Erythropoietin Amplifies Stroke-Induced Oligodendrogenesis in the Rat

Background

Erythropoietin (EPO), a hematopoietic cytokine, enhances neurogenesis and angiogenesis during stroke recovery. In the present study, we examined the effect of EPO on oligodendrogenesis in a rat model of embolic focal cerebral ischemia.

Methodology and Principal Findings

Recombinant human EPO (rhEPO) at a dose of 5,000 U/kg (n = 18) or saline (n = 18) was intraperitoneally administered daily for 7 days starting 24 h after stroke onset. Treatment with rhEPO augmented actively proliferating oligodendrocyte progenitor cells (OPCs) measured by NG2 immunoreactive cells within the peri-infarct white matter and the subventricular zone (SVZ), but did not protect against loss of myelinating oligodendrocytes measured by cyclic nucleotide phosphodiesterase (CNPase) positive cells 7 days after stroke. However, 28 and 42 days after stroke, treatment with rhEPO significantly increased myelinating oligodendrocytes and myelinated axons within the peri-infarct white matter. Using lentivirus to label subventricular zone (SVZ) neural progenitor cells, we found that in addition to the OPCs generated in the peri-infarct white matter, SVZ neural progenitor cells contributed to rhEPO-increased OPCs in the peri-infarct area. Using bromodeoxyuridine (BrdU) for birth-dating cells, we demonstrated that myelinating oligodendrocytes observed 28 days after stroke were derived from OPCs. Furthermore, rhEPO significantly improved neurological outcome 6 weeks after stroke. In vitro, rhEPO increased differentiation of adult SVZ neural progenitor cells into oligodendrocytes and enhanced immature oligodendrocyte cell proliferation.

Conclusions

Our in vivo and in vitro data indicate that EPO amplifies stroke-induced oligodendrogenesis that could facilitate axonal re-myelination and lead to functional recovery after stroke.     

Phosphorylatable and epitope-tagged human erythropoietins: Utility and purification of native baculovirus-derived forms

The hematopoietic glycopeptide erythropoietin (EPO) is a prime regulator of red cell production in mammals, yet the precise nature of its interaction with specific cell surface receptors is poorly understood. Towards defining domains of EPO that are involved in receptor activation, we have developed (i) conditions for the expression of recombinant human EPO (rhEPO) at high levels in SF9 cells using modified 2- and 5-liter stirred reactors, (ii) a two-step procedure for the purification of this EPO without denaturation, and (iii) forms of EPO tagged with either a hemagglutinin influenza virus epitope or a consensus sequence for in vitro phosphorylation. Compared to EPO expressed in mammalian cells, rhEPO from SF9 cells in N-glycosylated with simple, neutral oligosaccharides of limited size, yet as purified presently using nondenaturing procedures, possesses exceptionally high in vitro activity (> or = 500,000 U/mg). Thus, this form of EPO should prove advantageous for direct physicochemical analyses. Regarding epitope-tagged and phosphorylatable EPOs, forms modified at the amino terminus (Ala1) fully retained receptor binding and in vitro biological activities. In contrast, forms modified at the carboxy terminus (Cys161) were inactive and did not compete for receptor binding, indicating that integrity of this domain is essential for receptor recognition. For active amino-terminal-modified forms, the specific binding of MAb 12CA5 to native HAI-EPO and the utility of 32P-labeled PHOS-EPO in receptor binding and internalization studies also were demonstrated. The development of these unique, highly active forms of human EPO should advance studies of essential interactions between this cytokine and its cell surface receptor.     

A statistical pattern recognition approach for determining cellular viability and lineage phenotype in cultured cells and murine bone marrow.

BACKGROUND: Cellular binding of annexin V and membrane permeability to 7-aminoactinomycin D (7AAD) are important tools for studying apoptosis and cell death by flow cytometry. Combining viability markers with cell surface marker expression is routinely used to study various cell lineages. Current classification methods using strict thresholds, or "gates," on the fluorescent intensity of these markers are subjective in nature and may not fully describe the phenotypes of interest. We have developed objective criteria for phenotypic boundary recognition through the application of statistical pattern recognition. This task was achieved using artificial neural networks (ANNs) that were trained to recognize subsets of cells with known phenotypes, and then used to determine decision boundaries based on statistical measures of similarity. This approach was then used to test the hypothesis that erythropoietin (EPO) inhibits apoptosis and cell death in erythroid precursor cells in murine bone marrow. METHODS: Our method was developed for classification of viability using an in vitro cell system and then applied to an ex vivo analysis of murine late-stage erythroid progenitors. To induce apoptosis and cell death in vitro, an EPO-dependent human leukemic cell line, UT-7(EPO) cells were incubated without recombinant human erythropoietin (rhEPO) for 72 h. Five different ANNs were trained to recognize live, apoptotic, and dead cells using a "known" subset of the data for training, and a K-fold cross validation procedure for error estimation. The ANNs developed with the in vitro system were then applied to classify cells from an ex vivo study of rhEPO treated mice. Tg197 (human tumor necrosis-alpha transgenic mice, a model of anemia of chronic disease) received a single s.c. dose of 10,000 U/kg rhEPO and femoral bone marrow was collected 1, 2, 4, and 8 days after dosing. Femoral bone marrow cells were stained with TER-119 PE, CD71 APC enable identification of erythroid precursors, and annexin V FITC and 7AAD to identify the apoptotic and dead cells. During classification forward and side angle light scatter were also input to all pattern recognition systems. RESULTS: Similar decision boundaries between live, apoptotic, and dead cells were consistently identified by the neural networks. The best performing network was a radial basis function multi-perceptron that produced an estimated average error rate of 4.5% +/- 0.9%. Using these boundaries, the following results were reached: depriving UT-7(EPO) cells of rhEPO induced apoptosis and cell death while the addition of rhEPO rescued the cells in a dose-dependent manner. In vivo, treatment with rhEPO resulted in an increase of live erythroid cells in the bone marrow to 119.8% +/- 9.8% of control at the 8 day time point. However, a statistically significant transient increase in TER-119(+) CD71(+) 7AAD(+) dead erythroid precursors was observed at the 1 and 2 day time points with a corresponding decrease in TER-119(+) CD71(+) 7AAD(-) Annexin V(-) live erythroid precursors, and no change in the number of TER-119(+) CD71(+) annexin V(+) 7AAD(-) apoptotic erythroid precursors in the bone marrow. CONCLUSIONS: A statistical pattern recognition approach to viability classification provides an objective rationale for setting decision boundaries between "positive" and "negative" intensity measures in cytometric data. Using this approach we have confirmed that rhEPO inhibits apoptosis and cell death in an EPO dependent cell line in vitro, but failed to do so in vivo, suggesting EPO may not act as a simple antiapoptotic agent in the bone marrow. Rather, homeostatic mechanisms may regulate the pharmacodynamic response to rhEPO.     

Intrinsic and extrinsic erythropoietin enhances neuroprotection against ischemia and reperfusion injury in vitro

This study was designed to investigate the neuroprotective effect of intrinsic and extrinsic erythropoietin (EPO) against hypoxia/ischemia, and determine the optimal time-window with respect to the EPO-induced neuroprotection. Experiments were conducted using primary mixed neuronal/astrocytic cultures and neuron-rich cultures. Hypoxia (2%) induces hypoxia-inducible factor-1alpha (HIF-1alpha) activity followed by strong EPO expression in mixed cultures and weak expression in neuron-rich cultures as documented by both western blot and RT-PCR. Immunoreactive EPO was strongly detected in astrocytes, whereas EPOR was only detected in neurons. Neurons were significantly damaged in neuron-rich cultures but were distinctly rescued in mixed cultures. Application of recombinant human EPO (rhEPO) (0.1 U/mL) within 6 h before or after hypoxia significantly increased neuronal survival compared with no rhEPO treatment. Application of rhEPO after onset of reoxygenation achieved the maximal neuronal protection against ischemia/reperfusion injury (6 h hypoxia followed 24 h reoxygenation). Our results indicate that HIF-1alpha induces EPO gene released by astrocytes and acts as an essential mediator of neuroprotection, prove the protective role of intrinsic astrocytic-neuronal signaling pathway in hypoxic/ischemic injury and demonstrate an optimal therapeutic time-window of extrinsic rhEPO in ischemia/reperfusion injury in vitro. The results point to the potential beneficial effects of HIF-1alpha and EPO for the possible treatment of stroke.     

EPO/EPOR 在非小性细胞肺癌发展中的作用研究

目的：研究促红细胞生成素（erythropoietin, EPO）及其受体（EPOR）在非小性细胞肺癌中的生物学作用。方法：收集27 例非小 性细胞肺癌（NSCLC）,免疫组织化学方法检测肺癌组织中EPO 和EPOR的表达;观察人源重组EPO（rhEPO）对HCC15 和 HCC1819 细胞活力和细胞周期的影响;分析缺氧对NSCLC细胞EPO 及EPOR 表达的影响。结果：27例非小细胞肺癌的组织标 本中13 例表达EPO,表达率为48 %,25 例表达EPOR,表达率为92 %。rhEPO明显增加了高表达EPOR 的HCC1819 细胞克隆 数,而对低表达EPOR 的HCC15 细胞的克隆形成没有影响。rhEPO增强了HCC1819 的细胞活力,但以siRNA干涉HCC1819 EPOR后,EPO对HCC1819 细胞活力增强作用消失。rhEPO 明显增加了HCC1819 细胞的细胞周期。缺氧促进了HCC1819 细胞的 EPO 的表达,增强了细胞活力。结论：EPO 和EPOR在非小性细胞肺癌中表达增高,EPO 通过EPOR 促进了NSCLC 细胞的增殖, 缺氧诱导了NSCLC 细胞EPO的表达。     

Development of a chemiluminescent imaging assay for the detection of anti‐erythropoietin antibody in human sera

Measuring low amounts of anti‐erythropoietin antibodies (anti‐EPO Abs) is important to evaluate the therapeutic safety of recombinant human erythropoietin (rhEPO). In this work, a simple, sensitive and high‐throughput chemiluminescent (CL) imaging assay was developed for the detection of anti‐EPO Abs in human sera. The influence of several physicochemical parameters, such as coating conditions, incubation time, detergent concentration and exposure time, were investigated. A calibration curve was established and the range of quantitative detection was 0.12–13.91 ng/mL. The limit of detection (LOD, 3σ) for the CL‐imaging assay was 0.033 ng/mL. Compared to conventional colorimetric enzyme‐linked immunosorbent assay (ELISA), the LOD of the CL‐imaging assay is 50‐fold lower. The recoveries of anti‐EPO Abs in the fortified serum were in the range 87.1–116.9% using the present method, which highlighted the validity of the CL‐imaging assay system to accurately determine the anti‐EPO Abs in serum samples. CL‐imaging assay was used to evaluate the presence of anti‐EPO Abs in serum samples obtained from chronic renal failure (CRF) patients treated with rhEPO. Contrary to what was expected, the sera from CRF patients did not contain anti‐EPO Abs. Copyright © 2008 John Wiley & Sons, Ltd.     

Erythropoietin therapy for acute stroke is both safe and beneficial

BACKGROUND: Erythropoietin (EPO) and its receptor play a major role in embryonic brain, are weakly expressed in normal postnatal/adult brain and up-regulated upon metabolic stress. EPO protects neurons from hypoxic/ ischemic injury. The objective of this trial is to study the safety and efficacy of recombinant human EPO (rhEPO) for treatment of ischemic stroke in man. MATERIALS AND METHODS: The trial consisted of a safety part and an efficacy part. In the safety study, 13 patients received rhEPO intravenously (3.3 X 10(4) IU/50 ml/30 min) once daily for the first 3 days after stroke. In the double-blind randomized proof-of-concept trial, 40 patients received either rhEPO or saline. Inclusion criteria were age <80 years, ischemic stroke within the middle cerebral artery territory confirmed by diffusion-weighted MRI, symptom onset <8 hr before drug administration, and deficits on stroke scales. The study endpoints were functional outcome at day 30 (Barthel Index, modified Rankin scale), NIH and Scandinavian stroke scales, evolution of infarct size (sequential MRI evaluation using diffusion-weighted [DWI] and fluid-attenuated inversion recovery sequences [FLAIR]) and the damage marker S100ss. RESULTS: No safety concerns were identified. Cerebrospinal fluid EPO increased to 60-100 times that of nontreated patients, proving that intravenously administered rhEPO reaches the brain. In the efficacy trial, patients received rhEPO within 5 hr of onset of symptoms (median, range 2:40-7:55). Admission neurologic scores and serum S100beta concentrations were strong predictors ofoutcome. Analysis of covariance controlled for these two variables indicated that rhEPO treatment was associated with an improvement in follow-up and outcome scales. A strong trend for reduction in infarct size in rhEPO patients as compared to controls was observed by MRI. CONCLUSION: Intravenous high-dose rhEPO is well tolerated in acute ischemic stroke and associated with an improvement in clinical outcome at 1 month. A larger scale clinical trial is warranted.     

人促红细胞生成素基因在家蚕体中的高效表达

人促红细胞生成素(EPO)是一种调控红系干细胞增殖、分化和成熟的糖蛋白激素。将合成的EPO cDNA插入杆状病毒转移载体pBlueBacⅢ,使其置于Ph基因强启动子控制之下,获得了转移载体pBlueBacEPO。将pBlueBacEPO DNA与野生型BmNPV DNA共转染BmN细胞,经空斑纯化,获插入EPO cDNA的重组病毒rBmNPVEPO。经Sonthern杂交和PCR扩增鉴定证明人EPO基因已正确组建于BmNPV的预定位置。将重组病毒rBmNPVEPO穿刺接种5龄幼虫和蛹,收集感染第3～5d的幼虫血淋巴和3～6.5d蛹血淋巴。用ELISA检测幼虫血淋巴中EPO表达量高达62800u/mL,蛹血淋巴中表达量达74000u/mL。Western blot结果显示幼虫血淋巴和蛹血淋巴均有一条明显的免疫杂交带,分子量均约为26kD。用TF1细胞对幼虫表达产物进行了生物活性测定,每毫升血淋巴中EPO活性约为63000u。     

Raves and risks for erythropoietin

Global use of erythropoietin (EPO) continues to increase as a proven agent for the treatment of anemia. Yet, EPO is no longer believed to have exclusive biological activity in the hematopoietic system and is now considered applicable for a variety of disorders such as diabetes, Alzheimer's disease, and cardiovascular disease. Treatment with EPO is considered to be robust and can prevent metabolic compromise, neuronal and vascular degeneration, and inflammatory cell activation. On the converse side, observations that EPO administration is not without risk have fueled controversy. Here we present recent advances that have elucidated a number of novel cellular pathways governed by EPO to open new therapeutic avenues for this agent and avert its potential deleterious effects.     

The role of erythropoietin and its receptor in growth,survival and therapeutic response of human tumor cells: From clinic to bench — a critical review

Recombinant human erythropoietin (rhEPO) has been used clinically to alleviate cancer- and chemotherapy-related anemia. However, recent clinical trials have reported that rhEPO also may adversely impact disease progression and survival. The expression of functional EPO receptors (EPOR) has been demonstrated in many human cancer cells where, at least in vitro, rhEPO can stimulate cell growth and survival and may induce resistance to selected therapies.     

Hematopoietic cytokines--on the verge of conquering neurology

Two hematopoietic cytokines are currently gaining increasing attention within neurological research. Erythropoietin (EPO) and granulocyte-colony stimulating factor (G-CSF) have long been known for their ability to induce the proliferation of certain populations of hematopoietic lineage cells. However, it has recently been found that EPO, G-CSF, and their respective receptors are also expressed in the human central nervous system (CNS) and may be an important part of the brain's endogenous system of protection. Both hematopoietic cytokines have been shown to have neuroprotective potential in a variety of animal disease models both in vitro and in vivo, through the inhibition of apoptosis, induction of angiogenesis, exertion of anti-inflammatory and neurotrophic effects, as well as by the enhancement of neurogenesis. EPO and G-CSF have been extensively studied in the context of hematological disorders and have recently been successfully applied in the first clinical trials in stroke patients. Intravenous high-dose EPO therapy was associated with an improvement in the clinical outcome and preclinical studies with intravenous high-dose G-CSF therapy have clearly shown that it has considerable neuroprotective potential in the acute, as well as in the chronic phase of stroke. In this review, the current knowledge of the neuroprotective mechanisms of EPO and G-CSF is summarized with regard to in vitro and in vivo data. Focus is placed on the role of EPO in neurological disease models with an emphasis on its influence on functional outcome. New experimental results are assessed in detail and correlated with the findings of recent clinical studies.     

Potential use of transgenic domestic pigs expressing recombinant human erythropoietin in diabetes translation research

Recently, diabetes mellitus (DM) has shown rapid global increases with about five million deaths annually. Animal models are imperative to understand disease mechanisms and develop diagnostic, preventive, and therapeutic interventions in translational research. Rodent and mini-pig models have been established and widely used for DM research. However, domestic pig models are limited in spite of advantages such as pharmacokinetic and physiopathological availability. This study examines the potential use of domestic pigs expressing recombinant human erythropoietin (rhEPO) as disease and therapeutic response models for DM. We previously generated transgenic pigs (n?=?16, EPO Tg) in which rhEPO was expressed and circulated in all organs. Thirty-two pigs, including 16 controls, were fed high fat (HF) diets for 42 weeks. Subsequently, blood samples for chemical and metabolic analysis were collected after fasting for 24?h and glucose loading for oral glucose tolerance tests (OGTTs). We found increased activation of the PI3?K/Akt signaling pathway under hypoxic conditions after rhEPO treatment, and HF diet-inducible-obesity in the EPO Tg and control pigs. OGTTs showed lower fasting glucose levels in the EPO Tg pigs than in controls before and after the HF diet, suggesting that rhEPO may affect glucose concentrations. Insulin and C-peptide concentrations responded slowly to glucose administration and returned to initial levels after 2?h. The blood test results suggest that EPO might affect metabolic and chemical components such as glucose, high-density lipoprotein, glucagon, triglyceride, and free fatty acid. Our findings support the use of rhEPO transgenic domestic pigs as model animals for translational DM research.     

A small nonerythropoietic helix B surface peptide based upon erythropoietin structure is cardioprotective against ischemic myocardial damage

Strong cardioprotective properties of erythropoietin (EPO) reported over the last 10 years have been difficult to translate to clinical applications for ischemic cardioprotection owing to undesirable parallel activation of erythropoiesis and thrombogenesis. A pyroglutamate helix B surface peptide (pHBP), recently engineered to include only a part of the EPO molecule that does not bind to EPO receptor and thus, is not erythropoietic, retains tissue protective properties of EPO. Here we compared the ability of pHBP and EPO to protect cardiac myocytes from oxidative stress in vitro and cardiac tissue from ischemic damage in vivo. HBP, similar to EPO, increased the reactive oxygen species (ROS) threshold for induction of the mitochondrial permeability transition by 40%. In an experimental model of myocardial infarction induced by permanent ligation of a coronary artery in rats, a single bolus injection of 60 μg/kg of pHBP immediately after coronary ligation, similar to EPO, reduced apoptosis in the myocardial area at risk, examined 24 h later, by 80% and inflammation by 34%. Myocardial infarction (MI) measured 24 h after coronary ligation was similarly reduced by 50% in both pHBP- and EPO-treated rats. Two wks after surgery, left ventricular remodeling (ventricular dilation) and functional decline (fall in ejection fraction) assessed by echocardiography were significantly and similarly attenuated in pHBP- and EPO-treated rats, and MI size was reduced by 25%. The effect was retained during the 6-wk follow-up. A single bolus injection of pHBP immediately after coronary ligation was effective in reduction of MI size in a dose as low as 1 μg/kg, but was ineffective at a 60 μg/kg dose if administered 24 h after MI induction. We conclude that pHBP is equally cardioprotective with EPO and deserves further consideration as a safer alternative to rhEPO in the search for therapeutic options to reduce myocardial damage following blockade of the coronary circulation.     

Induction of foetal haemoglobin synthesis in erythroid progenitor stem cells: mediated by water‐soluble components of Terminalia catappa

Current novel therapeutic agents for the treatment of sickle cell anaemia (SCA) focus on increasing foetal haemoglobin (HbF) levels in SCA patients. Unfortunately, the only approved HbF‐inducing agent, hydroxyurea, has long‐term unpredictable side effects. Studies have shown the potential of plant compounds to modulate HbF synthesis in primary erythroid progenitor stem cells. We isolated a novel HbF‐inducing Terminalia catappa distilled water active fraction (TCDWF) from Terminalia catappa leaves that induced the commitment of erythroid progenitor stem cells to the erythroid lineage and relatively higher HbF synthesis of 9.2‐ and 6.8‐fold increases in both erythropoietin (EPO)‐independent and EPO‐dependent progenitor stem cells respectively. TCDWF was differentially cytotoxic to EPO‐dependent and EPO‐independent erythroid progenitor stem cell cultures as revealed by lactate dehydrogenase release from the cells. TCDWF demonstrated a protective effect on EPO‐dependent and not EPO‐independent progenitor cells. TCDWF induced a modest increase in caspase 3 activity in EPO‐independent erythroid progenitor stem cell cultures compared with a significantly higher (P?0.05) caspase 3 activity in EPO‐dependent ones. The results demonstrate that TCDWF may hold promising HbF‐inducing compounds, which work synergistically, and suggest a dual modulatory effect on erythropoiesis inherent in this active fraction. Copyright © 2014 John Wiley & Sons, Ltd.     

聚乙二醇对PEG化重组人促红细胞生成素热稳定性和生物学活性的影响

为了研究聚乙二醇对PEG化rhEPO热稳定性和生物学活性的影响,用酶学方法切除rhEPO和PEGrhEPO分子中的N连接糖基,研究酶切产物在37℃时287nm和350nm吸光度随时间的变化;用MTT比色法,网织红细胞法和HCT法比较体内和体外生物学活性的改变。结果显示聚乙二醇修饰能显著减少无N连接糖基的rhEPO分子间的聚集,降低rhEPO体外生物学活性,但增高体内生物学活性;无N连接糖基的PEG化rhEPO具有与rhEPO相当的体内生物学活性。因此聚乙二醇能提高rhEPO热稳定性;聚乙二醇可替代糖基,维持rhEPO的体内生物学活性。用PEG修饰原核细胞表达的rhEPO是开发rhEPO制品的新思路。     

Nonhematopoietic erythropoietin derivatives prevent motoneuron degeneration in vitro and in vivo

Chronic treatment with asialo erythropoietin (ASIALO-EPO) or carbamylated erythropoietin (CEPO) improved motor behavior and reduced motoneuron loss and astrocyte and microglia activation in the cervical spinal cord of wobbler mice, an animal model of amyotrophic lateral sclerosis, but had no effect on hematocrit values. ASIALO-EPO and CEPO, like the parent compound EPO, protected primary motoneuron cultures from kainate-induced death in vitro. Both EPO receptor and the common CD131 beta chain were expressed in cultured motoneurons and in the anterior horn of wobbler mice spinal cord. Our results strongly support a role for the common beta chain CD131 in the protective effect of EPO derivatives on motoneuron degeneration. Thus CEPO, which does not bind to the classical homodimeric EPO receptor and is devoid of hematopoietic activity, could be effective in chronic treatment aimed at reducing motoneuron degeneration.     

Recombinant human erythropoietin attenuates hepatic injury induced by ischemia/reperfusion in an isolated mouse liver model

Introduction Apoptosis is a central mechanism of cell death following reperfusion of the ischemic liver. Recombinant human erythropoietin (rhEPO) have an important role in the treatment of myocardial ischemia/reperfusion (I/R) injury, by preventing apoptosis. The aim of the study was to investigate the effect of different regimens of rhEPO in preventing apoptosis following I/R-induced hepatic injury. Material and methods Isolated mouse livers were randomly divided into five groups: (1) control group, perfused for the whole study period (105 min); (2) 30-min perfusion followed by 90 min of ischemia and 15 min of reperfusion; (3), (4) and (5) like group 2, but with administration of rhEPO 5,000 units/kg i.p. at 30 min, 24 h, or both 30 min and 24 h respectively, before induction of ischemia. Perfusate liver enzyme levels and intrahepatic caspase-3 activity were measured, and apoptotic cells were identified by morphological criteria, TUNEL assay, and immunohistochemistry for caspase-3. Using immunoblot the expression of the proapoptotic JNK and inhibitor of NFκB (IκBα) were also evaluated. von Willebrand factor (vWF) immunohistochemistry was used as a marker of endothelial cells. Results Compared to the I/R livers, all 3 rhEPO pretreated groups showed: a significant reduction in liver enzyme levels (P < 0.05) and intrahepatic caspase-3 activity (P < 0.05), fewer apoptotic hepatocytes (P < 0.05) and positive vWF staining in numerous endothelial cells lining the sinusoids. EPO decreased JNK phosphorylation and the degradation of the inhibitor of NFκB (IκBα) during I/R. There was no added benefit of the multiple- over the single-dose rhEPO regimen. Conclusion Pretreatment with one dose of rhEPO can attenuate post-I/R hepatocyte apoptotic liver damage. NFκB and JNK activation is likely to play a pivotal role in the pathophysiology of I/R hepatic injury and might have a key role in EPO-mediated protective effects. This effect is associated with the increase in sinusoidal vWF immunostaining suggests an additional effect of rhEPO in liver angiogenesis recovery. These findings have important implications for the potential use of rhEPO in I/R injury during liver transplantation. Edith Hochhauser and Orit Pappo are first two coauthors.     

N-Glycosylation engineering of tobacco plants to produce asialoerythropoietin

Erythropoietin (EPO) is a glycoprotein hormone that displays both hematopoietic and tissue-protective functions by binding to two distinct receptors. Recombinant human EPO (rhuEPO) is widely used for the treatment of anemia, but its use for tissue protection is limited because of potentially harmful increases in red blood cell mass when higher doses of rhuEPO are used. Recent studies have shown that asialoerythropoietin (asialo-rhuEPO), a desialylated form of rhuEPO, lacks hematopoietic activity, but retains cytoprotective activity. Currently, a small amount of asialo-rhuEPO is produced by enzymatic desialylation of rhuEPO. The prohibitive cost of rhuEPO, however, is a major limitation of this method. Plants have the ability to synthesize complex N-glycans, but lack enzymatic activities to add sialic acid and β1,4-galactose to N-glycan chains. Plants could be genetically engineered to produce asialo-rhuEPO by introducing human β1,4-galactosyltransferase. The penultimate β1,4-linked galactose residues are important for in vivo biological activity. In this proof of concept study, we show that tobacco plants co-expressing human β1,4-galactosyltransferase and EPO genes accumulated asialo-rhuEPO. Purified asialo-rhuEPO binds to an Erythrina cristagalli lectin column, indicating that its N-glycan chains bear terminal β1,4-galactose residues and that the co-expressed GalT is functionally active. Asialo-rhuEPO interacted with the EPO receptor (EPOR) with similar affinity as rhuEPO, implying that it was properly folded. The strategy described here provides a straightforward way to produce asialo-rhuEPO for research and therapeutic purposes. KEY MESSAGE: N-glycosylation pathway in tobacco plants could be genetically engineered to produce a tissue-protective cytokine, asialoerythropoietin (a desialylated form of human hormone erythropoietin).