Endothelial damage and dysfunction in acute graft-versus-host disease

Altres ajuts: Deutsche Forschungsgemeinschaft (DFG) TRR221 (B11 Z02), TRR225 (B08)Clinical studies have suggested a potential involvement of endothelial 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 accompanied by reduced expression of endothelial tight junction proteins leading to increased endothelial leakage in aGvHD target organs. During intestinal aGvHD, colonic vasculature structurally changed, reflected by increased vessel branching and vessel diameter. As recent data demonstrated an association of endothelium-related factors and steroid refractory aGvHD (SR-aGvHD), we analyzed human biopsies and murine tissues from SR-aGvHD. We found extensive tissue damage but low levels of alloreactive T-cell infiltration in target organs, providing the rationale for T-cell independent SR-aGvHD treatment strategies. Consequently, we tested the endothelium-protective PDE5 inhibitor sildenafil, which reduced apoptosis and improved metabolic activity of endothelial cells in vitro. Accordingly, sildenafil treatment improved survival and reduced target organ damage during experimental SR-aGvHD. Our results demonstrate extensive damage, structural changes, and dysfunction of the vasculature during aGvHD. Therapeutic intervention by endothelium-protecting agents is an attractive approach for SR-aGvHD complementing current anti-inflammatory treatment options

Endothelial damage and dysfunction in acute graft-versus-host disease

Clinical studies suggested that endothelial dysfunction and damage could be involved in the development and severity of acute graft-versus-host disease (aGVHD). Accordingly, we found increased percentage of apoptotic Casp3+ 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 accompanied by reduced expression of endothelial tight junction proteins leading to increased endothelial leakage in aGVHD target organs. During intestinal aGVHD, colonic vasculature structurally changed, reflected by increased vessel branching and vessel diameter. Because recent data demonstrated an association of endothelium-related factors and steroid refractory aGVHD (SR-aGVHD), we analyzed human biopsies and murine tissues from SR-aGVHD. We found extensive tissue damage but low levels of alloreactive T cell infiltration in target organs, providing the rationale for T-cell independent SR-aGVHD treatment strategies. Consequently, we tested the endothelium-protective PDE5 inhibitor sildenafil, which reduced apoptosis and improved metabolic activity of endothelial cells in vitro. Accordingly, sildenafil treatment improved survival and reduced target organ damage during experimental SR-aGVHD. Our results demonstrate extensive damage, structural changes, and dysfunction of the vasculature during aGVHD. Therapeutic intervention by endothelium-protecting agents is an attractive approach for SR-aGVHD complementing current anti-inflammatory treatment options

The Green Tea Catechin Epigallocatechin Gallate Ameliorates Graft-versus-Host Disease.

Allogeneic hematopoetic stem cell transplantation (allo-HSCT) is a standard treatment for leukemia and other hematologic malignancies. The major complication of allo-HSCT is graft-versus-host-disease (GVHD), a progressive inflammatory illness characterized by donor immune cells attacking the organs of the recipient. Current GVHD prevention and treatment strategies use immune suppressive drugs and/or anti-T cell reagents these can lead to increased risk of infections and tumor relapse. Recent research demonstrated that epigallocatechin gallate (EGCG), a component found in green tea leaves at a level of 25-35% at dry weight, may be useful in the inhibition of GVHD due to its immune modulatory, anti-oxidative and anti-angiogenic capacities. In murine allo-HSCT recipients treated with EGCG, we found significantly reduced GVHD scores, reduced target organ GVHD and improved survival. EGCG treated allo-HSCT recipients had significantly higher numbers of regulatory T cells in GVHD target organs and in the blood. Furthermore, EGCG treatment resulted in diminished oxidative stress indicated by significant changes of glutathione blood levels as well as glutathione peroxidase in the colon. In summary, our study provides novel evidence demonstrating that EGCG ameliorates lethal GVHD and reduces GVHD-related target organ damage. Possible mechanisms are increased regulatory T cell numbers and reduced oxidative stress

Impact of EGCG on GVHD-related mortality and clinical GVHD symptoms.

<p>(a) Impact of EGCG on survival during GVHD. 2x10⁷ BM cells and 5x10⁶ T cells from C57BL/6 (H-2K^b) mice were injected into B6D2F1 (H-2K^d) mice after busulfan and cyclophosphamide conditioning (4 days 20mg/kg/day busulfan, 2 days 100mg/kg/day cyclophosphamide). Significant differences were seen between EGCG treated and untreated control group (***<i>p</i><0.001). (b) Impact of EGCG on clinical GVHD scores after BMT. 1,5x10⁷ BM and 2x10⁶ T cells from LP/J mice were injected into C57BL/6 mice after busulfan and cyclophosphamide conditioning (5 days 20mg/kg/day, 2 days 100mg/kg/day cyclophosphamide). EGCG +quercetin treated mice showed significantly decreased GVHD scores compared with control animals. N = 10 per group (*<i>p</i><0.05).</p

Impact of EGCG on oxidative stress during GVHD.

<p>(a) Glutathione levels during GVHD. Glutahione (GSH) was measured in blood, colon and the liver of EGCG treated and untreated allo-BMT recipients at day +17. A significantly decreased amount of glutathione in the blood was observed after treatment with EGCG. (b) GPO amounts in the colon and liver during GVHD. Sections of colon and liver at day +17 were stained with GPO antibody, and at least 6 representative pictures per animal were evaluated. A significantly decreased amount of glutathione peroxidase in the colon but not in the liver was observed after treatment with EGCG. <i>n</i> = 8 (ns = not significant, *<i>p</i> <0.05, **<i>p</i><0.01, ***<i>p</i><0.001).</p

Impact of quercetin and piperine on EGCG plasma levels.

<p>Mice were i.p. injected with 25mg/kg EGCG and 1mg/kg quercetin or 1mg/kg piperine, respectively. Blood was taken after 1, 2, 3, 4 and 5 hours and analyzed by LC-MC/MC by Pharmacelsus, Saarbrücken, Germany. A typical experiment is shown.</p

Impact of EGCG on systemic inflammation during GVHD.

<p>1,5x10⁷ BM and 2x10⁶ T cells from LP/J mice were injected into C57BL/6 mice after busulfan and cyclophosphamide conditioning. (a) Sections of colon and liver at day +17 were stained with CD4 or CD8 antibody, and at least 6 representative pictures per animal were evaluated. CD4 positive cells were increased and CD8 positive cells were decreased as a result of EGCG treatment. (b) Representative staining of colon with anti-CD4 antibody. Abundant CD4 positive cell infiltration in colon sections of mice treated with EGCG. Sections were counterstained with 4`, 6-Diamino-2-phenylindole. (c) Regulatory T cells during GVHD. FACS analysis of blood and lymph nodes and section analysis of colon of mice treated or non-treated with EGCG. Tregs were increased in blood and colon of EGCG treated mice. (d) Leukocytes during GVHD. FACS analysis of blood and spleens of myeloid and lymphoid cells during GVHD. Within analyzed cells (B cells, monocytes, granulocytes and NK cells), monocytes were significantly decreased in the blood of EGCG treated mice. Shown is data from one representative experiment, <i>n</i> = 8 (ns = not significant, *<i>p</i> <0.05, **<i>p</i><0.01 ***<i>p</i><0.001).</p

Impact of EGCG on target organ GVHD.

<p>1,5x10⁷ BM and 2x10⁶ T cells from LP/J mice were injected into C57BL/6 mice after busulfan and cyclophosphamide conditioning. (a) Histopathological scores in GVHD target organs at day +17. H&E-stained sections of colon, liver and skin were analyzed according to Lerner [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169630#pone.0169630.ref017" target="_blank">17</a>]. Significant differences between EGCG treated and untreated control group in all target organs. Data from one representative experiment is shown, <i>N</i> = 8 per group, ***<i>p</i><0.001. (b) Histopathological changes in GVHD target organs at day +17. GVHD tissue damage of untreated control group showed in the colon cryptal abscesses (CA) and crypt loss (CL), whereas EGCG treated group showed normal architecture. In the liver of the control group excessive lymphoid infiltration (LI) was observed. Liver veins of EGCG treated mice showed only minimal infiltrations around the veins. Skin of untreated control mice exhibited vacuolization of the epidermis (V), surface damage (SD) and infiltration of immune cells (LI). EGCG treated mice showed normal skin structure. Original magnification was 200x.</p