Bone Marrow Sinusoidal Endothelial Cells Are a Site of Fgf23 Upregulation in Iron Deficiency Anemia

Iron deficiency anemia (IDA) has been identified as a potent stimulator of FGF23 (fibroblast growth factor 23), a phosphaturic hormone classically thought to be produced by bone-embedded osteocytes. Recently, both phlebotomy and erythropoietin administration have been shown to upregulate FGF23 production in bone marrow. However, the cell type(s) mediating FGF23 upregulation in states of perturbed erythropoiesis require further clarification. Tmprss6 -/- mice exhibit hepcidin elevation leading to systemic iron deficiency and iron-restricted anemia. We previously reported that Tmprss6-/- mice exhibit altered phosphate balance, elevated circulating FGF23, and Fgf23 mRNA upregulation in bone marrow but not cortical bone. Here, we clarify the sites of Fgf23 promoter activity in Tmprss6 -/- bone marrow using a reporter allele in which the enhanced green fluorescent protein (eGFP) coding sequence has been knocked into the endogenous Fgf23 locus. We generated Tmprss6 +/+,Tmprss6 +/-, and Tmprss6 -/- littermates of both sexes that carried either one (Fgf23 +/eGFP) or zero (Fgf23 +/+) copies of the reporter allele. Tmprss6-/- mice showed hyperhepcidinemia, hypoferremia, microcytic anemia, and tissue iron deficiency, which were not altered by heterozygous Fgf23 disruption (Figure 1A-C). By ELISA, Tmprss6-/- Fgf23 +/eGFP mice showed plasma levels of "total" FGF23 (intact, active hormone and C-terminal cleaved fragments) that remained markedly elevated compared to Tmprss6+/+ littermates (Figure 1D). Total FGF23 elevation in Tmprss6-/- Fgf23 +/eGFP mice was slightly less pronounced than Tmprss6-/- Fgf23 +/+ mice, suggesting an effect of Fgf23 gene dosage. In mice with 2 intact Fgf23 alleles, serum erythropoietin showed a strong linear correlation with plasma total FGF23. By confocal imaging, femurs of mice carrying the Fgf23 eGFP allele showed green fluorescence in vascular regions of the bone marrow but not in the bone cortex. Green fluorescence was more intense in Tmprss6-/- Fgf23+/eGFP mice than non-anemic controls. By flow cytometry of enzymatically digested bone marrow, we observed bright green fluorescence in a subset of endothelial cells (CD45 - Ter119 - CD31 +) exclusively in mice carrying the Fgf23 eGFP reporter allele (Figure 1E). The percentage of endothelial cells that were GFP bright was higher in Tmprss6-/- Fgf23 +/eGFP versus non-anemic mice. To clarify the endothelial cell subtype that expresses Fgf23, we mined published transcriptomic datasets from mice of normal iron balance and discovered higher Fgf23 mRNA in bone marrow sinusoidal endothelial cells compared to other bone marrow endothelial cell populations. Accordingly, we used anti-GFP immunohistochemistry in formalin-fixed bone marrow sections to assess Fgf23 eGFP reporter allele expression in the context of tissue architecture. Tmprss6-/- Fgf23 +/eGFP mice showed GFP expression in bone marrow sinusoidal endothelial cells, which was more intense than in non-anemic controls (Figure 1F). GFP reporter expression was also detected in rare cells of the thymus but not in liver, spleen, heart, muscle, or kidney. Collectively, our data reveal that bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in chronic IDA. Because IDA in Tmprss6-/- mice results from pathologic hepcidin elevation, we also sought to determine if bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in anemic mice with intact hepcidin regulation. We therefore subjected Fgf23 +/eGFP mice (with 2 intact Tmprss6 alleles) to a 500µl phlebotomy regimen (with saline volume replacement) known to induce marked anemia and hepcidin suppression. Compared to non-phlebotomized Fgf23 +/eGFP controls, phlebotomized Fgf23 +/eGFP mice showed severe anemia, elevated serum erythropoietin, and elevated plasma FGF23 18 hours after blood loss. Additionally, immunohistochemistry revealed more intense GFP expression in bone marrow sinusoidal endothelial cells of phlebotomized Fgf23 +/eGFP mice than non-phlebotomized controls. Taken together, our results show for the first time that bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in both acute and chronic anemia. Given the serum erythropoietin elevation in both models, our findings suggest that erythropoietin may act directly or indirectly on sinusoidal endothelial cells to promote FGF23 production during anemia. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Endogenous erythropoietin at birth is associated with neurodevelopmental morbidity in early childhood

Background New biomarkers that predict later neurodevelopmental morbidity are needed. This study evaluated the associations between umbilical cord serum erythropoietin (us-EPO) and neurodevelopmental morbidity by the age of 2–6.5 years in a Finnish cohort. Methods This study included 878 non-anomalous children born alive in 2012 to 2016 in Helsinki University Hospitals and whose us-EPO concentration was determined at birth. Data of these children were linked to data from the Finnish Medical Birth Register and the Finnish Hospital Discharge Register. Neurodevelopmental morbidity included cerebral palsy, epilepsy, intellectual disability, autism spectrum disorder, sensorineural defects, and minor neurodevelopmental disorders. Results In the cohort including both term and preterm children, us-EPO levels correlated with gestational age (r = 0.526) and were lower in premature children. High us-EPO levels (>100 IU/l) were associated with an increased risk of severe neurodevelopmental morbidity (OR: 4.87; 95% CI: 1.05–22.58) when adjusted for the gestational age. The distribution of us-EPO levels did not differ in children with or without the later neurodevelopmental diagnosis. Conclusions Although high us-EPO concentration at birth was associated with an increased risk of neurodevelopmental morbidity in early childhood, the role of us-EPO determination in clinical use appears to be minor. Impact We determined whether endogenous umbilical cord serum erythropoietin would be a new useful biomarker to predict the risk of neurodevelopmental morbidity. This study evaluated the role of endogenous erythropoietin at birth in neurodevelopmental morbidity with a study population of good size and specific diagnoses based on data from high-quality registers. Although high umbilical cord serum erythropoietin concentration at birth was associated with an increased risk of neurodevelopmental morbidity in early childhood, the clinical value of erythropoietin determination appears to be minor.

Low haptoglobin and a positive direct antiglobulin test without haemolysis in pregnancy

Haemolysis is typically associated with low haptoglobin and elevated reticulocyte count, lactate dehydrogenase and indirect bilirubin. Positive direct antiglobulin testing is consistent with autoimmune haemolysis. A case of anaemia in pregnancy with low haptoglobin levels and positive direct antiglobulin testing in a woman with systemic lupus erythematosus is presented. The lack of response to intravenous immune globulin and absence of other markers of haemolysis prompted further investigation. In the setting of mild renal dysfunction, the woman’s serum erythropoietin was inappropriately low consistent with a failure of erythropoietin response to anaemia, and the woman’s haemoglobin improved rapidly with darbopoietin therapy. darbepoetin Health professionals need to be aware of the possibility of low haptoglobin and positive direct antiglobulin testing in the absence of haemolysis with autoimmune disease and anticardiolipin antibodies, and the possibility of anaemia due to failure of erythropoietin response with mild renal dysfunction in pregnancy.

How Does 2016 WHO Criteria for Polycythemia Vera Contribute to Our Daily Practice? A Single-Center Study from Turkey

Background: We evaluated the frequency of subnormal erythropoietin levels, JAK2V617F positivity and polycythemia vera (PV) in patients who did not meet WHO 2008 criterion for hemoglobin levels but were suggested to be investigated for PV in 2016 revision. Materials and Methods: We assessed the data of 92 patients, who were further evaluated with JAK2V617F mutation and serum erythropoietin (EPO) levels and bone marrow biopsy, if necessary. We also compared this patient group with 20 patients whose Hgb>18.5 g/dL for men and >16.5 g/dL for women. Results: Nine patients (45%) in the higher hemoglobin group were JAK2V617F positive, while 4 patients (4.3%) in the lower hemoglobin group were JAK2V617F positive (p<0.001). The number of patients with serum EPO levels <4.3 mIU/mL was significantly higher in the higher hemoglobin group (n=13, 65%) than the lower hemoglobin group (n=7, 7.6%) (p<0.001). Finally, the number of patients who received a diagnosis of PV was significantly higher in the higher hemoglobin group (n=13, 65%) than the lower hemoglobin group (n=9, 9.8%) (p<0.001). Conclusion: We found a substantial increase in patients who were candidates for testing for PV with the introduction of WHO 2016 criteria; these patients were diagnosed with PV with a rate (9.8%) that cannot be underestimated.

Epo receptor signaling in macrophages alters the splenic niche to promote erythroid differentiation

Anemic stress induces stress erythropoiesis, which rapidly generates new erythrocytes to restore tissue oxygenation. Stress erythropoiesis is best understood in mice where it is extramedullary and occurs primarily in the spleen. However, both human and mouse stress erythropoiesis use signals and progenitor cells that are distinct from steady-state erythropoiesis. Immature stress erythroid progenitors (SEPs) are derived from short-term hematopoietic stem cells. Although the SEPs are capable of self-renewal, they are erythroid restricted. Inflammation and anemic stress induce the rapid proliferation of SEPs, but they do not differentiate until serum erythropoietin (Epo) levels increase. Here we show that rather than directly regulating SEPs, Epo promotes this transition from proliferation to differentiation by acting on macrophages in the splenic niche. During the proliferative stage, macrophages produce canonical Wnt ligands that promote proliferation and inhibit differentiation. Epo/Stat5-dependent signaling induces the production of bioactive lipid mediators in macrophages. Increased production of prostaglandin J2 (PGJ2) activates peroxisome proliferator-activated receptor γ (PPARγ)-dependent repression of Wnt expression, whereas increased production of prostaglandin E2 (PGE2) promotes the differentiation of SEPs.