Background: Von Willebrand factor (vWF) is a prothrombotic mediator stored in intracytoplasmic granules within endothelial cells and is released by endothelial exocytosis, leading to microangiopathic lesions reminiscent of transplant glomerulopathy. The multimeric form of the protein makes it difficult to quantify accurately the amount of the molecule released. Because the exocytosis is calcium-dependent, we sought to determine if the use of a calcineurin inhibitor could allow intracellular measurement of vWF in vitro in endothelial cells.

Methods: We used an in vitro a model of antibody-mediated rejection (ABMR), in which a pan-HLA I antibody (that recognizes a monomorphic determinant on all class I molecules) was used to stimulate human microvascular glomerular endothelial cells (HMVEC). HMVECs (passage 4) were grown to confluence and then pre-incubated for 30min with pharmacological concentrations of tacrolimus (2, 5, 10 and 15 ng/mL), prior to stimulation by pan-HLA I antibody (2 [micro]g/mL) for 10min; unstimulated cells and histamine 1mM were used as negative and positive controls, respectively. vWF was stained using rabbit anti-human-vWF IgG and secondary Alexa 488 goat IgG and examined by fluorescent microscopy.

Results: As displayed in Figure 1, the negative control shows that in unstimulated conditions, vWF is scattered in the cytoplasm (A). HLA stimulation without tacrolimus resulted in an abundant exocytosis of vWF strings (B). In contrast, pre-incubation of HMVECs with tacrolimus 5 ng/mL, produced an intense perinuclear staining for vWF (C). Dose-response experiments showed an increasing effect of tacrolimus up to 10 ng/mL.

Conclusions: The use of tacrolimus allows for intracellular accumulation of vWF following stimulation of endothelial cells. Flow cytometric experiments are underway to optimize a quantitative intracellular assay to assess endothelial response to HLA stimulation.

: (A) Unstimulated. (B) HLA-I 2,0 [mu]g/mL. (C) HLA-I 2,0 [mu]g/mL, tacrolimus 5 ng/mL.

CITATION INFORMATION: Béland S, Vallin P, Désy O, De Serres S. Tacrolimus as a Tool for Intracellular Assessment of von Willebrand Factor Production by Activated Human Glomerular Endothelial Cells. Am J Transplant. 2016;16 (suppl 3).

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