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

Toll-like receptor 3 signalling mediates angiogenic response upon shock wave treatment of ischaemic muscle

Aims Shock wave therapy (SWT) represents a clinically widely used angiogenic and thus regenerative approach for the treatment of ischaemic heart or limb disease. Despite promising results in preclinical and clinical trials, the exact mechanism of action remains unknown. Toll-like receptor 3, which is part of the innate immunity, is activated by binding doublestranded (ds) RNA. It plays a key role in inflammation, a process that is needed also for angiogenesis. We hypothesize that SWT causes cellular cavitation without damaging the target cells, thus liberating cytoplasmic RNA that in turn activates TLR3. Methods and results SWT induces TLR3 and IFN-b1 gene expression as well as RNA liberation from endothelial cells in a time-dependant manner. Conditioned medium from SWT-treated HUVECs induced TLR3 signalling in reporter cells. The response was lost when the medium was treated with RNase III to abolish dsRNAs or when TLR3 was silenced using siRNAs. In a mouse hind limb ischaemia model using wt and TLR3 2/2 mice (n ¼ 6), SWT induced angiogenesis and arteriogenesis only in wt animals. These effects were accompanied by improved blood perfusion of treated limbs. Analysis of main molecules of the TLR3 pathways confirmed TLR3 signalling in vivo following SWT. Conclusion Our data reveal a central role of the innate immune system, namely Toll-like receptor 3, to mediate angiogenesis upon release of cytoplasmic RNAs by mechanotransduction of SWT. -

The early activation of toll-like receptor (TLR)-3 initiates kidney injury after ischemia and reperfusion

Acute kidney injury (AKI) is one of the most important complications in hospitalized patients and its pathomechanisms are not completely elucidated. We hypothesize that signaling via toll-like receptor (TLR)-3, a receptor that is activated upon binding of double-stranded nucleotides, might play a crucial role in the pathogenesis of AKI following ischemia and reperfusion (IR). Male adult C57Bl6 wild-type (wt) mice and TLR-3 knock-out (-/-) mice were subjected to 30 minutes bilateral selective clamping of the renal artery followed by reperfusion for 30 min 2.5h and 23.5 hours or subjected to sham procedures. TLR-3 down-stream signaling was activated already within 3 h of ischemia and reperfusion in post-ischemic kidneys of wt mice lead to impaired blood perfusion followed by a strong pro-inflammatory response with significant neutrophil invasion. In contrast, this effect was absent in TLR-3-/- mice. Moreover, the quick TLR-3 activation resulted in kidney damage that was histomorphologically associated with significantly increased apoptosis and necrosis rates in renal tubules of wt mice. This finding was confirmed by increased kidney injury marker NGAL in wt mice and a better preserved renal perfusion after IR in TLR-3-/- mice than wt mice. Overall, the absence of TLR-3 is associated with lower cumulative kidney damage and maintained renal blood perfusion within the first 24 hours of reperfusion. Thus, we conclude that TLR-3 seems to participate in the pathogenesis of early acute kidney injury

Prednisolone reduces hypoxia induced signaling and attenuates VEGF-A expression.

<p>. (<b>A</b>) Circulating carbonic anhydrase (CA) IX has been measured by ELISA in serum samples of all animals (values are expressed in pg/ml). (<b>B</b>) VEGF-A expression (mRNA, circulating and tissue protein) have been detected by RT-PCR (upper left graph, values expressed as % of Perfadex groups, Perfadex group has been set at 100%), ELISA (upper right graph, expression in pg/ml) and protein array (lower left graph, values expressed in mean pixel densities). Significance level was set to P<0.05. P<0.05: *, § and P<0.01: **, § § and P<0.001: § § §. * = significantly different from shams and § = significantly different from Perfadex-group. RT-PCR: experiments were performed in triplicate, protein array: pooled analysis in duplicate. ANOVA followed by Bonferroni’s post-hoc multiple comparisons test.</p

Prednisolone induces M2 macrophage polarization.

<p>(<b>A</b>) The gene expressions of the anti-inflammatory cytokine IL-13 and of a marker for M2 polarization, MRC-1, were analyzed by RT-PCR. (<b>B</b>) Selective immunohistochemical CD163 staining (brown cells) and counting of the cells (graph). N=3 fields per rat have been evaluated. Significance level was set to P<0.05. P<0.01: **, § § and P<0.001: § § §. * = significantly different from shams and § = significantly different from Perfadex-group. RT-PCR experiments were performed in triplicates and means are expressed as % of Perfadex group (Perfadex group has been set at 100%). ANOVA followed by Bonferroni’s post-hoc multiple comparisons test.</p

Prednisolone has no direct effect on the number of macrophages invading the grafts after LTX.

<p>(<b>A</b>) Quantification of the total number of macrophages per field. Total macrophage staining was performed by immunohistochemistry (micrographs) and macrophage counting was performed (left upper graph). The macrophage specific MMP-12 gene expression was quantified by RT-PCR (left lower graph). (<b>B</b>) Quantification of CD68 positive cells. CD68+ cells have specifically been stained for immunohistochemistry (right micrographs) and positively stained cells per field (brown) have been counted (left graph). Calibration bar for micrographs represents 100 µm (200 x magnification, upper micrographs) and 20 µm (1000 x magnification, lower micrographs). N=3 fields per slide have been evaluated. Significance level was set to P<0.05. P<0.05: *, P<0.01: **, § § and P<0.001: § § §. * = significantly different from shams and § = significantly different from Perfadex-group. RT-PCR: experiment was performed in triplicates and data are expressed as % of Perfadex group (Perfadex group has been set at 100%), protein array: pooled analysis in duplicate. ANOVA followed by Bonferroni’s post-hoc multiple comparisons test.</p

Prednisolone induces anti-inflammatory and inhibits pro-inflammatory pathways.

<p>(<b>A</b>) Tissue protein expression of TNF-α, CXCL7, MIP-1α, IL-1ra, RANTES and IL-4 were evaluated using protein arrays. Experiments using pooled samples from n=6 rats per group were performed in duplicates. (<b>B</b>) representative array pictures. Significance level was set to P<0.05. P<0.05: *, §; P<0.01: **, § § and P<0.001: § § §. * = significantly different from shams and § = significantly different from Perfadex-group Data are expressed as mean pixel densities. ANOVA followed by Bonferroni’s post-hoc multiple comparisons test.</p

Prednisolone treatment significantly increases short-term survival following orthotopic lung transplantation (LTX).

<p>Mean short-term survival in hours following LTX. Survival within the observation period of 48h following LTX was tracked. Values are expressed as means ± standard error of the mean (SEM); significance level was set to P<0.05,:*, significantly different from Perfadex-group. Transplantation procedure was repeated until n=6 survivors per group were obtained to ensure homogenic comparison between the groups. ANOVA followed by Bonferroni’s post-hoc multiple comparisons test.</p

Prednisolone-pretreatment decreases ICAM-1 related pro-inflammatory cell activation.

<p>(<b>A</b>) The cellular inflammation activation marker ICAM-1 was assessed by gene expression analysis using RT-PCR (left graph) and specific ICAM-1 staining (micrographs, arrows) and counting of the ICAM-1+ cells per field (right graph). A subdivision for low ICAM-1 positive cells (micrographs, encircled areas) was performed. (<b>B</b>) Representative immunofluorescence triple staining pictures on ICAM-1 (green), macrophages (RM-4, red) and cell-cores (DAPI, blue). ICAM-1 and RM-4 co-expressing cells are colored in purple. Calibration bar represents 20 µm. (<b>C</b>) CXCR4 expression analysis was performed by immunohistochemistry (micrographs), mRNA expression analysis by RT-PCR (upper right graph) and western blot analysis (lower graph, representative western blots (right picture)). Calibration bar represents 100 µm (200 x magnification, CXCR4 stainings), 50 µm (400 x magnification, upper ICAM stainings) respectively 20 µm (1000 x magnification, lower ICAM stainings). N=3 fields per slide have been evaluated. Significance level was set to P<0.05. P<0.05: *, P<0.01: **, § § and P<0.001: § § §. * = significantly different from shams and § = significantly different from Perfadex-group. RT-PCR and western blot analysis experiments were performed in triplicates and data are expressed as % of Perfadex group (Perfadex group has been set at 100%). For CXCR4 staining, data are expressed as the ratios of CXCR4 positive vs. CXCR4 negative areas per field. ANOVA followed by Bonferroni’s post-hoc multiple comparisons test.</p

Prednisolone inhibits neutrophil invasion after LTX.

<p>(<b>A</b>) Specific neutrophil staining (arrows, micrographs) and quantification (left graph). Data are expressed as number of neutrophils/field. (<b>B</b>) Measurement of tissue protein expression of neutrophil chemoattractants in transplanted lungs by protein array. Data are expressed in mean pixel densities. (<b>C</b>) Evaluation of MMP-2 mRNA expression (measured by RT-PCR, data are expressed as % of Perfadex group, Perfadex group has been set at 100%) and its Inhibitor TIMP-1 by protein analysis (protein array). (<b>D</b>) Representative protein array pictures. Calibration bar for micrographs (<b>A</b>) represents 100 µm (200 x magnification, upper micrographs) and 50 µm (400 x magnification, lower micrographs). Significance level was set to P<0.05. P<0.01: **, § § and P<0.001: § § §. * = significantly different from shams and § = significantly different from Perfadex-group. RT-PCR: experiments were performed in triplicate, protein array: pooled analysis in duplicate. ANOVA followed by Bonferroni’s post-hoc multiple comparisons test.</p