Surface modification of pig endothelial cells with a branched heparin conjugate improves their compatibility with human blood

Anjan K Bongoni,David L Ayares,Robert Rieben,Peetra U Magnusson,Peter J Cowan,Evelyn Salvaris,Eckhard Wolf,Nikolai Klymiuk,Sofia Nordling

doi:10.1038/s41598-017-04898-w

Abstract

Corline Heparin Conjugate (CHC), a compound of multiple unfractionated heparin chains, coats cells with a glycocalyx-like layer and may inhibit (xeno)transplant-associated activation of the plasma cascade systems. Here, we investigated the use of CHC to protect WT and genetically modified (GTKO.hCD46.hTBM) pig aortic endothelial cells (PAEC) in two pig-to-human in vitro xenotransplantation settings. Model 1: incubation of untreated or hTNFα-treated PAEC with 10% human plasma induced complement C3b/c and C5b-9 deposition, cellular activation and coagulation activation in WT and GTKO.hCD46.hTBM PAEC. Coating of untreated or hTNFα-treated PAEC with CHC (100 µg/ml) protected against human plasma-induced endothelial activation and damage. Model 2: PAEC were grown on microcarrier beads, coated with CHC, and incubated with non-anticoagulated whole human blood. Genetically modified PAEC significantly prolonged clotting time of human blood (115.0 ± 16.1 min, p < 0.001) compared to WT PAEC (34.0 ± 8.2 min). Surface CHC significantly improved the human blood compatibility of PAEC, as shown by increased clotting time (WT: 84.3 ± 11.3 min, p < 0.001; GTKO.hCD46.hTBM: 146.2 ± 20.4 min, p < 0.05) and reduced platelet adhesion, complement activation, coagulation activation and inhibition of fibrinolysis. The combination of CHC coating and genetic modification provided the greatest compatibility with human blood, suggesting that pre-transplant perfusion of genetically modified porcine organs with CHC may benefit post-transplant xenograft function.

Highlights

In organ transplantation, the vascular endothelium of the donor organ is the first point of contact with the recipient’s blood
We hypothesized that Corline Heparin Conjugate (CHC) can be used to coat pig aortic endothelial cells (PAEC) with a layer that will protect them from the pro-coagulant and pro-inflammatory environment induced by xenotransplantation
These results demonstrate that hTNFα at high concentration can trigger alterations of the endothelial glycocalyx in WT PAEC

Summary

Introduction

The vascular endothelium of the donor organ is the first point of contact with the recipient’s blood. Vascular endothelial cells normally express an anti-inflammatory and anti-coagulant surface, crucial for the natural anticoagulation of blood These properties are maintained by several mechanisms including the expression of membrane-bound regulatory molecules and the presence of the glycocalyx, a multicomponent layer of proteoglycans and glycoproteins covering the luminal surface of the endothelium. We hypothesized that CHC can be used to coat pig aortic endothelial cells (PAEC) with a layer that will protect them from the pro-coagulant and pro-inflammatory environment induced by xenotransplantation We tested this hypothesis by investigating whether coating of wild-type (WT) and GTKO.hCD46.hTBM PAEC with CHC inhibited coagulation and inflammation mediated by in vitro incubation with human plasma (model 1) or non-anticoagulated whole human blood (model 2)

Methods

Results

Conclusion