Abstract
AimsNADPH oxidase (NOX) is the primary source of reactive oxygen species (ROS) in vascular smooth muscle cells (SMC) and is proposed to play a key role in redox signaling involved in the pathogenesis of cardiovascular disease. Growth factors and cytokines stimulate coronary SMC (CSMC) phenotypic modulation, proliferation, and migration during atherosclerotic plaque development and restenosis. We previously demonstrated that increased expression and activity of intermediate-conductance Ca2+-activated K+ channels (KCNN4) is necessary for CSMC phenotypic modulation and progression of stenotic lesions. Therefore, the purpose of this study was to determine whether NOX is required for KCNN4 upregulation induced by mitogenic growth factors.Methods and ResultsDihydroethidium micro-fluorography in porcine CSMCs demonstrated that basic fibroblast growth factor (bFGF) increased superoxide production, which was blocked by the NOX inhibitor apocynin (Apo). Apo also blocked bFGF-induced increases in KCNN4 mRNA levels in both right coronary artery sections and CSMCs. Similarly, immunohistochemistry and whole cell voltage clamp showed bFGF-induced increases in CSMC KCNN4 protein expression and channel activity were abolished by Apo. Treatment with Apo also inhibited bFGF-induced increases in activator protein-1 promoter activity, as measured by luciferase activity assay. qRT-PCR demonstrated porcine coronary smooth muscle expression of NOX1, NOX2, NOX4, and NOX5 isoforms. Knockdown of NOX5 alone prevented both bFGF-induced upregulation of KCNN4 mRNA and CSMC migration.ConclusionsOur findings provide novel evidence that NOX5-derived ROS increase functional expression of KCNN4 through activator protein-1, providing another potential link between NOX, CSMC phenotypic modulation, and atherosclerosis.
Highlights
One of the central components of cardiovascular disease (CVD) is atherosclerosis, which is a slow degenerative process characterized by remodeling of the arterial wall and formation of atherosclerotic plaques [1,2]
Dihydroethidium micro-fluorography in porcine coronary SMC (CSMC) demonstrated that basic fibroblast growth factor increased superoxide production, which was blocked by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) inhibitor apocynin (Apo)
We have previously shown that platelet derived growth factor-BB (PDGF-BB) induced coronary smooth muscle cell (SMC) (CSMC) phenotypic modulation requires the functional upregulation of intermediate-conductance Ca2+-activated K+ channels (KCNN4) [7]
Summary
One of the central components of cardiovascular disease (CVD) is atherosclerosis, which is a slow degenerative process characterized by remodeling of the arterial wall and formation of atherosclerotic plaques [1,2]. Studies have shown that KCNN4 upregulation is required for mitogen-induced suppression of SMC markers as well as vascular SMC migration and proliferation, and has been shown to occur during atherosclerosis and restenosis indicating these channels play a key role in coronary plaque formation [7,15,17,18]. Studies have shown that in addition to being activated by growth factors, serum, and cytokines [24], the AP-1 components are increased following coronary angioplasty [18] These results support the idea that AP-1 is a critical component of signaling pathways involved in KCNN4 regulation [13]
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