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Increased F2-isoprostane oxidative stress in coronary artery disease (CAD) patients with poor aspirin-induced thromboxane B2 inhibition
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Abstracts / Atherosclerosis 252 (2016) e236ee264
Results: In platelets, GLP-1 increased the inhibitory effects of NO and reduced the agonist-induced activation; however, these effects were lower in T2DM compared with healthy subjects. In particular, the percent increases of the antiaggregating effects of SNP were 4.0±1.8 vs 28.2±4.8 (p<0.0001) with ADP, and 17.0±5.2 vs 36.8±5.1 (p<0.02) with collagen; the percent reductions of pAKT were 3.2±4.2 vs 43.1±3.5 (p<0.0001) with collagen, and 5.4±5.2 vs 32.1±6.8 (p<0.004) with arachidonic acic (AA); the percent reductions of pERK-2 were 3.5±2.2 vs 25±8.2 (p<0.004) with collagen, and 10.5±8.6 vs 40.2±9.5 (p<0.03) with AA; the percent reductions of ROS production were 15.1±5.2 vs 41.2±7.5 (p<0.006) with AA. Conclusions: GLP-1 abilities to increase the antiaggregating effects of NO and to reduce platelet activation are reduced in T2DM. These abnormalities could be involved in the platelet hyperreactivity and increased atherothrombotic risk described in T2DM.
EAS16-0828, IMAGING, BIOMARKERS AND CLINICAL EPIDEMIOLOGY. INCREASED F2-ISOPROSTANE OXIDATIVE STRESS IN CORONARY ARTERY DISEASE (CAD) PATIENTS WITH POOR ASPIRIN-INDUCED THROMBOXANE B2 INHIBITION L.R. Lopez 1, T. Bottiglieri 2, A. Vasudevan 3, C. Swift 4, R. Schiffmann 4, P.A. McCullough 5. 1 Corgenix e Inc., Medical Research, Broomfield, USA; 2 Institute of Metabolic Disease, Baylor Research Institute, Center for Metabolomics, Dallas, USA; 3 Baylor Research Institute, Epidemiology, Dallas, USA; 4 Institute of Metabolic Disease, Baylor Research Institute, Research, Dallas, USA; 5 Baylor Heart & Vascular Hospital, Baylor Research Institute, Dallas, USA Objectives: Aspirin (ASA) is commonly used in CVD prevention because irreversibly inhibits platelet cyclooxygenase-1 (COX-1) and thromboxanemediated platelet aggregation. However, some subjects fail to benefit from ASA treatment. Among the mechanisms to explain ASA failure, oxidative stress has emerged as a relevant factor. We investigated the association of oxidative stress (urinary 8-isoPGF2a) in a cohort of CAD patients on ASA treatment categorized by tertiles of urinary 11-dehydro thromboxane B2 (11dhTxB2) response. Methods: An ASA treated (81-325 mg) cohort of angiographically diagnosed CAD patients (n¼404) and non-CVD controls (n¼72) were categorized by data driven tertiles of urinary 11dhTxB2 and by applying a >1,500 pg/mg 11dhTxB2 cut-off for classification of good/poor ASA response. Urinary F2-isoprostane (8-isoPGF2a), a marker of in vivo oxidative stress, was measured by competitive ELISA in all subjects. Results: The median 8-isoPGF2a in females (1,669.7 pg/mg, [324.713,356.2]) was significantly higher than males (1,396.4 pg/mg [369.719,256.3], p¼0.003). A significant trend was noted with higher values of 8-isoPGF2a in the middle (1,566.8 pg/mg [324.8-5229.4]) and upper tertiles (1,906.2 pg/mg [366.8-15264.1], trend p<0.001) of urinary 11dhTxB2. Patients with poor ASA-induced thromboxane response (>1,500 pg/mg) had significant higher 8-isoPGF2a levels (1,774.7 [366.815,264.1]) compared to good ASA responders (1,292 pg/mg [324.719,256.3], p<0.0001). Conclusions: 8-isoPGF2a induced oxidative stress is associated with aspirin resistance. This phenomenon appears to be more significant in women compared to men. Future research is warranted into the relationships between oxidative stress, isoprostanes, and the therapeutic efficacy of aspirin on clinical outcomes.
EAS16-0583, INTRACELLULAR LIPID METABOLISM. AN UNEXPECTED ROLE OF AUTOPHAGY IN TARGETING FUNCTIONAL MITOCHONDRIAL ATP SYNTHASE TO THE PLASMA MEMBRANE: ITS ROLE IN HDL ENDOCYTOSIS L.O. Martinez, G. Cardouat, S. Fried, S. Najib. INSERM UMR1048, Universit e de Toulouse, Institute of Cardiovascular and Metabolic Diseases, Toulouse, France
Objectives: ATP synthase, classically known to be located in the mitochondrial inner membrane, has been unexpectedly found expressed at the plasma membrane (PM), as a receptor for apoA-I, playing a role in HDLcholesterol uptake by hepatocytes. Although the ectopic expression of F1ATPase at the PM (ecto-F1) has been related to several biological processes, the pathway responsible for its transport to the PM remain unknown. Autophagy is a cellular process by which intracellular components are sequestered in the autophagosome, a double-membrane vesicle, and degraded after fusion with lysosomes. Interestingly, autophagy was also shown to promote mitochondrial fusion and to target intracellular proteins at the PM, suggesting it could control ecto-F1 expression. Methods: Autophagy was induced in hepatoma cell line HepG2 by starvation or rapamycin treatment. The formation of autophagosome was inhibited by 3-methyladenine or by extinction of beclin1, a protein of autophagy initiation complex. The fusion of autophagosome with lysosomes was prevented by chloroquine or Lamp1/2 extinction. Ecto-F1 expression was quantified by PM proteins biotinylation and immunofluorescence. HDL endocytosis was assessed by measuring cellular uptake of fluo-HDL. Results: We observed that basal and induced autophagosome formation increased the expression of ecto-F1 at the PM of hepatocytes. Interestingly, we further demonstrated that the fusion of autophagosome with lysosomes was not required for this autophagy-dependent transport. Moreover, we showed that autophagy was essential for expression of functional ecto-F1 and HDL endocytosis by hepatocytes. Conclusions: Our data suggest a new atheroprotective function of autophagy, in recycling a functional mitochondrial complex to the PM and promoting hepatic HDL uptake.
EAS16-0398, INTRACELLULAR LIPID METABOLISM.
a/b HYDROLASE DOMAIN-CONTAINING 6 (ABHD6) DEGRADES THE LATE ENDOSOMAL/LYSOSOMAL LIPID BIS(MONOACYLGLYCERO)PHOSPHATE R. Zimmermann. Institute of Molecular Biosciences, Biochemistry, Graz, Austria Objectives: ABHD6 was originally identified as monoacylglycerol lipase playing a role in endocannabinoid metabolism. Recent investigations revealed that inactivation of ABHD6 protects mice from high-fat dietinduced obesity and hepatic steatosis. Furthermore, the enzyme acts as a negative modulator of glucose-stimulated insulin secretion implicating an important role of the enzyme in lipid and energy metabolism. Yet, the mechanistic link between gene function and disease is incompletely understood. Here, we aimed to further characterize the role of ABHD6 in lipid metabolism. Methods: Lipid substrates for ABHD6 were identified in activity screening assays against a variety of ester-bond containing lipids. Subcellular localization of ABHD6 was determined in cell fractionation and live cell imaging experiments. The effect of ABHD6 inactivation on lipid metabolism was investigated using ABHD6 antisense-oligonucleotides and the smallmolecule inhibitor KT182. Results: Activity screening assays revealed that mouse and human ABHD6 degrade bis(monoacylglycero)phosphate [BMP, also known as lysobisphosphatidic acid (LBPA)] with high specific activity. BMP is found in intraluminal vesicles of late endosomes/lysosomes. It is considered as key activator of cholesterol sorting and membrane digestion in acidic organelles. We demonstrate that ABHD6 is responsible for ~ 90% of the BMP hydrolase activity detected in liver. Knockdown of ABHD6 in mice strongly increases hepatic BMP levels. The enzyme co-localizes with late endosomes/lysosomes, is active at cytosolic pH, and lacks acid hydrolase activity. These observations implicate that ABHD6 degrades BMP exported from acidic organelles or counteracts de novo formation of BMP. Conclusions: ABHD6 controls BMP catabolism and is therefore part of the late endosomal/lysosomal lipid sorting machinery.
Abstracts / Atherosclerosis 252 (2016) e236ee264
Results: In platelets, GLP-1 increased the inhibitory effects of NO and reduced the agonist-induced activation; however, these effects were lower in T2DM compared with healthy subjects. In particular, the percent increases of the antiaggregating effects of SNP were 4.0±1.8 vs 28.2±4.8 (p<0.0001) with ADP, and 17.0±5.2 vs 36.8±5.1 (p<0.02) with collagen; the percent reductions of pAKT were 3.2±4.2 vs 43.1±3.5 (p<0.0001) with collagen, and 5.4±5.2 vs 32.1±6.8 (p<0.004) with arachidonic acic (AA); the percent reductions of pERK-2 were 3.5±2.2 vs 25±8.2 (p<0.004) with collagen, and 10.5±8.6 vs 40.2±9.5 (p<0.03) with AA; the percent reductions of ROS production were 15.1±5.2 vs 41.2±7.5 (p<0.006) with AA. Conclusions: GLP-1 abilities to increase the antiaggregating effects of NO and to reduce platelet activation are reduced in T2DM. These abnormalities could be involved in the platelet hyperreactivity and increased atherothrombotic risk described in T2DM.
EAS16-0828, IMAGING, BIOMARKERS AND CLINICAL EPIDEMIOLOGY. INCREASED F2-ISOPROSTANE OXIDATIVE STRESS IN CORONARY ARTERY DISEASE (CAD) PATIENTS WITH POOR ASPIRIN-INDUCED THROMBOXANE B2 INHIBITION L.R. Lopez 1, T. Bottiglieri 2, A. Vasudevan 3, C. Swift 4, R. Schiffmann 4, P.A. McCullough 5. 1 Corgenix e Inc., Medical Research, Broomfield, USA; 2 Institute of Metabolic Disease, Baylor Research Institute, Center for Metabolomics, Dallas, USA; 3 Baylor Research Institute, Epidemiology, Dallas, USA; 4 Institute of Metabolic Disease, Baylor Research Institute, Research, Dallas, USA; 5 Baylor Heart & Vascular Hospital, Baylor Research Institute, Dallas, USA Objectives: Aspirin (ASA) is commonly used in CVD prevention because irreversibly inhibits platelet cyclooxygenase-1 (COX-1) and thromboxanemediated platelet aggregation. However, some subjects fail to benefit from ASA treatment. Among the mechanisms to explain ASA failure, oxidative stress has emerged as a relevant factor. We investigated the association of oxidative stress (urinary 8-isoPGF2a) in a cohort of CAD patients on ASA treatment categorized by tertiles of urinary 11-dehydro thromboxane B2 (11dhTxB2) response. Methods: An ASA treated (81-325 mg) cohort of angiographically diagnosed CAD patients (n¼404) and non-CVD controls (n¼72) were categorized by data driven tertiles of urinary 11dhTxB2 and by applying a >1,500 pg/mg 11dhTxB2 cut-off for classification of good/poor ASA response. Urinary F2-isoprostane (8-isoPGF2a), a marker of in vivo oxidative stress, was measured by competitive ELISA in all subjects. Results: The median 8-isoPGF2a in females (1,669.7 pg/mg, [324.713,356.2]) was significantly higher than males (1,396.4 pg/mg [369.719,256.3], p¼0.003). A significant trend was noted with higher values of 8-isoPGF2a in the middle (1,566.8 pg/mg [324.8-5229.4]) and upper tertiles (1,906.2 pg/mg [366.8-15264.1], trend p<0.001) of urinary 11dhTxB2. Patients with poor ASA-induced thromboxane response (>1,500 pg/mg) had significant higher 8-isoPGF2a levels (1,774.7 [366.815,264.1]) compared to good ASA responders (1,292 pg/mg [324.719,256.3], p<0.0001). Conclusions: 8-isoPGF2a induced oxidative stress is associated with aspirin resistance. This phenomenon appears to be more significant in women compared to men. Future research is warranted into the relationships between oxidative stress, isoprostanes, and the therapeutic efficacy of aspirin on clinical outcomes.
EAS16-0583, INTRACELLULAR LIPID METABOLISM. AN UNEXPECTED ROLE OF AUTOPHAGY IN TARGETING FUNCTIONAL MITOCHONDRIAL ATP SYNTHASE TO THE PLASMA MEMBRANE: ITS ROLE IN HDL ENDOCYTOSIS L.O. Martinez, G. Cardouat, S. Fried, S. Najib. INSERM UMR1048, Universit e de Toulouse, Institute of Cardiovascular and Metabolic Diseases, Toulouse, France
Objectives: ATP synthase, classically known to be located in the mitochondrial inner membrane, has been unexpectedly found expressed at the plasma membrane (PM), as a receptor for apoA-I, playing a role in HDLcholesterol uptake by hepatocytes. Although the ectopic expression of F1ATPase at the PM (ecto-F1) has been related to several biological processes, the pathway responsible for its transport to the PM remain unknown. Autophagy is a cellular process by which intracellular components are sequestered in the autophagosome, a double-membrane vesicle, and degraded after fusion with lysosomes. Interestingly, autophagy was also shown to promote mitochondrial fusion and to target intracellular proteins at the PM, suggesting it could control ecto-F1 expression. Methods: Autophagy was induced in hepatoma cell line HepG2 by starvation or rapamycin treatment. The formation of autophagosome was inhibited by 3-methyladenine or by extinction of beclin1, a protein of autophagy initiation complex. The fusion of autophagosome with lysosomes was prevented by chloroquine or Lamp1/2 extinction. Ecto-F1 expression was quantified by PM proteins biotinylation and immunofluorescence. HDL endocytosis was assessed by measuring cellular uptake of fluo-HDL. Results: We observed that basal and induced autophagosome formation increased the expression of ecto-F1 at the PM of hepatocytes. Interestingly, we further demonstrated that the fusion of autophagosome with lysosomes was not required for this autophagy-dependent transport. Moreover, we showed that autophagy was essential for expression of functional ecto-F1 and HDL endocytosis by hepatocytes. Conclusions: Our data suggest a new atheroprotective function of autophagy, in recycling a functional mitochondrial complex to the PM and promoting hepatic HDL uptake.
EAS16-0398, INTRACELLULAR LIPID METABOLISM.
a/b HYDROLASE DOMAIN-CONTAINING 6 (ABHD6) DEGRADES THE LATE ENDOSOMAL/LYSOSOMAL LIPID BIS(MONOACYLGLYCERO)PHOSPHATE R. Zimmermann. Institute of Molecular Biosciences, Biochemistry, Graz, Austria Objectives: ABHD6 was originally identified as monoacylglycerol lipase playing a role in endocannabinoid metabolism. Recent investigations revealed that inactivation of ABHD6 protects mice from high-fat dietinduced obesity and hepatic steatosis. Furthermore, the enzyme acts as a negative modulator of glucose-stimulated insulin secretion implicating an important role of the enzyme in lipid and energy metabolism. Yet, the mechanistic link between gene function and disease is incompletely understood. Here, we aimed to further characterize the role of ABHD6 in lipid metabolism. Methods: Lipid substrates for ABHD6 were identified in activity screening assays against a variety of ester-bond containing lipids. Subcellular localization of ABHD6 was determined in cell fractionation and live cell imaging experiments. The effect of ABHD6 inactivation on lipid metabolism was investigated using ABHD6 antisense-oligonucleotides and the smallmolecule inhibitor KT182. Results: Activity screening assays revealed that mouse and human ABHD6 degrade bis(monoacylglycero)phosphate [BMP, also known as lysobisphosphatidic acid (LBPA)] with high specific activity. BMP is found in intraluminal vesicles of late endosomes/lysosomes. It is considered as key activator of cholesterol sorting and membrane digestion in acidic organelles. We demonstrate that ABHD6 is responsible for ~ 90% of the BMP hydrolase activity detected in liver. Knockdown of ABHD6 in mice strongly increases hepatic BMP levels. The enzyme co-localizes with late endosomes/lysosomes, is active at cytosolic pH, and lacks acid hydrolase activity. These observations implicate that ABHD6 degrades BMP exported from acidic organelles or counteracts de novo formation of BMP. Conclusions: ABHD6 controls BMP catabolism and is therefore part of the late endosomal/lysosomal lipid sorting machinery.