title: Tissue factor knockdown in porcine islets: an effective approach to suppressing the instant blood-mediated inflammatory reaction.
abstract: Tissue factor (TF) expression on islets has been shown to trigger instant blood-mediated inflammatory reaction (IBMIR), leading to rapid islet loss in portal vein islet transplantation. This study investigated whether antisense RNA-mediated TF gene knockdown in islets could suppress  IBMIR as a strategy to overcome IBMIR. Neonatal porcine islet cell clusters (NICCs) were transfected with or without TF-specific antisense RNA or a nonspecific RNA by a lipid-based method. Expression of both TF gene and protein in NICCs was analyzed after transfection by real-time PCR, Western  blot, and FACS, respectively. The impact of antisense RNA transfection on NICC viability and in vitro function was examined by FACS and insulin release test, respectively. The effect of TF knockdown in NICCs on IBMIR was assessed with an in vitro tubing loop assay using human blood. A significant  reduction in TF gene and protein expression was achieved in TF antisense RNA but not control RNA transfected NICCs, which did not affect NICCs' viability or their insulin secreting capacity. Incubation of TF antisense RNA transfected with human blood resulted in a considerable reduction in  blood clot formation, platelet consumption, and complement and coagulation activation compared to that observed in the loops containing human blood and untreated or control RNA transfected NICCs. Consistent with these findings, infiltrating neutrophils in the blood clots with entrapped TF  antisense RNA transfected NICCs was also reduced substantially compared to that seen in the clots containing untreated or control RNA transfected NICCs. This study presents a nontoxic TF antisense RNA-mediated TF knockdown in porcine islets that leads to an effective suppression of IBMIR,  suggesting a potentially new strategy to improve islet transplantation outcomes.
authors.affiliation: Cell Transplantation and Gene Therapy Institute, The Third Xiang Ya Hospital of Central South University, Changsha, Hunan, China.
authors.forename: Xiaoqian
authors.initials: X
authors.lastname: Ma
authors.forename: Bin
authors.initials: B
authors.lastname: Ye
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
authors.forename: Qi
authors.initials: Q
authors.lastname: Liang
authors.forename: Qiong
authors.initials: Q
authors.lastname: Dong
authors.forename: Yin
authors.initials: Y
authors.lastname: Liu
authors.forename: Pengfei
authors.initials: P
authors.lastname: Rong
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Shounan
authors.initials: S
authors.lastname: Yi
mesh_terms: D000818:Animals; D001777:Blood Coagulation; D001792:Blood Platelets; D002478:Cells, Cultured; D003167:Complement Activation; D005260:Female; D020868:Gene Silencing; D006801:Humans; D007249:Inflammation / Q000517:prevention & control*; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D016381:Islets of Langerhans Transplantation; D008297:Male; D009504:Neutrophils / Q000276:immunology; D016372:RNA, Antisense; D013552:Swine; D013925:Thromboplastin / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014183:Transplantation, Heterologous
chemical_list: D007328:Insulin; D016372:RNA, Antisense; D013925:Thromboplastin