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Am J Physiol Lung Cell Mol Physiol 292: L1073-L1084, 2007. First published January 5, 2007; doi:10.1152/ajplung.00347.2006
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Hyperoxia reduces bone marrow, circulating, and lung endothelial progenitor cells in the developing lung: implications for the pathogenesis of bronchopulmonary dysplasia

Vivek Balasubramaniam, Cela F. Mervis, Anne M. Maxey, Neil E. Markham, and Steven H. Abman

Pediatric Heart Lung Center, Department of Pediatrics, University of Colorado School of Medicine, Denver, Colorado

Submitted 6 September 2006 ; accepted in final form 1 January 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hyperoxia disrupts vascular and alveolar growth of the developing lung and contributes to the development of bronchopulmonary dysplasia (BPD). Endothelial progenitor cells (EPC) have been implicated in repair of the vasculature, but their role in lung vascular development is unknown. Since disruption of vascular growth impairs lung structure, we hypothesized that neonatal hyperoxia impairs EPC mobilization and homing to the lung, contributing to abnormalities in lung structure. Neonatal mice (1-day-old) were exposed to 80% O2 at Denver's altitude (= 65% at sea level) or room air for 10 days. Adult mice were also exposed for comparison. Blood, lung, and bone marrow were harvested after hyperoxia. Hyperoxia decreased pulmonary vascular density by 72% in neonatal but not adult mice. In contrast to the adult, hyperoxia simplified distal lung structure neonatal mice. Moderate hyperoxia reduced EPCs (CD45–/Sca-1+/CD133+/VEGFR-2+) in the blood (55%; P < 0.03), bone marrow (48%; P < 0.01), and lungs (66%; P < 0.01) of neonatal mice. EPCs increased in bone marrow (2.5-fold; P < 0.01) and lungs (2-fold; P < 0.03) of hyperoxia-exposed adult mice. VEGF, nitric oxide (NO), and erythropoietin (Epo) contribute to mobilization and homing of EPCs. Lung VEGF, VEGF receptor-2, endothelial NO synthase, and Epo receptor expression were reduced by hyperoxia in neonatal but not adult mice. We conclude that moderate hyperoxia decreases vessel density, impairs lung structure, and reduces EPCs in the circulation, bone marrow, and lung of neonatal mice but increases EPCs in adults. This developmental difference may contribute to the increased susceptibility of the developing lung to hyperoxia and may contribute to impaired lung vascular and alveolar growth in BPD.

lung vascular development

PULMONARY VASCULAR AND DISTAL air space growth are highly coordinated concomitant processes during the late canalicular and saccular periods of fetal lung development and continue into the alveolar stage after birth (16, 79). The alveolar stage of lung development is characterized by an exponential increase in vascular growth and septation, which are critical determinants of normal lung architecture in postnatal life (11, 31, 68). These complex processes are dependent on highly regulated, temporal, and spatial expression of multiple cytokines, growth factors, and other signaling peptides (59, 64, 74). Previous studies have shown that lung injury during the postnatal alveolar stage of lung development disrupts both alveolar and vascular growth (56, 65, 67, 76, 87). In addition, disruption of vascular growth during the alveolar stage impairs septation, leading to marked simplification of the distal lung structure (40, 54). The mechanisms that maintain or repair distal lung structure and regulate vascular growth during normal development and after neonatal lung injury are incompletely understood (71).

Premature birth and the treatment of respiratory distress syndrome lead to the development of chronic lung disease known as bronchopulmonary dysplasia (BPD) (60). BPD is characterized by persistent abnormalities of lung structure due to dysmorphic vascular growth and impaired alveolarization (20, 42). Structural lung abnormalities in infants with BPD include a reduction of lung surface area, resulting in abnormal gas exchange, exercise intolerance, and pulmonary hypertension (2, 20, 37, 41, 70, 72). Oxygen toxicity in the developing lung contributes to the pathogenesis of BPD, but mechanisms by which hyperoxia induces lung injury and impairs lung repair are unclear (85, 86, 88). Past studies suggest that inhibition of angiogenesis can impair alveolarization (40, 54), but the mechanisms that disrupt vascular and alveolar growth leading to the development of BPD are poorly understood.

Neonatal rodents are believed to be "tolerant" to severe levels of hyperoxia (>95%) compared with adults (29, 89). Adult rodents exposed to this level of hyperoxia die within days from pulmonary edema and interstitial inflammation. In contrast, neonatal rodents can survive prolonged exposure to this level of hyperoxia (29, 89). Although these neonatal rodents survive, a striking inhibition of distal lung growth persists during infancy, as evidenced by increased distal air space size with reduced septation and decreased vascular density (27, 28, 50, 55, 65, 67, 85, 87). Less extreme levels of hyperoxia have also been shown to inhibit lung growth in neonatal rats (18, 22, 35). These changes in lung structure closely mimic the histology of altered lung architecture observed in human infants with BPD (20, 37, 41, 42, 70, 72), thereby providing a useful experimental model for studying BPD. Past studies have shown that hyperoxia reduces the expression of lung vascular endothelial growth factor (VEGF), a potent proangiogenic and endothelial cell survival factor, and its receptor, VEGFR-2 (KDR/Flk-1) (50, 55). Blockade of VEGFRs after birth decreases vascular and alveolar growth in infant rats (40, 54, 77, 78). Hyperoxia also reduces lung endothelial nitric oxide synthase (eNOS) expression and NO production, a critical downstream effector of proangiogenic effects of VEGF (55). These studies suggest that disruption of vascular growth and alveolarization during neonatal hyperoxic lung injury contributes to abnormal lung structure.

Circulating endothelial progenitor cells (EPC) play a role in the repair of many organs after vascular injury by promoting neovascularization in the heart (5, 46), brain (93), and the ischemic hindlimb (6, 43) of adult animals. EPC have been defined as cells that express the surface markers CD34+, CD133+, and VEGFR-2+, and in mice, Sca-1+ (6, 32, 44, 62, 63, 92). Recently, increased circulating EPC levels have been noted in patients with pneumonia (90) and were strongly correlated with improved survival after acute lung injury in adult patients (15). In addition, decreased circulating EPCs have been reported in patients with chronic obstructive pulmonary disease and long-standing hypoxemia (25). In adult mouse models of acute lung injury, bone marrow-derived EPCs participate in the preservation and repair of lung structure after LPS-induced lung injury (1, 91). Although there is evidence for a role of EPCs in the repair of the adult lung after injury, little is known about the direct or indirect effects of neonatal hyperoxia on EPC function and the role of EPC in lung vascular growth.

Therefore, we hypothesized that mobilization of bone marrow-derived EPCs and homing of EPCs to the lung will be impaired in an experimental model of impaired vascular and alveolar growth caused by neonatal hyperoxia. Since the neonatal mouse is more susceptible than the adult to hyperoxia-induced changes in lung structure, we further hypothesized that circulating and lung EPC levels may be maintained in adult mice exposed to similar levels of hyperoxia.

To test these hypotheses, we measured changes in bone marrow, circulating and lung EPC number, and morphometric analysis of lung vascular growth and distal air space structure in neonatal and adult mice exposed to moderate hyperoxia. To further assess whether bone marrow-derived EPCs preserve lung structure after hyperoxia in adult mice, we studied the effects of suppression of the bone marrow with irradiation of adult mice before hyperoxia on lung structure. Finally, since VEGF, erythropoietin (Epo), and eNOS may contribute to homing of EPCs to the lung after injury, we measured lung VEGF, Epo, Epo receptor (EpoR), and eNOS protein content in neonatal and adult mice after hyperoxia. We report that hyperoxia reduces EPCs in the circulation, bone marrow, and lung in neonatal mice but has an opposite effect by increasing EPCs in the adult mouse. In addition, hyperoxia decreases vessel density, impairs air space structure, and downregulates lung VEGF and EpoR and eNOS expression in neonatal but not adult mice. Pretreatment of adult mice with irradiation increased their susceptibility to the hyperoxia-induced changes in lung structure. We speculate that this striking developmental difference in the EPC response to hyperoxia may contribute to the increased susceptibility of the developing lung to hyperoxia and may contribute to impaired lung vascular and alveolar growth in BPD.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Study animals and protocols. All procedures and protocols were reviewed and approved by the Animal Care and Use Committee at the University of Colorado Health Sciences Center. Mice (C57BL/6J) were obtained from Jackson Labs and bred inhouse.

Hyperoxia exposure. Litters from pregnant mice were allowed to deliver and recover in room air for 24 h. Mothers and litters were placed in chambers in which the oxygen concentration was maintained at a FIO2 = 0.8 (PO2 = 466 mmHg at Denver's altitude barometric pressure = 630 mmHg; equivalent to an FIO2 = 0.65 at sea level) for 10 days or maintained in room air. Exposure to hyperoxia was continuous, with a brief interruption for animal care (less than 10 min/day). The concentration of oxygen was controlled by the use of a ProOx (Reming Bioinstruments). To determine if there was a difference between the response of neonatal and adult mice, adult mice (8-wk-old) were studied in a similar manner as described above.

To determine if bone marrow-derived EPCs contribute to the maintenance of lung structure in the adult in response to hyperoxia, a group of adult mice (n = 8) were initially exposed to 450 cGy of radiation to suppress the bone marrow and were then exposed to hyperoxia. A control group (n = 8) was irradiated and then maintained in room air. At the end of 10 days of hyperoxia, lung tissue, blood, and bone marrow were collected from each study group for histology and FACS analysis.

Lung histology and morphometric analysis. Lungs were prepared for histology, as previously described (8). Briefly, mice were euthanized with intraperitoneal injections of pentobarbital (100 mg/kg). A catheter was placed into the trachea, and the lungs were inflated and maintained at 30 cmH2O pressure with 4% paraformaldehyde in PBS for at least 45 min. A ligature was tightened around the trachea to maintain pressure after removal of the tracheal cannula. Lungs were immersed in paraformaldehyde solution overnight, and the left lower lobe was embedded in paraffin. Sections were cut with a microtome set at 5 µm and mounted on RNase free slides for histochemical analysis.

Morphometric analysis. Morphometric analysis was performed using standard techniques in our lab and computer-assisted image analysis, as previously described (9). Briefly, six lung sections from each animal were selected for study in an unbiased fashion. The orientation of these samples was at random, creating isotropic uniform random plane sections of the lung tissue (10). Each section was stained with hematoxylin and eosin. Six lung sections were selected in unbiased fashion for assessment of skeletonization (82), mean linear intercept (MLI), average alveolar size, and internal surface area. Images of each section were captured with a QICAM digital camera on a Zeiss Axioscope2 with a x20 objective and were saved as TIFF files. These images were processed with a plug-in that utilizes ImageJ, a public-domain Java image processing program created by Wayne Rasband at the Research Services Branch, National Institute of Mental Health, Bethesda, MD (http://rsb.info.nih.gov/ij/), as previously described (9). The maximum and minimum diameter of each alveolus (feret length) is calculated, averaged, and displayed as average alveolar size (µm2). The internal surface area (septal membrane length) is measured for each image as µm/high-powered field (hpf). Images of lung parenchyma are transformed into a skeleton of curved and straight line segments with nodal and end points, as described by Tschanz and Burri (82). The number of end points, intersections (nodes), and internodal distances are quantified. The intra-alveolar distance was measured as the MLI by standard methods (10, 80, 81), utilizing the same plug-in by dividing the total length of 42 lines drawn across the lung section by the number of intercepts encountered (81). Lines that crossed large airways or vessels were excluded from analysis. MLI is inversely proportional to the surface area of the lung (80, 81). Radial alveolar counts (RAC) were assessed by standard methods, as previously described (10, 21).

Sections were stained for the presence of factor VIII [von Willebrand factor (vWF)] (Dako), an endothelial-specific marker. For assessment of vessel density, images of factor VIII-stained slides were captured with a x20 objective. The number of factor VIII-positive vessels (20–80 µm in size) was counted per each high-powered field.

Western blot analysis. Frozen lung samples were homogenized in ice-cold buffer containing 50 mM Tris·HCl (pH 7.4), EDTA (1 mM), EGTA (1 mM), 0.1% 2-mercaptoethanol, 4-(2-aminoethyl)-benzenenesulfoyl fluoride (1 mM), leupeptin (1 µM), and pepstatin A (1 µM). Samples were centrifuged at 1,500 g for 20 min at 4°C to remove cellular debris. Protein content in the supernatant was determined by the Bradford method (14), using bovine serum albumin as the standard. Briefly, 25 µg of protein sample per lane was resolved by SDS-PAGE, and proteins from the gel were transferred to PVDF membrane. Blots were blocked 1 h in 5% nonfat dry milk in PBS with 0.1% Tween 20. These blots were incubated for 1 h at room temperature with either rabbit anti-human polyclonal VEGF antibody (sc-152, 1:500; Santa Cruz Biotechnology), rabbit anti-human polyclonal VEGFR-2 antibody (KDR/flk-1; sc-504, 1:250; Santa Cruz Biotechnology), rabbit anti-human polyclonal EpoR (sc-5624, 1:200; Santa Cruz Biotechnology), goat anti-mouse polyclonal hepatocyte growth factor (HGF) (AF2207, 1:500; R&D Systems), rat anti-mouse monoclonal HGF receptor/c-Met (MAB527, 1:500; R&D Systems), mouse anti-human monoclonal SDF-1/CXCL-12 (MAB350, 1:1,000; R&D Systems), or mouse anti-human polyclonal eNOS (bd610297, 1:500; BD Biosciences) in 5% nonfat dry milk in PBS with 0.1% Tween 20. Blots were incubated for 1 h at room temperature with a goat anti-rabbit IgG horseradish peroxidase (HRP) antibody (sc-2054, 1:5,000; Santa Cruz Biotechnology) or goat anti-mouse IgG HRP antibody (AP124P, 1:10,000; Chemicon). After being washed, bands were visualized by enhanced chemiluminescence (ECL+ kit; Amersham Pharmacia Biotech, Buckinghamshire, UK). Adult mouse lung homogenate was run as a control, and the band that comigrated with the molecular size as identified by the manufacturer for the protein of interest was quantified by densitometry for VEGFR-2 and eNOS. For Western analysis of VEGF, recombinant mouse VEGF protein (Santa Cruz Biotechnology) was used as a control. Densitometry was performed using ImageJ (v1.33q).

Immunohistochemical staining. Lungs were fixed in 4% paraformaldehyde/PBS for 24 h and then stored in 70% ethanol. The left lower lobe was embedded in paraffin, cut into 5-µm-thick sections, and mounted on "plus" slides. Slides were deparaffinized in xylene and rehydrated by serial immersions in 100% ethanol; 95% ethanol; 70% ethanol and 100% water. Proteinase K (50 µg/ml) was placed on the sections for 5 min. The sections were washed with 1x PBS (2.7 mM KCl, 1.2 mM KH2PO4, 138 mM NaCl, 8.1 mM Na2HPO4). Endogenous peroxidase activity was quenched by immersion in 3% hydrogen peroxide in methanol and rinsed with PBS. The sections were incubated with 10% goat/2% mouse serum and rabbit anti-human polyclonal factor VIII antibody (Dako, A0082) or rabbit IgG diluted 1:1,000 in 1x PBS with 1% BSA and 0.1% sodium azide for 1 h at room temperature. After incubation, the sections were rinsed with PBS, incubated in 10% goat/2% mouse serum for 5 min, and then incubated with biotin-labeled goat anti-rabbit secondary antibody diluted 1:200 in 10% goat/2% mouse serum for 15 min at room temperature. After incubation with the secondary antibody, sections were rinsed with PBS. Sections were incubated with ABC complex (Vector) for 30 min at room temperature, rinsed in PBS, and developed with diaminobenzidine (DAB; Vector) and hydrogen peroxide. Washing with water stopped the DAB reaction. A light hematoxylin counterstain was applied. Sections were dehydrated by sequential immersion in 70% ethanol; 95% ethanol; 100% ethanol and then HemoDe before placing a coverslip on the section.

Isolation of EPCs. Blood was collected from the right ventricle in each animal. The pulmonary circulation was then thoroughly flushed with PBS to remove circulating cells. Lung tissue was harvested, minced finely, and digested for 1.5 h at 37°C in digestion buffer [DMEM-F-12, 80 U/ml DNase I (Sigma, 4263), 0.7% collagenase-I (V-Worthington,LS004194)]. The tissue was filtered through 70-µm mesh, centrifuged at 440 g for 10 min, and resuspended in RBC lysis buffer (150 mM NH4Cl, 2mM Na2-EDTA). Cells were centrifuged at 500 g for 4 min and then resuspended in staining buffer (PBS, 1% BSA, 100 U/ml DNase I, 1 mM MgCl2). Bone marrow was harvested by removing each end of the femur, which was then centrifuged at 1,300 g for 2 min to remove the marrow. Bone marrow was incubated for 10 min in RBC lysis buffer, centrifuged at 500 g for 3 min, filtered through a 70-µm mesh, and resuspended in staining buffer. Blood was incubated with 3x vol of RBC lysis buffer and centrifuged at 440 g. The pellet resuspended in RBC lysis buffer, filtered through a 70-µm mesh filter, and resuspended in staining buffer. Since EPCs represent relatively "rare events" when sorted by FACS, we followed a modified ISHAGE protocol that has been described to isolate CD34+ cells (73). This procedure has been used successfully to isolate and characterize CD133+ cells from cord blood, bone marrow, and apheresis products, utilizing small samples (100 µl) for analysis (23). This method has been previously described to allow the reliable quantification of rare events at frequencies that were much lower than we report (33). A minimum of 100,000 cells were counted from each sample, and a minimum of 100 of the triple-positive events that would generate a coefficient of variation of <1% was counted for the lung and bone samples from neonatal and adult mice. In blood samples, as many triple-positive events as possible were counted in 200 µl of blood from each mouse. The lung, bone marrow, and blood cells were then incubated with Ter-119 (1:35; Caltag; MTER00) antibody for 10 min and goat anti-rat PE-Texas Red (1:35; Caltag; R40117 [GenBank] ) secondary antibody for 10 min. The samples were washed with PBS and resuspended in staining buffer. The samples were then incubated with the following pregconguated antibodies to CD45-APC (1:13, BD Pharmingen, 559864), CD133-FITC (1:20; e-Biosciences, 11-1331-82), VEGFR-2-PE (1:20, BD Pharmingen, 555308), and Sca-1-Biotin (1:20, BD Pharmingen, 557404) for 20 min. Finally, Sav-PeCy-7 (1:35, BD Pharmingen, 557598) was added to activate the Sca-1 antibody signal. The cells were analyzed at the University of Colorado Health Sciences Flow Cytometry Core with a Beckman (Miami, FL) Epics-XL Flow Cytometer. Single-stain controls (positive control) and appropriate isotype IgG controls (negative controls) were used to set the gates. Voltage compensation for fluorescent intensity was performed with the use of CompBeads (BD Pharmingen, 552844). All samples were stained with propidium iodide (PI) to detect dead cells, and these were gated out of the analysis. To set the gates for VEGFR-2, we used a fluorescence minus one control sample of lung, bone marrow, and blood cells incubated with all of the above antibodies except VEGFR-2. The gating strategy was as follows: the first gate was set to exclude PI-positive and Ter-119-positive cells; the cells were then gated on the CD45-negative and dim population; these cells were then gated for cells that were both CD133 positive and Sca-1 positive; these cells were then finally gated on the VEGFR-2-positive population, yielding a subset of cells that are CD45–/dim/ Sca-1+/CD133+/VEGFR-2+.

In vitro EPC studies. To collect sufficient numbers of cells for in vitro studies of EPCs, bone marrow was harvested from fetal lambs, as described above. EPCs were characterized by staining for CD133 (mouse anti-human mononuclear CD133-PE; Miltenyi Biotech 130–080-801) and VEGFR-2 (mouse anti-human monoclonal VEGFR-2-biotin; Sigma B0555) and sorted on a 3 Laser MoFlo high performance cell sorter (DakoCyotomation). In culture, isolated cells develop a spindle-shaped appearance typical of EPCs (5, 6, 38, 39) and subsequently differentiate into cells that express PECAM, vWF, and eNOS, as assayed by immunofluorescence staining and take up acetylated-LDL. Cells were cultured on fibronectin-coated plates for 10 days in either room air (16% O2/5% CO2/balance N2) or hyperoxia (80% O2/5% CO2/balance N2) and assayed for growth and survival.

Statistical analysis. Data are presented as means ± SE. Statistical analysis was performed with the Prism software package (GraphPad Software, San Diego, CA). Statistical comparisons were made between groups using an unpaired t-test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hyperoxia exposure impairs lung structure in neonatal but not adult mice. Lung histology of neonatal mice exposed to 10 days of 80% oxygen demonstrate enlargement of distal air spaces with a simplification of lung structure (Fig. 1, A and B). In contrast, hyperoxia did not alter the lung structure of adult mice (Fig. 1, C and D). Vessel density is reduced by 72% in neonatal mice after hyperoxia compared with room air controls (Fig. 2; P < 0.001). In addition, there is a striking decrease in vascular growth as shown by a reduction in the number of small vessels seen by vWF staining (Fig. 2, A vs. B). Morphological measurements of lung structure in neonatal mice (Table 1) show that hyperoxia increased MLI by 11% (P < 0.02), reduced RAC by 57% (P < 0.001), reduced internal surface area by 17% (P < 0.001), reduced nodal point density by 23% (P < 0.001), and increased alveolar size by 49% (P < 0.001). There was no effect of moderate hyperoxia on the body weight of the infant mice or the mothers.


Figure 1
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  		Fig. 1. Moderate hyperoxia impairs lung structure in neonatal but not adult mice. Lung structure is markedly abnormal, with enlarged air spaces and simplified structure, in neonatal mice exposed to hyperoxia (FIO2 = 0.8 at Denver's altitude) for 10 days (B) compared with room air controls (A). Lung structure is not different in adult mice exposed to hyperoxia for 10 days (D) compared with room air controls (C). All images were taken with x10 objective (n = 5 animals/group). Scale bar, 100 µm.

 

Figure 2
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  		Fig. 2. Vessel density of neonatal mice after moderate hyperoxia. The density of vessels identified by von Willebrand factor staining (arrows) is decreased in neonatal mice exposed to hyperoxia (A) compared with room air (B). The number of vessels per high-powered field (hpf) is significantly reduced in mice exposed to hyperoxia (filled bar) compared with room air controls (open bar). *P < 0.001; n = 5 animals/group. All images were taken with x20 objective. Scale bar, 100 µm.

 

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  		Table 1. Morphometric analysis of lung structure in neonatal mice after moderate hyperoxia

 
Contrasting effects of hyperoxia on EPC number in neonatal and adult mice. There was no significant difference in the number of mononuclear cells recovered from the blood, bone marrow, or lungs of mice exposed to moderate hyperoxia compared with room air. Blood, bone marrow, and lung tissue was subjected to FACS analysis to gate for CD45dim/–/Sca-1+/CD133+/VEGFR-2+ to define the EPC population in each tissue type (Fig. 3). The total number of mononuclear cells analyzed in the blood, bone marrow, and lung was unchanged by exposure to hyperoxia in either the neonatal or adult mouse. The percent of EPCs was reduced in the neonatal animal exposed to hyperoxia by 55% in the blood (Fig. 4A; P < 0.03), by 48% in the bone marrow (Fig. 4B; P < 0.001), and by 66% in the lung (Fig. 4C; P < 0.001). In the adult mouse exposed to hyperoxia, EPC number was not changed in the blood (Fig. 5A; P = 0.13) but was increased by 2.5-fold in bone marrow samples (Fig. 5B; P < 0.001) and 2-fold in lung samples (Fig. 5C; P < 0.03).


Figure 3
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  		Fig. 3. FACS analysis for endothelial progenitor cells (EPC). An example of the gating strategy used for bone marrow is shown. Live cells were first gated by dim or absent staining for CD45 (A). Cells were then gated for double positive staining for CD133 (x-axis) and Sca-1 (y-axis) (B). EPCs were quantified by positive staining for VEGF receptor-2 (VEGFR-2) (KDR/Flk-1) (C). The gate for VEGFR-2 was set with the use of a fluorescence minus one control lacking only the VEGFR-2 antibody (blue line) compared with fully stained bone marrow (red line).

 

Figure 4
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  		Fig. 4. EPC number is reduced in the blood, bone marrow, and lung of neonatal mice exposed to hyperoxia. In neonatal mice exposed to hyperoxia (filled bars), EPC number is decreased in the blood (A), bone marrow (B), and lung (C) compared with room air controls (open bars). *P < 0.03; n = 6 animals/group.

 

Figure 5
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  		Fig. 5. EPC number is increased in the bone marrow and lung of adult mice exposed to hyperoxia. In adult mice exposed to hyperoxia (filled bars), EPC number is unchanged in the circulation (A) but increased in the bone marrow (B) and lung (C) compared with room air controls (open bars). *P < 0.03; n = 6 animals/group.

 
In vitro exposure of EPCs to hyperoxia impairs survival. Fetal bone marrow-derived EPCs cultured in 80% oxygen for 10 days failed to grow and remained small round cells as seen in Fig. 6B. In vitro hyperoxia resulted in the death of >97% of the EPCs. In contrast, EPCs grown in room air continued to grow and maintained their characteristic appearance.


Figure 6
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  		Fig. 6. Hyperoxia impairs EPC survival in vitro. Fetal bone marrow EPCs were cultured in either room air or hyperoxia (FIO2 = 0.8) for 10 days. In room air, EPCs survived and proliferated (A). Hyperoxia resulted in the death of >97% of the EPCs (B).

 
Functional bone marrow is necessary for the maintenance of lung structure in the adult mouse exposed to moderate hyperoxia. Adult mice whose bone marrow was supressed with sublethal irradiation were exposed to hyperoxia or kept in room air. Lung structure of irradiated mice kept in room air was unchanged from non-irradiated controls (Fig. 7C). Irradiated mice exposed to hyperoxia had enlargement of air spaces and a simplification of lung structure (Fig. 7, A vs. B). There was no change in MLI of adult mice exposed to irradiation after 10 days, but there was a 6% increase in MLI of adult mice irradiated and exposed to hyperoxia compared with mice kept in room air after irradiation (Fig. 7C). As expected, there was a marked reduction in the number of mononuclear cells in the circulation (decreased by 86%; P < 0.001) and bone marrow (decreased by 95%; P < 0.001) in irradiated mice compared with non-irradiated controls. Yet there was no difference in the percentage of EPCs in the circulation or bone marrow. There was a 75% reduction in mononuclear cells in the lungs of irradiated mice compared with non-irradiated mice (P < 0.001). In the irradiated mice kept in room air, the percentage of mononuclear cells that were EPCs were increased fivefold compared with non-irradiated mice. In irradiated mice exposed to hyperoxia, the percent of mononuclear cells that were EPCs was reduced by 98% compared with irradiated mice kept in room air (P < 0.001). This reduction in EPCs in the lung in the irradiated mice exposed to hyperoxia is in stark contrast to the effect of hyperoxia on adult mice with intact bone marrow.


Figure 7
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  		Fig. 7. Adult mice exposed to hyperoxia after bone marrow suppression have abnormal lung structure. Adult mice (8-wk-old) were subjected to sublethal irradiation to suppress their bone marrow. After this, they were placed in hyperoxia for 10 days. Lung structure shows enlargement of air spaces in the mice exposed to hyperoxia after irradiation (B) compared with animals kept in room air (A). Mean linear intercept is increased in mice exposed to hyperoxia after bone marrow suppression (C). *P < 0.05; n = 5 animals/group. All images were taken with x20 objective. Scale bar, 100 µm. RAD, radiation exposure.

 
Effect of hyperoxia on lung VEGF, VEGFR-2, eNOS, HGF, SDF-1, and EpoR protein in neonatal and adult mice. Western blot analysis revealed that exposure of the neonatal mouse to hyperoxia resulted in a 56% decrease in lung VEGF protein (Fig. 8A; P < 0.05) and a 41% decrease in lung VEGFR-2 protein (Fig. 8B; P < 0.01), whereas there was no change in lung VEGF or VEGFR-2 protein seen in the adult mouse with hyperoxia exposure (Fig. 8, F and G). In the neonatal mouse, there was a 40% reduction in lung eNOS protein (Fig. 8C; P < 0.05), but there was a 1.5-fold increase in lung eNOS protein in the adult mouse exposed to hyperoxia (Fig. 8H; P < 0.03). Hyperoxia exposure of the neonate resulted in a 45% reduction in lung EpoR protein (Fig. 8D; P < 0.03) but no change in the adult mouse exposed to hyperoxia (Fig. 8I). Lung SDF-1/CXCL-12 protein was unchanged in the neonate exposed to moderate hyperoxia (Fig. 8E), but there was a 76% increase in lung SDF-1 protein in the adult mouse exposed to moderate hyperoxia (Fig. 8J; P < 0.05). There was no change in lung HGF or HGF receptor as assayed by Western blot analysis.


Figure 8
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  		Fig. 8. Lung VEGF and VEGFR-2 protein is reduced in neonatal mice exposed to hyperoxia but unchanged in adult mice. Lung endothelial nitric oxide synthase (eNOS) protein is reduced in neonatal mice exposed to hyperoxia and increased in adult mice. Western blot analysis of lung tissue from neonatal mice exposed to hyperoxia (filled bars) shows a reduction in lung VEGF (A), VEGFR-2 (B), eNOS (C), and erythropoietin receptor (EpoR) (D) protein compared with room air controls (open bars). Lung SDF-1 protein was unchanged by neonatal hyperoxia exposure (E). In adult mice exposed to hyperoxia, there was no change in lung VEGF (F), VEGFR-2 (G), or EpoR (I) protein, but there was an increase in lung eNOS protein (H) and SDF-1 protein (J). *P < 0.05; n = 4 animals/group.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Mobilization and engraftment of EPCs play a key role in the repair of the pulmonary vasculature after lung injury in adult mice after elastase and LPS injury (1, 91) and in adult humans with pneumonia and acute respiratory distress syndrome (15, 90). However, the role of EPCs during lung vascular development and in response to neonatal hyperoxia exposure is unknown. In very low birth weight human infants (<1,000 g) a "new" form of BPD has been recognized that is primarily characterized by impaired vascular growth and alveolarization (20, 41, 42). To study the new BPD, models exposing neonatal animals to moderate hyperoxia of 60–65% oxygen (alveolar PAO2 = 428–463 mmHg; equivalent to 80% oxygen at Denver's altitude where this study was performed) have been developed (18, 22, 35).

In this study, we report that moderate hyperoxia reduces the number of circulating, bone marrow, and lung EPCs in neonatal mice. In contrast, moderate hyperoxia increases the number of EPCs in the bone marrow and lung and maintains the number of circulating EPCs in adult mice. In addition, there is a developmental difference in the response to hyperoxia between neonatal and adult mice. Moderate hyperoxia exposure has profound effects in the neonate resulting in a marked reduction in lung vascular density and alveolarization. In contrast, in adult mice, moderate hyperoxia does not affect lung structure. To determine the role of bone marrow-derived EPCs in the maintenance of lung structure during exposure of adult mice to moderate hyperoxia, we irradiated adult mice to decrease bone marrow cellularity and subsequently exposed these mice to moderate hyperoxia. Irradiation alone did not have an effect on adult lung structure, but we report that pretreatment with irradiation increases the susceptibility of the lung to hyperoxia.

These findings are interesting because previous work has focused on the effect of impaired angiogenesis as the primary mechanism for impaired vascular growth in models of alveolar simplification in the developing lung (40, 53, 54, 65, 67, 71, 87). Vasculogenesis, or the in situ growth and differentiation of EPCs into endothelial cells and blood vessels (19, 64), was initially thought to be limited to vascular development during the prenatal period. During the prenatal lung growth, the distal lung microvasculature expands by vasculogenesis and connects with the invading angiogenic sprouts from the main pulmonary artery (34). The possibility of a role for EPC-mediated vasculogenesis in the postnatal period was first demonstrated by an increase in the numbers of bone marrow-derived circulating EPCs in response to VEGF treatment and the participation of these bone marrow-derived cells subsequent neovascularization in the corneal micropocket injury model (7). The precise roles of EPCs may be uncertain, but past studies in other settings have shown their ability to induce neovascularization or repair vessels after injury (44). This is the first study to suggest that EPC-mediated vasculogenesis, outside of the early embryonic period of lung growth, may play an important role in normal postnatal lung vascular and alveolar growth. Furthermore, disruption of lung vascular and alveolar growth by neonatal hyperoxia may be exacerbated by a reduction in vasculogenesis secondary to impaired mobilization and homing of bone marrow-derived EPCs. In contrast, adult mice exposed to moderate hyperoxia increased bone marrow and lung EPCs, which may contribute to the preservation of lung structure during hyperoxia exposure. We also must emphasize that the level of hyperoxia used in this study (80% oxygen at Denver's altitude = 65% oxygen at sea level) should be considered moderate and did not result in an increase in mortality of adult or newborn mice, in contrast to the reported increased mortality of adult rodents with exposure to more severe hyperoxia (>95% O2) (29, 89). The specific roles of EPCs in this model remain uncertain, and future studies are needed to demonstrate the function of EPCs after neonatal lung injury. Overall, these findings suggest that bone marrow-derived cells, such as EPCs, play a role in the protection of the lung from sustained injury from hyperoxia in adult mice and contribute to the developmentally different response to moderate hyperoxia.

VEGF is a potent endothelial cell mitogen involved in vasculogenesis and is an important mediator of EPC mobilization, homing, and engraftment. VEGF administered to rodents resulted in the mobilization of EPCs from the bone marrow (7, 43, 57). Blockade of VEGF receptor activity in the neonatal period impairs lung growth and structure, which is similar to the lung histology of infants with BPD (40, 54). In the baboon model of BPD, lung VEGF and VEGFR-2 expression are reduced as observed in human neonates who die from BPD (13, 51, 75). In this current study, we have observed that hyperoxia reduces lung VEGF and VEGFR-2 protein in neonatal mice. In contrast, the adult mouse exposed to moderate hyperoxia maintains lung VEGF and VEGFR-2 protein expression. We are unsure of the mechanism by which a reduction in VEGF and VEGFR-2 in the neonatal lung results in impaired vessel growth but speculate that there may be local effects on endothelial cell growth, survival, and function, or the effect may be related to a reduction in homing and engraftment of EPCs to the lung. This developmentally different response suggests a mechanism by which moderate hyperoxia impairs vasculogenesis and lung growth through impaired EPC mobilization and engraftment in the neonate but not the adult.

One of the downstream effectors of VEGF signaling is the activation of eNOS and the generation of NO (17, 24, 30, 47, 58, 69, 84). Mice genetically deficient in eNOS or wild-type mice in which eNOS has been pharmacologically blocked have impaired EPC mobilization from the bone marrow in response to exercise (52). In the ischemic hindlimb neovascularization model, the eNOS-deficient mouse has an impaired maturation and mobilization of EPCs from the bone marrow in response to VEGF (3). Furthermore, the number of endothelial cell colonies that will grow from bone marrow of eNOS-deficient mice is also reduced (3). These studies suggest that the generation of endothelial-derived NO is necessary for the maturation, mobilization, and differentiation of EPCs. In our present study, hyperoxia reduces lung eNOS protein expression in the neonate but increases lung eNOS protein in the adult mouse. This is a novel observation of a developmentally different response to hyperoxia. We speculate that the reduction in lung eNOS protein may result in alterations in local NO production and may contribute to an impairment of EPC homing and engraftment in the neonatal lung. In contrast, increased lung eNOS protein may promote circulating EPC homing, engraftment, and differentiation in the adult, thus preserving lung vascular and alveolar structure during exposure to moderate hyperoxia.

Epo is also known to play a role in the mobilization and engraftment of EPCs (66). The EpoR is highly expressed in the developing lung (26) and may act through upregulation of eNOS expression (12, 83). Epo treatment of neonatal mice exposed to hyperoxia improves lung vascular and alveolar growth (61). In this study, we report that there is a reduction in the lung EpoR in the neonate, but not adult mouse, exposed to hyperoxia that parallels the changes in EPC numbers. This suggests another mechanism, perhaps acting in synergy with VEGF and NO, through which hyperoxia exposure of the neonate reduces EPC engraftment resulting in abnormal lung structure during development.

Stromal derived factor-1 (SDF-1/CXCL12) was originally isolated from bone marrow stromal cells and is a chemotactic cytokine for progenitor cells (4, 36, 45). Recent evidence suggests that SDF-1 may act synergistically with VEGF to promote neoangiogenesis through the recruitment of EPCs (48) and may induce endothelial cell proliferation by increasing the production of NO (49). In this study, we report that in the neonate exposed to moderate hyperoxia, there is no increase in SDF-1 protein in the lung, but in the adult exposed to moderate hyperoxia, lung SDF-1 increases significantly, and there is a preservation of lung alveolar and vascular structure. The ability of the adult to increase SDF-1 expression in response to moderate hyperoxia exposure may contribute to the increase in lung EPC number and preservation of lung structure that is not observed in the neonate.

Previous studies have shown that functional bone marrow is necessary for the preservation of lung structure after intratracheal LPS-induced lung injury in adult mice (1, 91). In the present study, we found that lung EPC content in the lung increased after irradiation and that there was no change in lung structure in adult mice. Exposure to moderate hyperoxia after irradiation markedly decreased the percent of EPCs in the lung and resulted in alveolar enlargement and simplification of lung structure. This suggests that hyperoxia has a direct effect on lung EPC survival and thus affects the preservation of lung structure. In addition, hyperoxic exposure of the adult mouse stimulates the recruitment and homing of EPCs from the bone marrow to the lung, which is critical in the maintenance of normal lung structure in the adult.

Potential limitations of this study include potential controversy regarding the definition of EPCs. The definition used in this study is based on extensive work by others in which EPCs can be defined as CD45dim/–, Sca-1+, CD133+, and VEGFR-2+ (6, 32, 38, 39, 44, 62, 63, 92). Although these results show clear differences in our measurements of EPCs between control and hyperoxia conditions and between neonatal and adult mice, it is possible that this is dependent on our definition of EPC. We have not attempted to determine whether the use of other markers for defining EPC would have led to the same results. In addition, although lung VEGF and eNOS expression are decreased in the lung, we did not measure changes in these proteins in bone marrow or blood to determine if these mechanisms may also lead to altered EPC mobilization or survival. Maternal factors, such as milk production or changes in the quality of the milk produced, may have been affected by exposure of the mothers to hyperoxia. Changes in infant nutrition can affect lung development, and we did not quantify these changes, but we also did not observe an effect of hyperoxia on the weight of the infant mice. Finally, moderate hyperoxia may also alter other growth factors and cytokines that may be involved in the mobilization and recruitment of EPCs to the lung during exposure to moderate hyperoxia.

We conclude that there are developmentally different responses, in terms of EPC number and lung structure, in response to exposure to moderate hyperoxia between the neonate and adult mouse. Preservation of lung structure in the adult animal during exposure to moderate hyperoxia is dependent on the mobilization, recruitment, and engraftment of EPCs to the lung, and this process is impaired in the neonatal animal exposed to moderate hyperoxia. The effects of moderate hyperoxia on lung structure are more pronounced during the neonatal period of alveolar and vascular growth. Furthermore, we conclude developmental differences may be due to a reduction in lung VEGF, VEGFR-2, EpoR, and eNOS in the neonate exposed to moderate hyperoxia compared with the adult. We speculate that impaired EPC mobilization, recruitment, and engraftment in premature infants exposed to moderate hyperoxia may result in impaired alveolar and vascular growth that is seen in BPD.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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This work was supported by National Heart, Lung, and Blood Institute Grant 1 K08 HL-073893 and an unrestricted grant from iNO Therapeutics (to V. Balasubramaniam). The University of Colorado Cancer Center Flow Cytometry Core is supported by National Institutes of Health Grant P30 CA-046934.


   ACKNOWLEDGMENTS
 
We thank Karen Helm at the University of Colorado Cancer Center Flow Cytometry Core for help.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. Balasubramaniam, Pediatric Pulmonary Medicine, Dept. of Pediatrics, UCHSC at Fitzsimmons, Pediatrics 8317, PO Box 6511, Aurora, CO 80045 (e-mail: vivek.balasubramaniam{at}uchsc.edu)

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


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