Effect of Telmisartan on Nitric Oxide–Asymmetrical Dimethylarginine System by Fortunato Scalera, Jens Martens-Lobenhoffer, Alicja Bukowska, Uwe Lendeckel, Michael Täger, and Stefanie M. Bode-Böger Hypertension Volume 51(3):696-703 March 1, 2008 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Telmisartan delayed EC senescence. Figure 1. Telmisartan delayed EC senescence. A through C, ECs were incubated with telmisartan, eprosartan, Ang II, and GW9662 starting from the fourth passage (young) and replaced every 48 hours until the twelfth passage (old). SA-β-gal staining was performed as described in Methods, and the number of SA-β-gal–positive cells was counted. Each point represents mean±SEM of 5 separate experiments. *P<0.05 vs old control; #P<0.05 vs young. D, Old ECs were transfected with control or PPARγ siRNA for 18 hours before exposing the cells to telmisartan for up to 48 hours. The data represent mean±SEM of 4 separate experiments. *P<0.05 vs control siRNA. Fortunato Scalera et al. Hypertension. 2008;51:696-703 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Telmisartan downregulated AT1R (A and B) and upregulated PPARγ (C and D) protein expression. Figure 2. Telmisartan downregulated AT1R (A and B) and upregulated PPARγ (C and D) protein expression. Whole-cell lysates (30 μg) prepared from ECs treated with telmisartan, eprosartan, Ang II, and GW9962 or transfected with control or PPARγ siRNA were examined to detect expression of AT1R and PPARγ. Protein expression of AT1R and PPARγ was standardized on the basis of GAPDH expression, and the relative levels of expression are plotted in the graphs. The corrected value in young control or control siRNA-treated cells was designated as 1.0. *P<0.05 vs old control or control siRNA; #P<0.05 vs young (n=4). Fortunato Scalera et al. Hypertension. 2008;51:696-703 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. Telmisartan decreased ROS (A and B) and 8-iso-PGF2α (C) level. Figure 3. Telmisartan decreased ROS (A and B) and 8-iso-PGF2α (C) level. ECs were incubated with telmisartan, Ang II, and GW9662 starting from the fourth passage (young) and replaced every 48 hours until the twelfth passage (old). Endogenous ROS formation was measured with DHR using a fluorescence-activatedcellsorter (FACS) analysis and 8-iso-PGF2α using HPLC-tandem mass spectrometry. *P<0.05 vs old control; #P<0.05 vs young (n=5). Fortunato Scalera et al. Hypertension. 2008;51:696-703 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. Telmisartan upregulated the activity (A) and protein expression (B, C) of DDAH II. ECs were incubated with telmisartan, eprosartan, Ang II, and GW9662 starting from the fourth passage (young) and replaced every 48 hours until the twelfth passage (old) or transfected with control or PPARγ siRNA for 18 hours before exposing the cells to telmisartan for up to 48 hours. Figure 4. Telmisartan upregulated the activity (A) and protein expression (B, C) of DDAH II. ECs were incubated with telmisartan, eprosartan, Ang II, and GW9662 starting from the fourth passage (young) and replaced every 48 hours until the twelfth passage (old) or transfected with control or PPARγ siRNA for 18 hours before exposing the cells to telmisartan for up to 48 hours. DDAH activity in cell lysates from each condition is expressed as percentage of amount of ADMA metabolized by young control, which is defined as 100% for every experiment. Whole-cell lysates (30 μg) were examined to detect expression of DDAH II. Protein expression of DDAH II was standardized on the basis of GAPDH expression, and the relative levels of expression are plotted in the graphs. The corrected value in young control- or control siRNA-treated cells was designated as 1.0. *P<0.05 vs old control or control siRNA; #P<0.05 vs young (n=4). *P<0.05 vs old control; #P<0.05 vs young (n=4). Fortunato Scalera et al. Hypertension. 2008;51:696-703 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. Telmisartan induced NO synthesis by decreasing concentration of ADMA. ECs were incubated with telmisartan, eprosartan, Ang II, and GW9662 starting from the fourth passage (young) and replaced every 48 hours until the twelfth passage (old) or transfected with control or PPARγ siRNA for 18 hours before exposing the cells to telmisartan for up to 48 hours. Figure 5. Telmisartan induced NO synthesis by decreasing concentration of ADMA. ECs were incubated with telmisartan, eprosartan, Ang II, and GW9662 starting from the fourth passage (young) and replaced every 48 hours until the twelfth passage (old) or transfected with control or PPARγ siRNA for 18 hours before exposing the cells to telmisartan for up to 48 hours. ADMA concentration (A and B) was measured by HPLC-mass spectrometry and NO metabolites (NOx) concentration (C and D) by gas chromatography-mass spectrometry. *P<0.05 vs old control or control siRNA; #P<0.05 vs young (n=5). Fortunato Scalera et al. Hypertension. 2008;51:696-703 Copyright © American Heart Association, Inc. All rights reserved.