Abstract
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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / pharmacology
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Animals
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Arachidonate 12-Lipoxygenase / genetics*
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Arachidonate 12-Lipoxygenase / metabolism
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Arachidonate 15-Lipoxygenase / genetics*
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Arachidonate 15-Lipoxygenase / metabolism
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Base Sequence
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Blotting, Western
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Cell Adhesion
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Cell Line
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Cell Movement
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Cells, Cultured
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Chemokine CCL2 / metabolism
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Chemokines / metabolism
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DNA Primers / chemistry
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DNA, Complementary / metabolism
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Down-Regulation
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Endothelium, Vascular / metabolism
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Ethidium / analogs & derivatives*
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Ethidium / pharmacology
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Fibronectins / chemistry
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Fibronectins / metabolism
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Gene Silencing*
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Green Fluorescent Proteins / metabolism
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Humans
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Immunoblotting
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Inflammation
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Lipoproteins, LDL / metabolism
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Macrophages / metabolism
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Mice
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Microscopy, Fluorescence
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Molecular Sequence Data
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Monocytes / metabolism
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Myocytes, Smooth Muscle / cytology
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Oxidants / metabolism
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Oxidative Stress
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Polymerase Chain Reaction
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Promoter Regions, Genetic
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Protein Binding
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RNA, Messenger / metabolism
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RNA, Small Interfering / metabolism*
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Rats
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Recombinant Fusion Proteins / metabolism
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Reverse Transcriptase Polymerase Chain Reaction
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Superoxides / metabolism
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Transfection
Substances
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12-15-lipoxygenase
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Chemokine CCL2
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Chemokines
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DNA Primers
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DNA, Complementary
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Fibronectins
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Lipoproteins, LDL
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Oxidants
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RNA, Messenger
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RNA, Small Interfering
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Recombinant Fusion Proteins
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dihydroethidium
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Superoxides
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Green Fluorescent Proteins
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12-Hydroxy-5,8,10,14-eicosatetraenoic Acid
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Arachidonate 12-Lipoxygenase
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Arachidonate 15-Lipoxygenase
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Ethidium