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Nebivolol and its 4-keto derivative increase nitric oxide in endothelial cells by reducing its oxidative inactivation

Luciano Cominacini, MD^*,*, Anna Fratta Pasini, MD^*, Ulisse Garbin, MD^*, Cristina Nava, MD^*, Anna Davoli, BSc^*, Marco Criscuoli, BSc, Attilio Crea, BSc, Tatsuya Sawamura, MD and Vincenzo Lo Cascio, MD^*

^* Department of Biomedical and Surgical Sciences (Medicina D), University of Verona, Verona, Italy
Department of Preclinical Development of Menarini Ricerche SpA, Firenze, Italy
Department of Pharmacokinetics and Metabolism of Menarini Ricerche SpA, Pomezia, Italy
Department of Bioscience, National Cardiovascular Center Research Institute, Osaka, Japan

View larger version (33K): [in a new window]   Figure 1 Effect of native low-density lipoprotein (LDL), oxidized LDL (ox-LDL), and ox-LDL plus anti-human LOX-1 monoclonal antibody (hLOX-1 mAb), SB203580 and bisindolylmaleimide on the translocation of p47phox and Rac1 to the plasma membrane of human umbilical vein endothelial cells. The cells were preincubated with hLOX-1 mAb, SB203580, and bisindolylmaleimide for 30 min at 37°C and then stimulated with native LDL (control) and ox-LDL (50 µg protein/ml) for 5 min at 37°C. The figure shows representative blots of six different experiments and the average data obtained by densitometric analysis. Results (means ± SD) are expressed as density in arbitrary units (AU). *p < 0.001 versus control; ¶p < 0.001 versus ox-LDL; p < 0.05 versus ox-LDL. Solid bars = p47phox; open bars = Rac1.

View larger version (27K): [in a new window]   Figure 2 (A) effect of increasing amounts of nebivolol, its 4-keto derivative, and atenolol on intracellular concentration of superoxide (O2·–) induced by oxidized low-density lipoprotein (ox-LDL) in bovine aortic endothelial cells. The cells were stimulated with ox-LDL (50 µg protein/ml) for 10 min at 37°C. Results are expressed as mean fluorescence intensity (MFI) and are means ± SD of experiments performed in triplicate in six separate occasions. *p < 0.001 versus control. Open bars = nebivolol; solid bars = 4-keto derivative; striped bars = atenolol. (B) Effect of increasing amounts of nebivolol on the translocation of p47phox and Rac1 to the plasma membrane of human umbilical vein endothelial cells. The cells were preincubated with increasing amounts of nebivolol for 30 min at 37°C and then stimulated with ox-LDL (50 µg protein/ml) for 10 min at 37°C. The figure shows representative blots of six different experiments and the average data obtained by densitometric analysis. Results (means ± SD) are expressed as density in arbitrary units (AU). *p < 0.001 versus control. Solid bars = p47phox; open bars = Rac1.

View larger version (23K): [in a new window]   Figure 3 Effect of nebivolol, its 4-keto derivative, and anti-bovine LOX-1 monoclonal antibody (bLOX-1 mAb) on oxidized low-density lipoprotein (ox-LDL)-induced variation of superoxide (O2·–) in bovine aortic endothelial cells (BAECs), in Chinese hamster ovary-K1 (CHO) cells, and CHO-K1 cell line stably expressing bovine LOX-1 (BLOX-1-CHO). Nebivolol (25 µmol/l), its 4-keto derivative (25 µmol/l), and bLOX-1 mAb (30 µg/ml) were preincubated with cells for 30 min at 37°C. The cells were then stimulated with ox-LDL (50 µg protein/ml) for 10 min at 37°C. Results are expressed as mean fluorescence intensity (MFI) and are means ± SD of experiments performed in triplicate in six separate occasions. *p < 0.001 versus ox-LDL.

View larger version (25K): [in a new window]   Figure 4 Effect of nebivolol, its 4-keto derivative, and atenolol on oxidized low-density lipoprotein (ox-LDL)-induced variations of nitric oxide (NO) in basal and bradykinin-stimulated bovine aortic endothelial cells. The cells were then stimulated with ox-LDL (50 µg protein/ml) for 10 min at 37°C. Results are expressed as mean fluorescence intensity (MFI) and are means ± SD of experiments performed in triplicate in six separate occasions. *p < 0.001 versus control.

View larger version (21K): [in a new window]   Figure 5 Effect of bovine anti-LOX-1 monoclonal antibody (bLOX-1 mAb), nebivolol, and its 4-keto derivative alone or in combination with bLOX-1 mAb on oxidized low-density lipoprotein (ox-LDL)-induced variations of nitric oxide (NO) in bradykinin-stimulated bovine aortic endothelial cells (BAECs). Nebivolol (25 µmol/l), its 4-keto derivative (25 µmol/l), and bLOX-1 mAb (30 µg/ml) were preincubated with BAECs for 30 min at 37°C. The cells were then stimulated with ox-LDL (50 µg protein/ml) for 10 min at 37°C. Results are expressed as mean fluorescence intensity (MFI) and are means ± SD of experiments performed in triplicate on six separate occasions. *p < 0.001 versus control; p < 0.001 versus ox-LDL; ¶p < 0.01 versus bLOX-1 mAb.

View larger version (23K): [in a new window]   Figure 6 Effect of oxidized low-density lipoprotein (ox-LDL), nitro-L-, and D-arginine methyl ester hydrochloride (L- and D-NAME), bradykinin, nebivolol, and atenolol on endothelial nitric oxide synthase (eNOS) activity in bovine aortic endothelial cells (BAECs). Ox-LDL (50 µg protein/ml), L- and D-NAME (2 mmol/l), bradykinin (100 nmol/l), nebivolol (25 µmol/l), and atenolol (25 µmol/l) were preincubated with BAECs for 5 min at 37°C. Results are expressed as pmol citrulline/mg protein/min and are means ± SD of experiments performed in triplicate in six separate occasions. *p < 0.001 versus control.