HDACi Valproic Acid (VPA) and Suberoylanilide Hydroxamic Acid (SAHA) Delay but Fail to Protect against Warm Hepatic Ischemia-Reperfusion Injury

<div><p>Background</p><p>Histone deacetylases (HDAC) catalyze N-terminal deacetylation of lysine-residues on histones and multiple nuclear and cytoplasmic proteins. In various animal models, such as trauma/hemorrhagic shock, ischemic stroke or myocardial infarction, HDAC inhibitor (HDACi) application is cyto- and organoprotective and promotes survival. HDACi reduce stress signaling, cell death and inflammation. Hepatic ischemia-reperfusion (I/R) injury during major liver resection or transplantation increases morbidity and mortality. Assuming protective properties, the aim of this study was to investigate the effect of the HDACi VPA and SAHA on warm hepatic I/R.</p><p>Material and Methods</p><p>Male Wistar-Kyoto rats (age: 6–8 weeks) were randomized to VPA, SAHA, vehicle control (pre-) treatment or sham-groups and underwent partial no-flow liver ischemia for 90 minutes with subsequent reperfusion for 6, 12, 24 and 60 hours. Injury and regeneration was quantified by serum AST and ALT levels, by macroscopic aspect and (immuno-) histology. HDACi treatment efficiency, impact on MAPK/SAPK-activation and Hippo-YAP signaling was determined by Western blot.</p><p>Results</p><p>Treatment with HDACi significantly enhanced hyperacetylation of Histone H3-K9 during I/R, indicative of adequate treatment efficiency. Liver injury, as measured by macroscopic aspect, serum transaminases and histology, was delayed, but not alleviated in VPA and SAHA treated animals. Importantly, tissue destruction was significantly more pronounced with VPA. SAPK-activation (p38 and JNK) was reduced by VPA and SAHA in the early (6h) reperfusion phase, but augmented later on (JNK, 24h). Regeneration appeared enhanced in SAHA and VPA treated animals and was dependent on Hippo-YAP signaling.</p><p>Conclusions</p><p>VPA and SAHA delay warm hepatic I/R injury at least in part through modulation of SAPK-activation. However, these HDACi fail to exert organoprotective effects, in this setting. For VPA, belated damage is even aggravated.</p></div

VPA and SAHA reinforce I/R-mediated hyperacetylation of Histone H3.

<p><b>A)</b> Time-schedule of the performed experiments. VPA: valproic acid; SAHA: suberoylanilide hydroxamic acid; DMSO: vehicle dimethylsulfoxide. <b>B)</b> Western blot for acetylated Lysine 9 of Histone H3. β-actin served as loading control. Representative blots for 6h and 24h of reperfusion are shown. Bar graphs depict quantification of at least four experiments. NI: non-ischemic right lobes; I: ischemia-reperfusion left lobes. ***: p<0.001; **: p<0.01.</p

VPA and SAHA modulate I/R-dependent activation of p38- and JNK-signaling.

<p><b>A)</b> Representative Western blots for (phospho-)JNK (T183/T185) and (phospho-)p38 (T180/Y182). β-actin served as loading control. <b>B)</b> Densitometric blot-quantifications for indicated treatment groups and reperfusion periods of at least three experiments. NI: non-ischemic right lobes; I: post-ischemic I/R left lobes. **: p<0.01; *: p<0.05.</p

Impact of HDAC-inhibition on regeneration.

<p><b>A)</b> Ki67 immunohistochemistry. Representative micrographs of non-damaged and damaged areas in sections of post-ischemic left liver lobes and of non-ischemic control lobes from animals treated as indicated. Scale bars: 200μm. <b>B)</b> Quantification of Ki67-positive hepatocytes in I/R unaffected areas. At least 20 HPFs of areas without massive inflammatory infiltrates and scar tissue were counted per slide. <b>C)</b> Representative Western blots with (phospho-)YAP (S127) antibodies and <b>D)</b> Graphs of densitometric quantifications of at least three experiments. Values were set in relation to the samples of sham treated animals. <b>E)</b> YAP immunohistochemistry. Representative micrographs of non-damaged and damaged areas in sections of post-ischemic left liver lobes and of non-ischemic right control lobes from animals treated as indicated. White arrows indicate nuclear staining in the images for non-ischemic lobes and the non-damaged areas of post-ischemic lobes. Arrows were omitted in images for damaged areas of post-ischemic lobes. Scale bars: 100μm. <b>F)</b> Quantification of nuclear YAP-positive hepatocytes. At least 20 HPFs were counted per slide. NI: non-ischemic right lobes; I: post-ischemic I/R left lobes. **: p<0.01; *: p<0.05. AUC: area under the curve.</p

VPA- and SAHA-treatment delays I/R-damage but fails to exert protective properties.

<p><b>A)</b> Serum levels of aspartate (AST) and alanine (ALT) aminotransferases at different time points. <b>B)</b> Representative H/E micrographs of left lateral liver lobe sections from animals treated as indicated. On the images with lower magnification (x40, scale bars 200μm) damaged areas are encircled. The images with higher magnification (lower row of 6h reperfusion; x400, scale bars 20μm) depict sinusoidal jamming (star) and signs of hepatocyte cell death like karyopyknosis (thick arrow), karyorrhexis (arrowhead), karyolysis (fine arrow) and loss of nuclei (raindrop) for all treatment options as indicated. <b>C)</b> Histological quantification of tissue damage. One whole representative horizontal section per animal was analyzed. Percentage of damaged area (includes: sinusoidal jamming, cell death/scar and inflammatory infiltrates) is given. <b>D)</b> Representative photographs of whole liver (top row), and dissected left lateral and left part of median liver lobe (bottom row) of animals subjected to the indicated treatments and sacrificed at indicated timepoints. <b>E)</b> Quantification of I/R-damage by macroscopic aspect of the left lateral liver lobe. Integrated volume of necrosis/scar was set in relation to the calculated total volume of the lobe. *: p<0.05. TTP: time to peak; AUC: area under the curve.</p