Neuroprotective effects of erythropoietin on neurodegenerative and ischemic brain diseases: the role of erythropoietin receptor

Erythropoietin （Epo） is a fundamental hormone in the regulation of hematopoiesis, and other secondary roles mediated by the binding of the hormone to its specific receptor （EpoR）, which leads to an activation of key signaling pathways that induce an increase in cell differentiation, apoptosis control and neuroprotection. It has been suggested that their ftmction depends on final conformation of glycosylations, related with affinity to the receptor and its half-life. The presence of EpoR has been reported in different tissues including central nervous system, where it has been demonstrated to exert a neuroprotective function against oxidative stress conditions, such as ischemic injury and neurodegenerative diseases. There is also evidence of an increase in EpoR expression in brain cell lysates of Alzheimer＇s patients with respect to healthy patients. These results are related with extensive in vitro experimental data of neuroprotection obtained from cell lines, primary cell cultures and hippocampal slices. Additionally, this data is correlated with in vivo experiments （water maze test） in mouse models of Alzheimer＇s disease where Epo treatment improved cognitive function. These stud- ies support the idea that receptor activation induces a neuroprotective effect in neurodegenerative disorders including dementias, and especially Alzheimer＇s disease. Taken together, available evidence suggests that Epo appears to be a central element for EpoR activation and neuroprotective properties in the central nervous system. In this review, we will describe the mechanisms associated with neuroprotection and its relation with the activation of EpoR in order with identify new targets to develop pharmacological strategies.

Erythropoietin -induced proliferation of gastric mucosal cells

AIM： To analyze the localization of erythropoietin receptor on gastric specimens and characterize the effects of erythropoietin on the normal gastric epithelial proliferation using a porcine gastric epithelial cell culture model. METHODS： Erythropoietin receptor was detected by RT-PCR, Western blotting and immunohistochermistry. Growth stimulation effects of erythropoietin on cultured gastric mucosal cells were determined by ELISA using bromodeoxyuridine （BrdU）. RESULTS： Erythropoietin receptor was detected on cultured porcine gastric mucosal epithelial cells. Erythropoietin receptor was also detected histochemically at the base of gastric mucosal epithelium. BrdU assay demonstrated a dose-dependent increase in growth potential of cultured porcine gastric mucosal epithelial cells by administration of erythropoietin, as well as these effects were inhibited by administration of antierythropoietin antibody （P〈 0.01）. CONCLUSION： These findings indicate that erythropoietin has a potential to proliferate gastric mucosal epithelium via erythropoietin receptor.

Bone marrow mesenchymal stem cells transplantation promotes the release of endogenous erythropoietin after ischemic stroke

This study investigated whether bone marrow mesenchymal stem cell（BMSC） transplantation protected ischemic cerebral injury by stimulating endogenous erythropoietin. The model of ischemic stroke was established in rats through transient middle cerebral artery occlusion. Twenty-four hours later, 1 × 106 human BMSCs（h BMSCs） were injected into the tail vein. Fourteen days later, we found that h BMSCs promoted the release of endogenous erythropoietin in the ischemic region of rats. Simultaneously, 3 μg/d soluble erythropoietin receptor（s EPOR） was injected into the lateral ventricle, and on the next 13 consecutive days. s EPOR blocked the release of endogenous erythropoietin. The neurogenesis in the subventricular zone was less in the h BMSCs ＋ s EPOR group than in the h BMSCs ＋ heat-denatured s EPOR group. The adhesive-removal test result and the modified Neurological Severity Scores（m NSS） were lower in the h BMSCs ＋ s EPOR group than in the heat-denatured s EPOR group. The adhesive-removal test result and m NSS were similar between the h BMSCs ＋ heat-denatured s EPOR group and the h BMSCs ＋ s EPOR group. These findings confirm that BMSCs contribute to neurogenesis and improve neurological function by promoting the release of endogenous erythropoietin following ischemic stroke.

Erythropoietin Receptor Gene (EPOR) Polymorphisms are Associated with Sow Litter Sizes

The erythropoietin receptor （EPOR） has shown to play an important role in fetal survival by promoting the maturation of red blood cells in many studies of uterine capacity and litter size in swine. In this study, we screened the porcine EPOR gene for mutations and identified five single nucleotide polymorphisms （SNPs）： g.705G〉T in intron 1, g.2 373C〉T in intron 4, and g.2 882C〉T, g.3 035A〉G, and g.3 132A〉T in intron 6. We then genotyped 247 Beijing Black （BB） sows and compared the polymorphism data with the litter sizes of 1 375 parities among the sows. At first parity, there was no association of g.2 882C〉T and g.3 132A〉T with litter sizes. However, the CT sows in g.2 882C〉T had 2.13 higher total number born （TNB） （P〈0.01） and 1.81 higher number born alive （NBA） （P〈0.01） than the CC sows and the heterozygous sows in g.3 132A〉T had the highest litter size when compared to the two homozygotes for the later parities （P〈0.05）. In the g.3 035A〉G SNP, for the later parities, the TNB of the sows with the GG genotype was 3.81 higher （P〈0.01） and the NBA was 2.75 higher （P〈0.01） than that with the AA genotype but no difference at first parity. The G allele of the EPOR g.705G〉T SNP was associated with a greater litter size at both the first parity （P〈0.05） and later parities （P〈0.01）. Furthermore, we determined the allele frequencies for this SNP among five Chinese indigenous pig breeds （Erhualian, Laiwu Black, Meishan, Min, and Rongchang） and three western commercial pig breeds （Duroc, Landrace, and Large White）. The G allele of the EPOR g.705G〉T SNP was significantly more common in the more prolific Chinese breeds. These results indicated that the EPOR could be an important candidate gene for litter size and g.705G〉T can serve as a useful genetic marker for improving litter size in both first and later parities in swine.

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

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

Prognostic significance of erythropoietin and erythropoietin receptor in gastric adenocarcinoma

AIM: To investigate the expression of Erythropoietin (Epo) and its receptor (EpoR) in gastric adenocarcinoma (GAC) and the correlation with angiogenesis and clinicopathological features. METHODS: The expressions of Epo, EpoR and vascular endothelial growth factor (VEGF), as well as mi-crovessel density were evaluated in 172 GAC biopsies by immunohistochemical staining. The correlations between these parameters and patient’s clinicopathological features were analyzed statistically. RESULTS: The proportion of Epo and EpoR alterations in GAC was higher than that in adjacent normal mucosa (P = 0.035 and 0.030). Epo high-expression was associ-ated with EpoR high-expression, Lauren type, extensivelymph node metastasis and advanced stage of GAC (P = 0.018, 0.018, 0.004 and 0), while EpoR expression was linked with older age, World Health Organization type, extensive lymph node metastasis and advanced stage (P = 0.001, 0.013, 0.008 and 0.001). VEGF high expression was significantly correlated with EpoR low-expression, Lauren type, extensive lymph node metastasis and advanced stage (P = 0.001, 0.001, 0.001 and 0.007). The expression of Epo or EpoR was associated with microvessel density (P = 0.004 and 0.046). On multivariate analysis, only lymph node metastasis, abnormal Epo expression and tumor nodes metastases stage were independently associated with survival. In addition, a strong association with the immunohistochemical expression of EpoR and the angiogenic protein, VEGF, was noted. CONCLUSION: Increased expression of Epo and EpoR may play a signif icant role in the carcinogenesis, angiogenesis and progression of GAC. Epo may be an inde-pendent prognostic factor.

Erythropoietin receptor antibody inhibits oxidative stress induced retinal neovascularization in mice

AIM: To observe the effect of erythropoietin receptor antibody (EpoRA) on oxygen-induced retinal neovascularization. METHODS: C57BL / 63 mice, newly born 7 days, were exposed in high oxygen for 5 days and then placed in normal air for another 5 days, thus the animal models of retinal neovascularization were made. Experimental animals were allocated into 3 groups: normal, experimental and therapeutic. The normal group was fed in the normal environment. Into the vitreous cavity of mice in the therapeutic group were injected 2 mu L of EpoRA for 5 successive days. And the experimental group was injected the same amount of normal saline. Mice were sacrificed 17 days after birth and their eyeballs were removed for detection of malonaldehyde (MDA) content in the retina and by HE staining endothelial cells were counted the breaking through internal limiting membrane. RESULTS: In the experimental group, MDA content in the retina was 25.11 +/- 3.46 mu mol/g, which was obviously less than those in the normal group (5.34 +/- 1.79 mu mol/g, P<0.01) and those in the therapeutic group (12.04 +/- 1.91 mu mol/g). Pathological sections showed the nuclear number of the endothelial cells breaking through internal limiting membrane was 0.7 +/- 0.2 in normal group, and 46.2 +/- 6.5 in high oxygen induced experimental group. In the therapeutic group injected with EpoRA, it was lowered to 24.0 +/- 5.0(P < 0.01). CONCLUSION: EpoRA can effectively inhibit oxygen-induced neovascularization in retina of mouse by reducing oxidative damage.

IMMUNOHISTOCHEMICAL DISTRIBUTION OF ERYTHROPOIETIN AND ITS RECEPTOR EXPRESSION IN POSTNATAL RAT RETINA DEVELOPMENT

Objective To investigate the distribution of erythropoietin （EPO） and erythropoietin receptor （ EPOR ） expression in the postnatal rat retina development. Methods Forty-two male Sprague-Dawley rats were divided into 7 groups according to their various postnatal days： postnatal 1 d （D1 group）, 3 d （D3 group）, I week （W1 group）, 2 weeks （W2 group）, 3 weeks （W3 group）, 4 weeks （W4 group） and8 weeks （W8 group） （ n = 6 ）. Single eye was randomly chosen from each rat for the study. The retinal sections were stained with hematoxylin and eosin （HE） and used for the retina development observation. Immunohistochemical staining was used to localize EPO and EPOR expressions in retinas.of differentstages of development, and the expression intensities were determined by an image plus 4 program~~ Results The retinal inner nuclear layer （INL） and outer nuclear layer （ONL） were mixed together and had not yet fully differentiated in D1 and D3 groups. The INL and ONL formed their own independent regions and the outer plexiform layer （OPL） appeared between two layers in W1 group. With the postnatal retinal development, the inner plexiform layer （ IPL ） , rods and cones layer （ RCL ）, and OPL were gradually widened and stabilized in W2 to W3 groups. EPO/EPOR expressions located prominently in the inner part of the postnatal rat developing retinas. The expression of EPO in GCL and INL gradually increased from DI to W4, then the expression decreased in W8. Expression of EPOR in GCL gradually increased from DI to WI , then decreased in W2 ; and it gradually increased again from W3 to W8. Expression of EPOR in INL gradually increased from D1 to W1, then decreased in W2 ; and it continued to decrease from W3 to W8. Expression of EPOR in the external segment of RCL gradually increased from D1 to W8. However, expression in the internal segment of RCL gradually decreased from D1 to W3 , then no obvious expression was seen in the internal segment of RCL in W4 and W8. Conclusion EPO/EPOR expressions locate prominently in the inner part of the postnatal rat developing retina. And EPO/EPOR expressions in the rat retinas exist the dynamic changes during the postnatal retina development period.

Use of agents stimulating erythropoiesis in digestive diseases

Anemia is the most common complication of inflammatory bowel disease (IBD). Control and inadequate treatment leads to a worse quality of life and increased morbidity and hospitalization. Blood loss, and to a lesser extent, malabsorption of iron are the main causes of iron def iciency in IBD. There is also a variable component of anemia related to chronic inflammation. The anemia of chronic renal failure has been treated for many years with recombinant human erythropoietin (rHuEPO), which significantly improves quality of life and survival. Subsequently, rHuEPO has been used progressively in other conditions that occur with anemia of chronic processes such as cancer, rheumatoid arthritis or IBD, and anemia associated with the treatment of hepatitis C virus. Erythropoietic agents complete the range of available therapeutic options for treatment of anemia associated with IBD, which begins by treating the basis of the inflammatory disease, along with intravenous iron therapy as f irst choice. In cases of resistance to treatment with iron, combined therapy with erythropoietic agents aims to achieve near-normal levels of hemoglobin/hematocrit (11-12 g/dL). New formulations of intravenous iron (iron carboxymaltose) and the new generation of erythropoietic agents (darbepoetin and continuous erythropoietin receptor activator) will allow better dosing with the same eff icacy and safety.

Expression of EPO Receptor in Pancreatic Cells and Its Effect on Cell Apoptosis

In order to explore the expression of erythropoietin receptor （EPOR） in pancreatic cell line NIT-1 and its effect on cell apoptosis after binding with erythropoietin （EPO）, NIT-1 cells were cultured and expanded. The expression of EPOR was detected using electrophoresis. NIT-1 apoptosis was induced by cytokines and their effects on cell apoptosis and cell insulin secretion were assayed after binding of EPO to EPOR. The results showed that EPOR was expressed in NIT-1 cells. Recombinant human EPO （rHuEPO） had no effect on cell apoptosis but significantly inhibited apoptosis induced by cytokines, rHuEPO had no effect on cell insulin secretion but significantly improved insulin secretion inhibited by cytokines. From these findings, it was concluded that EPOR was expressed in NIT- 1 cells and EPO could protect NIT- 1 cells from apoptosis induced by cytokines.

Knockout of the tumor necrosis factor a receptor 1 gene can up-regulate erythropoietin receptor during myocardial ischemia-reperfusion injury in mice

Background Tumor necrosis factor a receptor 1 （TNFaR1） plays an important role in the signal pathway of apoptosis. The objective of this study was to investigate the effects of TNFaR1 knockout on the up-regulation of erythropoietin receptor （Epo-R） and the coordinated anti-apoptosis functions during myocardial ischemia-reperfusion injury in mice. Methods The ischemia-reperfusion injury model for cardiomyocytes was performed by ligating the left circumflex branch artery of TNFaR1 knockout （P55/） C17 B6 mice, as well as wild-type （P55^＋/^＋） C17 B6 mice. Triphenyltetrazolium chloride （TTC） staining was performed to observe the damaged area of the heart. TUNEL staining and DNA fragmentation were used to identify apoptosis. Mitochondrial Bcl-2 and Bax as well as expression of Epo-R and its downstream genes （Jak-2, stat-5, Akt, lkB-a, HIF-1a） were measured by Western blotting. The gene knockout mice were assigned into those undergoing the apoptosis surgical model group （KO group）, and those subjected to sham operation （KOs group）. Similarly, wild-type mice were either exposed to the surgical model （WT group） or subject to a sham operation （WTs group）. Results The myocardial damage ratio of the wild-type group after the operation was significantly higher than that of the knockout group, （50.5±6.4）% vs （36.9±6.9）%, P 〈0.01. Similarly, TUNEL positive ratio of the wild-type group was significantly higher than that of the knockout group, （63.1±5.6）% vs （42.1±4.7）%, P〈0.01. The gray value ratios of Epo-R, Jak-2, stat-5, Akt, IkB-a, HIF-1 and mitochondrial Bcl-2 in the KO group were significantly higher than those of the WT group, P 〈0.05; however, mitochondrial Bax was significantly lower than that of the WT group significantly （P 〈0.05）. Conclusions Using the ischemia-reperfusion injury model in mice, cardiomyocytes of TNFaR1 knockouts exhibited anti-apoptotic characteristics. This information could be used to coordinate the prevention of myocardial apoptosis by up-regulating and activating the Epo-R pathway.

Upregulation of erythropoietin and erythropoietin receptor in castration-resistant progression of prostate cancer

Hypoxia-induced erythropoietin signaling plays an important role in tumor growth and invasion.In the present study,we investigated the contribution of erythropoietin signaling pathway to castration-resistant prostate cancer and the development of a neuroendocrine phenotype.Immunohistochemical staining showed that the erythropoietin and erythropoietin receptor scores in castration-resistant prostate cancer and androgen-dependent prostate cancer were 7.55 versus 4.5 and 7.45 versus 5.9,respectively(P<0.001).Furthermore,a cell proliferation assay was conducted,and the differential expression of erythropoietin and erythropoietin receptor in LNCaP cells and hypoxia-induced LNCaP cells was evaluated using western blot and quantitative real-time PCR.The proliferation capacity of hypoxia-induced LNCaP cells was similar in cultures of both fetal bovine serum and charcoal-stripped fetal bovine serum,suggesting that LNCaP cells acquired hypoxia-induced androgen-independent growth.After 2 weeks of hypoxic culture,LNCaP cells showed a neuroendocrine cell change and increased expression of neuron-specific enolase,erythropoietin,and erythropoietin receptor;knockdown of erythropoietin receptor reversed the hypoxia-induced upregulation of neuron-specific enolase in the LNCaP cells.In conclusion,the concurrent upregulation of erythropoietin and erythropoietin receptor in castration-resistant prostate cancer suggests that the erythropoietin/erythropoietin receptor autocrine loop plays an important role in the progression of castration resistance and is responsible for the development of a neuroendocrine phenotype.