St. Vincent's Hospital
Attenuation of high mobility group box 1 (HMGB1)-mediated proinflammatory responses by transgenic expression of human thrombomodulin on pig cells
Anjan Bongoni1, Nikolai Klymiuk2, Eckhard Wolf2, David Ayares3, Robert Rieben4, Peter Cowan1,5.
1Immunology Research Centre, St. Vincent’s Hospital Melbourne, Victoria, Melbourne, Australia; 2Institute of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, Munich, Germany; 3Revivicor, Inc., Blacksburg, VA, United States; 4Department of Clinical Research, University of Bern, Bern, Switzerland; 5Department of Medicine, St. Vincent’s Hospital Melbourne, Victoria, Melbourne, Australia
Background: Coagulation dysregulation due to immune responses and cross-species molecular incompatibilities represents a major obstacle to successful pig-to-human xenotransplantation. Thrombomodulin (TM) is an important vascular anti-coagulant that binds thrombin and blocks its procoagulant activity. Transgenic expression of human TM on alpha-1,3-galactosyltransferase knockout (GalTKO)/hCD46 transgenic porcine aortic endothelial cells (PAEC) prolongs the clotting time of non-anticoagulated whole human blood in vitro. Recent studies have revealed that the lectin-like domain of TM can modulate inflammatory responses by sequestering the pro-inflammatory cytokine high-mobility group box 1 (HMGB1), which can then be cleaved and inactivated by TM-bound thrombin. HMGB1, a chromosomal protein released from injured cells, initiates potent innate immune responses in vascular endothelial cells. However, HMGB1-mediated activation of PAEC has not been explored in depth, and the protective effect of hTM against HMGB1’s actions has not been examined in xenotransplantation.
Aims: (1) To investigate HMGB1-mediated pro-inflammatory and procoagulant effects on wild-type (WT) PAEC. (2) To test the capacity of transgenic expression of hTM on PAEC to cleave HMGB1 and neutralise its effects.
Methods: PAEC from WT or GalTKO/hCD46/hTM pigs were treated with HMGB1 (50-200 ng/ml) or positive control hTNFα (10 ng/ml) for 4-24h and analysed for expression of the adhesion molecules E-selectin and ICAM-1, the pro-coagulant fibrinogen-like protein 2 (FGL2), and the anti-fibrinolytic molecule PAI-1 by cell ELISA. Formation of the fluid phase anti-fibrinolytic marker tPA/PAI-1 complexes and production of cytokines were measured by ELISA of culture supernatants. Use of rabbit anti-HMGB1 antibody to block HMGB1 function was also tested. In addition, degradation of HMGB1 (10 µg/ml) by PAEC in the presence of human thrombin (5 U/ml) was analysed by immunoblot/Image J. Statistical significance was determined using one-way Analysis of Variance (ANOVA) with Bonferroni correction (GraphPad Prism).\
Results: HMGB1 increased the expression of E-selectin, ICAM-1 and PAI-1 as well as the production of pTNFα and pMCP-1 by WT PAEC in a dose- and time-dependent manner; FGL2 expression was also increased, although this was significant only at the highest HMGB1 concentration (200 ng/ml: p<0.05). Treatment of WT PAEC with 100 ng/ml HMGB1 for 24h increased the formation of tPA/PAI-1 complexes (p<0.0001). Rabbit anti-HMGB1 antibody (5 µg/ml) decreased hTNFα-induced expression of E-selectin (p<0.0001) and secretion of pTNFα and pMCP-1 (both, p<0.001). Cleavage of HMGB1 by thrombin-treated PAEC was significantly higher with GalTKO/hCD46/hTM cells than with WT cells (p<0.005). Upregulation of E-selectin by HMGB1 or hTNFα was significantly reduced in GalTKO/hCD46/hTM PAEC compared to WT PAEC (p<0.001).
Conclusion: HMGB1 exerts its proinflammatory and procoagulant effects in WT PAEC, upregulating the expression of adhesion molecules to promote leukocyte interaction, activate coagulation and inhibit fibrinolysis. Transgenic expression of hTM on genetically modified PAEC increased thrombin-mediated cleavage of HMGB1 to a less proinflammatory form. These results indicate that transgenically expressed hTM is capable of inhibiting xenotransplantation-induced pro-inflammatory and pro-coagulant responses and may thus support the survival and function of porcine xenograft.
11:00 - 12:30
|Inflammation & Coagulation||Effect of C1 esterase inhibitor treatment of wild-type and multi-transgenic (GalTKO/hCD46) porcine aortic endothelial cells in an in vitro pig-to-human transplantation model||Room 109|
11:00 - 12:30
|Inflammation & Coagulation||Attenuation of high mobility group box 1 (HMGB1)-mediated proinflammatory responses by transgenic expression of human thrombomodulin on pig cells||Room 109|