Increased apoptosis and depletion of smooth muscle cells (SMCs) plays an important role in atherosclerotic plaque vulnerability to rupture, a major cause of acute coronary events. We previously reported that the ability of oxidized low-density lipoprotein (oxLDL) to down-regulate insulin-like growth factor 1 receptor (IGF-1R) expression is a critical mediator of oxLDL-induced SMC apoptosis. OxLDL-induced IGF-1R down-regulation was dependent on the generation of reactive oxygen species (ROS), namely peroxide and superoxide as measured by H2DCFDA and dihydroethidium fluorescence. However, an oxidative stress-inducing agent, paraquat (0-800 μM, up to 24 hours), did not stimulate IGF-1R down-regulation, suggesting that another downstream event was critical for receptor down-regulation. To explore mechanisms, we assessed the ability of oxLDL to induce nitric oxide (NO) generation in human aortic SMC culture using DAF-FM fluorescence. OxLDL (60 μg/mL, 4 hours) enhanced NO generation by 2.4-fold vs native LDL (nLDL) control (n = 8, p < .01). As an enzymatic source, we detected NO synthase 2 by Western blot analysis. To evaluate the significance of oxLDL-induced NO generation in mediating IGF-1R down-regulation, we incubated SMCs with 60 μg/mL oxLDL plus NO-scavenger, carboxy-PTIO (40 μM). Carboxy-PTIO completely prevented OxLDL induced IGF-1R down-regulation. Consistent with this observation, the NO-donor, S-nitroso-N-acetylpenisillamine (SNAP), in combination with paraquat, decreased IGF-1R protein level by 60% (400 μM paraquat and 1 mM SNAP, n = 4, p < .01), whereas SNAP or paraquat alone did not down-regulate IGF-IR. RNase protection assay, real-time PCR, and pulse-chase labeling experiments indicated that oxLDL decreased IGF-1R mRNA expression by 40% and reduced IGF-1R protein half-life from 20 hours (nLDL) to 12 hours (oxLDL). In summary, oxLDL enhanced NO and ROS generation in human SMC, and these radicals acted coordinately to down-regulate IGF-1R via a decrease in gene expression and in protein stability. These findings provide novel insights into OxLDL-triggered oxidant signaling and mechanisms of SMC depletion that contribute to plaque destabilization and acute coronary events.
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