Cell Therapy & Immunotherapy ARTICLE Endothelial damage and dysfunction in acute Ferrata Storti Foundation graft-versus-host disease Steffen Cordes,1 Zeinab Mokhtari,2 Maria Bartosova,3 Sarah Mertlitz,1 Katarina Riesner,1 Yu Shi,1 Jörg Mengwasser,1,4 Martina Kalupa,1 Aleixandria McGeary,1 Johanna Schleifenbaum,5,6 Jens Schrezenmeier,1 Lars Bullinger,1 Maribel Diaz- Ricart,7 Marta Palomo,7,8 Enric Carreras,8 Gernot Beutel,9 Claus Peter Schmitt, 3 Andreas Beilhack2 and Olaf Penack1 1 Department of Hematology, Oncology and Tumor Immunology, Charité Universitätsmedizin Berlin, Campus Virchow Clinic, Berlin, Germany; 2Department of Medicine II, Würzburg University Hospital, Interdisciplinary Center for Clinical Research Haematologica 2021 (IZKF), Laboratory for Experimental Stem Cell Transplantation, Würzburg, Germany; Volume 106(8):2147-2160 3 Pediatric Nephrology, Center for Pediatric and Adolescent Medicine, University Hospital Heidelberg, Heidelberg, Germany; 4Department of Surgery, Charité Universitätsmedizin Berlin, Campus Charité Mitte/Campus Virchow Clinic, Berlin, Germany; 5Experimental and Clinical Research Center (ECRC) - a joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine (MDC), Berlin, Germany; 6 Charité Universitätsmedizin Berlin, Institute of Vegetative Physiology, Berlin, Germany; 7 Department of Hematopathology, Hospital Clinic of Barcelona, Biomedical Diagnosis Centre (CDB), Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain; 8Josep Carreras Leukemia Research Institute, Hospital Clinic/University of Barcelona Campus, Barcelona, Spain and 9Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany ABSTRACT C linical studies have suggested a potential involvement of endothe- lial dysfunction and damage in the development and severity of acute graft-versus-host disease (aGvHD). Accordingly, we found an increased percentage of apoptotic caspase 3 positive blood vessels in duodenal and colonic mucosa biopsies of patients with severe aGvHD. In murine experimental aGvHD, we detected severe microstructural endothelial damage and reduced endothelial pericyte coverage accompa- nied by reduced expression of endothelial tight junction proteins leading to increased endothelial leakage in aGvHD target organs. During intes- tinal aGvHD, colonic vasculature structurally changed, reflected by Correspondence: increased vessel branching and vessel diameter. As recent data demon- OLAF PENACK strated an association of endothelium-related factors and steroid refrac- olaf.penack@charite.de tory aGvHD (SR-aGvHD), we analyzed human biopsies and murine tis- sues from SR-aGvHD. We found extensive tissue damage but low levels Received: March 27, 2020. of alloreactive T-cell infiltration in target organs, providing the rationale for T-cell independent SR-aGvHD treatment strategies. Consequently, Accepted: July 13, 2020. we tested the endothelium-protective PDE5 inhibitor sildenafil, which Pre-published: July 16, 2020. reduced apoptosis and improved metabolic activity of endothelial cells in vitro. Accordingly, sildenafil treatment improved survival and reduced https://doi.org/10.3324/haematol.2020.253716 target organ damage during experimental SR-aGvHD. Our results demonstrate extensive damage, structural changes, and dysfunction of the vasculature during aGvHD. Therapeutic intervention by endotheli- ©2021 Ferrata Storti Foundation um-protecting agents is an attractive approach for SR-aGvHD comple- Material published in Haematologica is covered by copyright. menting current anti-inflammatory treatment options. All rights are reserved to the Ferrata Storti Foundation. Use of published material is allowed under the following terms and conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode. Copies of published material are allowed for personal or inter- Introduction nal use. Sharing published material for non-commercial pur- poses is subject to the following conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode, Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only cur- sect. 3. Reproducing and sharing published material for com- ative treatment option for many patients suffering from hematological malignan- mercial purposes is not allowed without permission in writing cies. A major complication of allo-HSCT is acute graft-versus-host disease from the publisher. (aGvHD), an inflammatory condition primarily affecting the skin, liver, and intes- tines. aGvHD occurs in more than two thirds of patients undergoing allo-HSCT.1 haematologica | 2021; 106(8) 2147 S. Cordes et al. Steroid treatment is successful in most of these patients the models described above. Recipient mice were treated (75%-80%). However, the 20%-25% of patients who fail intraperitoneal with 0.5 mg/kg/day dexamethasone beginning at initial treatment with steroids (steroid-refractory aGvHD day+4 after HSCT (Merck, Darmstadt, Germany).22,23 We used [SR-aGvHD]) have very high mortality.2,3 No standard dexamethasone because of its longer lasting effects compared to treatment for SR-aGvHD is currently available, and its methyl-prednisolone or prednisolone enabling once daily dosing pathobiology is poorly understood, thereby hindering the in the murine models. The rationale for starting at day+4 is that development of novel therapeutic approaches. during this time leukocytes start to infiltrate target organs during The endothelium is the first contact for immunological aGvHD.8,21,24 Clinical scores, weight loss and survival in the B6- effector cells in the blood and a key regulator in various BDF SR-aGVHD model are given in the Online Supplementary inflammatory processes. In early complications after allo- Figure S1. HSCT such as transplantation-associated-microangiopa- thy,4 veno-occlusive disease,5 capillary leak syndrome6 Histology of murine tissues and diffuse alveolar hemorrhage7 the endothelium was Tissue samples were cryoembedded and scored as previously shown to be relevant. Recent studies also suggest a criti- described.19 cal role of the endothelium in aGvHD. Angiogenesis appears to be a very early event during aGvHD, occurring Evans blue assay before immune cell infiltration in target organs.8 During For assessment of endothelial leakage, Evans blue assay was later phases of aGvHD, endothelial apoptosis has been performed as described in detail elsewhere.25 described in cutaneous aGvHD.9,10 Clinical studies demonstrated that soluble markers of endothelial damage Immunolabeling against VE-cadherin for light sheet such as von Willebrand factor,11 thrombomodulin,12-14 fluorescence microscopy micro particles released by endothelial cells (EC)15 as well 25 mg/mouse anti-VE-cadherin antibody (Thermo as the CD40/CD40 ligand axis,16 can be used as biomark- Scientific, eBioBV13-eFluor660) was intravenously (i.v.) ers during aGvHD. Furthermore, factors of endothelial injected in mice from B6→BDF aGvHD model. Mice damage were correlated with the mortality rate of were sacrificed and perfused with phosphate buffered patients suffering from SR-aGvHD.12,13,17 In addition to saline (PBS) followed by 4% paraformaldehyde in PBS. endothelial pathology in the microvasculature, arterial Sample preparation and imaging of whole organs were vessels were described to suffer from endothelial damage performed by light sheet fluorescence microscopy as pre- during aGvHD with consequences for their physiological viously described.26 Analysis of vasculature and its seg- properties.18 mentation was performed using Imaris 8.1 software The characterization of endothelial function during (Bitplane, Concord, MA, USA). Branch level was deter- aGvHD and SR-aGvHD is still incomplete and effective mined by branching point and diameter changes of the pharmacologic strategies aiming at the normalization of vasculature.27 endothelial dysfunction to ameliorate aGvHD and SR- aGvHD are lacking. Hepatic endothelial cell isolation Single cell suspensions were generated via digestion with 2 mg/mL collagenase D and 5 mL deoxyribonuclease. Hepatic EC Methods cell fraction was enriched by gradient centrifugation using 30% histodenz (Sigma Aldrich). For gene expression analysis, the Patient material and histology of human biopsies obtained single cell suspension was further enriched for EC Collection of human samples was approved by the institu- (CD11b-, CD45dim/-, CD31+) by flow cytometry using a Bio-Rad tional ethics committees of Charité Berlin and Medical S3 cell Sorter. EC purity was determined via flow cytometry University Hannover and was in accordance with the analysis of ICAM1+ and CD31+ cells. Declaration of Helsinki. From the Charité cohort, we included intestinal biopsies with aGvHD versus no aGvHD after allo- Statistics HSCT performed between 2007 and 2015. We identified 12 duo- Survival data were analyzed using the Kaplan–Meier method denal and 11 colon biopsies from patients with aGvHD grade III- and compared with the Mantel–Cox log-rank test. For statistical IV. As a control, we used 19 duodenal and ten colon biopsies analysis of all other data, Student’s t-test was used, unless indi- from allo-HSCT recipients without histological evidence of cated otherwise. Values are presented as mean ± standard error aGvHD.19 From the Hannover cohort colon biopsies from 11 of the mean (SEM). Values of P ≤ 0.05 were considered statisti- patients with aGvHD were included. From each patient, biop- cally significant. All statistical analyses were performed using sies were taken at two time points: at diagnosis of aGvHD and GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, later at diagnosis of SR-aGvHD. Detailed clinical data from both USA). cohorts are given in the Online Supplementary Tables S1, S2, S4 Additional experimental procedures are described in the and S6. Online Supplementary Appendix. Mice and acute graft-versus-host disease experiments aGVHD models were used as described previously.8,20,21 Results Control groups (no aGvHD) were transplanted with the same bone marrow (BM) cell numbers and T-cell numbers from syn- Endothelial apoptosis during severe intestinal acute geneic donors. In order to mimic SR-aGvHD, we used the graft-versus-host disease in human biopsies chemotherapy based murine models 129→B6 major histocom- We stained colon and duodenum biopsies from allo- patibility complex (MHC)-matched and B6→B6D2F1 (hap- HSCT recipients with the apoptotic cell marker caspase 3 loidentical) and the radiation based murine model BALB/c→B6 (Casp3). We found that Casp3 positive (Casp+) EC were MHC-mismatch with conditioning and cell dosages analogue to rare events in biopsies of allo-HSCT recipients without 2148 haematologica | 2021; 106(8) aGvHD impact on endothelium aGvHD. In contrast, Casp3+ EC were frequent in intestin- HSCT recipients without aGvHD is not affected. The al biopsies of patients diagnosed for grade III-IV aGvHD. endothelial monolayer as well as the EC-cell contacts Figure 1 shows exemplary pictures of colon sections in were intact (Figure 2A and B), and we observed a normal human allo-HSCT recipients without aGvHD (Figure 1A) endothelial monolayer in hepatic sinusoids (Figure 2B). In versus grade III-IV intestinal aGvHD (Figure 1B). contrast, the hepatic sinusoidal endothelium during Quantification revealed a significant increase in percent- aGvHD was severely damaged with close immune cell- age of Casp3+ vessels in duodenal (Figure 1C) and colonic EC interactions (Figure 2C). Furthermore, we observed a mucosa (Figure 1D) of grade III-IV aGvHD. Patient char- discontinuous endothelial monolayer at the EC-immune acteristics and clinical data are given in the Online cell contact zone during hepatic aGvHD (Figure 2D). In Supplementary Table S1 (for Figure 1C) and the Online addition, we found blistering of the endothelial monolay- Supplementary Table S2 (for Figure 1D). Our data demon- er (Figure 2E) as well as platelet adhesion on the sinu- strate endothelial apoptosis during severe intestinal soidal endothelial monolayer (Figure 2E) and in the blis- aGvHD in human allo-HSCT recipients. tered endothelium (Figure 2F) during hepatic aGvHD. In colonic mucosa, vessels were again normal in allo-HSCT Micro-structural endothelial changes during recipients without aGvHD (Online Supplementary Figure experimental acute graft-versus-host disease S2A to F). The endothelial monolayer was well-struc- in transmission electron microscopy tured, smooth and surrounded by pericytes (Online In order to further investigate micro-structural changes Supplementary Figure S2A) with intact tight junctions of the endothelium we used experimental aGvHD mod- (Online Supplementary Figure S2B). In contrast, during els. We first performed transmission electron microscopy intestinal aGvHD the endothelial monolayer was ruffled (TEM) of liver (Figure 2A to F) and colon (Online (Online Supplementary Figure S2C) with perivascular fib- Supplementary Figure S2A to F) at day+15 after allo-HSCT. rinogen deposits (Online Supplementary Figure S2A, C, D We found that the hepatic sinusoidal endothelium in allo- and F). Endothelial cytoplasm was enriched with vesicles A B C D Figure 1. Endothelial damage in human intestinal biopsies. (A) Exemplary picture of a colon biopsy of a patient after allogeneic hematopoietic stem cell transplan- tation (allo-HSCT) without histologic evidence of acute graft-versus-host disease (aGvHD) and low level of endothelial apoptosis. The white dotted lines indicate ves- sel lumen and the arrow indicates one apoptotic caspase 3 positive (Casp3+) endothelial cell. (B) Exemplary picture of a colon biopsy of a patient with grade III-IV intestinal aGvHD and increased endothelial apoptosis. The white dotted lines indicate vessel lumen and the arrows indicate apoptotic Casp3+ endothelial cells. (C) Quantification of Casp3+ events in duodenal endothelium of allo-HSCT recipients given in percent of vessels in high-power fields (HPF). (D) Quantification of Casp3+ events in colonic endothelium of allo-HSCT recipients given in percent of vessels in HPF. Percentage of Casp3+ vessels was tested for significance by Student’s t-test (***P<0.001; n=7-11 patients per group). Error bars indicate mean ± standard error of the mean. haematologica | 2021; 106(8) 2149 S. Cordes et al. and the endothelial monolayer was disrupted (Online expression of the tight junction protein ZO-1 and the Supplementary Figure S2D and E). During intestinal intercellular junction protein VE-cadherin in vessels of aGvHD, clotting was present in the colonic microvascula- aGvHD target organs a shown in the representative pic- ture as well as convolution of the endothelial monolayer tures in Figure 3C and I. Immunostaining revealed (Online Supplementary Figure S2E). In conclusion, TEM reduced endothelial ZO-1 abundance during aGvHD, revealed an extensive endothelial damage in target organs which correlates with reduced numbers of intact tight during aGvHD. junction (Figure 3D and J). Moreover, colonic microvas- cular and hepatic sinusoidal endothelium VE-cadherin Pericyte coverage, tight junctions and endothelial abundance was reduced (Figure 3E and K). leakiness during acute graft-versus-host disease In order to investigate, if the reduced expression of As a method to quantify endothelial damage in exper- tight junction and intercellular connection proteins of the imental aGvHD, we analyzed pericyte-coverage of endothelium during aGvHD have functional conse- CD31+ vessels in the colon and liver by fluorescence quences in vivo, we injected Evans blue solution intra- microscopy. Representative pictures are shown in Figure venously and analyzed the penetration into the organs. 3A and G. We found reduced pericyte-coverage of ves- In allo-HSCT recipients with aGvHD endothelial leakage sels in hepatic sinusoids (Figure 3B) and in colonic was significantly increased in liver (Figure 3F) and colon mucosa (Figure 3H) during aGvHD, indicating a dam- (Figure 3L) as compared to HSCT recipients without aged endothelial monolayer. In order to address the aGvHD. question whether the endothelial barrier function is Next, we were interested if the detected endothelial altered during aGvHD, we assessed the endothelial changes occurred exclusively in the target organs during A B C D Figure 2. Visualization of acute graft-versus-host dis- ease-associated ultrastructural changes in the liver by transmission electron microscopy. Representative pictures of sections from liver taken at day+15 after experimental hematopoietic stem cell transplantation (HSCT) in the chemotherapy based B6→BDF model. (A and B) Liver sinusoidal endothelial monolayer after syngeneic-HSCT (syn-HSCT) without acute graft-ver- sus-host disease (aGvHD). (A) Normal, fenestrated sinusoidal blood vessel completely covered with endothelial monolayer. (B) Higher magnification of a 100 nm large fenestration of the endothelium in the liver. (C to-F) Sinusoidal liver endothelial monolayer after allo-HSCT during aGvHD. (C) Liver sinusoidal E F vessel with destroyed and unregularly shaped endothelial monolayer in contact with an immune cell. (D) Higher magnification of contact zone between immune cell and endothelial cell. (E) Blistering of the endothelial monolayer with a platelet in the region of injury. (F) Higher magnification of endothelial blistering. The perivascular space is marked by a red triangle. V: vessel lumen; EM: endothelial monolayer; F: fenestrated endothelium; IC: immune cell; E: erythrocyte; P: platelet; red circle: loss of endothelium; red triangle: endothelial blister- ing. Control groups (no aGvHD) were transplanted with the same bone marrow cell numbers and T-cell numbers from syngeneic donors. 2150 haematologica | 2021; 106(8) aGvHD impact on endothelium A G B H C I D J E K F L Figure 3. Pericyte coverage, tight junctions and endothelial leakiness during acute graft-versus-host disease. Organs were harvested at day+15 after experimental hematopoietic stem cell transplantation (HSCT) in the chemotherapy based LP/J→B6 model. Control groups (no acute graft-versus-host disease [aGvHD]) were trans- planted with the same bone marrow cell numbers and T-cell numbers from syngeneic donors. (A, B, and G to H) Quantification of pericyte coverage of vessels. (A and G) Representative pictures of staining for pericyte marker α smooth muscle actin (αSMA) in red and endothelial cell marker CD31 in green. Right organs of animals with- out aGvHD and left organs of animals suffering from aGvHD. (B and H) Ratio of αSMA positive area and CD31 positive area in (B) liver sinusoidal endothelium and (H) in colonic mucosal vessels in aGvHD versus no aGvHD. (C, D, I and J) Quantification of endothelial tight junction protein expression ZO-1. (C and I) Representative pic- tures of staining ZO-1 in green and CD31 in red. Right organs of animals without aGvHD and left organs of animals suffering from aGvHD. (D and J) Percentage of ZO- 1+ CD31+ area in (D) liver sinusoidal endothelium and (J) colonic mucosal vessels in aGvHD versus no aGvHD. (E and K) Quantification of endothelial adherence junction protein expression VE-cadherin. Percentage of VE-cadherin positive area in (E) liver sinusoidal endothelium and (K) colonic mucosal vessels in aGvHD versus no aGvHD. (F and L) Measurement of Evans blue extravasation in ng Evans blue per mg at day+15 after experimental allo-HSCT in the chemotherapy based B6→BDF model. (F) Liver and (L) colon of aGvHD versus no aGvHD. Significance was tested with Student’s t-test (*P<0.05; **P<0.01; n=5 animals per group). All experiments were repro- duced in a biological independent experiment and shown are representative results of one experiment. Error bars indicate mean ± standard error of the mean. haematologica | 2021; 106(8) 2151 S. Cordes et al. aGvHD or if this is a systemic effect. We therefore ana- nificant endothelial gene expression changes in comple- lyzed endothelial leakage in the kidney and skeletal mus- ment activation, apoptosis, oxidative damage, IL-1 sig- cle, which are typically not affected in human aGvHD. naling and cell cycle during aGvHD (Online We have previously confirmed these organs to be unaf- Supplementary Figures S5, S6 and S7; Online Supplementary fected by aGvHD in our models.8,21 The Evans blue assay Table S3). Furthermore, barrier function and cytoskeleton revealed no significant changes of endothelial leakage in pathways were differentially regulated, including expres- these non-target organs during aGvHD (Online sion changes of Rho GTPases (Online Supplementary Table Supplementary Figure 3A and B) suggesting that increased 3). We performed interaction analysis of selected endothelial leakiness predominantly occurs in aGvHD endothelium-specific genes (Online Supplementary Figure target organs. 7) and found differential expression of Cdh1 and Cdh13 Taken together, we found disturbed endothelial tight (coding for cadherins), Thbd (coding for thrombomod- junctions and intercellular connections leading to ulin), Vegfc (coding for vascular endothelial growth factor increased vascular leakiness in target organs of aGvHD. c) and Anpgt1/2 (coding for angiopoietin 1 and 2). In sum- mary, we detected endothelial gene expression changes Structural changes of vasculature in target organs in different clinically relevant pathways during aGvHD during aGvHD providing possible targets for future therapeutic inter- In order to get a better overview of the three-dimen- ventions. sional (3D) changes of the vasculature, we compared aGvHD and non-GvHD colonic vessel structure and Inflammatory activity and endothelial damage during organization with light sheet fluorescence microscopy steroid-refractory acute graft-versus-host disease after in vivo perfusion with an anti-VE-cadherin antibody. Since recent clinical data has provided evidence on the Figure 4A and C show normal VE-cadherin staining and importance of endothelial pathology specifically in SR- vessel branching in a 3D model in colon at d+15 after aGVHD,12,17,28-31 we decided to characterize both inflam- HSCT without aGvHD. During aGvHD, we found con- matory infiltrates and endothelial damage in this setting. siderably different staining patterns for VE-cadherin Based on previous data,22 we first performed dose-finding (Figure 4B) and increased vessel branching in colon studies with steroids in different aGvHD models to estab- (Figure 4D). Using this 3D model to quantify vessel lish a protocol with progressive aGvHD despite high dose branch levels of vasculature, we found significantly steroid treatment. Treatment with dexamethasone start- increased total vessel branching in colonic vasculature ing at day+4 after allo-HSCT, showed progressive during aGVHD as compared to HSCT recipients without aGvHD in our models (Online Supplementary Figure S1; aGvHD (Figure 4E). Accordingly, analysis of branching Figure 5). levels from vessel segments (Figure 4F) and vessel diame- We analyzed vascular pericyte coverage as well as ter (Figure 4G) revealed a significant increase during intes- infiltration by CD4+ donor T cells in dexamethasone- tinal aGvHD. We conclude that aGvHD is associated treated SR-aGvHD versus untreated aGvHD at day+15 with structural changes in colonic vasculature. after allo-HSCT. We found reduced inflammatory T-cell infiltrates in SR-aGvHD versus untreated aGvHD (Figure Dysfunction of the macrovasculature during acute 5A to D). The predominant remaining cell population in graft-versus-host disease colon during SR-aGvHD were CD4+ and CD8+ T cells As aGvHD is associated with severe signs of microvas- (Figure 5 C and D) as well as CD11b+ and F4/80+ myeloid cular dysfunction, we became interested if aGvHD also cells (Online Supplementary Figure S8A and B). Endothelial influences the physiological functions of macrovascula- damage as quantified by pericyte coverage reduction in ture as suggested previously.18 We examined vessel con- colon vessels was equally severe in SR-aGvHD and traction and relaxation in mouse mesenteric arteries dur- untreated aGvHD (Figure 5E). In order to connect the ing aGvHD versus no aGVHD after HSCT by myographic experimental findings to the clinical context of SR- measurements. We found that maximum contraction of aGvHD, we quantified leukocyte infiltration in intestinal mesenteric arteries in response to high doses of noradren- biopsies of patients at diagnosis of aGvHD and at later aline (NA) and phenylephrine (Phe) was not significantly time points of SR-aGvHD in two independent cohorts. changed (Online Supplementary Figure S4A and B). Figure panels 5F to J demonstrate significant lower infil- However, partial contraction at lower doses of NA and tration of CD45+ leukocytes in colon biopsies as well as Phe was moderately increased during aGvHD versus no duodenum biopsies (Figure 5I). The leukocyte reduction aGvHD (Online Supplementary Figure S4C and D). In addi- was mainly due to significantly lower infiltration by tion, fractional relaxation after maximum contraction in CD3+ T cells in colon biopsies (Figure 5K, L, M and O) as response to ACh was slower in arteries from allo-HSCT well as duodenum biopsies (Figure 5N) during SR- recipients with aGvHD (Online Supplementary Figure S4E). aGvHD versus aGvHD at time of diagnosis. Figure 5K Taken together we found increased contraction and gives a typical example of colonic aGvHD at onset with reduced relaxation potential of mesenteric arteries during high levels of inflammatory infiltrates by CD3+ T cells aGvHD indicating systemic hypertension. and Figure 5L a representative colonic section after steroid treatment and diagnosis for SR-aGvHD, demon- Gene expression changes of endothelial cells during strating reduced inflammatory infiltrates. In addition, we acute graft-versus-host disease performed caspase 3 staining and demonstrated a high In order to better understand the molecular pathways level of endothelial apoptosis during aGvHD both at involved in endothelial dysfunction during aGvHD we diagnosis as well as during SR-aGVHD (Figure 5P). A performed gene array expression analysis of flow cytom- typical example of high inflammatory activity at aGvHD etry sorted hepatic EC from HSCT recipients with diagnosis time point and low level of inflammatory aGvHD versus without aGvHD at day+15. We found sig- activity at diagnosis of SR-GvHD in combination with 2152 haematologica | 2021; 106(8) aGvHD impact on endothelium considerable tissue damage in hematoxylin and eosin Endothelial protection by PDE5 inhibition in acute (H&E) staining is given in the Online Supplementary Figure graft-versus-host disease (aGvHD) and S8, panels C and D. Patient characteristics and clinical steroid-refractory aGvHD information is given in the Online Supplementary Tables S4 Based on our results demonstrating extensive endothe- to S6. Our data indicates that inflammatory activity in lial damage and reduced inflammatory T-cell infiltration intestinal tissues is reduced after steroid treatment, while during SR-aGvHD, we hypothesized that pharmacologic the endothelial damage is not influenced by steroid treat- protection of the endothelium would have stronger pro- ment during SR-aGvHD. tective effects on SR-aGvHD versus treatment of aGvHD Figure 4. Structural changes of vasculature in tar- get organs during acute graft-versus-host disease by scanning light sheet fluorescence microscopy. A B We analyzed organs at day+15 after experimental hematopoietic stem cell transplantation (HSCT) in the chemotherapy based B6→BDF model. Control groups (no aGvHD) were transplanted with the same bone marrow (BM) cell numbers and T-cell numbers from syngeneic donors. (A and B) VE-cad- herin signal of vasculature in colon of (A) allogene- ic-HSCT (allo-HSCT) recipients without aGvHD and (B) allo-HSCT recipients with aGvHD. (C-D) Computed three-dimensional (3D) model of colonic vasculature with number of branches (blue= low branch levels; red= high branch levels) of (C) allo-HSCT recipients without aGvHD and (D) allo-HSCT recipients with aGvHD. (E to G) Analysis of colonic vasculature parameters. Assessment of C D (E) total number of branches (F) distribution of vessels branching level and (G) vessel diameter distribution in allo-HSCT recipients with aGvHD versus without aGvHD. Significance of total num- ber of branches was tested by Student’s t-test (***P<0.001; n=3 animals per group) and signifi- cance of vessel branching level was tested by two- way ANOVA with Tukey’s multiple comparison test (***P<0.001; n=3 animals per group). Error bars indicate mean ± standard error of the mean. E F G haematologica | 2021; 106(8) 2153 2154 A B C D E S. Cordes et al. F G H I J P K L M N O Figure 5. Inflammatory infiltration and endothelial damage in human steroid-refractory acute graft-versus-host disease (SR-aGvHD) and in an experimental model of SR-aGvHD. (A to E) Immune cell infiltration in colon samples at day+15 after experimental allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the chemotherapy based B6→BDF model. Exemplary pictures of CD4+ infiltrates (green) in colon mucosa of (A) control phosphate buffered saline (PBS) treated (untreated) aGvHD and (B) Dexamethasone treated aGvHD (SR-aGvHD, 0.5mg/kg/day Dexamethasone starting at day+4). (C) Quantification of CD4+ area in colonic mucosa of untreated aGvHD versus SR-aGvHD. (D) Quantification of CD8+ area in colonic mucosa of untreated aGvHD versus SR-aGvHD. (E) Ratio of pericyte marker α smooth muscle actin (αSMA) positive area and endothelial cell marker CD31 positive area in in colonic mucosa of untreated aGvHD versus SR-aGvHD. Significance was tested by Student’s t-test (***P<0.001; n=5 animals per group). Error bars indicate mean ± standard error of the mean. (F to P) Human intestinal biopsies at aGvHD diagnosis and during the course of SR-aGvHD. (F to J) Intestinal biopsies at aGvHD diagnosis and SR-aGvHD stained against pan leukocyte marker CD45 (F) Colon biopsy stained against CD45 at aGvHD diagnosis with massive infiltrates. (G) Colon biopsy stained against CD45 during SR-aGvHD with low infiltrates. (H to J) Quantification of CD45 infiltrates at aGvHD diagnosis versus SR-aGvHD in (H) colon and (K) duodenum biopsies of cohort 1 and (J) in colon biopsies of cohort 2. (K-O) Intestinal biopsies at aGvHD diagnosis and SR-aGvHD stained against T-cell receptor marker CD3 (K) Colon biopsy stained against CD3 at aGvHD diagnosis with massive infiltrates. (L) Colon biopsy stained against CD3 during SR-aGvHD with low infiltrates. (M to O) Quantification of CD3 infiltrates at aGvHD diagnosis versus SR-aGvHD in (M) colon and (N) duodenum biopsies of cohort 1 and (O) in colon biopsies of cohort 2. (P) Quantification of capspase 3 positive (Casp3+) events in colonic endothelium of allo-HSCT recipients of cohort 2 at aGvHD diagnosis and during SR-aGvHD given in percent of vessels per high power field (HPF). Significance was tested by Student’s t- test (*P<0.05; **P<0.01; ***P<0.001; n= 4-6 patients per group). Error bars indicate mean ± standard error of the mean. haematologica | 2021; 106(8) aGvHD impact on endothelium A B C Figure 6. Reduction of endothelial apoptosis and endothelial activation by sildenafil in vitro. Mouse cardiac endothelial cells (MCEC) were incubated with either phosphate buffered saline (PBS)/ 0,1% dimethylsulfoxide (DMSO) (control [ctr]), 100 nm etoposide (eto), an inducer of cell death, or with 100 nm etoposide and 34 nm sildenafil (eto+sil) for 24 hours before analysis. (A) MTT assay showed higher optical density of eto+sil group versus eto-only group. (B) Staining for apoptotic cell marker caspase 3 (Casp3) showed reduced Casp3+ cells per high-power field (HPF) in eto+sil group compared to eto-only group. (C) Flow cytometry analysis of CD86, a costimulatory and endothelial activation marker, showed reduced percentage of endothelial CD86high cells in eto+sil group compared to eto-only group. Significance was tested by Student’s t-test (*P<0.05; n=2-3 experiments with at least triplicates per condition). Error bars indicate mean ± standard error of the mean. at its initiation stage. As a first example for such an ings in another murine aGvHD model (129→B6, MHC- approach, we used the PDE5 inhibitor sildenafil, which matched, Online Supplementary Figure S10). We found has been demonstrated to normalize endothelial dysfunc- non-significant trends towards increased vascular density tion in vivo in different settings.32-39 First, we tested silde- (Online Supplementary Figure S9C) and lymphatic vascular nafil in vitro for EC protection from cytotoxic damage, density in sildenafil-treated SR-GvHD versus controls mediated by etoposide. Sildenafil protected EC from (Online Supplementary Figure S9D). Next, we analyzed the etoposide-induced reduction of endothelial metabolic effect of sildenafil on the endothelium during SR-aGvHD activity and proliferation (Figure 6A). Sildenafil signifi- with electron microscopy analyses of liver and colon tis- cantly reduced etoposide-induced endothelial apoptosis, sues (Figure 7M to P). We found endothelial damage in as quantified by Casp3+ staining (Figure 6B). For co-stim- the liver and colon in untreated and sildenafil-treated SR- ulatory capacity, we checked CD86high expression of EC aGvHD. However, the sildenafil treated SR-aGvHD by flow cytometry. Sildenafil treatment resulted in a sig- group showed reduced endothelial ruptures in liver nificant reduction of etoposide-induced CD86 expression (Figure 7N) and less prominent fibrinogen deposits in on EC (Figure 6C). Our data demonstrate a protective colonic biopsies (Figure 7P). effect of sildenafil on etoposide-induced endothelial dys- In order to analyze for effects of sildenafil on T-cell pro- function in vitro. liferation, we performed in vivo proliferation assays with We tested the effect of sildenafil in our experimental carboxyfluorescein succinimidyl ester (CFSE) labeled allo- models of aGvHD (Figure 7A to F) and SR-aGvHD (Figure geneic T cells. In irradiated mice, sildenafil had no signif- 7G to L). In the aGvHD model without steroid treatment, icant effects on in vivo proliferation of allogeneic T cells we found that sildenafil treatment had no significant effect (Online Supplementary Figure S11A to C). We next ana- on survival (Figure 7A). However, sildenafil-treated allo- lyzed and quantified the impact on sildenafil on immune HSCT recipients with aGvHD had significantly lower clin- cell subsets in peripheral blood during SR-aGvHD and ical scores at different time points (Figure 7B) as well as found no significant differences (Online Supplementary lower histopathological aGvHD scores in the liver (Figure Figure S11D to M). Our data suggest that the observed 7C) and colon (Figure 7D) as compared to untreated allo- positive effect of sildenafil on SR-aGvHD is predominant- HSCT recipients with aGvHD. In addition, we found a ly mediated by its effect on EC as opposed to effects on non-significant trend towards reduced costimulatory immune cells. capacity and antigen presentation potential of hepatic EC under sildenafil treatment (Figure 7E and F). The vascular density (Online Supplementary Figure S9A) as well as the Discussion density of lymphatic vessels was not significantly affected by sildenafil treatment (Online Supplementary Figure S9B). The standardized and well-described aGvHD mice During SR-aGvHD, sildenafil treatment significantly models provide the unique opportunity to experimentally improved survival (Figure 7G). Due to high mortality, address the role of endothelial dysfunction after HSCT. clinical scoring was only significant in the early phase We found that severe endothelial damage, structural (Figure 7H). Histopathological aGvHD scores in the iver changes of the vasculature and endothelial dysfunction (Figure 7I) and colon (Figure 7J) were significantly reduced occur during aGvHD. Due to lack of suitable animal mod- in sildenafil-treated allo-HSCT recipients with SR- els, less detailed data is available for other important aGvHD. Additionally, we found a trend towards reduced endothelium-related complications of allo-HSCT, such as co-stimulatory capacity as well as significantly reduced veno-occlusive disease and transplantation-associated antigen presentation potential of hepatic EC in sildenafil- microangiopathy. In line with the experimental results, treated SR-aGvHD, demonstrated by lower MHC class I we found that endothelial damage is present in the colon and II expression (Figure 7K). We confirmed these find- and duodenum of patients with severe aGvHD. This find- haematologica | 2021; 106(8) 2155 S. Cordes et al. ing is consistent with a previously published study, result in restructuring of the vasculature as visualized and demonstrating increased endothelial injury in human skin quantified in Figure 4. Interestingly, increased branching biopsies during aGvHD.10 Additionally, a series of clinical levels of vasculature were first reported during pathologi- studies have collected supporting evidence on increased cal angiogenesis in malignant tumors.48 This finding led to endothelial dysfunction during aGvHD, such as high the concept of ‘vascular normalization’ to improve anti- numbers of circulating EC40-43 and elevated serum levels of tumor therapies.49 However, the significance of pathologi- the endothelial stress markers ST2, von Willebrand factor, cal vascular organization with increased branching level angiopoietin 2 and thrombomodulin.31,44-46 for the pathophysiology of aGvHD and possible therapeu- We have previously shown that aGvHD is associated tic implications remains to be determined. with increased angiogenesis in target organs.8,47 In the cur- We found that the expression of tight junction- and rent study, we used light sheet fluorescence microscopy to adherence junction proteins were reduced during demonstrate that target organ aGvHD is associated with aGVHD, leading to increased vascular permeability in vascular structural changes in terms of higher branching aGvHD target organs after allo-HSCT. The role of tight levels and larger diameter of the vasculature. Taking into junction- and adherence junction proteins for vascular account the available evidence from our previous studies barrier maintenance50-52 as well as leukocyte transmigra- as well as the experiments from this manuscript, it tion53,54 has been described previously. Additionally, the becomes clear that vascular destruction as well as patho- promotion of the adherence junction protein VE-cadherin logical angiogenesis occur in parallel in the early phase in tumor vasculature increased T-cell infiltration into the during aGvHD in target organs. These processes finally tumor.55 These findings may open a new window for A B C D E F Figure 7. In vivo treatment of acute graft-versus-host disease (aGvHD) and steroid-refractory-aGvHD with sildenafil. (A to G) Treatment of aGvHD with 10 mg/kg/d sildenafil after experimental allogeneic hematopoietic stem cell transplantation (allo-HSCT) at day+15 after experimental allo-HSCT in the radiation based B6→BALB/c model. (A) Survival analysis and (B) clinical aGvHD manifestations of sildenafil (sil) treated allo-HSCT recipients with aGvHD versus control substance phosphate buffered saline/dimethylsulfoxide (PBS/DMSO, control [ctr]) treated allo-HSCT recipients with aGvHD. Histopathological assessment of aGvHD manifes- tations in (C) liver and (D) colon in sildenafil versus control substance treated allo-HSCT recipients at day+15. Flow cytometry quantification of (E) major histocom- patibility complex I (MHCI) and MHCII expression and (F) CD80 and CD86 expression of isolated liver sinusoidal endothelial cells of sildenafil versus control substance treated allo-HSCT recipients at day+15. (G to L) Treatment with 10 mg/kg/d sildenafil in a murine model of SR-aGvHD at day+15 after experimental allo-HSCT in the radiation based B6→BALB/c model. (continued on next page.) 2156 haematologica | 2021; 106(8) aGvHD impact on endothelium G H I J L Figure 7. (G) Survival analysis and (H) clin- K ical aGvHD manifestations of sildenafil (SR-aGvHD+sil) versus control substance treated (SR-aGvHD+ctr) SR-aGvHD. Histopathological assessment of aGvHD severity in (I) liver and (J) colon in SR- aGvHD+sil and SR-aGvHD+ctr at day+15 after allo-HSCT. Flow cytometry quantifi- cation of (K) MHCI and MHCII expression and (L) CD80 and CD86 expression of iso- lated liver sinusoidal endothelial cells of SR-aGvHD+sil and SR-aGvHD+ctr at day+15 after allo-HSCT. Significance was tested by Kaplan-Meier method and com- pared with the Mantel-Cox log-rank test (*P<0.05; n=8-10 animals per group) and Student’s t-test (*P<0.05; **P<0.01; M N n=6 animals per group). Error bars indi- cate mean ± standard error of the mean. Survival data was pooled from two experi- ments. All experiments were reproduced in a biological independent experiment and shown are representative results of one experiment. (M to P) Visualization of SR-aGvHD and sildenafil treated SR- aGvHD associated ultrastructural changes in the liver and the colon by transmission electron microscopy. Shown are typical pictures of sections from liver and colon taken at day+15 after experi- mental allo-HSCT in the chemotherapy based 129→B6 model. (M and N) Sinusoidal liver endothelial monolayer during SR-aGvHD and sildeanfil (sil) treat- ed SR-aGvHD. (M) Liver sinusoidal vessel of untreated SR-aGvHD with destroyed and unregularly shaped endothelial O P monolayer, marked by a red circle. (N) Liver sinusoidal vessel of Sildenafil treat- ed SR-aGvHD with small ruptures of the endothelial monolayer, marked by a red circle. (O and P) Colonic mucosa endothe- lium during SR-aGvHD and sildeanfil treated SR-aGvHD. (O) The vessel of untreated SR-aGvHD is surrounded by massive perivascular fibrinogen deposits marked by a red rhombus. (P) Occasionally little perivascular fibrinogen deposits, marked by a red rhombus, could be detected in sildenafil treated SR- aGvHD. V: vessel lumen; E: erythrocyte; N: nucleus; red rhombus: perivascular fib- rinogen deposits; red circle: loss of endothelium; red trapezoid: endothelial convolution). haematologica | 2021; 106(8) 2157 S. Cordes et al. aGvHD therapies aiming at reducing vascular permeabil- such as nitric oxide synthase (Nos3) and BCL-2, was ity and stabilization of vascular tight-junctions as well as increased in target organ EC during aGvHD. Sildenafil has adherence-junctions. Vestweber and colleagues have gen- been shown to have cyto-protective effects by regulation erated mice with stabilized endothelial junctions; these of nitric oxide synthase 3 signaling and BCL-2 expres- mice had strongly reduced neutrophil and lymphocyte sion.63 In addition, sildenafil may have positive effects by recruitment into inflamed tissues.56 Examples for a thera- stabilizing vessel surrounding pericytes and thereby vas- peutic approach derive from the field of sepsis where cular integrity: PDE5 targets cGMP, which has been reduction of vascular permeability with the αVb3 antag- demonstrated to inhibit the proliferation of smooth mus- onist cilengitide57 or with an antibody binding to cle cells by causing a delay in G /S transition in the cell 1 angiopoietin 2 and TIE258 which led to substantial biolog- cycle.64 Interestingly, we detected differentially regulated ical benefits. cell cycle genes in EC during aGvHD. Of note, we found Recent clinical studies suggest a role of endothelial that sildenafil treatment led to reduction of aGvHD-asso- pathology particularly in SR-aGvHD13,14,17,28,29,31 and a high ciated expression of co-stimulatory molecules on target medical need for development of respective therapies. organ EC. However, our finding that sildenafil had no sig- We now demonstrate in two independent clinical cohorts nificant effects on in vivo proliferation of allogeneic donor as well as in experimental models that inflammatory infil- T cells in irradiated hosts argue against the biologic signif- tration in target organs is considerably reduced compared icance of the observed impact of sildenafil on EC co-stim- to aGvHD at diagnosis. Consequently, we found that ulatory molecule expression during aGvHD. dexamethasone treatment did not significantly reduce the In conclusion, our results demonstrate extensive dam- severity of endothelial damage during SR-aGvHD. Pavan age, structural changes, and dysfunction of the vascula- Reddy’s group recently published the results of extensive ture during aGvHD, and our findings suggest a novel con- experiments in murine models of SR-GvHD. In line with cept of therapeutic intervention by endothelium-protect- our findings, their results point towards the existence of ing agents as an attractive treatment approach for SR- T-cell independent mechanisms in the pathophysiology aGvHD. A likely future perspective is to test endotheli- of SR-GvHD.23 This may explain the historically disap- um-targeting approaches complementing treatment pointing clinical results of immunosuppressive treatments options with proven efficacy in SR-aGvHD, such as JAK- for SR-aGvHD. The low inflammatory status in connec- 2 inhibition.65 tion with considerable endothelial pathology provides a rationale for therapies protecting the endothelium during Disclosures SR-aGvHD. Based on previous encouraging data on nor- No conflicts of interest to disclose. malization of endothelial dysfunction,32-39 we used the PDE5 inhibitor sildenafil as a first attempt to treat SR- Contributions aGvHD by an ‘endothelium-protective’ approach. SC, AB, EC, MD-R, JM, LB, CPS and OP designed the Sildenafil treatment ameliorated aGvHD and the effect study; SC, ZM, MB, YS, SM, KR, JM, MK, AM, JS, MP and was more pronounced in case of SR-aGvHD compared GB performed experiments and analyzed results; SC and OP with previously untreated aGvHD. The latter observation wrote the manuscript. All authors read, edited and approved the may be explained by the high level of tissue inflammation manuscript. in non-glucocorticoid treated aGvHD mice superimpos- ing the beneficial effects of sildenafil on EC. However, we Acknowledgments cannot exclude the possibility of a synergistic effect of This work was supported by the José Carreras Leukämie- sildenafil with steroids in our models. Stiftung (11R2016, 03R\2019), Deutsche Krebshilfe The direct mechanisms of sildenafil treatment on the (70113519), Deutsche Forschungsgemeinschaft (PE 1450/7-1), course of SR-aGvHD remain unclear. However, improve- Monika-Kutzner-Stiftung, and Wilhelm-Sander-Stiftung ment of vascular barrier function and reduction of EC (2014.150.1). apoptosis probably contribute to the beneficial effects of sildenafil. During aGvHD we found increased vascular Funding permeability in target organs and alterations of Rho MB was funded by the European Training and Research in GTPases, who are critical regulators of endothelial barrier Peritoneal Dialysis Program, funded by the European Union function.59 Sildenafil-mediated stabilization of endothelial within the Marie Curie Scheme (287813). AB received funding barrier function60,61 as well as impact of sildenafil on regu- from the German Research Foundation (DFG), collaborative lation of Rho GTPases62 have been described previously. research center TRR221 (B11, Z02) and ZM was funded by the We found that apoptosis and expression of relevant genes, DFG collaborative research center TRR225 (B08). References al. Circulating dsDNA, endothelial injury, 7. Ueda N, Chihara D, Kohno A, et al. and complement activation in thrombotic Predictive value of circulating angiopoietin- 1. Garnett C, Apperley JF, Pavlu J. Treatment microangiopathy and GVHD. Blood. 2 for endothelial damage-related complica- and management of graft-versus-host dis- 2017;130(10):1259-1266. tions in allogeneic hematopoietic stem cell ease: improving response and survival. 5. Fan CQ, Crawford JM. Sinusoidal obstruc- transplantation. Biol Blood Marrow Ther Adv Hematol. 2013;4(6):366-378. tion syndrome (hepatic veno-occlusive dis- Transplant. 2014;20(9):1335-1340. 2. Deeg HJ. How I treat refractory acute ease). J Clin Exp Hepatol. 2014;4(4):332- 8. Riesner K, Shi Y, Jacobi A, et al. Initiation of GVHD. Blood. 2007;109(10):4119-4126. 346. acute graft-versus-host disease by angio- 3. Westin JR, Saliba RM, De Lima M, et al. 6. Xie Z, Ghosh CC, Patel R, et al. Vascular genesis. Blood. 2017;129(14):2021-2032. Steroid-refractory acute GVHD: predictors endothelial hyperpermeability induces the 9. Murata H, Janin A, Leboeuf C, et al. Donor- and outcomes. Adv Hematol. 2011;2011: clinical symptoms of Clarkson disease (the derived cells and human graft-versus-host 601953. systemic capillary leak syndrome). Blood. disease of the skin. Blood. 2007;109(6): 4. Gloude NJ, Khandelwal P, Luebbering N, et 2012;119(18):4321-4332. 2663-2665. 2158 haematologica | 2021; 106(8) aGvHD impact on endothelium 10. Dumler JS, Beschorner WE, Farmer ER, Di 25. Radu M, Chernoff J. An in vivo assay to 42. Medinger M, Heim D, Gerull S, et al. Gennaro KA, Saral R, Santos GW. test blood vessel permeability. J Vis Exp. Increase of endothelial progenitor cells in Endothelial-cell injury in cutaneous acute 2013(73):e50062. acute graft-versus-host disease after allo- graft-versus-host disease. Am J Pathol. 26. Brede C, Friedrich M, Jordan-Garrote AL, et geneic haematopoietic stem cell transplan- 1989;135(6):1097-1103. al. Mapping immune processes in intact tis- tation for acute myeloid leukaemia. Leuk 11. Mir E, Palomo M, Rovira M, et al. sues at cellular resolution. J Clin Invest. Res. 2016;47:22-25. Endothelial damage is aggravated in acute 2012;122(12):4439-4446. 43. Yan Z, Zeng L, Jia L, Xu S, Ding S. Increased GvHD and could predict its development. 27. Wertheimer T, Velardi E, Tsai J, et al. numbers of circulating ECs are associated Bone Marrow Transplant. 2017;52(9):1317- Production of BMP4 by endothelial cells is with systemic GVHD. Int J Lab Hematol. 1325. crucial for endogenous thymic regenera- 2011;33(5):507-515. 12. Rachakonda SP, Penack O, Dietrich S, et al. tion. Sci Immunol. 2018;3(19):eaal2736. 44. Tatekawa S, Kohno A, Ozeki K, et al. A Single-nucleotide polymorphisms within 28. Dietrich S, Okun JG, Schmidt K, et al. High novel diagnostic and prognostic biomarker the thrombomodulin gene (THBD) predict pre-transplant serum nitrate levels predict panel for endothelial cell damage-related mortality in patients with graft-versus-host risk of acute steroid-refractory graft-versus- complications in allogeneic transplantation. disease. J Clin Oncol. 2014;32(30): 3421- host disease in the absence of statin thera- Biol Blood Marrow Transplant. 3427. py. Haematologica. 2014;99(3):541-547. 2016;22(9):1573-1581. 13. Dietrich S, Falk CS, Benner A, et al. 29. Luft T, Dietrich S, Falk C, et al. Steroid- 45. Lindas R, Tvedt TH, Hatfield KJ, Reikvam Endothelial vulnerability and endothelial refractory GVHD: T-cell attack within a H, Bruserud O. Preconditioning serum lev- damage are associated with risk of graft- vulnerable endothelial system. Blood. els of endothelial cell-derived molecules versus-host disease and response to steroid 2011;118(6):1685-1692. and the risk of posttransplant complica- treatment. Biol Blood Marrow Transplant. 30. Rotz SJ, Dandoy CE, Davies SM. ST2 and tions in patients treated with allogeneic 2013;19(1):22-27. Endothelial injury as a link between GVHD stem cell transplantation. J Transplant. 14. Andrulis M, Dietrich S, Longerich T, et al. and microangiopathy. N Engl J Med. 2014;2014:404096. Loss of endothelial thrombomodulin pre- 2017;376(12):1189-1190. 46. Biedermann BC, Tsakiris DA, Gregor M, dicts response to steroid therapy and sur- 31. Vander Lugt MT, Braun TM, Hanash S, et Pober JS, Gratwohl A. Combining altered vival in acute intestinal graft-versus-host al. ST2 as a marker for risk of therapy-resis- levels of effector transcripts in circulating T disease. Haematologica. 2012;97(11):1674- tant graft-versus-host disease and death. N cells with a marker of endothelial injury is 1677. Engl J Med. 2013;369(6):529-539. specific for active graft-versus-host disease. 15. Pihusch V, Rank A, Steber R, et al. 32. Balarini CM, Leal MA, Gomes IB, et al. Bone Marrow Transplant. 2003;32(11): Endothelial cell-derived microparticles in Sildenafil restores endothelial function in 1077-1084. allogeneic hematopoietic stem cell recipi- the apolipoprotein E knockout mouse. J 47. Penack O, Henke E, Suh D, et al. Inhibition ents. Transplantation. 2006;81(10):1405- Transl Med. 2013;11:3. of neovascularization to simultaneously 1409. 33. Bivalacqua TJ, Sussan TE, Gebska MA, et ameliorate graft-vs-host disease and 16. Rachakonda SP, Dai H, Penack O, et al. al. Sildenafil inhibits superoxide formation decrease tumor growth. J Natl Cancer Inst. Single nucleotide polymorphisms in and prevents endothelial dysfunction in a 2010;102(12):894-908. CD40L predict endothelial complications mouse model of secondhand smoke 48. Less JR, Skalak TC, Sevick EM, Jain RK. and mortality after allogeneic stem-cell induced erectile dysfunction. J Urol. Microvascular architecture in a mammary transplantation. J Clin Oncol. 2018;36(8): 2009;181(2):899-906. carcinoma: branching patterns and vessel 789-800. 34. Aksu V, Yuksel V, Chousein S, et al. The dimensions. Cancer Res. 1991;51(1):265- 17. Luft T, Benner A, Jodele S, et al. EASIX in effects of sildenafil and n-acetylcysteine on 273. patients with acute graft-versus-host dis- ischemia and reperfusion injury in gastroc- 49. Huang Y, Goel S, Duda DG, Fukumura D, ease: a retrospective cohort analysis. Lancet nemius muscle and femoral artery endothe- Jain RK. Vascular normalization as an Haematol. 2017;4(9):e414-e423. lium. Vascular. 2015;23(1):21-30. emerging strategy to enhance cancer 18. Schmid PM, Bouazzaoui A, Doser K, et al. 35. Valatsou A, Briasoulis A, Vogiatzi G, et al. immunotherapy. Cancer Res. 2013;73(10): Endothelial dysfunction and altered Beneficial effects of sildenafil on tissue per- 2943-2948. mechanical and structural properties of fusion and inflammation in a murine model 50. Tornavaca O, Chia M, Dufton N, et al. ZO- resistance arteries in a murine model of of limb ischemia and atherosclerosis. Curr 1 controls endothelial adherens junctions, graft-versus-host disease. Biol Blood Vasc Pharmacol. 2017;15(3):282-287. cell-cell tension, angiogenesis, and barrier Marrow Transplant. 2014;20(10):1493- 36. Fahning BM, Dias AT, Oliveira JP, et al. formation. J Cell Biol. 2015;208(6):821-838. 1500. Sildenafil improves vascular endothelial 51. Fanning AS, Little BP, Rahner C, 19. Lerner KG, Kao GF, Storb R, Buckner CD, structure and function in renovascular Utepbergenov D, Walther Z, Anderson JM. Clift RA, Thomas ED. Histopathology of hypertension. Curr Pharm Biotechnol. The unique-5 and -6 motifs of ZO-1 regu- graft-vs.-host reaction (GvHR) in human 2015;16(9):823-831. late tight junction strand localization and recipients of marrow from HL-A-matched 37. McLaughlin K, Lytvyn Y, Luca MC, Liuni A, scaffolding properties. Mol Biol Cell. sibling donors. Transplant Proc. Gori T, Parker JD. Repeated daily dosing 2007;18(3):721-731. 1974;6(4):367-371. with sildenafil provides sustained protec- 52. Umeda K, Ikenouchi J, Katahira-Tayama S, 20. Mertlitz S, Shi Y, Kalupa M, et al. tion from endothelial dysfunction caused et al. ZO-1 and ZO-2 independently deter- Lymphangiogenesis is a feature of acute by ischemia and reperfusion: a human in mine where claudins are polymerized in GVHD, and VEGFR-3 inhibition protects vivo study. Am J Physiol Heart Circ tight-junction strand formation. Cell. against experimental GVHD. Blood. Physiol. 2014;307(6):H888-894. 2006;126(4):741-754. 2017;129(13):1865-1875. 38. Yin J, Kukucka M, Hoffmann J, et al. 53. Reyat JS, Chimen M, Noy PJ, Szyroka J, 21. Riesner K, Kalupa M, Shi Y, Elezkurtaj S, Sildenafil preserves lung endothelial func- Rainger GE, Tomlinson MG. ADAM10- Penack O. A preclinical acute GVHD tion and prevents pulmonary vascular interacting tetraspanins Tspan5 and mouse model based on chemotherapy con- remodeling in a rat model of diastolic heart Tspan17 regulate VE-cadherin expression ditioning and MHC-matched transplanta- failure. Circ Heart Fail. 2011;4(2):198-206. and promote T lymphocyte transmigration. tion. Bone Marrow Transplant. 39. Yaguas K, Bautista R, Quiroz Y, et al. J Immunol. 2017;199(2):666-676. 2016;51(3):410-417. Chronic sildenafil treatment corrects 54. Vockel M, Vestweber D. How T cells trig- 22. Bouazzaoui A, Spacenko E, Mueller G, et endothelial dysfunction and improves ger the dissociation of the endothelial al. Steroid treatment alters adhesion mole- hypertension. Am J Nephrol. 2010;31(4): receptor phosphatase VE-PTP from VE-cad- cule and chemokine expression in experi- 283-291. herin. Blood. 2013;122(14):2512-2522. mental acute graft-vs.-host disease of the 40. Almici C, Skert C, Bruno B, et al. 55. Zhao Y, Ting KK, Li J, et al. Targeting vas- intestinal tract. Exp Hematol. 2011;39(2): Circulating endothelial cell count: a reliable cular endothelial-cadherin in tumor-associ- 238-249 e231. marker of endothelial damage in patients ated blood vessels promotes T-cell-mediat- 23. Toubai T, Rossi C, Tawara I, et al. Murine undergoing hematopoietic stem cell trans- ed immunotherapy. Cancer Res. 2017;77 models of steroid refractory graft-versus- plantation. Bone Marrow Transplant. (16):4434-4447. host disease. Sci Rep. 2018;8(1):12475. 2017;52(12):1637-1642. 56. Schulte D, Kuppers V, Dartsch N, et al. 24. Beilhack A, Schulz S, Baker J, et al. In vivo 41. Woywodt A, Scheer J, Hambach L, et al. Stabilizing the VE-cadherin-catenin com- analyses of early events in acute graft-ver- Circulating endothelial cells as a marker of plex blocks leukocyte extravasation and sus-host disease reveal sequential infiltra- endothelial damage in allogeneic vascular permeability. EMBO J. 2011;30 tion of T-cell subsets. Blood. 2005;106(3): hematopoietic stem cell transplantation. (20):4157-4170. 1113-1122. Blood. 2004;103(9):3603-3605. 57. McHale TM, Garciarena CD, Fagan RP, et haematologica | 2021; 106(8) 2159 S. Cordes et al. al. Inhibition of vascular endothelial cell Inhibition of phosphodiesterase 5 restores Phosphodiesterase-5 inhibitor sildenafil leak following Escherichia coli attachment endothelial function in renovascular hyper- preconditions adult cardiac myocytes in an experimental model of sepsis. Crit tension. J Transl Med. 2014;12:250. against necrosis and apoptosis. Essential Care Med. 2018;46(8):e805-e810. 61. Chen W, Oberwinkler H, Werner F, et al. role of nitric oxide signaling. J Biol Chem. 58. Han S, Lee SJ, Kim KE, et al. Amelioration Atrial natriuretic peptide-mediated inhibi- 2005;280(13):12944-12955. of sepsis by TIE2 activation-induced vascu- tion of microcirculatory endothelial Ca2+ 64. Fukumoto S, Koyama H, Hosoi M, et al. lar protection. Sci Transl Med. and permeability response to histamine Distinct role of cAMP and cGMP in the cell 2016;8(335):335ra355. involves cGMP-dependent protein kinase I cycle control of vascular smooth muscle 59. Wojciak-Stothard B, Tsang LY, Haworth and TRPC6 channels. Arterioscler Thromb cells: cGMP delays cell cycle transition SG. Rac and Rho play opposing roles in the Vasc Biol. 2013;33(9):2121-2129. through suppression of cyclin D1 and regulation of hypoxia/reoxygenation- 62. Guilluy C, Sauzeau V, Rolli-Derkinderen cyclin-dependent kinase 4 activation. Circ induced permeability changes in pul- M, et al. Inhibition of RhoA/Rho kinase Res. 1999;85(11):985-991. monary artery endothelial cells. Am J pathway is involved in the beneficial effect 65. Zeiser R, von Bubnoff N, Butler J, et al. Physiol Lung Cell Mol Physiol. of sildenafil on pulmonary hypertension. Br Ruxolitinib for glucocorticoid-refractory 2005;288(4):L749-760. J Pharmacol. 2005;146(7):1010-1018. acute graft-versus-host disease. N Engl J 60. Dias AT, Cintra AS, Frossard JC, et al. 63. Das A, Xi L, Kukreja RC. Med. 2020;382(19):1800-1810. 2160 haematologica | 2021; 106(8)