Iron Overload Damages The Endothelial Mitochondria Via ROS/ADMA/DDAHI?/eNOS/NO Pathway

Objective:Previously it has been recognized that iron overload may harm the body's health.Vascular endothelial cells(VECs)are one of the main targets of iron overload injury,and the mechanism involved was initially thought to be related to the excessive generation of reactive oxygen species(ROS).However,the subcellular region,temporal characteristics of ROS generation,downstream underlying mechanisms,target organelles of ROS of iron overload-induced VECs injury,and the role of asymmetric dimethylarginine(ADMA)/dimethylarginine dimethylaminohydrolase ?(DDAH ?)/endothelial nitric oxide synthase(eNOS)/nitric oxide(NO)pathway are yet to be elucidated.In this study,we elucidated the above-mentioned issues through in vitro experiments.Method:In this study,Human umbilical vein endothelial cells were treated with 50 ?M iron dextran for 48 hours to establish the injury model.The experimental design as follows.Phase 1.We confirmed whether iron overload could induce HUVECs injury.HUVECs were randomly divided into four groups.(1)Control group(Control);(2)iron dextran group(50 ?M Iron-D);(3)pAD/DDAH?pretreatment group(50 ?M Iron-D+pAD/DDAH?);(4)L-Arg pretreatment group(50 ?M Iron-D+1mM L-Arg).At the end of experiments,cell viability,LDH and caspase-3 activities,and apoptosis of HUVECs were determined.Phase 2.we investigated whether iron overload in HUVECs could induce an excess in intracellular ROS generation.HUVECs were randomly divided into five groups.(1)Control group(Control);(2)iron dextran group(50 ?M Iron-D);(3)L-Arg pretreatment group(50 ?M Iron-D+1 mM L-Arg);(4)Eda pretreatment group(50 ?M Iron-D+100 ?M Eda);(5)CsA pretreatment group(50 ?M Iron-D+1?M CsA).At 4,8,16,24,and 48 hours after the addition of Iron-D,intracellular and mitochondrial ROS generation of cells in each group were determined,respectively.At the end of experiments,the cell viability and LDH activity were determined.Phase 3.We further investigated how excessive ROS generation impairs VECs,and we explored the ADMA/DDAH?/eNOS/NO signaling pathway.HUVECs were randomly divided into six groups.(1)Control group(Control);(2)iron dextran group(50 ?M Iron-D);(3)pAD/DDAH ? pretreatment group(50 ?M Iron-D+pAD/DDAH?);(4)L-Arg pretreatment group(50 ?M Iron-D+1 mM L-Arg);(5)pAD/DDAH?-shRNA pretreatment group(50 ?M Iron-D+pAD/DDAH?-shRNA);(6)pAD/DDAH ? +L-NAME pretreatment group(50 ?M Iron-D+pAD/DDAH?+10 ?M L-NAME).At the end of experiments,cell viability and the LDH activity,levels of ADMA and NO in the culture medium,the expression of eNOS,p-eNOS,and DDAH ? in the lysate of HUVECs,and the DDAH ?activity in the lysate of HUVECs were determined.Phase 4.we investigated the iron overload damage-inducing effector.HUVECs were randomly divided into five groups.(1)Control group(Control);(2)iron dextran group(50 ?M Iron-D);(3)pAD/DDAH ? pretreatment group(50 ?M Iron-D+pAD/DDAH?);(4)L-Arg pretreatment group(50 ?M Iron-D+1 mM L-Arg);(5)CsA pretreatment group(50 ?M Iron-D+1 ?M CsA).At the end of experiments,the oxygen consumption rate(OCR),mitochondrial membrane potential(MMP),mPTP opening,and the Cyt C release from mitochondria to the cytoplasm in HUVECs were determined.Results:1.Compared with the control group,the cell viability,NO content,DDAH ?expression and activity,and phosphorylation of eNOS decreased,lactate dehydrogenase(LDH)and caspase-3 activity,ADMA content,and the ratio of apoptotic cells significantly increased after treatment with iron dextran.After addition of L-arginine(L-Arg)or pAD/DDAH?,the above-mentioned changes were reversed.2.Free radical scavenger edaravone(Eda),or ADMA,the competitive substrate L-Arg,mPTP closing agent ciclosporin A(CsA)reduced the generation of intracellular or mitochondrial ROS.ROS originates from the cytoplasm and activates the ROS-induced ROS release(RIRR)mechanism,leading to the level of mitochondrial ROS boost.3.Using edaravone,L-Arg,mPTP,ciclosporin A,orN-nitro-l-arginine methylester,upregulated or downregulated DDAH ? expression can change the activity of DDAH?,ADMA and NO and the expression of p-eNOS.4.Compared with the control group,the OCR declined,loss of the MMP occurred and mPTP opening was induced in iron dextran group.The Cyt C release from mitochondria to the cytoplasm was markedly increased.Co-treatment with pAD/DDAH?,L-Arg and ciclosporin A ameliorated these changes.Conclusion:1.Iron overload can induce excessive ROS burst and cause vascular endothelial cell(VECs)injury.2.ADMA /DDAH ? /eNOS/NO pathway plays an important role in VECs injury by iron overload.3.Mitochondria are the target organelles of VECs damage induced by iron overload.