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Respiratory distress syndrome (RDS) and development of bronchopulmonary dysplasia (BPD) are characterized by endothelial cell damage. Persistent pulmonary hypertension of the newborn (PPHN) is a disorder that alters the pulmonary microvasculature. Immunohistochemistry for VEGFA165, an endothelial cell mitogen, and its receptor Flt-1, was performed on lung tissues from autopsies from four fetuses, three preterm infants, four term infants without primary lung disease, four infants with BPD, and four infants with PPHN. VEGF was measured in tracheal aspirates from 31 preterm infants, 5 intubated term infants without primary lung injury, and 12 infants with PPHN during the first 10 postnatal days, and from 8 infants with BPD. Immunohistochemistry for VEGF and Flt-1 was similar in fetuses, preterm infants, and term infants: for VEGF mostly in bronchial epithelium and alveolar macrophages, and for Flt-1 mostly in vascular endothelial cells and bronchial epithelial cells. In patients with BPD, and PPHN, staining for VEGF and Flt-1 appeared also in Type II pneumocytes. Preterm infants with more severe RDS had lower VEGF than those who recovered. The persistent expression of VEGF and Flt-1 during the fetal and neonatal period supports a physiological role for VEGF in human lung development. The lower pulmonary VEGF in preterm infants with more severe RDS may contribute to the pathophysiology of the acute lung injury. In BPD, the expression of VEGF in alveolar epithelium may represent a compensatory increase after the acute phase of the lung disease. In PPHN, that more cell types express VEGF and Flt-1, and the tendency toward a higher concentration of pulmonary VEGF may represent enhanced production of VEGF in response to impaired endothelial function.
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Keywords: bronchopulmonary dysplasia; immunohistochemistry; respiratory distress syndrome; tracheal aspirate fluid; VEGF
Vascular endothelial growth factor (VEGF) is a specific endothelial cell mitogen that regulates endothelial cell differentiation, angiogenesis, and maintenance of existing vessels (1). Two high-affinity receptors for VEGF have been characterized, Flt-1 (fms-like tyrosine kinase 1) and KDR/Flk-1 (fetal liver kinase 1), which are expressed mainly in the endothelium during embryonic development (3, 5, 6). In human fetal lung, VEGF is localized in alveolar epithelial cells and myocytes, suggesting a paracrine role for VEGF in modulating activities in adjacent vascular endothelium (4). Mice deficient in VEGF, Flt-1, or Flk-1 show abnormal angiogenesis or vasculogenesis and die in utero, indicating a crucial role for the VEGF/Flt-1/ Flk-1 system in vascular development (7).
In preterm infants with respiratory distress syndrome (RDS), barotrauma, inflammation, and toxic effects of oxygen are associated with development of bronchopulmonary dysplasia (BPD) (10). The lungs of preterm infants are prone to injury and have a limited ability for repair because of immaturity of the pulmonary structures (10). RDS and the development of BPD in preterm infants are characterized by early endothelial cell damage (13). Destruction of the pulmonary microvasculature is characteristic of experimental lung injury, and during hyperoxic exposure endothelial regeneration is impaired (14). Integral to recovery from experimental lung injury is repair of the microvascular endothelium, which correlates with increased expression of VEGF in alveolar epithelial cells (15). In newborn rabbits, hyperoxic lung injury reduces pulmonary expression of VEGF mRNA and protein, a reduction suggested to contribute to impaired microvascular repair of the injury (16).
Persistent pulmonary hypertension of the newborn (PPHN) is a syndrome of acute respiratory failure characterized by systemic hypoxemia and elevated pulmonary artery pressure. PPHN is most common in infants with underlying diseases such as perinatal asphyxia, meconium aspiration, respiratory distress syndrome, or lung hypoplasia, or it may be idiopathic (17, 18). PPHN is characterized by vascular intimal thickening, which is related to increased migration and proliferation of vascular smooth muscle cells (19). In PPHN, with its damage and functional changes in endothelium, accompanied by release of growth factors, pulmonary vascular endothelial cells may play a central role in the vascular responses in such pathogenesis (19, 20).
We asked the following questions: (1) Where do VEGF and Flt-1 proteins localize in the lungs of fetuses and neonates, and does their expression differ between fetuses and preterm and term infants, or infants with BPD? (2) Because infants with more severe RDS have less VEGF in their tracheal aspirate fluid (TAF) during the early postnatal period (21), what is the concentration of VEGF in TAF in infants with prolonged RDS or with BPD? (3) As PPHN is a disorder that alters the pulmonary microvasculature, and VEGF is a specific endothelial cell mitogen, we wanted to determine the following: Where do VEGF and Flt-1 proteins localize in the lungs of infants with PPHN? Does their expression differ between those with PPHN and those without lung disease? Do the circulating and pulmonary concentrations of VEGF differ between infants with PPHN and infants without lung diseases?
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All studies were done with the approval of the Ethics Committee of the Hospital for Children and Adolescents, University Central Hospital (Helsinki, Finland).
Immunohistochemistry
Four fetuses that were aborted because of major extrapulmonary anomalies between October 1998 and January 1999, 14 infants who died between 1985 and 1992, and 1 infant with a lung biopsy in 1997 in the University Central Hospital (Helsinki, Finland) were included. All the fetuses had microscopically and macroscopically normal lungs. Of the 14 infants, 7 were prematurely born. Three of these seven died of respiratory distress syndrome (RDS), and four died of bronchopulmonary dysplasia (BPD). Of the seven term infants, four with macroscopically and microscopically normal lungs died of congenital cardiac anomalies, and three died of PPHN. The lung biopsy was obtained from an infant suffering alveolar- capillary dysplasia (ACD). None of these infants had infections at the time of death. Autopsies were performed within 2 d of death (Table 1).
Tracheal Aspirate and Blood Samples
Preterm infants Thirty-one preterm infants with RDS were enrolled. None of them had any major anomalies or fulminant infections. All were intubated at birth because of failure to establish spontaneous ventilation and underwent mechanical ventilation during the study period (duration of intubation from 8 to 69 d; median, 16 d). Antenatal steroids were given in 12 pregnancies as 12 mg of betamethasone twice with a 12-h interval. To facilitate weaning from mechanical ventilation, six infants received treatment with dexamethasone during the study period at a dose of 0.5 mg/kgd for 3 d, followed by 0.25 mg/kg/d for 5 to 7 d and 0.125 mg/kg/d for 7 d, starting from 3 to 15 d (median, 9 d). Of the 31 infants, 16 developed BPD, defined as the need for supplemental oxygen at the age of 36 gestational weeks, in association with chest radiographic findings typical of BPD (22) (Table 2).
BPD infants From 8 of the 31 preterm infants, samples were collected also during postnatal weeks 3 to 5. All these infants developed BPD, and they were all intubated for more than 3 wk (duration of intubation, from 26 to 69 d; median, 41 d) (Table 2).
Term infants without primary lung injury Thirty-five healthy term infants from normal pregnancies were studied. A TAF sample was collected after delivery from 22 of these 35 infants; these infants were intubated for tracheal suctioning because of meconium-stained amniotic fluid. In none of them was any significant amount of meconium found in the trachea. Blood samples were taken from the remaining 13 infants during the first postnatal week. Five other term infants (three males and two females) who had cardiac anomalies without pulmonary pathology, and had cardiac surgery during the first 10 postnatal days, were enrolled between June 1998 and February 1999. A TAF sample was collected before the surgical operation. These infants had no prenatal complications, and none had infections (Table 2).
PPHN infants Twenty-three infants with PPHN were enrolled between January 1993 and January 1997. PPHN was diagnosed as Doppler ultrasound-confirmed iso- or suprasystemic pulmonary artery pressure and persistent hypoxia despite optimal conventional therapy. Infants whose pulmonary artery pressure could not be assessed were excluded. All infants were intubated at birth (duration of intubation, from 4 to 21 d; median, 8 d). All received inhaled nitric oxide (NO), and six received intravenous prostacyclin treatment (Flolan; Wellcome, London, UK) before the start of inhaled NO. From these 23 infants, tracheal aspirate fluid (TAF) samples were collected from 12, and blood samples from 11 infants, during the first 10 postnatal days (Table 2).
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Immunohistochemistry
The lung samples were obtained at autopsy and from one infant with ACD at lung biopsy. The samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and kept in dry storage at room temperature. After sectioning, the slides were used within 2 wk. Five-micrometer sections were deparaffinized, rehydrated, and either treated with trypsin (VEGF) or microwaved (Flt-1). Subsequently, the sections were incubated in 0.33% hydrogen peroxidase in methanol and in 5% normal hose serum at room temperature. The sections were incubated overnight at 4° C with the primary antibody. A subsequent incubation in biotinylated anti-rabbit serum was performed with reagents of the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Peroxidase activity was developed with 3-amino-9-ethyl carbazole, and finally the sections were stained with hematoxylin. VEGF antibody was used at a 1:100 dilution (A-20; Santa Cruz Biotechnology, Santa Cruz, CA), and Flt-1 at a 1:100 dilution (C17; Santa Cruz Biotechnology). A negative control was included in each staining round. In these slides, staining was performed by the identical protocol without primary antibody.
Tracheal Aspirate Samples
From 31 preterm infants, 152 TAF samples were collected during the first 10 postnatal days (median time of sample collection, 6 d) (from each infant, from 3 to 8 samples; median, 5 samples). Of these 31 infants, from 8 who developed BPD, 27 samples were also collected during postnatal weeks 3 to 5 (range, 23 to 35 d; median, 27 d) (from each infant, 3 or 4 samples; median, 3 samples). From 22 healthy term infants, 22 samples were collected at birth. From 5 intubated term infants with cardiac anomalies and without primary lung injury, 9 samples were collected (range, 2 to 8 d; median, 5 d) (from each infant, 1 or 2 samples; median, 2 samples). From 12 PPHN infants, 54 samples were collected during the first 10 postnatal days (range, 1 to 10 d; median, 5 d) (from each infant, 3 to 6 samples; median, 5 samples). Our center is a tertiary hospital to which infants with PPHN are referred mostly from other hospitals, and a first-day value was available from one infant with PPHN.
Samples of TAF from preterm infants and infants with PPHN were
collected once daily by routine tracheal lavage, from healthy term infants after delivery, and from intubated term infants through the intubation catheter before the cardiac operation. One milliliter of sterile
isotonic saline was instilled into the endotracheal tube, the infant was
manually ventilated for three breaths, and the trachea was suctioned
twice, each time for 5 s. For analysis of tracheal aspirates, secretions
were collected into a trap and transferred into tubes containing 500 IU of aprotinin (Trasylol; Bayer, Leverkusen, Germany) and 5 mg of
deferoxiamine (Desferal; Ciba, Basel, Switzerland). The tubes were
stored at 
20° C until analysis.
Analysis of VEGF and of the Secretory Component of IgA in Tracheal Aspirate Samples
VEGF in TAF was analyzed by the Quantikine human VEGF immunoassay (R&D Systems, Oxon, UK). To eliminate the effect of dilution of TAF samples, the concentration of the secretory component of IgA (IgA-SC) was determined by direct ELISA. 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 gestational or postnatal age (23). Secretory IgA isolated from human colostrum served as the standard. The results were standardized by B Götze-Speer and C. Speer (Kinderklinik, Tübingen, Germany). Microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4° C with 100-µl aliquots of 1:2,000 diluted anti-human secretory component (Dako, Glostrup, Denmark) in 50 mM sodium bicarbonate, pH 9.5. After washing with 200 µl of 20 mM Tris-500 mM NaCl, pH 7.5 (TBS), the plates were blocked for unspecific protein binding by incubation with 200 µl of 2% bovine serum albumin (BSA) in TBS and washed with 0.05% Tween 20 in TBS (TTBS). TAF samples were diluted to between 1:10 to 1:500 in diluting buffer (1% BSA in TTBS), and 100-µl aliquots were added to the wells. After incubation overnight at room temperature, the plates were washed three times with TTBS; 100 µl of peroxidase-conjugated rabbit anti-human SC (Dako), diluted 1:400 in diluting buffer, was added, and the plates were incubated for 4 h at room temperature. After washing with TTBS, the plates were developed with 100 µl of substrate solution containing 8 mg of orthophenylenediamine (Dako) and 5 µl of 30% H2O2 in 12 ml of water. After 30 min at room temperature, the optical densities of the plates were read at 450 nm.
Blood Samples
During the first 10 postnatal days, 13 samples were collected from 13 healthy term infants (range, 1 to 8 d; median, 4 d), and 29 samples
were collected from 11 infants with PPHN (range, 1 to 10 d; median, 3 d)
(from each infant, 2 to 4 samples; median, 3 samples). From healthy
term infants, blood samples were taken from peripheral veins, and
from infants with PPHN, samples were drawn through peripheral arterial catheters into EDTA tubes. The tubes were centrifuged (3,000 rpm for 10 min), and plasma was stored at 20° C until analysis.
Statistical Analysis
Infant data are given as median and range, and results as means ± SEM. Comparisons between groups were performed with the one-way ANOVA, and the Bonferroni correction served for the post hoc comparisons. Comparisons between unpaired items were performed with the Student t test. Simple regression analysis served for continuous variables. Logarithmic transformation of the data was performed when appropriate. p Values less than 0.05 were considered significant. All calculations were done with StatView 4.1 (Abacus Concepts, Berkeley, CA).
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Immunohistochemistry for VEGF
In fetuses and in premature infants, positive staining was detected in bronchial epithelial cells, in cuboidal cells in the alveolar epithelium, and in vascular endothelial cells, and in addition some alveolar macrophages were positive. In term infants, staining was seen in bronchial epithelial cells and in alveolar macrophages, and in infants with BPD, staining was seen in bronchial epithelial cells and alveolar macrophages, in vascular endothelium, and in Type II pneumocytes. In infants with PPHN, positive staining was detected in bronchial epithelium and vascular endothelial cells. In addition, staining was also seen in Type II pneumocytes in alveolar epithelium. In the infant with ACD, strong staining was seen in bronchial epithelium and vascular endothelial cells; in addition, alveolar macrophages were also positive (Figure 1).
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= vascular endothelial cells; 
= pneumocytes; * = alveolar macrophages.
Immunohistochemistry for Flt-1
In fetuses, endothelial cells in capillaries, veins, and small arteries, as well as in the intima of the small arteries, were strongly positive, and a staining reaction was apparent in bronchial epithelial cells and cuboidal cells in alveolar epithelium. In premature infants, a similar staining reaction was seen in vessels and bronchi. In term infants, positive staining was visible in the endothelial lining of veins and capillaries and arteries, as well as in the intima of the small arteries, and in bronchial epithelial cells. In infants with BPD, a positive staining reaction was visible throughout the walls of small arteries, and in the endothelial lining of veins and capillaries; bronchial epithelium was positive, as were Type II pneumocytes in the alveoli, and alveolar macrophages. In infants with PPHN and in the infant with ACD, a staining reaction was seen throughout the vascular walls in capillaries, arteries, and veins. Bronchial epithelium, and Type II pneumocytes in alveolar epithelium, were positive, as well as alveolar macrophages (Figure 1).
VEGF in Tracheal Aspirates
IgA-SC, the secretory component of immunoglobulin A, was measured and data were corrected for dilution by adjustment with IgA-SC before analysis. The dilution-adjusted mean concentration of VEGF at birth for preterm infants was 5.5 ± 5.4 pg/ml/IgA-SC unit, and for healthy term infants it was 0.5 ± 0.2 pg/ml/IgA-SC unit. The dilution-adjusted mean concentration of VEGF during the first 10 postnatal days for preterm infants was 54.2 ± 9.1 pg/ml/IgA-SC unit, for intubated term infants it was 13.7 ± 8.6 pg/ml/IgA-SC unit, for infants with BPD it was 33.4 ± 3.6 pg/ml/IgA-SC unit, and for infants with PPHN it was 19.6 ± 3.5 pg/ml/IgA-SC unit (Figure 2).
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Preterm infants with an initial arteriolar-alveolar ratio of less than 0.22 had lower VEGF (p = 0.0011), and negative correlations existed between VEGF and the number of surfactant doses required (r = 0.26, p = 0.0077), and between VEGF and duration of intubation (r = 0.25, p = 0.01) (Table 2). No differences appeared in VEGF concentrations between patients receiving glucocorticoid treatment (antenatal or postnatal) or not. Preterm infants who developed BPD had lower VEGF in TAF during the first 10 postnatal days than did those who survived without it (45.9 ± 7.3 versus 64.1 ± 17.2 pg/ml/IgA-SC unit, respectively) but this difference did not reach significance (p = 0.28). For those eight infants with BPD, there was a tendency toward lower VEGF concentrations during Weeks 3 to 5 than during the first 10 postnatal days (33.4 ± 3.6 versus 49.6 ± 9.1 pg/ ml/IgA-SC unit, p = 0.061). For PPHN infants, no correlations existed between clinical data and mean VEGF.
VEGF in Plasma
The mean concentration of VEGF for healthy term infants was 138.5 ± 39.4 pg/ml, and for infants with PPHN it was 43.5 ± 6.7 pg/ml (p = 0.039). For infants with PPHN, no correlations existed between clinical data and mean VEGF (Figure 2).
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In human fetuses and adults, VEGF mRNA can be detected in multiple tissues, with most abundant expression in lung, kidney, and spleen, and with VEGF peptide localization in epithelial cells and myocytes (4). As a paracrine mediator, VEGF is secreted by nonendothelial cells and modulates activity in adjacent vascular endothelium (4). This is in accordance with our findings of strong staining for VEGF in all cases in bronchial epithelium and alveolar macrophages, and in addition, in fetuses and preterm infants also in cuboidal cells in the alveolar epithelium. We also observed staining in the vascular endothelium in fetuses, and in preterm infants. In term infants, no staining of vascular endothelium was apparent, which is in line with previous findings (24). Endothelial cells are the major target of action for VEGF, which has been shown to bind to them with high affinity (25). In mice, Flt-1 is expressed in endothelium during vascular development similarly in embryos and in adults (3). In the present data, fetuses, preterm infants, and term infants showed positive staining for Flt-1 in endothelial cells lining capillaries, veins, and small arteries. VEGF is known to induce proliferation almost exclusively in vascular endothelial cells, but for Flk-1, upregulation induced by VEGF may represent a positive feedback mechanism for VEGF action (26, 27). We found also, in all groups, positive staining for Flt-1 in bronchial epithelial cells. This may represent an autocrine feedback mechanism for VEGF in vivo, although this observation still needs to be resolved. VEGF is expressed at high levels in lungs of normal human adults undergoing physiological angiogenesis, and loss of a single VEGF allele is lethal in the mouse embryo from impaired angiogenesis and several developmental anomalies (9, 28). The persistent appearance of VEGF and Flt-1 that we have observed during the perinatal period from gestational week 16 therefore also suggests a physiological role for VEGF in the development of the human lung.
The concentration VEGF in the TAF of preterm and term infants was low after birth, but increased during the first 10 postnatal days. Previously, a rise in VEGF has been reported in the plasma of term infants during the first postnatal days (29). Our previous finding of severalfold higher concentrations in TAF than in plasma suggests that this postnatal increase in VEGF concentration is produced locally, as VEGF acts as a paracrine mediator (21). The concentration of VEGF in TAF was higher at birth and during the first 10 postnatal days in preterm than in term infants, which may reflect the effect of gestation. The higher increase in postnatal VEGF in preterm infants could be a physiological phenomenon and belong to this developmental period. Those preterm infants who had lower VEGF suffered prolonged and more severe respiratory distress. Hyperoxia itself may be a factor behind this decrease in VEGF. On the other hand, infants with severe pulmonary injury may be incapable of responding to the inflammatory stimuli with an increase in VEGF. In animal studies, with induced pulmonary inflammation or after prolonged exposure to hyperoxia, VEGF mRNA and protein both decrease, a phenomenon that has been suggested to contribute to the pathophysiology of oxygen-induced lung injury, and to impaired vascular repair in such injury (15, 16, 30, 31). Consequently, in preterm infants with severe RDS the lower pulmonary VEGF may contribute to the pathophysiology of their acute lung injury.
VEGF and Flt-1 staining was seen in Type II pneumocytes in alveolar epithelium only in those infants with BPD. In rabbits, after recovery from hyperoxic lung injury, Type II pneumocytes exclusively express VEGF mRNA, suggesting a role for VEGF in the regulation of microvascular endothelial cell proliferation after oxidant injury (15). Analogously, the presence of VEGF in alveolar epithelium of infants with BPD may, on the one hand, be associated with the healing process after acute RDS. On the other hand, it may also play a role in the pathogenesis of BPD.
In PPHN infants we observed positive staining for VEGF in bronchial epithelium and also in Type II pneumocytes in alveolar epithelium and in vascular endothelium. PPHN is characterized by vascular intimal thickening, which is related to increased migration and proliferation of vascular smooth muscle cells; and by elevated pulmonary artery pressure, which is associated with vascular intimal thickening of arteries with a diameter less than 200 µm, arteries that play an important role in pulmonary blood pressure and vascular resistance regulation (17, 32). In infants with PPHN, we observed Flt-1 expression as in term infants, in bronchial epithelium and the vascular wall, but also in Type II-resembling pneumocytes in alveolar epithelium, as well as in alveolar macrophages. More cell types expressing Flt-1 and VEGF may represent enhanced production of VEGF due to impaired endothelial function in PPHN. As in other cases, we observed in PPHN, similar coexpression of VEGF and its receptor Flt-1 in the same cell types. VEGF in the normal lung is important for regulation of endothelial cell turnover, differentiation, and vascular repair (3). In infants with PPHN we demonstrated a tendency to higher concentration of pulmonary VEGF protein. This may be due to stimuli induced by endothelial injury, as increased VEGF expression is associated with repair of microvascular endothelium in animals (15). VEGF is a paracrine factor affecting adjacent endothelium, and we have previously noticed that VEGF in plasma is markedly lower than pulmonary VEGF, suggesting that VEGF leaks from different tissues into the circulation (4, 21). We have previously reported lower plasma VEGF concentrations in preterm infants than those found in this study in healthy term infants (21). This is surprising because pulmonary VEGF concentrations are higher in preterm than in term infants. One possible explanation is that in healthy term infants, VEGF is produced in higher amounts in another organ(s) than lung and shed from there into the circulation. In infants with PPHN circulatory VEGF was lower than in term infants with normal lungs. The lower VEGF in the former may reflect an overall disturbance in vascular development. Alternatively, in normal circumstances the pulmonary bed may be an important source of circulating VEGF. In severe PPHN, there is a right-to-left shunt through the foramen ovale and also via the ductus arteriorus if it remains open. Consequently, pulmonary blood flow is diminished, which may contribute to low plasma concentrations in these infants.
The condition known as misalignment of the pulmonary vessels or alveolar capillary dysplasia (ACD) has been reported as a cause of "idiopathic" PPHN (33). Microscopically, there is failure of formation and ingrowth of alveolar capillaries that make no contact with the alveolar epithelium, medial muscular thickening of small pulmonary arterioles with extension of muscularization to the smallest, intra-acinar arterioles, and thickened alveolar walls (34). In ACD, staining for VEGF and Flt-1 was similar to that seen in infants with PPHN, but the intensity of the staining was higher than in other cases. The high expression of VEGF/Flt-1 seen here may be associated with the muscular thickening of small pulmonary arterioles seen in ACD. An explanation that cannot be excluded for this higher intensity staining is that the sample was obtained from a lung biopsy and fixed instantly, whereas the other samples were obtained from autopsies.
In conclusion, the persistent expression of VEGF in bronchial epithelium and in alveolar macrophages, and of Flt-1 in vascular endothelial cells during the fetal and neonatal period, suggests a physiological role for each of them in human lung development. The consistent presence of Flt-1 in bronchial epithelial cells may represent a feedback mechanism for VEGF. Higher VEGF in TAF was seen in preterm than in term infants. Those preterm infants who suffered more severe respiratory distress had lower VEGF, which may contribute to the pathophysiology of their acute lung injury. In patients with BPD, VEGF occurred also in Type II pneumocytes, which may represent a mechanism potentially indicative of a compensatory increase after the acute phase of the lung disease. In infants with PPHN, we observed positive staining for VEGF located, in addition to bronchial epithelium, also in alveolar epithelium and vascular endothelium, and for Flt-1 throughout the vascular wall, as well as in bronchial and alveolar epithelium. The additional cell types expressing Flt-1 and VEGF, and the tendency to a higher concentration of VEGF in TAF seen in infants with PPHN, may represent an enhanced production of VEGF due to the impaired endothelial function characteristic of PPHN.
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Correspondence and requests for reprints should be addressed to Patrik Lassus, M.D., Hospital for Children and Adolescents, University of Helsinki, Stenbäckinkatu 11, 00290 Helsinki, Finland. E-mail: patrik.lassus{at}helsinki.fi
(Received in original form December 8, 2000 and accepted in revised form September 27, 2001).
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: Kristina von Boguslawski, Helena Huotarinen, Marjatta Vallas, and Merja Lahtinen for excellent technical assistance; Dr. B Götze-Speer and Prof. Ch. Speer (Kinderklinik, Tübingen, Germany) for generous help with IgA-SC standardization; and Carolyn Norris, Ph.D., for linguistic revision of the manuscript.
Supported by Finska Läkaresällskapet, and the Helsinki University Center Hospital Research Fund.
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