Viral vector-mediated 12/15-lipoxygenase overexpression in vascular smooth muscle cells enhances inflammatory gene expression and migration.
Increased expression and activity of 12/15-lipoxygenase (12/15-LO) in vascular smooth muscle cells (VSMCs) play a key role in the pathogenesis of diabetes and vascular complications. However, the consequences of 12/15-LO overexpression for VSMC migration and inflammatory gene expression are not known. In this study, 12/15-LO was overexpressed using adeno- and baculoviral vectors in human VSMC (HVSMCs) and proatherogenic responses compared with control enhanced green fluorescent protein (EGFP)-expressing cells. HVSMCs transduced with 12/15-LO viruses expressed high levels of enzymatically active protein and produced increased levels of the LO product, 12(S)-hydroxyeicosatetraenoic acid. 12/15-LO-overexpressing HVSMCs exhibited increased oxidant stress, activation of p38 mitogen-activated protein kinase, migration and inflammatory gene expression relative to HVSMCs expressing EGFP. Furthermore, inflammatory gene expression induced by 12/15-LO overexpression was abolished by anti-oxidants, siRNAs targeting p65 (nuclear factor-kappaB), or new-generation baculoviruses expressing inhibitory IkappaBalpha or IkappaBalpha superrepressor mutant. Thus, we have used novel viral vector delivery systems, including baculoviruses, for the first time to deliver foreign genes into VSMCs and thereby demonstrated that 12/15-LO overexpression increases oxidant stress, mitogen-activated protein kinase activation, migration and inflammatory genes in VSMCs and that NF-kappaB is a key downstream effector. Enhanced proatherogenic responses in VSMCs triggered by increased 12/15-LO levels under pathological conditions may contribute to vascular dysfunction.
Dwarakanath
Roopashree S
RS
Department of Diabetes, Beckman Research Institute of City of Hope, Duarte, Calif., USA.
Sahar
Saurabh
S
Lanting
Linda
L
Wang
Nanping
N
Stemerman
Michael B
MB
Natarajan
Rama
R
Reddy
Marpadga A
MA
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D001093:Arachidonate 15-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism; D007249:Inflammation / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D018836:Inflammation Mediators / Q000378:metabolism; D051379:Mice; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D000072000:NF-KappaB Inhibitor alpha; D018384:Oxidative Stress; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000975:Antioxidants; D020672:I-kappa B Proteins; D018836:Inflammation Mediators; D009097:Multienzyme Complexes; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D034741:RNA, Small Interfering; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; C521960:Alox15 protein, mouse; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase; D048051:p38 Mitogen-Activated Protein Kinases