Arterioscler Thromb Vasc Biol TNF-α Contributes to Endothelial Dysfunction by Upregulating Arginase in Ischemia/Reperfusion Injury by Xue Gao, Xiangbin Xu, Souad Belmadani, Yoonjung Park, Zhonghua Tang, Arthur M. Feldman, William M. Chilian, and Cuihua Zhang Arterioscler Thromb Vasc Biol Volume 27(6):1269-1275 June 1, 2007 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. In WT mice, mRNA expression of TNFR1 increased about 2-fold in isolated coronary arterioles in I/R vs WT-Sham, but I/R did not affect mRNA expression of TNFR2 in WT mice (Figure 1A). Figure 1. In WT mice, mRNA expression of TNFR1 increased about 2-fold in isolated coronary arterioles in I/R vs WT-Sham, but I/R did not affect mRNA expression of TNFR2 in WT mice (Figure 1A). NOS activity was higher in WT-Sham mice vs WT-I/R, TNF++/++-Sham, and TNF++/++-I/R mice, but NOS activity was not altered in TNF−/− mice before and after I/R (B). Arginase activity (ie, urea production) was lower in TNF−/− mice than that in WT mice. I/R increased arginase activity in WT mice but not in TNF−/− mice, and anti–TNF-α (Ab) decreased arginase activity in WT mice (C). n=5, n=number of experiments. Data represent mean±SD; *P<0.05 vs WT-Sham. Xue Gao et al. Arterioscler Thromb Vasc Biol. 2007;27:1269-1275 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Arginase protein expression was higher in TNF++/++ mice and was lower in TNF−/− mice than that in WT mice. Figure 2. Arginase protein expression was higher in TNF++/++ mice and was lower in TNF−/− mice than that in WT mice. I/R increased the arginase protein expression in WT and TNF++/++ mice, but did not affect the arginase expression in TNF−/− mice (Figure 2A and 2B). Anti–TNF-α decreased arginase expression in WT-I/R mice (Figure 2C and 2D). The blot was replicated on 4 mice in each group for each protein. *P<0.05 vs WT-Sham; # P<0.05 vs I/R. Xue Gao et al. Arterioscler Thromb Vasc Biol. 2007;27:1269-1275 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. The protein expression of eNOS was decreased in I/R vs sham, in WT and TNF++/++ mice. Figure 3. The protein expression of eNOS was decreased in I/R vs sham, in WT and TNF++/++ mice. The blot was replicated on 6 mice in each group for each protein. *P<0.05 vs WT-Sham. Xue Gao et al. Arterioscler Thromb Vasc Biol. 2007;27:1269-1275 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. Double immunostaining of MPO and arginase or arginase and von Willebrand factor (vWF) using specific antibodies followed by fluorescent-labeled secondary antibodies in mouse heart. Figure 4. Double immunostaining of MPO and arginase or arginase and von Willebrand factor (vWF) using specific antibodies followed by fluorescent-labeled secondary antibodies in mouse heart. A–C, Dual labeling of MPO (green) and arginase (red) in sham heart tissue. D–F, Dual labeling of MPO (green) and arginase (red) in heart tissue after I/R. G–I, Dual labeling of MPO (green) and arginase (red) in heart tissue pretreated with NOHA in I/R. J–L, Dual labeling of arginase (red) and vWF (green) in I/R. The white arrow shows a specific staining of MPO. The pink arrow shows a specific staining of arginase in heart tissue, and the dashed white arrow shows the specific staining of Von Willebrand Factor. M–O, Negative control (=negative CTL) panel shows an absence of staining in coronary vessels using only the secondary antibodies; these results indicate the specificity of our immunostaining. The size bar in A is the same for all panels. Original magnification ×63. Data shown are representative of 3 separate experiments. Xue Gao et al. Arterioscler Thromb Vasc Biol. 2007;27:1269-1275 Copyright © American Heart Association, Inc. All rights reserved.