Evidence of Defibrotide Internalization and Its Protective Effect in a Hepatic Endothelial in Vitro model

INTRODUCTION: Defibrotide (DF) interferes with several steps of the coagulation-inflammation cycle. Last October (2013), DF received EMA authorization for its use in the treatment of severe hepatic veno-occlusive disease (VOD) after hematopoietic cell transplantation (HCT). Since endothelial dysfunction has been associated with the development of VOD, among other HCT complications, we decided to explore the mechanisms of action of DF in a hepatic endothelial in vitromodel.

OBJECTIVES: To investigate the interaction of DF with a hepatic endothelial cell line from human origin (SK-HEP-1), the mechanisms involved in its traffic and its potential protective effect on fludarabine induced apoptotic mechanisms.

METHODS: SK-HEP-1 cells were exposed to DF (20µg/ml), previously labelled with ULYSIS® Nucleic Acid Labeling Kit, for up to 24 hours. Confocal microscopy (CM, Leica TCS SP5) was used to localize DF into the different cell compartments. Additional experiments were performed to assess colocalization of DF with different endocytosis pathways, by using specific antibodies. MTT assay to measure cell viability was performed in SK-HEP-1 cells after incubation with DF at different concentrations (10 to 200µg/mL, for 24h) followed by fludarabine exposure (at 20µg/mL, for 24h).

RESULTS: CM analysis revealed a significant interaction of DF with endothelial cell membranes followed by internalization and redistribution to the cytoplasm. Mechanisms involved seem to be dependent on endosomes and cytoskeletal assembly. DF did not reach the cell nucleus even after 24h of exposure. In addition, MTT assay demonstrated the protective effect of DF (at concentrations >20µg/mL) on the fludarabine damage on endothelial cells.

CONCLUSIONS: Our studies show that DF interacts with endothelial cell membranes, becoming internalized and redistributed into the endothelial cell cytoplasm without evidence in the nucleus. Moreover, DF protects hepatic endothelial cells from the toxic effect of fludarabine. Our findings may contribute to a better understanding of the precise mechanisms of action of DF as a therapeutic and potential preventive agent on the endothelial damage underlying different pathological situations.

Partially supported by grant SAF2011-28214 (Spanish MINECO and FEDER).