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Prostacyclin Signalling Promotes Cytoprotection and Angiogenesis Via Up-Regulation of NADPH Oxidase 4
dihydroethidium and amplex red, respectively. Results showed that high glucose exposure (i) elevated ROS levels (p<0.05) and (ii) resulted in time-dependent gene expression level change. The gene expression level changes indicated a cell trying to maintain homeostasis under high glucose exposure. These results further the current understanding of hyperglycemia induced changes in endothelial cell homeostasis and dysfunction. Supported by NIH R01 HL084337.
doi:10.1016/j.freeradbiomed.2012.10.472
432 Prostacyclin Signalling Promotes Cytoprotection and Angiogenesis Via Up-Regulation of NADPH Oxidase 4 1,2
Hitesh M Peshavariya , Guei-Sheung Liu1,2, Catherine W. T. Chang1,2, Fan Jiang3, Elsa Chan1, and Gregory J Dusting1,2 1 2 University of Melbourne, Australia, O’Brien Institute, Australia, 3 Shandong University, China Prostacyclin (PGI2) released from the vascular endothelium plays an important role in vasodilatation and thromboresistance, and has long been suspected to protect cell survival in the vasculature. How it does so has never been clear. Overproduction of superoxide in ischaemic and inflammatory conditions compromises production of vascular cytoprotective mediators and impairs endothelial functions. Previously, we and others have shown that NADPH oxidase type 4 (Nox4) improves endothelial functions and promotes angiogenesis in vitro and in vivo, but how this cellular signaling is regulated is poorly understood. We now reveal how the selective and stable prostacyclin receptor agonist cicaprost increases expression of Nox4 (n=4; P<0.01) in human endothelial cells of several types, including endothelial progenitor cells. Cicaprost signaling was blocked by the IP receptor antagonist (CAY10441). Cicaprost enhanced phosphorylation of cAMP response element-binding (CREB) via the PKA-dependent pathway and either treatment with PKA inhibitor or over-expression of mutant CREBM1 attenuated cicaprost-induced Nox4 expression suggesting Nox4 up-regulation acts via the IP/PKA/CREB pathway. Cicaprostinduced Nox4 and H2O2 production were blocked by Nox4 gene silencing. Importantly, up-regulation of Nox4 by cicaprost protects endothelial cells from apoptosis measured by caspase3/7 activity, and enhances their proliferation, migration and angiogenic responses (n= 4 to 8; P<0.01), all effects being substantially decreased by Nox4 gene silencing (n= 4 to 8; P<0.01). Finally, cicaprost enhanced the blood vessel formation into subcutaneous sponges as indicated by a two-fold increase in haemoglobin content (n=4; P<0.01) in C57BL/6 mice. This response to cicaprost was substantially reduced (n=4; P<0.01) in Nox4 silencing mice. This is the first evidence of PGI2-mediated upregulation of Nox4, which opens up a novel strategy to protect and enhance endothelial cell functions in cardiovascular diseases, such as after myocardial infarction or other ischaemic and inflammatory conditions.
doi:10.1016/j.freeradbiomed.2012.10.473
433 Impaired Diastolic Relaxation in Cys42Ser PKGIalpha Mutant Mice 1
Diastolic dysfunction is a significant predictor of the development of heart failure, and increases the risk of cardiovascular events independently of hypertrophy or blood pressure. Protein kinase G (PKG) can modulate myocardial relaxation and diastolic tone by 2+ 2+ altering cytoplasmic Ca concentration and myofilament Ca sensitivity. PKGIα forms an interprotein disulfide between its two subunits in response to oxidants and this activates the kinase independently of the classical NO-cGMP pathway. To investigate the impact of oxidative activation of PKGIα on the regulation of myocardial ‘contractility’ we compared the cardiac function of Cys42Ser PKGIα knock-in (KI) mice with wild-type (WT) littermate controls. Age-matched WT and KI male mice with equal body weight between genotypes were assessed by echocardiography, demonstrating that cardiac mass and ejection fraction (68.5±1.4% and 65.2±1.5% respectively, n=13-14 per group) were identical. The systolic volumes of WT (25.5±1.9μl) and KI (23.6±1.5 μl) were also identical between genotypes. However, the diastolic volume of KI mice (65.6±2.3μl) was significantly (p<0.01) smaller than WT (80.1±3.0 μl). This lead to ~20% decrease of stroke volume and cardiac output in KI. Pulse wave Doppler analysis of mitral inflow velocity showed that the E/A ratio (an important predictor of adverse cardiac events) was smaller (p<0.001) in KI (1.35±0.03) than WT (1.84±0.05). The phosphorylation of key 2+ regulators of myocardial Ca homeostasis were compared between genotypes, showing phospholamban pSer16 status was decreased in KI compared to WT. Complementary studies comparing contractile function in isolated buffer-perfused Langendorff WT or KI hearts were carried out. Hypoxia (n=5 per group) or global ischemia (n=7 per group) both increased end diastolic pressure in WT. KI hearts also increased end diastolic pressure, but this was significantly potentiated (~2-fold at any time point, p<0.05) during hypoxia or ischemia compared to WT. Western immunoblotting showed both hypoxia and ischemia increased PKGIα disulfide dimerisation (activation) in WT, which was accompanied by increased Ser16 PLB phosphorylation. We conclude PKGIα disulfide dimerisation is necessary for maintaining diastolic relaxation during myocardial hypoxia or ischemia.
doi:10.1016/j.freeradbiomed.2012.10.474
434 Bicarbonate Modulates Oxidative and Functional Damage in Ischemia-Reperfusion 1
Bruno Barros Queliconi , Thire B M Marazzi1, Sandra M Vaz1, Paul S. Brookes2, Keith W Nehrke2, Ohara Augusto1, and Alicia J Kowaltowski1 1 2 Universidade de São Paulo, Brazil, University of Medicine and Dentistry of Rochester The carbon dioxide/bicarbonate (CO2/HCO3Ǧ) pair is the main biological buffer. However, its influence on biological processes, and in particular redox processes, is still poorly explored. Here we study the effect of CO2/HCO3Ǧ on ischemic injury in three distinct models (cardiac HL-1 cells, perfused rat heart and C. elegans). We found that, while different concentrations of CO2/HCO3Ǧ do not affect function under basal conditions, ischemia-reperfusion in the presence of higher CO2/HCO3Ǧ resulted in greater functional loss associated with higher oxidative damage in all models. Since the effect of CO2/HCO3Ǧ was observed in all models tested, we believe this buffer is an important determinant of oxidative damage following ischemia-reperfusion.
doi:10.1016/j.freeradbiomed.2012.10.475
Oleksandra Prysyazhna , Friederike Cuello1,2, and Philip Eaton1 1 2 King's College London,UK, University Medical Center Hamburg-Eppendorf, Germany
S172
SFRBM 2012
doi:10.1016/j.freeradbiomed.2012.10.472
432 Prostacyclin Signalling Promotes Cytoprotection and Angiogenesis Via Up-Regulation of NADPH Oxidase 4 1,2
Hitesh M Peshavariya , Guei-Sheung Liu1,2, Catherine W. T. Chang1,2, Fan Jiang3, Elsa Chan1, and Gregory J Dusting1,2 1 2 University of Melbourne, Australia, O’Brien Institute, Australia, 3 Shandong University, China Prostacyclin (PGI2) released from the vascular endothelium plays an important role in vasodilatation and thromboresistance, and has long been suspected to protect cell survival in the vasculature. How it does so has never been clear. Overproduction of superoxide in ischaemic and inflammatory conditions compromises production of vascular cytoprotective mediators and impairs endothelial functions. Previously, we and others have shown that NADPH oxidase type 4 (Nox4) improves endothelial functions and promotes angiogenesis in vitro and in vivo, but how this cellular signaling is regulated is poorly understood. We now reveal how the selective and stable prostacyclin receptor agonist cicaprost increases expression of Nox4 (n=4; P<0.01) in human endothelial cells of several types, including endothelial progenitor cells. Cicaprost signaling was blocked by the IP receptor antagonist (CAY10441). Cicaprost enhanced phosphorylation of cAMP response element-binding (CREB) via the PKA-dependent pathway and either treatment with PKA inhibitor or over-expression of mutant CREBM1 attenuated cicaprost-induced Nox4 expression suggesting Nox4 up-regulation acts via the IP/PKA/CREB pathway. Cicaprostinduced Nox4 and H2O2 production were blocked by Nox4 gene silencing. Importantly, up-regulation of Nox4 by cicaprost protects endothelial cells from apoptosis measured by caspase3/7 activity, and enhances their proliferation, migration and angiogenic responses (n= 4 to 8; P<0.01), all effects being substantially decreased by Nox4 gene silencing (n= 4 to 8; P<0.01). Finally, cicaprost enhanced the blood vessel formation into subcutaneous sponges as indicated by a two-fold increase in haemoglobin content (n=4; P<0.01) in C57BL/6 mice. This response to cicaprost was substantially reduced (n=4; P<0.01) in Nox4 silencing mice. This is the first evidence of PGI2-mediated upregulation of Nox4, which opens up a novel strategy to protect and enhance endothelial cell functions in cardiovascular diseases, such as after myocardial infarction or other ischaemic and inflammatory conditions.
doi:10.1016/j.freeradbiomed.2012.10.473
433 Impaired Diastolic Relaxation in Cys42Ser PKGIalpha Mutant Mice 1
Diastolic dysfunction is a significant predictor of the development of heart failure, and increases the risk of cardiovascular events independently of hypertrophy or blood pressure. Protein kinase G (PKG) can modulate myocardial relaxation and diastolic tone by 2+ 2+ altering cytoplasmic Ca concentration and myofilament Ca sensitivity. PKGIα forms an interprotein disulfide between its two subunits in response to oxidants and this activates the kinase independently of the classical NO-cGMP pathway. To investigate the impact of oxidative activation of PKGIα on the regulation of myocardial ‘contractility’ we compared the cardiac function of Cys42Ser PKGIα knock-in (KI) mice with wild-type (WT) littermate controls. Age-matched WT and KI male mice with equal body weight between genotypes were assessed by echocardiography, demonstrating that cardiac mass and ejection fraction (68.5±1.4% and 65.2±1.5% respectively, n=13-14 per group) were identical. The systolic volumes of WT (25.5±1.9μl) and KI (23.6±1.5 μl) were also identical between genotypes. However, the diastolic volume of KI mice (65.6±2.3μl) was significantly (p<0.01) smaller than WT (80.1±3.0 μl). This lead to ~20% decrease of stroke volume and cardiac output in KI. Pulse wave Doppler analysis of mitral inflow velocity showed that the E/A ratio (an important predictor of adverse cardiac events) was smaller (p<0.001) in KI (1.35±0.03) than WT (1.84±0.05). The phosphorylation of key 2+ regulators of myocardial Ca homeostasis were compared between genotypes, showing phospholamban pSer16 status was decreased in KI compared to WT. Complementary studies comparing contractile function in isolated buffer-perfused Langendorff WT or KI hearts were carried out. Hypoxia (n=5 per group) or global ischemia (n=7 per group) both increased end diastolic pressure in WT. KI hearts also increased end diastolic pressure, but this was significantly potentiated (~2-fold at any time point, p<0.05) during hypoxia or ischemia compared to WT. Western immunoblotting showed both hypoxia and ischemia increased PKGIα disulfide dimerisation (activation) in WT, which was accompanied by increased Ser16 PLB phosphorylation. We conclude PKGIα disulfide dimerisation is necessary for maintaining diastolic relaxation during myocardial hypoxia or ischemia.
doi:10.1016/j.freeradbiomed.2012.10.474
434 Bicarbonate Modulates Oxidative and Functional Damage in Ischemia-Reperfusion 1
Bruno Barros Queliconi , Thire B M Marazzi1, Sandra M Vaz1, Paul S. Brookes2, Keith W Nehrke2, Ohara Augusto1, and Alicia J Kowaltowski1 1 2 Universidade de São Paulo, Brazil, University of Medicine and Dentistry of Rochester The carbon dioxide/bicarbonate (CO2/HCO3Ǧ) pair is the main biological buffer. However, its influence on biological processes, and in particular redox processes, is still poorly explored. Here we study the effect of CO2/HCO3Ǧ on ischemic injury in three distinct models (cardiac HL-1 cells, perfused rat heart and C. elegans). We found that, while different concentrations of CO2/HCO3Ǧ do not affect function under basal conditions, ischemia-reperfusion in the presence of higher CO2/HCO3Ǧ resulted in greater functional loss associated with higher oxidative damage in all models. Since the effect of CO2/HCO3Ǧ was observed in all models tested, we believe this buffer is an important determinant of oxidative damage following ischemia-reperfusion.
doi:10.1016/j.freeradbiomed.2012.10.475
Oleksandra Prysyazhna , Friederike Cuello1,2, and Philip Eaton1 1 2 King's College London,UK, University Medical Center Hamburg-Eppendorf, Germany
S172
SFRBM 2012