Red Cell Production Research Articles

L'érythropoïèse : un paradigme pour l'étude du rôle des caspases dans la mort et la différenciation cellulaire

Erythroid differentiation involves the transcription factor GATA-1 that positively regulates promoters of erythroid genes (including haemoglobin, glycophorin, erythropoietin receptor) and of erythropoietin. Terminal erythroid differentiation is characterized by major morphological changes that include chromatin condensation and cell size reduction. The morphological changes are partially similar at least to those observed during apoptosis. The production of red cells depends on the apoptosis rate of erythroid progenitors and precursors. Upon erythropoietin starvation or engagement of the death receptor Fas, caspases are activated in erythroid precursors and cleave GATA-1, thus inducing maturation arrest and apoptosis of immature erythroblasts. We have recently demonstrated that, upon erythropoietin stimulation, caspase-3 was also activated, an event required for human terminal erythroblast maturation. Proteins cleaved by caspases in erythroid cells undergoing terminal differentiation include Lamin B and Acinus, which are involved in chromatin condensation. In contrast, despite caspase-3 activation neither GATA-1 degradation nor apoptosis was observed. Thus, the fate of erythroid precursors is determined downstream of caspase activation by the pattern of cleaved targets. Therefore, there are some mechanisms underlying the selective protection of caspase-3 targets during erythropoiesis. This model in which caspases activation is required for differentiation may apply to other haematopoietic or non haematopoietic cellular systems which are described in this review.

Manipulation and Visualization of the Erythroid Lineage - In Vivo Models for Erythroid Disorders.

Red blood cells have a short half-life and are continuously renewed in a tightly controlled growth process. Dysregulation of erythropoiesis results in erythroleukemias or more frequently in anemias. To elucidate molecular mechanisms regulating red cell production in an animal model, we generated a mouse line (ErGFPcre) that simultaneously facilitates erythroid specific gene manipulation via the CreloxP recombination system and visualization of erythroid progenitor cells. To avoid site of integration effects frequently observed for randomly integrated transgenes, we used a knock-in strategy targeting the genomic EpoR locus that ensures a reliable EpoR promoter controlled transgene expression of our GFPcre fusion protein.The flow cytometric analysis of GFP fluorescence in different hematopoietic subpopulations of adult and embryonic ErGFPcre mice revealed a strictly erythroid-specific expression pattern for GFPcre. Further studies on GFP positive erythroid progenitor cells indicated a developmental switch in lineage progression from the hematopoietic stem cell compartment to early erythroid progenitor cells that are Sca-1 negative and c-kit high. To monitor previous and persistent GFPcre expression during development and to determine the efficiency of Cre-mediated recombination in our mouse model, we crossed the ErGFPcre mouse line with the LacZ reporter strain R26R. The spatial and temporal analysis of GFPcre-mediated LacZ expression confirmed that within the hematopoietic system GFPcre expression is limited to the erythroid lineage. Surprisingly, non-hematopoietic expression of GFPcre is restricted to the vascular system. It is possible that the observed differential transcriptional activity of the knock-in and the wild-type allele is linked to the absence of intron 2 to 7 in the knock-in locus. The quantitative analysis of the recombination frequency confirmed that GFPcre-mediated recombination is limited to erythroid progenitor cells and showed that it occurs in the adult bone marrow at a frequency of up to 80% and in the fetal liver with an efficiency close to 100%.Thus, our ErGFPcre mouse model offers the possibility to study regulatory mechanisms in erythroid progenitor cells and facilitates the establishment of mouse models for erythroid disorders.

Death Associated Protein Kinase 2 (DAPK2): A Novel Epo-Modulated Apoptotic Regulator of Late Erythropoiesis.

Death-associated kinase-1 (DAPK1) is a broadly expressed pro-apoptotic cytoplasmic S/T protein kinase that regulates cell proliferation and survival, modulates a p53 apoptotic checkpoint, and contributes to ALL, AML, and myelodysplastic syndromes. DAPK2, by comparison, is an under-studied (but likewise pro-apoptotic) DAPK1 orthologue that we have discovered to be expressed (via Affymetrix gene profiling) at high-levels in late-stage erythroid progenitor cells. In particular, and as examined in purified and developmentally-staged proerythroblasts, basophillic erythroblasts and orthrochromatic erythroblasts, DAPK2 gene expression was sharply up-modulated as progenitor cells developed to a Ter119(+), CD71-high stage. Using a DAPK2-specific antibody, this was confirmed at the protein level, and initial analyses of expression in cell lines further indicated lineage-restricted DAPK2 expression (e.g. high level in erythroid GIE2 cells but undetectable in pluripotent EML cells). To advance an initial understanding of DAPK2's role in erythroid cells, its possible regulation via the Epo receptor (EpoR) also was studied. Here, primary erythroid progenitor cells from mice with knocked-in minimal EpoR alleles were used (PY-null and PY343-only EpoR-HM and EpoR-H forms, respectively). Interestingly, the point-mutation of a PY3434/Stat5 specific binding site (to F343) (EpoR-HM allele) resulted in a clear multi-fold de-repression of DAPK2 that also correlated with a decreased survival potential for EpoR-HM erythroblasts. This repressive effect of EpoR/PY343/Stat5 signaling on DAPK2 expression was highly stage-specific, and was exerted distinctly in advance of an observed EpoR/PY343/Stat5-dependent up-modulation of Bcl-xl expression. In addition, in wild-type EpoR pro-erythroblasts, DAPK2 levels increased with decreasing Epo concentrations. Investigations therefore reveal DAPK2 to be a late-stage erythroid- restricted, (and EpoR/PY343/Stat5- modulated) novel candidate regulator of red cell production.

Effects of Hepcidin, Ferritin, and Iron Deprivation on Erythroid Colony Formation in Vitro.

The anemia of chronic disease (ACD) results from a combination of three pathologic processes. In ACD, a modest shortening of red cell survival creates an increased demand for red cell production, which is not met because of an impaired erythropoietic response and defects in reticuloendothelial iron mobilization and utilization. The impaired erythropoietic response, in turn, has two components: a blunted erythropoietin response, and an impaired response of erythroid progenitors to erythropoietin. Recombinant human erythropoietin (rhEPO) can reverse this impaired progenitor response in vitro, and can also correct ACD in patients. These processes have generally been considered effects of the cytokines which mediate the immune and inflammatory response, such as tumor necrosis factor, interleukin-1, and the interferons. It has recently been proposed that hepcidin, a mediator of innate immunity with the iron regulatory properties, is the factor responsible for ACD. If this is the case, then hepcidin should be able to induce the pathophysiologic mechanisms implicated in ACD. We therefore evaluated the effects of hepcidin and associated phenomena on human CFU-E colony formation in vitro. All CFU-E cultures were performed in plasma clots in serum-containing medium with rhEPO 1 U/mL. Hepcidin at concentrations 10 ng/mL -10 μg/mL had no effect on CFU-E colony formation. A number of studies have demonstrated that increased hepcidin message expression and protein production are strongly associated with increases in serum ferritin concentrations, and so the effect of added ferritin on erythroid colony formation was studied. Neither ferritin nor apo-ferritin 10 – 1000 ng/mL had inhibitory effects on CFU-E colony formation. The effect of iron deprivation on erythroid colony formation was evaluated with using desferrioxamine. Desferrioxamine 0.01mM decreased CFU-E colony formation to 60% of control values, while higher concentrations completely ablated colony growth. In summary, hepcidin does not appear to inhibit CFU-E colony formation directly or indirectly through ferritin. It may exert such an effect by decreasing availability of iron for erythropoiesis; however, such a finding would be difficult to reconcile with the observed clinical response of ACD to rhEPO, given that iron availability is typically a limiting factor in the erythropoietic response to rhEPO. The role of hepcidin in the overall pathogenesis of ACD remains to be fully determined.

Transfusion-transmitted tick-borne infections: A cornucopia of threats

Over the past several decades, the frequency of contact between humans and ticks has increased dramatically. Concomitantly, several newly recognized tick-borne pathogens have emerged joining those already known to be transmitted by ticks. Together these factors have led to an enhanced public health awareness of ticks, tick-borne agents, and their associated diseases. Reports that several of these agents are transmitted by blood transfusion have raised concerns about blood safety. The primary agents of interest are members of the genus Babesia, but Anaplasma phagocytophilum, Rickettsia rickettsii, Colorado tick fever virus, and tick-borne encephalitis virus also have been transmitted by transfusion. In many cases, these agents and their diseases share common features including vectors, symptoms, and diagnosis. Unfortunately, they also share the common problem of insufficient epidemiologic and transmissibility data necessary for making informed decisions regarding potential blood safety interventions. Although further surveillance and epidemiologic studies of tick-borne agents are clearly needed, at present only the Babesia warrant consideration for active intervention; because donor management strategies based on risk-factor questions are inadequate, leukoreduction not effective for agents found in red cells and pathogen inactivation remains problematic for red cell products. Despite the present unavailability of screening assays, some form of serologic and nucleic acid testing may be justified for the Babesia. Given that interactions between humans and ticks are likely to increase in the future, vigilance is required as new and extant tick-borne agents pose potential threats to transfusion safety.

The Rb tumor suppressor is required for stress erythropoiesis.

The retinoblastoma tumor suppressor gene plays important roles in cell cycle control, differentiation and survival during development and is functionally inactivated in most human cancers. Early studies using gene targeting in mice suggested a critical role for pRb in erythropoiesis, while more recent experiments have suggested that many of the abnormal embryonic phenotypes in the Rb null mouse result from a defective placenta. To address this controversy and determine whether Rb has cell intrinsic functions in erythropoiesis, we examined the effects of Rb loss on red cell production following acute deletion of pRb in vitro and under different stress conditions in vivo. Under stress conditions, pRb was required to regulate erythroblast expansion and promote red cell enucleation. Acute deletion of Rb in vitro induced erythroid cell cycle and differentiation defects similar to those observed in vivo. These results demonstrate a cell intrinsic role for pRb in stress erythropoiesis and hematopoietic homeostasis that has relevance for human diseases.

Beneficial and ominous aspects of the pleiotropic action of erythropoietin.

The primary function of the glycoprotein hormone erythropoietin (Epo) is to promote red cell production by inhibiting apoptosis of erythrocytic progenitors in hemopoietic tissues. However, functional Epo receptors (Epo-R) have recently been demonstrated in various nonhemopoietic tissues indicating that Epo is a more pleiotropic viability and growth factor. Herein, in vitro and in vivo effects of Epo in the brain and the cardiovascular system are reviewed. In addition, the therapeutic impact of Epo in oncology is considered, including the question of whether Epo might promote tumor growth. Convincing evidence is available that Epo acts as a neurotrophic and neuroprotective factor in the brain. Epo prevents neuronal cells from hypoxia-induced and glutamate-induced cell death. Epo-R is expressed by neurons and glia cells in specific regions of the brain. Epo supports the survival of neurons in the ischemic brain. The neuroprotective potential of Epo has already been confirmed in a clinical trial on patients with acute stroke. With respect to the vasculature, Epo acts on both endothelial and smooth muscle cells. Epo promotes angiogenesis and stimulates the production of endothelin and other vasoactive mediators. In addition, Epo-R is expressed by cardiomyocytes. The role of Epo as a myocardial protectant is at the focus of present research. Epo therapy in tumor patients is practiced primarily to maintain the hemoglobin concentration above the transfusion trigger and to reduce fatigue. In addition, increased tumor oxygenation may improve the efficacy of chemotherapy and radiotherapy. However, tumor cells often express Epo-R. Therefore, careful studies are required to fully exclude that recombinant human Epo (rHuEpo) promotes tumor growth.

Erythropoietin and the hypoxic brain.

Normal tissue function in mammals depends on adequate supply of oxygen through blood vessels. A discrepancy between oxygen supply and consumption (hypoxia) induces a variety of specific adaptation mechanisms at the cellular, local and systemic level. These mechanisms are in part governed by the activation of hypoxia-inducible transcription factors (HIF-1, HIF-2), which in turn modulate expression of hypoxically regulated genes such as those encoding vascular endothelial growth factor (VEGF) and erythropoietin (EPO). EPO is a glycoprotein that is produced mainly by interstitial fibroblasts in the kidneys of the adult and in hepatocytes in the foetus. Released into the circulation, EPO makes its way to the bone marrow, where it regulates red cell production by preventing apoptosis of erythroid progenitor cells. Recently, EPO has emerged as a multifunctional growth factor that plays a significant role in the nervous system. Both EPO and its receptor are expressed throughout the brain in glial cells, neurones and endothelial cells. Hypoxia and ischaemia have been recognised as important driving forces of EPO expression in the brain. EPO has potent neuroprotective properties in vivo and in vitro and appears to act in a dual way by directly protecting neurones from ischaemic damage and by stimulating endothelial cells and thus supporting the angiogenic effect of VEGF in the nervous system. Thus, hypoxia-induced gene products such as VEGF and EPO might be part of a self-regulated physiological protection mechanism to prevent neuronal injury, especially under conditions of chronically reduced blood flow (chronic ischaemia). In this review, I will briefly summarize the recent findings on the molecular mechanisms of hypoxia-regulated EPO expression in general and give an overview of its expression in the central nervous system, its action as a growth factor with non-haematopoietic functions and its potential clinical relevance in various brain pathologies.

Erythropoietin attenuates post-traumatic injury in organotypic hippocampal slices.

Recent experimental evidence indicates that erythropoietin (Epo), in addition to its hormonal role in regulating red cell production, operates as a neuroprotective agent. So far, the neuroprotective effect of human recombinant Epo (rhEpo) has been mainly demonstrated in models of cerebral ischemia/hypoxia and in selected in vivo studies of traumatic neuronal injury. To further investigate the potential role of this multifunctional trophic factor in post-traumatic cell death, we examined the protective effects of rhEpo in a newly developed model of mechanical trauma in organotypic hippocampal slices. Organotypic rat hippocampal slices were subjected to traumatic injury by allowing a stylus to impact on the CA1 area with an energy of 6 microJ. Hippocampal damage was identified and measured 24 and 48 h later with the fluorescent dye propidium iodide (PI). In untreated slices, the impact induced a significant increase in the mean hippocampal PI fluorescence, co-localized with the area of impact at 24 h (primary post-traumatic injury) and progressively spread to the whole slice between 24 and 48 h (secondary post-traumatic injury). Addition of rhEpo (1-100 UI/mL) or of the NMDA antagonist MK-801 (30 microM) immediately after the traumatic injury reduced hippocampal damage by approximately 30% when observed 24 h later. At 48 h after trauma, the protective effect of rhEpo was greater (by about 47%) and significantly more pronounced than that of MK-801 (28%). Our results suggest that the neuroprotective activity of rhEpo is particularly effective against delayed, secondary post-traumatic damage. This well tolerated agent could provide a therapeutic benefit in pathologies involving post-traumatic neurodegeneration.

Hypoxia‐induced erythropoietin expression in human neuroblastoma requires a methylation free HIF‐1 binding site

The glycoprotein hormone Erythropoietin (EPO) stimulates red cell production and maturation. EPO is produced by the kidneys and the fetal liver in response to hypoxia (HOX). Recently, EPO expression has also been observed in the central nervous system where it may be neuroprotective. It remained unclear, however, whether EPO is expressed in the peripheral nervous system and, if so, whether a neuronal phenotype is required for its regulation. Herein, we report that EPO expression was induced by HOX and a HOX mimetic in two cell lines derived from neuroblastoma (NB), a tumor of the peripheral nervous system. Both cell lines with inducible EPO expression, SH-SY5Y and Kelly cells, expressed typical neuronal markers like neuropeptide Y (NPY), growth-associated protein-43 (GAP-43), and neuron-specific enolase (ENO). NB cells with a more epithelial phenotype like SH-SHEP and LAN-5 did not show HOX inducible EPO gene regulation. Still, oxygen sensing and up-regulation of hypoxia-inducible factor-1 (HIF-1) were intact in all cell lines. We found that CpG methylation of the HIF binding site (HBS) in the EPO gene 3' enhancer was only present in the SH-SHEP and LAN-5 cells but not in SH-SY5Y and Kelly cells with regulated EPO expression. The addition of recombinant EPO to all NB cells, both under normoxic and hypoxic conditions, had no effect on cell proliferation. We conclude that the ability to respond to HOX with an increase in EPO expression in human NB may depend on CpG methylation and the differentiation status of these embryonic tumor cells but does not affect the proliferative characteristics of the cells.

Effects of androgenic-anabolic steroids in athletes.

Androgenic-anabolic steroids (AAS) are synthetic derivatives of the male hormone testosterone. They can exert strong effects on the human body that may be beneficial for athletic performance. A review of the literature revealed that most laboratory studies did not investigate the actual doses of AAS currently abused in the field. Therefore, those studies may not reflect the actual (adverse) effects of steroids. The available scientific literature describes that short-term administration of these drugs by athletes can increase strength and bodyweight. Strength gains of about 5-20% of the initial strength and increments of 2-5 kg bodyweight, that may be attributed to an increase of the lean body mass, have been observed. A reduction of fat mass does not seem to occur. Although AAS administration may affect erythropoiesis and blood haemoglobin concentrations, no effect on endurance performance was observed. Little data about the effects of AAS on metabolic responses during exercise training and recovery are available and, therefore, do not allow firm conclusions. The main untoward effects of short- and long-term AAS abuse that male athletes most often self-report are an increase in sexual drive, the occurrence of acne vulgaris, increased body hair and increment of aggressive behaviour. AAS administration will disturb the regular endogenous production of testosterone and gonadotrophins that may persist for months after drug withdrawal. Cardiovascular risk factors may undergo deleterious alterations, including elevation of blood pressure and depression of serum high-density lipoprotein (HDL)-, HDL2- and HDL3-cholesterol levels. In echocardiographic studies in male athletes, AAS did not seem to affect cardiac structure and function, although in animal studies these drugs have been observed to exert hazardous effects on heart structure and function. In studies of athletes, AAS were not found to damage the liver. Psyche and behaviour seem to be strongly affected by AAS. Generally, AAS seem to induce increments of aggression and hostility. Mood disturbances (e.g. depression, [hypo-]mania, psychotic features) are likely to be dose and drug dependent. AAS dependence or withdrawal effects (such as depression) seem to occur only in a small number of AAS users. Dissatisfaction with the body and low self-esteem may lead to the so-called 'reverse anorexia syndrome' that predisposes to the start of AAS use. Many other adverse effects have been associated with AAS misuse, including disturbance of endocrine and immune function, alterations of sebaceous system and skin, changes of haemostatic system and urogenital tract. One has to keep in mind that the scientific data may underestimate the actual untoward effects because of the relatively low doses administered in those studies, since they do not approximate doses used by illicit steroid users. The mechanism of action of AAS may differ between compounds because of variations in the steroid molecule and affinity to androgen receptors. Several pathways of action have been recognised. The enzyme 5-alpha-reductase seems to play an important role by converting AAS into dihydrotestosterone (androstanolone) that acts in the cell nucleus of target organs, such as male accessory glands, skin and prostate. Other mechanisms comprises mediation by the enzyme aromatase that converts AAS in female sex hormones (estradiol and estrone), antagonistic action to estrogens and a competitive antagonism to the glucocorticoid receptors. Furthermore, AAS stimulate erythropoietin synthesis and red cell production as well as bone formation but counteract bone breakdown. The effects on the cardiovascular system are proposed to be mediated by the occurrence of AAS-induced atherosclerosis (due to unfavourable influence on serum lipids and lipoproteins), thrombosis, vasospasm or direct injury to vessel walls, or may be ascribed to a combination of the different mechanisms. AAS-induced increment of muscle tissue can be attributed to hypertrophy and the formation of new muscle fibres, in which key roles are played by satellite cell number and ultrastructure, androgen receptors and myonuclei.

Universal prestorage leukoreduction in Canada decreases platelet alloimmunization and refractoriness

AbstractRandomized controlled trials have shown a reduction in platelet alloimmunization and refractoriness in patients with acute leukemia (AL) with the use of poststorage leukoreduction of blood products. Universal prestorage leukoreduction (ULR) of red cell and platelet products has been performed in Canada since August 1999. We conducted a retrospective analysis of 13 902 platelet transfusions in 617 patients undergoing chemotherapy (CT) for AL or stem cell transplantation (SCT) before (n = 315) and after (n = 302) the introduction of ULR. Alloimmunization was significantly reduced (19% to 7%, P < .001) in the post-ULR group. Alloimmune platelet refractoriness was similarly reduced (14% to 4%, P < .001). Fewer patients in the post-ULR group received HLA-matched platelets (14% vs 5%, P < .001). Alloimmunization and alloimmune refractoriness in the 318 patients who were previously pregnant and/or transfused were also reduced after ULR (P = .023 and P = .005, respectively). In a Cox regression model, the 3 independent factors that predicted for alloimmune refractoriness were nonleukoreduced blood products (relative risk [RR], 2.2 [95% CI, 1.2-4.3]), a history of pregnancy and/or transfusion (RR, 2.3 [95% CI, 1.3-4.2]), and receipt of 13 or more platelet transfusions (RR, 6.0 [95% CI, 2.4-15.3]). In conclusion, ULR reduces alloimmunization, refractoriness, and requirements for HLA-matched platelets when applied as routine transfusion practice to patients receiving CT or SCT.

Erythropoietin: a new tool for muscle disorders?

The main action of erythropoietin (EPO) is to regulate the production of red cells. However both experimental evidence and clinical experience suggest that erythropoietin has a positive effect on skeletal and cardiac muscle. Mice lacking EPO or its receptors suffer from hearth hypoplasia and have a reduced number of proliferating cardiac myocytes. EPO receptors are expressed on mouse primary satellite cells and in cultured myoblasts, and their stimulation appears to enhance proliferation and reduce the differentiation of both cell types. Moreover EPO is capable of promoting angiogenesis in muscle cells, which provides an additional route to increase oxygen supply to active muscles. In men, the effects of EPO on muscle cells are suggested by the illegal use of EPO by agonistic and amateur athletes to enhance their performances. In some athletes EPO improved their long-duration muscular performances much more than expected on the basis of the increment of the blood hemoglobin alone. Our proposal is to investigate the effect of EPO treatment in various animal models of muscular dystrophies (MD), which are common hereditary primary muscle disorders characterized by muscle damage and wasting, to date without any effective treatment. The ability of EPO to induce the proliferation of satellite cells in the presence of differentiating conditions, typical of the damaged muscle, may represent a tool to expand the cellular population competent for muscle repair. This would lengthen the period when muscles can be efficiently repaired. In the presence of positive results, the possibility could be considered of selecting some of the human forms of MD and treating the patients with EPO.

Anemia in the newborn.

In neonatal period anemia is a complex problem owing to the unique blood picture. The erythrocytic system undergoes serial adaptation to meet progressively changing demands of oxygen in the embryo, the fetus and neonate. This leads to rapid change in normal hematological change in post-birth period. Definition of anemia is difficult because as described earlier, several important factors influence normal blood in the newborn infants. The etiology of neonatal anemia can be classified into i) hemorrhage (ii) hemolysis (iii) failure of red cell production. Severe fetal hemorrhage may accompany various placental anomalies like placenta praevia, abruptio placenta and accidental incision of placenta during the caesarian section. It is reported that 10% of all infants born following placenta praevia and 4% of infants born following abruptio placenta present with severe anemia. The passage of fetal erythrocytes in maternal circulation occurs commonly during pregnancy. In 50% of pregnancies some fetal cells are passed in maternal circulation sometimes during gestation or during birth process. Treatment of a neonate with anemia due to blood depends on the degree of hypovolemia or anemia and whether the blood loss has been acute or chronic. Newborn with pale skin should be differentiated from an asphyxiated baby.

Vacuum-assisted venous return reduces blood usage

ObjectiveTo determine whether vacuum-assisted venous return has clinical advantages over conventional gravity drainage apart from allowing the use of smaller cannulas and shorter tubing. MethodsA total of 150 valve operations were performed at our institution between February and July 1999 using vacuum-assisted venous return with small venous cannulas connected to short tubing. These were compared with (1) 83 valve operations performed between April 1997 and January 1998 using the initial version of vacuum-assisted venous return, and (2) 124 valve operations performed between January and April of 1997 using conventional gravity drainage. Priming volume, hematocrit value, red blood cell usage, and total blood product usage were compared multivariably. These comparisons were covariate and propensity adjusted for dissimilarities between the groups and confirmed by propensity-matched pairs analysis. ResultsPriming volume was 1.4 ± 0.4 L for small-cannula vacuum-assisted venous return, 1.7 ± 0.4 L for initial vacuum-assisted venous return, and 2.0 ± 0.4 L for gravity drainage (P < .0001). Smaller priming resulted in higher hematocrit values both at the beginning of cardiopulmonary bypass (27% ± 5% compared with 26% ± 4% and 25% ± 4%, respectively, P < .0001) and at the end (30% ± 4% compared with 28% ± 4% and 27% ± 4%, respectively, P < .0001). Red cell transfusions were used in 17% of the patients having small-cannula vacuum-assisted venous return, 27% of the initial patients having vacuum-assisted venous return, and 37% of the patients having gravity drainage (P = .001); total blood product usage was 19%, 27%, and 39%, respectively (P = .002). Although ministernotomy also was associated with reduced blood product usage (P < .004), propensity matching on type of sternotomy confirmed the association of vacuum-assisted venous return with lowered blood product usage. ConclusionsVacuum-assisted venous return results in (1) higher hematocrit values during cardiopulmonary bypass and (2) decreased red cell and total blood product usage.

Theophylline treatment may adversely affect the anoxia-induced erythropoietic response without suppressing erythropoietin production.

To investigate the influence of theophylline on erythropoiesis in chronic obstructive pulmonary disease (COPD) and explore the potential underlying mechanisms. We evaluated the haematological parameters and erythropoietin (EPO) values in 38 COPD patients, 18 of which had been treated with theophylline (8 mg/kg daily) for at least 1 year, and the other 20 had never received this drug; 38 sex- and age-matched healthy volunteers served as controls. We further studied the development of BFU-E (bursts forming units of erythrocyte precursors) -derived colonies in semisolid methylcellulose cultures in blood samples from 7 patients randomly selected from both groups. In addition, we studied the effects of theophylline on the erythroid cell development by adding this agent to erythroid cell cultures from 6 healthy volunteers at various concentrations. Haemoglobin values were found to be significantly lower in COPD patients treated with theophylline than in those untreated ( P<0.05). Both groups of patients exhibited significantly higher haemoglobin values than normal subjects ( P<0.01 and P<0.001 for treated and untreated patients, respectively). Serum EPO levels did not differ among the three studied groups. Unlike untreated patients and controls, the serum of the theophylline-treated patients produced a significant growth inhibition of erythroid bursts ( P<0.05); the in vitro use of theophylline showed a concentration-dependent inhibition ( P<0.001). Our findings confirm the decrease of red cell production, which occurs following administration of theophylline, exclude the possibility of decreased EPO synthesis and suggest a direct inhibitory action of theophylline on erythropoiesis.

Activation of extracellular signal‐regulated kinases ERK1 and ERK2 induces Bcl‐xL up‐regulation via inhibition of caspase activities in erythropoietin signaling

Erythropoietin (EPO) can rescue erythroid cells from apoptosis during erythroid development, leading to red cell production. However, the detailed mechanism of how EPO protects erythroid cells from apoptosis is still open to question. To address this problem, we used a human EPO-dependent leukemia cell line UT-7/EPO and normal erythroid progenitor cells. After deprivation of EPO, UT-7/EPO cells underwent apoptosis, accompanied by down-regulation of the Bcl-xL protein. In addition, the cleaved products of caspase-3, p11 and p21, and a few cleaved forms of inhibitor of caspase-activated DNase (ICAD) were detected in these cells. When the cells were pre-treated with the pancaspase inhibitor Z-VAD-FMK, the ratio of apoptotic cells was significantly reduced, suggesting that EPO protects the UT-7/EPO cells from apoptosis via inhibition of caspase activities. When an MEK 1/2 inhibitor U0126 inhibited activities of extracellular signal-regulated kinases (ERKs), the expression of Bcl-xL protein was down-regulated and subsequently apoptosis was induced. Interestingly, Z-VAD-FMK blocked U0126-induced down-regulation of Bcl-xL protein and apoptosis, strongly suggesting that Bcl-xL expression is regulated by caspases which lies downstream of ERK activation pathway in EPO signaling. Importantly, these findings were also observed in normal erythroid progenitor cells. In conclusion, the activation of ERKs by EPO up-regulates Bcl-xL expression via inhibition of caspase activities, resulting in the protection of erythroid cells from apoptosis.

Proposal for scoring severity in chronic mountain sickness (CMS). Background and conclusions of the CMS Working Group.

Living above 2500 m are millions of persons who are at risk for Chronic Mountain Sickness (CMS), a disorder of excessive red cell and hemoglobin production, which threatens health and even life itself. The CMS Working Group was founded in Matsumoto, Japan and meets regularly at international meetings when a quorum of interested scientists is present. The overall goals of the CMS Working Group are to lay the foundation for better understanding of the description, pathogenesis and treatment of CMS by developing consensus among interested scientists from around the world. Toward that end this chapter presents the latest efforts of the CMS Consensus Working Group.

Non-erythroid functions of erythropoietin.

The oxygen-dependent, renal cytokine eythropoietin (Epo) is well known to increase red cell production. Binding of Epo to the Epo receptor (EpoR) represses apoptosis of erythroid progenitor cells, thereby allowing their final maturation. We and others showed that Epo and its receptor are expressed in many other tissues, including brain, spinal cord, retina and testis. The presence of a blood barrier suggests that Epo plays a local role in these organs. Indeed, therapeutically applied or hypoxically induced Epo has been shown to reduce the infarct volume in various stroke animal models, to prevent retinal degeneration, and to ameliorate spinal cord injury. In a study conducted by Ehrenreich and colleagues, stroke patients treated with Epo showed reduced infarct volume, fast neurological recovery and improved clinical outcome. In analogy to its function on erythroid progenitor cells, this neuroprotective effect of Epo might be explained by repression of programmed cell death. Apart from neuroprotection, there is an assumption that Epo present in breast milk has the potential to protect against mother-to-infant transmission of HIV. When using Epo at high doses for longer time periods; however, care has to be taken to control the resulting chronic polycythemia that most probably caused enlarged cerebral infarct volumes in a transgenic mouse model that due to Epo-overexpression reached hematocrit levels of about 0.8. Overall, these data strongly support the notion that Epo will soon find new applications in the clinic.

Erythropoietin: physiology and pharmacology update.

This minireview is an update of a 1997 review on erythropoietin (EPO) in this journal. EPO is a 30,400-dalton glycoprotein that regulates red cell production. In the human, EPO is produced by peritubular cells in the kidneys of the adult and in hepatocytes in the fetus. Small amounts of extra-renal EPO are produced by the liver in adult human subjects. EPO binds to an erythroid progenitor cell surface receptor that includes a p66 chain, and, when activated, the p66 protein becomes dimerized. EPO receptor activation induces a JAK2 tyrosine kinase, which leads to tyrosine phosphorylation of the EPO receptor and several proteins. EPO receptor binding leads to intracellular activation of the Ras/mitogen-activated kinase pathway, which is involved with cell proliferation, phosphatidylinositol 3-kinase, and STATS 1, 3, 5A, and 5B transcriptional factors. EPO acts primarily to rescue erythroid cells from apoptosis (programmed cell death) to increase their survival. EPO acts synergistically with several growth factors (SCF, GM-CSF, 1L-3, and IGF-1) to cause maturation and proliferation of erythroid progenitor cells (primarily colony-forming unit-E). Oxygen-dependent regulation of EPO gene expression is postulated to be controlled by a hypoxia-inducible transcription factor (HIF-1alpha). Hypoxia-inducible EPO production is controlled by a 50-bp hypoxia-inducible enhancer that is approximately 120 bp 3' to the polyadenylation site. Hypoxia signal transduction pathways involve kinases A and C, phospholipase A(2), and transcription factors ATF-1 and CREB-1. A model has been proposed for adenosine activation of EPO production that involves protein kinases A and C and the phospholipase A(2) pathway. Other effects of EPO include a hematocrit-independent, vasoconstriction-dependent hypertension, increased endothelin production, upregulation of tissue renin, change in vascular tissue prostaglandins production, stimulation of angiogenesis, and stimulation of endothelial and vascular smooth muscle cell proliferation. Recombinant human EPO (rHuEPO) is currently being used to treat patients with anemias associated with chronic renal failure, AIDS patients with anemia due to treatment with zidovudine, nonmyeloid malignancies in patients treated with chemotherapeutic agents, perioperative surgical patients, and autologous blood donation. A novel erythropoiesis-stimulating factor (NESP, darbepoetin) has been synthesized and when compared with rHuEPO, NESP has a higher carbohydrate content (52% vs 40%), a longer plasma half-life, the amino acid sequence differs from that of native human EPO at five positions, and has been reported to maintain hemoglobin levels just as effectively in patients with chronic renal failure as rHuEPO at less frequent dosing. The use of rHuEPO and darbepoetin to enhance athletic performance is officially banned by most sports-governing bodies because the excessive erythrocytosis can lead to increased thrombogenicity and can cause deep vein, coronary, and cerebral thromboses.