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Am J Physiol Lung Cell Mol Physiol 293: L941-L951, 2007. First published August 10, 2007; doi:10.1152/ajplung.00054.2007
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Neonatal lung side population cells demonstrate endothelial potential and are altered in response to hyperoxia-induced lung simplification

D. Irwin,1,3 K. Helm,4 N. Campbell,1 M. Imamura,1 K. Fagan,1,3 J. Harral,1,3 M. Carr,1,3 K. A. Young,1,2 D. Klemm,1,3 S. Gebb,6 E. C. Dempsey,1,3,5 J. West,1,3 and S. Majka1,2

1Department of Medicine, Cardiovascular Pulmonary Research Section, 2Division of Cardiology, 3Division of Pulmonary and Critical Care Medicine, University of Colorado Health Sciences Center, Denver; 4Cancer Center, Flow Cytometry Core, 5Denver Veterans Administration Medical Center, Denver, Colorado; and 6Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, Alabama

Submitted 7 February 2007 ; accepted in final form 1 August 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Lung side population (SP) cells are resident lung precursor cells with both epithelial and mesenchymal potential that are believed to play a role in normal lung development and repair. Neonatal hyperoxic exposure impairs lung development leading to a long-term decrease in gas exchange surfaces. The hypothesis that lung SP cells are altered during impaired lung development has not been studied. To address this issue, we characterized the endothelial potential of neonatal lung SP and subsets of lung SP from neonatal mice following hyperoxic exposure during room air recovery. Lung SP cells were isolated and sorted on the basis of their capacity to efflux Hoechst 33342. The lung SP was further sorted based on expression of Flk-1 and CD45. In vitro, both CD45pos/Flk-1pos and CD45neg/Flk-1pos bind isolectin B4 and incorporate LDL and form networks in matrigel, indicating that these populations have endothelial cell characteristics. Hyperoxic exposure of neonatal mice resulted in subtle changes in vascular and alveolar density on P13, which persisted with room air recovery to P41. During room air recovery, a decrease in lung SP cells was detected in the hyperoxic-exposed group on postnatal day 13 followed by an increase on day 41. Within this group, the lung SP subpopulation of cells expressing CD45 increased on day 21, 41, and 55. Here, we show that lung SP cells demonstrate endothelial potential and that the population distribution changes in number as well as composition following hyperoxic exposure. The hyperoxia-induced changes in lung SP cells may limit their ability to effectively contribute to tissue morphogenesis during room air recovery.

bronchopulmonary dysplasia; microvessel density; alveolarization

THE ROLE AND FATE of resident stem cells during development vs. disease in the lung is an area of intense study. Resident lung stem cells include the alveolar type II pneumocyte and the Clara cell (6, 16, 46) as well as a recently described population of primitive adult precursor cells termed the side population (SP) (3, 19, 54, 55). SP cells are found in many adult tissues including the lung and are distinguished by their ability to efflux the DNA dye Hoescht 33342 (3, 20, 38, 62). The lung SP is a primitive multipotent population defined by its lack of differentiated lineage markers and the ability to differentiate into hematopoietic, epithelial, and mesenchymal cells (19, 54, 55). In the lung, the CD45pos SP fraction originates from bone marrow and retains limited hematopoietic ability (3, 49, 50). The origin of the CD45neg SP population is unclear (3, 49, 50). Further in vivo and in vitro characterization of this specialized subpopulation of lung cells is required to fully assess the role these cells play in lung development, disease, and repair.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease occurring in premature newborns requiring mechanical ventilation and supplemental oxygen. BPD occurs in up to 60% of low-birth weight newborns (<1,000 g) and affects ~33% of premature surviving infants (1, 9, 10, 23, 32, 52, 61). The advent of surfactant therapy and use of lung-protective ventilation strategies has markedly decreased the lung injury previously associated with BPD; however, persistent lung hypoplasia continues as a hallmark of the "new" BPD and contributes to continuing mortality and morbidity in affected neonates. The severity of lung simplification corresponds to the gestational stage of lung development at the time of delivery and is characterized by disrupted alveolar and vascular morphogenesis (1, 15, 29, 30, 32). Thus, transient hyperoxic exposure in the developing lung results in BPD with lasting effects such as a loss of complexity in the distal lung, including decreased alveolarization and vascularization. The mechanisms contributing to this pathology have been studied extensively. However, little is known about the effect of oxygen on stem cell populations within the developing lung.

The developing lung is most vulnerable to injury before the final stage of lung development, alveolarization. During human lung development, alveoli are present by 36 wk of gestation; however, greater than 85–90% of alveoli are formed after birth (15, 32). Alveogenesis involves the septation of alveolar sacs and formation of alveoli, which increases the surface area of the lung for oxygen exchange via the pulmonary circulation (14). Vascular surface area also increases at this time and is rate limiting for alveolarization (18, 27). The mechanisms regulating this vascular development are unclear; however, both the disruption of angiogenesis and the loss of coordinated vascular endothelial growth factor (VEGF-A) signaling between the epithelium and endothelium result in lung hypoplasia (5, 18, 24, 27, 33, 34, 40, 50, 57–59). This illustrates the importance of preventing injury and maintaining homeostasis in not only the alveolar compartment but also the pulmonary vascular compartment. In neonatal rats, hyperoxia reduces the proportion of arteries to alveoli and decreases septation, therefore compromising gas exchange (10, 48). Since functional vasculature and angiogenesis are required for normal alveogenesis, deficiencies in pulmonary vascular endothelial precursors likely play a key role in the developmental defects produced by hyperoxia manifested in BPD.

The goal of these studies was to characterize both resident lung SP cells as vascular precursors and examine changes in this population following hyperoxic exposure. Alveolarization and vascular development are intimately linked. Therefore, in our murine model of moderate hyperoxic exposure and recovery, we examined properties of lung simplification. We found decreased alveolarization and microvessel density in the lungs of hyperoxia-exposed animals. Neonatal lung SP were isolated and their vascular potential was confirmed in vitro. Changes in the lung SP were also identified using flow cytometry. Understanding how transient hyperoxia affects pulmonary endothelial precursors such as lung SP is vital to understanding the pathology of sustained vascular and alveolar defects found in BPD.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of lung SP. Lung SP cells were isolated from neonatal mice at ages postnatal day (P)13, 21, 41, and 55 using a 0.2% collagenase digest of lung tissue to obtain a single-cell suspension from control and hyperoxia-exposed lungs. Hoechst staining was performed on cell suspensions as previously described (20, 38). SP counts may vary from animal to animal therefore we obtained and reported the standard error. The SP gates were set based on a bone marrow SP control. The lung SP in these experiments was also analyzed for CD45 and expression of additional cell surface antigens as detailed below.

Briefly, freshly isolated and Hoechst 33342-stained cells were suspended at a concentration of 107 cells/ml and incubated with primary antibodies (1:100 dilution). We used specific primary antibodies directly conjugated to the indicated fluorophores (all from Pharmingen, San Diego, CA): ScaI (clone D7), CD44 (clone IM7 APC), CD45 (clone 30-F11; APC or PE), c-kit (clone 2B8 APC), CD34 (clone 49E8 FITC), CD11b (clone M1/70 FITC), CD14 (clone rmC5–3 PE), and Flk-1 (clone Avas 12a1 PE). Lung SP cells were identified and selected by flow cytometry based on differential Hoechst 33342 red and blue fluorescence for an SP (20). Nonviable cells were excluded with propidium iodide (PI). Verapamil was used to ensure specificity of the SP population. Before Hoechst staining, cells were incubated for 10 min with verapamil and over the entire 90 min with Hoechst. With verapamil slight changes in peak channel for 2N populations can be due to instrumentation, staining methodology, cell concentration, and/or dye concentration as well as actual variations in 2N DNA content for different cell populations. The voltage was adjusted to maintain the same localization on the scale. Seven color analyses were performed to maximize the number of cell surface antigens detected for a small sample size as well as to characterize populations expressing multiple antigens. Whole bone marrow was used as a positive control for all cytometry analyses. Specific isotype control antibodies (IgG2akappa, clone R35–95; IgG2bkappa, clone A95–1) were used as negative controls. Data represent means ± standard deviation of four independent experiments. Analysis was performed on a triple laser MoFlow instrument (DAKO, Fort Collins, CO) and analyzed using Summit (DAKO).

In vitro examination of endothelial potential. The neonatal lung SP cells were also sorted based on CD45 expression into positive and negative fractions. A small sample of the sorted cells was reanalyzed immediately following the collection, showing that the purity of the CD45 subpopulations was greater than 95%. The abort rate is typically 10% of the total events. These sorted subpopulations were expanded in culture and analyzed for endothelial properties from passage 3 (4 wk postsort) to passage 6 (6 wk postsort). Culture conditions were as follows: 21% O2 in a-MEM, 20% FBS, 1% Pen/strep. To determine DiLDL uptake, Alexa 488-labeled AcDiLDL (Molecular Probes) was added to the culture media and incubated with expanded SP cells under normal conditions for 3 h according to the manufacturer's guidelines. Gates for sorting AcDiLDL-positive cells were based on control staining of positive and negative controls including pulmonary artery endothelial cells and lung mesenchyme. In vitro, angiogenesis assay was a 96-well matrigel-based kit (Chemicon, Temecula, CA) following the manufacturer's instructions using cultured SP cells as above.

Mouse model of BPD. Timed pregnant C57Bl/6-ROSA mice were purchased from Jackson Laboratory (Bar Harbor, ME) and housed in the University of Colorado Health Sciences Center central vivarium under pathogen-free conditions. All procedures and protocols were approved by Institutional Animal Care and Use Committees at the UCHSC. Newborn mice (P0-P1 days of age) were exposed to 70% oxygen for 10 days and normal atmospheric CO2 (<1%) (60). Age-matched control animals remained in room air (RA). The environment of 70% was maintained by a pro-ox system that delivered oxygen (100%) into a specially designed Plexiglas chamber housing the cages. The chamber was constantly ventilated to RA. The mice were observed daily for proper ambulation and appearance. Following 10 days of hyperoxic exposure, dams and neonates were placed at RA (relative normoxia at Denver altitude). Animals were studied at postnatal day 13, 21, 41, and 55 (that is 3, 11, 31, and 45 days after hyperoxic exposure). We chose to begin analyses on P13 to avoid potential acute stress response to changes in oxygen tension (PO2). At the time of death, age and body weights were recorded. Lung SP cells were assessed at days 13, 21, 41, and 55; lungs from days 13 and 41 were fixed, inflated, and used for morphometric analysis.

Hemodynamic measurements. At the end of the treatment period, mice were anesthetized by intramuscular injection with Ketamine-Rompun (100 and 15 mg/kg; Fort Dodge and Miles Laboratories) and weighed. After calibration of the pressure transducer (Statham), closed-chest measurements of right ventricular systolic pressure (an indirect index of pulmonary arterial pressure) were recorded on spontaneously breathing mice as previously described under normoxic conditions (37).

Morphometric analysis. Morphometric analysis was performed to characterize lung simplification. Animals were euthanized with an overdose of inhaled isofluorane by a catheter placed in the trachea. The lungs were then perfused, fixed under constant pressure for 45 min-1 h with a ligature placed around the trachea to maintain pressure. The hearts were removed, dissected, and right ventricle and left ventricle + septal weights were obtained. The fixed lung was paraffin-embedded and 10-µm sections were cut for histochemical analysis. Hematoxylin and eosin-, pentachrome-, and anti-factor VIII-stained lung sections for each animal were selected in an unbiased fashion. The orientation of these samples was at random, creating isotropic uniform random plane sections of the lung tissue. Images of each section were digitally captured and analyzed by counting alveoli and vessels in six fields per section at x200 magnification (12). Ten high-powered fields were used for alveoli counting per animal. Five to seven animals were used per quantitation (P13C, n = 5; P13H, n = 6; P41C, n = 6; P41H, n = 7). Factor VIII-positive vessels were also counted in this manner to determine vessel density (50, 60). To determine vessel and alveolar density, counting was performed by a blinded observer, in randomly selected high-powered fields of distal lung. Fields including large airways or large vessels were excluded.

Statistical analysis. All experiments followed a randomized block design with the use of cells from at least two different animals. Assays were completed from at least four independent experiments consisting of two to six replicates in each. Data were expressed as means ± SE, and significance between groups was determined by ANOVA or Student's t-test, using the statistical software package JMP5 (SAS Institute, Cary, NC). A two-way ANOVA was performed to detect significance of in vivo analyses. Statistical significance was set at P < 0.05, {alpha} = 0.05. * Denotes significance for P < 0.05; ** for P < 0.01; *** for P < 0.001.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and identification of neonatal lung SP of cells as endothelial precursors. Freshly isolated lung SP cells were identified and selected by flow cytometry based on the differential Hoechst 33342 red and blue fluorescence for an SP (20). Nonviable cells were excluded with PI. These cells represented less than 1% of the total viable lung cells selected by PI uptake (Fig. 1A). Viability was typically greater than 90%. This number was consistent in greater than 10 independent experiments. Verapamil was used to inhibit the multidrug resistance pump, which specifies a large percentage of the SP population (Fig. 1B) (51, 56). Verapamil treatment significantly decreased the percentage of SP. With the addition of verapamil to the staining procedure, slight changes in peak channel for 2N populations resulted. This may be due to cell concentration and/or dye concentration as well as actual variations in 2N DNA content for different cell populations.


Figure 1
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  		Fig. 1. Identification of neonatal lung side population (SP) cells and distribution of CD45 subpopulations. Lung tissue was digested with type II collagenase to prepare a single-cell suspension. Lung cells were costained with Hoescht 33342 and an antibody to detect CD45 expression. A: flow cytometric dot plot of Hoescht 33342 to identify % SP. B: verapamil control. C: histogram representation of CD45 staining of the lung SP [postnatal day 55 (P55)]. By coloring the 2 subpopulations based on CD45 expression (CD45neg = red R3; CD45pos = blue R4), their distribution in the SP arm as well as the forward (FSC)/side scatter (SSC) plot (D) were visualized. These data are representative of 10 independent experiments.

 
The cytometric characterization of our lung SP population confirmed it to be similar to the population reported by Summer et al. (54, 55). Lung SP lacked markers of differentiated hematopoietic cell lineages (lin- and c-kit) and expressed the marker CD45 (see GoTable 2). Both CD11b- and CD34-positive lung SP cell populations were more than 99% copositive for CD45.


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  		Table 1. Total number of cells analyzed in lung SP cytometric analyses

 

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  		Table 2. Normalization of lung SP and CD45 subpopulations per 100,000 total cells

 
We then sorted the lung SP into two distinct subpopulations based on coexpression of CD45 and Flk-1, collected cells, and analyzed the differences in phenotype and endothelial cell potential. Previous studies suggested that the lung SP CD45 subpopulations have distinct origins and lineage potential (3, 19, 38, 54, 55). The origin of the CD45pos lung SP was bone marrow while the CD45neg cells were mesenchymal-like with an unidentified origin (50). Both of these populations are distributed throughout the SP (Fig. 1, A and C). However, their cellular characteristics of size are slightly different, as illustrated in forward side scatter cytometric analysis (Fig. 1D). Flk-1 is a marker of both hemangioblast and mesenchymal lung endothelial cell precursors during development (18). We costained freshly isolated lung SP for both CD45 and Flk-1 (Fig. 2, A-C) and found that the Flk-1pos lung SP cells residing in lung tissue were also greater than 95% positive for CD45 (Fig. 2B). Here, we confirm that similar to whole lung tissue, the levels of Flk-1 detection in the lung SP are greatest at P10 and decrease steadily as the mouse lungs mature (Fig. 2C). The Flk-1-positive lung SP cells were negative for the endothelial cell marker VE-Cadherin (Fig. 1D).


Figure 2
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  		Fig. 2. Identification of neonatal lung SP CD45/Flk-1 subpopulations. Cell suspensions from lung tissue on P13 were stained with Hoechst 33342 to detect an SP as well as Flk-1 and CD45 isotype controls (A) and corresponding CD45/Flk-1 antibodies (B). C: Flk-1 expression on lung SP was quantitated over time. D: Flk-1-positive (R35) or -negative (R34) SP did not express the endothelial marker VE-Cadherin.

 
In our studies, neonatal lung SP cells were sorted to specifically select for endothelial cell precursors based on CD45 and Flk-1pos expression and expanded in culture. There was a clear separation between the CD45-positive and -negative populations (Fig. 2C). This separation allowed the collection of relatively pure populations based on selection using conservative gates and a 10% abort rate. A postsort purity check confirmed greater than 95% purity for the subpopulations collected. The CD45pos/Flk-1pos and CD45neg/Flk-1pos lung SP exhibited distinct phenotypes and growth characteristics. The CD45neg/Flk-1pos cells were more readily adherent, visible at 24 h (Fig. 3, A and B). CD45pos/Flk-1pos cells maintained a small, round, bright, and less-adherent phenotype longer in culture (Fig. 3, A and B). After 2 wk in culture cells were passed and allowed to reach confluence. By passage 3 (4 wk postsort), both populations assumed a cobblestone morphology characteristic of endothelial cell phenotypes (Fig. 4, A and B) and the functional ability to take up AcDiLDL (Fig. 4, C and D). Angiogenic ability of both subpopulations was confirmed by the ability to form tube structures in matrigel in vitro (Fig. 4, E and F). Both populations bound isolectin B4 (Fig. 4, G and H).


Figure 3
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  		Fig. 3. Isolation of neonatal lung SP CD45 subpopulations. Lung SP were sorted into CD45neg (A) or CD45pos (B) populations. Representative light micrographs were taken at 24 h postsort. Distinct phenotypes were observed with x200 magnification. C and D: respective 300% enlargements. Scale bar = 50 µm.

 

Figure 4
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  		Fig. 4. Endothelial potential of neonatal lung SP CD45 subpopulations. Lung SP cells cultured at passage 3 (4 wk) postisolation develop a cobblestone-like endothelial morphology. Magnification x200 (A and B). C and D: AcDiLDL uptake demonstrated in vitro functional endothelial cell properties of both CD45 lung SP subpopulations. The uptake was quantitated by cytometric analysis to detect green fluorescence. Magnification x100; n = 2. E and F: in vitro angiogenic activity of CD45neg and CD45pos lung SP confirmed by matrigel tube forming assay. Cells were seeded in matrigel and tubes were visualized 20 h later. Magnification x100. G and H: cultured lung SP also bind isolectin B4 (R26), a property specific to endothelial cells. Scale bar = 50 µm.

 
Moderate hyperoxia results in lung simplification: a murine model of BPD. Newborn mice (P1) were exposed to RA or 70% oxygen for 10 days, a period corresponding to the late terminal saccular/early alveolar stages of lung development (Fig. 5A). Following hyperoxic exposure, animals were placed at RA and analyzed at P13 and P41 to analyze changes in lung architecture and precursors during a recovery period when functional tissue alveolar development and repair would likely occur, as well as avoid potential acute stress response to changes in oxygen tension. Morphometric analysis was performed on lungs from P13 and P41 for histological documentation of lung simplification. Moderate exposure of neonatal mice to hyperoxia resulted in subtle histological changes by P13 (Fig. 5, B and C). Lung simplification, including decreased mean alveolar (Fig. 5) and microvessel density, was most evident by P41. At P41 enlarged airspaces were evident (Fig. 5, D and E). Alveolar density (P < 0.0001) was decreased following hyperoxic exposure, corresponding with decreased microvascular density (P < 0.01) per area of lung tissue (Fig. 5, H and I). There was a significant difference in body weight at P13 (RA 7.8 g; Hyperoxia 5.9 g; P < 0.0001) but not at P41 (RA 17.9 g; Hyperoxia 16.5 g; P < 0.22). The systolic right ventricular pressure (RVSP) was quantified on P41 and no significant difference was detected (RA 23.6 mmHg; Hyperoxia 23.8 mmHg; P < 0.82).


Figure 5
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  		Fig. 5. Histological pulmonary changes following neonatal hyperoxic exposure at P13 and P41. A: murine model of bronchopulmonary dysplasia (BPD). B: P13 lung tissue from room air (RA) mice or hyperoxic-exposed mice (C). Hematoxylin and eosin (H&E) stain, magnification x200; n = 4–5 per group. D and E: P41 lung tissue from RA mice or hyperoxic-exposed mice. H&E stain, magnification x200. F and G: representative light micrographs of P41 lung tissue following factor VIII immunohistochemistry, magnification x400. H: P41 alveolar density and microvessel density (I) presented as mean counts per field of view ± SE. Arrows indicate positive factor VIII microvessels (brown), n = 4–5 per group. Scale bar = 50 µm.

 
Changes in the neonatal lung SP population of endothelial precursors in response to in vivo hyperoxia. Flow cytometric analyses were performed to evaluate the percent of lung SP cells relative to total lung cells (Fig. 6B) as well as the composition of the CD45 subpopulations of the SP following hyperoxic exposure in a murine model of BPD (Fig. 6C). Neonatal lung SP cells were isolated from mice at P13, P21, P41, and P55 days of age. These ages correspond to the alveolar (P13 and P21) and growth (P41 and P55) stages of lung development. SP cells are rare events so to determine changes in this small population of cells when comparing large cell numbers per sample, we collected analysis files based on the cells of interest, the SP. This methodology was developed to conform to Poisson statistics for rare event analysis (20a, 35, 51, 56). Increasing the number of SP cells collected increased precision in the percentage values of the SP subsets. To maximize analysis of SP cell parameters, we gated the SP and analyzed samples based on SP cell numbers. We used a live gate to collect 2,000–5,000 events within the SP gate (Fig. 6A, R3) and collect 10% of the events outside the SP gate (Fig. 6A, R2). This required the analysis of 8–10 x 105 total lung cells (Table 1). This method allowed us to analyze the SP cells of interest in relation to the non-SP cells within the sample while keeping the listmode files a manageable size. Numbers of cells isolated, analyzed, and corresponding percentages are presented in Table 1. There were similar numbers of cells analyzed per group (Table 1). The numbers of total cells isolated in RA compared with hyperoxic exposed were not significantly different. Once reliable percentages of SP cells were collected, the numbers of SP cells and CD45 subpopulations were normalized to 100,000 total cells (Table 2).


Figure 6
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  		Fig. 6. Alterations in lung SP population result from neonatal hyperoxic exposure. A: single lung cell suspensions from each mouse were analyzed based on events within the SP gate (R3) and while 10% of the events outside the SP gate were collected (R2); 8–10 x 105 total lung cells were analyzed (Table 1). B: quantification of lung SP percentages and CD45 subpopulations (C) of lung SP over time following neonatal hyperoxic exposure. Corresponding CD45-positive and -negative values are presented in Table 2. *Significant changes in CD45pos populations following hyperoxic exposure compared with RA, n = 7–18. See METHODS for details.

 
A developmentally related increase in the quantity of lung SP at RA from P13 to P21 and P41 was identified (P21, P < 0.0003; P41, P < 0.01; compared with P13), which decreased as the mice reached adulthood by P55 (P < 0.002; Fig. 6B; Tables 1 and 2). Conversely, following hyperoxic exposure, at P13 there was a decrease in the lung SP compared with RA (Fig. 6B; Tables 1 and 2; P < 0.0001) and an increasing trend by P41 (P < 0.031; Fig. 6B; Tables 1 and 2). There was no significant difference between groups by P55. Circulating blood lacks a detectable SP fraction, therefore these changes are not due to measurement of contaminating blood-borne SP cells (Majka, unpublished observations, 56).

We concurrently analyzed changes in the lung SP CD45 subpopulations, in this case to distinguish between what was previously reported to be hematopoietic-like cells [CD45pos (3, 54, 55)] and mesenchymal-like cells (CD45neg). There were subtle differences when comparing the percentages of CD45pos lung SP between RA and hyperoxic-exposed groups (Table 3). However, when normalized by cell numbers, the trend remained the same but the differences between groups became more significant (Fig. 6C). During RA recovery on P21, P41, and P455, there was an increase in the proportion of CD45pos lung SP cells in response to hyperoxic exposure compared with RA (Fig. 6C; Table 2). We also examined the proportion of lung SP cells that coexpressed CD45 and CD11b, stem cell antigen I (ScaI), or CD34. Sca1/CD11b markers have been used to detect putative stem cells in adult tissues (7). We did not observe any change in the lung SP CD45pos/CD11bpos cells following hyperoxic treatment (Table 3). There was no significant change following hyperoxic exposure of CD45pos/ScaI, CD11b, CD34, CD45neg/ScaI, or ScaI/CD11b populations (Table 3). c-kit was also used as a marker of bone marrow-derived hematopoietic stem cells in these experiments; however, the lack of detection was likely due to nonspecific proteolytic cleavage during lung SP cell isolation (44, 54, 55) (Table 3).


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  		Table 3. Flow cytometric characterization of lung SP cell surface determinants

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates the consequences of hyperoxic exposure of mice to lung SP cells. Alterations in lung SP coincided with decreased pulmonary microvessel density in response to hyperoxic exposure during a RA recovery period. We therefore used a murine model of BPD to begin to address the hypothesis that the lung SP are a source of endothelial precursor cells which may be altered during BPD. Lung simplification as a result of hyperoxic exposure was confirmed by morphometric analyses, which detected a decrease in both alveolar and microvessel density. This lung simplification was coupled to initial decreases in the percentage of lung SP. An understanding of the mechanisms by which transient hyperoxia affects precursors and impairs functional pulmonary tissue architecture is vital to understanding the pathology of BPD.

Bone marrow SP cells have been shown to contribute to collateral vascular regrowth following ischemic injury in the heart in a bone marrow transplant model (25). In the skeletal muscle, the SP cells were found to differentiate into vascular lineages following injury (38). Therefore, we hypothesized that this population in the lung would likely be affected in BPD, a disease with vascular defects. The adult lung SP cell population has been previously characterized for expression of markers indicative of stem cell, epithelial and mesenchymal lineages (19, 36, 54, 55). These studies determined that the adult mouse lung SP population has epithelial, endothelial, and mesenchymal potential that resides within a CD45neg mesenchymal subpopulation, as well as limited hematopoietic ability that resides in the bone marrow-derived CD45pos subpopulation (19, 36, 54, 55). In these studies, we analyzed the developmental kinetics of the endothelial precursor portion of lung SP using Flk-1 (VEGF receptor 2) detection and demonstrated the vascular endothelial potential for both the CD45pos Flk-1pos and CD45neg Flk-1pos lung SP populations in vitro. The kinetics of Flk-1 expression parallel what has previously been reported for mesenchymal-derived endothelial cell precursors during development (18).

To perform in vivo analysis of lung SP during RA recovery following hyperoxic exposure, we first validated our model of BPD and quantified vascular and alveolar defects. Consistent with BPD, we detected an initial decrease in body weight following hyperoxic exposure, which was correlated to subtle histological changes on P13. However, by P41, developmental arrest was indicated by significant lung simplification with enlarged airspaces and decreased microvessel density. Interestingly, despite the lung simplification observed, by P41 no change in RVSP was evident, suggesting the absence of resting pulmonary hypertension at this time (28, 39, 53).

Hyperoxic injury to the developing lung alters normal patterns of epithelial, endothelial, and mesenchymal cell differentiation, proliferation, and migration, which result in the simplified tissue architecture (45, 60). The quantity and composition of the resident lung SP population were altered in response to hyperoxia. The changes in lung SP illustrate a dynamic response of precursor cells posthyperoxic exposure during recovery. Because the phenotype of lung simplification continues into adulthood, with notable defects in vascular density and alveolarization at P41, these cells are obviously not participating in functional tissue repair. However, the difference of percent lung SP may be in the CD45 composition and/or functionality of the population. The cells may also be responding to the altered tissue microenvironment in BPD.

We quantified the developmental kinetics of the lung SP and found an increase in the percentage of lung SP from P13 to P41, which then decreased by P55, as the mice reached adulthood. This temporal change in lung SP may represent tissue maturation. Both Flk-1-positive endothelial cell precursors in lung and putative stem cell populations in other tissues tend to decrease in the adult compared with developing tissues (18). Following hyperoxia, the lung SP were decreased compared with control animals; however, the changes noted in the CD45 analyses do not appear to be permanent. This is evident from the increases in CD45pos bone marrow-derived cells following hyperoxic injury. The requirement for tissue injury and turnover to induce hematopoietic precursor recruitment and engraftment has previously been established using genetically marked donor cells and bone marrow transplantation studies as well as the presence of hematopoietic bone marrow-derived cells resident in the SP of adult tissues (3, 7, 38). The purpose and role of these cells are unknown. However, a significant change in CD45pos SP was evident. However, increases in SP precursor cells with time do not rescue the decrease in factor VIII-positive microvessels and alveoli. Future studies will focus on whether the hyperoxic environment alters the endothelial cell differentiation potential of precursor cells at a crucial point in vascular lung development. Whether these effects on precursors are permanent or reversible is unknown. Future studies will be performed to define the fate and function of these cells in lung vascular repair.

Our studies did not address aspects of lung SP contribution to tissue remodeling evident in this BPD model. However, it will be important for future studies to track these precursor cells in vivo. Subsequently, we may be able to identify microenvironmental factors, which play a part in fates of these cells. Therefore, our ability to identify lung vascular precursor cells allows us to further study the potential of these cells and how microenvironmental changes affect their role in the regulation of tissue homeostasis and response to injury.

In conclusion, we demonstrated that both the CD45pos and CD45neg neonatal lung SP are vascular precursor cells. We also demonstrated that in vivo hyperoxic exposure of neonatal mice during the late terminal sac and early alveolar stages of lung development resulting in lung simplification alters these populations of lung SP cells. These changes are both in quantity as well as composition. An understanding of the mechanisms by which transient hyperoxia affects endothelial cell precursor differentiation to pulmonary vasculature is vital to understanding the pathology of BPD. This has broader applications to any lung disease in which functional regeneration of tissue by precursors does not occur. The success of cell-based reparative therapies largely depends on our ability to understand how the therapeutic benefit of the host's endogenous stem cell populations is altered during disease.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded by grants to S. M. Majka from American Heart Association (AHA; SDG-0335052N), the Denver Childrens Hospital Pilot Award and the American Physiological Society Giles Filley Award; D. Irwin from National Institutes of Health (NIH) National Research Service Award 5-T32-HL07171; J. West from NIH-National Heart, Lung, and Blood Institute (NHLBI) R01, K. Fagan from AHA-EIA 0340122N and NIH R01-HL-066328; the University of Colorado Cancer Center Flow Cytometry Core (supported by NIH Grant 5-P30-CA-46934–15), and D. Klemm from NIH NHLBI P01-HL-014985 and a Veterans Affairs MERIT Award.


   ACKNOWLEDGMENTS
 
We thank Drs. A. Das and J. Bhattacharya for critical review of the manuscript, Dr. T. Vincent Shankey for input on verapamil and cell cycle dye staining, Drs. I. Douglas, S. Flores, D. Wagner, M. Oka, and N. Markham for logistical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Majka, SON 3928, Mail Stop B-133, 4200 East 9th Ave., Denver, CO 80262 (e-mail: Susan.majka{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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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