Bronchopulmonary dysplasia (BPD), characterized by impaired alveolarization and vascularization in association with lung inflammation and apoptosis, often occurs after mechanical ventilation with oxygen-rich gas (MV-O2). in ventilated TNF-?/? mice. Preterm infants who went on to develop BPD showed significantly lower TNF- levels at birth. Our results suggest a critical balance between TNF- and TGF- signaling in the developing lung, and underscore the critical importance of these key pathways in the pathogenesis of BPD. Future treatment strategies need to weigh the potential benefits of inhibiting pathologic cytokine expression against the potential of altering key developmental pathways. = 6C8/group) were fixed intratracheally with buffered 4% paraformaldehyde overnight at 20 cmH2O, as previously described (3). Volume of fixed lungs was measured by fluid displacement (28). After paraffin embedding and isotropic uniform random sectioning (28), quantitative assessment of alveolar area and number of incomplete and complete alveolar walls (septal density) was performed in 2C3 independent random tissue sections (4 m, hematoxylin and eosin) per animal (CAST-Grid 2.1.5, Olympus, Ballerup, Denmark). Radial alveolar counts were assessed 30 fields of view in 2C3 independent random tissue sections per animal (13). Assessment of PDGF-r positive cells and related apoptosis in distal lung. Paraformaldehyde-fixed lung tissue sections were stained for PDGF-R (C-20) (Santa Cruz Biotechnology, No. sc-338), cleaved Caspase-3 (Cell Signaling Technology, No. 9661S), and DAPI (Sigma Aldrich, No. D8417) in combination. Double-positive cells were quantified in eight different fields of view/animal (400 magnification) with the Imaris Software (Imaris C646 Software, Zurich, Switzerland). Protein extraction and immunoblot analysis. Lungs from 8-h studies (= 4/group) were excised, weighed, and stored at ?80C for C646 later protein extraction by using high urea buffer (KPO4, Urea, AppliChem, Darmstadt, Germany) and Halt Protease Inhibitor Nrp2 Cocktail (Thermo Fisher Scientific, No. 1861280). After measurement of protein concentrations (BCA, No. 23227, Pierce Scientific Rockford, IL), immunoblots were performed using a Bis-Tris (Life Technologies, No. NP0321BOX, Darmstadt, Germany) or a Tris-Acetate (Life Technologies, No. EA0375BOX) gel as published previously (15) using the following antibodies: Caspase-3 (Cell Signaling, No. 9662S), cleaved caspase-3 (Cell Signaling Technology, No. 9661), cleaved caspase-6 (Cell Signaling, No. 9761S), caspas-8 (Bio Vision, 3259-100), pSMAD 2 (Cell Signaling, No. 3101S), SMAD 2/3 (Cell Signaling, No. 3102S), SMAD 7 (Santa Cruz Biotechnology, No. sc-9183) -actin (Santa Cruz Biotechnology, No. sc-81178); secondary antibody goat anti-mouse IgG (Santa Cruz Biotechnology, No. 2060) secondary antibody goat anti-rabbit IgG (Santa Cruz Biotechnology, No. 2301), or donkey anti-goat IgG-HRP (Santa Cruz Biotechnology, No. 2020) conjugated to horseradish peroxidase. Images were detected by chemiluminescence (GE Healthcare, No. RPN2232, Buckinghamshire, Great Britain) and quantified by densitometry (Bio Rad, Munich, Germany). RNA extraction and quantitative real-time PCR. After mRNA extraction (Carl Roth, No. A979.1) and purification (Peqlab, No. 12C6834-01, Erlangen, Germany) quantitative real-time C646 PCR was applied to measure lung mRNA expression of IL-1, CXCL-1, and MCP-1 using proprietary primer-probes (Eurofins mwg operon, Ebersberg, Germany). In Vitro Studies Mouse primary MFBs. Mouse MFBs were extracted from PBS-flushed lungs of 5- to 7-day-old C57B/6J WT mice and cultured on a petridish (Corning, No. 430167, Tewksbury MA) in media (Gibco, No. 41966-029, Darmstadt, Germany) containing Pen/Strep (Gibco, No. 15140-122) and Gentamycin (Lonza, No. BE02-012E, Basel, Switzerland). FACS C646 analysis of primary mouse lung MFBs showed the following characterization: 77.2 14% PDGF-R+Vimentin+, 16.7 12% Vimentin+, 77.6 27% SMA+, 32 8.6% CD90+, and 8.5 4.5% CD105+. In addition, the analysis showed a negligible amount of leucocytes (0.6 0.5% CD45+). Mechanical stretch experiments. Primary mouse lung MFBs were seeded on flexible-bottomed laminin-coated culture plates (Flex Cell International, No. BF-3001L) to undergo in vitro stretch in room air at 70C80% confluence (cyclic strain by vacuum pressure: shape/sine; elongation min 2%, max 8%; frequency 2 Hz; duty cycle 50%; cycles 43,216; duration 24 h) for 24 h. Treatment with TNF- was performed with 100 ng/ml recombinant TNF- (Pepro Tech, No. 300-01A). The stretch experiment was started right after adding TNF- treatment. At the end of each experiment, cells were harvested in 60 l of RIPA buffer [150 mM NaCl (AppliChem, No. A2942), 10 mM Tris-buffer pH 7.2, (AppliChem, No. A1379), 0.1% SDS, (AppliChem, No. A1502), 1% Triton X 100, (Carl Roth, No. 3051.2), 1% sodium deoxycholate (Sigma, No. D6750),.