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Donor genetic modification for xenotransplantation 1

Thursday October 26, 2023 - 12:45 to 13:45

Room: Indigo D

108.6 Overexpression of hCD31 in porcine endothelial cells reduces neutrophil extracellular trap formation in human and non-human primates

Award Winner

Suchen Kumar Yadav, Korea has been granted the TTS-IXA Congress Scientific Award

Suchen Kumar Yadav, Korea

Graduate Research Student
Department of Medicine (Transnational Medicine Major)
Seoul National University


Overexpression of hCD31 in porcine endothelial cells reduces neutrophil extracellular trap formation in human and non-human primates

Suchen Yadav5,8, Sunghoon Hurh1, Boem Seok Kim6, Joon Young Jang6, Ji Jin Yan6, Ji Young Kim6, Sungjoo Kim2, Curie Ahn7, Jaeseok Yang6, Jong Cheol Jeong8.

1Department of Medicine, Korea University, Seoul, Korea; 2Department of Surgery, GenNbio Inc., Pyeongtaek, Korea; 3Department of Surgery, Seoul National University Hospital, Seoul, Korea; 4Department of Medicine, Seoul National University Hospital, Seoul, Korea; 5Graduate School of Translational Medicine, Seoul National University, Seoul, Korea; 6Department of Internal Medicine, Yonsei University, Seoul, Korea; 7Department of Internal Medicine, National Medical Center, Seoul, Korea; 8Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Korea

Introduction: Neutrophil extracellular traps (NETs) formation, or NETosis, is a type of programmed cell death that involves the release of extracellular traps, which plays an important role in the immune response against pathogens. Still, it can also contribute to the development of various pathological conditions. There was some evidence that NETs play a significant role in the rejection of allotransplantation; NETs were released in lung transplantation, and disruption of NETs increased graft survival (1). In a study of pig-to-baboon xenotransplants, researchers observed a significant increase in NET markers like IL-8 and cell-free histone in plasma, which were associated with the development of graft rejection (2-3). In addition to their role in rejecting grafts, NETs have also been shown to contribute to developing transplant-associated thrombosis. This is a serious complication that can occur after transplantation, in which blood clots form in the blood vessels of the transplanted organ. NETs have been shown to activate platelets and promote clot formation, which can lead to thrombosis (4).

The human cluster of differentiation 31 (hCD31), also known as platelet endothelial cell adhesion molecule 1 (PECAM-1), is a transmembrane glycoprotein expressed on the surface of endothelial cells and leukocytes, including neutrophils. hCD31 has been shown to play a crucial role in regulating neutrophil activation and migration and has been proposed as a potential therapeutic target for various inflammatory disorders. However, there are incompatibilities between the porcine cluster of differentiation 31 (pCD31) and hCD31 (5). We hypothesized that hCD31 overexpression in porcine endothelial cells would prevent NETosis and hence increase graft survival.

Method: After cloning the coding sequence of the hCD31 gene into the pCAG1.2 vector, the gene was introduced into the porcine aortic endothelial cell (PAEC), or MPN3. Human and non-human primate neutrophils were isolated from venous blood. The neutrophil extracellular traps (NETs) were induced in vitro by coculturing human or monkey neutrophils with MPN3, GGTA1, and B4GALNT2 knockout MPN3 (DKO.MPN3)  or hCD31-transfected MPN3 or DKO.MPN3 cells in microfluidic chips and plates. NETs were quantified by measuring the DNA-histone complex and IL-8 using an Elisa kit from the supernatant of coculture, and FACS was used to assess the oxidative burst and tissue factor expression.

Results: We confirmed the NET formation in coculture using the colocalization of DNA and citrullinated histones both in microfluidic chips and plates. Even DKO.MPN3 cells, independent of plasma and other cellular components can induce NETs. We observed using fluorescent imaging that overexpression of hCD31 decreased NET formation in coculture. We found tissue factor expression and oxidative burst in human neutrophils, assessed using FACS, were reduced by overexpression of hCD31. hCD31 overexpression also reduced DNA-histone complex and IL-8 secretion in coculture.

Conclusion: Current study results suggest that porcine aortic endothelial cells can independently induce NETs formation in vitro coculture. overexpression of hCD31 on porcine endothelial cells suppresses NETosis. Further studies can be aimed at the role of hCD31 overexpression in porcine endothelial cells on other leukocytes, as most of them constitutively express the protein.


[1] Sayah DM, Mallavia B, Liu F, et al. Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation. Am J Respir Crit Care Med. 2015;191(4):455-463. doi:10.1164/rccm.201406-1086OC
[2] Iwase H, Liu H, Li T, et al. Therapeutic regulation of systemic inflammation in xenograft recipients. Xenotransplantation. 2017;24(2):10.1111/xen.12296. doi:10.1111/xen.12296
[3] Li T, Lee W, Hara H, et al. An Investigation of Extracellular Histones in Pig-To-Baboon Organ Xenotransplantation. Transplantation. 2017;101(10):2330-2339. doi:10.1097/TP.0000000000001676
[4] Esmon CT. Molecular circuits in thrombosis and inflammation. Thromb Haemost. 2013;109(3):416-420. doi:10.1160/TH12-08-0634
[5] Nasu K, Whyte A, Green SJ, Evans PC, Kilshaw PJ. Alpha-galactosyl-mediated activation of porcine endothelial cells: studies on CD31 and VE-cadherin in adhesion and signaling. Transplantation. 1999;68(6):861-867. doi:10.1097/00007890-199909270-00020

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