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Cardiovascular Research Advance Access [Accepted Manuscript] published online on May 15, 2009
Cardiovascular Research, doi:10.1093/cvr/cvp154
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2009. For permissions please email: journals.permissions@oxfordjournals.org.

Shear Flow Increases S-nitrosylation of Proteins in Endothelial Cells

Bin Huang1, Shih Chung Chen2 and Danny Ling Wang1,*

1 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 11529.
2 Division of Cardiology, Taipei Medical University, Wan Fang Hospital, Taipei, Taiwan 11696.

* Corresponding author: Dr. Danny Ling Wang, Cardiovascular Division, Institute of Biomedical Sciences, Academia Sinica, 128 sec. 2 Academia Rd. NanKang, Taipei, Taiwan 11529, TEL: +886-2-23699132, FAX: +886-2-27899143, E-MAIL: lingwang{at}ibms.sinica.edu.tw

Aims: Endothelial cells constantly exposed to shear flow increase nitric oxide production via the activation of endothelial nitric oxide synthase. Nitric oxide-mediated S-nitrosylation has recently been identified as an important posttranslational modification that may alter signaling and/or protein function. S-Nitrosylation of endothelial proteins after shear flow treatment has not been fully explored. In this study, the CyDye switch method was utilized to examine S-nitrosylated proteins in endothelial cells after exposure to shear flow.

Methods: Human umbilical vein endothelial cells were subjected to shear flow for 30 minutes, and S-nitrosylated proteins were detected by the CyDye switch method. In principle, free thiols in proteins become blocked by alkylation, the S-nitrosylated bond is reduced by ascorbate, and then CyDye labels proteins. Proteins that separately labeled with Cy3 or Cy5 were mixed and subjected to two-dimensional gel electrophoresis for further analysis.

Results: More than 100 S-nitrosoproteins were detected in static and shear-treated endothelial cells. Among these, 12 major proteins of heterogeneous function showed a significant increase in S-nitrosylation following shear flow. The S-nitrosylated residues in tropomyosin and vimentin, which were localized in the hydrophobic motif of each protein, were identified as Cys170 and Cys328, respectively.

Conclusion: Posttranslational S-nitrosylation of proteins in endothelial cells can be detected by a reliable CyDye switch method. This flow-induced S-nitrosylation of endothelial proteins may be essential for the adaptation and remodeling of endothelial cells under flow conditions.

KEYWORDS Shear flow; S-nitrosylation; 2-D DIGE; CyDye switch; endothelial cells

Time for primary review: 24 Days


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