Saturday September 25, 2021 - 19:00 to 20:00
Immunosuppressive effect of MCP and TBM expressions against human serum in aortic endothelial cells of GTKO/MCP/TBM pig
Haesun Lee1, In-Sul Hwang1, Mi-Ryung Park1, Jingu No1, Yeong-ji Kim1, Tae-Uk Kwak1, Keon Bong Oh1.
1Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun, Korea
Introduction We generated genetically engineered pig designed to express membrane cofactor protein (MCP) ubiquitously and thromobomodulin (TBM) endothelium-specifically under the background of homozygous alpha 1,3 galactosyltransferase knockout (GTKO/MCP/TBM). We aimed to investigate protective effects of MCP and TBM in aorta endothelial cells (pAECs) of GTKO/MCP/TBM pig against human serum attack.
Method Quantitative real-time PCR (qPCR) and western blot analyses were performed to identify expressions of MCP and TBM in various tissues of GTKO/MCP/TBM pig. The pAECs isolated from GTKO and GTKO/MCP/TBM pigs were cultured with medium supplemented with pig and human serum, respectively, and subsequently performed WST-1 cell proliferation assay. qPCR was performed to analyze expression level of IL1α, IL8, MCP-1, ICAM-1, VCAM-1, E-selectin, TF and TFPI genes in the pAECs.
Results We confirmed that GTKO/MCP/TBM pig expressed MCP efficiently in all tissues analyzed and TBM specifically in vascular endothelium. GTKO/MCP/TBM pAECs exhibited significantly decreased cytotoxicity compared with GTKO pAECs under the culture condition with 10% human serum. Notably, human serum compared with pig serum supplemented in culture medium induced upregulation of IL1α, IL8, MCP-1, ICAM-1, VCAM-1, E-selectin, and TF genes. Among those, a cytokine IL1α, cell adhesion molecules VCAM-1 and E-selectin, and a coagulant TF genes in GTKO/MCP/TBM pAECs were shown to significantly lower than those in GTKO pAECs. Interestingly, expression of anticoagulant TFPI gene showed higher level in GTKO/MCP/TBM pAECs than GTKO pAECs.
Conclusion MCP and TBM expression with GTKO resulted in downregulation of pro-inflammatory cytokine, adhesion molecules and coagulant in response to xenogeneic human serum stimulation. These results suggest that GTKO/MCP/TBM pig developed in this study may attenuate rejection mechanisms occurring in a xeno-graft by inter-species molecular incompatibility between donor tissue and recipient.
This research was supported by the National Institute of Animal Science (project no. PJ01422802), Rural Development Administration, Republic of Korea.