Congestive heart failure induces endothelial cell apoptosis: protective role of carvedilol
Lothar Rössig, MD*,
Judith Haendeler, PhD*,
Ziad Mallat, MD ,
Benedicte Hugel, PhD ,
Jean-Marie Freyssinet, PhD,
Alain Tedgui, PhD,
Stefanie Dimmeler, PhD* and
Andreas M. Zeiher, MD*
* Division of Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Frankfurt, Germany
INSERM U141, IFR "Circulation," Hôpital Lariboisiere, Paris, France
INSERM U143, Le Kremlin-Bicetre, Strasbourg, France
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Figure 1 Induction of apoptosis in HUVECs by the serum of patients with CHF. A, Apoptosis rates of HUVECs after exposure to 10% serum of patients with CHF as compared with healthy volunteers (control group) for 18 h. Box plots show median, 75% quartile and total range of values (*p < 0.001 vs. control group). B, Deoxyribonucleic acid fragmentation in HUVECs after 18-h incubation with the serum of a representative patient with CHF. Left lane, Serum of a healthy volunteer (control). Upper panel, Ethidium bromide staining of the gel to demonstrate equal loading of DNA. C, Cell viability after incubation with CHF or control serum for 24 h (*p = 0.002 vs. control group). D, Correlation of apoptosis induction by the serum of patients with CHF symptoms according to the NYHA classification (*p < 0.05 vs. NYHA class II; **p < 0.001 vs. control group). Data are presented as the mean value ± SEM. E, Shed membrane particles detected in human plasma derived from control subjects as compared with patients with CHF (mean ± SEM) (*p < 0.001).
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Figure 2 Serum levels of TNF-alpha and sTNF-R1. A, Serum levels of TNF-alpha and sTNF-R1 in patients with CHF (n = 15) and in control subjects (n = 11). *p < 0.01 vs. control group. **p < 0.005 vs. control group. B, Correlation of TNF-alpha serum levels with apoptosis rates in HUVECs after exposure to serum for 18 h (n = 26).
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Figure 3 Involvement of caspases in apoptosis induction in CHF and apoptosis suppression by carvedilol. A, Effect of carvedilol (5 µmol/liter) and the caspase inhibitor ZVAD-fmk (100 µmol/liter) on HUVEC apoptosis induced by the serum of control subjects (n = 7) or patients with CHF (n = 10). *p < 0.001 vs. CHF serum in the absence of the compounds; **p < 0.001 vs. control group. B, Suppression of CHF serum-induced DNA fragmentation by carvedilol (5 µmol/liter). C, Effect of carvedilol (5 µmol/liter) on DNA laddering induced by TNF-alpha (50 ng/ml; 18 h). Upper panel, amounts of DNA loaded. A representative autoradiographic study, out of three experiments, is shown. D, Dose-dependent inhibition of TNF-alpha–induced apoptosis by carvedilol, its analogue BM91.0228 and propranolol. 100% HUVEC apoptosis induced by TNF-alpha (50 ng/ml; 18 h). *p < 0.001 vs. carvedilol or BM91.0228 at the respective concentration (n = 4 to 6).
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Figure 4 Effect of carvedilol on TNF-alpha–induced MAPK activation in HUVECs. A, Activity of JNK and (B) phosphorylation of p38 after stimulation with TNF-alpha (50 ng/ml) in the presence or absence of carvedilol (5 µmol/liter). Anisomycin-treated and native C-6 glioma cell extracts as positive and negative control studies, respectively. Representative blots, out of three experiments, are shown.
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Figure 5 Effect of carvedilol on caspase-mediated apoptosis signal transduction in HUVECs. A, Caspase-3–like activity after treatment with TNF-alpha (50 ng/ml, 18 h) alone and in combination with carvedilol (5 µmol/liter), its analogue BM91.0228 (5 µmol/liter) or propranolol (5 µmol/liter). *p < 0.001 vs. TNF-alpha group (n = 4). B, Apoptosis rates of HUVECs cotransfected with pcDNA3.1 caspase-8 or pcDNA3.1 without insert (mock) and pcDNA3.1 LacZ in the presence or absence of carvedilol (5 µmol/liter), BM91.0228 (5 µmol/liter) or propranolol (5 µmol/liter). *p < 0.001 vs. caspase-8. C, Cytosolic cytochrome c levels after incubation with TNF-alpha (50 ng/ml) and carvedilol (5 µmol/liter) for 18 h. Equal loading of the blot was confirmed by reprobing against actin. A representative blot, out of three independent experiments, is shown.
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