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Plasma Allantoin Reveals Oxidative Status in Subclinical Atherosclerosis
339 Reactive Oxygen Species via Activation of Bruton Tyrosine Kinase Mediate Cholesterol CrystalsInduced CD36 Expression and Foam Cell Formation Sivareddy Kotla1, Nikhlesh K Singh1, and Gadiparthi N Rao1 University of Tennessee Health Science Center, Memphis, USA
1
In understanding the mechanisms of cholesterol in the pathogenesis of atherosclerosis, previous studies from other laboratories have demonstrated that cholesterol crystals (CCs) induce scavenger receptor CD36 expression and NLRP3-mediated inflammasome formation. In understanding the mechanisms by which CCs could enhance CD36 expression and foam cell formation, here we report that CCs via NADPH oxidase and xanthine oxidase-mediated ROS production activates BTK, a nonreceptor tyrosine kinase. In addition, CCs induce p300 tyrosine phosphorylation and activation in a BTK-dependent manner, which in turn, leads to STAT1 acetylation and its interaction with PPAR in CD36 expression, oxLDL uptake and foam cell formation. Furthermore, p300, STAT1 and PPAR bound to a STAT binding site at 107 nt in CD36 promoter and enhanced its activity in ROSmediated BTK activation-dependent manner. Disruption of this STAT binding site by site-directed mutagenesis abolished CCsinduced CD36 promoter activity. Together these results reveal that CCs via producing ROS and activating BTK causes p300-mediated STAT1 acetylation and its interaction with PPAR in CD36 expression, oxLDL uptake and foam cell formation in macrophages.
doi: 10.1016/j.freeradbiomed.2016.10.380 340 Bromine Inhalation in Pregnant Mice Induces Systemic and Pulmonary Hypertension, Fetal Growth Restriction, Heart Failure, and Death James Lambert1, Matthew Carlisle1, Rakesh Patel1, David Ford2, Tamas Jilling1, and Sadis Matalon1 1 University of Alabama at Birmingham, USA, 2Saint Louis University, USA Inhalation of oxidant gas and vapor like the halogen bromine (Br2) may pose an increased threat to pregnant women and their unborn fetuses. C57Bl/6 pregnant and non-pregnant mice were exposed to 600ppm Br2 for 30 minutes (P600 and NP600) at embryonic day 15 (E15) and were compared to air controls (P-AIR and NP-AIR). At E19, P600 exhibited 75% mortality vs 36% seen in NP600. We hypothesized that exposure to Br2 via inhalation results in systemic endothelial dysfunction and a vasoconstrictive environment. We further hypothesized the ensuing placental injury in pregnant females induces production of anti-angiogenic molecules and inflammation, leading to escalation of vascular dysfunction, increased pulmonary and systemic blood pressures, fetal growth restriction (FGR), and death that may be mitigated by tadalafil (TAD) therapy. FGR was observed following Br2 exposure and mitigated after 2mg/kg/day TAD treatment. Placentae had reduced junctional zones and absent glycogen containing cells in P600 vs P-AIR. Placentae also expressed increased levels of short FMSlike tyrosine kinase 1 (sFLT1) in P600. At E19, cGMP levels in the lungs decreased in P600, and were mitigated by TAD. Similar changes were seen in the aorta. Additionally, bronchoalveolar lavage fluid protein content, glutathionylated lipids, and inflammatory cytokines (IL-1Β, IL-6, KC) in plasma were elevated
in P600 and reduced in P600+TAD. Right ventricle pressures were increased in P600 and mitigated by TAD. Systemic blood pressures measured by intra-arterial catheter also increased in P600 and were mitigated by TAD. We have identified circulating brominated fatty acids and fatty acid aldehydes (2-BrPA and 2BrPALD) as potential mediators of Br2-inhalation induced systemic injury. When treated with 2-BrPA and 2-BrPALD ex vivo, arteries of pregnant mice exhibited increased sensitivity to phenylephrineinduced vasoconstriction as well as a decreased sensitivity to acetylcholine-induced vasodilation. This study implicates Br2 inhalation-induced vascular dysfunction augmented in pregnancy as a significant contributor to increased mortality. Additionally, tadalafil diminishes deleterious effects of Br2 inhalation on mother and fetus. Tadalafil is a potentially safe and effective countermeasure for Br2 inhalation injury in pregnancy.
doi: 10.1016/j.freeradbiomed.2016.10.381 341 Plasma Allantoin Reveals Oxidative Status in Subclinical Atherosclerosis Marli Stela Maciel1, Karolline Pereira Nascimento1, Paulo Lotufo1, and Flavia Carla Meotti1 1 Universidade de São Paulo, Brazil The role of uric acid as an independent factor of risk for cardiovascular disease is a matter of intense debate. Uric acid can be oxidized by the inflammatory enzyme myeloperoxidase to urate free radical, urate hydroperoxide and allantoin. The present study investigated whether patients with subclinical atherosclerosis would be more prone to oxidation of uric acid than healthy individuals and the role of myeloperoxidase in this event. A total of 35 subjects with carotid intima-media thickness in the 75th percentile (c-IMT P>75, cut off 1 mm) and 35 healthy subjects (cIMT P<75), male gender and aged 45-60 years, were selected from the ELSA-Brasil project, a multicenter cohort study in Brazil. Exclusion criteria included the use of uricosuric drugs, body mass index (BMI) > 30, glomerular filtration rate ≥ 70. The levels of xanthine/hypoxanthine, uric acid, allantoin and myeloperoxidase were higher in individuals with c-IMT P>75, but only allantoin levels were significantly different between groups. Retinol and betacarotene were slightly decreased in c-IMT P>75 individuals. A simple logistic regression showed a positive and significant association between c-IMT and allantoin. Pearson’s correlation revealed that allantoin had a positive and significant correlation with the pro-inflammatory and pro-oxidant enzyme myeloperoxidase only for c-IMT P>75 patients. No correlation was found between allantoin and its precursor uric acid. A significant negative correlation was found between allantoin and the antioxidant retinol in plasma of healthy individuals. Uric acid was prompt oxidized to allantoin when purified myeloperoxidase and hydrogen peroxide were added to the plasma. This study demonstrated that uric acid is more prone to oxidation in patients with subclinical atherosclerosis. The vascular inflammation in these patients is likely to oxidize uric acid to urate free radical and urate hydroperoxide that, at last, breaks down to allantoin. The oxidation of uric acid by myeloperoxidase in subclinical atherosclerosis may propagate vascular damage and inflammation in these patients. The present study suggests that the measurement of allantoin and the inflammatory status would be more reliable than uric acid to the prognosis of subclinical atherosclerosis. Supported by: FAPESP CEPID-REDOXOMA 2013/07937-8 and 2011/18106-4, CNPq, USP.
doi: 10.1016/j.freeradbiomed.2016.10.382
SfRBM / SFRRI 2016
S145
1
In understanding the mechanisms of cholesterol in the pathogenesis of atherosclerosis, previous studies from other laboratories have demonstrated that cholesterol crystals (CCs) induce scavenger receptor CD36 expression and NLRP3-mediated inflammasome formation. In understanding the mechanisms by which CCs could enhance CD36 expression and foam cell formation, here we report that CCs via NADPH oxidase and xanthine oxidase-mediated ROS production activates BTK, a nonreceptor tyrosine kinase. In addition, CCs induce p300 tyrosine phosphorylation and activation in a BTK-dependent manner, which in turn, leads to STAT1 acetylation and its interaction with PPAR in CD36 expression, oxLDL uptake and foam cell formation. Furthermore, p300, STAT1 and PPAR bound to a STAT binding site at 107 nt in CD36 promoter and enhanced its activity in ROSmediated BTK activation-dependent manner. Disruption of this STAT binding site by site-directed mutagenesis abolished CCsinduced CD36 promoter activity. Together these results reveal that CCs via producing ROS and activating BTK causes p300-mediated STAT1 acetylation and its interaction with PPAR in CD36 expression, oxLDL uptake and foam cell formation in macrophages.
doi: 10.1016/j.freeradbiomed.2016.10.380 340 Bromine Inhalation in Pregnant Mice Induces Systemic and Pulmonary Hypertension, Fetal Growth Restriction, Heart Failure, and Death James Lambert1, Matthew Carlisle1, Rakesh Patel1, David Ford2, Tamas Jilling1, and Sadis Matalon1 1 University of Alabama at Birmingham, USA, 2Saint Louis University, USA Inhalation of oxidant gas and vapor like the halogen bromine (Br2) may pose an increased threat to pregnant women and their unborn fetuses. C57Bl/6 pregnant and non-pregnant mice were exposed to 600ppm Br2 for 30 minutes (P600 and NP600) at embryonic day 15 (E15) and were compared to air controls (P-AIR and NP-AIR). At E19, P600 exhibited 75% mortality vs 36% seen in NP600. We hypothesized that exposure to Br2 via inhalation results in systemic endothelial dysfunction and a vasoconstrictive environment. We further hypothesized the ensuing placental injury in pregnant females induces production of anti-angiogenic molecules and inflammation, leading to escalation of vascular dysfunction, increased pulmonary and systemic blood pressures, fetal growth restriction (FGR), and death that may be mitigated by tadalafil (TAD) therapy. FGR was observed following Br2 exposure and mitigated after 2mg/kg/day TAD treatment. Placentae had reduced junctional zones and absent glycogen containing cells in P600 vs P-AIR. Placentae also expressed increased levels of short FMSlike tyrosine kinase 1 (sFLT1) in P600. At E19, cGMP levels in the lungs decreased in P600, and were mitigated by TAD. Similar changes were seen in the aorta. Additionally, bronchoalveolar lavage fluid protein content, glutathionylated lipids, and inflammatory cytokines (IL-1Β, IL-6, KC) in plasma were elevated
in P600 and reduced in P600+TAD. Right ventricle pressures were increased in P600 and mitigated by TAD. Systemic blood pressures measured by intra-arterial catheter also increased in P600 and were mitigated by TAD. We have identified circulating brominated fatty acids and fatty acid aldehydes (2-BrPA and 2BrPALD) as potential mediators of Br2-inhalation induced systemic injury. When treated with 2-BrPA and 2-BrPALD ex vivo, arteries of pregnant mice exhibited increased sensitivity to phenylephrineinduced vasoconstriction as well as a decreased sensitivity to acetylcholine-induced vasodilation. This study implicates Br2 inhalation-induced vascular dysfunction augmented in pregnancy as a significant contributor to increased mortality. Additionally, tadalafil diminishes deleterious effects of Br2 inhalation on mother and fetus. Tadalafil is a potentially safe and effective countermeasure for Br2 inhalation injury in pregnancy.
doi: 10.1016/j.freeradbiomed.2016.10.381 341 Plasma Allantoin Reveals Oxidative Status in Subclinical Atherosclerosis Marli Stela Maciel1, Karolline Pereira Nascimento1, Paulo Lotufo1, and Flavia Carla Meotti1 1 Universidade de São Paulo, Brazil The role of uric acid as an independent factor of risk for cardiovascular disease is a matter of intense debate. Uric acid can be oxidized by the inflammatory enzyme myeloperoxidase to urate free radical, urate hydroperoxide and allantoin. The present study investigated whether patients with subclinical atherosclerosis would be more prone to oxidation of uric acid than healthy individuals and the role of myeloperoxidase in this event. A total of 35 subjects with carotid intima-media thickness in the 75th percentile (c-IMT P>75, cut off 1 mm) and 35 healthy subjects (cIMT P<75), male gender and aged 45-60 years, were selected from the ELSA-Brasil project, a multicenter cohort study in Brazil. Exclusion criteria included the use of uricosuric drugs, body mass index (BMI) > 30, glomerular filtration rate ≥ 70. The levels of xanthine/hypoxanthine, uric acid, allantoin and myeloperoxidase were higher in individuals with c-IMT P>75, but only allantoin levels were significantly different between groups. Retinol and betacarotene were slightly decreased in c-IMT P>75 individuals. A simple logistic regression showed a positive and significant association between c-IMT and allantoin. Pearson’s correlation revealed that allantoin had a positive and significant correlation with the pro-inflammatory and pro-oxidant enzyme myeloperoxidase only for c-IMT P>75 patients. No correlation was found between allantoin and its precursor uric acid. A significant negative correlation was found between allantoin and the antioxidant retinol in plasma of healthy individuals. Uric acid was prompt oxidized to allantoin when purified myeloperoxidase and hydrogen peroxide were added to the plasma. This study demonstrated that uric acid is more prone to oxidation in patients with subclinical atherosclerosis. The vascular inflammation in these patients is likely to oxidize uric acid to urate free radical and urate hydroperoxide that, at last, breaks down to allantoin. The oxidation of uric acid by myeloperoxidase in subclinical atherosclerosis may propagate vascular damage and inflammation in these patients. The present study suggests that the measurement of allantoin and the inflammatory status would be more reliable than uric acid to the prognosis of subclinical atherosclerosis. Supported by: FAPESP CEPID-REDOXOMA 2013/07937-8 and 2011/18106-4, CNPq, USP.
doi: 10.1016/j.freeradbiomed.2016.10.382
SfRBM / SFRRI 2016
S145