Shu-Whei Tsai

Effects of Mechanical Forces on Lung-Specific Gene Expression

Fetal breathing movements (FBM) are necessary for fetal lung growth and maturation. The authors analyzed fetal rat lungs cultured with or without lung distension and tracheal ligation, and examined the effects of mechanical stretch on a human pulmonary epithelial cell line (NCI-H441) that shows regulated expression of surfactant proteins (SP-A, SP-B). Cells were grown on silastic membranes and mounted in a Flexercell Strain Unit. Cyclic deformation simulating FBM was achieved by applying a vacuum of 22 kPa (5%-15% radial deformation) at 50 cycles per minute for 2 to 24 hours. Results indicate that static distension for as little as 4 hours decreased steady-state SP-A and SP-B mRNA levels in whole lung (n = 5-6, P < .01). In contrast, cyclic stretching of H441 cells for 24 hours increased SP-B and SP-A expression 2- to 4-fold over controls. Cyclic deformation also significantly enhanced 3H-choline incorporation into saturated phosphatidylcholine. Dynamic mechanodeformation may be a critical stimulus for fetal lung development.

Arrested pulmonary alveolar cytodifferentiation and defective surfactant synthesis in mice missing the gene for parathyroid hormone-related protein

Parathyroid hormone‐related protein (PTHrP) and PTH/PTHrP receptor expression are developmentally regulated in lung epithelium and adepithelial mesenchyme, respectively. To test the hypothesis that PTHrP is a developmental regulator of terminal airway development, we investigated in vivo and in vitro models of alveolar cytodifferentiation using mice in which the gene encoding PTHrP was ablated by homologous recombination. We have determined that fetal and newborn PTHrP(−/−) lungs showed delayed mesenchymal–epithelial interactions, arrested type II cell differentiation, and reduced surfactant lamellar body formation and pulmonary surfactant production. Embryonic PTHrP(−/−) lung buds cultured in the absence of skeletal constriction or systemic compensating factors also exhibited delayed alveolar epithelial (type II cell) and mesenchymal cytodifferentiation, as well as a >40% inhibition of surfactant phospholipid production (n = 3–5). Addition of exogenous PTHrP to embryonic PTHrP(−/−) lung cultures normalized interstitial cell morphology and surfactant phospholipid production. The importance of PTHrP as an endogenous regulatory molecule in mammalian lung development is supported by the findings that ablation of PTHrP expression in isolated developing lung is sufficient to disrupt normal development of the alveolar ducts and the centriacinar regions. Developmental Dynamics 230:278–289, 2004.

Atrial natriuretic peptide attenuates agonist-induced pulmonary edema in mice with targeted disruption of the gene for natriuretic peptide receptor-A

Atrial natriuretic peptide (ANP) inhibits agonist-induced pulmonary edema formation, but the signaling pathway responsible is not well defined. To investigate the role of the particulate guanylate cyclase-linked receptor, natriuretic peptide receptor-A (NPR-A), we measured acute lung injury responses in intact mice and pulmonary microvascular endothelial cells (PMVEC) with normal and disrupted expression of NPR-A. NPR-A wild-type (NPR-A+/+), heterozygous (NPR-A+/-), and knockout (NPR-A-/-) mice were anesthetized and treated with thrombin receptor agonist peptide (TRAP) or lipopolysaccharide (LPS). Lung injury was assessed by lung wet-to-dry (W/D) weight and by protein and cell concentration of bronchoalveolar lavage (BAL) fluid. No difference in pulmonary edema formation was seen between NPR-A genotypes under baseline conditions. TRAP and LPS increased lung W/D weight and BAL fluid cell counts more in NPR-A-/- mice than in NPR-A+/- or NPR-A+/+ mice, but no genotype-related differences were seen in TRAP-induced increases in bloodless lung W/D weight or LPS-induced increases in BAL protein concentration. Pretreatment with ANP infusion completely blocked TRAP-induced increases in lung W/D weight and blunted LPS-induced increases in BAL cell counts and protein concentration in both NPR-A-/- and NPR-A+/+ mice. Thrombin decreased transmembrane electrical resistance in monolayers of PMVECs in vitro, and this effect was attenuated by ANP in PMVECs isolated from both genotypes. Administration of the NPR-C-specific ligand, cANF, also blocked TRAP-induced increases in lung W/D weight and LPS-induced increases in BAL cell count and protein concentration in NPR-A+/+ and NPR-A-/- mice. We conclude that ANP is capable of attenuating agonist-induced lung edema in the absence of NPR-A. The protective effect of ANP on agonist-induced lung injury and pulmonary barrier function may be mediated by NPR-C.

The parathyroid hormone-related protein (PTHrP) knockout mouse shows delayed lung development. • 1583

The parathyroid hormone-related protein (PTHrP) knockout mouse shows delayed lung development. • 1583

Hypoxia Decreases Surfactant Synthesis, Alters Cell Cycle Progression and Activates Apoptosis in Airway Epithelial Cells • 1729

Hypoxia Decreases Surfactant Synthesis, Alters Cell Cycle Progression and Activates Apoptosis in Airway Epithelial Cells • 1729

Expression of Pro- and Anti-Apoptotic Proteins during Placental (Trophoblast) Differentiation

Expression of Pro- and Anti-Apoptotic Proteins during Placental (Trophoblast) Differentiation

Effects of Mechanical Forces on Pulmonary Epithelial Gene Expression and Differentiation • 1738

Fetal breathing movements (FBM) are necessary for normal fetal lung growth and maturation. Absence of FBM or lung constriction results in lung hypoplasia. The molecular mechanisms by which FBM stimulate lung growth are not known. We analyzed fetal rat lungs (E19) cultured ± lung distension and tracheal ligation. We also examined the effects of mechanical stretch on rat fetal lung type II cells, human pulmonary adenocarcinoma A549 cells, and a human pulmonary epithelial cell line (NCI-H441) that shows regulated expression of surfactant proteins SP-A and SP-B. Cells were grown to confluence on matrix-coated silastic membranes and mounted in a FX-3000 Strain Unit (Flexcell). Cyclic deformation simulating FBM was achieved by applying a vacuum of 22 kPa (5-15% radial deformation) at 50 cycles/min for 2-24h. Unstretched cells were used as controls. We separately evaluated the effects of fluid shear forces (without cellular radial elongation) and cell deformation. SP-A and SP-B mRNA abundance was assayed by northern blot. Parathyroid hormone-related protein (PTHrP, a stretch-dependent lung developmental regulator), and cyclin-dependent kinase (cdk) inhibitors were analyzed by RNase protection assay (RPA). Our results indicate that static distension for as brief as 4h decreased steady-state SP-A and SP-B mRNA levels in whole lung to 51% and 31%, respectively, of control levels (n=5-6; p SP-A expression 2-4 fold over controls and PTHrP expression by 30-60%. These effects were not mediated by fluid shear but were partially inhibited by lanthanum (a stretch-activated channel blocker). Cyclic deformation also significantly enhanced 3H-choline incorporation into saturated phosphatidylcholine in the different epithelial culture models. Cyclic stretching for 48h altered cell cycle phase distribution, apparently by inducing a block in SG2 progression, and did not activate apoptosis(assayed by DNA flow cytometry). The cell cycle effects do not appear to be due to transcriptional regulation of cdk inhibitor proteins. Our results in lung organ culture are consistent with observations made in vivo in tracheal ligation models, and suggest that continuous lung distension may interfere with airway epithelial cytodifferentiation. In contrast, dynamic mechanodeformation (distension-relaxation) may be a critical stimulus for fetal lung maturation. The molecular mechanisms by which mechanotransduction alters gene expression and cytodifferentiation are under investigation.