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277 IDENTIFICATION OF 5-, 12/15-LIPOXYGENASE AND NADPH OXIDASE AS KEY MEDIATORS OF OXIDIZED LOW DENSITY LIPOPROTEIN-INDUCED APOPTOSIS OF HUMAN AORTIC SMOOTH MUSCLE CELLS: IMPLICATIONS FOR ATHEROSCLEROTIC PLAQUE DESTABILIZATION.
  1. L. Semprun-Prieto1,
  2. S. Sukhanov1,
  3. Y. Higashi1,
  4. J. Titterington1,
  5. P. Delafontaine1
  1. 1Tulane University School of Medicine, New Orleans, LA

Abstract

Apoptosis of smooth muscle cells (SMCs) is a prominent feature of advanced atherosclerotic plaque. We have previously shown that the proatherogenic molecule oxidized low-density lipoprotein (OxLDL) is associated with apoptotic SMCs in advanced plaques of human aorta and that OxLDL produces marked SMC apoptosis in vitro. In human aortic smooth muscle cells (HASMCs), the antioxidant Tiron (1 mM) suppressed superoxide production (by 18 ± 2%, DHE assay) and peroxide generation (by 88 ± 4%, CDC assay) induced by OxLDL (80 μg/mL, 16 hours) and completely blunted HASMC apoptosis (by 89 ± 2.6%, CELL DEATH ELISA), indicating that OxLDL-induced apoptosis was mediated by a redox-sensitive mechanism. The lipoxygenase (LOX) inhibitors AA-861, 5 μM and NDGA, 10 μM decreased OxLDL-induced superoxides (by 15 ± 2% and 25 ± 4%, respectively) and peroxides (by 48 ± 3% and 62 ± 6%, respectively) and NDGA completely blocked HASMC apoptosis (flow cytometry with annexin V staining). AA-861, 10 μM, another LOX inhibitor, Baicalein, 10 μM, and the NADPH oxidase-specific blocker apocynin, 100 μM suppressed OxLDL-induced HASMC apoptosis (by 93.0 ± 7%, 94 ± 0.4%, and 83 ± 3%, respectively) as was measured using cell death ELISA. To identify the oxidases involved in OxLDL-induced apoptosis, we studied the effect of OxLDL on SMC isolated from the aorta of 5-LOX- and 12/15-LOX-deficient and wild-type (WT) mice. 12/15-LOX deficiency markedly reduced OxLDL-induced superoxides and peroxides (by 31 ± 3% and 62 ± 7%, respectively, compared with WT). OxLDL-induced apoptosis was completely inhibited in 5-LOX- or 12/15-LOX-deficient SMC (by 82.6 ± 0.5% and 94.1 ± 0.7%, respectively); however, OxLDL produced 90.8 ± 4.4% apoptosis of WT SMC compared with control (0.5 μM staurosporine). In summary, we have identified 5-, 12/15-lipoxygenase and NADPH oxidase as key mediators of OxLDL-induced oxidative stress and SMC apoptosis. These data establish a novel functional relationship between different oxidases and together with our previous findings have major implications for understanding the mechanism of SMC depletion in advanced atherosclerotic plaque leading to plaque destabilization and acute coronary events.

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