We therefore hypothesized selleckchem that i the PDE6 subunits potentiality can be expressed in the lung, ii the subunits are differentially regulated in IPF and iii the specific subunit of PDE6, PDE6D, modulates the proliferation rate of AECs. To this end, we achieved our aim to eluci date previously unrecognized PDE6 expression in nor mal human lungs, significant alterations of the PDE6D and PDE6G H subunits in IPF derived lungs and char acterize the functional role of PDE6D in AEC proliferation. Materials and methods Ethics Statement The study protocol for tissue donation was approved by the Ethics Committee of the Justus Liebig University School of Medicine. Informed consent was obtained from each individual patient or the patients next of kin.

Human Tissues Explanted lung tissues from IPF subjects or donor were obtained during lung transplantation at the Department of Cardiothoracic Surgery, University of Vienna, Austria. Diagnosis was established on the basis of a proof of a usual interstitial pneumonitis pattern in the explanted lungs from lung transplant reci pients. Apart from IPF subjects, tissue was also obtained from 6 donor lungs, which could not be uti lized due to size limitations between donor and putative recipient or due to incompatibility between donor and recipient. Isolation of human ATII cells Primary human ATII cells were isolated, as previously described. Briefly, the lung was digested and minced. The cell rich fraction was filtered, layered onto a Percoll density gradient, and centrifuged. The cells were then incubated with anti CD14 antibody coated magnetic beads.

The remaining cell suspension was incubated in human IgG coated tissue culture dishes at 37 C in a 5% CO2, 95% O2 atmosphere. The purity of isolated human ATII cells was examined by Papanico laou staining. The purity and viability of ATII cell pre parations was consistently between 90 and 95%. Cell culture The A549 human AEC line was maintained in Dulbeccos modified Eagles F12 medium supplemented with 10% heat inactivated fetal bovine serum, 100 units ml penicillin, 0. 1 mg ml streptomycin, and 2 mM L glutamine at 37 C in a 5% CO2, 95% O2 atmosphere. For cytokine stimula tion A549 cells were cultured in the absence or presence of TGF b1 for 12 h and 24 h.

For studies with inhibitors A549 cells were cultured Drug_discovery in the absence or presence of ERK inhibitor, U 0126 or p38a b inhibitor, SB 203580, details are specified in Measurement of Cell proliferation section from Materi als and Methods. RNA isolation, cDNA synthesis and mRNA quantification by qRT PCR or semi quantitative RT PCR Total RNA was isolated from frozen human lung tissues and cell pellets using Trizol reagent. cDNA synthesis was carried out with an ImProm II reverse transcription system by incubating 5 ug of RNA, following the manufacturers protocol.