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Am J Physiol Lung Cell Mol Physiol 291: L588-L595, 2006. First published May 5, 2006; doi:10.1152/ajplung.00098.2006
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Enhancement of angiogenic effectors through hypoxia-inducible factor in preterm primate lung in vivo

Tiina M. Asikainen,1 Nahid S. Waleh,2 Barbara K. Schneider,1 Ronald I. Clyman,2,3 and Carl W. White1

1Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado; 2SRI International, Menlo Park, and 3Cardiovascular Research Institute, University of California, San Francisco, California

Submitted 17 March 2006 ; accepted in final form 28 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Development of lung microvasculature is critical for distal airway formation. Both processes are arrested in the lungs of preterm newborns with bronchopulmonary dysplasia (BPD), a chronic form of lung disease. We hypothesized that activation of hypoxia-inducible factors (HIFs) augments lung vascular development. Pulmonary angiogenic factors were assessed by quantitative real-time PCR, Western blot, and immunohistochemistry in preterm baboons (125 days + 14 days pro re nata O2 model) treated for 14 days with intravenous FG-4095, an inhibitor of prolyl hydroxylase domain-containing proteins (PHDs) that initiates HIF degradation. HIF-1{alpha}, but not HIF-2{alpha}, mRNA and protein were increased (8- and 3-fold, respectively) in FG-4095-treated baboons relative to untreated controls. Expression of PHD-1, -2, and -3 was unchanged. Of note, mRNA and/or protein for platelet-endothelial cell adhesion molecule 1 (PECAM-1) and vascular endothelial growth factor (VEGF) were increased by FG-4095. Moreover, PECAM-1-expressing capillary endothelial cells detected by immunohistochemistry were augmented in FG-4095-treated baboons to levels comparable to those in fetal age-matched controls. Alveolar septal cell expression of Ki67, a proliferative marker, and VEGF were similar in untreated controls and FG-4095-treated neonates. These results indicate that HIF stimulation by PHD inhibition enhances lung angiogenesis in the primate model of BPD.

prolyl hydroxylase domain-containing protein; angiogenesis; alveolization; bronchopulmonary dysplasia; prematurity

THE LUNG VASCULATURE HAS TRADITIONALLY been thought to develop by both central angiogenesis (sprouting of new vessels from existing ones) and by distal mesenchymal vasculogenesis (formation of new blood vessels from angioblasts) followed by fusion of these two systems (35). Precise interactions between the mesenchymal and epithelial compartments are needed for coordinated development of mature airways and vessels (18). Recently, a new form of lung vascular growth termed distal angiogenesis has been identified (34). It differs from the other model in that the capillary networks surround the terminal buds and, importantly, are postulated to expand by formation of new capillaries from the existing ones as the terminal bud expands (34). Accumulating evidence underscores the importance of vascular development to distal airway development (39). However, the regulatory mechanisms linking development of microvasculature and distal airway formation are not fully understood.

Both vascular and alveolar growth are disrupted in lungs of prematurely born babies with the chronic lung disease bronchopulmonary dysplasia (BPD) (29). The pathogenesis of BPD is multifactorial, but ventilatory therapy combined with supraphysiological O2 concentrations are considered to play a significant role (28). The fetal lung normally develops in hypoxia in utero and is exposed to sudden hyperoxia due to premature birth and initiation of pulmonary respiration. This dramatic shift in O2 homeostasis likely disturbs the hypoxia-mediated signaling necessary for lung development (42). Most hypoxia-induced regulatory events are mediated by hypoxia-inducible factors (HIFs) (19), which are known to control >500 genes, many of these being involved in endothelial cell proliferation and survival (32). HIF-1{alpha} is downregulated in lungs of preterm baboons after preterm birth and O2 breathing (3). Furthermore, expression of several angiogenic factors is modified after premature birth and in BPD (reviewed in Ref. 39). With this background in mind, we are now asking whether stimulation of HIFs could restore lung growth in prematurely born babies.

HIFs are heterodimers composed of the helix-loop-helix/Per-Arnt-Sim protein HIF-{alpha} and the aryl hydrocarbon receptor nuclear translocator (ARNT or HIF-1beta) (46). The HIF-heterodimer accumulates in the nucleus and binds to hypoxia response elements of target genes, thereby regulating their transcription (27). HIF expression is mainly regulated at the posttranslational level by prolyl hydroxylase domain-containing proteins (PHDs), although other mechanisms have also been described (7, 15, 26, 31). The PHDs have been suggested to be the cellular O2 sensors (23, 24). In addition to O2, they require {alpha}-ketoglutarate, iron, and ascorbate for enzyme activity. In the presence of O2, the product of von Hippel-Lindau tumor suppressor gene, pVHL, binds HIF-{alpha} subunits and targets them for polyubiquitination and proteosomal destruction (12, 22). Interaction of pVHL and the O2-dependent domain of HIF-{alpha} subunit is regulated by hydroxylation of certain proline residues by the PHDs (23, 24). To date, three classes of HIF-PHDs are known (9, 17, 20). PHD activity, in turn, can be modulated by Siah1a/2 E3 ubiquitin ligases (33) and OS-9 (6).

We have previously demonstrated that HIF activation through PHD inhibition in lung endothelial and epithelial cells in vitro and in fetal lung explants ex vivo is effective and causes increased expression of angiogenic factors and enhancement of in vitro angiogenesis (2, 5). The aim of the current study was to investigate the potential of this approach in vivo using the baboon model of prematurity.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
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Premature primate model. All animal studies were performed at the Southwest Foundation for Biomedical Research Primate Center (San Antonio, TX), and protocols and procedures were approved by the Institutional Animal Care and Use Committee. Fetal baboons (gestational controls) of gestational ages 125 ± 2 days (n = 6) and 140 ± 2 days (n = 5) were delivered by hysterotomy and killed at birth before the first breath. The baboon gestation, 125 and 140 days, corresponds to 27 and 32 human gestational weeks, respectively (baboon term gestation is 185 days). The preterm animals (125 days ± 2 days), also delivered by hysterotomy, were intubated and received surfactant (4 ml/kg Survanta, a gift from Ross Laboratories, Columbus, OH) before being placed on ventilatory support for 14 days with pro re nata (PRN) O2 support. For PRN treatment, FIO2 varied between 0.21 and 0.80 (usually 0.21–0.50), with the goal to maintain arterial PO2 at 55–70 mmHg (7.3–9.3 kPa). The preterm animals were randomly assigned to either the control group (n = 10) receiving routine care or to the FG-4095 (n = 5) group receiving the experimental compound (FG-4095; FibroGen, South San Francisco, CA) in addition to the routine care. The baboons first received an intravenous loading dose of FG-4095 (5 mg·kg–1·2–1 h) starting at age 2 h, and the continuous, intravenous drip (0.6 mg·kg–1·h–1) was started at age 4 h. FG-4095 was diluted in routine glucose solutions. All baboons received antenatal steroids, either dexamethasone or betamethasone (both were given at 6 mg, 48 and 24 h before hysterotomy). Proper fluid and nutritional and antimicrobial therapy were given to all animals as described (11). At necropsy, the tissues were immediately fixed with 4% paraformaldehyde or frozen in liquid N2 for further analysis. All samples were collected during the years 2003–2005.

Cell culture. Human lung microvascular endothelial cells (HLMVEC, primary cell line) were purchased from Cambrex BioScience (Walkersville, MD) and grown as described (2). Cells were exposed to experimental compounds at either 21 or 95% O2.

PHD inhibitors. For in vivo studies, only HIF-PHD selective FG-4095 (FibroGen) was used. For in vitro studies, both FG-4095 (125 µM) and a more nonselective PHD inhibitor, dimethyloxaloylglycine (DMOG, 1 mM; a kind gift of Dr. C. Pugh, Univ. of Oxford, Oxford, UK), were diluted in phosphate-buffered saline and used as described (2).

mRNA analyses by quantitative PCR. mRNA for HIF-1{alpha} and -2{alpha}, platelet-endothelial cell adhesion molecule 1 (PECAM-1), endothelial and inducible nitric oxide synthase (eNOS and iNOS, respectively), vascular endothelial growth factor (VEGF), fms-like tyrosine kinase 1 (Flt-1), and kinase insert domain-containing receptor/fetal liver kinase (KDR) were measured by real-time PCR as described (43). Total RNA was isolated from lung tissue and reverse transcribed using random primers and Moloney murine leukemia virus RNase H-reverse transcriptase (Invitrogen, Carlsbad, CA). An ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA) was used for thermal cycling. TaqMan probes and primers were designed using the Primer Express version 3.0 program (Applied Biosystems). The probes were synthesized using the chromophores 6-carboxy-fluorescein and 6-carboxy-tetramethyl-rhodamine as reporter and quencher dyes, respectively (Integrated DNA Technologies, Coralville, IA). Reactions were carried out in 96-well plates in triplicate, and human malate dehydrogenase gene was used as internal control to normalize the test results. Data were analyzed using Sequence Detector System version 1.3 program (Applied Biosystems).

Protein analyses by Western blot and ELISA. Several angiogenic proteins were assessed from either nuclear or cytosolic extracts by Western blotting (2, 3) using the following anti-human antibodies: mouse monoclonal anti-HIF-1{alpha} and anti-eNOS (both 1:500; BD Biosciences, San Diego, CA), rabbit polyclonal anti-HIF-2{alpha} (1:750), anti-PHD-1 (1:500), anti-PHD-2 (1:500), and anti-PHD-3 (1:750; Novus Biologicals, Aurora, CO), goat polyclonal anti-angiopoietin 1 (1:200) and -4 (Ang-1 and Ang-4, respectively; 1:200), anti-Tie-2 (angiopoietin receptor; 1:200), anti-KDR (1:200; R&D Systems, Minneapolis, MN), rabbit polyclonal anti-Flt-1 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), and mouse monoclonal anti-PECAM-1 (1:3,000; Dako, Glostrup, Denmark). Mouse monoclonal anti-beta-actin (Sigma, St. Louis, MO) was used as a loading control. VEGF protein was quantitated by ELISA as described (3).

Immunohistochemistry. PECAM-1, VEGF, and Ki67 were detected by immunohistochemistry. Paraffin-embedded sections (5 µm) were dewaxed, rehydrated, and exposed to antigen retrieval (for VEGF: 72°C, pH 10, overnight; for PECAM-1/Ki67: 95°C, pH 6, 40 min). After quenching of endogenous peroxidase and alkaline phosphatase (Dual Blocker; Dako, Carpinteria, CA), the nonspecific binding was blocked (Serum Free Protein Block; Open Biosystems, Huntsville, AL). The sections were then incubated overnight with anti-human antibodies against VEGF (mouse monoclonal; 1:200, Santa Cruz), PECAM-1 (mouse monoclonal, 1:40, DakoCytomation), or with protein concentration-matched mouse IgG (BD Pharmingen, San Diego, CA) for negative controls. After incubation with secondary horseradish peroxidase-labeled polymer antibody (EnVision +HRP, Dako) for 60 min, color was developed by 3,3-diaminobenzidine (BioCare Medical, Walnut Creek, CA) combined with H2O2 for both VEGF and PECAM-1. The PECAM-1 sections were then incubated for 2 h with rabbit monoclonal anti-human Ki67 (1:200; NeoMarkers/LabVision, Fremont, CA), and after exposure to secondary alkaline phosphatase-labeled polymer antibody for 45 min, color was developed with Fast Red combined with alkaline phosphatase (BioCare Medical). Counterstaining was performed with hematoxylin (Open Biosystems). The methods were developed in collaboration with and stains performed by IHCtech, Aurora, CO.

For quantitation, a viewer blinded to the identity of the sections took images (9–16/baboon) of representative areas of alveolar parenchyma, small vessels, and bronchial/bronchiolar epithelium. Two blinded reviewers then independently scored in a semiquantitative manner the staining (0 = negative, 1 = mild, 2 = moderate, and 3 = intense staining) using preprepared tables listing the following structures and cell types: alveolar epithelium, capillary endothelium, matrix cells (fibroblasts and smooth muscle cells), bronchiolar/bronchial epithelium, endothelium of muscular arteries, arterioles, veins, and venules. The mean scores per animal of the two reviewers were similar (differences in mean scores <1).

Statistical analyses. Two groups or multiple groups were compared by Student's t-test or ANOVA with Fisher's protected least significant difference for post hoc comparisons. Statistical significance was accepted at P ≤ 0.05. Results are shown as means (SD), and statistical analyses were performed using StatView 4.51 (Abacus Concepts, Berkeley, CA).


   RESULTS
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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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In vivo mRNA analyses of angiogenic factors. Pulmonary mRNA expression for HIFs and HIF-regulated, selected angiogenic markers was analyzed to estimate effectiveness of FG-4095 treatment in the preterm baboon. Control and FG-4095-treated baboons (125 days +14 days PRN O2 model) were compared with two groups of fetal controls, 125 and 140 days, the latter group being an age-matched control for the preterm animals (125 + 14 days = 139 days). HIF-1{alpha} mRNA increased 8.7-fold from 125 to 140 days of gestation (Fig. 1A). This increase was preserved in FG-4095-treated, but not in control, baboons at 14 days of age (Fig. 1A). There were no clear differences in HIF-2{alpha} mRNA expression among the four groups of baboons (Fig. 1B).


Figure 1
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  		Fig. 1. mRNA for hypoxia-inducible factors (HIFs) and selected important angiogenic markers. HIF-1{alpha} (A), HIF-2{alpha} (B), platelet-endothelial cell adhesion molecule 1 (PECAM-1; C), endothelial nitric oxide synthase (eNOS; D), VEGF (E), fms-like tyrosine kinase 1 (Flt-1; F), and kinase insert domain-containing receptor (KDR; G) were measured by real-time PCR in lungs of preterm baboons (125 days) treated for 14 days with pro re nata (PRN) O2 with or without FG-4095. 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, **P ≤ 0.01, and ***P ≤ 0.001 vs. 125-day gestational control (GC); #P ≤ 0.05, ##P ≤ 0.01, and ###P ≤ 0.001 vs. 140-day GC; ^^P ≤ 0.01 FG-4095 vs. control.

 
Lung PECAM-1 mRNA expression increased 2.5-fold from 125 to 140 days of gestation and appeared better conserved in FG-4095-treated animals relative to controls, although the difference was not statistically significant (P = 0.07; Fig. 1C). eNOS (Fig. 1D) and VEGF (Fig. 1E) expression was similar in 125- and 140-day fetal baboons, but both were downregulated in control baboons compared with 125-day (eNOS 2.3-fold, VEGF 3.5-fold) or 140-day (eNOS 2.8-fold, VEGF 4.7-fold) fetal control. Expression of VEGF, but not of eNOS, was higher (1.7-fold) in the FG-4095-treated animals than in controls (Fig. 1, D and E). iNOS mRNA could not be detected by real-time PCR.

Lung Flt-1 mRNA was elevated (2.2-fold) in 140-day relative to 125-day fetuses (Fig. 1F). Both groups of preterm animals had decreased levels of Flt-1 compared with 140-day gestational controls (FG-4095 2.5-fold, control 3.8-fold; Fig. 1F). Lung KDR mRNA expression was low in all baboons, and there were no clear changes in expression among any of the groups (Fig. 1G).

In vivo protein analyses of angiogenic factors. Protein expression of pulmonary HIFs and selected important angiogenic markers in FG-4095-treated baboons and untreated controls were analyzed next. HIF-1{alpha} protein was enhanced threefold by FG-4095 (Fig. 2A). Expression of HIF-2{alpha} was the same for the treated and untreated animals with variability between individual animals (Fig. 2B).


Figure 2
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  		Fig. 2. Proteins for HIFs and selected important angiogenic factors. HIF-1{alpha} (A), HIF-2{alpha} (B), PECAM-1 (C), eNOS (D), VEGF (E), Flt-1 (F), KDR (G), Ang-1 (H), Ang-4 (I), and Tie-2 (J) were measured by Western blot analysis (A–D, F–J) or ELISA (E) in lungs of preterm baboons (125 days) treated for 14 days with PRN O2 with or without FG-4095. For proteins measured by Western blot, each lane represents an individual animal, and the results are shown as percent of control. Data are shown as means (SD), n = 4–6. **P ≤ 0.01.

 
Similarly to PECAM-1 mRNA expression, PECAM-1 protein was also higher (2.5-fold) in FG-4095-treated baboons relative to untreated controls (Fig. 1C). Protein expression for eNOS (Fig. 2D), VEGF (Fig. 2E), Flt-1 (Fig. 2F), and KDR (Fig. 2G) was the same for FG-4095-treated and untreated animals. Protein expression for Ang-1 and -4 (Fig. 2, H and I, respectively) and their receptor Tie-2 (Fig. 2J) was also assessed, and the expression of each was found to be similar for control and FG-4095-treated baboons.

In vivo analysis of PHD proteins. Our previous studies with human pulmonary cells (2) and fetal baboon explants (5) indicated that HIF stimulation through PHD inhibition could be limited due to enhancement of PHD-2 and/or -3 proteins that target HIFs for degradation. Therefore, we sought to determine whether PHDs were enhanced following continuous treatment with FG-4095. At age 14 days, the expression of none of the three PHDs (PHD-1, -2, -3) was altered by treatment with FG-4095 (Fig. 3, A–C).


Figure 3
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  		Fig. 3. Pulmonary HIF-degrading prolyl hydroxylase domain-containing proteins (PHDs) in preterm baboons. PHD-1 (A), PHD-2 (B), and PHD-3 (C) were assessed by Western blot analysis in control or FG-4095-treated baboons (125 days + 14 days PRN O2). Results are percent of control [means (SD), n = 5].

 
In vitro analysis of HIF-1{alpha} and HIF-2{alpha} mRNA. FG-4095 augments HIF expression by inhibiting the degradation pathway of HIF protein. Therefore, it was of great interest that the HIF-1{alpha} mRNA expression in vivo was also induced (Fig. 1A). This finding led us to investigate HIF-1{alpha} and -2{alpha} mRNA expression in an in vitro model (HLMVEC) where we had previously (5) characterized HIF-1{alpha} and -2{alpha} protein stimulation through PHD inhibition. In HLMVEC, HIF-1{alpha} mRNA decreased (1.8- to 3-fold) and HIF-2{alpha} mRNA increased (1.4- to 2-fold) in response to treatment with FG-4095 or the positive control DMOG, both in 21 and 95% O2.

In vivo localization and expression of PECAM-1 and VEGF. Staining for lung PECAM-1 was intense in alveolar parenchyma in 140-day fetal baboons (Fig. 4A) but was weak in preterm baboons with BPD (Fig. 4B). However, after FG-4095 treatment, PECAM-1 expression in capillary and vascular endothelium was nearly restored to the fetal levels (Table 1 and Fig. 4, C and D). Staining for Ki67 in bronchial/bronchiolar epithelium (not shown) and alveolar membrane was weak to moderate in 140-day fetal animals and was weak in both treated and untreated preterm baboons (Fig. 4, A–C). Like PECAM-1, VEGF was also highly expressed in the fetal 140-day lung (Table 1, Fig. 4E), with moderate-to-intense positivity in alveolar epithelial and endothelial cells. Relative to fetal animals, both preterm controls and FG-4095-treated baboons had weaker VEGF staining in alveolar membrane (Table 1, Fig. 4, F and G). VEGF expression was low or absent in bronchial/bronchiolar epithelium and in endothelium of arterioles/venules in all samples (not shown). A common feature for both PECAM-1 and VEGF staining in all samples was that the pattern of staining was heterogeneous. There was no staining in negative controls for any of the primary antibodies (not shown).


Figure 4
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  		Fig. 4. Immunohistochemical stains for angiogenic factors in lungs of preterm baboons after FG-4095 treatment. PECAM-1 (brown) and Ki67 (red) (A–D) and VEGF (brown; E–H) are shown in lungs of 140-day fetal controls (A and E), preterm controls (B and F), and FG-4095-treated baboons (C, D, G, and H). At 140 days gestation, staining for endothelial cell marker PECAM-1 (A), proliferating cell marker Ki67 (A; closed arrowheads), and angiogenic factor VEGF (E; open arrowheads, VEGF-positive alveolar epithelial cells; arrows, VEGF-positive capillary endothelial cells) was intense in both primary septae and secondary crests (asterisks). Staining for both PECAM-1 (B) and VEGF (F) was weak in bronchopulmonary dysplasia (B and F), whereas following FG-4095 treatment, PECAM-1 expression in capillary endothelium was nearly restored (C and D). Original magnification, x40 (A–C and E–G) and x100 (D and H), counterstaining with hematoxylin.

 

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  		Table 1. Immunohistochemical assessment of PECAM-1 and VEGF in fetal and preterm baboon lung

 

   DISCUSSION
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Disrupted vascular development at critical stages of lung development leads to impaired development of distal airways and later alveolization (25, 39, 42). Indeed, arrested vascular and airway development are principal histopathological features of BPD, a chronic lung disease affecting approximately two-thirds of human preterm babies born before 32 gestational weeks (16). We and others (2, 5, 30, 39, 40, 42) have previously hypothesized that augmenting angiogenic factors in this clinical situation could provide a valuable tool for restoring arrested lung growth. In proof of principal studies, we have demonstrated that expression of selected angiogenic factors, as well as angiogenesis, can be augmented both in vitro and ex vivo through stimulation of HIFs via PHD inhibition (2, 5). The goal of the present study was to assess the feasibility of HIF activation in enhancing angiogenesis in vivo using the well-characterized baboon model of prematurity (11). This report focuses on the biochemical findings in these baboons, whereas the clinical course of the animals is described separately (4).

Most of the knowledge regarding HIFs has originated from cancer research involving abnormal angiogenesis (36, 37). However, HIFs play essential roles during development as well (10). HIF-1{alpha} and -2{alpha} appear to have individual, nonredundant roles in hypoxic gene regulation (21, 38). Regulation of, for example, glycolytic enzymes has been ascribed to HIF-1{alpha} and of erythropoietin to HIF-2{alpha} (21, 44). Recently, a differential regulatory mechanism between HIF-1{alpha} and -2{alpha} involving protein-protein interaction has been revealed (8). Specifically, NF-{kappa}B essential modulator has been found to enhance HIF-2{alpha}, but not HIF-1{alpha}, transcriptional activity, suggesting a specific functional role for HIF-2{alpha} (8).

In the present study, expression of pulmonary HIF-1{alpha}, but not HIF-2{alpha}, was modified during fetal development and also in FG-4095-treated baboons. As we studied whole lung homogenate, we cannot rule out the possibility that HIF-2{alpha} expression could have been altered in a subpopulation of cells. The current findings extend those from a previous study showing that HIF-1{alpha}, but not HIF-2{alpha}, was downregulated in the baboon model of BPD (3). HIF-1{alpha} mRNA increased eightfold during the early third trimester in the current study. HIF-1{alpha} mRNA increase did not occur in untreated control baboons but was fully preserved in FG-4095-treated animals. Of note, increased HIF-1{alpha} mRNA was associated with enhanced protein expression in FG-4095-treated baboons relative to untreated controls. In addition, the previously described induction of one or more of the PHDs in response to hypoxia (13) or PHD inhibition (2, 5) did not occur in lungs of preterm baboons treated with FG-4095 in vivo when assessed on day 14 of life.

Because FG-4095 enhances HIFs by blocking their degradation through inhibition of PHDs, it was unexpected to find that FG-4095 increased HIF-1{alpha} mRNA in addition to protein in baboon lung. On the other hand, others have found elevated HIF-1{alpha} mRNA expression in rodent lung under hypoxic conditions (45). We next determined whether FG-4095 affected HIF mRNA levels in HLMVEC where we have previously shown (5) strong increases of both HIF-1{alpha} and -2{alpha} proteins following PHD inhibition. Remarkably, FG-4095 and the positive control DMOG both had opposite effects on HIF-1{alpha} and HIF-2{alpha} mRNA in vitro as compared in vivo in that mRNA for HIF-1{alpha} was downregulated and for HIF-2{alpha} upregulated in the endothelial cells. Others have described reduced HIF-1{alpha} mRNA expression in A549 cells in hypoxia due to decreased mRNA stability (41), supporting our findings of diminished HIF-1{alpha} mRNA expression in vitro. Furthermore, in the present study, the upregulation of HIF-1{alpha} mRNA by FG-4095 in vivo could be due to, for example, growth factors not present in cell culture, timing (14 days in vivo vs. 24 h in vitro), or, alternatively, could have occurred in nonendothelial cells. Finally, the mode of action for FG-4095 is stabilizing HIF at the posttranslational level and, therefore, the downregulation of HIF-1{alpha} mRNA by FG-4095 in vitro might represent a physiological, homeostatic response of cells to excessive HIF or its downstream targets, such as VEGF (10-fold induction in these cells). However, specific upregulation of HIF-2{alpha} mRNA under the same circumstances suggests a specific effect of FG-4095 and further emphasizes the different roles of HIF-1{alpha} and -2{alpha}.

Expression of selected HIF-driven angiogenic factors was assessed in lungs of preterm baboons following FG-4095 treatment. In vivo expression of some of these factors was altered by FG-4095, but mRNA expression did not always correlate with protein expression. For example, VEGF mRNA, but not protein, was enhanced in FG-4095-treated relative to untreated controls. Of note, the method used for mRNA quantitation detects all VEGF isoforms, whereas antibodies used for protein analyses detect VEGF121 and VEGF165, and, therefore, differing mRNA and protein results may occur from methodological factors. eNOS expression, which is typically low in BPD (1), was the same for both treated and untreated baboons at age 14 days. However, pulmonary expression of eNOS and other factors remain unknown at earlier time points since such studies could not be performed without death of the valuable model. Alternatively, failure to detect changes in some endothelial markers assessed from whole lung might possibly indicate dilution of signals originating in the microvasculature. Relative to untreated controls, PECAM-1 protein was increased, and PECAM-1-expressing capillary endothelial cells were augmented by FG-4095, suggesting enhanced angiogenesis. Notably, lung function and growth were also improved in FG-4095-treated baboons (4). These results support the concept that vascular and alveolar development were linked and are also in line with a recent study underscoring the importance of PECAM-1 for postnatal lung development in newborn rats (14). Furthermore, two groups of investigators have shown improvement of lung vascular and/or alveolar development through intratracheal and intramuscular VEGF therapy at the time of evolving O2-induced lung injury (40), and interestingly, also after that injury has developed (30). Collectively, these studies indicate that angiogenic therapy may hold promise for future treatment of BPD.

To summarize, we have shown that in vivo treatment of preterm baboons with FG-4095, a PHD inhibitor, stimulates mRNA and/or protein expression of pulmonary HIF-1{alpha} and of selected angiogenic factors, notably PECAM-1 and VEGF. Furthermore, PECAM-1-positive endothelial cells in the alveolar membrane are augmented by PHD inhibition.


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Financial support was obtained from National Institutes of Health Grants U01 HL-56263 (C. W. White), HL-52636 (BPD Resource Center), P51-RR-13986 (Facility Support at Southwest Foundation), Academy of Finland (T. M. Asikainen), and Foundation for Pediatric Research in Finland (T. M. Asikainen).


   DISCLOSURES
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No monetary contributions beyond provision of the study compound were received from FibroGen, and neither the authors nor their institutions hold equity in FibroGen.


   ACKNOWLEDGMENTS
 
The authors thank the personnel supporting the BPD Resource Center (San Antonio, TX), especially Jacqueline Coalson, Vicki Winter, Donald McCurnin, and Bradley Yoder (Univ. of Texas Health Sciences Center). Xiaoling Guo (National Jewish Medical and Research Center) is thanked for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. W. White, Dept. of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Rm. J-318, Denver, CO 80206 (e-mail: whitec{at}njc.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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  1. Afshar S, Gibson LL, Yuhanna IS, Sherman TS, Kerecman JD, Grubb PH, Yoder BA, McCurnin DC, and Shaul PW. Pulmonary NO synthase expression is attenuated in a fetal baboon model of chronic lung disease. Am J Physiol Lung Cell Mol Physiol 284: L749–L758, 2003.[Abstract/Free Full Text]
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