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Proteomic analysis of shear stress-mediated protection from TNF-alpha in endothelial cells. Microcirculation 2010 May;17(4):259-70 PMID: 20536739 PMCID: PMC3712086

Abstract

Previous studies have shown that physiological levels of shear stress can protect endothelial cells (ECs) from apoptotic stimuli. Here, we differentiate between acute and chronic protection and demonstrate the use of proteomic technologies to uncover mechanisms associated with chronic protection of ECs. We hypothesized that changes in abundance of proteins associated with the TNF-alpha signaling cascade orchestrate shear stress-mediated protection from TNF-alpha when cells are preconditioned with shear prior to the exposure of apoptotic stimuli. Detection of cleaved caspase 3 through Western blot analysis confirmed chronic shear stress-mediated protection from TNF-alpha. In the presence of the nitric oxide synthase inhibitor, LNMA (N(omega)-monomethyl-l-arginine), chronic protection remained. Treatment with a de novo protein synthesis inhibitor, cycloheximide, eliminated this protective effect. Isotopic-labeling experiments, coupled with LC-MS/MS (liquid chromatography-tandem mass spectrometry) of isolated components of the TNF-alpha pathway revealed that CARD9, a known activator of the NF-kappaB pathway, was increased (60%) in sheared cells versus nonsheared cells. This result was confirmed through Western blot analysis. Our data suggest that de novo formation of proteins is required for protection from TNF-alpha in ECs chronically exposed to shear stress, and that CARD9 is a candidate protein in this response.

Author List

Freed JK, Greene AS

Authors

Julie K. Freed MD, PhD Assistant Professor in the Anesthesiology department at Medical College of Wisconsin
Andrew S. Greene PhD Interim Vice Chair, Chief, Professor in the Biomedical Engineering department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Apoptosis
CARD Signaling Adaptor Proteins
Cells, Cultured
Endothelial Cells
Enzyme Inhibitors
Fas-Associated Death Domain Protein
I-kappa B Proteins
Models, Biological
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase
Proteomics
Rats
Signal Transduction
Stress, Mechanical
TNF Receptor-Associated Death Domain Protein
Tumor Necrosis Factor-alpha
omega-N-Methylarginine



View this publication's entry at the Pubmed website PMID: 20536739
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