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S. Cordes et al.
aGvHD or if this is a systemic effect. We therefore ana- lyzed endothelial leakage in the kidney and skeletal mus- cle, which are typically not affected in human aGvHD. We have previously confirmed these organs to be unaf- fected by aGvHD in our models.8,21 The Evans blue assay revealed no significant changes of endothelial leakage in these non-target organs during aGvHD (Online Supplementary Figure 3A and B) suggesting that increased endothelial leakiness predominantly occurs in aGvHD target organs.
Taken together, we found disturbed endothelial tight junctions and intercellular connections leading to increased vascular leakiness in target organs of aGvHD.
Structural changes of vasculature in target organs during aGvHD
In order to get a better overview of the three-dimen- sional (3D) changes of the vasculature, we compared aGvHD and non-GvHD colonic vessel structure and organization with light sheet fluorescence microscopy after in vivo perfusion with an anti-VE-cadherin antibody. Figure 4A and C show normal VE-cadherin staining and vessel branching in a 3D model in colon at d+15 after HSCT without aGvHD. During aGvHD, we found con- siderably different staining patterns for VE-cadherin (Figure 4B) and increased vessel branching in colon (Figure 4D). Using this 3D model to quantify vessel branch levels of vasculature, we found significantly increased total vessel branching in colonic vasculature during aGVHD as compared to HSCT recipients without aGvHD (Figure 4E). Accordingly, analysis of branching levels from vessel segments (Figure 4F) and vessel diame- ter (Figure 4G) revealed a significant increase during intes- tinal aGvHD. We conclude that aGvHD is associated with structural changes in colonic vasculature.
Dysfunction of the macrovasculature during acute graft-versus-host disease
As aGvHD is associated with severe signs of microvas- cular dysfunction, we became interested if aGvHD also influences the physiological functions of macrovascula- ture as suggested previously.18 We examined vessel con- traction and relaxation in mouse mesenteric arteries dur- ing aGvHD versus no aGVHD after HSCT by myographic measurements. We found that maximum contraction of mesenteric arteries in response to high doses of noradren- aline (NA) and phenylephrine (Phe) was not significantly changed (Online Supplementary Figure S4A and B). However, partial contraction at lower doses of NA and Phe was moderately increased during aGvHD versus no aGvHD (Online Supplementary Figure S4C and D). In addi- tion, fractional relaxation after maximum contraction in response to ACh was slower in arteries from allo-HSCT recipients with aGvHD (Online Supplementary Figure S4E). Taken together we found increased contraction and reduced relaxation potential of mesenteric arteries during aGvHD indicating systemic hypertension.
Gene expression changes of endothelial cells during acute graft-versus-host disease
In order to better understand the molecular pathways involved in endothelial dysfunction during aGvHD we performed gene array expression analysis of flow cytom- etry sorted hepatic EC from HSCT recipients with aGvHD versus without aGvHD at day+15. We found sig-
nificant endothelial gene expression changes in comple- ment activation, apoptosis, oxidative damage, IL-1 sig- naling and cell cycle during aGvHD (Online Supplementary Figures S5, S6 and S7; Online Supplementary Table S3). Furthermore, barrier function and cytoskeleton pathways were differentially regulated, including expres- sion changes of Rho GTPases (Online Supplementary Table 3). We performed interaction analysis of selected endothelium-specific genes (Online Supplementary Figure 7) and found differential expression of Cdh1 and Cdh13 (coding for cadherins), Thbd (coding for thrombomod- ulin), Vegfc (coding for vascular endothelial growth factor c) and Anpgt1/2 (coding for angiopoietin 1 and 2). In sum- mary, we detected endothelial gene expression changes in different clinically relevant pathways during aGvHD providing possible targets for future therapeutic inter- ventions.
Inflammatory activity and endothelial damage during steroid-refractory acute graft-versus-host disease
Since recent clinical data has provided evidence on the importance of endothelial pathology specifically in SR- aGVHD,12,17,28-31 we decided to characterize both inflam- matory infiltrates and endothelial damage in this setting. Based on previous data,22 we first performed dose-finding studies with steroids in different aGvHD models to estab- lish a protocol with progressive aGvHD despite high dose steroid treatment. Treatment with dexamethasone start- ing at day+4 after allo-HSCT, showed progressive aGvHD in our models (Online Supplementary Figure S1; Figure 5).
We analyzed vascular pericyte coverage as well as infiltration by CD4+ donor T cells in dexamethasone- treated SR-aGvHD versus untreated aGvHD at day+15 after allo-HSCT. We found reduced inflammatory T-cell infiltrates in SR-aGvHD versus untreated aGvHD (Figure 5A to D). The predominant remaining cell population in colon during SR-aGvHD were CD4+ and CD8+ T cells (Figure 5 C and D) as well as CD11b+ and F4/80+ myeloid cells (Online Supplementary Figure S8A and B). Endothelial damage as quantified by pericyte coverage reduction in colon vessels was equally severe in SR-aGvHD and untreated aGvHD (Figure 5E). In order to connect the experimental findings to the clinical context of SR- aGvHD, we quantified leukocyte infiltration in intestinal biopsies of patients at diagnosis of aGvHD and at later time points of SR-aGvHD in two independent cohorts. Figure panels 5F to J demonstrate significant lower infil- tration of CD45+ leukocytes in colon biopsies as well as duodenum biopsies (Figure 5I). The leukocyte reduction was mainly due to significantly lower infiltration by CD3+ T cells in colon biopsies (Figure 5K, L, M and O) as well as duodenum biopsies (Figure 5N) during SR- aGvHD versus aGvHD at time of diagnosis. Figure 5K gives a typical example of colonic aGvHD at onset with high levels of inflammatory infiltrates by CD3+ T cells and Figure 5L a representative colonic section after steroid treatment and diagnosis for SR-aGvHD, demon- strating reduced inflammatory infiltrates. In addition, we performed caspase 3 staining and demonstrated a high level of endothelial apoptosis during aGvHD both at diagnosis as well as during SR-aGVHD (Figure 5P). A typical example of high inflammatory activity at aGvHD diagnosis time point and low level of inflammatory activity at diagnosis of SR-GvHD in combination with
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haematologica | 2021; 106(8)