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Patients with Apert syndrome (AS) display a wide range of congenital malformations including tracheal stenosis, which is a disease characterized by a uniform cartilaginous sleeve in place of a normally ribbed cartilagenous trachea. We have studied the cellular and molecular basis of this phenotype in a mouse model of AS (Fgfr2c/mice), which shows ectopic expression of Fgfr2bin mesenchymal tissues. Here we report that tracheal stenosis is associated with increased proliferation of mesenchymal cells, where the expression of Fgf10and its upstream regulators Tbx4and Tbx5are abnormally elevated. We show that Fgf10has a critical inductive role in tracheal stenosis, as genetic knockdown of Fgf10in Fgfr2c/mice rescues this phenotype. These novel findings demonstrate a regulatory role for Fgf10in tracheal development and shed more light on the underlying cause of tracheal defects in AS.

The “hard wiring” encoded within the genome that determines the emergence of the laryngotracheal groove and subsequently early lung branching morphogenesis is mediated by finely regulated, interactive growth factor signaling mechanisms that determine the automaticity of branching, interbranch length, stereotypy of branching, left-right asymmetry, and finally gas diffusion surface area. The extracellular matrix is an important regulator as well as a target for growth factor signaling in lung branching morphogenesis and alveolarization. Coordination not only of epithelial but also endothelial branching morphogenesis determines bronchial branching and the eventual alveolar-capillary interface. Improved prospects for lung protection, repair, regeneration, and engineering will depend on more detailed understanding of these processes. Herein, we concisely review the functionally integrated morphogenetic signaling network comprising the critical bone morphogenetic protein, fibroblast growth factor, Sonic hedgehog, transforming growth factor-β, vascular endothelial growth factor, and Wnt signaling pathways that specify and drive early embryonic lung morphogenesis.

Burkholderia cepaciais a prevalent pulmonary pathogen in patients with cystic fibrosis (CF). The lung pathology observed in patients with CF is postulated to be due to an overexpression of chemokines. This study investigated the induction of the neutrophil chemoattractant chemokine IL-8 and the signaling pathways activated by B. cepacia–infected human lung epithelial A549 (HLE) cells. Cells were infected with B. cepacia(genomovar III of the B. cepaciacomplex), and reverse transcriptase–PCR and ELISA for the cytokines were performed. B. cepacia(multiplicity of infection ≥41) induced HLE cells to significantly secrete IL-8 in a more potent manner than the predominant CF pathogen Pseudomonas aeruginosa(multiplicity of infection ≥641). IL-8 secretion by B. cepacia–infected HLE cells was abrogated by the gene transcription inhibitor actinomycin D and the protein translation inhibitor cycloheximide, confirming that B. cepacia–induced IL-8 secretion was mediated through de novoprotein synthesis. Treatment of B. cepaciawith proteinase K failed to down-regulate IL-8 secretion; furthermore, IL-8 secretion by B. cepacia–infected HLE cells was abrogated by ≥80 in the presence of anti-CD14 specific lipopolysaccharide (LPS) receptor antibody, thus suggesting that the IL-8–inducing component of B. cepaciawas LPS and therefore dependent on CD14. The p38 mitogen-activated protein kinase (MAPK) inhibitor and the extracellular signal-regulated kinase MAPK inhibitor significantly abrogated IL-8 secretion by B. cepacia–infected HLE cells (SB203580, ≥80 inhibition; PD98059, ≥30 inhibition). In conclusion, B. cepacia–induced IL-8 secretion in A549 airway epithelial cells is more potent than P. aeruginosa; is mediated through LPS, which is CD14 dependent; and involves activation of the p38 and ERK MAPK pathways.

The profound intestinal epithelial defects in the newborn epidermal growth factor receptor (EGFR) knockout mouse suggests that EGFR signaling plays important roles in embryonic gut development. Herein, we further elucidated the function of EGFR signaling on early embryonic gut development by comparing the effects of 1–10 ng/mL of exogenous epidermal growth factor (EGF) or 10–25 M of the tyrphostin 3,4,5 trihydroxybenzene malononitrile, a specific inhibitor of EGFR tyrosine kinase, on intact E12 Swiss-Webster mouse midgut grown in chemically defined organ culture using Fitton-Jackson BGJb medium for 4 or 6 d. Intestinal development during culture was assayed by morphometry, histology, reverse transcription/competitive PCR for villin and intestinal fatty acid binding protein mRNA, and immunohistochemistry for epithelial proliferative markers. During organ culture, control specimens grew in length, developed smooth muscle, simple columnar epithelial and goblet cell phenotypes, showed early villus formation in the proximal intestine, and increased expression of villin and intestinal fatty acid binding protein mRNA. EGF failed to significantly alter small intestinal lengthening, whereas EGF 10 ng/mL inhibited colonic length growth. Tyrphostin 25 M resulted in regional losses of stromal and smooth muscle cells in the small intestine and absent colonic goblet cells. In controls, cellular proliferation initially occurred throughout the small intestinal epithelium but became increasingly localized to the intervillus crypt regions. This sequestration of epithelial proliferation into crypts was much more apparent in EGF-treated versustyrphostin-treated specimens. EGFR activation, therefore, appears to accelerate the maturation rate of goblet cells and the differential crypt/villus proliferation pattern in early embryonic mouse gut.

Many of the signaling pathways regulating fetal lung mesenchymal cell proliferation are mediated by the Shc intracellular signaling proteins. Shc is expressed as three isoforms: 52 kD and 46 kD proteins (Shc 52 and Shc 46, respectively) translated from the same mRNA, and a 66 kD form (Shc 66) translated from a separate mRNA. Shc 52 is an activator of Ras and mitogen-activated protein kinase, whereas Shc 66 antagonizes Ras activation. The function of Shc 46 is unclear. We hypothesized that the Shc isoforms are differentially regulated during fetal mouse lung morphogenesis. Relative Shc 66 and Shc 46 protein expression are high until parturition (term = 18.5 d), when a dramatic decrease begins; by postconceptual d 20, relative Shc 66 and Shc 46 expression have fallen by 75 and 69%, respectively. A similar pattern of decreasing Shc 66 mRNA expression in the peripartum period was detected by reverse transcription and competitive polymerase chain reaction during the same period. By isoform-specific immunohistochemistry, Shc 66 is widely distributed in the embryonic lung but becomes restricted to the bronchial smooth muscle and overlying epithelia, periarterial smooth muscle, and the interlobar pleura late in gestation. After parturition, Shc 66 is virtually absent from the lung. All three Shc isoforms are phosphorylated by epidermal growth factor stimulation in fetal lung mesenchymal cells, indicating that Shc 66 is functional in these cells. These data indicate that Shc isoforms are differentially regulated during lung development.

Posthepatic insulin availability has been evaluated by steadystate insulin turnover studies with 131I-insulin. Spontaneously delivered term (age 3.3 ± 0.8 days) (mean ± S.E.) and prematurely delivered lambs (betamathasone treated at 132 days) (age, 1.1 ± 0.2 days) were compared with 4− to 5-month-old sheep. After a 7-hr fast, animals received 0.45 saline or 5.7 mg/kg/min glucose (0.06 ml/kg/min) for 6-hr followed by the tracer insulin infusion for 110 min. Plasma glucose, insulin, and immunoprecipitable insulin were measured sequentially during the steady state. Endogenous posthepatic insulin secretion and metabolic clearance rates were derived. Neither endogenous posthepatic insulin secretion rates nor metabolic clearance rates were different among the three groups of animals when either 0.45 saline or 5.7 mg/kg/min exogenous glucose infusions were compared. Secretory response of the pancreatic βcell to continuous glucose infusion seems similar for the term and preterm lamb when compared to adult sheep.

The early fetus responds to cutaneous wounds in a fundamentally different way from the adult; fetal wounds heal without scars. Wound contraction is a vital component of wound healing. The cytokine transforming growth factor(TGF)-β promotes wound contraction and can be activated by the serine protease plasmin. Herein, we explored whether murine skin fibroblast contractile properties, TGF-β, and plasmin formation are developmentally regulated. Our results showed that early fetal mouse embryonic day 15 skin fibroblasts contracted a collagen gel less, secreted less active and total TGF-β, and generated less plasmin than either late fetal (embryonic day 17) or adult skin fibroblasts. Furthermore, there was a slight positive correlation between the formation of plasmin and the level of activation of TGF-β. We conclude that early fetal mouse skin fibroblasts contract a collagen gel and secrete and activate TGF-β to a lesser extent than do late fetal and adult skin fibroblasts. We speculate that the fetal skin fibroblast undergoes a developmental transition that causes wounds in mouse to contract at or after embryonic day 17. Further, this developmental transition is influenced by growth factor-fibroblast interactions and coincides with the emergence of the skin fibroblast's ability to generate plasmin and activate TGF-β.

Forty-three twin lamb fetuses of 121 ± 1 d gestation were catheterized and received i.v. saline (n= 8), 0.75 mgjkg/h cortisol for 60 h (n= 15), 5 μg/kg thyrotropin-releasing hormone (TRH) every 12 h for five doses (n= 9), or cortisol and TRH (n= 11) before delivery at 128 ± 1 d. After delivery, the lambs were randomized for natural sheep surfactant treatment or sham treatment, ventilated for 75 min, and killed. Superoxide dismutase, catalase, and glutathione peroxidase activities were measured in fetal lung tissue. Superoxide dismutase and catalase activities were increased in both the corticosteroid (p< 0.001) and the corticosteroid with TRH (p< 0.01) groups. Glutathione peroxidase activity was higher after prenatal corticosteroid treatment, but statistical significance was not reached (p= 0.06). Although prenatal exposure to corticosteroids increased superoxide dismutase, catalase, and glutathione peroxidase activities, TRH alone or TRH added to corticosteroids provided no additional benefit. Lambs treated with surfactant had higher lung catalase activities than lambs that did not receive surfactant, probably secondary to the presence of catalase activity in the surfactant preparation. Increased pulmonary antioxidant enzyme activity may be an additional feature of the overall beneficial effect of corticosteroids on fetal lung development. (Pediatr Res30: 518–521, 1991)

We studied the interactions of corticosteroids, thyroid hormones, and β-agonist on surfactant phospholipids, pulmonary mechanics, and β-receptor binding in fetal lambs. We infused cortisol (450 μg/h for 48 h), thyrotropin-releasing hormone (TRH) (25 μg/h for 48 h), and ritodrine (1.3 μg/kg/min for 24 h) independently, and in double (cortisol plus β-agonist, cortisol plus TRH), and in triple (cortisol plus TRH plus β-agonist) combinations into chronically catheterized fetal lambs between 0.88 and 0.90 gestation. Infusion of the triple combination of cortisol plus TRH plus β-agonist resulted in a 20.9-fold increase in the saturated phosphatidylcholine content of fetal lung lavage, in a 5.8-fold increase in the saturated phosphatidylcholine content of whole fetal lung, and in a 13.3-fold increase in the saturated phosphatidylcholine content of fetal tracheal fluid. In addition, lung stability to inflation increased 3-fold, and lung stability to deflation increased 8-fold. The increases in the saturated phosphatidylcholine content of fetal lung lavage and tracheal fluid were greater than the effects of each hormone acting independently, or in the double combinations. The β-receptor maximal binding capacity was increased 30 by the combined infusion of cortisol and TRH. In addition, the maximal binding capacity after cortisol plus TRH plus β-agonist infusion was 54 greater than the maximal binding capacity after β-agonist infusion. We conclude that the triple combination of cortisol, TRH, and β-agonist increases fetal lamb lung surfactant phospholipids better than do any of the hormones acting either independently, or in double combinations, and also improves pulmonary mechanics better than single hormones or the double combination of TRH and cortisol (p < 0.01). We speculate that the interaction of cortisol and TRH enhances the capacity of the fetal lamb lung to respond to β-adrenergic stimulation, and that the triple combination of cortisol plus TRH plus β-agonist might prepare the fetal lung for air breathing more effectively than each of the hormones acting either independently, or in the double combinations. (Pediatr Res 24:166–170,1988)

We used chronic fetal glucose infusion to test the hypothesis that chronic fetal hyperglycemia and hyperinsulinemia inhibit the development of β-receptor binding capapcity (Bmax) in fetal lamb lung. Glucose was infused (14 ± 4 mg/kg/h, mean ± SD) into eight twin and four singleton fetuses from 112 days gestation until death between 118–145 days gestation. The other eight twins and eleven additional singleton fetuses served as controls. Serum glucose levels were elevated 2-fold and serum insulin levels were elevated 3-fold in the glucose-infused fetuses. In the control fetuses β-receptor Bmax increased 2.5-fold between 130 days gestation and term. However, this increase was attenuated to 1.5-fold in the glucose infused fetuses, p < 0.01. The 50 inhibition of Bmax was similar in both male and female fetuses, except that the Bmax fell to 30 lower levels in males, p < 0.01. Chronic glucose infusion also resulted in an 80 reduction in lung lavage saturated phosphatidylcholine content, and an 85 reduction in tracheal fluid saturated phosphatidylcholine content, p < 0.001. Lung lavage and tracheal fluid saturated phosphatidylcholine content correlated significantly with beta receptor Bmax (r = 0.9, r = 0.85). We conclude that chronic glucose infusion inhibits the development of β-receptor binding in fetal lamb lung, and that this effect is greater in males than females. Such a mechanism could be a factor in the predisposition of infants of diabetic mothers to develop respiratory distress and could contribute to a male disadvantage in respiratory morbidity. (Pediatr Res 24: 171–174, 1988)

We studied the effect of hyperviscosity on plasma glucose concentration, endogenous glucose production, and plasma insulin concentration in 12 term mixed breed newborn lambs. After a 7-hr fast, 0.45 saline was infused at a constant rate for 6 hr during which time hourly plasma glucose and 3-hourly plasma insulin concentrations were determined. A glucose turnover determination by the prime-constant infusion technique using 3H6radiolabeled glucose was performed at a steady state. An exchange transfusion was then carried out using maternal packed red blood cells in six lambs to produce hyperviscosity and using maternal whole blood in six other lambs to maintain normoviscosity. After the exchange transfusion, a second study identical to that in the pre-exchange period was carried out. There was no significant difference in the mean plasma glucose concentration (mg/dl; mean ± S.E.) between groups during the first turnover period (pre-exchange transfusion) [103 ± 4.9 (Normoviscous) versus96 ± 4.8 (Hyperviscous)), but during the postexchange transfusion turnover period, the plasma glucose concentration was lower in the hyperviscous than in the normoviscous group [89 ± 4.3 (Normoviscous) versus76 ± 4.0 (Hyperviscous)] (P<0.05.) Endogenous glucose production declined from the first to the second glucose turnover determination in both groups, but there was no significant difference between the normoviscous and hyperviscous groups. Plasma insulin concentrations were similarly low in both groups suggesting suppression in the fasted state. The data suggest that the hyperviscosity acts as an independent variable to depress plasma glucose concentration, the mechanism of which is still undefined.

We sought to test the hypothesis that hyperinsulinemia per sealters the flux of surface active material (SAM) into tracheal fluid by continuously infusing insulin (0.24 ± 0.04 units/kg/hr, mean ± S.E.) from 112 through 135 days gestation into five chronically catheterised fetal lambs, from which tracheal fluid could be collected.

To determine the effects of fetal β-2 agonist exposure on fetal hepatic glycogen metabolism, we infused ritodrine at a rate of 1.3 ± 0.4 μg/kg/min (mean ± SD) for 24 h into six chronically catheterized twin fetal lambs starting between 128 and 134 days gestation. The control twins received 0.9 saline at 1.2 ± 0.12 ml/kg/h. In addition, 15 uncatheterized fetuses were killed between 115 and 148 days gestation as unoperated controls. Ritodrine infusion produced a 1.7-fold elevation in fetal serum glucose level, from 23 ± 5 to 42 ± 15 mg/dl, and a 2-fold elevation in serum insulin level, from 16 ± 5 to 34 ± 8 mg/ml, p< 0.01. Hepatic glycogen content increased 7-fold in the uncatheterized controls between 115 and 148 days gestation (r= 0.9, p< 0.001). Ritodrine infusion reduced hepatic glycogen content by 50 from 179 ± 19 μg/mg in twin controls to 90 ± 25 μg/mg in the ritodrine-infused twins, p< 0.001. Hepatic glycogen phosphorylase kinase activity was elevated 1.3-fold from 0.149 ± 0.100 mU/mg protein in control twins to 0.186 ± 0.007 mU/mg protein in the ritodrine infused twins, p< 0.001. Glycogen phosphorylase a activity was also increased 1.4-fold from 8.60 ± 0.76 nM NADPH/min/mg protein in control twins to 11.85 ± 0.68 nM NADPH/min/mg protein in the ritodrine infused twins, p< 0.001. There were no significant differences in hepatic total glycogen phosphorylase (a + b) activity, active (I) or total I + D) glycogen synthase activity, protein, or DNA content between the ritodrine treated and control twins or between the control twins and the uncatheterized control fetuses. We conclude that β-2 agonist depletes hepatic glycogen in fetal lambs through activation of glycogen phosphorylase by glycogen phosphorylase kinase. Depletion of hepatic glycogen may be an unrecognized side effect of fetal β-2 agonist exposure. (Pediatr Res24: 330–332, 1988)

Diaphragm strength was measured as maximal transdiaphragmatic pressure (Pdi) during airway occlusion in 38 infants aged 11.6 ± 0.5 (S.E.) months postconception (mpc), range 8–21 mpc. All infants were asymptomatic at the time of study and required no mechanical ventilatory assistance. Ten infants had previous surgical correction of abdominal wall defects (gastroschisisomphalocele); 10 infants had previous surgical correction of congenital diaphragmatic hernia; and 18 infants had no thoracic or abdominal surgery. The mean maximal Pdi for all infants was 72 ± 3 cmH2O. There were no significant differences between the three groups. All infants with a maximal Pdi of less than 60 cmH2O were aged less than 10 mpc. After 13 mpc there was no significant increase in maximal Pdi. Between the ages 8–13 mpc there was a significant positive correlation between maximal Pdi and age postconception (r0.87, P< 0.0005), reflecting a developmental pattern of increasing maximal transdiaphragmatic pressure in infants during crying.

Protein phosphatase (PP) 1 and 2A activities were measured in soluble and particulate fractions of rat lung homogenates, obtained from 18− to 22-day gestation fetal rats, from neonates, and from adults. PP 1 activity in the particulate fraction increased 7-fold from 15 ± 2 to 102 ± 12 (mean ± SD) U/g lung between 18 and 22 days gestation, increased a further 1.5-fold to 152 ± 18 U/g in newborns, with no further increase in adults. PP 2A activity in the particulate fraction increased 3-fold from 10 ± 2 to 32 ± 5 U/g between 21 and 22 days gestation and did not change postnatally. PP 1 activity in the soluble fraction increased 7.8-fold from 4 ± 2 to 31 ± 4 U/g lung between 18 and 22 days gestation, increased a further 1.8-fold in newborns to 54 ± 8 U/g lung and increased 1.5-fold to 81 ± 6 U/g lung in adults. PP 2A activity in the soluble fraction increased 2.2-fold from 12 ± 4 to 28 ± 8 U/g lung between 18 and 22 days gestation, increased a further 1.7-fold in newborns to 47 ± 9 U/g lung, and increased 1.6-fold to 75 ± 10 U/g lung in adults. PP 1 activity was 1.6-to 3-fold higher in the particulate than the soluble fraction. PP 2A activity was 2-fold higher in the soluble than the particulate fraction after 21 days gestation, in neonates and adults. We conclude that protein phosphatase activity is regulated developmentally during the critical perinatal period of fetal lung ontogeny and suggest that these enzymes play an important role in regulating phosphorylation reactions in the developing lung. (Pediatr Res 24: 25–27, 1988)