Elevated Erythropoietin Levels Research Articles

Serum erythropoietin levels, breast cancer and breast cancer-initiating cells

BackgroundCancer is frequently associated with tumor-related anemia, and many chemotherapeutic agents impair hematopoiesis, leading to impaired quality of life for affected patients. The use of erythropoiesis-stimulating agents has come under scrutiny after prospective clinical trials using recombinant erythropoietin to correct anemia reported increased incidence of thromboembolic events and cancer-related deaths. Furthermore, previous preclinical reports indicated expansion of the pool of breast cancer-initiating cells when erythropoietin was combined with ionizing radiation.MethodsUsing four established breast cancer cell lines, we test the effects of recombinant human erythropoietin and the number of breast cancer-initiating cells in vitro and in vivo and study if recombinant human erythropoietin promotes the phenotype conversion of non-tumorigenic breast cancer cells into breast cancer-initiating cells. In a prospective study, we evaluate whether elevated endogenous serum erythropoietin levels correlate with increased numbers of tumor-initiating cells in a cohort of breast cancer patients who were scheduled to undergo radiation treatment.ResultsOur results indicate that recombinant erythropoietin increased the number of tumor-initiating cells in established breast cancer lines in vitro. Irradiation of breast cancer xenografts caused a phenotype conversion of non-stem breast cancer cells into induced breast cancer-initiating cells. This effect coincided with re-expression of the pluripotency factors c-Myc, Sox2, and Oct4 and was enhanced by recombinant erythropoietin. Hemoglobin levels were inversely correlated with serum erythropoietin levels, and the latter were correlated with disease stage. However, tumor sections revealed a negative correlation between serum erythropoietin levels and the number of ALDH1A3-positive cells, a marker for breast cancer-initiating cells.ConclusionsWe conclude that physiologically slow-rising serum erythropoietin levels in response to tumor-related or chemotherapy-induced anemia, as opposed to large doses of recombinant erythropoietin, do not increase the pool of breast cancer-initiating cells.

Polyglobulia in patients with hemangioblastomas is related to tumor size but not to serum erythropoietin

BackgroundHemangioblastomas are associated with elevated hemoglobin (Hb) levels (polyglobulia), which is associated with a higher risk for cerebral stroke, cardiac infarction and pulmonary embolism. The pathomechanism of polyglobulia remains unclear and different theories have been postulated. Among those are elevated serum erythropoietin (EPO) levels caused by secretion of the tumor or associated tumor cyst.MethodsTo elucidate the pathomechanism, we systematically investigated the relation between polyglobulia, serum EPO level, size of the solid tumor and associated cyst in hemangioblastomas. We prospectively evaluated hemoglobin and EPO levels in a series of 33 consecutive patients operated on hemangioblastomas in our center. We measured the size of the solid tumor and associated cyst in magnetic resonance imaging. Statistical evaluations were performed using the Fisher’s exact test and student’s t-test.ResultsAs a result five patients had elevated hemoglobin levels. Only one of these had an elevated serum EPO level. Of 26 patients with normal hemoglobin levels, 4 patients had elevated EPO levels.Patients with low or normal hemoglobin levels (84%) had an average tumor size of 0.8 cm3, which differed significantly from patients with elevated hemoglobin levels (16%), who had an average solid tumor size of 8.0 cm3 (p < 0.05). We did not observe a significant correlation between EPO levels or polyglobulia and associated cysts.ConclusionsWe therefore conclude that in contrast to previous case reports and interpretations, our data show no correlation between polyglobulia and EPO levels or associated cysts in patients with hemangioblastomas. In fact, it is the size of the solid tumor that correlates with polyglobulia.The study was retrospectively registered in the German Clinical Trial Registry on 10 July 2014; Trial registration: DRKS00006310.

Erythroferrone is not required for the glucoregulatory and hematologic effects of chronic erythropoietin treatment in mice.

Erythropoietin (EPO) acts on erythroid progenitor cells to promote their survival and differentiation to mature erythrocytes. Along with this canonical role, EPO is also reported to modulate energy metabolism, resulting in improved glucose tolerance and insulin sensitivity. EPO also stimulates the production of the hormone erythroferrone (ERFE) which acts to suppress hepcidin production, thus increasing dietary iron absorption and mobilizing stored iron for use in erythropoiesis. ERFE (initially termed myonectin) was also reported have an effect on systemic lipid metabolism by promoting the clearance of nonesterifed fatty acids (NEFA) from circulation. As increased levels of circulating NEFA blunt insulin sensitivity and impair glucose tolerance, ERFE‐induced clearance of NEFA after EPO administration would have a beneficial effect on glucose metabolism. The aim of this study was to determine if the known metabolic effect of EPO treatment on glucose homeostasis is mediated by ERFE, produced in response to EPO. Mice lacking Erfe did not differ from wild‐type mice in blood lipid parameters, blood glucose, and glucose or insulin tolerance at baseline or after chronic EPO treatment. Additionally, hepcidin suppression and the response of erythrocyte parameters to chronic EPO treatment were unaffected by the absence of Erfe. These findings suggest that the known beneficial effects of EPO on glucose metabolism are not attributable to an accompanying increase in ERFE production, and that Erfe is dispensable for normal glucose homeostasis. Furthermore, our data indicate that ERFE‐independent mechanisms can suppress hepcidin in response to chronically elevated EPO levels.

The Metabolic Effect of Chronic Elevated Erythropoietin in Tg6 Mice

Erythropoietin (EPO) is the cytokine required for erythrocyte production. However, EPO receptor (EpoR) expression is not restricted to erythroid tissue and non-hematopoietic EPO action has been associated with the regulation of glucose homeostasis and fat mass accumulation in animal models. The hemizygous transgenic tg6 mouse (PDGF- promoter/human EPO cDNA) overexpresses EPO, allowing for the study of chronic elevated levels of EPO. Tg6 mice exhibit a hematocrit of 80-90%, almost twice that of their wild type (wt) littermates. The elevated EPO also alters their glucose metabolism. In both males and females, tg6 mice exhibit significantly lower fasting glucose and improved glucose tolerance with significantly reduced blood glucose levels during a 2-hour glucose tolerance test (GTT). EPO treatment in male wt mice reduces fat mass and body weight compared to saline treatment. Similarly, tg6 mice with chronic elevated EPO have been reported to exhibit reduced body mass compared with control mice. We find that male tg6 mice tend to have reduced body weight, becoming significant with age and due to an associated decrease in fat mass, and a trend toward reduced food intake. In contrast, while female tg6 mice show significant decreases in fat mass, their total body weight is similar to control mice due in part to an increase in lean body mass. These data suggest that metabolically, tg6 mice show improved glucose metabolism compared to control mice associated with a decrease fat store in both male and female mice, and with a sex-specific reduction in body weight in male mice. In addition, the increase in lean body mass in female mice counters the weight loss associated with decrease fat mass resulting in no apparent change in body weight. Disclosure H. Rogers: None. O. Gavrilova: None. C.T. Noguchi: None.

A Gain-of-Function Mutation in EPO in Familial Erythrocytosis

SummaryFamilial erythrocytosis with elevated erythropoietin levels is frequently caused by mutations in genes that regulate oxygen-dependent transcription of the gene encoding erythropoietin (EPO). We identified a mutation in EPO that cosegregated with disease with a logarithm of the odds (LOD) score of 3.3 in a family with autosomal dominant erythrocytosis. This mutation, a single-nucleotide deletion (c.32delG), introduces a frameshift in exon 2 that interrupts translation of the main EPO messenger RNA (mRNA) transcript but initiates excess production of erythropoietin from what is normally a noncoding EPO mRNA transcribed from an alternative promoter located in intron 1. (Funded by the Gebert Rüf Foundation and others.)

Active Smoking and Hematocrit and Fasting Circulating Erythropoietin Concentrations in the General Population

Cigarette smoking continues to be one of the major risk factors for increased morbidity and mortality worldwide. Among many adverse health effects, smoking can induce erythrocytosis, which is commonly believed to result from elevated serum erythropoietin (EPO) levels. Currently, however, this notion is only alleged, without data available to substantiate it. Hence, we analyzed data from the Prevention of Renal and Vascular End-Stage Disease study, a prospective population-based cohort study. Smoking behavior was quantified as number of cigarettes smoked per day and as 24-hour urinary cotinine excretion levels, an objective and quantitative measure of nicotine exposure. In 6808 community-dwelling participants, the prevalence of nonsmokers, former smokers, and current smokers were 29%, 43%, and 28%, respectively. Hematocrit levels were higher in current smokers (41.4%±3.6%) than in nonsmokers (40.3%±3.6%) (P<.001). In contrast, median EPO levels were lower in current smokers (7.5 IU/L; interquartile range [IQR], 5.7-9.6 IU/L) than in nonsmokers (7.9 IU/L; IQR, 6.0-10.7 IU/L) (P<.001). In multivariate linear regression analysis, current smoking, compared with nonsmoking, was independently positively associated with hematocrit levels (β=.12; P<.001) and hemoglobin levels (β=.11; P<.001), but inversely associated with EPO levels (β=−.09; P<.001). In sensitivity analyses, we observed a dose-dependent inverse association of smoking exposure reflected by 24-hour urinary cotinine excretion levels with EPO levels. Contrary to common belief, we identified that in the general population, smoking is inversely associated with EPO levels. Future mechanistic insight is needed to unravel the currently identified association, and if reproduced in other studies, guidelines for diagnosis of secondary erythrocytosis may need to be revisited.

Translational repression of HIF2α expression in mice with Chuvash polycythemia reverses polycythemia.

Chuvash polycythemia is an inherited disease caused by a homozygous germline VHLR200W mutation, which leads to impaired degradation of HIF2α, elevated levels of serum erythropoietin, and erythrocytosis/polycythemia. This phenotype is recapitulated by a mouse model bearing a homozygous VhlR200W mutation. We previously showed that iron-regulatory protein 1-knockout (Irp1-knockout) mice developed erythrocytosis/polycythemia through translational derepression of Hif2α, suggesting that IRP1 could be a therapeutic target to treat Chuvash polycythemia. Here, we fed VhlR200W mice supplemented with Tempol, a small, stable nitroxide molecule and observed that Tempol decreased erythropoietin production, corrected splenomegaly, normalized hematocrit levels, and increased the lifespans of these mice. We attribute the reversal of erythrocytosis/polycythemia to translational repression of Hif2α expression by Tempol-mediated increases in the IRE-binding activity of Irp1, as reversal of polycythemia was abrogated in VhlR200W mice in which Irp1 was genetically ablated. Thus, a new approach to the treatment of patients with Chuvash polycythemia may include dietary supplementation of Tempol, which decreased Hif2α expression and markedly reduced life-threatening erythrocytosis/polycythemia in the VhlR200W mice.

Umbilical cord blood Erythropoietin as a biomarker of subacute and chronic hypoxia in deliveries complicated by meconium stained amniotic fluid

Background: The presence of meconium stained amniotic fluid (MSAF) during labour is often associated with a lot of adverse outcomes and historically has been considered to be a predictor of bad neonatal outcome. It is associated with fetal hypoxia and leads to emergency C/S, instrumental vaginal delivery for fetal distress, low Apgar score at birth, meconium aspiration syndrome and neuro-developmental handicaps. Umbilical cord serum erythropoietin levels have been claimed to be elevated in response to fetal hypoxia. Objective: To evaluate the umbilical cord serum erythropoietin levels in neonates born through MSAF and to determine whether it is influenced by gestational age. Study Design: Prospective case control study. Materials & Methods: Women with term uncomplicated singlet on pregnancies at gestational age between (37-41 +6 ) weeks admitted to the labour room in the active phase of labour were enrolled to participate in this study. The studied population has been divided into two groups, women with thick meconium stained amniotic fluid (study group) and women with clear amniotic fluid (control group). Umbilical cord serum Erythropoietin levels will be measured for both groups by ELISA, after delivery and Apgar score will be assessed for neonates of both groups then statistical analysis will be performed. Results: A significant relationship was found between MSAF and elevated umbilical cord serum erythropoietin levels since the p value was P < 0.001. Apgar score was lower significantly after 1 minute in study group, but lower none significantly after 5 minutes, as P <0.001and 0.109 respectively. Conclusions: The elevated umbilical cord serum Erythropoietin levels in neonates born through MSAF suggest subacute or chronic fetal Hypoxia, independent of gestational age.

Daily propranolol administration reduces persistent injury-associated anemia after severe trauma and chronic stress.

After severe trauma, patients develop a norepinephrine-mediated persistent, injury-associated anemia. This anemia is associated with suppression of bone marrow (BM) erythroid colony growth, along with decreased iron levels, and elevated erythropoietin (EPO) levels, which are insufficient to promote effective erythropoiesis. The impact of norepinephrine on iron regulators, such as ferroportin, transferrin, and transferrin receptor-1 (TFR-1), is unknown. Using a clinically relevant rodent model of lung contusion (LC), hemorrhagic shock (HS), and chronic stress (CS), we hypothesize that daily propranolol (BB), a nonselective β blocker, restores BM function and improves iron homeostasis. Male Sprague-Dawley rats were subjected to LCHS ± BB and LCHS/CS ± BB. BB was achieved with propranolol (10 mg/kg) daily until the day of sacrifice. Hemoglobin, plasma EPO, plasma hepcidin, BM cellularity and BM erythroid colony growth were assessed. RNA was isolated to measure transferrin, TFR-1 and ferroportin expression. Data are presented as mean ± SD; *p < 0.05 versus untreated counterpart by t test. The addition of CS to LCHS leads to persistent anemia on posttrauma day 7, while the addition of BB improved hemoglobin levels (LCHS/CS: 10.6 ± 0.8 vs. LCHS/CS + BB: 13.9 ± 0.4* g/dL). Daily BB use after LCHS/CS improved BM cellularity, colony-forming units granulocyte, erythrocyte, monocyte megakaryocyte, burst-forming unit erythroid and colony-forming unit erythroid cell colony growth. LCHS/CS + BB significantly reduced plasma EPO levels and increased plasma hepcidin levels on day 7. The addition of CS to LCHS resulted in decreased liver ferroportin expression as well as decreased BM transferrin and TFR-1 expression, thus, blocking iron supply to erythroid cells. However, daily BB after LCHS/CS improved expression of all iron regulators. Daily propranolol administration after LCHS/CS restored BM function and improved anemia after severe trauma. In addition, iron regulators are significantly reduced after LCHS/CS, which may contribute to iron restriction after injury. However, daily propranolol administration after LCHS/CS improved iron homeostasis.

Low level arsenite exposures suppress the development of bone marrow erythroid progenitors and result in anemia in adult male mice

Epidemiological studies report an association between chronic arsenic (As) exposure and anemia in men, and women who are predisposed to anemia. The purpose of these studies was to determine whether a 60 d drinking water exposure of adult male C57BL/6J mice to 0, 100, and 500ppb arsenite (As+3) results in anemia due to alterations in erythroid progenitor cell development in the bone marrow. Exposure to 500ppb As+3 for 60 d resulted in a reduction of mean corpuscular hemoglobin (MCH) levels, but did not significantly alter red blood cell (RBC) counts, hemoglobin (Hgb) levels, mean corpuscular Hgb concentrations (MCHC), or mean corpuscular volumes (MCV). Attenuation of burst-forming unit-erythroid (BFU-E) colony formation was observed in bone marrow cells of mice exposed to 500ppb As+3. The differentiation of late-stage bone marrow erythroblasts as defined by CD71 and Ter119 surface marker expression was reduced with the 500ppb As+3 exposure. Mice exposed to 500ppb As+3 also had elevated serum levels of erythropoietin (EPO). Collectively, these results show that exposure to low levels of As+3 attenuate the development of early BFU-E cells and reduce the differentiation of late-stage erythroblasts. This suppression of bone marrow erythropoiesis may be a contributing factor to the mild hypochromic anemia observed in 500ppb As+3 exposed mice.

Direct Current Helium Plasma for In vivo Delivery of Plasmid DNA Encoding Erythropoietin to Murine Skin.

The use of electric fields in vivo to deliver DNA, called electroporation, has the potential to broadly impact vaccination and disease treatment. The evidence for this has emerged from a large number of recently completed and ongoing clinical trials. The methods for applying electric fields to tissues traditionally involve contact between metal electrodes and the tissue. In this study, we investigated the use of helium plasma as a noncontact method for electrically treating tissue in a manner that results in the uptake and expression of foreign DNA in murine skin. More specifically, our goal was to demonstrate that DNA encoding a model-secreted protein could be delivered, detected in the blood, and remain functional to produce its known biological effect. Murine erythropoietin (EPO) was the model-secreted protein. Results clearly demonstrated that an intradermal DNA injection followed by plasma treatment for 2 min resulted in elevated levels of EPO in the blood and corresponding hemoglobin increases that were statistically significant relative to DNA injection alone.

Erythroferrone Regulates Hepcidin Expression Independently of Matriptase 2

Introduction: The iron-regulatory hormone hepcidin regulates the body iron stores and its expression is repressed when erythropoietic activity intensifies to meet the iron requirements for erythropoiesis (e.g. during anemia). Under the influence of erythropoietin (EPO), the hormone erythroferrone (ERFE) is secreted by erythroid precursors in the bone marrow and the spleen, and suppresses hepcidin synthesis to facilitate the recovery from anemia. However, the mechanism by which ERFE suppresses hepcidin is unknown. In contrast with forms of anemia in which hepcidin is suppressed, patients with mutations in transmembrane serine protease 6 (TMPRSS6) have iron-refractory iron deficiency anemia (IRIDA) but increased hepcidin production despite a severe anemia and elevated EPO levels. Recently, it has been suggested that matriptase-2 activity facilitates ERFE-mediated suppression of hepcidin. We therefore investigated the potential crosstalk between ERFE and Matriptase 2.Methods: We first measured serum ERFE concentration in Tmprss6-/- mice. To assess the contribution of ERFE to the phenotype of Tmprss6-/-mice, we next generated Tmprss6-/- mice with disrupted Erfe (Erfe+/- Tmprss6-/-; Erfe-/- Tmprss6-/- and Erfe+/+ Tmprss6-/-). To determine whether ERFE requires TMPRSS6 to regulate hepcidin production, we treated freshly isolated hepatocytes from wild-type (WT) or Tmprss6-/- mice with conditioned medium from cells expressing recombinant ERFE or not.Results: While wild-type mice have undetectable plasma ERFE (below the 500 pg/ml limit of detection), plasma ERFE concentration was elevated in Tmprss6-/- to levels comparable to those of WT animals 24 hours after phlebotomy (~3 ng/ml) but was lower than ERFE levels in thalassemic mice (~10 ng/ml). Ablation of Erfe in Tmprss6-/- mice did not result in any change in hematological parameters, hepcidin expression and iron levels compared to Tmprss6-/- animals at 6 weeks of age. However, treatment of WT and Tmprss6-/-hepatocytes with ERFE resulted in a comparable suppression of hepcidin mRNA expression.Conclusion: Although matriptase-2 may dampen the BMP signaling under the influence of EPO, it is not part of the ERFE signaling pathway. DisclosuresGanz:Intrinsic Lifesciences: Other: shareholder and scientific advisor; Merganser Biotech: Other: shareholder and scientific advisor; Silarus therapeutics: Other: shareholder and scientific advisor; Keryx Biopharmaceuticals: Consultancy. Nemeth:Intrinsic Lifesciences: Other: shareholder and scientific advisor; Merganser Biotech: Other: shareholder and scientific advisor; Silarus therapeutics: Other: shareholder and scientific advisor.

Heme-Regulated eIF2α Kinase Coordinates Translational Repression of eIF2αP and mTORC1 Signaling during Iron Deficiency to Mitigate Ineffective Erythropoiesis

Iron deficiency anemia (IDA) is a prevalent disease, yet molecular mechanisms by which iron and heme regulate erythropoiesis are not well understood. Heme-regulated eIF2α kinase (HRI) is a hemoprotein highly expressed in erythroid precursors. HRI senses intracellular heme concentrations to ensure that globin synthesis does not exceed the amount of heme available for hemoglobin production. In heme deficiency, HRI is activated and phosphorylates eIF2α, which inhibits the translation of globin mRNAs as well as enhances selectively the translation of activating transcription factor 4 (ATF4) mRNA to induce transcription of stress response genes. Previously, we reported that HRI is also necessary to promote erythroid differentiation during iron deficiency (ID). To further interrogate the molecular mechanism of HRI-eIF2αP-ATF4 signaling in erythropoiesis, we generated a novel mouse model (eAA mice) that lacks of eIF2α phosphorylation (eIF2αP) by substituting serine 51 residue with alanine specifically in the erythroid lineage. eAA mice did not display significant erythroid phenotypes during normal adult steady-state erythropoiesis. However, eAA mice developed ineffective erythropoiesis with splenomegaly and macrocytic hyperchromic anemia in ID, similar to Hri-/- mice. In contrast, iron deficient Atf4-/- mice developed more severe anemia, and were microcytic hypochromic. Thus, regulation of cell size and hemoglobin content of RBCs by HRI is mediated through eIF2αP and not ATF4. Consistent with the regulation of globin translation by eIF2αP, globin inclusions were present in reticulocytes of eAA mice, but not Atf4-/- mice. Both eAA and Atf4-/- mice exhibited inhibition of terminal erythroid differentiation starting at basophilic erythroblasts in ID. The ROS levels in RBCs and reticulocytes were elevated, demonstrating that HRI-eIF2αP-ATF4 signaling contributes significantly to mitigation of oxidative stress in late stage erythroid cells. Our results demonstrate for the first time that both eIF2αP and ATF4 are necessary for iron-restricted erythropoiesis.mTORC1 (mechanistic target of rapamycin complex 1) signaling in blood cells was recently reported to be down regulated in ID. Furthermore, constitutively activated mTORC1 signaling in the hematopoietic lineage results in macrocytic hyperchromic anemia with splenomegaly (similar to our ID Hri-/- and eAA mice) whereas inactivation of mTORC1 signaling results in microcytic hyperchromic anemia, characteristics of IDA in Wt mice (Knight et al. eLife 2014). We therefore investigated whether HRI stress signaling was necessary to down-regulate mTORC1 in ID. mTORC1 signaling increases protein synthesis by phosphorylating eIF4E binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (S6K), which phosphorylates S6. We found that mTORC1 signaling activities as measured by pS6 and p4E-BP1, were higher in iron deficient Hri-/-, eAA and Atf4-/-erythroid precursors in comparison to Wt cells. Treatment of iron deficient Hri-/-, eAA and Atf4-/-mice with rapamycin, an mTORC1 inhibitor, increased RBC counts and hemoglobin content in the blood, improved erythroid differentiation and reduced splenomegaly significantly. However, globin inclusions remained visible in Hri-/- and eAA reticulocytes, indicating a non-redundant role of HRI-eIF2αP in inhibiting globin translation. Dietary iron repletion of the iron deficient mice rapidly and completely reversed the anemia in 10 days by promoting erythroid differentiation and decreasing HRI and mTORC1 signaling in Hri-/-, eAA and Atf4-/-mice. Furthermore, the elevated serum erythropoietin (EPO) levels in our ID anemic mice were decreased significantly upon rapamycin treatment or iron repletion. Together, our results demonstrate that HRI is indispensible for both the activation of the eIF2αP-ATF4 signaling and the repression of mTORC1 signaling during ID to circumvent ineffective erythropoiesis and macrocytic anemia. Our findings also indicate that HRI stress signaling could serves as a feedback mechanism for EPO signaling by inhibiting mTORC1. This study provides molecular insights into the role of heme and translation in the regulation of erythropoiesis and may provide novel targets for the treatment of hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

Salmonella Infection Enhances Erythropoietin Production by the Kidney and Liver, Which Correlates with Elevated Bacterial Burdens.

Salmonella infection profoundly affects host erythroid development, but the mechanisms responsible for this effect remain poorly understood. We monitored the impact of Salmonella infection on erythroid development and found that systemic infection induced anemia, splenomegaly, elevated erythropoietin (EPO) levels, and extramedullary erythropoiesis in a process independent of Salmonella pathogenicity island 2 (SPI2) or flagellin. The circulating EPO level was also constitutively higher in mice lacking the expression of signal-regulatory protein α (SIRPα). The expression level of EPO mRNA was elevated in the kidney and liver but not increased in the spleens of infected mice despite the presence of extramedullary erythropoiesis in this tissue. In contrast to data from a previous report, mice lacking EPO receptor (EPOR) expression on nonerythroid cells (EPOR rescued) had bacterial loads similar to those of wild-type mice following Salmonella infection. Indeed, treatment to reduce splenic erythroblasts and mature red blood cells correlated with elevated bacterial burdens, implying that extramedullary erythropoiesis benefits the host. Together, these findings emphasize the profound effect of Salmonella infection on erythroid development and suggest that the modulation of erythroid development has both positive and negative consequences for host immunity.

Prognostic significance of endogenous erythropoietin in long-term outcome of patients with acute decompensated heart failure.

Although previous reports suggest that an elevated endogenous erythropoietin (EPO) level is associated with worse clinical outcomes in chronic heart failure (HF) patients, the prognostic implication of EPO in patients with acute decompensated HF (ADHF) and underlying mechanisms of the high EPO level in severe HF patients who have a poor prognosis remain unclear. We examined 539 consecutive ADHF patients with EPO measurement on admission from our registry. During a median follow-up period of 329 days, a higher EPO level on admission was independently associated with worse clinical outcomes [hazard ratio (HR) 1.25, 95% confidence interval (CI) 1.06-1.48, P = 0.008], and haemoglobin level was the strongest determinant of EPO level (P < 0.001), whereas estimated glomerular filtration rate (eGFR) was not significant in multivariate regression analysis. In the anaemic subgroup of 318 patients, a higher EPO level than expected on the basis of their haemoglobin level was related to increased adverse events (HR 1.63, 95% CI 1.05-2.49, P = 0.028). Moreover, estimated plasma volume excess rate was positively associated with EPO level (P = 0.003), and anaemic patients with a higher than expected EPO level tended to have a higher estimated plasma volume excess rate and plasma lactate level, and lower systemic oxygen saturation level with the preservation of the reticulocyte production index than those with a lower than expected EPO level. A high EPO level predicts long-term worse clinical outcomes in ADHF patients, independent of anaemia and impaired renal function. Anaemia and hypoxia due to severe congestion may synergistically contribute to a high EPO level in high-risk HF patients.

Chronic restraint stress after injury and shock is associated with persistent anemia despite prolonged elevation in erythropoietin levels

Following severe traumatic injury, critically ill patients have a prolonged hypercatacholamine state that is associated with bone marrow (BM) dysfunction and persistent anemia. However, current animal models of injury and shock result in a transient anemia. Daily restraint stress (chronic stress [CS]) has been shown to increase catecholamines. We hypothesize that adding CS following injury or injury and shock in rats will prolong the hypercatecholaminemia and prolong the initial anemia, despite elevated erythropoietin (EPO) levels. Male Sprague-Dawley rats (n = 6-8 per group) underwent lung contusion (LC) or combined LC/hemorrhagic shock (LCHS) followed by 6 days of CS. CS consisted of a 2-hour restraint period interrupted with repositioning and alarms every 30 minutes. At 7 days, urine was assessed for norepinephrine (NE) levels, blood for EPO and hemoglobin (Hgb), and BM for erythroid progenitor growth. Animals undergoing LC or combined LCHS predictably recovered by Day 7; urine NE, EPO, and Hgb levels were normal. The addition of CS to LC and LCHS models was associated with a significant elevation in NE on Day 6. The addition of CS to LC led to a persistent 20% to 25% decrease in the growth of BM hematopoietic progenitor cells. These findings were further exaggerated when CS was added following LCHS, resulting in a 20%q to 40% reduction in BM erythroid progenitor colony growth and a 20% decrease in Hgb when compared with LCHS alone. Exposing injured animals to CS results in prolonged elevation of NE and EPO, which is associated with worsening BM erythroid function and persistent anemia. Chronic restraint stress following injury and shock provides a clinically relevant model to further evaluate persistent injury-associated anemia seen in critically ill trauma patients. Furthermore, alleviating CS after severe injury is a potential therapeutic target to improve BM dysfunction and anemia.

Pyruvate Is Superior to Citrate in Oral Rehydration Solution in the Protection of Intestine via Hypoxia-Inducible Factor-1 Activation in Rats With Burn Injury.

Recent studies have suggested that pyruvate-enriched oral rehydration solution (Pyr-ORS) may be superior to the standard bicarbonate-based ORS in the protection of intestine from ischemic injury. The aim of this study was to compare the effects of Pyr-ORS with citrate-enriched ORS (Cit-ORS) on the intestinal hypoxia-inducible factor-1 (HIF-1)-erythropoietin (EPO) signaling pathway for enteral rehydration in a rat model of burn injury. Rats were randomly assigned to 4 groups (N = 20, 2 subgroups each: n = 10): scald sham (group SS), scald with no fluid resuscitation (group SN), scald and resuscitation with enteral Cit-ORS (group SC), and scald and resuscitation with enteral Pyr-ORS (group SP). At 2.5 and 4.5 hours after a 35% total body surface area (TBSA) scald, intestinal mucosal blood flow (IMBF), contents of HIF-1, EPO, endothelial nitric oxide synthase (eNOS), nitric oxide (NO), barrier protein (ZO-1), levels of serum diamine oxidase (DAO), and intestinal mucosal histology injury score were determined. Serum DAO activities in the scalded groups were significantly elevated, but less raised in group SP than in group SC, at 2.5 hours and at 4.5 hours after the scald. Further, group SP more profoundly preserved intestinal HIF-1 expression compared with group SC at the 2 time points. Compared with group SC, group SP had markedly elevated intestinal EPO, eNOS, and NO levels at the same time points, respectively (P < .05). Similarly, IMBF and ZO-1 levels were significantly higher in group SP than in group SC. Intestinal mucosal histopathological scores were statistically higher at 2.5 hours and 4.5 hours after scalding but were more attenuated in group SP than in group SC (P < .05). Immunofluorescence expression of intestinal mucosal ZO-1 was consistent with the above changes. The above parameters were also significantly different between groups SC and SN (all P < .05). Pyr-ORS provides a superior option to Cit-ORS for the preservation of intestinal blood flow and barrier function and the attenuation of histopathological alterations in enteral resuscitation of rats with burn injury. Its underlying mechanism may be closely related to the pyruvate in activation of intestinal HIF-1-EPO signaling cascades.

Bone marrow findings of the newly described TEMPI syndrome: when erythrocytosis and plasma cell dyscrasia coexist

TEMPI syndrome (telangiectasias, elevated erythropoietin level and erythrocytosis, monoclonal gammopathy, perinephric fluid collections, and intrapulmonary shunting) is a recently described syndrome that, owing to erythrocytosis, may be confused with polycythemia vera. It is best classified as a type of plasma cell dyscrasia with paraneoplastic manifestations, similar to POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin abnormalities). To date, 11 patients have been identified. This is the first morphologic review of TEMPI syndrome bone marrow samples, in order to define pathologic features that may aid in the recognition of the syndrome and to identify post-therapy changes. Seven bone marrow aspirates and biopsies from three patients, including two post-treatment marrows, were examined. Patients were 36, 49, and 49 years old at time of diagnosis. In all cases, erythropoietin levels were extremely elevated at >5000 IU/l, the paraprotein was IgG kappa, JAK2 V617F was negative and vascular endothelial growth factor levels were normal. In one case, the increase in clonal plasma cells reached levels of smoldering myeloma (18%), but remaining marrows showed few monoclonal plasma cells (<5%). All pre-treatment biopsies showed erythroid hyperplasia, with mild nonspecific megakaryocytic, and erythroid cytologic atypia in one marrow. Prominent plasma cell vacuolization and reactive-appearing lymphoid aggregates were noted in one case. Findings of myeloproliferative neoplasms, including megakaryocyte clusters and fibrosis, were not identified. In conclusion, TEMPI syndrome should be considered when erythrocytosis and plasma cell dyscrasia coexist. The bone marrow findings, although nonspecific, differ significantly from polycythemia vera. Peculiar clinical and laboratorial findings of TEMPI, including elevated erythropoietin and normal vascular endothelial growth factor level, allow the diagnosis and distinction from POEMS syndrome. Significant decrease in erythropoietin level following treatment suggests a role of erythropoietin in monitoring therapeutic response.

Erythropoietin directly stimulates osteoclast precursors and induces bone loss.

Erythropoietin (EPO) primarily regulates red blood cell formation, and EPO serum levels are increased on hypoxic stress (e.g., anemia and altitude). In addition to anemia, recent discoveries suggest new therapeutic indications for EPO, unrelated to erythropoiesis. We investigated the skeletal role of EPO using several models of overexpression (Tg6 mice) and EPO administration (intermittent/continuous, high/low doses) in adult C57Bl6 female mice. Using microcomputed tomography, histology, and serum markers, we found that EPO induced a 32%-61% trabecular bone loss caused by increased bone resorption (+60%-88% osteoclast number) and reduced bone formation rate (-19 to -74%; P < 0.05 throughout). EPO targeted the monocytic lineage by increasing the number of bone monocytes/macrophages, preosteoclasts, and mature osteoclasts. In contrast to the attenuated bone formation in vivo, EPO treatment in vitro did not inhibit osteoblast differentiation and activity, suggesting an indirect effect of EPO on osteoblasts. However, EPO had a direct effect on preosteoclasts by stimulating osteoclastogenesis in isolated cultures (+60%) via the Jak2 and PI3K pathways. In summary, our findings demonstrate that EPO negatively regulates bone mass and thus bears significant clinical implications for the potential management of patients with endogenously or therapeutically elevated EPO levels.

Elevated Serum Levels of Erythropoietin after Xenon Anesthesia in Cardiac Surgery: A Secondary Analysis of a Randomized Controlled Trial

Objectives: The noble gas xenon has recently been amended to the list of prohibited substances because it is supposed to trigger the production of HIF-1α and subsequently erythropoietin. While any performance-enhancing treatments are strictly prohibited in sport competitions researcher and clinician have opened a debate about the potential transfer of these promising findings into clinical context. Methods: The objective of this study was to evaluate the effect of xenon anesthesia on serum levels of erythropoietin in patients that underwent cardiac surgery. Therefore we performed a secondary analysis of serum samples and dataset of patients from a prospective randomized controlled trial in cardiac surgery. Serum levels of erythropoietin and testosterone were measured in serum samples that were collected prior to surgery, at admission to the ICU and 24 hours thereafter. In 30 patients, we evaluated the effect of xenon anesthesia on the perioperative serum levels of erythropoietin and its association with testosterone and postoperative hemoglobin levels. Results: Patients in the xenon group showed a significant increase of circulating serum levels of erythropoietin 24 hours after xenon anesthesia (12.9 ± 7.4 versus 8.7 ± 4.3 at baseline; p = 0.017) The testosterone levels were comparable between the xenon and sevoflurane group. The postoperative hemoglobin levels trended to be higher in the xenon group, whereas no difference was found with respect to the intraoperative administration of fluids or blood products (Fig. 1). Conclusion: This study provides first evidence for a xenon triggered effect on circulating serum levels of erythropoietin during cardiac surgery. Further validation in a larger trial is needed.