FOXO3A elicits vascular smooth muscle cell apoptosis through release of MMP-13 and subsequent degradation of the extracellular matrix

Abstract

s / Atherosclerosis 241 (2015) e32ee71 e57 apolipoprotein B100 modification in MGmin-LDL.A computed structural model predicted that methylglyoxal modification of apolipoprotein B100 induces distortion, increasing exposure of the N-terminal proteoglycan binding domain on the surface of LDL. Conclusion: Methylglyoxal modification of LDL forms small, dense LDL with increased atherogenicity. This non-oxidative process may explain escalation of cardiovascular risk in diabetes and renal failure, and resistance of early stage atherosclerosis to antioxidant therapy. Quantitation of methylglyoxal-modified LDL may improvecardiovascular disease risk models and therapeutics to decrease methylglyoxal may improve preventive treatment of cardiovascular disease. EAS-0611. FOXO3A ELICITS VASCULAR SMOOTH MUSCLE CELL APOPTOSIS THROUGH RELEASE OF MMP-13 AND SUBSEQUENT DEGRADATION OF THE EXTRACELLULAR MATRIX A. Fellows, Z. Yang, M.R. Bennett, H. Yu. Department of Cardiovascular Medicine, Addenbrooke's Hospital, Cambridge, United Kingdom Aims: Vulnerable atherosclerotic plaques are characterised by heightened vascular smooth muscle cell (VSMC) apoptosis and degradation of the extracellular matrix (ECM) within the fibrous cap. The ECM can be degraded by matrix metalloproteinase enzymes (MMPs), thereby promoting VSMC apoptosis. We have previously found that VSMCs in human atherosclerotic plaques show impaired inhibition of the pro-apoptotic transcription factor FOXO3a and we investigated the precise mechanisms underlying this VSMC apoptosis. Methods: We generated rat VSMCs that stably express an inducible mutant of FOXO3a (FOXO3aA3ER) which cannot be inhibited by Akt. We usedmicroarrays, qPCR andWestern blots to identify FOXO3a target genes, flow cytometry and MTT assay to determine cell death, and in situ fluorescence gelatin zymography to assay MMP activity. Results: FOXO3a activation induced profound cell death in FOXO3aA3ER cells (48.0±9.0% vs. 11.8±3.7% control, p<0.01). In addition to known pro-apoptotic genes such as BIM (6.24-fold), FOXO3a potently increased mRNA expression of MMP-13 (371-fold) and potentiated the expression and cleavage of MMP-13 protein, indicating greater release as well as activation. FOXO3a-induced MMP-13 secretion triggered significant enzymatic digestion of fluorescent gelatin surrounding VSMCs, and supplementation of gelatin provided substantial protection against FOXO3a-mediated apoptosis (19.3% vs. 68.0%). Finally, apoptosis of FOXO3aER cells was rescued by MMP-13-selective small molecule inhibitors, and to a greater extent than the broad-spectrum MMP inhibitor GM6001. Conclusions: FOXO3a induces VSMC apoptosis both directly and via MMP13-mediated degradation of the protective ECM.We predict that FOXO3a is a potent inducer of vulnerable plaques in atherosclerosis through this mechanism. EAS-0804. EFFECT OF ENGINEERED NANOPARTICLES ON PLATELET ACTIVATION AND THROMBUS FORMATION IN MAN J.B. Raftis , R. Duffin , A. Hunter , N. Joshi , A. Lucking , S. Alam, D.E. Newby , K. Donaldson , N.L. Mills . Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom; Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom Aims: Engineered nanoparticles, developed to facilitate imaging of atherosclerosis and as a drug delivery tool, share properties with combustion-derived particles in air pollution that have been associated with thrombosis and acute myocardial infarction. We hypothesize that engineered nanoparticles will exert similar pro-thrombotic effects in man. Methods: We assessed the effects of ultra-small superparamagnetic iron oxide nanoparticles (USPIO) nanoparticles on platelet activation, aggregation, and thrombus formation using flowcytometric techniques and the Badimon chamber (an ex vivo model of thrombosis). In a randomised, double-blind, cross-over study 12 healthy volunteers received saline control or USPIO (150 mg/mL & 300 mg/mL) added to whole blood in the extra-corporeal circuit of the Badimon chamber. In 8 patients with abdominal aortic aneurysms platelet activation and thrombus formation were measured both before and 1-hour after intravenous USPIO (4 mg/kg) bolus injection. Results: In healthy volunteers extra-corporeal infusion of USPIO at 300mg/mL increased thrombus area compared to saline control (16,916 ± 1,766 mm2, versus 11,678 ± 1,045 mm2; P1⁄40.001), but not at 150 mg/mL. In patients, intravenous bolus injection of USPIO increased thrombus formation compared to baseline (27,670 ± 709 mm2 versus 12,156 ± 1156 mm2; P1⁄40.006), and increased platelet p-selectin and CD36 expression (P Conclusions: We demonstrate that engineered nanoparticles cause platelet activation and have prothrombotic effects in the circulation of healthy persons and patients with vascular disease. The clinical consequences of these findings are unknown but warrant further investigation. Nutrition, nutraceuticals and cardiovascular disease in diabetes, myocardial infarction and chronic renal disease EAS-0083. FOOD SUPPLEMENTATION WITH RICE BRAN ENZYMATIC EXTRACT PREVENTS VASCULAR SENESCENCE AND ATHEROGENESIS IN APOE-/MICE C. Perez Ternero , C. Werner , J. Parrado , M. Alvarez de Sotomayor , M.D. Herrera , U. Laufs . Departamento de Farmacologia, Universidad de Sevilla, Sevilla, Spain; Klinik fur Innere Medizin III (Kardiologie Angiologie und Internistische Intensivmedizin), Universit€ atsklinikum des Saarlandes, Homburg, Germany; Departamento de Bioquimica y Biologia Molecular, Universidad de Sevilla, Sevilla, Spain We aimed to evaluate the effects of g-oryzanol-, phytosteroland tocolrich rice bran enzymatic extract (RBEE) on apoptosis and telomere shortening in aorta and spleen-derived mononuclear cells (MNC) in relation to macrophage infiltration and atherosclerotic plaque development in the ApoE-/model of high fat diet (HFD)-induced atherogenesis. Male ApoE-/mice were fed HFD or isocaloric HFD supplemented with 5% (w/w) RBEE for 30 weeks. C57BL6/J wild-type mice (WT) were kept as healthy controls under standard chow diet for the same period. Diet supplementation with RBEE improved serum lipid profile showing lower total cholesterol (744±63 vs 947±66 mg/dL) and triglycerides (97±16 vs 155±20 mg/dL) and higher HDL-cholesterol (69±8 vs 35±8 mg/ dL). Compared to WT, ApoE-/mice on HFD were characterized by significant telomere shortening in aorta (11±2%) and MNC (73±7%), which was reduced by co-treatment with RBEE (aorta: 40±7%; MNC: 105±10%). While telomerase activity and expression of TERT remained unchanged, expression of telomere repeat-binding factor 2 was increased in RBEE mice. In the aorta, RBEE treatment reduced expression of apoptosis markers (p16, p53 and bax/bcl2-ratio) and apoptosis of aortic endothelial cells (38±38% of ApoE-/HFD animals; hairpin oligonucleotide assay). In contrast, MNC of RBEE-fed mice exhibited enhanced apoptosis with increased expression of p53 and bax/bcl2. Importantly, plaque development in the aortic sinus was potently attenuated (7,737±836 vs 12,040±1,001 mm2) and macrophage infiltration was reduced by 52±5% by RBEE treatment. In conclusion, treatment of ApoE-/on high fat diet with rice bran enzymatic extract prevented atherogenesis involving mechanisms related to vascular apoptosis and senescence.