Cells were cultured in 48-well plates in DMEM with 5% FBS, may take one night sown t and then stand for 48 hours so Rum-free DMEM. The cells were then placed in fresh medium containing 0.25 Ci / well were incubated in the presence of thymidine PDGF BB. PDE inhibitors R788 and / or analogs of prostacyclin was added 30 45 min before the addition of a mitogen, and thymidine, and thymidine incorporation was determined by Flssigszintillationsz COOLING analysis as described above. To determine cell proliferation, cells were sown in 24-well plates in DMEM with 5% FBS T and adhere overnight. The media were then incubated with fresh medium containing drugs and replaced ver Changed every 3 2 days for 13 days. The adh Pensions cells were trypsinized, counted Hlt and Lebensf Ability By trypan blue Ausschlu assessed.
The effects of the inhibition of PDE4 and cAMP signaling on apoptosis was defined using AM-1241 the Hoechst 33342 F Staining nuclear chromatin and morphology to determine Cell Death Detection ELISA kit to cytoplasmic histone associated DNA fragments. The cells were either in a medium containing 5% FBS or serum deprived for 48 hours and treated with either iloprost or roflumilast for 48 h. PASMCs were Kammerobjekttr Fond bred 8 and Permanox for Hoechst 33342 F Coloration and individual nuclei were at least 5 ZUF Llig Selected Selected fields for each also counted Hlt. The number of apoptotic cells with condensed nuclear fluorescence was determined and expressed as a percentage of the total cells. DNA fragmentation was in accordance with cells in 24-well plates developed the manufacturer’s instructions. Gelatin zymography and the cells of the matrix metalloproteinase production, sown in 24-well plates T been in a medium.
10% FBS for at least 2 days before it deprived of serum 24 h cultured The cells were incubated in fresh medium without serum and stimulated with 10 ng / ml recombinant human tumor necrosis factor, interleukin-1 and transforming growth factor-1, phorbol 12-myristate 13-acetate or inactive phorbol ester 4 PMA, in the absence and indicated the presence of drug concentrations. The medium was collected after 48 hours and gelatinase activity t zymography measured and visualized with MMP 2 and MMP 9 Biotrack man ELISA systems, according to the manufacturer’s instructions. Conditioned medium was removed, in a non-reducing conditions in a 8% SDS-polyacrylamide gel containing 1 mg / ml gelatin, 4 After electrophoresis, the gels were incubated in 2.
5% Triton X-100, SDS, washed with water and incubated overnight at 37 in a buffer containing 50 mM Tris-HCl, 5 mM CaCl2 remove, 1 M ZnCl2 and 0.1% triton X-100. After fixing in 25% isopropanol and 10% acetic Acid for 10 min, the gels were stained with Coomassie blue 0.25% for 1 h Fnd 2 Rbt. and N rbt in fixation / Bleichl solution until the bands of T activity were clearly visible. The presence of MMP activity T was by inhibition with 10 mM EDTA, and the use of gelatinases CONFIRMS after activation with 1.5 mM aminophenyl mercuric acetate p purified best. Statistical Analysis Data were expressed as mean SEM or a confidence interval of 95% and 4.0 using GraphPad Prism version. Comparisons were appropriate with analysis of variance with post hoc Tukey test or Student t test. A value of P 0.05 was considered significant.