Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation. 2005;112:2477-2486
doi: 10.1161/CIRCULATIONAHA.105.541524
CLINICAL PERSPECTIVE
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
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 Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thébaud, B.
Right arrow Articles by Archer, S. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thébaud, B.
Right arrow Articles by Archer, S. L.
Related Collections
Right arrow Pulmonary circulation and disease
Right arrow Gene therapy
Right arrow Other Vascular biology
Right arrow Angiogenesis
Right arrow Animal models of human disease
Right arrow Growth factors/cytokines

(Circulation. 2005;112:2477-2486.)
© 2005 American Heart Association, Inc.

Vascular Medicine

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

Bernard Thébaud, MD, PhD; Faruqa Ladha, BSc; Evangelos D. Michelakis, MD; Monika Sawicka, BSc; Gavin Thurston, MD; Farah Eaton, BSc; Kyoko Hashimoto, BSc; Gwyneth Harry, MSc; Alois Haromy, BSc; Greg Korbutt, PhD; Stephen L. Archer, MD

From the Vascular Biology Research Group (B.T., F.L., E.D.M., M.S., F.E., K.H., G.H., A.H., S.L.A.), the Department of Pediatrics, Division of Neonatology (B.T.), the Department of Medicine (E.D.M., S.L.A.), the Surgical-Medical Research Institute (G.K.), and the Department of Physiology (S.L.A.), University of Alberta, Edmonton, Canada; and Regeneron Pharmaceuticals Inc, Tarrytown, NY (G.T.).

Correspondence to Dr Bernard Thébaud, MD, PhD, University of Alberta, HMRC 407, Edmonton, Alberta, T6G 2S2, Canada. E-mail bthebaud{at}ualberta.ca

Received February 8, 2005; revision received June 6, 2005; accepted July 5, 2005.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background— Bronchopulmonary dysplasia (BPD) and pulmonary emphysema, both significant global health problems, are characterized by a loss of alveoli. Vascular endothelial growth factor (VEGF) is a trophic factor required for endothelial cell survival and is abundantly expressed in the lung.

Methods and Results— We report that VEGF blockade decreases lung VEGF and VEGF receptor 2 (VEGFR-2) expression in newborn rats and impairs alveolar development, leading to alveolar simplification and loss of lung capillaries, mimicking BPD. In hyperoxia-induced BPD in newborn rats, air space enlargement and loss of lung capillaries are associated with decreased lung VEGF and VEGFR-2 expression. Postnatal intratracheal adenovirus-mediated VEGF gene therapy improves survival, promotes lung capillary formation, and preserves alveolar development in this model of irreversible lung injury. Combined VEGF and angiopoietin-1 gene transfer matures the new vasculature, reducing the vascular leakage seen in VEGF-induced capillaries.

Conclusions— These findings underscore the importance of the vasculature in what is traditionally thought of as an airway disease and open new therapeutic avenues for lung diseases characterized by irreversible loss of alveoli through the modulation of angiogenic growth factors.


Key Words: angiogenesis • lung • gene therapy • pediatrics • oxygen


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Preterm delivery is a major health care problem, affecting 10% of all births and accounting for more than 85% of all perinatal complications and deaths.1 Survival of extremely premature newborns (those born at <28 weeks of gestation) has increased because of improvements in perinatal care.2 These infants, however, are at high risk for long-term injury to both lung and brain.3 Each year, 5000 to 10 000 newborns suffer from bronchopulmonary dysplasia (BPD) (http://www.nhlbi.nih.gov/health/dci/Diseases/Bpd/Bpd_WhoIsAtRisk.html), a chronic lung disease that follows ventilator and oxygen therapy for acute respiratory failure after premature birth.4 BPD has long-term respiratory5 and neurodevelopmental6,7 consequences that reach beyond childhood and result in increased health care costs.8 Further advances are required to increase survival free of neonatal morbidity or neurodevelopmental impairment. Emphysema, defined as airspace enlargement distal to terminal bronchioles, is a major component of chronic obstructive pulmonary disease, the fourth leading cause of death in the United States.9 BPD and emphysema are characterized by interrupted development and loss of alveolar structures, and therapy is palliative.

Editorial p 2383 Clinical Perspective p 2486

Perinatal lung injury in neonates born during the late canalicular stage of lung development disrupts the normal sequence of lung growth, resulting in a histological pattern of "alveolar simplification" (fewer and larger alveoli with fewer septae), loss of small pulmonary arteries, and decreased capillary density. Structural abnormalities closely mimicking human BPD have been demonstrated in a newborn rat model of BPD caused by exposure to hyperoxia.10–12 The mechanisms and signal-transduction pathways that regulate the normal alveolar development remain poorly understood, and even less is known about how these pathways are altered in disease. Interactions between airways and blood vessels are critical for normal lung development, suggesting that a coordinated and timely release of vascular-specific growth factors from respiratory epithelial cells promotes alveolar development.

Vascular endothelial growth factor (VEGF) is crucial for blood vessel formation.13–16 In the lung, VEGF mRNA and protein are localized to distal airway epithelial cells and the basement membrane subjacent to the airway epithelial cells.17 This suggests that translocation of VEGF protein occurs after its synthesis in the epithelium. VEGF is present in alveolar type II cells in the developing mouse lung, and its expression peaks during the canalicular stage, when most of the vessel growth occurs in the lung, then decreases until day 10 postnatal (P10), when it plateaus at adult levels.18 VEGF receptor 1 (VEGFR-1) and VEGFR-2 mRNA expression also increases during normal mouse lung development,19,20 and these receptors are localized on pulmonary endothelial cells, closely apposed to the developing epithelium.18 This spatial relationship suggests that VEGF plays a role in the development of the alveolar capillary bed. Consistent with this, infants who die of BPD have little or no VEGF in their lung epithelium, and their pulmonary vasculature lacks VEGF receptors.21 On the basis of the normal interactions between airways and blood vessels in lung development and the observed abnormalities in both the vasculature and airways in BPD, we hypothesized that (1) VEGF-driven angiogenesis is crucial for normal alveolarization; (2) oxygen-induced experimental BPD impairs angiogenesis and decreases alveolarization by decreasing lung VEGF expression; and (3) overexpression of VEGF enhances lung angiogenesis, thereby promoting alveolarization and reversing established BPD.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Pharmacological VEGF Inhibition
VEGF-TrapR1/R2 (VEGF-Trap) (25 mg/kg; Regeneron Pharmaceuticals), a soluble combined truncated form of VEGFR-1 and VEGFR-2 fused to the Fc portion of immunoglobulin G,22 was administered subcutaneously to rat pups at P4, P7, and P10.

Endothelial Network Formation Assay
The formation of cord-like structures by human pulmonary artery endothelial cells (HPAECs) was assessed on Matrigel-coated wells. HPAECs (80 000 cells/well) were seeded into 24-well plates coated with Matrigel (BD Biosciences). The media (m200, Cascade Biologics) were supplemented with the agents (VEGF-Trap, hFc, or adenoviral constructs) and incubated at 37°C for 8 to 12 hours in normoxia (21%) or hyperoxia (95%). Cord-like structures were observed with an inverted phase contrast microscope (Olympus) and quantified by measuring the number of intersects and the length of structures in random fields from each well using OpenLab (Quorum Technologies Inc).

Oxygen-Induced Lung Injury
All procedures were approved by the Animal Health Care Committee of the University of Alberta. Rat pups were exposed to normoxia (21%, control group) or hyperoxia (95% O2, BPD group) from birth to P14 in sealed Plexiglas chambers (BioSpherix) with continuous O2 monitoring. Dams were switched every 48 hours between the hyperoxic and normoxic chambers to prevent damage to their lungs23 and to provide equal nutrition to each litter. Litter size was adjusted to 11 pups to control for effects of litter size on nutrition and growth. Rat pups were euthanized at various ages with intraperitoneal pentobarbital, and lungs and heart were processed according to the experiments performed.

Lung Morphometry
Lungs were fixed with a 4% glutaraldehyde solution through the trachea under a constant pressure of 20 cm H2O. The trachea was then ligated, and the lungs were immersed in fixative overnight at 4°C. Lung volume was measured by water displacement.24 Lungs were processed and embedded in paraffin. Serial step sections, 4 µm in thickness, were taken along the longitudinal axis of the lobe. The fixed distance between the sections was calculated so as to allow systematic sampling of 10 sections across the whole lobe. Lungs were stained with hematoxylin and eosin (H&E). Alveolar structures were quantified on a motorized microscope stage using the mean linear intercept method.25

Scanning Electron Microscopy on Lung Vascular Casts
A freshly prepared mixture of Mercox catalyst and resin (Ladd Research Industries) at a 50:1 ratio was injected into the main pulmonary artery through a 30-gauge needle until the left cardiac atrium was filled with Mercox. To clear soft tissue, the injected lungs were placed in 20% KOH for 2 days, with daily changes of solution. The vascular casts were rinsed in distilled water, air-dried in an oven at 40°C for 1 hour, and mounted on a stub. After having been sputter-coated with gold (Edwards S150B; Edwards), the samples were examined by a Hitachi SEM S-2500.

Lectin Staining of Lung Endothelium
FITC Lycopersicon esculentum lectin (100 µg/20 g in 0.5 mL, Vector Laboratories) was injected into the pulmonary artery, followed by a perfusion with 1% PFA in PBS, pH 7.4, at 30 mm Hg for 2 minutes. Lungs were then inflated through a tracheotomy with warmed 1% low-melt agarose, cooled with iced PBS, and embedded in warmed 3% agarose for sectioning. Thick sections (80 to 120 µm) were mounted in Vectashield and visualized under a confocal microscope.

Barium-Gelatin Angiograms and Arterial Density Counts
A barium-gelatin mixture (60°C) was infused at 70 mm Hg pressure into the main pulmonary artery catheter for 3 to 4 minutes until surface filling of vessels with barium was seen uniformly over the surface of the lung.10 At this pressure, the barium evenly filled arteries and capillaries. The main pulmonary artery was tied off under pressure, and the lungs were inflation-fixed with formalin. The right upper lobe was embedded in paraffin, and sections were cut and stained with H&E. Barium-filled pulmonary capillaries were counted per high-powered field (x100 magnification). Four to 5 lungs per animal, 5 sections per lung, and 10 high-power fields per section were counted.

Immunoblotting
Protein expression in whole lungs was measured with immunoblotting using available antibodies, as previously described.26 The intensity of the bands was normalized to the intensity of a reporter protein (actin) using the Kodak Gel-doc system.

Laser Capture Microdissection
To quantify mRNA in various lung compartments, laser capture microdissection (LCM) was performed on frozen sections by use of the microdissector PixCell II (Arcturus Engineering).27

Quantitative Real-Time Polymerase Chain Reaction
LCM specimens were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) using specific primers. Primers for each gene were designed by use of Primer Express software. Total RNA was extracted using an RNEasy Mini Kit (Qiagen). The TaqMan One-Step RT-PCR Master Mix reagent kit (Applied Biosystems) was used to quantify the copy number of cDNA targets. Levels of mRNA were normalized to a housekeeping gene (18S rRNA) and expressed as 2{Delta}{Delta}Ct, as described previously.27

Preparation of Recombinant Adenoviral Vector
We used a recombinant adenovirus (serotype 5) vector that was rendered replication-deficient by virtue of deletions of the E1 and E3 genes.28 Ad5-encoding genes, each under a cytomegalovirus promoter, were generously provided by GenVec, Gaithersburg, MA (for green fluorescent protein [GFP] and for VEGF145-GFP) and by Regeneron Pharmaceuticals (for angiopoietin-1 [Ang*1]).

In Vivo Ad5 Intratracheal Administration
Gene delivery into the airways of newborn rats was performed through intratracheal puncture at P4 or P21. After halothane anesthesia, the trachea was exposed through a neck incision. The gene of interest (25 µL) was delivered through a tracheal puncture with a short, 30-gauge needle (Becton-Dickinson). After closure of the incision with biological glue (Vetbond, 3 mol/L), rats were allowed to recover.

Statistics
Values are expressed as the mean±SEM. Intergroup differences were assessed by Student’s paired t test or a factorial ANOVA, as appropriate. Post hoc analysis used a Fisher’s probable least significant difference test (Statview 5.1, Abacus Concepts). Survival curves were derived by the Kaplan-Meier method, and differences evaluated by log-rank tests. A value of P<0.05 was considered statistically significant.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
VEGF Inhibition Decreases Lung Angiogenesis and Impairs Alveolarization
In an in vitro endothelial network formation assay, inhibition of VEGF signaling by VEGF-Trap decreased cord-like structure formation by HPAECs (Figure 1A). In vivo, subcutaneous injection of VEGF-Trap in rat pups between postpartum days 4 and 14 (P4–14, during the alveolar period of lung development) reduced overall body weight and decreased the ratio of lung weight to body weight, highlighting the importance of VEGF for proper organ development in general and lung development in particular (Figure 1B). VEGF-Trap decreased lung VEGF and VEGFR-2 protein expression (Figure 1B), and this was associated with a decreased number of lung capillaries (Figure 1C) and enlarged air spaces, reminiscent of BPD/emphysema (Figure 1D).



View larger version (81K):
[in this window]
[in a new window]
  		Figure 1. VEGFR inhibition decreases lung angiogenesis in vitro and in vivo and impairs alveolarization. A, HPAECs seeded on Matrigel form a delicate endothelial network that is detectable within 2 to 3 hours and are fully developed after 8 to 12 hours. Quantitative assessment of cord-like structure formation show a significant decrease in number of intersects and total length of cord-like structures in VEGF-Trap–exposed HPAECs compared with controls and inactive hFc-treated HPAECs. B, In vivo, VEGF-Trap decreased overall body weight and the ratio of lung weight to body weight (LW/BW) compared with control and hFc-treated animals. VEGF-Trap decreased lung VEGF and VEGFR-2 expression. C, Scanning electron microscopy of Mercox casts of the pulmonary vasculature show that VEGF-Trap–treated lungs have a significantly less dense vascular network compared with control and hFc-treated lungs. Decreased capillary density in VEGF-inhibited lungs is confirmed by the quantification of barium angiograms. D, Representative H&E–stained lung sections showing larger and fewer alveoli in VEGF-Trap–treated lungs. Decreased alveolarization is quantified by use of the mean linear intercept (Lm), which was significantly increased in VEGF-Trap versus control and hFc.

Impaired Alveolarization in Oxygen-Induced Lung Injury Is Associated With Decreased Lung Angiogenesis and VEGF Expression
Exposure of newborn rats to hyperoxia (95%) from birth to P14 impaired alveolar growth, as assessed by the mean linear intercept (Lm) (Figure 2A). Lung capillaries were visualized by labeling the pulmonary endothelium with FITC-L. esculentum lectin injected into the pulmonary artery at P14. Pups housed in room air showed a dense capillary network, whereas those exposed to hyperoxia had a rarified capillary network (Figure 2B). The quantification of arterial density with barium angiograms confirmed decreased numbers of pulmonary vessels in hyperoxia (Figure 2B). In this model, enlarged air spaces and decreased lung angiogenesis were associated with decreased lung VEGF and VEGFR-2 expression (Figure 2C).



View larger version (60K):
[in this window]
[in a new window]
  		Figure 2. Hyperoxia impairs alveolar and vascular growth and decreases lung VEGF and VEGFR-2 expression. A, Exposure of rat pups to hyperoxia during the critical period of alveolar development results in arrested alveolar growth. Representative H&E–stained lung sections show larger and fewer alveoli in hyperoxia-exposed lungs, resulting in a significantly higher Lm than in control-room air–exposed animals. B, Fluorescent lung angiography using FITC-lectin shows that hyperoxia exposure is associated with a much less dense capillary network compared with room-air–housed animals. Barium angiogram quantification confirms decreased lung capillary density in hyperoxia-treated animals. C, Hyperoxia-induced decreased alveolarization and angiogenesis is associated with diminished lung VEGF and VEGFR-2 expression.

In Vivo Intratracheal VEGF145 Gene Therapy Enhances Survival and Promotes Alveolarization in Irreversible Oxygen-Induced BPD
First, we showed the effect of VEGF gene therapy in vitro in the endothelial network formation assay. Hyperoxia decreased endothelial cord-like structure formation by HPAECs; VEGF gene transfer significantly counteracted the effect of oxygen and promoted endothelial network formation (Figure 3A). Because VEGF inhibition decreases alveolarization and because oxygen-induced hypoalveolarization is associated with decreased VEGF expression, we investigated the therapeutic potential of VEGF gene therapy to restore normal alveolarization in the rat BPD model. Exposure of newborn rats from birth to P14 to hyperoxia (95%) results in impaired alveolar growth and vascular abnormalities that persist into "adult" life.10–12 Animals were randomized into 4 groups: (1) control, room air; (2) hyperoxia (O2); (3) O2+intratracheal administration of a replication-deficient adenovirus carrying genes for the human VEGF145 isoform and the GFP reporter (O2+Ad5-VEGF145) administered at P4; and (4) O2+Ad5 carrying GFP only (O2+Ad5-GFP). Intratracheal gene transfer resulted in efficient GFP expression in the distal lung (Figure 3B). VEGF145 gene therapy significantly increased lung VEGF protein expression (Figure 3B). VEGF145 gene expression was highest 4 days after infection and declined gradually over a period of 21 days (Figure 3B). Laser capture microdissection showed compartmentalized VEGF145 gene expression, with maximal transgene expression in bronchi and lower expression in pulmonary arteries and alveoli (Figure 3C). No VEGF145 mRNA was detected in the other groups. VEGF gene transfer was also associated with increased alveolar endothelial nitric oxide synthase (eNOS) expression (Figure 3C).



View larger version (74K):
[in this window]
[in a new window]
  		Figure 3. Intratracheal gene therapy with VEGF145 results in efficient distal lung gene expression. A, Hyperoxia decreases lung endothelial network formation on Matrigel. Quantitative assessment of cord-like structure formation shows a significant decrease in number of intersects and total cord-structure length in hyperoxia-exposed HPAECs. Ad5-VEGF145 but not Ad5-GFP restores the number of intersects and total cord-structure length to control values. B, Intratracheal injection of Ad5-GFP at P4 results in efficient distal lung expression as visualized by confocal microscopy 5 days after injection. No green fluorescence is seen in a saline-injected lung. Ad5-mediated gene therapy of VEGF145 restored deficient VEGF expression in hyperoxic lungs. Lung mRNA for VEGF145 was maximal 4 days after gene therapy and declined gradually over the next 3 weeks. C, qRT-PCR on different lung compartment retrieved by laser capture microdissection revealed that intratracheal gene transfer resulted in maximal VEGF145 mRNA expression in bronchi (Br) and lower expression in pulmonary vessels (V) and alveoli (Alv).

In vivo, VEGF gene transfer at P4 improved survival (Figure 4A) compared with O2 and O2+Ad5-GFP–exposed animals. Ad5-VEGF145–treated animals had increased lung capillary density (Figure 4B) and preserved alveolarization (Figure 4C), compared with O2- and O2+Ad5-GFP–exposed animals. Because this model is thought to produce an irreversible lung injury,10–12 we also tested the ability of VEGF gene transfer to reverse established lung hypoalveolarization by overexpressing VEGF after the insult. In this rescue experiment, VEGF gene therapy performed at P21 significantly improved the lung architecture (Lm=39±2 µm) compared with O2-exposed (Lm=44±4, P<0.01) and O2+Ad5-GFP–exposed (Lm=43±1, P<0.01) animals 2 weeks after treatment and was not different from control (Lm=36±2, P=0.08) (Figure 4 D).



View larger version (98K):
[in this window]
[in a new window]
  		Figure 4. In vivo VEGF145 gene therapy enhances survival, promotes lung angiogenesis, and restores alveolar growth in experimental BPD. A, Kaplan-Meier curve showing increased survival in Ad5-VEGF145–treated animals compared with O2- and O2+Ad5-GFP–exposed animals (log-rank test, P<0.02). B, Representative barium angiograms at P21 show decreased capillary density in oxygen-exposed lungs. In vivo intratracheal Ad5-VEGF145 gene therapy at P4 but not Ad5-GFP restores arterial density to control values. C, Oxygen-exposed lungs display the characteristic features of alveolar simplification (larger and fewer air sacs with decreased septation). Lungs exposed to oxygen and injected with Ad-GFP show the same histological characteristics. Conversely, in vivo intratracheal Ad5-VEGF145 gene therapy resulted in smaller and more numerous alveoli. Quantification of alveoli structures using the Lm confirms improved alveolarization in VEGF-treated animals compared with the other oxygen-exposed groups. D, Representative H&E–stained lung sections of P35 old rats shows that even when administered after the insult, VEGF145 gene therapy at P21 rescues the loss of alveoli and improves alveolarization within 2 weeks.

Scanning electron microscopy revealed that VEGF-induced lung capillaries, although more numerous than in BPD lungs, are fenestrated and appear immature (Figure 5). Because the angiogenic growth factor Ang-1 is crucial for subsequent vessel stabilization and maturation, we hypothesized that combined VEGF and Ang-1 gene transfer would alleviate the enhanced permeability of VEGF-induced angiogenesis. Combined Ad5-VEGF145+Ang*1 gene therapy given at P4 improved survival (88%), increased lung capillary formation (Figure 6A), and improved lung architecture (Figure 6B) compared with hyperoxia. Pulmonary vessels were less fenestrated compared with Ad5-VEGF145–treated animals (Figure 6A). Combined Ad5-VEGF145+Ang*1 gene therapy was also associated with a decreased wet/dry lung weight compared with O2-, O2+Ad5-GFP–, and O2+Ad5-VEGF145–exposed animals (Figure 6C), suggesting decreased vessel permeability.



View larger version (102K):
[in this window]
[in a new window]
  		Figure 5. Hyperoxia-induced decrease in capillary density is restored by VEGF therapy. Mercox casts of the lung capillary bed show that intratracheal gene therapy with VEGF145 promotes lung angiogenesis and restores a denser capillary network in BPD rats. At higher magnification, capillaries in VEGF145-treated lungs are fenestrated and display an immature, leaky phenotype.



View larger version (76K):
[in this window]
[in a new window]
  		Figure 6. Combined VEGF and Ang gene therapy creates a more mature angiogenic phenotype and improves alveolarization. A, Mercox casts of the lung capillary bed examined under the scanning electron microscope show that oxygen-exposed lungs treated with combined Ad5-VEGF145 and Ang*1 gene therapy have a dense capillary network with no or little fenestration (in contrast to the effects of VEGF alone, Figure 5). B, Combined Ad5-VEGF145 and Ang*1 gene therapy also improved alveolarization. C, Combined Ad5-VEGF145 and Ang*1 gene therapy decreased the lung wet/dry weight ratio compared with Ad5-VEGF145 lungs, suggesting a beneficial effect of Ang-1 on permeability.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Here, we show that VEGF-driven angiogenesis is critical for normal lung alveolar development and demonstrate the therapeutic potential of postnatal, intratracheal gene transfer for lung diseases that are currently viewed as having irreversible loss of alveoli. As proof of concept, we showed that inhibition of VEGF with a very potent high-affinity blocker impaired alveolarization, mimicking BPD/emphysema. Likewise, irreversible oxygen-induced hypoalveolarization was associated with decreased vascular growth and VEGF expression. Furthermore, VEGF overexpression through intratracheal VEGF145 gene therapy improved survival and was able to preserve and restore normal alveolarization in this model, even when given late in the disease.

In 1959, Liebow observed that the alveolar septa in centrilobular emphysema were remarkably thin and almost avascular.29 He postulated that a reduction in the blood supply of the small precapillary blood vessels might induce the disappearance of alveolar septa. Despite this early observation, pulmonary vessels were thought for many years to be passive bystanders in lung development, following the branching pattern of the airways. Histological observations showed that endothelial tubes line up around the terminal buds of the airways, suggesting an inductive influence on the part of the epithelium.30 Recent data suggest that angiogenesis is actively involved in distal lung development. Inactivation studies of VEGF alleles13,16 and knockouts of VEGFR-114 and -215 result in lethal embryonic phenotypes characterized by deficient organization of endothelial cells. Mice with a targeted deletion of the VEGF heparin-binding isoforms VEGF164 and VEGF188, however, are viable and display a variety of vascular defects, including a significant reduction in the formation of air spaces and capillaries, resulting in delayed airway maturation.31 Conversely, excessive expression of VEGF in the normal developing lung disrupts lung architecture and causes pulmonary hemorrhage and hemosiderosis.32 VEGF-induced angiogenesis is in part mediated by NO. Lungs of eNOS-deficient mice exhibit a paucity of distal arteriolar branches and misalignment of pulmonary veins, characteristic features of alveolar capillary dysplasia.33 In this study, we used VEGF-Trap, a potent and specific VEGF blocker,22 to study the role of VEGF-driven angiogenesis on normal alveolar development. Rat pups treated during the alveolar period of lung development (P4–14) with VEGF-Trap displayed decreased vascular growth associated with enlarged airspaces reminiscent of BPD/emphysema (Figure 1B and 1C). In our study, chronic VEGF inhibition was also accompanied by a strong downregulation of VEGFR-2 protein. Our interpretation is that VEGF has a feedback to induce VEGFR-2. Prolonged lack of VEGFR-2 stimulation by VEGF (14 days) may have resulted in a downregulation of VEGFR-2. Our results are consistent with previous reports showing that a VEGFR-2 aptamer or antiangiogenic agents (such as fumagillin and thalidomide) given to rat pups impair alveolar and lung vascular development.34 In adult rats, disruption of VEGF signaling (using the VEGFR-1 and -2 blocker SU5416) leads to enlargement of the air spaces, indicative of emphysema,35 suggesting that VEGF is required for the maintenance of both the pulmonary vasculature and alveolar structures throughout adulthood. We show that the reverse is also true: in this model of established hypoalveolarization, ie, the hyperoxic BPD model, vascular growth is arrested and VEGF signaling is impaired (Figure 2). This is consistent with experimental BPD in other species36,37 and with findings in humans.21 Whether inhibition of vascular growth may be a cause rather than simply a consequence of impaired alveolarization remains controversial. Nonetheless, these data suggest a therapeutic potential for angiogenic growth factor modulation in lung diseases characterized by alveolar damage.

To explore this possibility, we used the hyperoxic BPD model of lung injury,10–12 hypothesizing that Ad5-mediated overexpression of VEGF would promote lung angiogenesis and thereby stimulate alveolarization. Traditionally, the transgene expression induced by Ad5-mediated gene transfer lasts &2 weeks. The transient nature of transgene expression is ideal in our setting because it covers exactly the target period of alveolarization in rats. Gene delivery into the airways of newborn rats was performed intratracheally at P4. Intratracheal injection of the virus has the advantage of locally administering the gene of interest to the target site, ie, the distal airways and small pulmonary arteries, while avoiding dissemination and induction of angiogenesis in extrapulmonary sites, as we showed previously.38 Another strength of this approach of postnatal treatment is that it mimics the clinical setting in which exogenous surfactant is routinely administered to premature infants with hyaline membrane disease.39 Hence, concomitant administration of lung protective/regenerative agents with surfactant through the endotracheal tube in premature infants likely to develop BPD is appealing and clinically relevant.40 The LCM technique allowed quantification of mRNA expression of the gene of interest in each compartment of the lung, being maximal in the bronchi but also expressed to a lesser extent, but equally, in the small pulmonary arteries and alveoli (Figure 3C). VEGF gene transfer also increased alveolar eNOS expression, suggesting that part of the beneficial effect of VEGF might be NO-mediated. Recent data suggest that inhaled NO treatment preserves alveolar architecture in experimental BPD41 and decreases the incidence of BPD in premature infants with respiratory failure.42 VEGF gene therapy given at P4 improved survival and increased lung capillary density and promoted alveolarization (Figure 4). These lung capillaries seen in scanning electron microscopy were dramatically fenestrated, consistent with a leaky phenotype of immature vessels lacking pericyte coating (Figure 5). Similar features have been observed in the lung32 and in other organs in which VEGF was overexpressed.28,43 Normally, VEGF and Ang function together during vascular development, with VEGF acting early during vessel formation and Ang-1 acting later during vessel remodeling, maturation, and stabilization. In the skin, concomitant overexpression of VEGF and Ang-1 alleviates VEGF-induced vascular permeability.28,43 In the present study, lung coexpression of Ang-1 and VEGF also enhanced angiogenesis, and scanning electron microscopic images provide evidence that the combined gene therapy approach resulted in more mature capillaries (Figure 6A) that were less permeable (Figure 6C).

Hypoxia is a major stimulator of VEGF expression.44 Premature exposure of the developing lung to a hyperoxic environment is expected to downregulate VEGF expression. Even ambient O2 levels (21%), ie, premature birth per se, may interfere with normal lung vascular development.45 Hypoxia upregulates VEGF gene transcription by activating the hypoxia-inducible transcription factors HIF-1 and HIF-2, which bind the hypoxia-response element in the VEGF promotor.46,47 HIF-2 is expressed in fetal type 2 pneumocytes.46 Interestingly, newborn mice lacking HIF-2 have deficient lung surfactant and die of respiratory failure.48 The possibility that this pathology may be the result of diminished VEGF expression is suggested by experiments in which administration of VEGF protein resulted in thinning of the alveolar walls, restoration of surfactant production, and reduction of respiratory distress in wild-type mice.48 The study suggests that the pneumotrophic effect of VEGF might have a therapeutic potential for lung maturation in preterm infants at risk for respiratory distress syndrome.

With the use of antenatal steroids and the introduction of exogenous surfactant administration, few premature infants die as a result of acute respiratory distress caused by biochemical lung immaturity (ie, surfactant deficiency); conversely, management of the structural lung immaturity remains problematic, and with improved perinatal care, a new form of lung disease has emerged that lacks specific treatment, resulting in a chronic form of respiratory distress, ie, BPD. The remaining challenge in perinatal medicine is to decrease the incidence/severity of BPD. In this respect, we show that VEGF treatment induces formation of new lung blood vessels, and this treatment improves lung development in an experimental model of chronic lung disease after premature birth.49 Our data provide strong evidence that angiogenesis in general, and VEGF in particular, is necessary for alveolarization during normal lung development and that inhibition of VEGF during a critical period of lung growth contributes to the late sequelae of BPD. Furthermore, our data suggest that modulation of vascular growth factors may have therapeutic potential for lung diseases characterized by irreversible loss of alveolar structures.


*    Acknowledgments
 
Drs Thébaud, Michelakis, and Archer are supported by the Canada Foundation for Innovation, the Alberta Heart and Stroke Foundation, the Alberta Heritage Foundation for Medical Research, the Canadian Institutes for Health Research, and ABACUS. Dr Archer holds a Canada Research Chair in Oxygen Sensing and Translational Cardiovascular Research and is supported by National Institutes of Health grant NIH-RO1-HL071115. Dr Michelakis holds a Canada Research Chair in Pulmonary Hypertension. Dr Thébaud is supported by the Stollery Children’s Hospital Foundation.

Disclosure

Dr Gavin Thurston is an employee of and has stock options in Regeneron Pharmaceuticals, which has a proprietary interest in VEGF trap.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Goldenberg RL, Hauth JC, Andrews WW. Intrauterine infection and preterm delivery. N Engl J Med. 2000; 342: 1500–1507.[Free Full Text]
  2. Goldenberg RL, Jobe AH. Prospects for research in reproductive health and birth outcomes. JAMA. 2001; 285: 633–639.[Abstract/Free Full Text]
  3. Vohr BR, Wright LL, Dusick AM, Mele L, Verter J, Steichen JJ, Simon NP, Wilson DC, Broyles S, Bauer CR, Delaney-Black V, Yolton KA, Fleisher BE, Papile LA, Kaplan MD. Neurodevelopmental and functional outcomes of extremely low birth weight infants in the national institute of child health and human development neonatal research network, 1993–1994. Pediatrics. 2000; 105: 1216–1226.[Abstract/Free Full Text]
  4. Northway WH Jr, Rosan RC, Porter DY. Pulmonary disease following respirator therapy of hyaline-membrane disease: bronchopulmonary dysplasia. N Engl J Med. 1967; 276: 357–368.[Medline] [Order article via Infotrieve]
  5. Allen J, Zwerdling R, Ehrenkranz R, Gaultier C, Geggel R, Greenough A, Kleinman R, Klijanowicz A, Martinez F, Ozdemir A, Panitch HB, Nickerson B, Stein MT, Tomezsko J, Van Der Anker J. Statement on the care of the child with chronic lung disease of infancy and childhood. Am J Respir Crit Care Med. 2003; 168: 356–396.[Free Full Text]
  6. Lemons JA, Bauer CR, Oh W, Korones SB, Papile LA, Stoll BJ, Verter J, Temprosa M, Wright LL, Ehrenkranz RA, Fanaroff AA, Stark A, Carlo W, Tyson JE, Donovan EF, Shankaran S, Stevenson DK. Very low birth weight outcomes of the national institute of child health and human development neonatal research network, January 1995 through December 1996. NICHD Neonatal Research Network. Pediatrics. 2001; 107: E1.[CrossRef][Medline] [Order article via Infotrieve]
  7. Chiriboga CA, Kuban KC, Durkin M, Hinton V, Kuhn L, Sanocka U, Bellinger D. Factors associated with microcephaly at school age in a very-low-birthweight population. Dev Med Child Neurol. 2003; 45: 796–801.[CrossRef][Medline] [Order article via Infotrieve]
  8. McLeod A, Ross P, Mitchell S, Tay D, Hunter L, Hall A, Paton J, Mutch L. Respiratory health in a total very low birthweight cohort and their classroom controls. Arch Dis Child. 1996; 74: 188–194.[Abstract]
  9. Shapiro SD. Vascular atrophy and VEGFR-2 signaling: old theories of pulmonary emphysema meet new data. J Clin Invest. 2000; 106: 1309–1310.[Medline] [Order article via Infotrieve]
  10. Wilson WL, Mullen M, Olley PM, Rabinovitch M. Hyperoxia-induced pulmonary vascular and lung abnormalities in young rats and potential for recovery. Pediatr Res. 1985; 19: 1059–1067.[Medline] [Order article via Infotrieve]
  11. Shaffer SG, O’Neill D, Bradt SK, Thibeault DW. Chronic vascular pulmonary dysplasia associated with neonatal hyperoxia exposure in the rat. Pediatr Res. 1987; 21: 14–20.[Medline] [Order article via Infotrieve]
  12. Randell SH, Mercer RR, Young SL. Neonatal hyperoxia alters the pulmonary alveolar and capillary structure of 40-day-old rats. Am J Pathol. 1990; 136: 1259–1266.[Abstract]
  13. Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, Powell-Braxton L, Hillan KJ, Moore MW. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996; 380: 439–442.[CrossRef][Medline] [Order article via Infotrieve]
  14. Fong GH, Rossant J, Gertsenstein M, Breitman ML. Role of the flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature. 1995; 376: 66–70.[CrossRef][Medline] [Order article via Infotrieve]
  15. Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu XF, Breitman ML, Schuh AC. Failure of blood-island formation and vasculogenesis in flk-1-deficient mice. Nature. 1995; 376: 62–66.[CrossRef][Medline] [Order article via Infotrieve]
  16. Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature. 1996; 380: 435–439.[CrossRef][Medline] [Order article via Infotrieve]
  17. Healy AM, Morgenthau L, Zhu X, Farber HW, Cardoso WV. VEGF is deposited in the subepithelial matrix at the leading edge of branching airways and stimulates neovascularization in the murine embryonic lung. Dev Dyn. 2000; 219: 341–352.[CrossRef][Medline] [Order article via Infotrieve]
  18. Ng YS, Rohan R, Sunday ME, Demello DE, D’Amore PA. Differential expression of VEGF isoforms in mouse during development and in the adult. Dev Dyn. 2001; 220: 112–121.[CrossRef][Medline] [Order article via Infotrieve]
  19. Gebb SA, Shannon JM. Tissue interactions mediate early events in pulmonary vasculogenesis. Dev Dyn. 2000; 217: 159–169.[CrossRef][Medline] [Order article via Infotrieve]
  20. Kalinichenko VV, Lim L, Stolz DB, Shin B, Rausa FM, Clark J, Whitsett JA, Watkins SC, Costa RH. Defects in pulmonary vasculature and perinatal lung hemorrhage in mice heterozygous null for the forkhead box f1 transcription factor. Dev Biol. 2001; 235: 489–506.[CrossRef][Medline] [Order article via Infotrieve]
  21. Bhatt AJ, Pryhuber GS, Huyck H, Watkins RH, Metlay LA, Maniscalco WM. 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. 2001; 164: 1971–1980.[Abstract/Free Full Text]
  22. Holash J, Davis S, Papadopoulos N, Croll SD, Ho L, Russell M, Boland P, Leidich R, Hylton D, Burova E, Ioffe E, Huang T, Radziejewski C, Bailey K, Fandl JP, Daly T, Wiegand SJ, Yancopoulos GD, Rudge JS. VEGF-trap: a VEGF blocker with potent antitumor effects. Proc Natl Acad Sci U S A. 2002; 99: 11393–11398.[Abstract/Free Full Text]
  23. Frank L, Bucher JR, Roberts RJ. Oxygen toxicity in neonatal and adult animals of various species. J Appl Physiol. 1978; 45: 699–704.[Abstract/Free Full Text]
  24. Scherle W. A simple method for volumetry of organs in quantitative stereology. Mikroskopie. 1970; 26: 57–60.[Medline] [Order article via Infotrieve]
  25. Tschanz SA, Makanya AN, Haenni B, Burri PH. Effects of neonatal high-dose short-term glucocorticoid treatment on the lung: a morphologic and morphometric study in the rat. Pediatr Res. 2003; 53: 72–80.[CrossRef][Medline] [Order article via Infotrieve]
  26. Thebaud B, Michelakis E, Wu XC, Harry G, Hashimoto K, Archer SL. Sildenafil reverses O2 constriction of the rabbit ductus arteriosus by inhibiting type 5 phosphodiesterase and activating BK(Ca) channels. Pediatr Res. 2002; 52: 19–24.[Medline] [Order article via Infotrieve]
  27. Michelakis E, Rebeyka I, Wu XC, Hashimoto K, Nsair A, Thebaud B, Dyck JRB, Haromy A, Harry G, Barr A, Archer SL. O2 sensing in the human ductus arteriosus: regulation of voltage-gated K channels in smooth muscle cells by a mitochondrial redox sensor. Circ Res. 2002; 91: 478–486.[Abstract/Free Full Text]
  28. Thurston G, Rudge JS, Ioffe E, Zhou H, Ross L, Croll SD, Glazer N, Holash J, McDonald DM, Yancopoulos GD. Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat Med. 2000; 6: 460–463.[CrossRef][Medline] [Order article via Infotrieve]
  29. Liebow AA. Pulmonary emphysema with special reference to vascular changes. Am Rev Respir Dis. 1959; 80: 67–93.[Medline] [Order article via Infotrieve]
  30. Hall SM, Hislop AA, Pierce CM, Haworth SG. Prenatal origins of human intrapulmonary arteries: formation and smooth muscle maturation. Am J Respir Cell Mol Biol. 2000; 23: 194–203.[Abstract/Free Full Text]
  31. Galambos C, Ng YS, Ali A, Noguchi A, Lovejoy S, D’Amore PA, DeMello DE. Defective pulmonary development in the absence of heparin-binding vascular endothelial growth factor isoforms. Am J Respir Cell Mol Biol. 2002; 27: 194–203.[Abstract/Free Full Text]
  32. Le Cras TD, Spitzmiller RE, Albertine KH, Greenberg JM, Whitsett JA, Akeson AL. VEGF causes pulmonary hemorrhage, hemosiderosis, and air space enlargement in neonatal mice. Am J Physiol. 2004; 287: L134–L142.
  33. Han RN, Babaei S, Robb M, Lee T, Ridsdale R, Ackerley C, Post M, Stewart DJ. Defective lung vascular development and fatal respiratory distress in endothelial NO synthase-deficient mice:a model of alveolar capillary dysplasia? Circ Res. 2004; 94: 1115–1123.[Abstract/Free Full Text]
  34. Jakkula M, Le Cras TD, Gebb S, Hirth KP, Tuder RM, Voelkel NF, Abman SH. Inhibition of angiogenesis decreases alveolarization in the developing rat lung. Am J Physiol. 2000; 279: L600–L607.
  35. Kasahara Y, Tuder RM, Taraseviciene-Stewart L, Le Cras TD, Abman S, Hirth PK, Waltenberger J, Voelkel NF. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest. 2000; 106: 1311–1319.[Medline] [Order article via Infotrieve]
  36. Maniscalco WM, Watkins RH, D’Angio CT, Ryan RM. Hyperoxic injury decreases alveolar epithelial cell expression of vascular endothelial growth factor (VEGF) in neonatal rabbit lung. Am J Respir Cell Mol Biol. 1997; 16: 557–567.[Abstract]
  37. Maniscalco WM, Watkins RH, Pryhuber GS, Bhatt A, Shea C, Huyck H. Angiogenic factors and alveolar vasculature: development and alterations by injury in very premature baboons. Am J Physiol. 2002; 282: L811–L823.
  38. Pozeg ZI, Michelakis ED, McMurtry MS, Thebaud B, Wu XC, Dyck JR, Hashimoto K, Wang S, Moudgil R, Harry G, Sultanian R, Koshal A, Archer SL. In vivo gene transfer of the O2-sensitive potassium channel Kv1.5 reduces pulmonary hypertension and restores hypoxic pulmonary vasoconstriction in chronically hypoxic rats. Circulation. 2003; 107: 2037–2044.[Abstract/Free Full Text]
  39. Soll RF, Morley CJ. Prophylactic versus selective use of surfactant in preventing morbidity and mortality in preterm infants. Cochrane Database Syst Rev. 2001; 2: CD000510.[Medline] [Order article via Infotrieve]
  40. Tanswell AK, O’Brodovich HM. The present and future role of gene therapy in the newborn. Curr Opin Pediatr. 1997; 9: 141–145.[Medline] [Order article via Infotrieve]
  41. 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. 2005; 288: L450–L459.
  42. Schreiber MD, Gin-Mestan K, Marks JD, Huo D, Lee G, Srisuparp P. Inhaled nitric oxide in premature infants with the respiratory distress syndrome. N Engl J Med. 2003; 349: 2099–2107.[Abstract/Free Full Text]
  43. Thurston G, Suri C, Smith K, McClain J, Sato TN, Yancopoulos GD, McDonald DM. Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science. 1999; 286: 2511–2514.[Abstract/Free Full Text]
  44. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992; 359: 843–845.[CrossRef][Medline] [Order article via Infotrieve]
  45. Hjalmarson O, Sandberg K. Abnormal lung function in healthy preterm infants. Am J Respir Crit Care Med. 2002; 165: 83–87.[Abstract/Free Full Text]
  46. Ema M, Taya S, Yokotani N, Sogawa K, Matsuda Y, Fujii-Kuriyama Y. A novel BHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1{alpha} regulates the VEGF expression and is potentially involved in lung and vascular development. Proc Natl Acad Sci U S A. 1997; 94: 4273–4278.[Abstract/Free Full Text]
  47. Tian H, McKnight SL, Russell DW. Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells. Genes Dev. 1997; 11: 72–82.[Abstract/Free Full Text]
  48. Compernolle V, Brusselmans K, Acker T, Hoet P, Tjwa M, Beck H, Plaisance S, Dor Y, Keshet E, Lupu F, Nemery B, Dewerchin M, Van Veldhoven P, Plate K, Moons L, Collen D, Carmeliet P. Loss of HIF-2{alpha} and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice. Nat Med. 2002; 8: 702–710.[Medline] [Order article via Infotrieve]
  49. Rabinovitch M, Bland R. Novel notions on newborn lung disease. Nat Med. 2002; 8: 664–666.[CrossRef][Medline] [Order article via Infotrieve]
 

CLINICAL PERSPECTIVE

Lung diseases characterized by impaired development/loss of alveoli, such as bronchopulmonary dysplasia (BPD) in premature infants or emphysema in adults, are significant global health problems. Currently, there is no specific treatment for these debilitating and life-threatening diseases. Understanding the mechanisms that regulate alveolar development is crucial for developing new treatment strategies. Here, we report that the vascular endothelial growth factor, VEGF, crucial for blood vessel development, regulates normal alveolar development, prevents alveolar damage, and regenerates alveoli after lung injury. We show that VEGF blockade during the critical period of alveolar development in newborn rats leads to air space enlargement and loss of lung capillaries, mimicking BPD. In experimental BPD in newborn rats induced by hyperoxia, air space enlargement and loss of lung capillaries are associated with decreased lung VEGF and VEGFR-2 expression. Intratracheal adenovirus-mediated VEGF gene therapy improves survival, promotes lung capillary formation, and preserves/restores alveolar development in this model of irreversible lung injury. Combined VEGF and angiopoietin-1 (another angiogenic growth factor crucial for blood vessel maturation) gene transfer matures the new vasculature, reducing the vascular leakage seen in VEGF-induced capillaries. Our findings underscore the importance of the vasculature in what is traditionally thought of as an airway disease and open new therapeutic avenues for lung diseases characterized by irreversible loss of alveoli through the modulation of angiogenic growth factors.




This article has been cited by other articles:


Home page
 Am. J. Respir. Crit. Care Med.Home page
S. H. Abman
The Dysmorphic Pulmonary Circulation in Bronchopulmonary Dysplasia: A Growing Story
Am. J. Respir. Crit. Care Med., July 15, 2008; 178(2): 114 - 115.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
E. Purevdorj, K. Zscheppang, H. G. Hoymann, A. Braun, D. von Mayersbach, M.-J. Brinkhaus, A. Schmiedl, and C. E. L. Dammann
ErbB4 deletion leads to changes in lung function and structure similar to bronchopulmonary dysplasia
Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L516 - L522.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
D. Xu, R. E. Perez, I. I. Ekekezie, A. Navarro, and W. E. Truog
Epidermal growth factor-like domain 7 protects endothelial cells from hyperoxia-induced cell death
Am J Physiol Lung Cell Mol Physiol, January 1, 2008; 294(1): L17 - L23.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
J. Gien, G. J. Seedorf, V. Balasubramaniam, N. Markham, and S. H. Abman
Intrauterine Pulmonary Hypertension Impairs Angiogenesis In Vitro: Role of Vascular Endothelial Growth Factor Nitric Oxide Signaling
Am. J. Respir. Crit. Care Med., December 1, 2007; 176(11): 1146 - 1153.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
M. Subramaniam, C. Bausch, A. Twomey, S. Andreeva, B. A. Yoder, L. Chang, J. D. Crapo, R. A. Pierce, F. Cuttitta, and M. E. Sunday
Bombesin-like Peptides Modulate Alveolarization and Angiogenesis in Bronchopulmonary Dysplasia
Am. J. Respir. Crit. Care Med., November 1, 2007; 176(9): 902 - 912.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
R. D. Bland, L. M. Mokres, R. Ertsey, B. E. Jacobson, S. Jiang, M. Rabinovitch, L. Xu, E. S. Shinwell, F. Zhang, and M. A. Beasley
Mechanical ventilation with 40% oxygen reduces pulmonary expression of genes that regulate lung development and impairs alveolar septation in newborn mice
Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1099 - L1110.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
J.-R. Tang, G. Seedorf, V. Balasubramaniam, A. Maxey, N. Markham, and S. H. Abman
Early inhaled nitric oxide treatment decreases apoptosis of endothelial cells in neonatal rat lungs after vascular endothelial growth factor inhibition
Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1271 - L1280.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
D. Irwin, K. Helm, N. Campbell, M. Imamura, K. Fagan, J. Harral, M. Carr, K. A. Young, D. Klemm, S. Gebb, et al.
Neonatal lung side population cells demonstrate endothelial potential and are altered in response to hyperoxia-induced lung simplification
Am J Physiol Lung Cell Mol Physiol, October 1, 2007; 293(4): L941 - L951.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
D. R. Prows, A. P. Hafertepen, A. V. Winterberg, W. J. Gibbons Jr., C. Liu, and T. G. Nick
Genetic analysis of hyperoxic acute lung injury survival in reciprocal intercross mice
Physiol Genomics, August 20, 2007; 30(3): 271 - 281.
[Abstract] [Full Text] [PDF]

Home page