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Pericardial fluid from patients with unstable angina induces vascular endothelial cell apoptosis

Atsushi Iwakura, MD^a, Masatoshi Fujita, MD, FACC, Koji Hasegawa, MD, Tatsuya Sawamura, MD, Ryuji Nohara, MD, Shigetake Sasayama, MD, FACC and Masashi Komeda, MD^a

^a Departments of Cardiovascular Surgery, Kyoto University, Graduate School of Medicine, Kyoto, Japan
College of Medical Technology, Kyoto University, Kyoto, Japan
Department of Cardiovascular Medicine, Kyoto University, Kyoto, Japan
Department of Bioscience, National Cardiovascular Center Research Institute, Osaka, Japan

View larger version (13K): [in a new window]   Figure 1 Pericardial fluid from patients with UA induces loss of F2 cells. F2 cells were plated in 96 well plates and maintained for 48 h in the presence of 10% pericardial fluid obtained during coronary artery bypass surgery. Cell viability was assayed using 3-(4,5-dimethyl thiaziazol-2-yl)-2,5-diphenyl tetrazolium bromide as described under "Methods." In each experiment, the viability of the well incubated with 10% FBS was set to 100%. All of the experiments were performed in triplicate for each patient sample and repeated at least three times. Results are the mean ± SD of each patient group.

View larger version (45K): [in a new window]   Figure 2 Fragmentation of genomic DNA in F2 cells incubated with pericardial fluid from a patient with UA. Genomic DNA was isolated from F2 cells maintained for 48 h in the presence of 10% pericardial fluid from a patient with UA (left lane) or stable angina (right lane) and loaded on a 2% agarose gel. The ladder assays were performed in all patients, and the data presented are representative.

View larger version (55K): [in a new window]   Figure 3 Induction of apoptosis in F2 cells by pericardial fluid from a patient with UA. A representative photograph of F2 cells incubated with pericardial fluid from patients with stable angina (A) and UA (B). These cells were stained with hematoxylin. Arrow heads show cells with evidence of apoptosis, including chromatin condensation.

View larger version (15K): [in a new window]   Figure 4 The effect of a neutralizing antibody against Fas ligand on the cell death–inducing activity of pericardial fluid from patients with UA. F2 cells were incubated with 10% pericardial fluid from patients with UA in the presence or absence of a neutralizing antibody against Fas ligand (1 µg/ml). Cell survival assays were performed as described in the legend for Figure 1. In each experiment, the viability of the well incubated with 10% fetal bovine serum was set to 100%. All of the experiments were performed in triplicate for each patient sample and repeated at least three times. Results are the mean ± SD of each group.

View larger version (30K): [in a new window]   Figure 5 Cell death–inducing activities exist in the Centricon C-100 retentates. Pericardial fluid was fractionated with Centricon C-100 as described under "Methods." A) A representative photograph showing a retentate (R) and a filtrate (F) electrophoresced by an SDS-polyacrylamide (2% to 15%) gradient gel and stained with Coomassie Brilliant Blue. MW = molecular weight. B) In each retentate and filtrate, F2 cell survival assays were performed as described in the legend for Figure 1. In each experiment, the viability of the well incubated with 10% FBS was set to 100%. All of the experiments were performed in triplicate for each patient sample and repeated at least three times. Results are the mean ± SD of each patient group.