Endogenous protective effects of arachidonic acid epoxygenase metabolites, epoxyeicosatrienoic acids, in cardiovascular system / 生理学报

The morbidity and mortality of cardiovascular diseases are increasing annually, which is one of the primary causes of human death. Recent studies have shown that epoxyeicosatrienoic acids (EETs), endogenous metabolites of arachidonic acid (AA) via CYP450 epoxygenase, possess a spectrum of protective properties in cardiovascular system. EETs not only alleviate cardiac remodeling and injury in different pathological models, but also improve subsequent hemodynamic disturbances and cardiac dysfunction. Meanwhile, various studies have demonstrated that EETs, as endothelial-derived hyperpolarizing factors, regulate vascular tone by activating various ion channels on endothelium and smooth muscle, which in turn can lower blood pressure, improve coronary blood flow and regulate pulmonary artery pressure. In addition, EETs are protective in endothelium, including inhibiting inflammation and adhesion of endothelial cells, attenuating platelet aggregation, promoting fibrinolysis and revascularization. EETs can also prevent aortic remodeling, including attenuating atherosclerosis, adventitial remodeling, and aortic calcification. Therefore, it is clinically important to study the physiological and pathophysiological effects of EETs in the cardiovascular system to further elucidate the mechanisms, as well as provide new strategy for the prevention and treatment of cardiovascular diseases. This review summarizes the endogenous cardioprotective effects and mechanisms of EETs in order to provide a new insight for research in this field.

CYP2C8-derived epoxyeicosatrienoic acids decrease oxidative stress-induced endothelial apoptosis in development of atherosclerosis: Role of Nrf2 activation / 华中科技大学学报（医学）（英德文版）

The aim of the present study is to investigate how cytochrome P450 enzymes (CYP) 2C8-derived epoxyeicosatrienoic acids (EETs) regulate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and protect against oxidative stress-induced endothelial injuries in the development and progression of atherosclerosis. In this study, cultured human umbilical vein endothelial cells (HUVECs) were transfected with CYP2C8 or pretreated with exogenous EETs (1 μmol/L) before TNF-α (20 ng/mL) stimulation. Apoptosis and intracellular ROS production were determined by flow cytometry. The expression levels of ROS-associated NAD(P)H subunits gp91 and p47, the anti-oxidative enzyme catalase (CAT), Nrf2, heme oxygenase-1 (HO-1) and endothelial nitric oxide synthase (eNOS) were detected by Western blotting. The results showed that CYP2C8-derived EETs decreased apoptosis of HUVECs treated with TNF-α. Pretreatment with 11, 12-EET also significantly blocked TNF-α-induced ROS production. In addition, 11, 12-EET decreased oxidative stress-induced apoptosis. Furthermore, the ability of 11, 12-EET to protect cells against TNF-α-induced apoptosis via oxidative stress was abrogated by transient transfection with Nrf2-specific small interfering RNA (siRNA). In conclusion, CYP2C8-derived EETs prevented TNF-α-induced HUVECs apoptosis via inhibition of oxidative stress associated with the Nrf2 signaling.

Protective effects of 11,12-epoxyeicosatrienoic acid preconditioning and postconditioning on myocardial ischemia/reperfusion injury in rats / 生理学报

To explore the effects of 11,12-epoxyeicosatrienoic acid (11,12-EET) preconditioning and postconditioning on myocardial ischemia/reperfusion (IR) injury in rats, the IR injury model was built by stopping perfusion for 40 min followed by reperfusion for 30 min, and the changes of mitochondrial functions, myocardial metabolism and function were measured. Langendorff-perfused isolated rat hearts were divided into 4 groups: control group, persistently perfused with Krebs-Henseleit (K-H) fluid for 100 min; IR group, stopped perfusion for 40 min followed by reperfusion for 30 min; Pre-EET group, preconditioned with 6.24×10(-9) mol/L 11,12-EET for 5 min twice before subjected to ischemia; Post-EET group, postconditioned with 6.24×10(-9) mol/L 11,12-EET for 30 s twice before reperfusion. The computer-based electrophysiological recording system was used to measure the changes of maximal rate of the pressure increase in contract phase (+dp/dt(max)), maximal rate of the pressure decrease in diastole phase of heart (-dp/dt(max)), left ventricular end-diastolic pressure (LVEDP) and difference of left ventricular pressure (DLVP). The activities of lactate dehydrogenase (LDH) in effluent, Ca(2+)-ATPase, Na(+)-K(+)-ATPase and succinate dehydrogenase (SDH) in mitochondria were measured with colorimetry method; superoxide dismutase (SOD) activity was measured with hydroxylamine method and malondialdehyde (MDA) content in myocardial tissues was measured with TBA method. The results showed that: (1) Compared with that in the control group, the myocardial functions, the values of SOD, SDH and Na(+)-K(+)-ATPase were decreased in IR group (P<0.05); the values of LDH, MDA and Ca(2+)-ATPase were increased (P<0.05) in IR group. (2) Compared with that in IR group, the values of SDH and Na(+)-K(+)-ATPase were increased (P<0.05) and the value of Ca(2+)-ATPase was decreased (P<0.05) in both Pre-EET and Post-EET groups. But no significant differences were detected between Pre-EET and Post-EET groups. (3) Compared with IR treatment, both 11,12-EET preconditioning and postconditioning caused significant decreases in MDA content and leakage of LDH, amendment of heart functions and increases in SOD activity (P<0.05). But there were no significant differences between 11,12-EET preconditioning and postconditioning. These results indicate that 11,12-EET preconditioning and postconditioning can protect myocardium from IR injury by improving mitochondrial functions, up-regulating the activities of Na(+)-K(+)-ATPase and SDH, and down-regulating the activity of Ca(2+)-ATPase in mitochondria. Moreover, 11,12-EET preconditioning and postconditioning also elevate the activity of SOD and reduce the content of MDA, suggesting that 11,12-EET can depress the oxidative stress in IR rat heart.

Effects of 11, 12-epoxyeicosatrienoic acid preconditioning and postconditioning on Ca(2+)- handling proteins in myocardial ischemia/reperfusion injury in rats / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the effects of 11, 12-epoxyeicosatrienoic acid (11, 12-EET) preconditioning and postconditioning on Ca(2+)-handling proteins in myocardial ischemia/reperfusion (IR) injury in rats and reveal the effects and mechanism of 11, 12-EET on cardioprotection. METHODS The IR injury model was built by stopping perfusion for 40 minutes followed by reperfusion for 30 minutes. The isolated Langendorff-perfused rat hearts were divided into 4 groups: control group, IR group, EET preconditioning (Pre-EET) group and EET postconditioning (Post-EET) group. The computer-based electrophysiological recorder system was used to measure the changes of the maximal rate of pressure increased in the contraction phase (+dp/dt(max)), the maximal rate of pressure decreased in the diastole phase (-dp/dt(max)), the left ventricular end diastolic pressure (LVEDP) and the difference of left ventricular pressure (delta LVP). The activity of Ca(2+)-ATPase in sarcoplasmic reticulum was measured with colorimetric method. Reverse transcription-polymerase chain reaction was used to assess the gene expression of C(a2+)-handling protein [sarcoplasic reticulum Ca(2+)-ATPase (SERCA), phospholamban (PLB), ryanodine receptor type 2 (RyR,), and 1, 4, 5-trisphosphate inositol receptor type 2 (IP3 R2) ] mRNAs level.</p><p><b>RESULTS</b>Compared with IR group, the myocardial functions, the value of Ca(2+)-ATPase, and the expressions of IP3 R2 mRNA were significantly increased and the expression of PLB mRNA was significantly decreased in both Pre-EET group and Post-EET group (P < 0.05, P < 0.01). And the expression of SERCA mRNA was significantly increased in Pre-EET group (P < 0. 05). However, no significant differences were detected between Pre-EET and Post-EET groups. Moreover, the expression of RyR2 mRNA was not significantly different among all groups.</p><p><b>CONCLUSIONS</b>11, 12-EET preconditioning and post-conditioning can protect myocardium from IR injury by elevating the activity of Ca(2+)-ATPase in sarcoplasmic reticulum, up-regulating the expression of IP3 R2 mRNA, and down-regulating the expression of PLB mRNA. Moreover, up-regulating the expression of SERCA mRNA maybe one of mechanisms of 11, 12-EET preconditioning on cardio protection against IR injury.</p>

Effect of 11, 12-epoxyeicosatrienoic acids on hypoxia/reoxygenation injury in the human umbilical vein endothelial cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the effects of 11, 12-epoxyeicosatrienoic acids (11, 12-EET) on the degree of hypoxia/reoxygenation injury in human umbilical vein endothelial cells ( HUVECs), and reveal the possible pathway of EET on protection.</p><p><b>METHODS</b>Primary cultured HUVECs were randomly divided into control group, hypoxia/reoxygenation group, 11, 12-EET control group, 11, 12- EET hypoxia/reoxygenation group, inhibition of extracellular signal-regulated kinase (ERKI/2) group, and inhibition of nitric oxide synthase (NOS) group. Hypoxia/reoxygenation injury model in HUVECs was established by exposure to hypoxia (2% O2, 5% CO2 and 93% N2) for 3 hours, followed by reoxygenation (95% air and 5% CO2) for 1 hour. The evaluation of the endothelial cells were made by immunohistochemistry. The cell viability was monitored by MTT assay. Colorimetry method was used to assay the lactate dehydrogenase (LDH) , malondialdehyde (MDA) and activity of superoxide dismutase (SOD) in culture medium. Western blot was used to detect the expressions of endothelial nitric oxide synthase (eNOS) and phosphorylated ERK1/2 in HUVECs.</p><p><b>RESULTS</b>11, 12-EET caused minor injury in normal oxygen incubated HUVECs; however, in hypoxia/reoxygenation HUVECs, it raised the cell viability markedly, decreased the LDH release and MDA content, and increased the activity of SOD and the expressions of eNOS and phosphorylated ERK1/2.</p><p><b>CONCLUSIONS</b>11, 12-EET may prevent against endothelial cell hypoxia/reoxygenation injury. The mechanism may be related to the increased activity of SOD, elimination of oxygen-derived free radicals, and reduction of eNOS and phosphorylated ERK1/2 lesion caused by hypoxia/reoxygenation.</p>

Effect of 11, 12-epoxyeicosatrienoic acid on nitric oxide synthase of ischemia-reperfusion myocardium in rats / 中国医学科学院学报

<p><b>OBJECTIVE</b>To observe the effect of 11,12-epoxyeicosatrienoic acid (11,12-EET) on nitric oxide synthase (NOS) in myocardial ischemia/reperfusion injury and explore the protective role of NOS in myocardium.</p><p><b>METHODS</b>Rat myocardial ischemia/reperfusion model was produced by ischemia for 60 minutes and reperfusion for 30 minutes. Rats were divided into 5 groups: 11,12-EET ischemia/reperfusion groups (including EET1, EET2, and EET3 groups), EET control group, ischemia/reperfusion group, sham operation group, and control group. Changes of the maximal rates of rise and decrease of left ventricular pressure (+/-dp/dtmax) were observed. Activities of inducible nitric oxide synthase (iNOS) and constrictive nitric oxide synthase (cNOS) in myocardium were measured with chemocolorimetry.</p><p><b>RESULTS</b>During both ischemia period (60 min) and reperfusion period (30 min), +/-dp/dtmax was significantly lower in ischemia/reperfusion group than in sham operation group (P < 0.01), and was significantly higher in EET1, EET2 and EET3 groups than in ischemia/reperfusion group (P < 0.01). cNOS level was significantly lower in ischemia/reperfusion group than in sham operation group, was significantly higher in EET1, EET2 and EET3 groups than in sham operation group (P < 0.01), and was significantly higher in EET2 group than in EET group (P < 0.01). iNOS level was significantly higher in sham operation group than in EET control group (P < 0.05), was significantly higher in ischemia/ reperfusion group than in sham operation group (P < 0.01), and was significantly lower in EET1, EET2 and EET3 groups than in ischemia/reperfusion group (P < 0.01).</p><p><b>CONCLUSION</b>Exogenous 11,12-EET can improve ischemia/reperfusion injury, which may be related with the changes of NOS isozymes.</p>

Arachidonic acid epoxygenases and their metabolites promote angiogenesis / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the angiogenetic effects of endogenous and exogenous epoxyeicosatrienoic acids (EET) and the relevant signaling mechanisms involved.</p><p><b>METHODS</b>Bovine aortic endothelial cells (BAEC) were incubated with synthetic EETs or infected with recombinant adeno-associated viruses (rAAV) containing CYP2C11-CYPOR, CYP2J2 or CYP102 F87V mutant to increase endogenous expression levels of EETs. BAEC proliferation measured by cell counting and chromatometry, migration assessed by transwell analysis, and capillary formation determined by chicken embryo chorioallantoic membrane assays (CAM) and tube formation tests on matrigel and angiogenesis were analysed in vivo. The potential involvement of various signaling pathways were explored using selective inhibitors.</p><p><b>RESULTS</b>Transfection with rAAV-2C11OR, rAAV-2J2 or rAAV-F87V promoted BAEC proliferation, migration, and capillary tubule formation. However, the effects of EETs on proliferation, migration and capillary tubule formation were attenuated by inhibitors of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3 kinase)/Akt pathways, and partially attenuated by endothelial nitric oxide synthase (eNOS) inhibitor, but not by a protein kinase C inhibitor. In a rat ischemic hind limb model, rAAV-mediated epoxygenase transfection induced angiogenesis.</p><p><b>CONCLUSIONS</b>Arachidonic acid epoxygenase and its metabolites can promote angiogenesis through activating MAPK and PI3 kinase/Akt signaling pathways, and to some extent, the eNOS pathway, and the angiogenic effects may provide protection to ischemic tissues.</p>

Effects of 11,12-epoxyeicosatrienoic acid on cardioplegia and reperfusion arrhythmias in the isolated immature rabbit hearts / 中华外科杂志

<p><b>OBJECTIVE</b>The aim of the present study was to study the Effects of 11,12-epoxyeicosatrienoic acid (11,12-EET) on cardioplegia and reperfusion arrhythmias in the isolated perfused immature rabbit hearts.</p><p><b>METHODS</b>Isolated immature rabbit hearts were randomly divided into two groups: group 1 (St. Thomas No.2 solution control n = 8) and group 2 (St. Thomas No.2 solution plus 11,12-EET n = 8). By means of Langendorff technique, these isolated rabbit hearts underwent (15 degrees C) hypothermia, 2 hours of ischemia after infusion of cardioplegic solution and 1 hour of reperfusion (37 degrees C). The mean times until the cessation of both electrical and mechanical activity were measured after infusion of cardioplegia. The same index until occurrence of both electrical and mechanical activity after reperfusion was observed too. We also measured the arrhythmias score, heart rate, coronary blood flow during the reperfusion and the myocardial water content, myocardial calcium content at the endpoint of the reperfusion period.</p><p><b>RESULTS</b>The times until electrical [(9.3 +/- 0.9) s vs (13.6 +/- 1.9) s, P < 0.01] and mechanical [(4.5 +/- 1.7) vs (7.3 +/- 2.1) s, P < 0.05] activity arrest were significantly shorter in the group 2 than those in the control group. 11,12-EET also provided significantly better myocardial water content [(84 +/- 4)% vs (90 +/- 5)%, P < 0.01], arrhythmia scores (2.03 +/- 0.83 vs 3.88 +/- 1.25, P < 0.01), coronary blood flow and myocardial calcium content [(3.22 +/- 0.33) micro mol/gram dry weight (gdw) vs (3.97 +/- 0.26) micro mol/gdw, P < 0.01] compared with control. There were no significant changes with heart rate and the mean times until occurrence of both electrical and mechanical activity after reperfusion.</p><p><b>CONCLUSIONS</b>These data suggest that 11,12-EET added to the cardioplegic solution of St. Thomas No.2 has better cardioplegia effects and lower incidence of reperfusion arrhythmias.</p>

Interactions of Nitric Oxide and Antioxidants in Hypoxia-Reoxygenation Injury

PURPOSE: An ischemia-reperfusion injury leads to profound functional and structural alterations of vascular smooth muscle cells (VSMC). It is still not clear whether hypoxia- reoxygenation and antioxidants affect the nitric oxide (NO) synthesis of VSMC. This study tried to investigate the effects of antioxidants on NO production, inducible nitric oxide synthase (iNOS) and the expression of NFkappaB p65, during the hypoxia-reoxygenation of VSMC cultures. METHODS: The VSMCs were primarily cultured from rat aortae, and confirmed by immunoreaction with the anti- smooth muscle myosin antibody. The condition of the hypoxia was verified by measuring the PO2 and PCO2 of the culture media. The concentrations of nitrite in the culture media were measured by the Griess reaction. Western blottings for the iNOS and NFkappaB p65 proteins were performed. L-NAME was used as an NOS inhibitor. Vitamins C and E, Glutathione (GSH), lipoic acid and dihydrolipoic acid (DHLA) were used as antioxidants. RESULTS: The iNOS protein was induced in the VSMC by 24 hours of hypoxia, which increased the nitrite in the VSMC culture medium. The reoxygenation profoundly increased the iNOS protein expression and nitrite concentration. The L- NAME, vitamins C and E, GSH, lipoic acid and DHLA decreased the nitrite productions during hypoxia and the hypoxia-reoxygenation, whereas, the expressions of the iNOS and NFkappaB p65 proteins were not influenced. CONCLUSION: We concluded that hypoxia-reoxygenation induced the iNOS protein, and the subsequent production of NO in the VSMC. The antioxidants and the NOS inhibitor decreased the NO production during the hypoxia-reoxygenation, but did not affect the expressions of the iNOS and NFkappaB p65 proteins

Cytoprotective effects of dihydrolipoic acid and lipoic acid on the oxidative stress in cultured rat cortical neurons

In brain hypoxic-ischemia, an excess release of glutamate and a marked production of reactive oxygen species (ROS) occur in neuronal and non-neuronal cells. The present study investigated the effect of the biological antioxidants dihydrolipoic acid (DHLA) and lipoic acid (LA) on N-methyl-D-aspartate (NMDA)and ROS-induced neurotoxicity in cultured rat cortical neurons. DHLA enhanced NMDA-evoked rises in intracellular calcium concentration ((Ca2+)i). In contrast, LA did not alter the NMDA-evoked calcium responses but decreased after a brief treatment of dithiothreitol (DTT), which possesses a strong reducing potential. Despite the modulation of NMDA receptor-mediated rises in (Ca2+)i, neither DHLA nor LA altered the NMDA receptor-mediated neurotoxicity, as assessed by measuring the amount of lactate dehydrogenase released from dead or injured cells. DHLA, but not LA, prevented the neurotoxicity induced by xanthine/xanthine oxidase-generated superoxide radicals. Both DHLA and LA decreased the glutathione depletion-induced neurotoxicity. The present data may indicate that biological antioxidants DHLA and LA protect neurons from ischemic injuries via scavenging oxygen free radicals rather than modulating the redox modulatory site(s) of NMDA receptor.