Resveratrol Induces Erythroid Maturation by Activating FOXO3 and Improves in Vivo Erythropoiesis in Normal and Beta -Thalassemic Mice

Resveratrol is a polyphenolic stilbene with anti-oxidant, anti-inflammatory and anti-tumoral bioactivities . High concentrations of resveratrol (50 \u3bcM) have been reported to induce HbF synthesis in an in vitro model of normal and beta-thalassemic erythropoiesis (Fibach E. Int J Mol Med 2012; Rodrigue CM. BJH 2001) and to improve erythropoiesis in a mouse model for Fanconi Anemia (Zhang Q. Blood 2010). Beta thalassemia (b-thal) is characterized by ineffective erythropoiesis and increased cellular oxidative stress. We studied the effects of resveratrol (5 \ub5M) on erythropoiesis in vitro from peripheral CD34+ cells of healthy and b-thal subjects. Erythroid maturation was evaluated at 7, 9, 11 and 14 days of culture by cytofluorimetric analysis using the CD71-GPA-CD36 strategy that allows to separate CFU-E, Pro-E, Int-E and Late-Erythroblasts (Merryweather-Clarke AT. Blood 2011). Resveratrol reduced cell growth in both cell types, with a reduction of CFU-E, increased Int-E at day 7 and 9, and increased Int-E and Late-E at 11 and 14 days. The early maturation of erythroid progenitors was confirmed by morphological analysis of the cells. We sorted CFU-E cells (at 7 days) from resveratrol treated and untreated cells and analyzed the cell cycle, cyclinD1 and p21 expression. In both cell types resveratrol induced increased frequency of S-G2/M cells compared to untreated cells with increased p21 levels, suggesting decreased cycling of CFU-E with increased maturation of erythroblasts. No changes of gamma chain mRNA levels were present in cells treated with resveratrol (5 \ub5M). Since FOXO3 is a key regulator of erythroid redox required for normal erythroid maturation (Marinkovic D. JCI 2007), FOXO3 expression and activity was assessed in sorted CFU (7day) and Int-E (11 day) with and without resveratrol. FOXO3a mRNA levels were increased in resveratrol treated cells in both sorted cell populations. We used nuclear localization as a surrogate assay for FOXO3a activity and found resveratrol increased the overall expression of FOXO3 protein in the nucleus without impacting significantly the nuclear/cytoplasmic ratio. Interestingly, resveratrol did not appear to modify FOXO1 expression or subcellular localization. These results suggest that resveratrol enhances specifically expression of FOXO3 in human erythroblasts. Dietary resveratrol supplementation (2.4 mg/Kg) was studied in wild-type and Hbb3th+/- mice (2 months of age) for 6 months. In resveratrol Hbb3th+/- treated mice increased Hb levels (8.3\ub10.6 vs 10.3\ub10.5 g/dL, n=12; P<0.05) and decreased reticulocyte count (33.9\ub10.8 vs 23.7\ub1 8.2 %, n=12; P<0.05) were observed. Significant increased MCV (34.6\ub10.6 vs 41.6\ub1 5.4 fL, n=12; P<0.05) and MCH ( 9.7\ub1 0.6 vs 12.8 \ub1 2.1 pg, n=12; P<0.05) were also noted. Flow cytometric evidence of decreased ineffective erythropoiesis and reduced spleen/ body weight ratio were also observed. These data indicate that resveratrol affects erythroid maturation both in vitro and in vivo, and that these effects have possible therapeutic relevance for the treatment of thalassemias

Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1

Autosomal dominant dehydrated hereditary stomatocytosis (DHSt) usually presents as a compensated hemolytic anemia with macrocytosis and abnormally shaped red blood cells (RBCs). DHSt is part of a pleiotropic syndrome that may also exhibit pseudohyperkalemia and perinatal edema. We identified PIEZO1 as the disease gene for pleiotropic DHSt in a large kindred by exome sequencing analysis within the previously mapped 16q23-q24 interval. In 26 affected individuals among 7 multigenerational DHSt families with the pleiotropic syndrome, 11 heterozygous PIEZO1 missense mutations cosegregated with disease. PIEZO1 is expressed in the plasma membranes of RBCs and its messenger RNA, and protein levels increase during in vitro erythroid differentiation of CD341 cells. PIEZO1 is also expressed in liver and bone marrow during human and mouse development. We suggest for the first time a correlation between a PIEZO1 mutation and perinatal edema. DHSt patient red cells with the R2456H mutation exhibit increased ion-channel activity. Functional studies of PIEZO1 mutant R2488Q expressed in Xenopus oocytes demonstrated changes in ion-channel activity consistent with the altered cation content of DHSt patient red cells. Our findings provide direct evidence that R2456H and R2488Q mutations in PIEZO1 alter mechanosensitive channel regulation, leading to increased cation transport in erythroid cells

Non-Invasive Prenatal Screening: The First Report of Pentasomy X Detected by Plasma Cell-Free DNA and Karyotype Analysis

Pentasomy X is a sex chromosome anomaly caused by the presence of three extra X chromosomes in females (49,XXXXX instead of 46,XX) and is probably due to a nondisjunction during the meiosis. So far, only five cases prenatally diagnosed were described. The main features in 49,XXXXX karyotype include severe intellectual disability with delayed speech development, short stature, facial dysmorphisms, osseous and articular abnormalities, congenital heart malformations, and skeletal and limb abnormalities. Prenatal diagnosis is often difficult due to the lack of a clear echographic sign like nuchal translucency (NT), and mostly cases were postnatally described. We report the first case of a 49,XXXXX female that was detected by non-invasive prenatal screening (NIPS), quantitative fluorescence polymerase chain reaction (QF-PCR) and a fetal karyotype

Molecular basis of inherited microcytic anemia due to defects in iron acquisition or heme synthesis

This review article examines our present knowledge on the molecular basis of inherited microcytic anemia due to defects in iron acquisition or heme synthesis. The conditions examined include congenital sideroblastic anemias due to mutations in ALAS2, ABCB7 or GRX5, microcytic anemias due to DMT1 mutations, and the microcytic anemia associated with TMPRSS6 mutations

How I Diagnose Non-thalassemic Microcytic Anemias

Microcytic anemia is the most common form of anemia, characterized by reduced hemoglobin (Hb) synthesis associated with decreased red blood cell volume (MCV). It is a very heterogeneous group of diseases that may be either acquired or inherited. Microcytic hypochromic anemia can result from defects in globin (hemoglobinopathies or thalassemias) or heme synthesis or in iron availability, or acquisition by the erythroid precursors. Diagnosis of microcytic anaemia appears to be important in children/adolescents, especially to set, where possible, a treatment plan on the basis of the etiology and pathogenesis. After excluding the acquired causes of microcytic anemia that represent the most frequent etiology, according to the differential diagnosis, the analysis of genetic causes, mostly hereditary, must be considered. This review will consider acquired and hereditary microcytic anemias due to heme synthesis or to iron metabolism defects and their diagnosis

Regulation of divalent metal transporter 1 (DMT1) non-IRE isoform by the microRNA Let-7d in erythroid cells

BACKGROUND: Divalent metal transporter 1 (DMT1) is a widely expressed metal-iron transporter gene encoding four variant mRNA transcripts, differing for alternative promoter at 5' (DMT1 1A and 1B) and alternative splicing at 3' UTR, differing by a specific sequence either containing or lacking an iron regulatory element (+IRE and -IRE, respectively). DMT1-IRE might be the major DMT1 isoform expressed in erythroid cells, although its regulation pathways are still unknown. DESIGN AND METHODS: The microRNA (miRNA) Let-7d (miR-Let-7d) was selected by the analysis of four miRNAs, predicted to target the DMT1-IRE gene in CD34(+) hematopoietic progenitor cells, in K562 and in HEL cells induced to erythroid differentiation. Using a luciferase reporter assay we demonstrated the inhibition of DMT1-IRE by miR-Let-7d in K562 and HEL cells. The function of miR-Let-7d in erythroid cells was evaluated by the flow cytometry analysis of erythroid differentiation markers, by benzidine staining and by iron flame atomic absorption for the evaluation of iron concentration in the endosomes from K562 cells over-expressing miR-Let-7d. RESULTS: We show that in erythroid cells, DMT1-IRE expression is under the regulation of miR-Let-7d. DMT1-IRE and miR-Let-7d are inversely correlated with CD34(+) cells, K562 and HEL cells during erythroid differentiation. Moreover, overexpression of miR-Let-7d decreases the expression of DMT1-IRE at the mRNA and protein levels in K562 and HEL cells. MiR-Let-7d impairs erythroid differentiation of K562 cells by accumulation of iron in the endosomes. CONCLUSIONS: Overall, these data suggest that miR-Let-7d participates in the finely tuned regulation of iron metabolism by targeting DMT1-IRE isoform in erythroid cells