Recombinant human erythropoietin alpha (rHu-EPO; stocks: commercial ERYPO® Product Line, 10,000 or 40,000 U/ml; 5.0 mg glycine/ml, preservative free without albumin and citrate) was kindly provided by Janssen-Cilag (Neuss, Germany). Primers for real-time polymerase chain reactions (PCRs) were obtained from Invitrogen (Karlsruhe, Germany), the RC DC Protein Assay® was purchased from Bio-Rad (Munich, Germany), and avian myeloblastosis virus reverse transcriptase from Promega (Heidelberg, Germany). Fetal calf serum, 2,2'-dipyridyl, Leibovitz L-15 medium, and Ponceau S came from Sigma-Aldrich (Taufkirchen, Germany). The fluorescent dye SYBR Green® (for real time PCR) was obtained from Eurogentec (Verviers, Belgium). The Primer Express software and the Gene Amp 5700 Sequence Detection System were obtained from Applied Biosystems (Weiterstadt, Germany). For the determination of total and phosphorylated STAT-3 and STAT- 5 and phosphorylated JAK-2 the PhosphoPlus®Stat3 (Tyr705), the PhosphoPlus®Stat5 (Tyr694) and the Phospho-Jak2 (Tyr1007/1008) antibody kits from Cell Signalling Technology® (Frankfurt, Germany) were used, respectively. The NE-PER® nuclear and cytoplasmic extraction kit was purchased from Pierce Biotechnology (Schwerte, Germany), EPOR antibodies C-20 (sc695) were from Santa Cruz (Heidelberg, Germany) and gas mixtures from Messer Griesheim (Krefeld, Germany).
Male Wistar rats (300–350 g) were obtained from the Zentrales Tierlaboratorium (Universitätsklinikum Essen). Animals were kept under standard conditions with free access to food and water. All animals received humane care in compliance with the institutional guidelines.
Perfusion of the rat liver in situ
Rat livers were perfused as previously described  with some modifications. Briefly, subsequent to ketamine/xylazine anaesthesia (80 mg and 6 mg, respectively, per kg body weight via intraperitoneal injection) animals underwent median laparotomy. Then the portal vein was cannulated and a peristaltic pump used to start perfusion of the liver at 30 ml/min with warm (37°) modified Krebs-Henseleit (KH) buffer (115 mM NaCl, 25 mM NaHCO3, 5.9 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 1.2 mM Na2SO4, 2.5 mM CaCl2, 20 mM HEPES, pH 7.4) that had been equilibrated with 95% O2/5% CO2 (carbogen); to avoid congestion of the liver, the Vena cava inferior was cut distally from the Venae renalis immediately after perfusion had started. Subsequent to thoracotomy the perfusate was drained through a second cannula that had been placed within the suprahepatic Vena cava inferior via the right atrium and the infrahepatic Vena cava inferior was ligated between the liver and the Venae renalis to avoid loss of the perfusate. After a short (2–3 minutes) perfusion period that was required to completely remove the blood within the liver, rHu-EPO was added to the reservoir bottle and rapidly mixed with the KH buffer to gain 8.9 U/ml; in controls no rHu-EPO was added. Then non-recirculating in situ liver perfusion was continued for further 15 minutes. Afterwards, the complete liver was removed and rapidly frozen in liquid nitrogen until tissue processing for analytic assays.
Isolation of the hepatocytes, culture and pre-treatment with rHu-EPO
Hepatocytes were isolated from male Wistar rats, seeded onto collagen-coated culture flasks or glass coverslips and cultured as described previously . Experiments were started 20–22 hours after isolation of the cells. Hepatocytes were pre-incubated with rHu-EPO (0.2–100 U/ml) for 15 minutes, 2, 3, 18 or 20 hours (over night, subsequent to their isolation) in L-15 medium at 74% N2/21% O2/5% CO2 (37°C, humidified atmosphere).
Culture of UT-7 cells with rHu-EPO
UT-7 cells, an erythropoietin-responsive hematopoietic cell line , which was generously provided by P. Mayeux (INSERM 152, Hopital Cochin, Paris, France), served as a positive control for EPOR signalling. UT-7 cells were grown in suspension in RPMI 1640 cell culture medium from BioWitthaker (Verviers, Belgium) supplemented with 10% fetal bovine serum from Biochrom (Berlin, Germany) and 1 U rHu-EPO/ml; cells were splitted twice/week. For experiments, cells were used at a density of 106 cells/ml and kept in medium without rHu-EPO for 1 hour before the start of the experiment.
Induction of hypoxia/reoxygenation in cultured hepatocytes
At the beginning of the experiments, hepatocytes were washed three times with Hanks' balanced salt solution (137.0 mM NaCl, 5.4 mM KCl, 1.0 mM CaCl2, 0.5 mM MgCl2, 0.4 mM KH2PO4, 0.4 mM MgSO4, 0.3 mM Na2HPO4, 25.0 mM HEPES, pH 7.4) and then covered with KH buffer at 37°C. Normoxic incubations (controls) were performed in a humidified atmosphere of 74% N2/21% O2/5% CO2. Hypoxic conditions were established by saturating the incubation solution with 95% N2/5% CO2 before adding it to the cells, followed by gentle flushing of the culture flasks with the gas mixture through cannulae piercing the rubber stoppers of the flasks, as described previously [21, 23–24]. After locking the flasks, cells were incubated for 5–6.5 hours within an incubator at 37°C. The flasks were again flushed with the respective gas mixtures each time a sample was taken. RHu-EPO (0.2–100 U/ml) was added to the KH buffer just prior to its addition to the cells. Some experiments were performed with rHu-EPO that had been heat-inactivated by boiling for 30 minutes. Where indicated, glycine (66 μM, 0.66 mM or 10 mM) was added to the cells prior to the start of the hypoxic treatment. Reoxygenation of the cells was performed after different hypoxic periods by gassing the flasks with 74% N2/21% O2/5% CO2 for 2 minutes followed by an incubation in this atmosphere within an incubator at 37°C. Moderate hypoxia was achieved by placing the culture dishes in an incubator with 1% or 3% O2 (5% CO2 and N2 as balance) for the indicated time periods.
For fluorescence microscopy, 6-well cell culture plates, containing collagen-coated coverslips with adherent hepatocytes that had been pre-treated or not with rHu-EPO for 20 hours, washed and supplied with KH buffer ± rHu-EPO (10 or 100 U/ml), were placed in air-tight vessels that were then flushed for 10 minutes either with 74% N2/21% O2/5% CO2 or 95% N2/5% CO2. After locking the vessels, cells were incubated for 2–4 hours at 37°C; note that hepatocytes died more slowly in the hypoxic vessel than in culture flasks (see above), as pre-equilibration of the medium with 95% N2/5% CO2 was not possible. Afterwards, the buffer was removed and the cells were incubated for 24 hours in L-15 medium (37°C) again with or without rHu-EPO at 74% N2/21% O2/5% CO2.
Induction of cold-induced apoptosis
Hypothermic injury was induced according to refs. [25, 26]. Cultured hepatocytes were washed with Hanks' balanced salt solution and cells covered with University of Wisconsin solution or L-15 medium with or without rHu-EPO (2, 10, 50 or 100 U/ml; medium supplemented as described previously ) at room temperature. Incubations in cell culture medium were performed in air-tight vessels which were flushed with 74% N2/21% O2/5% CO2, cells in University of Wisconsin solution were exposed to room air; cells were incubated at 4°C for 24 hours. In some experiments, the iron chelator 2,2'-dipyridyl (100 μM) was added at the beginning of the cold incubation as a positive control for protection from cold-induced injury in University of Wisconsin solution and, in other experiments, in order to assess an effect of rHu-EPO to the weaker iron-independent component of cold-induced apoptosis to hepatocytes in L-15 medium [27, 28]. Increases in the hepatocyte chelatable iron pool were provoked in cell culture medium at 74% N2/21% O2/5% CO2 by the addition of the membrane-permeable Fe(III)/8-hydroxyquinoline complex (; prepared as described previously ).
Assessment of cellular and nuclear alterations (apoptotic vs. necrotic cell death)
Hepatocyte morphology after various periods of hypoxia and reoxygenation, respectively, was assessed by phase contrast microscopy. Nuclear morphology was assessed by fluorescence microscopy according to ref. . Twenty fields of vision (original magnification: × 400) à 15–30 hepatocytes were visually evaluated (blinded) per condition and experiment.
Loss of cell viability was assessed by the determination of extracellular, i.e. released, lactate dehydrogenase (LDH) activity; released LDH activity is given as percentage of total LDH activity .
Detection and quantification of EPOR, EPO and Bcl-2 mRNA
Isolation of total RNA from cultured hepatocytes and perfused liver, reverse transcription into cDNA and real-time PCR were performed as described . Primers for real-time PCR were designed to yield amplicon sizes of 150 bp, annealing temperature of 60°C and CG content of about 60%. Primers for real-time PCRs were: EPOR upstream EPOR1 5'-ccg gga tgg gct tca act ac-3' and downstream EPOR2 5'-tcc agt ggc aca aaa ctc gac-3' spanning nucleotides 291 – 441 or for detection of full length EPOR mRNA EPOR3 5'-ggg cta cat cat gga cca act c-3'and EPOR4 5'-ggc tgg agt cct agg agc agg cc-3' spanning nucleotides 71 – 1616 of rat EPO receptor sequence NM_017002.2. Primers for EPO mRNA were upstream 5'-ggt cac ctg tcc cct ctc ct -3' and downstream 5'-ctg gag tgt cca tgg gac ag-3' and for Bcl-2 upstream 5'-gga cgc gaa gtg cta ttg g-3' and downstream 5'-ccg aac tca aag aag gcc ac-3', respectively. The identities of the amplification products for EPOR were verified by sequencing (SEQLAB; Göttingen, Germany).
Determination of activated JAK-2, STAT-3 and STAT-5
Cultured rat hepatocytes and UT-7 cells were treated with 10 U rHu-EPO/ml for 15 minutes and rat livers perfused in situ with 8.9 U/ml rHu-EPO (for 15 minutes). Cellular extracts were prepared using the NE-PER® nuclear and cytoplasmic extraction kit and used for Western blotting to detect nuclear phosphorylated and thus activated JAK-2 (pJAK-2), STAT-3 (pSTAT-3) and STAT-5 (pSTAT-5) exactly as described in the protocol from Cell Signalling Technology®. As positive controls, extracts from the EPO-responsive cell line UT-7 were used. Western blotting and detection were performed as described in Frede et al. .
All experiments were performed in duplicate and repeated at least 3 times. Traces and blots shown in the figures are representative for all the corresponding experiments carried out. Data are expressed as mean values ± or + SD. Data obtained from two groups were compared by means of Student's t test (matched values, two-tailed, paired) and comparisons among multiple groups were performed using an analysis of variance (ANOVA). A p-value of < 0.05 was considered significant.