[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] A key risk factor for the development of atherosclerosis is familial hypercholesterolemia (FH), a genetic disease characterized by elevated levels of low density lipoprotein (LDL). Studies have shown that oxidative stress and vascular smooth muscle cell (VSMC) phenotypic modulation play critical roles in the development and stability of atherosclerotic plaques. The key source of reactive oxygen species (ROS) contributing to oxidative stress in the vasculature is the enzymatic complex nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Upregulation of intermediateconductance Ca[superscript 2 +]-activated K[superscript +] channels (K[subscript Ca]3.1) and modification of NADPH oxidase activity have been shown to mediate phenotypic modulation of coronary smooth muscle cells (CSMC). It remains unclear, however, whether K[subscript Ca]3.1 expression and activity are altered in atherosclerotic coronary smooth muscle of individuals with FH, and whether NADPH oxidase plays a role in atherosclerosis through regulation of K[subscript Ca]3.1 channels. Our objective was thus twofold, 1) to determine whether K[subscript Ca]3.1 expression and activity are increased in CSMCs isolated from FH swine, and 2) to determine if NADPH oxidase plays a role in growth factor-induced upregulation of K[subscript Ca]3.1. Right coronary artery (RCA) sections from 2 year old FH swine showed a [approximate sign]15 fold increase in artery stenosis accompanied by significantly elevated triglyceride and cholesterol values. In the media of the diseased FH coronaries, there was a trend for increased K[subscript Ca]3.1mRNA expression and K[subscript Ca]3.1 protein expression was elevated [approximate sign]20% compared to control coronaries. In addition, K[subscript Ca]3.1 channel activity increased almost 2- fold in coronary artery cells isolated from FH swine compared to control animals. In cultured CSMCs, basic fibroblast growth factor (bFGF) increased superoxide (O[subscript 2] [superscript .-]) production which was inhibited by treatment with the NADPH oxidase inhibitor apocynin (Apo). Treatment with bFGF increased K[subscript Ca]3.1 mRNA levels [aproximate sign]2.5 fold in both right coronary artery (RCA) sections and CSMCs, while addition of Apo prevented the increase. Furthermore, inhibition of NADPH oxidase abolished the bFGF-induced increase in coronary smooth muscle K[subscript Ca]3.1 protein expression and CSMC K[subscript Ca]3.1 channel activity. Treatment with bFGF significantly increased activator protein-1 (AP-1) promoter activity which was inhibited by addition of Apo. RCA and CSMC express all four cardiovascular Nox isoforms (Nox1, Nox2, Nox4, Nox5) with Nox4 being the predominant isoform. Treatment with bFGF decreased Nox1, Nox2, and Nox4 CSMC message, while treatment with Apo increased the mRNA expression of all four isoforms. Knock down of Nox2 and Nox4 did not affect the K[subscript Ca]3.1 message response to bFGF or Apo. Consistent with our earlier findings of increased medial K[subscript Ca]3.1 expression in FH coronaries; whole vessel K[subscript Ca]3.1 mRNA expression was increased in FH coronary smooth muscle and Nox2 rather than Nox4 was the predominant Nox isoform. Our findings demonstrate that K[subscript Ca]3.1 is upregulated in coronary smooth muscle of FH swine and support previous research indicating K[subscript Ca]3.1 plays a key role in the development and progression of atherosclerosis. Our findings also provide novel evidence that NADPH oxidase contributes to VSMC phenotypic modulation associated with atherosclerosis through AP-1 transcriptional upregulation of K[subscript Ca]3.1.
Upregulation of Intermediate-Conductance Ca2+-Activated K+ Channels (KCNN4) in Porcine Coronary Smooth Muscle Requires NADPH Oxidase 5 (NOX5)
