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Published ahead of print on May 11, 2006, doi:10.1164/rccm.200508-1291OC
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American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 326-330, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200508-1291OC

Original Article

Pulmonary Vascular Endothelial Growth Factor-C in Development and Lung Injury in Preterm Infants

Joakim Janér, Patrik Lassus, Caj Haglund, Karri Paavonen, Kari Alitalo and Sture Andersson

Hospital for Children and Adolescents, Department of Surgery, Helsinki University Central Hospital, and Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, University of Helsinki, Helsinki, Finland; and Angiogenesis Laboratory, Melbourne Branch of Tumour Biology, Ludwig Institute for Cancer Research, Melbourne, Australia

Correspondence and requests for reprints should be addressed to Joakim Janér, B.M., The Hospital for Children and Adolescents, POB 281, 00029 HUS, Helsinki, Finland. E-mail: joakim.janer{at}helsinki.fi


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: In mice, vascular endothelial growth factor-C (VEGF-C) plays an important role in development of the lymphatic system and in pathogenesis of pulmonary inflammation. Its role in development of the lymphatic system in human lung and in lung injury in newborns remains unclear.

Objectives: We studied the role of VEGF-C in developing human lung, and in acute and chronic lung injury in preterm infants.

Methods: Included in the immunohistochemistry study were 10 fetuses, 15 control neonates without primary lung disease, 15 preterm infants with respiratory distress syndrome, and 8 infants with bronchopulmonary dysplasia. Tracheal aspirate fluid samples of intubated very-low-birth-weight infants during Postnatal Weeks 1–5 were analyzed with ELISA.

Results: Bronchiolar staining for VEGF-C was observed in all 48 samples. Alveolar epithelial staining was seen in most fetuses (8/10). In addition, staining was observed in alveolar macrophages in bronchopulmonary dysplasia (4/8), and late respiratory distress syndrome (2/7). VEGF receptor-3 (VEGFR-3) staining was observed in lymphatic endothelium adjacent to vascular endothelium. VEGF-C was expressed consistently in tracheal aspirate fluid, being highest during the first 2 postnatal days. Antenatal administration of glucocorticoids was associated with higher VEGF-C in tracheal aspirate fluid.

Conclusions: The pattern of pulmonary VEGF-C and VEGFR-3 protein expression and consistent VEGF-C protein appearance in tracheal aspirate fluid in human preterm infants indicate a role for VEGF-C in the physiologic development of the lymphatic system of the lung.

Key Words: fetal development • lung • respiratory distress • VEGF-C

An infant born at the early third trimester of gestation has poorly developed lungs; the alveoli are just forming, surfactant production has only recently begun, and the capillary bed is poorly developed. Because birth at this stage interrupts the normal development of the lung, it has been suggested that in infants with very low birth weight (VLBW), born between 26 and 32 wk of gestation (1), the pathogenesis of bronchopulmonary dysplasia (BPD) is caused primarily by this arrest in development (2, 3). As has been previously shown (4–6), vascular endothelial growth factor-A (VEGF-A) plays a role in the alveolarization in preterm infants with respiratory distress.

VEGF-C is a member of the VEGF family of vascular endothelial growth factors, which comprises VEGF-A, placenta growth factor (PlGF), VEGF-B, VEGF-C, and VEGF-D (7). VEGF-C and VEGF-D have been identified as lymphangiogenic growth factors (8, 9). VEGF-C is produced as a 61-kD prepropeptide and is proteolytically processed to form a homodimer of 21 kD, which has a high binding affinity for VEGF receptor-2 (VEGFR-2/kinase domain-containing receptor [KDR]/fetal liver kinase-1 [Flk-1]) and VEGFR-3 (fms-like tyrosine kinase 4 [Flt-4]) (10, 11). During development VEGF-C and its receptor VEGFR-3 are localized particularly to regions of lymphatic vessels sprouting from embryonic veins, such as the developing lung (12). In the adult, VEGFR-3 is located primarily on lymphatic endothelial cells (12). VEGF-C is essential for the embryonic development of the lymphatic system as gene-targeted mice lacking the VEGF-C gene are embryonically lethal due to tissue fluid accumulation in tissues (13).

In preterm infants, respiratory distress syndrome (RDS) is characterized by pulmonary inflammation and edema (14). Immaturity in combination with volutrauma, the effects of reactive oxygen species, and inflammatory processes may result in BPD and increased morbidity and mortality (15). We therefore studied the role of VEGF-C in the developing human lung, and in the development of acute and chronic lung injury in the preterm infant. To do this, we determined the concentration of VEGF-C in tracheal aspirate fluid from VLBW infants during the first postnatal week, as well as during Postnatal Weeks 2–5. In addition, VEGF-C and VEGFR-3 protein expression was localized by immunohistochemistry during fetal development and in VLBW infants with lung injury, as well as in preterm and term infants with macroscopically and microscopically normal lungs.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All studies were approved by the ethics committee of the Hospital for Children and Adolescents, Helsinki University Central Hospital, Helsinki, Finland.

Patients in Immunohistochemistry Study
Lung samples from 48 subjects collected between March 1991 and June 2000 were stained for VEGF-C and VEGFR-3. All fetuses and control subjects had macroscopically and microscopically normal lungs. One of the subjects in the late-RDS group had systemic candidiasis; otherwise, no infections were evident at the time of death. Autopsies were performed within 2 d post mortem. (Table 1, see also Tables E1 and E2 of the online supplement).


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  		TABLE 1. PATIENTS IN IMMUNOHISTOCHEMISTRY STUDY

 
Patients in Tracheal Aspirate Sample Study
Twenty-seven patients who subsequently developed BPD (16) and 27 gestational-age– and birth-weight–matched patients who survived without BPD were selected (Table E3).

Immunohistochemistry for VEGF-C and VEGFR-3
Lung samples were obtained and treated as previously described (17). In addition, sections to be stained for VEGF-C were treated with Trizmabase-HCl (pH 8.5) in four 5-min cycles and microwaved, and for VEGFR-3 staining, sections were microwaved. VEGF-C antibody was used at 1:100 dilution (Z-CVC7 polyclonal VEGF-C antibody; Zymed Laboratories, Inc., San Francisco, CA) and VEGFR-3 antibody at 1:300 dilution (SC-321; Santa Cruz Biotechnology, Santa Cruz, CA). Negative controls were done by omission of the primary antibody. Known positive sections from human mammary–gland ductal carcinoma were included as positive controls for both antibodies.

Tracheal Aspirate Sample Collection
Samples of tracheal aspirate fluid were collected by standardized routine tracheal lavage as previously described (18). A total of 191 samples were collected during the first postnatal week. In addition, 13 samples during Week 2 and 23 samples during Weeks 3 to 5 were collected from six patients who later developed BPD. Fifty-six samples from 15 patients collected during the first postnatal week were also analyzed for VEGF-A.

Analysis of VEGF-C, VEGF-A, VEGF-D, and Secretory Component of IgA in Tracheal Aspirate Fluid
VEGF-C was analyzed with the VEGF-C ELISA Kit (Zymed), VEGF-A with the Human VEGF Immunoassay Kit (R&D Systems, Inc., Minneapolis, MN), and VEGF-D with the Human VEGF-D Immunoassay Kit (R&D Systems, Inc.). For further details, see the online supplement. To estimate the in situ pulmonary concentration of VEGF-C, the secretory component of IgA (IgA-SC) in tracheal aspirate fluid was used (19). Secretory IgA isolated from human colostrum was used as standard. The method was standardized by Dr. Götze-Speer and Professor Speer (University Children's Hospital, Würzburg, Germany) (20).

ELISA Assaying
We assayed for ability of the VEGF-C ELISA kit to recognize human full-length VEGF-C and human {Delta}N{Delta}C–VEGF-C by using the conditioned media from the adenoviral infections with full-length VEGF-C, {Delta}N{Delta}C–VEGF-C, and beta-galactosidase. We also assayed for recognition of recombinant human full-length VEGF-C and {Delta}N{Delta}C–VEGF-C. Finally, we used conditioned media from cells stably transfected with full-length VEGF-C, {Delta}N{Delta}C–VEGF-C, or mock vector. The ELISA kit recognized all full-length human VEGF-C proteins tested but not the mature form of VEGF-C ({Delta}N{Delta}C–VEGF-C).

Additional information on method verification can be found in the online supplement.

Statistical Analysis
Statistical comparisons were performed with StatView 5.1 (StatView, Berkeley, CA). 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 in the text and tables are nontransformed. Values of p less than 0.05 were considered statistically significant. Student's t test was used to test differences between unpaired items, and between-group comparisons were performed with one-way analysis of variance with Bonferroni posthoc test. Frequency distributions between the groups were compared with {chi}2 test. Correlations were calculated with simple and multiple regression analyses.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunohistochemistry for VEGF-C
In all 10 fetuses and 38 infants, VEGF-C staining was seen in bronchial epithelium. In most cases, positivity was observed in all bronchial structures in which most of the bronchial cells stained positively. Uniform and strong intensity staining was seen apically and less intense and more scattered staining was seen in areas not adjacent to lumen (Figures 1 and 2).


Figure 1
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  		Figure 1. Immunohistochemistry for vascular endothelial growth factor-C (VEGF-C) and VEGF receptor-3 (VEGFR-3) in lung tissues obtained at autopsy. Top left: Fetus, gestational age (GA) 15 wk. VEGF-C staining in bronchial epithelial cells and cuboidal cells in alveolar epithelium. Top right: Respiratory distress syndrome (RDS), GA 24 + 2 wk. VEGF-C staining in bronchial epithelial cells. Center left: Control, GA 37 wk. VEGF-C staining in bronchial epithelial cells. Center right: Bronchopulmonary dysplasia (BPD), GA 26 + 3 wk. VEGF-C staining in bronchial epithelial cells, pneumocytes in alveolar epithelium, and macrophages. Lower left: Fetus, GA 15 wk. VEGFR-3 staining in lymphatic endothelium. a = alveolar epithelium; b = bronchial epithelium; c = cuboidal epithelium; l = lymphatic endothelium; and m = macrophages.

 

Figure 2
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  		Figure 2. Localization of VEGF-C protein. Percentage of subjects with positive VEGF-C staining. Fetus (n = 10), early RDS (n = 8), late RDS (n = 7), BPD (n = 8), preterm control (n = 5), term control (n = 10). *p < 0.05.

 
In eight fetuses, one preterm and one term control infant, one infant with late RDS, and two infants with BPD, VEGF-C staining was observed in alveolar epithelium. In most of the positive cases, staining was visible in most of the alveoli in which staining was seen in type II pneumocyte–resembling cells (Figures 1 and 2).

In two infants with late RDS and in four infants with BPD, staining was also seen in alveolar macrophages. In these samples, macrophages were found in clusters and more than 50% of the macrophages stained positively for VEGF-C (Figures 1 and 2).

Thus, bronchial epithelium staining was positive for VEGF-C in all 48 samples. Staining of alveolar epithelium was seen mostly in fetuses. Staining of alveolar macrophages was seen only in late RDS and BPD groups (Figure 2).

Immunohistochemistry for VEGFR-3
Staining for VEGFR-3 was observed in all samples in lymphatic endothelium adjacent to vascular endothelium (Figure 1).

VEGF-C in Tracheal Aspirate Fluid
Mean VEGF-C concentration was 94.2 ± 20.7 pg/ml/IgA-SC unit (mean ± SEM) on Day 1 and 25.7 ± 4.8 pg/ml/IgA-SC unit on Day 7. VEGF-C was also measured from six of the subjects during Week 2 (mean VEGF-C, 31.3 ± 4.7 pg/ml/IgA-SC unit) and during Weeks 3 to 5 (mean VEGF-C, 14.3 ± 2.5 pg/ml/IgA-SC unit). Mean VEGF-C during the first postnatal week was used in statistical analysis (Figure 3).


Figure 3
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  		Figure 3. VEGF-C concentrations in tracheal aspirate fluid. VEGF-C concentrations in tracheal aspirate fluid (pg/ml/IgA-sc unit) in 54 preterm infants during the first postnatal week and in 6 infants during Postnatal Week 2 and Weeks 3 to 5. Numbers inside the plot indicate amount of samples.

 
Of 54 infants, 43 had received antenatal betamethasone. The administration of antenatal betamethasone was associated with higher VEGF-C concentrations in tracheal aspirate fluid (63.0 ± 5.9 vs. 36.2 ± 6.5 pg/mL/IgA-SC unit, p = 0.046). Also, higher number of antenatal betamethasone doses (1 dose, n = 32, vs. 2 doses, n = 11; mean VEGF-C 40.7 ± 11.7 vs. 64.1 ± 6.7 pg/mL/IgA-SC unit, p = 0.01), and administration of betamethasone closer to birth correlated with higher VEGF-C concentrations (R = –0.22, p = 0.046; see Figure E1).

Low birth weight, but not gestational age, correlated with higher concentrations of VEGF-C in tracheal aspirate fluid (R = –0.21, p = 0.004; Figure E2). Lactocyl ceramide (LC), which has been demonstrated in large amounts in granulocytes and in inflamed fetal membranes (21), was measured in 27 infants. Presence of LC in tracheal aspirate fluid (46.0 ± 8.7 vs. 80.0 ± 10.9 pg/mL/IgA-SC unit, p = 0.002) associated with lower VEGF-C concentrations.

Indomethacin was given to 40 of 54 infants due to patent ductus arteriosus. Indomethacin treatment associated with lower VEGF-C concentrations (56.0 ± 5.9 vs. 71.0 ± 9.7 pg/mL/IgA-SC unit, p = 0.016).

Higher umbilical cord pH (R = 0.26, p = 0.038), but not base excess, correlated with higher concentrations of VEGF-C in tracheal aspirate fluid. Subsequent development of BPD did not associate with VEGF-C concentrations (Table E3).

Multiple regression analysis was performed by inclusion of all significant parameters after which all nonsignificant parameters were withdrawn. Three significant parameters remained: time elapsed between antenatal betamethasone administration and birth (p = 0.013), presence of LC (p = 0.047), and treatment with indomethacin (p = 0.007).

VEGF-A in Tracheal Aspirate Fluid
Concentrations of VEGF-A in addition to VEGF-C were analyzed in 56 samples from 15 infants (7 who later developed BPD and 8 who did not) during the first postnatal week. No significant correlation existed between VEGF-A and VEGF-C concentrations (p = 0.28).

VEGF-D in Tracheal Aspirate Fluid
VEGF-D concentrations were measured in 17 samples from 11 infants. Concentration of VEGF-D in tracheal aspirate fluid of all but one sample was too low to be measured.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We describe here a consistent pattern of pulmonary VEGF-C and VEGFR-3 protein expression perinatally and VEGF-C protein appearance in tracheal aspirate fluid in human preterm infants. In immunohistochemistry, all fetuses and infants exhibited staining in bronchial epithelium, whereas alveolar staining was seen mostly in fetuses. VEGF-C protein was found consistently in tracheal aspirate fluid in infants with VLBW. Staining for VEGFR-3 protein was observed in all samples from fetuses to term infants in lymphatic endothelium adjacent to vascular structures. These data indicate a role for VEGF-C in the physiologic development of the lymphatic system of the lung.

VEGF-C and VEGFR-3 expression is critical for the developing lymphatic system in mouse embryos. VEGF-C+/– heterozygous knockout mice lacking one VEGF-C allele develop cutaneous lymphatic hypoplasia and lymphedema and VEGF-C–/– homozygous knockout mice lacking both VEGF-C alleles do not survive until birth and exhibit chylous fluid accumulation in tissues (13). The development of blood vascular endothelial cells into lymphatic endothelial cells requires VEGFR-3 expression (22, 23), and VEGFR-3 expression may determine the function and fate of lymphatic endothelial cells (24). Moreover, signaling through VEGFR-3, not VEGFR-1 or VEGFR-2, is required for the formation of the lymphatic vessel sprouts from the embryonic veins (13). The concentration of VEGF-C in tracheal aspirate fluid was highest during the first 2 postnatal days, after which VEGF-C levels decreased during the first postnatal week. Our data of changing pattern of VEGF-C expression and the decreasing VEGF-C protein levels postnatally suggest that prematurely born infants may have a disturbance in lymphangiogenic development of the lung after the decrease in VEGF-C levels.

In preterm infants, treatment with antenatal glucocorticoids was associated with higher VEGF-C concentrations in tracheal aspirate fluid. In addition, a higher number of doses and the administration closer to birth both correlate with higher VEGF-C. In the preterm infant, administration of glucocorticoids before birth induces lung maturation. This involves increasing surfactant production, removing excess fluid, and structural maturation (25). The increase in VEGF-C levels may be part of the accelerated maturation process, whereas a decrease of VEGF-C may hinder lung maturation in these infants.

In the early postnatal period, VEGF-C staining was found in bronchial and in some extent in alveolar epithelium. However, positive VEGF-C staining in macrophages was seen only in late RDS and BPD. This is in accordance with experimental data demonstrating that lymphangiogenesis seems to be driven by VEGF-C and VEGF-D derived from inflammatory cells that migrate into the airways (26). In the present study, infants with maternal chorioamnionitis were associated with lower VEGF-C in tracheal aspirate fluid. Moreover, the presence of lactocyl ceramide in tracheal aspirate fluid, a phospholipid derived from neutrophils in the airways that is associated with maternal chorioamnionitis (21), was also associated with lower VEGF-C. Prenatal inflammation in utero has been associated with accelerated lung development (21, 27), whereas postnatal inflammation contributes to development of chronic lung injury and is associated with an arrest of lung development (2, 28–30). We conclude that it may be that inflammation in utero versus postnatally influences VEGF-C levels differently.

Infants treated with indomethacin had lower concentration of VEGF-C in tracheal aspirate fluid. This may be due either to patency of ductus arteriosus or to the pharmacologic effects of indomethacin. The latter possibility is supported by recent data demonstrating that cyclooxygenase 2 (COX-2) up-regulates the expression of VEGF-C in adenocarcinoma of human lung (31). COX-2 is present in the alveolar epithelium of the human preterm infant (32). Therefore, indomethacin may decrease pulmonary concentrations of VEGF-C through inhibition of COX-2.

In conclusion, the consistent appearance of VEGF-C perinatally supports the role for VEGF-C in the development of the lymphatic system in the human lung. Prematurely born infants have a clear decrease in the levels of VEGF-C postpartum. In these infants, decrease in pulmonary VEGF-C levels may impair development of lung lymphatics and hence interfere with normal lung fluid homeostasis.


   Acknowledgments
 
The authors thank the personnel of the Neonatal Intensive Care Unit and the Neonatal Nursery of the Hospital for Children and Adolescents for their kind cooperation, Marjatta Vallas and Elina Laitinen for excellent technical assistance, and Dr. Götze-Speer and Professor Speer (University Children's Hospital, Würzburg, Germany) for generous help with IgA-SC standardization.


   FOOTNOTES
 
Supported by the Sigrid Juselius Foundation, Finska Läkaresällskapet, Nylands Nation, Helsinki University Central Hospital Research Fund, the Foundation for Pediatric Research, Helsingin Sanomat Centennial Foundation, the Finnish Medical Society Duodecim, Orion-Pharma, Aarne Koskelo Foundation, and the Maud Kuistila Memorial Foundation.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200508-1291OC on May 11, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form August 19, 2005; accepted in final form May 10, 2006


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