Arterioscler Thromb Vasc Biol Peroxisome Proliferator-Activated Receptor-γ Coactivator 1-α Overexpression Prevents Endothelial Apoptosis by Increasing ATP/ADP Translocase Activity by Jong Chul Won, Joong-Yeol Park, Yun Mi Kim, Eun Hee Koh, Somi Seol, Byeong Hwan Jeon, Jin Han, Jung Ran Kim, Tae-Sik Park, Cheol Soo Choi, Woo Je Lee, Min-Seon Kim, In-Kyu Lee, Jang Hyun Youn, and Ki-Up Lee Arterioscler Thromb Vasc Biol Volume 30(2):290-297 February 1, 2010 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Time-dependent changes in intracellular (A) and mitochondrial ROS (B) generation in response to LA treatment (60 μmol/L). *P<0.05 vs baseline; **P<0.05 vs cells infected with adenoviruses carrying β-gal (Ad-β-gal). Figure 1. Time-dependent changes in intracellular (A) and mitochondrial ROS (B) generation in response to LA treatment (60 μmol/L). *P<0.05 vs baseline; **P<0.05 vs cells infected with adenoviruses carrying β-gal (Ad-β-gal). C, Apoptosis measured at 16 hours by the cell death enzyme-linked immunosorbent assay kit. *P<0.05 vs control; **P<0.05 vs Ad-β-gal (n=5, each). Jong Chul Won et al. Arterioscler Thromb Vasc Biol. 2010;30:290-297 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Effect of LA and Ad-PGC-1α on the expression of PGC-1α and antioxidant genes. Figure 2. Effect of LA and Ad-PGC-1α on the expression of PGC-1α and antioxidant genes. HAEC were infected with either Ad-β-gal or Ad-PGC-1α. After 48 hours, cells were treated with control vehicle, 60 μmol/L LA, or 1.5 mU/mL glucose oxidase for 4 hours. A, Representative Western blots of PGC-1α protein expression. B, Real-time polymerase chain reaction analysis of the mRNA expression of manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (Cu/Zn SOD1), catalase, glutathione peroxidase, and UCP-2. *P<0.05 vs Ad-β-gal; **P<0.05 vs control (cells not treated with LA or glucose oxidase) (n=5, each). Jong Chul Won et al. Arterioscler Thromb Vasc Biol. 2010;30:290-297 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. PGC-1α prevents LA-induced mitochondrial membrane hyperpolarization by increasing ATP/ADP translocase activity. Figure 3. PGC-1α prevents LA-induced mitochondrial membrane hyperpolarization by increasing ATP/ADP translocase activity. A, Effect of Ad-PGC-1α on LA-induced changes in Δψm. *P<0.05 vs baseline; **P<0.05 vs Ad-β-gal. B–D, Effect of Ad-PGC-1α on ADP/ATP translocase activity, fatty acid oxidation, and intracellular levels of triglycerides (TG), ceramide and DAG. HAEC preinfected with Ad-β-gal or Ad-PGC-1α were treated with control vehicle or 60 μmol/L LA for 4 hours. *P<0.05 vs Ad-β-gal; **P<0.05 vs control. E, Effect of 500 nM triascin and 500 nM myriocin on LA-induced change in ADP/ATP translocase activity measured at 4 hours. *P<0.05 vs untreated cells; **P<0.05 vs LA-treated cells (n=5, each). Jong Chul Won et al. Arterioscler Thromb Vasc Biol. 2010;30:290-297 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. ANT1 expression is induced by ROS and reduces it. Figure 4. ANT1 expression is induced by ROS and reduces it. A, Effects of LA and PGC-1α on ANT1 mRNA expression. HAEC preinfected with Ad-β-gal or Ad-PGC-1α were treated with control vehicle or 60 μmol/L LA for 4 hours. *P<0.05 vs Ad-β-gal. B and C, Effect of N-acetylcysteine (10 mmol/L) on ANT1 mRNA expression and ATP/ADP translocase activity. *P<0.05 vs untreated cells. **P<0.05 vs LA-treated cells. D–F, Effect of ANT1 siRNA on ANT1 protein expression, cellular apoptosis, and ROS generation. HAEC were treated with 50 nM of ANT1 siRNA or control siRNA. ANT1 protein and ROS generation were measured at 24 hours. Apoptosis was measured at 48 hours. *P<0.05 vs control siRNA (n=5, each). Jong Chul Won et al. Arterioscler Thromb Vasc Biol. 2010;30:290-297 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. Effect of ANT1 siRNA on ATP/ADP translocase activity (A), apoptosis (B, C), intracellular ROS (D), and time-dependent changes in Δψm (E) (n=5, each); 50 nM of ANT1 siRNA or control siRNA was transfected into HAEC. After 24 hours, cells were infected with Ad-β-gal or Ad-PGC-1α (5×106 pfu/mL). Figure 5. Effect of ANT1 siRNA on ATP/ADP translocase activity (A), apoptosis (B, C), intracellular ROS (D), and time-dependent changes in Δψm (E) (n=5, each); 50 nM of ANT1 siRNA or control siRNA was transfected into HAEC. After 24 hours, cells were infected with Ad-β-gal or Ad-PGC-1α (5×106 pfu/mL). After 48 hours, cells were treated with 60 μmol/L of LA or vehicle. ATP/ADP translocase activity and ROS generation were measured at 4 hours, and apoptosis was measured at 16 hours. A–D, *P<0.05 vs control (without LA); **P<0.05 vs Ad-β-gal; #P<0.05 vs control siRNA. E, *P<0.05 vs baseline; **P<0.05 vs Ad-β-gal; #P<0.05 vs control siRNA. Jong Chul Won et al. Arterioscler Thromb Vasc Biol. 2010;30:290-297 Copyright © American Heart Association, Inc. All rights reserved.

Figure 6. Effect of Ad-PGC-1α or Ad-ANT1 shRNA on LA-induced impairment in vasorelaxation. Figure 6. Effect of Ad-PGC-1α or Ad-ANT1 shRNA on LA-induced impairment in vasorelaxation. Adenoviral overexpression of rat aortic ring was achieved by infection with Ad-PGC-1α aor Ad-ANT1 shRNA. Endothelium-dependent vasorelaxation was measured by the method described in Materials and Methods. A, Western blots showing changes in PGC-1α and ANT1 expressions. B, Effect of Ad-PGC-1α on LA-induced (60 μmol/L) impairment of vascular dilatory response. *P<0.05 vs control (Ad-β-gal alone); **P<0.05 vs LA plus Ad-β-gal. C, Effect of Ad-ANT1 shRNA on Ad-PGC-1α–induced changes in the vasodilatory response. *P<0.05 vs LA plus Ad-β-gal plus Ad-control shRNA; **P<0.05 vs LA plus Ad-PGC-1α plus Ad-control shRNA (n=5, each). Jong Chul Won et al. Arterioscler Thromb Vasc Biol. 2010;30:290-297 Copyright © American Heart Association, Inc. All rights reserved.