Published online December 11, 2006
PEDIATRICS Vol. 119 No. 1 January 2007, pp. e241-e246 (doi:10.1542/peds.2005-3039)
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ARTICLE

Pulmonary Endostatin Perinatally and in Lung Injury of the Newborn Infant

Joakim Janér, MDa, Sture Andersson, MD, PhDa, Caj Haglund, MD, PhDb and Patrik Lassus, MD, PhDa,c

a Hospital for Children and Adolescents
b Departments of Surgery
c Plastic Surgery, University of Helsinki, Helsinki, Finland


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
OBJECTIVE. Endostatin is a potent angiogenesis inhibitor. Angiogenesis is central for the development of the human lung. The role of endostatin in the development of the human lung and its connection to chronic lung disease remain unclear. We set out to study the role of endostatin in the developing human lung and in acute and chronic lung injury in the preterm infant.

METHODS. Nine fetuses, 14 control neonates without primary lung disease, 14 preterm infants with respiratory distress syndrome, and 8 infants with bronchopulmonary dysplasia were included in the immunohistochemistry study. Tracheal aspirate-fluid samples of intubated very low birth weight infants during postnatal weeks 1 through 5 were analyzed with enzyme-linked immunosorbent assay.

RESULTS. Endothelial cell staining was positive for endostatin in all 45 samples. Staining of epithelial cells (cuboidal, bronchiolar, and alveolar) was seen mostly in fetuses, as well as in infants with late respiratory distress syndrome and bronchopulmonary dysplasia. Staining in alveolar macrophages was most abundant in infants with late respiratory distress syndrome and bronchopulmonary dysplasia. Endostatin was expressed consistently in tracheal aspirate fluid, being highest during the first postnatal day. Higher endostatin concentrations correlated with parameters reflecting lower lung maturity.

CONCLUSIONS. The pattern of pulmonary endostatin protein expression in immunohistochemistry and consistent endostatin protein appearance in tracheal aspirate fluid in human preterm infants indicate a role in the physiologic development of the lung. Preterm birth influences pulmonary endostatin protein expression, which may alter normal lung development and response to lung injury.

Key Words: lung development • endostatin • bronchopulmonary dysplasia • respiratory distress syndrome

Abbreviations: VLBW—very low birth weight • BPD—bronchopulmonary dysplasia • VEGF—vascular endothelial growth factor • GA—gestational age • Ig—immunoglobulin • SC—secretory component • FIO2—fraction of inspired oxygen • L/S—lecithin/sphingomyelin

Endostatin is a 20-kilodalton (kd) angiogenesis inhibitor that is a proteolytic fragment of the C-terminal non–triple-helical (NC1) domain of collagen XVIII.1 The inhibitory effect of endostatin on endothelial cells includes inhibition of proliferation,1 migration,2,3 and induction of cell apoptosis.3,4

An infant born at the early third trimester of gestation has poorly developed lungs; the alveoli are just forming, surfactant production has only just begun, and the capillary bed is poorly developed. Birth at this stage interrupts normal development of the lung in very low birth weight (VLBW) infants (born at <32 weeks of gestation).5 The development of bronchopulmonary dysplasia (BPD) may be caused by disruption of vascular development by premature birth.6,7

In animal models, vascular endothelial growth factor A (VEGF-A) has been shown to participate in alveolarization.8,9 Moreover, preterm infants with high pulmonary concentration of VEGF-A have lower incidence of BPD.10 Endostatin antagonizes effects of VEGF-A and other proangiogenic factors11,12; thus, we hypothesized that endostatin may play a role in the development of the lung and pathogenesis of lung injury in the preterm infant. To study this we determined the concentration of endostatin in tracheal aspirates from VLBW infants during the first postnatal week. We also localized pulmonary endostatin protein expression by immunohistochemistry during fetal development and in VLBW infants with lung injury, as well as in VLBW and term infants with macroscopically and microscopically normal lungs.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
All studies were approved by the ethics committee of the Hospital for Children and Adolescents.

Patients in the Immunohistochemistry Study
Forty-five subjects were included in the study of immunohistochemistry for endostatin. Samples were collected between March 1991 and June 2000. Fetuses and controls had macroscopically and microscopically normal lungs. One of the subjects in the late-RDS group had systemic candidiasis, and 1 subject in the BPD group had pneumonia; otherwise, no infections were evident at the time of death. Autopsies were performed within 3 days postmortem (Table 1).


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  		TABLE 1 Patients in Immunohistochemistry Study

 
Patients in the Tracheal Aspirate Sample Study
Samples from 59 preterm infants were collected. Twenty-seven patients who subsequently developed BPD and age- (32 weeks' gestational age [GA]) and birth weight–matched patients who survived without BPD were selected. BPD was defined as a need for supplemental oxygen at the age of 36 gestational weeks in addition to chest radiograph findings typical of BPD.13 All of the infants included were intubated at birth as a result of failure to establish spontaneous ventilation.

Three infants died after the study period, 2 as a result of BPD and 1 as a result of cerebral hemorrhage (Table 2).


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  		TABLE 2 Patient Data and Results in the Tracheal Aspirate Fluid Study

 
Immunohistochemistry for Endostatin
Lung samples were obtained as described previously14 and treated with EDTA and Tris-HCl. Endostatin antibody was used at 1:200 dilution (PK-6105, Vectastain Elite ABC kit [goat immunoglobulin G (IgG)]; Vector Laboratories Inc, Burlingame, CA). Negative controls were performed by omission of the primary antibody, and a known positive section for the antibody was included as a positive control.

Tracheal Aspirate Sample Collection
Samples of tracheal aspiration fluid were collected once daily by standardized routine tracheal lavage as described previously.15 A total of 223 samples from 59 patients during the first postnatal week in addition to 23 samples during week 2 and 22 samples during weeks 3 to 5 collected from 6 patients who later developed BPD were used for analysis.

Analysis of Endostatin and Secretory Component of IgA in Tracheal Aspirate Fluid
Endostatin was analyzed with the human endostatin immunoassay kit (R&D Systems Inc, Minneapolis, MN). To estimate the in situ pulmonary concentration of endostatin, a correction for dilution of the tracheal aspirate sample was calculated by use of concentration of secretory component of IgA (IgA-SC) in tracheal aspirate fluid. Concentration of IgA-SC in lung secretions is independent of capillary leak, and the concentration of IgA-SC in tracheal aspirates is independent of respiratory distress or GA.16 IgA-SC concentration was determined by direct enzyme-linked immunosorbent assay. Secretory IgA isolated from human colostrum was used as standard. The method was standardized by using IgA-SC standards (kindly provided by Dr B. Götze-Speer and Prof C. Speer [University Children's Hospital, Würzburg, Germany]).17

Statistical Analysis
Statistical comparisons were performed with StatView 5.1 (SAS Institute, Inc, Cary, NC). Values represent mean ± SD for patient data, mean ± SEM for experimental results, and frequencies for categorical variables. Variables with skewed distribution were log10-transformed before analyses, but values shown in the text and tables are nontransformed. P values of <.05 were considered statistically significant. The Student's t test was used to test differences between unpaired items, and between-group comparisons were performed with 1-way analysis of variance with the Bonferroni posthoc test. Frequency distributions between groups were compared with the {chi}2 test. Correlations were calculated with simple and multiple regression analyses.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Immunohistochemistry for Endostatin
In all 9 fetuses endostatin-positive staining endothelial cells and in 7 fetuses cuboidal epithelial cells were detected. In all 5 preterm and 9 term controls, positive-staining endothelial cells were observed. In addition, alveolar macrophage–positive staining was seen in preterm controls in 2 and in term controls in 3 cases. In all 7 early-RDS and 7 late-RDS cases, positive-staining endothelial cells were observed. In addition, positive-staining bronchiolar epithelial cells in 2 cases and alveolar epithelial cells in 2 cases, as well as alveolar macrophages in 5 cases, were seen in late-RDS cases. In all 8 BPD cases, positive-staining endothelial cells, in 3 cases alveolar epithelial cells, and in 5 cases alveolar macrophages were detected (Fig 1).


Figure 1
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  		FIGURE 1 Immunohistochemistry for endostatin in lung tissues obtained at autopsy. Fetus (GA of 15 weeks), endostatin staining in endothelial cells (arteries and veins); term control (GA of 37 weeks), endostatin staining in endothelial cells (artery); RDS early (GA of 24 weeks), endostatin staining in endothelial cells (artery); BPD (GA of 26 weeks 3 days), endostatin staining in endothelial cells (artery), as well as in alveolar epithelial cells. a indicates artery; v, vein; ep, epithelium.

 
Thus, endothelial cell staining was positive for endostatin in all 45 samples. Staining of epithelial cells (cuboidal, bronchiolar, and alveolar) was seen mostly in fetuses, where staining was restricted to cuboidal epithelium as well as late-RDS and BPD cases, where staining was observed in bronchiolar and alveolar epithelium. Staining in alveolar macrophages was most abundant in late-RDS and BPD cases (Fig 2).


Figure 2
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  		FIGURE 2 Localization of endostatin protein. A, Endothelium; B, epithelium; C, macrophages (fetus: n = 9; control: n = 14; early RDS: n = 7; late RDS: n = 7; BPD: n = 8). a P < .05, fetus versus preterm control, term control, and early RDS; b P < .05, late RDS versus preterm control, term control, and early RDS; c P < .05, BPD versus term control; d P < .05, fetus versus preterm control; e P < .05, late RDS vs fetus and early RDS; f P < .05, BPD versus fetus and early RDS.

 
Endostatin in Tracheal Aspirate Fluid
The mean endostatin concentration was 0.61 ± 0.12 ng/mL per IgA-SC unit (mean ± SEM) on day 1 and 0.14 ± 0.04 ng/mL per IgA-SC unit on day 7. During week 2, the mean endostatin concentration was 0.30 ± 0.08 ng/mL per IgA-SC unit and during weeks 3 to 5 was 0.10 ± 0.01 ng/mL per IgA-SC unit. Mean endostatin concentration during the first postnatal week was used in the statistical analysis (Fig 3).


Figure 3
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  		FIGURE 3 Endostatin concentrations in tracheal aspirate fluid (ng/mL per IgA-sc unit) in 59 preterm infants during the first postnatal week and in 6 patients during postnatal week 2 and weeks 3 to 5. The numbers in parentheses indicate the amount of samples.

 
Of 59 infants, 46 had received antenatal betamethasone. It was associated with a higher endostatin concentration in tracheal aspirate fluid. The number of antenatal betamethasone doses or administration of betamethasone closer to birth did not correlate with endostatin concentrations.

Chorionamnionitis was diagnosed in 11 cases. Chorioamnionitis was associated with a lower concentration of endostatin in tracheal aspirate fluid. These cases were compared with subjects diagnosed with proteinuric preeclampsia, as well as to those diagnosed with neither chorioamnionitis nor proteinuric preeclampsia. Cesarean section was performed in 37 cases. Delivery by cesarean section was associated with a higher concentration of endostatin in tracheal aspirate fluid.

Low birth weight, but not GA, correlated with a higher concentration of endostatin in tracheal aspirate fluid. In 33 patients, lung maturity was estimated by measuring the lecithin/sphingomyelin (L/S) ratio from a tracheal aspirate-fluid sample taken postnatally before treatment with surfactant. A negative correlation existed between the L/S ratio and endostatin concentration.

A higher fraction of inspired oxygen (FIO2) correlated with a higher endostatin concentration in tracheal aspirate fluid (Table 2).

Multiple regression analysis was performed by inclusion of all significant parameters. In this analysis, no parameter remained significant in comparison to other parameters.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
The pattern of pulmonary endostatin protein expression and consistent protein appearance in tracheal aspirate fluid in human preterm infants indicate a role for endostatin in the physiologic development of the human lung. In immunohistochemistry, fetuses at early second trimester exhibited endostatin staining in endothelium as well as in cuboidal epithelium. In contrast, at term, endostatin staining was found only in endothelium with no staining in epithelium. Endostatin protein was found consistently in tracheal aspirate fluid in VLBW infants. The concentration was highest during the first postnatal day, after which endostatin levels decreased during the first postnatal week. Endostatin knock-out mice have a normal life span and, apart from ocular abnormalities, exhibit no major vascular abnormalities. However, aortic explants from these mice show a twofold increase in the number and length of microvessels, suggesting a more proangiogenic environment.18 Our data indicate that in the human lung, endostatin is expressed constantly from a GA of at least 15 weeks and during the neonatal period. In addition, fetuses expressed endostatin in more cell types than controls, and endostatin levels decreased postnatally. Thus, endostatin seems to play a role in fetal lung development.

In this study, significantly lower endostatin in tracheal aspirate fluid was found in subjects with maternal chorioamnionitis. Preterm birth is often associated with at least subclinical chorioamnionitis, and neonatal care in itself involves many proinflammatory components.19–21 Thus, the presence of chorioamnionitis in correlation with lower endostatin may indicate accelerated lung maturation in these subjects. Higher endostatin levels correlated with a lower L/S ratio and lower birth weight. Taken together, these results indicate that immature lungs exhibit higher levels of endostatin than more mature lungs do.

The need for higher FIO2 correlated with higher endostatin concentration in tracheal aspirate fluid. In the Supplemental Therapeutic Oxygen for Prethreshold Retinopathy of Prematurity (STOP-ROP) trial,22 initiated to evaluate the effects of higher blood oxygenation on retinopathy of prematurity, it was found that increased oxygenation was associated with a tendency for increased risk for chronic lung injury. Endostatin downregulates several important signaling pathways in human microvascular endothelium that are associated with proangiogenic activity.11 Importantly for lung development and development of BPD, in 1 of these pathways endostatin antagonizes the physiologic angiogenic actions of VEGF-A through hypoxia-inducible transcription factor-{alpha} (HIF-{alpha}). Endostatin also antagonizes VEGF-A by inhibiting the expression of VEGFR-2 (Flk-1),12 although the effect could be mediated through HIF-{alpha}. VEGF-A has been suggested to participate in normal alveolarization and prevent development of BPD.6,7,10 Moreover, VEGF-A is also known as a permeability factor, and endostatin suppresses vascular permeability.23 This may be of physiologic significance; in the preterm lung, both endostatin and VEGF-A are needed for continued normal vascular development, with endostatin guiding the continued vascular growth induced by VEGF-A and other proangiogenic mediators. Excess expression of endostatin could disturb the equilibrium between endostatin and VEGF-A and hinder normal lung development. In our study, in subjects with late RDS and BPD, bronchial and alveolar epithelial cells, as well as macrophages, stained positive for endostatin protein. This pattern of protein expression could be seen neither in controls nor in early-RDS cases. It seems that with the progression of lung injury, the protein expression of endostatin is upregulated and appears in cells that are normally dormant for endostatin expression at that point in time. This further suggests that increased postnatal expression of endostatin may have an adverse effect in the premature lung.


   CONCLUSIONS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
We suggest that endostatin plays a physiologic role in the development of the human lung. Endostatin expression is observed mostly prenatally, and it decreases during the first postnatal days. A disturbance in the equilibrium between endostatin and VEGF-A in the preterm infant could interfere with the continued physiologic development of the lung. Postnatally, higher pulmonary concentrations as well as wider protein expression of endostatin may contribute to the pathogenesis of chronic lung injury.


   ACKNOWLEDGMENTS
 
We acknowledge the Sigrid Juselius Foundation, Finska Läkaresällskapet, Nylands Nation, Helsinki University Central Hospital Research Fund, and Foundation for Pediatric Research for support of this study.

We thank the personnel of the NICU and the neonatal nursery of the Hospital for Children and Adolescents for their kind cooperation. Marjatta Vallas, Elina Laitinen, and Päivi Peltokangas are thanked for excellent technical assistance. Dr Götze-Speer and Prof Speer (University Children's Hospital, Würzburg, Germany) are thanked for generous help with IgA-SC standardization.


   FOOTNOTES
 
Accepted Jul 7, 2006.

Address correspondence to Joakim Janér, MD, Hospital for Children and Adolescents, POB 281, 00029 HUS, Helsinki, Finland. E-mail: joakim.janer{at}helsinki.fi

The authors have indicated they have no financial relationships relevant to this article to disclose.


   REFERENCES
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 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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PEDIATRICS (ISSN 1098-4275). ©2007 by the American Academy of Pediatrics

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