Erythropoietin Variants Research Articles

Reduction of Laser-Induced Choroidal Neovascularization in Mice With Erythropoietin RNA Interference

PurposeThe purpose of this study was to evaluate the pathological involvement of erythropoietin (EPO) in experimental choroidal neovascularization (CNV) and its association with neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) in the Chinese population.MethodsTreatment effect of recombinant EPO protein were assessed by human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation, and ex vivo choroid-sprouting ability. The effect of intravitreal injection of Epo siRNA against neovascularization was evaluated in the laser-induced CNV mouse model. In addition, the association of EPO variants with neovascular AMD and PCV was determined.ResultsExogenous supplementation of EPO significantly enhanced the migration and tube formation of HUVECs and promoted ex vivo choroid sprouting in mouse retinal pigment epithelium (RPE)-choroid-sclera complex culture. In the experimental CNV mouse model, Epo expression was found to be significantly upregulated by 3.5-folds in RPE-choroid-sclera complex at day 10 after laser induction as compared to the baseline. Immunofluorescence analysis showed that Epo was mainly expressed around the vascular endothelial cells in the RPE-choroid-sclera complex. Intravitreal injection of siRNA targeting Epo reduced 40% Epo expression and 40% CNV lesion areas as compared to the scramble control. However, EPO variants were not associated with neovascular AMD nor PCV in the Chinese population.ConclusionsThis study revealed the promotion of human endothelial cell tube formation in vitro and choroid sprouting ex vivo by EPO, and the reduction of laser-induced CNV in vivo by Epo RNA interference.Translational RelevanceTargeting EPO could be a potential additional treatment for CNV-related diseases.

Glycoform analysis of intact erythropoietin by MALDI FT-ICR mass spectrometry

Recombinant human erythropoietin (EPO) is a complex therapeutic glycoprotein with three N- and one O-glycosylation sites. Glycosylation of EPO influences its safety and efficacy and is defined as a critical quality attribute. Thus, analytical methods for profiling EPO glycosylation are highly demanded. Owing to the complexity of the intact protein, information about EPO glycosylation is commonly derived from released glycan and glycopeptide analysis using mass spectrometry (MS). Alternatively, comprehensive insights into the glycoform heterogeneity of intact EPO are obtained using ESI MS-based methods with or without upfront separation of EPO glycoforms. MALDI MS, typically performed with TOF mass analyzers, has been also used for the analysis of intact EPO but, due to the poor glycoform resolution, has only provided limited glycoform information. Here, we present a MALDI FT-ICR MS method for the glycosylation profiling of intact EPO with improved glycoform resolution and without loss of sialic acid residues commonly observed in MALDI analysis. Three EPO variants were characterized in-depth and up to 199 glycoform compositions were assigned from the evaluation of doubly-charged ions, without any deconvolution of the mass spectra. Key glycosylation features such as sialylation, acetylation, and N-acetyllactosamine repeats were determined and found to agree with previously reported data obtained from orthogonal analyses. The developed method allowed for a fast and straightforward data acquisition and evaluation and can be potentially used for the high-throughput comparison of EPO samples throughout its manufacturing process.

Synthesis of Erythropoietins Site‐Specifically Conjugated with Complex‐Type N‐Glycans

The biological activity of the glycoprotein hormone erythropoietin (EPO) is dependent mainly on the structure of its N-linked glycans. We aimed to readily attach defined N-glycans to EPO through copper-catalyzed azide alkyne cycloaddition. EPO variants with an alkyne-bearing non-natural amino acid (Plk) at the N-glycosylation sites 24, 38, and 83 were obtained by amber suppression followed by protein purification and refolding. Click conjugation of the alkynyl EPOs with biantennary N-glycan azides provided biologically active site-specifically modified EPO glycoconjugates.

Recombinant Human Erythropoietin Proteins Synthesized in Escherichia coli Cells: Effects of Additional Domains on the in vitro and in vivo Activities.

The aim of this work was to compare biological activities of three variants of bacterially expressed human recombinant erythropoietin (EPO) with additional protein domains: 6His-s-tag-EPO protein carrying the s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) at the N-terminus and HBD-EPO and EPO-HBD proteins containing heparin-binding protein domains (HBD) of the bone morphogenetic protein 2 from Danio rerio at the N- and C-termini, respectively. The commercial preparation Epostim (LLC Pharmapark, Russia) produced by synthesis in Chinese hamster ovary cells was used for comparison. The EPO variant with the C-terminal HBD domain connected by a rigid linker (EPO-HBD) possesses the best properties as compared to HBD-EPO with the reverse domain arrangement. It was ~13 times more active in vitro (i.e., promoted proliferation of human erythroleukemia TF-1 cells) and demonstrated a higher rate of association with the erythropoietin receptor. EPO-HBD also exhibited the greatest binding to the demineralized bone matrix (DBM) and more prolonged release from the DBM among the four proteins studied. Subcutaneous administration of EPO-HBD immobilized on DBM resulted in significantly more pronounced vascularization of surrounding tissues in comparison with the other proteins and DBM alone. Therefore, EPO-HBD displayed better performance with regard to all the investigated parameters than other examined EPO variants, and it seems promising to study the possibility of its medical use.

Direct quality control of glycoengineered erythropoietin variants

Recombinant production of glycoprotein therapeutics like erythropoietin (EPO) in mammalian CHO cells rely on the heterogeneous N-glycosylation capacity of the cell. Recently, approaches for engineering the glycosylation capacities of mammalian cells for custom designed glycoforms have been developed. With these opportunities there is an increasing need for fast, sensitive, and global analysis of the glycoproteoform landscape produced to evaluate homogeneity and consistency. Here we use high-resolution native mass spectrometry to measure the glycoproteoform profile of 24 glycoengineered variants of EPO. Based on the unique mass and intensity profiles of each variant, we classify them according to similarities in glycosylation profiles. The classification distinguishes EPO variants with varying levels of glycan branchingand sialylation, which are crucial parameters in biotherapeutic efficacy. We propose that our methods could be of great benefit in the characterization of other glycosylated biopharmaceuticals, ranging from the initial clonal selection to batch-to-batch controls, and the assessment of similarity between biosimilar/biobetter products.

Point of View about Erythropoietin in Treatment of Central Nervous System Diseases

Spinal Cord Injury (SCI) is a complex process which leads to destruction of neuronal tissue and also vascular structure. Recent many studies have shown erythropoietin (EPO) has neuro-protective effects in acute SCI animal models. Alzheimer’s disease (AD), Parkinson’s disease (PD) and SCI belong to central nervous system diseases, a few studies also indicated that EPO may be effective for AD and PD. In this a short review article, we provide a short overview of the animal and human studies on rhEPO in SCI, AD and PD. Further clinical studies should reveal whether derivatives and variants of EPO provide any benefits over the clinical application EPO to against SCI, AD and PD in the experimental studies.

Post-translational modification of a chimeric EPO-Fc hormone is more important than its molecular size in defining its in vivo hematopoietic activity

BackgroundRecombinant erythropoietin (EPO) has been marketed as biopharmaceutical for anemia and chronic renal failure. Long-acting EPO variants that aimed at achieving less frequent dosing have been generated, either by the addition of glycosylation sites or increasing its molecular weight. MethodsThe hEPO cDNA linked to the human IgG Fc fragment was cloned as a single codifying gene on the pAdtrack-CMV vector, yielding the recombinant adenoviral genome. For in vitro and in vivo expression assays cervical cancer cell line (SiHa) and nulliparous goats were used, respectively. The hematopoietic activity of EPO-Fc, expressed as the differential increment of hematocrit was evaluated in B6D2F1 mice. NP-HPLC of the 2AB-labeled N-glycan was carried out to profile analysis. ResultsThe direct transduction of mammary secretory cells with adenoviral vector is a robust methodology to obtain high levels of EPO of up to 3.5mg/mL in goat's milk. SiHa-derived EPO-Fc showed significant improvement in hematopoietic activity compared to the commercial hEPO counterpart or with the homologous milk-derived EPO-Fc. The role of the molecular weight seemed to be important in enhancing the hematopoietic activity of SiHa-derived EPO-Fc. However, the lack of sialylated multi-antennary glycosylation profile in milk-derived EPO-Fc resulted in lower biological activity. ConclusionsThe low content of tri- or tetra-antennary sialylated N-glycans linked to the chimeric EPO-Fc hormone, expressed in the goat mammary gland epithelial cells, defined its in vivo hematopoietic activity. General significanceThe sialylated N-glycan content plays a more significant role in the in vivo biological activity of hEPO than its increased molecular weight.

Agonists of the tissue-protective erythropoietin receptor in the treatment of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease affecting more than a million people in the USA alone. While there are effective symptomatic treatments for PD, there is an urgent need for new therapies that slow or halt the progressive death of dopaminergic neurons. Significant progress has been made in understanding the pathophysiology of PD, which has substantially facilitated the discovery efforts to identify novel drugs. The tissue-protective erythropoietin (EPO) receptor, EPOR/CD131, has emerged as one promising target for disease-modifying therapies. Recombinant human EPO (rhEPO), several variants of EPO, EPO-mimetic peptides, cell-based therapies using cells incubated with or expressing EPO, gene therapy vectors encoding EPO, and small molecule EPO mimetic compounds all show potential as therapeutic candidates. Agonists of the EPOR/CD131 receptor demonstrate potent anti-apoptotic, antioxidant, and anti-inflammatory effects and protect neurons, including dopaminergic neurons, from diverse insults in vitro and in vivo. When delivered directly to the striatum, rhEPO protects dopaminergic neurons in animal models of PD. Early-stage clinical trials testing systemic rhEPO have provided encouraging results, while additional controlled studies are required to fully assess the potential of the treatment. Poor CNS availability of proteins and challenges related to invasive delivery limit delivery of EPO protein. Several variants of EPO and small molecule agonists of the EPO receptors are making progress in preclinical studies and may offer solutions to these challenges. While EPO was initially discovered as the primary modulator of erythropoiesis, the discovery and characterization of the tissue-protective EPOR/CD131 receptor offer an opportunity to selectively target the neuroprotective receptor as an approach to identify disease-modifying treatments for PD.

Nasal Neuro EPO Could be a Reliable Choice for Neuroprotective Stroke Treatment

The most common cause of stroke is cerebral ischemia, where blood flow to the brain is interrupted due to a thrombus in a major cerebral artery. Currently, the only therapeutic approach available is thrombolysis. A more recent approach that has started to gain attention is neuroprotection, the ability to prevent neuronal death and enhance endogenous protective mechanisms. Several studies have shown the neuroprotective action of Erythropoietin (EPO). A potential problem in the use of EPO for neurodegenerative disorders is the undesirable erythropoietic side effects. In this context, investigations have been focused to develop derivatives of EPO lacking erythropoietic activity but retaining neuroprotective potential. Low sialic acid-containing EPO (Neuro EPO) is very similar to the one that occurs in the mammalian brain and is rapidly degraded by the liver. Similar neuroprotective effects had been observed with neuro EPO, original recombinant human EPO and EPO variants in ischemia models. Intranasal route could be safe and hematological side effects could be avoided. Neuro EPO that constitutes a new agent has retained the neuroprotective effects without stimulating the EPOR in the bone marrow and can therefore be used without increasing the hematocrit. This review gives a brief introduction to the no hematopoietic effects of EPO, the evidence of neuroprotective effect, the alternatives for obtaining an EPO derivate without hematological side effects and discusses the advantages of nasal administration of Neuro EPO for neuroprotective stroke treatment.

Erythropoietin Attenuates Neurological and Histological Consequences of Toxic Demyelination in Mice

Erythropoietin (EPO) reduces symptoms of experimental autoimmune encephalomyelitis in rodents and shows neuroregenerative effects in chronic progressive multiple sclerosis. The mechanisms of action of EPO in these conditions with shared immunological etiology are still unclear. Therefore, we used a model of toxic demyelination allowing exclusion of T cell-mediated inflammation. In a double-blind (for food/injections), placebo-controlled, longitudinal four-arm design, 8-wk-old C57BL/6 mice (n = 26/group) were assigned to cuprizone-containing (0.2%) or regular food (ground chow) for 6 wks. After 3 wks, mice were injected every other day with placebo or EPO (5,000 IU/kg intraperitoneally) until the end of cuprizone feeding. Half of the mice were exposed to behavioral testing, magnetic resonance imaging (MRI) and histology immediately after treatment cessation, whereas the other half were allowed a 3-wk treatment-free recovery. Immediately after termination of cuprizone feeding, all toxin-exposed mice were compromised regarding vestibulomotor function/coordination, with EPO-treated animals performing better than placebo. Likewise, ventricular enlargement after cuprizone, as documented by MRI, was less pronounced upon EPO. After a 3-wk recovery, remarkable spontaneous improvement was observed in all mice with no measurable further benefit in the EPO group ("ceiling effect"). Histological analysis of the corpus callosum revealed attenuation by EPO of the cuprizone-induced increase in microglial numbers and amyloid precursor protein accumulations as a readout of inflammation and axonal degeneration. To conclude, EPO ameliorates neurological symptoms in the cuprizone model of demyelination, possibly by reduction of inflammation-associated axonal degeneration in white matter tracts. These findings underscore the value of future therapeutic strategies for multiple sclerosis based on EPO or EPO variants.

The Nasal Route as a Potential Pathway for Delivery of Erythropoietin in the Treatment of Acute Ischemic Stroke in Humans

Intranasal delivery provides a practical, noninvasive method of bypassing the blood-brain barrier (BBB) in order to deliver therapeutic agents to the brain. This method allows drugs that do not cross the BBB to be delivered to the central nervous system in a few minutes. With this technology, it will be possible to eliminate systemic administration and its potential side effects. Using the intranasal delivery system, researchers have demonstrated neuroprotective effects in different animal models of stroke using erythropoietin (EPO) as a neuroprotector or other different types of EPO without erythropoiesis-stimulating activity. These new molecules retain their ability to protect neural tissue against injury and they include Asialoerythropoietin (asialoEPO) carbamylated EPO (CEPO), and rHu-EPO with low sialic acid content (Neuro-EPO). Contrary to the other EPO variants, Neuro-EPO is not chemically modified, making it biologically similar to endogenous EPO, with the advantage of less adverse reactions when this molecule is applied chronically. This constitutes a potential benefit of Neuro-EPO over other variants of EPO for the chronic treatment of neurodegenerative illnesses. Nasal administration of EPO is a potential, novel, neurotherapeutic approach. However, it will be necessary to initiate clinical trials in stroke patients using intranasal delivery in order to obtain the clinical evidence of its neuroprotectant capacity in the treatment of patients with acute stroke and other neurodegenerative disorders. This new therapeutic approach could revolutionize the treatment of neurodegenerative disorders in the 21 century.

Erythropoietin Protects Cardiac Myocytes Against Anthracycline-Induced Apoptosis by PI3K-Akt-Mediated Phosphorylation of Glycogen Synthase Kinase-3β.

Anthracycline antibiotics are an important component of chemotherapy regimens for hematologic malignancies and solid tumors despite their well-documented potential for adverse cardiac effects. Identification of mechanisms of anthracycline-induced cardiotoxicity has been important to develop strategies to prevent the cardiotoxic effects of anthracyclines without interfering with their anti-tumor effects. The hematopoietic cytokine erythropoietin (EPO), frequently used in anemic cancer patients, exhibits significant tissue-protective effects in various non-hematopoietic organs. Recent studies of EPO variants that retain tissue-protective properties of EPO in non-hematopoietic tissues without stimulating erythropoiesis suggested fundamental differences between EPO-mediated cellular signaling in hematopoietic versus non-hematopoietic cells as well as novel clinical applications for recombinant EPO and its derivatives in disorders other than anemia. In the isolated, Langendorff-perfused heart, we previously showed that EPO improves left ventricular function and reduces infarct size during ischemia-reperfusion injury. These protective effects were associated with preservation of myocardial ATP and EPO-mediated activation of specific signal transduction pathways in the isolated, perfused heart. In this study, we investigated the mechanisms of EPO-mediated protection of cardiac myocytes against apoptotic cell death induced by doxorubicin (DOX). Cultures of primary cardiac myocytes isolated from neonatal rat ventricles (NRVM) were established and cells were treated with DOX in the presence or absence of EPO. Using immunofluorescence microscopy of cardiomyocytes double-labeled with TUNEL and DAPI to quantify apoptotic nuclei, we found that EPO treatment significantly reduced by 35% DOX-induced apoptosis in a dose-dependent manner (p<0.001 by ANOVA). The ability of EPO to protect against DOX-mediated apoptosis was also confirmed by a cell-death ELISA that quantifies mono- and oligo-nucleosomes associated with apoptosis demonstrating a significant 33% reduction in cell death. In addition, EPO treatment significantly reduced caspase-3 activation during DOX-induced apoptosis. EPO treatment of cardiomyocytes induced the rapid phosphorylation of PI3K substrate Akt (serine 473 and threonine 308) and the p44/42 MAP kinases Erk 1/2 in a time-dependent fashion. The PI3K inhibitor LY294002 and Akt inhibitor Akti 1/2 or the MEK inhibitor U0126 inhibited the EPO-induced phosphorylation of Akt and Erk1/2, respectively. Treatment of cells with LY294002 or Akti 1/2 abolished the anti-apoptotic effect of EPO during during DOX treatment, whereas treatment with U0126 did not. EPO induced the phosphorylation of GSK-3β, a downstream target of PI3K-Akt. The ability of EPO to phosphorylate GSK-3β was blocked by LY294002 or Akti 1/2. Because phosphorylation is known to inactivate GSK-3β, we investigated whether GSK-3β inhibition is protective. We used two different inhibitors of GSK-3β including lithium chloride and SB216763. Using TUNEL and DNA fragmentation assays, we found that treatment with lithium chloride or SB216763 exerts a significant protective effect in cardiomyocytes during DOX-induced apoptosis (p<0.001), in a manner similar to EPO. These data suggest that erythropoietin may serve as a novel cardioprotective agent against anthracycline-induced cardiotoxicity by inhibiting cardiomyocyte apoptosis through the PI3K-Akt pathway and inactivation of GSK-3β by phosphorylation.

Erythropoietin Protects Cardiomyocytes Not Only during Hypoxic Injury but Also Against Doxorubicin-Induced Apoptosis: A Novel Effect Mediated by Phosphatidylinositol 3-Kinase Pathway.

Erythropoietin (EPO) is a tissue-protective cytokine that exerts cytoprotective effects against various types of injury in non-hematopoietic tissues. Our previous work using Langendorff-perfused adult rat hearts demonstrated that EPO improves left ventricular function and reduces infarct size during ischemia-reperfusion injury. Recent studies of EPO variants that retain tissue-protective properties of EPO in non-hematopoietic tissues without stimulating erythropoiesis suggested fundamental differences between EPO-mediated cellular signaling in hematopoietic versus non-hematopoietic cells as well as novel clinical applications for recombinant EPO and its derivatives in disorders other than anemia. In this study, we characterized the activation of EPO-mediated intracellular signal transduction in primary cardiac myocytes and determined the specific pathways required for protection of cardiomyocytes against hypoxic injury. We also investigated the ability of EPO to protect against cell death induced by doxorubicin (DOX), a chemotherapeutic agent associated with well-established cardiotoxic effects. We hypothesized that protective effects of EPO during hypoxic or DOX-mediated cardiomyocyte injury may be inhibited by specific kinase inhibitors targeting pathways activated by EPO. Primary cardiac myocytes were isolated from neonatal rat ventricles (NRVM) establishing cultures containing >97% cardiac myocytes as determined by immunostaining of cells with anti-cardiac muscle sarcomeric actinin. We found that EPO induces the rapid phosphorylation of PI3K substrate Akt and p44/42 MAP kinases Erk 1/2 in a time-dependent fashion. Constitutive tyrosine phosphorylation of Jak2 was observed that did not increase following EPO treatment of cardiomyocytes. Pre-incubation of cells with PI3K inhibitor LY294002 or MEK inhibitor U0126 prior to EPO treatment inhibited the increased phosphorylation of Akt and Erk1/2, respectively. EPO treatment of NRVMs significantly reduced hypoxia-induced apoptosis by 44% assessed by immunoflurescence microscopy of cardiomyocytes double-labeled with TUNEL and DAPI to quantify apoptotic nuclei (P<0.001 by ANOVA; hypoxia-treated versus hypoxia+EPO, n=12 fields of ~4,000 cells in each group). The ability of EPO to protect against hypoxia-mediated apoptosis was also confirmed by a cell-death ELISA that quantifies mono- and oligo-nucleosomes associated with apoptosis. Treatment of cells with LY294002 or protein kinase C inhibitor chelerythrine abolished the anti-apoptotic effect of EPO during hypoxia, whereas treatment with U0126 or Jak2 inhibitor AG490 did not. We also found that EPO exerts a significant protective effect during DOX-induced cell death in a dose-dependent manner, reducing apoptosis by 36% in TUNEL assays (P<0.001 by ANOVA; DOX-treated versus DOX+Epo, n= 6 fields of ~2,000 cells in each group) and by 29% in ELISA assays (P<0.001 by ANOVA, DOX-treated versus DOX+Epo, n=14). Treatment of cells with LY294002 abrogated the anti-apoptotic effect of EPO in NVRMs treated with DOX. These findings demonstrate that 1)-EPO induces rapid phosphorylation of Akt and Erk1/2 but not Jak2 in primary cardiac myocytes in a time-dependent fashion; 2)-EPO protects cardiomyocytes against apoptosis induced by hypoxia or DOX; 3)-The protective effect of EPO during hypoxia-mediated injury of cardiomyocytes requires the PI3K and PKC pathways, but not MAP kinase or Jak2 pathways; 4) The protective effect of EPO during DOX treatment of cardiomyocytes requires the PI3K pathway.

Glycosylation of erythropoietin affects receptor binding kinetics: role of electrostatic interactions.

Erythropoietin (EPO) is a cytokine produced by the kidney whose function is to stimulate red blood cell production in the bone marrow. Previously, it was shown that the affinity of EPO for its receptor, EPOR, is inversely related to the sialylation of EPO carbohydrate. To better understand the properties of EPO that modulate its receptor affinity, various glycoforms were analyzed using surface plasmon resonance. The system used has been well characterized and is based on previous reports employing an EPOR-Fc chimera captured on a Protein A surface. Using three variants of EPO containing different levels of sialylation, we determined that sialic acid decreased the association rate constant (k(on)) about 3-fold. Furthermore, glycosylated EPO had a 20-fold slower k(on) than nonglycosylated EPO, indicating that the core carbohydrate also negatively impacted k(on). The effect of electrostatic forces on EPO binding was studied by measuring binding kinetics in varying NaCl concentrations. Increasing NaCl concentration resulted in a slower k(on) while having little impact on k(off), suggesting that long-range electrostatic interactions are primarily important in determining the rate of association between EPO and EPOR. Furthermore, the glycosylation content (i.e., nonglycosylated vs glycosylated, sialylated vs desialylated) affected the overall sensitivities of k(on) to [NaCl], indicating that sialic acid and the glycan itself each impact the overall effect of these electrostatic forces.

Role of glycosylation on the secretion and biological activity of erythropoietin.

The erythropoietin (EPO) molecule contains four carbohydrate chains. Three contain N-linkages to asparagines at positions 24, 38, and 83, and one contains an O-linkage to a serine at position 126. We constructed human EPO variants that eliminated the three N-glycosylation sites by replacing the asparagines with glutamines singly or in combination. The O-linked carbohydrate chain was removed by replacing the serine with glutamine, valine, histidine, or alanine. A variant with a double mutation (Gln38,83) and another with a triple mutation (Gln24,38,83) were secreted poorly from COS1 and CHO cells even though RNA encoding these variants was present. All other variants with mutations in N-linked glycosylation sites were secreted normally. Removal of any of the N-glycosylation sites reduced the in vivo but not the in vitro biological activity of the EPO molecule. All the mutations at Ser126, the O-glycosylation site, were secreted normally. In vitro activity was also unaffected except for Ala126 which had a 50-fold decrease. The Val126 variant was tested in vivo, and its specific activity was only slightly less than that of the native EPO, which indicates that the O-linked carbohydrate is not essential for activity.