Distinct Deleterious Effects of Cyclosporine and Tacrolimus and Combined Tacrolimus– Sirolimus on Endothelial Cells: Protective Effect of Defibrotide

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Distinct Deleterious Effects of Cyclosporine and Tacrolimus and Combined Tacrolimus– Sirolimus on Endothelial Cells: Protective Effect of Defibrotide Alba Carmona, Maribel Díaz-Ricart, Marta Palomo, Patricia Molina, Marc Pino, Montserrat Rovira, Ginés Escolar, Enric Carreras Biology of Blood and Marrow Transplantation Volume 19, Issue 10, Pages 1439-1445 (October 2013) DOI: 10.1016/j.bbmt.2013.07.001 Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Morphology and growth kinetics of endothelial cells exposed to different immunosuppressants. Micrographs correspond to untreated HMEC-1 monolayers (C) and cells exposed to cyclosporine A (CSA), tacrolimus (TAC), or sirolimus (SIR). In the presence of SIR, HMEC-1 monolayers were rarely confluent and fewer dividing cells were seen. Images are representative of 5 different experiments. Biology of Blood and Marrow Transplantation 2013 19, 1439-1445DOI: (10.1016/j.bbmt.2013.07.001) Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Expression of ICAM-1 on the surface of microvascular endothelial cells (HMEC-1) induced by different immunosuppressive drugs: effect of DF. Micrographs show ICAM-1 expression on untreated HMEC-1 cells (C) or in cells exposed to cyclosporine A (CSA), tacrolimus (TAC), sirolimus (SIR), or the combination of TAC and SIR (TAC+SIR) in the absence of DF. The bar diagram shows ICAM-1 expression on HMEC-1 cells in response to the immunosuppressive drugs in the absence (black bars) and presence (gray bars) of DF. Data are expressed as mean ± SEM; *P < .05 versus control and #P < .05 versus the drug in the absence of DF. Biology of Blood and Marrow Transplantation 2013 19, 1439-1445DOI: (10.1016/j.bbmt.2013.07.001) Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Reactivity of the ECM from microvascular endothelial cells grown in the presence of immunosuppressive drugs: effect of DF. (A) Light micrographs show platelet adhesion and aggregate formation on ECM generated by untreated HMEC-1 cells (C) and cells treated with cyclosporine A (CSA) in the absence and presence of 100 μg/mL defibrotide (CSA + DF). (B) The bar diagram shows the percentages of surface covered by platelets (%SC) on the ECM from untreated HMEC-1 cells and on cells exposed to the immunosuppressive drugs in the absence (black bars) and presence (gray bars) of DF. Data are expressed as mean ± SEM; *P < .05 versus control and #P < .05 versus the drug in the absence of DF. Biology of Blood and Marrow Transplantation 2013 19, 1439-1445DOI: (10.1016/j.bbmt.2013.07.001) Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Modifications of the viscoelastic properties of clots. Bar diagrams show the coagulation time (CT) (A) and clot formation time (CFT) (B) of untreated whole-blood samples (C) or blood samples treated with cyclosporine A (CSA), tacrolimus (TAC), sirolimus (SIR), or the combination of TAC and SIR (TAC + SIR) in the absence (black bars) and presence (gray bars) of DF. Data are expressed as mean ± SEM, n = 5. None of the differences was significant. Biology of Blood and Marrow Transplantation 2013 19, 1439-1445DOI: (10.1016/j.bbmt.2013.07.001) Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions