Erythropoietin in the intensive care unit: beyond treatment of anemia

Erythropoietin (EPO) is the major hormone stimulating the production and differentiation of red blood cells. EPO is used widely for treating anemia of critical illness or anemia induced by chemotherapy. EPO at pharmacological doses is used in this setting to raise hemoglobin levels (by preventing the apoptosis of erythroid progenitor cells) and is designed to reduce patient exposure to allogenic blood through transfusions. Stroke, heart failure, and acute kidney injury are a frequently encountered clinical problem. Unfortunately, in the intensive care unit advances in supportive interventions have done little to reduce the high mortality associated with these conditions. Tissue protection with EPO at high, nonpharmacological doses after injury has been found in the brain, heart, and kidney of several animal models. It is now well known that EPO has anti-apoptotic effects in cells other than erythroid progenitor cells, which is considered to be independent of EPOs erythropoietic activities. This review article summarizes what is known in preclinical models of critical illness and discusses why this does not correlate with randomized, controlled clinical trials

Erythropoietin Receptor Signaling Is Membrane Raft Dependent

Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3±1.4(SE) vs. 25.6±3.2 aggregates/cell; p≤0.001), accompanied by a >3-fold increase in cluster size (p≤0.001). Raft fraction immunoblotting showed Epo-R translocation to MR after Epo stimulation and was confirmed by fluorescence microscopy in Epo stimulated UT7 cells and primary erythroid bursts. Receptor recruitment into MR was accompanied by incorporation of JAK2, Lyn, and STAT5 and their activated forms. Raft disruption by cholesterol depletion extinguished Epo induced Jak2, STAT5, Akt and MAPK phosphorylation in UT7 cells and erythroid progenitors. Furthermore, inhibition of the Rho GTPases Rac1 or RhoA blocked receptor recruitment into raft fractions, indicating a role for these GTPases in receptor trafficking. These data establish a critical role for MR in recruitment and assembly of Epo-R and signal intermediates into discrete membrane signaling units

Sox6 Is Necessary for Efficient Erythropoiesis in Adult Mice under Physiological and Anemia-Induced Stress Conditions

BACKGROUND: Definitive erythropoiesis is a vital process throughout life. Both its basal activity under physiological conditions and its increased activity under anemia-induced stress conditions are highly stimulated by the hormone erythropoietin. The transcription factor Sox6 was previously shown to enhance fetal erythropoiesis together and beyond erythropoietin signaling, but its importance in adulthood and mechanisms of action remain unknown. We used here Sox6 conditional null mice and molecular assays to address these questions. METHODOLOGY/PRINCIPAL FINDINGS: Sox6fl/flErGFPCre adult mice, which lacked Sox6 in erythroid cells, exhibited compensated anemia, erythroid cell developmental defects, and anisocytotic, short-lived red cells under physiological conditions, proving that Sox6 promotes basal erythropoiesis. Tamoxifen treatment of Sox6fl/flCaggCreER mice induced widespread inactivation of Sox6 in a timely controlled manner and resulted in erythroblast defects before reticulocytosis, demonstrating that impaired erythropoiesis is a primary cause rather than consequence of anemia in the absence of Sox6. Twenty five percent of Sox6fl/flErGFPCre mice died 4 or 5 days after induction of acute anemia with phenylhydrazine. The others recovered slowly. They promptly increased their erythropoietin level and amplified their erythroid progenitor pool, but then exhibited severe erythroblast and reticulocyte defects. Sox6 is thus essential in the maturation phase of stress erythropoiesis that follows the erythropoietin-dependent amplification phase. Sox6 inactivation resulted in upregulation of embryonic globin genes, but embryonic globin chains remained scarce and apparently inconsequential. Sox6 inactivation also resulted in downregulation of erythroid terminal markers, including the Bcl2l1 gene for the anti-apoptotic factor Bcl-xL, and in vitro assays indicated that Sox6 directly upregulates Bcl2l1 downstream of and beyond erythropoietin signaling. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that Sox6 is necessary for efficient erythropoiesis in adult mice under both basal and stress conditions. It is primarily involved in enhancing the survival rate and maturation process of erythroid cells and acts at least in part by upregulating Bcl2l1

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Spry1 Plays Selective Hematopoietic Roles as An Erythropoietin - Modulated Positive Regulator of Erythropoiesis.

Abstract Abstract 777 The four vertebrate Sprouty (Spry1-4) proteins are molecular adaptors, best known as negative regulators of MAP kinase activation mediated by FGFR, VEGFR and RET. Prior studies in human hematopietic stem cells and zebrafish implicated Spry proteins in stem cell development. Presently, we have ascertained the role of Spry1 in erythroid development using cellular models and in knockout mice. Treatment of UT7 erythropoietin-responsive cells led a strong increase in phosphorylation of Spry1 and Spry2 on critical N-terminal tyrosine sites of these proteins (Y53 and Y55, respectively). UT7 cells engineered to constitutively express Spry1 also demonstrated decreased ERK activation in response to erythropoietin treatment. Spry expression was measured in developing primary bone marrow (pro)erythroblasts by real time PCR. Spry1 was expressed most prominently in erythroblasts at a level 40 times higher than Spry 2-4. Furthermore, Spry1 expression rose markedly as erythroblasts matured. To determine the role of Spry1 in murine hematopoiesis, conditional, LoxP flanked allele of Spry1 was crossed with Mx1-Cre transgenic mice and Spry1 was deleted in murine marrow by injection of mice with poly pIpC. Efficient deletion of the Spry1 gene in murine marrow did not affect lymphocytes or granulocytes and selectively led to an increased reticulocyte count (8.9% +/- 0.2% in Spry1 deleted vs. 4.9 +/- 0.5% in control mice, p&lt;0.002). Deletion of Spry1 led to activation of splenic erythropoiesis with a four fold enrichment of CD71high, Ter119pos precursors in Mx1-Cre; Spry1flox/flox animals compared to Mx1-Cre;Spry1flox/+ animals. In ex vivo expansion cultures, however, erythroid progenitors, were significantly compromised in their intrinsic capacity to form KitnegCD71highTer119neg and KitnegCD71highTer119pos erythroblasts. Collectively these data suggest that during hematopoiesis, SPRY1 acts selectively as a non-redundant novel positive effector of EPO- dependent red cell formation. Disclosures: No relevant conflicts of interest to declare. </jats:sec

JAK2/Y343/STAT5 signaling axis is required for erythropoietin-mediated protection against ischemic injury in primary renal tubular epithelial cells

Erythropoietin has emerged as a potential therapy for the treatment of ischemic tissue injury. In erythroid cells, the JAK2/Y343/STAT5 signaling axis has been shown to be necessary for stress but not steady-state erythropoiesis. The requirement for STAT5 activation in erythropoietin-mediated protection from ischemic injury has not been well-studied. To answer this question, we induced reproducible necrotic ischemic injury in primary mouse renal tubular epithelial cells (RTEC) in vitro. Using RTEC from erythropoietin receptor mutant mice with differential STAT5 signaling capabilities, we demonstrated first, that EPO administration either before or during injury significantly protects against mild-moderate but not severe necrotic cell death; and second, the JAK2/Y343/STAT5 signaling axis is required for protection against ischemic injury in primary mouse RTEC. In addition, we identified Pim-3, a prosurvival STAT5 target gene, as responsive to EPO in the noninjured kidney both in vitro and in vivo