Departments of 1 Pediatrics and 2 Molecular Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611-3008
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ABSTRACT |
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Recent studies implicate reactive oxygen species (ROS) such as superoxide anions and H2O2 in the proliferation of systemic vascular smooth muscle cells (SMCs). However, the role of ROS in SMC proliferation within the pulmonary circulation remains unclear. We investigated the effects of endothelin-1 (ET-1), a potential SMC mitogen, on ROS production and proliferation of fetal pulmonary artery SMCs (FPASMCs). Exposure to ET-1 resulted in increases in superoxide production and viable FPASMCs after 72 h. These increases were prevented by pretreatment with PD-156707. Treatment with pertussis toxin blocked the effects of ET-1, whereas cholera toxin stimulated superoxide production and increased viable cell numbers even in the absence of ET-1. Wortmannin, LY-294002, diphenyleneiodonium (DPI), 4-(2-aminoethyl)benzenesulfonyl fluoride, and apocynin also prevented the ET-1-mediated increases in superoxide production and viable cell numbers. Exposure to H2O2 or diethyldithiocarbamate increased viable cell number by 37% and 50%, respectively. Conversely, ascorbic acid and DPI decreased viable cell number, which appeared to be due to an increase in programmed cell death. Our data suggest that ET-1 exerts a mitogenic effect on FPASMCs via an increase in ROS production and that antioxidants can block this effect via induction of apoptosis. Antioxidant treatment may therefore represent a potential therapy for pulmonary vascular diseases.
endothelin-1; free radicals; mitogenesis; hypertension; apoptosis
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INTRODUCTION |
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IN PERSISTENT PULMONARY HYPERTENSION of the newborn (PPHN), pulmonary vascular resistance does not decrease normally at birth, which results in pulmonary hypertension, right-to-left shunting, and hypoxemia (35). Newborns who die of PPHN exhibit an increase in both the thickness of the smooth muscle layer within small pulmonary arteries and an extension of this muscle to nonmuscular arteries (13). Often, microvascular thrombi occlude these arteries, and there is also proliferation of adventitial tissue (24). These structural changes indicate that in utero events have altered the pulmonary circulation.
We and others have developed an animal model of PPHN whereby prolonged compression or ligation of the ductus arteriosus in utero in the lamb produces fetal and neonatal pulmonary hypertension (1, 3, 26, 41). Similar to newborns who die of PPHN, these lambs have an increase in the thickness of smooth muscle within the small pulmonary arteries, complete muscularization of normally partially muscularized pulmonary arteries, and extension of muscle to nonmuscularized arteries. One of our previous studies demonstrated that similar to what is observed in newborns who die of PPHN (32), endothelin-1 (ET-1) gene expression is increased in this ductal ligation model of fetal pulmonary hypertension (3).
ET-1 is a 21-amino acid polypeptide that is produced by vascular endothelial cells with potent vasoactive properties that have been implicated in the pathophysiology of pulmonary hypertensive disorders (42). It has been suggested that ET-1 plays an important role in the vascular smooth muscle cell (SMC) proliferation that is seen in animal models of pulmonary hypertension (26). Although its precise mode of action remains controversial, ETA receptor blockade has been shown to prevent the vascular remodeling normally associated with ductal ligation (17). However, the role of ET-1 in the systemic circulation is less clear; some experiments carried out in SMCs from adult aortic vessels have found potent ET-1-mediated mitogenic effects (14, 19), whereas others have found none (18, 34).
Reports have indicated that reactive oxygen species (ROS) such as superoxide anions and hydrogen peroxide (H2O2) are capable of stimulating vascular SMC proliferation (30, 37). These ROS appear to be rapidly produced by SMCs after exposure to growth factors that are known to cause SMC proliferation (9, 37). In contrast, other experiments have shown that treatment with antioxidants (40) or overexpression of catalase (4) reduced viability and induced apoptosis in vascular SMCs. Overall, these findings suggest that ROS modulate both proliferation and survival of vascular SMCs. However, these studies have thus far been restricted to cells from systemic systems, and, therefore, the proliferative roles of ET-1 and ROS remain to be established in the pulmonary system. This is of particular importance in understanding the causes of PPHN to enable the development of new and improved prevention and treatment strategies. In this study, we determined whether ET-1 is, in fact, mitogenic with respect to fetal pulmonary arterial SMCs (FPASMCs) and whether this proliferative effect occurs via a signaling pathway that leads to an increase in the generation of ROS.
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MATERIALS AND METHODS |
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Cell culture. Primary cultures of FPASMCs from sheep were isolated by the explant technique. Briefly, a segment of the main pulmonary artery from 136-day-old fetal lambs was excised and placed in a sterile 10-cm dish containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 1 g/l glucose. The segment was stripped of adventitia with a sterile forceps. The main pulmonary artery segment was then cut longitudinally to open the vessel, and the endothelial layer was removed by gentle rubbing with a cell scraper. The vessel was then cut into 2-mm segments, inverted, and placed on a collagen-coated 35-mm tissue culture dish. A drop of DMEM containing 10% fetal bovine serum (FBS; HyClone), antibiotics (MediaTech), and antimycotics (Mediatech) was then added, and the cells were grown overnight at 37°C in a humidified atmosphere with 5% CO2-95% air. The next day, an additional 2 ml of complete medium were added. The growth medium was subsequently changed every 2 days. When SMC islands could be observed under the microscope, the tissue segment was removed and the individual cell islands were subcloned. Identity was confirmed as FPASMCs by immunostaining (>99% positive) with SMC actin. This was taken as evidence that cultures were not contaminated with fibroblasts or endothelial cells. All cultures for subsequent experiments were maintained in DMEM supplemented with 10% FBS, antibiotics, and antimycotics at 37°C in a humidified atmosphere with 5% CO2-95% air.
Cell proliferation assays. FPASMCs were seeded onto 96-well plates (Costar) at 25% confluence and allowed to adhere for at least 18 h. Cells were washed with PBS and incubated with serum-free medium containing (where required) ET-1 (1 µM; Sigma) and the ETA receptor agonist PD-156707 (1 µM); these concentrations were found to exert maximal effects in previous studies. Pertussis toxin (PTX; 100 ng/ml; Calbiochem) (38), cholera toxin (CTX; 6 nM; Calbiochem) (38), wortmannin (100 nM; Sigma) (27), LY-294002 (50 µM; Calbiochem) (6), H2O2 (100 µM; Fisher), diethyldithiocarbamate (DETC; 1 mM; Sigma) (7), diphenyleneiodonium (DPI; 0-10 µM; Sigma) (36), apocynin (0-1 µM; Acros Organics) (15), 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF; 0-100 µM; Sigma) (8), and ascorbic acid (1 mM; Sigma) (39) were added where required. Where dose responses were not carried out, pharmacological agents were used at concentrations found to be effective as reported in the previous studies that are referenced. After 72 h, the number of viable cells was determined using the CellTiter 96 AQueous One Solution kit (Promega). This reagent is bioreduced to a colored product, the quantity of which is proportional to the number of metabolically active cells. Reagent (20 µl) was added directly to cells in 100 µl of medium, and after a 4-h incubation period at 37°C, the absorbance at 492 nm was read using a Labsystems Multiskan EX plate reader (Fisher).
Fluorescence analysis. Cells were seeded onto 96-well plates and allowed to adhere for at least 18 h. Cells were then washed in PBS and incubated in serum-free DMEM with ET-1 and inhibitors added as described. At the appropriate time, dihydroethidium (DHE; 20 µM; Molecular Probes) or dichlorofluorescein diacetate (DCF-DA; 20 µM) was added to the medium, and the incubation was continued for an additional 15 min. Cells were washed with PBS and imaged using a Nikon Eclipse TE-300 fluorescence microscope. DHE-stained cells were observed after excitation at 518 nm and emission at 605 nm. DCF-DA-stained cells were observed using excitation at 485 nm and emission at 530 nm. Fluorescence images were captured with a Photometrix digital camera, and the average fluorescence intensities (to correct for differences in cell number) were quantified using Metamorph imaging software (Fryer). Statistical analyses between treatments were carried out as detailed (see Statistical analysis).
Terminal deoxynucleotidyltransferase dUTP nick end labeling analysis. Terminal deoxynucleotidyltransferase (TdT) dUTP nick end labeling analysis (TUNEL) (20) was performed on ovine FPASMCs using a kit obtained from Promega. Briefly, cells were grown on eight-well Permanox slides in DMEM (plus ET-1, H2O2, DETC, DPI, and ascorbic acid where required) for 3 days, washed in sterile PBS, and fixed in 4% (vol/vol) paraformaldehyde for 25 min at room temperature. Slides were washed twice in PBS and then incubated with TdT and reaction mix including fluorescein-12-dUTP for 1 h at 37°C. Slides were washed for 30 min in 2× saline-sodium citrate buffer and then incubated in PBS + 4',6-diamidino-2-phenylinodole (DAPI; 5 µM) for 15 min at room temperature. DAPI is a blue fluorescent nuclear stain, and this step ensured that approximately equal numbers of cells were imaged in each slide. Coverslips were mounted in ProLong antifade mounting medium (Molecular Probes), and then the slides were imaged by indirect immunofluorescence.
Caspase activation analysis. Cells were seeded onto 96-well plates, treated with ET-1, H2O2, DETC, and ascorbic acid as described, and incubated for 6 h at 37°C. Caspase activation was visualized by cotreating cells with 1 µM CaspACE FITC-VAD-FMK in situ marker (Promega). This is a fluorescent analog of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD-FMK), which readily enters cells and binds irreversibly to activated caspases. Fluorescent cells were observed using excitation at 485 nm and emission at 530 nm.
The activity of caspase-3 was determined using the CaspACE assay system (Promega). This uses a colorimetric substrate that is cleaved by caspase-3 to yield a yellow color that is proportional to the enzymatic activity in the sample. After 6 h, cells were lysed directly on 96-well plates and incubated with 200 µM substrate. After an additional 4-h incubation at 37°C, the absorbance at 405 nm was determined using a Labsystems Multiskan EX plate reader, and statistical analyses were performed.Statistical analysis. The relative average fluorescence intensities were calculated for DHE, DCF-DA, and FITC-VAD-FMK and are expressed as means ± SD. The relative changes in cell number and caspase-3 activity were calculated for the treatment groups and are expressed as means ± SD. Comparisons between treatment groups were made by the unpaired t-test using the GB-STAT software program. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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In cell proliferation assays, we initially demonstrated that ET-1
stimulated a 26% increase in the number of viable FPASMCs compared
with an 87% increase observed with 10% serum over a 72-h period (Fig.
1). Because the ETA receptor
is predominant on SMCs, cells were incubated with the ETA
receptor-specific inhibitor PD-156707. The ET-1-induced increase in
viable cell number was prevented by coexposure to PD-156707 (Fig. 1).
Although PD-156707 alone had no effect on the number of viable FPASMCs
cultured in serum-free medium, in the presence of 10% serum, the
number of viable cells was reduced by 14% (Fig. 1). This suggests that
at least part of the stimulatory effect on FPASMCs is via the presence of ET-1 in serum. Neither basic fibroblast growth factor (FGF; 10 ng/ml), platelet-derived growth factor (PDGF; 1 ng/ml), nor transforming growth factor- (TGF-
; 1 ng/ml) was able to increase the viable FPASMC number alone and in combination with ET-1; no comitogenic effects were observed (data not shown).
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Next, we determined whether ET-1 was stimulating ROS production in
FPASMCs. ET-1-mediated increases in superoxide production by FPASMCs
were determined using DHE (which is a superoxide-sensitive fluorescent
dye) and fluorescence microscopy. Preliminary studies indicated that
ET-1-induced increases in DHE fluorescence were prevented in cells
pretreated with polyethylene glycol-superoxide dismutase (PEG-SOD),
which suggests that DHE was specifically detecting superoxide in these
cells. Results obtained demonstrated that exposure of FPASMCs to ET-1
for 72 h resulted in a time-dependent increase in superoxide
production up to a maximum of 7.8-fold at 72 h compared with
control cells (Fig. 2A). This
increase was blocked in cells pretreated with PD-156707 (Fig. 2,
B and C). Incubation of cells with PD-156707
alone had no effect on the superoxide production in FPASMCs (Fig. 2,
B and C).
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Utilizing pharmacological agents, we next delineated the signal
transduction pathway linking ET-1, superoxide generation, and FPASMC
proliferation. The results obtained revealed that both the ET-1-induced
increases in viable FPASMCs and superoxide production were blocked by
PTX, a Gi protein inhibitor (Fig.
3). Increases in both viable cell number
and superoxide production could be stimulated by CTX, an activator of
the -subunit of G proteins, in the absence of ET-1; however, these
CTX-mediated increases were not enhanced in the presence of ET-1 (Fig.
3, A and B). Also, we found that the
phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and
LY-294002 reduced both viable FPASMC numbers and superoxide production,
although there were differences between the two agents (Fig. 3,
A and B). Differences in the extent of inhibition
may be expected because these agents inhibit PI3K via different
mechanisms (6, 27). Finally, we found that coexposure of
FPASMCs to the NADPH oxidase inhibitors AEBSF, apocynin, and DPI dose
dependently prevented the ET-1-mediated increase in proliferation, with
viable cell numbers being decreased relative to control at higher
inhibitor concentrations (Fig.
4A). When we examined DHE fluorescence, we demonstrated that at low inhibitor concentrations, ET-1-induced superoxide production was blocked, but surprisingly, at
higher inhibitor concentrations, DHE fluorescence was markedly increased independent of ET-1 exposure (Fig. 4B). Increases
in DHE fluorescence have been demonstrated in cells undergoing
apoptosis (22). To investigate the possibility
that NADPH oxidase inhibition could induce programmed cell death, we
performed TUNEL. The results obtained demonstrated that apoptotic
nuclei were found in cells exposed to the NADPH oxidase inhibitor DPI
(10 µM) but not in control cells, which indicates that NADPH oxidase
inhibition can induce SMC apoptosis (Fig. 4C).
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To more clearly define the link between ROS generation and FPASMC
proliferation, we exposed cells to both pro- and antioxidants. The
results obtained indicated that viable FPASMCs were increased by 37%
and 50%, respectively, after a 72-h exposure to
H2O2 and the Cu/Zn SOD inhibitor DETC.
Conversely, a 72-h exposure to ascorbic acid (vitamin C) decreased
FPASMC numbers by 30% (Fig.
5A).
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We next verified that the changes in proliferation were associated with changes in DHE fluorescence (Fig. 5B) or DCF-DA (Fig. 5C) as a measure of superoxide and H2O2 levels, respectively. We found a time-dependent increase in superoxide production in cells in which Cu/Zn SOD was inhibited with DETC at times up to 24 h. This was then followed by a decrease back to baseline at 48 h. H2O2 levels peaked at 30 min and returned to baseline by 4 h (Fig. 5C). Exposure to vitamin C resulted in an initial decrease in fluorescence at 30 min and a progressive increase at later time points (Fig. 5B).
To investigate whether the vitamin C-induced decrease in cell viability
was caused by an induction of apoptosis, we again performed
TUNEL analysis. The results obtained indicated that apoptotic
nuclei were detected in cells exposed vitamin C but not in control
cells or those exposed to the prooxidants DETC, H2O2, or ET-1 (Fig.
6A). We
then measured caspase activation to confirm the apoptotic effect of
vitamin C on FPASMCs. We found that vitamin C treatment increased the
fluorescence of the FITC-VAD-FMK peptide, which indicates an increase
in binding to activated caspases (Fig. 6, B and
C) and increased caspase-3 activity (Fig. 6D). Caspase-3 activity was not increased in control cells or those exposed
to the prooxidants DETC, H2O2, or ET-1 (Fig.
6D).
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DISCUSSION |
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It is clear that the proliferation of vascular SMCs contributes to the pathophysiology of both pulmonary and systemic hypertension, atherosclerosis, coronary artery restenosis after angioplasty, and stent placement (33). In addition, the production of ROS in the vessel wall is increased in models associated with vascular remodeling such as hypercholesterolemia, hypertension, diabetes, and balloon injury to the coronary arteries (11, 21, 28). Thus a better understanding of the mechanisms involved may lead to additional treatments for such diseases. Studies using animal models or human tissue suggest a role for ET-1 in the vascular structural changes that occur in proliferative cardiovascular disease. However, conflicting in vitro data have masked the exact contribution of ET-1 to the vascular remodeling. The role of ROS in SMC proliferation is also controversial. Moreover, previous studies have been carried out using systemic SMCs and may have limited relevance to the events that lead to the pulmonary vascular remodeling associated with PPHN. Here we determined the effects of ET-1 and ROS on proliferation of ovine FPASMCs to examine the roles that these factors may play in the vascular remodeling observed in infants with PPHN.
Patients with PPHN display elevated levels of ET-1
(5). Furthermore, increased levels of ET-1 mRNA and
decreased levels of ETB receptor mRNA have been observed in
an ovine model of PPHN (3). Studies with human aortic SMCs
showed that ET-1 was unable to stimulate proliferation alone, although
there was a marked growth-potentiating effect of ET-1 in conjunction
with PDGF, which was mainly via ETA-receptor activation
(43). Our results indicate that in ovine FPASMCs, ET-1 can
stimulate proliferation via ETA-receptor activation, and
this mitogenic effect occurs even in the absence of additional growth
factors. It has been suggested that ET-1 can potentiate the stimulatory
effects of other growth factors (12, 43). This could
further contribute to the increased muscularization seen in PPHN
(12, 43). However, in our experiments, basic FGF, PDGF,
and TGF- were unable to increase viable FPASMC numbers alone and had
no comitogenic effects in combination with ET-1. This can be explained
in two ways: either FPASMCs do not have the receptors for these growth
factors or these factors are not mitogens for these particular cells.
Furthermore, studies will be required to distinguish between these
possibilities. It has been postulated that inconsistent results
regarding ET-1-induced SMC proliferation may arise due to different
culture conditions [particularly regarding the components of the
culture medium such as growth factors in serum (18, 43)].
The data presented here suggest that the mitogenic effects of ET-1 may
also depend on the species, age, and location of the source vessels.
Our discovery that superoxide production by FPASMCs is increased in response to ET-1 treatment identifies ROS as a potential link between the elevated levels of ET-1 and the increased SMC proliferation seen in PPHN. These results are supported by studies where the ETA receptor blockade prevented the vascular remodeling normally associated with ductal ligation (17). In particular, a lack of SMC hypertrophy was observed in the ligated lambs exposed to the ETA receptor antagonist (17). Previous studies have also demonstrated that SMCs prepared from the systemic circulation can respond to exogenous growth factor stimulation by increasing intracellular production of ROS. For example, PDGF stimulates the production of H2O2 in vascular SMCs and leads to SMC growth (37). Conversely, if the PDGF-stimulated rise in H2O2 is prevented, the proliferative response to PDGF is blunted (37). Furthermore, thrombin stimulates both superoxide and H2O2 production in SMCs, and treatment with SOD or catalase to reduce the levels of ROS inhibits thrombin-induced proliferation (29). Taken together, these studies strongly suggest that both superoxide and H2O2 can mediate the proliferative phenotype in systemic SMCs. To confirm the importance of ROS on FPASMC proliferation, we increased the levels of ROS independently of ET-1 and showed that the direct addition of exogenous H2O2 or treatment with the Cu/Zn SOD inhibitor DETC induced cell proliferation. Previous reports suggest that the addition of exogenous H2O2 or pharmacological agents that can increase ROS generation appear to stimulate the activation of mitogen-activating protein kinases and stimulate adult systemic SMC growth (2, 30, 31, 37). Whether the same proliferative pathways are stimulated by H2O2 and superoxide remains to be elucidated. Because DETC-treated cells have elevated levels of superoxide at the expense of H2O2 production, it is possible that superoxide has a more potent effect on FPASMC growth, particularly when intracellular concentrations are increased above a required threshold. DETC treatment elevated superoxide levels until at least 24 h, whereas the H2O2 was removed within 4 h. Furthermore, ET-1 treatment resulted in a prolonged increase in superoxide production that peaked after 3 days. Thus differences in the onset, duration, and magnitude of ROS production may influence the proliferative signaling pathway. The use of ROS donors and scavengers administered to cells at different times and concentrations may provide further insight into the mechanisms involved in ROS-induced SMC proliferation.
In parallel to their importance in SMC proliferation, ROS also seem to be necessary for SMC survival. Suppression of endogenous intracellular H2O2 through overexpression of catalase not only inhibits proliferation but also promotes apoptosis in SMCs (4, 40). Tsai and colleagues (40) have also observed reduced viability and induced apoptosis in aortic SMCs but not in aortic endothelial cells in response to treatment with the antioxidants N-acetylcysteine and pyrrolidinedithiocarbamate. Our results complement these studies on the adult systemic vasculature by demonstrating a reduction in viability and an induction of apoptosis in FPASMC after antioxidant treatment. Furthermore, we found that inhibitors of NADPH oxidase also reduce viability and induce apoptosis at high concentrations, which suggests that this enzyme complex may play an important role in FPASMC survival by maintaining a required level of superoxide within the cell. Antioxidant therapy may therefore selectively inhibit vascular SMC proliferation in both pulmonary and systemic SMCs without adversely affecting the endothelium, although the effects of antioxidants on FPAEC proliferation remain to be established.
Using pharmacological agents, we have begun to elucidate the signaling
pathway leading from ETA receptor activation to superoxide production. Our results suggest that ET-1-induced superoxide production and proliferation in FPASMCs occurs via the activation of PTX- and
CTX-sensitive G proteins and PI3K. In addition, we found that the
inhibition of NADPH oxidase reduced the ET-1-mediated mitogenic signal
and the increase in ROS. The expression of dominant negative and
constitutively active forms of these molecules may help to confirm
their roles in the ET-1-mediated signaling pathway, leading to
superoxide production and FPASMC proliferation. The vascular NADPH
oxidase enzyme complex is membrane associated and appears to respond to
external signals to generate superoxide (10). Activation
of the oxidase has been demonstrated for angiotensin II
(9), thrombin (29), PDGF (23),
and tumor necrosis factor- (25), as well as by
biomechanical forces (16). However, it remains unclear as
to how this activation is mediated. Our data add ET-1 to the list of
external stimuli that activate the vascular NADPH oxidase and suggest
that G proteins and PI3K are involved in the activation pathway. In
addition, our data demonstrate that NADPH oxidase inhibition can
decrease SMC numbers by inducing an apoptotic pathway. This
indicates that ROS generation is required for the survival as well as
the proliferation of FPASMCs.
In summary, this study demonstrated that the proliferative effects of ET-1 on FPASMCs likely involves an increase in the production of ROS. Further study of the signaling pathways involved may identify additional points at which ET-1-induced proliferation can be inhibited. Because antioxidants or NADPH oxidase inhibition induced apoptosis in ovine FPASMCs, these agents may be of benefit in preventing the SMC proliferation associated with pulmonary vascular diseases such as PPHN.
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ACKNOWLEDGEMENTS |
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This research was supported in part by National Heart, Lung, and Blood Institute Grant HL-60190; National Institute of Child Health and Human Development Grant HD-398110; March of Dimes Grant FY00-98 (to S. M. Black); and American Heart Association, Midwest Affiliate Grants 0051409Z (to S. M. Black) and 0030412Z (to R. W. Dettman).
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. M. Black, Division of Neonatology, Northwestern Univ. Medical School, Ward 12-191, 303 E. Chicago Ave., Chicago, IL 60611-3008 (E-mail: steveblack{at}northwestern.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 February 2001; accepted in final form 31 July 2001.
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