The Combination Of Homocysteine And Copper Ion Induces Oxidative Damage In Vascular Endotheial Cells Through Metabolic Compensation-mediated Nadph Oxidase Activation

Part 1The role of Cu²⁺in Hcy-induced endothelial cell injuryObjective:To explore the role of Cu²⁺in Hcy-induced endothelial cell injury.Methods:Primary human umbilical vein endothelial cells（HUVEC）and primary mouse aortic endothelial cells（MAEC）were isolated and cultured in vitro.Flow cytometry and immunofluorescence were used to characterize the CD31phenotype of endothelial cells.Cell viability was assessed by MTT assay after treatment with Hcy alone,Cu²⁺alone,Hcy and Cu²⁺/Fe²⁺/Mg²⁺/Ca²⁺,and the combination of Hcy,Cu²⁺and a Cu²⁺chelator bathocuproine.Results:Hcy or Cu²⁺alone or Hcy and Fe²⁺/Mg²⁺/Ca²⁺had no effect on cell death.The combination of Hcy and Cu²⁺decreased primary HUVEC and MAEC viability.Cu²⁺chelating agent bathocuproine inhibited Hcy and Cu²⁺mediated cell death.Conclusion:Hcy damages vascular endothelial cells in the presence of Cu²⁺,and Hcy and Cu²⁺have a synergistic effect on endothelial cell damage.Part 2The combination of Hcy and Cu²⁺ induces apoptosis in endothelial cell through producing ROS and RNSObjective: To investigate the mechanisms of vascular endothelial cell damage induced by the combination of Hcy and Cu²⁺.Methods: Primary HUVEC and MAEC were treated with the combination of Hcy and Cu²⁺ for varying periods of time.Flow cytometry was used to quantify intracellular ROS,apoptotic cells,mitochondrial membrane potential and phospholipid oxidation levels.The ultrastructural morphology of the cell and mitochondria in primary HUVEC and MAEC was assessed by transmission electron microscopy.Total protein was extracted for the measurement of caspase 3 and 3-NT expressions by western blot.Isolated cytoplasm to detected the cyt C expression by western blot.Primary MAEC was treated with the combination of Hcy,Cu²⁺,and/or NAC,or SOD,or catalase.Flow cytometry was used to quantify intracellular ROS,apoptotic cells.Total protein was extracted for the measurement of 3-NT expression by western blot.Cell viability was assessed by MTT assay after treatment with Hcy in combination with Cu²⁺,H₂O₂,and ONOO^-,respectively.Results: The combination of Hcy and Cu²⁺ increased the levels of H₂O₂ and ONOO^-in primary endothelial cells.Hcy in combination with Cu²⁺ induced mitochondrial damage,cytochrome c release from mitochondria to cytoplasm,and caspase 3 cleavage in primary HUVEC and MAEC.Pan caspase inhibitor z VADfmk inhibited primary HUVEC death.Chromatin condensation and apoptotic bodies appearance were observed under transmission electron microscope.Antioxidants decreased cellular levels of H₂O₂ and ONOO^-in primary MAEC,and inhibited Hcy and Cu²⁺ mediated apoptosis.Equal concentration of ONOO^-significantly decreased primary HUVEC viability than H₂O₂ treatment.Conclusion: The combination of Hcy and Cu²⁺ produces a large amount of ROS and RNS in primary endothelial cells in the forms of H₂O₂ and ONOO^-,respectively.The combination of Hcy and Cu²⁺ damages mitochondria in primary HUVEC and MAEC and induces mitochondrial apoptosis pathway.ONOO^-may play an important role in Hcy and Cu²⁺ mediated endothelial cells apoptosis.Part 3Mechanisms of Hcy in combination with Cu²⁺-induced H₂O₂ and ONOO^-Objective: To explore the mechanisms of Hcy in combination with Cu²⁺-induced H₂O₂ and ONOO^-.Methods: Primary HUVEC and MAEC were treated with the combination of Hcy,Cu²⁺,and/or NADPH for varying periods of time.Flow cytometry was used to quantify intracellular ROS,mitochondrial ROS,mitochondrial membrane potential and phospholipid oxidation levels,and NO levels.The activity of the respiratory chain complexes,SOD and catalase and the levels of lactate in culture medium were measured by spectrophotometry.Cellular oxygen consumption rate was measured by a Clark electrode.The intracellular ATP contents were measured using luminescence ATP detection assay.The expressions of GPx-1,e NOS,i NOS and e NOS dimer/ monomer were detected by western-blot.NOX activity was measured in the isolated cell membrane and cytoplasm using lucigenin enhanced chemi-luminescence.Cell viability was assessed by MTT assay after treatment with the combination of Hcy,Cu²⁺,and/or NOX inhibitor DPI or VAS2870.Results: The combination of Hcy and Cu²⁺ increased NOX activity for almost 2000-5000 fold in primary HUVEC and MAEC,which was inhibited by DPI or VAS2870.When added to NADPH,Hcy in combination with Cu²⁺-induced ROS was further increased and mitochondrial membrane potential was decreased in HUVEC.However,NOX inhibitors failed to prevent Hcy and Cu²⁺ mediated cell death.On the contrary,NOX inhibitors increased primary HUVEC and MAEC apoptosis.Hcy and Cu²⁺ increased lactate production in HUVEC.The combination of Hcy and Cu²⁺ rapidly decreased the activity of mitochondrial electron transport chain complex IV,and then decreased complex I-III activities.Mitochondrial inner membrane cardiolipins were oxidized and mitochondrial membrane potential,oxygen consumption rate and intracellular ATP contents were decreased correspondently.The combination of Hcy and Cu²⁺ increased NO levels and e NOS expression and then decreased both of them,increased i NOS expression,and induced e NOS uncoupling in HUVEC.Hcy and Cu²⁺ increased SOD activity,and decreased activity of catalase in HUVEC.The combination of Hcy and Cu²⁺ decreased the expression of GPx-1 in primary HUVEC and MAEC.Conclusion: The combination of Hcy and Cu²⁺ increases NOX activity in endothelial cells through metabolic energy compensation,and thus increases the levels of ROS and RNS.