–8 M (Figure 4). The
hormone-induced upregulation of VEGF mRNA was completely blocked by the
application of the receptor antagonists ICI and RU 486 (Figure 4). The single or combined treatment with ICI and/or RU 486 did not influence the basal expression of
investigated proteins as determined by rt-PCR analysis (data not shown). Using ELISA
analysis, we could confirm the transcriptional regulation of VEGF by E2 and P.
Only the combined application increased extracellular VEGF protein levels in
fibroblasts (Figure 5). Pretreatment with the receptor antagonists again abrogated
this effect.Figure 3
Quantitative
analysis of VEGF gene expression in central lung fibroblasts treated for 48 hours
with E2-8 M and  P-8 M alone or in combination. Values were
normalized against a housekeeping gene (HPRT) and expressed as % of controls.
Note that only the combined application of both hormones significantly
increased VEGF expression in central lung fibroblasts. Also note that the
application of dexamethasone (D) had similar effects on VEGF expression.P*<.01 control versus
E2/P-8 M, P**<.01 control versus D-8 M.Figure 4
Quantitative
analysis of VEGF gene expression in central lung fibroblasts treated for 48 hours
with increasing concentrations of both E2 and P and with ICI/RU 486. Values
were normalized against a housekeeping gene (HPRT) and expressed as % of
controls. Note that the application of receptor antagonists 1 hour prior to
hormone application (ICI/RU 486) antagonizes hormonal effects.P<.01 control versus E2/P-8 M; P**<.01 control versus
E/P-6 M.Figure 5
Quantification
of VEGF protein release of lung fibroblasts treated for 48 hours with E2-8 M and P-8 M alone or in combination determined by ELISA. Note
that corresponding with results obtained by gene expression analysis (Figure3)
VEGF protein is increased in central lung fibroblast cultures only by combined treatment
with E2 and P. Pretreatment with ICI/RU 486 abrogated this effect. P*<.05 control versus E2/P-8 M.


### 3.3. Hormonal Effects on AT-LI Cells

As
shown for fibroblasts only the simultaneous exposure to E2-8 M  and  P-8 M
significantly enhanced the expression of VEGF (Figure6) and this could be
confirmed at the transcriptional level (Figure 7). Dexamethasone also increased
VEGF amount in AT-II cells (Figure 6). Combined application of E2 and P increased
mRNA expression of SP-B and SP-C to a similar extent as dexamethasone (Figure 8).
Pretreatment with the receptor antagonists ICI and RU 468 abrogated this
effect. SP-A was not found in AT-II cells, however was expressed in mature lung
tissue which did serve as a positive control (not shown).Figure 6
Quantitative
analysis of VEGF gene expression in alveolar cells type II treated for 48 hours
with E2-8 M and P-8 M alone or in combination. Values were
normalized against a housekeeping gene (HPRT) and expressed as % of controls.
Note that only the combined application of both hormones significantly
increased VEGF expression in central lung fibroblasts. Also note that the
application on dexamethasone had similar effects on VEGF expression.P*<.01 control versus E2/P-8 M, P**<.01 control versus D-8 M.Figure 7
Quantification
of VEGF protein release of alveolar cells type II treated for 48 h with E2-8 M and  P-8 M alone or in combination determined by
ELISA. Note that corresponding with results obtained by gene
expression analysis (Figure6) VEGF protein is increased in alveolar type II
cell cultures only by combined treatment with E2 and P.  Pretreatment with ICI/RU 486 abrogated
this effect.  P*<.05 control versus E2/P-8 M.Quantitative
analysis of SP-B and SP-C gene expression in alveolar cells type II treated for
48 hours with E2-8 M/P-8 M in combination or with dexamethasone-8 M.
Values were normalized against a housekeeping gene (HPRT) and expressed as % of
controls. Note that
combined hormonal treatment increased expression of both surfactant proteins to
a similar extent like dexamethasone. Further note that pretreatment with the receptor
antagonists ICI and RU 486 antagonized the hormonal effects.P*<.01 control versus E2/P-8 M; P**<.01 control versus D-8 M.
(a)(b)


## 3.1. Cell Culture Purity and Receptor Expression

The culturing protocol for
lung fibroblast yielded a greater than 98% homogeneity of vimentin-positive fibroblasts.
The culturing protocol for AT-II cells yielded a greater than 95% homogeneity
of cytokeratin-positive cells. Cross contamination of purified fibroblasts cell
cultures was excluded by immunohistochemical staining of fibroblast cell
cultures with anti-cytokeratin antibody (Figure1). No expression of SP-B and
SP-C was detected in lung fibroblasts further proving purity of cell cultures. Semiquantitative
PCR revealed that all three hormone receptors, namely, estrogen receptor alpha
(ER-α), estrogen receptor beta (ER-β), and the progesterone receptor (PR) are
expressed in purified central lung fibroblast and AT-II cell cultures (Figure
2).Alevolar
cells type II (AT-II) and central lung fibroblasts stained with cytokeratin
antibody. Note that AT-II stain positive for cytokeratin whereas fibroblasts do
not. Therefore, contamination of AT-II cells in fibroblast cell cultures can be
excluded.
(a)(b)Semiquantitative
analysis of estrogen receptor alpha (ER-α), estrogen receptor beta (ER-β), and the
progesterone receptor (PR) expression in central lung fibroblast and AT-II cell
cultures. Note that
all three hormone receptors
are expressed in both cell cultures.
(a)(b)


## 3.2. Hormonal Effects on Central Lung Fibroblasts

The
treatment of cultured fibroblast with either E2-8 M or P-8 M alone for
48 hours had no significant influence on VEGF mRNA expression (Figure3). However,
a combined application with both steroids resulted in a significant increase by
approximately 100% compared to controls in VEGF mRNA levels (P < .01, Figure 3). Dexamethasone was applied as positive control (P < .01, Figure 3). The
combined application at concentrations lower than 10–8 M did not significantly affect
VEGF mRNA expression (Figure 4), and higher concentrations (10–6 M) did not
further promote VEGF mRNA expression compared to 10–8 M (Figure 4). The
hormone-induced upregulation of VEGF mRNA was completely blocked by the
application of the receptor antagonists ICI and RU 486 (Figure 4). The single or combined treatment with ICI and/or RU 486 did not influence the basal expression of
investigated proteins as determined by rt-PCR analysis (data not shown). Using ELISA
analysis, we could confirm the transcriptional regulation of VEGF by E2 and P.
Only the combined application increased extracellular VEGF protein levels in
fibroblasts (Figure 5). Pretreatment with the receptor antagonists again abrogated
this effect.Figure 3
Quantitative
analysis of VEGF gene expression in central lung fibroblasts treated for 48 hours
with E2-8 M and  P-8 M alone or in combination. Values were
normalized against a housekeeping gene (HPRT) and expressed as % of controls.
Note that only the combined application of both hormones significantly
increased VEGF expression in central lung fibroblasts. Also note that the
application of dexamethasone (D) had similar effects on VEGF expression.P*<.01 control versus
E2/P-8 M, P**<.01 control versus D-8 M.Figure 4
Quantitative
analysis of VEGF gene expression in central lung fibroblasts treated for 48 hours
with increasing concentrations of both E2 and P and with ICI/RU 486. Values
were normalized against a housekeeping gene (HPRT) and expressed as % of
controls. Note that the application of receptor antagonists 1 hour prior to
hormone application (ICI/RU 486) antagonizes hormonal effects.P<.01 control versus E2/P-8 M; P**<.01 control versus
E/P-6 M.Figure 5
Quantification
of VEGF protein release of lung fibroblasts treated for 48 hours with E2-8 M and P-8 M alone or in combination determined by ELISA. Note
that corresponding with results obtained by gene expression analysis (Figure3)
VEGF protein is increased in central lung fibroblast cultures only by combined treatment
with E2 and P. Pretreatment with ICI/RU 486 abrogated this effect. P*<.05 control versus E2/P-8 M.


## 3.3. Hormonal Effects on AT-LI Cells

As
shown for fibroblasts only the simultaneous exposure to E2-8 M  and  P-8 M
significantly enhanced the expression of VEGF (Figure6) and this could be
confirmed at the transcriptional level (Figure 7). Dexamethasone also increased
VEGF amount in AT-II cells (Figure 6). Combined application of E2 and P increased
mRNA expression of SP-B and SP-C to a similar extent as dexamethasone (Figure 8).
Pretreatment with the receptor antagonists ICI and RU 468 abrogated this
effect. SP-A was not found in AT-II cells, however was expressed in mature lung
tissue which did serve as a positive control (not shown).Figure 6
Quantitative
analysis of VEGF gene expression in alveolar cells type II treated for 48 hours
with E2-8 M and P-8 M alone or in combination. Values were
normalized against a housekeeping gene (HPRT) and expressed as % of controls.
Note that only the combined application of both hormones significantly
increased VEGF expression in central lung fibroblasts. Also note that the
application on dexamethasone had similar effects on VEGF expression.P*<.01 control versus E2/P-8 M, P**<.01 control versus D-8 M.Figure 7
Quantification
of VEGF protein release of alveolar cells type II treated for 48 h with E2-8 M and  P-8 M alone or in combination determined by
ELISA. Note that corresponding with results obtained by gene
expression analysis (Figure6) VEGF protein is increased in alveolar type II
cell cultures only by combined treatment with E2 and P.  Pretreatment with ICI/RU 486 abrogated
this effect.  P*<.05 control versus E2/P-8 M.Quantitative
analysis of SP-B and SP-C gene expression in alveolar cells type II treated for
48 hours with E2-8 M/P-8 M in combination or with dexamethasone-8 M.
Values were normalized against a housekeeping gene (HPRT) and expressed as % of
controls. Note that
combined hormonal treatment increased expression of both surfactant proteins to
a similar extent like dexamethasone. Further note that pretreatment with the receptor
antagonists ICI and RU 486 antagonized the hormonal effects.P*<.01 control versus E2/P-8 M; P**<.01 control versus D-8 M.
(a)(b)


## 4. Discussion

The role of
angiogenic growth factors for developmental processes is increasingly
recognized [25]. A decreased
expression may be a potential mechanism of alveolar capillary dysmorphogenesis
in BPD. In animal models of bronchopulmonary dysplasia and in preterm infants
dying from BPD, diminished VEGF mRNA expression is evident (for a review see [25]). VEGF
stimulates the growth of lung epithelial cells in vitro [26] and is
important for pulmonary vascular development [27]. Mice with a deficiency of
VEGF die from RDS [17], and VEGF
knockout results in embryonic lethality [15]. To our
knowledge no data on the influence of E2 and P on VEGF expression in the
developing lung is available. In primary lung fibroblasts and AT-II cells only
the combined application of E2 and P resulted in increased expression levels of
VEGF mRNA and VEGF protein. This effect was abolished by pretreatment with the specific
E2 and P antagonists ICI and RU 486, respectively. Assuming that E2 and P
increase VEGF expression in the developing lung in vivo, the withdrawal of the
placental supply of E2 and P in preterm infants may explain the disturbed lung
development and function. Postnatal replacement of E2 and P in preterm infants
was associated with a
trend toward reduced incidence of BPD [5, 7].Disturbed
lung function in RDS of preterm infants is due to surfactant deficiency. A
regulative role of E2 in surfactant synthesis is supported by enhanced mRNA
expression for SP-B found in fetal rabbit lung cells [28]. Our data supports a
regulative role of E2 and P for surfactant synthesis as in AT-II cells combined
treatment with E2 and P resulted not only in increased VEGF but also in
increased mRNA expression of SP-B and SP-C. We did not perform experiments to
evaluate the role of VEGF for surfactant synthesis. However, data from the
literature implicates a trigger function of VEGF for
surfactant synthesis. Intra-amniotic
injection of VEGF in preterm rats resulted in increased SP-B mRNA expression [18].
Furthermore, type II pneumocytes respond to VEGF by enhancing their expression of SP-B
and SP-C [17]. We
speculate that E2 and P promote
VEGF production and, thereby, surfactant synthesis.One major finding of our study is that only the combination of E2 and P
was effective in the upregulation of VEGF, SP-B, and SP-C expression. This is
in accordance with findings about the epithelial Na+-channel
which plays a critical role in the active reabsorption of alveolar fluid at the
time of birth and during pulmonary oedema. In rats, only the combined application
of E2 and P promoted mRNA levels of the epithelial Na+-channel in
the lung suggesting complex interactions between the intracellular E2 and P
signalling [29]. It appeared that only the administration of both hormones
is fully effective in preventing demyelination in a multiple sclerosis animal
model [30]. It is well known that ER and PR are coexpressed in the same cells in
several areas of the target tissues [31, 32].
In addition, a number of reports demonstrated that ER and PR can have
synergistic or inhibitory cross-talk in their transcriptional regulation in
promoter type- and PR subtype-specific manners [33, 34].
Also interactions on well-known nongenomic levels are assumed. For example, in
breast cancer cells, estrogens activate the Src/Erk pathway through an
interaction of the ER with the SH2 domain of c-Src. Progestins have been
reported to activate also this pathway either via an interaction of the PR with
ER, which itself activates c-Src, or by direct interaction of PR with the SH3
domain of c-Src [35].
Future studies have to show the underlying mechanisms for combined positive
hormonal effects. The preterm infant is deprived of both E2
and P, simultaneously. Our study adds evidence that only combined replacement
of E2 and P may be effective to prevent BPD in preterm infants [5, 7].Treatment
with dexamethasone increased VEGF mRNA expression and VEGF protein in lung
fibroblasts and AT-II cells. In contrast, in vitro studies have shown that
dexamethasone downregulates VEGF expression in cells derived from alveolar
epithelial cells [36]. However, in mid-trimester
fetal human lung explants dexamethasone increased VEGF mRNA expression [37] and this at
least at the translational level was also found in vivo [38].
Interestingly, treatment of preterm infants with dexamethasone was associated
with increased VEGF levels in deep pulmonary lavages [39].The embryos were not stratified by gender. Therefore, we cannot exclude
that by chance some experiments were conducted in cell cultures from
predominant male or female embryos. The steroid concentrations being most
effective in our experiments, that is, 10–8 M, appear at first glance to be above the
physiological plasma levels found in rodents during the estrous cycle. However,
it is generally accepted that under in vitro conditions higher steroid levels
are required to yield cellular effects. In previous in vitro studies, these
high estrogen concentrations were applied to generate physiological effects [40, 41]. Another
more intriguing point is that tissues themselves produce steroids. Thus, tissue
intrinsically synthesized steroids may contribute together with plasma steroids
to reach higher local tissue steroid concentrations. In support for this view
is the recent observation that in rodent hippocampal tissue local estrogen
production yields tissue steroid levels at approximately 10–9 M which are
greater than those
in the plasma [42].In
conclusion, combined use of E2 and P enhanced expression of VEGF and surfactant
proteins in primary embryonic lung cells. These proteins are known to be key
factors for the prenatal lung development and postnatal lung function. Further
research about the effects of E2 and P on lung development may open
therapeutic perspectives for preterm infant prone to develop lung disease.



---

*Source: 170491-2009-03-22.xml*