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

Abstract

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. Porcine aortic endothelial cells were treated with HMGB1, human (h)TNFα 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, TNFα, 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. 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 TNFα, IL-8, and monocyte chemotactic protein-1 and the formation of PAI-1/tissue plasminogen activator complexes. Human TNFα- 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 hTNFα, 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. 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 hTNFα and LPS. Human thrombomodulin transgenic PAECs are less sensitive to activation by either HMGB1 or hTNFα, an effect that appears to be dependent on the lectin-like domain of TBM.