Red Cell Formation Research Articles

Oxygen sensing in the kidney.

The kidneys fulfil several essential homeostatic functions for the body. One of them is the maintenance of sufficient oxygen supply to the organs. For this purpose, the kidneys control the formation of red blood cells by the production of the hormone erythropoietin. This control of red cell formation is not only relevant to prevent states of oxygen deficiency but also to prevent an unwanted increase of red cell numbers causing thromboembolic risks. The adequate production of erythropoietin requires a sensing of the arterial oxygen content and transduction to hormone production. This oxygen sensing is a two-step process which includes a translation of the arterial oxygen content to respective oxygen tension in the tubulointerstitium and a perception of the resulting local interstitial oxygen tension to translate them into specific cellular responses such as the production of erythropoietin. This contribution will describe these steps of oxygen sensing for the healthy kidney and for the changes occurring during states of chronic renal disease, which are commonly associated with anemia. In this context a special focus will also be set on intrarenal hypoxia and oxygen sensing in the diabetic kidney including the treatment with tubular glucose transport (SGLT-2) inhibitors which might influence the oxygen sensing in the kidney. Finally, we will consider the effects of prolylhydroxylase inhibitors (PHD-I), which fundamentally interfere with the cellular oxygen sensing and which are meanwhile treatment options in renal anemia.

Cardiac complications in thalassemia throughout the lifespan: Victories and challenges.

Thalassemias are among the most common hereditary diseases in the world because heterozygosity offers protection against malarial infection. Affected individuals have variable expression of alpha or beta chains that lead to their unbalanced utilization during hemoglobin formation, oxidative stress, and apoptosis of red cell precursors prior to maturation. Some individuals produce sufficient hemoglobin to survive but suffer the vascular stress imposed by chronic anemia and ineffective erythropoiesis. In other patients, mature red cell formation is insufficient, and chronic transfusions are required-suppressing anemia and ineffective erythropoiesis but at the expense of iron overload. The cardiovascular consequences of thalassemia have changed dramatically over the previous five decades because of evolving treatment practices. This review summarizes this evolution, focusing on complications and management pertinent to modern patient cohorts.

Anemia in children: a pediatrician’s view

Anemia is defined as a hemoglobin level that is two standard deviations below the mean for age. After children reach the age of 12, the hemoglobin norm can be further divided into gender-specific ranges. When a patient presents with anemia, it is important to establish whether the abnormality is isolated to a single cell line [red blood cells (RBC) only] or whether it is part of a multiple cell line abnormality. In children, anemia is usually caused by decreased RBC production or increased RBC turnover. Anemia is usually classified based on the size of RBC (microcytosis, normocytosis, or macrocytosis) as measured by the mean corpuscular volume. Although iron deficiency anemia is usually microcytic, some patients may have normocytic blood cells. From a practical point of view, it is better to use in children the etiologic classification of anemia which includes impaired red cell formation, blood loss and hemolytic anemia. Most children with anemia are asymptomatic, and the condition is detected on screening laboratory evaluation. Iron deficiency can be treated with oral iron, intravenous iron, and/or blood transfusion, depending on the patient`s hemoglobin levels, tolerance and co-morbidity. Oral iron salts are usually the first line of treatment for uncomplicated iron deficiency, but are poorly absorbed and lead to gastrointestinal side effects. In some cases, iron refractory iron deficiency anemia (IRIDA), a hereditary recessive anemia refractory to oral iron, occurs. IRIDA shows a slow response to intravenous iron and partial correction of anemia.

“RHEX” / C1ORF186 Governs JAK2 Activation, Differentially Modulates STAT-5 and -3, Balances Human Erythroblast Development, and Becomes Essential during Red Cell Formation

Introduction: Via phospho-PTM-targeted LC/MS proteomics, we identified RHEX as an EPO- and pY-regulated integral plasma membrane adaptor protein that associates with the h-EPOR and supports early stage human* (pro)erythroblast expansion (dexamethasone system) (JEM 211:1715-22). [*RHEX has no orthologues, is conserved in H sapiens and primates, but is not represented among mouse, rat or lower vertebrates]. Presently, by employing a 3-phase HSC-to-RBC ex vivo system, shRNA lentivirus mediated knockdowns (KD's), cytohistomorphology imaging, and molecular phenotyping (including RNA seq), we have now discovered and defined three major novel effects that RHEX exerts on erythroblast formation. As an introduction to findings: 1] RHEX supports erythroblast proliferation, balances differentiation, and is essential for the formation of maturing late-stage erythroblasts. 2] RHEX governs EPO activation of not only p-AKT, p-ERK1/2, p-STAT5 and p-STAT3 but also p-JAK2, while also inversely modulating total STAT5 and STAT3 levels. 3] RNA-seq analyses reveal that the knockdown of RHEX sharply alters factors networked within "signal transduction", "plasma membrane" and "protein binding" as top GO terms. KEGG analysis defines adhesion proteins, and oxidative factors that are strongly modulated upon RHEX KD. RHEX therefore coordinates EPO/EPOR/JAK2 signals that dynamically regulate erythroblast maturation. Results: In primary HSC- derived erythroid progenitor cells (EPCs), RHEX knockdown (KD) attenuated (pro)erythroblast proliferation up to 50% of control cultures (shNT EPCs), especially during culture phases II (SCF, EPO) and III (EPO only). During development, erythroblast phenotypes were altered in three impacting ways. During early phase-II, RHEX KD heightened the frequency of polychromatic erythroblasts (200 % over controls) (Fig panel A). During late phase-II, this increase in sh-RHEX polychromatic erythroblast frequencies persisted, while parallel control shNT erythroblasts advanced to orthochromatic erythroblasts at frequencies significantly above sh-RHEX EPCs. The orthochromatic stage therefore marks a developmental point that is first compromised due to RHEX KD. During phase-III, the late-stage development of sh-RHEX EPCs became corrupted due to RHEX KD to the extent that terminally differentiating erythroblasts became markedly mis-shapen, and failed to efficiently convert to reticulocytes and erythrocytes (Fig panel B). By d6 of phase-III, in fact, intact cells were challenging to identify in sh-RHEX EPC cultures. When EPO/EPOR/JAK2 signaling modules were analyzed, RHEX's KD in UT7epo cells dysregulated EPO- activation of not only p-ERK1/2, p-AKT, p-STAT5, p-STAT3 but also pY-JAK2 (each multi-fold). In UT7epo RHEX KD cells, total STAT5 levels were elevated, while total STAT3 levels decreased (up to10-fold). Compartmentalized signaling within membrane and cytoplasmic cell fractions was also analyzed. In control shNT cells, activated pY-JAK2 was predominantly membrane-associated. RHEX KD, however, resulted in aberrantly heightened levels of pY-JAK2 in cytoplasm. This displacement effect was also observed for activated pY-STAT5. Findings suggest that RHEX may promote the membrane association of activated pY-JAK2. In addition, RHEX KD proved to markedly limit UT7epo cell-cell aggregation. To gain insight into cellular pathways that RHEX supports, RNAseq was performed using P-I d6 erythroblasts (unaffected by RHEX KD in morphologies). For factors differentially expressed due to RHEX KD plasma membrane adhesion proteins were highly represented, with high confidence. Transcripts altered 2 to >10-fold, include ITGB2, ITGaX, ITGaM, ICAM3, SEL-L, CD38, CD84, PLEXIND, PANNEXIN2, ADGRG3 - and each exhibited patterned erythroid expression. Select oxidizing enzymes were also elevated in RHEX KD erythroblasts as MPO, EPX, and the NADPH oxidase subunits NCF1, NCF4 and NCF2 (50+ fold). Future investigations will assess the extent to which such factors underlie RHEX's pro-erythropoietic roles. Summary: As an EPO/EPOR/JAK2 target, RHEX governs EPO- activation of JAK2, pY-JAK2's membrane vs cytoplasmic localization, and levels of total STAT5 and STAT3. During late erythropoiesis, RHEX's actions become essential for the integrity of maturing erythroblasts. Associated mechanisms may involve RHEX's effects on cell adhesion and/or oxidation. Figure Disclosures No relevant conflicts of interest to declare.

3085 – THE EPOR STIMULATES RAPID CYCLING AND FORMATION OF LARGER RED CELLS DURING MOUSE AND HUMAN ERYTHROPOIESIS

EpoR signaling is essential for the survival of erythroid precursors during terminal differentiation, when these cells undergo 3-5 cell divisions that are coupled to a rapid loss in cell size. The absolute dependence of erythroblasts on EpoR signaling for survival makes it challenging to identify other essential functions of EpoR in these cells. Key open questions include a role for EpoR in cell cycle regulation; and a potential role for EpoR in the induction of erythroid genes. To address these gaps, we developed a genetic system that allowed us to examine essential non-survival functions of EpoR signaling in erythroid differentiation. We rescued mouse Epor-/- fetal liver erythroid progenitors from apoptosis by transduction with the anti-apoptotic protein bcl-xL, comparing their ensuing differentiation with that of Epor-/- progenitors that were rescued by re-introduction of the EpoR. We found that the bcl-xL survival signal, in the absence of any EpoR signaling, was sufficient to allow erythroid terminal differentiation and formation of anucleated red cells. However, key features were abnormal. First, erythroblasts underwent slower and fewer cell cycles, generating fewer than half the number of red cells, showing an essential role for EpoR signaling in the regulation of cell cycle speed and number. Second, we found that EpoR signaling unexpectedly increased red cell size, in spite of the concurrent increase in the number of cell divisions. Using mice doubly deleted for both EpoR and HRI, we found that EpoR regulation of red cell size is independent of the well-described red cell size regulation by iron and heme. Healthy human volunteers that were administered Epo showed an increase in mean corpuscular volume (MCV) that persisted long after Epo and reticulocyte levels returned to baseline, supporting a role for the regulation of red cell size by Epo and suggesting new diagnostic interpretations for this common clinical test. Together, our findings reveal novel functions for the EpoR in erythroid differentiation including an unexpected role in the regulation of red cell size.

Biological basis for efficacy of activin receptor ligand traps in myelodysplastic syndromes.

Signaling by the TGF-β superfamily is important in the regulation of hematopoiesis and is dysregulated in myelodysplastic syndromes (MDSs), contributing to ineffective hematopoiesis and clinical cytopenias. TGF-β, activins, and growth differentiation factors exert inhibitory effects on red cell formation by activating canonical SMAD2/3 pathway signaling. In this Review, we summarize evidence that overactivation of SMAD2/3 signaling pathways in MDSs causes anemia due to impaired erythroid maturation. We also describe the basis for biological activity of activin receptor ligand traps, novel fusion proteins such as luspatercept that are promising as erythroid maturation agents to alleviate anemia and related comorbidities in MDSs and other conditions characterized by impaired erythroid maturation.

An Unexpectedly High Frequency of Sptb Gene Mutation (SPTB exon 29: c.6055T>C (p.Ser2019Pro; Spectrin Thai)) with a Single Origin in Thailand Suggesting a New Model of Red Blood Cell Trait Against Malarial Pressure

Mutations in SPTB gene coding β-spectrin can lead to congenital hemolytic anemia (HA) or red cell membranopathy. Most patients presented with mild to moderate HA with either spherocytes or ovalo-elliptocytes. Only one report in the literature described a family with non-immune hydrops fetalis (HF) due to homozygous SPTB: c.6055T>C (p.Ser2019Pro). Recently, we have identified the second patient presented with HF in Thailand with the same genotypes through genomic approach. This finding has driven extensive study to determine the impact of SPTB mutation in our cohort of unexplained HA and HF.From 107,947 live births at our center between 2005 and 2016, 83 non-immune HF were identified. Nineteen cases from 42 unexplained HF (45%) had FFPE DNA available from autopsy (Fig 1). In addition, 7 patients who were transfusion dependent anemia due to unknown cause were recruited. These were subjects of our Whole Exome Analysis (WES) using Illumina HiSeq4000 capturing by SureSelect V5 plus UTR covering 21,522 genes. Moreover, we studied WES in 120 Thai individuals without anemia as controls. Surprisingly, 6 out of 26 patients (23%) were homozygous SPTB: c.6055T>C and none in controls. We confirmed this finding by direct genomic Sanger sequencing and set up a PCR-RFLP to detect this further in other cohorts. Interestingly, we found additional 13 heterozygotes of this mutation in our thalassemia patients (1 in 361) and general population (12 in 2,576) (Fig 1) making an unexpectedly high carrier rate of this “so-called rare genetic disease” into 0.47% in Thai population. Using Hardy-Weinberg principle, homozygous SPTB: c.6055T>C would be 5.42 per million or expected 358 cases in Thailand.Since most reported SPTB mutations are sporadic or familial specific, we wondered whether this affected allele has a single or multiple origins. We generated a haplotype analysis of SPTB on chromosome 14q23 using 23 informative SNPs at the gene locus and adjacent region from WES data. Total 23 different haplotypes were generated and the SPTB c.6055T>C originated from the same ancestral chromosome as it was found only in haplotype IA (Fig 2). Therefore, we proposed to name this as spectrin Thai, instead of spectrin Providence which was named after the place of the first case reported (Gallagher PG, et al. J Clin Invest 1995), but not related to its native population.All 4 survived patients due to homozygous spectrin Thai (one received intrauterine transfusion) had severe anemia at birth and neonatal jaundice requiring therapy and became transfusion dependent afterward. All had marked hepatosplenomegaly with blood picture of pyropoikilocytosis. Three patients underwent splenectomy without improvement on anemia, this finding contradicted those in patients with red cell membranopathies. Of note, hematology from 25 heterozygotes including 12 parents showed no apparently abnormal indices and normal EMA binding assay. Using shortgun proteomics to analyze red cell membrane proteins, hemolysate and plasma proteins, the only difference was we could detect unbound free β-spectrin in plasma from controls but not from spectrin Thai traits. This might be useful for future screening of heterozygotes at population level.To explore whether red cells with spectrin Thai trait has any protection against malaria, the most important selective pressure in the past of our region we studied in vitro growth of P. falciparum and kinetics of erythroid invasion using live-cell microscopy. We found no significant differences between normal and spectrin Thai traits on these aspects. Since β-spectrin is involved on knob formation of malarial infected red cells that plays an important role on sequestration and cerebral manifestation, a further study using scanning electron microscopy comparing between malarial infected normal against spectrin Thai red cells is underway.For the first time, we showed that red cell membranopathy might be endemic in the tropics than we expected. Homozygous spectrin Thai is one of the most common causes of unexplained HF and HA in our region. The possibility that red cell membranopathy traits might be under malarial pressure throughout human history has completed the whole red cell quantitative trait genetic polymorphism in our population. This is in consistent with those of hemoglobinopathy (thalassemia) and enzymopathy (G6PD), both are highly prevalent in our region due to the same biological advantage against malaria. [Display omitted] DisclosuresViprakasit:Agios: Consultancy, Research Funding; Protagonist Therapeutics: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding.

Thyroid hormone receptor beta and NCOA4 regulate terminal erythrocyte differentiation

An effect of thyroid hormone (TH) on erythropoiesis has been known for more than a century but the molecular mechanism(s) by which TH affects red cell formation is still elusive. Here we demonstrate an essential role of TH during terminal human erythroid cell differentiation; specific depletion of TH from the culture medium completely blocked terminal erythroid differentiation and enucleation. Treatment with TRβ agonists stimulated premature erythroblast differentiation in vivo and alleviated anemic symptoms in a chronic anemia mouse model by regulating erythroid gene expression. To identify factors that cooperate with TRβ during human erythroid terminal differentiation, we conducted RNA-seq in human reticulocytes and identified nuclear receptor coactivator 4 (NCOA4) as a critical regulator of terminal differentiation. Furthermore, Ncoa4-/- mice are anemic in perinatal periods and fail to respond to TH by enhanced erythropoiesis. Genome-wide analysis suggests that TH promotes NCOA4 recruitment to chromatin regions that are in proximity to Pol II and are highly associated with transcripts abundant during terminal differentiation. Collectively, our results reveal the molecular mechanism by which TH functions during red blood cell formation, results that are potentially useful to treat certain anemias.

The effects of macrocyclic dinaphtho diamide on the oxidative states and stimulating the CSF production on lung tissue and colony formation of bone marrow cells

Objective Colony stimulating factors (CSFs) are endogenous cytokines that have key roles in proliferation and differentiation of hematopoietic progenitor cells and in regulation of mature blood cells performance. The CSFs families members are widely used for therapeutic purposes in many field include microbial infections, in cancer chemotherapy, alzheimer disease, hematopoiesis process, and for some neutropenia- related pathologies. Crown ethers are chemical compounds with therapeutic application that can affect the colony formation in vitro. The primary objective of the present study is to evaluate the effect of TDN (novel crown ether) on colony formation of red bone marrow cells in incubation with lung tissues cells.

"C1ORF150", a Novel Mediator of EPO/EPOR/JAK2 Dependent Human Erythroid Progenitor Cell Formation

An understanding of cellular events that are propagated within erythroid progenitor cells upon HGF-R / JAK complex activation is of basic importance for generating new insight into regulated red cell formation, anemia and myeloproliferative disease. Using the EPO/EPOR/JAK2 system as a paradigm, our group is successfully applying post-translational modification-based proteomics to uncover important new mediators of EPO-dependent human erythropoiesis (certain of which may also relate to EPO's untoward effects on hypertension and cancer progression). Here, we report on the discovery of a novel ORF, "C1ORF150", that is strongly tyrosine phosphorylated in response to EPO, possesses several unique features, and modulates EPO- dependent erythroid progenitor cell formation.In human erythroid progenitor UT7epo cells, EPOR ligation leads to C1ORF150 phosphorylation at tandem tyrosine p-Y69, p-Y89 and p-Y110, p-Y129 sites (up to 10-fold within 15 minutes). For each PTM site, EPOR/JAK2 mediated- phosphorylation was validated in independent LC-MS/MS experiments using Hematide as an EPOR agonist. p-Y69 and p-Y89 are predicted SFK sites, while p-Y110 and p-Y129 are predicted RTK sites (including KIT). Notably, C1ORF150 is conserved in H. sapiens and primates, but is not represented in mouse, rat or lower vertebrates. In addition, C1ORF150 has no obvious orthologues, but within EPO-regulated pY regions exhibits sequence homology with HGAL, an important factor for B cell receptor signaling.To assess C1ORF150’s functional effects, we used a lentiviral shRNA loss-of-function approach (80% knockdown efficiency). At physiological EPO levels, the knockdown of C1ORF150 substantially compromised UT7epo erythroid progenitor cell (EPC) survival, including 200% increases in apoptosis observed relative to control sh-NT transduced EPCs (p < 0.01). The ectopic expression of C1ORF150, in contrast, heightened baseline JAK2 activation, and potentiated STAT5 activation following EPO challenge. C1ORF150’s subcellular localization proved to be predominantly membrane associated.With regards to expression profiles, C1ORF150 levels were markedly elevated in bone marrow (among 30 human tissues), and during erythroid development were maximal at a CFUe stage. Furthermore, transcriptome profiles of myelodysplastic syndrome (MDS) CD34pos hematopoietic progenitor cells revealed elevated C1ORF150 expression in MDS refractory anemia (p=0.005) and refractory anemia-blast patients (p=0.05) compared to normal controls. In summary, via PTM-proteomics we have discovered "C1ORF150" as a major new pY- regulated EPOR/JAK2 target and membrane associated phosphoprotein that is proposed to have evolved in human erythroid progenitor cells to support EPO's cytoprotective effects, and red cell formation, in part by reinforcing JAK2 and STAT5 activation. In anemia and pre-leukemic contexts, attention also is brought to possible roles for C1ORF150 in the onset and progression of MDS. DisclosuresNo relevant conflicts of interest to declare.

Thyroid Hormone Receptor Beta and NCOA4 Regulate Terminal Erythrocyte Differentiation

An effect of thyroid hormone on erythropoiesis has been known for more than a century but the molecular mechanism(s) by which thyroid hormone affects red cell formation are still elusive. Our study demonstrates an essential role of thyroid hormone during terminal human erythroid cell differentiation; specific depletion of thyroid hormone from the culture medium completely blocked human erythroid differentiation. Genome wide analysis showed that thyroid hormone receptor β (TRβ) occupies many gene loci encoding transcripts abundant during terminal erythropoiesis, including globin genes, and cooperates with GATA-1 and RNA polymerase II (Pol II) to regulate their expression. Treatment with TRβ agonists enhanced erythroblast differentiation in vivo and alleviated anemic symptoms in a chronic anemia mouse model. To identify factors that cooperate with TRβ during human erythroid terminal differentiation, we conducted RNA-Seq in human reticulocytes and identified NCOA4 as a critical regulator of terminal differentiation. Furthermore, Ncoa4-/- mice are anemic in both the embryonic and perinatal periods and fail to respond to thyroid hormone by enhanced erythropoiesis. Genome wide analysis suggests that thyroid hormone promotes NCOA4 recruitment to chromatin regions that are in proximity to Pol II and are highly associated with transcripts abundant during terminal differentiation. Collectively, our results reveal the molecular mechanism of thyroid hormone function on red blood cell formation and are potentially useful to treat certain anemias. DisclosuresNo relevant conflicts of interest to declare.

From GWAS to function: lessons from blood cells.

Haematopoiesis, or the process of formation of mature blood cells from committed progenitors, represents an accessible and well‐studied paradigm of cell differentiation and lineage specification. Genetic association studies provide a powerful approach to discover new genes, biological pathways and mechanisms underlying haematopoietic development. Here, we highlight recent findings of genomewide association studies (GWAS) linking 145 genomic loci to traits affecting the formation of red and white cells and platelets in European and other ancestries. We present strategies to address the main challenges in GWAS discoveries, particularly to find functional and regulatory effects of genetic variants, and to identify genes through which these genetic variants affect haematological phenotypes. We argue that studies of haematological trait variation provide an ideal paradigm for understanding the function of GWAS‐associated variants owing to the accessible nature of cells, simple cellular phenotype and focused efforts to characterize the genetic and epigenetic factors influencing the regulatory landscape in highly pure mature cell populations.

PPAR-α and glucocorticoid receptor synergize to promote erythroid progenitor self-renewal.

SummaryMany acute and chronic anemias, including hemolysis, sepsis, and genetic bone marrow failure diseases such as Diamond-Blackfan Anemia (DBA), are not treatable with erythropoietin (Epo), because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production 1,2,3–5,6,7,8,9. Treatment of these anemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently we showed that glucocorticoids specifically stimulate self-renewal of the early erythroid progenitor, the burst-forming unit erythroid (BFU-E), and increase the production of terminally differentiated erythroid cells 10,11. Here we demonstrate that activation of the peroxisome proliferator-activated receptor alpha (PPARα) by PPARα agonists, GW7647 and fenofibrate, synergizes with glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures both of mouse fetal liver BFU-Es and of mobilized human adult CD34+ peripheral blood progenitors, the latter employing a new and effective culture system that generates normal enucleated reticulocytes. While PPARα−/− mice show no hematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis, PPARα agonists facilitate recovery of wild-type mice, but not PPARα−/− mice, from PHZ-induced acute hemolytic anemia. We also showed that PPARα alleviates anemia in a mouse model of chronic anemia. Finally, both in control and corticosteroid-treated BFU-E cells PPARα co-occupies many chromatin sites with GR; when activated by PPARα agonists, additional PPARα is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPARα agonists in stimulating self-renewal of early erythroid progenitor cells suggests that the clinically tested PPARα agonists we used may improve the efficacy of corticosteroids in treating Epo resistant anemias.

Erythropoietin-directed erythropoiesis depends on serpin inhibition of erythroblast lysosomal cathepsins

Erythropoietin (EPO) and its cell surface receptor (EPOR) are essential for red blood cell production and exert important cytoprotective effects on select vascular, immune, and cancer cells. To discover novel EPO action modes, we profiled the transcriptome of primary erythroid progenitors. We report Serpina3g/Spi2A as a major new EPO/EPOR target for the survival of erythroid progenitors. In knockout mice, loss of Spi2A worsened anemia caused by hemolysis, radiation, or transplantation. EPO-induced erythropoiesis also was compromised. In particular, maturing erythroblasts required Spi2A for cytoprotection, with iron and reactive oxygen species as cytotoxic agents. Spi2A defects were ameliorated by cathepsin-B/L inhibition, and by genetic co-deletion of lysosomal cathepsin B. Pharmacological inhibition of cathepsin B/L enhanced EPO-induced red cell formation in normal mice. Overall, we define an unexpected EPO action mode via an EPOR-Spi2A serpin-cathepsin axis in maturing erythroblasts, with lysosomal cathepsins as novel therapeutic targets.

Renal complications of sickle cell anemia in Zaria, Nigeria: An ultrasonographic assessment

Background: Vaso-occlusion in the kidney is a capillary phenomenon. Renal medullary hyperosmolalrity and low oxygen tension encourage the maximum formation of sickled red cells. In addition, the glomerular loops constitute an aggregating point for the sickled red cells with subsequent obstruction to the blood flow. These factors, together with the large volume of blood flowing through the kidneys make renal complications inevitable in sickle cell anemia (SCA). Aim: The purpose of this prospective study was to report the renal sonographic findings of a sample of patients with hemoglobin electrophoretic pattern consistent with sickle cell anemia Materials and Methods: A cross-sectional prospective study of 74 patients with the diagnosis of SCA, as documented by electrophoresis and who attended the adult sickle cell clinic, Hematology Department of the Ahmadu Bello University Teaching Hospital, Zaria, Nigeria and 20 age-matched controls with a normal hemoglobin (HbAA) phenotype and with no history of renal disease, was carried out between April and December, 2010. None of the patients had any clinical evidence of acute sickle episode (crisis) at the time of ultrasonographic examination. B-mode ultrasonography with Aloka SSD-3500 was used to assess the kidneys. The hematological parameters were determined by multiparameter analyzer Sysmex XT 2000i, whereas creatinine and urea of the patients were also analyzed using the Selectra XL chemistry autoanalyzer. Results: Renal size of the study group was compared with that of the control group and it showed a significant increase in the adult patients with SCA (P<0.05). The mean right renal length in the study group and control group was 10.65±0.97 cm and 9.95±0.80 cm (P<0.001), respectively, whereas the mean left renal length in the study group and control group was 10.70±1.02 cm and 10.00±0.66 cm (P<0.001), respectively. Statistical relationships between renal length and some hematological indices (packed cell volume (PCV), red blood cell count, and reticulocyte count) showed no correlation but renal length was positively correlated with reticulocyte count, especially high reticulocyte count. Other findings documented and discussed include echogenicity of renal parenchyma, hydronephrosis, and papillary necrosis. Conclusion: Renal ultrasound imaging of patients with SCA showed a high incidence of renal abnormalities.

Plasma cell leukemia of IgG secreting type: A rare case

Plasma cell leukemia is very rare comprising 2% plasma cell dyscrasias. We report a case of a 49-year-old male with complaints of chest pain, breathlessness, and acute attacks of gastroenteritis. On examination, there was marked pallor and hepatosplenomegaly. X-ray and computed tomography (CT) scan revealed multiple paratracheal lymph node enlargements. However, there was no diffuse osteopenia or multiple osteolytic lesions. Laboratory analysis revealed high serum calcium 11.5 mg/dL, low hemoglobin 5 g/dL, normal WBC count, thrombocytopenia 22000/mm³ and elevated erythrocyte sedimentation rate (ESR). Peripheral smear showed many atypical cells (41%) with blastoid morphology, giving a blood picture of acute leukemia. However, on closer inspection many of them appeared plasmacytoid. There was marked rouleaux formation of red cells in the background. Bone marrow aspirate and biopsy also revealed near total replacement by such atypical cells with plasmacytoid morphology. Serum electrophoresis revealed a prominent M spike and immunofixation revealed IgG heavy chain and kappa light chain immunostaining ruling out the possibility of Waldenström’s macroglobulinemia. Plasma cell leukemias are predominantly IgD and IgE secreting type. This case is rare in that it is IgG secreting type. Plasma cell leukemias are very aggressive and with higher incidence of organomegaly, lymphadenopathy, thrombocytopenia, hypercalcemia, renal impairment and surprisingly lesser occurrence of lytic bony lesions as in our case.

Erythropoietin-Directed Erythropoiesis Depends Upon Serpin Inhibition of Erythroblast Lysosomal Cathepsins

Abstract 981EPO is used to treat anemia associated with chronic kidney disease, and cancer chemotherapy. However, EPO has hypertensive and thrombolytic side-effects and can impact on innate immunity, diabetes, vasculogenesis, and the progression of certain cancers. These observations, together with the clinical emergence of new EPOR agonists provide compelling reasons to better understand key EPO/EPOR actions. This includes effects on erythroid progenitor cells (EPC's) as a prime target in which EPO/EPOR actions remain incompletely understood. To discover novel EPO action modes we profiled the transcriptome of primary EPC's. We report Serpina3g/Spi2A as a major EPO/EPOR target, and a key EPC survival factor. In knockout mice, loss of Spi2A sharply worsened anemia due to hemolysis, radiation or transplantation (p = 0.008; p = 0.0001; p = 0.010, respectively). EPO-induced red cell formation also was limited to 8.6 + 0.7% of wild-type control responses. Unexpectedly, maturing erythroblasts proved to require Spi2A for cytoprotection, with iron and ROS defined as cytotoxic agents. Notably, Spi2A defects were ameliorated by cathepsin-B/L inhibition, and by the genetic co-deletion of lysosomal cathepsin-B. In vivo, pharmacological inhibition of cathepsin B/L enhanced EPO's effects on hematocrits by 5+ points (d4 and d6 post- EPO). Overall, we define an unexpected EPO action mode via an EPOR- Spi2A serpin- cathepsin axis in maturing erythroblasts, with oxidants (and iron) as cytotoxic potentiators, and lysosomal cathepsins as novel therapeutic targets. Disclosures:No relevant conflicts of interest to declare.

From Hematopoietic Stem Cell to Erythroblast: Regulation of Red Cell Production At Multiple Levels

Abstract SCI-1 Hematopoietic stem cells (HSC) undergo self-renewal and also generate all types of blood cells, including red cells, myeloid cells, and all immune system cells. Formation of red cells from HSC involves multiple cellular stages, including early erythroid-specific progenitors (burst-forming unit-erythroid [BFU-E]) that respond to several growth factors and an erythropoietin (Epo)-responsive progenitor, and the colony-forming unit-erythroid (CFU-E) that in 3 days generates ∼30 reticulocytes. In adult animals most CFU-E cells undergo apoptosis. A short-term need for red cells is met by increased Epo production by the kidney, which rescues increasing numbers of CFU-Es from apoptosis and increases red cell production over a few days. Chronic stress, such as in certain types of anemias and inflammatory diseases, leads to a marked increase in numbers of HSC and many types of progenitor cells. I will focus most of my talk on stress-triggered BFU-E proliferation and formation of CFU-Es, since our very recent work showed how glucocorticoids – an important class of stress hormones – as well as HIF-1α stimulate red cell production. Acting synergistically these allow ∼300 times more CFU-Es and erythroblasts to be formed from each BFU-E. More specifically, these molecules stimulate limited self-renewal of BFU-Es during cell division, thus maintaining progenitor immaturity and allowing over time increased numbers of CFU-E cells and thus mature red cells to be formed. This explains why certain corticosteroids are useful in treating some non-Epo responsive anemias, and I will present a molecular dissection of glucocorticoid and HIF-1α action on BFU-E progenitors that has led to the identification of genes critical for self-renewal and that suggest other possible avenues for treatment of chronic anemias. Disclosures: No relevant conflicts of interest to declare.

EPO-Dependent Recovery of Late-Stage Erythroid Progenitors in the Marrow Precedes Splenic Expansion: Insights From a Sublethal Radiation Model

Abstract 180Erythropoiesis is a robust process of cellular expansion and maturation that occurs in the bone marrow and spleen of mice. Following clastogenic injury such as total body irradiation (TBI), erythroblasts are severely depleted in these organs, resulting in loss of reticulocyte output and the development of a mild anemia (Peslak et al., Exp. Hematol. 2011). However, the mechanistic and microenvironmental factors underlying erythroid recovery following sublethal TBI are poorly understood. To this end, we utilized colony assays to quantify erythroid progenitors, which consist of immature d7 erythroid burst-forming units (BFU-E) and more mature d3 BFU-E and erythroid colony forming units (CFU-E). Imaging flow cytometry was used to quantify erythroblast precursors. We found that d7 BFU-E undergo a slow, incomplete recovery during the first 10 days post-4 Gy TBI of C57Bl/6 mice. In contrast, d3 BFU-E exhibit a robust recovery beginning at 4 days post-TBI that is immediately followed by a rapid increase in CFU-E numbers to over 200 percent of steady-state levels. This initial erythroid progenitor recovery is followed by a wave of erythroid precursor maturation and red cell formation that occurs in close association with macrophages in the bone marrow. These erythroblast islands undergo a rapid synchronous expansion that peaks at 6 days post-TBI, suggesting that the bone marrow microenvironment plays a role in the recovery of the erythron from sublethal TBI. We hypothesized that erythropoietin (EPO), the primary regulator of erythroid survival and proliferation, mediates the rapid, specific expansion of late-stage erythroid progenitors following radiation injury. We found that plasma EPO levels increase 13-fold 4 days after 4 Gy TBI, temporally correlated with expansion of d3 BFU-E. Furthermore, maintenance of steady-state hematocrit levels following TBI prevented EPO induction and blocked expansion of late-stage erythroid progenitors, while exogenous EPO administered at 1 hour post-radiation specifically advanced recovery of late-stage progenitors. These data indicate that EPO is required for expansion of d3 BFU-E and CFU-E following radiation-induced marrow depletion.During times of acute hypoxia, such as the severe anemia induced by bleeding or phenylhydrazine exposure, EPO production is rapidly upregulated and splenic stress erythropoiesis is induced. Surprisingly, splenic erythropoiesis is absent during the rapid initial recovery of erythropoiesis in the bone marrow at 4–6 days post-TBI. However, a massive expansion of CFU-E begins at 7–8 days post-4 Gy TBI in spleen. EPO administration at 4 days following 4 Gy TBI significantly enhances late-stage progenitor recovery exclusively in the marrow, indicating that erythroid progenitors are not present in spleen at the time of rapid bone marrow expansion and that late-stage erythroid progenitor recovery initiates in the marrow and subsequently proceeds to the spleen. Furthermore, we found that erythroid progenitors transiently emerge in the bloodstream at 6–8 days post-TBI, following marrow recovery and prior to initiation of splenic erythropoiesis. These data are consistent with endogenous migration of the erythron from the bone marrow to the spleen during recovery from radiation-induced erythroid injury. Taken together, our data indicate that recovery from sublethal irradiation injury is regulated primarily by the EPO-induced expansion of late-stage erythroid progenitors in the bone marrow. This form of clastogenic injury is critically different from bleeding or hemolysis, which preserve bone marrow and splenic erythroblasts and induce expansion of splenic erythroid stress progenitors. Sublethal irradiation injury thus provides a unique model for the in vivo study of endogenous erythroid recovery. This model may be clinically useful for the functional evaluation of therapeutic factors that regulate or modulate erythroid cell maturation. Disclosures:Bemis:Litron Laboratories: Employment, Patents & Royalties.

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.