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. Author manuscript; available in PMC: 2012 Dec 1.
Published in final edited form as: Liver Transpl. 2011 Dec;17(12):1457–1466. doi: 10.1002/lt.22415

CARBON MONOXIDE INDUCES HYPOTHERMIA TOLERANCE IN KUPFFER CELLS AND ATTENUATES LIVER ISCHEMIA/REPERFUSION INJURY IN RATS

Lung-Yi Lee 1, Takashi Kaizu 1, Hideyoshi Toyokawa 1, Matthew Zhang 1, Mark Ross 2, Donna Beer Stolz 2, Chao Huang 1, Chandrashekhar Gandhi 1, David A Geller 1, Noriko Murase 1
PMCID: PMC3222745  NIHMSID: NIHMS317781  PMID: 21850691

Abstract

Ischemia/reperfusion (I/R) injury in liver grafts, initiated by cold preservation and augmented by reperfusion, is a major problem complicating graft quality, post-transplant patient care, and outcomes of liver transplantation (LTx). Kupffer cells (KC) play important roles in I/R injury; however, little is known about their changes during cold preservation. We examined whether pretreatment with carbon monoxide (CO), a cytoprotective product of heme degradation, would influence KC activity during cold storage and protect the liver graft against LTx-induced I/R injury. In vitro, primary rat KC were stimulated for 24 hrs with hypothermia (4°C, 20% O2), LPS, or hypoxia (37°C, 5% O2) with and without CO pretreatment. When exposed to hypothermia, rat KC produced ROS, but not TNF-α or NO. Preincubation of KC with CO upregulated HSP70 and inhibited ROS generation. When liver grafts obtained from donor rats exposed to CO (250 ppm) for 24 hrs were transplanted after 18 hrs cold preservation in UW solution, HSP70 expression in the grafts increased, and serum AST/ALT levels as well as necrotic area and inflammatory infiltrates were significantly reduced after LTx, when compared to control grafts. CO-pretreated liver grafts showed less TNF-α, ICAM-1 and iNOS mRNA upregulation, as well as reduced pro-apoptotic Bax mRNA, cleaved caspase-3 and PARP expressions. Thus, donor pretreatment with CO ameliorates I/R injury associated with LTx, with an increased hepatic HSP70 expression, particularly in KC population.

INTRODUCTION

Ischemia/reperfusion (I/R) injury is one of the major problems complicating post-transplant liver graft function and the subsequent long-term care and outcomes for the patients after liver transplantation (LTx). As cold preservation and reperfusion of liver grafts are requisite processes of LTx, I/R injury of the graft is inherent in every LTx procedure. Although I/R injury has been a documented problem, it has become even more significant due to the recent expansion of the potential donor pool to meet the current growing demands (1). Liver grafts from the marginal donor pool are likely to have higher risks of initial poor function or primary nonfunction, as well as late graft loss. However, these liver grafts could provide a survival advantage over dying while waiting for LTx, and effective therapeutic methods to minimize I/R injury will have a significant impact on transplant outcomes and support safer use of marginal organs.

During cold preservation of the liver graft, sinusoidal endothelial cells (SEC) are the crucial site of tissue damage, and SEC in cold preserved liver grafts develop significant ultrastructual changes, including retraction and detachment, leading to SEC death (2, 3). These SEC changes were significantly reduced in liver grafts depleted of Kupffer cells (KC), suggesting that KC activation and regulation play crucial roles in SEC viability during hypothermic condition (4, 5). As KC reside in the sinusoidal space of the liver, adherent to SEC, they are the first cell population that comes in contact with the portal blood from the gastrointestinal tract. Thus, KC are constantly activated by bacterial endotoxins and microbial debris, and activated KC are known to release a plethora of inflammatory factors such as cytokines, reactive oxygen species (ROS), nitrogen oxides, and chemokines (6, 7). Liver graft KC can be activated by stress during the process of hypoxic condition, brain death, and organ procurement procedure, as well as hypothermic storage, and contribute to I/R injury. While much work has indicated roles of KC in tissue damage after warm reperfusion, information about the role of KC during cold preservation is less defined.

Heme oxygenase-1 (HO-1), also known as a heat shock protein (HSP) 32, is the rate-limiting enzyme, which degrades heme protein to equimolar amounts of biliverdin (BV)-IXα and carbon monoxide (CO), while the central iron is released (8). HO-1 induction has been shown to function as a body’s defense system against oxidative stress due to catalysis of potentially pro-oxidant and cytotoxic heme (9), and generation of physiologically antioxidant and cytoprotective byproducts (10). In particular, endogenous CO has been shown to function as a physiological regulator (8, 11). Exogenously provided CO protects endothelial cells and hepatocytes against cytotoxic agents in culture experiments, and in vivo inhaled CO ameliorates I/R injury in various injury models (12). In the rodent LTx-induced I/R injury model, we have previously shown that in vivo recipient treatment with inhaled CO or ex vivo liver graft treatment with CO in preservation solution ameliorates hepatic injury and improves graft survival (4, 13).

Here, we show that cold preservation of liver grafts promotes KC production of ROS; however, CO pretreatment inhibited ROS generation by KC with an upregulation of HSP70. Furthermore, donor pretreatment with inhaled CO before graft retrieval inhibited LTx-induced cold I/R injury with an upregulation of hepatic HSP70 expression.

MATERIALS & METHODS

Reagents

Collagenase (type IV), bovine serum albumin (BSA), EDTA, EGTA, Histodenz, LPS, ethidium bromide and acridine orange were obtained from Sigma (St Louis, MO). L-glutamine and gentamicin were from Life Technologies (Grand Island, NY). Mouse monoclonal antibodies (mAb) specific for HSP70 (HSP72), HSP60, HO-1 (Stressgen Biotech, Victoria, BC, Canada), CD68 (ED1, Serotec, Oxford, UK), iNOS (Transduction Laboratories, Lexington, KY), and ATP synthase (Abcam, Inc., Cambridge, MA), as well as rabbit polyclonal antibodies for cleaved caspase-3, and cleaved PARP (Cell Signaling Technology, Beverly, MA) were used.

Animals

Male Lewis (LEW, RT.1l) rats (8-10 weeks old) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). Animals were maintained in laminar flow, specific-pathogen-free atmosphere at the University of Pittsburgh with a standard diet and water supplied ad libitum.

LTx and experimental design

Liver procurement and orthotopic transplantation without hepatic arterial reconstruction were performed as previously described (14, 15). Donor animals were given inhaled CO (250 ppm) for 24 hrs before liver graft retrieval. Normal room air was used as control. Liver grafts were stored in UW solution at 4°C for 18 hrs and transplanted into syngenic recipients. Recipient animals were sacrificed at 1, 3, 6, 24 and 48 hrs, and liver graft samples (n=4-7 for each time point) were obtained for analyses described below. All procedures in this experiment were performed according to the guidelines of the National Research Council’s Guide for the Care and Use of Laboratory Animals and approved by Institutional Animal Care and Use Committee at the University of Pittsburgh.

Kupffer cell (KC) isolation and culture

KC were isolated from the liver by the collagenase digestion method (16) with some modifications. Briefly, the liver was perfused in situ with Ca+Mg+-free HBSS containing 5 mM EGTA and 10 mM HEPES, then with HBSS containing 0.05% collagenase (type IV) and 5 mM HEPES. Non-parenchymal cells (NPC) were liberated from the liver, filtrated through a 70 μm nylon mesh, and separated by low-speed centrifugation (five times at 50 g for 5 min) and washed by high-speed centrifugation (at 150 g for 7 min). KC were then purified by density gradient centrifugation on a 17.5% (wt/vol) Histodenz gradient, followed by centrifugal elutriation (15, 17). Purified KC were suspended in William’s medium E, containing 2 mM L-glutamine, 10 μg/mL gentamicin, and 10% FBS. After overnight culturing, KC were subjected to various experimental conditions, including hypothermia (4 °C, 20% O2), hypoxia (37 °C, 5% O2), LPS (100 ng/ml), or without stimulation (37 °C, 20% O2) with and without pretreatment with 20% CO in medium.

KC were stained with ethidium bromide (4 μg/ml) and acridine orange (4 μg/ml) to determine their viability. They also were fixed in PFA and stained for ATP synthase. Scanning electron microscopy was used to examine the ultrastructure of KC after glutaraldehyde fixation.

CO administration

For in vivo CO inhalation, CO 1% in air was mixed with air (21% oxygen) in a stainless steel mixing cylinder and then directed into a 3.70 ft3 glass exposure chamber at a flow rate of 12 L/min. CO concentration was maintained at 250 ppm using a CO analyzer (Interscan, Chatsworth, CA), and animals were housed in the chamber. For in vitro culture, culture medium was bubbled with 100% CO gas for 5 minutes and immediately diluted with complete culture medium to make 20% CO saturated medium. KC were exposed to 20% CO-containing medium for 6 hrs in a humidified atmosphere in an incubator maintained at 37°C before being subjected to various experimental conditions.

Liver Enzymes and inflammatory mediators

Hepatic injury following LTx was assessed by AST and ALT levels in the clinical lab at the University of Pittsburgh Medical Center. TNF-α protein levels were determined using rat TNF-α ELISA kit (BioSource International, Camarillo, CA). Nitrite (NO2−) and nitrate (NO3−) levels were measured using a commercially available kit (Cayman Chemical, Ann Arbor, MI).

ROS detection

Oxidative activity in KC was measured using the cell-permeant 5-(and-6)-carboxy-2′,7′-difluorodihydrofluorescein diacetate (carboxy-H2DFFDA, Invitrogen, Carlsbad, CA). Briefly, primary rat KC were cultured on collagen-coated cover slips and exposed to various stimuli. KC were then washed with PBS and loaded with 10 μM of H2DFFDA in PBS for 20 min. H2DFFDA was hydrolyzed by intracellular esterase and oxidized to the impermeable fluorescent form. KC were then fixed with 2% PFA and stained with Hoechst dye. The cover slips were mounted with gelvatol and visualized under Olympus Provis AX70 microscope (Olympus, Center Valley, PA). ROS producing KC were quantified by blindly counting green fluorescent-labeled KC in 4 randomly selected fields. H2O2 levels in KC culture medium were determined by a commercially available kit (Amplex Red, Invitrogen).

Real-Time RT-PCR

Total RNA was extracted from the liver tissues and mRNA expression was quantified by SYBR Green, real-time RT-PCR using primers for TNF-α, iNOS, ICAM-1 and GAPDH as previously described (15). Each sample was analyzed on an ABI PRISM 7000 Sequence Detection System (PE Applied Biosystems). The expression of each gene was normalized to GAPDH mRNA content and calculated relative to normal control.

Protein Extraction and Western blot

KC were detached from culture plate using trypsin, washed twice with ice cold PBS, and incubated with RIPA buffer on ice for 30 min with intermittent agitation. Cell lysate was then sonicated twice on ice and centrifuged. Hepatic cytoplasmic proteins (20-100 μg) were isolated from graft liver samples as previously described (13). Protein samples were prepared with SDS sample buffer and analyzed in 8%-12% SDS-PAGE gel and transferred to nitrocellulose membranes (Scleicher & Schuell, Keene, NH). After blocking nonspecific binding with non-fat dry milk, membranes were incubated with primary rabbit polyclonal or mouse monoclonal antibody. After incubation with secondary goat anti-rabbit or anti-mouse antibody, membranes were developed with the SuperSignal detection systems (Pierce Chemical) and exposed to x-ray film. The band densities were measured by NIH Image analysis software, and presented in comparison to fresh normal liver.

Immunohistochemistry and image analysis

Liver graft tissues were fixed in 10% formalin, embedded in paraffin, sectioned into 6 μm thickness and stained with H&E. In histopathological analysis, the percentage of the necrotic area was determined by the morphometric analysis of 10 randomly selected low-power fields (40×) per section using NIH image analysis software in a blinded fashion. The neutrophils were stained using a naphthol AS-D chloroacetate esterase staining kit (Sigma Diagnostics, St. Louis, MO). The macrophages were stained by immunohistochemistry for ED1 as previously described (15). Positively stained cells were blindly counted in 20 high-power fields (x 200) per each section and expressed as the number of cells per 1 mm2.

Statistical analyses

The results were expressed as mean ± SD. Statistical analysis was performed using unpaired Student’s t-test or analysis of variance (ANOVA) where appropriate. A probability level of p<0.05 was considered statistically significant

RESULTS

KC generates ROS during hypothermic condition

Staining of isolated rat primary KC with mAb ED1 revealed high purity of KC in the culture (Figure 1A). To determine the responses of KC to hypothermia, rat KC were exposed to 4 °C, and generation of NO, TNF-α, and H2O2 was compared to that at 37 °C culture condition with and without LPS. While LPS stimulation led to a significant increase in both TNF-α and NO production and release, hypothermia did not generate TNF-α or NO when compared to the control group at 37 °C without LPS (Figure 1B). Hypoxia (37 °C 5% O2) did not stimulate KC to generate TNF-α or NO (data not shown). Levels of hydrogen peroxide in the culture medium also increased with LPS, but KC did not generate hydrogen peroxide during hypothermia. Hypothermia did not alter the viability of KC, and KC showed the presence of intact mitochondrial ATP synthase after exposed to 4 °C for 24 hrs (Figure 1C). Ethidium bromide/acridine orange stain showed bright green nucleus and red orange cytoplasm, indicating that KC were viable after exposure to 4 °C for 24 hrs.

Figure 1. Responses of cultured primary rat KC to hypothermic condition.

Figure 1

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A) Immunocytochemical staining of isolated primary rat KC culture (48 hr) with ED1 (Red) with nuclear Hoechst dye (Blue).

B) After overnight culture, primary rat KC were cultured without stimulation (37°C, none), under LPS stimulation (37°C, LPS), or hypothermia (4°C, none) for 24 hrs. All groups were then cultured under 37°C without LPS for 24 hrs, and medium samples were analyzed for TNF-α, NO, and H2O2. N=3 for each group.

(C) Rat primary KC were kept at 4 °C for 24 hrs and stained for anti-mitochondrial ATP synthase (left panels, red) with nuclear DAPI (blue). KC also were stained with ethidium bromide (red)/acridine orange (green) (right panels). All KC showed bright green chromatin in the nucleus and red orange cytoplasm. There was no nonviable cell with ethidium bromide uptake (nuclear red stain).

In ultrastructual analysis with TEM, KC exposed to hypothermia showed many large lysosomes with diluted contents. In contrast, lysosomes in KC cultured at 37 °C were in various stages of engulfing and processing (Figure 2). The findings suggested that hypothermia arrested KC’s activity to phagocyte and process the lysosomal contents. Diluted materials in the lysosomes seen in KC during hypothermia might suggest that lyosomal digestive enzymes slowly broke up the contents that were already present prior to exposure to 4 °C. Additional morphological signs of activation, such as round-shape with ruffles and degranulation, were not observed.

Figure 2. Ultrastructure of KC after 24 hrs exposure to 4 °C.

Figure 2

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Isolated rat KC were exposed to hypothermia for 24 hrs, fixed with glutaraldehyde and analyzed with TEM. KC cultured at 4 °C showed many large lysosomes (L) with diluted contents (right panel). In contrast, lysosomes in KC cultured at 37 °C showed various stages of engulfing and processing.

When KC were placed at 4 °C, intracellular ROS generation was promptly increased. As early as 1 hr of hypothermia, ~15% of KC showed ROS production, and the similar frequency of ROS positive KC were also seen at 24 hrs. No detectable ROS generation was observed in normoxic culture of KC at 37 °C. In contrast, stimulation with LPS did not induce prompt ROS generation by KC at 1 hr (Figure 3), but 17.8% of KC produced ROS at 24 hrs. These results suggest that KC in liver grafts could produce ROS soon after they were placed in cold preservation for transplantation.

Figure 3. ROS generation by KC during hypothermic condition is inhibited by CO pre-exposure.

Figure 3

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For ROS detection, KC were preloaded with H2DFFDA, exposed to LPS (37°C, LPS) or hypothermia (4 °C, none) or without stimulation (37°C, none) for 1 hr, and examined the conversion of carboxy-H2DFFDA to a fluorescent (green) deacetylated and oxidized carboxy-DFF due to oxidation within the cell. KC were also cultured in 20% CO containing medium for 6 hrs, loaded with H2DFFDA, and exposed to hypothermia (4°C, CO) for 1 hr. The conversion of H2DFFDA to a fluorescent derivative due to ROS generation was detected, and numbers of ROS producing KC (green) were quantified and shown as the percentages of total numbers of KC. For the positive control, KC were cultured for 24 hrs with LPS. N=3 for each group.

CO inhibits hypothermia-induced ROS generation from KC

As CO has been shown to regulate ROS generation at 37 °C (18), we next determined if exposure to CO prior to hypothermic stimulation can influence ROS generation from KC. When KC were treated with 20% CO containing medium for 6 hrs then exposed to hypothermia, prompt ROS generation at 1 hr in KC was reduced (Figure 3). In contrast, when KC were exposed to CO and maintained at 37 °C, the frequency of ROS positive KC increased to 17.2 ± 9.6%, as shown previously (19, 20).

CO-pretreatment increases HSP70 expression in liver grafts and isolated KC

To explore the possible mechanisms by which CO inhibited intracellular ROS generation in KC during hypothermic storage, we examined if CO induced the expression of stress inducible HSPs. After in vivo treatment of animals with inhaled CO, HSP protein expression in the liver was analyzed by Western blot. CO exposure significantly increased HSP70, but not HSP60 or HSP32 (HO-1), protein expression in the liver (Figure 4A).

Figure 4. HSP70 expression in the liver and KC with CO.

Figure 4

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(A) Western blot for HSP70, HSP60 and HO-1 was conducted using liver samples obtained from naïve and CO (250 ppm for 24 hrs) exposed animals. CO significantly increased HSP70, but not HSP60 or HO-1, expression compared to naïve livers. *P < 0.05 vs. air-control (n=4 for each group).

(B) KC were isolated from naïve animals and cultured for 3-6 hrs with and without 20% CO in the medium. Proteins were isolated and Western blot was performed. Ex vivo exposure of KC with CO increased HSP70 protein expression.

To examine if HSP70 upregulation with CO was seen in KC, isolated KC were pretreated with 20% CO in culture medium. HSP70 protein expression started to increase at 3 hrs, and significant HSP70 expression was seen at 6 hrs after CO exposure (Figure 4B).

Liver grafts with in vivo CO pretreatment show less hepatic cold I/R injury

Based on in vitro findings, we hypothesized that CO pretreatment and upregulation of HSP70 in liver grafts, in particular in KC, could have beneficial effects on hepatic I/R injury. LTx experiment was conducted with donor pretreatment with 250 ppm CO for 24 hrs and cold preservation for 18 hrs in UW solution. After transplantation into syngenic recipients, CO-treated grafts showed significantly less serum AST and ALT levels at 6-24 hrs as compared to untreated liver grafts (Figure 5A). Histopathological analysis of liver grafts at 48 hrs showed reduced necrotic areas in liver grafts pretreated with CO as compared to controls (Figure 5B). At 48 hrs, infiltrating neutrophils were mostly found in the area of liver necrotic foci and their surroundings. This was significantly reduced in CO exposed liver grafts as compared to the control group (Figure 5C).

Figure 5. Hepatic I/R injury after transplant is inhibited by CO donor pretreatment.

Figure 5

Figure 5

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A) Serum AST and ALT levels were measured after liver transplantation. Liver grafts were obtained from animals treated with 250 ppm CO for 24 hrs, preserved for 18 hrs in UW solution, and transplanted into syngenic recipients. *P < 0.01, #P < 0.05 vs. air-control (n=7-10 for each group).

B) Necrotic area was quantified in H&E stained sections at 48 hrs after reperfusion. Consistent with the improvement in liver function tests, necrotic area was decreased in CO exposed liver grafts. *P < 0.01 vs. air-control (n=5 for each group).

C) Neutrophils in liver grafts were visualized with naphthol AS-D chloroacetate esterase staining, and the number of positively stained cells at 48 hrs were counted and expressed as the number per 1 mm2 field. Original magnification; x400. *P < 0.001 vs. air-control (n=5 for each group).

D) Immunohistochemistry of liver grafts for FD1+ cells. The number of positively stained cells at 48 hrs were counted and expressed as the number per 1 mm2 field. Original magnification; x400. *P < 0.05 vs. air-control (n=5 for each group).

When donor rats were exposed to 250 ppm CO for 24 hrs before liver graft retrieval, COHb levels increased to ~24% in donors by the time of graft retrieval. However, recipient COHb levels after transplantation did not elevate, and COHb levels were within the normal level in both groups of recipients.

KC in liver grafts

In normal liver, resident KC were stained with ED1. In the control liver grafts at 48 hrs after LTx, ED-1 expression increased due to graft ED1 upregulation and host macrophage infiltration. CO-treated liver grafts had significantly less ED1+ cells (Figure 5D).

Liver grafts exposed to CO before transplantation show less TNF-α and iNOS

Real-time RT-PCR assay was next conducted to examine gene expressions of TNF-α and iNOS in early post-transplant liver grafts. In the control grafts, hepatic mRNA levels for TNF-α and iNOS promptly increased and peaked at 1-3 hrs after reperfusion (Figure 6). CO exposed liver grafts had significantly lower mRNA expression for TNF-α and iNOS. Hepatic iNOS protein expression and its product NO in the serum also increased during I/R injury. CO-pretreated liver grafts showed markedly suppressed hepatic iNOS protein expressions and significantly decreased serum NO levels at 6 and 24 hrs after reperfusion as compared with control liver grafts (Figure 6).

Figure 6. TNF-α and iNOS upregulation in hepatic I/R injury.

Figure 6

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A) TNF-α and iNOS mRNA levels in liver grafts after transplant were assessed by real-time RT-PCR. Prompt increases in TNF-α and iNOS mRNA expressions in control grafts were significantly reduced in liver grafts which were exposed to CO before cold storage. NL; fresh normal liver. *P < 0.001, #P < 0.05 vs. air-control (n=4 for each group).

B) Hepatic iNOS protein expression was assessed by Western blot. CO exposure markedly suppressed hepatic iNOS protein expression during reperfusion period. Each lane represents different liver graft sample. Blots shown are representative of 3 similar experiments.

C) After transplantation of CO pretreated liver grafts, serum NO levels were significantly lower than those with control liver grafts. NL; normal rats. *P < 0.01 vs. air-control (n=3-4 for each group).

Apoptotic pathways

Although the major cause of cell death during hepatic I/R injury is necrosis, ROS can induce apoptosis during the injury process. Bax and Bcl-2 mRNA levels increased at 1 hr after reperfusion in the control group (Figure 7A). CO exposed liver grafts showed significantly decreased pro-apoptotic Bax mRNA expression at 1 hr after reperfusion without affecting anti-apoptotic Bcl-2 mRNA expression. The increase in cleaved caspase-3 peaked at 3 hrs in untreated controls (21). Both cleaved caspase-3 and cleaved PARP expressions were significantly less in CO-exposed liver grafts at 3 hrs (Figure 7B).

Figure 7. Anti-apoptotic effects of CO donor pretreatment.

Figure 7

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A) Bax and Bcl-2 mRNA levels in liver grafts were assessed by real-time RT-PCR. CO-pretreated liver grafts showed significantly reduced mRNA levels for Bax, but not Bcl-2, at 1 hr after reperfusion. NL; fresh normal liver. *P < 0.01 vs. air-control (n=4 for each group).

B) Cleaved caspase-3 and cleaved PARP protein expressions in liver grafts were measured at 3 hrs after reperfusion. CO-pretreated liver grafts showed significantly decreased cleaved caspase-3 and cleaved PARP expressions at 3 hrs. NL; fresh normal liver. Each lane represents a different animal. Band density was expressed in comparison to normal liver. *P < 0.05 vs. air-control (n=4 for each group).

DISCUSSION

Using the rat primary KC culture system, we demonstrate that KC promptly release ROS, but generate only negligible amounts of NO or TNF-α when exposed to hypothermic condition. When KC are exposed to LPS at 37 °C, they can generate NO, TNF-α, and ROS; however, ROS generation was only seen after culturing for 24 hrs. These results suggest that KC might have an unique feature in promptly responding to hypothermia and play key roles in initiating the injury process during the cold storage period. In fact, during cold storage, KC develop morphological changes suggestive of activation, such as rounding, lysosomal polarization, and vacuolization as shown here and previously (22). We have also shown that when KC are pretreated with CO, HSP70 expression is upregulated, and decreased amount of ROS is produced after exposed to hypothermia. Further, in in vivo liver transplant experiments, liver grafts obtained from donors exposed to CO in vivo show increased expression of HSP70 and protection against I/R injury; serum AST/ALT levels are significantly lower, and histopathological changes are also attenuated with less severe necrosis and reduced neutrophil recruitment. These beneficial effects of donor CO pretreatment and hepatic HSP70 upregulation are associated with downregulation of TNF-α and iNOS. Grafts expressing HSP70 also show decreased activation of pro-apoptotic bax and caspase-3 during early post-transplant period. These results suggest that donor pretreatment with CO can inhibit transplant-induced hepatic I/R injury. The hepatic protection with CO-pretreatment in this study associated with hepatic HSP 70 upregulation; however, it remains to be determined whether hepatic HSP70 expression, in particular on Kupffer cells, plays a causative role in protecting the liver graft from I/R injury.

ROS are known to react with macromolecules and cause cellular damage via lipid peroxidization, DNA oxidation, and protein denaturation (23). Although ROS generation is a feature of a wide range of normal cell metabolism, excess ROS generation in the liver has been known to be associated with various forms of injuries, including alcoholic liver disease, cirrhosis, viral hepatitis, non-alcoholic fatty liver, and I/R injury (24-28). A major source of ROS is mitochondrial electron gradient and the resulting uncoupling of oxidative phosphorylation; however, many other cellular enzymes (e.g. cytochrome P450, xanthine oxidoreductase, nitric oxide synthases, lipoxygenase) and membrane enzymes (e.g. NADPH oxidase) also contribute to ROS generation in KC, hepatocytes and neutrophils. Many of these are hemeproteins whose functions can be affected by CO.

HSPs are a ubiquitous family of highly inducible stress proteins, which are markedly expressed in various tissues in a wide variety of stress conditions in order to confer protection against such stressors. Among HSPs, the 70 kDa family of stress proteins is the most inducible and abundant HSP in mammalian cells, and includes several isoforms. HSP73 (constitutive isoform) and HSP72 (stress-inducible isoform) are the most studied HSP70 members. The protective function of HSP70 is thought to be related to its ability to promote refolding of aberrantly folded, mutated, or denatured polypeptides, to prevent protein aggregate formation, and to remove damaged proteins (29, 30). Hence, HSP70 has been shown to prevent caspase activation through binding to cytochrome c and other proapoptotic proteins (31, 32). In fact, in numerous experimental settings, overexpression of HSP70 can provide protection against a wide range of stresses, including hyperthermia, oxidative stress, apoptotic stimuli, and ischemia. Pre-exposure of the liver to transient ischemia, hyperthermia, or mild oxidative stress, known as hepatic preconditioning, increases the tolerance against subsequent I/R injury (33, 34). One of the proposed mechanisms by which hepatic preconditioning affords hepatoprotection is induction of HSPs such as HO-1 and HSP70 (35-38). In the liver, HSP70 upregulation is seen with brief ischemia, hyperthermia, or pharmacological agents and affords hepatoprotection against warm liver I/R injury (35, 36, 38). In the LTx setting, HSP70 induction in donor livers by heat preconditioning or transient ischemia attenuated cold I/R injury and improved survival following rat LTx (39, 40). Although these studies do not show cause-effect relationships between HSP70 expression and hepatic protection, HSP70 upregulation might be an important mechanism in preventing hepatic I/R injury.

CO has been shown to provide cytoprotective effects in a variety of injury models such as hyperoxic lung injury, acute inflammation, acute or chronic rejection, and septic shock via anti-inflammatory, anti-apoptotic, and vasodilative functions. These protective actions of CO have been linked to activation of several molecular pathways including soluble guanylate cyclase (sGC) and mitogen-activated protein kinases (MAPK). Using the same I/R injury model, we have previously shown that CO does not inhibit hepatic I/R injury-induced NF-kB activation (41), but inhibits ERK1/2 MAPK pathway activation (13). CO also inhibits ICAM expression in liver grafts during I/R injury (4, 41). These experiments use recipient or graft treatment with CO, and it remains undetermined if donor pretreatment with CO could also involve these pathways. CO has been shown to induce HSP70 expression in the lung and other organs in mice, and provide the cytoprotective effects in the mouse endotoxemia model (42). Donor pretreatment with inhaled CO in this study also upregulates hepatic HSP70 and protect liver grafts against I/R injury without elevating recipient COHb levels or causing any adverse event. Further studies are warranted to determine the molecular targets of CO and how CO regulates KC activity for the ROS and HSP70 production.

Although tissue necrosis is a major pathophysiological finding of established I/R injury, apoptosis of sinusoidal endothelial cells and hepatocytes has been shown to represent a feature of hepatic I/R injury (43, 44). Apoptosis and necrosis are usually considered two largely independent processes; however, apoptotic cell death is shown to be a crucial event in initiating inflammation and subsequent tissue injury (45). In fact, anti-apoptotic therapy with adenoviral bcl-2 or Bag-1 gene transfer, or caspase inhibitors have been shown to ameliorate hepatic I/R injury (46-48). Inhibition of early I/R-induced apoptosis, as shown in Figure 7, could prevent the development of inflammation and liver graft dysfunction.

In summary, in vitro, CO inhibits hepatic KC production of ROS in the hypothermic condition with an upregulation of HSP70. In vivo, donor pretreatment with inhaled CO induced hepatic HSP70 upregulation and caused significant inhibition of cold I/R injury after LTx in association with mitigated expression of proinflammatory and pro-apoptotic mediators. These results strongly support the possibility that donor pretreatment with inhaled CO could be one of the promising strategies in combating cold I/R injury after organ transplantation.

ACKNOWLEDGEMENT

We would like to thank Lifang Shao and Rita Sico for their excellent technical support, and Carla Forsythe for the preparation and organization of the manuscript.

This work was supported by DK071753, P01AI081678, Ruth L. Kirschstein National Research Service Award (T35 DK 065521).

Abbreviations

CO

carbon monoxide

HO-1

heme oxygenase-1

HSP

heat shock protein

I/R

ischemia/reperfusion

KC

Kupffer cells

LTx

liver transplantation

PARP

poly ADP-ribose polymerase

SEC

sinusoidal endothelial cells

UW

University of Wisconsin

Footnotes

*

Lung-Yi Lee and Takashi Kaizu equally contributed to this study.

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