Background: Thrombotic microangiopathy (TMA) is a fatal complication that occurs after pancreas-kidney transplantation leading to allograft dysfunction and graft loss. Immunosuppressants, e.g. calcineurine inhibitors, have been implicated in the development of nonimmune TMA. Tacrolimus has been shown to be associated with decreased NO-production, thus contributing to thromosis. ADMA acts as an endogenous NO-synthase inhibitor and is considered to be a marker of endothelial dysfunction.Aim of this study was to analyse the influence of different immunosuppressants on microvascular thrombus formation in vivo and to further examine the underlying endothelial function.
Methods: Using the skin fold chamber in C57BL/6J mice, microvascular thrombus formation was induced photochemically and quantitatively analyzed by intravital fluorescence microscopy. Mice were treated with Tacrolimus (TAC: 10 mg/kg/d; n=7), Cyclosporine (CYA: 5 mg/kg/d) or Sirolimus (RAPA: 1,5 mg/kg/d ip) on 3 consecutive days. Control-mice received NaCl 0,9% (10 ml/kg/d ip; n=5). Drug plasma levels were examined to ensure therapeutic doses. Additionally, sP-, sE-Selectin and ADMA-plasma levels were measured by ELISA.
Results: Application of immunosuppressants produced clinically relevant plasma levels (TAC: 8±2 ng/ml, CYA: 214±16 ng/ml; RAPA: 9±1 ng/ml). Microvascular thrombus formation was significantly accelerated in mice receiving TAC compared to control-mice (arteriolar and venular occlusion: 251±101s and 179±27s vs. control 818±221s and 749±231s; p<0,05). Application of CYA significantly increased thrombus formation only in venules (276±49s; p<0,05 vs. control), whereas RAPA had no significant effect on thrombosis induction. Plasma concentrations of sP- and sE-Selectin were slightly reduced after TAC and CYA application. ADMA-levels however were significantly increased in mice receiving TAC (1,28±0,16 µmol/l vs. control: 0,74±0,13 µmol/l; p<0,05).
Conclusions: The impact of immunosuppressants on TMA as well as the underlying mechanisms have not been clearly delineated. Our results show that clinically relevant plasma levels of Cyclosporine enhanced thrombus formation only in venules whereas Tacrolimus significantly accelerated thrombus formation in arterioles and venules. This effect could be explained by increased ADMA-levels in plasma resulting in impaired NO-bioavailability and increased thrombogenicity, thus presenting a new molecular mechanism in the development of transplant-thrombosis. Preliminary results in post-ischaemic tissue confirm the effect of Tacrolimus on microvascular thrombus formation, further underlining the significant role of calcineurine inhibitors in TMA.