Research On The Role And Mechanisms Of Tissue Factor In Cultured Human Umbilical Vein Endothelial Cells Subjected To Anoxia-reoxygenation

Anoxia-reoxygenation (A/R) is a process in which oxygen is given after a period of ischemia in cells. In the early stage of reoxygenation, cells injury due to ischemia are not improved, but aggravated. Many ischemic disorders in heart and brain are accompanied with anoxia-reoxygenation or ischemia-reperfusion injury, which is the common pathophysiological pathogenesis. Ischemia-reperfusion depicts a tissue or an organ with blood supply, while anoxia-reoxygenation describes a cell without blood supply. The main disturbance in anoxia-reoxygenation is the generation of large amounts of reactive oxygen species (ROS), which brings about lipid peroxidation, cell injury, even the whole cell destruction.Apart from lipid peroxidation, both blood coagulation and inflammation are involved in anoxia-reoxygenation or ischemia-reperfusion. As the initiator of extrinsic coagulation system, tissue factor (TF) is the only transmembrane protein in blood coagulation system. Recent investigations reveal that besides taking part in blood coagulation, TF mediates tumorigenesis, embryonic development, neogenesis of blood vessels, inflammation and atherogenesis as a signal transduction molecule. Therefore, TF may play a role in the genesis and development of anoxia-reoxygenation or ischemia-reperfusion injury. Up to now, there is no report on how TF performs durinn anoxia-reoxygenation or ischemia-reperfusion process. In this study, the role and mechanisms of TF in cultured human umbilical vein endothelial cells (HUVECs) subjected to anoxia-reoxygenation were investigated.【Objective】The role and mechanisms of TF in cultured HUVECs subjected to anoxia-reoxygenation were investigated.【Methods】HUVECs were divided randomly into control group, A/R group, aODN/TF+A/R group, sense oligodeoxynucleotide (sODN/TF)+A/R group and mismatched oligodeoxynucleotide (mODN/TF)+A/R group, in the latter 3 groups, HUVECs were transfected with aODN/TF, sODN/TF and mODN/TF respectively. HUVECs in all A/R groups underwent 3 hrs of anoxia and followed by 2 hrs of reoxygenation. Then, HUVECs were collected for TF mRNA determination by using Run-on assay, tissue factor procoagulant activity (TF:C) and tissue factor antigen (TF:Ag) detection with one stage coagulation method and enzyme linked immunosorbent assay (ELISA) respectively. In addition, HUVECs viability, concentrations of lactate dehydrogenase (LDH), malonaldehyde (MDA), superoxide dismutase (SOD), glutathion peroxidase (GSH-PX), nitric oxide (NO) and endothelin-1 (ET-1) were investigated simultaneously. In order to investigate the potential mechanism of how increased TF may contribute to A/R injury, HUVECs were randomly divided into control group, A/R group and human recombinant active site blocked factor VII (FVIIai) plus A/R group (FVIIai+A/R group). HUVECs in A/R group and FVIIai+A/R group underwent the same anoxia-reoxygenation mentioned above, but 1,5-dansyl-Glu-Gly-Arg chloromethyl ketone (DEGRck) inactivated human FVIIa was added into the cell culture medium in FVIIai+A/R group with the final concentration of 200 nM before anoxia-reoxygenation. The cells in control group were incubated under normoxia at 37℃for totally 5 hrs. The relative concentrations of reactive oxygen species (ROS), protease activated receptor-1 (PAR-1), protease activated receptor-2 (PAR-2), phospho-p38 mitogen activated protein kinases (phospho-p38 MAP kinase) and phospho-p42/44 mitogen activated protein kinases (phospho-p42/44 MAP kinase) were determined by using flow cytometry.【Results】After A/R, TF expression at both mRNA and protein level was increased, furthermore, cell viability and the concentrations of SOD, GSH-PX and NO were declined, while LDH, MDA and ET-1 were overproduced (P<0.05 to 0.001 versus control group). In HUVECs of aODN/TF+A/R group, however, TF expression was inhibited, while the declined cell viability and the concentrations of SOD, GSH-PX, NO as well as the enhanced LDH, MDA and ET-1 levels occurred during A/R were ameliorated and reversed effectively (P<0.05 to 0.01 versus those in other A/R groups). The results also showed that ROS was increased and PAR-1, PAR-2, p38 MAP kinase and p42/44 MAP kinase were all activated after A/R (P<0.001 versus HUVECs under normoxia), while FVIIai inhibited the increment of ROS, PAR-1, PAR-2, p38 MAP kinase and p42/44 MAP kinase, and improved the changes of TF:C, MDA, SOD, GSH-PX, cell viability and LDH occurred during A/R (P<0.05 to 0.001 versus HUVECs without FVIIai treatment).【Conclusion】During anoxia-reoxygenation, not only TF mediates blood coagulation, but also induces inflammation via activating PAR-1, PAR-2, p38 MAP kinase and p42/44 MAP kinase, and aggravates cells injury. Therefore, TF is the "central molecule" in anoxia-reoxygenation, which connects anoxia-reoxygenation, blood coagulation and inflammation, and constructs a complicated network. Our findings in the present study provide important data to enrich the mechanisms of anoxia-reoxygenation and ischemia-reperfusion injury.