Carbamylated Erythropoietin Research Articles

Combined use of hair follicle stem cells and CEPO (carbamylated erythropoietin)-Fc in a rat model of chronic cerebral hypoperfusion: A behavioral, electrophysiological, and molecular study

BackgroundIn dementia, synaptic dysfunction appears before neuronal loss. Stem cell therapy could potentially provide a promising strategy for the treatment of dementia models. The carbamylated erythropoietin fusion protein (CEPO-Fc) has shown synaptotrophic effects. This study aimed to determine the efficiency of the combined use of hair follicle stem cells (HFSC) and CEPO-Fc in the basal synaptic transmission (BST) and long-term plasticity (LTP) of chronic cerebral hypoperfusion (CCH) rats. MethodsWe divided 64 adult rats into control, sham, CCH+vehicle, CCH+CEPO, CCH+HFSC, and CCH+HFSC+CEPO groups. The CEPO-Fc was injected three times/week for 30 days. HFSC transplantation was done on days 4, 14, and 21 after surgery. The Morris water maze test and passive avoidance were used to assess memory. BST and LTP were assessed by a field-potential recording of the CA1 region. The hippocampal mRNA expression of IGF-1, TGF-β1, β1-Catenine, NR2B, PSD-95, and GSk-3β was evaluated by quantitative RT-PCR. ResultsFollowing combination therapy, spatial memory retention, and BST showed significant improvement relative to HFSC and CEPO-Fc groups. These effects were also confirmed by recovered mRNA expression of β1-catenin, TGF-β1, and NR2B. GSK-3β expression was downregulated in all treatment groups. The upregulated PSD-95 was identified in HFSC and combination groups compared to the vehicle group. ConclusionsThese findings indicate that the combined use of HFSC and CEPO-Fc may be more advantageous for treating memory disruption in the CCH model than CEPO-Fc or HFSC alone. This type of combination therapy may hopefully lead to a new approach to treatment for dementia.

Carbamylated Erythropoietin Alleviates Kidney Damage in Diabetic Rats by Suppressing Oxidative Stress.

The oxidative stress response plays an important role in the occurrence and development of diabetic kidney disease (DKD). It has become a new treatment target for DKD. In the current study, the effects of carbamylated erythropoietin (CEPO) on renal oxidative stress and damage in diabetic rats were examined. Thirty Sprague Dawley rats were intraperitoneally administered with 60 mg/kg streptozotocin to establish the diabetes model. The diabetic rats were randomly allocated into 4 groups (n=6 each): diabetes model group (DM group), DM + CEPO treatment group (DC group), DM + CEPO + EPO receptor (EPOR) blocking peptide treatment group (DCEB group), and DM + CEPO + CD131 blocking peptide treatment group (DCCB group). Meanwhile, a normal control group (NC group, n=6) was set up. Kidney tissues and blood samples were obtained for evaluation of oxidative stress and renal function. The results showed that diabetic rats exhibited increased oxidative stress in the kidney and early pathological changes associated with DKD. Treatment with CEPO reduced oxidative stress and attenuated renal dysfunction. However, diabetic rats treated with the combination of CEPO and EPOR blocking peptide or CD131 blocking peptide showed increased oxidative stress and reduced renal function when compared with CEPO treatment alone group. These results suggested that CEPO can protect against kidney damage in DKD by inhibiting oxidative stress injury via EPOR-CD131 heterodimers.

The analytical approach for detection of carbamylated erythropoietin for doping control purposes

Erythropoietin (EPO) has protective effects in several tissues and could be used for therapeutic purposes, but the doses of EPO that can be beneficial in case of hypoxic-ischemic conditions due to overinduced erythropoiesis could be detrimental in treated patients. Carbamylation of erythropoietin maintains the tissue-protective effects of EPO but without erythropoietic effects. Carbamylated EPO (CEPO) is listed in WADA Prohibited List in class S2 as "Innate repair receptor agonists." The CEPO was synthesized using the method described previously. Digestion with endoproteinase Lys-C was used to distinguish rhEPO from CEPO. The digested samples containing recombinant EPO, urinary EPO (uEPO), or CEPO were analyzed by the SAR-PAGE method (sarcosyl polyacrylamide gel electrophoresis-PAGE). Endoproteinase Lys-C breaks the peptide chains of lysine. Lysine residues, converted to homocitrulline by carbamylation, cannot be cleaved by endoproteinase Lys-C. Therefore, the CEPO protein chain remained unchanged in contrast to rhEPO and uEPO, which allows for easily differentiation of them.

Carbamylated erythropoietin regulates immune responses and promotes long-term kidney allograft survival through activation of PI3K/AKT signaling

Modulation of alloimmune responses is critical to improving transplant outcome and promoting long-term graft survival. To determine mechanisms by which a nonhematopoietic erythropoietin (EPO) derivative, carbamylated EPO (CEPO), regulates innate and adaptive immune cells and affects renal allograft survival, we utilized a rat model of fully MHC-mismatched kidney transplantation. CEPO administration markedly extended the survival time of kidney allografts compared with the transplant alone control group. This therapeutic effect was inhibited when the recipients were given LY294002, a selective inhibitor of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway or anti-EPO receptor (EPOR) antibody, in addition to CEPO. In vitro, CEPO inhibited the differentiation and function of dendritic cells and modulated their production of pro-inflammatory and anti-inflammatory cytokines, along with activating the PI3K/AKT signaling pathway and increasing EPOR mRNA and protein expression by these innate immune cells. Moreover, after CD4+ T cells were exposed to CEPO the Th1/Th2 ratio decreased and the regulatory T cell (Treg)/Th17 ratio increased. These effects were abolished by LY294002 or anti-EPOR antibody, suggesting that CEPO regulates immune responses and promotes kidney allograft survival by activating the PI3K/AKT signaling pathway in an EPOR-dependent manner. The immunomodulatory and specific signaling pathway effects of CEPO identified in this study suggest a potential therapeutic approach to promoting kidney transplant survival.

CEPO (carbamylated erythropoietin)-Fc protects hippocampal cells in culture against beta amyloid-induced apoptosis: considering Akt/GSK-3β and ERK signaling pathways.

The tissue-protective properties of erythropoietin (EPO) have been described in several neurodegenerative diseases models, but erythrocytosis following EPO treatment may lead to deleterious effects. Carbamylated erythropoietin, an EPO derivative lacking hematopoietic side effects, has shown protective properties in some studies. However, it is not known if CEPO protects primary hippocampal cells against Aβ25-35 toxicity. The present study aimed to investigate the effect of CEPO-Fc on biochemical alterations in Akt, GSK-3β, and ERK signaling and cell death induced by Aβ25-35 in isolated hippocampal cell culture. The embryonic hippocampal cells were obtained from 18-19 day rat embryos. The cells were exposed with Aβ25-35 (20 µM) in the absence or presence of CEPO-Fc (1 or 5 IU) and PI3k and ERK inhibitors. CEPO-Fc at the dose of 5 IU significantly prevented the cell loss and caspase-3 cleavage caused by Aβ25-35. Additionally, CEPO-Fc noticeably reversed Aβ mediated decrement of Akt and GSK-3β phosphorylation. With exposure to LY294002, PI3 kinase inhibitor, Akt phosphorylation diminished and CEPO-Fc protective effects disappeared. Furthermore, while CEPO-Fc nullified Aβ-induced increment of phospho-ERK, inhibition of ERK activity by PD98059, had no effect on Aβ25-35-mediated caspase-3 cleavage and cell toxicity. These results imply that protective effects of CEPO-Fc seem to be mainly exerted through the PI3K/Akt pathway rather than ERK signaling. This study suggested that CEPO-Fc prevents Aβ-induced cell toxicity as well as Akt/GSK-3β and ERK alterations in isolated hippocampal cells. These findings might provide a new perspective on CEPO-Fc protective properties as a prospective remedial factor for neurodegenerative diseases like AD.

Effect of carbamylated erythropoietin on neuronal apoptosis in fetal rats during intrauterine hypoxic-ischemic encephalopathy

BackgroundHypoxic-ischemic encephalopathy (HIE) is a common disease that occurs during the perinatal period. The primary cause of neonatal HIE is related to fetal intrauterine anoxia. Carbamylated erythropoietin (CEPO), a derivative of erythropoietin (EPO), does not exert any erythropoietic effect; however, the neuroprotective effects resemble those of EPO. Previous studies have shown the potential benefits of CEPO on the central nervous system. The present study aimed to investigate the role of CEPO in neuronal apoptosis during intrauterine HIE and the underlying mechanisms.ResultsTo validate our hypothesis, we established an intrauterine HIE model by occluding the bilateral utero-ovarian arteries of pregnant Sprague–Dawley rats. Compared to the I/R group, neuronal apoptosis in the CEPO group was significantly lower at 4, 12, 24, and 48 h (P < 0.05). CEPO significantly inhibited CC3 expression (P < 0.05) during the early-stages after ischemia–reperfusion (0.5, 4, 8, 12 and 24 h), upregulated Bcl-2 expression, and downregulated Bax expression at 4, 8, 12, and 24 h (P < 0.05).ConclusionsCarbamylated erythropoietin pretreatment inhibited the expression of proapoptotic protein CC3 in brain and regulated the Bcl-2/Bax ratio, resulting in reduced neuronal apoptosis and thus resulting in a protective effect on intrauterine HIE.

The role of PI3-K/Akt signal pathway in the antagonist effect of CEPO on CHF rats.

The possible role of phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt) signal pathway in the antagonist effect of carbamylated erythropoietin (CEPO) on chronic heart failure (CHF) in rats was investigated. Twenty of 120 rats were randomly selected as the control group, and the remaining rats as the model group. Rats in the model group received intraperitoneal injection of isoproterenol, those in the control group underwent intraperitoneal injection of equivalent normal saline. Rats with successful model establishment were divided into 4 groups, i.e. CHF group, CEPO group, LY294002 (LY) group and CEPO + LY group. Rats in the CEPO group underwent intraperitoneal injection of CEPO, while those in the CHF group received intraperitoneal injection of equivalent normal saline at the same time, those in the LY group received intraperitoneal injection of LY after model establishment, and those in the CEPO + LY group received the combined intraperitoneal injection of CEPO and LY simultaneously. Indicators for hemodynamics were determined using BL-410S bio-functional experiment system, including heart rate (HR), left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP) and maximal increased rate of left ventricular pressure (LVP)/maximal reduced rate of LVP (±dp/dtmax). Western blotting assay was utilized to determine the changes in activity of PI3-K/Akt signal pathway. LVSP and ±dp/dtmax in the CHF, the CEPO, the CEPO + LY and the LY groups were significantly lower than those in the control group (P<0.05); LVSP and ±dp/dtmax in the CEPO group were also elevated significantly compared with CHF, LY and CEPO + LY groups (P<0.05) with significant decreases in LVEDP and HR (P<0.05); compared with the CHF group, LVSP and ±dp/dtmax in the LY group were each significantly decreased (P<0.05), in the LY group, pAkt level was significantly lower than that in the CHF group (P<0.05). In conclusion, CEPO can generate the antagonist effect on CHF in rats through activation of PI3-K/Akt signal pathway.

Carbamylated Erythropoietin Decreased Proliferation and Neurogenesis in the Subventricular Zone, but Not the Dentate Gyrus, After Irradiation to the Developing Rat Brain.

Cranial radiotherapy for pediatric brain tumors causes progressive, debilitating late effects, including cognitive decline. Erythropoietin (EPO) has been shown to be neuroprotective and to promote neuroregeneration. Carbamylated erythropoietin (CEPO) retains the protective properties of EPO but is not erythrogenic. To study the effects of CEPO on the developing brain exposed to radiotherapy, a single irradiation (IR) dose of 6 Gy was administered to the brains of postnatal day 9 (P9) rats, and CEPO (40 μg/kg s.c.) was injected on P8, P9, P11, P13, and P15. To examine proliferation, 5-Bromo-2-deoxyuridine (BrdU) was injected on P15, P16, and P17. CEPO administration did not affect BrdU incorporation in the granule cell layer (GCL) of the hippocampus or in the subventricular zone (SVZ) as quantified 7 days after the last BrdU injection, whereas IR decreased BrdU incorporation in the GCL and SVZ by 63% and 18%, respectively. CEPO did not affect BrdU incorporation in the GCL of irradiated brains, although it was reduced even further (to 31%) in the SVZ. To evaluate the effect of CEPO on neurogenesis, BrdU/doublecortin double-positive cells were quantified. CEPO did not affect neurogenesis in non-irradiated brains, whereas IR decreased neurogenesis by 58% in the dentate gyrus (DG) but did not affect it in the SVZ. In the DG, CEPO did not affect the rate of neurogenesis following IR, whereas in the SVZ, the rate decreased by 30% following IR compared with the rate in vehicle-treated rats. Neither CEPO nor IR changed the number of microglia. In summary, CEPO did not promote neurogenesis in non-irradiated or irradiated rat brains and even aggravated the decreased neurogenesis in the SVZ. This raises concerns regarding the use of EPO-related compounds following radiotherapy.

Molecular mechanism of the role of carbamyl erythropoietin in treating diabetic retinopathy rats.

The aim of the present study was to investigate the therapeutic effects of carbamyl erythropoietin (CEPO) and safflor yellow (SY) in the treatment of rats with diabetic retinopathy (DR) as well as exploring the mechanism of action. Male SD rats were used to establish a diabetes model and streptozotocin-induced retinopathy was also performed in rats. A total of 126 rats with DR were obtained, and model rats were randomly divided into the model (n=42), experimental (n=42) and control (n=42) groups. The rats in the model group were injected with saline, the rats in the experimental group were treated with CEPO, and the rats in the control group were treated with SY. After treatment for 2 weeks, the retinas were harvested for quantitative analysis of the mRNA expression levels of angiogenesis-promoting and -inhibiting molecules, apoptosis-promoting and -inhibiting molecules, and oxidative stress pathway-related factors by Reverse transcription-quantitative PCR (RT-qPCR). No significant differences in expression levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), angiopoietin (Ang-1), tissue kallikrein (TKLK) and pigment epithelium-derived factor (PEDF) were observed between the experimental and model groups (P>0.05). The expression levels of apoptosis-promoting molecules Bcl-2 related X protein (Bax) and cysteine aspartate specific protease (caspase-3) mRNA in the retina of the experimental group was significantly lower than those in the control group (P<0.05). The expression levels of Bcl-2 and survivin mRNA were significantly higher in the experimental group than in the control group (P<0.05). The expression levels of the oxidative stress pathway nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1) mRNA were significantly higher in the experimental group than in the control group. Therefore, the therapeutic effects of CEPO in treating DR are better than those of SY. As a result, CEPO may inhibit apoptosis and oxidative stress damage of retinal tissue cells in DR rats without affecting angiogenesis.

Study on the neuroprotection and mechanism of C-EPO and EPO in MCAO mice models

Background: Brain ischemia can be treated using two methods, namely thrombolysis and neuroprotection. The therapeutic time window for thrombolysis is less than 6 h, while that for most neuroprotective agents ranges from several hours to several weeks. Many stroke survivors can profit from neuroprotective treatment. Erythropoietin (EPO) is an interesting candidate neuroprotective agent in human patients with acute ischemic stroke, in addition to playing a role in red blood cell propagation. Carbamylated EPO (C-EPO) plays no role in red blood cell propagation, and its underlying mechanism of action in neuroprotection is unknown.Objective: To understand the mechanisms underlying the neuroprotective effects of EPO and C-EPO in stroke.Methods: Twenty one male mice with a Middle Cerebral Artery Occlusion (MCAO) were equally divided into 3 subgroups (n = 7 per subgroup): A1: MCAO; A2: 2500U/ kg EPO-MCAO; A3:C-EPO 25 μg/kg. MCAO models were developed by introducing a silicon-coated nylon monofilament into the common carotid artery and distally advancing it by 9 mm toward the carotid bifurcation. Ninety minutes following reperfusion, the behavior of the mice was observed. Finally, the mice from all the groups were decapitated, their brains were removed and sectioned, and the sections were stained with cresyl violet. Western blotting and immunohistochemistry were used to measure the expression of the IP3, ERK1/2, JNK, P38, JAK, STAT5, BCL-XL, and Caspase3.Results: EPO and C-EPO increased the BCL-XL protein expression and decreased the Caspase3 protein expression in different manners. EPO can reduce apoptosis through activating IP3, MAPK, and JAK-STAT pathways, while C-EPO can reduce apoptosis only by activating the IP3 pathway.Conclusion: EPO and C-EPO play a role in neuroprotection in the MCAO mouse model through similar mechanisms, possibly mediated by the modulation of the IP3 pathway.

Carbamylated erythropoietin can promote neural regeneration after cerebral ischemia

Objective To investigate any effects of carbamylated erythropoietin (CEPO) on the expression of LINGO-1, growth-associated protein-43 (GAP-43) and the infarcted volume after cerebral ischemia, so as to explore the effect of CEPO on neural regeneration after cerebral ischemia. Methods Forty-eight Sprague-Dawley rats were randomly divided into a sham operation group, an ischemia control group and a CEPO treatment group, each of 16. Middle cerebral artery occlusion (MCAO) was used to simulate focal cerebral ischemia in all except the rats in the sham operation group. Then the CEPO group was injected with 0.5 ml of CEPO, while the other two groups were given a 0.5 ml injection of normal saline daily for 7 days before they were sacrificed to prepare slices of brain tissue. Western blotting was used to detect the expression of LINGO-1 and activated caspase-3. Immunohistochemical staining was applied to observe the expression of GAP-43. The slices of brain tissue were stained with cresyl violet and the volume of infarction and edema were quantified with the Image J software. Results The average expression of LINGO-1 in the sham operation group, the ischemia control group and the CEPO treatment group were (0.25±0.02), (1.22±0.06) and (0.66±0.05) respectively, with significant differences among the 3 groups. There was no expression of activated caspase-3 in the sham operation group. However, the expression of activated caspase-3 increased significantly (to 86.6±10.2)% in the ischemia control group and increased significantly less (to 40.3±8.7)% in the CEPO treatment group. The average positive expression of GAP-43 in the sham operation group, the ischemia control group and the CEPO treatment group were 0, (55.02±1.62) and (72.11±3.23)/HP, respectively, with significant differences among them. Moreover, the average volumes of cerebral infarction and brain edema in the CEPO treatment group were significantly lower than those in the ischemia control group. Conclusions CEPO can inhibit the expression of LINGO-1 and activated caspase-3, promote the expression of GAP-43, reduce infarct volume and limit cerebral edema so as to promote neural regeneration after cerebral ischemia, at least in rats. Key words: Cerebral ischemia; Carbamylated erythropoietin; Growth-associated protein 43; Caspase-3; Neural regeneration

Neuroprotection and CD131/GDNF/AKT Pathway of Carbamylated Erythropoietin in Hypoxic Neurons.

Carbamylated erythropoietin (CEPO), an EPO derivative, is attracting widespread interest due to neuroprotective effects without erythropoiesis. However, little is known about molecular mechanisms behind CEPO-mediated neuroprotection. In primary neurons with oxygen-glucose deprivation (OGD) and mice with hypoxia-reoxygenation, the neuroprotection and possible molecular mechanism of CEPO were performed by immunohistochemistry and immunocytochemistry, Western blot, RT-PCR, and ELISA. The comparisons were analyzed by ANOVA followed by unpaired two-tailed Student's t test. Both CEPO and EPO showed the neuroprotective effects in OGD model and hypoxic brain. CEPO did not trigger JAK-2 but activated AKT through glial cell line-derived neurotrophic factor (GDNF). It has been shown that CEPO acts upon a heteroreceptor complex comprising both the EPO receptor and the common β receptor subunit (βcR, also known as CD131). The blockage of CD131 reduced CEPO-mediated GDNF production, while GFR receptor blockage and GDNF neutralization inhibited CEPO-induced neurogenesis. Addition of GDNF to cultured neurons increased phosphorylation of AKT. CEPO protects neurons possible through the CD131/GDNF/AKT pathway.

Carbamylated erythropoietin mediates retinal neuroprotection in streptozotocin-induced early-stage diabetic rats.

The neuroprotective effect of carbamylated erythropoietin (CEPO), an erythropoietin (EPO) derivative, in diabetic retinopathy (DR) has not been clearly verified. We conducted this study to investigate the potential neuroprotective role of CEPO in a streptozotocin-induced diabetic rat model. Streptozotocin-induced diabetic rats and blank controls were treated with or without CEPO and EPO for 4 weeks. Retinal functional and histological changes were quantified by electroretinogram, light microscopy, and terminal dUTP nick end labeling assay. Gene and protein levels of colony-stimulating factor 2 receptor beta, low-affinity (CD131), EPO receptor (EPOR), THY1, glial fibrillary acidic protein (GFAP), and vascular endothelial growth factor (VEGF-A) in retinal tissues were determined by real-time PCR and western blotting, respectively. Vascular penetration was assessed by fluorescein retinal angiography. Diabetic rats had decreased retinal thickness, decreased ganglion cells, and increased retinal neuron apoptosis. CEPO increased CD131 and THY1 expression, while EPO increased EPOR expression. High glucose increased GFAP expression in the diabetic group, but both CEPO and EPO attenuated the trend for increase. CEPO downregulated VEGF-A expression. The amplitudes of b-wave and oscillatory potentials were decreased in the untreated diabetic group, whereas neither parameter decreased in diabetic rats after CEPO or EPO treatment. Vascular leakage and microaneurysms in the diabetic group were significantly improved following CEPO treatment. CEPO has similar anti-apoptotic effects to EPO in DR, but CEPO does not induce neovascularization. CEPO may exert neuroprotective effects via its receptor CD131.

The lack of CD131 and the inhibition of Neuro-2a growth by carbamylated erythropoietin.

Recombinant human erythropoietin (EPO), a glycohormone, is one of the leading biopharmaceutical products, while carbamylated erythropoietin (CEPO), an EPO derivative, is attracting widespread interest due to its neuroprotective effects without erythropoiesis in several cells and animal models. However, exogenous EPO promotes an angiogenic response from tumor cells and is associated with tumor growth, but knowledge of CEPO on tumor growth is lacking. Here we show that CEPO, but not EPO, inhibited Neuro-2a growth and viability. As expected, CEPO--unlike EPO--did not activate JAK-2 either in primary neurons or in Neuro-2a cells. Interestingly, CEPO did not induce GDNF expression and subsequent AKT activation in Neuro-2a cells. Before CEPO/EPO treatment, glial cell line-derived neurotrophic factor (GDNF) neutralization and GFR receptor blocking decreased the viability of EPO-treated Neuro-2a cells but did not influence CEPO-treated Neuro-2a cells. As compared to primary neurons, the expression of CD131, as a receptor complex binding to CEPO, is almost lacking in Neuro-2a cells. In BABL/C-nu mice, CEPO did not promote the growth of Neuro-2a cells nor extended the survival time compared to mice treated with EPO. The results indicate that CEPO did not promote tumor growth because of lower expression of CD131 and subsequent dysfunction of CD131/GDNF/AKT pathway in Neuro-2a cells, revealing its therapeutic potential in future clinical application.

Further improving the cognitive effect profile of electroconvulsive therapy (ECT): The case for studying carbamylated erythropoietin

Electroconvulsive therapy (ECT) remains the most effective acute treatment for severe depression and several other psychiatric illnesses. However, its use has been limited by concerns about cognitive adverse effects. ECT may cause temporary cognitive impairment in some patients, typically anterograde amnesia for 1–2weeks after a course of treatment, and circumscribed retrograde amnesia. These cognitive effects largely disappear within days to weeks after treatment. Efforts to find a pharmacological agent to reduce the cognitive effects of ECT have largely been unsuccessful, with the possible exception of thyroid hormone. We review the literature on pharmacological attempts to attenuate ECT’s cognitive effects, and propose a novel neuroprotective and neurotrophic agent, carbamylated erythropoietin (CEPO), for this indication.

Low Oxygen Tension Primes Aortic Endothelial Cells to the Reparative Effect of Tissue-Protective Cytokines.

Erythropoietin (EPO) has both erythropoietic and tissue-protective properties. The EPO analogues carbamylated EPO (CEPO) and pyroglutamate helix B surface peptide (pHBSP) lack the erythropoietic activity of EPO but retain the tissue-protective properties that are mediated by a heterocomplex of EPO receptor (EPOR) and the β common receptor (βCR). We studied the action of EPO and its analogues in a model of wound healing where a bovine aortic endothelial cells (BAECs) monolayer was scratched and the scratch closure was assessed over 24 h under different oxygen concentrations. We related the effects of EPO and its analogues on repair to their effect on BAECs proliferation and migration (evaluated using a micro-Boyden chamber). EPO, CEPO and pHBSP enhanced scratch closure only at lower oxygen (5%), while their effect at atmospheric oxygen (21%) was not significant. The mRNA expression of EPOR was doubled in 5% compared with 21% oxygen, and this was associated with increased EPOR assessed by immunofluorescence and Western blot. By contrast, βCR mRNA levels were similar in 5% and 21% oxygen. EPO and its analogues increased both BAECs proliferation and migration, suggesting that both may be involved in the reparative process. The priming effect of low oxygen tension on the action of tissue-protective cytokines may be of relevance to vascular disease, including atherogenesis and restenosis.

P.1.g.031 The neuroprotective and anti-amnestic effects of carbamylated erythropoietin derivates in ischemic brain injury

Purpose: To compare the neuroprotection of carbamylated erythropoietin (CEPO) and EPO fusion protein containing TR domain from glycoprotein MUC1 (CEPO-TR), and EPO fusion protein with modified Fc fragments of IgG1 (CEPO-Fc) against ischemic brain injury, including behavioral disturbances in a bilateral focal ischemic infarction of the rat prefrontal cortex. Methods: The carbamylated erythropoietin and erythropoietin derivates were produced by treatment of purified proteins with potassium cyanate in borate buffer. The resulting carbamylated erythropoietin and derivates exhibit no erythropoietic activity in UT-7/EPOR cell viability assay. Bilateral focal ischemic infarction of the prefrontal cortex (areas Frl and Fr2) was induced by the method of photochemical thrombosis in rats. After passive avoidance training and testing rats were treated, respectively, with following regimens: saline, 50mg/kg CEPO, 50mg/kg CEPO-TR, 50mg/kg CEPO-Fc. The substance was injected intraperitoneally in 1 h after operation. Neurological deficit scores and infarct volume were assessed at 4 and 7 days after operation. Functional state of CNS was determined by conditioned passive avoidance response (PA), i.e. by the latency of transition from light compartment to dark compartment (in sec). Morphometric measurements of the areas and volumes of the ischemic focus on serial slices were carried out on animal brain fixed by plunging into formalinethanol-acetic acid mixture (2:7:1). The data were statistically processed using Statistica 6.0 software. Summary of results: Additional oligosaccharides, linked to the erythropoietin, prolong its half-life and increase bioactivity in vivo. For this purpose we, for the first time, used TR domain from glycoprotein MUC1, bearing 5 additional sites for O-glycosylation. Our result indicated that ligation of TR-domain to the coding sequence of EPO did not affect secretion of the chimeric protein into the medium, receptor binding affinity in vitro bioactivity, compared with EPO wild type. However, both the in vitro potency and half-life in circulation of EPO bearing TR or Fc fragments were significantly enhanced. After photothrombosis and injection of saline, the latency of entry into the dark compartment decreased from 300 s to 76 s. Treatment of rats with CEPO, CEPOTR, and CEPO-Fc after photothrombosis restored latency of PA to 243, 258, 227 s, respectively, on day 4. Only injection of CEPOTR retains passive avoidance latency 207 s on the day 7. Ischemic damage volume in animals treated with CEPO-TR on 7th day after operation was 15.8±5.4 against 24.22±4.7 with control injection of saline (P< 0.05). This phenomenon on 7th day was observed only with CEPO-TR. Protection efficiency coefficient, calculated from these experiments represented 34%. Another erythropoietin derivate did not give statistically significant results. Conclusion: Treatment of rats with CEPO and CEPO fusion proteins after photothrombosis of cortex resulted in restoration of passive avoidance response and diminishing of volume of ischemic damage. Those, our study indicates that CEPO-Fc and CEPO-TR display neuroprotectionand anti-amnestic activity, while CEPO-TR demonstrates prolongation ability.

Carbamylated erythropoietin attenuates cardiomyopathy via PI3K/Akt activation in rats with diabetic cardiomyopathy

The aim of the present study was to investigate the protective effect of carbamylated erythropoietin (CEPO) against cardiomyopathy in high-fat, high-carbohydrate diet-fed rats with streptozotocin (STZ)-induced diabetic cardiomyopathy (DCM). Healthy male Wistar rats were fed a high-fat, high-carbohydrate diet for four weeks, and then were injected with STZ twice (50 mg/kg, intraperitoneally). Once DCM was confirmed, the rats were divided randomly into the following groups: DCM without treatment, CEPO treatment at different dosages (500, 1,000 or 2,000 IU/kg) or recombinant human erythropoietin (rhEPO) treatment (1,000 IU/kg), for a four-week short intervention or an eight-week long intervention protocol. Healthy rats were used as normal controls. Venous blood samples were drawn for routine hematological examinations, and heart tissues were collected for histological analysis, as well as the determination of myocardial apoptosis and phosphatidylinositol-3-kinase (PI3K)/Akt signaling. CEPO treatment had no significant effect on the erythrocyte or hemoglobin levels in the rats with DCM; however, it reduced myocardial cell apoptosis in the rats and protected the cellular ultrastructure. In addition, CEPO treatment inhibited caspase-3 and increased Bcl-xl protein expression (P<0.05). It also increased PI3K (p85) and Akt1 expression at the mRNA and protein levels in the hearts of the rats with DCM, with a dose-response relationship. An eight-week treatment using CEPO, in comparison with a four-week protocol, marginally increased PI3K (p85) and Akt1 expression, and did not demonstrate significant benefit. The study indicated that CEPO protects against DCM, without markedly affecting erythropoiesis, and that the activation of PI3K/Akt may be a key mechanism in the protection conferred by CEPO.

Glutaraldehyde erythropoietin protects kidney in ischaemia/reperfusion injury without increasing red blood cell production

Recombinant human erythropoietin (rHuEPO) is currently the mainstay of renal anaemia treatment. Recently, rHuEPO has been shown to provide pleiotrophic tissue protection in various pathological conditions. However, the benefits of rHuEPO beyond anaemia treatment are limited because it increases red blood cell mass. Carbamylated erythropoietin (CEPO) is the first rHuEPO derivative that lacks erythropoietic activity but retains tissue protection properties. Since carbamylation targets lysine residues on rHuEPo, we hypothesized that targeted lysine modifications of rHuEPO may result in a novel non-erythropoietic erythropoietin. rHuEPO was subjected to various targeted lysine modifications. In vitro cytoprotection and apoptosis were evaluated using P19 and HEK293 cells. In vivo erythropoiesis was performed by administering the derivatives to animals for 2 weeks. Renoprotection was tested on an ischaemia/reperfusion (I/R) model. We synthesized a novel derivative, a glutaraldehyde erythropoietin (GEPO). This construct abolished in vivo erythropoiesis. Biochemical characterization showed that GEPO was more electrostatically negative than rHuEPO. Immunoprecipitation experiments revealed that GEPO bound to the IL3RB/EPOR heterotrimeric receptor and ameliorated cellular apoptosis via the activation of Bcl-2. Notably, Bcl-2 activation was suppressed by the JAK2 inhibitor, tyrphostin AG490. In vivo experiments showed that GEPO also ameliorated kidney damage due to I/R injury both functionally and histologically. Herein, we describe a novel lysine-modified rHuEPO, glutaradehyde-EPO (GEPO), obtained from a simple reaction. This derivative has no erythropoietic properties but retains cell-protective characteristics both in vitro and in vivo, with promise for future use as an adjunctive treatment of kidney disease.

Carbamylated erythropoietin ameliorates hypoxia‐induced cognitive and behavioral defects with the generation of choline acetyltransferase‐positive neurons

Carbamylated erythropoietin (CEPO) is attracting widespread interest because of its neuroprotective effects without influencing erythropoiesis. Here we show that CEPO, unlike EPO, does not stimulate erythropoiesis. Both CEPO and EPO inhibit the death/apoptosis of neurons in the hypoxic model of primary neurons and induce neuron proliferation and differentiation in hypoxic mice. Hypoxic mice show apparent memory deficits at 3 and 30 days after hypoxia. The administration of CEPO/EPO significantly improves cognitive and behavioral defects after hypoxic insults. Further investigation shows that CEPO/EPO induces neuron proliferation and differentiation and promotes the generation of choline acetyltransferase (ChAT)(+) neurons in hypoxic mice. Phosphorylated AKT was colabeled with ChAT(+) neurons and coexpressed in bromodeoxyuridine-positive cells, suggesting that the PI3K/AKT pathway may play a pivotal role in CEPO/EPO-cholinergic neuron generation. These results reveal that CEPO/EPO ameliorates hypoxia-induced cognitive and behavioral defects possibly through the generation of ChAT-positive neurons.