postnatal estradiol up-regulates lung nitric oxide synthases and improves lung function in...

Postnatal Estradiol Upregulates Lung

NO Synthases and Improves Lung Function in

Bronchopulmonary Dysplasia

Donald C. McCurnin2,3, Richard A. Pierce4, Brigham C. Willis1, Ling Yi Chang5,

Bradley A. Yoder6, Ivan S. Yuhanna1, Philip L. Ballard7, Ronald I. Clyman7, Nahid Waleh8

William Maniscalco9, James D. Crapo5, Peter H. Grubb10, and Philip W. Shaul1

Division of Pulmonary and Vascular Biology1, Department of Pediatrics,

University of Texas Southwestern Medical Center Department of Pediatrics2, University of Texas Health Science Center at San Antonio,

and the Southwest Foundation for Biomedical Research3 Department of Internal Medicine4, Washington University School of Medicine

Department of Medicine5, National Jewish Medical and Research Center Department of Pediatrics6, University of Utah School of Medicine

Department of Pediatrics7, University of California San Francisco School of Medicine Life Sciences Division8, SRI International, Menlo Park, CA

Department of Pediatrics9, University of Rochester School of Medicine and Dentistry Department of Pediatrics10, Vanderbilt University School of Medicine

Running Head: Estradiol and Bronchopulmonary Dysplasia

Address correspondences to: Philip W. Shaul, Department of Pediatrics, University of

Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX. Tel. 214-648-2015, Fax 214-648-2098, email: [email protected].

Support: NIH grants HL63399 and HD30276 (PWS), HL63387 (RAP), HL63397 (JDC),

and HL46691 and HL56061 (RIC). Additional backing was provided by HL52636 (BPD Resource Center) and P51RR13986 for facility support.

Postnatal therapies to prevent bronchopulmonary dysplasia (BPD) in preterm infants are limited. These experiments in a primate model indicate that postnatal estradiol treatment has favorable cardiovascular impact, it enhances pulmonary function and it lowers requirements for ventilatory support in BPD. These effects occur in association with an upregulation of lung NO synthases. Estradiol may be an efficacious postnatal therapy to improve lung function and outcome in preterm infants.

This article has an online data supplement, which is accessible from this issue’s table of

contents online at www.atsjournals.org. 95. Neonatal Lung Disease Word Count: 4397

of 56 AJRCCM Articles in Press. Published on January 16, 2009 as doi:10.1164/rccm.200805-794OC

Copyright (C) 2009 by the American Thoracic Society.

ABSTRACT Rationale: Nitric oxide (NO) plays an important role in lung development and perinatal lung

function, and pulmonary NO synthases (NOS) are decreased in bronchopulmonary dysplasia

(BPD) following preterm birth. Fetal estradiol levels increase during late gestation and estradiol

upregulates NOS, suggesting that after preterm birth estradiol deprivation causes attenuated lung

NOS resulting in impaired pulmonary function. Objective: Test the effects of postnatal estradiol

administration in a primate model of BPD over 14 days after delivery at 125 days gestation (term

= 185 days). Methods: Cardiopulmonary function was assessed by echocardiography and whole

body plethysmography. Lung morphometric and histopathologic analyses were performed, and

NOS enzymatic activity and abundance were measured. Measurements and Main Results:

Estradiol caused an increase in blood pressure, and it caused ductus arteriosus closure.

Expiratory resistance and lung compliance were also improved, and this occurred prior to

spontaneous ductal closure. Furthermore, both oxygenation and ventilation indices were

improved with estradiol, and the changes in lung function and ventilatory support requirements

persisted throughout the study period. Whereas estradiol had negligible effect on indicators of

lung inflammation and lung structure assessed after the initial 14 days of ventilatory support, it

caused an increase in lung neuronal and endothelial NOS enzymatic activity. Conclusions: In a

primate model of BPD, postnatal estradiol treatment had favorable cardiovascular impact, it

enhanced pulmonary function, and it lowered requirements for ventilatory support in association

with an upregulation of lung NOS. Estradiol may be an efficacious postnatal therapy to improve

lung function and outcome in preterm infants.

Abstract Word Count: 250 Key Words: airway resistance, compliance, patent ductus arteriosus

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INTRODUCTION

The signaling molecule nitric oxide (NO), generated by nitric oxide synthase (NOS),

plays a key role in multiple processes in the mature lung(1, 2). In the developing lung, NO

participates in pulmonary vascularization, alveolarization, and airway branching, and NO also

counteracts apoptosis in multiple lung cell types(3-6). In the perinatal period, epithelium-derived

NO is critically involved in the regulation of lung liquid production and of peripheral contractile

elements(7, 8), and NO also mediates pulmonary vasomotor tone(9). In studies of lungs from

fetal baboons, we showed that all three NOS isoforms, neuronal NOS (nNOS), endothelial NOS

(eNOS) and inducible NOS (iNOS), are principally expressed in proximal respiratory epithelium,

and that there are maturational increases in their abundance and in NO production during the

early third trimester(10). Thus, pulmonary NOS expression is upregulated during fetal

development in the primate, and this process may be critical to lung structural development and

airway, parenchymal and pulmonary vascular function in the early postnatal period.

Bronchopulmonary dysplasia (BPD) is a devastating primary complication of premature

birth that develops in the preterm human lung following ventilatory and oxygen support. This

disorder results in disrupted lung maturation and postnatal pulmonary maladaptation. We

previously determined whether there are alterations in NOS in proximal lung and accompanying

changes in NO production in a model of BPD in baboon fetuses delivered at 125d gestation (term

= 185d) and ventilated for 14d. This model best exemplifies the current form of BPD observed

in extremely preterm human infants(11). In contrast to the normal 73% increase in NOS activity

which occurs over the same developmental period in utero, there was an 83% decline in activity

due to decreases in nNOS and eNOS expression. In addition, exhaled NO levels at the time of

preterm birth at 125d gestation were one-third those observed at birth later in the third trimester,

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and they remained depressed until day of life 11(12). In addition, the administration of inhaled

NO gas (iNO) in the same model improved early pulmonary function. However, with iNO these

improvements were transient and there was negligible impact on ventilatory support

requirements(13). Studies of NOS and iNO in the primate and other animal models of BPD led

to trials of iNO therapy in preterm human infants. Collectively these important trials have

indicated that certain subsets of preterm infants benefit from iNO. However, others do not, and

new postnatal strategies to improve lung function and prevent BPD continue to be needed(14-

17).

The hormone estrogen upregulates nNOS and eNOS and activates NOS enzymatic

activity in diverse tissues and cell types(18-20), including in ovine fetal lung in vivo and in

cultured bronchial epithelial cells and pulmonary artery endothelial cells(20, 21). Fetal plasma

estradiol (E2) levels increase progressively during late gestation, they rise further with the onset

of parturition at term, and then fall in the early postnatal period due to the loss of the placentally-

derived hormone(22, 23). These cumulative observations suggest that there is relative estrogen

deprivation following preterm birth that may adversely impact lung NOS and thereby impair

pulmonary development and function.

In the present investigation we determined the effects of postnatal E2 administration in

the baboon model of BPD that mirrors the current form of the disease in extremely preterm

human infants(11). The baboons were born by cesarean section at 0.67 gestation, which is

comparable to 27 weeks post-conceptual age in humans, and E2 was administered by

subcutaneous pellet beginning at one hour of age. We tested the hypothesis that E2 improves the

pulmonary dysfunction that is due to prematurity and the development of BPD, and that this is

associated with an upregulation of pulmonary NOS. Since estrogen has numerous

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cardiovascular actions(24), we also determined the effects of postnatal E2 administration on the

pulmonary and systemic circulation and on the status of the ductus arteriosus. In addition,

because the current form of BPD is characterized by abnormal elastin deposition and fewer and

larger alveoli(25, 26), we evaluated the effect of E2 on pulmonary growth and structure.

Furthermore, since estrogen modulates inflammatory responses(27), we assessed the effect of E2

on markers of lung inflammation.

METHODS

Animal Model

Fetal baboons were delivered at 125±2 d gestation (term = 185d) by cesarean section. At

birth the baboons were weighed, sedated, intubated, given 4 cc/kg of surfactant (Survanta,

courtesy of Ross Laboratories, Columbus, OH), and ventilator support was provided for 14d.

Animals were randomly assigned to either the control group which received routine care and

control treatment, or to the estrogen group which received routine care plus E2. Additional detail

on the management of the animals is provided in an online data supplement.

Echocardiography and Pulmonary Function Testing

Echocardiographic studies were performed at 1 and 6 h of age and at 24 h intervals

thereafter. The echocardiograms were done by one of the authors (DM) coincident to the

pulmonary function tests which were performed using the VT1000 body plethysmograph

(Vitaltrends Technology, New York, NY)(13). Dynamic lung compliance and resistance

measurements were made during controlled mechanical breaths, and they were of the respiratory

system as a whole. Since tidal volume can impact measurements of compliance, tidal volume

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was controlled at 4 - 6 ml/kg, and measured tidal volumes did not differ between study groups.

For data analysis compliance was corrected for body weight.

Postmortem Pressure-Volume Measurements

Immediately prior to termination the animals breathed 100% oxygen for 5 min and the

lungs were degassed by clamping the trachea at end expiration for 2 min. Following the removal

of the lungs from the thoracic cavity en bloc, postmortem quasi-static inflation pressure-volume

measurements were carried out. Additional detail on the method employed is provided in an

online data supplement.

Morphometric-histopathologic Analyses and Assessments of Lung Inflammation

Analyses were performed using the methods previously reported(13, 28). Additional

details are provided in an online data supplement.

Pulmonary Surfactant Analysis

At the end of study a bronchoalveolar lavage (BAL) was done on the left lower lobe with

25 ml of normal saline. Cells were removed by centrifugation and the supernatant was

centrifuged (27,000 × g, 60 min) to yield a large aggregate surfactant pellet and supernatant, and

pulmonary surfactant analysis was performed. Additional details are provided in an online data

supplement.

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NOS Enzymatic Activity and Expression

NOS enzymatic activity and expression were evaluated in lung parenchymal samples

taken from the proximal 1/3 of the respiratory tree. This approach was taken because the three

NOS isoforms are primarily expressed in the respiratory epithelium of the proximal airways of

the developing primate(10). Additional details are provided in an online data supplement.

Estrogen Receptor Expression

Using previously described methods(29), immunoblot analysis was performed to evaluate

ERα and ERβ expression in the proximal lung of 125d and 140d gestational control animals and

in proximal lung from control- and E2-treated animals. Positive controls consisted of lysates of

COS-7 cells transfected with cDNA for either human ERα or human ERβ.

Statistical Analysis

Differences between gestational control groups were compared by one-way analysis of

variance (ANOVA) followed by Newman-Keuls post hoc testing. Longitudinal between-group

differences in pulmonary and cardiac function parameters over the full course of study were

compared by two-way ANOVA. Repeated measures was not performed for these endpoints

because values for individual animals were occasionally unobtainable due to technical

difficulties or unavailability of the echocardiographer. The inflation and deflation limbs of the

postmortem pressure-volume curves were assessed by separate two-way repeated measures

ANOVAs. OI and VI were assessed by repeated measures ANOVA. Single comparisons

between two groups were performed with nonpaired Student's t tests or Mann-Whitney

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(nonparametric) for continuous data and by Fisher’s exact test for categorical data. Significance

was accepted at the 0.05 level of probability. All results are expressed as mean±SEM.

RESULTS

Study Groups

To first evaluate developmental changes in fetal serum E2 concentrations during the third

trimester in the baboon, levels were initially measured in additional fetal baboons at 125d, 140d,

160d, or 180d gestation upon euthanization immediately at delivery. E2 levels were determined

by radioimmunoassay as previously described(30). Fetal serum E2 concentrations rose 5-fold

between 125-140d gestation and 160-180d gestation to achieve mean levels of 250 pg/ml (Fig.

1A). Seeking to attain the upper range of the concentrations observed in the latter third trimester,

in the studies of postnatal E2 administration the hormone was provided by placement of a 0.5 mg,

21d extended release pellet subcutaneously in the left axilla at 1h of life. Control animals

received a placebo pellet, and a second E2 or control pellet was placed subcutaneously in the

right axilla on day of life 7. The subcutaneous route was selected for E2 administration due to

favorable stability and metabolic fate compared with intravenous or oral forms of estrogen(31,

32).

Nine animals were randomized to the control group and 10 to the E2 treatment group.

There was one death in control animals which occurred at 10d of age, and there was one death in

E2-treated animals at 11d of age, and these were related to coagulase negative staph sepsis.

These animals were excluded from longitudinal analyses. Six of the 8 animals in the control

group and 5 of the 9 in the E2-treated group were born following prenatal betamethasone

treatment, and the remainder were born after prenatal dexamethasone. The birthweights,

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gestational ages at delivery and number of males versus females were similar in the control and

E2-treated animals (Table 1). There were no differences between groups for daily fluid intake,

daily urine output or daily weights over the course of the study (data not shown).

Serum E2 levels achieved in the control and E2-treated groups are shown in Fig. 1B. With

E2 administration the initial E2 levels were 1000 pg/ml at 6h of life, and they fell to 600 pg/ml on

day 2. By day of life 7 in the treated animals E2 levels were 230 pg/ml and a second E2 pellet

was placed. E2 then rose to concentrations of 400 to 500 pg/ml during the second week of life.

At all timepoints except day of life 7, E2 levels were greater in treated animals than control

animals.

Effect of E2 on Systemic and Pulmonary Hemodynamics

The administration of E2 immediately after birth and continuously throughout the study

period caused an increase in mean systemic BP (Fig. 2A). Whereas systolic BP did not differ

between study groups, diastolic BP was greater with E2. This led to a lower requirement for

pressor support in the estrogen group in which only 2 of 9 animals required therapy versus 7 of 8

animals in the control group (P=0.01). There were no demonstrable changes in the ratio of the

estimated pulmonary artery pressure to systemic BP or the ratio of pulmonary to systemic blood

flow with E2 administration (See Figure E1A and B, respectively, in the online data supplement).

E2 also altered the incidence of patent ductus arteriosus. Whereas none of the control

animals had spontaneous ductal closure during the 14d study period, 4 of the 9 E2-treated

animals had spontaneous ductal closure by echocardiography (Fig. 2B). In one of these animals

closure was apparent at 4d of age, and in the other 3 animals ductal closure was found at 8 to 11d

of age. The increases in systemic BP with E2 occurred prior to the time of ductal closure (Fig.

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2A), indicating that the change in BP was due to processes other than the loss of left to right

shunting across the ductus with its spontaneous closure.

Effect of E2 on Pulmonary Function

The effect of E2 on pulmonary function is shown in Fig. 3. Expiratory resistance was

lower across the study period in the E2 group (Fig. 3A), and dynamic lung compliance was

increased with E2 (Fig. 3B). To provide an additional assessment of pulmonary function,

postmortem PV curves were performed. During the procedure air leaks occurred in lungs from 3

control animals and from 4 E2-treated animals, and complete analysis was therefore available in

lungs from 5 animals per study group. There was a directional change in both the inflation and

deflation limbs of the PV curves, with shift upward and to the left with E2, and P values were

0.10 and 0.21, respectively (See Figure E2 in the online data supplement). The increase in lung

volume at 35 cm H2O with E2 also approached but did not achieve statistical significance (34±4

versus 48±6 ml/kg for lungs from control and E2 treatment groups, respectively, P=0.07).

Effect of E2 on Ventilatory Support Requirements

The requirements for ventilatory support are shown in Fig. 4. Oxygenation index over

the course of the 14d study was decreased in the E2-treated animals compared with controls (Fig.

4A), frequently by 30-50%. As impressively, the ventilation index was also lowered throughout

the postnatal period by E2 administration, typically by 40% or more (Fig. 4B).

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Effect of E2 on Lung Weight, Structure and Inflammation

The weights of the lungs from control and E2-treated animals at the end of the study were

similar (3.6±0.2 and 3.8±0.2% of body weight, respectively). The wet-to-dry weight ratios were

also similar, being 5.13±0.16 and 4.96±0.30, respectively. Representative lungs from 125d

gestation, 140d gestation and control- and E2-treated animals are shown in Fig. 5. With in utero

development between 125d and 140d gestation (Fig. 5A, B), there was thinning of alveolar walls

and increased septation. Compared with fetal lungs at 140d gestation, which is at the same

postconceptual age (Fig. 5B), lungs from control-treated animals on ventilatory support for 14d

had areas with thickened alveolar walls and simplification of the alveolar structures (Fig. 5C): E2

administration did not alter overall lung histology compared to control treatment (Fig. 5D).

Pulmonary morphology was quantitatively compared in the two treatment groups by

skeletonization of alveolar structures and computer-assisted measurements. E2 did not alter

alveolar surface area, the number or length of end segments indicative of secondary crests, or

other determined parameters (Table 2). E2 also did not alter lung vascularization evaluated by

the distribution of PECAM-1 immunostaining of endothelial cells or PECAM-1 abundance, and

VEGF expression or distribution was unchanged (Table E1 in the online data supplement, and

data not shown).

Elastin distribution and levels of expression were evaluated by multiple strategies. Hart’s

staining indicated that elastic fibers were localized to both alveolar walls and septal tips in lungs

of control-treated animals (Fig. 6A) and primarily to emerging septal tips in E2-treated lungs

(Fig. 6B). In situ hybridization on serial sections detected often intense elastin mRNA

expression within alveolar walls and at emerging septae in control-treated animals (Fig. 6C), and

expression was localized primarily to emerging septae in E2-treated animals (Fig. 6D).

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However, quantitative RT-PCR revealed equal total elastin mRNA abundance in control and E2-

treated lungs (Fig. 6E). Thus, E2 had a modest impact on elastin distribution favoring emerging

septae, but did not alter its overall expression in the lung.

The impact of E2 on lung inflammation was also assessed. TGF-β1 abundance in

tracheal aspirates and terminal BAL samples did not change over the course of development of

BPD in the primate, and it was also similar between control and E2 treatment groups (See Figure

E3 in the online data supplement). In addition, levels of mRNA expression of genes that regulate

inflammation in the lung and are altered in the primate BPD model(28) were not affected by E2

treatment (See Table E1 in the online data supplement).

Effect of E2 on Pulmonary Surfactant

In an effort to understand the basis for changes in pulmonary function with E2, surfactant-

related parameters were measured in terminal bronchoalveolar lavage samples (See Figure E4 in

the online data supplement). The total protein and phospholipid contents of the surfactant pellet

were similar in the control and E2 groups (E4A, B, respectively). Mean values for minimal

surface tension were also similar, but there was a directional change suggesting improved surface

tension properties in surfactant from E2-treated animals, with surfactant from 44% of the animals

achieving a normal minimal surface tension of less than 5 mN/m versus 25% in the control group

(Fig. E4C). SP-A, SP-B and SP-C content in the surfactant pellet were unaffected by E2

administration (Fig.E4D).

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Effect of E2 on Pulmonary NOS and ER

The impact of E2 on pulmonary NOS was first evaluated by determinations of total,

calcium-dependent and calcium-independent NOS enzymatic activity in samples of proximal

lung. Total NOS activity was greater in lungs from E2-treated animals versus controls, and this

was entirely due to greater calcium-dependent activity whereas calcium-independent activity was

unchanged (Fig. 7A). nNOS- and eNOS-derived activity were determined, and nNOS activity

was increased 2-fold with E2 and eNOS activity was increased by over 7-fold (Fig. 7B).

Whereas iNOS protein was not detected by immunoblot analysis in lungs from either study

group (data not shown), nNOS protein was detected and was similar in abundance in lungs from

control and E2-treated animals (Fig. 7C). In contrast, eNOS protein expression was increased by

more than 3-fold with E2 treatment (Fig. 7D), and eNOS mRNA abundance was also increased

by E2 (See Table E1 in the online data supplement).

Having previously shown that estrogen upregulation of eNOS expression is ER-

dependent(33), and that estrogen modifies ERα and ERβ abundance in primary fetal pulmonary

artery endothelial cells(34), the impact of E2 on lung estrogen receptor expression was assessed

by immunoblot analysis (See Figure E5 in the online data supplement). ERβ protein was

detected in 125d gestation control lung, and the level of expression did not change between 125d

and 140d gestation; ERα protein was not detected (data not shown). ERβ was also readily

detected in lungs from both control and E2-treated animals (Fig. E5) and ERα protein was not

(data not shown), and E2 administration did not modify ERβ abundance.

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DISCUSSION

With preterm birth early in the third trimester, there is an obligatory decline in circulating

E2 levels that otherwise would increase dramatically during the latter part of gestation in utero.

Since pulmonary NOS is deficient following preterm birth, since E2 upregulates NOS expression

and activity in diverse tissues and cell types, and since NO plays an important role in lung

development and perinatal lung function, we determined the impact of postnatal E2

administration on early postnatal pulmonary status in preterm baboons. We found that the

provision of E2 enhanced pulmonary function and caused a persistent decrease in ventilatory

support requirements. These benefits were associated with an upregulation of both nNOS- and

eNOS-derived NOS enzyme activity in the lung. Thus, postnatal E2 administration has a potent

positive impact on pulmonary status following preterm birth in the primate.

Along with the pulmonary studies, the effect of E2 on systemic hemodynamic status was

evaluated. Postnatal E2 administration caused a persistent elevation in mean systemic BP related

to increased diastolic BP. The E2-treated animals also required pressor support less frequently

than controls, and the disparities in BP between the two groups remained apparent despite the

differences in pressor support. Both endogenous and exogenous estrogens stimulate the hepatic

synthesis of angiotensin that raises aldosterone via activation of the renin-angiotensin system,

and aldosterone causes renal sodium resorption, and these mechanisms may underlie the

hypertension that can occur with oral contraceptive use(35, 36). However, these processes are

unlikely in the present study in which first-pass hepatic metabolism of E2 was avoided with

cutaneous delivery of the hormone(36), and daily weights and urine output were not affected by

E2 treatment. Preterm human infants are at significant risk of hypotension and this is mimicked

in the preterm baboon(37). Although the underlying mechanism is yet to be elucidated, the

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increase in systemic BP and the lowered requirements for pressor support observed with E2 in

the present study would be of considerable potential clinical benefit.

An additional cardiovascular response observed with postnatal E2 administration was an

increase in the rate of spontaneous closure of the ductus arteriosus. The increases in systemic BP

with E2 occurred considerably earlier than the ductal closure evaluated daily by

echocardiography, indicating that differences in ductal shunting between study groups are most

likely not the cause for the disparities in systemic BP. Although a genetic polymorphism of ERα

has been associated with a lower likelihood of patent ductus arteriosus in preterm male

infants(38), further in-depth investigation will be required to elucidate the mechanisms by which

E2 and ER influence ductal patency. The beneficial impact of E2 on ductal patency would

decrease the need for pharmacologic or surgical closure of the ductus and also the risk of the

multiple significant potential complications that can accompany these interventions(39).

The primary physiologic effects of E2 on the lung were to cause improvements in both

dynamic lung compliance and expiratory resistance in the early postnatal period. The degrees

and durations of these improvements surpass those obtained previously in the preterm baboon

model with interventions including the administration of a superoxide dismutase mimetic, a

modulator of prolyl hydrolase that impacts HIF-related processes, and inhaled NO gas(13, 40,

41). Although attempts to generate postmortem PV curves were complicated by air leaks in the

lungs of some animals, the available data provide further evidence of improved lung function

with postnatal E2 administration.

The improvements in pulmonary function caused by postnatal E2 administration likely

underlie the decline in ventilatory support requirements reflected by long-term diminutions in

both the oxygenation and ventilation indices. It is notable that this beneficial impact of postnatal

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E2 was apparent in the setting of maternal prenatal steroid treatment, which is the current clinical

strategy in routine use that best optimizes the pulmonary status of the preterm infant(42).

E2 had negligible impact on pulmonary morphology, specifically secondary crest/end

segment formation and vascularization. However, this may be due to the timing of the

assessment of these parameters at 2 weeks of life, which is early in the postnatal period. Later

impact on lung structure is possible because there were albeit modest but consistent E2-induced

changes in elastin distribution favoring localization to emerging septae at 2 weeks of age.

Studies in postnatal rats and mice indicate that E2 modulates alveolar formation and

regeneration(43, 44), and ER and progesterone receptor blockade during late gestation in piglets

causes impaired alveolar formation and fluid clearance(45). However, aromatase inhibition

during the latter half of baboon pregnancy that lowered umbilical venous E2 levels by 95% did

not alter fetal lung growth or alveolarization(46). Considering the improvements in pulmonary

function and support requirements that we observed in the first two weeks with E2 treatment, and

the benefits on lung structure that can be found after 28d in this model if ventilatory support is

lessened(11, 47), additional studies including long-term assessments of lung morphology are

now warranted to determine the ultimate impact of postnatal E2 administration on the developing

lung.

Lung inflammation was also evaluated to determine the potential basis for the observed

effects of E2 on lung function. TGF-β1 levels in tracheal aspirates and terminal BAL were

unchanged during the course of BPD development in the primate and were also unaffected by E2,

and there were no effects of E2 on the expression of genes regulating lung inflammation.

Surfactant-related parameters were also assessed in terminal BAL, and no changes were apparent

with E2. The latter findings are consistent with prior observations that aromatase inhibition

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during the latter half of baboon pregnancy did not alter lung SP-A or SP-B expression(46).

Thus, there were no observed changes in lung inflammation or surfactant status with postnatal E2

treatment.

Since E2 upregulates NOS expression and enzymatic activity in numerous tissues and cell

types(18-20), changes in lung NOS activity and expression that would be favorable to pulmonary

function were anticipated. Increases in calcium-dependent nNOS-derived enzymatic activity

were found in the lungs of E2-treated animals, and there were even greater increases in eNOS-

derived activity. In contrast, iNOS-derived, calcium-independent activity was unaltered. There

was no demonstrable change in nNOS protein expression with E2, and this may reflect the

greater sensitivity of the enzyme activity assay to discern alterations in enzyme abundance.

However, eNOS protein and mRNA expression were increased by E2 paralleling the rise in

eNOS-derived activity, and this mirrors the known capacity of the hormone to upregulate eNOS

gene expression in numerous model systems(20). Thus, the beneficial changes in lung function

induced by E2 were associated with upregulation of NOS enzyme activity, and the pleiotropic

functions of NO in pulmonary cells may be operative in this intervention. From a therapeutic

standpoint, the ability to upregulate endogenous pulmonary NOS may be more favorable that the

provision of exogenous NO, which benefits only certain subpopulations of preterm infants at risk

for BPD(14-17). Additional potential mechanism of action of E2, including the nongenomic

activation of eNOS that is independent in changes in enzyme abundance and other processes that

do not involve NO(20), should be queried in future experiments.

BPD occurs in over 20% of the more than 50,000 preterm infants born in the U.S. each

year with birthweights < 1500g(48), causing considerable morbidity and mortality, and

additional strategies are needed to combat the disorder. In the animal model that best

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exemplifies the human condition, we have found that postnatal transcutaneous E2 administration

following preterm birth caused persistent improvements in pulmonary function and a decrease in

ventilatory support requirements in association with lung NOS upregulation. In a recently-

reported small trial by Trotter and colleagues, intravenous E2 and progesterone treatment in

preterm infants tended to decrease the incidence of BPD. In addition, their work to date suggests

potential added benefits on bone mineralization, retinopathy of prematurity, and neurologic

outcome(49-51), lending further credence to the concept of postnatal estrogen treatment. In our

model there were also favorable impacts on hypotension and PDA that are also key

complications of prematurity. As such, estrogen-based therapies for BPD and other

complications of prematurity should now be further developed.

As future studies of postnatal E2 treatment for BPD are contemplated either in animal

models or in humans, full consideration must be given to the possible effects of estrogen on

nonpulmonary development including that related to reproductive health(52). Fortunately, in the

studies of E2 and progesterone replacement in preterm infants, Trotter and coworkers found that

changes in vaginal cytology and mammary and uterine growth were no greater than those that

would have occurred in utero, and they ceased when replacement was discontinued(53).

Furthermore, if necessary, systemic actions of estrogen can potentially be obviated by the use of

E2 in aerosolized form(54). Thus, after decades of consideration of estrogen treatment to prevent

diseases in the postmenopausal period, the hormone now has the potential to ameliorate a

devastating condition at the extreme opposite end of the age spectrum.

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ACKNOWLEDGMENTS

The authors are indebted to Dr. J. Coalson and V. Winter, without whom the studies

could not have been performed. The authors also thank all the personnel that support the BPD

Resource Center: the animal husbandry group led by Drs. D. Carey and M. Leland, the NICU

staff (H. Martin, S. Ali, D. Correll, L. Kalisky, L. Nicley, R. Degan, S. Gamez), the Wilford Hall

Medical Center neonatal fellows and D. Catland, NNP, who assist in the care of the animals, and

the UTHSCSA pathology staff (L. Buchanan, H. Dixon, A. Schreiner) who perform necropsies

and obtain biological specimens. The authors also thank M. Dixon for assistance in the

preparation of the manuscript.

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48. Fanaroff AA, Stoll BJ, Wright LL, Carlo WA, Ehrenkranz RA, Stark AR, Bauer CR,

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low birthweight infants. Am. J. Obstet. Gynecol. 2007;196:147-148.

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50. Trotter A, Bokelmann B, Sorgo W, Bechinger-Kornhuber D, Heinemann H, Schmucker G,

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51. Trotter A, Maier L, Kron M, Pohlandt F. Effect of oestradiol and progesterone replacement

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52. Jones LA, Hajek RA. Effects of estrogenic chemicals on development. Environ. Health

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53. Trotter A, Maier L, Kohn T, Bohm W, Pohlandt F. Growth of the uterus and mammary

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54. Studd J, Pornel B, Marton I, Bringer J, Varin C, Tsouderos Y, Christiansen C. Efficacy and

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response study. Aerodiol Study Group. Lancet 1999;353:1574-1578.

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FIGURE LEGENDS

Figure 1. A. Fetal E2 levels increase in the latter half of the third trimester of primate pregnancy.

Serum E2 was measured in fetal baboons at 125d, 140d, 160d, or 180d gestation upon

euthanization immediately at delivery. Values are mean±SEM, n=6/group. *P<0.05 vs 125d

gestation. B. Subcutaneous E2 administration raises serum levels in the immediate postnatal

period. The hormone was provided postnatally to preterm baboons delivered by cesarean section

at 125d gestation by placement of a 0.5 mg, 21d extended release pellet subcutaneously in the

left axilla at 1h of life. Control animals received a placebo pellet, and a second E2 or control

pellet was placed subcutaneously in the right axilla on day of life 7. Serum levels were

determined at 6h of life and at 1, 2, 3, 7, 10 and 14d of age. Values are mean±SEM, n=8 and 9

for control and E2-groups, respectively. *P<0.05 vs control.

Figure 2. Postnatal E2 administration increases systemic blood pressure and causes closure of the

ductus arteriosus. A. Systemic mean, systolic and diastolic blood pressures were measured via

an arterial catheter. B. Ductal patency was determined by echocardiography. Green indicates an

open ductus, red indicates a closed ductus, and white indicates that an echocardiogram was not

performed. Values are mean±SEM, n=8 and 9 for control and E2-groups, respectively.

Statistical comparisons of BP between groups were made by repeated measures ANOVA, and

ductal patency was compared by two-way ANOVA. P<0.05 for mean BP, diastolic BP and

ductal patency.

Figure 3. Postnatal E2 administration causes improvements in pulmonary function. Expiratory

resistance (A, cmH2O/ml/s) and compliance (B, ml/cm H2O/kg) were measured by whole body

plethysmography. Reported values are for the respiratory system as a whole. Values are

mean±SEM, n = 8 and 9 for control and E2 groups, respectively. Statistical comparisons were

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made between groups by two-way ANOVA. P<0.05 for expiratory resistance and for

compliance.

Figure 4. Postnatal E2 administration causes improvements in oxygenation index (A) and

ventilation index (B). Values are mean±SEM, n = 8 and 9 for control and E2 groups,

respectively. Statistical comparisons were made between groups by two-way ANOVA (p<0.05

for Panel A and for Panel B).

Figure 5. Representative lungs from 125d gestation (A), 140d gestation (B) and control- and E2-

treated animals (C and D, respectively). Magnification = 20X, bar = 100 um.

Figure 6. Impact of postnatal E2 administration on elastin distribution and expression. Hart’s

staining indicated that elastic fibers were localized to both alveolar walls and septal tips (arrows)

in lungs of control-treated animals (A) and primarily to emerging septal tips in E2-treated lungs

(B). In situ hybridization on serial sections similarly detected often intense elastin mRNA

expression within alveolar walls and at emerging septae in control-treated animals (C), and

expression was localized primarily to emerging septae in E2-treated animals (D). Findings in A-

D are representative of those in 4-5 animals/group, magnification = 200X, bar = 50 um. E.

Elastin mRNA expression was evaluated by quantitative RT-PCR. Values are mean±SEM,

n=6/group.

Figure 7. Postnatal E2 administration upregulates lung nNOS and eNOS enzymatic activity.

Using arginine-to-citrulline conversion, total, calcium-dependent and calcium-independent NOS

activity was measured in proximal lung (A). nNOS-derived and eNOS-derived enzymatic

activity was also quantified (B). nNOS (C) and eNOS (D) protein abundance was evaluate by

immunoblot analysis. In C and D, upper panels display representative immunoblots for NOS and

actin, and cumulative findings for NOS abundance relative to actin in lungs from 6 animals per

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group are shown in lower panels. Values are mean±SEM, *P<0.05 vs control.

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Table 1- Study Populations

Birthweight (g) 387±48 370±39

Gestation (d) 125±1 124±1

Gender (M/F) 7/1 6/3

Control Estradiol

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Table 2- Morphometric Analysis of Alveolar Structure

Lung volume (cm3)

Mean length of primary septa (um)Number of branch points (#/mm2)Surface density of primary septa (cm2/cm3)Total surface area of primary septa (cm2)

Mean length of end segments (um)Number of end segments (#/mm2)Surface density of end segments (cm2/cm3)Total surface area of end segments (cm2)

#end segment/mm internodal segmentLength ratio of internodal/end segment

Total alveolar surface area (cm2)

Control Estradiol

6.6±0.6

11.2±0.51232±115269±171736±127

6.7±0.2200±1726.3±1.8170±13

15.0±1.210.5±0.9

1905±132

7.5±0.7

13.3±0.6865±107222±211645±194

7.0±0.2198±1327.5±1.3206±18

18.3±1.18.0±0.5

1851±208

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0

100

200

300

400 *

125 140 160 180Postconceptual Age (d)

Estr

adio

l(pg

/ml) *

Fig. 1

0 2 4 6 8 10 12 140

500

1000

1500EstrogenPlacebo

Estr

adio

l (pg

/ml)

Postnatal Age (d)

**

** * *

ControlEstradiolControlEstradiol

A

B

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0 2 4 6 8 10 12 14

30

35

40

45

50

Mea

n B

P (m

m H

g)0 2 4 6 8 10 12 14

30

35

40

45

50

Mea

n B

P (m

m H

g)

Days0 2 4 6 8 10 12 14 16

Syst

olic

BP

(mm

Hg)

35.0

37.5

40.0

42.5

45.0

47.5

50.0

52.5

55.0

57.5

60.0

62.5

65.0

67.5

70.0

0 2 4 6 8 10 12 143540455055606570

Syst

olic

BP

(mm

Hg)

Days0 2 4 6 8 10 12 14 16

Syst

olic

BP

(mm

Hg)

35.0

37.5

40.0

42.5

45.0

47.5

50.0

52.5

55.0

57.5

60.0

62.5

65.0

67.5

70.0

0 2 4 6 8 10 12 143540455055606570

Syst

olic

BP

(mm

Hg)

0 2 4 6 8 10 12 14

20.022.525.027.530.032.535.037.5

Dia

stol

ic B

P (m

m H

g)

0 2 4 6 8 10 12 14

20.022.525.027.530.032.535.037.5

Dia

stol

ic B

P (m

m H

g)

Postnatal Age (d)

A

B


P<0.05

P<0.05

Fig. 2

Age (d)Control

E2

14131211109876543210 14131211109876543210

of 56

0 2 4 6 8 10 12 140

100

200

300

400

500

Exp.

Res

.

0 2 4 6 8 10 12 140

100

200

300

400

500

Exp.

Res

.

0 2 4 6 8 10 12 140.0

0.2

0.4

0.6

0.8

1.0

Com

plia

nce/

Kg

0 2 4 6 8 10 12 140.0

0.2

0.4

0.6

0.8

1.0

Com

plia

nce/

Kg

Fig. 3

A

BPostnatal Age (d)

Postnatal Age (d)


P<0.05

P<0.05

Com

plia

nce

(ml/c

m H

2O/k

g)Ex

p. R

es.

(cm

H2O

/ml/s

)

of 56

0 2 4 6 8 10 12 140

20

40

60

80

100

Vent

ilatio

n In

dex

0 2 4 6 8 10 12 140

20

40

60

80

100

Vent

ilatio

n In

dex

0 2 4 6 8 10 12 140

5

10

Oxg

enat

ion

Inde

x

0 2 4 6 8 10 12 140

5

10

Oxg

enat

ion

Inde

xA

Fig. 4

Postnatal Age (d)

Postnatal Age (d)

B


P<0.05

P<0.05

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A B

C D

Fig. 5

of 56

0

0.5

1.0

1.5

2.0

2.5

Control Estradiol

Elas

tinm

RN

A/G

APD

H

E

Fig. 6

of 56

0

20

40

60

80

100

120

140EstrogenControl

Total Calcium Calcium depenedent independent

*

NO

S A

ctiv

ity

(fm

ole/

mg

prot

/min

) *

0

20

40

60

80

100 EstrogenControl

nNOS eNOS

*

NO

S A

ctiv

ity

(fm

ole/

mg

prot

/min

)

*

0

5 0

1 0 0

1 5 0

2 0 0

Con E2

Rel

ativ

e A

bund

ance

nNOS

Actin - 43 kDA

Control Estradiol

- 155 kDA

0

5 0

1 0 0

1 5 0

2 0 0

Con E2

Rel

ativ

e A

bund

ance

nNOS

Actin - 43 kDA

Control Estradiol

- 155 kDA

Actin

eNOS - 135 kDa

- 43 kDa

Rel

ativ

e A

bund

ance

0

1 0 0

2 0 0

3 0 0*

Con E2

Control Estradiol

Actin

eNOS - 135 kDa

- 43 kDa

Rel

ativ

e A

bund

ance

0

1 0 0

2 0 0

3 0 0*

Con E2

Control Estradiol

Fig. 7

A B

C D

NO

S A

ctiv

ity(fm

ole/

mg

prot

/min

)

NO

S A

ctiv

ity(fm

ole/

mg

prot

/min

)

Total Calcium-dependent

Calcium-independent nNOS eNOS

C E2 C E2

C E2 C E2

200150100500

300200100

0

Rel

ativ

eA

bund

ance

Rel

ativ

eA

bund

ance

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Postnatal Estradiol Upregulates Lung

NO Synthases and Improves Lung Function in

Bronchopulmonary Dysplasia

Donald C. McCurnin, Richard A. Pierce, Brigham C. Willis, Ling Yi Chang,

Bradley A. Yoder, Ivan S. Yuhanna, Philip L. Ballard, Ronald I. Clyman, Nahid Waleh

William Maniscalco, James D. Crapo, Peter H. Grubb, and Philip W. Shaul

ONLINE DATA SUPPLEMENT

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ONLINE DATA SUPPLEMENT

METHODS

Animal Model

All animal studies were performed at the Southwest Foundation for Biomedical Research

Primate Center in San Antonio, TX. Pregnancies in baboons (Papio papio) were timed with

cycle dates, and fetal growth parameters were obtained from prenatal ultrasound examinations

performed at 70 and 100d estimated fetal gestation. Mirroring current clinical practice in

humans, all animals received antenatal steroids. Initially 12 mg of betamethasone was given IM

every 24h beginning 48h prior to delivery. However, due to a world-wide shortage of

betamethasone which restricted its use to humans only, dexamethasone (6 mg IM every 12h

beginning 48h prior to delivery) was substituted until betamethasone again became available.

Following birth at 125±2 d gestation (term = 185d) by cesarean section and initial

stabilization, ventilation was provided for 14d with a humidified, pressure-limited, time-cycled

infant ventilator (InfantStar, Infrasonics, San Diego, CA) or a 3100A high frequency oscillatory

ventilator (kindly provided by Sensormedics, Yorba Linda, CA) when necessary. Pulse

oximeters were kindly provided by Masimo Corporation (Irving, CA). Details of animal care

have been published elsewhere(1, 2).

Ventilatory Management

The ventilatory approach entailed a strategy to maintain tidal volumes at 4 - 6 ml/kg as

determined by a VitalTrends body plethysmograph system (VT1000, Vitaltrends Technology,

New York, NY) and to generate adequate chest motion by clinical examination. There was

1

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rigorous targeting of arterial blood gas parameters to PaCO2 values ranging from 45 to 55 torr

and PaO2 levels between 55 and 70 torr. In an attempt to minimize exposure to high FiO2, if the

PaO2 level was above target goals, FiO2 was weaned until < 0.40 and then modifiers of mean

airway pressure or FiO2 were decreased as tolerated. If PaO2 was below target guidelines, a

chest radiograph was obtained to evaluate lung inflation. Adjustments in mean airway pressure

were made to minimize underinflation or overinflation of the lung. If lung inflation was deemed

adequate, FiO2 alone was adjusted.

Levels of ventilatory and oxygen support were assessed by determinations of

oxygenation index and ventilation index. The formula utilized for oxygenation index (OI) was:

OI = mean airway pressure (cm/H2O) X FiO2 X 100/ PaO2

The formula for ventilation index (VI) was:

VI = peak inspiratory pressure X ventilator rate X PaCO2/ 1000

Hemodynamic Support

Significant hypotension was defined as a transduced mean blood pressure < 28 mm Hg

accompanied by either increasing base deficit or decreasing urine output. Hypotension was

initially treated with additional volume supplementation (20 ml/kg over 1h) and the use of

dopamine (5-20 ug/kg/min). Dobutamine was added (4-10 ug/kg/min) if mean pressure was not

restored to > 28 mm Hg. In those animals where dobutamine appeared to exacerbate

hypotension, epinephrine (0.2-1.0 ug/kg/min) was used in lieu of or as an additional inotrope. If

this approach failed to improve mean blood pressure within 2 h, then a stress dose of

hydrocortisone (1.0 mg/kg) was administered at 6 h intervals until either mean blood pressure

increased to > 28 mm Hg or a maximum of 4 doses of hydrocortisone were received. Once mean

2

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blood pressure was stable for > 12 h, pressor support was weaned in reverse to the order it was

initiated.

Echocardiography

2-D, m-mode and Doppler echo studies were performed with a ND 256-8 Biosound

echocardiographic system (Genoa, Italy) equipped with an 8 MHz mechanical transducer.

Postmortem Pressure-Volume Measurements

Postmortem quasi-static inflation pressure-volume measurements were carried out by

inflating the lungs in a stepwise manner (5 cm H2O increments) to a pressure of 20 cm H2O. At

each increment, the pressure was held for 30 sec and volume was recorded. The lungs were then

inflated to 35 cm H2O for 1 min and maximal lung volume was recorded. A deflation limb

pressure-volume curve was also generated by reducing pressure in steps of 5 cm H2O, with

stabilization at each step, and recording of the corresponding volumes(2).

Morphometric-histopathologic Analyses

To quantifiably assess alveolarization, digital image analysis of the lung parenchyma was

performed using the methods previously reported(2). The number and length of primary septal

segments and of secondary crests that develop in alveoli and are quantified as end segments were

tallied and analyzed. The following specific parameters were also evaluated: number of branch

points, numerical frequency of primary septal segments and end segments per mm2 of alveolar

area, total surface area of primary septal segments and end segments, the number of end

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segments per mm of internodal segment, the length ratio of internodal segment to end segment,

and total alveolar surface area.

To evaluate capillary density, lung sections were immunostained for PECAM-1 (platelet

endothelial cell adhesion molecule, CD31, Dako Corporation, Via Real, CA), an endothelial cell

marker, and PECAM immunoblot analysis was also performed. VEGF mRNA was also

quantified by real-time quantitative PCR and riboprotection assay, and its localization was

assessed by in situ hybridization(3, 4).

To evaluate elastin deposition, sections of paraformaldehyde-fixed, paraffin-embedded

lung were deparaffinized and hydrated, and stained by Hart’s method as previously described.

Counterstaining was with tartrazine (yellow)(5). Elastin mRNA distribution was assessed by in

situ hybridization, and abundance was evaluated by quantitative RT-PCR (5).

Analyses of Lung Inflammation

TGF-β1 abundance in tracheal aspirates and terminal BAL samples obtained at postnatal

ages of 1, 3, 6-7, 9-11 and 14 d was quantified by ELISA (R&D System, Minneapolis, MN) and

was normalized to total protein content measured using the NanoDrop 1000 Protein A280

Application (Thermo Scientific, Wilmington, DE). mRNA expression of genes involved in

inflammation and remodeling in the lung, as well as vascular behavior, was evaluated at 14d by

quantitative RT-PCR as previously described(6). Total RNA was isolated from lung samples

and the TaqMan Universal PCR master mix of PE Applied Biosystems (Foster City, CA) was

used to quantify gene expression. Taqman probes were designed using the Primer Express

program and labeled with fluorophores FAM (6-caboxy-fluorescein) and TAMRA (6 carboxy-

tetramethyl-rhodamine) as reporter and quencher dyes, respectively. An ABI PRISM 7700

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Sequence detection system was used to determine the cycle threshold (CT). Reactions were

carried out in triplicates, and data were analyzed using the Sequence Detector version 1.6.3

program. Malate dehydrogenase (MDH) was used as an internal control to normalize the data.

We previously compared transcript abundance in lungs from BPD animals at 14d postnatal age

and 140d gestation fetuses of equal postconceptual age, and found that there were significant

differences in RNA expression for COX2, TGF β3, TNFα and VEGF, but not for several other

genes involved with lung inflammation, remodeling and vascular development and function

(e.g., CD14, CD154, COX1, E-selectin, ET-1, IL-1. IL-6, IL-8, interferon gamma, MMP-2, -3,

or -9, TGFβ1, VCAM-1). We therefore analyzed COX2, TGF β3, TNFα and VEGF mRNA

expression in lungs from140d gestation fetuses and control and E2 treatment groups. In addition,

since eNOS protein abundance was altered by E2 treatment in the current study, eNOS mRNA

abundance was also evaluated.

Pulmonary Surfactant Analysis

Analyses were performed as previously described(7). Protein and PL content were

determined and the remaining pellet was diluted to 1.5 mg PL/mL for pulsating bubble

surfactometery, with minimal surface tension (STmin, <5 mN/m defined as normal) of primary

interest. Due to the limited amount of available BAL, a single determination of surfactant

function was made for each animal; the coefficient of variation for 10 replicate determinations of

STmin in a pooled sample was 6%. SP-A, SP-B, and SP-C content were analyzed by Western

immunoblotting and quantitative immunodot assays.

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NOS Enzymatic Activity and Expression

NOS enzymatic activity was measured in proximal lung samples from control and E2-

treated animals. Prior studies demonstrated that the majority of NOS expression and activity in

fetal baboon lung resides in the proximal respiratory epithelium(8). Activity derived from

calcium-dependent nNOS and eNOS versus calcium-independent inducible NOS (iNOS) was

distinguished. In addition, nNOS- and eNOS-related enzymatic activity was delineated by

measurement of calcium-dependent activity in the absence versus presence of the nNOS-specific

antagonist S-methyl-L-thiocitrulline (SMTC, 10 8M). Immunoblot analyses were also

performed to evaluate NOS isoform abundance, using alpha actin as the loading control(9).

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REFERENCES

E1. Coalson JJ, Winter VT, Siler-Khodr T, Yoder BA. Neonatal chronic lung disease in

extremely immature baboons. Am. J Respir. Crit Care Med. 1999;160:1333-1346.

E2. McCurnin DC, Pierce RA, Chang LY, Gibson LL, Osborne-Lawrence S, Yoder BA,

Kerecman JD, Albertine KH, Winter VT, Coalson JJ, Crapo JD, Grubb PH, Shaul PW.

Inhaled NO improves early pulmonary function and modifies lung growth and elastin

deposition in a baboon model of neonatal chronic lung disease. Am J Physiol Lung Cell

Mol. Physiol 2005;288:L450-L459.

E3. Maniscalco WM, Watkins RH, Roper JM, Staversky R, O'Reilly MA. Hyperoxic ventilated

premature baboons have increased p53, oxidant DNA damage and decreased VEGF

expression. Pediatr. Res. 2005;58:549-556.

E4. Pierce RA, Joyce B, Officer S, Heintz C, Moore C, McCurnin D, Johnston C, Maniscalco

W. Retinoids increase lung elastin expression but fail to alter morphology or angiogenesis

genes in premature ventilated baboons. Pediatr. Res. 2007;61:703-709.

E5. Pierce RA, Albertine KH, Starcher BC, Bohnsack JF, Carlton DP, Bland RD. Chronic lung

injury in preterm lambs: disordered pulmonary elastin deposition. Am. J. Physiol

1997;272:L452-L460.

E6. McCurnin D, Seidner S, Chang LY, Waleh N, Ikegami M, Petershack J, Yoder B,

Giavedoni L, Albertine KH, Dahl MJ, Wang ZM, Clyman RI. Ibuprofen-induced patent

ductus arteriosus closure: physiologic, histologic, and biochemical effects on the premature

lung. Pediatrics 2008;121:945-956.

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E7. Ballard PL, Gonzales LW, Godinez RI, Godinez MH, Savani RC, McCurnin DC, Gibson

LL, Yoder BA, Kerecman JD, Grubb PH, Shaul PW. Surfactant composition and function

in a primate model of infant chronic lung disease: effects of inhaled nitric oxide. Pediatr.

Res 2006;59:157-162.

E8. Shaul PW, Afshar S, Gibson LL, Sherman TS, Kerecman JD, Grubb PH, Yoder BA,

McCurnin DC. Developmental changes in nitric oxide synthase isoform expression and

nitric oxide production in fetal baboon lung. Am. J. Physiol Lung Cell Mol. Physiol

2002;283:L1192-L1199.

E9. Afshar S, Gibson LL, Yuhanna IS, Sherman TS, Kerecman JD, Grubb PH, Yoder BA,

McCurnin DC, Shaul PW. Pulmonary NO synthase expression is attenuated in a fetal

baboon model of chronic lung disease. Am. J. Physiol Lung Cell Mol. Physiol

2003;284:L749-L758.

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Supplemental Table E1: Real Time PCR measurements of genes involved in inflammation,

lung remodeling and vascular behavior. ΔCT(MDH-gene) = difference in cycle threshold (CT)

between the expression of the housekeeping gene Malate dehydrogenase (MDH) and the gene of

interest. Each unit of ΔCT(MDH-gene) represents a 2-fold increase in a gene’s mRNA

abundance. The more negative the ΔCT(MDH-gene), the fewer the number of starting copies of

a gene (mRNA). n= number of separate animals used. Values are mean±SEM, n=11, 8 and 7 for

140d gestatation fetus, control and estradiol groups. * p<0.05 versus 140d gestation fetus. #

p<0.05 versus control group.

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SUPPLEMENTAL FIGURE LEGENDS

Figure E1: Postnatal E2 administration does not modify pulmonary artery pressure (PAP) or the

ratio of pulmonary to systemic blood flow (Qp/Qs). A. PAP was estimated noninvasively by

measuring the peak velocity of the patent ductus arteriosus jet, and the ratio of PAP to systemic

arterial pressure (SAP) was calculated. B. Left and right ventricular output was calculated from

the mean velocity and used to determine Qp/Qs.

Figure E2: Impact of postnatal E2 administration on postmortem pressure-volume

measurements. Quasi-static inspiratory pressure-volume measurements were performed up to 20

cm H2O, lung volume at maximal distending pressure of 35 cm H2O was recorded, and

expiratory curves were also generated. Values are mean±SEM, n = 5 for control and E2 groups.

Separate two-way ANOVA analysis of the inflation and deflation limbs yielded P = 0.10 and

0.21, respectively. Comparison of the lung volumes at 35 cm H2O yielded P=0.07.

Figure E3: Bronchoalveolar lavage TGFβ1 levels are not altered during the development of

BPD in the primate model, and they are not affected by E2 treatment. Values normalized to total

protein content are shown as mean±SEM, and n=8 and 9 for control (open bars) and E2 groups

(closed bars), respectively.

Figure E4: Postnatal E2 administration does not modify pulmonary surfactant. A large

aggregate surfactant pellet was prepared from terminal BAL, protein (A) and PL content (B)

were quantified, and the remaining pellet underwent pulsating bubble surfactometery to

determine minimal surface tension (C). Normal minimal surface tension, defined as <5 mN/m,

was observed in 25% and 44% of control and E2 treated animals, respectively. SP-A, SP-B, and

SP-C content were also analyzed (D). Values are mean±SEM, n=8 and 9 for control and E2

groups, respectively.

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Figure E5: Postnatal E2 administration does not modify lung ER expression. Immunoblot

analyses were performed for ERα and ERβ on lungs from 125d gestation and 140d gestation

control lung and from lungs from both control- and E2-treated animals. Equal protein loading

was confirmed by immunoblotting for GAPDH. ERβ protein was detected in all samples, and its

abundance did not change with fetal development between 125 and 140d gestation or with E2

treatment. ERα protein was not detected in any groups. Results shown are representative of 4

independent experiments.

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Supplemental Table E1- Genes Involved in Inflammation,Lung Remodeling and Vascular Behavior

∆CT(MDH-gene)140d Gest. Fetus Control Estradiol

-7.06±0.13* -5.07±0.19* -3.88±0.11* -9.11±0.27* +0.04±0.13*

-6.58±0.12*# -5.23±0.21* -3.70±0.13* -8.77±0.34* +0.11±0.13*

eNOS COX-2 TGFβ3 TNFα VEGF

-5.45±0.11 -6.90±0.13 -2.81±0.10 -10.51±0.20 +1.60±0.11

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postnatal estradiol up-regulates lung nitric oxide synthases and improves lung function in...

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