Circulating Erythropoietin Levels Research Articles

Defining ketone supplementation: the evolving evidence for postexercise ketone supplementation to improve recovery and adaptation to exercise.

Over the last decade, there has been a growing interest in the use of ketone supplements to improve athletic performance. These ketone supplements transiently elevate the concentrations of the ketone bodies acetoacetate (AcAc) and d-β-hydroxybutyrate (βHB) in the circulation. Early studies showed that ketone bodies can improve energetic efficiency in striated muscle compared with glucose oxidation and induce a glycogen-sparing effect during exercise. As such, most research has focused on the potential of ketone supplementation to improve athletic performance via ingestion of ketones immediately before or during exercise. However, subsequent studies generally observed no performance improvement, and particularly not under conditions that are relevant for most athletes. However, more and more studies are reporting beneficial effects when ketones are ingested after exercise. As such, the real potential of ketone supplementation may rather be in their ability to enhance postexercise recovery and training adaptations. For instance, recent studies observed that postexercise ketone supplementation (PEKS) blunts the development of overtraining symptoms, and improves sleep, muscle anabolic signaling, circulating erythropoietin levels, and skeletal muscle angiogenesis. In this review, we provide an overview of the current state-of-the-art about the impact of PEKS on aspects of exercise recovery and training adaptation, which is not only relevant for athletes but also in multiple clinical conditions. In addition, we highlight the underlying mechanisms by which PEKS may improve exercise recovery and training adaptation. This includes epigenetic effects, signaling via receptors, modulation of neurotransmitters, energy metabolism, and oxidative and anti-inflammatory pathways.

EPOtential target for endurance performance: the effect of exogenous ketone supplementation on circulating erythropoietin levels.

EPOtential target for endurance performance: the effect of exogenous ketone supplementation on circulating erythropoietin levels.

Administration of α-Klotho Does Not Rescue Renal Anemia in Mice.

Renal anemia is a common complication in chronic kidney disease (CKD), associated with decreased production of erythropoietin (EPO) due to loss of kidney function, and subsequent decreased red blood cell (RBC) production. However, many other factors play a critical role in the development of renal anemia, such as iron deficiency, inflammation, and elevated fibroblast growth factor 23 (FGF23) levels. We previously reported that inhibition of FGF23 signaling rescues anemia in mice with CKD. In the present study we sought to investigate whether α-Klotho deficiency present in CKD also contributes to the development of renal anemia. To address this, we administered α-Klotho to mice with CKD induced by an adenine-rich diet. Mice were sacrificed 24 h after α-Klotho injection, and blood and organs were collected immediately post-mortem. Our data show that α-Klotho administration had no beneficial effect in mice with CKD-associated anemia as it did not increase RBC numbers and hemoglobin levels, and it did not stimulate EPO secretion. Moreover, α-Klotho did not improve iron deficiency and inflammation in CKD as it had no effect on iron levels or inflammatory markers. Interestingly, Klotho supplementation significantly reduced the number of erythroid progenitors in the bone marrow and downregulated renal Epo and Hif2α mRNA in mice fed control diet resulting in reduced circulating EPO levels in these mice. In addition, Klotho significantly decreased intestinal absorption of iron in control mice leading to reduced serum iron and transferrin saturation levels. Our findings demonstrate that α-Klotho does not have a direct role in renal anemia and that FGF23 suppresses erythropoiesis in CKD via a Klotho-independent mechanism. However, in physiological conditions α-Klotho appears to have an inhibitory effect on erythropoiesis and iron regulation.

Activation of Sympathetic Nervous System Contributes to Erthroprotein Gene Upregulation by Hypobaric Hypoxia

Hypobaric hypoxia (HH) experienced at high altitude initiates a series of physiological adaptations to maintain homeostasis. One such adaptation is the activation of the Epo gene which encodes the glycoprotein hormone erythropoietin (EPO). Increased circulating EPO levels defend against HH by facilitating blood O2 transport through increasing red blood cell production. HH also increases blood pressure (BP) through activation of the sympathetic nervous system and the ensuing elevation of circulating catecholamine levels. The present study examined the role of sympathetic nerve activation in HH‐evoked Epo gene activation and elevated circulating levels of EPO. Studies were performed on adult, male wild‐type (WT) mice treated with 18 hours of HH (0.4 atmospheres), simulating low O2 condition experienced at high altitude. Our results showed increased renal Epo gene abundance and elevated plasma EPO levels. Remarkably, these responses were either markedly attenuated or absent in mice with selective ablation of adrenal medulla as well as after treatment with propranolol, a non‐selective beta‐adrenergic receptor blocker. Moreover, infusion of epinephrine alone activated renal Epo gene and elevated plasma EPO levels. These results suggest that sympathetic nerve activation and the ensuing increase in plasma epinephrine contribute to Epo gene activation by HH.

Exposure to high levels of oxygen in neonatal rats induce a decrease in hemoglobin levels.

Anemia of prematurity is common in extremely preterm neonates, and oxygen exposure may participate to anemia by inhibiting erythropoietin secretion. We aimed to determine whether hyperoxia exerts an independent role in the occurrence of the anemia of prematurity. Sprague-Dawley pups were exposed to 80% oxygen or room air from days 3 to 10 of life. Main outcome was the difference in hemoglobin and circulating erythropoietin levels in animals exposed to hyperoxia at 10 days of life. We performed a complete blood count analysis using fluorescent laser flow cytometry and measured circulating erythropoietin levels using ELISA. We found lower hemoglobin in the hyperoxia group, compared to the normoxia group, both in males (70 ± 3 versus 78 ± 2 g/l) and in females (71 ± 2 versus 81 ± 3 g/l) at 10 days of life. Reticulocyte count was not increased in the hyperoxia group. Circulating erythropoietin levels were lower at 10 days of life in the animals exposed to hyperoxia, both in males (33 ± 7 versus 73 ± 6 pg/ml) and in females (37 ± 5 versus 66 ± 3 pg/ml), but were similar at 28 days of life. Neonatal exposure to hyperoxia decreases hematopoiesis in rats. Mechanisms leading to anemia of prematurity are not well known and their study in humans is complicated due to multiple confounders. This study shows for the first time that exposure to high concentrations of oxygen in the neonatal period decreases hematopoiesis in rats, providing insight on the pathophysiological mechanisms of the anemia of prematurity. This research paves the way for future therapeutic developments aiming to reduce the burden of anemia of prematurity and the necessity of red blood cell transfusions in extremely preterm neonates.

Proteinuric chronic kidney disease is associated with altered red blood cell lifespan, deformability and metabolism

Anemia is a common complication of chronic kidney disease, affecting the quality of life of patients. Among various factors, such as iron and erythropoietin deficiency, reduced red blood cell (RBC) lifespan has been implicated in the pathogenesis of anemia. However, mechanistic data on invivo RBC dysfunction in kidney disease are lacking. Herein, we describe the development of chronic kidney disease-associated anemia in mice with proteinuric kidney disease resulting from either administration of doxorubicin or an inducible podocin deficiency. In both experimental models, anemia manifested at day 10 and progressed at day 30 despite increased circulating erythropoietin levels and erythropoiesis in the bone marrow and spleen. Circulating RBCs in both mouse models displayed altered morphology and diminished osmotic-sensitive deformability together with increased phosphatidylserine externalization on the outer plasma membrane, a hallmark of RBC death. Fluorescence-labelling of RBCs at day 20 of mice with doxorubicin-induced kidney disease revealed premature clearance from the circulation. Metabolomic analyses of RBCs from both mouse models demonstrated temporal changes in redox recycling pathways and Lands' cycle, a membrane lipid remodeling process. Anemic patients with proteinuric kidney disease had an increased proportion of circulating phosphatidylserine-positive RBCs. Thus, our observations suggest that reduced RBC lifespan, mediated by altered RBC metabolism, reduced RBC deformability, and enhanced cell death contribute to the development of anemia in proteinuric kidney disease.

Nonclinical Characterization of the Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Roxadustat, a Novel Treatment of Anemia of Chronic Kidney Disease.

Anemia of chronic kidney disease (CKD) is a multifactorial disorder caused by impaired erythropoietin (EPO) production and altered iron homeostasis associated with inflammation. Hypoxia-inducible factor (HIF) is a transcription factor that stimulates erythropoiesis via a coordinated response involving increased EPO production and enhanced iron availability for Hb synthesis. HIF degradation is regulated by HIF-prolyl hydroxylase (HIF-PH) enzymes. We hypothesized that roxadustat, an orally available small-molecule inhibitor of HIF-PH, would increase EPO production and promote erythropoiesis in animal models of anemia. In cells, roxadustat increased both HIF-1α and HIF-2α proteins, leading to an increase in EPO production, even in the presence of EPO-suppressing inflammatory cytokines. Roxadustat administered intermittently to healthy rats and cynomolgus monkeys increased circulating EPO levels, reticulocytes, blood Hb, and hematocrit in a dose-dependent manner. Roxadustat corrected anemia in a rat model of CKD after five-sixth nephrectomy and in a rat model of anemia of inflammation with impaired iron metabolism induced by peptidoglycan-polysaccharide (PG-PS). In the PG-PS model, roxadustat significantly decreased hepatic expression of hepcidin, a hormone responsible for iron sequestration and functional iron deficiency, and increased expression of two genes involved in duodenal iron absorption: divalent metal transporter 1 and duodenal cytochrome b. In conclusion, by activating the HIF pathway, roxadustat increased EPO production, elevated Hb, corrected anemia, and improved iron homeostasis. The coordinated erythropoietic response stimulated by roxadustat, involving both EPO production and mobilization of iron stores, makes this compound a promising treatment of anemia of CKD and anemia associated with functional iron deficiency. SIGNIFICANCE STATEMENT: Roxadustat is a novel orally available small-molecule inhibitor of HIF prolyl hydroxylase enzymes that reversibly stabilizes HIF-α, thus activating transcription of HIF-dependent genes, including EPO and regulators of iron homeostasis. Activation of the HIF pathway by roxadustat induces erythropoiesis in healthy rats and monkeys and corrects experimentally induced anemia in rats. The coordinated erythropoietic response that increases EPO production and mobilizes iron stores makes roxadustat a promising treatment for anemia of chronic kidney disease and anemia associated with functional iron deficiency.

Single Cell Resolution of Glucocorticoid Effects on Erythroid Progenitor Cells

To generate the billions of new erythrocytes required on a daily basis, erythroid progenitor cells must exponentially increase in number before undergoing terminal differentiation. A limited number of cell divisions occur during erythropoietin (EPO)-regulated erythroid terminal differentiation, but the principal regulation of erythroid transit-amplification occurs earlier in erythropoiesis between the burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E) stages of development. The importance of this EPO-independent early erythropoietic process is highlighted in Diamond-Blackfan Anemia (DBA). DBA is characterized by pure red cell aplasia, loss of BFU-E and CFU-E progenitors in the bone marrow, and severe anemia despite high circulating EPO levels. The only known effective medical therapy for DBA also provides insight into the regulation of erythroid transit-amplification. In patients responsive to glucocorticoid treatment, there are increased numbers of BFU-E and CFU-E progenitors in the bone marrow, and ex vivo culture studies indicate that the synthetic glucocorticoid dexamethasone (Dex) predominantly increases proliferative capacity of BFU-Es, with minimal effect on proliferative capacity of CFU-Es. These findings led to a prevailing model that glucocorticoids increase BFU-E proliferative capacity by stimulating several rounds of self-renewal cell divisions. However, a limitation of this model is that there is no mechanistic explanation for how BFU-Es regulate the fate choice of undergoing a self-renewal cell division versus a differentiation cell division in the presence or absence of glucocorticoids. In this study, we address this question by examining progression of early erythroid progenitor development at single cell resolution, and subsequently elucidate the true mechanistic nature of glucocorticoid-induced erythroid progenitor proliferative capacity amplification. By performing single cell transcriptome profiling (scRNAseq) of primary-isolated mouse fetal liver BFU-Es, CFU-Es, and their developmental intermediates, we identify a continuum of transcriptomic states during erythroid transit-amplification when performing principle component analysis (PCA) on transcriptomes of individual cells. We show that ex vivo culture of primary-isolated BFU-Es in serum free media supplemented with stem cell factor, insulin-like growth factor 1, and EPO results in developmental progression along the transcriptome continuum when performing scRNAseq and PCA on cultured BFU-Es. The addition of Dex into this culture system does not result in self-renewal of BFU-Es at the transcriptome level, but rather still results in developmental progression, albeit to less of a degree per cell division than BFU-Es cultured without Dex. We additionally show that the continuum of transcriptome states in erythroid transit-amplification is reflective of a continuum of functional states, with developmental progression characterized by decreasing proliferative capacity and decreasing glucocorticoid-responsiveness. Lastly, through manual separation of daughter cells resulting from a BFU-E cell division, we demonstrate that BFU-E cell division is a symmetric process at the transcriptome level, both with and without the addition of Dex. Our results clarify the nature of how glucocorticoids amplify BFU-E proliferative capacity. As opposed to stimulating a finite number of BFU-E self-renewal cell divisions, glucocorticoids decrease the extent of progression through the erythroid transit-amplifying developmental continuum per cell division. Thus, a decreased rate of progression through the developmental continuum is associated with an increased number of transit-amplifying cell divisions prior to terminal differentiation. These findings are important not only for the rational development of glucocorticoid-alternatives for treating DBA, but also for all bone marrow failure syndromes characterized by progenitor cell hypoplasia. DisclosuresNo relevant conflicts of interest to declare.

Off-tumor targets compromise antiangiogenic drug sensitivity by inducing kidney erythropoietin production

Anti-VEGF drugs are commonly used for treatment of a variety of cancers in human patients, and they often develop resistance. The mechanisms underlying anti-VEGF resistance in human cancer patients are largely unknown. Here, we show that in mouse tumor models and in human cancer patients, the anti-VEGF drug-induced kidney hypoxia augments circulating levels of erythropoietin (EPO). Gain-of-function studies show that EPO protects tumor vessels from anti-VEGF treatment and compromises its antitumor effects. Loss of function by blocking EPO function using a pharmacological approach markedly increases antitumor activity of anti-VEGF drugs through inhibition of tumor angiogenesis. Similarly, genetic loss-of-function data shows that deletion of EpoR in nonerythroid cells significantly increases antiangiogenic and antitumor effects of anti-VEGF therapy. Finally, in a relatively large cohort study, we show that treatment of human colorectal cancer patients with bevacizumab augments circulating EPO levels. These findings uncover a mechanism of desensitizing antiangiogenic and anticancer effects by kidney-produced EPO. Our work presents conceptual advances of our understanding of mechanisms underlying antiangiogenic drug resistance.

Polycythemia is associated with bone loss and reduced osteoblast activity in mice.

Increased fragility has been described in humans with polycythemia vera (PV). Herein, we describe an osteoporotic phenotype associated with decreased osteoblast activity in a mouse model of PV and another mouse of polycythemia and elevated circulating erythropoietin (EPO). Our results are important for patients with PV or those treated with recombinant EPO (rEPO). PV and other myeloproliferative syndromes have been recently associated with an increased risk for fractures. However, the presence of osteoporosis in these patients has not been well documented. EPO, a hormone primarily known to stimulate erythropoiesis, has been shown recently to regulate bone homeostasis in mice. The aim of this study was to examine the bone phenotype of a mouse model of PV and compare it to that of animals with polycythemia caused by elevated circulating EPO. Bone mass and remodeling were evaluated by micro-computed tomography and histomorphometry. The JAK2(V617F) knock-in mouse, a model of human PV, manifests polycythemia and low circulating EPO levels. Results from this mouse were compared to wild type (wt) controls and the tg6 transgenic mouse that shows polycythemia caused by increased constitutive expression of EPO. Compared to wt, both JAK2(V617F) and tg6 mice had a decrease in trabecular bone mass. Tg6 mice showed an additional modest decrease in cortical thickness and cortical bone volume per tissue volume (P < 0.01) suggesting a more severe bone phenotype than JAK2(V617F). Decreased osteoblast numbers and bone formation along with normal osteoclast numbers and activity were found in both mice. This study indicates that PV is associated with low bone mass and decreased osteoblast activity in mice. Our results support future studies of osteoporosis in affected humans. Polycythemia caused by chronically elevated circulating EPO also results in bone loss, and implications on patients treated with rEPO should be evaluated.

Correlation between erythropoietin serum levels and erythrocyte susceptibility to lipid peroxidation in elderly with type 2 diabetes.

Erythropoietin (EPO), a key hormone involved in red blood cell formation has been recently acknowledged for its pleiotropic actions and protective role in ageing and various pathological conditions concurrent with oxidative stress, vascular diseases and metabolic abnormalities such as diabetes mellitus. The aim of the study was to evaluate the relationship between circulating erythropoietin levels and oxidative stress biomarkers, in elderly with type 2 diabetes (T2DM). The study was carried out in 67 subjects with T2DM (69 ± 5 years; n = 37) without anemia, and aged-matched controls (70 ± 6 years; n = 30). EPO serum levels, erythrocyte susceptibility to lipid peroxidation (ESP) and total antioxidant capacity (TAC) were evaluated. Lower EPO levels (p < 0.01) and higher ESP values (p < 0.001) were found in T2DM group, compared to healthy subjects. EPO levels showed significant negative associations with ESP, both in T2DM subjects (r = -0.565; p < 0.001) and in all study population (r = -0,600; p < 0,001; n = 67). In conclusion, we provide new data regarding the cytoprotective effect of EPO exerted at systemic level on erythrocyte membrane, in the particular state of impaired glucose metabolism associated with oxidative stress, in the elderly.

Pharmacological Characterization of ZYAN1, a Novel Prolyl Hydroxylase Inhibitor for the Treatment of Anemia.

Prolyl hydroxylase (PHD) inhibitors stabilize hypoxia inducible factor (HIF), and exert antianemic effect by potentiating erythropoietin (EPO) expression and down-regulation of hepcidin. ZYAN1 is a novel PHD inhibitor under clinical development for the treatment of anemia. The pharmacodynamic effects of acute and chronic dosing of ZYAN1 were assessed in normal and 5/6 nephrectomized Wistar rats. The effect of ZYAN1 was also investigated in cisplatin-induced anemia using C57 mice. Acute treatment with ZYAN1 increased circulating EPO levels (10.3 ± 3.7 and 40.0 ± 8.5 fold rise at 15 and 30 mg/kg, respectively), reticulocyte count (4.2 ± 0.5 and 6.0 ± 0.2 fold rise at 15 and 30 mg/kg, respectively) and stabilized HIF (28% increase at 45 mg/kg) in normal rats. Nephrectomized rats showed similar dose-related pharmacodynamic effects. In a 28-day study in nephrectomized rats, ZYAN1 administered every alternate day, caused increase in hemoglobin (1.9 ± 0.3 and 2.5 ± 0.4 g/dL) and RBC count (10.7 ± 4.0 and 14.0 ± 4.1%) at 15 and 30 mg/kg respectively. In cisplatin-treated mice also an increase in hemoglobin (3.4 ± 0.2 and 5.9 ± 0.2 g/dL) and RBC count (22.5 ± 2.2 and 37.3 ± 1.7%) at 15 and 30 mg/kg respectively was observed. ZYAN1's effects on hemoglobin and RBC count were distinct from darbepoietin. ZYAN1 demonstrated hematinic potential by combined effects on EPO release and efficient iron utilization. The efficacy of ZYAN1 in disease models of different etiologies suggests that it will be useful in treating wide spectrum of anemia patients.

RNAi-Mediated Inhibition of Tmprss6 Ameliorates Anemia and Secondary Iron Overload in a Mouse Model of β-Thalassemia Intermedia and Decreases Iron Overload in Hfe−/− Mice

AbstractAbstract 1018β-Thalassemia intermedia (TI), an inherited hemoglobinopathy caused by partial loss of β-globin synthesis, is characterized by anemia, extramedullary hematopoiesis and ineffective erythropoiesis as well as secondary iron overload. Hereditary hemochromatosis (HH) is most frequently caused by mutations in HFE and is marked by excess uptake of dietary iron with concomitant tissue iron overload. In both diseases, increased iron absorption is due to inappropriately low levels of the liver hormone, hepcidin (encoded by Hamp1). The membrane serine protease Matriptase-2 (encoded by Tmprss6) attenuates BMP-mediated Hamp1 induction by cleaving the BMP co-receptor, hemojuvelin. Previously, it has been shown that elevating Hamp1 expression by genetic inactivation of Tmprss6 reduces disease severity in the Hbbth3/+ mouse model of TI and prevents iron overload in Hfe−/− mice. Therefore, a therapeutic approach comprising specific inhibition of Tmprss6 could prove efficacious in TI and HH.Here we show that systemic administration of a potent lipid nanoparticle (LNP) formulated siRNA directed against Tmprss6 leads to >80% inhibition of Tmprss6 mRNA in the livers of Hbbth3/+ and Hfe−/− mice with concomitant >2-fold elevation in Hamp1 expression. In the TI model, Tmprss6 silencing leads to ∼30% reductions in serum iron and non-heme liver iron. In Hfe−/− mice, serum iron and non-heme liver iron are similarly reduced, and Perls staining of peri-portal iron is diminished. Remarkably, the partial iron restriction induced by Tmprss6 inhibition in Hbbth3/+ mice leads to dramatic improvements in the hematological aspects of the disease phenotype: the severity of the anemia is decreased as evidenced by an approximately 1 g/dL increase in total hemoglobin and a 50% decrease in circulating erythropoietin levels. As in the human disease, Hbbth3/+ mice exhibit the hallmarks of ineffective erythropoiesis including splenomegaly, decreased erythrocyte survival and marked reticulocytosis. Treatment with LNP formulated Tmprss6 siRNA leads to a dramatic 2–3 fold decrease in spleen size, a 3–4 fold decrease in reticulocyte counts and a >7-day increase in RBC half-life. Histological analysis of spleens from Tmprss6 siRNA treated animals demonstrates restoration of normal splenic architecture, as well as a reduction in the number of Tfr1-positive erythrocyte precursors in the spleen. Furthermore, as evidenced by the near normalization of blood smears, the overall quality of erythropoiesis in treated animals is vastly improved.Taken together, these data demonstrate that RNAi-mediated silencing of liver Tmprss6 elevates Hamp1 expression and reduces iron overload in both TI and HH model mice. More significantly, Tmprss6 siRNA treatment ameliorates all aspects of the disease phenotype in the TI mouse model. These results support the development of an RNAi therapeutic targeting TMPRSS6 for the treatment of TI, HH and potentially other disorders characterized by excess iron absorption due to physiologically inappropriately low levels of hepcidin. Disclosures:Racie:Alnylam Pharmaceuticals: Employment. Butler:Alnylam Pharmaceuticals, Inc.: Employment, Equity Ownership. Bumcrot:Alnylam Pharmaceuticals, Inc.: Employment, Equity Ownership.

Abstract 5011: Potential tumor suppressor role of PHD2 : functional study of mutations identified in germline DNA of patients with congenital polycythemia with or without paraganglioma

Abstract The Hypoxia Inducible Factor (HIF) plays a major role in tissue response to hypoxia. HIF induces expression of many genes involved in anaerobic glycolysis, angiogenesis, erythropoiesis and cell proliferation. HIF-alpha subunit is tightly regulated by a family of specific hydroxylases in normoxia. PHD (prolyl-hydroxylase domain) proteins hydroxylate HIF-alpha, thereby allowing its attachment to the VHL protein, a component of an E3 ubiquitin ligase complex involved in protein ubiquitination and degradation. Mutations in HIF regulators have been described and lead to the stabilization of HIF, activation of target genes and development of either tumors or polycythemia. Heterozygous germline mutations in the VHL gene predispose to the von Hippel-Lindau disease characterized by a panel of highly vascularized tumors (central nervous system and retinal hemangioblastomas, pheochromocytomas, renal cell carcinoma, pancreatic endocrine tumors) whereas homozygous or compound heterozygous VHL mutations are responsible for isolated polycythemia (”Chuvash polycythemia”) with high circulating erythropoietin levels. Recently, heterozygous germline mutations in the PHD2 gene have been described in few patients with such congenital polycythemia and a particular mutation (H374R) has been identified in a patient with a polycythemia and a recurrent paraganglioma1. This observation raised the question of a possible role of PHD2 loss of function in the genesis of tumors usually seen in the VHL disease. We report here four new heterozygous PHD2 germline mutations in patients with familial or apparently sporadic polycythemia. We performed a functional study of these mutants compared to the H374R mutant and we showed differences in the capacity of the PHD2 mutants to downregulate HIF-alpha. These differences, tested in various conditions, are highly reproducible and suggest that there may be a genotype/phenotype correlation for the PHD2 mutants like the VHL mutants with their different ability to regulate HIF associated with different phenotypes in the VHL disease. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5011.

Bone marrow fat accumulation after 60 days of bed rest persisted 1 year after activities were resumed along with hemopoietic stimulation: the Women International Space Simulation for Exploration study

Immobility in bed and decreased mobility cause adaptations to most human body systems. The effect of immobility on fat accumulation in hemopoietic bone marrow has never been measured prospectively. The reversibility of marrow fat accumulation and the effects on hemopoiesis are not known. In the present study, 24 healthy women (age: 25-40 yr) underwent -6 degrees head-down bed rest for 60 days. We used MRI to noninvasively measure the lumbar vertebral fat fraction at various time points. We also measured hemoglobin, erythropoietin, reticulocytes, leukocytes, platelet count, peripheral fat mass, leptin, cortisol, and C-reactive protein during bed rest and for 1 yr after bed rest ended. Compared with baseline, the mean (+/-SE) fat fraction was increased after 60 days of bed rest (+2.5+/-1.1%, P<0.05); the increase persisted 1 yr after the resumption of regular activities (+2.3+/-0.8%, P<0.05). Mean hemoglobin levels were significantly decreased 6 days after bed rest ended (-1.36+/-0.20 g/dl, P<0.05) but had recovered at 1 yr, with significantly lower mean circulating erythropoietin levels (-3.8+/-1.2 mU/ml, P<0.05). Mean numbers of neutrophils and lymphocytes remained significantly elevated at 1 yr (+617+/-218 neutrophils/microl and +498+/-112 lymphocytes/microl, both P<0.05). These results constitute direct evidence that bed rest irreversibly accelerated fat accumulation in hemopoietic bone marrow. The 2.5% increase in fat fraction after 60 days of bed rest was 25-fold larger than expected from historical ambulatory controls. Sixty days of bed rest accelerated by 4 yr the normal bone marrow involution. Bed rest and marrow adiposity were associated with hemopoietic stimulation. One year after subjects returned to normal activities, hemoglobin levels were maintained, with 43% lower circulating erythropoietin levels, and leukocytes remained significantly elevated across lineages. Lack of mobility alters hemopoiesis, possibly through marrow fat accumulation, with potentially wide-ranging clinical consequences.

Development of Efficent Gene Therapy for the Treatment of Erythrocyte Pyruvate Kinase Deficiency.

Human erythrocyte R-type pyruvate kinase deficiency (PKD) is an autosomal recessive disorder produced by mutations in the PKLR gene. Haemolytic anaemia is the major symptom of the disease. A severe deficiency in PKD causes ATP depletion in the RBC metabolism, which ultimately leads to haemolysis that may require periodical blood transfusion, splenectomy, and in some cases bone marrow transplantation. These clinical features make this disease a good candidate for gene therapy. With this aim, we have developed different gammaretroviral and lentiviral vectors expressing the human RPK and have characterized the functionality of the erythroid specific expression regulatory sequences of the human RPK gene in a lentiviral backbone. The transduction of mouse hematopoietic stem cells from a mouse strain deficient in the pklr gene [AcB55: pklr269A/269A mice], which mainly resembles the human RPK deficiency, with a retroviral vector expressing the human RPK, followed by the transplantation into irradiated syngenic recipients completely recovered the red cell parameters in peripheral blood, spleen and bone marrow. Also, intracellular values of ATP, plasmatic iron and circulating erythropoietin levels were recovered to normal values. After 100 days of transplantation, treated mice did not show any clinical symptom of the disease. Secondary pklr269A/269A recipients were also transplanted and their hematological symptoms were also reverted, demonstrating the stable therapeutic efficacy of the vector. To specifically express the RPK in the erythroid lineage, a lentiviral vector expressing the EGFP marker gene under the above RPK promoter (LVpRPKEG) was constructed to test its specificity. EGFP expression was detected in erythroid, but not in non-erythroid cell lines, transduced with this vector. Human CD34+ cells were also transduced with the LVpRPKEG vector and transplanted into NOD/SCID mice. Forty days post-transplantation human bone marrow cells were obtained and seeded in semisolid media. Significantly, only human erythroid colonies expressed the EGFP protein, demonstrating the efficacy of the RPK promoter to specifically express proteins in the erythroid lineage. Experiments using a lentiviral vector expressing the human RPK gene under the control of the human RPK promoter are now in progress. Overall, the transfer of lentiviral vectors harbouring the hRPK cDNA driven by its own promoter in PKLR mutated hematopoietic stem cells could represent an efficient therapeutic treatment of severe clinical cases of human erythrocyte PKD.

Circulating Erythropoietin Levels and Prognosis in Patients With Congestive Heart Failure

Considerable morbidity and mortality are still associated with congestive heart failure (CHF) syndromes, despite improvement in therapy. Activation of neurohormonal, inflammatory, and oxidative mechanisms has been shown to contribute to the significant morbidity and mortality. Erythropoietin (EPO) is a cytokine known to regulate erythroid proliferation, attenuate apoptosis and oxidative stress, and promote angiogenesis. We prospectively evaluated the predictive value of baseline EPO, N-terminal pro-B-type natriuretic peptide, and C-reactive protein levels in patients with clinically controlled chronic CHF. One hundred eighty-eight outpatients from a CHF clinic had baseline assessment of EPO, N-terminal pro-B-type natriuretic peptide, and C-reactive protein levels and a complete clinical data profile. These patients were followed up for 24 months for any hospitalization due to CHF or mortality. Circulating EPO levels were higher in CHF patients and increased in subjects with higher New York Heart Association scores. Levels of EPO (at a cutoff of 23 mU/mL) and N-terminal pro-B-type natriuretic peptide (cutoff at the median of 1556 pg/mL) were found to be strong predictors of mortality and CHF hospitalization, whereas C-reactive protein levels (cutoff of 10 mg/L) predicted CHF hospitalizations but not mortality. Left ventricular ejection fraction was found to be a predictor of mortality but not of CHF hospitalizations. Serum levels of EPO were significantly correlated with N-terminal pro-B-type natriuretic peptide and C-reactive protein levels but not with left ventricular ejection fraction. If confirmed in large-scale clinical studies, determination of circulating EPO levels may aid in predicting morbidity and mortality in patients with clinically controlled congestive CHF.

“Living high − training low” altitude training improves sea level performance in male and female élite runners

Acclimatization to moderate high altitude accompanied by training at low altitude (living high–training low) has been shown to improve sea level endurance performance in accomplished, but not élite, runners. Whether élite athletes, who may be closer to the maximal structural and functional adaptive capacity of the respiratory (i.e. oxygen transport from environment to mitochondria) system, may achieve similar performance gains is unclear. To answer this question, we studied 14 élite men and eight élite women before and after 27 days of living at 2500 m while performing high-intensity training at 1250 m. The altitude sojourn began 1 week after the USA Track and Field National Championships, when the athletes were close to their season's fitness peak. Sea level 3000-m time trial performance was significantly improved by 1.1% (95% confidence limits 0.3–1.9%). One-third of the athletes achieved personal best times for the distance after the altitude training camp. The improvement in running performance was accompanied by a 3% improvement in maximal oxygen uptake (72.1 ± 1.5–74.4 ± 1.5 ml kg− 1 min− 1). Circulating erythropoietin levels were near double initial sea level values 20 h after ascent (8.5 ± 0.5–16.2 ± 1.0 IU ml−1). Soluble transferrin receptor levels were significantly elevated on the 19th day at altitude, confirming a stimulation of erythropoiesis (2.1 ± 0.7–2.5 ± 0.6 μ g ml-1). Hb concentration measured at sea level increased 1 g dl−1 over the course of the camp (13.3 ± 0.2–14.3 ± 0.2 g dl−1). We conclude that 4 weeks of acclimatization to moderate altitude, accompanied by high-intensity training at low altitude, improves sea level endurance performance even in élite runners. Both the mechanism and magnitude of the effect appear similar to that observed in less accomplished runners, even for athletes who may have achieved near maximal oxygen transport capacity for humans.

"Living high-training low" altitude training improves sea level performance in male and female elite runners.

Acclimatization to moderate high altitude accompanied by training at low altitude (living high-training low) has been shown to improve sea level endurance performance in accomplished, but not elite, runners. Whether elite athletes, who may be closer to the maximal structural and functional adaptive capacity of the respiratory (i.e., oxygen transport from environment to mitochondria) system, may achieve similar performance gains is unclear. To answer this question, we studied 14 elite men and 8 elite women before and after 27 days of living at 2,500 m while performing high-intensity training at 1,250 m. The altitude sojourn began 1 wk after the USA Track and Field National Championships, when the athletes were close to their season's fitness peak. Sea level 3,000-m time trial performance was significantly improved by 1.1% (95% confidence limits 0.3-1.9%). One-third of the athletes achieved personal best times for the distance after the altitude training camp. The improvement in running performance was accompanied by a 3% improvement in maximal oxygen uptake (72.1 +/- 1.5 to 74.4 +/- 1.5 ml x kg(-1) x min(-1)). Circulating erythropoietin levels were near double initial sea level values 20 h after ascent (8.5 +/- 0.5 to 16.2 +/- 1.0 IU/ml). Soluble transferrin receptor levels were significantly elevated on the 19th day at altitude, confirming a stimulation of erythropoiesis (2.1 +/- 0.7 to 2.5 +/- 0.6 microg/ml). Hb concentration measured at sea level increased 1 g/dl over the course of the camp (13.3 +/- 0.2 to 14.3 +/- 0.2 g/dl). We conclude that 4 wk of acclimatization to moderate altitude, accompanied by high-intensity training at low altitude, improves sea level endurance performance even in elite runners. Both the mechanism and magnitude of the effect appear similar to that observed in less accomplished runners, even for athletes who may have achieved near maximal oxygen transport capacity for humans.

Serum thrombopoietin and erythropoietin levels in patients with acute promyelocytic leukaemia during all‐trans retinoic acid treatment

Endogenous serum thrombopoietin (TPO) and various cytokines including erythropoietin (EPO), interleukin (IL)-3, IL-6, IL-11, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF) and stem cell factor (SCF) levels were measured in five patients with acute promyelocytic leukaemia (APL) during all-trans retinoic acid (RA) treatment. During differentiation-inducing therapy, platelet counts slowly increased and reached a peak between days 29 and 46 (median day 35). Serum TPO levels increased parallel to the increasing platelet counts and reached a maximum level during the first 10-20 d of all-trans RA treatment. The circulating TPO levels then decreased in inverse correlation to the platelet counts. These unique changes in serum TPO levels revealed that TPO levels were not regulated by platelet or megakaryocyte mass in patients with APL during differentiation-inducing therapy, and it would appear that TPO levels are directly regulated by all-trans RA during the first 10-20 d of treatment. In addition, the change in circulating EPO levels and reticulocyte counts were similar to that of the TPO levels and platelet counts during all-trans RA treatment, suggesting a close relationship between TPO and EPO signalling.