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Research Communications |
a Institut für Physiologische Chemie, Universitätsklinikum, D-45122 Essen, Germany
b Abteilung für Anatomie und Embryologie, Ruhr-Universität, D-44780 Bochum, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol provided complete protection in both cell types. During cold incubation, and especially during rewarming, lipid peroxidation occurred. These results suggest that the formation of reactive oxygen species (ROS) is a key mediator of cold-induced apoptosis, with ROS formation being completely iron-mediated in liver endothelial cells and partially iron-mediated in hepatocytes.—Rauen, U., Polzar, B., Stephan, H., Mannherz, H. G., de Groot, H. Cold-induced apoptosis in cultured hepatocytes and liver endothelial cells: mediation by reactive oxygen species. FASEB J. 13, 155–168 (1999)
Key Words: hypothermia • iron • hydroxyl radical • free radical • cold preservation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On the basis of morphological features, two forms of cell death, necrosis and apoptosis, can be distinguished (6–9). The necrotic form of cell death is characterized by swelling of the whole cell, its nucleus and organelles, and by an early disintegration of the plasma membrane (6, 8, 10). In contrast, apoptosis is characterized by cellular and nuclear shrinkage as well as by pronounced budding or blebbing, which finally leads to the pinching off of blebs giving rise to `apoptotic bodies' (6–9, 11). These events are accompanied by alterations of nuclear morphology such as chromatin condensation and margination, ruffling of the nuclear membranes, and nuclear fragmentation. In addition, increased nuclear stainability with dyes such as H33342 can be observed (7). In contrast to necrosis, the blebs occurring during apoptosis often contain organelles (8); and in apoptosis, the functional integrity of the plasma membrane is long maintained (7, 8, 10). Frequently, apoptosis is accompanied by internucleosomal DNA fragmentation, giving rise to the classical `ladder' pattern on DNA electrophoresis (7–9, 11).
Hypothermia injury is usually attributed to disturbances in cellular ion homeostasis and to alterations in membrane fluidity (1, 3, 12–14). The most prominent (and decisive) alterations under hypothermic conditions are thought to be an influx of sodium and chloride (3, 15) leading to secondary alterations of the cellular calcium homeostasis and, particularly, to cell swelling (4, 5, 13, 15, 16). Thus, hypothermia and preservation injury are thought to occur in the typical setting for a `necrotic' type of cell injury. However, after exposing cultured cells to hypothermia, followed by rewarming, we observed morphological changes that were strongly suggestive of apoptosis. We therefore set out to verify whether hypothermia per se can induce apoptosis in cultured liver cells and, if so, to delineate some of the intracellular signals involved in this process.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol, 2,2'-dipyridyl, 4,4'-dipyridyl, the dye Hoechst 33342 (H33342), propidium iodide, tert-butylhydroperoxide, and triphenylphosphine were from Sigma (Deisenhofen, Germany). Collagen R and glutaraldehyde were purchased from Serva (Heidelberg, Germany), 1,10-phenanthroline and 4,7-phenanthroline from Aldrich (Sigma-Aldrich, Steinheim, Germany). 1,1,3,3-Tetramethoxy-propane was obtained from Fluka (Neu-Ulm, Germany), fibronectin from Boehringer (Mannheim, Germany), ebselen from Calbiochem (Bad Soden, Germany), and deferoxamine mesylate (Desferal) from Ciba-Geigy (Wehr, Germany).
Cell culture
Hepatocytes were isolated from male Wistar rats (250–310 g) as described previously (17). The cells were seeded onto collagen-coated 12.5 cm2 or 25 cm2 culture flasks (Falcon, Heidelberg, Germany) or onto collagen-coated 6.2 cm2 glass coverslips (Assistent, Röhn, Germany) and cultured in L-15 medium supplemented with 5% fetal calf serum (Sigma), L-glutamine (2 mM), glucose (8.3 mM), bovine serum albumin (0.1%), NaHCO3 (14.3 mM), gentamycin (50 µg/ml), and dexamethasone (1 µM). Two hours after seeding, adherent cells were washed three times with Hanks' balanced salt solution (HBSS) and supplied with fresh medium. Experiments were started 20–24 h after the isolation of the cells.
A rat liver endothelial cell line, derived from the liver of a male Wistar rat, was used for additional experiments. The cells had been isolated and characterized as described previously (18). Briefly, cells were isolated from rat liver by collagenase perfusion. The endothelial cells were separated from hepatocytes and Kupffer cells by differential centrifugation and selective adherence methods. Cells were cultured in RPMI 1640 medium supplemented with fetal calf serum (20%), L-glutamine (2 mM), gentamycin (100 µg/ml), and dexamethasone (1 µM) at 37°C in a 100% humidified atmosphere containing 5% CO2/95% air. Cultured cells were identified as liver endothelial cells by immunological demonstration of von Willebrand factor and by scanning electron microscopic demonstration of sieve plates in primary cultures. Subcultures were obtained by trypsinization.
In this study, 6th–22nd passage cultures were used. In these passages, the cells were still able to form tubular structures in vitro upon prolonged culturing, confirming the endothelial origin of the cells and the maintenance of endothelial cell features in the higher passages (19, 20). For the experiments, the cells were split 1:3 and seeded onto collagen-coated 12.5 cm2 or 25 cm2 culture flasks or onto fibronectin-coated 6.2 cm2 glass coverslips for fluorescence microscopy. The cells were used for experiments on day 5 or 6 after subcultivation. By this time, cells were in the confluent state.
Experimental procedures
The cells were washed three times with HBSS (37°C) at the beginning of the experiments and then covered with cell culture medium (Leibovitz L-15 medium for hepatocytes and RPMI 1640 medium for liver endothelial cells, both supplemented as described above) at room temperature. The cells were then incubated either at 4°C or at 37°C. Aerobic incubations were performed in an atmosphere of 95% air/5% CO2. Hypoxic conditions were established by saturating the incubation solution with 95% N2/5% CO2 before addition to the cells, followed by gentle flushing of the culture flasks with 95% N2/5% CO2 through cannulae piercing the rubber stoppers of the flasks, as described in ref 17. The flasks were again flushed with the respective gas mixtures each time a sample was taken, and otherwise at least every 12 h. To avoid diffusion of oxygen or carbon dioxide through the plastic walls of the culture flasks during the long incubation periods, flasks were placed in air-tight vessels that were also flushed with the respective gas mixtures. In some experiments, DMSO (final concentration 10%, v/v), DMPO (50 mM), TEMPOL (1 and 50 mM), BHT (20 µM), BHA (100 µM), or ebselen (500 µM), or the transition metal chelators 2,2'-dipyridyl (100 µM) or 1,10-phenanthroline (100 µM) or their nonchelating analogs 4,4'-dipyridyl (100 µM) or 4,7-phenanthroline (100 µM) were added to the medium at the beginning of the cold incubation. Some cultures were preincubated with deferoxamine (10 mM; in cell culture medium, 30 min at 37°C) prior to the start of the experiments. Preincubation of the cells with -tocopherol was performed according to Martin et al. (21). Briefly, cells were incubated for 20 h in cell culture medium containing 10% fetal calf serum and -tocopherol added from a stock solution (10 mg/ml in ethanol) to achieve a final concentration of 60 µM -tocopherol and 0.26% ethanol. Deferoxamine and -tocopherol were not added to the solutions during the cold incubations. Solvent controls were included.
After various periods of cold incubation, the cells were rewarmed to 37°C in an incubator containing a humidified atmosphere of 95% air/5% CO2. Hypoxic incubations were flushed with 95% N2/5% CO2 prior to rewarming, as described above, and flushing with N2/CO2 was repeated when a sample was taken.
Assays
Nuclear staining with H33342 and propidium iodide
Nuclei of cells grown on glass coverslips were stained with the DNA binding fluorochrome H33342. At the time points indicated, the usual cell culture medium (see above) was replaced by cell culture medium containing 5% fetal calf serum. H33342 was added to the supernatants to give a final concentration of 1 µg/ml and cells were incubated for 12 min at 37°C (or 20 min at 4°C for cold incubations). Thereafter, cells were immediately assessed on a Zeiss Axiovert 135 TV microscope equipped with epifluorescence optics (Zeiss, Oberkochen, Germany). Maintaining the incubation temperature at either 4°C or 37°C using a liquid-cooled microscope stage (Zeiss) connected to a cryostat/water bath (2219 Multitemp II; Pharmacia Biosystems, Freiburg, Germany), nuclear morphology was evaluated using an excitation wavelength of 380 ±10 nm and a bandpass filter of 460–490 nm for emission. For double staining with H33342 and propidium iodide, cells were treated as before with H33342. Two minutes before the end of the incubation with H33342 (or 5 min at 4°C), propidium iodide was added in a final concentration of 5 µg/ml and mixed thoroughly. The excitation wavelength was unchanged while a longpass filter 
520 nm was used for emission. Nuclear staining and morphology were evaluated optically and documented photographically.
In situ end labeling (TUNEL test)
At the times indicated, cells cultured on glass coverslips were washed three times with phosphate-buffered saline (PBS), fixed 10 min in 4% paraformaldehyde and again washed with PBS. Thereafter, in situ end labeling was performed as described in ref 22. Liver endothelial cells grown on glass coverslips tended to detach after cold incubation/rewarming and could not be kept adherent during washing/fixation. Therefore, this test was performed only on hepatocytes.
Oligonucleosome enzyme-linked immunosorbent assay (ELISA)
Alternatively, DNA fragmentation was determined by a commercial sandwich ELISA for low molecular weight, histone-associated DNA, i.e., mono- and oligonucleosomes, using a microtiter plate coated with a primary anti-histone antibody and using a secondary, peroxidase-coupled anti-DNA antibody (Boehringer). The cells were washed twice with HBSS, then lysed, and the lysate was centrifuged for 10 min at 4°C at 16,000 x g. The supernatant was diluted 1:100 (hepatocytes) or 1:10 (liver endothelial cells) with Tris-EDTA buffer and then used in the ELISA according to the manufacturer's instructions. From the absorbance values at 405 nm (vs. reference at 490 nm), the percentage of fragmentation in comparison to controls was calculated according to the following formula:
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Transmission electron microscopy
For transmission electron microscopy, cells were cultured on 78.5 cm2 culture dishes (Falcon) and exposed to experimental conditions. At the times indicated, cells were washed with PBS and fixed overnight (16 h) with glutaraldehyde (2% in PBS). After fixation, cells were scraped off with a rubber policeman, centrifuged, and the pellet was resuspended in PBS and treated with osmium tetroxide (1%) overnight (16 h). Thereafter, cells were embedded in Epon resin according to the manufacturer's instructions (Fluka, Neu-Ulm, Germany). Ultrathin sections (60 nm) were contrasted serially with 1% uranyl acetate and 0.66% lead acetate for 10 min each. Electron microscopic examination was performed using a Philips EM 420 (Philips, Eindhoven, The Netherlands) operated at 80 kV.
Thiobarbituric acid-reactive substances
Thiobarbituric acid-reactive substances (TBARS) were determined in the supernatant incubation solution after various incubation times using the assay described in ref 23 with minor modifications. The amounts of TBARS formed were expressed as malondialdehyde equivalents using 1,1,3,3-tetramethoxy-propane as a standard.
Determination of lipid hydroperoxides
Lipid hydroperoxides were determined with the ferrous oxidation-xylenol orange assay, version 2, described by Wolff (24). For these measurements, cultures were washed three times with HBSS at the respective temperatures, and the reagent (containing 90% methanol) was added directly to the cells. After incubation for 30 min at room temperature, the reagent was centrifuged at 10.000 rpm for 5 min to remove particulate material and the absorbance was read at 560 nm. To increase the specificity of the test, parallel incubations were treated with triphenylphosphine (1 mM in methanol) for 30 min at room temperature prior to the addition of the reagent as described by Nourooz-Zadeh (25). Because triphenylphosphine specifically reduces lipid hydroperoxides (26), triphenylphosphine-inhibitable color development was taken as a measure of lipid hydroperoxides and expressed as nanomoles of lipid hydroperoxides per 106 cells, using tert-butylhydroperoxide as a standard.
Lactate dehydrogenase (LDH) release
Extracellular, i.e., released, LDH activity was measured using a standard assay. At the end of the incubation period, cellular LDH activity was determined after lysis of the cells with the detergent Triton X-100 (1% in HBSS, 30 min at 37°C). LDH values were corrected for the change in the volume of incubation medium resulting from repetitive sampling, and released LDH activity was given as a percentage of total LDH activity.
Statistics
All experiments were performed in duplicate and repeated three to seven times. Data are expressed as means ±SE. Data obtained from two groups were compared by means of Student's t test and comparisons among multiple groups were performed using an analysis of variance with Student-Newman-Keuls post hoc comparisons. A P value of <0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fig. 1).
Small bleb-like vesicles without contact to cells (apoptotic bodies) appeared ( Fig. 1C) and an array of nuclear changes, including the appearance of rim-like structures in the nuclear periphery, occurred ( Fig. 1B) as assessed after staining with H33342, the changes in nuclear morphology appeared to include chromatin condensation and margination ( Fig. 2B, C),
ruffling of the nuclear membranes ( Fig. 2D), nuclear condensation ( Fig. 2B), and increased stainability with H33342. Some cells extruded their nuclei ( Fig. 1D), but nuclear fragmentation was rarely seen. All these changes in nuclear morphology occurred within the first hour of rewarming, typically giving rise to a pleomorphic picture with more than 70% of nuclei showing an altered morphology after rewarming. Double staining with H33342 and propidium iodide clearly demonstrated that the nuclear alterations shown occurred before the cells took up propidium iodide ( Fig. 2C, D). One hour after rewarming, after 24 h of cold incubation, more than 70% of hepatocytes took up propidium iodide (data not shown). Transmission electron microscopy of hepatocytes exposed to hypothermia/rewarming revealed chromatin condensation and margination ( Fig. 3B)
as well as bleb formation (with blebs containing intact organelles, Fig. 3C). Numerous convoluted and a few fragmented nuclei could be observed ( Fig. 3C, D). At later stages (2 h after rewarming) the plasma membrane had lost its continuity in a substantial proportion of the cells and swollen mitochondria appeared, indicating the occurrence of secondary necrosis ( Fig. 3D).
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The morphological alterations were accompanied by DNA fragmentation. There was little DNA fragmentation in control cells, and after 24 h of hypothermia there was progressive DNA fragmentation upon rewarming, as assessed by in situ end labeling as well as by an ELISA for mono-/oligonucleosomes ( Fig. 4
and Fig. 5).
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Although most of the morphological alterations developed during rewarming, their occurrence depended on the duration of the cold incubation (with the most extensive morphological alterations occurring after 18 h of cold incubation; data not shown). Similarly, the occurrence, extent, and velocity of the uptake of propidium iodide and the secondary release of lactate dehydrogenase depended on the duration of the cold incubation period ( Fig. 6).
The same basic pattern of dependence was found for DNA fragmentation (
Fig. 5). The apoptotic cell injury did not occur in cells incubated for the same periods (i.e., up to 36 h) at 37°C (data not shown), underscoring the absolute dependence on the hypothermic period.
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Hypothermia-induced alterations of liver endothelial cells
When confluent cultures of liver endothelial cells were incubated in cell culture medium at 4°C for 24 h, cells still appeared morphologically normal (data not shown). After rewarming, the cells developed multiple blebs, some containing organelles, and the cells appeared to retract their processes, giving rise to intercellular gaps ( Fig. 7).
As observed by phase-contrast microscopy, the nuclei became dark and prominent and appeared to shrink considerably. Staining with H33342 revealed some chromatin condensation and margination, but shrinkage of the large oval endothelial cell nuclei soon became the prominent feature (see Fig. 9C). This nuclear condensation, which could be seen in >70% of nuclei after 24 h cold incubation/2 h rewarming, was associated with increased stainability with H33342. Later, the nuclei showed some indentation but fragmentation was seen only occasionally. As in hepatocytes, these alterations in nuclear morphology occurred before the cells took up propidium iodide.
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Transmission electron microscopy revealed nuclear shrinkage as well as chromatin condensation and margination in cells exposed to hypothermia/rewarming (data not shown). Considerable extensions of the perinuclear cisterna were frequent. In contrast to these pronounced nuclear changes, the mitochondria appeared to be largely unchanged (in particular, they were not swollen) and the plasma membrane maintained its continuity.
In contrast to hepatocytes, the results on DNA fragmentation obtained with the oligonucleosome ELISA were variable, but even in positive experiments the absolute absorbance values were more than one order of magnitude lower than in hepatocytes (data not shown), suggesting that DNA fragmentation to oligonucleosomal fragments is not a prominent feature in these cells.
As in hepatocytes, the occurrence, extent, and velocity of the morphological alterations as well as the uptake of propidium iodide and the secondary release of lactate dehydrogenase depended on the duration of the cold incubation period (data not shown), and the alterations did not occur in cells incubated at 37°C for the whole duration of the experiment (data not shown).
Participation of reactive oxygen species
Surprisingly, it was possible to decrease the percentage of apoptotic hepatocytes after hypothermia/rewarming by performing the cold incubation and rewarming under hypoxic conditions ( Table 1).
Taking this inhibitory effect of hypoxia as an indication for a potential involvement of reactive oxygen species in cold-induced apoptosis, we tested the effects of an array of antioxidants/free radical scavengers on the apoptotic features. In hepatocytes, the lipophilic antioxidants -tocopherol, butylated hydroxytoluene, and butylated hydroxyanisole strongly inhibited morphological alterations, the uptake of propidium iodide, and the release of LDH ( Fig. 8A
and Fig. 9B,
Table 1). Similar results were obtained with the glutathione peroxidase mimetic and free radical scavenger ebselen (27) and with the combination of the hydroxyl radical scavenger dimethyl sulfoxide with the spin-trap TEMPOL [the need for both dimethyl sulfoxide and TEMPOL to be present might be explained by the fact that the methyl radical, formed when hydroxyl radicals are scavenged by dimethyl sulfoxide (28), still exerts some toxicity unless it is scavenged by another substance such as TEMPOL]. Similarly, these lipophilic antioxidants afforded strong protection to cultured liver endothelial cells ( Fig. 8B, Table 1). In addition, in liver endothelial cells, transition metal chelators such as 1,10-phenanthroline and 2,2'-dipyridyl or the chelator deferoxamine, which is relatively specific for iron(III), almost completely inhibited cold-induced apoptosis ( Fig. 8D and Fig. 9D, Table 1), whereas in hepatocytes they provided partial protection ( Fig. 8C, Table 1) but—in contrast to DMSO/TEMPOL—appeared to be able to inhibit cold-induced DNA fragmentation ( Fig. 5). These results suggest that the formation of reactive oxygen species (ROS) is a key mediator of cold-induced apoptosis. In line with the formation of ROS, lipid peroxidation was found to occur; lipid hydroperoxides were already formed during cold incubation of hepatocytes and further increased during rewarming ( Table 2).
This was confirmed by an increase in the amount of released TBARS ( Fig. 10).
During warm incubation, in contrast, there was almost no lipid peroxidation ( Table 2, Fig. 10). As in hepatocytes, a release of TBARS—although at lower levels than in hepatocytes—could be observed during cold incubation/rewarming of liver endothelial cells (data not shown). In both cell types, the lipophilic antioxidant BHT as well as the iron chelator deferoxamine completely inhibited the formation of TBARS.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figs. 1–3), ruffling of the nuclear membranes ( Fig. 2D), increased nuclear stainability, late loss of plasma membrane integrity ( Fig. 2C, D), pronounced bleb formation ( Figs. 1 and 3; with blebs containing organelles, Fig. 1C, D and Fig. 3C), leading to the formation of `apoptotic bodies' ( Fig. 1C), and DNA fragmentation ( Figs. 4 and 5).
Experiments with cultured liver endothelial cells yielded largely similar results. Hypothermia/rewarming induced an injury that was characterized by marked condensation of the nucleus ( Fig. 7and Fig. 9C) associated with increased nuclear stainability, pronounced bleb formation ( Fig. 7) with blebs containing organelles, and a late loss of the integrity of the plasma membrane ( Fig. 9C). It was possible to demonstrate chromatin condensation and margination by using electron microscopy. Only the results on DNA fragmentation were not clear-cut. This might be due either to cleavage of the chromatin to 50 kb fragments, which are not consistently detected by the ELISA method used, or to a minor role of DNA fragmentation in this injury to cultured liver endothelial cells. However, although DNA fragmentation has long been the hallmark of apoptosis (6, 7), it is now known that apoptosis can also occur in the absence of DNA fragmentation (7–9, 11, 29, 30). This is especially true for established cell lines (31). It is therefore now generally accepted that morphological criteria and not the absence of DNA fragmentation should govern the decision as to whether a cell injury is necrotic or apoptotic in nature (8, 10, 11). Using these criteria, the hypothermia/rewarming injury in cultured liver endothelial cells also appears to be an apoptotic form of cell death.
This apoptosis occurred under normal cell culture conditions, but only when the cells had been exposed to hypothermia for several hours ( Fig. 6; Table 1). In both cell types, the occurrence and extent of the apoptotic injury depended on the duration of the cold incubation ( Fig. 6). These results show that apoptosis was triggered by hypothermia as such, although most of the features indicative of apoptosis did not develop until the cells were returned to 37°C. Thus, the injury to cultured hepatocytes and liver endothelial cells in this setting is a cold-induced apoptosis. The rapidity with which this apoptotic process occurred after rewarming is fairly unusual for apoptotic forms of cell death, but is probably explained by part of the apoptotic pathway having already been covered during the cold incubation.
Cold or hypothermia, although often used as a means of reducing cellular alterations or injury during periods of storage or transport of biological material, has been described in the past as provoking injury in its own right (1–3, 12, 14). This hypothermia injury has been attributed to disturbances in cellular ion homeostasis, especially sodium homeostasis: An influx of sodium—no longer counteracted by the Na+-K+-ATPase paralyzed by hypothermia and followed by an influx of chloride and subsequently of water—was thought to lead to cellular swelling (3–5, 13, 15, 32), i.e., hypothermia injury was believed to occur in the typical setting of a necrotic type of cell injury. Against this background, the induction of apoptosis by hypothermia is an astonishing finding. However, recent results show that these old concepts concerning cellular ion homeostasis under hypothermic conditions might need some reevaluation. In liver endothelial cells and hepatocytes, there was no increase in the cellular sodium content within 3 h of cold incubation, but an early and massive efflux of sodium and chloride upon initiation of cold incubation (33). Furthermore, hypothermia injury occurred independent of whether cold incubation was performed in sodium-rich or sodium-poor solutions (34–36).
One of the characteristics of apoptosis is that it is an active process executed by the cell, involving distinct cellular signal transduction pathways (6, 7). These pathways are known to differ depending on the cell type and the inducer of apoptosis (7, 8, 37). However, cellular calcium homeostasis and the release of reactive oxygen species have turned out to be key signaling pathways, involved in many different forms of apoptotic cell death and in many diverse cell types (7, 37, 38). Having observed that hypoxia can partially inhibit cold-induced apoptosis ( Table 1) and that cultured liver endothelial cells and hepatocytes suffer a free radical-mediated injury when they are incubated for prolonged periods (for some days) under hypothermic conditions (without rewarming; ref 36), we considered reactive oxygen species to be a very likely mediator of cold-induced apoptosis. We tested this hypothesis by using an array of cell-permeable antioxidants and free radical scavengers. The lipophilic antioxidants almost completely inhibited cold-induced apoptosis in both cell types ( Figs. 8 and 9, Table 1). Similarly, the iron chelators provided complete protection in liver endothelial cells; in hepatocytes they also provided protection, but were not as effective as the other antioxidants. Furthermore, as an indication of the action of reactive oxygen species, we observed the accumulation of products of lipid peroxidation during cold incubation and also upon rewarming; there was a marked increase in the amount of lipid hydroperoxides formed and of thiobarbituric acid-reactive substances (an indicator of the formation of malondialdehyde) released ( Table 2, Fig. 10). Controls kept at 37°C for the whole incubation time hardly showed any evidence of lipid peroxidation, confirming that this process was cold induced. Together, these results suggest that ROS are decisively involved in the signaling and/or execution of cold-induced apoptosis. The key role of ROS in this model contrasts with its role in some other models of apoptosis where ROS release is secondary to other apoptotic changes and/or constitutes one of several events during the apoptotic effector or degradation phase, but is not obligatory for apoptosis to occur (7, 39).
Although both hepatocytes and liver endothelial cells showed cold-induced apoptosis that was mediated by reactive oxygen species, some differences still seem to exist between the two cell types. In liver endothelial cells, all inhibitors used provided complete inhibition and the hydroxyl radical appears to be virtually the only reactive species involved, as evidenced by the complete inhibition achieved by transition metal/iron chelators. In hepatocytes, on the other hand, other reactive oxygen species might also be involved: lipophilic antioxidants/scavengers inhibited cold-induced apoptosis completely, transition metal chelators only partially. It remains to be established whether these differences are due to the differences in mitochondrial oxygen metabolism known to exist between hepatocytes and microvascular endothelial cells or whether other factors are involved.
It is well known that apoptosis is a very complex phenomenon involving many cellular components and mechanisms interacting in the signaling and the effector phase (7, 8, 39). Of these, mitochondrial alterations and the activation of caspases have emerged as very general features involved in many models of apoptosis (7, 39, 40). Further study of the mechanism of cold-induced apoptosis might prepare the ground for additional interventions, e.g., inhibition of the apoptotic proteolytic machinery.
Free radical-mediated, cold-induced apoptosis affected cells as different as hepatocytes and microvascular liver endothelial cells, and as different as primary cells and a cell line. This renders it likely that cold-induced apoptosis might also affect other cells, especially since we found that free radical-mediated hypothermia injury can also be observed in isolated rabbit proximal tubules (41) and in rat alveolar macrophages (N. M. Murawski, U. Rauen, H. de Groot, unpublished result) upon prolonged cold incubation. Free radicals might also be involved in `cold shock'-induced apoptosis, an experimental model considered to be clearly different from the model of prolonged hypothermia used here (42). In cold shock-induced apoptosis, especially drastic procedures have been used for cooling and rewarming, and the cells have been kept in the cold only for short periods (in most cases, 1 h; refs 42–46) since prolonged cold incubation was considered to induce necrotic injury (42, 46, 47). Alterations in the cellular calcium homeostasis have been suggested to be the decisive mediator of this apoptosis in synovial cells (44), although this could not be reproduced in thymoma cells (43). In Burkitt lymphoma cells, a failure to inhibit their constitutively active apoptotic program after cold shock has been suggested as the causative factor (46); the involvement of ROS has not been studied in any of these models. Thus, it remains to be established whether free radical-mediated, cold-induced apoptosis and cold shock-induced apoptosis are different entities or two facets of the same type of injury. However, though the very drastic procedures used for cooling and rewarming in cold shock were considered by some authors to have no biological relevance (44) [cold shock-induced apoptosis was regarded solely as a model for studying the mechanisms of apoptosis (44, 45)], a potential relevance of cold-induced apoptosis as described here is much more obvious.
Clinical organ preservation for transplantation is usually performed at about 2–4°C (48). The cooling and rewarming of the whole organ are not instantaneous and cold preservation times for the liver are usually in the range of 5 to 24 h (48, 49), similar to the times used in this study. Organ preservation is usually done by `simple cold storage' (16), involving hypothermia and ischemia at the same time, both possibly contributing to tissue injury. Recently, it was reported that an apoptosis of hepatocytes can be observed after cold preservation and reperfusion of pig livers (50), and that apoptotic hepatocytes and liver endothelial cells can be observed after cold preservation and reperfusion of human livers (51). According to current thinking, this apoptosis has been attributed to ischemia/reperfusion, although the experimental conditions did not allow any distinction to be drawn between the effects of ischemia and those of hypothermia.
The entity ischemia/reperfusion is usually considered to be the decisive factor for preservation injury (10, 13, 15, 16). In contrast, we previously demonstrated that in a cell culture model designed to mimic the conditions of clinical organ preservation (cold hypoxic incubation in the organ preservation solution `University of Wisconsin solution', followed by normoxic rewarming in cell culture medium), cultured liver endothelial cells suffered an injury that was cold preservation-induced rather than being due to hypoxia/reoxygenation (52). The notion that cold ischemia cannot simply be delayed ischemia, but that hypothermia has its own (injurious) aspects under the conditions of organ preservation, is reinforced by experimental reports suggesting that the liver injury occurring after cold ischemia differs from the injury during and after warm ischemia in terms of the mechanisms predominating (10, 53, 54). Our results and these previous data together suggest that the cold-induced apoptosis described here might contribute to preservation injury of the liver. Indeed, taking account of the results obtained with other cell types (see above), it might also apply to other organs. Thus, cold-induced apoptosis might be of considerable clinical significance. A rational antioxidant therapy, taking into account the mechanistic differences between the different cells, might offer considerable improvement of organ quality.
For scientific purposes, too, cold-induced apoptosis might be a relevant issue. When data obtained from cold-stored cells/tissues are interpreted, it should be kept in mind that apoptotic features can appear within a few minutes after rewarming. Furthermore, cold storage or handling of material used for the isolation of cells should be reconsidered, especially when hypoxia does not pose a problem. Cold-induced apoptosis is also one of the rare forms of apoptosis due to alterations of the physical environment of the cells, similar only to heat- or cold shock-induced apoptosis (9, 55); since it is not linked to a receptor, it might provide a straightforward model for assessing endogenous differences in the apoptotic pathways of different cell types (independent of receptor status, etc.).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbant assay; H33342, Hoechst 33342; HBSS, Hanks' balanced salt solution; LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; ROS, reactive oxygen species; TBARS, thiobarbituric acid-reactive substances; TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl.
Received for publication June 18, 1998.
Revision received August 13, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 K. Thorlacius, J. E. Slotta, M. W. Laschke, Y. Wang, M. D. Menger, B. Jeppsson, and H. Thorlacius Protective effect of fasudil, a Rho-kinase inhibitor, on chemokine expression, leukocyte recruitment, and hepatocellular apoptosis in septic liver injury J. Leukoc. Biol., May 1, 2006; 79(5): 923 - 931. [Abstract] [Full Text] [PDF]
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 L. Richert, M. J. Liguori, C. Abadie, B. Heyd, G. Mantion, N. Halkic, and J. F. Waring GENE EXPRESSION IN HUMAN HEPATOCYTES IN SUSPENSION AFTER ISOLATION IS SIMILAR TO THE LIVER OF ORIGIN, IS NOT AFFECTED BY HEPATOCYTE COLD STORAGE AND CRYOPRESERVATION, BUT IS STRONGLY CHANGED AFTER HEPATOCYTE PLATING Drug Metab. Dispos., May 1, 2006; 34(5): 870 - 879. [Abstract] [Full Text] [PDF]
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 M. Bartels-Stringer, J. F. M. Wetzels, A. C. Wouterse, E. Steenbergen, F. G. M. Russel, and C. Kramers Iron chelators do not reduce cold-induced cell injury in the isolated perfused rat kidney model Nephrol. Dial. Transplant., December 1, 2005; 20(12): 2646 - 2653. [Abstract] [Full Text] [PDF]
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X. Li, D. Klintman, Q. Liu, T. Sato, B. Jeppsson, and H. Thorlacius Critical role of CXC chemokines in endotoxemic liver injury in mice J. Leukoc. Biol., March 1, 2004; 75(3): 443 - 452. [Abstract] [Full Text] [PDF]
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 M. L Riess, A. K.S Camara, L. G Kevin, J. An, and D. F Stowe Reduced reactive O2 species formation and preserved mitochondrial NADH and [Ca2+] levels during short-term 17 {degrees}C ischemia in intact hearts Cardiovasc Res, February 15, 2004; 61(3): 580 - 590. [Abstract] [Full Text] [PDF]
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 D. Klintman, X. Li, and H. Thorlacius Important Role of P-Selectin for Leukocyte Recruitment, Hepatocellular Injury, and Apoptosis in Endotoxemic Mice Clin. Vaccine Immunol., January 1, 2004; 11(1): 56 - 62. [Abstract] [Full Text] [PDF]
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 H. Martin, L. Richert, and A. Berthelot Magnesium Deficiency Induces Apoptosis in Primary Cultures of Rat Hepatocytes J. Nutr., August 1, 2003; 133(8): 2505 - 2511. [Abstract] [Full Text] [PDF]
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 A. Parolari, P. Rubini, A. Cannata, L. Bonati, F. Alamanni, E. Tremoli, and P. Biglioli Endothelial damage during myocardial preservation and storage Ann. Thorac. Surg., February 1, 2002; 73(2): 682 - 690. [Abstract] [Full Text] [PDF]
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 M. J. Gomez-Lechon, E. O'Connor, J. V. Castell, and R. Jover Sensitive Markers Used to Identify Compounds That Trigger Apoptosis in Cultured Hepatocytes Toxicol. Sci., February 1, 2002; 65(2): 299 - 308. [Abstract] [Full Text] [PDF]
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 U. RAUEN, F. PETRAT, T. LI, and H. DE GROOT Hypothermia injury/cold-induced apoptosis--evidence of an increase in chelatable iron causing oxidative injury in spite of low O2-/H2O2 formation FASEB J, October 1, 2000; 14(13): 1953 - 1964. [Abstract] [Full Text]
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M. Shimura, Y. Osawa, A. Yuo, K. Hatake, F. Takaku, and Y. Ishizaka Oxidative stress as a necessary factor in room temperature-induced apoptosis of HL-60 cells J. Leukoc. Biol., July 1, 2000; 68(1): 87 - 96. [Abstract] [Full Text]
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 T. Stefanec Endothelial Apoptosis: Could It Have a Role in the Pathogenesis and Treatment of Disease? Chest, March 1, 2000; 117(3): 841 - 854. [Abstract] [Full Text] [PDF]
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 F. Dignat-George, A. Blann, and J. Sampol Circulating endothelial cells in acute coronary syndromes Blood, January 15, 2000; 95(2): 728 - 728. [Full Text] [PDF]
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T. Stefanec Circulating Apoptotic Endothelial Cells Blood, August 15, 1999; 94(4): 1482 - 1483. [Full Text] [PDF]
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