William M. Maniscalco

Development of glycogen and phospholipid metabolism in fetal and newborn rat lung

Glucose, a major metabolic substrate for the mammalian fetus, probably makes significant contributions to surface active phospholipid synthesis in adult lung. We examined the developmental patterns of glycogen content, glycogen synthase activity, glycogen phosphorylase activity and glucose oxidation in fetal and newborn rat lung. These patterns were correlated with the development of phosphatidylcholine synthesis, content and the activities of enzymes involved in phosphatidylcholine synthesis. Fetal lung glycogen concentration increased until day 20 of gestation (term is 22 days) after which it declined to low levels. Activity of both glycogen synthase I and total glycogen synthase (I + D) in fetal lung increased late in gestation. Increased lung glycogen concentration preceded changes in enzyme activity. Glycogen phosphorylase a and total glycogen phosphorylase (a + b) activity in fetal lung increased during the period of prenatal glycogen depletion. The activity of the pentose phosphate pathway, as measured by the ratio of CO2 derived from oxidation of C1 and C6 of glucose, declined after birth. Fetal lung total phospholipid, phosphatidycholine and disaturated phosphatidylcholine content increased by 60, 90 and 180%, respectively, between day 19 of gestation and the first postnatal day. Incorporation of choline into phosphatidylcholine and disaturated phosphatidylcholine increased 10-fold during this time. No changes in phosphatidylcholine enzyme activities were noted during gestation, but both choline phosphate cytidylyltransferase and phosphatidate phosphatase activity increased after birth. The possible contributions of carbohydrate derived from fetal lung glycogen to phospholipid synthesis are discussed.

Effects of betamethasone on phospholipid content, composition and biosynthesis in the fetal rabbit lung.

Administration of betamethasone (0.2 mg/kg, intramuscularly) to pregnant rabbits had the following effects on the fetal lung at 26--27 days gestation. It increased the amount of phosphatidylcholine in lung lavage by 70% and almost doubled the phosphatidylcholine/sphingomyelin ratio, it increased the rate of incorporation of choline into phosphatidylcholine in fetal lung slices by up to 90%, it increased the activities of pulmonary cholinephosphate cytidylyltransferase and phosphatidate phosphatase by 50% and it reduced the amount of lung glycogen to 60% of the amount in the controls. Betamethasone had no effect on the activities of pulmonary cholinephosphotransferase or lysolecithin: lysolecithin acyltransferase but it slightly decreased the activity of choline kinase. Betamethasone administration to the doe did not increase the amount of surfactant phospholipid in fetal lung lavage to as great an extent as did direct administration of cortisol to the fetuses. Neither did betamethasone stimulate the activity of pulmonary cholinephosphotransferase. These data suggest that agents other than glucocorticoids mediate the stress-induced acceleration of fetal lung maturation and surfactant production.

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·kg−1·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.

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.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.

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-XL. 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-XL 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-XL, 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-XL 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-XL, 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.

De novo fatty acid synthesis in developing rat lung

The rate of de novo fatty acid synthesis in developing rat lung was measured by the rate of incorporation of 3H from 3H2O into fatty acids in lung slices and by the activity of acetyl-CoA carboxylase in fetal, neonatal and adult lung. Both tritium incorporation and acetyl-CoA carboxylase activity increased sharply during late gestation, peaked on the last fetal day, and declined by 50% 1 day after birth. In the adult, values were only one-half the peak fetal rates. In vitro regulation of acetyl-CoA carboxylase activity in fetal lung was similar to that described in adult non-pulmonary tissues: activation by citrate and inhibition by palmitoyl-CoA. Similarly, incubation conditions that favored enzyme phosphorylation inhibited acetyl-CoA carboxylase activity in lung while dephosphorylating conditions stimulated activity. Incorporation of [U-14 C]glucose into lung lipids during development was influenced heavily by incorporation into fatty acids, which generally paralleled the rate of tritium incorporation into fatty acids. The relative utilization of acetyl units from exogenous glucose for overall fatty acid synthesis was greater in adult lung than in fetal or neonatal lung, suggesting that other substrates may be important for fatty acid synthesis in developing lung. In fetal lung explants, de novo fatty acid synthesis was inhibited by exogenous palmitate. Taken together, these data suggest that de novo synthesis may be an important source of saturated fatty acids in fetal lung but of lesser importance in the neonatal period. Furthermore, the regulation of acetyl-CoA carboxylase activity and fatty acid synthesis in lung may be similar to non-pulmonary tissues.

Properties of freshly isolated type II alveolar epithelial cells

The biochemical characteristics of type II alveolar epithelial cells dissociated from adult rabbit lung by instillation of low concentrations of an elastase trypsin mixture are reported. Cells studied immediately (within 4 h) after isolation were found to incorporate the radioactively labelled precursors [U-14C]glucose, [methyl-3H]choline and [3H]palmitate into cellular phosphatidylcholine at rates 2-10-fold higher than previously reported for cells not subject to short-term cell culture. Secretion of phosphatidylcholine was stimulated by beta-adrenergic agonists. Measurement of specific activities of enzymes of phospholipid biosynthesis in subcellular fractions of isolated lung cells showed a significant enrichment of acyl coenzyme A-lysophosphatidylcholine acyltransferase, an enzyme believed to be involved in pulmonary surfactant phosphatidylcholine remodeling, in the endoplasmic reticulum of type II cells. These observations support the utility of freshly isolated type II cells as a model system for the study of the functions of the alveolar epithelium.

Hyperoxic Ventilated Premature Baboons Have Increased p53, Oxidant DNA Damage and Decreased VEGF Expression

Hyperoxia is implicated in the pathogenesis of bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants. High levels of supplemental oxygen can result in microvascular endothelial cell death and may disrupt lung development. In postnatal animals, hyperoxia inhibits expression of vascular endothelial growth factor (VEGF), which is required for normal vascular development. A potential mechanism of oxygen effects on VEGF is induction of p53, a transcription factor that represses VEGF gene transcription. Oxidant DNA damage can increase p53. We used a moderately premature baboon model of hyperoxia to examine p53, oxidant DNA damage, and VEGF expression. Fetal baboons delivered at 140 d of gestation (75% of term) were ventilated with 100% oxygen or oxygen as needed for 6 or 10 d. Lungs from the 10-d 100% oxygen animals had increased nuclear p53, compared with the oxygen as needed animals. The mechanism of increased p53 was probably related to oxidant DNA damage, which was documented by increased oxidized guanine. Dual fluorescent confocal microscopy found increased oxidized guanine in mitochondrial DNA of distal lung epithelial cells. Distal epithelial cell VEGF expression was decreased and p21, another downstream target of p53, was increased in the distal epithelium of the hyperoxic animals. These data show that p53 is induced in hyperoxic fetal lung epithelium and are consistent with p53 repression of VEGF expression in these cells. The findings suggest that oxidant DNA damage may be a mechanism of increased p53 in hyperoxic fetal lung.

Decreased cholestasis with enteral instead of intravenous protein in the very low-birth-weight infant

Thirty to 50% of very low-birth-weight infants have parenteral nutrition-associated cholestasis. To test the hypothesis that the incidence of cholestasis would be decreased if parenteral amino acids were avoided and protein given enterally, infants with a gestational age of less than 30 weeks were randomized to two groups. One group received amino acid-free parenteral nutrition and whey protein enterally with added premature infant formula. The control group received standard parenteral nutrition with amino acids and enteral premature formula. At the end of 3 weeks of parenteral nutrition, infants who had a direct serum bilirubin level of greater than 3 mg/dl were considered to have significant cholestasis. Twenty-nine infants required parenteral nutrition for 3 weeks, 17 in the whey group and 12 in the control group. No instances of significant cholestasis were observed in the whey group (0/17), whereas seven of 12 infants (58%) in the amino acid control group had cholestasis (p less than 0.001).

De novo fatty acid synthesis by freshly isolated alveolar type II epithelial cells

Fatty acid synthesis was studied in freshly isolated type II pneumocytes from rabbits by 3H2O and (U-14C)-labeled glucose, lactate and pyruvate incorporation and the activity of acetyl-CoA carboxylase. The rate of lactate incorporation into fatty acids was 3-fold greater than glucose incorporation; lactate incorporation into the glycerol portion of lipids was very low but glucose incorporation into this fraction was approximately equal to incorporation into fatty acids. The highest rate of de novo fatty acid synthesis (3H2O incorporation) required both glucose and lactate. Under these circumstances lactate provided 81.5% of the acetyl units while glucose provided 5.6%. Incubations with glucose plus pyruvate had a significantly lower rate of fatty acid synthesis than glucose plus lactate. The availability of exogenous palmitate decreased de novo fatty acid synthesis by 80% in the isolated cells. In a cell-free supernatant, acetyl-CoA carboxylase activity was almost completely inhibited by palmitoyl-CoA; citrate blunted this inhibition. These data indicate that the type II pneumocyte is capable of a high rate of de novo fatty acid synthesis and that lactate is a preferred source of acetyl units. The type II pneumocyte can rapidly decrease the rate of fatty acid synthesis, probably by allosteric inhibition of acetyl-CoA carboxylase, if exogenous fatty acids are available.