Proliferation Of Erythroid Progenitor Cells Research Articles

Treatment of Chronic Kidney Diseases with Darpeboetin can Ameliorate Kidney Functions: A Possible Clinical Relevance

Background: Erythropoietin (EPO) has been used clinically for the treatment of anemia as it was the first characterized hematopoietic factor. Erythropoietin is produced primarily by the kidneys (renal cortical fibroblasts) which induces proliferation and differentiation of erythroid progenitor cells. Previous studies suggested that erythropoietin may directly stimulates the regeneration of damaged tubular cells due to presence of erythropoietin receptors on renal tubular epithelial, mesangial and endothelial cells. EPO directly prevent glomerulosclerosis as a consequence of amelioration of podocyte injury. Therefore, to improve kidney function in chronic kidney disease (CKD), it is of critical importance to investigate clinically the beneficial reno-protective effects of EPO in patients with CKD. Methodology: The study was conducted at King Abdulaziz Medical City and included patients with chronic kidney diseases. Patients were under medical treatment with Darpeboetin for the first time. The administration frequency of Darpeboetin was single dose weekly. Kidney function tests including serum creatinine, serum urea, alkaline phosphatase, serum calcium, serum phosphorus, serum albumin and albuminuria were collected from the hospital electronic medical record system (BestCareR). Test values before and after treatment were compared. of the patients before starting treatment and at the end of treatment. The week before starting treatment was considered week 0 (baseline) Results: The results indicated that, treatment with a weekly single dose of Darpeboetin reduces blood urea nitrogen (BUN p=0.002) and serum uric acid level (p=0.009). The effect of treatment on serum calcium and potassium, showed a significant elevation of serum potassium (P<0.001), serum calcium (P<0.001) and adjusted calcium (P<0.001) levels parallel with significant decrease in serum phosphorus (p=0.004). Also treatment of chronic kidney disease patients significantly ameliorates serum albumin (P<0.001). Conclusion: Treatment with Darpeboetin may confirm the reno-protective potency. Darpeboetin is a promising drug to prevent or attenuate tissues kidney damage especially in chronic kidney disease

Hypoxia-inducible factors and essential hypertension: narrative review of experimental and clinical data.

Hypoxia-inducible factor (HIFs) is a new class of drug developed for the management of anemia in chronic kidney disease (CKD) patients. HIFs increase the production of erythropoietin in the kidney and liver, enhance the absorption and utilization of iron, and stimulate the maturation and proliferation of erythroid progenitor cells. Besides, HIFs regulate many physiologic processes by orchestrating the transcription of hundreds of genes. Essential hypertension (HT) is an epidemic worldwide. HIFs play a role in many biological processes involved in the regulation of blood pressure (BP). In the current review, we summarize pre-clinical and clinical studies investigating the relationship between HIFs and BP regulation in patients with CKD, conflicting issues, and discuss future potential strategies.

Iron status in chronic inflammatory disease: therapeutic implications.

Anemia of inflammation (AI) is a very frequent clinical condition affecting globally more than a billion people with chronic inflammatory disorders, such as chronic kidney disease, heart failure, and inflammatory bowel disease. It is usually associated with iron deficiency (ID), which imposes a severe additional burden on the recovery from the primary disease. The pathophysiology of iron dysregulation that may ultimately lead to absolute iron deficiency anemia (IDA) during inflammation is multifactorial and includes reduced iron absorption in the bowel, iron retention in macrophages of the reticuloendothelial system, reduction in circulatory half‑life of erythrocytes, inadequate production and activity of erythropoietin, and impaired proliferation and differentiation of erythroid progenitor cells. These result in hypoferremia and iron-restricted erythropoiesis. AI is mostly mild to moderate, normochromic and normocytic with normal or even increased ferritin levels. The current treatment options for AI include iron replacement therapy, treatment with erythropoiesis‑stimulating agents, and red blood cell transfusion. ID management is based on oral or intravenous iron preparations. Given the pathophysiology, treatment with oral iron, although widely used, presents several limitations that impact its effectiveness in patients with chronic inflammatory conditions. Instead, intravenous iron preparations are a valuable option for patients with chronic inflammatory diseases, as they overcome reduced bowel absorption. Novel therapeutic approaches include downregulation of hepcidin synthesis and function, and stabilization of the hypoxia‑inducible factor via inhibition of prolyl hydroxylase domain. Several studies in vitro and in vivo are ongoing; however, the results in humans are still elusive.

Immunophenotypical profiling of myeloid neoplasms with erythroid predominance using mass cytometry (CyTOF).

Acute erythroid leukemia (AEL) is a disease continuum between Myelodysplastic syndrome (MDS) and Acute myeloid leukemia (AML) with the cellular hallmark of uncontrolled proliferation and impaired differentiation of erythroid progenitor cells. First described by Giovanni di Guglielmo in 1917 AEL accounts for less than 5% of all de novo AML cases. There have been efforts to characterize AEL at a molecular level, describing recurrent alterations in TP53, NPM1 and FLT3 genes. A genomic analysis of AEL cases confirmed its complexity. Despite these advances, the biology underlying erythroid proliferations remains unclear and the prognosis is dismal with a median survival of only 3months for pure erythroid leukemia (PEL). Marker combinations suitable for the identification and characterization of leukemic stem cell (LSC) candidates, monitoring measurable residual disease (MRD) during chemotherapy treatment and the development of innovative targeted therapies are missing. Here, we developed a mass cytometry panel for an in-depth characterization of erythroid and myeloid blast cell populations from human AEL bone marrow samples in comparison to other AML subtypes and healthy counterparts. A total of 8 AEL samples were analyzed and compared to 28 AML samples from different molecular subtypes, healthy bone marrow counterparts (n=5) and umbilical cord blood (n=6) using high-dimensional mass cytometry. Identification of erythroid and myeloid blast populations in high-dimensional mass cytometry data enabled a refined view on erythroblast differentiation stages present in AEL erythroid blasts and revealed immunophenotypical profiles specific to AEL. Profiling of phenotypic LSCs revealed aberrant erythroid marker expression in the CD34+ CD38- stem cell compartment. In addition, the identification of novel candidate surface marker combinations and aberrancies might enhance clinical diagnostics of AEL. We present a high-parameter mass cytometry approach feasible for immunophenotypical analysis of blast and stem cell populations in myeloid neoplasms with erythroid predominance laying the foundation for more precise experimental approaches in the future.

Exploring the Functional Role of an Alternative mRNA Transcript of the EPO Gene

Erythropoietin (EPO) is the key regulator of erythropoiesis and stimulates the proliferation and differentiation of erythroid progenitor cells into mature erythrocytes. In a previous study, we identified a frame-shift mutation in the EPO gene that causes familial erythrocytosis with elevated erythropoietin (EPO) levels by initiating excess production of EPO protein from an alternative EPO mRNA [1]. This alternative non-coding mRNA is transcribed from a promoter (P2) located in intron 1, whereas the full length mRNA that encodes the EPO protein is transcribed from the P1 promoter located upstream of exon 1. Large parts of intron 1 sequences, including the P2 transcript are conserved among different species (Figure 1A), suggesting that the P2 transcript could have an as yet unknown physiological function. To study the putative function of the P2 transcript, we performed experiments in a human cell line, Hep3B, which can produce EPO. We used the CRISPR/Cas9 system to generate mutant Hep3B clones with targeted deletions in intron 1 of the EPO gene. Close to the P2 transcription start site in intron 1, we identified three GATA-consensus motifs that might act as promoter elements for the P2 transcript. In the first round of CRISPR/Cas9 mutagenesis, we targeted the first two GATA elements and expected to knock down the expression of the P2 transcript. Targeted Hep3B cells were sorted and deposited as single cells to derive clones that were genotyped by next generation sequencing. We obtained 2 clones with homozygous deletions in EPO intron 1 and one clone that was compound heterozygous (Figure 1A). In these Hep3B clones we assessed EPO mRNA expression by qPCR and EPO protein levels in the supernatant by ELISA (Figure 1B). Unexpectedly, the P2 transcript was still detectable in all three clones carrying deletions in intron 1 that eliminated GATA boxes 1 and 2 (data not shown). EPO P1 mRNA expression and EPO protein levels in the supernatant were slightly reduced in most of the clones. These data indicate that the GATA box 1 and 2 motifs are not required for generating the P2 transcript. We have now performed a second round of CRISPR/Cas9 mutagenesis. In one series we targeted all three GATA-motifs through a 186 bp deletion, and in a second series we targeted a highly conserved region upstream of the GATA boxes by a 93 bp deletion (Figure 1A). We obtained 4 clones homozygous for the 93 bp deletion and the clones for the 186 bp deletion are currently being expanded. The analysis of EPO mRNA and protein in these additions CRISPR/Cas9 clones will be presented. Our study shows that the GATA motifs 1 and 2 are not required for function of the P2 promoter in intron 1 of the EPO gene. However, deleting these two motifs in two different cell clones decreased the P1 full-length EPO mRNA expression and EPO protein production in Hep3B cells by 70.2% and 37.2%, respectively. Additional clones with deletion of all three GATA motifs and a highly conserved region upstream of the GATA boxes will likely provide more insights into the physiological role of the P2 transcript.

Novel Immediate Direct Targets of EPO Signalling in Human Erythropoiesis

Introduction: Erythropoietin (EPO) regulates expression of genes that drive proliferation, survival and differentiation of erythroid progenitor cells into mature erythrocytes. The EPO receptor signals via JAK2 and STAT, PI3K and MAPK pathways. Only a few direct target genes of these pathways have been identified to date. Aim: To determine the repertoire of EPO-induced target genes in human erythroid cells Methods: We employed a well characterized conditionally immortalized EPO-dependent human erythroid progenitor model (HUDEP-2 cells). The cells require stem cell factor and EPO for growth, but can be starved of both and then re-challenged with either growth factor. A multi-omics approach was employed to find direct target genes of EPO stimulation. We performed ChIP-seq for pSTAT5, ATAC-seq, and a new method of primary RNA-seq based on SLAM-seq but using full length cDNA libraries. Results: Three hours of starvation followed EPO (5U/ml) stimulation for 1 hour resulted in rapid phosphorylation of STAT5, but not STAT1 or STAT3. ChIP-seq or pSTAT5 identified 3128 EPO-induced binding sites. The majority of peaks contain a palindromic 'GAS' motif (TTCYXRGAA), and are located at intronic (50%), distal (29%) and intergenic (15%) enhancers; only 3% are located at promoters. De novo motif discovery identified significant enrichment of DNA-binding motifs for GATA and KLF transcription factors (TFs), suggesting co-operativity between EPO signalling and the essential basal erythroid TFs, GATA1 and KLF1. We show GATA1 and KLF1 bind most of these by ChIP-seq. We found 14,535 new ATAC-seq peaks after EPO stimulation, at both enhancers and promoters. Only ~8% overlap with pSTAT5 ChIP-seq peaks, suggesting EPO-mediated phosphorylation and DNA-binding of undiscovered TFs. By searching for enriched motifs within these regions, we identified binding sites for TFs of the NFY, EGR, and NRF1 families and others, suggesting a hidden complexity of transcription factors that mediate responses to EPO. ChIP-seq for EGR1 is in progress and will be reported. We developed a novel metabolic labelling technique and bio-informatic pipeline, called BodySLAM-seq, to determine the immediate transcriptional targets of EPO. We used this to find ~100 differentially transcribed genes (DTGs) as well as differentially expressed genes (DEGs) in immediate response to EPO (45 min labelling period). Some are direct pSTAT5 target genes such as BCL2L1, PIM1, and CISH; others are novel targets involved in transcription regulation, erythropoiesis and signalling. Some are known immediate early genes such as EGR1. The RNAs for these are rapidly degraded in the absence of EPO and rapidly induced. Other genes are not degraded in the absence of EPO but are nevertheless rapidly induced. These were only detectable by BodySLAM-seq. Conclusions: We have discovered new pSTAT5 direct target genes, and also new DNA motifs that mediate responses to EPO, in human erythropoiesis. We have discovered a set of immediate early genes (IEGs) that are rapidly degraded in the absence of an EPO stimulus and rapidly induced again by EPO. These are involved in negative regulation of JAK-STAT signalling, cell proliferation, cell survival, and transcription. They are not involved in erythroid cell differentiation specifically. This suggests the main function of EPO is to induce cell proliferation and survival rather than differentiation.

Erythropoietin-driven dynamic proteome adaptations during erythropoiesis prevent iron overload in the developing embryo.

Erythropoietin (Epo) ensures survival and proliferation of colony-forming unit erythroid (CFU-E) progenitor cells and their differentiation to hemoglobin-containing mature erythrocytes. A lack of Epo-induced responses causes embryonic lethality, but mechanisms regulating the dynamic communication of cellular alterations to the organismal level remain unresolved. By time-resolved transcriptomics and proteomics, we show that Epo induces in CFU-E cells a gradual transition from proliferation signature proteins to proteins indicative for differentiation, including heme-synthesis enzymes. In the absence of the Epo receptor (EpoR) in embryos, we observe a lack of hemoglobin in CFU-E cells and massive iron overload of the fetal liver pointing to a miscommunication between liver and placenta. A reduction of iron-sulfur cluster-containing proteins involved in oxidative phosphorylation in these embryos leads to a metabolic shift toward glycolysis. This link connecting erythropoiesis with the regulation of iron homeostasis and metabolic reprogramming suggests that balancing these interactions is crucial for protection from iron intoxication and for survival.

Association of Vitamin D in Different Trimester with Hemoglobin during Pregnancy.

The association between vitamin D and hemoglobin has been suggested. Vitamin D can affect erythropoiesis by the induction of erythroid progenitor cell proliferation and enhance iron absorption by regulating the iron-hepcidin-ferroportin axis in monocytes. However, this relationship in pregnant women is scarce. The purpose of this study was to investigate the association between plasma vitamin D levels with hemoglobin concentration in pregnant women considering each trimester and iron supplementation. The data were obtained from Zhoushan Pregnant Women Cohort, collected from 2011 to 2018. Plasma 25(OH)D was measured in each trimester using liquid chromatography–tandem mass spectrometry. Generalized estimating equations and multiple linear regressions were performed. Finally, 2962 pregnant women and 4419 observations in the first trimester were included in this study. Plasma 25(OH)D in first trimester (T1) (β = 0.06, p = 0.0177), second trimester (T2) (β = 0.15, p < 0.0001), and third trimester (T3) (β = 0.12, p = 0.0006) were positively associated with Hb. Association between plasma 25(OH)D levels in T1 and Hb concentration was positively associated with gestational age (β = 0.005, p = 0.0421). Pregnant women with VD deficiency in T1 (OR = 1.42, 95% CI: 1.07–1.88) or T2 (OR = 1.94, 95% CI: 1.30–2.89) presented an increased risk of anemia, compared with women without VD deficiency. Moreover, the significant relationship between VD and Hb was only observed among women with iron supplementation during pregnancy. Plasma 25(OH)D levels in each trimester were positively associated with Hb concentration. Iron supplementation might be an important factor affecting the relationship between VD and Hb.

Carbon Dots as a Potential Therapeutic Agent for the Treatment of Cancer‐Related Anemia

AbstractDue to the adverse effects of erythropoietin (EPO) on cancer patient survival, it is necessary to develop new agents that can be used to efficiently manage and treat cancer‐related anemia. In this study, novel distinctive carbon dots, J‐CDs, derived from jujube are designed, synthesized, and characterized. Based on the obtained results, this material comprises sp2 and sp3 carbon atoms, as well as oxygen/nitrogen‐based groups, and it specifically promotes the proliferation of erythroid cells by stimulating the self‐renewal of erythroid progenitor cells in vitro and in vivo. Moreover, J‐CDs have no discernible effects on tumor proliferation and metastasis, unlike EPO. Transcriptome profiling suggests that J‐CDs upregulate the molecules involved in hypoxia response, and they also significantly increase the phosphorylation levels of STAT5, the major transducer of signals for erythroid progenitor cell proliferation. Overall, this study demonstrates that J‐CDs effectively promote erythrocyte production without affecting tumor proliferation and metastasis; thus, they may be promising agents for the treatment of cancer‐related anemia.

Hematology Profile of Tuberculosis Lymphadenitis Patients at Siti Rahmah Hospital, Padang, Indonesia

Tuberculosis (TB) is a progressive granulomatous infectious disease caused by Gram-positive acid-resistant bacilli classified in the genus Mycobacterium. Tuberculosis in humans is caused by Mycobacterium tuberculosis (MTB) and it mainly infects the lungs, although it can also infect intestines, meninges, bones, lymph nodes, skin that cause extra-pulmonary TB. Tuberculous lymphadenitis is an inflammatory process in lymph nodes as a result of MTB activity. The inflammatory process caused by MTB activity is often associated with anemia as the most common complication. Anemia in TB is caused by an inflammatory process associated with bone marrow suppression due to pro-inflammatory cytokines, thus inhibiting the proliferation and differentiation of erythroid progenitor cells. This chronic TB infection also affects iron hemostasis, thus further affecting the hematological profile of TB patients. This study aimed to determine the hematology profile of TB lymphadenitis patients in the Siti Rahmah Hospital, Padang, Indonesia. This was a descriptive study, with a population of patients diagnosed with TB lymphadenitis based on the results of a fine needle biopsy in the Colli region. The samples of this study consisted of 24 patients who have tested the hematology profile. Tuberculosis lymphadenitis patients in females were higher than males, the mean age was 26.75±19.53 years old dominated by an adult (54%). The mean of 3 hemoglobin levels was 11.8±1.522 g/dL, with 62.5% anemia patients. The mean leukocyte count was 10400±3018.926/mm , 3 with 54.2% of patients had normal levels of leukocyte. The mean of thrombocyte count was 334750±74440.668/mm , with 83.3% in normal levels and the mean of Erythrocyte Sedimentation Rate (ESR) was 35.25±31.489 mm/h, with 54.2% patients in high ESR. Hematology profile in TB lymphadenitis patient was anemia, normal levels of leukocytes, thrombocyte, and increased levels of ESR.

SIRT1 Increases the Expression of Fetal Hemoglobin Genes in Human Adult and Cord Blood Erythroblasts

Introduction: High fetal hemoglobin (HbF, α2γ2) levels ameliorate the clinical manifestations of sickle cell disease and β thalassemia. The mechanisms that repress HbF expression and silence γ-globin genes (HBG) in adults are incompletely characterized and only a single HbF inducer, hydroxyurea, is approved for treatment, and only in patients with sickle cell disease. Many efforts have been made to develop new HbF inducers. HDAC1 and HDAC2, the class I histone deacetylases, suppress HbF gene transcription, and histone deacetylase inhibitors induce HbF expression. We therefore wanted to study whether SIRT1, a NAD-dependent class III histone deacetylase, also played a role in suppressing HBG expression. Unexpectedly, we found that SIRT1 was an inducer of HBG expression.Methods: We evaluated the role of SIRT1 in fetal globin gene regulation by ectopic expression or knockdown of SIRT1 and treatment with small molecule SIRT1 activators SRT2104 and SRT1720, in primary erythroid progenitor cells isolated from both cord blood and adult peripheral blood, CD34+ HUDEP-1 and HUDEP-2 cell lines that express fetal and adult hemoglobins, and K562 cells, using qRT-PCR, flow cytometry, immunoblotting, chromatin immunoprecipitation, and chromatin conformation capture assays.Results: We identified SIRT1 as a new inducer of γ-globin. Ectopic expression of SIRT1 in cord blood CD34+ cells increased HBG mRNA 4-fold and the g-globin protein level 2.5-fold compared with vector control (p<0.05). SIRT1 knockdown in cord blood CD34+ cells decreased HBG mRNA 8-fold and γ-globin protein level 5-fold compared with a scrambled shRNA control (p<0.05). Stable SIRT1 knockdown in K562 cells decreased HBG mRNA levels by 11-fold (p<0.01).To evaluate the effect of small molecule SIRT1 agonists on HbF gene expression, we treated erythroid progenitor cells from cord blood or normal adult blood and HUDEP-1 and HUDEP-2 cells and analyzed HBG mRNA, HbF-cells and g-globin protein levels. In cord erythroid progenitor cells, SRT12104 at 100nM, 500nM, and 2uM concentrations induced a mean increase of 2.4-fold, 3.4-fold and 8.1-fold in HBG mRNA, respectively, compared with vehicle treated controls (p<0.01). SRT1720 at 500nM and 5uM concentrations induced a mean increase of 2.1-fold and 4.4-fold in HBG mRNA levels above the controls, respectively (p<0.05). In adult progenitor cells, SRT2104 at 500nM and 2uM induced a mean increase of 2.6-fold and 4.1-fold HBG mRNA respectively (p<0.05), and induced an increase of 2.7% and 6.5% HbF-cells respectively (p<0.01). g-Globin was increased 5-fold and 3-fold by exposure to SRT2104 at 2uM and SRT1720 at 2.5uM respectively. Incubation of HUDEP-1 cells with 500nM and 2uM SRT2104 induced HBG expression 3.3-fold (p<0.01) and 6.2-fold (p<0.05) respectively; SRT1720 at 2.5uM induced HBG expression 8.4-fold (p<0.01), compared with controls. F-cells were elevated by 15.0 % and 17.4 % with SRT2104 treatment at 500nM and 2uM respectively (p<0.05), compared with controls. HUDEP-2 cells treated with SRT2104 at 500 nM and 2 uM had 1.8-fold and 2.5-fold induction of HBG expression (p<0.05). Treatment with SRT1720 at 2.5uM induced HBG expression 4.2-fold (p<0.05) compared with controls. These findings show that SIRT1 is an HBG inducer and that SIRT1 activators can reactivate silenced HBG expression in adult progenitor cells.SIRT1 binds in the β-globin gene cluster locus control region (LCR) and HBG promoters, and promotes the looping of the LCR to HBG promoter, and increases the binding of RNA polymerase II and H4K16Ac in the HBG promoter. SIRT1 suppressed the expression of the HBG suppressors BCL11A, KLF1, HDAC1 and HDAC2 . SIRT1 did not change the proliferation of human erythroid progenitor cells or the expression of differentiation marker CD235a.Conclusions: SIRT1 is an activator of HBG transcription. Our data suggest that SIRT1 activates HBG expression through facilitating LCR looping to the HBG promoter, inhibiting the expression of transcriptional suppressors of HBG, and indirectly increasing histone acetylation in the HBG promoter. SIRT1 is a potential therapeutic target for γ-globin induction, and small molecule SIRT1 activators might serve as a lead compound for the development of new HbF inducers. DisclosuresNo relevant conflicts of interest to declare.

Modulators of histone demethylase JMJD1C selectively target leukemic stem cells.

Leukemic stem cells (LSCs) comprise a very rare cell population that results in the development of acute myeloid leukemia. The selective targeting of drivers in LSCs with small molecule inhibitors holds promise for treatment of acute myeloid leukemia. Recently, we reported the identification of inhibitors of the histone lysine demethylase JMJD1C that preferentially kill MLL rearranged acute leukemia cells. Here, we report the identification of jumonji domain modulator #7 (JDM-7). Surface plasmon resonance analysis showed that JDM-7 binds to JMJD1C and its family homolog JMJD1B. JDM-7 did not significantly suppress cell proliferation in liquid cell culture at higher doses, although it led to a significant decrease in semi-solid colony formation experiments at lower concentrations. Moreover, low doses of JDM-7 did not suppress the proliferation of erythroid progenitor cells. We identified that JDM-7 downregulates the LSC self-renewal gene HOXA9 in leukemia cells. We further found that the structure of JDM-7 is similar to that of tadalafil, a drug approved by the US Food and Drug Administration. Molecular docking and surface plasmon resonance analysis showed that tadalafil binds to JMJD1C. Moreover, similar to JDM-7, tadalafil suppressed colony formation of leukemia cells in semi-solid cell culture at a concentration that did not affect primary umbilical cord blood cells. In summary, we have identified JDM-7 and tadalafil as potential JMJD1C modulators that selectively inhibit the growth of LSCs.

Erythropoietin attenuates high glucose-induced oxidative stress and inhibition of osteogenic differentiation in periodontal ligament stem cell (PDLSCs)

Diabetes mellitus and periodontitis have long been considered to be biologically linked. Erythropoietin (EPO) has multiple biological functions, such as stimulating the proliferation and differentiation of erythroid progenitor cells and reducing glucose-induced oxidative stress via different mechanisms, acting as a direct antioxidant. The purposes of the study to examine the anti-oxidative effect of EPO on reducing high glucose-induced oxidative stress of hPDLSCs and provide a better understanding of the mechanism of these processes. PDLSCs were induced by highglucose (HG, 30 mM) in the presence or absence of EPO. Cell proliferation was measured by MTT assay. The reactiveoxygen species (ROS) level, malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were detected to evaluate oxidative stress. qRT-PCR and western blot analysis were used to examine the expression of osteogenic related genes and protein (Runx2 and Osterix). Alizarin Red-S staining was used to detect mineralized nodule formation. The results showed that EPO promote the proliferation of PDLSCs, which was suppressed by high glucose (30 mM). Moreover, EPO attenuated high glucose (30 mM) induced oxidative stress by reducing the levels of ROS and MDA, and increasing the SOD activity. Furthermore,EPO alleviate high glucose(30 mM) induced suppression of osteogenic differentiation ability in PDLSCs, as evidenced by the up-regulated mRNA and protein expression of Runx2 and Osterix and increased ALP activity. In conclusion, EPO attenuates high glucose-induced oxidative stress, inhibitory effect of proliferation and inhibition of osteogenic differentiation in periodontal ligament stem cell (PDLSCs).

SIRT1 activates the expression of fetal hemoglobin genes.

High fetal hemoglobin (HbF, α2 γ2 ) levels ameliorate the clinical manifestations of sickle cell disease and β thalassemia. The mechanisms that repress HbF expression and silence γ-globin genes in adults are incompletely characterized and only a single HbF inducer, hydroxyurea, is approved for treatment, and only in patients with sickle cell disease. We identified SIRT1, a protein deacetylase, as a new inducer of γ-globin. SIRT1 knockdown decreased, while SIRT1 ectopic expression upregulated γ-globin gene (HBG) expression in primary human erythroid cells and in K562 cells. The small molecule SIRT1 activators SRT2104 and SRT1720 enhanced HBG expression in cord blood human erythroblasts and reactivated silenced HBG in adult human erythroblasts. Furthermore, SIRT1 binds in the β-globin gene cluster locus control region (LCR) and HBG promoters, promotes the looping of the LCR to HBG promoter, and increases the binding of RNA polymerase II and H4K16Ac in the HBG promoter. SIRT1 suppressed the expression of the HBG suppressors BCL11A, KLF1, HDAC1 and HDAC2. Lastly, SIRT1 did not change the proliferation of human erythroid progenitor cells or the expression of differentiation marker CD235a. These data suggest that SIRT1 activates HBG expression through facilitating LCR looping to the HBG promoter, inhibiting the expression of transcriptional suppressors of HBG, and indirectly increasing histone acetylation in the HBG promoter. SIRT1 is a potential therapeutic target for γ-globin gene induction, and small molecule SIRT1 activators might serve as a lead compound for the development of new HbF inducers.

Visualization of 57Fe-Labeled Heme Isotopic Fine Structure and Localization of Regions of Erythroblast Maturation in Mouse Spleen by MALDI FTICR-MS Imaging

Epoetin beta pegol (continuous erythropoiesis receptor activator; C.E.R.A.), or methoxy-polyethylene glycol-modified epoetin beta, is a long-acting erythropoiesis stimulating agent (ESA) that effectively maintains hemoglobin levels. It promotes proliferation of erythroid progenitor cells in hematopoietic organs and leads to increased reticulocyte and hemoglobin levels. However, the detailed erythropoietic effects of various ESAs on their target organs have yet to be clarified, and new approaches are needed to analyze tissue iron localization with structural information. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) techniques are widely used in basic pharmaceutical research. High-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) imaging enables the spatial mapping and identification of biomolecules. In this study, mice administered with C.E.R.A. were fed a diet containing the stable iron isotope 57Fe. The 57Fe-heme+ isotopic fine structure peak (m/z 617.1772) was separated from the non-labeled heme+ isotopic peak (Δ0.0029) by FTICR-MS with a resolving power of more than 500,000. We optimized the platform to analyze the distribution of 57Fe-heme in the spleen using MALDI FTICR-MS imaging. The combination of the ultrahigh resolution power of FTICR-MS and a stable isotope labeling technique has the potential to be very effective in basic pharmaceutical research.Graphical ᅟ

Direct targets of pSTAT5 signalling in erythropoiesis

Erythropoietin (EPO) acts through the dimeric erythropoietin receptor to stimulate proliferation, survival, differentiation and enucleation of erythroid progenitor cells. We undertook two complimentary approaches to find EPO-dependent pSTAT5 target genes in murine erythroid cells: RNA-seq of newly transcribed (4sU-labelled) RNA, and ChIP-seq for pSTAT5 30 minutes after EPO stimulation. We found 302 pSTAT5-occupied sites: ~15% of these reside in promoters while the rest reside within intronic enhancers or intergenic regions, some >100kb from the nearest TSS. The majority of pSTAT5 peaks contain a central palindromic GAS element, TTCYXRGAA. There was significant enrichment for GATA motifs and CACCC-box motifs within the neighbourhood of pSTAT5-bound peaks, and GATA1 and/or KLF1 co-occupancy at many sites. Using 4sU-RNA-seq we determined the EPO-induced transcriptome and validated differentially expressed genes using dynamic CAGE data and qRT-PCR. We identified known direct pSTAT5 target genes such as Bcl2l1, Pim1 and Cish, and many new targets likely to be involved in driving erythroid cell differentiation including those involved in mRNA splicing (Rbm25), epigenetic regulation (Suv420h2), and EpoR turnover (Clint1/EpsinR). Some of these new EpoR-JAK2-pSTAT5 target genes could be used as biomarkers for monitoring disease activity in polycythaemia vera, and for monitoring responses to JAK inhibitors.

Anemia of chronic&#x0D; diseases as a systemic manifestation of chronic pulmonary obstructive disease

Anemia of chronic disease (ACD) is one of the most frequent forms of anemia is often observed in patients with infections, cancer and chronic inflammatory or autoimmune diseases. The underlying mechanisms are complex and include dysregulation of iron homeostasis and erythropoietin production, impaired proliferation of erythroid progenitor cells and reduced life span of red blood cells. Moreover, ACD is often superimposed by malnutrition, bleeding and renal failure. ACD is mediated through inflammatory cytokines and characterized by low serum iron (hypoferremia) and often increased reticuloendothelial stores of iron. ACD is usually normocytic, normochromic anemia, but it can become microcytic and hypochromic as the disease progresses. Hepcidin, the main regulator of iron homeostasis and its synthesis, is inhibited by iron deficiency and stimulated by inflammation. In many patients the disease is associated with several extrapulmonary manifestations regarded as the expression of the systemic inflammatory state of chronic obstructive pulmonary disease (COPD). Recent studies showed that anemia in patients with COPD is more frequent than expected, with its prevalence ranging from 8 to 33%. Systemic inflammation may be an important pathogenic factor, but anemia in COPD can also be the result of a number of factors, such as the treatment with certain drugs (angiotensin-converting enzyme inhibitors or theophylline), endocrine disorders, acute exacerbations and oxygen therapy. Anemia in COPD patients is strongly associated with increased functional dyspnea, decreased exercise capacity and is an independent predictor of mortality. Treatment options to correct anemia used in other chronic diseases, such as congestive heart failure, cancer or chronic kidney disease have not been explored in COPD (i.e. erythropoietic agents, iron supplements or combined therapy). It is not known whether treating the underlying inflammation could improve hematological characteristics. It is important to develop basic diagnostic modalities for this group of patients and formulate methods of anemia correction.

Effect of Tumor Necrosis Factor-Alpha on Erythropoietin and Erythropoietin Receptor-Induced Erythroid Progenitor Cell Proliferation in β-Thalassemia/Hemoglobin E Patients.

Objective:Thalassemia is one of the genetic diseases that cause anemia and ineffective erythropoiesis. Increased levels of several inflammatory cytokines have been reported in β-thalassemia and might contribute to ineffective erythropoiesis. However, the mechanism by which tumor necrosis factor-alpha (TNF-α) is involved in ineffective erythropoiesis in thalassemic patients remains unclear. The objective of this study is to investigate the effect of TNF-α on the erythropoietin (EPO) and erythropoietin receptor (EPOR) expression involved in proliferation of β-thalassemia/hemoglobin (Hb) E erythroid progenitor cells compared with cells from healthy subjects.Materials and Methods:CD34-positive cells were isolated from heparinized blood by using the EasySep® CD34 selection kit. Cells were then cultured with suitable culture medium in various concentrations of EPO for 14 days. The effect of TNF-α on percent cell viability was analyzed by trypan blue staining. In addition, the percentage of apoptosis and levels of EPOR protein were measured by flow cytometry.Results:Upon EPO treatment, a higher cell number was observed for erythroid progenitor cells from both healthy participants and β-thalassemia/Hb E patients. However, a reduction of apoptosis was found in EPO-treated cells especially for β-thalassemia/Hb E patients. Interestingly, TNF-α caused higher levels of cell apoptosis and lower levels of EPOR protein in thalassemic erythroid progenitor cells.Conclusion:TNF-α caused a reduction in the level of EPOR protein and EPO-induced erythroid progenitor cell proliferation. It is possible that TNF-α could be involved in the mechanism of ineffective erythropoiesis in β-thalassemia/Hb E patients.

Epobis is a Nonerythropoietic and Neuroprotective Agonist of the Erythropoietin Receptor with Anti-Inflammatory and Memory Enhancing Effects.

The cytokine erythropoietin (EPO) stimulates proliferation and differentiation of erythroid progenitor cells. Moreover, EPO has neuroprotective, anti-inflammatory, and antioxidative effects, but the use of EPO as a neuroprotective agent is hampered by its erythropoietic activity. We have recently designed the synthetic, dendrimeric peptide, Epobis, derived from the sequence of human EPO. This peptide binds the EPO receptor and promotes neuritogenesis and neuronal cell survival. Here we demonstrate that Epobis in vitro promotes neuritogenesis in primary motoneurons and has anti-inflammatory effects as demonstrated by its ability to decrease TNF release from activated AMJ2-C8 macrophages and rat primary microglia. When administered systemically Epobis is detectable in both plasma and cerebrospinal fluid, demonstrating that the peptide crosses the blood-brain barrier. Importantly, Epobis is not erythropoietic, but systemic administration of Epobis in rats delays the clinical signs of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, and the peptide has long-term, but not short-term, effects on working memory, detected as an improved social memory 3 days after administration. These data reveal Epobis to be a nonerythropoietic and neuroprotective EPO receptor agonist with anti-inflammatory and memory enhancing properties.

ОСОБЛИВОСТІ ПРОЛІФЕРАЦІЇ ТА ДИФЕРЕНЦІЮВАННЯ КЛІТИН-ПОПЕРЕДНИКІВ ЕРИТРОПОЕЗУ ПРИ ХРОНІЧНІЙ МІЄЛОЇДНІЙ ЛЕЙКЕМІЇ В КУЛЬТУРІ КЛІТИН IN VIVO

The article presents a study of proliferation and differentiation features of erythroid progenitor cells of patients with chronic myeloid leukemia during the treatment with tyrosine kinase inhibitors – imatinib and nilotinib. The cultivation results showed an increase in proliferative activity of erythropoiesis progenitor cells both in the case of patients with leukemia, diagnosed for the first time, and in the case of bone marrow samples of patients with a resistance of leukemic cells clone to treatment with tyrosine kinase inhibitors. Moreover, the results showed an inhibition of erythroid progenitor cell differentiation and acquisition of resistance to tyrosine kinase inhibitors by leukemic cells clone.