Abstract
Bovine aortic endothelial cells (ECs) respond to nitric oxide (NO) donors by activating the redox-sensitive NF-E2-related factor 2/antioxidant response element pathway and up-regulating heme oxygenase (HO)-1 expression. EC exposure to steady laminar shear stress causes a sustained increase in NO, a transient increase in reactive oxygen species (ROS), and activation of the HO-1 gene. Because steady laminar flow increases the mitochondrial superoxide (
) production, we hypothesized that mitochondria-derived ROS play a role in shear-induced HO-1 expression. Flow (10 dynes/cm2, 6 h)-induced expression of HO-1 protein was abolished when BAECs were preincubated and sheared in the presence of either NG-nitro-l-arginine methyl ester or N-acetyl-l-cysteine, suggesting that either NO or ROS up-regulates HO-1. Ebselen and diphenylene iodonium blocked HO-1 expression, and uric acid had no effect. The mitochondrial electron transport chain inhibitors, myxothiazol, rotenone, or antimycin A, and the mitochondria-targeted antioxidant peptide, Szeto-Schiller (SS)-31, which scavenges 
, hydrogen peroxide (H2O2), peroxynitrite, and hydroxyl radicals, markedly inhibited the increase in HO-1 expression. These data collectively suggest that mitochondrial H2O2 mediates the HO-1 induction. MitoSOX and 2′,7′-dichlorofluorescin (DCF) fluorescence showed that mitochondrial 
levels and intracellular peroxides, respectively, are higher in sheared ECs compared with static controls and, in part, dependent on NO. SS-31 significantly inhibited both the shear-induced MitoSOX and DCF fluorescence signals. Either phosphatidylinositol 3-kinase or mitogen-activated protein kinase cascade inhibitors blocked the HO-1 induction. In conclusion, under shear, EC mitochondria-derived H2O2 diffuses to the cytosol, where it initiates oxidative signaling leading to HO-1 up-regulation and maintenance of the atheroprotective EC status.
Footnotes
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This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants HL91417, HL38324, HL63744, HL65608]; the National Institutes of Health National Institute on Drug Abuse [Grant DA08924]; and the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK73595].
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Patent applications have been filed by Cornell Research Foundation Inc. for the technology (SS peptides) described in this article. H.H.S. is the inventor. Cornell Research Foundation Inc., on behalf of Cornell University, has licensed the technology for further research and development to a commercial enterprise in which Cornell Research Foundation Inc. and H.H.S. have financial interests.
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doi:10.1124/jpet.108.145557.
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ABBREVIATIONS: HO, heme oxygenase; Nrf2, NF-E2-related factor 2; ARE, antioxidant response element; Keap1, Kelch-like erythroid-derived Cap-N-Collar-Homology-associated protein 1; 15d-PGJ2, 15-deoxy-Δ12,14-prostaglandin J2; EC, endothelial cell; NO, nitric oxide;
, superoxide; ROS, reactive oxygen species; HUVEC, human umbilical vein EC; SNO, S-nitrosothiol; ONOO-, peroxynitrite; ETC, electron transport chain; BAEC, bovine aortic EC; NOS, NO synthase; l-NAME, NG-nitro-l-arginine methyl ester; NAC, N-acetyl-l-cysteine; UA, uric acid; SS, Szeto-Schiller; H2O2, hydrogen peroxide; OH., hydroxyl radical; GSH, glutathione; DPI, diphenylene iodonium; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; PD98059, 2′-amino-3′-methoxyflavone; ERK, extracellular signal-regulated kinase; SB202190, 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]phenol; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; DAPI, 4′,6-diamidino-2-phenylindole; DCF, 2′,7′-dichlorofluorescin. -
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The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. - Received August 31, 2008.
- Accepted January 7, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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