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Erythropoietin (ЕPО) is a glycoprotein hormone, mainly known for its haemopoietic function. For orthopaedics, its pleiotropic effects – osteogenic and angiogenic potential, are of primary interest. The exact mechanism of EPO action is still unclear. The effects of EPO on bone healing were investigated through experiments with rats, mice, rabbits and pigs. Each of used models for experimental bone defects (calvarial models, long bone segmental defects, posterolateral spinal fusion and corticosteroid-induced femoral head osteonecrosis) has specific advantages and flaws. Obtaining specific and correct results is largely dependent on the used model. The brief evaluation of models could serve for standardisation of preclinical studies on bone regeneration.

Potent beneficial immunomodulatory and anti-inflammatory effects of whole-molecule erythropoietin have been demonstrated in a variety of animal disease models including experimental autoimmune encephalomyelitis (EAE); however, excessive hematopoiesis limits its use in clinical applications. Our group previously generated an Epo-derived small peptide JM4 that is side-effect free and has strong neuroprotective activity without hematologic effects. Here, we investigated the long-term clinical effects of brief treatment with JM4 in chronic relapsing EAE using bioluminescence imaging (BLI) in transgenic mice containing the luciferase gene driven by the murine GFAP promoter. EAE mice treated with JM4 exhibited marked improvement in clinical scores and showed fewer disease flareups than control animals. JM4 therapy concomitantly led to markedly decreased GFAP bioluminescence in the brain and spinal cord in both acute and chronic relapsing EAE mouse models. We found a marker for toxic A1 astrocytes, complement component C3, that is upregulated in the brain and cord of EAE mice and sharply reduced in JM4-treated animals. In addition, an abnormally leaky neurovascular unit permeability was rapidly normalized within 5 days by JM4 therapy. The prolonged therapeutic benefit seen following brief JM4 treatment in EAE mice closely resemble that recently described in humans receiving pulsed immune reconstitution therapy with the disease-modifying compounds, alemtuzumab and cladribine. Our study suggests that JM4 therapy may have widespread clinical applicability for long-term treatment of inflammatory demyelinating diseases and that BLI is a useful noninvasive means of monitoring murine disease activity of the central nervous system. Electronic supplementary material The online version of this article (10.1007/s13311-020-00923-5) contains supplementary material, which is available to authorized users.

Despite advances in neonatal intensive care in the recent decade, a large number of very preterm infants (VPIs) remain at risk for significant neurodevelopmental impairment (NDI). Given that there are many interventions need to be implemented during the critical perinatal period so that complications of these vulnerable VPIs could be minimized, it is urgent to develop multi-discipline strategies based on evidence to be carried out. The objective of this new term evidence-based perinatal critical strategies (EBPCS), is to provide beneficial intervention towards better neurodevelopmental outcomes, specifically for preterm infants below 28 weeks gestational age. EBPCS is defined as the management of the VPIs during the perinatal period which would include antenatal counseling with team briefing and share decision making, treat the chorioamnionitis, antenatal MgS04, antenatal steroid, delayed cord clamping/milking, neonatal resuscitation team preparation, prevention of hypothermia, immediate respiratory support with continuous positive airway pressure at delivery room, less invasive surfactant administration, early surfactant with budesonide therapy, support of cardiovascular system, early initiate of probiotics administration, early caffeine, early parenteral and enteral nutrition, promptly initiating antibiotics. These critical strategies will be discussed detail in the text; nonetheless, standardized protocols, technical skills and repeated training are the cornerstones of successful of EBPCS. Further experience from different NICU is needed to prove whether these very complicate and comprehensive perinatal critical strategies could translate into daily practice to mitigate the incidence of NDI in high-risk VPIs.

Cardiac dysfunction is an important prognostic predictor of cardiovascular mortality in patients on hemodialysis (HD). Erythropoietin (EPO) has been reported to improve cardiac function by binding to the EPO receptor (EPOR) on cardiomyocytes. This study investigated whether anti-EPOR antibodies were associated with left ventricular cardiac function in patients undergoing HD. This multicenter, cross-sectional observational study included 377 patients (median age, 70 years; 267 (70.8%) males) with chronic kidney disease (CKD) undergoing stable maintenance HD. Serum levels of anti-EPOR antibodies were measured, and echocardiography was used to assess the left ventricular mass index (LVMI) and left ventricular ejection fraction (LVEF). Anti-EPOR antibodies were found in 17 patients (4.5%). LVMI was greater (median of 135 g/m2 vs. 115 g/m2, p = 0.042), and the prevalence of LVEF < 50% was higher (35.3% vs. 15.6%, p = 0.032) in patients with anti-EPOR antibodies than in those without. Multivariable linear regression and logistic regression analysis (after adjusting for known risk factors of heart failure) revealed that anti-EPOR antibodies were independently associated with LVMI (coefficient 16.2%; 95% confidence interval (CI) 1.0–35.0%, p = 0.043) and LVEF

Background The purpose of periodontal treatments is to reduce inflammation, restore gingival health and clinical attachment level gain by controlling microbial plaque formation and other etiological factors. One of the drugs that has been tested in many areas and shown good anti-inflammatory properties is erythropoietin (EPO). We evaluated the effect of this drug on the improvement of periodontitis after the phase I treatment. Methods This study was conducted on 30 patients with stage III periodontitis who had at least two bilateral teeth with CAL of ≥ 5 mm and PPD ≥ 6 mm at ≥ 2 non‐adjacent teeth and bleeding on probing. After oral hygiene instruction and scaling and root planning (SRP), EPO gel containing a solution of 4000 units was applied deeply in the test group and placebo gel was deeply administered in the control pockets (5 times, every other day). The clinical parameters of the plaque index (PI), gingival index (GI), clinical attachment level (CAL), probing depth (PD) and bleeding index (BI) were measured at baseline and after three months of follow up. The P-value was set at 0.05. Results All clinical variables improved after treatment in both groups. The BI and GI scores (which reflects the degree of gingival inflammation) showed statistically more reduction in test group. The CAL decreased from 5.1 ± 4.1 to 3.40 ± 2.71 mm; and 5.67 ± 4.32 to 4.33 ± 3.19 mm in test and control group, respectively (P P Conclusion Local application of EPO gel in adjunct to SRP can improve clinical inflammation and CAL gain in periodontitis. Trial registration: This study was registered at 2017-11-06 in IRCT. All procedures performed in this study were approved with ID number of IR.TUMS.DENTISTRY.REC.1396.3139 in Tehran University of medical science.

The enhancement of recombinant therapeutic protein production in mammalian cell culture has been regarded as an important issue in the biopharmaceutical industry. Previous studies have reported that the addition of the recombinant 30Kc19 protein, a silkworm-derived plasma protein with simultaneous cell-penetrating and mitochondrial enzyme-stabilizing properties, can enhance the recombinant protein expression in Chinese hamster ovary (CHO) cell culture. Here, we produced an α-helix N-terminal domain of 30Kc19, called (30Kc19α), and investigated its effects on the production of human erythropoietin (EPO), a widely used therapeutic protein for the treatment of anemia, in recombinant CHO cell culture. Similar to the full-length 30Kc19, 30Kc19α was able to be mass-produced in a form of recombinant protein through an Escherichia coli expression system and delivered into EPO-producing CHO (EPO–CHO) cells. Supplementing the medium of EPO–CHO cell culture with 30Kc19α increased the intracellular NADPH/NADP+ ratio related to the flux of metabolic reducing power for protein biosynthesis, subsequently enhancing EPO production in serum-free culture. 30Kc19α is considered to have certain advantages in the downstream purification process of therapeutic protein production when it is used as a medium supplement due to its small size and low isoelectric point compared to the full-length 30Kc19. These results suggest that 30Kc19α has potential use for manufacturing biopharmaceutical proteins.

Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood–brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000™ was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.

The aim of this study was to optimize the formulation of erythropoietin (EPO) using amino acids instead of human serum albumin (HSA) and to evaluate its in vivo stability in order to avoid the risk of viral contamination and antigenicity. Different EPO formulations were developed in such a way as to allow studying the effects of amino acids and surfactants on the EPO stability profile. The main techniques applied for EPO analysis were ELISA, Bradford method, and SDS gel electrophoresis. The in vivo stability was evaluated in a Balb-c mouse animal model. The results showed that the presence of surfactant was very useful in preventing the initial adsorption of EPO on the walls of vials and in minimizing protein aggregation. Amino acid combinations, glycine with glutamic acid, provided maximum stability. Formulation F4 (containing glycine, glutamic acid and Tween 20) showed minimum aggregation and degradation and in vivo activity equivalent to commercially available HSA-stabilized EPO (Eprex®).

Background: Curcumae Radix (Yujin) has been widely used to treat Qi stagnation due to Liver depression (LDQS) in traditional Chinese medicine (TCM) for a long time and is good at dispelling melancholy by soothing liver to regulate qi and relieving pain by promoting blood circulation for removing blood stasis. Yujin stir-frying with vinegar can strengthen the effect of dispersing stagnated hepatoqi to stop pain by injecting medicine into the liver and stir-frying with wine can strengthen the effect of promoting blood circulation for removing blood stasis. Because the reason for the enhancement of clinical efficacy by processing is unclear, it is difficult to select and use processed products in the clinic. Aim/Hypothesis: In this study, from the point of view of the platelet function, we start to investigate the mechanism for promoting blood circulation, removing blood stasis, and enhancing clinical efficacy by processing. Methods: This study mainly takes Curcumae Longae Radix (HSYJ) and Curcuma phaeocaulis Radix (LSYJ) as the research subjects. They are genuine drugs in Sichuan Province, China. A high-performance liquid chromatography (HPLC) is used to analyze the main chemical constituents of Yujin and its processed products, and show the difference between the two species, with and without processing. A rat model of blood stasis induced by LDQS is established by giving the tail clamp stimulation, injecting epinephrine hydrochloride subcutaneously, and treating with an effective dose (0.9 g⋅kg-1) according to the conversion of human clinical dosage for 2 weeks. After the experiment, the serum levels of erythropoietin are measured by ELISA. Furthermore, RT-PCR and WB are used to detect EpoR mRNA and protein expression in the hepatic tissue. Flow cytometry is used to measure peripheral blood CD62p expression. Results: There is a great difference between the chemical compositions of the two species, the number of chromatographic peaks of Curcumae Longae Radix is more than that of Curcuma phaeocaulis Radix. Curcuminoids is the main component of HSYJ, while curcuminoids is almost free from LSYJ. Curcuminoids is almost insoluble in water. After stir-frying with vinegar or wine, it can increase the dissolution of curcuminoids in water. In rat models, the levels of Epo, EpoR, and CD62p are significantly increased. After intragastric administration of Yujin, indicators show varying degrees of callback. HSYJ is better than LSYJ, and the processed product by stir-frying with wine is better than other processed products. Conclusion: The results show that the mechanism of promoting blood circulation for removing blood stasis in Yujin may be able to inhibit the activation and aggregation of platelets by intervening the Epo/EpoR pathway and regulating CD62p down. Stir-frying with wine can enhance this effect.

In adult’s burn injuries belong to the top 15 causes of injury. Annually more than a million patients receive specialized treatment. Improving burned patients’ outcomes is still a challenge. Effects of erythropoietin (EPO) are reported to be pro-angiogenic, pro-regenerative, anti-inflammatory, immunomodulatory and hypoxia/ischemia protective. Study objectives were to demonstrate cytoprotective and regenerative effects of EPO in burned patients in terms of improved wound healing, reduced morbidity and mortality. This was a prospective, placebo-controlled, randomized, double-blind trial. The trial was conducted in 13 specialized burn care centers in Germany. Adult Patients with 2b° or 3° burn injuries were included. Patients received state of the art burn care including obligatory split skin graft transplantation. Study medication was EPO or placebo every other day for 21 days. Between 12/08 and 06/14, 116 patients were randomized, 84 received study medication (EPO 45, Placebo 39). Primary endpoint analysis revealed inconclusive results, as only a minority of patients reached the primary endpoint [100% re-epithelialization: EPO: 23% (9/40); Placebo 30% (11/37)]. Several secondary endpoints such as SOFA score (morbidity), EPO level in blood and wound healing onset revealed clinical, and statistically significant results in favor of the EPO group. Adverse Events (AEs) and Severe Adverse Events (SAEs) were in expected ranges; AEs EPO: 80%, (36/45), Placebo: 77%, (30/39); SAEs EPO: 24%, (11/45), Placebo: 24%, (8/39). Out of 84 patients two died, one per group, thus mortality was lower than expected. Results (SOFA score) indicate a lower morbidity of the EPO group, suggesting pro-regenerative effects of EPO in burned patients. Higher EPO levels might influence the faster onset of re-epithelialization in the first 10 days of the treatment. Both effects could reveal new therapeutic options. Clinical Trial Registration: ISRCT Number: ISRCTN95777824 and EudraCT Number: 2006-002886-38, Protocol Number: 0506.

In patients with chronic kidney disease (CKD), adverse outcomes such as systemic inflammation and anemia are contributing pathologies which increase the risks for cardiovascular mortality. Amongst these complications, abnormalities in mineral metabolism and the metabolic milieu are associated with chronic inflammation and iron dysregulation, and fibroblast growth factor 23 (FGF23) is a risk factor in this context. FGF23 is a bone-derived hormone that is essential for regulating vitamin D and phosphate homeostasis. In the early stages of CKD, serum FGF23 levels rise 1000-fold above normal values in an attempt to maintain normal phosphate levels. Despite this compensatory action, clinical CKD studies have demonstrated powerful and dose-dependent associations between FGF23 levels and higher risks for mortality. A prospective pathomechanism coupling elevated serum FGF23 levels with CKD-associated anemia and cardiovascular injury is its strong association with chronic inflammation. In this review, we will examine the current experimental and clinical evidence regarding the role of FGF23 in renal physiology as well as in the pathophysiology of CKD with an emphasis on chronic inflammation and anemia.

Abstract The quantitative real-time polymerase chain reaction (qPCR) is a sensitive technique for examining the influence of erythropoietin (Epo) on gene expression. A critical and fundamental step for data analysis is the selection of and normalization to the optimal reference gene(s). We identified appropriate reference gene(s) among 32 genes during chronic recombinant human Epo (rHuEpo) treatment of SH-SY5Y cells using TaqMan human Express Endogenous Control Plate. Expression stability of the selected reference gene (RPLP) was retested with qPCR, together with two commonly used reference genes (GAPDH, ACTB) and six genes of interest (EPOR, EPO, STAT5B, STAT5A, JUN, AKT). In PC12 cells, three commonly used reference genes (Gapdh, CycA and Ywhaz) and seven genes of interest (EpoR, Epo, Stat5b, Stat5a, Jun, Akt, Fos) were evaluated. For the evaluation of expression stability, geNorm, NormFinder and BestKeeper software were used. All three gave similar results. We demonstrated that among the housekeeping genes, RPLP in SH-SY5Y and CycA and Ywhaz in PC12 are the most stable genes. Additionally, we showed that normalization with GAPDH gave misleading results compared to normalization with geNorm. In conclusion, selection of the appropriate normalization gene(s) is crucial for correct interpretation of rHuEpo treatment results. Graphical abstract

It has been recently shown that the biological effects of erythropoietin (EPO) are not limited to the hematopoietic compartment but, as pleiotropic glycoprotein, this hormone can exert pro-angiogenic and tissue-protective functions also in a wide range of non-hematopoietic organs. The role of EPO and the effective functionality of its receptor in solid tumors are still a controversial point of debate. In the present work we analyzed the gene expression of EPO and its cognate receptor (EpoR) in a rat model of thioacetamide-induced damage and tumor. An analysis of the EPO/EpoR axis was also performed on human cholangiocarcinoma (CC) cell lines. A progressive increase of EPO and EpoR mRNA can already be observed during the fibrotic–cirrhotic development with a peak of expression rising at tumor formation (24.7 ± 9.9-fold increase and 15.5 ± 1.1-fold increase, respectively, for the two genes). Co-localization studies by immunofluorescence revealed hepatocytes in the regenerative cirrhotic nodules (Hep Par-1+) and in the dysplastic bile duct cells (CK19+) as the major EPO producers in this specific condition. The same cell populations, together with endothelial cells, exhibited an increased expression of EpoR, although all the non-parenchymal cell populations in the liver exhibited modest basal mRNA levels. Challenging human CC cells, Mz-Cha-2, with a combination of EPO and SCF resulted in a synergistic effect on the gene expression of EPO, CyclinD1 and PCNA. This study suggests that the autocrine and paracrine release of endogenous EPO in the microenvironment may contribute to the development and maintenance of the CC possibly in cooperation with other signaling pathways. Electronic supplementary material The online version of this article (doi:10.1007/s00418-012-1037-x) contains supplementary material, which is available to authorized users.

International audience; Cloning of the human erythropoietin () gene and development of the first recombinant human erythropoietin (rHuEPO) drug were truly breakthroughs. This allowed a deeper understanding of the structure and pharmacology of rHuEpo, which in turn inspired the discovery and development of additional erythropoiesis-stimulating agents (ESAs). In vivo specific activity and serum half-life of rHuEPO are influenced by the amount and structure of the attached carbohydrate. Increased numbers of sialic acids on carbohydrate attached to rHuEPO correlated with a relative increase in in-vivo-specific activity and increased serum half-life. The effect of increasing the number of sialic-acid-containing carbohydrates on in-vivo-specific activity was explored. Initial research focused on solving the problem of how the protein backbone could be engineered so a cell would add more carbohydrate to it. Additional work resulted in darbepoetin alfa, a longer-acting molecule with two additional carbohydrate chains.

Erythropoietin (EPO), a glycoprotein hormone of ∼34 kDa, is an important hematopoietic growth factor, mainly produced in the kidney and controls the number of red blood cells circulating in the blood stream. Sensitive and rapid recombinant human EPO (rHuEPO) detection tools that improve on the current laborious EPO detection techniques are in high demand for both clinical and sports industry. A sensitive aptamer-functionalized biosensor (aptasensor) has been developed by controlled growth of gold nanostructures (AuNS) over a gold substrate (pAu/AuNS). The aptasensor selectively binds to rHuEPO and, therefore, was used to extract and detect the drug from horse plasma by surface enhanced Raman spectroscopy (SERS). Due to the nanogap separation between the nanostructures, the high population and distribution of hot spots on the pAu/AuNS substrate surface, strong signal enhancement was acquired. By using wide area illumination (WAI) setting for the Raman detection, a low RSD of 4.92% over 150 SERS measurements was achieved. The significant reproducibility of the new biosensor addresses the serious problem of SERS signal inconsistency that hampers the use of the technique in the field. The WAI setting is compatible with handheld Raman devices. Therefore, the new aptasensor can be used for the selective extraction of rHuEPO from biological fluids and subsequently screened with handheld Raman spectrometer for SERS based in-field protein detection. Electronic supplementary material The online version of this article (doi:10.1186/s12951-015-0102-8) contains supplementary material, which is available to authorized users.

In order to determine the initial values and dynamic changes of EPO (erythropoietin) after therapy, 57 consecutively presenting, typical COPD (chronic obstructive pulmonary disease) patients with chronic hypoxia and acute exacerbated serum EPO levels were serially measured. Initial mean EPO levels were slightly above the normal range (41.4±83.5 units/l), but in the majority of patients the initial EPO levels were significantly reduced. Following the correction of hypoxaemia, mean EPO levels decreased to 14.1±16.9 units/l (P=0.0093). However, not all COPD patients showed this pattern; in an important subset of patients (36.8%), who had initially lower EPO levels and lower erythrocyte count, EPO levels were significantly increased (by more than 60%; P=0.0028) on the second day of treatment, despite correction of the hypoxaemia. This finding was unexpected and paradoxical when compared with physiological studies addressing the same issue. The data presented support previous reports of variable EPO levels in severely hypoxic COPD patients and suggest that the haematological response is already hampered at an early stage, at the level of EPO production, and much less likely at later steps in the haemopoietic response by failure to respond to elevated EPO levels. Our data are consistent with recent discoveries that the O2 sensing and regulation of EPO production is a complex process in which multiple factors, including cytokines and therapeutic agents, play a role by enhancing or inhibiting the response. We believe that further studies on this clinical condition are complementary to basic physiological research and may help to elucidate the role of cytokines and other individual factors in complex clinical hypoxic situations.

Human erythropoietin (hEPO), an endogenously produced glycoprotein, plays a fun- damental role in erythropoiesis controlling the formation of red blood cells. Production of recombinant erythropoietin (rEPO) by molecular biology techniques has made pos- sible its therapeutic use together with its abuse in competitive sports. Since the glyco- sylation pattern of rEPO depends, among other things, of the cell line used for pro- duction, it presents a glycoform scheme different from manufacturer to manufacturer and different also from endogenous hEPO. The separation of these different glyco- forms is interesting since it could be useful: i) for determining the glycoform distribu- tion in recombinant products from different batches and/or manufacturers, ii) for studying the link between glycosylated forms and their activity, and iii) for helping to discriminate between endogenous and recombinant EPO. This paper desribes the development of three different Capillary Electrophoresis methods that use UV detec- tion to separate rEPO glycoforms. The special features of the three methods are dis- cussed in terms of resolution of bands of rEPO glycoforms, method reproducibility, and compatibility with other more sensitive detection systems such as laser induced fluorescence using on-column derivatization protocols or more selective ones such as mass-spectrometry., The authors are grateful to European Pharmacopoeia for providing rEPO BRP used as sample in this work and to M.T. Veledo for carrying out the CE-LIF experiments. This work was supported by an International Olympic Commit- tee project (1767/2000/HMS/sls) and by a CICYT project (AGL2000-1480).

Objective To investigate the effectiveness of comprehensive medical intervention in improving the nutritional status of elderly hemodialysis patients. Methods A total of 21 elderly patients on hemodialysis were treated with diet guidance, erythropoietin, adequate dialysis, hemodiafiltration, and correction of metabolic acidosis. Quality of life, nutritional status, and plasma amino acids were assessed in these patients before treatment, and at 6 and 12 months after treatment. Results The general clinical situation of the patients was much improved, and the nutritional status of the patients was also improved after treatment, especially 12 months later. The serum levels of fibronectin, hematocrit, and hemoglobin were significantly increased after 12 months. After the treatment, the pattern of plasma amino acids was also largely changed. Conclusion Comprehensive medical intervention should be taken to improve the nutritional status of elderly patients on hemodialysis.

OBJECTIVES: Exogenously administered recombinant human erythropoietin (rhEPO) has been reported to exhibit neuroprotective effects in animal models. However, there are still have some controversies that combination of EPO and tissue plasminogen activator (tPA) in acute ischemic stroke. In the present study, we investigated the effects of local intra-arterial infusion of low-dose EPO in combination with tPA on focal cerebral ischemic stroke. MATERIALS AND METHODS: Sixty adult male Sprague-Dawley rats were randomly divided into five groups, including sham, vehicle, EPO, tPA, and EPO+tPA groups. Rats were subjected to middle cerebral artery occlusion (MCAO) and administrated with EPO (800 U/kg, middle cerebral artery injection), tPA (10 mg/kg, tail vein injection), EPO+tPA, or saline (vehicle) onset of reperfusion. Neurobehavioral deficits, infarct volume, brain edema, the expression of tight junction proteins (Claudin-5, Occludin), and AQP4 were assessed following 2 h ischemia and 24 h reperfusion. The number of apoptotic cells in the periinfarct region was detected by the terminal deoxyribonucleotide transferase dUTP nick end labeling (TUNEL) staining. RESULTS: The neurobehavioral deficits, brain infarct volume, edema volume, TUNEL-positive cells and downregulation of Claudin-5 and Occludin were alleviated by EPO or EPO plus tPA, following the ischemia/reperfusion (I/R) in rats. The EPO and EPO plus tPA both reduced the upregulation of AQP4 in the ischemic brain tissue. CONCLUSION: Our data demonstrate local intra-arterial infusion of low-dose EPO in combination with tPA protected against focal cerebral ischemia in rats manifested by a decrease in brain edema and blood-brain barrier (BBB) disruption after 2 h ischemia and 24 h reperfusion.

Purpose: Myelodysplastic syndromes (MDS) are heterogeneous clonal diseases characterized by cytopenias as a result of ineffective hematopoiesis. Little is known about alterations in signal transduction pathways in MDS. Experimental Design: Multiparameter flow cytometry was used to evaluate the proteolytic activation of caspase-3 and the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and STAT5 specifically in defined CD34+, CD45+, or CD71+CD45− bone marrow (BM) cells from 60 MDS cases and normal controls, both at baseline and following stimulation with granulocyte colony-stimulating factor (G-CSF) and erythropoietin. Results: In CD71+CD45− cells from a subpopulation of 36 MDS cases who were predicted to be responsive by clinical parameters (endogenous erythropoietin levels, transfusion dependency, percentage of blasts in the BM), erythropoietin failed to activate ERK1/2 or STAT5 in 23 of 36 cases, but it was effective in 13 of 36 cases, although to a significantly lower degree than in CD71+CD45− cells from healthy donor BM. The erythropoietin response in vivo correlated with in vitro erythropoietin-dependent STAT5 activation in 20 of 22 cases. STAT5 was significantly activated at baseline in MDS cells compared with normal controls, whereas caspase-3 was activated in CD34+ and CD45+ MDS cells, and was activated more often in the RA and RAEB-1 MDS subtypes. G-CSF stimulation activated ERK1/2 and STAT5 equally in MDS and normal CD34+ cells. Conclusions: Abnormalities in the response to growth factors are restricted to erythropoietin stimulation in CD71+CD45− cells and correlate with the clinical response to erythropoietin. Activation of baseline signal transduction for proliferative and apoptotic signals is altered in MDS but with different patterns among the various BM subpopulations. Clin Cancer Res; 18(11); 3079–89. ©2012 AACR.

Erythropoietin has been shown to promote tissue regeneration after ischaemic injury in various organs. Here, we investigated whether Erythropoietin could ameliorate ischaemic spinal cord injury in the mouse and sought an underlying mechanism. Spinal cord ischaemia was developed by cross-clamping the descending thoracic aorta for 7 or 9 min. in mice. Erythropoietin (5000 IU/kg) or saline was administrated 30 min. before aortic cross-clamping. Neurological function was assessed using the paralysis score for 7 days after the operation. Spinal cords were histologically evaluated 2 and 7 days after the operation. Immunohistochemistry was used to detect CD34+ cells and the expression of brain-derived neurotrophic factor and vascular endothelial growth factor. Each mouse exhibited either mildly impaired function or complete paralysis at day 2. Erythropoietin-treated mice with complete paralysis demonstrated significant improvement of neurological function between day 2 and 7, compared to saline-treated mice with complete paralysis. Motor neurons in erythropoietin-treated mice were more preserved at day 7 than those in saline-treated mice with complete paralysis. CD34+ cells in the lumbar spinal cord of erythropoietin-treated mice were more abundant at day 2 than those of saline-treated mice. Brain-derived neurotrophic factor and vascular endothelial growth factor were markedly expressed in lumbar spinal cords in erythropoietin-treated mice at day 7. Erythropoietin demonstrated neuroprotective effects in the ischaemic spinal cord, improving neurological function and attenuating motor neuron loss. These effects may have been mediated by recruited CD34+ cells, and enhanced expression of brain-derived neurotrophic factor and vascular endothelial growth factor.

EPO (erythropoietin) has long been identified as a primary regulator of erythropoiesis. Subsequently, EPO has been recognized as playing a role in a broad variety of processes in cardiovascular pathophysiology. In particular, the tight interactions of EPO with the nitric oxide pathway, apoptosis, ischaemia, cell proliferation and platelet activation appear of great interest. Although enhanced EPO synthesis is viewed as an appropriate compensatory mechanism in the cardio–renal syndrome, which features CHF (congestive heart failure) and CRF (chronic renal failure), maladaptative excessive EPO synthesis in the advanced stages of these diseases appears to be predictive of higher mortality. Clinical trials based on the use of EPO in both heart and renal failure have so far produced contradictory results, whereas treatment targeted to restore low Hb levels appears rational and is supported by regulatory authorities. New areas for therapeutic use of EPO, such as acute coronary syndromes, are under investigation, and they are discussed in the present review together with other clinical applications in cardiovascular diseases. The revisited concept of a potential use of endogenous EPO levels as a predictor of CHF severity, as well as in the monitoring of responses to treatment, deserves appropriate investigation, as this may identify EPO as a useful biomarker in the clinical management of cardiovascular diseases.

Background The enhancement of glycosylation by applying glycoengineering approaches has become widely used to boost properties for protein therapeutics. The objective of this work was to engineer a new hyperglycosylated analog of erythropoietin (EPO) with appropriately targeted N-linked carbohydrates through bioinformatics tools. Materials and methods The EPO protein sequence was retrieved from NCBI protein sequence database. Prediction of N-glycosylation sites for the target protein was done using the prediction server, NetNGlyc. The three-dimensional model of glycoengineered EPO (named as kypoetin) was constructed using the homology modeling program. Ramchandran plot obtained from PROCHECK server was used to check stereochemical property. Meanwhile, 3D model of kypoetin with attached N-carbohydrates was built up using the GlyProt server. Results In the new modified analog, three additional N-glycosylation sites at amino-acid positions 30, 34 and 86 were inserted. Ramchandran plot analysis showed 81.6% of the residues in the most favored region, 15.6% in the additional allowed, 1.4% in the generously allowed regions and 1.4% in the disallowed region. 3D structural modeling showed that attached carbohydrates were on the proper spatial position. The whole solvent accessible surface areas of kypoetin (15132.69) were higher than EPO (9938.62). Conclusions Totally, various model evaluation methods indicated that the glycoengineered version of EPO had considerably good geometry and acceptable profiles for clinical studies and could be considered as the effective drug.

Erythropoietin (Epo) is a pleiotropic agent, that is to say, it can act on several cell types in different ways. An independent system Epo/Epo receptor (EpoR) was detected in brain, leading to the hypothesis that this hormone could be involved in cerebral functions. Epo/EpoR expression changes during ontogenesis, thus indicating the importance of this system in neurodevelopment. Moreover, the hypoxia-induced production of Epo in the adult brain suggests that it could exert a neurotrophic and neuroprotective effect in case of brain injury. Epo could also influence neuro- transmission, inducing neurotransmitters (NT) release. Epo therapy in anemic cancer patients is still a controversial issue, because of its possible action as a growth and an angiogenic factor. In our speculative hypothesis Epo could be involved in a ‘‘two steps process’’ that, after a neo- vascularization phase, leads to its down regulation. Moreover, Epo-activated signaling pathways could be modulated as possible targets to interfere in neoplastic cells cycle. In conclusion, treatment with rHuEpo could change therapeutical perspectives in different pathological condi- tions, such as central nervous system (CNS) diseases, but further studies are needed to clarify its physiopathological activities in different clinical fields.

Erythropoietin (EPO), already known as the stimulating hormone for erythropoiesis, has shown different and interesting pleiotropic actions. It does not only affect erythroid cells, but also myeloid cells, lymphocytes and megakaryocytes. This hormone can also enhance phagocytic function of the polymorphonuclear cells and reduce the activation of macrophages, thus modulating the inflammatory process.Moreover, hematopoietic and endothelial cells probably have the same cellular origin, and the discovery of erythropoietin receptors (EPO-R) also on mesangial and myocardial cells, smooth muscle fibrocells and neurons has prompted the study of the non-erythropoietic functions of this hormone.The interaction between EPO and VEGF may be of particular importance in neovascularization and wound healing.Different studies have demonstrated that EPO has an important direct hemodynamic and vasoactive action, which does not depend exclusively on any increase in hematocrit and viscosity. Moreover EPO showed protective effects on myocardial cells against apoptosis induced by ischemia/repefusion injury, but it could negatively affect pulmonary hypertension in patient with chronic cor pulmonale.This review aims to stress the importance of the increasing interest in EPO applications and the necessity of further studies to gain a deeper knowledge of this hormone and its pleiotropic and complex actions. © 2006 Bentham Science Publishers Ltd.

In a previous study EPO values were significantly lower in exposed subjects (PbBor =30 mcg/dl) than in controls (PbB" 20 mcg/dl). The aim of the study was to verify if high PbB cause the reduction of EPO in connection with the serum concentration of this hormone expected considering the Hct and hemoglobin (Hb) levels. Both in Pb exposed workers and controls blood levels of EPO, PbB, Hb, Hct were measured. It was not observed any line relation between Hct e log EPO (p = 0.01) and any multiple regression relation between log EPO, Hct and PbB into the total studied population. In our subjects PbB does not change physiologic relation between EPO and Hct and between EPO and Hb. Maybe because normal levels of Hct and Hb reduce EPO production in the healthy population. In physiologic condition of erythropoiesis, EPO does not seem influenced by PbB.