Tuesday May 02, 2023 - 14:00 to 14:25
1999 - MD, Charles university, Prague, CZ
2005 - board exam - Internal Medicine
2006 - Ph.D. in Cellular Biology and Pathology
2006-2008 PostDoc Fellowship at University of Western Ontario, London, ON, CA
2011 - board exam - Diabetology
2020 - associate professor at Charles university, Prague, CZ
2023 - deputy head of Diabetes Center in Institute for Clinical and Experimental Medicine, Prague, CZ
Clinical interests: Type-1 diabetes mellitus, Pancreas organ and Pancreatic Islet transplantation
Research interests: islet imaging, RNA interference in transplantation, artificial site for islet transplantation
The temporary inhibition of tissue factor in pancreatic islets using a synthetic siRNA can reduce the post transplant liver ischemia
Jan Kriz1, Lucie Kosinova2, Ivan Leontovyč2, Eva Fabryova2, Zuzana Berkova2, Veronika Tomsovska2, Daniel Jirak3, Peter Girman1, Tomas Koblas2.
1Diabetes Center, Institute for Clinical and Experimental Medicine , Prague, Czech Republic; 2Pancreatic islet laboratory, Institute for Clinical and Experimental Medicine , Prague, Czech Republic; 3MR spectroscopy unit, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
Background: The intensity of IBMIR (Instant Blood-Mediated Inflammatory Reaction) depends on the amount of tissue factor (TF) molecules on the surface of pancreatic islet cells in time of transplantation to the portal vein. At the same time, TF is an important growth factor stimulating islet graft revascularization. In order to reduce the intensity of IBMIR and improve the early function of the graft, we tested the possibility of the temporary inhibition of TF synthesis in islet cells using RNA interference (specific siRNA).
Methods: Male Brown Norway rats (250-270g) served both as pancreatic islet (PI) donors and recipients. Diabetes was induced by a single injection of streptozotocin (50 mg/kg, i.p.) and verified by three consecutively measured glycemias over 18 mmol/l. PI were isolated using collagenase digestion according to standard protocol. After overnight cultivation PIs were transfected with anti-TF siRNA (s130189, ThermoFisher Scientific, USA) using lipofection (RNAiMAX, 100nM) or microporation (200nM, Neon, 2 pulses, 950 V, 20 ms). After 24 hours, the treated PIs (microporation n=6, lipofection n=6) were transplanted to portal vein of diabetic animals in marginal dose (2 PI/g). The volume of hypoperfused liver tissue was quantified 2 hours after transplantation of 1000 PIs into the portal vein using magnetic resonance imaging (Fig. 1).
Results: The microporation of siRNA reduced the amount of mRNA by 76/54% and protein by 100/24% for 24/48 hours. The lipofection of siRNA reduced the amount of mRNA by 77/65% and protein by 100/40% for 24/48 hours. PIs normalized glycemias in all recipients transplanted with lipofected PIs and in none with microporated PIs. The transplantation of 1000 PIs caused impairment of perfusion in 28% of liver tissue. If islets were transfected with either a specific siRNA using microporation or lipofection, the total volume of hypoperfused tissue declined to 11% or 0%, respectively.
Conclusions: AntiTF-siRNA transfected by microporation efficently reduced the amount of TF for 24 hours but did not improve the function of transplanted PIs. AntiTF-siRNA transfected by lipofection efficiently reduced amount of TF for 24 hours and significantly improve the function of transplanted PIs. Microporation likely caused the Off-target effect in PI graft.
Funded by the project National Institute for Research of Metabolic and Cardiovascular Diseases (Programme EXCELES, Project No. LX22NPO5104) - Funded by the European Union - Next Generation EU. Supported by MH CZ - DRO ("Institute for Clinical and Experimental Medicine - IKEM, IN 00023001") .
 Kosinova L, Patikova A, Jirak D, Galisova A, Vojtiskova A, Saudek F, Kriz J. A novel model for in vivo quantification of immediate liver perfusion impairment after pancreatic islet transplantation. Islets. 2019;11(6):129-140. doi: 10.1080/19382014.2019.1651164. Epub 2019 Sep 9. PMID: 31498024; PMCID: PMC6930024.