Desferoxamine preconditioning protects against cerebral ischemia in rats by inducing expressions of hypoxia inducible factor 1α and erythropoietin

Objective To investigate whether desferoxamine （DFO） preconditioning can induce tolerance against cerebral ischemia and its effect on the expression of hypoxia inducible factor 1 α （HIF- 1α） and erythropoietin （EPO） in vivo and in vitro. Methods Rat model of cerebral ischemia was established by middle cerebral artery occlusion with or without DFO administration. Infarct size was examined by TTC staining, and the neurological severity score was evaluated according to published method. Cortical neurons were cultured under ischemia stress which was mimicked by oxygen-glucose deprivation （OGD）, and the neuron damage was assessed by MTT assay. Immunofluorescent staining was employed to detect the expressions of HIF-1 and EPO. Results The protective effect induced by DFO （decreasing the infarction volume and ameliorating the neurological function） appeared at 2 d after administration ofDFO （post-DFO）, lasted until 7 d and disappeared at 14 d （P 〈 0.05）; the most effective action was observed at 3 d post-DFO. DFO induced tolerance of cultured neurons against OGD： neuronal viability was increased 23%, 34%, 40%, 48% and 56% at 8 h, 12 h, 24 h, 36 h, and 48 h, respectively, post-DFO （P 〈 0.05）. Immunofluorescent staining found that HIF-1 α and EPO were upregulated in the neurons of rat brain at 3 d and 7 d post-DFO; increase of HIF-1 α and EPO appeared in cultured cortex neurons at 36 h and 48 h post-DFO. Conclusion DFO induced tolerance against focal cerebral ischemia in rats, and exerted protective effect on OGD cultured cortical neurons. DFO significant induced the expression of HIF- 1 α and EPO both in vivo and in vitro. DFO preconditioning can protect against cerebral ischemia, which may be associated with the synthesis of HIF- 1 α and EPO.

Protective effect of erythropoietin against 1-methyl-4-phenylpyridinium-induced neurodegenaration in PC12 cells

Objective The neuroprotective effect of erythropoietin （EPO） against 1-methyl-4-phenylpyridinium （MPP^＋）- induced oxidative stress in cultured PC12 cells, as well as the underlying mechanism, were investigated. Methods PC12 ceils impaired by MPP^＋ were used as the cell model of Parkinson＇s disease. Methyl thiazolyl tetrazolium （MTT） was used to assay the viability of the PC12 cells exposed to gradient concentrations of EPO, and the terminal deoxynucleotidyl transferase dUTP nick end labelling （TUNEL） assay was used to analyze the apoptosis ratio of PC 12 cells. The expression of Bcl-2 and Bax in PC 12 cells were examined by Western blot, and the reactive oxygen species （ROS）, the mitochondrial transmembrane potential and the activity of caspase-3 in each group were detected by spectrofluorometer. Results Treatment of PC12 cells with MPP^＋ caused the loss of cell viability, which may be associated with the elevation in apoptotic rate, the formation of ROS and the disruption of mitochondrial transmembrane potential. It was also shown that MPP＋ significantly induced the upregulation of Bax/Bcl-2 ratio and the activation of caspase-3. In contrast, EPO significantly reversed these responses and had the maximum protective effect at 1 U/mL. Conclusion The inhibitive effect of EPO on the MPP^＋ -induced cytotoxicity may be ascribed to its anti-oxidative property and anti-apoptotic activity, and EPO may provide a useful therapeutic strategy for treatment of neurodegenerative diseases such as Parkinson＇s disease.

Plasma erythropoietin levels in anaemic and non-anaemic patients with chronic liver diseases

AIM: To investigate the serum erythropoietin (Epo) levels in patients with chronic liver diseases and to compare to subjects with iron-deficiency anaemia and healthy controls.METHODS: We examined 31 anaemic (ALC) and 22 non-anaemic (NALC) cirrhotic patients, 21 non-anaemic subjects with chronic active hepatitis (CAH), 24 patients with iron-deficiency anaemia (ID) and 15 healthy controls. Circulating Epo levels (ELISA; R&D Systems, Europe Ltd, Abingdon,UK) and haemoglobin (Hb) concentration were determined in all subjects.RESULTS: Mean±SD of Epo values was 26.9±10.8 mU/mL in ALC patients, 12.5±8.0 mU/mL in NALC subjects,11.6±6.3 mU/mL in CAH patients, 56.4±12.7 mU/mL in the cases of ID and 9.3±2.6 mU/mL in controls. No significant difference (P>0.05) was found in Epo levels between controls, CAH and NALC patients. ALC individuals had higher Epo levels (P<0.01) than these groups whereas ID subjects had even higher levels (P<0.001) than patients suffering from ALC.CONCLUSION: Increased Epo values in cirrhotics, are only detectable when haemoglobin was lesser than 12 g/dL.Nevertheless, this rise in value is lower than that observed in anaemic patients with iron-deficiency and appears blunted and inadequate in comparison to the degree of anaemia.

Stimulating erythropoiesis in inflammatory bowel disease associated anemia

Anemia is a frequent complication in patients with inflammatory bowel disease (IBD), and is associated with decreased quality of life and increased rate of hospitalization. The primary therapeutic targets of IBD- associated anemia are iron deficiency and anemia of chronic disease. An important prognostic parameter of the success or failure of therapy is the outcome of the underlying disease. Iron deficiency should be appropriately managed with iron supplementation. However, the use of oral iron therapy is limited by several problems, the most important being gastrointestinal side effects leading occasionally to disease relapse and poor iron absorption. Intravenous iron preparations are more reliable, with iron sucrose demonstrating the best efficacy and tolerability. Treatment with erythropoietin or darbepoetin has been proven to be effective in patients with anemia, who fail to respond to intravenous iron. Patients with ongoing inflammation have anemia of chronic disease and may require combination therapy comprising of intravenous iron sucrose and erythropoietin. After initiating treatment, careful monitoring of hemoglobin levels and iron parameters is needed in order to avoid recurrence of anemia. In conclusion, anemia in the setting of IBD should be aggressively diagnosed, investigated, and treated. Future studies should define the optimal dose and schedule of intravenous iron supplementation and appropriate erythropoietin therapy in these patients.

Classification of anemia for gastroenterologists

Most anemia is related to the digestive system by dietary deficiency, malabsorption, or chronic bleeding. We review the World Health Organization definition of anemia, its morphological classifi cation (microcytic, macrocytic and normocytic) and pathogenic classi-fication (regenerative and hypo regenerative), and integration of these classifications. Interpretation of laboratory tests is included, from the simplest (blood count, routine biochemistry) to the more specific (iron metabolism, vitamin B12, folic acid, reticulocytes, erythropoietin, bone marrow examination and Schilling test). In the text and various algorithms, we propose a hierarchical and logical way to reach a diagnosis as quickly as possible, by properly managing the medical interview, physical examination, appropriate laboratory tests, bone marrow examination, and other comple-mentary tests. The prevalence is emphasized in all sections so that the gastroenterologist can direct the diagnosis to the most common diseases, although the tables also include rare diseases. Digestive diseases potentially causing anemia have been studied in preference, but other causes of anemia have been included in the text and tables. Primitive hematological diseases that cause anemia are only listed, but are not discussed in depth. The last section is dedicated to simplifying all items discussed above, using practical rules to guide diagnosis and medical care with the greatest economy of resources and time.

Anemia and inflammatory bowel diseases

Too often anemia is considered a rare or unimportant manifestation in inflammatory bowel disease (IBD). However, over the last 10 years a number of studies have been conducted and the most relevant conclusions obtained are: (1) anemia is quite common in IBD; (2) although in many cases anemia parallels the clinical activity of the disease, many patients in remission have anemia, and iron, vitamin B12 and/or folic acid deficiency; (3) anemia, and also iron def iciency without anemia, have important consequences in the clinical status and quality of life of the patient; (4) oral iron can lead to gastrointestinal intolerance and failure of treatment; (5) intravenous iron is an effective and safe way to treat iron deficiency; (6) erythropoietin is needed in a significant number of cases to achieve normal hemoglobin levels. Thus, the clinician caring for IBD patients should have a comprehensive knowledge of anemia, and apply recently published guidelines in clinical practice.

Functional significance of erythropoietin receptor on tumor cells

Erythropoietin (Epo) is the regulator of red blood cell formation. Its receptor (EpoR) is now found in many cells and tissues of the body. EpoR is also shown to occur in tumor cells and Epo enhances the proliferation of these cells through cell signaling. EpoR antagonist can reduce the growth of the tumor in vivo. In view of our current knowledge of Epo, its recombinant forms and receptor, use of Epo in cancer patients to enhance the recovery of hematocrit after chemotherapy treatment has to be carefully evaluated.

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 erythropoietin （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 without diabetes （n=7）, non-prolif- erative DR （NPDR, n=7）, proliferative DR （PDR, n=8）, and PDR complicated with diabetic nephropathy （PDR-DN, n=7）. Results The numbers of EPOR^＋ CPCs and EPOR^＋ EPCs were reduced remarkably in NPDR corn pared with the control group （both P（0.01）, whereas rebounded in PDR and PDR-DN groups in varying degrees. Similar changes were observed in respect of the proportion of EPOR^＋ CPCs in CPCs （NPDR vs. control, P（0.01） and that of EPOR^＋ EPCs in EPCs （NPDR vs. control, P〈0.05）. 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.

Protective effects of erythropoietin pretreatment on myocardium with hypoxia/reoxygenation injury in rats

Objective: To establish the rat model with myocardial hypoxia/reoxygenation (H/R) injury, and investigate the protective effect of EPO pretreatment on the myocardium. Methods: Sixty male adult Wistar rats were randomly divided into 3 groups: control group, H/R group, and EPO group, 20 in each group. The rats in EPO group accepted injection of 5 000 U/kg recombinant human erythropoietin (RHuEPO) through vein, and the other rats accepted the injection of the same volume of saline. Twenty-four hours after the injection, rats in the EPO and H/R groups were put into the hypoxia environment for 12 h and then returned to the normoxic environment for 2 h, and then the samples of blood and myocardium were collected. Serum myocardial enzyme activity, apoptosis, ultrastructure, myocardial MDA contents, EPO receptor (EPOR) expression in cardiac myocytes and cardiac functions were tested. Results: EPOR expression was positive in cardiac myocytes of adult rat according to the result of immunonistochemitry assaying. Compared to those in H/R group, rats in EPO group presented lighter injury of myocardial ultrastructure, the reduction of serum myocardial enzyme activity, inhibition of apoptosis, the better recovery of cardiac functions, and the less production of oxygen-derived free radicals. Conclusion: Adult rat cardiac myocytes could express EPOR, and EPO pretreatment produced protective effects on myocardium with H/R injury.

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.

Anemia and digestive diseases: An update for the clinician

Anemia and iron deficiency are so common in digestive diseases that often are underestimated and undertreated. Our goal is to review from classif ication to treatment of the diverse types of anemias in different digestive diseases to update our knowledge on diagnosis and treatment. With the goal of improving the prognosis and quality of life of digestive diseases patients, we will review current transfusion, intravenous iron, and erythropoietin roles in the treatment of anemia.

Is iron overload in alcohol-related cirrhosis mediated by hepcidin?

In this case report we describe the relationship between ferritin levels and hepcidin in a patient with alcohol-related spur cell anemia who underwent liver transplantation.We demonstrate a reciprocal relation-ship between serum or urinary hepcidin and serum ferritin,which indicates that inadequate hepcidin production by the diseased liver is associated with elevated serum ferritin.The ferritin level falls with increasing hepcidin production after transplantation.Neither inflammatory indices(IL6)nor erythropoietin appear to be related to hepcidin expression in this case.We suggest that inappropriately low hepcidin production by the cirrhotic liver may contribute substantially to elevated tissue iron stores in cirrhosis and speculate that hepcidin replacement in these patients may be of therapeutic benefi t in the future.

The effect of erythropoietin to the proliferation of cancer cell lines

Objective： To investigated whether erythropoietin （EPO） would promote the proliferation of human cancer cell line MCF-7, Panc-1 and HepG2 so as to determine whether EPO would result a worse prognosis to the patients with malignant diseases. Methods： We examined the growth inhibitory or promotional effect of EPO to MCF-7, Panc-1 and HepG2 by MTT assay. Then we examined the cell cycle of those cancer cell lines which was cultured with EPO and without EPO by FCM. Results： The results showed there was no significant growth inhibitory or promotional effect of EPO to MCF-7, Panc-1 and HepG2. The cell cycle of those cancer cell lines cultured with EPO also has no significant different to that of those cancer cell lines cultured without EPO. Conclusion： It is concluded that EPO has no promotion effect to the cancer cell lines＇ proliferation, and maybe will not result a worse prognosis to the patients with malignant diseases.

Effects of angiopoietin-1 on attachment and metastasis of human gastric cancer cell line BGC-823

AIM： To evaluate the effects of angiopoietin-1 （Ang-1） on adhesion of gastric cancer cell line BGC-823 and expression of integrin β1, CD44V6, urokinase-type plasminogen activator （uPA） and matrix metalloproteinase-2 （MMP-2）. METHODS： BGC-823 cells were transfected transiently with adenovirus-Ang-1 （Ad-Ang-1）. Cells transfected transiently with adenovirus-green fluorescent protein （Ad-GFP） and untransfected cells were used as a negative and blank control group, respectively. The cell adhesion rate between cell and extracellular matrix （ECM） was determined by cell adhesion assay. To investigate whether Ang-1 could reinforce gastric carcinoma metastasis, we performed migration and invasion assays in BGC-823 cells. The mRNA and protein expression of integrin β1, CD44V6, uPA and MMP-2 were detected by reverse transcription polymerase chain reaction and Western blotting, respectively. The expression of integrin β1 and CD44V6 was measured by immunohistochemistry. RESULTS： BGC-823 cells were transfected successfully. The adhesion rate increased significantly in the Ad-Ang-1 group （P 〈 0.05）. The Ad-Ang-1-transfected group had a significant increase in migration and invasion compared with that of the mock-transfected and Ad-GFP groups. The mRNA and protein expression of integrin β1, CD44V6, uPA and MMP-2 in the Ad- Ang-1 group was higher than that in the Ad-GFP and blank control groups （P 〈 0.05）. Compared with mocktransfected and Ad-GFP groups, integrin 131 and CD44V6 expression intensity greatly increased （P 〈 0.05）. CONCLUSION： Transfection of Ang-1 into human gastric cancer cell line BGC-823 can significantly increase expression of integrin β1 and CD44V6, by which cell adhesion and metastasis to the ECM are promoted.

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.

Effects of nutrients and light intensity on the growth and biochemical composition of a marine microalga Odontella aurita

Algal biotechnology has advanced greatly in the past three decades. Many microalgae are now cultivated to produce bioactive substances. Odontella aurita is a marine diatom industrially cultured in outdoor open ponds and used for human nutrition. For the first time, we have systematically investigated the effects of culture conditions in cylindrical glass columns and fiat-plate photobioreactors, including nutrients （nitrogen, phosphorus, silicon, and sulfur）, light intensity and light path, on O. aurita cell growth and biochemical composition （protein, carbohydrate, β-1,3-glucan, lipids, and ash）. The optimal medium for photoautotrophic cultivation of O. aurita contained 17.65 mmol/L nitrogen, 1.09 mmol/L phosphorus, 0.42 mmol/L silicon, and 24.51 mmol/L sulfur, yielding a maximum biomass production of 6.1-6.8 g/L and 6.7-7.8 g/L under low and high light, respectively. Scale-up experiments were conducted with fiat-plate photobioreactors using different light-paths, indicating that a short light path was more suitable for biomass production of O. aurita. Analyses of biochemical composition showed that protein content decreased while carbohydrate （mainly composed of 15-1,3-glucan） increased remarkably to about 50% of dry weight during the entire culture period. The highest lipid content （19.7% of dry weight） was obtained under 0.11 mmol/L silicon and high light conditions at harvest time. Fatty acid profiles revealed that 80% were Cx4, C^6, and C20, while arachidonic acid and eicosapentaenoic acid （EPA） accounted for 1.6%-5.6% and 9%-20% of total fatty acids, respectively. High biomass production and characteristic biochemical composition profiles make O. aurita a promising microalga for the production ofbioactive components, such as EPA and D-1,3-glucan.

Does recombinant human erythropoietin accelerate correction of post-ulcer-bleeding anaemia?A pilot study

AIM:Anaemia caused by acute upper gastrointestinal bleeding is treated with blood transfusion or iron,but patients usually face a two-month recovery period from post- haemorrhage anaemia.This prospective,randomised,open, pilot study was designed to investigate whether recombinant human erythropoietin(Epoetin)therapy accelerate haematocrit increase in the post-bleeding recovery period. METHODS:We studied hospitalised patients admitted because of acute ulcer bleeding or haemorrhagic gastritis, who had a haematocrit of 27-33% and did not receive blood transfusions.One day after the endoscopic confirmation of cessation of bleeding,they were randomised either to erythropoietin(20 000 IU Epoetin alfa subcutaneously,on days 0,4 and 6)plus iron(100 mg im,on days 1-6,(G_1)or iron only(G_2).Haematocdt was measured on days 0,6,14, 30,45,and 60,respectively. RESULTS:One patient from G_1 and two from G_2 were lost to follow-up.Therefore,14 and 13 patients from G_1 and G_2 respectively were analysed.Demographic characteristics,serum iron,ferritin,total iron binding capacity,reticulocytes,and haernatoait were not significantly different at entry to the study. Median reticulocyte counts were significantly different between groups on day six(G_1:4.0,3.0-6.4 vs G_2:3.5,2.1-4.4%, P=0.03)and median haematocrit on day fourteen [G_1:35.9, 30.7-41.0 vs G_2:32.5,29.5-37.0%(median,range),P=0.04]. CONCLUSION:Erythropoietin administration significantly accelerates correction of anemia after acute ulcer bleeding. The haematocrit gain is equivalent to one unit of transfused blood two weeks after the bleeding episode.

Regulation of plasma erythropoietin in chronic liver disease

In a May-issue of the World Journal of Gastroenterology, there is a very interesting study by Bruno et al. on erythropoietin (EPO) levels in patients with chronic liver disease. We have very recently reported a similar, but much larger study by Tacke et al. on the role of EPO in chronic liver disease. By comparing Bruno's results with our patient cohort and applying their

STAT1 is involved in signal transduction in the EPOinduced HEL cells

Erythropoietin (EPO) is the ma jor regulator of mammalian erythropoisis, which stimulates the growth and differentiation of hematopoietic cells through interaction with its receptor (EPO-R). Here we use HEL cells (a human erythro-leukemia cell line) as a model to elucidate the pathway of signal transduction in the EPO-induced HEL cells. our data show that the EPOR (EPO receptor) on the surface of HEL cells interacts with the Janus tyrosine protein kinase (Jak2) to transduce intracellular signals through phosphorylation of cytoplasmic proteins in EPO-treated HEL cells. Both STAT1 and STAT5 in this cell line are tyrosine-phosphorylated and translocated to nucleus following the binding of EPO to HEL cells.Furthermore, the binding of both STAT1 and STAT5 proteins to specific DNA elements (SIE and PIE elements) is revealed in an EPO-dependent manner. Our data demonstrate that the pathway of signal transduction following the binding of EPO to HEL cells is similar to immature erythroid cell from the spleen of mice infected with anemia strain of Friend virus.

Erythropoietin in liver cirrhosis: Two questions without answers

TO THE EDITORIn a recent paper[1], and in a subsequent letter[2], Tacke et al. reported the investigation of plasma erythropoietin (Epo) levels in patients affected by chronic liver disease of various aetiologies. The authors also compared[2] their data to our previous work[3]. The results show a substantial agreement but also some important differences between the two works. We would like to highlight, from our point of view, this issue. Both the papers demonstrated increased Epo values in anaemic cirrhotic subjects when compared to healthy controls and non-anemic patients with liver disease.