Prednisolone as preservation additive prevents from ischemia reperfusion injury in a rat model of orthotopic lung transplantation

The lung is, more than other solid organs, susceptible for ischemia reperfusion injury after orthotopic transplantation. Corticosteroids are known to potently suppress pro-inflammatory processes when given in the post-operative setting or during rejection episodes. Whereas their use has been approved for these clinical indications, there is no study investigating its potential as a preservation additive in preventing vascular damage already in the phase of ischemia. To investigate these effects we performed orthotopic lung transplantations (LTX) in the rat. Prednisolone was either added to the perfusion solution for lung preservation or omitted and rats were followed for 48 hours after LTX. Prednisolone preconditioning significantly increased survival and diminished reperfusion edema. Hypoxia induced vasoactive cytokines such as VEGF were reduced. Markers of leukocyte invasiveness like matrix metalloprotease (MMP)-2, or common pro-inflammatory molecules like the CXCR4 receptor or the chemokine (C-C motif) ligand (CCL)-2 were downregulated by prednisolone. Neutrophil recruitment to the grafts was only increased in Perfadex treated lungs. Together with this, prednisolone treated animals displayed significantly reduced lung protein levels of neutrophil chemoattractants like CINC-1, CINC-2α/β and LIX and upregulated tissue inhibitor of matrix metalloproteinase (TIMP)-1. Interestingly, lung macrophage invasion was increased in both, Perfadex and prednisolone treated grafts, as measured by MMP-12 or RM4. Markers of anti-inflammatory macrophage transdifferentiation like MRC-1, IL-13, IL-4 and CD163, significantly correlated with prednisolone treatment. These observations lead to the conclusion that prednisolone as an additive to the perfusion solution protects from hypoxia triggered danger signals already in the phase of ischemia and thus reduces graft edema in the phase of reperfusion. Additionally, prednisolone preconditioning might also lead to macrophage polarization as a beneficial long-term effect

Deguelin Attenuates Reperfusion Injury and Improves Outcome after Orthotopic Lung Transplantation in the Rat

The main goal of adequate organ preservation is to avoid further cellular metabolism during the phase of ischemia. However, modern preservation solutions do rarely achieve this target. In donor organs hypoxia and ischemia induce a broad spectrum of pathologic molecular mechanisms favoring primary graft dysfunction (PGD) after transplantation. Increased hypoxia-induced transcriptional activity leads to increased vascular permeability which in turn is the soil of a reperfusion edema and the enhancement of a pro-inflammatory response in the graft after reperfusion. We hypothesize that inhibition of the respiration chain in mitochondria and thus inhibition of the hypoxia induced mechanisms might reduce reperfusion edema and consecutively improve survival in vivo. In this study we demonstrate that the rotenoid Deguelin reduces the expression of hypoxia induced target genes, and especially VEGF-A, dose-dependently in hypoxic human lung derived cells. Furthermore, Deguelin significantly suppresses the mRNA expression of the HIF target genes VEGF-A, the pro-inflammatory CXCR4 and ICAM-1 in ischemic lungs vs. control lungs. After lung transplantation, the VEGF-A induced reperfusion-edema is significantly lower in Deguelin-treated animals than in controls. Deguelin-treated rats exhibit a significantly increased survival-rate after transplantation. Additionally, a downregulation of the pro-inflammatory molecules ICAM-1 and CXCR4 and an increase in the recruitment of immunomodulatory monocytes (CD163+ and CD68+) to the transplanted organ involving the IL4 pathway was observed. Therefore, we conclude that ischemic periods preceding reperfusion are mainly responsible for the increased vascular permeability via upregulation of VEGF. Together with this, the resulting endothelial dysfunction also enhances inflammation and consequently lung dysfunction. Deguelin significantly decreases a VEGF-A induced reperfusion edema, induces the recruitment of immunomodulatory monocytes and thus improves organ function and survival after lung transplantation by interfering with hypoxia induced signaling

Anti-inflammatory macrophages are recruited to Deguelin treated lungs.

<p>Figure representing micrographs of cellular invasion into transplanted lungs at the end of the reperfusion phase. The left micrographs represent control animals and the right micrographs represent animals that received Deguelin treatment. From top to bottom, micrographs represent DAB immunostainings from RM-4 (pan-macrophage), ICAM-1, CXCR4, CD68 and CD163. Magnification was set at 40× and 100× for pictures in picture. CXCR4 micrographs are represented in 20× magnification. Calibration bar represents 50 µm (40×). The graphs represent the statistical evaluation of each cell type. The upper graph represents a total macrophage count (RM-4+ cells), followed by ICAM-1+ cell count and CXCR4staining. The last two graphs represent CD68+ cell count, and finally a CD 163+ cell count. Arrowheads mark positively stained cells. Evaluation for CXCR4 is performed by calculating the ratio between positively and negatively stained amount of cells to avoid bias from alveoli that contain no cells. Arrowheads mark positively stained cells. Columns and error bars represent means ± SEM. * indicates significance level vs. control. ***, P<0.0001; one-way ANOVA and unpaired t test.</p

Layout of the first experimental stage.

<p>Layout of the first experimental stage.</p

Layout of the second experimental stage.

<p>Layout of the second experimental stage.</p

Deguelin prevents from ischemia induced edema and loss of lung microstructure.

<p>From the explantation experiment, micrographs are analyzed to detect structural edema as sign for organ damage. (A) Representative H&E micrographs (purple) from native lungs (sham), ischemic lungs without treatment (w.i.) and ischemic lungs with deguelin treatment (w.i.D). The blue graphs represent planimetric evaluation of the H&E stains to evaluate the area/field occupied by tissue as measurement for edema. Magnifications used: 10× and 40×. Bar in the 10× magnified micrographs represent 100 µm and in the 40× magnified micrographs represent 50 µm. From each H&E stain representing always one animal, 3 different areas were photographed and evaluated. (B) Graph representing the evaluation of the planimetric measurements. Columns and error bars represent means ± SEM. * indicates significance level vs. sham, † indicates significance level vs. w.i.; ***, P<0.0005; †††, P<0.0001; one-way ANOVA and unpaired t test.</p

VEGF-A activity correlates with edema formation.

<p>The transplantation experiments compromise two groups, one control (perfadex only) and one Deguelin treated group. Animals receiving Deguelin (donor and recipient) are pretreated 3 days prior transplantation and treatment is kept upright in the recipients until the end of the experiment after 48 hours after transplantation. (A) Graphs and representative western blot images representing tissue VEGF-A mRNA and protein levels after transplantation and reperfusion. Animals that received Deguelin are compared vs. controls. (B) Right graph representing the wet-to-dry ratio evaluating the extent of tissue edema. Left graph representing the mean survival expressed in hours of both groups (controls vs. animals that received Deguelin). (C) Corresponding micrographs (10× magnification, H&E) show microstructural changes that occur after reperfusion. Calibration bar represents 100 µm. Columns and error bars represent means ± SEM. * indicates significance level vs. control. ***, P<0.0005; one-way ANOVA and unpaired t test.</p

Technique of lung transplantation.

<p>(A) For perfusion, a venous catheter is placed through an incision in the right ventricle into the pulmonary artery. After perfusion, the heart-lung complex is removed <i>in toto</i>, suspended on the trachea that is ligated in maximal inspiration to avoid atelectasis. B) Cuffs are prepared using venous catheters. The vessel is slipped through the lumen and fixed with a 6–0 polypropylene suture. C) Donor (cuffed) blood vessels and bronchus were slipped (sleeved) into the recipient’s artery, bronchus and vein. When no twisting of the corresponding structure was observed, the anastomosis was secured with a 5–0 Silk suture placed around the cuff and sleeved recipient’s structure.</p

Deguelin effectively inhibits pro-edema and pro-inflammatory genes during hypoxia <i>in vivo</i>.

<p>Lungs from animals pretreated with or without Deguelin are explanted. After 1 hour incubation at 37°C, simulating warm ischemia, the ischemic lungs are processed for further analysis. (A) Scheme representing the ischemia experiment. (B) Gene expression of VEGF-A, CXCR4 and ICAM-1 in ischemic lungs treated with or without Deguelin. Beta actin served as negative control. Groups are compared to native Lungs (sham). Sham  =  native lungs without ischemia, w.i.  =  ischemic lungs without treatment, w.i.D  =  ischemic lungs with Deguelin treatment. Measurements were performed in triplicate. Columns and error bars represent means ± SEM. * indicates significance level vs. sham, † indicates significance level vs. w.i.; *, P<0.05; ***, P<0.0005; ††, P<0.005; †††, P<0.0005; one-way ANOVA and unpaired t test.</p

Specificity of Deguelin.

<p>Western blot analysis of total AKT (tAKT), phospho AKT (pAKT) and ACTB of transplanted lungs derived from animals that were either treated with Deguelin or without (control). However a trend to lower levels of pAKT (n.s.; P = 0.2403) may be seen, no significant differences are observed between the two groups, underlining HIF-1 downregulating potency of Deguelin.</p