Increased human tumor necrosis factor-α levels induce procoagulant change in porcine endothelial cells in vitro.
BACKGROUND
Intravascular thrombosis and systemic coagulation abnormalities are major hurdles to successful xenotransplantation and are signs of acute humoral rejection. Increased expression of tissue factor (TF) is associated with the development of microvascular thrombosis in xenografts. To develop an effective strategy to prevent accelerated coagulation in xenografts, we investigated the mechanism by which porcine endothelial cells (PECs) become procoagulant after contact with human blood.


METHODS
The changes in TF mRNA levels and activity in PECs after incubation with 20% human serum or human bioactive molecules, including C5a, tumor necrosis factor-α (TNFα) and interleukin (IL)-1α, were evaluated using real-time PCR and the factor Xa chromogenic assay, respectively. The procoagulant changes in PECs by these agonists were evaluated by measuring the coagulation time of human citrated plasma suspended with PECs pretreated with each agonist. TF expression and coagulation times were also assessed in PECs transfected with short interfering RNA (siRNA) designed to knock down porcine TF. We also examined the production of proinflammatory cytokines in human whole-blood or plasma after contact with PECs, which were screened using the cytometric bead array system. TNFα levels were measured using ELISA in whole-blood after contact with PECs, with or without the addition of xenoreactive antibodies or C1 esterase inhibitor.


RESULTS
Porcine TF mRNA and activity in PECs were up-regulated in response to human TNFα and IL-1α but were not affected by C5a or 20% human serum. Up-regulation of TF expression by human TNFα or IL-1α shortened PEC-induced coagulation time, while siRNA-mediated knockdown of TF expression prolonged coagulation time. The incubation of PECs with human whole-blood led to a significant increase in human TNFα levels in the blood, which was promoted by the addition of xenoreactive antibodies and prevented by C1 esterase inhibitor.


CONCLUSIONS
Human TNFα level increases in human blood after contact with PECs, which is attributed to xenoreactive antibody binding and subsequent complement activation. Human TNFα induces procoagulant changes in PECs with increased TF expression. This study suggests that human TNFα may be one of the mediators linking complement activation with procoagulant changes in the xenoendothelium.
Division of Biotechnology, Korea University College of Life Sciences and Biotechnology, Seoul, Korea.
Kyoung Geun
KG
Lee
Haneulnari
H
Lee
Jeong Mi
JM
Ha
Young Kyung
YK
Lee
Hee Jung
HJ
Kang
Chung-Gyu
CG
Park
Sang Joon
SJ
Kim
D000818:Animals; D001011:Aorta / Q000166:cytology; D001777:Blood Coagulation / Q000276:immunology; D001779:Blood Coagulation Factors / Q000276:immunology* / Q000378:metabolism; D002478:Cells, Cultured; D003167:Complement Activation / Q000276:immunology; D042783:Endothelial Cells / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D006084:Graft Rejection / Q000276:immunology / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D053582:Interleukin-1alpha / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D013552:Swine; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism; D013927:Thrombosis / Q000276:immunology; D014183:Transplantation, Heterologous / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology* / Q000378:metabolism* / Q000494:pharmacology
D001779:Blood Coagulation Factors; D053582:Interleukin-1alpha; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; C045071:leukocyte procoagulant activity; D013925:Thromboplastin