EpoR-tdTomato-Cre mice enable identification of EpoR expression in subsets of tissue macrophages and hematopoietic cells.

The erythropoietin receptor (EpoR) has traditionally been thought of as an erythroid-specific gene. Notably, accumulating evidence suggests that EpoR is expressed well beyond erythroid cells. However, the expression of EpoR in non-erythroid cells has been controversial. In this study, we generated EpoR-tdTomato-Cre mice and used them to examine the expression of EpoR in tissue macrophages and hematopoietic cells. We show that in marked contrast to the previously available EpoR-eGFPcre mice, in which a very weak eGFP signal was detected in erythroid cells, tdTomato was readily detectable in both fetal liver (FL) and bone marrow (BM) erythroid cells at all developmental stages and exhibited dynamic changes during erythropoiesis. Consistent with our recent finding that erythroblastic island (EBI) macrophages are characterized by the expression of EpoR, tdTomato was readily detected in both FL and BM EBI macrophages. Moreover, tdTomato was also detected in subsets of hematopoietic stem cells, progenitors, megakaryocytes, and B cells in BM as well as in spleen red pulp macrophages and liver Kupffer cells. The expression of EpoR was further shown by the EpoR-tdTomato-Cre-mediated excision of the floxed STOP sequence. Importantly, EPO injection selectively promoted proliferation of the EpoR-expressing cells and induced erythroid lineage bias during hematopoiesis. Our findings imply broad roles for EPO/EpoR in hematopoiesis that warrant further investigation. The EpoR-tdTomato-Cre mouse line provides a powerful tool to facilitate future studies on EpoR expression and regulation in various non-hematopoietic cells and to conditionally manipulate gene expression in EpoR-expressing cells for functional studies.

New insights into erythropoietin and erythropoietin receptor in laryngeal cancer tissue.

ABSTRACT: To investigate whether laryngeal cancer cells express erythropoietin (Epo) and erythropoietin receptor (EpoR) and what is their possible relationship with clinical and pathological features of the tumor.We performed immunohistochemical analysis of Epo and EpoR expression on 78 tissue samples of invasive and in situ squamous cell laryngeal carcinoma.The statistical analysis showed a weak positive and statistically significant correlation of EpoHS and EpoR HS expression levels. Epo HS and EpoR HS levels did not correlate with patient sex or age, type of diagnosis, cancer stage, histological tumor grade, presence or absence of disease recurrence, type of oncologic cancer therapy provided, or results of selected laboratory blood work. The results show a statistically significant difference in Epo expression with respect to survival.We confirmed the presence of Epo an EpoR in malignant laryngeal tumors and demonstrated the correlation between Epo expression and survival. Further studies are needed to more precisely define the role of Epo and EpoR in treatment of patients with laryngeal cancer.

Erythropoetin and erythropoetin receptor systems, and oxidative status in patients with basal cell.

INTRODUCTION: Erythropoietin (Epo) controls a variety of signal transduction pathways during oxidative stress. The main function of Epo and its receptor (EpoR) is the stimulation of erythropoiesis. AIM: The role of Epo and EpoR on non-hematopoietic normal and cancerous tissues is still poorly understood. This is the first report in which we aimed to investigate the role of Epo and EpoR systems at oxidative condition in human basal cell carcinoma (BCC), which is the most common tumour in the world.Materials and methods: Fresh normal and cancerous skin paired tissue was obtained from 63 patients who underwent curative BCC resection in Kahramanmaras, Turkey. Preliminary diagnosis of BCC was made in the dermatology clinic by excision and then the diagnosis was confirmed as histopathologic findings. Oxidative stress biomarkers such as superoxide dismutase (SOD) and catalase (CAT) activities, and malondialdehyde (MDA) levels in biopsy samples were measured spectrophotometrically, and also the levels of Epo and EpoR were measured by ELISA. RESULTS: While the levels of MDA in cancerous tissue of patients with skin BCC were significantly higher than normal neighbouring skin tissue (p<0.05), SOD and CAT activities decreased (p<0.05). Furthermore, a remarkable increase was found in the Epo level ofpatients with skin BCC in comparison with the normal neighbouring skin tissue (p<0.05). However, we found that EpoR levels decreased (p<0.05). CONCLUSIONS: Results indicate that there is an active oxidative process in BCC biopsies. The levels of increased Epo and decreased EpoR in oxidative condition due to hypoxia may aggravate tumour growth by its angiogenic activity.

Age-associated expression of erythropoietin and its receptor in rat spiral ganglion neurons and its association with neuronal apoptosis and hearing alterations.

The present study aimed to determine the expression of erythropoietin (EPO) and the EPO receptor (EPOR) in spiral ganglion neurons (SGNs) in the inner ear of rats of various ages, and the associated neuronal apoptosis and hearing alterations. A total of 15 healthy rats (n=30 ears), were divided into three groups: i) A nominated infant group at post­natal day (PND) 12­14, ii) an adult group at PND 60 and iii) a 3­year postnatal aged group. Auditory brainstem response (ABR) measurements were performed on all rats. EPO and EPOR expression in the inner ear was detected by immunohistochemistry. In situ terminal deoxynucleotidyl transferase dUTP nick end labeling assays were performed to detect the apoptosis of SGNs. The average hearing thresholds of the ABR (decibels above normal hearing level) were 5.625±4.955 in the infant, 15.000±8.498 in the adult and 23.500±13.134 in the aged groups. Hearing thresholds for aged and adult rats increased significantly compared with infant rats. However, the difference in latencies of peak I was not significant (P>0.05). EPO in SGNs was detected during different developmental periods without significant alterations, but were reduced compared with Corti's organ or the stria vascularis. EPOR expression increased significantly from infant to adult stage, and this increased expression was maintained in the aged group. An age­associated increase in the apoptosis of SGNs was detected in all three groups (P=0.0347). The potential neuroprotective effects of EPO in SGNs may not be revealed during the aging process under natural conditions, and may be associated with spontaneous neuronal apoptosis and consequently, hearing diminution. However, the age­associated increase in EPOR in SGNs may exert a role in neuroprotection when necessary, for example in presbycusis.

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

BACKGROUND: 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. OBJECTIVES: 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). MATERIAL AND METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Erythropoietin (EPO)-receptor signaling induces cell death of primary myeloma cells in vitro.

BACKGROUND: Multiple myeloma is an incurable complex disease characterized by clonal proliferation of malignant plasma cells in a hypoxic bone marrow environment. Hypoxia-dependent erythropoietin (EPO)-receptor (EPOR) signaling is central in various cancers, but the relevance of EPOR signaling in multiple myeloma cells has not yet been thoroughly investigated. METHODS: Myeloma cell lines and malignant plasma cells isolated from bone marrow of myeloma patients were used in this study. Transcript levels were analysed by quantitative PCR and cell surface levels of EPOR in primary cells by flow cytometry. Knockdown of EPOR by short interfering RNA was used to show specific EPOR signaling in the myeloma cell line INA-6. Flow cytometry was used to assess viability in primary cells treated with EPO in the presence and absence of neutralizing anti-EPOR antibodies. Gene expression data for total therapy 2 (TT2), total therapy 3A (TT3A) trials and APEX 039 and 040 were retrieved from NIH GEO omnibus and EBI ArrayExpress. RESULTS: We show that the EPOR is expressed in myeloma cell lines and in primary myeloma cells both at the mRNA and protein level. Exposure to recombinant human EPO (rhEPO) reduced viability of INA-6 myeloma cell line and of primary myeloma cells. This effect could be partially reversed by neutralizing antibodies against EPOR. In INA-6 cells and primary myeloma cells, janus kinase 2 (JAK-2) and extracellular signal regulated kinase 1 and 2 (ERK-1/2) were phosphorylated by rhEPO treatment. Knockdown of EPOR expression in INA-6 cells reduced rhEPO-induced phospo-JAK-2 and phospho-ERK-1/2. Co-cultures of primary myeloma cells with bone marrow-derived stroma cells did not protect the myeloma cells from rhEPO-induced cell death. In four different clinical trials, survival data linked to gene expression analysis indicated that high levels of EPOR mRNA were associated with better survival. CONCLUSIONS: Our results demonstrate for the first time active EPOR signaling in malignant plasma cells. EPO-mediated EPOR signaling reduced the viability of myeloma cell lines and of malignant primary plasma cells in vitro. Our results encourage further studies to investigate the importance of EPO/EPOR in multiple myeloma progression and treatment. TRIAL REGISTRATION: [Trial registration number for Total Therapy (TT) 2: NCT00083551 and TT3: NCT00081939 ].

Isoforms of the Erythropoietin receptor in dopaminergic neurons of the Substantia Nigra.

Erythropoietin receptor (EpoR) regulates erythrocytes differentiation in blood. In the brain, EpoR has been shown to protect several neuronal cell types from cell death, including the A9 dopaminergic neurons (DA) of the Substantia Nigra (SN). These cells form the nigrostriatal pathway and are devoted to the control of postural reflexes and voluntary movements. Selective degeneration of A9 DA neurons leads to Parkinson's disease. By the use of nanoCAGE, a technology that allows the identification of Transcription Start Sites (TSSs) at a genome-wide level, we have described the promoter-level expression atlas of mouse A9 DA neurons purified with Laser Capture Microdissection (LCM). Here, we identify mRNA variants of the Erythropoietin Receptor (DA-EpoR) transcribed from alternative TSSs. Experimental validation and full-length cDNA cloning is integrated with gene expression analysis in the FANTOM5 database. In DA neurons, the EpoR gene encodes for a N-terminal truncated receptor. Based on STAT5 phosphorylation assays, we show that the new variant of N-terminally truncated EpoR acts as decoy when co-expressed with the full-length form. A similar isoform is also found in human. This work highlights new complexities in the regulation of Erythropoietin (EPO) signaling in the brain.

Identification of Cell Type-Specific Differences in Erythropoietin Receptor Signaling in Primary Erythroid and Lung Cancer Cells.

Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC), is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO). However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR) and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid) and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR) in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR). The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in erythroid progenitor cells unaffected. Thus, the proposed modeling strategy can be employed as a general procedure to identify cell type-specific parameters and to recommend treatment strategies for the selective targeting of specific cell types.

Lenalidomide Stabilizes the Erythropoietin Receptor by Inhibiting the E3 Ubiquitin Ligase RNF41.

In a subset of patients with non-del(5q) myelodysplastic syndrome (MDS), lenalidomide promotes erythroid lineage competence and effective erythropoiesis. To determine the mechanism by which lenalidomide promotes erythropoiesis, we investigated its action on erythropoietin receptor (EpoR) cellular dynamics. Lenalidomide upregulated expression and stability of JAK2-associated EpoR in UT7 erythroid cells and primary CD71+ erythroid progenitors. The effects of lenalidomide on receptor turnover were Type I cytokine receptor specific, as evidenced by coregulation of the IL3-Rα receptor but not c-Kit. To elucidate this mechanism, we investigated the effects of lenalidomide on the E3 ubiquitin ligase RNF41. Lenalidomide promoted EpoR/RNF41 association and inhibited RNF41 auto-ubiquitination, accompanied by a reduction in EpoR ubiquitination. To confirm that RNF41 is the principal target responsible for EpoR stabilization, HEK293T cells were transfected with EpoR and/or RNF41 gene expression vectors. Steady-state EpoR expression was reduced in EpoR/RNF41 cells, whereas EpoR upregulation by lenalidomide was abrogated, indicating that cellular RNF41 is a critical determinant of drug-induced receptor modulation. Notably, shRNA suppression of CRBN gene expression failed to alter EpoR upregulation, indicating that drug-induced receptor modulation is independent of cereblon. Immunohistochemical staining showed that RNF41 expression decreased in primary erythroid cells of lenalidomide-responding patients, suggesting that cellular RNF41 expression merits investigation as a biomarker for lenalidomide response. Our findings indicate that lenalidomide has E3 ubiquitin ligase inhibitory effects that extend to RNF41 and that inhibition of RNF41 auto-ubiquitination promotes membrane accumulation of signaling competent JAK2/EpoR complexes that augment Epo responsiveness. Cancer Res; 76(12); 3531-40. ©2016 AACR.

Erythropoietin and erythropoietin receptor in hepatocellular carcinoma: correlation with vasculogenic mimicry and poor prognosis.

To evaluate erythropoietin (Epo) and erythropoietin receptor (EpoR) expression, its relationship with vasculogenic mimicry (VM) and its prognostic value in human hepatocellular carcinoma (HCC), we examined Epo/EpoR expression and VM formation using immunohistochemistry and CD31/PAS (periodic acid-Schiff) double staining on 92 HCC specimens. The correlation between Epo/EpoR expression and VM formation was analyzed using two-tailed Chi-square test and Spearman correlation analysis. Survival curves were generated using Kaplan-Meier method. Multivariate analysis was performed using Cox regression model to assess the prognostic values. Results showed positive correlation between Epo/EpoR expression and VM formation (P < 0.05). Patients with Epo or EpoR expression exhibited poorer overall survival (OS) than Epo-negative or EpoR-negative patients (P < 0.05). Epo-positive/VM-positive and EpoR-positive/VM-positive patients had the worst OS (P < 0.05). In multivariate survival analysis, age, Epo and EpoR were independent prognostic factors related to OS. These results will provide evidence for further research on HCC microcirculation patterns and also will provide new possible targets for HCC diagnosis and treatment.

Erythropoietin receptor and tissue factor are coexpressed in human breast cancer cells.

PURPOSE: Erythropoiesis-stimulating agents (ESAs) are recommended for treating chemotherapy-induced anemia in breast cancer patients. Reduced survival rates in ESAs-treated patients have been reported, possibly due to thromboembolic complications, however the exact mechanism remains obscure. The principal activator of blood coagulation in cancer is tissue factor (TF). There are data that erythropoietin receptor (EPO-R) is expressed in tumor cells. The purpose of this study was to evaluate the expression of EPO-R and TF in loco in breast cancer. METHODS: The expression of EPO-R and TF was investigated in 24 invasive breast carcinoma specimens. Immunohistochemical (IHC) methodologies according to ABC technique and double-staining IHC procedure were employed utilizing antibodies against EPO-R and TF. RESULTS: Expression of EPO-R and TF was demonstrated in the tumor cells in all breast cancer specimens. No staining for EPO-R and TF was visualized in normal breast tissue. Double staining studies revealed co-expression of both EPO-R and TF in breast cancer cells and endothelial cells. CONCLUSIONS: EPO-R and TF expression and their coexpression in breast cancer cells suggest a possibility that EPO-R might be responsible for some adverse effects and reduced survival observed in ESAs-treated breast cancer patients with anemia, possibly due to the interaction with TF. Further experimental studies are warranted to determine the role of both EPO-R and TF in the treatment with ESAs of breast cancer patients with chemotherapy-induced anemia.

Biologic factors associated with tumor oxygenation are prognostic in patients with stage III esophageal cancer: long-term results.

BACKGROUND/AIM: Long-term results of a study investigating potential prognostic factors for treatment outcomes in patients with stage III esophageal cancer are presented. PATIENTS AND METHODS: In 64 patients, the impact of tumor cell expression of erythropoietin (EPO) and erythropoietin-receptor (EPO-R) and ten additional factors (age, gender, performance status, tumor length, tumor stage (T-stage), nodes (N-stage), histology/grading, hemoglobin levels during radiotherapy, surgery) on survival and loco-regional control was evaluated up to 10 years following radio-chemotherapy. RESULTS: On multivariate analysis, improved survival was associated with low EPO-R expression (p=0.034) and hemoglobin levels during radiotherapy ≥ 12 g/dl (p=0.026). Low EPO expression was associated with survival on univariate (p=0.010) but not on multivariate analysis (p=0.42). On multivariate analysis, improved loco-regional control was significantly associated with hemoglobin levels during radiotherapy ≥ 12 g/dl (p<0.001). CONCLUSION: The long-term results confirm that hemoglobin levels during radiotherapy and tumor cell expression of EPO-R are significant prognostic factors in patients with locally advanced esophageal cancer.

Potential role of erythropoietin receptors and ligands in attenuating apoptosis and inflammation in critical limb ischemia.

OBJECTIVE: Managing critical limb ischemia (CLI) is challenging. Furthermore, ischemic myopathy prevents good functional outcome after revascularization. Hence, we have focused on limiting the tissue damage rather than angiogenesis, which has traditionally been the motivation to develop nonsurgical treatments for CLI. Erythropoietin (EPO) protects ischemic tissue, and this property may also benefit CLI. The objective of this study was to examine the expression of the tissue-protective EPO receptor complex (EPOR-CD131 [ß-chain of interleukin (IL)-3/IL-5/granulocyte macrophage colony-stimulating factor receptor]) in skeletal muscle obtained from humans with CLI. Because native EPO is thrombogenic, the antiapoptotic and anti-inflammatory effects of a nonhematopoietic helix-B peptide of EPO (ARA 290) were investigated on ischemic myotubes in vitro. METHODS: Tissue was obtained from gastrocnemius muscle of 12 patients undergoing amputation for CLI and from 12 patients without limb ischemia. The expression of EPOR and CD131 was demonstrated by immunohistochemistry and Western blot. A validated in vitro model of myotube ischemia was used in which mature C2C12 myotubes were cultured 6 to 12 hours in a depleted media and gas mixture (20% CO2 and 80% N2). The myotubes were pretreated with EPO or ARA 290 before exposure to simulated ischemia. Apoptosis and cell death were determined by cleaved caspase-3 assay and lactate dehydrogenase release assay. Enzyme-linked immunosorbent assay measured the inflammatory cytokines. RESULTS: EPOR and CD131 were expressed and significantly upregulated in CLI (average optical density [OD] in Western blot [control vs CLI] EPOR, 0.05 U vs 0.1 U; CD131, 0.10 U vs 0.22 U; P < .01). There was colocalization of EPOR and CD131 in the sarcolemma (cell membrane) of the skeletal myofiber. There was no difference in the distribution of colocalization between the CLI and the normal muscle. The ischemic myotubes treated by ARA 290 in vitro had a significantly decreased number of apoptotic cells (ischemia vs ischemia plus ARA 290: 71.1% vs 55.1%; P < .01), cleaved caspase-3 (OD of ischemia vs ischemia plus ARA 290: 0.15 U vs 0.02 U; P < .01), lactate dehydrogenase release (ischemia vs ischemia plus ARA 290: 32.5 U/L vs 21.3 U/L; P < .01), and IL-6 release (OD at 450 nm, ischemia vs ischemia plus ARA 290: 0.18 vs 0.13; P < .01). CONCLUSIONS: This study demonstrates the expression and the upregulation of EPOR and CD131 in CLI and also shows that EPOR and CDI are colocalized in the cell membrane of both ischemic and control muscle fiber. The in vitro experiments demonstrate that ARA 290 decreases inflammation and apoptosis of ischemic myotubes. ARA 290 may potentially be used as adjunctive treatment for CLI.

Epo receptors are not detectable in primary human tumor tissue samples.

Erythropoietin (Epo) is a cytokine that binds and activates an Epo receptor (EpoR) expressed on the surface of erythroid progenitor cells to promote erythropoiesis. While early studies suggested EpoR transcripts were expressed exclusively in the erythroid compartment, low-level EpoR transcripts were detected in nonhematopoietic tissues and tumor cell lines using sensitive RT-PCR methods. However due to the widespread use of nonspecific anti-EpoR antibodies there are conflicting data on EpoR protein expression. In tumor cell lines and normal human tissues examined with a specific and sensitive monoclonal antibody to human EpoR (A82), little/no EpoR protein was detected and it was not functional. In contrast, EpoR protein was reportedly detectable in a breast tumor cell line (MCF-7) and breast cancer tissues with an anti-EpoR polyclonal antibody (M-20), and functional responses to rHuEpo were reported with MCF-7 cells. In another study, a functional response was reported with the lung tumor cell line (NCI-H838) at physiological levels of rHuEpo. However, the specificity of M-20 is in question and the absence of appropriate negative controls raise questions about possible false-positive effects. Here we show that with A82, no EpoR protein was detectable in normal human and matching cancer tissues from breast, lung, colon, ovary and skin with little/no EpoR in MCF-7 and most other breast and lung tumor cell lines. We show further that M-20 provides false positive staining with tissues and it binds to a non-EpoR protein that migrates at the same size as EpoR with MCF-7 lysates. EpoR protein was detectable with NCI-H838 cells, but no rHuEpo-induced phosphorylation of AKT, STAT3, pS6RP or STAT5 was observed suggesting the EpoR was not functional. Taken together these results raise questions about the hypothesis that most tumors express high levels of functional EpoR protein.

Prognosis of patients with locally advanced squamous cell carcinoma of the head and neck. Impact of tumor cell expression of EPO and EPO-R.

BACKGROUND AND PURPOSE: This study investigated the prognostic role of tumor cell expression of erythropoietin (EPO) and its receptor (EPO-R) in patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN) treated with surgery plus radiotherapy. PATIENTS AND METHODS: The impact of EPO, EPO-R, and 11 additional factors on locoregional control (LRC), metastases-free survival (MFS), and overall survival (OS) was retrospectively evaluated in 144 patients. Additional factors were age, gender, performance status, preradiotherapy (pre-RT) hemoglobin levels, tumor site, histologic grade, T category, N category, human papillomavirus (HPV) status, extent of resection, and chemotherapy. Univariate analyses were performed with the Kaplan-Meier method and the log-rank test, multivariate analyses with the Cox proportional hazard model. RESULTS: On multivariate analysis, improved LRC was significantly associated with no EPO expression (risk ratio [RR] 3.72; 95 % confidence interval [CI] 1.35-15.42; p = 0.008), lower T category (RR 1.60; 95 %CI 1.14-2.32; p = 0.005), and oropharynx or larynx cancer (RR 1.23; 95 %CI 1.02-1.49; p = 0.033). Improved MFS was significantly associated with no EPO expression (RR 5.45; 95 %CI 1.13-97.81; p = 0.031), lower T category (RR 1.66; 95 %CI 1.11-2.65; p = 0.013), lower N category (RR 2.44; 95 %CI 1.04-6.66; p = 0.039), HPV positivity (RR 3.14; 95 %CI not available; p = 0.034), and oropharynx or larynx cancer (RR 1.28; 95 %CI 1.01-1.61; p = 0.041). Improved OS was significantly associated with no EPO expression (RR 4.77; 95 %CI 1.63-20.68; p = 0.003), no EPO-R expression (RR 2.36; 95 %CI 1.22-4.92; p = 0.010), lower T category (RR 1.44; 95 %CI 1.04-2.04; p = 0.027), oropharynx or larynx cancer (RR 1.30; 95 %CI 1.08-1.57; p = 0.007), and pre-RT hemoglobin ≥ 12 g/dl (RR 1.94; 95 %CI 1.03-3.65; p = 0.042). CONCLUSION: EPO expression of tumor cells was an independent prognostic factor for LRC, MFS, and OS. EPO-R expression was an independent prognostic factor for OS.

Expression and functional significance of the erythropoietin receptor in hepatocellular carcinoma.

BACKGROUND: Erythropoietin, through its specific receptor (EpoR), may induce responses in a variety of non-haematopoietic tissues including malignant cells. The purpose of this study was to examine the expression of EpoR in hepatocellular carcinoma (HCC) and to correlate the levels of EpoR expression with the clinicopathological properties of HCC and tumour recurrence. METHODS: The study included 134 patients who underwent curative hepatectomy for hepatitis B virus (HBV)-related primary HCC. The clinical, laboratory and pathological data from these patients were retrospectively collected. The expression of EpoR mRNA and protein were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. RESULTS: Expression of EpoR mRNA in the cirrhotic liver was positively correlated with tumour cell differentiation and 1-year disease-free survival (74.8% in the high expression group versus 46.9% in the low expression group; P = 0.001), as it was for EpoR mRNA expression in HCC (64.4% in the high expression group versus 52.7% in the low expression group; P = 0.044). Tumour recurrence showed stronger dependence on the expression of EpoR protein in non-malignant cirrhotic livers than in HCC. CONCLUSION: In HBV-related HCC, the levels of EpoR mRNA and protein in non-tumour cirrhotic livers were positively correlated with tumour cell differentiation, which is a favourable predictor of disease-specific survival.

Skin regeneration with conical and hair follicle structure of deep second-degree scalding injuries via combined expression of the EPO receptor and beta common receptor by local subcutaneous injection of nanosized rhEPO.

BACKGROUND: Acceleration of skin regeneration is still an unsolved problem in the clinical treatment of patients suffering from deep burns and scalds. Although erythropoietin (EPO) has a protective role in a wide range of organs and cells during ischemia and after trauma, it has been recently discovered that EPO is not tissue-protective in the common ß subunit receptor (ßCR) knockout mouse. The protective capacity of EPO in tissue is mediated via a heteroreceptor complex comprising both the erythropoietin receptor (EPOR) and ßCR. However, proof of coexpression of these heterogenic receptors in regenerating skin after burns is still lacking. METHODS: To understand the role of nanosized recombinant human erythropoietin (rhEPO) in wound healing, we investigated the effects of subcutaneous injections of EPO on skin regeneration after deep second-degree scalding injuries. Our aim was to determine if joint expression of EPOR and ßCR is a prerequisite for the tissue-protective effect of rhEPO. The efficiency in wound regeneration in a skin scalding injury mouse model was examined. A deep second-degree dermal scald injury was produced on the backs of 20 female Balb/c mice which were subsequently randomized to four experimental groups, two of which received daily subcutaneous injections of rhEPO. At days 7 and 14, the mice were sacrificed and the effects of rhEPO were analyzed with respect to grade of re-epithelialization (wound closure) and stage of epidermal maturation. This was investigated using different histological parameters of epithelial covering, such as depth of the epidermal layer, epidermal stratification, and presence of conical and hair follicle structures. RESULTS: Expression of EPOR, ßCR, and growth hormone receptor at the mRNA and protein levels was demonstrated with reverse transcriptase polymerase chain reaction and Western blot analysis. After rhEPO treatment, the rate of re-epithelialization of the scalding injury was increased and the time to final wound closure was reduced. In addition, the quality of regenerated skin was improved. In this investigation, for the first time, we demonstrated coexpression of EPOR and ßCR at the RNA and protein levels in vivo using a deep second-degree scalding injury mouse model. These results highlight the potential role of rhEPO in the improved treatment of burns patients, which might be crucial for the development of innovative new therapy regimes. CONCLUSION: Local injection of nanosized rhEPO directly to the injury site rather than systemic administration for deep second-degree scalding injuries achieved complete skin regeneration with conical and hair follicle structure via combined expression of EPOR and ßCR.

Beneficial nutraceutical modulation of cerebral erythropoietin expression and oxidative stress: an experimental study.

The main object of this study is to examine the effect of Klamin®, a nutraceutical containing phenylethylamine, phycocyanins, mycosporine-like aminoacids and aphanizomenon flos aquae-phytochrome on the learning and memory ability, the oxidative status and cerebral erythropoietin and its receptor EPO/EPOR system in prematurely senescent (PS) mice. A total of 28 PS mice, selected according to a prior T-maze test, and 26 non-prematurely senescent mice (NPS) mice were chosen. PS animals were divided into 3 groups and followed for 4 weeks: A) normal chow diet; B) added with Klamin® at 20 mg/kg/day (low dose); C) added with Klamin® at 100mg/kg/day (high dose). A further group of NPS mice given either normal food (group D) or high dose Klamin® (group E) was also considered. The behavioral procedures of spatial learning ability (Morris test) showed that PS mice had significantly longer learning time as compared to their NPS counterpart (p<0.01), but this effect was prevented especially in mice supplemented with high-dose Klamin® (p<0.05) which improved performances in NPS mice (p<0.05). High-dose Klamin® supplementation restored the depleted total thiol concentration in the brain observed in PS mice while normalizing their increased malonildialdehyde level (p<0.05). Moreover, the high-dosage only caused a significant upregulation of EPO/EPOR system both in PS and in NPS animals (p<0.05). Taken together, these data suggest that this specific alga Klamath extract has considerable antioxidant and adaptogenic properties, also through a stimulatory effect of cerebral EPO/EPO system.

Experimental evidence of the potential use of erythropoietin by intranasal administration as a neuroprotective agent in cerebral hypoxia.

Stroke is a major human health problem without efficient available therapeutics. Ischemic brain injury can induce cell death as well as upregulation of endogenous adaptive mechanisms depending on the severity and duration of hypoxia, and the activity of transcription factors, such as hypoxia inducible factor 1-α (HIF-1α). HIF-1α induces gene expression as multidrug resistance (MDR-1) gene associated with drug-refractory phenotype, as well as erythropoietin (Epo) and erythropoietin receptor (Epo-R) associated with O(2) supply. The spontaneous stimulation of the Epo/Epo-R system is not enough for brain protection. Therefore, administration of exogenous recombinant human Epo (rHu-Epo) was suggested as an alternative therapy in stroke. In several experimental models of brain hypoxia, Epo and Epo variants, including rHu-Epo, showed neuroprotective effects. Intranasal administration of these Epo-compounds can reach the central nervous system and protect the brain against ischemia, avoiding hematopoietic effects. However, it has been reported that high expression of Epo-R in neurons must be available to be activated by Epo. According to these considerations, intranasal delivery of rHu-Epo could be an interesting approach in the treatment of cerebral hypoxias avoiding both (i) adverse peripheral effects of treatment with Epo in stroke, and (ii) the pharmacoresistant phenotype depending on MDR-1 expression.

Erythropoietin receptor positive circulating progenitor cells and endothelial progenitor cells in patients with different stages of diabetic retinopathy.

OBJECTIVE: To investigate the possible involvement of erythr opoietin (EPO)/erythropoietin receptor (EPOR) system in neovascularization and vascular regeneration in diabetic retinopathy (DR). METHODS: EPOR positive circulating progenitor cells (CPCs: CD34(+)) and endothelial progenitor cells (EPCs: CD34(+)KDR(+)) were assessed by flow cytometry in type 2 diabetic patients with different stages of DR. The cohort consisted of age- and sex-matched control patients with out diabetes ( n=7),non-proliferative DR (NPDR, n=7),non-proliferative DR (PDR, n=8), and PDR complicated with diabetic nephr opathy (PDR-DN, n=7). RESULTS: The numbers of EPOR(+) CPCs and EPOR(+) EPCs were reduced remarkably in NPDR compared with the control group (both Pü0.01), whereas rebounded in PDR and PDR-DN groups in varyingdegrees. Similar changes were observed in respect of the proportion of EPOR(+)CPCs in CPCs (NPDR vs. control, Pü0.01) and that of EPOR(+) EPCs in EPCs (NPDR vs. control, Pü0.05). CONCLUSION: Exogenous EPO, mediated via the EPO/EPOR system of EPCs, may alleviate the impaired vascular regeneration in NPDR, whereas it might aggravate retinal neovascularization in PDR due to a rebound of EPOR(+)EPCs associated with ischemia.