High Concentrations Of Erythropoietin Research Articles

Neuronal nitric oxide synthase is required for erythropoietin stimulated erythropoiesis in mice.

Introduction: Erythropoietin (EPO), produced in the kidney in a hypoxia responsive manner, is required for red blood cell production. In non-erythroid tissue, EPO increases endothelial cell production of nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) that regulates vascular tone to improve oxygen delivery. This contributes to EPO cardioprotective activity in mouse models. Nitric oxide treatment in mice shifts hematopoiesis toward the erythroid lineage, increases red blood cell production and total hemoglobin. In erythroid cells, nitric oxide can also be generated by hydroxyurea metabolism that may contribute to hydroxyurea induction of fetal hemoglobin. We find that during erythroid differentiation, EPO induces neuronal nitric oxide synthase (nNOS) and that neuronal nitric oxide synthase is required for normal erythropoietic response. Methods: Wild type (WT) mice and mice with targeted deletion of nNOS (nNOS-/-) and eNOS (eNOS-/-) were assessed for EPO stimulated erythropoietic response. Bone marrow erythropoietic activity was assessed in culture by EPO dependent erythroid colony assay and in vivo by bone marrow transplantation into recipient WT mice. Contribution of nNOS to EPO stimulated cell proliferation was assessed in EPO dependent erythroid cells and in primary human erythroid progenitor cell cultures. Results: EPO treatment increased hematocrit similarly in WT and eNOS-/- mice and showed a lower increase in hematocrit nNOS-/- mice. Erythroid colony assays from bone marrow cells were comparable in number from wild type, eNOS-/- and nNOS-/- mice at low EPO concentration. Colony number increased at high EPO concentration is seen only in cultures from bone marrow cells of wild type and eNOS-/- mice but not from nNOS-/- mice. Colony size with high EPO treatment also exhibited a marked increase in erythroid cultures from wild type and eNOS-/- mice but not from nNOS-/- mice. Bone marrow transplant from nNOS-/- mice into immunodeficient mice showed engraftment at comparable levels to WT bone marrow transplant. With EPO treatment, the increase in hematocrit was blunted in recipient mice that received with nNOS-/- donor marrow compared with recipient mice that received WT donor marrow. In erythroid cell cultures, addition of nNOS inhibitor resulted in decreased EPO dependent proliferation mediated in part by decreased EPO receptor expression, and decreased proliferation of hemin induced differentiating erythroid cells. Discussion: EPO treatment in mice and in corresponding cultures of bone marrow erythropoiesis suggest an intrinsic defect in erythropoietic response of nNOS-/- mice to high EPO stimulation. Transplantation of bone marrow from donor WT or nNOS-/- mice into recipient WT mice showed that EPO treatment post-transplant recapitulated the response of donor mice. Culture studies suggest nNOS regulation of EPO dependent erythroid cell proliferation, expression of EPO receptor and cell cycle associated genes, and AKT activation. These data provide evidence that nitric oxide modulates EPO dose dependent erythropoietic response.

Protection of insect neurons by erythropoietin/CRLF3-mediated regulation of pro-apoptotic acetylcholinesterase

Cytokine receptor-like factor 3 (CRLF3) is a conserved but largely uncharacterized orphan cytokine receptor of eumetazoan animals. CRLF3-mediated neuroprotection in insects can be stimulated with human erythropoietin. To identify mechanisms of CRLF3-mediated neuroprotection we studied the expression and proapoptotic function of acetylcholinesterase in insect neurons. We exposed primary brain neurons from Tribolium castaneum to apoptogenic stimuli and dsRNA to interfere with acetylcholinesterase gene expression and compared survival and acetylcholinesterase expression in the presence or absence of the CRLF3 ligand erythropoietin. Hypoxia increased apoptotic cell death and expression of both acetylcholinesterase-coding genes ace-1 and ace-2. Both ace genes give rise to single transcripts in normal and apoptogenic conditions. Pharmacological inhibition of acetylcholinesterases and RNAi-mediated knockdown of either ace-1 or ace-2 expression prevented hypoxia-induced apoptosis. Activation of CRLF3 with protective concentrations of erythropoietin prevented the increased expression of acetylcholinesterase with larger impact on ace-1 than on ace-2. In contrast, high concentrations of erythropoietin that cause neuronal death induced ace-1 expression and hence promoted apoptosis. Our study confirms the general proapoptotic function of AChE, assigns a role of both ace-1 and ace-2 in the regulation of apoptotic death and identifies the erythropoietin/CRLF3-mediated prevention of enhanced acetylcholinesterase expression under apoptogenic conditions as neuroprotective mechanism.

Effect of erythropoietin on angiogenic potential of dental pulp cells.

Erythropoietin (EPO) is a 34-kDa glycoprotein that possesses the potential for angiogenesis, as well as anti-inflammatory and anti-apoptotic properties. The present study aimed to examine the effect of EPO on the angiogenesis of dental pulp cells (DPCs) and to explore the underlying mechanisms of these effects. It was demonstrated that EPO not only promoted DPCs proliferation but also induced angiogenesis of DPCs in a paracrine fashion. EPO enhanced the angiogenic capacity by stimulating DPCs to secrete a series of angiogenic cytokines. ELISA confirmed that high concentrations of EPO increased the production of MMP-3 and angiopoietin-1 but decreased the secretion of IL-6. Furthermore, EPO activated the ERK1/2 and p38 signaling pathways in DPCs, while inhibition of these pathways diminished the angiogenesis capacity of DPCs. The present study suggested that EPO may have an important role in the repair and regeneration of dental pulp.

Endogenous erythropoietin concentrations and association with retinopathy of prematurity and brain injury in preterm infants.

BackgroundEndogenous erythropoietin (EPO) concentrations vary widely in preterm infants and may be associated with perinatal risk factors and neurological outcomes. Erythropoietin is elevated in fetal hypoxia but is also a potential neuroprotectant.MethodsIn a prospective study of 27 infants ≤ 30 weeks gestation, serum erythropoietin concentrations were measured during the first month of life, on day 1 and weeks 1, 2, and 4, and related to perinatal risk factors and outcomes including retinopathy of prematurity and cerebral injury evaluated near term-equivalent post menstrual age using magnetic resonance imaging with quantitative scoring.ResultsLower birth weight was associated with higher EPO concentrations throughout the first 2 weeks of life (r = -0.6, p < 0.01). Higher day 1 and week 1 EPO concentrations were associated with lower Apgar score at 1 minute (r = - 0.5) and 5 minutes (r = -0.7), respectively (p < 0.01). Higher day 1 EPO concentrations and 2-week area under the curve were associated with increased risk (p = 0.01) and severity (r = 0.5, p < 0.02) of retinopathy of prematurity. Higher EPO concentrations at 2 weeks were associated with increased total brain injury score (r = 0.5, p < 0.05).ConclusionElevated endogenous erythropoietin concentrations in the first two weeks of life are associated with lower birth weight and increased risk of adverse outcomes.

Effects of 100-km ultramarathon on erythropoietin variation in runners with hepatitis B virus carrier.

Completing an ultramarathon leads to an immediate postrace surge of erythropoietin (EPO). Patients with chronic liver disease may have high plasma EPO concentrations. This study aims to explore whether plasma EPO concentrations vary between hepatitis B virus carrier (HBVc) and non-HBVc runners during long distance running. Blood samples were collected from 8 HBVc and 18 non-HBVc runners at 3 different time points: 1 week before, immediately following, and then 24 h after the 100-km ultramarathon race. Samples were analyzed for plasma EPO levels. EPO concentration had a statistically significant rise immediately after the race (8.7 [7.1-11.9] mU·mL-1 to 23.7 [14.8-37.2] mU·mL-1, P < 0.001) and maintained the high levels 24 h after the race finished (16.7 [11.5-21.0] mU·mL-1, P < 0.001) in all participants. The mean of EPO concentration was 8.9 (5.7-13.2) mU·mL-1 in HBVc runners and was 8.7 (7.7-11.2) mU·mL-1 in non-HBVc runners in the prerace. In HBVc runners, plasma EPO levels were no different at baseline (P = 0.657) and increased in the same fashion in response to ultramarathon compared with non-HBVc runners. Plasma EPO levels between the two groups were not statistically different at any time point. Prolonged endurance exercise led to a significant increase in EPO. A comparable increase in EPO levels was observed in HBVc and non-HBVc runners during and 24 h after 100-km ultramarathon. However, a small sample size might have affected the ability to detect a difference if it does exist.

Intranasal administration of erythropoietin rescues the photoreceptors in degenerative retina: a noninvasive method to deliver drugs to the eye

Inherited retinopathies typically lead to photoreceptor loss and severe visual impairments in the subjects. Intranasal administration is an efficient approach to deliver therapeutic agents to the targeted tissue. The present study is designed to deliver the erythropoietin (EPO) into the N-methyl-N-nitrosourea (MNU) induced mice, a pharmacological retinopathy model via intranasal or intravenous route. The mice were then subjected to bioavailability assay and therapeutic effects evaluation. Our results showed that the intranasal delivery of EPO is effective to alleviate the morphological disruptions in the MNU induced mice. The intranasal delivery of EPO also ameliorated the visual impairments in the MNU induced mice. Immunostaining experiment showed that both the M-cone and S-cone populations in the degenerative retinas are rescued by the intranasal delivery of EPO. In particular, the M-cone photoreceptors in dorsal-temporal (DT) quadrant and the S-cone photoreceptors in ventral-nasal (VN) quadrant were preferentially preserved by the intranasal delivery of EPO. Mechanism studies showed that the intranasal delivery of EPO could the modulate apoptosis and restrict oxidation in the degenerative retina. Compared with intravenous delivery, the intranasal delivery led to the significantly higher EPO concentration in the retina. The intranasal delivery resulted in more potent protection and had less erythropoiesis-stimulating activity than the intravenous delivery. Our results suggest that the intranasal administration is a noninvasive and efficient approach to deliver EPO into the retinas. These findings lay the groundwork for further intranasal administration of EPO in ophthalmological practice.

The Hypoxic Response and Altered Gene Expression in Patients with Sickle Cell Disease

AbstractAbstract 3245Sickle cell disease (SCD) and Chuvash polycythemia (CP) are both monogenic hematologic disorders, the first resulting in hemolytic anemia and the second in polycythemia related to an upregulated hypoxic response at normoxia. Specifically, homozygosity for the VHLR200W mutation leads to increased levels of the transcription factors hypoxia inducible factor (HIF)-1 and HIF-2 at normoxia and altered transcription of many genes. Much attention in the pathophysiology of SCD has focused on the adverse effects of chronic inflammation, enhanced cellular adhesion pathways and hemolytic rate. However, the chronic anemia of SCD is associated with an upregulation of the hypoxic response as evidenced by high erythropoietin concentrations. In this prospective study, we compared gene expression alterations in peripheral blood mononuclear cells in SCD and CP. Our pre-specified hypothesis was that we could identify hypoxia-mediated gene expression alterations in SCD through a comparison with altered gene expression in CP and reveal molecular pathways that are potentially shared by these two conditions. We prospectively compared gene expression profiles in two independent cohorts, an SCD cohort comprised of 22 SCD patients and 19 African American controls and a CP cohort comprised of 8 VHLR200W homozygotes and 17 Chuvash controls with wildtype VHL. Because iron deficiency can influence the hypoxic response, we excluded iron-deficient subjects from each cohort. We used the identical Affymetrix exon array platform for both cohorts.Differential gene expression was highly correlated between the two conditions, with Spearman correlation ρ = 0.75 between their expression profiles across 16,642 genes, suggesting that 56% of expression variation triggered by beta hemoglobin mutation in SCD may be explained by hypoxic transcriptional responses. A small portion of differential genes were highly induced in both conditions. Among 54 genes up-regulated >1.5-fold in SCD patients, 24 (44%) overlapped with the 31 genes up-regulated >1.5-fold in VHLR200W homozygotes. The genes highly induced in both conditions included FAM46C (family with sequence similarity 46 member C), SELENBP1 (selenium binding protein 1), IL1B (interleukin 1, beta), MOP-1, SNCA (synuclein, alpha), GMPR (guanosine monophosphate reductase), BPGM (2,3-bisphosphoglycerate mutase), SLC25A37 (solute carrier family 25 member 37), CA1 (carbonic anhydrase I), DCAF12 (DDB1 and CUL4 associated factor 12), EPB42 (erythrocyte membrane protein band 4.2), AHSP (alpha hemoglobin stabilizing protein), SLC4A1 (solute carrier family 4 member 1), HBB (hemoglobin, beta), HBD (hemoglobin, delta), CSDA (cold shock domain protein A), FECH (ferrochelatase), BCL2L1 (BCL2-like 1), OSBP2 (oxysterol binding protein 2), APOBEC3A (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3A), IFIT1 (interferon-induced protein with tetratricopeptide repeats 1), IFIT3 (interferon-induced protein with tetratricopeptide repeats 3), IFI44L (interferon-induced protein 44-like), and IFI27 (interferon, alpha-inducible protein 27). Three genes were down-regulated >1.5-fold in SCD patients. One of these, GIMAP7 (GTPase, IMAP family member 7), was among the 11 genes down-regulated >1.5-fold in VHLR200Whomozygotes.These results suggest that there is a broad upregulation of the hypoxic response in SCD and that the hypoxic response may underlie or interact with an important proportion of the clinical and pathophysiologic manifestations of SCD. Disclosures:No relevant conflicts of interest to declare.

A case of anaplastic clear-cell ependymoma presenting with high erythropoietin concentration and 1p/19q deletions

We describe a 19-year-old woman with onset of epileptic seizure, and a small mural nodule and multicystic lesions with severe brain edema located in the right frontal lobe. At surgery, the tumor and a clear margin was removed, and symptoms improved postoperatively. Extended local radiotherapy (60Gy) was performed. Histopathological examination revealed oligodendroglioma-like tumor cells with a perinuclear halo. The tumor cells extended processes toward CD34-positive proliferating vessels, which resemble a basement membrane. These proliferating vessels formed a tumor membrane so that there was a clear margin between the tumor and brain tissue. Tumor cells were positive for epithelial membrane antigen in a dot-like pattern. MIB-1 staining index was 50.6%. Electron microscopy showed cilia and zipper-like junctions, and anaplastic clear-cell ependymoma grade III was diagnosed. A characteristic of the case was formation of a tumor membrane by proliferating tumor blood vessels. Fluorescence in situ hybridization showed 1p/19q deletions, and the concentration of erythropoietin in the cyst fluid was abnormally high, at 1,859.4mIU/ml. Erythropoietin and erythropoietin receptors were verified with immunohistochemical staining.

Study of Two Subjects with Absence of Severe Anemia with Homozygous β0 Thalassemia Due to Disrupted γ-to-β Switch

AbstractAbstract 5172Sickle cell disease (SCD) and β thalassemia due to defects of the HBB (β globin gene) are among the most common inherited genetic disorders. At birth, there is a switch of γ globin transcription to β and d, with replacement of HbF by HbA and HbA2 virtually completed by six months of age. At that time, serious inherited disorders of the β gene, such as sickle cell disease and Cooley's anemia (homozygosity for β0 thalassemia mutations), become clinically apparent. Cooley's anemia is a life-threatening disorder wherein, in most patients, chronic transfusions or bone marrow transplantations are needed to sustain life. Rare patients with homozygosity or compound heterozygosity for β0 have no or only mild anemia. These patients maintain a high level of γ globin synthesis, apparently from a disrupted γ-to-β switch, thus attenuating their disease state. Recent work has demonstrated that BCL11A plays an important role in the suppression of γ-globin expression, as do polymorphisms of the gene that remain to be fully elucidated at a functional level. We recruited two unrelated subjects with homozygous β0 thalassemia mutations with no or only mild anemia (Patient #1, IVS2+1 G>A; Hb 14.2 Gm%; 97.2% HbF, 2.8% HbA2, Patient #2, IVS 2 G-T; Hb 11.2 Gm%; 92/5% HbF, 6.8% HbA2, 0.7% HbA). We sequenced transcripts and genomic loci of BCL11A from these patients. No mutations or splicing variants on transcripts were found. However, when the ≂f102 kb of genomic material from patient #1 was sequenced, 5 single nucleotide changes at intron II were found (2 known and 3 previously unpublished), while no genomic changes were found in patient #2. We then performed in vitro erythroid expansion from peripheral blood utilizing high erythropoietin concentrations and analyzed the cell proliferation and expression of globin and BCL11A genes. Interestingly, detectable amounts of β-globin transcripts were present in both patients during expansion, although protein levels were not detectable by the conventional HPLC method, probably due to limited sensitivity of this assay. Patient #1 showed mild in vitro induction of β-globin expression, which is lower than the control group, but no apparent cell proliferation. Patient #2 showed no induction of β-globin expression and hyperproliferation at a later stage of expansion (See Figure); however, the levels of BCL11A and γ-globin transcripts were indistinguishable from controls. [Display omitted] Although we were unable to detect any abnormality of the BCL11A transcript as a cause of high fetal hemoglobin expression in these patients, we cannot rule out the possibility that the intronic mutations in patient #1 may interfere with BCL11A gene translation, perhaps by interference with non-coding RNA. The potential molecular mechanism of γ-to-β switch is being explored by gene expression profiling and microRNA analyses. Disclosures:No relevant conflicts of interest to declare.

Erythropoietin Is Increased in Aqueous Humor of Glaucomatous Eyes

Purpose: To determine the aqueous concentration of erythropoietin (EPO) in patients with and without glaucoma.Methods: Prospective, comparative control study. Concentrations of EPO in the aqueous humor were measured using ELISA kits from 75 patients, of whom 55 had glaucoma (14 primary acute angle-closure glaucoma, PAACG; 26 primary chronic angle-closure glaucoma, PCACG; 11 primary open-angle glaucoma, POAG; 4 neovascular glaucoma, NVG), and 20 had cataract only as control.Results: EPO concentrations in eyes with glaucoma (71.0 ± 12.0 mU/mL) were significantly higher than those in eyes with cataract (6.4 ± 0.8 mU/mL, P < 0.001). There were no significant differences (P = 0.421) between PCACG (28.84 ± 3.9 mU/mL) and POAG (20.2 ± 2.0 mU/mL); however, EPO concentrations in eyes with PAACG (118.5 ± 14 mU/mL) were significantly higher than these two chronic subtypes of glaucoma (P < 0.001, respectively). Unusually high EPO concentrations were detected in four eyes with NVG (319.5 ± 47.7 mU/mL). No effect of age, gender, different eyes, body mass index of the aqueous humor EPO concentration could be detected (P > 0.05). No significant correlation was found between aqueous humor and plasma EPO concentrations in PAACG and control group (P = 0.285, 0.500, respectively).Conclusion: Our prospective study suggests that the aqueous humor EPO concentrations are increased in eyes with glaucoma.

EPO‐mediated reduction in Hamp expression in vivo corrects iron deficiency anaemia in TMPRSS6 deficiency

Hepcidin, a 25 amino acid liver-derived peptide, has been recognized as a regulator of iron homeostasis. Hepcidin levels are negatively correlated with erythropoietic expansion, consistent with erythrocytes representing the major compartment that utilizes iron and hepcidin the major regulator for limiting iron availability (Nemeth, 2008). However, the molecular pathway transmitting the signal from proliferating erythrocytes to the regulation of hepcidin expression in hepatocytes has not been clearly defined.

Effect of Phlebotomy Therapy On Hemoglobin Concentration and Tricuspid Regurgitation Velocity in Chuvash Polycythemia.

Abstract 1897Poster Board I-920 Background:Chuvash polycythemia is caused by homozygosity for the VHL598C>T mutation, which leads to up-regulation of HIF-1a and HIF-2a in normoxia. As the result, circulating concentrations of erythropoietin are elevated. Chuvash polycythemia patients suffer from cardiovascular abnormalities that include pulmonary arterial hypertension, thrombosis and stroke. Phlebotomy is a common therapy for patients to decrease symptoms such as plethora and headache. However, the outcomes of phlebotomy have not been assessed for these patients. The objective of this analysis is to evaluate the effect of phlebotomy on hemoglobin concentration, serum concentrations of ferritin and erythropoietin, and echocardiographically-determined tricuspid regurgitation velocity, which reflects systolic pulmonary artery pressure. Methods:One hundred twenty patients homozygous for VHL598C>T and 38 controls of comparable age and gender from Chuvash Republic of the Russian Federation were studied. Clinical and demographic characteristics were determined and echocardiography was performed. Serum ferritin and erythropoietin concentrations were measured by ELISA. Results:The median (interquartile) age for Chuvash polycythemia cases was 36 (22–48) years. They included 68 females (56%). Chuvash polycythemia patients had higher serum erythropoietin concentration (medians of 46 versus 8 mIU/ml, P = 0.0001) and lower serum ferritin concentration (medians of 12 versus 48 ng/ml, P = 0.0001) compared to controls. Tricuspid regurgitation velocity was higher in cases than controls (medians of 2.5 vs. 2.3 m/sec, P = 0.007). Among the cases, 87 (71%) had a history of phlebotomy and 54 of these had phlebotomy within the last year. Phlebotomy was associated with higher erythropoietin concentration (P = 0.033) and lower ferritin concentration (P = 0.024) but no significant difference in hemoglobin concentration (P = 0.9) (Table 1). After adjusting for the effect of age, phlebotomy was associated with significantly higher odds of tricuspid regurgitation velocity ≥2.5 (m/sec) (odds ratio: 3.3; 95% CI: 1.1–9.9).Table 1Iron status and hematologic markers in Chuvash polycythemia patients by history of phlebotomy. Results are in mean (95% confidence interval).No phlebotomy (N = 28)Phlebotomy >1 year ago (N = 25)Phlebotomy in last year (N = 42)P for trendResultsResultsResultsFerritin (ng/ml)*17.6 (11.7–26.3)12.6 (9.9–15.8)8.9 (6.3–12.6)0.024Erythropoietin (mIU/ml)39 (26–54)50 (41–61)64 (49–80)0.033Hemoglobin (g/dL)*18.1 (17.2–19.0)17.9 (17.5–18.5)17.9 (17.1–18.6)0.9*Values are adjusted for age and gender Conclusion:Patients with Chuvash polycythemia tend to mobilize iron stores and increase erythropoietin production to maintain a constant, elevated hemoglobin concentration despite phlebotomy therapy. In this process, estimated pulmonary systolic blood pressure appears to increase. Therefore, phlebotomy therapy might be a risk factor for pulmonary hypertension in the context of Chuvash polycythemia. Disclosures:No relevant conflicts of interest to declare.

Relationship of erythropoietin, fetal hemoglobin, and hydroxyurea treatment to tricuspid regurgitation velocity in children with sickle cell disease

AbstractHydroxyurea and higher hemoglobin F improve the clinical course and survival in sickle cell disease, but their roles in protecting from pulmonary hypertension are not clear. We studied 399 children and adolescents with sickle cell disease at steady state; 38% were being treated with hydroxyurea. Patients on hydroxyurea had higher hemoglobin concentration and lower values for a hemolytic component derived from 4 markers of hemolysis (P ≤ .002) but no difference in tricuspid regurgitation velocity compared with those not receiving hydroxyurea; they also had higher hemoglobin F (P < .001) and erythropoietin (P = .012) levels. Hemoglobin F correlated positively with erythropoietin even after adjustment for hemoglobin concentration (P < .001). Greater hemoglobin F and erythropoietin each independently predicted higher regurgitation velocity in addition to the hemolytic component (P ≤ .023). In conclusion, increase in hemoglobin F in sickle cell disease may be associated with relatively lower tissue oxygen delivery as reflected in higher erythropoietin concentration. Greater levels of erythropoietin or hemoglobin F were independently associated with higher tricuspid regurgitation velocity after adjustment for degree of hemolysis, suggesting an independent relationship of hypoxia with higher systolic pulmonary artery pressure. The hemolysis-lowering and hemoglobin F–augmenting effects of hydroxyurea may exert countervailing influences on pulmonary blood pressure in sickle cell disease.

Searching new biomarkers in nipple aspirate fluid: peroxidation status and adhesive molecules to early identify breast cancer

During the last two years (with the support of the DSLRF Grant Award 2007), we have mainly focused our attention on both 1) the involvement of oxidative stress in breast microenvironment and 2) the function of adhesion molecules in the breast cells of the ductal-lobular unit. 1) Starting from our previous evidence that active lipid peroxidation may have a physiologic role in the normal mammary gland and that lower levels of isoprostanes in NAF of BC patients suggest altered arachidonate metabolism (Mannello et al, Int J Cancer 2007;120:1971–6), we have recently discovered that in NAF Phospholipase A2-IIa activity (key enzyme regulating arachidonate metabolism) may play a dual role in breast microenvironment during cancer development and progression. It shows a beneficial role releasing low Arachidonate levels in physiologic status, while its up-regulation has detrimental functions allowing the conversion of high arachidonate levels into leukotrienes and prostaglandins, promoting BC progression. Noteworthy, the inhibition of this pathway through COX-inhibitors leads to induction of apoptosis and tumor growth reduction (Figure ​(Figure11). Figure 1 2) Based on our hypothesis that peculiar junctional complexes may differently seal breast ductal cells leading them to different evolution (Mannello et al, Breast Cancer Res Treat 2007;102:125), we have recently discovered that higher levels of soluble form of P-cadherin were found in NAF from cancer patients. We found a positive correlation of sP-cad with disease stages and tumor grade, and an inverse relationship with estrogen/progesterone receptor status. High levels of sP-cad in cancer NAF suggest its release via proteolytic processing, favouring cancer cell detachment from breast duct. 3) Moreover, we have found that breast epithelial cells floating in NAF of cancer patients contain higher concentrations of Erythropoietin which may be shed in NAF, triggering intracellular signal cascade with subsequent breast cancer initiation. These results may explain the controversies of preclinical/clinical studies about the use of Epo, (i.e., beneficial role improving patient' survival and negative impact promoting tumor growth progression) 4) We overviewed how Diet nutrients may regulate in breast microenvironment both NAF protein expression and cancer etiology. We also reviewed how the Intraductal approaches assessing metabolic changes in breast microenvironment may help to identify early breast cancer biomarkers in NAF. Finally, we critically overviewed and discussed how the Protein analyses of NAF (mirroring the breast microenvironment) may differentiate healthy women from breast cancer patients.

Temporal and spatial distribution of erythropoietin in transgenic tobacco plants

Plants have shown promise as bioreactors for the large-scale production of a wide variety of recombinant proteins. To increase the economic feasibility of this technology, numerous molecular approaches have been developed to enhance the production yield of these valuable proteins in plants. Alternatively, we chose to examine the temporal and spatial distribution of erythropoietin (EPO) accumulation during tobacco plant development, in order to establish the optimal harvesting time to further maximize heterologous protein recovery. EPO is used extensively worldwide for the treatment of anaemia and is currently the most commercially valuable biopharmaceutical on the market. Our results indicate that the concentration of recombinant EPO and endogenous total soluble protein (TSP) declined significantly for every leaf of the plant during maturation, although the rate of these declines was strongly dependent on the leaf's position on the plant. As a result, the amount of EPO produced in leaves relative to TSP content remained essentially unchanged over the course of the plant's life. Decreasing levels of recombinant protein in leaves was attributed to proteolytic degradation associated with tissue senescence since transgene silencing was not detected. We found that significantly higher concentrations of EPO within younger leaves more than compensated for their smaller size, when compared to their low-expressing, fully-grown counterparts. This suggests that fast-growing, young leaves should be periodically harvested from the plants as they continue to grow in order to maximize recombinant protein yield. These findings demonstrate that EPO accumulation is highly influenced by the plant's physiology and development.

Increased plasma EPO and MIP-1α are associated with recruitment of vascular progenitors but not CD34(+) cells in autologous peripheral blood stem cell grafts

Increased levels of endothelial-like vascular progenitor cells (VPCs) in peripheral blood stem cell (PBSC) products have been associated with reduced transplant-related toxicity following autologous hematopoietic stem cell transplantation. In this study, a panel of angiogenic and inflammatory plasma proteins were quantitatively analyzed in patients undergoing PBSC collection for autologous hematopoietic stem cell transplantation to identify profiles associated with greater VPC recruitment. A panel of 16 candidate plasma factors were quantified using multianalyte fluorescence and/or enzyme-linked immunosorbent assay. VPC clusters were enumerated using a standard cell culture assay. Thirty-six patients (mean age=51 years, 42% female) had plasma collected at baseline prior to PBSC mobilization and on the day of PBSC collection. Only erythropoietin (EPO) levels increased significantly on the day of PBSC collection in comparison with baseline plasma levels (2.2-fold increase; p=0.003). Interleukin-2, -10, epidermal growth factor, interferon-alpha, and angiopoietin-1 all decreased significantly between baseline and the day of PBSC collection (p<0.02). The remaining cytokine levels did not change appreciably (p=NS). The cohort was divided into "low" graft VPCs (<2.0 x 10(3)/kg) and "high" graft VPCs (>or=2.0 x 10(3)/kg) and cytokine levels were compared between the groups. At baseline, increased levels of macrophage inflammatory protein-1 alpha (MIP-1 alpha) were associated with increased graft VPCs (p=0.05) while higher EPO concentrations on the day of PBSC collection predicted higher graft VPC levels (p=0.02). These cytokines were not associated with CD34(+) cell mobilization. The association of different plasma proteins with graft VPC and CD34(+)-cell levels suggests that mobilization of vascular and hematopoietic progenitors occurs through independent mechanisms. Patients with low levels of MIP-1 alpha at baseline may be candidates for interventions aimed at increasing graft VPC levels. Strategies that increase plasma EPO concentrations may be most promising to augment the regenerative properties of PBSC products.

Novel Red Cell Indices Indicating Reduced Availability of Iron are Associated With High Erythropoietin Concentration and Low pH Level in the Venous Cord Blood of Newborns

There is evidence that an elevated erythropoietin (EPO) concentration is associated with signs of iron deficient erythropoiesis. The aim of this study was to evaluate the iron status by means of novel cellular indices and serum iron markers and to determine whether these are associated with EPO and pH in the venous cord blood of 193 full-term newborns. There were positive correlations between EPO and the percentage of hypochromic red blood cells (%HYPOm) and reticulocytes (%HYPOr) [r = 0.45 (p < 0.001) and r = 0.56 (p < 0.001), respectively]. %HYPOm and %HYPOr also had negative correlations with pH [r = -0.53 (p = 0.001) and r = -0.46 (p = 0.001), respectively]. Newborns who had low pH (pH < or =7.15, n = 16) had significantly higher %HYPOm, %HYPOr, and serum transferrin receptor and transferrin concentrations in their cord blood than newborns with normal pH. Thus, in newborn cord blood, the higher number of red cells and reticulocytes with lower Hb content may have impaired the oxygen carrying capacity that has been a trigger for EPO production. Furthermore, signs of lower hemoglobinization of red cells are associated with low umbilical vein pH in the newborns, indicating an increased risk of birth asphyxia.

Podocalyxin Is a Selective Marker of Erythroid Progenitors but Is Dispensable for Anemia Recovery.

Podocalyxin, a member of the CD34-family of anti-adhesins, is induced on erythroid cells in the spleen and bone marrow following administration of high concentrations of erythropoietin (Epo), or, phenylhydrazine (PHz)-induced anemia. Notably, Podocalyxin is not expressed on committed erythroid progenitors during homeostatic red cell turnover. Our previous work has suggested that stress erythropoiesis in the mouse draws on a specific population of resident splenic erythroid progenitors which respond to a distinct set of signals during anemic recovery in contrast to the erythroid populations residing primarily in the marrow responsible for maintaining normal homeostasis (Lenox et al., 2005, Blood; Perry et al., 2007, Blood). During stress erythropoiesis, Podocalyxin expression is upregulated, in part, via a Stat5-dependent pathway in response to Epo (Sathyanarayana et al., 2007, Blood) and Podocalyxin expression has been postulated to play a key role in the release of reticulocytes into the periphery. In this work, we have addressed this hypothesis and further characterized the expression pattern of Podocalyxin during stress erythropoiesis. Since Podocalyxin (Podxl) gene deletion results in perinatal lethality (Doyonnas et al., 2001, J. Exp. Med), we used hematopoietic cell-reconstituted chimeric mice lacking Podocalyxin expression in their hematopoietic compartment. Ten weeks after transplantation, chimeric mice were demonstrated to have normal peripheral blood red cell and platelet homeostasis. Chimeric mice were subjected to Epo stimulation or chemically-induced models of anemia. We found that during stress erythropoiesis, Podocalyxin is rapidly upregulated on early erythroid precursors, with expression on populations with BFU-e and CFU-e, although not CFU-GM, potential. Podocalyxin expression continues through a proerythroblast stage of erythroid development and is maintained on immature reticulocytes in the periphery. While Podocalyxin is highly expressed on erythroblasts and progenitors during anemic stress recovery, we found that loss of Podocalyxin has no major influence on the proportion of erythroid progenitors and staged erythroblasts in the spleen and marrow in response to Epo and, further, Podocalyxin is dispensable for efficient recovery from chemically-induced models of anemia. Our findings suggest that Podocalyxin expression is not critical for reticulocyte release or efficient stress erythroid differentiation. We speculate that Podocalyxin may play a subtle role in early erythroid development during anemic recovery, population of bone marrow and spleen during late embryonic development or establishment of neo-natal homeostasis. Furthermore, we suggest Podocalyxin may be used as a highly specific marker to sort stress-induced BFU-e and CFU-e progenitors from lineage-marker depleted bone marrow, spleen or peripheral blood. MRH and SM contributed equally to this work and are fellows of the Strategic Training Program in Transfusion Science funded by the Canadian Institutes for Health Research (CIHR) and the Heart and Stroke Foundation of Canada at the University of British Columbia Centre for Blood Research(CBR). RFP holds a research grant from the National Blood Foundation(USA). KMM is a Michael Smith Foundation for Health Research Scholar and CBR Member.

Production of erythropoietin and multiple cytokines by metanephric adenoma results in erythrocytosis

This is the first report of direct evidence that metanephric adenoma cells produce erythropoietin and other types of cytokines, which may be the cause of the high incidence of erythrocytosis in patients with this tumor. The purpose of the study was to establish a metanephric adenoma cell line in vitro from nephrectomized tumor tissue in order to investigate the ability of metanephric adenoma cells to produce erythropoietin and other types of cytokines. The tumor tissue was obtained from a 16-year-old boy who had developed metanephric adenoma with erythrocytosis and was served for cell culture. Significantly high concentrations of erythropoietin, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and IL-8 were detected in the cell culture supernatant. Southern hybridization showed specific positive signals for GM-CSF, G-CSF, IL-6, IL-8 and erythropoietin. The number of chromosomes was 46-XY without any structural abnormalities in cytogenetic analysis of the cultured cells.

Mouse embryonic stem cells that express a NUP98–HOXD13 fusion protein are impaired in their ability to differentiate and can be complemented by BCR-ABL

NUP98-HOXD13 (NHD13) fusions have been identified in patients with myelodysplastic syndrome, acute myelogenous leukemia and chronic myeloid leukemia blast crisis. We generated 'knock-in' mouse embryonic stem (ES) cells that express a NHD13 fusion gene from the endogenous murine NUP98 promoter, and used an in vitro differentiation system to differentiate the ES cells to hematopoietic colonies. Replating assays demonstrated that the partially differentiated NHD13 ES cells were immortal, and two of these cultures were transferred to liquid culture. These cell lines are partially differentiated immature hematopoietic cells, as determined by morphology, immunophenotype and gene expression profile. Despite these characteristics, they were unable to differentiate when exposed to high concentrations of erythropoietin (Epo), granulocyte colony-stimulating factor or macrophage colony-stimulating factor. The cell lines are incompletely transformed, as evidenced by their dependence on interleukin 3 (IL-3), and their failure to initiate tumors when injected into immunodeficient mice. We attempted genetic complementation of the NHD13 gene using IL-3 independence and tumorigenicity in immunodeficient mice as markers of transformation, and found that BCR-ABL successfully transformed the cell lines. These findings support the hypothesis that expression of a NHD13 fusion gene impairs hematopoietic differentiation, and that these cell lines present a model system to study the nature of this impaired differentiation.