Defibrotide, an anti-atheroslerotic and anti-apoptotic drug, prevents leukotriene B4 (LTB4) expression and caspase 3 activation in human microvascular endothelial cells
Aims: Defibrotide (DF) is a polydisperse mixture of 90% single-stranded polydeoxyribonucleotides with anti-ischemic and thrombolytic functions. Since reperfusion-induced cell death is critical in heart and lung transplantation, DF could be of value for the protection of the vasculature in cardiopulmonary bypass and transplant settings.
Methods: Human microvascular endothelial cells (HMEC) were seeded in y-shaped flow chambers mimicking atherogenic vessel branches. A continous arterial flow of 10dyn/cm2 was applied for 48 hours. Subsequently, atherogenic proteins (5LOX, FLAP) and apoptosis, as well as deformation grade of HMEC were determined by flow cytometry, nuclear DNA staining and computer-assisted cell tracking, respectively. Leukotriene B4 (LTB4) was measured via ELISA. Stress-induced caspase3 activation was assessed by cytometric bead arrays and intracellular flow cytometry.
Results: Shear stress results in expression of 5-LOX and FLAP in >90% of HMEC. As a consequence of this, LTB4 could be detected in the supernatants of stressed cells. DF (10µM) down-regulated 5-LOX and FLAP expression as well as LTB4 release by 50%. In addition, DF attenuated deformation of cells at sites of pro-atherogenic flow. In another set of experiments, HMEC were treated with Fludarabine (35µM) to induce caspase3 activation. Co-administration of DF (10µM) led to a complete abrogation of caspase3 activation.
Conclusion: Our data suggest that the anti-atherosclerotic function of DF can be explained by LTB4 down-regulation. The anti-apoptotic effect is based on an inhibition of the extrinsic apoptosis pathway. Its anti-ischemic and thrombolytic function qualifies DF for a prophylactic and therapeutic use in cardiopulmonary bypass and heart transplant settings.