Defibrotide Prevents the Activation of Macrovascular and Microvascular Endothelia Caused by Soluble Factors Released to Blood by Autologous Hematopoietic Stem Cell Transplantation  Marta Palomo, Maribel Diaz-Ricart, Montserrat Rovira, Ginés Escolar, Enric Carreras  Biology of Blood and Marrow Transplantation  Volume 17, Issue 4, Pages 497-506 (April 2011) DOI: 10.1016/j.bbmt.2010.11.019 Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Expression of ICAM-1 on endothelial cells induced by autologous HSCT: effect of defibrotide. Micrographs show ICAM-1 expression on HMEC exposed to sera from autologous HSCT recipients, corresponding to day 14, in the absence (HMEC) and in the presence of 100 μg/mL of defibrotide (HMEC + DF). Bar diagrams represent levels of ICAM-1 expression on HUVEC (A) and on HMEC (B). Filled bars correspond to levels of ICAM-1 (pixels/μm2) in response to the sera from autologous recipients along the period of study in the absence of defibrotide. Open bars correspond to the same conditions but in the presence of defibrotide. Dotted line represents the mean value for control results. Data are expressed as mean ± SEM, n = 6, being ∗P < .05 versus day Pre, #P < .05 in the presence versus the absence of defibrotide. Biology of Blood and Marrow Transplantation 2011 17, 497-506DOI: (10.1016/j.bbmt.2010.11.019) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Reactivity of the extracellular matrices from macrovascular and microvascular endothelial cells grown in the presence of autologous HSCT sera: effect of defibrotide. Micrographs show platelet adhesion and aggregate formation on ECM generated by HMEC grown with culture media containing sera from autologous recipients, at day 0, in the absence (HMEC) and in the presence of 100 μg/mL of defibrotide (HMEC + DF). Bar diagrams represent percentages of surface covered by platelets (%SC) on ECM from HUVEC (A) and HMEC (B). Filled bars correspond to %SC in response to the sera from autologous recipients along the period of study in the absence of defibrotide. Open bars correspond to the same conditions but in the presence of defibrotide. Dotted line represents the mean value for control results. Data are expressed as mean ± SEM, n = 6, being ∗P < .05 versus day Pre, #P < .05 in the presence versus the absence of defibrotide. Biology of Blood and Marrow Transplantation 2011 17, 497-506DOI: (10.1016/j.bbmt.2010.11.019) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Expression of VWF and tissue factor on the extracellular matrix from HMEC cells exposed to autologous HSCT sera: effect of defibrotide. Micrographs show VWF and TF expression on the ECM from HMEC exposed to sera from autologous HSCT recipients, corresponding to day 0, in the absence (upper micrographs VWF and TF) and in the presence of 100 μg/mL of defibrotide (lower micrographs VWF + DF and TF + DF). Bar diagrams represent levels of VWF (A) and TF (B) expression on ECM from HMEC. Filled bars correspond to levels of VWF or TF, respectively, in response to the sera from autologous recipients along the period of study in the absence of defibrotide. Open bars correspond to the same conditions but in the presence of defibrotide. Dotted line represents the mean value for control results. Data are expressed as mean ± SEM, n = 6, being ∗P < .05 versus day Pre, #P < .05 in the presence versus the absence of defibrotide. Biology of Blood and Marrow Transplantation 2011 17, 497-506DOI: (10.1016/j.bbmt.2010.11.019) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Reactivity of the extracellular matrices from microvascular endothelial cells grown in the presence of autologous HSCT sera: effect of defibrotide. Micrographs show platelet adhesion and aggregate formation on ECM generated by HMEC grown with culture media containing sera from autologous recipients, at day 0, in the absence (HMEC) and in the presence of antibodies to VWF (HMEC + anti-VWF) and to TF (HMEC + anti-TF). Biology of Blood and Marrow Transplantation 2011 17, 497-506DOI: (10.1016/j.bbmt.2010.11.019) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Activation of the signaling molecules p38 MAPK and Akt in HUVEC and HMEC by autologous HSCT sera: effect of defibrotide. Activation of p38 MAPK and Akt was measured in HUVEC and HMEC cells in presence or absence of 100 μg/mL of DF during the previous 24 hours, and activated with the sera samples from autologous recipients collected at days 0 and 14. Exposure was carried out for 15 and 30 minutes to assess the kinetics of activation. Images are representative of n = 6 different experiments. Biology of Blood and Marrow Transplantation 2011 17, 497-506DOI: (10.1016/j.bbmt.2010.11.019) Copyright © 2011 American Society for Blood and Marrow Transplantation Terms and Conditions