Erythropoietin Stimulation Research Articles

Activating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entry

BackgroundCalcium (Ca2+) signaling regulates various vital cellular functions, including integrin activation and cell migration. Store-operated calcium entry (SOCE) via calcium release-activated calcium (CRAC) channels represents a major pathway for Ca2+ influx from the extracellular space in multiple cell types. The impact of JAK2-V617F and CALR mutations which are disease initiating in myeloproliferative neoplasms (MPN) on SOCE, calcium flux from the endoplasmic reticulum (ER) to the cytosol, and related key signaling pathways in the presence or absence of erythropoietin (EPO) or thrombopoietin (TPO) is poorly understood. Thus, this study aimed to elucidate the effects of these mutations on the aforementioned calcium dynamics, in cellular models of MPN.MethodsIntracellular Ca2+ levels were measured over a time frame of 0–1080 s in Fura-2 AM labeled myeloid progenitor 32D cells expressing various mutations (JAK2-WT/EpoR, JAK2-V617F/EpoR; CALR-WT/MPL, CALR-ins5/MPL, and del52/MPL). Basal Ca2+ concentrations were assessed from 0–108 s. Subsequently, cells were stimulated with EPO/TPO in Ca2+-free Ringer solution, measuring Ca2+ levels from 109–594 s (store depletion). Then, 2 mM of Ca2+ buffer resembling physiological concentrations was added to induce SOCE, and Ca2+ levels were measured from 595–1080 s. Fura-2 AM emission ratios (F340/380) were used to quantify the integrated Ca2+ signal. Statistical significance was assessed by unpaired Student's t-test or Mann–Whitney-U-test, one-way or two-way ANOVA followed by Tukey's multiple comparison test.ResultsFollowing EPO stimulation, the area under the curve (AUC) representing SOCE significantly increased in 32D-JAK2-V617F cells compared to JAK2-WT cells. In TPO-stimulated CALR cells, we observed elevated Ca2+ levels during store depletion and SOCE in CALR-WT cells compared to CALR-ins5 and del52 cells. Notably, upon stimulation, key components of the Ca2+ signaling pathways, including PLCγ-1 and IP3R, were differentially affected in these cell lines. Hyper-activated PLCγ-1 and IP3R were observed in JAK2-V617F but not in CALR mutated cells. Inhibition of calcium regulatory mechanisms suppressed cellular growth and induced apoptosis in JAK2-V617F cells.ConclusionsThis report highlights the impact of JAK2 and CALR mutations on Ca2+ flux (store depletion and SOCE) in response to stimulation with EPO and TPO. The study shows that the JAK2-V617F mutation strongly alters the regulatory mechanism of EpoR/JAK2-dependent intracellular calcium balance, affecting baseline calcium levels, EPO-induced calcium entry, and PLCγ-1 signaling pathways. Our results reveal an important role of calcium flux in the homeostasis of JAK2-V617F positive cells.

The Role of Zinc and Copper in Anemia and Erythropoietin Responsiveness in Patients on Hemodialysis and Peritoneal Dialysis

Introduction: Imbalance in copper (Cu) and zinc (Zn) homeostasis is a frequent finding in patients undergoing dialysis. These patients have higher serum Cu and lower serum Zn levels compared to controls. This study is designed to investigate the relationship between blood Cu and Zn levels and Cu/Zn ratio and, ESA (erythropoietin stimulating agent) dosage, erythropoietin responsiveness and anemia in dialysis patients. Methods: Clinical and demographic data for 151 participants (men=79) with a median dialysis vintage of 69.7 months [101 on Hemodialysis (HD), 50 on Peritoneal Dialysis (PD)] and mean age of 56.25±15.6 years was collected in November 2022. Zn and Cu levels were measured with atomic absorption. Erythropoietin responsiveness was evaluated by the erythropoietin resistance index (ERI) and ESA dosing. Results: In HD patients, mean serum Zn and Cu levels were 61.33 ± 15.77 μg/dL and 95.1 ± 21.1 µg/dL, respectively, with a Cu/Zn ratio of 1.58 ± 0.69. In PD patients, mean serum Zn and Cu levels were 54.8 ± 10.3 µg/dL and 97 ± 20.2 µg/dL, respectively, with a Cu/Zn ratio of 1.72 ± 0.55. In Spearman analyses serum Cu was negatively correlated with hemoglobin (p=0.018), serum iron (p=0.03), transferrin saturation (p=0.019), and iron supplementation (p <0.01) in the HD patient group. Serum Cu was found to be positively correlated with erythropoietin (EPO) dosage (p=0.03), ERI (p=0.04), high sensitive C reactive protein (hsCRP) (p=0.01), and Kt/V (p=0.033). Cu/Zn ratio was positively correlated with EPO dosage (p=0.043), ERI (p=0.051) and hsCRP (p=0.015). Conversely, it was negatively correlated with iron supplementation (p=0.014) and serum albumin levels (p=0.049). Serum Zn levels was solely correlated with albumin level (p=0.039). In PD, patient’s serum Cu level was correlated positively with hsCRP (p=0.036); serum Zn levels correlated positively (p=0.01), and Cu/Zn ratio was correlated negatively with albumin (p=0.042). Conclusion: This study has shown a possible association between both serum Cu and Cu/Zn ratio, ESA dosage and hsCRP in HD patients.

Role of Cytokine-Inducible SH2 Domain-Containing (CISH) Protein in the Regulation of Erythropoiesis.

The cytokine-inducible SH2 domain-containing (CISH) protein was the first member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators discovered, being identified in vitro as an inducible inhibitor of erythropoietin (EPO) signaling. However, understanding of the physiological role played by CISH in erythropoiesis has remained limited. To directly assess the function of CISH in this context, mice deficient in CISH were characterized with respect to developmental, steady-state, and EPO-induced erythropoiesis. CISH was strongly expressed in the fetal liver, but CISH knockout (KO) mice showed only minor disruption of primitive erythropoiesis. However, adults exhibited mild macrocytic anemia coincident with subtle perturbation particularly of bone marrow erythropoiesis, with EPO-induced erythropoiesis blunted in the bone marrow of KO mice but enhanced in the spleen. Cish was expressed basally in the bone marrow with induction following EPO stimulation in bone marrow and spleen. Overall, this study indicates that CISH participates in the control of both basal and EPO-induced erythropoiesis in vivo.

FRI431 Erythrocytosis Associated With Oral Testosterone Replacement

Abstract Disclosure: A.A. Engel: None. J.L. Sarvaideo: None. Background: A growing prevalence in male hypogonadism has led to increased interest in the most common adverse event, erythrocytosis. Defined by hematocrit (HCT) > 49-51%, the incidence varies depending on the testosterone formulation.3 The formulations with the lowest risk of erythrocytosis are intranasal testosterone (0-2%) and oral testosterone (0.03%).3 A phase 3 clinical trial of testosterone undecanoate (Jatenzo) reported 4.8% patients experienced an increase in hematocrit, although not severe enough to warrant therapy discontinuation.2 We present a case of erythrocytosis with oral testosterone undecanoate that prompted a change in therapy. Clinical Case: Our patient is a 55-year-old male with secondary hypogonadism potentially related to obesity (BMI 37 kg/m²). AM total testosterone (TT) 151 (348-1197 ng/dL), free testosterone 4.6 pg/mL (6.8-21.5 pg/mL) and LH 3.8 mIU/mL. Labs confirmed. Secondary work-up negative. HCT 43.6% prior to any therapy. Patient treated with injectable testosterone. Despite decreasing to testosterone cypionate 80 mg weekly his HCT increased to 52.7% with a TT of 544 ng/dL. He was switched to oral testosterone undecanoate 237 mg twice daily given presumably lower risk of erythrocytosis. However, HCT increased to 55.1% and TT to 1542 ng/dL. Patient donated blood and oral testosterone dose decreased to 158 mg twice daily with starting HCT 48.9%, but this increased to 53.7%. TT 522 ng/dL. Therapy stopped with improvement of HCT to 45.8%-48.9%, TT 192 ng/dL, and SHBG 20 g/dL. Erythropoietin remained normal. Patient does not smoke cigarettes nor has OSA. Conclusion: We present a case of oral testosterone undecanoate complicated by persistent erythrocytosis despite dose reduction. Testing for contributing etiology was unrevealing. Oral testosterone replacement is considered the formulation least likely to cause erythrocytosis. Certain patient characteristics like OSA, advanced age, obesity, type II diabetes mellitus and an elevated HCT > 50% can predict the likelihood of erythrocytosis although its specific etiology is unclear.3 Proposed mechanisms include elevation in dihydrotestosterone, erythropoietin stimulation, suppression of hepcidin and relation to androgen receptor CAG repeat length.1 Providers should be aware of the possibility of erythrocytosis on oral testosterone. Further research is needed to determine predictive characteristics for erythrocytosis and etiology. Reference: 1. Aghazadeh, M, et al. Elevated dihydrotestosterone is associated with testosterone induced erythrocytosis. J Urol. 2015; 194(1): 160-165. 2. Swerdloff, S, et al. A new oral testosterone undecanoate formulation restores testosterone to normal concentrations in hypogonadal men. J Clin Endocrinol Metab. 2020; 105)8): 1-17. 3. White, J, et al. Testosterone therapy and secondary erythrocytosis. IJIR: Your Sexual Medicine Journal. 2022; 34: 693-697. Presentation: Friday, June 16, 2023

BK viraemia as a cause of anaemia in after ABO-incompatible liver transplant: a case report

BackgroundWhile anaemia following liver transplant is common, anaemia in the context of BK viraemia is not a commonly recognised phenomenon.Case presentationWe present the case of 59-year old gentleman with severe anaemia in the context of BK viraemia and nephropathy following ABO incompatible liver transplant. Severity of anaemia appeared to correlate with high titres of BK virus in the serum. Bone marrow biopsy revealed hypocellular marrow with normal cytogenetics. Anaemia improved with treatment with cidofovir, intravenous immunoglobulin, reduction in immunosuppression and erythropoietin stimulating agent.ConclusionTo our knowledge, this is the first case of anaemia post liver transplant contributed to by BK viraemia.

#5534 HIFS AND BUTS: A NETWORK META-ANALYSIS OF HIF-PH INHIBITORS AND ESAS IN NDD-CKD PATIENTS WITH ANEMIA COMPARING THEIR SAFETY AND EFFICACY

Abstract Background and Aims Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are a new class of oral medicines for the management of anemia in CKD patients. These agents are found to be efficacious in the management of renal anemia, however safety is still under scrutiny. While, ESAs (Erythropoietin stimulating agents) are available since long with a well-established clinical profile. This network meta-analysis aimed to compare the safety and efficacy of HIF-PHI vs ESA in CKD patients with anemia not undergoing dialysis. Method An electronic database search was carried out in EBM (Evidence Based Medicine) Reviews, Cochrane Library and PubMed from inception to July 2022 for phase III clinical trials comparing six different HIF-PHIs and ESA for treating anemia in non-dialysis-dependent (NDD) CKD patients. The outcomes included the serious adverse events (SAEs) and change in hemoglobin (Hb) levels. Results Total 163 records were identified out of which 6 studies involving a total of 6847 patients were eligible for analysis. Compared to ESA, all HIF-PHIs increased risk for SAEs (daprodustat of RR, 1.21[95% CI, 0.825–1.77]; desidustat, 1.38[95% CI, 0.695–2.76]; enarodustat, 1.29[95% CI, 0.568–3.02]; molidustat, 1.02[95% CI, 0.585–1.74]; roxadustat, 1.29[95% CI, 0.674–2.57] and vadadustat, 1.01[95% CI, 0.693–1.47]) although these differences were statistically non-significant. When change in Hb was analyzed, there was no statistically significant difference between the two groups. The mean difference in change in HB with various HIFs as compared to ESA were: daprodustat (MD: 0.0796, 95% CI −0.676–0.838), desidustat (MD: 0.120, 95% CI: −0.626–0.883), enarodustat (MD: -0.600, 95% CI: −1.36–0.157), molidustat (MD: -0.309, 95% CI: −1.07–0.449), roxadustat (MD: -0.0702, 95% CI: −0.831–0.692) and vadadustat (MD: -0.0402, 95% CI: −0.802–0.720). Conclusion In terms of safety, HIF PHIs were associated with more SAEs compared to ESA although these differences are not statistically significant. Comparing efficacy, HIF-PHIs and ESA both effectively increase Hb level in NDD-CKD patients without any significant difference. Results of our network meta-analysis suggest the need for larger and long-term studies comparing HIF-PHIs with ESAs to have better understanding of their comparative efficacy and safety profiles.

Exogenous ketosis elevates circulating erythropoietin and stimulates muscular angiogenesis during endurance training overload.

De novo capillarization is a primary muscular adaptation to endurance exercise training and is crucial to improving performance. Excess training load, however, impedes such beneficial adaptations, yet we recently demonstrated that such downregulation may be counteracted by ketone ester ingestion (KE) post-exercise. Therefore, we investigated whether KE could increase pro-angiogenic factors and thereby stimulate muscular angiogenesis during a 3-week endurance training-overload period involving 10 training sessions/week in healthy, male volunteers. Subjects received either 25g of a ketone ester (KE, n=9) or a control drink (CON, n=9) immediately after each training session and before sleep. In KE, but not in CON, the training intervention increased the number of capillary contacts and the capillary-to-fibre perimeter exchange index by 44% and 42%, respectively. Furthermore, KE also substantially increased vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) expression both at the protein and at the mRNA level. Serum erythropoietin concentration was concomitantly increased by 26%. Conversely, in CON the training intervention increased only the protein content of eNOS. These data indicate that intermittent exogenous ketosis during endurance overload training stimulates muscular angiogenesis. This likely resulted from a direct stimulation of muscle angiogenesis, which may be at least partly due to stimulation of erythropoietin secretion and elevated VEGF activity, and/or an inhibition of the suppressive effect of overload training on the normal angiogenic response to training. This study provides novel evidence to support the potential of exogenous ketosis to benefit endurance training-induced muscular adaptation. KEY POINTS: Increased capillarization is a primary muscular adaptation to endurance exercise training. However, excess training load may impede such response. We previously observed that intermittent exogenous ketosis by post-exercise and pre-sleep ketone ester ingestion (KE) counteracted physiological dysregulations induced by endurance overload training. Therefore, we investigated whether KE could increase pro-angiogenic factors thereby stimulating muscular angiogenesis during a 3-week endurance training overload period. We show that the overload training period in the presence, but not in the absence, of KE markedly increased muscle capillarization (+40%). This increase was accompanied by higher circulating erythropoietin concentration and stimulation of the pro-angiogenic factors vascular endothelial growth factor and endothelial nitric oxide synthase in skeletal muscle. Collectively, our data indicate that intermittent exogenous ketosis may evolve as a potent nutritional strategy to facilitate recovery from strenuous endurance exercise, thereby stimulating beneficial muscular adaptations.

Kindlin-3 deficiency leads to impaired erythropoiesis and erythrocyte cytoskeleton.

Kindlin-3 (K3) is critical for the activation of integrin adhesion receptors in hematopoietic cells. In humans and mice, K3 deficiency is associated with impaired immunity and bone development, bleeding, and aberrant erythrocyte shape. To delineate how K3 deficiency (K3KO) contributes to anemia and misshaped erythrocytes, mice deficient in erythroid (K3KO∖EpoR-cre) or myeloid cell K3 (K3KO∖Lyz2cre), knockin mice expressing mutant K3 (Q597W598 to AA) with reduced integrin-activation function (K3KI), and control wild-type (WT) K3 mice were studied. Both K3-deficient strains and K3KI mice showed anemia at baseline, reduced response to erythropoietin stimulation, and compromised recovery after phenylhydrazine (PHZ)-induced hemolytic anemia as compared with K3WT. Erythroid K3KO and K3 (Q597W598 to AA) showed arrested erythroid differentiation at proerythroblast stage, whereas macrophage K3KO showed decreased erythroblast numbers at all developmental stages of terminal erythroid differentiation because of reduced erythroblastic island (EBI) formation attributable to decreased expression and activation of erythroblast integrin α4β1 and macrophage αVβ3. Peripheral blood smears of K3KO∖EpoR-cre mice, but not of the other mouse strains, showed numerous aberrant tear drop-shaped erythrocytes. K3 deficiency in these erythrocytes led to disorganized actin cytoskeleton, reduced deformability, and increased osmotic fragility. Mechanistically, K3 directly interacted with F-actin through an actin-binding site K3-LK48. Taken together, these findings document that erythroid and macrophage K3 are critical contributors to erythropoiesis in an integrin-dependent manner, whereas F-actin binding to K3 maintains the membrane cytoskeletal integrity and erythrocyte biconcave shape. The dual function of K3 in erythrocytes and in EBIs establish an important functional role for K3 in normal erythroid function.

Mechanisms of enhanced renal and hepatic erythropoietin synthesis by sodium-glucose cotransporter 2 inhibitors.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce the risk of major heart failure events, an action that is statistically linked to enhanced erythropoiesis, suggesting that stimulation of erythropoietin and cardioprotection are related to a shared mechanism. Four hypotheses have been proposed to explain how these drugs increase erythropoietin production: (i) renal cortical reoxygenation with rejuvenation of erythropoietin-producing cells; (ii) counterregulatory distal sodium reabsorption leading to increased tubular workload and oxygen consumption, and thus, to localized hypoxia; (iii) increased iron mobilization as a stimulus of hypoxia-inducible factor-2α (HIF-2α)-mediated erythropoietin synthesis; and (iv) direct HIF-2α activation and enhanced erythropoietin gene transcription due to increased sirtuin-1 (SIRT1) signaling. The first two hypotheses assume that the source of increased erythropoietin is the interstitial fibroblast-like cells in the deep renal cortex. However, SGLT2 inhibitors do not alter regional tissue oxygen tension in the non-diabetic kidney, and renal erythropoietin synthesis is markedly impaired in patients with anemia due to chronic kidney disease, and yet, SGLT2 inhibitors produce an unattenuated erythrocytic response in these patients. This observation raises the possibility that the liver contributes to the production of erythropoietin during SGLT2 inhibition. Hypoxia-inducible factor-2α and erythropoietin are coexpressed not only in the kidney but also in hepatocytes; the liver is a major site of production when erythropoietin stimulation is maintained for prolonged periods. The ability of SGLT2 inhibitors to improve iron mobilization by derepressing hepcidin and ferritin would be expected to increase cytosolic ferrous iron, which might stimulate HIF-2α expression in both the kidney and liver through the action of iron regulatory protein 1. Alternatively, the established ability of SGLT2 inhibitors to enhance SIRT1 might be the mechanism of enhanced erythropoietin production with these drugs. In hepatic cell lines, SIRT1 can directly activate HIF-2α by deacetylation, and additionally, through an effect of SIRT in the liver, peroxisome proliferator-activated receptor-γ coactivator-1α binds to hepatic nuclear factor 4 to promote transcription of the erythropoietin gene and synthesis of erythropoietin. Since SIRT1 up-regulation exerts direct cytoprotective effects on the heart and stimulates erythropoietin, it is well-positioned to represent the shared mechanism that links erythropoiesis to cardioprotection during SGLT2 inhibition.

The novel GATA1-interacting protein HES6 is an essential transcriptional cofactor for human erythropoiesis.

Normal erythropoiesis requires the precise regulation of gene expression patterns, and transcription cofactors play a vital role in this process. Deregulation of cofactors has emerged as a key mechanism contributing to erythroid disorders. Through gene expression profiling, we found HES6 as an abundant cofactor expressed at gene level during human erythropoiesis. HES6 physically interacted with GATA1 and influenced the interaction of GATA1 with FOG1. Knockdown of HES6 impaired human erythropoiesis by decreasing GATA1 expression. Chromatin immunoprecipitation and RNA sequencing revealed a rich set of HES6- and GATA1-co-regulated genes involved in erythroid-related pathways. We also discovered a positive feedback loop composed of HES6, GATA1 and STAT1 in the regulation of erythropoiesis. Notably, erythropoietin (EPO) stimulation led to up-regulation of these loop components. Increased expression levels of loop components were observed in CD34+ cells of polycythemia vera patients. Interference by either HES6 knockdown or inhibition of STAT1 activity suppressed proliferation of erythroid cells with the JAK2V617F mutation. We further explored the impact of HES6 on polycythemia vera phenotypes in mice. The identification of the HES6-GATA1 regulatory loop and its regulation by EPO provides novel insights into human erythropoiesis regulated by EPO/EPOR and a potential therapeutic target for the management of polycythemia vera.

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

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

Enteral administration of a simulated amniotic fluid in preventing feeding intolerance in very low birthweight neonates: A randomized controlled trial

ObjectivesFeeding intolerance (FI) is a common finding in preterm neonates. Enteral administration of different forms of amniotic fluid (AF) has been tried for treating FI in high-risk neonates. Simulated amniotic fluid (SAF) is a solution with a similar electrolyte composition to human AF. The aim of this study was to examine whether enteral administration of SAF would improve feeding tolerance in very low birthweight (VLBW) neonates. MethodsForty VLBW neonates were randomized to either SAF or placebo (total daily dose 20 mL/kg/d–1 divided every 3 h) to their milk for a maximum of 7 d. Neonates with major congenital anomalies, those in whom early feeding was contraindicated, and those treated with parental erythropoietin and/or human granulocyte stimulating factor were excluded. The primary outcome was the total amount of enteral feeds reached by day 7. Secondary outcomes were incidence of FI and necrotizing enterocolitis (NEC). Study intervention was stopped on completing 7 d, reaching enteral feeds of 100 mL/kg/d–1, or the appearance of any sign of FI or NEC. ResultsAll neonates tolerated the test solution well. The SAF group reached significantly larger volume and higher calories on days 3 and 7 (P < 0.05 for all). No statistical differences were seen between the two groups in incidence of FI (P = 0.311), NEC (P = 0.429), mortality (P = 0.632), length of stay (P = 0.744), or weight gain on day 10 (P = 0.389). Baseline hematologic parameters showed no statistical differences before or after enteral administration (P > 0.05). ConclusionResults of the present study demonstrated that SAF solution might improve feeding tolerance in VLBW babies without evidence of its systemic absorption. Larger multicenter randomized studies are recommended.

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 Administration Delays B Cell Maturation and Promotes B Cell to Erythroid Transdifferentiation in Mice

Background: Erythropoietin (EPO) is a glycoprotein hormone which serves as a key regulator of erythropoiesis. Previous studies have shown that it also has a myriad of extraerythropoietic effects including on immune and skeletal systems. Specifically, it was shown that EPO reduces bone marrow (BM) B cell population, in part, by inducing an egress of IgM+ cells out of BM to the peripheral blood (PB). However, the effect of EPO on early B cells (IgM- Pro- and Pre-B cells) has not been studied. Moreover, a process of transdifferentiation of early B cells into erythroid cells under EPO stimulation has not been addressed. Methods: We used MB1-Cre;R26-eYFP mice for B-cell-specific lineage tracing as well as MB1-Cre; EPO-Rfl/fl and Osx-Cre; EPO-Rfl/fl mouse models for B-cell and osteoblast-specific EPO-R knockdown, respectively. Mice (10-12 week old females) were treated with either diluent or human recombinant EPO (hereafter, EPO) for one week (180IU x3/week). Immunophenotyping of BM cells was performed by multi-color flow cytometry (mFC) and immunofluorescence microscopy (IF) was used to demonstrate B cell-derived erythropoiesis in BM sections. Expression of PAX5 and IL-7 in the BM was determined by real-time quantitative PCR. Results: EPO treatment led to a significant 2.4-fold reduction in BM B cells (B220+/CD19+) manifested by a decrease in both IgM+ and IgM- cell fractions. While the IgM+ B cell fraction in PB after one week of EPO did not change, we did observe an almost 2-fold increase in IgM- B cells in EPO-treated as compared to diluent-treated mice (1.4±0.8% vs 0.7±0.5%, respectively, p<0.05). There was no change in IgM+ or IgM- B cells in the spleen, suggesting that IgM- B cells in PB originate from the BM. Concurring with these data, expression of CXCR4 adhesion molecule by BM Pro-B (IgM-) cells was significantly reduced (1.8-fold) upon EPO treatment. Since a decrease in BM B cells could not be solely explained by their egress from BM, we hypothesized that EPO induced a maturation delay at the level of Pre/Pro-B cell stages. Using both MB1-Cre; EPO-Rfl/fl and Osx-Cre; EPO-Rfl/fl mouse models we showed that this maturation delay is independent of EPO-EPO-R signaling in both early B cells and osteoblasts, i.e. following EPO treatment the amount and subpopulation distribution of BM B cells were similar in the control and corresponding EPO-R knockdown mice. However, we found significant perturbations in the key regulators of B cell lymphopoiesis in EPO-treated mice (EPO-R+/+): a 4-fold and 1.7-fold decrease in the expression of PAX5 and IL-7, respectively, while expression of IL-7 receptor by BM cells (both B220+ and B220-)was modestly but significantly reduced (as measured by mFC). Finally, applying IF on BM sections, we could detect eYFP+ BM cells (derived from MB1-Cre;R26-eYFP mice) that co-expressed the specific erythroid marker TER119 (Figure 1). These findings demonstrate that a fraction of BM B cells can transdifferentiate into erythroid cells upon EPO stimulation. Conclusions: Besides its effect on erythropoiesis, EPO compromises BM B cell compartment by a multifaceted mechanism, including BM egress, delay in maturation as well as transdifferentiation to the erythroid lineage. An understanding of the biological and clinical significance of this phenomenon requires further research. For example, the transdifferentiation might explain an improved erythropoiesis in patients with anemia who already have high EPO levels. This research was funded by Israel Science Foundation (ISF) grant number 1188/21 to DN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

An Ancient Transcriptional Hub Couples Developmentally Regulated Gene Expression with Metabolism during Erythropoiesis

The α-globin genes and their regulation by the α-globin superenhancer have been extensively characterised. An adjacent gene, Nprl3, contains 4 of the 5 α-globin enhancers in mice, and 3 of 4 in humans. The neighbouring genomic positions of Nprl3 and α-globin, which share a topologically associated domain, have been maintained for >500 million years. However, the potential functional requirement of this genomic linkage has not been investigated until now. Nprl3 is conserved across Animalia and Fungi, and has been characterised to provide negative regulation of mTORC1, a critical controller of cellular metabolism. Here we report phenotypes of Nprl3-deficient erythropoiesis in mouse fetal liver and bone marrow, and in human peripheral blood. We provide molecular mechanistic insight that Nprl3 is essential for optimal erythropoiesis and for responding to fluctuating nutrient and growth factor concentrations. Finally, we show that erythroid Nprl3 function depends on transcriptional support arising from the α-globin enhancers. Analysis of Nprl3-/- fetal livers revealed severe impairment of erythroid development. On day E13.5, erythroid cells fail to develop beyond the proerythroblast stage. Loss of Nprl3 is accompanied by elevated mTORC1 signalling, confirming the canonical signalling role of Nprl3 in erythroblasts. Active mTORC1 typically inhibits autophagy; in consistence with this, we observed suppressed autophagic activity in Nprl3-/- erythroblasts. We next investigated whether the erythroid defect results from haematopoietic-intrinsic Nprl3 requirements. This was tested using competitive bone marrow - fetal liver chimera experiments, in which Nprl3-/- fetal liver cells contributed poorly to erythroid lineage reconstitution of irradiated adult bone marrow. Thus, Nprl3 regulates baseline erythropoiesis in the mouse. To discover whether NPRL3 is also required for human erythropoiesis, NPRL3-knockout was induced in primary human CD34+ progenitors using a CRISPR-Cas9 RNP-editing system. These progenitors had reduced ability to produce enucleated erythroid cells compared to their negative control counterparts. NPRL3-knockout erythroblasts also demonstrated defective mTORC1 signalling responses to iron deficiency, amino acid withdrawal and erythropoietin stimulation. These results indicate that NPRL3 has a critical role in interpreting fluctuating nutritional environments, and in tuning the metabolic response of developing erythroid cells to the extracellular milieu. Importantly, Nprl3 expression is known to be elevated in erythroid cells, increasing ~30-fold during erythroid lineage commitment due to α-globin enhancer activity. We sought to discover whether this erythroid-specific increase in Nprl3 expression is required for the functionality outlined above. Using genetic engineering to delete the Nprl3 promoter on one allele, and to delete all α-globin enhancers on the other, we eliminated all interactions between Nprl3 and α-globin regulatory elements. Embryos heterozygous for both alleles (Nprl3+/-α-globin-enhancers+/-,'Nalph') retain non-erythroid 'un-enhanced' Nprl3 expression levels, and enhancer-regulated α-globin expression. Remarkably, at E13.5, the Nalph genotype presented impaired erythropoiesis reminiscent of that observed in Nprl3-/- embryos, with development inhibited at the same stage of differentiation. Thus, we conclude that the genomic contact hub Nprl3 shares with α-globin and its enhancers bestows the erythroid-specific transcriptional upregulation required for Nprl3 to perform its important erythropoietic role. This finding suggests that the deep evolutionary coupling of these two genes has enabled the α-globin enhancers to control metabolism, as well as α-globin production, in developing erythroid cells.

New insights and advances of sodium-glucose cotransporter 2 inhibitors in heart failure

Sodium-glucose cotransporter 2 inhibitors (SGLT2is) are newly emerging insulin-independent anti-hyperglycemic agents that work independently of β-cells. Quite a few large-scale clinical trials have proven the cardiovascular protective function of SGLT2is in both diabetic and non-diabetic patients. By searching all relevant terms related to our topics over the previous 3 years, including all the names of agents and their brands in PubMed, here we review the mechanisms underlying the improvement of heart failure. We also discuss the interaction of various mechanisms proposed by diverse works of literature, including corresponding and opposing viewpoints to support each subtopic. The regulation of diuresis, sodium excretion, weight loss, better blood pressure control, stimulation of hematocrit and erythropoietin, metabolism remodeling, protection from structural dysregulation, and other potential mechanisms of SGLT2i contributing to heart failure improvement have all been discussed in this manuscript. Although some remain debatable or even contradictory, those newly emerging agents hold great promise for the future in cardiology-related therapies, and more research needs to be conducted to confirm their functionality, particularly in metabolism, Na+-H+ exchange protein, and myeloid angiogenic cells.

Erythroblastic islands foster granulopoiesis in parallel to terminal erythropoiesis

AbstractThe erythroblastic island (EBI), composed of a central macrophage surrounded by maturing erythroblasts, is the erythroid precursor niche. Despite numerous studies, its precise composition is still unclear. Using multispectral imaging flow cytometry, in vitro island reconstitution, and single-cell RNA sequencing of adult mouse bone marrow (BM) EBI-component cells enriched by gradient sedimentation, we present evidence that the CD11b+ cells present in the EBIs are neutrophil precursors specifically associated with BM EBI macrophages, indicating that erythro-(myelo)-blastic islands are a site for terminal granulopoiesis and erythropoiesis. We further demonstrate that the balance between these dominant and terminal differentiation programs is dynamically regulated within this BM niche by pathophysiological states that favor granulopoiesis during anemia of inflammation and favor erythropoiesis after erythropoietin stimulation. Finally, by molecular profiling, we reveal the heterogeneity of EBI macrophages by cellular indexing of transcriptome and epitope sequencing of mouse BM EBIs at baseline and after erythropoietin stimulation in vivo and provide a searchable online viewer of these data characterizing the macrophage subsets serving as hematopoietic niches. Taken together, our findings demonstrate that EBIs serve a dual role as niches for terminal erythropoiesis and granulopoiesis and the central macrophages adapt to optimize production of red blood cells or neutrophils.

Kinome Analysis to Define Mechanisms of Adjuvant Action: PCEP Induces Unique Signaling at the Injection Site and Lymph Nodes.

Understanding the mechanism of action of adjuvants through systems biology enables rationale criteria for their selection, optimization, and application. As kinome analysis has proven valuable for defining responses to infectious agents and providing biomarkers of vaccine responsiveness, it is a logical candidate to define molecular responses to adjuvants. Signaling responses to the adjuvant poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP) were defined at the site of injection and draining lymph node at 24 h post-vaccination. Kinome analysis indicates that PCEP induces a proinflammatory environment at the injection site, including activation of interferon and IL-6 signaling events. This is supported by the elevated expression of proinflammatory genes (IFNγ, IL-6 and TNFα) and the recruitment of myeloid (neutrophils, macrophages, monocytes and dendritic cells) and lymphoid (CD4+, CD8+ and B) cells. Kinome analysis also indicates that PCEP’s mechanism of action is not limited to the injection site. Strong signaling responses to PCEP, but not alum, are observed at the draining lymph node where, in addition to proinflammatory signaling, PCEP activates responses associated with growth factor and erythropoietin stimulation. Coupled with the significant (p < 0.0001) recruitment of macrophages and dendritic cells to the lymph node by PCEP (but not alum) supports the systemic consequences of the adjuvant. Collectively, these results indicate that PCEP utilizes a complex, multi-faceted MOA and support the utility of kinome analysis to define cellular responses to adjuvants.

Absence of Erythropoietin and Cardio‐respiratory Responses to Hypobaric Hypoxia in Olfactory Receptor 78 Null mice

Hypobaric hypoxia (HH) evokes a series of physiological adaptations, including carotid body (CB)–dependent ventilatory acclimatization to hypoxia (VAH), elevated blood pressure (BP), and stimulation of erythropoietin (Epo) gene in the kidney. Olfactory receptor 78 (Olfr78) is a G‐protein coupled receptor that has been implicated in CB response to acute hypoxia. Given that both CB and kidney express Olfr78, we tested the hypothesis that Olfr78 plays a role in the cardio‐respiratory and renal Epo gene responses to HH. Studies were performed on wild type (WT) and Olfr78 null mice reared in room air (control) or exposed to 18h of HH (0.4 atmospheres). HH treated WT exhibited: 1) VAH manifested as increased baseline breathing, and enhanced hypoxic ventilatory response (HVR; 12% O2); 2) elevated BP and plasma norepinephrine levels; and 3) increased baseline CB sensory nerve activity and augmented CB sensory nerve response to subsequent acute hypoxia‐ all these responses to HH are either markedly attenuated or absent in Olfr78 null mice. WT mice responded to HH with activation of the renal Epo gene expression and elevated plasma Epo levels, and these effects were attenuated or absent in Olfr78 null mice. The attenuated Epo activation by HH was accompanied with markedly reduced hypoxia‐inducible factor (HIF)‐2α protein and reduced activation of HIF‐2 target gene Sod‐1 in Olfr78 null mice, suggesting impaired transcriptional activation of HIF‐2 contributes to attenuated Epo responses to HH. These results suggest a role for Olfr78 in physiological adaptations to HH experienced at high altitude

The HIFα-Stabilizing Drug Roxadustat Increases the Number of Renal Epo-Producing Sca-1+ Cells

Inhibition of the prolyl-4-hydroxylase domain (PHD) enzymes, leading to the stabilization of hypoxia-inducible factor (HIF) α as well as to the stimulation of erythropoietin (Epo) synthesis, is the functional mechanism of the new anti-anemia drug roxadustat. Little is known about the effects of roxadustat on the Epo-producing cell pool. To gain further insights into the function of PHD inhibitors, we characterized the abundance of mesenchymal stem cell (MSC)-like cells after roxadustat treatment of mice. The number of Sca-1+ mesenchymal cells following roxadustat treatment increased exclusively in the kidneys. Isolated Sca-1+ cells demonstrated typical features of MSC-like cells, including adherence to tissue culture plates, trilineage differentiation potential, and expression of MSC markers. Kidney-derived Sca-1+ MSC-like cells were cultured for up to 21 days. Within the first few days in culture, cells stabilized HIF-1α and HIF-2α and temporarily increased Epo production upon incubation in hypoxia. In summary, we have identified a Sca-1+ MSC-like cell population that is involved in renal Epo production and might contribute to the strong anti-anemic effect of the PHD inhibitor roxadustat.