Transgenic Expression of Human Thrombomodulin Inhibits HMGB1-Induced Porcine Aortic Endothelial Cell Activation.

Background: Transgenic expression of human thrombomodulin (hTBM), which has the potential to solve the problem of coagulation dysregulation in pig-to-primate xenotransplantation, may have additional benefits by neutralizing the proinflammatory cytokine high-mobility group box 1 (HMGB1). The aim of this study was to investigate HMGB1-mediated effects on porcine aortic endothelial cells (PAEC) from wild-type (WT) and hTBM transgenic pigs. Methods: Porcine aortic endothelial cells were treated with HMGB1, human (h)TNF[alpha] or lipopolysaccharide (LPS). Procoagulant and proinflammatory responses were assessed by measuring expression of cell surface markers (adhesion molecules, fibrinogen-like protein 2, plasminogen activator inhibitor (PAI)-1), secretion of porcine cytokines and chemokines (HMGB1, TNF[alpha], IL-8, monocyte chemotactic protein-1), and formation of PAI-1/tissue plasminogen activator complexes. Thrombin-mediated degradation of HMGB1 in the presence of PAEC was examined by Western blot and functional assay. Results: High-mobility group box 1 potently activated WT PAEC, increasing the expression of E-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, fibrinogen-like protein 2, and PAI-1, the secretion of TNF[alpha], IL-8, and monocyte chemotactic protein-1 and the formation of PAI-1/tissue plasminogen activator complexes. Human TNF[alpha]- or LPS-induced activation of WT PAEC was inhibited by treatment with rabbit anti-HMGB1 antibody. Transgenic expression of hTBM significantly reduced the activation of PAEC by HMGB1 or hTNF[alpha], and significantly enhanced thrombin-induced HMGB1 cleavage. Chemically induced shedding of the lectin-like domain of TBM resulted in significantly increased HMGB1-induced PAEC activation. Conclusions: High-mobility group box 1 exerts powerful proinflammatory and procoagulant effects on WT PAEC, and appears to be an important downstream mediator for the actions of hTNF[alpha] and LPS. Human thrombomodulin transgenic PAECs are less sensitive to activation by either HMGB1 or hTNF[alpha], an effect that appears to be dependent on the lectin-like domain of TBM. Copyright (C) 2016 Wolters Kluwer Health, Inc. All rights reserved.

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