The inhibition of inflammation-associated angiogenesis ameliorates inflammatory diseases by reducing the recruitment of tissue-infiltrating leukocytes. However, it is not known if angiogenesis has an active role during the initiation of inflammation or if it is merely a secondary effect occurring in response to stimuli by tissue-infiltrating leukocytes. Here, we show that angiogenesis precedes leukocyte infiltration in experimental models of inflammatory bowel disease and acute graft-versus-host disease (GVHD). We found that angiogenesis occurred as early as day+2 after allogeneic transplantation mainly in GVHD typical target organs skin, liver, and intestines, whereas no angiogenic changes appeared due to conditioning or syngeneic transplantation. The initiation phase of angiogenesis was not associated with classical endothelial cell (EC) activation signs, such as / upregulation or increased adhesion molecule expression. During early GVHD at day+2, we found significant metabolic and cytoskeleton changes in target organ ECs in gene array and proteomic analyses. These modifications have significant functional consequences as indicated by profoundly higher deformation in real-time deformability cytometry. Our results demonstrate that metabolic changes trigger alterations in cell mechanics, leading to enhanced migratory and proliferative potential of ECs during the initiation of inflammation. Our study adds evidence to the hypothesis that angiogenesis is involved in the initiation of tissue inflammation during GVHD.

Animal disease models have been criticized for lack of resemblance to human illnesses, hampering transfer of knowledge from preclinical research to clinical medicine. In the field of allogeneic hematopoietic stem cell transplantation (allo-HSCT), it is standard practice to study GVHD in lethal TBI-based murine models. Frequently, MHC-mismatched donors are used in GVHD models. In contrast, in clinical allo-HSCT conditioning with chemotherapy (+/ − TBI) is common and donors are often MHC-matched. Aiming at a more clinically oriented situation, we established and characterized a murine MHC-matched, minor histocompatibility antigen mismatched GVHD model (LP/J [H2k b ] → C57BL/6 [H2k b ]) using busulfan and cyclophosphamide conditioning. We found typical clinical and histological features of acute GVHD. T-cell infiltration, GVHD-specific damage and systemic inflammation were similar to observations made in patients after allo-HSCT. In survivors of acute GVHD, we found expansion of CD4+ T cells and the development of scleroderma-like chronic GVHD. The use of chemotherapy-based, minor histocompatibility antigen (miHA)-mismatched GVHD animal models may be a good option when studying clinically relevant questions in the field of allo-HSCT.

Angiogenesis and endothelial activation and dysfunction have been associated with acute graft-vs.-host disease (aGVHD), pointing to the endothelium as a potential target for pharmacological intervention. Defibrotide (DF) is a drug with an endothelium-protective effect that has been approved for the treatment of veno-occlusive disease/sinusoidal obstruction syndrome after allogeneic hematopoietic cell transplantation. Clinical data suggest that DF also reduces the incidence of aGVHD; however, the mechanisms of DF-mediated aGVHD regulation have not been examined. To investigate possible DF-mediated prophylactic and therapeutic mechanisms in aGVHD, we performed in vitro studies using endothelial cell (EC) lines. We found that DF significantly and dose-dependently suppressed EC proliferation and notably reduced their ability to form vascular tubes in Matrigel. To explore whether DF administered prophylactically or therapeutically has a significant effect on aGVHD endothelial dysfunction, ECs were exposed to media containing sera from patients with aGVHD (n = 22) in the absence or presence of DF and from patients that did not develop aGVHD (n = 13). ECs upregulated adhesion molecules (vascular cell adhesion molecule 1, intercellular adhesion molecule 1), the adherence junction protein VE-cadherin, von Willebrand factor (VWF), and Akt phosphorylation in response to aGVHD sera. These responses were suppressed upon treatment with DF. In summary, DF inhibits vascular angiogenesis and endothelial activation induced by sera from aGVHD patients. Our results support the view that DF has notable positive effects on endothelial biology during aGVHD.

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