Erythropoietin Receptor Expression Research Articles

Imaging and serum biomarkers reflecting the functional efficacy of extended erythropoietin treatment in rats following infantile traumatic brain injury.

OBJECTIVE Traumatic brain injury (TBI) is a leading cause of death and severe morbidity for otherwise healthy full-term infants around the world. Currently, the primary treatment for infant TBI is supportive, as no targeted therapies exist to actively promote recovery. The developing infant brain, in particular, has a unique response to injury and the potential for repair, both of which vary with maturation. Targeted interventions and objective measures of therapeutic efficacy are needed in this special population. The authors hypothesized that MRI and serum biomarkers can be used to quantify outcomes following infantile TBI in a preclinical rat model and that the potential efficacy of the neuro-reparative agent erythropoietin (EPO) in promoting recovery can be tested using these biomarkers as surrogates for functional outcomes. METHODS With institutional approval, a controlled cortical impact (CCI) was delivered to postnatal Day (P)12 rats of both sexes (76 rats). On postinjury Day (PID)1, the 49 CCI rats designated for chronic studies were randomized to EPO (3000 U/kg/dose, CCI-EPO, 24 rats) or vehicle (CCI-veh, 25 rats) administered intraperitoneally on PID1-4, 6, and 8. Acute injury (PID3) was evaluated with an immunoassay of injured cortex and serum, and chronic injury (PID13-28) was evaluated with digitized gait analyses, MRI, and serum immunoassay. The CCI-veh and CCI-EPO rats were compared with shams (49 rats) primarily using 2-way ANOVA with Bonferroni post hoc correction. RESULTS Following CCI, there was 4.8% mortality and 55% of injured rats exhibited convulsions. Of the injured rats designated for chronic analyses, 8.1% developed leptomeningeal cyst-like lesions verified with MRI and were excluded from further study. On PID3, Western blot showed that EPO receptor expression was increased in the injured cortex (p = 0.008). These Western blots also showed elevated ipsilateral cortex calpain degradation products for αII-spectrin (αII-SDPs; p < 0.001), potassium chloride cotransporter 2 (KCC2-DPs; p = 0.037), and glial fibrillary acidic protein (GFAP-DPs; p = 0.002), as well as serum GFAP (serum GFAP-DPs; p = 0.001). In injured rats multiplex electrochemiluminescence analyses on PID3 revealed elevated serum tumor necrosis factor alpha (TNFα p = 0.01) and chemokine (CXC) ligand 1 (CXCL1). Chronically, that is, in PID13-16 CCI-veh rats, as compared with sham rats, gait deficits were demonstrated (p = 0.033) but then were reversed (p = 0.022) with EPO treatment. Diffusion tensor MRI of the ipsilateral and contralateral cortex and white matter in PID16-23 CCI-veh rats showed widespread injury and significant abnormalities of functional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD); MD, AD, and RD improved after EPO treatment. Chronically, P13-P28 CCI-veh rats also had elevated serum CXCL1 levels, which normalized in CCI-EPO rats. CONCLUSIONS Efficient translation of emerging neuro-reparative interventions dictates the use of age-appropriate preclinical models with human clinical trial-compatible biomarkers. In the present study, the authors showed that CCI produced chronic gait deficits in P12 rats that resolved with EPO treatment and that chronic imaging and serum biomarkers correlated with this improvement.

Expression of Hypoxia-Inducible Factor 1α in Invasive Breast Cancer with Metastasis to Lymph Nodes: Correlation with Steroid Receptors, HER2 and EPO-R.

Expression of hypoxia-inducible factor 1α (HIF-1α) reflects the degree of cell hypoxia and its increased expression was found in most neoplasms, their metastasis as well as in some precancerous lesions. The study aimed to investigate the expression HIF-1α in invasive breast cancer with metastasis to lymph nodes in correlation with steroid receptors (ER-estrogen receptor, PR-progesterone receptor), HER2 (human epidermal growth factor receptor 2) and EPO-R (erythropoietin receptor). A total of 58 cases of invasive breast cancer with metastasis to lymph nodes were assessed for expression of HIF-1α, EPO-R, ER, PR and HER2. In our study, among all invasive breast cancers, 36.2% exhibited HIF-1α expression in the nuclei of neoplastic cells. We also assessed the correlation between histological type of cancer and basic immunohistochemical profile that included HIF-1α expression and statistical significance was noted only in the case of PR-/ER-/HER2-/ /HIF-1α-/ and PR+/ER+/HER2-/HIF-1α-/ (p = 0.028 and p = 0.008, respectively). However, only in the case of the PR+/ER+/HER2-/HIF-1α+/ immunohistochemical profile and histological grading did we note a statistical significance (p = 0.006). Expression of HIF-1α was most often noted in cancers exhibiting expression of HER2 protein (57.14%). Our study also assessed the relationship between the expression of HIF-1α in invasive breast cancers and the expression of EPO-R and areas of necrosis, demonstrating a statistically significant dependence (p = 0.003). Expression of HIF-1α was more often noted in invasive HER2+ cancers characterized by high degree of aggressiveness and poorer prognosis, which might suggest that presence of HIF-1α protein expression in HER2+ cancers could be an additional prognostic factor, the frequent occurrence of the phenotype of HIF-1α and EPO-R in cancers invasive HER2 +, in the absence of ER and PR, may suggest that HIF-1α and EPO-R may be an indicator of the aggressiveness of invasive breast cancers, indicating the need for a specific forms of treatment in this group of patients.

Impact of Erythropoietin Modulation Using Hyperbaric Oxygen on Umbilical Cord Blood CD34+ Cell Homing

Background: Previously, we demonstrated that host pre-treatment with hyperbaric oxygen (HBO) improves engraftment of umbilical cord blood (UCB) CD34+ cells in a transplant murine model. Recent data suggest that erythropoietin (EPO) determines the fate of hematopoietic progenitor cells in favor of erythroid differentiation. Herein, we evaluated mechanisms underlying the favorable effect of HBO therapy on UCB CD34+ cell engraftment. We hypothesized that HBO modulates EPO which in turn enhances UCB CD34 homing to the marrow, thereby enhancing engraftment. Materials/methods: We examined EPO receptor (EPOR) expression on UCB CD34+ cells by flow cytometry and western blot. Transmigration assays were used to evaluate EPO effects on UCB CD34+ cell migration toward Stromal Cell-Derived Factor 1 (SDF-1; CXCL12) which has been shown to mediate CD34+ chemotaxis to the marrow. Using an NSG transplant murine model, we evaluated serum EPO levels by ELISA and early UCB bone marrow/spleen homing by flow cytometry. Bone marrow UCB cell differentiation was examined 1 and 2 weeks post-transplant using CFU-assay. Results: We found that on average 17.6 (4.9-38.7) % of CD34+ enriched UCB cells express EPOR. EPOR expression was confirmed by western blot. The percentage of EPOR positive cells was significantly higher in hematopoietic stem cells (HSC) defined as Lin- CD34+ CD38- CD45RA- CD90+ CD49f+ (45.7+/-1.4) compared to hematopoietic progenitor cells defined as Lin- CD34+ CD38+ (25.1+/-0.7) and multipotent progenitors defined as Lin- CD34+ CD38- CD45RA- CD90- CD49f- (27.2+/-0.3). Also, exposure of UCB CD34+ cells to EPO during cell culture inhibited their migration toward SDF-1 (Figure-1). HBO treated mice demonstrated significantly lower serum EPO blood levels compared to control mice (727.4+/- 42.02 versus1576 +/- 80.90 pg/mL, p <0.0001) measured 3-hours post-transplant. A higher percentage of human CD34+ was seen in the bone marrow of HBO treated mice 3-hours post-transplant (6.8+/-1.0 versus 3.4+/-0.6), p=0.01). HBO treated mice demonstrated significantly lower numbers of BFU-E ( 26.7+/-8.7 versus 75.3+/-19.3, p =0.043) and increasing numbers of CFU-G/M (166.0+/-27.9 versus 74.8+/-32.6, p =0.05). Conclusions: EPOR is enriched in UCB HSC. HBO lowers host blood EPO and as a result improves UCB CD34+ cell early bone marrow homing and enhances myeloid differentiation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Local HIF-2α/EPO Pathway in the Bone Marrow is Associated with Excessive Erythrocytosis and the Increase in Bone Marrow Microvessel Density in Chronic Mountain Sickness.

Chronic mountain sickness (CMS) is characterized by excessive erythrocytosis, and angiogenesis may be involved in the pathogenesis of this disease. The bone marrow niche is the primary site of erythropoiesis and angiogenesis. This study was aimed at investigating the associations of the levels of hypoxia-inducible factors (HIFs), erythropoietin (EPO), and erythropoietin receptor (EPOR), as well as microvessel density (MVD) in the bone marrow with CMS. A total of 34 patients with CMS and 30 control subjects residing in areas at altitudes of 3000-4500 m were recruited for this study. The mRNA and protein expression of HIF-2α and EPO in the bone marrow cells was significantly higher in the CMS patients than in the controls. Moreover, changes in HIF-2α expression in CMS patients were significantly correlated with EPO and hemoglobin levels. In contrast, the expression of mRNA and protein expression of HIF-1α and EPOR did not differ significantly between the CMS and control patients. Increased MVD was observed in the bone marrow of the patients with CMS and it was significantly correlated with hemoglobin. Bone marrow cells of CMS patients may show enhanced activity of the HIF-2α/EPO pathway, and EPO may regulate the erythropoiesis and vasculogenesis through autocrine or/and paracrine mechanisms in the bone marrow niche. The increased MVD in the bone marrow of CMS patients appears to be involved in the pathogenesis of this disease.

Nanoparticle facilitated inhalational delivery of erythropoietin receptor cDNA protects against hyperoxic lung injury

Our goals were to develop and establish nanoparticle (NP)-facilitated inhalational gene delivery, and to validate its biomedical application by testing the hypothesis that targeted upregulation of pulmonary erythropoietin receptor (EpoR) expression protects against lung injury. Poly-lactic-co-glycolic acid (PLGA) NPs encapsulating various tracers were characterized and nebulizated into rat lungs. Widespread NP uptake and distribution within alveolar cells were visualized by magnetic resonance imaging, and fluorescent and electron microscopy. Inhalation of nebulized NPs bearing EpoR cDNA upregulated pulmonary EpoR expression and downstream signal transduction (ERK1/2 and STAT5 phosphorylation) in rats for up to 21days, and attenuated hyperoxia-induced damage in lung tissue based on apoptosis, oxidative damage of DNA, protein and lipid, tissue edema, and alveolar morphology compared to vector-treated control animals. These results establish the feasibility and therapeutic efficacy of NP-facilitated cDNA delivery to the lung, and demonstrate that targeted pulmonary EpoR upregulation mitigates acute oxidative lung damage. From the Clinical EditorAcute lung injury often results in significant morbidity and mortality, and current therapeutic modalities have proven to be ineffective. In this article, the authors developed nanocarrier based gene therapy in an attempt to upregulate the expression of pulmonary erythropoietin receptor in an animal model. Inhalation delivery resulted in reduction of lung damage.

Correlation of adrenomedullin with the erythropoietin receptor and microvessel density in hepatocellular carcinoma.

IntroductionUncontrolled angiogenesis plays an essential role in the occurrence, metastasis and malignant progression of hepatocellular carcinoma (HCC). This study aimed to investigate the expression of adrenomedullin (ADM) in human HCC and its correlation with the expression of erythropoietin receptor (EPOR), microvessel density (MVD) and the tumor pathological characteristics.Material and methodsFresh tumor tissues were obtained from 30 HCC patients after hepatectomy. Ten cirrhotic and 10 normal liver tissues were included as controls. Expression of ADM and EPOR was determined by real-time PCR. The MVD was determined by counting the number of microvessels.ResultsThe MVD and the mRNA levels of ADM and EPOR in cancer tissues were significantly higher than those in the non-cancer tissues (p < 0.05). Expression of ADM was significantly correlated with the MVD and EPOR (r = 0.68 and 0.74, p < 0.01). Adrenomedullin and EPOR mRNA levels in HCC tissues were correlated with capsule invasion, pathological differentiation and tumor metastasis (p < 0.05).ConclusionsOur findings suggest that ADM and EPOR may serve as new regulatory factors involved in angiogenesis of HCC and represent novel targets for the treatment of HCC.

New genetic and physiological factors for excessive erythrocytosis and Chronic Mountain Sickness.

In the last few years, genetic and functional studies have provided important insight on the pathophysiology of excessive erythrocytosis (EE), the main sign of Chronic Mountain Sickness (CMS). The recent finding of the association of the CMS phenotype with a single-nucleotide polymorphism (SNP) in the Sentrin-specific Protease 1 (SENP1) gene, and its differential expression pattern in Andean highlanders with and without CMS, has triggered large interest in high-altitude studies because of the potential role of its gene product in the control of erythropoiesis. The SENP1 gene encodes for a protease that regulates the function of hypoxia-relevant transcription factors such as Hypoxia-Inducible Factor (HIF) and GATA, and thus might have an erythropoietic regulatory role in CMS through the modulation of the expression of erythropoietin (Epo) or Epo receptors. The different physiological patterns in the Epo-EpoR system found among Andeans, even among highlanders with CMS, together with their different degrees of erythropoietic response, might indicate specific underlying genetic backgrounds, which in turn might reflect different levels of adaptation to lifelong high-altitude hypoxia. This minireview discusses recent genetic findings potentially underlying EE and CMS, and their possible physiological mechanisms in Andean highlanders.

Abstract 4072: Embryonic rest hypothesis of cancer development revisited: functional gonadotropic hormone receptors are expressed by normal and malignant hematopoietic cells and functional erythropoietin receptor is expressed by germline-derived tumors

Abstract Background: The existence in adult tissues of developmentally early stem cells with broader specification potential may suggest the presence of embryonic primordial germ cell (PGC) remnants in post-natal organs. To support this small, quiescent stem cells (VSELs) that express several markers of PGCs reside in adult murine bone marrow (BM) (Leukemia 2010;24:1450), and like PGCs, are kept quiescent by erasure of imprinting on paternally imprinted genes (Leukemia 2009;23:2042). As reported hematopoietic stem/progenitor cells (HSPCs) can become specified from a population of migrating PGCs isolated from embryos (Blood 1995;86:463) as well as from adult bone marrow VSELs (Leukemia 2011;25,1278). In support of this intriguing possibility, HSPCs and PGCs are both highly migratory populations of stem cells, and specification of the first primitive HSPCs in yolk sac blood islands as well as the origin of definitive HSPCs in the aorta-gonado-mesonephros region are chronologically and anatomically correlated with the developmental migration of PGCs in extra- and intra-embryonic tissues on their way to the genital ridges. Hypothesis: Based on these observations, we have hypothesized that PGC-derived cells as well as HSPCs share some common receptors, and we tested the expression of gonadotropic hormone receptors (GHR) and erythropoietin receptor (EpoR) on HSPCs and PGC-derived cells, respectively. Materials and Methods. We employed RT-PCR studies to evaluate the expression of GHR on normal and malignant HSPCs, whereas we evaluated the expression of EpoR on teratocarcinoma and ovarian cancer cell lines. The functionality of these receptors was tested by chemotaxis, adhesion, and proliferation assays, and we performed signal transduction studies employing specific ligands for gonadal receptors to stimulate HSPCs and erythropoietin (EPO) to stimulate germline-derived cells. Results. We observed the expression of functional FSH, LH, PRL, estrogen, and androgen receptors on normal murine and human HSPCs and in leukemia cell lines. At the same time, we observed the presence of functional EpoRs on murine and human teratocarcinoma cells and ovarian cancer cell lines. Conclusions. Our data provide further evidence for the existence of a developmental link between germline and hematopoiesis and shed new light on the developmental hierarchy of the stem cell compartment in adult tissues and possibility that some malignancies may develop from embryonic remnants. These observations also have important practical implications: i) pituitary gonadal hormones could be employed in selected cases of BM failure to stimulate hematopoiesis and ii) EPO treatment (e.g., because of anemia after chemotherapy) should be avoided in patients with germline malignancies. Citation Format: Malwina Suszynska, Katarzyna Mierzejewska, Agata Poniewierska-Baran, Ahmed Abdelbasit Ismail, Gabriela Schneider, Pranesh Gunjal, Janina Ratajczak, Sham S. Kakar, Magda Kucia, Mariusz Z. Ratajczak. Embryonic rest hypothesis of cancer development revisited: functional gonadotropic hormone receptors are expressed by normal and malignant hematopoietic cells and functional erythropoietin receptor is expressed by germline-derived tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4072. doi:10.1158/1538-7445.AM2015-4072

The systemic vascular resistance response: a cardiovascular response modulating blood viscosity with implications for primary hypertension and certain anemias.

Without an active regulatory feedback loop, increased blood viscosity could lead to a vicious cycle of ischemia, increased erythropoiesis, further increases of blood viscosity, decreased tissue perfusion with worsened ischemia, further increases in red cell mass, etc. We suggest that an increase in blood viscosity is detected by mechanoreceptors in the left ventricle which upregulate expression of cardiac natriuretic peptides and soluble erythropoietin receptor. This response normalizes systemic vascular resistance and blood viscosity at the cost of producing 'anemia of chronic disease or inflammation' or 'hemolytic anemia' both of which are better described as states of compensated hyperviscosity. Besides its role in disease, this response is also active in the physiologic adaptation to chronic exercise. Malfunction of this response may cause primary hypertension.

Expression of platelet-derived growth factor BB, erythropoietin and erythropoietin receptor in canine and feline osteosarcoma

The discovery of expression of the erythropoietin receptor (EPO-R) on neoplastic cells has led to concerns about the safety of treating anaemic cancer patients with EPO. In addition to its endocrine function, the receptor may play a role in tumour progression through an autocrine mechanism. In this study, the expression of EPO, EPO-R and platelet-derived growth factor BB (PDGF-BB) was analysed in five feline and 13 canine osteosarcomas using immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR).EPO expression was positive in all tumours by IHC, but EPO mRNA was only detected in 38% of the canine and 40% of the feline samples. EPO-R was expressed in all samples by quantitative RT-PCR (RT-qPCR) and IHC. EPO-R mRNA was expressed at higher levels in all feline tumours, tumour cell lines, and kidney when compared to canine tissues. PDGF-BB expression was variable by IHC, but mRNA was detected in all samples. To assess the functionality of the EPO-R on tumour cells, the proliferation of canine and feline osteosarcoma cell lines was evaluated after EPO administration using an alamarBlue assay and Ki67 immunostaining. All primary cell lines responded to EPO treatment in at least one of the performed assays, but the effect on proliferation was very low indicating only a weak responsiveness of EPO-R. In conclusion, since EPO and its receptor are expressed by canine and feline osteosarcomas, an autocrine or paracrine tumour progression mechanism cannot be excluded, although in vitro data suggest a minimal role of EPO-R in osteosarcoma cell proliferation.

Retrospective outcomes in patients with BCR-ABL positive Acute Lymphoid Leukemia without Allogeneic stem cell transplantation at the Houston Methodist

S176 Introduction: Ph-like acute lymphoblastic leukemia (ALL) is characterized by a variety of rearrangements and other genetic alterations activating cytokine receptor and kinase signaling and poor outcome. We recently identified recurring rearrangements of the erythropoietin receptor (EPOR) into the immunoglobulin heavy (IGH) or kappa (IGK) chain loci. The goal of this study was to examine the role of these alterations in kinase signaling and lymphoid transformation.Methods:Wild-type or EPOR rearranged alleles were expressed in interleukin-3 (IL-3)-dependent mouse hematopoietic Ba/F3 cells and interleukin-7 (IL-7)-dependent pre-B cells harboring alterations of Arf and/or the dominant negative IKZF1 allele IK6 observed in EPOR-rearranged ALL. Proliferation and signaling were examined in the absence or presence of EPO. EPOR expression and signaling in cell lines and primary leukemic cells were examined by immunofluorescence, flow cytometry and immunoblotting. Luciferase-marked xenografts of human EPORrearranged ALL were established in NOD-SCID-IL2R gamma (NSG) null mice, and signaling, EPO-dependent proliferation and sensitivity to the JAK inhibitor ruxolitinib were assessed ex vivo and in vivo. Results: Eight cases out 154 Ph-like ALL (5.2%) harbored rearrangements of EPOR into either the IGH or IGK loci with two consequences: i) inversion and insertion of EPOR 5’ untranscribed region into the the promoter and enhancer region of IGH/IGK; ii) truncation of the last coding exon of EPOR, resulting in overexpression of a C-terminal truncated receptor that retains the phosphorylation site required for STAT5 activation, but lacking multiple residues required for negative EPOR regulation. Notably, the location of the truncation sites is similar to those arising from inherited mutations in primary familial congenital polycythemia. The truncated alleles were expressed at higher levels than wild-type EPOR in IL-3-dependent Ba/F3 and IL-7-dependent Arf-/mouse pre-B cells, and sustained cell proliferation and increased STAT5 phosphorylation following stimulation with exogenous EPO. Xenograft leukemic cells from EPOR-rearranged cases exhibited high levels of mutant EPOR on the cell surface, constitutive pSTAT5 and noteworthy ex vivo and in vivo sensitivity to the JAK2 inhibitor ruxolitinib. Conclusions: Rearrangements of truncated EPOR into the IGH/IGK chain loci define a new subgroup of Phlike ALL and unexpectedly implicate EPOR signaling as an important factor influencing B-lymphoid malignancies. Finally, these findings suggest that aberrant EPOR signaling is amenable to JAK-STAT5 inhibition in cases harboring these rearrangements.

EPOR expression by peritoneal macrophages and B1b cells (TUM6P.1010)

Abstract Erythropoietin (EPO) is used in the treatment of chemotherapy-related anemia. A higher mortality rate among cancer patients treated with EPO has been documented. Research in this area has focused upon EPOR expression by cancer cells, either cell lines or ex vivo tumor tissue. We find that addition of exogenous EPO suppresses the activation of peritoneal cavity (PerC) T cells. Via FACs analysis of cultured C57BL/6J (wildtype,WT) PerC cells, we detected the EPO receptor (EPOR) on F4/80+, CD11b+ macrophages (Mfs). The specificity of the anti-EPOR pAb was validated by inhibition of staining with soluble EPOR. The percentage of Mfs expressing the EPOR increased with culture duration. EPOR+ Mfs also increased expression of the immunoinhibitory molecule PDL1/B7H1. Interestingly, EPOR expression also appeared on cultured B1b (IgMhi, CD11blo) cells. Although less evident in WT mice assessed ex vivo, EPOR+ PerC Mfs were found in IL10KO mice. Considerably more EPOR+ B1b cells were observed in the inflammatory ascites of mice with the ovarian cancer that develops following injection of the ID8 mouse epithelial carcinoma cell line. We are currently investigating how EPO contributes to T cell suppression in vitro and impacts Mf and B1b cell function and phenotype. Our data support a link between inflammation and EPOR expression by PerC Mfs and B1b cells.

Erythropoietin attenuates intestinal inflammation and promotes tissue regeneration

Background. The prevalence of inflammatory bowel disease (IBD) is increasing. Since patients usually need long-term treatment and suffer from reduced quality of life, there is a need to develop new therapeutic strategy. The aim of this study was to investigate the therapeutic potential of erythropoietin (EPO) for the treatment of IBD. Methods. Murine colitis was induced by 3.0% Dextran Sulfate Sodium (DSS). Recombinant human EPO (rhEPO) was given to evaluate the anti-inflammatory and regenerative effects on intestinal inflammation. The effect of rhEPO on human colon epithelial cells was also evaluated. Immunohistochemical analysis of EPO receptor was performed in human IBD tissues. Results. While about 62% of control mice with severe colitis induced by 5-day DSS died, 85% of mice treated with rhEPO survived. Histological analysis confirmed that EPO treatment reduced the colonic inflammation. Furthermore, EPO treatment significantly downregulated the local expressions of IFN-γ, TNF-α and E-selectin in the colon, suggesting that the effect was associated with inhibiting local immune activation. In a 4-day DSS-induced colitis model, rhEPO significantly improved the recovery of body weight loss compared to controls. Furthermore, proliferating cell nuclear antigen expression was significantly upregulated in the colon tissue from mice treated with rhEPO compared to controls. In addition, rhEPO increased the growth of cultured human colon epithelial cells in a dose-dependent manner. Furthermore, EPO-receptor expression was confirmed in human IBD colon tissues. Conclusion. Three major functions of EPO, hematopoiesis, anti-inflammation and regeneration, may produce significant effects on intestinal inflammation, therefore suggesting that rhEPO might be useful for IBD.

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.

Widespread Expression of Erythropoietin Receptor in Brain and Its Induction by Injury.

Erythropoietin (EPO) exerts potent neuroprotective, neuroregenerative and procognitive functions. However, unequivocal demonstration of erythropoietin receptor (EPOR) expression in brain cells has remained difficult since previously available anti-EPOR antibodies (EPOR-AB) were unspecific. We report here a new, highly specific, polyclonal rabbit EPOR-AB directed against different epitopes in the cytoplasmic tail of human and murine EPOR and its characterization by mass spectrometric analysis of immuno-precipitated endogenous EPOR, Western blotting, immunostaining and flow cytometry. Among others, we applied genetic strategies including overexpression, Lentivirus-mediated conditional knockout of EpoR and tagged proteins, both on cultured cells and tissue sections, as well as intracortical implantation of EPOR-transduced cells to verify specificity. We show examples of EPOR expression in neurons, oligodendroglia, astrocytes and microglia. Employing this new EPOR-AB with double-labeling strategies, we demonstrate membrane expression of EPOR as well as its localization in intracellular compartments such as the Golgi apparatus. Moreover, we show injury-induced expression of EPOR. In mice, a stereotactically applied stab wound to the motor cortex leads to distinct EpoR expression by reactive GFAP-expressing cells in the lesion vicinity. In a patient suffering from epilepsy, neurons and oligodendrocytes of the hippocampus strongly express EPOR. To conclude, this new analytical tool will allow neuroscientists to pinpoint EPOR expression in cells of the nervous system and to better understand its role in healthy conditions, including brain development, as well as under pathological circumstances, such as upregulation upon distress and injury.

Erythropoietin Receptors and IgG Autoantibody Expression on Nucleated Erythrocytes in Some Cases of Immuno-Related Pancytopenia.

In recent years, we have detected autoantibodies in bone marrow (BM) hemopoietic cells in some patients with idiopathic cytopenia of undetermined significance (ICUS), termed immunorelated pancytopenia (IRP). However, we know little about the targets of these autoantibodies. Twenty-six newly diagnosed IRP patients with IgG autoantibody on nucleated erythrocytes and 20 healthy donors as controls were enrolled in this study. The serum erythropoietin (EPO) level was examined by ELISA. Expression of EPO receptor (EPOR) and IgG autoantibody on the membrane of nucleated erythrocytes were detected by flow cytometry before and after autoantibody stripping. The serum EPO level of the untreated patients was 199.9 ± 106.4 mIU/mL, which was significantly higher than that of normal controls (13.2 ± 8.41 mIU/mL, p < 0.01). EPOR expression on nucleated erythrocytes in the patients was 1.38 ± 0.73%, lower than that of normal controls (2.33 ± 1.73%), but there was no significant difference; EPOR in these patients was inversely correlated with IgG autoantibody on erythrocytes (r = -0.479, p = 0.013). The regression equation was Y = 0.116-0.479X; EPOR expression on the membrane increased significiantly (5.63 ± 4.99%, p < 0.01) after stripping the autoantibodies. After immunosuppressive treatment, median hemoglobin increased from 72 g/L to 98 g/L, and median reticulocytes increased from 1.46% to 3.56%. IgG autoantibodies might block or competitively inhibit EPOR on nucleated erythrocytes in some cases of ICUS.

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.

Erythropoietin and cancer: the unintended consequences of anemia correction.

Until 1990, erythropoietin (EPO) was considered to have a single biological purpose and action, the stimulation of red blood cell growth and differentiation. Slowly, scientific and medical opinion evolved, beginning with the discovery of an effect on endothelial cell growth in vitro and the identification of EPO receptors (EPORs) on neuronal cells. We now know that EPO is a pleiotropic growth factor that exhibits an anti-apoptotic action on numerous cells and tissues, including malignant ones. In this article, we present a short discussion of EPO, receptors involved in EPO signal transduction, and their action on non-hematopoietic cells. This is followed by a more detailed presentation of both pre-clinical and clinical data that demonstrate EPO’s action on cancer cells, as well as tumor angiogenesis and lymphangiogenesis. Clinical trials with reported adverse effects of chronic erythropoiesis-stimulating agents (ESAs) treatment as well as clinical studies exploring the prognostic significance of EPO and EPOR expression in cancer patients are reviewed. Finally, we address the use of EPO and other ESAs in cancer patients.

In-vivo detection of the erythropoietin receptor in tumours using positron emission tomography.

Recombinant human erythropoietin (rhuEpo) is used clinically to treat anaemia. However, rhuEpo-treated cancer patients show decreased survival rates and erythropoietin receptor (EpoR) expression has been found in patient tumour tissue. Thus, rhuEpo application might promote EpoR(+) tumour progression. We therefore developed the positron emission tomography (PET)-probe (68)Ga-DOTA-rhuEpo and evaluated its performance in EpoR(+) A549 non-small-cell lung cancer (NSCLC) xenografts. (68)Ga-DOTA-rhuEpo was generated by coupling DOTA-hydrazide to carbohydrate side-chains of rhuEpo. Biodistribution was determined in tumour-bearing mice 0.5, 3, 6, and 9 h after probe injection. Competition experiments were performed by co-injecting (68)Ga-DOTA-rhuEpo and rhuEpo in five-fold excess. Probe specificity was further evaluated histologically using Epo-Cy5.5 stainings. The blood half-life of (68)Ga-DOTA-rhuEpo was 2.6 h and the unbound fraction was cleared by the liver and kidney. After 6 h, the highest tumour to muscle ratio was reached. The highest (68)Ga-DOTA-rhuEpo accumulation was found in liver (10.06 ± 6.26%ID/ml), followed by bone marrow (1.87 ± 0.53%ID/ml), kidney (1.58 ± 0.39%ID/ml), and tumour (0.99 ± 0.16%ID/ml). EpoR presence in these organs was histologically confirmed. Competition experiments showed significantly (p < 0.05) lower PET-signals in tumour and bone marrow at 3 and 6 h. (68)Ga-DOTA-rhuEpo shows favourable pharmacokinetic properties and detects EpoR specifically. Therefore, it might become a valuable radiotracer to monitor EpoR status in tumours and support decision-making in anaemia therapy. • PET-probe (68) Ga-DOTA-rhuEpo was administered to assess the EpoR status in vivo • (68) Ga-DOTA-rhuEpo binds specifically to EpoR positive organs in vivo • Tumour EpoR status determination might enable decision-making in anaemia therapy with rhuEpo.

Erythropoietin Accelerates the Regeneration of Ureteral Function in a Murine Model of Obstructive Uropathy

Unilateral ureteral obstruction halts ureteral peristalsis, and may cause pain and lead to infection. Ureteral ability to recover after obstruction removal remains unclear. Erythropoietin has protective effects in nonhematopoietic organs and restores peristalsis in hypocontractile intestinal smooth muscle cells. We investigated the role of erythropoietin in ureteral smooth muscle function and its therapeutic value for unilateral ureteral obstruction. Unilateral ureteral obstruction was created for 24, 48 and 72 hours in 22 mice per group using a nontraumatic microclip via laparotomy. We determined erythropoietin, erythropoietin receptor and β-common receptor expression in obstructed and unobstructed ureters by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Ten mice per group received 20 IU erythropoietin for 4 days and controls received saline. Hydronephrosis regression after obstruction removal was assessed by ultrasound. Peristalsis was determined microscopically before and after obstruction removal. Erythropoietin, erythropoietin receptor and β-common receptor were expressed in the unobstructed and obstructed ureters of untreated mice. Erythropoietin mRNA was up-regulated in response to obstruction and erythropoietin expression was identified in ureteral smooth muscle. After obstruction removal hydronephrosis and ureteral dysfunction correlated with obstruction duration. Hydronephrosis resolution and ureteral peristalsis restoration were significantly accelerated in erythropoietin treated mice compared to controls. Erythropoietin treatment significantly promoted functional recovery of the ureter after obstruction removal. Erythropoietin may be a helpful strategy for ureteral motility recovery and hydronephrosis resolution in ureteral obstruction.