St. Vincent's Hospital
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
Anjan K Bongoni1,2, Pavan Garimella2, Nikolai Klymiuk3, Eckhard Wolf3, David Ayares4, Rolf Spirig5, Robert Rieben2.
1Immunology Research Centre, St. Vincent’s Hospital Melbourne, Melbourne, Australia; 2Department of Clinical Research, University of Bern, Bern, Switzerland; 3Institute of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, Munich, Germany; 4Revivicor, Inc., , Blacksburg, Virginia, VA, United States; 5CSL Behring AG, CSL Behring AG, Bern, Switzerland
Background: Prevention of endothelial activation by transgenic (over) expression of human membrane-bound complement regulators, in addition to Gal-alpha1,3-Gal knockout (GalTKO), was shown to be insufficient to avoid thrombosis formation in pig-to-primate xenotransplantation. Additional strategies to prevent coagulation are therefore needed. C1 esterase inhibitor (C1inh) is a main regulator of the plasmatic cascade systems complement, contact phase, coagulation, and fibrinolysis. The aim of this study was to test the effect of C1inh on regulation of xenotransplantation-induced activation of complement as well as coagulation in vitro.
Methods: Porcine aortic endothelial cells (PAEC) from wild-type (WT) and GalTKO/hCD46 pigs were treated with normal human serum (NHS, 1:10) in the presence or absence of C1inh (10 U/ml) and analyzed for complement deposition as well as adhesion molecule expression (E-selectin, VCAM-1). Subsequently, the anti-coagulant properties of C1inh were tested in a microcarrier based coagulation assay with WT PAEC and non-anticoagulated human blood.
Results: Deposition of C1q, MBL and Factor Bb on WT PAEC treated with NHS demonstrated the involvement of all the three pathways of complement in our xenotransplantation model. However, PAEC from GalTKO/hCD46 pigs showed significantly reduced deposition of C4b/c (p<0.0001), C3b/c (p=0.045) and C5b-9 (p=0.0009) as well as expression of E-selectin (p=0.001) and VCAM-1 (p=0.072) when treated with NHS as compared to WT PAEC. In addition, formation of tissue plasminogen activator (tPA)/plasminogen activator inhibitor (PAI)-1 complexes, measured as fluid phase anti-fibrinolytic marker, were also significantly lower (p<0.0001) in NHS treated GalTKO/hCD46 than in WT PAEC. Co-incubation of WT PAEC with NHS/C1inh induced significantly decreased C4b/c (p=0.008), C3b/c (p=0.0005) and C5b-9 deposition (p<0.0001) as well as E-selectin expression (p=0.004) and tPA/PAI-1 complex formation (p=0.002) as compared to NHS-only treated PAEC. However, no differences were observed for these markers between NHS-only and NHS/C1inh treated GalTKO/hCD46 PAEC.
In further experiments, C1inh treatment of WT PAEC prolonged clotting of whole, non-anticoagulated human blood (23.5 ± 2.1 min) as compared to untreated PAEC (14.5 ± 0.7 min, p=0.048, n=2). The formation of sC5b-9 (p=0.010) and tPA/PAI-1 (p=0.025) complexes in EDTA-plasma samples collected from coagulation assays was significantly lower in C1inh treated WT PAEC as compared to untreated PAEC.
Conclusion: C1inh treatment significantly reduced complement deposition and complement mediated endothelial activation in WT PAEC. However, the effect of C1inh treatment was no longer significant when multi-transgenic GalTKO/hCD46 PAEC were used, most probably because of the dramatic reduction of complement activation by hCD46.
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|