Fgl2 induction in porcine endothelial cells through xenogeneic CD40-CD40L interaction

Abstract

Background: Fibrinogen-like protein 2 (fgl2) is a direct prothrombinase that generates thrombin from prothrombin in the absence of a classical prothrombinase complex. Fgl2 is known to be involved in experimental xenograft rejection by mediating coagulation, fibrin deposition and microthrombus formation, leading to classical pathological changes of acute vascular rejection. We tried to investigate whether interaction between CD40 on porcine endothelial cells (PECs) and CD40L on human cells mediates induction of fgl2 on PECs. Methods: Fgl2 mRNA expression was assessed using reverse-transcriptase polymerase chain reaction (RT-PCR). Protein expression and enzymatic activity of fgl2 in PECs were assessed using western blot and fgl2 thrombin generation assay, respectively. Both porcine fgl2 and CD40 knockdowned stable cell lines were established using sifgl2 and siCD40 RNAi system. Results: Fgl2 mRNA expression was induced in PECs at 30 min after PECs had been cultured with Jurkat D1.1 and its expression was suppressed by anti-CD40L antagonistic antibody treatment. Protein expression of fgl2 in PECs was up-regulated at 4 h after treatment of anti-CD40 agonistic antibody as well as TNF-a. The enzymatic activity of fgl2 on PECs was increased by about twofold at 6 h after stimulation of anti-CD40 agonistic antibody. Stable cell lines for either sifgl2 or siCD40 were established. The enzymatic activity of fgl2 was diminished by both sifgl2 and siCD40, but not affected by a control siEGFP. Conclusion: Fgl2 can be up-regulated and mediate coagulation response in activated PECs through xenogeneic CD40-CD40L interaction. Therefore, it might be helpful for the successful xenotransplantation to control fgl2 induction through development of porcine CD40 knockout pigs or antagonistic anti-CD40 treatment.