FASEB J. Cell Migration Consortium
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.06-5887fje.
This Article
Right arrow Abstract Freely available
Right arrow Summary
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
fj.06-5887fjev1
20/10/1698    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Asikainen, T. M.
Right arrow Articles by White, C. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Asikainen, T. M.
Right arrow Articles by White, C. W.
(The FASEB Journal. 2006;20:1698-1700.)
© 2006 FASEB

Improved lung growth and function through hypoxia-inducible factor in primate chronic lung disease of prematurity

Tiina M. Asikainen*, Ling-Yi Chang{dagger}, Jacqueline J. Coalson{dagger}, Barbara K. Schneider*, Nahid S. Waleh§, Machiko Ikegami||, John M. Shannon||, Vicki T. Winter{ddagger}, Peter Grubb, Ronald I. Clyman§, Bradley A. Yoder{ddagger}, James D. Crapo{dagger} and Carl W. White*,1

* Department of Pediatrics and

{dagger} Internal Medicine, National Jewish Medical and Research Center, Denver, Colorado, USA;

{ddagger} University of Texas Health Science Center and Southwest Foundation for Biomedical Research, San Antonio, Texas, USA;

§ SRI International, Menlo Park, and Cardiovascular Research Institute, University of California San Francisco, California, USA;

|| Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, USA; and

Division of Neonatology, Vanderbilt University Medical Center, Nashville, Tennessee, USA

1Correspondence: Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Rm. J-318, Denver, CO 80206, USA. E-mail: whitec{at}njc.org

ABSTRACT

Bronchopulmonary dysplasia (BPD), a chronic lung disease affecting preterm neonates, is associated with significant childhood and adult health problems. Histopathologic features of BPD include impaired vascular and distal airway development. We previously showed that activation of hypoxia-inducible factors (HIFs) by inhibition of prolyl hydroxylase domain-containing proteins (PHDs) is feasible and that it stimulates vascular endothelial growth factor (VEGF) -dependent angiogenesis in vitro. We tested the hypothesis that enhancement of angiogenesis by activation of HIFs improves lung growth and function in prematurely born neonates in vivo. Preterm baboons (125 day+14 day pro re nata O2 model, corresponding to 27 human gestational weeks) were treated for 14 days with intravenous (i.v.) FG-4095, a PHD inhibitor. Notably, 77% of diminished total alveolar surface area in untreated controls was recovered by FG-4095 treatment. Functional significance of the structural changes was indicated by improved oxygenation and lung compliance in FG-4095-treated newborns. Surfactant proteins B and C and saturated phosphatidylcholine were unchanged. Incidence of spontaneous ductus arteriosus closure was increased, likely contributing to lower ratio of pulmonary to systemic blood flow in FG-4095 group. These findings indicate that HIF stimulation by PHD inhibition ameliorates pathological and physiological consequences of BPD.—Asikainen, T. M., Chang, L.-Y., Coalson, J. J., Schneider, B. K., Waleh, N. S., Ikegami, M., Shannon, J. M., Winter, V. T., Grubb, P., Clyman, R. I., Yoder, B. A., Crapo, J. D., White, C. W. Improved lung growth and function through hypoxia-inducible factor in primate chronic lung disease of prematurity.


Key Words: bronchopulmonary dysplasia • preterm neonate • angiogenesis • alveolization • lung development

PULMONARY ALVEOLAR DEVELOPMENT is an elaborate process requiring defined interactions between epithelial, mesenchymal, and interstitial components of the lung (1 , 2) . The foundation for successful postnatal alveolization in humans is established during late canalicular to early saccular development, usually starting at 26–28 gestational weeks (3) . Premature birth at this stage of development inevitably perturbs lung structural development. Indeed, 77% of preterm neonates born at less than 32 gestational weeks are ultimately diagnosed with bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by arrested vascular and alveolar development (4 , 5) . Since the description of BPD almost 40 yr ago (6) , substantial research has been directed at finding ways to prevent, ameliorate, and treat this clinical problem (7 , 8) . Major advances in perinatal care (antenatal steroid and postnatal surfactant therapy) have lowered mortality rates previously associated with BPD but have also introduced a new form of BPD characterized by structural immaturity of the lung in surviving babies. None of the new, currently clinically available experimental treatment strategies, with the possible exception of inhaled NO (9 , 10) , have been successful in restoring the impeded lung development and diminished lung function in ventilated preterm infants with evolving BPD (8 , 11) .

Normal fetal development occurs in hypoxia, and the fetal blood O2 fraction is ~3%. Hypoxia, relative to normoxia, promotes development of lung microvasculature and epithelial branching morphogenesis (12) . On the other hand, blocking lung vascular development impairs branching morphogenesis, and therefore pulmonary vascular development appears to be critical for distal epithelial development (12 , 13) . Most (14) , but not all (15) , studies have supported the view that normal, hypoxia-associated angiogenic signaling pathways are interrupted by preterm birth and onset of O2 breathing. This is further reflected in the altered levels of angiogenic factors, such as VEGF and its receptors, in lungs of preterm neonates with respiratory distress syndrome or BPD (16 17 18 19) .

Hypoxia-inducible factors (HIFs) are important transcriptional regulators of angiogenesis, both in disease and during development (20 , 21) . In endothelial cells they are known to up- or down-regulate nearly 600 target genes, including ones important for cell growth, proliferation, and survival (22) . Both HIF-1{alpha} and -2{alpha} are essential for normal embryonic development as shown by gene ablation models (23 24 25) . Specifically, homozygous knockouts for Hif1{alpha} or Epas1 (encoding HIF-2{alpha}) die either during embryogenesis or the neonatal period with various cardiovascular malformations, mesenchymal cell death, neural tube defects, and/or multiorgan failure (23 24 25) . Of note, the Epas1 null mutant mice that survive until term succumb to respiratory failure soon after birth (24) . HIF activation could be expected to produce more balanced angiogenesis compared to enhancement of only single angiogenic factors (26 27 28) .

We previously showed that pulmonary HIF-1{alpha} protein is down-regulated in BPD (29) . Given that angiogenic processes are disrupted after premature birth, HIF stabilization might have advantageous effects on lung development in the preterm neonate. Proof-of-principle studies using HIF-stabilizing compounds (inhibitors of prolyl hydroxylase domain-containing proteins, PHDs, which degrade HIFs) have been carried out in relevant cell types in vitro and in fetal lung explants ex vivo, and have demonstrated that HIFs and selected angiogenic factors can be enhanced by this approach (30 , 31) . The aim of the current study was to test the hypothesis that activation of HIFs through PHD inhibition restores normal lung development in vivo. This report focuses on the clinical findings of these studies carried out using the baboon model of prematurity, a well-characterized experimental model of BPD with close applicability to human disease (16) , whereas the biochemical endpoints, including mRNA and protein analyses for several angiogenic markers (e.g., HIFs, PECAM-1, vascular endothelial growth factor (VEGF) and its receptors, angiopoietins, eNOS), are characterized elsewhere (32) .

MATERIALS AND METHODS

Baboon model of prematurity
Animal studies were carried out at the Southwest Foundation for Biomedical Research Primate Center (San Antonio, TX, USA) and protocols were approved by the Institutional Animal Care and Use Committee. Baboon pregnancies were timed and antenatal ultrasound examination was performed between 110 and 115 days of estimated term gestation (185 days). Preterm baboons (125±2 days corresponding to 27 human gestational weeks) were delivered by cesarean section, weighed, sedated, intubated, and given surfactant (Survanta 4 ml/kg, courtesy of Ross Laboratories, Columbus, OH, USA) prior to placement on ventilatory support. Baboons were randomly placed to either control group receiving routine care or to FG-4095 (FibroGen, Inc., South San Francisco, CA, USA) group receiving experimental therapy with FG-4095 in addition to routine management. General treatment protocols, including nutritional and antimicrobial care, have been described (16) . Sepsis was common (Table 1 ) and was defined as either positive bacterial or fungal blood or autopsy tissue culture and/or septic emboli with or without bacteria or fungus in histopathologic assessment, confirmed with tissue Gram stain if necessary. For some endpoints (lung morphometry and surfactant mRNA analyses), additional studies were performed using lung tissue from 125 and 140 days fetal baboons delivered by hysterotomy and euthanized at birth before onset of breathing. 140 days of baboon gestation corresponds to 32 human gestational weeks and serves as an age-matched fetal control for the 125 + 14 days (139 days) preterm baboon. All animals received maternal antenatal steroids (6 mg, dexamethasone or betamethasone) 48 and 24 h prior to delivery (Table 1) .


View this table:
[in this window]
[in a new window]
  		Table 1. Clinical characteristics of control and FG-4095-treated baboonsa

Inclusion and exclusion criteria
The study was designed to evaluate lung growth in the critical 14 day neonatal period of 125 days preterm baboons. Inclusion and exclusion criteria for baboons were established before the study and/or data analyses. General criterium for inclusion was birth between 11/2003 and 2/2005 ± 1 month (15 FG-4095-treated and 26 control baboons). For assessment of lung growth and function, 14 day survival was required. The most common reasons for nonsurvival were early cardiovascular instability, sepsis, and/or iatrogenic causes. To establish exclusion criteria, a panel of six principal researchers evaluated individual baboon records before data analyses. Following exclusions (6 FG-4095-treated animals and 8 controls; Supplemental Table 1), 9 FG-4095-treated and 18 control baboons remained (Table 1) . For data analyses, these included animals were separated into 14 days survivor FG-4095-treated (n=5) and control (n=12) subgroups, and early death (ED) FG-4095-treated (n=4) and control (n=6) subgroups. In addition, mortality and potential side effects were assessed using an intention-to-treat approach involving all animals.

FG-4095 treatment
FG-4095 was administered in routine glucose (Glc) solutions with an i.v. loading dose (5 mg/kg/2 h) at age 2 h and continuous i.v. dosing (0.6 mg/kg/h) started at age 4 h. For estimation of pharmacokinetics of FG-4095 (calculated by Dr. Charles Peloquin, Pharmacokinetics Laboratory, National Jewish Medical and Research Center), dosing was interrupted at age 170 h for 8 h for one animal. Pilot studies had demonstrated possible precipitation of FG-4095 when infused to the same i.v. line with nutrients, and therefore FG-4095 animals received a second femoral i.v. line for exclusive compound administration. Plasma FG-4095 levels were measured by HPLC from samples stored in liquid N2 for a maximum of one year (Dr. Charles Peloquin).

Ventilatory therapy
Ventilation was provided for 14 days with pressure-limited, time-cycled infant ventilator (InfantStar, Infrasonics, San Diego, CA, USA, or VIP Gold, VIASYS Healthcare Critical Care Division, Palm Springs, CA, USA) or when required due to worsening oxygenation, with high-frequency oscillatory ventilator (provided by Sensormedics, Yorba Linda, CA, USA). The ventilatory goals have been described elsewhere (9 , 16) . In short, treatment sought to maintain PaO2 and PaCO2 between 55 and 70 mmHg (7.3 to 9.3 kPa) and 45 to 55 mmHg (6 to 7.3 kPa), respectively, keeping tidal volumes between 4 to 6 ml/kg and minimizing exposure to high FiO2. Mean airway pressures were adjusted and chest radiographs were obtained as necessary. Degree of O2 and ventilatory support were determined by calculating oxygenation index [mean airway pressure (cmH2O)x FiO2x100/PaO2 (mmHg)], alveolar-arterial O2 gradient [{(PB–PH2O) (mmHg)xFiO2}–PaCO2 (mmHg)/R–PaO2 (mmHg), where (PB–PH2O)=713 and R (respiratory exchange ratio)=0.8], and ventilation index (peak inspiratory pressure (cmH2O)xventilator ratexPaCO2 (mmHg)/1000].

Hemodynamic support, echocardiographic evaluation, dynamic lung compliance
The principles of hemodynamic support, echocardiographic evaluation, and pulmonary function testing have been detailed elsewhere (9 , 33) .

Postmortem measurements
Pulmonary pressure-volume curve was obtained as described (9) . Individual lung lobes were weighed and bronchoalveolar lavage fluid (BALF) was collected from left lower lobe by pooling the material from five rinses with normal saline. Gross pathological assessment of various organs was performed. Gross bleeding into tissues (lungs, thymus, thyroid, heart, liver, gallbladder, spleen, pancreas, salivary glands, esophagus, stomach, intestine, kidneys, adrenals, testes/reproductive organs, urinary bladder, abdominal cavity, soft tissues, brains) was evaluated using a semiquantitative score from 0 to 3 as follows: 0 = none; 1 = mild bleeding in one organ, typically lung; 2 = bleeding in two or three lung lobes and/or organs, pulmonary and extrapulmonary organs required; 3 = bleeding in more than three lung lobes and/or organs, pulmonary and extrapulmonary organs required. Plasma and tissues were collected for subsequent histopathologic and biochemical analyses.

Lung histopathology
Hematoxylin/eosin and Movat’s pentachrome stains were obtained and histopathologic evaluation using semiquantitative scoring was performed. None of the 14 day survivors and early death animals exhibited a consolidative bronchopneumonia or lobar pneumonia, and therefore scores of 0 to 3 were used to reflect the following findings: 0 = no pneumonitis; 1 = rare polymorphonuclear (PMN) cells, no intra-alveolar infiltrate; 2 = PMN cells in several sites intermixed with other intra-alveolar cells (mostly alveolar macrophages); 3 = more frequent microscopic foci in which intra-alveolar aggregates of PMN cells were present. Similarly, none of the animals showed diffuse consolidating bleeding, and scores of 0 to 3 were used to describe the degree of microscopic extravasation of red blood cells (RBC) as follows: 0 = none; 1 = one to three foci of RBC extravasation; 2 = RBC extravasation in more than three areas; 3 = more frequent intra-alveolar RBC and subpleural bleeding. Collagen and elastin estimates were done in a blinded manner on the Movat-stained preparations utilizing a 1 to 3 categorization. To assess thickness of arterial smooth muscle cell layer, small muscular arteries (6–14 per section) were photographed and measurements carried out in a blinded manner using the NIH Image 1.63 program.

Lung morphometry
Blinded lung morphometric analyses were performed by digital image analysis system (34) validated for the baboon model of BPD (9) . The right lower lobe main stem bronchus was cannulated and the lobe was fixed by instillation of 4% paraformaldehyde in 0.1 M phosphate buffer at 20 cm water pressure and fixed for 24 h. The same lobe (right lower lobe) was used in the control animals, and the same tissue blocks were used for both histopathology and morphometry.

The random sampling method was as follows. Right lower lobe was cut into four pieces of equal thickness at a random orientation. Three sections were obtained from cut faces (either lower cut faces of blocks 1, 2, and 3 or upper cut faces of blocks 2, 3, and 4 by a random choice). Total tissue area occupied by the three sections were determined by digitizing the areas to determine the number of 10x fields that were encompassed by the sections. That number was divided by 30 to determine the sample interval number (N) to achieve ~30 sampling fields per animal. Photographic samples were obtained using a motorized stage controlled by StagePro software (Media Cybernetics, Silver Spring, MD, USA). Upper, lower, left, and right boundaries of the section were defined first. Sampling started from upper left corner (field #1). A random number, X, between 1 and N, was generated. Software was instructed to take the first picture at field #X and every N fields afterward. Field and interval counts continued uninterrupted from section 1 to section 2 and then 3 (without starting from field #1 with the new section).

The following indices were measured/calculated: 1) lung volume (cm3) by water replacement, 2) mean length (µm) and number (#/mm2) of internodal or end segments, 3) surface density (cm2/cm3, or cm–1) of primary septae or secondary crests [surface density indicates the alveolar surface area contributed by the alveolar septum specified (primary or secondary) in a unit volume of parenchyma. Surface density was estimated by the ratio of total septal length (of primary or secondary septae from all pictures) divided by total alveolar tissue area from all pictures multiplied by "2" (the multiplier "2" is added because each septum has two alveolar surfaces)], 4) surface area (cm2) of primary septae or of secondary crests (Surface area is calculated by the equation: Lung volume (cm3) x fraction of lung that is alveolar x surface density (cm–1), and 5) total alveolar surface area (cm2) consisting of surface area of both primary septae and secondary crests.

NO measurement
Exhaled NO was measured in accordance with ATS guidelines as described previously (35) . Briefly, three exhaled tidal volumes were diverted via a nonrestricting, low dead space valve system to an NO chemiluminescence detection system (EcoPhysics, Durnten, Switzerland) at a constant exhalation flow rate. Fraction of exhaled NO was measured and minute ventilation of NO was calculated as published (35) .

Cell culture
Human lung microvascular endothelial (HLMVE) and alveolar epithelial-like (A549) cells were purchased from Cambrex Bio Science (Walkersville, MD, USA) and American Type Culture Collection (ATCC) (Rockville, MD, USA), respectively, and cultured as reported (29 , 30) . FG-4095 (125 µM) and positive control dimethyloxaloylglycine (DMOG, 1 mM) were used as described (30) .

ARNT detection
Cytosolic and nuclear extracts were prepared (29 ,30) . A mouse monoclonal antibody (mAb) against human ARNT was used at a dilution of 1:200 or 1:1,000 (Novus Biologicals, Aurora, CO, USA).

Surfactant analyses
Lung mRNA for surfactant protein B and C (SP-B and -C) were measured by real-time and conventional polymerase chain reaction (PCR) as described (36) . Saturated phosphatidylcholine (Sat PC) was quantitated by isolation from lipid extracts of BALF and lung homogenate using osmium tetroxide, followed by measurement of phosphorous (37) . SP-B and -C were assessed by Western blot of extracted lipids of BALF and lung homogenate containing the same amount of Sat PC (1–4 nmol depending on analysis) using antiserum against mature human SP-B peptide (Chemicon, Temecula, CA, USA) or high-titer anti-SP-C antibody (Ab) raised against a modified human recombinant, 34-amino acid SP-C peptide (38) . Goat anti-rabbit peroxidase-conjugate (Calbiochem, La Jolla, CA, USA) was utilized as the secondary Ab. Immunoreactive bands were quantitated by densitometric analyses in a range where the density of the bands was linearly related to the amount of Sat PC loaded. From the total volume of BALF and lung homogenate, left and right lung weights, body wt, and aliquot volume used for immunoblot, surfactant protein content in total BALF or lung tissue/kg body wt was calculated. BALF SP-B protein was also quantitated by ELISA (39) .

Statistical analyses
Repeated-measures ANOVA or 2-way ANOVA with Fisher’s protected least significant difference (PLSD) for post hoc testing at individual time points were used to assess differences between groups over the 14 day period. Multiple groups were also compared by ANOVA with Fisher’s PLSD. Survival was assessed by Kaplan-Meier analysis. Mortality, ductus arteriosus (DA) status, as well as other categorical data were analyzed by {chi}2 test with Fisher’s exact P. Single end point comparisons between groups involving continuous data were performed using Student’s unpaired t test or Mann-Whitney nonparametric test. Analyses were carried out using StatView 4.51 (Abacus Concepts Inc., Berkeley, CA, USA). Results are shown as mean ± SE or mean (SD) with P values of ≤0.05 indicating statistical significance.

RESULTS

Unless otherwise indicated, the results depict data analyses of the included 14 day survivor groups (5 FG-4095-treated and 12 control baboons).

Clinical characteristics of control and FG-4095-treated baboons
Mortality as analyzed from either all animals using intention-to-treat approach or only from included animals (Table 1) was not statistically significantly different between FG-4095-treated baboons and untreated controls. Further, cumulative survival by Kaplan-Meier analysis was similar between the groups. Pattern of body wt change did not differ between treated and untreated groups (not shown). To assess permeability, percent necropsy lung wet wt (LW) of birth wt (BW) or necropsy body wt (NW) was calculated, and neither differed between the treated (LW/BW 3.0±0.2%; LW/NW 3.4±0.3%) and untreated (LW/BW 3.5±0.1%; LW/NW 3.6±0.1%) 14 day baboons. BALF protein expressed as total protein per wt of left lower lobe was comparable in FG-4095-treated (7.2±1.0 µg/mg) and control (5.3±1.0 µg/mg) baboons. Routine blood chemistry parameters pertaining to kidney and liver function were measured and found to be similar in FG-4095-treated and control animals (Supplemental Table 2).

FG-4095 pharmacokinetics
Plasma FG-4095 accumulated for the first 3 to 4 days, remained high until age 5 to 6 days, and steadily declined thereafter (Fig. 1 ). Calculated circulating half-life for FG-4095 was 13.5 h. The highest (60 µg/ml) and lowest (20 µg/ml) plasma levels correspond to 198 and 66 µM, respectively, a concentration range shown to be effective for HIF stabilization (30 , 31) .


Figure 1
View larger version (7K):
[in this window]
[in a new window]
  		Figure 1. Plasma FG-4095 levels in preterm baboons treated with FG-4095 for 14 days. Plasma levels of FG-4095 were measured by HPLC and are shown for both 14 day survivors (n=5, black circles) and early death baboons (n=3, white circles) (mean, SD).

Lung morphometry
Lung morphometry of untreated controls relative to fetal controls (140 day gestational control, or GC) revealed many abnormalities, including loss of total alveolar surface area, and decreased number of internodal segments (branch points) and of end segments (secondary crests), suggestive of alveolar simplification and arrested alveolar septal budding (formation of new alveoli) (Table 2 ). Notably, several of these abnormalities were improved, or normalized, in FG-4095-treated baboons. Specifically, surface density and area of primary septae were significantly greater in FG-4095-treated baboons than in untreated newborns (Table 2 , representative pictures in Fig. 2 ). Moreover, total alveolar surface area, consisting of area of primary and secondary septae, was augmented in FG-4095-treated baboons relative to untreated controls (Table 2 , Fig. 2 ).


View this table:
[in this window]
[in a new window]
  		Table 2. Comparison of lung morphometry in preterm and fetal baboonsa


Figure 2
View larger version (31K):
[in this window]
[in a new window]
  		Figure 2. Qualitative illustration of typical lung morphometric findings in panel A) control and B) FG-4095-treated preterm baboons (125 days+14 day pro re nata O2 model). The general alveolar simplification in untreated controls (A) is improved in FG-4095-treated baboons (B) (see Table 2 for quantitative morphometry). Hematoxylin/eosin stain, original magnification 10x.

Lung histopathology
Approximately half of the 14 day controls exhibited a moderate pneumonitis; otherwise the FG-4095-treated and control groups did not differ greatly (see Supplemental Table 4). Movat’s pentachrome stains were performed to estimate pulmonary collagen and elastin, as expression of each could potentially be altered by expression of HIFs and/or PHD inhibition. Collagen and elastin content and distribution in proximal and distal lung preparations were generally low and comparable in the 14 day FG-4095-treated and control baboons (not shown). Microscopic blood extravasation was overall less frequent and milder in the 14 day FG-4095-treated animals compared with the 14 day control group.

Chronic hypoxia, in part through HIF stimulation, has been associated with pulmonary hypertension caused by proliferation of arterial smooth muscle cells and vasoconstriction. We therefore measured thickness of arterial medial smooth muscle layer as percent of wall thickness and found it to be identical for FG-4095-treated (43±3%) and control (40±2%) animals.

Pulmonary function
Alveolar-arterial O2 gradient and oxygenation index were improved for the last 5 to 6 days by FG-4095 treatment as assessed by repeated-measures ANOVA (Fig. 3 A, B). Corresponding analysis over the whole 14 day course was not statistically significant. Ventilation index appeared improved in FG-4095-treated animals compared with controls, but the difference was not statistically significant (Fig. 3C ). Dynamic lung compliance was enhanced during the last 4 days by FG-4095 treatment (Fig. 3D ). Postmortem pressure-volume curve was the same for treated and untreated animals (Supplemental Fig. 1). Two control early death animals were placed on high-frequency oscillatory ventilation due to worsening oxygenation, but that treatment was not required for any of the FG-4095 animals.


Figure 3
View larger version (12K):
[in this window]
[in a new window]
  		Figure 3. Lung function tests in FG-4095-treated preterm baboons relative to untreated controls. A) Alveolar-arterial PO2 gradient, B) oxygenation index, C) ventilation index, and D) dynamic lung compliance were measured in untreated controls (black) and baboons treated with FG-4095 (white) (125+14 days pro re nata O2 model). Data were pooled per animal per 12 h (A–C) or per 48 h (D) and statistical significance was estimated using repeated-measures ANOVA (A–C) or 2-way ANOVA (D) with Fisher’s PLSD for post hoc comparisons. Data are shown as mean ± SE (control n=10–12; FG-4095 n=3–5); *P ≤ 0.05; ***P ≤ 0.001 FG-4095 vs. control.

Surfactant expression
Expression of pulmonary SP-B mRNA did not differ between treated and untreated neonates (Fig 4 A). Pulmonary SP-C mRNA levels measured by semiquantitative PCR were low and not changed by FG-4095 treatment (not shown). BALF or lung Sat PC content was similar in FG-4095-treated and control animals (Fig. 4B, C ). BALF and lung SP-B and -C proteins by Western blot were unaltered in FG-4095-treated relative to control baboons (Fig. 4D, E ). BALF SP-B expression was confirmed by ELISA and results supported Western blot analysis (mean+SD) [control 1.7 (0.9) and FG-4095 1.5 (1.1) SP-B (µg) per left lower lobe wt (g)].


Figure 4
View larger version (15K):
[in this window]
[in a new window]
  		Figure 4. Surfactant analyses in preterm baboons treated with FG-4095 relative to untreated and fetal controls. A) Pulmonary SP-B mRNA was measured by real-time PCR in 125 day preterm baboons treated with 14 days of pro re nata O2 with or without FG-4095, as well as in 125 and 140 days fetal controls. The change in gene expression was determined by calculating {Delta}CT, where the threshold cycle (CT) value of the target gene was subtracted from that of the housekeeping gene malate dehydrogenase (MDH). Each unit of {Delta}CT represents a 2-fold change in the target gene mRNA expression. Data are shown as means, SD (n=5–10), *P ≤ 0.05 vs. 125 days GC. Sat PC was analyzed in both BALF (B) and lung homogenate (C). SP-B and -C proteins were assessed in BALF (D) and lung homogenate (E) by Western blot and quantitated by densitometry (black, control; white, FG-4095).

Exhaled NO
There were no clear differences in either the fraction of exhaled NO or minute ventilation of NO between FG-4095-treated and control baboons (not shown).

Hemodynamic assessment
Hemodynamic variables were assessed to address the possibility that HIF-augmenting therapy with FG-4095 caused alterations in left ventricular function, systemic or pulmonary vascular resistance, or incidence of ductus arteriosus closure. Left ventricular function was assessed by echocardiographic measurement of shortening fraction and velocity of circumferential fiber shortening, and was identical for control and FG-4095-treated baboons (Supplemental Fig. 2). Ratio of pulmonary to systemic blood flow was lower in the FG-4095 baboons relative to controls (Fig. 5 A). Systemic diastolic, but not systolic, and mean arterial (Fig. 5B ) blood pressure were higher in baboons receiving FG-4095 compared with controls. Total fluid intake and urine output were comparable in treated and untreated animals (not shown). Pharmacologic blood pressure support did not differ between FG-4095-treated baboons and untreated controls (Supplemental Table 3).


Figure 5
View larger version (13K):
[in this window]
[in a new window]
  		Figure 5. Hemodynamic evaluation of preterm baboons with and without FG-4095 treatment. A) Ratio of pulmonary to systemic blood flow (Qp/Qs), data were pooled per 48 h per animal and B) mean arterial blood pressure (MAP) after FG-4095 treatment (black squares, control; white squares, FG-4095). Statistical analyses were carried out using 2-way ANOVA with Fisher’s PLSD (A) or repeated-measures ANOVA (B). Data are shown as mean ± SE (n=4–12 for control and n=3–5 for FG-4095-treated baboons). *P ≤ 0.05; **P ≤ 0.01, and ***P ≤ 0.001.

Incidence of spontaneous ductus arteriosus (DA) closure, assessed by daily echocardiography, was higher (P<0.001) in all FG-4095-treated 14 day animals than respective controls both during the first (% days control DA open 75%, closed 1%, not recorded 24%; FG-4095 DA open 46%, closed 34%, not recorded 20%) and second (% days control DA open 62%, closed 1%, not recorded 37%; FG-4095 DA open 51%, closed 20%, not recorded 29%) week of life. To seek possible mechanisms for DA closure, we undertook studies of aryl hydrocarbon receptor nuclear translocator (ARNT, also called HIF-ß) expression, as it has been implicated in developmental closure of vascular structures (40) . In proof of principle studies using HLMVE and A549 cells, exposure to FG-4095 or DMOG caused nuclear translocation of ARNT (Supplemental Fig. 3). Another possible mechanism for DA closure could be prostaglandin (PG) -induced vasodilatation and decrease of pulmonary pressure. As plasma volumes available from preterm baboons are very low, thereby limiting in vivo measurements, we performed in vitro experiments to learn whether FG-4095 treatment was associated with increased PGE2 secretion. However, PGE2 in culture medium of HLMVEC was undetectable (<60 pg/ml).

Possible untoward effects
As noted above in the various analyses, FG-4095 generally was well tolerated and elicited some beneficial lung outcomes in the 14 day survivors. Further, as shown in Table 1 , the majority of the early deaths suffered by both the control and FG-4095-treated groups were sepsis-associated. However, when the early death subgroups were examined, several troublesome findings were identified. All four included early death FG-4095 animals and exhibited a skin rash (not shown) never previously observed in this model. Occurrence of rash, as analyzed using intention-to-treat principle, was statistically significant (P<0.001) in FG-4095-treated baboons (40%, n=15) relative to untreated controls (0%, n=26). This strikingly erythematous, papular rash was usually detected between days 4 and 6, was not exfoliative, involved primarily face and scalp, and was occasionally generalized. Even though overall mortality did not differ between the treated and untreated animals, there was a statistically significant (P=0.002) association between death and rash in FG-4095-treated animals vs. untreated controls. In addition, early death FG-4095 baboons had more moderate to severe graded pulmonary microscopic extravasation of RBC compared with early death controls, and at necropsy gross examination showed more moderate to severe graded bleeding in lungs and other organs when compared with early death controls that had none to mild grades (Supplemental Table 4). Of note, rash or bleeding were not noted in baboons completing the 14 day treatment with FG-4095.

DISCUSSION

BPD is associated with significant childhood and adult health problems, including exercise intolerance, increased risk of asthma, pulmonary hypertension, possible emphysema and chronic obstructive pulmonary disease, and neurodevelopmental impairment (11) . Arrested development of microvasculature and distal air spaces, both common features of BPD, contribute to many of these clinical abnormalities (14) . Since lung branching morphogenesis depends on vascular growth, and lung vascular development is disrupted by preterm birth and ventilatory therapy, we hypothesized that enhancement of angiogenesis by HIF stimulation could improve lung growth in prematurely born neonates. In vitro and ex vivo studies from our laboratory have shown that HIF stimulation via inhibitors of prolyl hydroxylase domain-containing proteins (PHDs) enhances expression of, for example, VEGF and platelet-endothelial adhesion molecule 1 (PECAM-1) as well as in vitro angiogenesis (30 , 31) . The goal of this study was to determine the therapeutic potential of this approach in an in vivo baboon model of prematurity. Treatment of preterm baboons with continuous, i.v. FG-4095 resulted in plasma levels shown to be effective for HIF stabilization (30 , 31) .

By lung morphometric analysis, untreated control baboons had decreased total alveolar surface area, alveolar simplification as demonstrated by reduced numbers of branch points, and arrested growth of new alveoli as implicated by fewer secondary crests. Remarkably, total alveolar surface area and number of branch points were normalized in FG-4095-treated baboons vs. untreated newborn and fetal controls, suggesting improved alveolar growth. We recently found that mRNA and/or protein for several angiogenic factors, including HIF-1{alpha}, PECAM-1, and VEGF are up-regulated in lungs of FG-4095-treated baboons compared with untreated controls (32) . Further, PECAM-1 expressing capillary endothelial cells are augmented by FG-4095 treatment (32) . These data are supported by recent investigations showing that postnatally administered VEGF, especially if combined with angiopoietin 1, preserves alveolar development in a term newborn rodent model of O2-induced lung injury (41 ,42) . Collectively, the data suggest that both vascular and alveolar growth are enhanced by FG-4095 in preterm baboon lung.

To assess whether the lung morphometric findings in FG-4095-treated newborns were associated with improved lung function, dependence on O2 and ventilatory support were assessed. Both control and FG-4095-treated baboons had typical worsening of oxygenation on days 2 to 5 due to severe respiratory distress syndrome. However, at age 8 to 9 days, oxygenation improved in FG-4095-treated relative to untreated baboons. The 2-fold reduction seen in oxygenation index and alveolar-arterial O2 gradient likely is physiologically significant. In addition, lung dynamic compliance increased on the final 4 days of life. These data indicate that the lung structural changes in FG-4095-treated baboons also allowed for improved gas exchange.

Possible mechanisms for improved oxygenation and lung compliance in FG-4095-treated animals were investigated. As the HIF-VEGF axis has been implicated to be important for surfactant production (24) , one potential responsible mechanism was augmented surfactant secretion due to FG-4095. Therefore, we measured Sat PC and mRNA and protein for SP-B and -C in BALF and lung tissue. However, none were altered by FG-4095 treatment. Thus, improved oxygenation and lung compliance in FG-4095-treated baboons could not be attributed to changes in surfactant production or secretion detectable at necropsy. We did not study surfactant secretion at earlier time points because BALF collection is poorly tolerated. Our next approach involved determining pulmonary NO, as accumulating evidence suggests NO to mediate some VEGF effects (43) and because expression and activity of NO synthases is deterred in BPD (35) . Utilizing a noninvasive method, we found no changes in exhaled NO between FG-4095-treated and untreated groups.

Hypotension is a common problem in preterm neonates. On the other hand, hypertension can cause severe complications in the prematurely born. Here, FG-4095-treated baboons had moderately increased diastolic, but not systolic, blood pressure. Because of increased diastolic pressure, mean arterial pressure also was higher, and pulse pressure lower, in FG-4095-treated baboons. Moreover, the ratio of pulmonary to systemic flow was reduced in the FG-4095-treated relative to control baboons. These positive hemodynamic effects likely resulted from the increased incidence of DA closure (44) in the FG-4095-treated animals.

After term birth, the DA normally closes within the first days of life through constriction and remodeling of the vessel wall (45) . After preterm birth, however, the DA often remains patent. Patent DA (PDA) is an independent factor complicating the clinical course of preterm neonates and increasing the risk of BPD, necrotizing enterocolitis, and intraventricular hemorrhage (46) . Clinically problematic PDA can be closed by indomethacin, ibuprofen, or surgery, but none of these are without risk (46) . Hypoxia, HIF-1{alpha}, and VEGF in the wall of DA have been implicated in its closure (45 , 47) . Therefore, it is possible, although so far not demonstrated, that FG-4095 could cause or contribute to DA closure through HIF-VEGF axis. Seeking other possible mechanisms, we determined whether FG-4095 altered ARNT expression. These experiments were considered because of involvement of ARNT in closure of fetal vascular structures, such as the hepatic ductus venosus and ocular hyaloid artery (40) . We found that FG-4095 enhanced nuclear expression of ARNT in vitro. However, ARNT expression in the DA of FG-4095-treated baboons is presently unknown to us, and therefore ARNT-induced DA closure remains speculative.

Possible untoward effects, including skin rash and bleeding, were noted in the FG-4095-treated early death baboons. Such effects have not been observed in preclinical and clinical studies of anemia and chronic kidney disease using related or unrelated HIF-PHD inhibitors (48 , 49) . However, the effects of these compounds in preterm neonate models have not been previously explored. Rash and bleeding were not found in animals completing the 14 day treatment. Further, heart, kidney, and liver function tests did not indicate toxicity. Currently, the mechanism responsible for skin rash and bleeding are unknown and the study population is too small to make definitive associations. A number of potential mechanisms might be operative and would need additional investigation. An initial consideration would be to see if there is a drug interaction with the presence of microbes and/or whether the drug may enhance some aspect of an infectious process. Many of the early death baboons were septic, but the untreated septic controls did not develop rash and had less bleeding. The rash appeared not to be associated with any single infectious agent. Second, immune effector molecules, abundant in the model due to respiratory distress syndrome, BPD, and/or sepsis, could play a role. Specifically, HIFs can modulate inflammatory responses through regulating neutrophil apoptosis and enhancing phagocyte bactericidal capacity (50 , 51) . Third, such effects could be related to interactions of the study compound with other routine medications used to treat preterm neonates. Fourth, the findings could be associated with increased vascular permeability and/or vasoproliferation. Namely, we have previously shown that VEGF mRNA and protein are induced many-fold by FG-4095 treatment (30 , 31) , and others have found that excess VEGF can cause pulmonary bleeding and edema indicative of vascular hyperpermeability (52) . Herein, pulmonary VEGF mRNA, but not protein, was elevated after FG-4095 treatment for 14 day (32) . It is possible, however, that elevated VEGF protein at earlier time points than 14 days may have increased vascular permeability contributing to bleeding. Plasma VEGF was undetectable in the current study. These systemic untoward effects observed with continuous, i.v. treatment may be circumvented with alternative treatment protocols, such as intermittent, intrapulmonary delivery of study compound, as suggested by a recent investigation using intratracheal VEGF delivery (41) .

To summarize, the findings from the current study reveal that in vivo treatment of preterm baboons with HIF-PHD inhibitor is associated with enhanced lung growth, improved oxygenation, and lung compliance, and increased incidence of DA closure. Thus, HIF stimulation by PHD inhibition ameliorates pathological and physiological consequences of BPD.

ACKNOWLEDGMENTS

We would like to express our gratitude to Donald McCurnin, Helen Martin, and all personnel supporting the BPD Resource Center (San Antonio, TX, USA), to James Murphy (Head, Division of Biostatistics, National Jewish Medical and Research Center, CO, USA) for statistical advice, and to Xiao-ling Guo (National Jewish Medical and Research Center) for technical assistance. The authors also thank Dr. Charles Peloquin and his laboratory personnel for HPLC assays of plasma FG-4095. Financial support was received from NIH U01 HL56263 (C.W.W.) and HL63397 (J.D.C.), NIH HL52636 (BPD Resource Center), P51RR13986 (facility support at Southwest Foundation), Academy of Finland (T.M.A.), and Foundation for Pediatric Research in Finland (T.M.A.). No monetary contributions beyond provision of the study compound were received from FibroGen, Inc. and none of the authors nor their institutions hold equity in FibroGen, Inc.

Received for publication February 2, 2006. Accepted for publication March 20, 2006.

REFERENCES

  1. Gebb, S. A., Shannon, J. M. (2000) Tissue interactions mediate early events in pulmonary vasculogenesis. Dev. Dyn. 217,159-169[CrossRef][Medline]
  2. Bourbon, J., Boucherat, O., Chailley-Heu, B., Delacourt, C. (2005) Control mechanisms of lung alveolar development and their disorders in bronchopulmonary dysplasia. Pediatr. Res. 57,38R-46R[CrossRef][Medline]
  3. Burri, P. H. (1984) Fetal and postnatal development of the lung. Annu. Rev. Physiol. 46,617-628[CrossRef][Medline]
  4. Ehrenkranz, R. A., Walsh, M. C., Vohr, B. R., Jobe, A. H., Wright, L. L., Fanaroff, A. A., Wrage, L. A., Poole, K. (2005) Validation of the National Institutes of Health consensus definition of bronchopulmonary dysplasia. Pediatrics 116,1353-1360[Abstract/Free Full Text]
  5. Jobe, A. H., Bancalari, E. (2001) Bronchopulmonary dysplasia. Am. J. Respir. Crit. Care Med. 163,1723-1729[Free Full Text]
  6. Northway, W. J., Rosan, R. C., Porter, D. Y. (1967) Pulmonary disease following respirator therapy of hyaline-membrane disease. Bronchopulmonary dysplasia. N. Engl. J. Med. 276,357-368[Medline]
  7. Asikainen, T. M., White, C. W. (2005) Anti-oxidant therapy for lung injury. Notter, R. H. Finkelstein, J. N. Holm, B. A. eds. Lung injury: Mechanisms, Pathophysiology, and Therapy 196,665-704 Taylor & Francis Group Boca Raton, FL, U. S. A..
  8. Bland, R. D. (2005) Neonatal chronic lung disease in the post-surfactant era. Biol. Neonate 88,181-191[CrossRef][Medline]
  9. McCurnin, D. C., Pierce, R. A., Chang, L. Y., Gibson, L. L., Osborne-Lawrence, S., Yoder, B. A., Kerecman, J. D., Albertine, K. H., Winter, V. T., Coalson, J. J., Crapo, J. D., Grubb, P. H., Shaul, P. W. (2005) 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. 288,L450-L459
  10. Schreiber, M. D., Gin-Mestan, K., Marks, J. D., Huo, D., Lee, G., Srisuparp, P. (2003) Inhaled nitric oxide in premature infants with the respiratory distress syndrome. N. Engl. J. Med. 349,2099-2107[Abstract/Free Full Text]
  11. Jobe, A. J. (1999) The new BPD: an arrest of lung development. Pediatr. Res. 46,641-643[Medline]
  12. van Tuyl, M., Liu, J., Wang, J., Kuliszewski, M., Tibboel, D., Post, M. (2005) Role of oxygen and vascular development in epithelial branching morphogenesis of the developing mouse lung. Am. J. Physiol. 288,L167-L178
  13. Jakkula, M., Le Cras, T. D., Gebb, S., Hirth, K. P., Tuder, R. M., Voelkel, N. F., Abman, S. H. (2000) Inhibition of angiogenesis decreases alveolarization in the developing rat lung. Am. J. Physiol. 279,L600-L607
  14. Stenmark, K. R., Abman, S. H. (2005) Lung vascular development: implications for the pathogenesis of bronchopulmonary dysplasia. Annu. Rev. Physiol. 67,623-661[CrossRef][Medline]
  15. De Paepe, M. E., Mao, Q., Powell, J., Rubin, S. E., Dekoninck, P., Appel, N., Dixon, M., Gundogan, F. (2006) Growth of pulmonary microvasculature in ventilated preterm infants. Am. J. Respir. Crit. Care Med. 173,204-211[Abstract/Free Full Text]
  16. Coalson, J. J., Winter, V. T., Siler-Khodr, T., Yoder, B. A. (1999) Neonatal chronic lung disease in extremely immature baboons. Am. J. Respir. Crit. Care Med. 160,1333-1346[Abstract/Free Full Text]
  17. Bhatt, A. J., Pryhuber, G. S., Huyck, H., Watkins, R. H., Metlay, L. A., Maniscalco, W. M. (2001) Disrupted pulmonary vasculature and decreased vascular endothelial growth factor, Flt-1, and TIE-2 in human infants dying with bronchopulmonary dysplasia. Am. J. Respir. Crit. Care Med. 164,1971-1980[Abstract/Free Full Text]
  18. Maniscalco, W. M., Watkins, R. H., Pryhuber, G. S., Bhatt, A., Shea, C., Huyck, H. (2002) Angiogenic factors and alveolar vasculature: development and alterations by injury in very premature baboons. Am. J. Physiol. 282,L811-L823
  19. Lassus, P., Turanlahti, M., Heikkilä, P., Andersson, L. C., Nupponen, I., Sarnesto, A., Andersson, S. (2001) Pulmonary vascular endothelial growth factor and Flt-1 in fetuses, in acute and chronic lung disease, and in persistent pulmonary hypertension of the newborn. Am. J. Respir. Crit. Care Med. 164,1981-1987[Abstract/Free Full Text]
  20. Pugh, C. W., Ratcliffe, P. J. (2003) Regulation of angiogenesis by hypoxia: role of the HIF system. Nat. Med. 9,677-684[CrossRef][Medline]
  21. Huang, L. E., Bunn, H. F. (2003) Hypoxia-inducible factor and its biomedical relevance. J. Biol. Chem. 278,19575-19578[Free Full Text]
  22. Manalo, D. J., Rowan, A., Lavoie, T., Natarajan, L., Kelly, B. D., Ye, S. Q., Garcia, J. G., Semenza, G. L. (2005) Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1. Blood 105,659-669[Abstract/Free Full Text]
  23. Iyer, N. V., Kotch, L. E., Agani, F., Leung, S. W., Laughner, E., Wenger, R. H., Gassmann, M., Gearhart, J. D., Lawler, A. M., Yu, A. Y., Semenza, G. L. (1998) Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev. 12,149-162[Abstract/Free Full Text]
  24. Compernolle, V., Brusselmans, K., Acker, T., Hoet, P., Tjwa, M., Beck, H., Plaisance, S., Dor, Y., Keshet, E., Lupu, F., et al (2002) Loss of HIF-2alpha and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice. Nat. Med. 8,702-710[Medline]
  25. Scortegagna, M., Ding, K., Oktay, Y., Gaur, A., Thurmond, F., Yan, L. J., Marck, B. T., Matsumoto, A. M., Shelton, J. M., Richardson, J. A., Bennett, M. J., Garcia, J. A. (2003) Multiple organ pathology, metabolic abnormalities and impaired homeostasis of reactive oxygen species in Epas1-/- mice. Nat. Genet. 35,331-340[CrossRef][Medline]
  26. Larcher, F., Murillas, R., Bolontrade, M., Conti, C. J., Jorcano, J. L. (1998) VEGF/VPF overexpression in skin of transgenic mice induces angiogenesis, vascular hyperpermeability and accelerated tumor development. Oncogene 17,303-311[CrossRef][Medline]
  27. Thurston, G., Suri, C., Smith, K., McClain, J., Sato, T. N., Yancopoulos, G. D., McDonald, D. M. (1999) Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science 286,2511-2514[Abstract/Free Full Text]
  28. Elson, D. A., Thurston, G., Huang, L. E., Ginzinger, D. G., McDonald, D. M., Johnson, R. S., Arbeit, J. M. (2001) Induction of hypervascularity without leakage or inflammation in transgenic mice overexpressing hypoxia-inducible factor-1alpha. Genes Dev. 15,2520-2532[Abstract/Free Full Text]
  29. Asikainen, T. M., Ahmad, A., Schneider, B. K., White, C. W. (2005) Effect of preterm birth on hypoxia-inducible factors and vascular endothelial growth factor in primate lungs. Pediatr. Pulmonol. 40,538-546[CrossRef][Medline]
  30. Asikainen, T. M., Ahmad, A., Schneider, B. K., Ho, W.-B., Arend, M., Brenner, M., Günzler, V., White, C. W. (2005) Stimulation of HIF-1alpha, HIF-2alpha, and VEGF by prolyl 4-hydroxylase inhibition in human lung endothelial and epithelial cells. Free Radic. Biol. Med. 38,1002-1013[CrossRef][Medline]
  31. Asikainen, T. M., Schneider, B. K., Waleh, N. S., Clyman, R. I., Ho, W. B., Flippin, L. A., Günzler, V., White, C. W. (2005) Activation of hypoxia-inducible factors in hyperoxia through prolyl 4-hydroxylase blockade in cells and explants of primate lung. Proc. Natl. Acad. Sci. U. S. A. 102,10212-10217[Abstract/Free Full Text]
  32. Asikainen, T. M., Waleh, N. S., Schneider, B. K., Clyman, R. I., White, C. W. (2006 May 5) Enhancement of angiogenic effectors through hypoxia-inducible factor in preterm primate lung in vivo. Am. J. Physiol. Lung Cell Mol. Physiol. [Epub ahead of print] PMID: 16679381
  33. Yoder, B., Martin, H., McCurnin, D. C., Coalson, J. J. (2002) Impaired urinary cortisol excretion and early cardiopulmonary dysfunction in immature baboons. Pediatr. Res. 51,426-432[Medline]
  34. Tschanz, S. A., Burri, P. H. (2002) A new approach to detect structural differences in lung parenchyma using digital image analysis. Exp. Lung Res. 28,457-471[CrossRef][Medline]
  35. Afshar, S., Gibson, L. L., Yuhanna, I. S., Sherman, T. S., Kerecman, J. D., Grubb, P. H., Yoder, B. A., McCurnin, D. C., Shaul, P. W. (2003) Pulmonary NO synthase expression is attenuated in a fetal baboon model of chronic lung disease. Am. J. Physiol. 284,L749-L758
  36. Waleh, N., Kajino, H., Marrache, A. M., Ginzinger, D., Roman, C., Seidner, S. R., Moss, T. J., Fouron, J. C., Vazquez-Tello, A., Chemtob, S., Clyman, R. I. (2004) Prostaglandin E2-mediated relaxation of the ductus arteriosus: effects of gestational age on G protein-coupled receptor expression, signaling, and vasomotor control. Circulation 110,2326-2332[Abstract/Free Full Text]
  37. Mason, R. J., Nellenbogen, J., Clements, J. A. (1976) Isolation of disaturated phosphatidylcholine with osmium tetroxide. J. Lipid Res. 17,281-284[Abstract]
  38. Ikegami, M., Le Cras, T. D., Hardie, W. D., Stahlman, M. T., Whitsett, J. A., Korfhagen, T. R. (2005) TGF-alpha perturbs surfactant homeostasis in vivo. Am. J. Physiol. 289,L34-L43
  39. Clark, J. C., Weaver, T. E., Iwamoto, H. S., Ikegami, M., Jobe, A. H., Hull, W. M., Whitsett, J. A. (1997) Decreased lung compliance and air trapping in heterozygous SP-B-deficient mice. Am. J. Respir. Cell Mol. Biol. 16,46-52[Medline]
  40. Walisser, J. A., Bunger, M. K., Glover, E., Harstad, E. B., Bradfield, C. A. (2004) Patent ductus venosus and dioxin resistance in mice harboring a hypomorphic Arnt allele. J. Biol. Chem. 279,16326-16331[Abstract/Free Full Text]
  41. Thébaud, B., Ladha, F., Michelakis, E. D., Sawicka, M., Thurston, G., Eaton, F., Hashimoto, K., Harry, G., Haromy, A., Korbutt, G., Archer, S. L. (2005) Vascular endothelial growth factor gene therapy increases survival, promotes lung angiogenesis, and prevents alveolar damage in hyperoxia-induced lung injury: evidence that angiogenesis participates in alveolarization. Circulation 112,2477-2486[Abstract/Free Full Text]
  42. Kunig, A. M., Balasubramaniam, V., Markham, N. E., Morgan, D., Montgomery, G., Grover, T. R., Abman, S. H. (2005) Recombinant human VEGF treatment enhances alveolarization after hyperoxic lung injury in neonatal rats. Am. J. Physiol. 289,L529-L535
  43. Dulak, J., Józkowicz, A. (2003) Regulation of vascular endothelial growth factor synthesis by nitric oxide: facts and controversies. Antioxid. Redox Signal. 5,123-132[CrossRef][Medline]
  44. McCurnin, D. C., Yoder, B. A., Coalson, J., Grubb, P., Kerecman, J., Kupferschmid, J., Breuer, C., Siler-Khodr, T., Shaul, P. W., Clyman, R. (2005) Effect of ductus ligation on cardiopulmonary function in premature baboons. Am. J. Respir. Crit. Care Med. 172,1569-1574[Abstract/Free Full Text]
  45. Levin, M., McCurnin, D., Seidner, S. R., Yoder, B., Waleh, N., Goldbarg, S., Roman, C., Liu, B. M., Boren, J., Clyman, R. I. (2006) Postnatal constriction, ATP depletion, and cell death in the mature and immature ductus arteriosus. Am. J. Physiol. 290,R359-R364
  46. Ohlsson, A., Walia, R., Shah, S. (2005) Ibuprofen for the treatment of patent ductus arteriosus in preterm and/or low birth weight infants. Cochrane Database Syst. Rev. CD003481
  47. Clyman, R. I., Seidner, S. R., Kajino, H., Roman, C., Koch, C. J., Ferrara, N., Waleh, N., Mauray, F., Chen, Y. Q., Perkett, E. A., Quinn, T. (2002) VEGF regulates remodeling during permanent anatomic closure of the ductus arteriosus. Am. J. Physiol. 282,R199-R206
  48. Günzler, V., Muthukrishnan, E., Neumayer, H. H., Sacherer, K., Schmidt, R., Mitzner, A., Wiecek, A., Piecha, G., Ignacy, W., Scigalla, P. (2006) FG-2216 increases hemoglobin concentration in anemic patients with chronic kidney disease. Keystone Symposia: Hypoxia and development, physiology and disease, A216 Breckenridge Colorado, U. S. A..
  49. Klaus, S., Arend, M., Flippin, L., Fourney, P., Gervasi, D., Günzler, V., Guo, G., Langsetmo, I., Lin, A., Lomongsod, E., Spong, S., Wang, Q., Liu, D. (2006) Induction of erythropoiesis and iron utilization by the HIF prolyl hydroxylase inhibitor FG-4592. Keystone Symposia: Hypoxia and development, physiology and disease, A241 Breckenridge Colorado, U. S. A..
  50. Peyssonnaux, C., Datta, V., Cramer, T., Doedens, A., Theodorakis, E. A., Gallo, R. L., Hurtado-Ziola, N., Nizet, V., Johnson, R. S. (2005) HIF-1alpha expression regulates the bactericidal capacity of phagocytes. J. Clin. Invest. 115,1806-1815[CrossRef][Medline]
  51. Walmsley, S. R., Print, C., Farahi, N., Peyssonnaux, C., Johnson, R. S., Cramer, T., Sobolewski, A., Condliffe, A. M., Cowburn, A. S., Johnson, N., Chilvers, E. R. (2005) Hypoxia-induced neutrophil survival is mediated by HIF-1alpha-dependent NF-kappaB activity. J. Exp. Med. 201,105-115[Abstract/Free Full Text]
  52. Le Cras, T. D., Spitzmiller, R. E., Albertine, K. H., Greenberg, J. M., Whitsett, J. A., Akeson, A. L. (2004) VEGF causes pulmonary hemorrhage, hemosiderosis, and air space enlargement in neonatal mice. Am. J. Physiol. 287,L134-L142



This article has been cited by other articles:


Home page
 BloodHome page
M. M. Hsieh, N. S. Linde, A. Wynter, M. Metzger, C. Wong, I. Langsetmo, A. Lin, R. Smith, G. P. Rodgers, R. E. Donahue, et al.
HIF prolyl hydroxylase inhibition results in endogenous erythropoietin induction, erythrocytosis, and modest fetal hemoglobin expression in rhesus macaques
Blood, September 15, 2007; 110(6): 2140 - 2147.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
F. A. Groenman, M. Rutter, J. Wang, I. Caniggia, D. Tibboel, and M. Post
Effect of chemical stabilizers of hypoxia-inducible factors on early lung development
Am J Physiol Lung Cell Mol Physiol, September 1, 2007; 293(3): L557 - L567.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
T. M. Asikainen and C. W. White
HIF stabilizing agents: shotgun or scalpel?
Am J Physiol Lung Cell Mol Physiol, September 1, 2007; 293(3): L555 - L556.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
T. R. Grover, T. M. Asikainen, J. P. Kinsella, S. H. Abman, and C. W. White
Hypoxia-inducible factors HIF-1{alpha} and HIF-2{alpha} are decreased in an experimental model of severe respiratory distress syndrome in preterm lambs
Am J Physiol Lung Cell Mol Physiol, June 1, 2007; 292(6): L1345 - L1351.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
B. Thebaud and S. H. Abman
Bronchopulmonary Dysplasia: Where Have All the Vessels Gone? Roles of Angiogenic Growth Factors in Chronic Lung Disease
Am. J. Respir. Crit. Care Med., May 15, 2007; 175(10): 978 - 985.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Summary
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
fj.06-5887fjev1
20/10/1698    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles