Sensitization by blood transfusion results in antibody production that correlates with C4d deposition and rejection

Abstract

vascular homeostasis and in multiple vascular pathologies. We analyzed the effects of the calcineurin inhibitors cyclosporin A (CyA) and tacrolimus (T), and the mTOR inhibitor sirolimus (S) on EPC survival. Methods: Peripheral blood mononuclear cells were isolated from healthy subjects by density gradient centrifugation, resuspended in endothelial growth medium and plated on to fibronectin-coated wells at a density of 0.5x10 cells/cm. At day 4 after plating, non-adherent cells were removed and cells were cultured to day 7. Cells were cultured with CyA (0.01 to 10000 ng/ml), T (0.001 to 1000 ng/ml), S (0.001 to 1000 ng/ml) or vehicle control (DMSO 0.1%). Immunosuppressants (IS) were added either at day 0 and maintained for 7 days or at day 4 after removal of non-adherent cells. At day 7, cell number and viability was quantified by XTT labeling. Human aortic endothelial cells (HAEC) were exposed to IS for the same times. Apoptosis was measured by annexin-FITC labeling. Results: S extensively reduced EPC viability. Clinically relevant concentrations of S reduced EPC viability by 90% compared with controls after 7 days of exposure. This effect was quite marked even at the sub-clinical doses. For CyA, there was a reduced viability with supra-clinical concentrations. For T, no effect was noticed at any dose. When HAEC were analyzed, S reduced viability, but to a much lower extent than with EPC. At clinical concentrations, S reduced EPC viability by 25%, whereas treatment with CyA or T did not significantly affect viability. EPC stained with annexin-V demonstrated a marked increase in apoptosis after S exposure. Conclusions: Sirolimus induces EPC apoptosis and greatly reduces EPC survival, even at low, sub-clinical concentrations. This effect may partially explain the findings that sirolimus inhibits angiogenesis, reduces postangioplasty restenosis and transplant vasculopathy.