Erythrocytes and Erythropoietin

In the present pilot study (56 patients), some red blood cell parameters in samples from patients with metabolic syndrome and subclinical atherosclerosis, but without any sign of coronary artery disease, have been analyzed. The main goal of this work was to determine, in this preclinical state, new peripheral gender-associated bioindicators of possible diagnostic or prognostic value. In particular, three di ﬀ erent “indicators” of red blood cell injury and aging have been evaluated: glycophorin A, CD47, and phosphatidylserine externalization. Interestingly, all these determinants appeared signiﬁcantly modiﬁed and displayed gender di ﬀ erences. These ﬁndings could provide novel and useful hints in the research for gender-based real-time bioindicators in the progression of metabolic syndrome towards coronary artery disease. Further, more extensive studies are, however, necessary in order to validate these ﬁndings. Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N, and 4.1B. Two isoforms of 4.1R (4.1R 135 and 4.1R 80 ), as well as 4.1G, are expressed in erythroblasts during terminal di ﬀ erentiation, but only 4.1R 80 is present in mature erythrocytes. One goal in the ﬁeld is to better understand the complex regulation of cell type and isoform-speciﬁc expression of 4.1 proteins. To start answering these questions, we are studying in depth the important functions of 4.1 proteins in the organization and function of the membrane skeleton in erythrocytes. We have previously reported that the binding proﬁles of 4.1R 80 and 4.1R 135 to membrane proteins and calmodulin are very di ﬀ erent despite the similar structure of the membrane-binding domain of 4.1G and 4.1R 135 . We have accumulated evidence for those di ﬀ erences being caused by the N-terminal 209 amino acids headpiece region (HP). Interestingly, the HP region is an unstructured domain. Here we present an overview of the di ﬀ erences and similarities between 4.1 isoforms and paralogs. We also discuss the biological signiﬁcance of unstructured domains. Missense mutations in the erythroid band 3 protein (Anion Exchanger 1) have been associated with hereditary stomatocytosis. Features of cation leaky red cells combined with functional expression of the mutated protein led to the conclusion that the AE1 point mutations were responsible for Na + and K + leak through a conductive mechanism. A molecular mechanism explaining mutated AE1-linked stomatocytosis involves changes in AE1 transport properties that become leaky to Na + and K + . However, another explanation suggests that point-mutated AE1 could regulate a cation leak through other transporters. This short paper intends to discuss these two alternatives. A new analog of EPO was designed by fusing one and two CTPs to the N -terminal and C -terminal ends of EPO (EPO-(CTP) 3 ), respectively. This analog was expressed and secreted e ﬃ ciently in CHO cells. The in vitro test shows that the activity of EPO-(CTP) 3 in TFI-1 cell proliferation assay is similar to that of EPO-WT and commercial rHEPO. However, in vivo studies indicated that treatment once a week with EPO-(CTP) 3 (15 μ g/kg) dramatically increased ( ∼ 8 folds) haematocrit as it was compared to rHuEPO. Moreover, it was found that EPO-(CTP) 3 is more e ﬀ ective than rHuEPO and Aranesp in increasing reticulocyte number in mice blood. The detected circulatory half-lives of rHuEPO, Aranesp, and EPO-(CTP) 3 following IV injection of 20 IU were 4.4, 10.8, and 13.1 h, respectively. These data established the rational for using this chimera as a long-acting EPO analog in clinics. The therapeutic e ﬃ cacy of EPO-CTP analog needs to be established in higher animals and in human clinical trials. Haemoglobinopathies such as thalassaemia and sickle cell disease present a major health burden. Currently, the main forms of treatment for these diseases are packed red blood cell transfusions and the administration of drugs which act to nonspeciﬁcally reactivate the production of foetal haemoglobin. These treatments are ongoing throughout the life of the patient and are associated with a number of risks, such as limitations in available blood for transfusion, infections, iron overload, immune rejection, and side e ﬀ ects associated with the drug treatments. The ﬁeld of cellular reprogramming has advanced signiﬁcantly in the last few years and has recently culminated in the successful production of erythrocytes in culture. This paper will discuss cellular reprogramming and its potential relevance to the treatment of haemoglobinopathies.

This special issue focuses on erythrocytes and erythropoietin including original research and review articles on the cellular physiology of erythrocytes and of erythrocyte-associated disorders. Moreover, the special issue includes papers on the action of erythropoietin and hypoxia-inducible factors on a cellular level. In the following, we present the original papers published in this special edition.
D. Barneaud-Rocca et al. deal with one of the most abundant proteins in red blood cells, the band 3 protein (Anion Exchanger 1). The paper reviews recent work hypothesizing the contribution of band 3 point mutations to a sodium and potassium leak. It is compared to alternative explanations suggesting that point mutations in band 3 regulate the cation leak through other transporters. The topic is driven by the fact that mutations in the band 3 protein have been associated with hereditary stomatocytosis. The molecular mechanisms discussed in the paper link the stomatocytosis and the sodium potassium leak of the mutated band 3 protein.
W. Nunomura et al. review the function of unstructured N-terminal domain of protein 4.1R and 4.1 G and characterize the binding profiles of proteins 4.1R80, 4.1R135 and protein 4.1G in erythropoiesis. The regulation of the binding profiles of these proteins by the presence or absence of the Nterminal 209 amino acid sequence (headpiece region (HP)) and unstructured domain of the protein as well as of 4.1R135 (which contains the HP) by both Ca 2+ and Ca 2+ /CaM is discussed. Knowing the different regulation and expression of the 4.1 protein isoforms will foster our understanding of erythropoiesis.
F. Fares et al. fused one Carboxyl-Terminal Peptide (CTP) of the human chorionic gonadotropin beta subunit to the N-terminal end and two CTPs to the C-terminal end of erythropoietin (Epo). This artificial erythropoiesisstimulating agent had increased in vivo activity as well as half-life compared with recombinant human Epo and the hyperglycosylated Epo analogue darbepoetin alfa (Aranesp). As erythropoiesis-stimulating agents often need to be injected repeatedly over long time periods, for example, for anemia treatment in kidney diseases or cancer, more effective and longer lasting Epo derivates improve the patients' quality of life.
E. Straface et al. present the results of a pilot study investigating new peripheral sex-associated markers in patients with metabolic syndrome and subclinical atherosclerosis. The metabolic syndrome, which is characteristic of hypertension, obesity, insulin resistance, hypertriglyceridemia, and hypercholesterolemia, is a major risk factor for cardiovascular mortality in the developed world. In their study, E. Straface et al. analyzed glycophorin A, CD47, and phosphatidylserine exposition at the cell surface as hallmarks of erythrocyte damage. They report significant gender differences of those parameters in patients with metabolic syndrome.
L. J. Norton et al. review the recent advance in cellular reprogramming with the particular emphasis on its potentially beneficial use for the future treatment of hemoglobinopathies. Typical and frequent hemoglobinopathies are genetic disorders such as thalassaemia and sickle cell disease. To date, blood transfusion is the principal therapy of those 2 International Journal of Cell Biology diseases. In their review article, L. J. Norton et al. outline the recent development in stem cell research such as classic cellular reprogramming and transdifferentiation and discuss their potential application for the treatment of anemia following hemoglobinopathies.
We hope that this special issue will alert researchers to some recent development in the field of erythrocytes and erythropoietin, particularly the correlation between protein alterations and clinical symptoms, and that a better understanding of this correlation can direct our efforts to the discovery of new therapeutic strategies for the treatment of anemia and metabolic disorders.