What is going on between defibrotide and endothelial cells? Snapshots reveal the hot spots of their romance

• Published in: Blood 2016-03, Vol.127 (13), p.1719-1727
• Main Authors: Palomo, Marta, Mir, Enrique, Rovira, Montse, Escolar, Ginés, Carreras, Enric, Diaz-Ricart, Maribel
• Format: Article
• Language: English
• Subjects: Anti-Inflammatory Agents - pharmacokinetics; Antioxidants - pharmacokinetics; Cell Membrane - drug effects; Cell Membrane - metabolism; Cells, Cultured; Dose-Response Relationship, Drug; Human Umbilical Vein Endothelial Cells - drug effects; Human Umbilical Vein Endothelial Cells - metabolism; Humans; Inflammation - pathology; Inflammation - prevention & control; Nitric Oxide Synthase Type III - metabolism; Pinocytosis - drug effects; Polydeoxyribonucleotides - pharmacokinetics; Temperature; Time Factors; Vascular Biology
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2015-10-676114

Defibrotide (DF) has received European Medicines Agency authorization to treat sinusoidal obstruction syndrome, an early complication after hematopoietic cell transplantation. DF has a recognized role as an endothelial protective agent, although its precise mechanism of action remains to be elucidated. The aim of the present study was to investigate the interaction of DF with endothelial cells (ECs). A human hepatic EC line was exposed to different DF concentrations, previously labeled. Using inhibitory assays and flow cytometry techniques along with confocal microscopy, we explored: DF-EC interaction, endocytic pathways, and internalization kinetics. Moreover, we evaluated the potential role of adenosine receptors in DF-EC interaction and if DF effects on endothelium were dependent of its internalization. Confocal microscopy showed interaction of DF with EC membranes followed by internalization, though DF did not reach the cell nucleus even after 24 hours. Flow cytometry revealed concentration, temperature, and time dependent uptake of DF in 2 EC models but not in other cell types. Moreover, inhibitory assays indicated that entrance of DF into ECs occurs primarily through macropinocytosis. Our experimental approach did not show any evidence of the involvement of adenosine receptors in DF-EC interaction. The antiinflammatory and antioxidant properties of DF seem to be caused by the interaction of the drug with the cell membrane. Our findings 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 pathologic situations. •Specific interaction of DF with EC membranes is followed by its internalization mainly through macropinocytic mechanisms.•DF attachment to the cell membrane is sufficient to perform its antiinflammatory and antioxidant effects on the endothelium.