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Journal of Lipid Research, Vol. 46, 220-229, February 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology
Gonda Diabetes Research Center, Beckman Research Institute of City of Hope, Duarte, CA 91010
2 To whom correspondence should be addressed. e-mail: rnatarajan{at}coh.org
The leukocyte-type 12/15-lipoxygenase (12/15-LO) has been implicated in the pathogenesis of atherosclerosis, hypertension, and diabetes. 12/15-LO and its products are associated with LDL oxidation, cellular growth, migration, adhesion, and inflammatory gene expression in monocytes/macrophages, endothelial cells, and vascular smooth muscle cells (VSMCs). Our objective, therefore, was to develop novel expression vectors for short interfering RNAs (siRNAs) targeting 12/15-LO to evaluate its functional relevance in macrophages and VSMCs. We used a PCR-based approach to rapidly identify effective siRNA target sites on mouse 12/15-LO and initially tested their efficacy on a fusion construct of 12/15-LO cDNA and enhanced green fluorescent protein. We then cloned these U6 promoter+siRNA PCR products into plasmid vectors [short hairpin siRNAs (shRNAs)] to knockdown endogenous 12/15-LO expression in mouse macrophages and also rat and mouse VSMCs. Furthermore, the functional effects of shRNA-mediated 12/15-LO knockdown were noted by the reduced oxidant stress and chemokine [monocyte chemoattractant protein-1 (MCP-1)] expression in a differentiated mouse monocytic cell line as well as by the reduced cellular adhesion and fibronectin expression in VMSCs. Knocking down 12/15-LO expression also reduced the expression of inflammatory genes, MCP-1, vascular cell adhesion molecule-1, and interleukin-6 in VSMCs.
Our results illustrate the functional relevance of 12/15-LO activation in macrophages and VSMCs and its relationship to oxidant stress and inflammation.
Supplementary key words vascular smooth muscle cells • monocytes • macrophages • atherosclerosis • RNA interference • inflammatory genes • monocyte chemoattractant protein-1 • arachidonic acid
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