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1 OXIDIZED LOW-DENSITY LIPOPROTEIN DOWN-REGULATES INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR VIA ENHANCED GENERATION OF NITRIC OXIDE AND REACTIVE OXYGEN SPECIES IN HUMAN AORTIC SMOOTH MUSCLE CELLS.
  1. Y. Higashi1,
  2. S. Sukhanov1,
  3. S. Parthasarathy2,
  4. P. Delafontaine1
  1. 1Tulane University School of Medicine, New Orleans, LA
  2. 2The Ohio State University College of Medicine, Columbus, OH.

Abstract

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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