The p53 protein plays a key role in the control of the cell response to various kinds of stress, with the activation of p53 resulting in the arrest of cell proliferation and/or apoptosis.13 PFT-α, a small molecule that reversibly blocks p53-dependent transcriptional activation and apoptosis, has been shown to protect mice from side effects of cancer therapy15 and neurons from death induced by ischemic and excitotoxic insults.24 Herein we report that PFT-α reduces procaspase-3 activation and both hepatocellular apoptosis and necrosis after cold storage and reperfusion, ensuing better liver function, metabolism, and tissue integrity.
Hepatic Cold Preservation Injury.
Although their compositions markedly differ, both University of Wisconsin and HTK solution are used for cold preservation of liver allografts. In the present study, liver preservation was accomplished with HTK solution. Although originally developed as a cardioplegic solution, the use of HTK solution has been extended and covers other organs in addition to the liver.25 HTK solution is thought to be equally as appropriate as University of Wisconsin solution for liver transplantation, even if cold ischemia extends to 15 hours.25 HTK solution imparts certain advantages, in particular the ability of histidine to enter the cells, affording effective intracellular buffering and preventing detrimental pH fall. Hydrogen ion and lactate accumulation from ATP breakdown and anaerobic glycolysis are known to suppress glycolytic enzymes, preventing ATP regeneration.26 Due to continuous removal of acidic products, HTK solution promotes anaerobic glycolysis and ATP preservation.25
Our aim was to investigate the effect of transient inhibition of p53 in reperfusion injury of cold ischemic livers. Ideally, such a study should be performed in a transplantation model, but too many confounding factors are present to conclusively identify specific mechanisms. Therefore, we used an ex vivo isolated liver perfusion model with a blood cell–free perfusate. Being aware that leukocytes and platelets play an enormous role in mediating reperfusion injury,27 it was our intent to simplify the model to exclusively study the individual p53-dependent pathway of hepatocellular apoptosis.
Flushing of livers before reperfusion allowed to collect effluent fluid for determination of K+ levels and transaminase activities. In parallel with other studies,28, 29 these measures portrayed a valuable tool of predicting organ damage, as indicated by significant correlations with morphological characteristics of final tissue injury. AST levels were approximately eightfold higher than corresponding values of ALT, indicating mitochondrial injury as a probable incentive for postischemic liver dysfunction.
The analysis of apoptotic cell injury in our model is primarily based on fluorescence microscopic assessment of nuclear morphology. In a previous study, we could demonstrate that condensation and fragmentation of nuclear chromatin, as visualized by fluorescence microscopy, indeed indicates apoptosis, corresponding with established criteria of apoptosis as assessed by scanning and transmission electron microscopy.30 The fact that z-VAD-fmk was capable of reducing the number of cells that we identified as apoptotic cells—because of their characteristic changes in nuclear chromatin morphology—underlines our finding that apoptosis is one mode of cell death after cold storage and reperfusion.
In contrast to most confirmed apoptosis models in which z-VAD-fmk completely abolished apoptotic cell death, the inhibitor was only partially effective in our preservation-reperfusion model. This may be due to the fact that z-VAD-fmk was given in the preservation solution during 4°C ischemia. Although there is no information on z-VAD-fmk action in 4°C liver storage, experiments analyzing apoptosis in cryopreserved hepatocytes using z-VAD-fmk in the cryopreservation solution also demonstrated an only partial reduction of apoptosis as indicated by a 30% diminution of caspase-3-like protease activity.31
There is an ongoing discussion not only on the predominant mode of cell death (i.e., apoptosis vs. necrosis), but also on the extent of apoptosis in postischemic hepatic reperfusion. In contrast to a recent study demonstrating quantitatively irrelevant numbers of apoptotic cells after partial no-flow warm ischemia reperfusion,32 we herein show 7% apoptotic cells after 24-hour storage and 17% after an additional 2 hours of reperfusion. This supports the view that (1) apoptosis occurs already during cold preservation, as also shown by Rauen et al.33 in in vitro systems, and (2) reperfusion represents its own pathogenic entity, enhancing preservation-induced damage either by aggravating or by unmasking the injury implicated during cold ischemia. In the present model, reperfusion of cold-stored livers was accomplished through machine perfusion forcing oxygenated perfusate into the liver, which to some extent differs from the in vivo situation in which no reflow, vasoconstriction, and plugging of sinusoids might have more impact on cellular ATP depletion, thus favoring necrotic instead of apoptotic cell death.
The amount of apoptotic cell death is nicely mirrored by the procaspase-3 activation, being 1.5- to 2-fold higher at the end vs. the beginning of reperfusion. Moreover, there is a marked increase of caspase-3 processing compared with sham-operated controls. The impact of caspase-mediated apoptosis has been emphasized in studies in which liver injury after ischemia reperfusion was prevented by application of caspase inhibitors,8, 34, 35 similarly as in the present study. In parallel to apoptotic cell death, cell membrane damage, as assessed by trypan blue uptake, occurred in dependency to storage time, ranging between 20% and 60%. Thus, in contrast to warm hepatic ischemia reperfusion injury, which is thought to preferentially occur through oncotic necrosis,32 both modes of cell death seem to substantially contribute to liver damage upon cold ischemia reperfusion. This is in line with the view of others, indicating that by inducing mitochondrial permeability transition, ischemia reperfusion causes both apoptosis and necrosis.36 In fact, apoptosis and necrosis may not be unrelated as initially thought, but rather may share common events, resulting in “necrapoptosis” or “aponecrosis.”37, 38 In necrapoptosis, mitochondrial permeability transition initiates a chain reaction that culminates in either apoptosis or necrosis, possibly depending on ATP supply.36 Rapid and complete cellular ATP depletion may direct cells toward necrosis, while apoptotic signaling may proceed if ATP depletion is delayed (cold ischemia) or restored (reperfusion). According to the definition of Lemasters,37 the mixture of apoptotic and necrotic cell death after cold preservation and reperfusion may represent a typical necrapoptotic response.
In addition, it should be kept in mind that cell membrane damage is characteristic not only of primary necrotic cell death, but also of secondary apoptosis. In this case, a considerable number of trypan blue–positive cells may have undergone apoptosis, which is supported by our result that apoptotic cell death, as determined by fluorescence microscopy and hematoxylin-eosin histomorphology, significantly correlated with trypan blue uptake. The observation that the majority of trypan blue uptake was located periportally might be due to the fact that ischemia reperfusion–induced sinusoidal perfusion failure is known to occur preferentially in periportal and midzonal segments,39 resulting in rapid ATP depletion and thus more pronounced cell death.
PFT-α and Hepatic Cold Preservation Injury.
In the present study, the 20-μM dose of PFT-α was chosen because it has demonstrated selective inhibition of p53 transcriptional activity and thus prevention of DNA damage–induced apoptosis.15 We further supplemented the preservation solution with PFT-α because this avoids special pretreatment of organ donors and can easily be done by the transplant surgeon, thus representing an attractive tool in clinical practice. A variety of in vivo studies have shown that PFT-α mediates antiapoptotic properties,24, 40–42 disproving the concern that the herein observed effect of PFT-α is specific for a buffer-perfused isolated tissue.
Being aware that the mechanisms of hepatic cold ischemia reperfusion injury are multifactorial,43 characteristic triggers (e.g., ATP depletion, hypoxia, acidosis, reactive oxygen species, and cytokines) are known to cause p53 activation with execution of p53-dependent cell apoptosis.44 Because apoptotic cell death may precede necrosis6 and aggravate the inflammatory response,45 targeting the apoptotic pathway using PFT-α in the preservation solution may constitute a valid strategy against cold storage–induced organ injury. This view may also explain why PFT-α–treated livers exhibited reductions in both apoptotic and necrotic cell death, finally resulting in improved hepatocellular metabolism and excretory function.
Others have shown that PFT-α does not alter phosphorylation or sequence-specific DNA binding of p53, but slightly lowers the levels of nuclear and not cytoplasmic p53.15 In line with this, we could recently confirm that PFT-α affects the nuclear/cytoplasmic ratio, thereby promoting antiapoptotic signals with proliferation and enhancement of wound healing.41 Assuming that this is not the only mechanism of PFT-α action, reduced activation of procaspase-3 in PFT-α–treated livers implies that antiapoptotic properties of PFT-α include the downstream caspase cascade. However, the connection between p53 and the caspase cascade is only beginning to be understood44 and is beyond the scope of this study.
In conclusion, we show that reduction of hepatocellular apoptosis and necrosis by targeting p53 using PFT-α causes a favorable effect on overall graft quality, as indicated by lower enzyme and K+ release as well as higher O2 consumption and bile flow upon reperfusion. Thus p53-targeting agents such as PFT-α may serve as a novel therapeutic adjuvant to improve liver preservation in hepatic transplantation.