Lack of EC-SOD worsens alveolar and vascular development in a neonatal mouse model of bleomycin-induced bronchopulmonary dysplasia and pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) worsens clinical outcomes in former preterm infants with bronchopulmonary dysplasia (BPD). Oxidant stress disrupts alveolar and vascular development in models of BPD. Bleomycin causes oxidative stress and induces BPD and PAH in neonatal rats. Disruption in the vascular endothelial growth factor (VEGF) and nitric oxide signaling pathways contributes to BPD. We hypothesized that loss of EC-SOD would worsen PAH associated with BPD in a neonatal mouse model of bleomycin-induced BPD by disrupting the VEGF/NO signaling pathway. METHODS: Neonatal wild-type mice (WT), and mice lacking EC-SOD (EC-SOD KO) received intraperitoneal bleomycin (2 units/kg) or phosphate-buffered saline (PBS) three times weekly and were evaluated at weeks 3 or 4. RESULTS: Lack of EC-SOD impaired alveolar development and resulted in PH (elevated right ventricular systolic pressures, right ventricular hypertrophy (RVH)), decreased vessel density, and increased small vessel muscularization. Exposure to bleomycin further impaired alveolar development, worsened RVH and vascular remodeling. Lack of EC-SOD and bleomycin treatment decreased lung total and phosphorylated VEGFR2 and eNOS protein expression. CONCLUSION: EC-SOD is critical in preserving normal lung development and loss of EC-SOD results in disrupted alveolar development, PAH and vascular remodeling at baseline, which is further worsened with bleomycin and associated with decreased activation of VEGFR2.

The NF-κB inhibitory proteins IκBα and IκBß mediate disparate responses to inflammation in fetal pulmonary endothelial cells.

Exposure to intrauterine inflammation impairs lung growth but paradoxically protects the neonatal pulmonary vasculature from hyperoxic injury. The mechanisms mediating these contradictory effects are unknown. The objective is to identify the role of NF-κB in mediating cytoprotective and proinflammatory responses to inflammation in the fetal pulmonary endothelium. In newborn rats exposed to intra-amniotic LPS, we found increased expression of the NF-κB target gene manganese superoxide dismutase (MnSOD) in the pulmonary endothelium. Supporting these in vivo findings, LPS induced NF-κB activation and MnSOD expression in isolated fetal pulmonary arterial endothelial cells. In addition, LPS exposure caused apoptosis and suppressed cellular growth and induced P-selectin expression. LPS-induced NF-κB activation that proceeded through specific isoforms of the inhibitory protein IκB mediated these diverse responses; NF-κB signaling through IκBα degradation resulted in MnSOD upregulation and preserved cell growth, whereas NF-κB signaling through IκBß degradation mediated apoptosis and P-selectin expression. These findings suggest that selective inhibition of NF-κB activation that results from IκBß degradation preserves the enhanced antioxidant defense and protects the developing pulmonary vascular endothelium from ongoing inflammatory injury.

Excess soluble vascular endothelial growth factor receptor-1 in amniotic fluid impairs lung growth in rats: linking preeclampsia with bronchopulmonary dysplasia.

Epidemiological studies have shown that maternal preeclampsia (PE) increases the risk of bronchopulmonary dysplasia (BPD), but the underlying mechanism is unknown. Soluble vascular endothelial growth factor receptor-1 (soluble VEGFR1, known as soluble fms-like tyrosine kinase 1, or sFlt-1), an endogenous antagonist of vascular endothelial growth factor (VEGF), is markedly elevated in amniotic fluid and maternal blood in PE. Therefore, we hypothesized that antenatal exposure to excess sFlt-1 disrupts lung development through impaired VEGF signaling in utero, providing a mechanistic link between PE and BPD. To determine whether increased sFlt-1 in amniotic fluid is sufficient to cause sustained abnormalities of lung structure during infancy, sFlt-1 or saline was injected into amniotic sacs of pregnant Sprague-Dawley rats at 20 days of gestation (term, 22 days). After birth, pups were observed through 14 days of age for study. We found that intra-amniotic sFlt-1 treatment decreased alveolar number, reduced pulmonary vessel density, and caused right and left ventricular hypertrophy in 14-day-old rats. In addition, intra-amniotic sFlt-1 treatment suppressed activation of lung VEGF receptor-2 and increased apoptosis in endothelial and mesenchymal cells in the newborn lung. We conclude that exposure to excess sFlt-1 in amniotic fluid during late gestation causes sustained reductions in alveolarization and pulmonary vascular growth during infancy, accompanied by biventricular hypertrophy suggesting pulmonary and systemic hypertension. We speculate that impaired VEGF signaling in utero due to exposure of high amniotic fluid levels of sFlt-1 in PE disrupts lung growth and contributes to the increased risk of BPD in infants born to mothers with PE.

Intrauterine growth restriction decreases pulmonary alveolar and vessel growth and causes pulmonary artery endothelial cell dysfunction in vitro in fetal sheep.

Intrauterine growth restriction (IUGR) increases the risk for bronchopulmonary dysplasia (BPD). Abnormal lung structure has been noted in animal models of IUGR, but whether IUGR adversely impacts fetal pulmonary vascular development and pulmonary artery endothelial cell (PAEC) function is unknown. We hypothesized that IUGR would decrease fetal pulmonary alveolarization, vascular growth, and in vitro PAEC function. Studies were performed in an established model of severe placental insufficiency and IUGR induced by exposing pregnant sheep to elevated temperatures. Alveolarization, quantified by radial alveolar counts, was decreased 20% (P < 0.005) in IUGR fetuses. Pulmonary vessel density was decreased 44% (P < 0.01) in IUGR fetuses. In vitro, insulin increased control PAEC migration, tube formation, and nitric oxide (NO) production. This response was absent in IUGR PAECs. VEGFA stimulated tube formation, and NO production also was absent. In control PAECs, insulin increased cell growth by 68% (P < 0.0001). Cell growth was reduced in IUGR PAECs by 29% at baseline (P < 0.01), and the response to insulin was attenuated (P < 0.005). Despite increased basal and insulin-stimulated Akt phosphorylation in IUGR PAECs, endothelial NO synthase (eNOS) protein expression as well as basal and insulin-stimulated eNOS phosphorylation were decreased in IUGR PAECs. Both VEGFA and VEGFR2 also were decreased in IUGR PAECs. We conclude that fetuses with IUGR are characterized by decreased alveolar and vascular growth and PAEC dysfunction in vitro. This may contribute to the increased risk for adverse respiratory outcomes and BPD in infants with IUGR.

Moderate postnatal hyperoxia accelerates lung growth and attenuates pulmonary hypertension in infant rats after exposure to intra-amniotic endotoxin.

To determine the separate and interactive effects of fetal inflammation and neonatal hyperoxia on the developing lung, we hypothesized that: 1) antenatal endotoxin (ETX) causes sustained abnormalities of infant lung structure; and 2) postnatal hyperoxia augments the adverse effects of antenatal ETX on infant lung growth. Escherichia coli ETX or saline (SA) was injected into amniotic sacs in pregnant Sprague-Dawley rats at 20 days of gestation. Pups were delivered 2 days later and raised in room air (RA) or moderate hyperoxia (O2, 80% O2 at Denver's altitude, ∼65% O2 at sea level) from birth through 14 days of age. Heart and lung tissues were harvested for measurements. Intra-amniotic ETX caused right ventricular hypertrophy (RVH) and decreased lung vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) protein contents at birth. In ETX-exposed rats (ETX-RA), alveolarization and vessel density were decreased, pulmonary vascular wall thickness percentage was increased, and RVH was persistent throughout the study period compared with controls (SA-RA). After antenatal ETX, moderate hyperoxia increased lung VEGF and VEGFR-2 protein contents in ETX-O2 rats and improved their alveolar and vascular structure and RVH compared with ETX-RA rats. In contrast, severe hyperoxia (≥95% O2 at Denver's altitude) further reduced lung vessel density after intra-amniotic ETX exposure. We conclude that intra-amniotic ETX induces fetal pulmonary hypertension and causes persistent abnormalities of lung structure with sustained pulmonary hypertension in infant rats. Moreover, moderate postnatal hyperoxia after antenatal ETX restores lung growth and prevents pulmonary hypertension during infancy.

Rehospitalization of extremely-low-birth-weight infants in first 2 years of life.

AIMS: To determine whether (1) chronic lung disease (CLD) is the prime reason for extremely-low-birth-weight (ELBW) infant readmission during the first 2 years of life, (2) surfactant and other advanced therapies have reduced ELBW infant readmissions, (3) home oxygen therapy (HOT) is efficacious for this group. STUDY DESIGN: The hospital records of these ELBW infants were reviewed retrospectively. Data on age, diagnosis, treatment, and duration of each hospitalization were compiled and analyzed for their association to CLD and to readmission for CLD and other reasons. SUBJECTS: All 60 surviving infants with a birth body weight of less than 1001 g (ELBW) born from January 1993 to February 1998 were followed up to 2 years (mean 20.4 +/- 7.4 months) to evaluate their respiratory outcome. RESULTS: Forty-two percent of these infants developed CLD. Upon discharge from the hospital, 28% (7/25) of the patients were given HOT for a median period of 60 days. Of the 47 ELBW infants who were studied the entire 2-year period, 72% were readmitted. Infants with CLD were readmitted more frequently (p=0.045) and had longer hospital stays during the first 2 years of life (p=0.034) than those without CLD. Respiratory illness was the main reason for readmission (55%) of these ELBW infants. The incidence of readmission due to respiratory tract infection was not significantly different in infants with CLD (61%) and infants without respiratory complications (44%) (p=0.159). CONCLUSIONS: Infants with CLD (whether receiving HOT or not) showed no higher readmission rate due to respiratory infection, but the HOT group did have higher morbidity. The premature lung itself rather than the presence of CLD, as we would expect, makes ELBW infants more prone to readmission for respiratory illness.

Neonatal outcome of infants born after in vitro fertilization at National Taiwan University Hospital.

BACKGROUND AND PURPOSE: This study compared the neonatal outcome between infants born after in vitro fertilization (IVF) and after natural conception at National Taiwan University Hospital. METHODS: All medical records of women who underwent IVF and gave birth at our hospital from January 1995 to December 1996 were reviewed. The charts of their offspring were also reviewed. We compared the neonatal outcome of infants born after IVF with that of infants born after natural conception. Neonatal outcome was evaluated based on preterm birth, very low birth weight (VLBW), perinatal morbidity, and neonatal mortality. RESULTS: A total of 75 women underwent IVF and gave birth to a total of 100 live newborns and two fetuses with intrauterine death during the 2-year study period. Among these newborns, the prevalence of preterm birth was 28%, of perinatal morbidity was 17%, and of neonatal mortality was 3%, which were significantly higher than those among the 7,736 neonates born after natural conception. However, the rate of VLBW was similar between the two groups. The rate of preterm birth for twin pregnancies were higher than that for singleton pregnancies in both groups. CONCLUSION: This study showed that infants born after IVF had a higher risk of preterm birth and higher perinatal morbidity and neonatal mortality.

A proposal of screening guideline for retinopathy of prematurity in Taiwan.

Retinopathy of prematurity (ROP) is one of the most important morbidity in premature infants. The latest American Academy of Pediatrics (AAP) screening guidelines for ROP are mandatory for infants with birth weights < or = 1500 g or gestational ages (GA) < or = 28 weeks. To determine the appropriate upper limits for ROP screening in Taiwan in terms of birth weight and gestational age, we performed a retrospective review of all 617 infants who were born at the National Taiwan University Hospital from January 1, 1994 to December 31, 1999, and survived beyond 28 days of life and received eye-ground evaluation under our current ROP screening guidelines. From the start of our study, the criteria for screening was birth weight <2000 g or gestational age < 35 weeks. The maximal stage of retinopathy observed was recorded. We found no ROP greater than stage I in infants with birth weights > 1500g and GA > 31 weeks. In comparing with our current guidelines, lowering the screening criteria to birth weight < or = 1500g or gestational age < or = 31 weeks reduced the number of infants requiring screening by 37.2%, while no case of ROP greater than stage 1 was missed. However, five cases of stage 2 ROP would have been missed in our study if the gestational age cut-off was < or = 28 weeks as recommended by AAP. Therefore, we recommend that in the tertiary nursery in Taiwan, the gestational age < or = 31 weeks or birth body weight < or = 1500 g should be screened for ROP regardless of clinical condition. The screening for larger infants with high risk of ROP requires further discretion of the attending physicians.

Ovarian cyst with torsion presenting as a wandering mass in a newborn.

A case of ovarian serous cystadenoma with torsion presenting as a wandering abdominal mass is reported. A full-term baby girl was noted to have a left pelvic cyst by prenatal ultrasound at the 34th gestational week. After delivery, a series of work-ups, including ultrasonography and CT scan, revealed a cyst with fluid-debris level in the right pelvis. An ovarian cyst with torsion was suspected and surgical intervention was done when she was 7 days old. The operative findings confirmed a left ovarian cystic mass with torsion. A left salpingo-oophorectomy was performed smoothly. Pathology revealed the typical microscopic findings of ovarian serous cystadenoma, a benign epithelial tumor of ovary. We suggest that a wandering ovarian cystic mass in female fetus and newborn should be considered an indication for surgical intervention due to its high risk of torsion.

Serum gamma-glutamyl transpeptidase activity and bile acids in normal Taiwanese infants.

To facilitate making a diagnosis of cholestatic liver disease in Taiwan, we have established reference ranges for serum gamma-glutamyl transpeptidase (gamma-GT) activity and bile acids in normal Taiwanese infants. The serum level of gamma-GT activity was assayed in 90 normal Taiwanese infants and children aged between 2 days and 2 years old. These data were analyzed in twenty-three 0-3-month-old, twenty-four 4-6-month-old, and forty-three 7-24-month-old infants. The mean values of serum gamma-GT activity were 47.4 +/- 26.6, 21.5 +/- 7.3, and 14.0 +/- 3.2 IU/L for the respective age groups. The highest reference gamma-GT values were 99.5, 35.8, and 20.3 IU/L for the respective age groups. The mean values of serum gamma-GT activity were highest in infants younger than 3 months, and these gradually decreased to the adult level with age (p < 0.01). Serum bile acids were measured in 24 premature and 56 full-term infants. The mean values of serum bile acids were 34.5 +/- 34.5 mumol/L in preterm infants and 18.7 +/- 21.9 mumol/L in full-term babies. The bile acid levels of preterm infants were higher than those in full-term babies (p < 0.01).

Necrotizing enterocolitis complicated with perforation in extremely low birth-weight premature infants.

This study determined the incidence, clinical characteristics, treatment and outcome in extremely low birth-weight (ELBW) premature infants with perforated necrotizing enterocolitis (NEC). We retrospectively reviewed the medical records of ELBW (birth weight <1000 g ) premature infants with perforated NEC diagnosed and managed at National Taiwan University Hospital (NTUH) from January 1993 through December 2000. A total of 8 ELBW premature infants with perforated NEC were collected. The incidence of perforated NEC in ELBW premature infants was 5.1% (8 out of 158). The average age at onset of perforated NEC was 26 days. The most common clinical features were abdominal distention, decreased bowel sound and poor activity level. Dilated and fixed bowel loops, bowel wall thickening and ascites with stool-like substance drainage out from penrose drain tube were the predominant signs at the time of diagnosis of perforated NEC. Thrombocytopenia, elevated C-reactive protein and anemia were the major laboratory findings. All infants received a primary penrose drain in the acute stage of disease. The overall survival rate was 37.5% (3 out of 8). Death occurred due to nosocomial infection with sepsis in 3 patients and due to perforated NEC in 2 patients. Two of the three surviving patients started enteral feeding 19 and 41 days after the diagnosis of perforated NEC and tolerated oral feedings well; the third patient still required total parenteral nutrition two years after diagnosis. Although the clinical characteristics and radiographic findings of perforated NEC in ELBW premature infants were variable, brown color ascites with stool-like substance may be considered a significant sign of perforated NEC despite the absence of free air on radiography at the early stage of disease. Close observation of clinical symptoms and signs, more aggressive surgical intervention and prevention of the following nosocomial infection may have the opportunity to reduce the mortality due to perforated NEC.

Early inhaled nitric oxide treatment decreases apoptosis of endothelial cells in neonatal rat lungs after vascular endothelial growth factor inhibition.

Vascular endothelial growth factor (VEGF) receptor blockade impairs lung growth and decreases nitric oxide (NO) production in neonatal rat lungs. Inhaled NO (iNO) treatment after VEGF inhibition preserves lung growth in infant rats by unknown mechanisms. We hypothesized that neonatal VEGF inhibition disrupts lung growth by causing apoptosis in endothelial cells, which is attenuated by early iNO treatment. Three-day-old rats received SU-5416, an inhibitor of VEGF receptor, or its vehicle and were raised in room air with or without iNO (10 ppm). SU-5416 reduced alveolar counts and lung vessel density by 28% (P < 0.005) and 21% (P < 0.05), respectively, as early as at 7 days of age. SU-5416 increased lung active caspase-3 protein by 60% at 5 days of age (P < 0.05), which subsided by 7 days of age, suggesting a transient increase in lung apoptosis after VEGF blockade. Apoptosis primarily colocalized to lung vascular endothelial cells, and SU-5416 increased endothelial cell apoptotic index by eightfold at 5 days of age (P <0.0001). iNO treatment after SU-5416 prevented the increases in lung active caspase-3 and in endothelial cell apoptotic index. There was no difference in alveolar type 2 cell number between control and SU-5416-treated rats. We conclude that neonatal VEGF receptor inhibition causes transient apoptosis in pulmonary endothelium, which is followed by persistently impaired lung growth. Early iNO treatment after VEGF inhibition reduces endothelial cell apoptosis in neonatal lungs. We speculate that enhancing endothelial cell survival after lung injury may preserve neonatal lung growth in bronchopulmonary dysplasia.

Inhaled nitric oxide enhances distal lung growth after exposure to hyperoxia in neonatal rats.

Exposure of newborn rats to hyperoxia impairs alveolarization and vessel growth, causing abnormal lung structure that persists during infancy. Recent studies have shown that impaired angiogenesis due to inhibition of vascular endothelial growth factor (VEGF) signaling decreases alveolar and vessel growth in the developing lung, and that nitric oxide (NO) mediates VEGF-dependent angiogenesis. The purpose of this study was to determine whether hyperoxia causes sustained reduction of lung VEGF, VEGF receptor, or endothelial NO synthase (eNOS) expression during recovery, and whether inhaled NO improves lung structure in infant rats after neonatal exposure to hyperoxia. Newborn rat pups were randomized to hyperoxia [fraction of inspired oxygen (Fio(2)), 1.00] or room air exposure for 6 d, and then placed in room air with or without inhaled NO (10 ppm) for 2 wk. Rats were then killed for studies, which included measurements of body weight, lung weight, right ventricular hypertrophy (RVH), morphometric analysis of alveolarization (by mean linear intercept (MLI), radial alveolar counts (RAC), and vascular volume (Vv), and immunostaining and Western blot analysis. In comparison with controls, neonatal hyperoxia reduced body weight, increased MLI, and reduced RAC in infant rats. Lung VEGF, VEGFR-2, and eNOS protein expression were reduced after hyperoxia. Inhaled NO treatment after hyperoxia increased body weight and improved distal lung growth, as demonstrated by increased RAC and Vv and decreased MLI. We conclude that neonatal hyperoxia reduced lung VEGF expression, which persisted during recovery in room air, and that inhaled NO restored distal lung growth in infant rats after neonatal hyperoxia.

Mild hypoxia impairs alveolarization in the endothelial nitric oxide synthase-deficient mouse.

In addition to its vasodilator properties, nitric oxide (NO) promotes angiogenesis in the systemic circulation and tumors. However, the role of NO in promoting normal lung vascular growth and its impact on alveolarization during development or in response to perinatal stress is unknown. We hypothesized that NO modulates lung vascular and alveolar growth and that decreased NO production impairs distal lung growth in response to mild hypoxia. Litters of 1-day-old mouse pups from parents that were heterozygous for endothelial nitric oxide synthase (eNOS) deficiency were placed in a hypobaric chamber at a simulated altitude of 12,300 ft (Fi(O(2)) = 0.16). After 10 days, the mice were killed, and lungs were fixed for morphometric and molecular analysis. Compared with wild-type controls, mean linear intercept (MLI), which is inversely proportional to alveolar surface area, was increased in the eNOS-deficient (eNOS -/-) mice [51 +/- 2 micro m (eNOS -/-) vs. 41 +/- 1 micro m (wild type); P < 0.01]. MLI was also increased in the eNOS heterozygote (+/-) mice (44 +/- 1 micro m; P < 0.03 vs. wild type). Vascular volume density was decreased in the eNOS -/- mice compared with wild-type controls (P < 0.03). Lung vascular endothelial growth factor (VEGF) protein and VEGF receptor-1 (VEGFR-1) protein content were not different between the study groups. In contrast, lung VEGFR-2 protein content was decreased from control values by 63 and 34% in the eNOS -/- and eNOS +/- mice, respectively (P < 0.03). We conclude that exposure to mild hypoxia during a critical period of lung development impairs alveolarization and reduces vessel density in the eNOS-deficient mouse. We speculate that NO preserves normal distal lung growth during hypoxic stress, perhaps through preservation of VEGFR-2 signaling.

Inhaled nitric oxide attenuates pulmonary hypertension and improves lung growth in infant rats after neonatal treatment with a VEGF receptor inhibitor.

VEGF plays a critical role during lung development and is decreased in human infants with bronchopulmonary dysplasia. Inhibition of VEGF receptors in the newborn rat decreases vascular growth and alveolarization and causes pulmonary hypertension (PH). Nitric oxide (NO) is a downstream mediator of VEGF, but whether the effects of impaired VEGF signaling are due to decreased NO production is unknown. Therefore, we sought to determine whether impaired VEGF signaling downregulates endothelial NO synthase (eNOS) expression in the developing lung and whether inhaled NO (iNO) decreases PH and improves lung growth after VEGF inhibition. Newborn rats received a single dose of SU-5416 (a VEGF receptor inhibitor) or vehicle by subcutaneous injection and were killed up to 3 wk of age for assessments of right ventricular hypertrophy (RVH), radial alveolar counts (RAC), lung eNOS protein, and NOx production in isolated perfused lungs (IPL). Neonatal treatment with SU-5416 increased RVH in infant rats and reduced RAC. Compared with controls, SU-5416 reduced lung eNOS protein expression by 89% at 5 days (P < 0.01). IPL studies from day 14 rats demonstrated increased baseline pulmonary artery pressure and lower perfusate NOx concentration after SU-5416 treatment. Importantly, iNO treatment prevented the increase in RVH and improved RAC after SU-5416 treatment. We conclude that treatment of neonatal rats with SU-5416 downregulates lung eNOS expression and that iNO therapy decreases PH and improves lung growth after SU-5416 treatment. We speculate that decreased NO production contributes to PH and decreases distal lung growth caused by impaired VEGF signaling.