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S. Cordes et al.
Steroid treatment is successful in most of these patients (75%-80%). However, the 20%-25% of patients who fail initial treatment with steroids (steroid-refractory aGvHD [SR-aGvHD]) have very high mortality.2,3 No standard treatment for SR-aGvHD is currently available, and its pathobiology is poorly understood, thereby hindering the development of novel therapeutic approaches.
The endothelium is the first contact for immunological effector cells in the blood and a key regulator in various inflammatory processes. In early complications after allo- HSCT such as transplantation-associated-microangiopa- thy,4 veno-occlusive disease,5 capillary leak syndrome6 and diffuse alveolar hemorrhage7 the endothelium was shown to be relevant. Recent studies also suggest a criti- cal role of the endothelium in aGvHD. Angiogenesis appears to be a very early event during aGvHD, occurring before immune cell infiltration in target organs.8 During later phases of aGvHD, endothelial apoptosis has been described in cutaneous aGvHD.9,10 Clinical studies demonstrated that soluble markers of endothelial damage such as von Willebrand factor,11 thrombomodulin,12-14 micro particles released by endothelial cells (EC)15 as well as the CD40/CD40 ligand axis,16 can be used as biomark- ers during aGvHD. Furthermore, factors of endothelial damage were correlated with the mortality rate of patients suffering from SR-aGvHD.12,13,17 In addition to endothelial pathology in the microvasculature, arterial vessels were described to suffer from endothelial damage during aGvHD with consequences for their physiological properties.18
The characterization of endothelial function during aGvHD and SR-aGvHD is still incomplete and effective pharmacologic strategies aiming at the normalization of endothelial dysfunction to ameliorate aGvHD and SR- aGvHD are lacking.
Methods
Patient material and histology of human biopsies
Collection of human samples was approved by the institu- tional ethics committees of Charité Berlin and Medical University Hannover and was in accordance with the Declaration of Helsinki. From the Charité cohort, we included intestinal biopsies with aGvHD versus no aGvHD after allo- HSCT performed between 2007 and 2015. We identified 12 duo- denal and 11 colon biopsies from patients with aGvHD grade III- IV. As a control, we used 19 duodenal and ten colon biopsies from allo-HSCT recipients without histological evidence of aGvHD.19 From the Hannover cohort colon biopsies from 11 patients with aGvHD were included. From each patient, biop- sies were taken at two time points: at diagnosis of aGvHD and later at diagnosis of SR-aGvHD. Detailed clinical data from both cohorts are given in the Online Supplementary Tables S1, S2, S4 and S6.
Mice and acute graft-versus-host disease experiments aGVHD models were used as described previously.8,20,21 Control groups (no aGvHD) were transplanted with the same bone marrow (BM) cell numbers and T-cell numbers from syn- geneic donors. In order to mimic SR-aGvHD, we used the chemotherapy based murine models 129→B6 major histocom- patibility complex (MHC)-matched and B6→B6D2F1 (hap- loidentical) and the radiation based murine model BALB/c→B6 MHC-mismatch with conditioning and cell dosages analogue to
the models described above. Recipient mice were treated intraperitoneal with 0.5 mg/kg/day dexamethasone beginning at day+4 after HSCT (Merck, Darmstadt, Germany).22,23 We used dexamethasone because of its longer lasting effects compared to methyl-prednisolone or prednisolone enabling once daily dosing in the murine models. The rationale for starting at day+4 is that during this time leukocytes start to infiltrate target organs during aGvHD.8,21,24 Clinical scores, weight loss and survival in the B6- BDF SR-aGVHD model are given in the Online Supplementary Figure S1.
Histology of murine tissues
Tissue samples were cryoembedded and scored as previously described.19
Evans blue assay
For assessment of endothelial leakage, Evans blue assay was performed as described in detail elsewhere.25
Immunolabeling against VE-cadherin for light sheet fluorescence microscopy
25 mg/mouse anti-VE-cadherin antibody (Thermo Scientific, eBioBV13-eFluor660) was intravenously (i.v.) injected in mice from B6→BDF aGvHD model. Mice were sacrificed and perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde in PBS. Sample preparation and imaging of whole organs were performed by light sheet fluorescence microscopy as pre- viously described.26 Analysis of vasculature and its seg- mentation was performed using Imaris 8.1 software (Bitplane, Concord, MA, USA). Branch level was deter- mined by branching point and diameter changes of the vasculature.27
Hepatic endothelial cell isolation
Single cell suspensions were generated via digestion with 2 mg/mL collagenase D and 5 mL deoxyribonuclease. Hepatic EC cell fraction was enriched by gradient centrifugation using 30% histodenz (Sigma Aldrich). For gene expression analysis, the obtained single cell suspension was further enriched for EC (CD11b-, CD45dim/-, CD31+) by flow cytometry using a Bio-Rad S3 cell Sorter. EC purity was determined via flow cytometry analysis of ICAM1+ and CD31+ cells.
Statistics
Survival data were analyzed using the Kaplan–Meier method and compared with the Mantel–Cox log-rank test. For statistical analysis of all other data, Student’s t-test was used, unless indi- cated otherwise. Values are presented as mean ± standard error of the mean (SEM). Values of P ≤ 0.05 were considered statisti- cally significant. All statistical analyses were performed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA).
Additional experimental procedures are described in the Online Supplementary Appendix.
Results
Endothelial apoptosis during severe intestinal acute graft-versus-host disease in human biopsies
We stained colon and duodenum biopsies from allo- HSCT recipients with the apoptotic cell marker caspase 3 (Casp3). We found that Casp3 positive (Casp+) EC were rare events in biopsies of allo-HSCT recipients without
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haematologica | 2021; 106(8)