Disrupted pulmonary vasculature and decreased vascular endothelial growth factor, Flt-1, and TIE-2 in human infants dying with bronchopulmonary dysplasia.

An abnormal pulmonary vasculature may be an important component of bronchopulmonary dysplasia (BPD). We examined human infant lung for the endothelial cell marker PECAM-1 and for angiogenic factors and their receptors. Lung specimens were collected prospectively at approximately 6 h after death. The right middle lobe was inflation fixed and part of the right lower lobe was flash frozen. We compared lungs from infants dying with BPD (n = 5) with lungs from infants dying from nonpulmonary causes (n = 5). The BPD group was significantly more premature and had more days of ventilator and supplemental oxygen support, but died at a postconceptional age similar to control infants. PECAM-1 protein and mRNA were decreased in the BPD group. PECAM-1 immunohistochemistry showed the BPD group had decreased staining intensity and abnormal distribution of alveolar capillaries. The dysmorphic capillaries were frequently in the interior of thickened alveolar septa. The BPD group had decreased vascular endothelial growth factor (VEGF) mRNA and decreased VEGF immunostaining, compared with infants without BPD. Messages for the angiogenic receptors Flt-1 and TIE-2 were decreased in the BPD group. We conclude that infants dying with BPD have abnormal alveolar microvessels and that disordered expression of angiogenic growth factors and their receptors may contribute to these abnormalities.

Maturation of carbonic anhydrase IV expression in rabbit kidney.

Carbonic anhydrase (CA) IV facilitates renal acidification by catalyzing the dehydration of luminal H(2)CO(3). CA IV is expressed in proximal tubules, medullary collecting ducts, and A-intercalated cells of the mature rabbit kidney (Schwartz GJ, Kittelberger AM, Barnhart DA, and Vijayakumar S. Am J Physiol 278: F894-F904, 2000). In view of the maturation of HCO transport in the proximal tubule and collecting duct, the ontogeny of CA IV expression was examined. During the first 2 wk, CA IV mRNA was expressed in maturing cortex and medulla at ~20% of adult levels. The maturational increase was gradual in cortex over 3-5 wk of age but surged in the medulla, so that mRNA levels appeared higher than those in the adult medulla. In situ hybridization showed very little CA IV mRNA at 5 days, with increases in deep cortex and medullary collecting ducts by 21 days. Expression of CA IV protein in the cortex and medulla was minimal at 3 days of age but then apparent in the juxtamedullary region, A-intercalated cells and medullary collecting ducts by 18 days; there was little labeling of the proximal straight tubules of the medullary rays. Thus CA IV expression may be regulated to accommodate the maturational increase in HCO absorption in the proximal tubule. In the medullary collecting duct, there is a more robust maturation of CA IV mRNA and protein, commensurate with the high rate of HCO absorption in the neonatal segment.

Expression of vascular endothelial growth factor and Flk-1 in developing and glucocorticoid-treated mouse lung.

Although the endothelial cell is the most abundant cell type in the differentiated lung, little is known about regulation of lung developmental vasculogenesis. Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen and angiogenic factor that has putative roles in vascular development. Mitogenic actions of VEGF are mediated by the tyrosine kinase receptor KDR/murine homologue fetal liver kinase Flk-1. HLF (hypoxia-inducible factor-like factor) is a transcription factor that increases VEGF gene transcription. Dexamethasone augments lung maturation in fetal and postnatal animals. However, in vitro studies suggest that dexamethasone blocks induction of VEGF. The objectives for the current study were to measure VEGF mRNA and Flk-1 mRNA in developing mouse lung and to measure the effects of dexamethasone treatment in vivo on VEGF and Flk-1 in newborn mouse lung. Our results show that VEGF and Flk-1 messages increase in parallel during normal lung development (d 13 embryonic to adult) and that the distal epithelium expresses VEGF mRNA at all ages examined. Dexamethasone (0.1-5.0 mg x kg(-1) x d(-1)) treatment of 6-d-old mice resulted in significantly increased VEGF, HLF, and Flk-1 mRNA. Dexamethasone did not affect cell-specific expression of VEGF, VEGF protein, or proportions of VEGF mRNA splice variants. These data suggest that the developing alveolar epithelium has an important role in regulating alveolar capillary development. In addition, unlike effects on cultured cells, dexamethasone, even in relatively high doses, did not adversely affect VEGF expression in vivo. The relatively high levels of VEGF and Flk-1 mRNA in adult lung imply a role for pulmonary VEGF in endothelial cell maintenance or capillary permeability.

p53-independent induction of GADD45 and GADD153 in mouse lungs exposed to hyperoxia.

Previous studies have shown that lungs of adult mice exposed to >95% oxygen have increased terminal deoxyribonucleotidyltransferase dUTP nick end-label staining and accumulate p53, the expression of which increases in cells exposed to DNA-damaging agents. The present study was designed to determine whether hyperoxia also increased expression of the growth arrest and DNA damage (GADD) gene 45 and GADD153, which are induced by genotoxic stress through p53-dependent and -independent pathways. GADD proteins have been shown to inhibit proliferation and stimulate DNA repair and/or apoptosis. GADD45 and GADD153 mRNAs were not detected in lungs exposed to room air but were detected after 48 and 72 h of exposure to hyperoxia. In situ hybridization and immunohistochemistry revealed that hyperoxia increased GADD45 and GADD153 expression in the bronchiolar epithelium and GADD45 expression predominantly in alveolar cells that were morphologically consistent with type II cells. Hyperoxia also increased GADD expression in p53-deficient mice. Terminal deoxyribonucleotidyltransferase dUTP nick end-label staining of lung cells from p53 wild-type and p53-null mice exposed to hyperoxia for 48 h revealed that hyperoxia-induced DNA fragmentation was not modified by p53 deficiency. These studies are consistent with the hypothesis that hyperoxia-induced DNA fragmentation is associated with the expression of GADD genes that may participate in DNA repair and/or apoptosis.

Early dexamethasone-attempting to prevent chronic lung disease.

BACKGROUND: We previously demonstrated improved survival and early outcomes in a pilot trial of 2 doses of intravenous dexamethasone for infants with surfactant-treated respiratory distress syndrome. (1) A multicenter, randomized, double-blind trial was undertaken to confirm these results. METHODS: Infants <30 weeks' gestation were eligible if they had respiratory distress syndrome, required mechanical ventilation at 12 to 18 hours of age, and had received at least 1 dose of exogenous surfactant. Infants were excluded if sepsis or pneumonia was suspected or if congenital heart disease or chromosomal abnormalities were present. A total of 384 infants were enrolled-189 randomized to dexamethasone (.5mg/kg birth weight at 12-18 hours of age and a second dose 12 hours later) and 195 to an equal volume of saline placebo. RESULTS: No differences were found in the dexamethasone versus placebo groups, respectively, regarding the primary outcomes of survival (79% vs 83%), survival without oxygen at 36 weeks' corrected gestational age (CGA; both 59%), and survival without oxygen at 36 weeks' CGA and without late glucocorticoid therapy (46% vs 44%). No significant differences between the groups in estimates from Kaplan-Meier survival analyses were found for median days on oxygen (50 vs 56 days), ventilation (20 vs 27 days), days to regain birth weight (15.5 vs 14 days), or length of stay (LOS; 88 vs 89 days). Infants given early dexamethasone were less likely to receive later glucocorticoid therapy for bronchopulmonary dysplasia during their hospitalization (27% vs 35%). No clinically significant side effects were noted in the dexamethasone group, although there were transient elevations in blood glucose and blood pressure followed by a return to baseline by study day 10. Among infants who died (40 vs 33), there were no differences in the median days on oxygen, ventilation, nor LOS. However, in survivors (149 vs 162), the following were observed: median days on oxygen 37 versus 45 days, ventilation 14 versus 19 days, and LOS 79 versus 81 days, for the dexamethasone versus placebo groups, respectively. CONCLUSIONS: This dose of early intravenous dexamethasone did not reduce the requirement for oxygen at 36 weeks' CGA and survival was not improved. However, early dexamethasone reduced the use of later prolonged dexamethasone therapy, and among survivors, reduced the median days on oxygen and ventilation. We conclude that this course of early dexamethasone probably represents a near minimum dose for instituting a prophylactic regimen against bronchopulmonary dysplasia.

Respiratory syncytial virus and premature infants born at 32 weeks' gestation or earlier: hospitalization and economic implications of prophylaxis.

OBJECTIVES: To assess the risk of hospitalization associated with respiratory syncytial virus (RSV) and to estimate the economic impact of RSV prophylaxis with either RSV immune globulin (RSV-Ig) or RSV monoclonal antibody (palivizumab) on a cohort of preterm infants born at 32 weeks' gestation or earlier. DESIGN: Historical cohort study. SETTING: A 12-county neonatal network served by the regional center in Rochester, NY. PARTICIPANTS: One thousand twenty-nine infants born at 32 weeks' gestation or earlier followed up until 1 year of corrected age. MAIN OUTCOME MEASURES: Rate of hospitalization with an RSV-associated illness; cost per hospitalization prevented resulting from either form of RSV prophylaxis. RESULTS: The probability of hospitalization with an RSV-associated illness for infants born at 32 weeks' gestation or earlier was estimated at 11.2%. The incidence of RSV hospitalization increased with decreasing gestational age (13.9% vs 4.4% for infants born at < or =26 weeks' gestation vs those born at 30-32 weeks' gestation). Infants requiring respiratory support at 36 weeks of postconceptual age (PCA) or older had a higher hospitalization rate (16.8% vs 6.2%), longer hospital stays, and higher hospital charges than infants requiring respiratory support at less than 36 weeks of PCA. For infants requiring respiratory support at less than 36 weeks of PCA, the incidence of RSV hospitalization still increased with decreasing gestational age (10.2% vs 4.3% for infants < or =26 weeks' gestation vs those 30-32 weeks' gestation). Analysis indicated that both forms of RSV prophylaxis would increase the net cost of care for all groups. Palivizumab was more cost-effective than RSV-Ig for preventing RSV hospitalization among infants who required respiratory support at less than 36 weeks of PCA, especially those born at 26 weeks' gestation or earlier. Overall, RSV-Ig was more cost-effective than palivizumab for infants requiring respiratory support at 36 weeks of PCA or older. CONCLUSIONS: This analysis suggests that available forms of RSV prophylaxis would increase the net cost of care not only for the entire cohort but for each of the subgroups studied. However, the RSV hospitalization rate and the cost-effectiveness of prophylaxis varied markedly by subgroup.

Bcl-2 family gene expression during severe hyperoxia induced lung injury.

Exposure of the lung to severe hyperoxia induces terminal transferase dUTP end-labeling (TUNEL) indicative of DNA damage or apoptosis and increases expression of the tumor suppressor p53 and of members of the Bcl-2 gene family. Because cell survival and apoptosis are regulated, in part, by the relative abundance of proteins of the Bcl-2 family, we hypothesized that lung cells dying during exposure would show increased expression of pro-apoptotic members, such as Bax, whereas surviving cells would have increased expression of anti-apoptotic members, such as Bcl-X(L). The hypothesis is tested in the current study by determining which Bcl-2 genes are regulated by hyperoxia, with specific focus on correlating expression of Bax and Bcl-X(L) with morphologic evidence of apoptosis or necrosis. Adult mice exposed to greater than 95% oxygen concentrations for 48 to 88 hours had increased whole-lung mRNA levels of Bax and Bcl-X(L), no change in Bak, Bad, or Bcl-2, and decreased levels of Bcl-w and Bfl-1. In situ hybridization revealed that hyperoxia induced Bax and Bcl-X(L) mRNA in uniform and overlapping patterns of expression throughout terminal bronchioles and parenchyma, coinciding with TUNEL staining. Electron microscopy and DNA electrophoresis, however, suggested relatively little classical apoptosis. Unexpectedly, Western analysis demonstrated increased Bcl-X(L), but not Bax, protein in response to hyperoxia. Bax and Bfl-1 were not altered by hyperoxia in p53 null mice; however, oxygen toxicity was not lessened by p53 deficiency. These findings suggest that oxygen-induced lung injury does not depend on the relative expression of these Bcl-2 members.

Vascular endothelial growth factor in pulmonary lavage fluid from premature infants: effects of age and postnatal dexamethasone.

Corticosteroids are used to ameliorate bronchopulmonary dysplasia (BPD). They also affect normal development, including the expression of growth factors such as vascular endothelial growth factor (VEGF). Deep pulmonary lavage specimens were collected on days 1, 3, 7 and 28 of life in 40 infants of <34 weeks of gestation at birth during a randomized controlled trial of two doses of dexamethasone (DEX) at 12 and 24 h of age for BPD prophylaxis. VEGF was measured by enzyme-linked immunosorbent assay. The 18 DEX and 21 control subjects had similar gestations, birth weights and oxygen requirements at study entry. Lavage VEGF tripled between day 1 and 3 in both groups. The day 7 levels were higher in DEX subjects than in controls. DEX and control values were similar on day 28. Higher lavage VEGF levels on days 1 and 3 were also correlated with lower gestational age at birth. Lavage VEGF levels were not associated with the development of BPD. We speculate that these DEX- and age-associated changes in VEGF may affect pulmonary angiogenesis.

Differential expression of VEGF mRNA splice variants in newborn and adult hyperoxic lung injury.

Lung development and repair of hyperoxic injury require closely regulated growth and regeneration of alveolar capillaries. Vascular endothelial growth factor (VEGF), a mitogen for endothelial cells, is expressed by alveolar epithelial cells. Alternative splicing of VEGF mRNA results in isoforms of varying mitogenicity and solubility. We examined changes in the proportions of the VEGF splice variant mRNAs in rabbit lung development and in control, oxygen-injured, and recovering newborn and adult rabbit lungs. The proportion of the 189-amino acid VEGF mRNA, which codes for an isoform that binds to the extracellular matrix, increased fivefold during development (from 8% of total VEGF message at 22 days gestation to 40% in 10-day newborn lungs; P < 0.001). During neonatal oxygen injury, its expression declined from 38 to 8% of VEGF message (P < 0.002) and returned to the control value in recovery. A similar pattern was observed in adults. VEGF protein in lung lavage fluid increased slightly during hyperoxia, declined to barely detectable levels at the 50% lethal dose time point, and increased 10-fold (newborn) or up to 40-fold (adult) in recovering animals. We conclude that alternative splicing may have important roles in the regulation of VEGF activity in developing and injured lungs.

Accumulation of p21(Cip1/WAF1) during hyperoxic lung injury in mice.

Hyperoxic lung injury results in decreased cell proliferation, DNA damage, and cell death. Because the cyclin-dependent kinase inhibitor p21(Cip1/WAF1) (p21) inhibits cell proliferation in G1/S, enhances DNA repair, and regulates apoptosis in some cells, we hypothesized that the expression of p21 would increase in lungs of C57Bl/6J male mice exposed to and recovered from > 95% oxygen. A low level of p21 messenger RNA (mRNA) expression was detected by Northern blot analysis of room air-exposed lungs. Exposure to hyperoxia resulted in a modest increase in p21 mRNA expression by 24 h, followed by a marked induction by 48 to 72 h. In situ hybridization revealed that p21 mRNA abundance increased in bronchiolar epithelium and in resident alveolar cells, but not in smooth-muscle cells or large airway epithelium. Hyperoxia increased the expression of p21 protein by 24 h and continued to increase at 48 and 72 h. Immunohistochemical staining showed that p21 protein accumulated in the bronchiolar epithelium and in alveolar regions that had increased p21 mRNA expression. In contrast, the expression of the cyclin-dependent kinase inhibitor p27(Kip1) was not altered by hyperoxia. To determine whether p21 expression was altered during the repair process, mice were exposed to hyperoxia for 64 h and allowed to recover for up to 4 d in room air. The abundance of p21 mRNA and protein decreased by 1 to 2 d of recovery and returned to room air-exposed levels by 3 to 4 d of recovery. These findings support the concept that bronchiolar epithelial and alveolar cells damaged by hyperoxia express molecules such as p21, which may participate in regulating cell proliferation, DNA repair, and cell death.

Comparison of two strategies for surfactant prophylaxis in very premature infants: a multicenter randomized trial.

INTRODUCTION: Previous trials of surfactant therapy in premature infants have demonstrated a survival advantage associated with prophylactic therapy as an immediate bolus, compared with the rescue treatment of established respiratory distress syndrome. The optimal strategy for prophylactic therapy, however, remains controversial. When administered as an endotracheal bolus immediately after delivery, surfactant mixes with the absorbing fetal lung fluid and may reach the alveoli before the onset of lung injury. This approach, however, causes a brief delay in the initiation of standard neonatal resuscitation, including positive pressure ventilation, and is associated with a risk for surfactant delivery into the right main stem bronchus or esophagus. As an alternative approach, surfactant prophylaxis may be administered in small aliquots soon after resuscitation and confirmation of endotracheal tube position. Although this strategy has substantial logistical advantages in clinical practice, its efficacy has not been established. OBJECTIVE: The purpose of this study was to determine whether the established benefits of the immediate bolus strategy for surfactant prophylaxis could still be achieved using a postventilatory aliquot strategy after initial standard resuscitation and stabilization. DESIGN: Multicenter randomized clinical trial with patients randomized before delivery to immediate bolus or postventilatory aliquot therapy. PARTICIPANTS: Inborn premature infants delivered to mothers at an estimated gestational age of 24[0/7] to 28[6/7] weeks. INTERVENTIONS: Those infants who were randomized to the immediate bolus strategy were intubated as rapidly as possible after birth, and a 3-mL intratracheal bolus of calf lung surfactant extract (Infasurf) was administered before the initiation of positive pressure ventilation. Those infants who were randomized to the postventilatory aliquot strategy received standard resuscitation measures with intubation by 5 minutes of age, if not required earlier. At 10 minutes after birth, 3 mL of surfactant was administered in 4 divided aliquots of 0.75 mL each. Patients in both groups were eligible to receive up to three additional doses of surfactant as rescue therapy in the neonatal intensive care unit, if needed. OUTCOME MEASURES: The primary outcome variable was survival to discharge to home. Secondary variables included neonatal complications and requirement for oxygen therapy at 36 weeks' postmenstrual age. RESULTS: Among three centers, 651 infants were enrolled and randomized before delivery. Survival to discharge to home was similar for the two strategies for surfactant therapy as prophylaxis: 76% for the immediate bolus group and 80% for the postventilatory aliquot group. In a secondary analysis, the rate of supplemental oxygen administration at 36 weeks' postmenstrual age was 18% for the immediate bolus group and 13% for the postventilatory aliquot group. CONCLUSIONS: Survival to discharge to home was similar with immediate bolus and postventilatory aliquot strategies for surfactant prophylaxis. Because of its logistical advantages in the delivery room and its beneficial effects on prolonged oxygen requirements, we recommend the postventilatory aliquot strategy for surfactant prophylaxis of premature infants delivered before 29 weeks' gestation.

Cell-specific expression of fibronectin and EIIIA and EIIIB splice variants after oxygen injury.

Cellular fibronectin (cFN) expression is characteristic of injured tissues. Unlike plasma FN, cFN mRNA often contains the EIIIA or EIIIB domains. We examined the lung cell-specific expression of total cFN mRNA and the EIIIA and EIIIB splice variants in rabbits after acute oxygen injury. By in situ hybridization, control lung had low cFN mRNA. After exposure to > 95% oxygen, mRNAs for total cFN and EIIIA were noted primarily in alveolar macrophages and large-vessel endothelial cells. By 3-5 days recovery, cFN and EIIIA mRNA abundance was increased in alveolar septal cells (i.e., alveolar epithelial, interstitial, or endothelial cells) and in some large-vessel endothelial cells but was low in bronchial epithelial cells. During recovery, EIIIB mRNA was low in alveolar septal cells but was noted mainly in chondrocytes. Immunostaining for EIIIA increased during recovery, paralleling the in situ hybridizations. Because FN may modulate alveolar type II cell phenotype, we investigated type II cell cFN mRNA expression in vivo. During recovery, neither isolated type II cells nor cells with surfactant protein C mRNA in vivo contained FN mRNA. In summary, these data suggest that cFN with the EIIIA domain has a role in alveolar cell recovery from oxygen injury and that type II cells do not express cFN during recovery.

Hyperoxic injury decreases alveolar epithelial cell expression of vascular endothelial growth factor (VEGF) in neonatal rabbit lung.

Normal neonatal lung growth requires a substantial increase in microvascular endothelial cells. Oxygen injury to neonatal lung destroys endothelial cells and alters the normal process of alveolarization, including development of the microvasculature. The mechanisms that regulate lung alveolar capillary growth and development are not known. Vascular endothelial growth factor (VEGF) is a specific mitogen for endothelial cells that is often expressed by epithelial cells in close proximity to capillary beds. VEGF expression is induced by hypoxia and may be inhibited by hyperoxia. We examined the cell-specific expression of VEGF during normal postnatal lung development and the effects of hyperoxic lung injury on VEGF mRNA and protein in vivo. Normal newborn rabbits between 1 day and 5 wk of age had VEGF transcripts located mainly in alveolar epithelial cells, with little or no VEGF mRNA noted in smooth muscle or endothelial cells. A subpopulation of freshly isolated, normal type II cells, but not mesenchymal cells, expressed VEGF mRNA. Newborn rabbits exposed to 100% oxygen for 4 days had no change in VEGF mRNA abundance, transcript location, or immunostaining. Animals exposed to 100% oxygen for an average of 9 days had an 80% decrease in lung VEGF mRNA abundance, decreased alveolar epithelial cell VEGF expression, and decreased VEGF immunostaining. Recovery of VEGF expression to control levels occurred during a 5-day recovery period. We conclude that alveolar epithelial cells in postnatal lung express VEGF, suggesting epithelial regulation of alveolar capillary formation. Furthermore, hyperoxic injury decreases neonatal lung VEGF mRNA and protein, which may be a contributory mechanism of impaired postnatal microvascular development in oxygen injury.

Changes in decorin expression with hyperoxic injury to developing rat lung.

Proteoglycans are extracellular matrix components that appear to play important roles in lung development and in the response to injury. Decorin, a small extracellular matrix-associated proteoglycan, is known to be involved in collagen fibrillogenesis and is a likely participant in the pathogenesis of lung injury. We hypothesized that chronic exposure of the developing lung to hyperoxia would result in temporal and spatial changes in decorin expression. To determine the expression of decorin in normal and oxygen-injured lung, newborn rats were exposed to hyperoxia for 6 wk. Decorin mRNA abundance was determined using Northern hybridization analyses, and decorin expression was localized by in situ hybridization and immunohistochemistry. Decorin mRNA expression in type II pneumocytes was studied using reverse transcription-polymerase chain reaction. Oxygen exposure is associated with a 77% reduction in decorin mRNA in whole lung and a decrease in decorin immunoreactivity in connective tissues surrounding large airways and blood vessels, but an increase in decorin mRNA and protein expression at the tips of alveolar septa. Studies using isolated cells indicate that macrophages and polymorphonuclear neutrophils contain decorin core protein but not decorin mRNA. Type II pneumocytes do not contain either decorin mRNA or core protein. These findings demonstrate that hyperoxic lung injury is associated with localized changes in decorin expression, changes that are not reflected in whole lung RNA studies. It is likely that regional changes in lung decorin expression are influenced by factors produced and acting locally, and that such changes may contribute to the morphologic alterations characteristic of oxygen-induced lung injury.

Expression of fibronectin mRNA splice variants by rabbit lung in vivo and by alveolar type II cells in vitro.

Fibronectin (FN) is a multidomain glycoprotein with putative functions in tissue development and repair. In repair of alveolar injury, FN may promote the transition of type II epithelial cells to type I epithelial cells. Alternative splicing of FN mRNA, including the EIIIA and EIIIB exons, results in protein isoforms that have cell, tissue, and developmental specificity. The present work found that FN mRNA with the EIIIA exon was in fetal, adult, and oxidant-injured lung. The EIIIB splice variant, however, was restricted to fetal lung and adult lung recovering from oxidant injury. Because alveolar type II cells in vitro express FN, we examined the splice variants in two conditions that induce FN [transforming growth factor-beta 1 (TGF-beta 1) treatment and time in culture]. TGF-beta 1 increased both EIIIA and EIIIB mRNA abundance by 10-fold. Increased EIIIA isoform immunostaining was also noted. Type II cells that spontaneously express FN at 72 h in vitro had increased EIIIA and EIIIB mRNA and increased immunostaining for EIIIA. Nuclear runoff showed induction of FN gene transcription at 72 h in vitro. Together, these data show differential FN splice variant expression in lung, with EIIIB mRNA restricted to fetal and recovering oxidant-injured lung. Furthermore, the transition of type II cells to a type I-like cell is accompanied by increased FN gene transcription and induction of both EIIIA and EIIIB mRNA.

Vascular endothelial growth factor mRNA increases in alveolar epithelial cells during recovery from oxygen injury.

Destruction of pulmonary endothelial cells is characteristic of hyperoxic lung injury. During recovery from hyperoxia, pulmonary endothelial cells proliferate to regenerate the vascular endothelium. Vascular endothelial growth factor (VEGF) is a peptide growth factor that is mitogenic specifically for endothelial cells. We hypothesized that VEGF messenger RNA (mRNA) increases during recovery from acute hyperoxic lung injury. Adult rabbits were exposed to 100% oxygen for 64 h and allowed to recover in air for 0, 1, 3, and 5 days. In situ hybridization showed increased VEGF expression in alveolar epithelial cells beginning at 1 day recovery. By 3 days recovery the message was in alveolar epithelial cells throughout the lung. Compared with alveolar epithelial cells, little or no expression was noted in large vessel endothelial cells, airway cells, or smooth muscle cells. Combined in situ hybridization for VEGF and immunostaining for macrophages and other mesenchymal cells found no VEGF message in those cell types. Isolated alveolar macrophages had no detectable VEGF message. Cells expressing VEGF mRNA were enriched in alveolar type II cell preparations from recovering lung. Double in situ hybridization for VEGF and surfactant protein-C (SP-C) showed co-expression in a population of type II cells, but with an inverse relationship: cells with abundant VEGF mRNA did not have abundant SP-C mRNA. Type II cells in vitro expressed VEGF message, but only when the SP-C message abundance was relatively low. We conclude that alveolar type II cells express increased VEGF mRNA during recovery from acute hyperoxia. These findings are consistent with a role for VEGF in regulating microvascular endothelial repair after oxidant injury.

Immunologic response of extremely premature infants to tetanus, Haemophilus influenzae, and polio immunizations.

OBJECTIVE: To determine whether extremely premature infants have immunologic responses to tetanus toxoid, Haemophilus influenzae type b polysaccharide and polio vaccines similar to those of full-term infants. INFANTS AND METHODS: Sixteen extremely premature (< 29 weeks, < 1000 g at birth) infants received separate diphtheria-tetanus-pertussis and H influenzae type b oligosaccharide-CRM197-conjugated (HbOC) vaccines at 2, 4 and 6 months of chronologic age, enhanced potency inactivated polio vaccine at 2 months, and oral polio vaccine at 4 months. Serum was obtained for anti-tetanus toxoid (TT), anti-Haemophilus b polysaccharide (HbPs) and polio neutralizing antibody assays before the 2-month vaccination and 4 to 6 weeks after the 6-month vaccination. Comparison sera were obtained from full-term infants immunized with the same lots of diphtheria-tetanus-pertussis (n = 46) and HbOC (n = 66) vaccines or the same sequence of polio vaccines (n = 10). RESULTS: Preterm and full-term infants had similar geometric mean titers of anti-TT antibodies, anti-HbPs antibodies, and neutralizing antibodies to polio serotypes 1, 2, and 3 after the completion of the primary series of vaccines. After vaccination, similar proportions of preterm and full-term infants had protective levels of antibody to TT (preterm 100% vs full-term 100% with levels > 0.01 IU/mL), HbPS (82% vs 87%, > 1.0 microgram/mL), and polio serotypes 1 (85% vs 80%, > or = 1:8) and 2 (100% vs 100%, > or = 1:8). Preterm infants were less likely than full-term infants to have protective levels of neutralizing antibody to polio serotype 3 (31% vs 90%, > or = 1:8). CONCLUSIONS: Extremely premature infants have adequate antibody responses to tetanus and HbOC antigens but may have diminished responsiveness to serotype 3 polio vaccine.

The use of capnography for recognition of esophageal intubation in the neonatal intensive care unit.

Failure to recognize esophageal intubation can result in severe hypoxia and permanent neurologic injury. Capnography is a standard monitoring modality in the operating room but has not been utilized fully in other environments. We used capnography at the time of endotracheal intubation in the neonatal intensive care unit (NICU) to determine whether capnography could more quickly and accurately identify endotracheal tube position than other clinical indicators of endotracheal tube position. One hundred intubation episodes were studied in 55 neonates. Capnograms were obtained 15 and 120 sec following tube placement. Intubating personnel were blinded to the capnographic data and determined endotracheal tube location (trachea vs. esophagus) by clinical criteria only. The sensitivity and specificity of capnography and clinical examination for identification of tube position were analyzed, and the time required for establishing by clinical confirmation whether the tube was in the trachea or not was compared to that required for capnography. Forty of 100 intubation attempts resulted in esophageal intubation. Capnography correctly identified these errant tube placements in 39 of 40 instances and did so in 1.6 sec (SD +/- 2.4). Capnography failed to identify successful endotracheal intubation on only one occasion. Clinical indicators of tube position required 97.1 sec (SD +/- 92.6) to identify an esophageal intubation and failed to identify successful endotracheal intubation in 5 of 60 cases. We conclude that capnography is a valuable adjunct to clinical examination to demonstrate whether an endotracheal tube is placed correctly in the trachea of neonates in the NICU.

Transforming growth factor-beta 1 modulates type II cell fibronectin and surfactant protein C expression.

Extracellular matrix (ECM) deposition by alveolar type II cells is important for repair of a damaged alveolar epithelium. Transforming growth factor-beta (TGF-beta) is abundant in injured lung and has profound effects on ECM production and cell differentiation. We determined the effects of TGF-beta 1 on type II cell expression of fibronectin and surfactant protein C (SP-C) in vitro. TGF-beta 1 increased the proportion of type II cells with detectable mRNA for fibronectin from 9 to 68%, as demonstrated by in situ hybridization, and increased the fibronectin mRNA levels 10-fold. TGF-beta 1-treated cultures had increased immunostaining for fibronectin and increased secretion of metabolically labeled fibronectin. A decreased proportion of type II cells treated with TGF-beta 1 had detectable mRNA for SP-C, and the abundance of this message per cell decreased to 25% of control values. No effects of TGF-beta 1 were noted on the proportion of cells that contained lamellar bodies, which was 87% in both groups. These data indicate that TGF-beta 1 regulates type II cell fibronectin protein an mRNA levels. In addition, the decreased abundance of SP-C mRNA suggests that TGF-beta 1 may also modulate type II cell differentiation in lung injury.