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. 2011 Sep 1;15(5):1379-88.
doi: 10.1089/ars.2010.3645. Epub 2011 Apr 14.

Oscillatory shear stress induces mitochondrial superoxide production: implication of NADPH oxidase and c-Jun NH2-terminal kinase signaling

Wakako Takabe  1 , Nelson Jen, Lisong Ai, Ryan Hamilton, Sky Wang, Kristin Holmes, Farhad Dharbandi, Bhavraj Khalsa, Steven Bressler, Mark L Barr, Rongsong Li, Tzung K Hsiai
Affiliations

Oscillatory shear stress induces mitochondrial superoxide production: implication of NADPH oxidase and c-Jun NH2-terminal kinase signaling

Wakako Takabe et al. Antioxid Redox Signal. .

Abstract

Fluid shear stress is intimately linked with vascular oxidative stress and atherosclerosis. We posited that atherogenic oscillatory shear stress (OSS) induced mitochondrial superoxide (mtO2•-) production via NADPH oxidase and c-Jun NH(2)-terminal kinase (JNK-1 and JNK-2) signaling. In bovine aortic endothelial cells, OSS (±3 dyn/cm2) induced JNK activation, which peaked at 1 h, accompanied by an increase in fluorescein isothiocyanate-conjugated JNK fluorescent and MitoSOX Red (specific for mtO2•- production) intensities. Pretreatment with apocynin (NADPH oxidase inhibitor) or N-acetyl cysteine (antioxidant) significantly attenuated OSS-induced JNK activation. Apocynin further reduced OSS-mediated dihydroethidium and MitoSOX Red intensities specific for cytosolic O2•- and mtO2•- production, respectively. As a corollary, transfecting bovine aortic endothelial cells with JNK siRNA (siJNK) and pretreating with SP600125 (JNK inhibitor) significantly attenuated OSS-mediated mtO2•- production. Immunohistochemistry on explants of human coronary arteries further revealed prominent phosphorylated JNK staining in OSS-exposed regions. These findings indicate that OSS induces mtO2•- production via NADPH oxidase and JNK activation relevant for vascular oxidative stress.

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Figures

FIG. 1.
FIG. 1.
Oscillatory shear stress (OSS) induced transient c-Jun NH2-terminal kinase (JNK) activation. Bovine aortic endothelial cells (BAEC) monolayers were exposed to static condition or OSS for 30 min, 1 h, or 2 h. Phosphorylated JNK was then analyzed by Western blot analysis, quantified by densitometry, and expressed as fold ratios relative to total JNK and static conditions. OSS induced a peaked JNK activation at 1 h (both JNK isoforms) (p < 0.01 vs. static condition). The experiments were performed in triplicates.
FIG. 2.
FIG. 2.
JNK activation in response to OSS. p-JNK was stained with fluorescein isothiocyanate-anti-p-JNK (green). Active cellular mitochondria were localized using MitoTracker Red (red). Nuclei were stained with DAPI (blue). (a) Under static conditions, JNK green fluorescence was hardly visible. (b) In response to OSS, a significant JNK green fluorescence developed after 30 min, accompanied by yellowish/orange signals as a result of merged spectra between fluorescein isothiocyanate and MitoTracker Red. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).
FIG. 3.
FIG. 3.
Apocynin or NAC attenuated OSS-induced JNK phosphorylation. (a) BAECs were pretreated with apocynin (1 mM) for 2 h before OSS exposure. Apocynin significantly reduced OSS-induced JNK phosphorylation expressed as fold change relative to total JNK in comparison with the untreated condition (*p < 0.01 vs. static conditions. #p < 0.01 vs. OSS, n = 3). (b) Pretreatment with 5 mM of N-acetyl cysteine (NAC) also significantly reduced OSS-induced JNK phosphorylation (*p < 0.01 vs. static conditions. #p < 0.01 vs. OSS, n = 3). All studies were performed in duplicates.
FIG. 4.
FIG. 4.
Apocynin attenuated oscillatory shear-induced dihydroethedium (DHE) intensities. OSS exposure increased DHE staining compared to the static conditions. This increase in DHE intensity was attenuated in response to Apocynin treatment (1 mM). (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).
FIG. 5.
FIG. 5.
Inhibition of JNK attenuated MitoSOX Red intensities. BAEC monolayers were pretreated with JNK inhibitor, SP600125, and mitochondrial superoxide (mtO2•−)-specific dye, MitoSOX Red, before flow exposure. Measurements were performed using BD LSR II flow cytometer. (Top) The data were presented by histograms in terms of the mean intensity of MitoSOX fluorescence normalized to those of the static conditions. (Bottom) OSS-induced MitoSOX intensity was significantly attenuated in response to SP600125 (p < 0.01 vs. static condition. #p < 0.01 vs. OSS, n = 3).
FIG. 6.
FIG. 6.
Knockdown of JNK reduced OSS-mediated MitoSOX Red intensities. (a) BAEC were transfected with siJNK or scramble (scr) siRNA for 48 h. Cell lysate was used to verify the efficiency of siJNK on the protein level of JNK. The blots were representative of two independent experiments with similar results. (b, top) The data were presented by histograms in terms of the mean intensity of MitoSOX fluorescence normalized to those of the static controls. (b, bottom) While scrambled JNK did not affect OSS-mediated MitoSOX intensity (p < 0.01 vs. static with scr siRNA, n = 3), transfecting BAECs with siJNK significantly reduced OSS-mediated MitoSOX intensity (#p < 0.01 vs. OSS with scr siRNA, n = 3).
FIG. 7.
FIG. 7.
Inhibition of NADPH oxidase attenuated OSS-induced MitoSOX Red intensities. Measurements were performed using BD LSR II flow cytometer. (Top) The data were presented by histograms in terms of the mean intensity of MitoSOX Red fluorescence normalized to those of the static conditions. (Bottom) While OSS induced an increase in MitoSOX Red fluorescence by 1.75 ± 0.2 (p < 0.01 vs. static conditions, n = 3), pretreatment with apocynin significantly attenuated OSS-induced MitoSOX Red intensity (#p < 0.01 vs. OSS, n = 3).
FIG. 8.
FIG. 8.
Immunohistochemistry of explants of human coronary arteries. The blue boxes indicate areas of interest that are subsequently magnified in successive panels. (a–c) In the OSS-exposed regions such as the left main coronary bifurcation, endothelial cells were stained positive for activated JNK. (c) Vaso vasorum from the same cross section also revealed prominent activated JNK staining (brown). (d–g) Cross section from the greater curvature of the right coronary artery revealed prominent activated JNK and cytochrome c staining in the endothelial cells. Positive von Willebrand factor (vWF) staining indicated presence of endothelial cells, while cytochrome c revealed presence of mitochondria. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).
FIG. 9.
FIG. 9.
Proposed mechanism of OSS-mediated mtO2•− production. OSS increased cytosolic superoxide production via NADPH oxidase. Cytosolic superoxide subsequently activated JNK, which in turn induced the production of mtO2•−.
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References

    1. Ai L. Rouhanizadeh M. Wu J. Takabe W. Yu H. Alavi M. Li R. Chu Y. Miller J. Heistad D. Hsiai T. Pulsatile and oscillatory shear stress influences spatial variations in vascular Mn-SOD expression and nitrotyrosine formation. Am J Physiol Cell Physiol. 2008;294:C1576–C1585. - PMC - PubMed
    1. Aoki H. Kang PM. Hampe J. Yoshimura K. Noma T. Matsuzaki M. Izumo S. Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes. J Biol Chem. 2002;277:10244–10250. - PubMed
    1. Arita Y. Harkness SH. Kazzaz JA. Koo HC. Joseph A. Melendez JA. Davis JM. Chander A. Li Y. Mitochondrial localization of catalase provides optimal protection from H2O2-induced cell death in lung epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2006;290:L978–L986. - PubMed
    1. Bochkov V. Kadl A. Huber J. Gruber F. Binder B. Leitinger N. Protective role of phospholipid oxidation products in endotoxin-induced tissue damage. Nature. 2002;419:77–81. - PubMed
    1. Chiu JJ. Chen LJ. Lee CI. Lee PL. Lee DY. Tsai MC. Lin CW. Usami S. Chien S. Mechanisms of induction of endothelial cell E-selectin expression by smooth muscle cells and its inhibition by shear stress. Blood. 2007;110:519–528. - PMC - PubMed

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