Secondary Logo

Journal Logo

Hepatocellular Heme Oxygenase-1: A Potential Mechanism of Erythropoietin-Mediated Protection After Liver Ischemia-Reperfusion Injury

Riehle, Kimberly J.†‡; Hoagland, Vicki*†; Benz, Whitney*†; Campbell, Jean S.; Liggitt, Denny H.§; Langdale, Lorrie A.*†

doi: 10.1097/SHK.0000000000000231
Basic Science Aspects
Free
Editor's Choice

ABSTRACT Hepatic ischemia-reperfusion (IR) results in progressive injury; initiated by oxidative stress during ischemia and compounded by cytokine-mediated inflammation during reperfusion. Recovery requires strict regulation of these events. Recombinant human erythropoietin (rhEPO) is thought to mitigate hepatocellular IR injury by altering the nonparenchymal liver microenvironment. This study sought to identify additional mechanisms whereby rhEPO is protective after liver IR injury. Mice were treated with rhEPO (4 units/g s.c.) at the onset of partial liver ischemia and assessed for transaminase and histologic injury at intervals after reperfusion. Induction of cytokines, activation of signal transducers and activators of transcription (STATs), suppressors of cytokine signaling (Socs1, Socs3, Cis), caspase-3 activation, and heme oxygenase-1 (HO-1) expression were assessed in postischemic liver. Effects of rhEPO stimulation were further characterized in whole-liver lysates from mice undergoing rhEPO injection alone and in cultured AML-12 hepatocytes. Recombinant human erythropoietin treatment at the onset of severe (90 min) hepatic IR confirmed commensurate biochemical and histological protection without affecting tissue cytokine levels. Although Socs3 and STAT5 activation were induced in normal liver after in vivo rhEPO injection, this treatment did not augment expression beyond that seen with IR alone, and neither was induced in cultured hepatocytes treated with rhEPO. Recombinant human erythropoietin inhibited caspase-3 activation in nonparenchymal cells, whereas hepatocellular HO-1 was rapidly induced both in vivo and in vitro with rhEPO treatment. These data suggest HO-1 as a potent mechanism of rhEPO-mediated protection after liver IR, which involves both direct hepatocellular and nonparenchymal mechanisms.

*Department of Surgery, Veterans Administration; and Departments of Surgery, Pathology, and §Comparative Medicine, University of Washington, Seattle, Washington

Received 30 Apr 2014; first review completed 16 May 2014; accepted in final form 26 Jun 2014

Address reprint requests to Lorrie A. Langdale, MD, Department of Surgery, Veterans Administration-Puget Sound Health Care, Bldg 1, Rm 314, 1660 South Columbian Way, Seattle, WA. E-mail: lorrie@u.washington.edu.

This study was supported by VA Merit Review 20559 (to L.A.L.), by NIH CA-127228 (to J.S.C.), by the Herbert Coe Foundation (to K.J.R.), by the American College of Surgeons Louis C. Argenta Fellowship, and by the American Surgical Association Foundation.

Back to Top | Article Outline

INTRODUCTION

Ischemia-reperfusion (IR) injury is the primary driver of acute liver dysfunction after elective liver resection and prolonged hemorrhagic shock. The initial hypoperfusion phase of any IR injury, including liver IR, is characterized by tissue hypoxia and cellular oxidative stress (1). Reperfusion initiates a complex chain of cytokine-mediated events that culminate in a distinct neutrophil-mediated “reperfusion injury.” If unchecked, this second phase of injury is dominated by tissue necrosis and sets the stage for a systemic response and remote organ failure, particularly when the primary ischemic injury is severe. Despite advances in critical care, a failing liver is difficult to support, short of transplantation. Although strategies to improve outcomes in the acute setting have aimed at blocking individual components of reperfusion injury, to date, none have translated to clinically relevant therapies. Aside from limiting the period of ischemia, or preconditioning with brief periods of portal occlusion (2), few strategies have focused on mitigating the oxidative stress phase of injury to improve tolerance of acute IR.

Erythropoietin (EPO) is a glycoprotein hormone vital to the differentiation of committed erythroid progenitor cells. A variety of nonhematopoietic properties of EPO have also been identified, suggesting additional potential clinical applications. Exogenous recombinant human EPO (rhEPO) has been shown to be protective after ischemia in a variety of tissues, including brain, heart, kidney, and liver (3,4). This protection has been observed in animal models using both preinjury and postinjury treatment strategies and has generally been attributed to induction of antiapoptotic mechanisms such as Bcl2 and Bclx. In addition, as is the case for a number of inflammatory mediators associated with acute hepatic IR injury, EPO signals through the signal transducer and activator of transcription (STAT) signaling pathway. Signal transducers and activators of transcription have been shown to regulate inflammatory cytokine function through induced expression of suppressor of cytokine signaling proteins (SOCS) in hematopoietic cell lines (5). It may therefore be postulated that rhEPO mitigates IR injury through both antiapoptotic and anti-inflammatory mechanisms.

The hepatocellular protective effect of rhEPO treatment in liver IR has been reported as being virtually entirely indirect through effects on nonparenchymal cells (NPCs) (6). We present studies of rhEPO protection after hepatic IR injury that challenge this belief. Postischemia treatment with rhEPO resulted in sustained protection after IR. Although rhEPO induces SOCS3 and STAT3 anti-inflammatory mechanisms in both injured and control whole livers, this induction did not occur in cultured hepatocytes treated with rhEPO and thus may require NPCs. By contrast, heme oxygenase-1 (HO-1), which is known to serve both anti-inflammatory and antiapoptotic functions in the face of liver injury, is induced by rhEPO in both whole liver and in cultured hepatocytes. These data indicate that rhEPO protection after hepatic IR can be attributed to a spectrum of both parenchymal (i.e., hepatocytic) and NPC mechanisms.

Back to Top | Article Outline

MATERIALS AND METHODS

Mice

All animal protocols were approved by the Institutional Animal Care and Use Committees of the Veterans Affairs and the University of Washington. C57/Bl6 male mice, free of Helicobacter species and 6 to 8 weeks of age, were purchased (Jackson Labs, Maine) and allowed to acclimate to their environment for 7 days before utilization in the experiments. All animals were housed in a specific pathogen-free facility, fed standard rodent chow with water ad libitum, and subjected to 12-h day/night light cycles.

Back to Top | Article Outline

Surgical preparation and sampling

Nonfasted mice underwent in situ partial liver ischemia followed by reperfusion (eight per group) under isoflurane anesthesia between 8:00 am and noon. Through a midline incision, an atraumatic vascular clip was applied to the porta hepatis such that the cephalad (median and left) lobes were ischemic while the posterior (right) and caudate lobes remained continuously perfused as previously described (7,8). Immediately after vascular clip placement, rhEPO (4 units/g in PBS; Amgen: commercial stock of 2,000 units/mL in solution containing 0.11 mg citric acid, 0.5 mg human albumin, 1.3 mg sodium citrate, 8.2 mg sodium chloride, 1% benzoyl alcohol preservative per milliliter) or an equivalent volume of PBS was s.c. administered. Animals remained under anesthesia on a heating pad at 39°C, with the abdomen temporarily closed to minimize fluid losses during the period of ischemia. After 90 min of ischemia, the clip was removed and reperfusion was visually confirmed. The abdomen was closed after infiltration of the wound with 0.25% marcaine, and the recovered animals were allowed ad libitum access to food and water. Animals undergoing laparotomy without vascular occlusion for similar periods of anesthesia served as sham-operated controls. All animals demonstrated normal activity and eating patterns by 24 h.

Cohorts of animals were euthanized 0.5, 1, 2, 4, or 24 h after reperfusion. At the time of CO2 inhalation euthanasia, serum was collected by cardiac puncture and stored at -80°C for serum aspartate aminotransferase (AST) and alanine transaminase (ALT) kinetic assay analysis (Sigma-Aldrich, St. Louis, Mo). Liver from sham, ischemic, and continuously perfused lobes was preserved in 10% formalin for histology or snap frozen in liquid nitrogen and stored at -80°C for future RNA and protein analyses. An additional cohort was treated with s.c. rhEPO (no surgery) and euthanized 30, 60, 90, 210, or 330 min after injection to determine the kinetic effects of rhEPO injection on normal liver (four mice per time point). These postinjection time points were chosen to correspond to major time points in the IR protocol: 90 min, severe ischemia time; 210 min, 2-h reperfusion; 330 min, 4-h reperfusion.

Back to Top | Article Outline

Cultured hepatocyte studies

AML-12 cells were plated and cultured as described (9) and treated with rhEPO (2 units/mL) after overnight serum starvation. Triplicate samples were harvested in Triton-X lysis buffer at the indicated time points and subjected to immunoblotting as described below.

Back to Top | Article Outline

RNA preparation and real-time polymerase chain reaction analysis

Total RNA was prepared from frozen liver tissue using Trizol/chloroform extraction according to the manufacturer’s recommendations (Invitrogen, Carlsbad, Calif). Total RNA purity and concentration were determined using a spectrophotometer (BioRad, Hercules, Calif). Wavelength ratios (260:280) between 1.7 and 2.0 indicated acceptable RNA quality. To confirm RNA integrity, 1-μg RNA samples were electrophoresed on 1% agarose/ethidium bromide gels.

DNase purified total RNA from ischemic, perfused, and sham-treated liver was reverse transcribed into cDNA (Invitrogen) and subjected to quantitative real-time polymerase chain reaction (RT-PCR) using FAM-labeled primers for Socs1, Socs3, and Cis on ABI Prism 7000 instrumentation, with reagents and software from Applied Biosystems (Foster City, Calif). Delta delta Ct (ΔΔCt) values were calculated by subtracting Ct values for the gene of interest from Ct values for β-actin (housekeeping gene) and then subtracting the ΔCt value obtained for each gene from nonoperated control mice. Fold change was calculated by normalizing all values to those of nonoperated wild-type mice.

Back to Top | Article Outline

Protein isolation, caspase analysis, and immunoblotting

Ischemic liver tissue samples were homogenized in Triton-X lysis buffer containing protease inhibitors as previously described (10). Protein concentrations were determined using Bradford analysis with bovine serum albumin as a standard. Caspase-3 activity in liver homogenates was determined using a fluorogenic substrate as previously described (11). Normalized lysate aliquots were stored in sample buffer at -80°C for future immunoblot analyses.

Tissue or cell lysate samples (30 – 50 μg) diluted in sample buffer were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Amersham Biosciences, Piscataway, NJ). Immunoblotting was performed using primary antibodies to phospho-STAT3, total STAT3, phospho-STAT5, total STAT5, β-actin (all Cell Signaling, Danvers, Mass), or HO-1 (Assay Designs). Membranes were incubated for 1 h at room temperature in appropriate horseradish peroxidase secondary antibodies, and signals were detected using ECL Western Blotting Detection Reagent (Amersham). Autoradiographic films representing each phospho- and total STAT3 blot were scanned, and NIH image analysis (version X; National Institutes of Health) was used to quantify the density of the appropriate bands.

Back to Top | Article Outline

Luminex protein analysis

Whole-liver lysates were tested for TNF-α, IL-1β, IL-4, IL-6, and IL-10 by the laboratory of James Lederer, PhD (Brigham and Women’s Medical Center, Boston, Mass) on a custom-made Luminex multiplex cytokine detection bead assay platform using a Luminex 200 instrument (Luminex Corporation, Austin, Tex). These Luminex bead multiplex cytokine assays have a detection sensitivity range of 5 to 25,000 pg/mL and were performed using 200 μL of cleared sample. Results were calculated using StarStation 3.0 Software (Applied Cytometry, Sheffield, UK).

Back to Top | Article Outline

Histologic scoring and Immunohistochemistry

Formalin-fixed samples from the right, middle, and left lobes of the ischemic liver were analyzed for injury severity by a Comparative Medicine pathologist, who was blinded to treatment groups. Samples were scored based on calculation of the area occupied by necrotic tissue in the following manner: images of all histologic sections of liver in perfused and control groups were captured using a Nikon Digital Sight D5-Fi1 camera and Nikon 80i microscope; surface areas occupied by the entire liver and individual or confluent necrotic areas within the liver were each digitally outlined using the area function of NIS-Elements Software (Nikon Instruments). The percentage of each liver section occupied by necrotic tissues was calculated using these data, and a severity score ranging from 0 (normal) to 4+ (15% or greater of combined liver sectional area affected) was constructed. Immunohistochemistry (IHC) for cleaved caspase-3 and HO-1 was performed using standard protocols and primary antibodies from Biocare Medical (cleaved caspase 3, catalog number CP 229B) and Assay Designs (HO-1, catalog number SPA-895).

Back to Top | Article Outline

Statistical analysis

Data are expressed as means ± SEM. Two-way analysis of variance and Bonferroni-Dunn post hoc testing were performed using GraphPad Prism 4.00 for Windows (San Diego, Calif). Values for each ischemic condition at intervals after reperfusion were compared, with significant differences accepted for values of P ≤ 0.05.

Back to Top | Article Outline

RESULTS

EPO is protective in hepatic IR

Recombinant human erythropoietin treatment before the onset of ischemia results in hepatic protection after mild or moderate IR (12,13). More severe degrees of ischemia followed by reperfusion have not been investigated. We confirmed rhEPO protection against IR injury by performing partial liver IR as described (14), with or without administration of s.c. rhEPO at the time of ischemia. Recombinant human erythropoietin treatment significantly decreased AST (Fig. 1A) and ALT levels (Fig. 1B) 4 h after severe (90 min) IR. Liver lobes subjected to 90 min of ischemia showed extensive areas of coagulative necrosis with hemorrhage and neutrophil infiltration, but rhEPO treatment at the onset of ischemia resulted in a comparatively less severe injury at both 4 and 24 h reperfusion (representative sections shown in Fig. 1C). Calculation of the percentage of necrotic parenchyma in sections of the right, middle, and left ischemic lobes from treated and untreated animals confirmed that rhEPO treatment decreased the severity of histologic injury by approximately 60%. Liver from perfused and sham-treated lobes had normal histology with or without rhEPO treatment (data not shown). The mean severity score in untreated mice was 3.25+ (8/8 section affected) compared with a mean score of 1.7+ (7/10 sections affected) for those mice receiving rhEPO treatment. These data support the hypothesis that rhEPO-mediated hepatocellular protection after IR occurs early after reperfusion.

Fig. 1

Fig. 1

Back to Top | Article Outline

rhEPO injection induces hepatic JAK-STAT and SOCS signaling mechanisms

Erythropoietin is known to activate JAK-STAT signaling and induce Socs expression in hematopoietic cells (15), suggesting a mechanism for modulation of activated inflammation after reperfusion. We first examined these pathways after rhEPO injections alone (without IR) to determine whether these mechanisms are induced in whole liver and thus may contribute to rhEPO-induced protection after IR. Using time points similar to harvest points after reperfusion in our IR protocol, we characterized the kinetics of rhEPO-induced whole-liver activation of phospho-STAT5, which peaked at 210 min (Fig. 2A), and phospho-STAT3, which was not significantly induced. Densitometric analyses confirmed statistically significant activation of STAT5 (Fig. 2B) but not of STAT3 (Fig. 2C). We then assayed expressions of Cis, Socs1, and Socs3 during the same time course of rhEPO treatment. Induction of Socs3 mRNA predominated, peaking within 90 min and declining to near basal levels during the ensuing 4 h (Fig. 2D). Induction of Socs1 was comparatively delayed, and Cis induction appeared to be biphasic, with an early peak at 90 min and a second at 330 min.

Fig. 2

Fig. 2

Back to Top | Article Outline

Expression of inflammatory mediators in ischemic liver is unaffected by rhEPO

Given our findings that rhEPO injection leads to activation of STAT5 and Socs3, we returned to our murine IR model to determine whether similar mechanisms are involved in the protection from IR by rhEPO. Similar to after injection alone, we found that IR + rhEPO lead to not only an increased activation of STAT5 (\A) but also an earlier activation of STAT3 (Fig. 3B) compared with IR alone. Similar to after rhEPO injection alone, Socs3 was the predominant SOCS family member induced after IR with rhEPO treatment (Fig. 3C). Given the activation of the STAT-Socs pathway, we investigated whether rhEPO’s protective effects reflected altered inflammatory mediator expression. Interestingly, Luminex assay of ischemic liver homogenates from rhEPO-treated and untreated mice showed similar induction of both proinflammatory and anti-inflammatory cytokines (TNF-α, IL-1β, IL-6; IL-4, IL-10) both 1 h (data not shown) and 4 h after reperfusion (Fig. 3D). These data indicate that altered cytokine expression is not a primary mechanism underlying rhEPO protection during liver IR.

Fig. 3

Fig. 3

Back to Top | Article Outline

Antiapoptotic effects of rhEPO after liver IR

Given that we could not identify significant changes in cytokine expression after IR because of rhEPO treatment, and that our findings of differential activation of STATs and Socs3 were subtle, we investigated effects of rhEPO on alternative protective pathways after IR. Previous investigators have suggested that inhibition of apoptosis within minutes of reperfusion is a dominant mechanism responsible for rhEPO-mediated protection after IR injury (12,13). We first performed IHC for activated caspase-3 in post-IR livers. As shown in Figure 4A, caspase-3 activity in untreated mice was most prominent in NPCs immediately adjacent to areas of patchy necrosis. Immunohistochemistry of rhEPO-treated ischemic liver demonstrated that this treatment abrogated the apoptotic response (Fig. 4B). To better quantify levels of apoptosis, activated caspase-3 levels were assayed using a fluorogenic assay 2, 4, and 24 h after reperfusion. As shown in Figure 4C, caspase-3 was activated at multiple time points after the 90-min IR, and rhEPO treatment led to a significant reduction in caspase-3 activation throughout the examined period of reperfusion. This suggests that the caspase-mediated antiapoptotic effects of rhEPO are in NPCs at the margins of injury, and that its contribution to parenchymal protection reflects an indirect limitation of local hepatocellular loss.

Fig. 4

Fig. 4

Back to Top | Article Outline

Protective mechanisms of rhEPO in hepatocytes in culture

Because both hepatocytes and NPCs use STAT5 and SOCS3 in the regulation of cytokine-signaling, EPO-mediated expression could reflect induction in either cellular compartment. Previous authors have reported a lack of JAK-STAT signaling induction by rhEPO in either whole rat liver or isolated hepatocytes, despite the constitutive expression of rhEPO receptor mRNA (6). However, given our in vivo observations, we reassessed those findings by evaluating effects of rhEPO on cultured hepatocytes. AML12 cells were cultured and stimulated with rhEPO, then assayed for STAT5 (Fig. 5A), STAT3 (Fig. 5B), and Socs induction (Fig. 5C) at various time points thereafter. These pathways indeed were not significantly induced by rhEPO treatment of cultured hepatocytes. These data suggest that the induction of STAT5 and Socs3 by rhEPO that we detected in whole liver is likely to require NPCs in vivo, and that, like caspase-3 limitation of apoptosis, hepatocellular protection via such mechanisms is indirect.

Fig. 5

Fig. 5

Back to Top | Article Outline

Hepatocellular induction of HO-1 by rhEPO

Ischemia-reperfusion injury and its progression can be curtailed through modulation of hepatocellular oxidative stress. Reduction of oxidative stress through induction of HO-1 has shown significant cytoprotection in models of acute and chronic liver damage, including IR injury (16,17). We found that rhEPO treatment of AML12 cells did induce HO-1 expression by immunoblotting, as shown in Figure 5D. This HO-1 induction in isolated hepatocytes provides evidence that, even in the absence of ischemic injury, rhEPO is capable of directly stimulating hepatocytes.

After determining that rhEPO could stimulate HO-1 expression in cultured hepatocytes, we returned to our model of rhEPO-treated severe hepatic IR. We performed IHC for HO-1 4 h after IR and confirmed that there is minimal expression of hepatocyte HO-1 in untreated mice undergoing IR (Fig. 6A). As shown in Figure 6B, rhEPO treatment at the time of IR injury leads to high levels of HO-1 expression in hepatocytes. Immunoblotting for HO-1 demonstrated that rhEPO induces significant HO-1 expression in lysates from ischemic liver after 90 min of ischemia followed by 4 h of reperfusion (Fig. 6, C and D). In summary, these data suggest that the abrogation of liver IR by rhEPO treatment is not limited to paracrine effects originating in NPCs as has previously been asserted. Rather, direct stimulation of hepatocyte HO-1 provides an additional potent mediator resulting in rhEPO-based parenchymal protection.

Fig. 6

Fig. 6

Back to Top | Article Outline

DISCUSSION

Although inflammation is a normal response to acute injury and crucial to injury resolution, it can also lead to unintended consequences. The events leading to injury after liver IR have been well described (18) and include the generation of reactive oxygen species during portal vascular occlusion and activation of the complement cascade. Activated Kupffer cells produce a spectrum of inflammatory cytokines, including IL-1β, IL-10, and TNF-α, which activate adjacent hepatocytes and endothelial cells. Activated hepatocytes in turn amplify the initial Kupffer cell response by expressing and releasing additional mediators (e.g., IL-6) to perpetuate injury through activation of neighboring cells. Neutrophils recruited during reperfusion adhere to the sinusoidal endothelium, contributing to microcirculatory vasoconstriction and setting the stage for injury progression to tissue necrosis. Control of injury and return to homeostasis hinge on a dynamic process of continuous subtle rebalancing of inflammatory signals across the hepatic microenvironment. Given the complexity of this system, the entire spectrum of inflammatory responses associated with IR must be highly regulated. Successful therapeutic strategies will need to impact more than one aspect of injury.

Erythropoietin is a glycoprotein hormone vital for the differentiation of committed erythroid progenitor cells. The recombinant human formulation has been used safely for many years as an effective means of recovering red blood cell mass, but nonhematopoietic functions of rhEPO have become increasingly apparent. Corwin and colleagues (19) reported that early treatment with rhEPO provides a survival advantage for trauma patients independent of hematopoietic impact. Studies in laboratory animal models of injury, including ours, further support this observed effect on outcome.

Our data offer further insight into rhEPO-mediated hepatic IR protection on several levels. We have shown that rhEPO-mediated protection is effective when treatment occurs at the onset reperfusion—when the primary effects of the ischemic injury phase are established. Alterations in cytokine expression have long been assumed to be a primary mechanism contributing to IR protection. We have shown that this is not the case for early reperfusion injury protection mediated through rhEPO. Our focus in this study was to explore early mechanisms that might further enhance our understanding of how rhEPO might be exploited in the management of IR injury.

Back to Top | Article Outline

EPO and apoptosis

To date, the protective nonhematopoietic effects of rhEPO in preinjury and/or postinjury treatment models of IR in a variety of tissues (3,4) have been largely attributed to upregulation of antiapoptotic genes (20–22). Pretreatment with rhEPO has been shown to ameliorate hepatic IR injury; inhibition of apoptosis has been cited as a primary mechanism of this protection (23). Warm hepatic IR leads to necrotic cell death (oncosis) and often occurs within minutes of reperfusion. Because the endogenous tissue receptor for EPO is upregulated by hypoxia (20–22), circulating rhEPO might be expected to have a rapid and intense effect on ischemic liver injury soon after reperfusion.

Although studies have suggested that apoptosis contributes to the early evolution of IR injury, strict interpretation of TUNEL assays leads to the conclusion that no more than 2% of the liver cells at risk are undergoing apoptosis (24). Nevertheless, caspase-dependent signaling pathways have been proposed as potential targets of rhEPO protection in IR (25,26). Although our data confirm that rhEPO significantly diminishes caspase activity initiated by IR, IHC localizes this alteration to NPCs (likely Kupffer cells) rather than hepatocytes. Given the relatively small number of Kupffer cells in the liver parenchyma, this cellular location would account for the low-level total caspase activation in our IR controls. These data indicate that rhEPO’s primary antiapoptotic effects after liver IR are centered on NPCs. We thus questioned whether additional mechanisms might be contributing to its protection in hepatic IR.

Back to Top | Article Outline

EPO signaling in liver

Like other inflammatory mediators associated with hepatic IR, rhEPO signals through the JAK-STAT pathway, predominantly through STAT5, but has been shown to induce SOCS proteins in erythroid cell lines (5). Induction of these potent regulators of inflammation might be expected to contribute to tissue protection through mitigation of the cytokine-mediated reperfusion injury. We have previously shown that expression levels of Socs1 and Socs3, signaling through STAT3, parallel the severity of hepatic IR injury (14). Because induction of mechanisms that regulate cytokine signaling is integral to the response to IR, these mechanisms might be treatment targets in the management of acute IR injury and recovery. The contribution of Socs gene induction to rhEPO-mediated protection after hepatic IR has not previously been explored.

Several models of IR have used rhEPO pretreatment strategies, and our data suggest that rhEPO has an effect on liver parenchyma in the absence of hypoxic injury. We have shown that Socs genes are induced in normal liver within 2 h of rhEPO injection, and that Socs3 is the dominant family member. To determine whether STAT/Socs activation is in NPCs or hepatocytes, we stimulated cultured hepatocytes with rhEPO and found that it failed to induce expression of either STAT3 or STAT5 in these cells (Figs. 5, A and B). These findings affirm that the contribution of JAK-STAT signaling associated with rhEPO-mediated protection in hepatic IR is through the NPC.

We hypothesized that early NPC STAT3 activation by rhEPO would rapidly induce sufficient Socs3 in whole liver to interrupt cytokine-mediated inflammation and inhibit reperfusion injury, even if rhEPO was injected after the onset of ischemia. Although rhEPO did induce early STAT3 phosphorylation in ischemic liver after IR when compared with untreated mice, circulating and whole-liver lysate levels of a spectrum of proinflammatory and anti-inflammatory mediators were unaffected by rhEPO treatment. These data suggest that induction of JAK-STAT regulatory mechanisms may not be the dominant feature of rhEPO-mediated protection after severe IR.

Back to Top | Article Outline

EPO, HO-1, and liver injury

Formation of reactive oxygen species and oxidative stress are central to the injury invoked by hepatic IR (1,27). The HO system and its role in the control of oxidative stress and subsequent inflammation have been the focus of considerable interest for more than a decade. Heme oxygenase-1 in particular has been identified as a potent defense mechanism induced by a varied of insults, including hypoxia, endotoxin, cytokines, heat shock, and shear stress in a variety of tissues and disease states (Wunder and Potter, 2003). HO-1 is a highly conserved molecule; its loss is characterized by growth retardation, susceptibility to oxidative stress, and chronic inflammation, particularly in liver and kidney (17). Chronic overexpression in the liver, with its attendant impaired apoptosis, has been associated with the development of hepatocellular carcinoma. Interestingly, Geuken et al have reported a range of HO-1 induction in human livers from brain-dead multi-organ donors. They found that those with initially low HO-1 expression showed significantly greater HO-1 induction during reperfusion, less injury, and improved overall function when compared with grafts expressing high levels of HO-1 prior to IR (28). This suggests that while the capacity for HO-1 induction in liver may be limited, a regimen that offers a controlled microenvironmental shift in HO-1 expression could offer a key to protection from hepatic injury.

The two isozymes of the heme oxygenase system, HO-1 and HO-2, show distinct topographical patterns of expression in normal and stressed liver (29). Heme oxygenase-2 is most abundant within hepatocytes, sinusoidal endothelial cells, and hepatic stellate (Ito cells), whereas Kupffer cells express both isoforms. In addition to redox pathways (Keap1/Nrf2, transcription repressor Bach1, AP-1, and NF-κB), multiple cell signaling pathways contribute to HO-1 gene regulation, including the omnipresent p38 MAPK, PI3K/Akt, TLR-4, IL-10, and JAK-STAT pathways (30). Heme oxygenase-1 is induced in stressed hepatocytes as a defense mechanism, but the majority of treatment strategies involving HO-1 have targeted Kupffer cell signaling, assuming hepatocyte protection to be the result of paracrine effects.

Luo et al. (31) previously described an association between rhEPO protection and HO-1 induction in whole rat liver after moderate liver IR (45 min) with 24 h rhEPO pretreatment. Our data build on these earlier observations, showing that protection by rhEPO is sustained in the face of a much more severe IR injury (90 min), even when reEPO treatment is initiated at the time of reperfusion. We have shown that rhEPO is a strong inducer of hepatocyte HO-1, particularly at the interface between necrotic and normal parenchyma after severe IR injury. We have further shown rhEPO is capable of direct hepatocyte stimulation, a finding that is contrary to prior assertions. We believe that these data, taken as a whole, show that the effects of a single postischemia administration of rhEPO on liver are multifaceted, directly inducing both parenchymal and NPC antioxidative and anti-inflammatory pathways.

Although we have shown a strong induction of HO-1 in cultured hepatocytes and documented its enhanced expression in tissue, we have not performed the studies that would be necessary to confirm that HO-1 is the sole or even primary mechanism underlying rhEPO-based liver IR protection. Possible areas for further exploration might include functional silencing of HO-1 with zinc protoporphyrin IX or hepatocyte-specific knockdown of Ho1 gene expression. Microarray analysis would offer an additional approach to investigate whether other proteins may also be important to the evolution of IR injury protection with rhEPO treatment. Such studies are the subject of an ongoing investigation.

Back to Top | Article Outline

CONCLUSIONS

A number of strategies have been proposed to reduce the risk of postoperative dysfunction in marginal livers that are more vulnerable to IR injury (1,27). Therapeutic interventions that target multiple pathways will likely hold the greatest promise. Properly applied, such strategies would also expand the potential donor pool for transplantation. It is clear that the actions of EPO extend well beyond erythropoiesis. This study emphasizes the spectrum of effects mitigated by the use of rhEPO as a therapy in hepatic IR. The inclusion of rhEPO as a component of postreperfusion support offers a safe and effective means of targeting multiple pathways inherent to liver recovery.

Back to Top | Article Outline

ACKNOWLEDGMENT

The authors thank James Lederer, PhD, and his laboratory at Brigham and Women’s Medical Center, Boston, Massachusetts, for Luminex analysis.

Back to Top | Article Outline

REFERENCES

1. Jaeschke H, Woolbright B: Current strategies to minimize hepatic ischemia-reperfusion injury by targeting reactive oxygen species. Transplant Rev 26: 103–114, 2012.
2. Teoh N, Farrell G: Hepatic ischemia-reperfusion injury: pathogenic mechanisms and basis for hepatoprotection. J Gastroenterol Hepatol 18: 891–902, 2003.
3. Brines M, Cerami A: Discovering erythropoietin’s extra-hematopoietic functions: biology and clinical promise. Kidney Int 70: 246–250, 2006.
4. Ghezzi P, Brines M: Erythropoietin as an anti-apoptotic, tissue-protective cytokine. Cell Death Diff 11: S37–S44, 2004.
5. Jegalian A, Wu H: Differential roles of SOCS family members in EpoR signal transduction. J Interferon Cytokine Res 22: 853–860, 2002.
6. Bramey T, Freitag P, Fandrey J, Rauen U, Pamp K, Erhard J, Frede S, deGroot H, Petrat F: No evidence for protective erythropoietin alpha signaling in rat hepatocytes. BMC Gastroenterol 9: 26, 2009.
7. Langdale L, Wilson L, Jurkovich G, Liggitt H: Effects of immunomodulation with interferon-gamma on hepatic ischemia-reperfusion injury. Shock 11: 356–361, 1999.
8. Langdale L, Kajikawa O, Frevert C, Liggitt H: Sustained tolerance to lipopolysaccharide after liver ischemia-reperfusion injury. Shock 19: 553–558, 2003.
9. Wu J, Merlino G, Fausto N: Establishment and characterization of differentiated, non-transformed hepatocyte cell lines derived from mice transgenic for transforming growth factor alpha. Proc Natl Acad Sci USA 91: 674–678, 1994.
10. Campbell J, Prichard L, Schaper F, Schmitz J, Stephenson-Famy A, Rosenfeld M, Argast G, Heinrich P, Fausto N: Expression of suppressors of cytokine signaling during liver regeneration. J Clin Invest 107: 1285–1292, 2001.
11. Chaisson M, Brooling J, Ladiges W, Tsai S, Fausto N: Hepatocyte-specific inhibition of NF-κB leads to apoptosis after TNF treatment, but not after partial hepatectomy. J Clin Invest 110: 193–202, 2002.
12. Schmeding M, MNeumann U, Boas-Knoop S, Spinelli A, Neuhaus P: Erythropoietin reduces ischemia-reperfusion injury in the rat liver. Eur Surg Res 39: 189–197, 2007.
13. Sepodes B, Maio R, Pinto R, Sharples E, Oliveira P, McDonald M, Yagoob M, Thiemermann C, Mota-Filipe H: Recombinant human erythropoietin protects the liver from hepatic ischemia-reperfusion injury in the rate. Transpl Int 19: 919–926, 2006.
14. Langdale L, Hoagland V, Benz W, Riehle K, Campbell J, Liggitt H, Fausto N: Suppressor of cytokine signaling expression with increasing severity of murine hepatic ischemia-reperfusion injury. J Hepatol 49: 198–206, 2008.
15. Sarna M, Ingley E, Busfield S, Cull V, Lepere W, McCarthy D, Wright M, Palmer G, Chappell D, Syer M, Klinken S, Tilbrook P: Differential regulation of SOCS genes in normal and transformed erythroid cells. Oncogene 22: 3221–3230, 2003.
16. Sass G, Barikbin R, Tiegs G: The multiple functions of heme oxygenase-1 in the liver. Z Gastrolenterol 50: 34–40, 2012.
17. Wunder C, Potter R: The heme oxygenase system: its role in liver inflammations. Curr Drug Targets Cardiovasc Haematol Disord 3: 199–208, 2003.
18. Jaeschke H, Lemasters J: Apoptosis versus oncotic necrosis in hepatic ischemia/reperfusion injury. Gastroenterology 125: 1246–1257, 2003.
19. Corwin H, Gettinger A, Fabian T, May A, Pearl R, Heard S, An R, Bowers P, Burton P, Klausner M, Corwin M: Efficacy and safety of Epoetin Alfa in critically ill patients. N Engl J Med 357: 965–976, 2007.
20. Paschos N, Lykissas MG, Beris AE: The role of erythropoietin as an inhibitor of tissue ischemia. Int J Biol Sci 4: 161–168, 2008.
21. Ratcliffe PJ, Ebert BL, Firth JD, Gleadle JM, Maxwell PH, Nagao M, O’Rourke JF, Pugh CW, Wood SM: Oxygen regulated gene expression: erythropoietin as a model system. Kidney Int 51: 514–526, 1997.
22. Wu H, Klingmuller U, Besmer P, Lodish HF: Interaction of the erythropoietin and stem-cell-factor receptors. Nature 377: 242–246, 1995.
23. Shawsky H, Younan S, Rashed L, Shoukry H: Effect of recombinant erythropoietin on ischemia-reperfusion-induced apoptosis in rat liver. J Physiol Biochem 68: 19–28, 2012.
24. Gujral J, Bucci T, Farhood A, Jaeschke H: Mechanism of cell death during hepatic ischemia-reperfusion in rats: apoptosis or necrosis? Hepatology 33: 397–405, 2001.
25. Faubel S, Edelstein C: Caspases as drug targets in ischemic organ injury. Curr Drug Targets Immune Endocrinol Metab Disord 5: 269–287, 2005.
26. Malhi H, Gores G, Lemasters J: Apoptosis and necrosis in the liver: a tale of two deaths? Hepatology 43( Suppl 1): S31–S44, 2006.
27. Elias-Miro M, Jimenex-Castro M, Rodes J, Peralta C: Current knowledge on oxidative stress in hepatic ischemia/reperfusion. Free Radic Res 47: 555–568, 2013.
28. Geuken E, Buis C, Visser D, Blokzijl H, Moshage H, Nemes B, Leuvenink H, de Jong K, Petters P, Slooff M, Porte R: Expression of heme oxygenase-1 in human livers before transplantation correlates with graft injury and function after transplantation. Am J Transplant 5: 1875–1885, 2005.
29. Bauer I, Wanner G, Rensing H, Alte C, Miescher E, Wolf B, Pannen B, MG C, Bauer M: Expression pattern of heme oxygenase isoenzymes 1 and 2 in normal and stress-exposed rat liver. Hepatology 27: 829–838, 1998.
30. Paine A, Eiz-Vesper B, Blasczyk R, Immenschuh S: Signaling to heme oxygenase-1 and its anti-inflammatory therapeutic potential. Biochem Pharmacol 80: 1985–1903, 2010.
31. Luo Y, Li Z, Liu L, Dong G: Pretreatment with erythropoietin reduces hepatic ischemia-reperfusion injury. Hepatobil Pancreat Dis Int 8: 294–299, 2009.
Keywords:

Hepatocyte; oxidative stress; inflammation; liver injury; JAK-STAT signaling; IR—ischemia-reperfusion; HO-1—heme oxygenase-1; rhEPO—recombinant human erythropoietin; Socs1, Socs3, and Cis—suppressors of cytokine signaling; STAT—signal transducer and activator of transcription; ALT—alanine transaminase; RT-PCR—real-time polymerase chain reaction; NPC—nonparenchymal cell; IHC—immunohistochemistry

© 2014 by the Shock Society