Epo Expression Research Articles

Increased Serum Erythropoietin despite Normalized Hb Concentration and Arterial O2 Saturation in Chronic Mountain Sickness after Isovolemic Hemodilution

Excessive erythrocytosis (EE) is a hallmark of Chronic Mountain Sickness (CMS), an incapacitating condition among high‐altitude residents. EE is associated with accentuated hypoxemia and neurological symptoms. Periodic hemodilution is a common treatment used to normalize excessive red blood cell count and relieve symptomatology. However, it is unclear whether relief results from an improvement of arterial O2 saturation (SaO2) and/or whether the normalization of Hb affects the erythropoietic stimulus. Thus, we sought to investigate the effect of isovolemic hemodilution on resting arterial PO2 (PaO2), SaO2, arterial O2 content (CaO2), serum EPO, iron profile, and CMS score in CMS subjects. Six CMS and eight non‐CMS male residents of Cerro de Pasco, Peru (4350m), of similar age participated in the study. The CMS group exhibited significantly higher Hb concentration (22.2 ± 1.2 vs 17.8 ± 1.3 g/dl, p<0.001) and CMS score (10.0 ± 6.6 vs 1.1 ± 1.1, p<0.01) with a lower pulse O2 saturation (SpO2; 84.7 ± 1.0 vs 88.3 ± 1.6%, p<0.05). Radial artery catheters were placed to obtain arterial blood measurements. For isovolemic hemodilution, a venous catheter was placed in the arm of CMS subjects to remove ~2–4 units of blood (1675 ± 442 ml) for a reduction of ~19% pre‐hemodilution Hb value. Isovolemia was maintained with an equivalent volume of plasma substitute (Haemaccel®). Baseline PaO2, SaO2, serum EPO, iron, ferritin, and transferrin saturation were similar in both non‐CMS and CMS groups, while CaO2 was higher in CMS (24.4 ± 1.9 vs 20.7 ± 1.6 mlO2/dl, p<0.001). At 36 hours post‐hemodilution, Hb in the CMS group decreased by 20.1 ± 1.6 % (from 22.2 ± 1.2 to 17.7 ± 0.6 g/dl, p<0.001), matching non‐CMS subject values. PaO2 and SaO2 remained essentially unchanged, whereas CaO2 decreased (from 24.4 ± 1.9 to 20 ± 0.8 mlO2/dl, p<0.001). A substantial drop was observed in serum iron (from 112.3 ± 44 to 60.8 ± 30.3 μg/dl, p<0.002) and transferrin saturation (from 28.0 ± 10.8 to 17.7 ± 8.2%, p<0.013). Serum EPO increased significantly after hemodilution (from 25.6 ± 7.6 to 68.2 ± 25.8 mIU/ml, p<0.006), resulting in higher values compared to pre‐hemodilution and non‐CMS subjects (15.9 ± 8.4 mIU/ml). After Hb normalization, CMS symptomatology improved (from 10.0 ± 6.6 to 1.2 ± 1.1, p<0.01) without a significant increase in SaO2. Despite this, serum EPO increased dramatically reflecting erythropoietic stimulus. It is possible that chronic EE determines a higher set‐point for Hb and CaO2 in CMS subjects, and thus even reduction to non‐CMS values is sensed as relative tissue hypoxia due to reduced O2 delivery. In addition, iron depletion could also contribute to EPO expression through inhibition of prolyl hydroxylase enzymes and HIF stabilization.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

VEGF-loaded mineral-coated microparticles improve bone repair and are associated with increased expression of epo and RUNX-2 in murine non-unions.

A poor vascular supply of the fracture gap is a key factor for the development of atrophic non-unions. Mineral-coated microparticles (MCM) represent a sophisticated carrier system for the delivery of vascular endothelial growth factor (VEGF). Hence, we investigated whether VEGF-loaded MCM improve bone repair in non-unions. For this purpose, we analyzed binding and release kinetics of MCM for VEGF in vitro. Moreover, we applied VEGF-loaded or -unloaded MCM in a murine non-union model in vivo and studied the process of bone healing by means of biomechanical, radiological, histomorphometric, and Western blot techniques. MCM-free non-unions served as controls. The binding efficiency of MCM for VEGF was 46 ± 3% and the release profile revealed an initial minor burst release followed by a sustained release over a 50-day study period, thus, mimicking the physiological expression profile of VEGF during bone healing. In vivo, bone defects treated with VEGF-loaded MCM exhibited a higher bending stiffness, a higher fraction of bone volume/tissue volume and a larger callus area on days 14 and 70 when compared to the other groups. Western blot analyses on day 14 revealed a higher expression of VEGF, erythropoietin (EPO), and runt-related transcription factor 2, but not of EPO-receptor in bone defects treated with VEGF-loaded MCM. These findings demonstrate that the use of MCM for VEGF delivery shows great potential due to the ability to maintain protein stability and functionality in vivo. Moreover, the application of VEGF-loaded MCM represent a promising strategy for the treatment of non-unions. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Ochratoxin A upregulates biomarkers associated with hypoxia and transformation in human kidney cells

Cellular adaptation to hypoxia is controlled by hypoxia-inducible factor 1α (HIF1α), a transcription factor activated in response to oxygen tension, reactive oxygen species (ROS) and inflammation. Overexpression of HIF1α and HSP90 has been associated with cancer induction. Ochratoxin A (OTA), a mycotoxin contaminant of food and beverages, has been linked to renal tumours and progressive nephropathies, inflammation and pro-oxidation. The aim of this study was to examine the effect of OTA on hypoxic and transformative regulators in human embryonic kidney (HEK293) cells. We evaluated the protein expression of HIF1α, HSP90 and PDK1 (western blotting), mRNA expression of HIF1α, VEGF, EPO and TGFβ (qPCR), and ATP levels (luminometry) in HEK293 cells exposed to a range of OTA concentrations (0.125 μM–0.5 μM) over two time periods (24 h and 48 h). After 24 h, OTA increased HIF1α protein (p < 0.005) and EPO gene expression (p < 0.05), while VEGF and TGFβ was significantly increased at 48 h. We also observed a correlation between PDK1 expression and ATP levels. In conclusion, OTA disrupts hypoxia regulation, modulates metabolic activity, and alters growth signalling (VEGF, TGFβ), which may lead to tumourigenesis.

The Recombinant Human Erythropoietin Administered in Neonatal Rats After Excitotoxic Damage Induces Molecular Changes in the Hippocampus.

In vitro and in vivo experimental evidence has contributed important knowledge regarding the antiapoptotic effect mediated by EPO signaling in the damaged brain, particularly through different models with a hypoxic component. However, little emphasis has been placed on the effectiveness of rhEPO administration against cellular alterations caused by in vivo excitotoxicity or on the molecular mechanism that regulates this effect. In this study, we investigated the effects of a single dose of rhEPO on hippocampal damage induced by subcutaneous application of monosodium glutamate (MSG) on postnatal days 1, 3, 5 and 7 in neonatal rats. We found that a dose of 1000 IU/kg of b.w. administered 24 h after MSG had the greatest protective effect. In addition, we analyzed changes in gene expression, particularly in 3 key molecules involved in EPO-mediated signaling (EPO, EPOR and βcR). We observed that the expression of EPO and EPOR was differentially modified at both the mRNA and protein levels under the evaluated conditions, while the expression of the βcR gene was substantially increased. Our data suggest that a low dose of rhEPO is sufficient to induce cellular protection under these experimental conditions and that the molecular changes could be a positive feedback mechanism, mediated by reactive astrocytes in association with in vivo neuroprotective mechanisms.

Upregulation of endogenous erythropoietin expression by DLBS6747, a bioactive fraction of Ipomoea batatas L. leaves, via increasing HIF1α transcription factor in HEK293 kidney cells

Ethnopharmacological relevanceIpomoea batatas L., locally known as ubi jalar, is widely used in Indonesia and other countries as a folk remedy for various chronic diseases, including anemia-associated chronic kidney disease by increasing hematological parameters such as packed cell volume, white blood cells and platelet counts. Aim of the studyThe aim of this study is to evaluate the effect of DLBS6747, a bioactive fraction of I. batatas L. leaves, on increasing EPO expression through the upregulation of HIF1α. Materials and methodsEffect of DLBS6747 on EPO expression and its transcription factor, HIFs, was evaluated in normoxia and hypoxia conditions. Effect of DLBS6747 on several genes involved in EPO expression were evaluated in a time-course manner using conventional and real-time PCR, while the protein level were revealed using western blot and ELISA. The involvement of HIF1α was also confirmed by HIF1α siRNA. ResultsAdministration of DLBS6747 increased transcriptional activity of EPO through the regulation of its transcriptional factors, which include HIF1α, HIF2α and NFᴋB. The effect was found to be dependent on oxygen availability, wherein DLBS6747-increased EPO expression was found to be more significant in hypoxic condition. In normoxia and hypoxia, 40 μg/mL DLBS6747 increased HIF1α and HIF2α expressions at mRNA level, wherein the peak appeared in 12 h treatment (up to 7.9- and 8.6-folds, respectively). On the other hand, increased protein level was only found in hypoxia, where the highest HIF1α expression was observed at 6 h (7.5-folds increase) and started to decrease after the hours, while HIF2α was found to be increased time-dependently (up to 13.8-folds in 24 h). The mechanism of action of DLBS6747 as erythropoietin stimulating agent is more likely to affect the regulation of HIF1α, as confirmed by HIF1α siRNA which showed that DLBS6747 failed to increase EPO expression during co-incubation with HIF1α siRNA. DLBS6747 treatment also decreased NFᴋB time-dependently in normoxia, while no NFᴋB was detected in hypoxia, which revealed mimicking hypoxia activity of DLBS6747 to increase EPO expression. ConclusionThese findings showed convincing evidences that DLBS6747 increases endogenous EPO production primarily via upregulation of its transcription factors, especially HIF1α, in human embryonic kidney HEK293 cells. This is the first molecular report that reveals the mechanism of action of natural-based erythropenia drug in different oxygen availability.

Clinicopathological significance of Epo, EpoR, Ki-67 and Bax expression in colorectal cancer

Introduction: Expression of Epo, a glycoprotein secreted by the fetal liver and the adult kidney in response to cellular hypoxia and its receptor have been described in human solid tumors, such as colon and breast cancer. Purpose: Since activation of Epo-EpoR signaling pathway in erythroid progenitor and precursor cells leads to promotion of proliferation and differentiation or prevention of programmed cell death through Bcl-xl and Bcl-2 it was of interest to investigate expression of Epo, EpoR, apoptosis regulator – Bax and marker of proliferating cells - Ki-67 and assess correlation between them, with regard to clinicopathological variables of colorectal cancer. Materials and methods: The correlations between expression of Epo, EpoR, Bax and Ki-67 in colorectal cancer were analyzed in regard to patient age, sex, primary localization, histopathological type, grading, staging and lymph node invasion. Statistical analyses were performed by using the Spearman rank correlation test applying a significance level of p&lt;0,05. Results: Correlation between Bax and EpoR is positive and statistically significant at all groups of patients except group pT1+pT2. Positive correlation between Bax and Epo is statistically significant at following groups of patients: all patients, age  60, age &gt;60, male, female , primary localization in rectum, primary localization in colon, adenocarcinoma, G2, G3. Statistical analysis revealed no significant correlations between expression of neither Ki-67 with Epo nor Ki-67 with EpoR in all groups of patients. Conclusions: Epo seems to be a pleiotropic cytokine, which can exert its biological effect on several cell types, including neoplastic cells. The effect of Epo-EpoR signaling can differ in various cells and conditions.

Effects of Abnormal Iron Metabolism on EPO-STAT5 Signaling Pathway in Anemia Patients

To study the effects of iron metabolism abnormality on EPO-STAT5 signaling pathway in anemia patients. According to diseases, the patients were divided into 3 groups: lower risk myelodysplastic syndrome (MDS) group (30 cases) including 14 cases of non-iron over load and 16 cases of iron over load, 12 cases of them were treated by iron chelation therapy; anemia of chronic disease (ACD) group (12 cases) and iron deficiency anemia (IDA) group (12 cases). In addition, the healthy control group was selected. The iron metaloslism index (SF, SI, TIBC), serum level of EPO, plasma level of P-STAT5 and STAT-5 mRNA expression in peripheral blood cells were detected and compared in different groups. Moreover, the effects of iron metabolism abnormality on the expression of EPO and STAT5 in anemia patients were analyzed. compared with non-iron over load group, the EPO level in iron over load group significantly increased (P<0.05), the expression of STAT5 mRNA and P-STAT5 significantly decreased (P<0.05). After iron chelation therapy, the EPO level in serum significantly decreased (P<0.05), the expression of STAT5 mRNA and P-STAT5 was up-regulated significantly (P<0.05). Compared with healthy control group, the expression of EPO in ACD group was down-regulated significantly, while the expression of STAT5 mRNA was not different, but the P-STAT5 expression was down-regulated significantly (P<0.05). Compared with the healtly control group, the EPO expression in IDA group was enhanced significantly (P<0.05), the expression of STAT5 mRNA and P-STAT5 were also significantly enhanced (P<0.05). The excessive iron load or chronic inflammation may inhibit the activation of EPO-STAT5 signaling pathway and aggravate the anemia.

Additive Effects of Decreased TfR1 and Ablated Erfe Improve Both Ineffective Erythropoiesis and Iron Overload in β-Thalassemic Mice

Patients with β-thalassemias manifest anemia, ineffective erythropoiesis, extramedullary hematopoiesis, splenomegaly, and systemic iron overload. Even in non-transfusion dependent patients, iron overload in β-thalassemia develops because of increased intestinal iron absorption, leading to multiple organ dysfunction if untreated and accounts for most of the deaths in this disease. The main regulator of body iron content and distribution is hepcidin, inhibiting iron absorption in duodenal enterocytes and release of stored iron from macrophages and hepatocytes. Despite iron overload in patients and mice with β-thalassemia, hepcidin levels are insufficiently increased, as ineffective erythropoiesis dominates hepcidin regulation. Relatively low hepcidin causes iron overload in β-thalassemia. Recent evidence demonstrates that erythroferrone (ERFE), an erythroid regulator of hepcidin, is increased in bone marrow and serum from β-thalassemic patients and th3/+ mice [Kautz Nat Gen 2014] and its loss results in increased hepcidin, partially reversing iron overload in th3/+ mice [Kautz Blood 2015]. In addition, bone marrow ERFE expression normalizes in TfR1 haploinsufficient th3/+ mice [Li Blood 2017]. We hypothesize that the loss of ERFE and TfR1 influences erythropoiesis and iron metabolism in complementary ways in th3/+ mice, and therefore aim to explore iron- and erythropoiesis-related parameters in th3/+ TfR1+/- ERFE-/- (triple mutant (TM)) mice. All models are on a C57BL6 background and have been crossed to generate 4-6 mice for analysis at 6 weeks of age. We confirm our previous reports [Li Blood 2017] that th3/+TfR1+/- mice have increased RBC count and hemoglobin, decreased MCV and reticulocyte count (Table I), and reduce splenomegaly (Fig 1a and 1b) relative to th3/+ mice. We also confirm that th3/+ ERFE-/- mice do not reverse splenomegaly or improve peripheral blood circulating erythroid parameters compared to th3/+ mice [Kautz Blood 2015] (Table I) but exhibit further increase in TfR1 in late stage erythroid precursors (Fig 1c). Analysis of the bone marrow reveals that total erythroid mass is unaltered in triple mutants relative to th3/+, th3/+ ERFE-/-, and th3/+ TfR1+/- mice, but the number of late erythroblasts (poly-E and ortho-E stages) is normalized to WT levels (Fig 1d), strongly suggesting that, unlike in th3/+ erythropoiesis, where the block in differentiation occurs at the poly-E stage, th3/+ TfR1+/- and especially triple mutant mice restore differentiation at this stage to generate a higher hemoglobin. No differences in erythroblast apoptosis or ROS concentration are evident in triple mutant relative to th3/+ ERFE-/- or th3/+ TfR1+/- mice. We also analyzed markers of Epo responsiveness and demonstrate that serum Epo and EpoR expression are increased in th3/+ relative to WT mice (Fig 1e and 1f), but while serum Epo is decreased, EpoR is further increased (Fig 1f). These findings suggest that Epo responsiveness is more optimized in triple mutant erythroblasts, enabling a smaller proportion of late stage erythroblasts to produce circulating RBCs with relatively less serum Epo. Remarkably, while neither th3/+ ERFE-/- and th3/+ TfR1+/- mice reverse iron overload or impact hepcidin expression at 6 weeks of age, triple mutant mice demonstrate fully normalized ratio of hepcidin expression relative to liver iron concentration (LIC) (Fig 1g). Taken together, these experiments provide evidence of the differential and additive effects of TfR1 and ERFE loss in th3/+ mice, with a predominantly erythropoietic benefit of TfR1 loss, a predominantly iron-homeostatic benefit of ERFE loss, and synergy of both in optimizing Epo responsiveness. DisclosuresGanz:Intrinsic LifeScience: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Silarus Pharma: Consultancy, Equity Ownership; Keryx Pharma: Consultancy, Research Funding; Gilead: Consultancy; Ablynx: Consultancy; Vifor: Consultancy; Akebia: Consultancy, Research Funding; La Jolla Pharma: Consultancy, Patents & Royalties: Patent licensed to La Jolla Pharma by UCLA.

Moringa oleifera extract attenuates the CoCl2 induced hypoxia of rat's brain: Expression pattern of HIF-1α, NF-kB, MAO and EPO

Hypoxia-induced oxidative stress and apoptosis are the major hallmark explanations underlying brain dysfunction. Hypoxia in the current study was induced by Cobalt chloride (CoCl2) treatment in rats. The aim of this experiment was to explore the potential ameliorative potency of Moringa oleifera ethanolic extract (MO) against experimentally induced hypoxia on the structure and function of the rat’s brain. Fifty male rats were allocated to five groups (10 rats each): a control group, a MO-treated group (400 mg/kg bw, orally), a CoCl2-treated group (40 mg/kg bw/day, orally), a prophylaxis group, and a therapeutic co-treated group. Oxidative stress biomarkers and monoamine neurotransmitter were evaluated in brain tissue. In addition, qRT-PCR for expression pattern of HIF-1α, EPO, CYTO, NF-kB, and MAO-A. Glial fibrillary acidic protein (GFAP), apoptotic markers (BCL-2 and caspase 3) were detected immunohistochemically in brain cells. The results revealed a significantly lower concentration of GABA, monoamine neurotransmitter in hypoxic rat’s brain. Moreover, an evident up-regulation of the mRNA expression of HIF-1α, EPO, CYTO, NF-kB, and MAO-A. There was marked encephalopathy manifested by pyknotic neurons with eosinophilic cytoplasm, vacuolations and cerebral congestions in the hypoxic rat brains. Additionally, the score of neuronal expression occupied by GFAP- positive astroglia, Caspase-3 and microglial CD68 were elevated but Bcl-2 expression was found decreased in the hypoxic group than control. The endpoints of this study clearly stated that MO ethanolic extract suggestively counteracted neurotoxic impacts caused by hypoxia, particularly when it administered prior to and concurrently with CoCl2 administration.

The protective effect of Hif3a RNA interference and HIF-prolyl hydroxylase inhibition on cardiomyocytes under anoxia-reoxygenation

The aim of this study was to investigate the molecular mechanisms underlying the protective effects of hypoxia-inducible factor (HIF) signaling pathway activation in cardiomyocytes under anoxia-reoxygenation (A/R) injury. In this study, rat neonatal cardiomyocytes were pretreated with anti-Hif3A/Hif-3α siRNA or HIF-prolyl hydroxylase inhibitor prior to A/R injury. Our results showed that both HIF3A silencing and HIF-prolyl hydroxylase inhibition effectively increased the cell viability during A/R, led to changes in mRNA expression of HIF1-target genes, and reduced the loss of mitochondrial membrane potential (Δψm). Furthermore, application of anti-Hif3a siRNA led to an increase in mRNA expression of Epo, Igf1, Slc2a1/Glut-1, and Slc2a4/Glut-4. Similar results were observed with HIF-prolyl hydroxylase inhibition, which additionally upregulated the mRNA expression of Epor, Tert, and Pdk1. Hif3a RNA-interference and application of HIF-prolyl hydroxylase inhibitor during A/R modelling led to an increase of Δψm on 11.5 and 11.9 mV respectively, compared to the control groups. Thus, Hif3a RNA interference and HIF-prolyl hydroxylase inhibition protect cardiomyocytes against A/R injury via the HIF signaling pathway.

An EGLN1 mutation may regulate hypoxic response in cyanotic congenital heart disease through the PHD2/HIF-1A pathway

Cyanotic congenital heart disease (CCHD), a term describing the most severe congenital heart diseases are characterized by the anatomic malformation of a right to left shunt. Although the incidence of CCHD are far less than the that of congenital heart diseases (CHD), patients with CCHD always present severe clinical features such as hypoxia, dyspnea, and heart failure. Chronic hypoxia induces hypoxemia that significantly contributes to poor prognosis in CCHD. Current studies have demonstrated that the prolyl-4-hydroxylase2 (PHD2, encoded by EGLN1)/hypoxia-inducible factor-1A (HIF-1A) pathway is a key regulator of hypoxic response. Thus, we aim to assess the associations of single polymorphisms (SNPs) of the EGLN1 gene and hypoxic response in CCHD. A missense variant of EGLN1 c.380G>C (rs1209790) was found in 46 patients (46/126), with lower hypoxia incidence and higher rate of collateral vessel formation, compared with the wild type (P < 0.05). In vitro experiments, during hypoxia, EGLN1 mutation reduced EGLN1 expression compared with the wild type, with higher HIF-1A, VEGF and EPO expression levels in the mutant. No difference in HK1 expression was observed between the mutant and wild type. CCHD patients with c.380G>C showed improved response to hypoxia compared with the wild-type counterparts. The EGLN1 c.380G>C mutation improves hypoxic response through the PHD2/HIF-1A pathway, which may provide a molecular mechanism for hypoxic response in CCHD. The effects of the EGLN1 c.380G>C mutation on CCHD prognosis deserve further investigation.

Protective effect of adenovirus-mediated erythropoietin expression on the spiral ganglion neurons in the rat inner ear.

The aim of the present study was to evaluate the expression of erythropoietin (Epo) and the Epo receptor (Epo-R) in the spiral ganglion neurons (SGNs) of the rat inner ear, and to assess the effect of Epo adenovirus vector (Ad-Epo) on the spontaneous apoptosis of SGNs. A total of 60 ears from 30 healthy neonatal (2-3 days postnatal) Sprague-Dawley rats were used to examine the expression of Epo in the SGNs. The rats were divided into three groups: The negative control group, the vector control group [infected with a green fluorescent protein expression vector (Ad-GFP)] and the Ad-Epo group (infected with Ad-Epo). The expression of Epo and Epo-R was detected by immunohistochemistry and dual immunofluorescence staining using polyclonal antibodies directed against Epo and Epo-R, followed by confocal laser-scanning microscopy. An adenovirus vector was constructed and used to transfect the cultured SGNs. Following adenovirus infection, apoptosis of the SGNs was evaluated and Epo protein expression was assessed. Epo and Epo-R were widely expressed in the plasma membrane and the cytoplasm of the SGNs, as well as in the organ of Corti and the stria vascularis within the inner ear. Epo protein expression was upregulated in the Ad-Epo group compared with that in the other two groups (P<0.05). Apoptotic cells were seldom observed at day 4 of SGN culture in the negative control group. At day 7, marked apoptotic cells were detected in the negative control group and the vector control group. The apoptosis level in the Ad-Epo group was significantly decreased compared with that in the negative control group or the vector control group at day 7 (P<0.05). In conclusion, Epo and Epo-R are expressed in the SGNs of the inner ear of the rat, and Ad-Epo can decrease the spontaneous apoptosis of SGNs, which may provide a basis for the prevention or alleviation of sensorineural hearing loss.

Spatial and Cellular Expression Patterns of Erythropoietin-Receptor and Erythropoietin during a 42-Day Post-Lesional Time Course after Graded Thoracic Spinal Cord Impact Lesions in the Rat.

Erythropoietin (Epo) exhibits promising neuroregenerative potential for spinal cord injury (SCI), and might be involved in other long-term sequelae, such as neuropathic pain development. The current studies investigated the time courses and spatial and cellular patterns of Epo and erythropoietin receptor (EpoR) expression along the spinal axis after graded SCI. Male Long Evans rats received 100 kdyn, 150 kdyn, and 200 kdyn thoracic (T9) contusions from an Infinite Horizon impactor. Sham controls received laminectomies. Anatomical and quantitative immunohistochemical analyses of the EpoR/Epo expression along the whole spinal axis were performed 7, 15, and 42 postoperative days (DPO) after the lesioning. Cellular expression was investigated by double- and triple-labeling for EpoR/Epo with cellular markers and proliferating cells in subgroups of 5-bromo-2-deoxyuridine pre-treated animals. Prolonged EpoR/Epo-expression was confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Quantified EpoR/Epo immunoreactivities in pain-related spinal cord regions and ventrolateral white matter (VLWM) were correlated with the mechanical sensitivity thresholds and locomotor function of the respective animals. EpoR and Epo were constitutively expressed in the ventral horn neurons and vascular and glial cells in the dorsal columns (DC) and the VLWM. After SCI, in addition to expression in the lesion core, EpoR/Epo immunoreactivities exhibited significant time- and lesion grade-dependent induction in the DC and VLWM along the spinal axis. EpoR and Epo immunoreactive cells were co-stained with markers for astroglial, neural precursor cell and vascular markers. In the VLWM, EpoR- and Epo-positive proliferating cells were co-stained with glial fibrillary acidic protein (GFAP) and nestin. The DC EpoR/Epo immunoreactivities exhibited linear relationships with the behavioral correlates of post-lesional chronic pain development at DPO 42. SCI leads to long-lasting multicellular EpoR/Epo induction beyond the lesion core in the spinal cord regions that are involved in central pain development and regenerative processes. Our studies provide a time frame to investigate the effects of Epo application on motor function or pain development, especially in the later time course after lesioning.

No erythropoietin-induced growth is observed in non-small cell lung cancer cells.

Lung cancer patients have the highest incidence of anemia among patients with solid tumors. The use of recombinant human erythropoietin (Epo) has consistently been shown to reduce the need for blood transfusions and to increase hemoglobin levels in lung cancer patients with chemotherapy-induced anemia. However, clinical and preclinical studies have prompted concerns that Epo and the presence of its receptor, EpoR, in tumor cells may be responsible for adverse effects and, eventually, death. The question has been raised whether Epo promotes tumor growth and inhibits the death of cancer cells. In this study, we investigated the presence and functionality of EpoR, as well as the implications of Epo upon the proliferation and survival of lung cancer cells. Since the protein expression of both Epo and EpoR is induced by hypoxia, which is frequently present in lung cancer, the cells were treated with Epo under both normoxic and hypoxic conditions (1% O2). By using quantitative (real-time) PCR, western blot analysis, and immunocytochemical staining, three non-small cell lung cancer (NSCLC) cell lines (A427, A549 and NCI-H358) were analyzed for the expression of EpoR and its specific downstream signaling pathways [Janus kinase2 (Jak2)-signal transducer and activator of transcription5 (STAT5), phosphatidylinositol-3-kinase (PI3K)-Akt, mitogen-activated protein (MAP) kinase]. The effects of 100U/ml Epo on cell proliferation and cisplatin-induced apoptosis were assessed. All NSCLC cell lines expressed EpoR mRNA and protein, while these levels differed considerably between the cell lines. We found the constitutive phosphorylation of EpoR and most of its downstream signaling pathways (STAT5, Akt and ERK1/2) independently of Epo administration. While Epo markedly enhanced the proliferation and reduced apoptosis of Epo-dependent UT-7/Epo leukemia cells, it did not affect tumor cell proliferation or the cisplatin-induced apoptosis of NSCLC cells. Thus, this invitro study suggests that there are no tumor-promoting effects of Epo in the NSCLC cell lines studied, neither under normoxic nor under hypoxic conditions.

Erythropoietin promoted the proliferation of hepatocellular carcinoma through hypoxia induced translocation of its specific receptor

BackgroundErythropoietin (EPO) is a hypoxia-inducible stimulator of erythropoiesis. Besides its traditional application in anemia therapy, it offers an effective treatment in the cancer patients, especially those who receive chemotherapy. Several reports indicated that it could promote the tumor cell proliferation through its specific receptor (EPOR). Unfortunately, the role of EPO/EPOR in hepatocellular carcinoma (HCC) progressing is still uncertain.MethodsProtein in tumor tissue from HCC patients or H22 tumor-bearing mice was detected with immunohistochemistry. Cells were cultured under 1% oxygen to establish hypoxia. RT-PCR and western blotting were used to measure mRNA and protein of EPO/EPOR, respectively. MTT, flow cytometry and PCNA staining were used to detect cell proliferation. Immunofluorescence staining was applied to study the expression and location of cellular EPOR. The EPOR binding studies were performed with 125I-EPO radiolabeling assay.ResultsEPO and EPOR protein were up-regulated in HCC tissue of patients and H22-bearing mice. These were positively correlated with hypoxia-inducible factor -1 α and ki-67. Hypoxia up-regulated the expression of EPO and EPOR in HepG2 cells. It also induced the proliferation and increased the percentage of divided cells after 24, 48 and 72 h treatment. These were inhibited in cells pre-treated with 0.5 μg/mL soluble-EPOR. Immunofluorescence staining presented that EPOR was obviously translocated from nucleus to cytoplasm and membrane under hypoxia. EPOR binding activity was also increased after exposure to hypoxia. Recombinant human erythropoietin obviously elevated cell proliferation rate and the percentage of divided under hypoxia but not normoxia, which were also inhibited by soluble-EPOR.ConclusionsOur result indicated for the first time that EPO promoted the proliferation of HCC cells through hypoxia induced translocation of it specific receptor.Trial registration TJC20141113, retrospectively registered

Erythropoietin signaling increases neurogenesis and oligodendrogenesis of endogenous neural stem cells following spinal cord injury both invivo and invitro.

Erythropoietin (Epo) promotes functional recovery following spinal cord injury (SCI); however, the exact underlying mechanisms are yet to be determined. Although endogenous neural stem cells (NSCs) in the adult spinal cord are a therapeutic target in SCI models, the effect of Epo on this NSC population remains unknown. The present study investigated the effects of Epo on endogenous NSCs in the adult spinal cord both in vitro and in vivo. For the in vivo analyses, normal rats (Normal) and SCI contusion model rats (SCI) received either recombinant human Epo or saline treatment for 7 days (5,000 U/kg), and spinal cords were subsequently analyzed at 2, 8, and 14 days. For in vitro analyses, NSCs harvested from adult rat spinal cords were exposed to Epo (10 U/ml). A significant increase in β-tubulin+ new neurons (P<0.01) was observed at all three time points and O4+ new oligodendrocytes (P<0.05) at days 8 and 14 in the SCI+Epo group compared with the SCI+Saline group. This was concomitant with a prolonged activation of Epo signaling; however, no effect on NSCs proliferation was observed. Similar results were also obtained in vitro. Motor functional recovery was also noted at days 8 and 14 only in the Epo-treated SCI rats. Although the expression of Epo and EpoR significantly increased in Normal+Epo rats compared with Normal+Saline rats (P<0.05), the cell numbers and phenotype were comparable between the two groups. To the best of the author's knowledge, this is the first study to demonstrate that Epo signaling promotes both neurogenesis and oligodendrogenesis following SCI and that these may represent the underlying mechanisms for the functional recovery and therapeutic effects of Epo following SCI.

Erythropoietin reduces experimental autoimmune encephalomyelitis severity via neuroprotective mechanisms

BackgroundTreatment with erythropoietin (Epo) in experimental autoimmune encephalomyelitis (EAE), the rodent model of multiple sclerosis (MS), has consistently been shown to ameliorate disease progression and improve overall outcome. The effect has been attributed to modulation of the immune response and/or preservation of the central nervous system (CNS) tissue integrity. It remains unclear, however, if (a) Epo acts primarily in the CNS or the periphery and if (b) Epo’s beneficial effect in EAE is mainly due to maintaining CNS tissue integrity or to modulation of the immune response. If Epo acts primarily by modulating the immune system, where is this modulation required? In the periphery, the CNS or both?MethodsTo address these questions, we used two well-characterized transgenic mouse strains that constitutively overexpress recombinant human Epo (rhEpo) either systemically (tg6) or in CNS only (tg21) in a MOG-induced EAE model. We assessed clinical severity, disease progression, immunomodulation, and CNS tissue integrity, including neuronal survival.ResultsAlthough disease onset remained unaffected, EAE progression was alleviated in transgenic animals compared to controls with both lines performing equally well showing that expression of Epo in the periphery is not required; Epo expression in the CNS is sufficient. Immunomodulation was observed in both strains but surprisingly the profile of modulation differed substantially between strains. Modulation in the tg21 strain was limited to a reduction in macrophages in the CNS, with no peripheral immunomodulatory effects observed. In contrast, in the tg6 strain, macrophages were upregulated in the CNS, and, in the periphery of this strain, T cells and macrophages were downregulated. The lack of a consistent immunomodulatory profile across both transgenic species suggests that immunomodulation by Epo is unlikely to be the primary mechanism driving amelioration of EAE. Finally, CNS tissue integrity was affected in all strains. Although myelin appeared equally damaged in all strains, neuronal survival was significantly improved in the spinal cord of tg21 mice, indicating that Epo may ameliorate EAE predominantly by protecting neurons.ConclusionsOur data suggests that moderate elevated brain Epo levels provide clinically significant neuroprotection in EAE without modulation of the immune response making a significant contribution.

Hypoxia-related biological markers as predictors of epirubicin-based treatment responsiveness and resistance in locally advanced breast cancer.

PurposeTo identify hypoxia-related biomarkers indicative of response and resistance to epirubicin treatment in patients with locally advanced breast cancer.Patients and MethodsOne hundred seventy-six women with T2-4 N0-1 breast tumours were randomly assigned to receive epirubicin 120 mg/m2/1-21 (EPI ARM), epirubicin 120 mg/m2/1-21 + erythropoietin 10.000 IU sc three times weekly (EPI-EPO ARM) and epirubicin 40 mg/m2/w-q21 (EPI-W ARM). Sixteen tumour proteins involved in cell survival, hypoxia, angiogenesis and growth factor, were assessed by immunohistochemistry in pre-treatment samples. A multivariate generalized linear regression approach was applied using a penalized least-square minimization to perform variable selection and regularization.ResultsVEGF and GLUT-1 expression were significantly positively associated with complete response (CR) to treatment in all leave-one-out iterations. Bcl-2 expression was inversely correlated with pCR, whilst EPO expression was positively correlated with pathological complete response (pCR). Haemaglobin and HIF-1a nuclear expression were inversely correlated with pCR. HB and HIF-1a expression were associated with a higher risk of relapse and overall survival.ConclusionHypoxic biomarkers determines the epirubicin resistance in breast cancer. Assessment of such biomarkers, may be useful for predicting chemosensitivity and also anthracycline-based treatment outcome.

Zebrafish slc30a10 deficiency revealed a novel compensatory mechanism of Atp2c1 in maintaining manganese homeostasis.

Recent studies found that mutations in the human SLC30A10 gene, which encodes a manganese (Mn) efflux transporter, are associated with hypermanganesemia with dystonia, polycythemia, and cirrhosis (HMDPC). However, the relationship between Mn metabolism and HMDPC is poorly understood, and no specific treatments are available for this disorder. Here, we generated two zebrafish slc30a10 mutant lines using the CRISPR/Cas9 system. Compared to wild-type animals, mutant adult animals developed significantly higher systemic Mn levels, and Mn accumulated in the brain and liver of mutant embryos in response to exogenous Mn. Interestingly, slc30a10 mutants developed neurological deficits in adulthood, as well as environmental Mn-induced manganism in the embryonic stage; moreover, mutant animals had impaired dopaminergic and GABAergic signaling. Finally, mutant animals developed steatosis, liver fibrosis, and polycythemia accompanied by increased epo expression. This phenotype was rescued partially by EDTA- CaNa2 chelation therapy and iron supplementation. Interestingly, prior to the onset of slc30a10 expression, expressing ATP2C1 (ATPase secretory pathway Ca2+ transporting 1) protected mutant embryos from Mn exposure, suggesting a compensatory role for Atp2c1 in the absence of Slc30a10. Notably, expressing either wild-type or mutant forms of SLC30A10 was sufficient to inhibit the effect of ATP2C1 in response to Mn challenge in both zebrafish embryos and HeLa cells. These findings suggest that either activating ATP2C1 or restoring the Mn-induced trafficking of ATP2C1 can reduce Mn accumulation, providing a possible target for treating HMDPC.

The Role of S100A8 and S100A9 in Differentiation of Human Eosinophilic Leukemia Cells, EoL-1

S100A8 and S100A9 are associated with myeloid cell differentiation, chemotactic activities, adhesion of neutrophils, and apoptosis. In this study, we investigated the contribution of S100A8 and S100A9 to differentiation of the human eosinophilic leukemia cell line, EoL-1. S100A8 and S100A9 increased the number of vacuole per one cell and the protein expression of EPO and MBP. Rottlerin, an inhibitor of protein kinase C delta (PKCδ), inhibited the EoL-1 cell differentiation induced by S100A8 and S100A9. These results suggest that S100A8 and S100A9 may regulate the differentiation of eosinophilic progenitors. Moreover, these findings may shed light on elucidation of eosinophil differentiation due to S100 proteins.