Erythropoietin (EPO) is a hematopoietic growth factor that stimulates proliferation and differentiation of erythroid precursor cells and is also known to exert neurotrophic activity in the central nervous system (CNS). However, little is known about expression of EPO and EPO receptor (EPOR) in human CNS tissues. In the present study, we investigated the effects of proinflammatory cytokines on EPO and EPOR expression in highly purified cultures of human neurons, astrocytes, microglia, and oligodendrocytes using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). EPO mRNA was demonstrated only in human astrocytes, while EPOR expression was found in human neurons, astrocytes, and microglia. Neither EPO nor EPOR expression was found in oligodendrocytes. In human astrocytes, EPO mRNA and secreted EPO protein levels were downregulated after exposure to proinflammatory cytokines (IL-1beta, IL-6, or TNF-alpha). In human neurons, TNF-alpha treatment markedly increased EPOR expression. These results suggest that proinflammatory cytokines regulate expression of EPO and EPOR in human neurons, astrocytes, and microglia and further facilitate interactions among different cell types in the human CNS.

The presence of a prevalent vertebral deformity increases the risk of a future vertebral fracture. The aim of this study was to determine whether certain characteristics of the prevalent deformity, including its shape and location in the spine, influenced this effect. The 3100 men and 3500 women who took part in this analysis were recruited from population registers for participation in the European Prospective Osteoporosis Study (EPOS). Subjects had lateral thoracic and lumbar spine x-rays at baseline, and again after a mean interval of 3.8 years. Prevalent morphometric vertebral deformities on the baseline film were identified by the McCloskey-Kanis method. Incident fractures were defined as vertebrae that also satisfied the McCloskey-Kanis criterion for prevalent deformities on the follow-up film, and in addition had at least one height (anterior, mid, or posterior) which had reduced by at least 20% between films. Poisson regression was used to assess the association between various characteristics of the prevalent deformity and the risk of an incident vertebral fracture, with generalised estimating equations used to allow for the fact that each subject contributed several vertebrae to the analysis. The risk of an incident fracture increased with the number of prevalent deformities: relative risk (RR) for one prevalent deformity 3.2 (95% confidence interval (CI); 2.1, 4.8); 9.8 (95% CI;6.1, 15.8) for 2; and 23.3 (95% CI;15.3, 35.4) for 3 or more. Relative risks differed significantly according to the shape of the prevalent deformity, ranging from 5.9 (95% CI; 4.1, 8.6) if the anterior and mid heights were reduced to 1.6 (95% CI;0.8, 3.2) if the posterior and mid heights were reduced. Risks varied also according to the severity of the deformity. There were fivefold differences in relative risk of incident fracture depending on the location of the prevalent deformity within the spine. Compared to vertebrae in subjects with no deformities at baseline, the relative risk of an incident fracture within three vertebrae of a prevalent deformity was greater (7.7 (95% CI;5.6, 10.5)) than the risk in more distant vertebrae (4.0 (95% CI;2.6, 6.0)). In summary, the risk of a subsequent vertebral fracture in individuals with preexisting deformities is importantly influenced by the characteristics of these deformities.

The Japanese Society for Dialysis Therapy (JSDT) guideline committee, chaired by Dr Y. Tsubakihara, presents the Japanese guidelines entitled "Guidelines for Renal Anemia in Chronic Kidney Disease." These guidelines replace the "2004 JSDT Guidelines for Renal Anemia in Chronic Hemodialysis Patients," and contain new, additional guidelines for peritoneal dialysis (PD), non-dialysis (ND), and pediatric chronic kidney disease (CKD) patients. Chapter 1 presents reference values for diagnosing anemia that are based on the most recent epidemiological data from the general Japanese population. In both men and women, hemoglobin (Hb) levels decrease along with an increase in age and the level for diagnosing anemia has been set at <13.5 g/dL in males and <11.5 g/dL in females. However, the guidelines explicitly state that the target Hb level in erythropoiesis stimulating agent (ESA) therapy is different to the anemia reference level. In addition, in defining renal anemia, the guidelines emphasize that the reduced production of erythropoietin (EPO) that is associated with renal disorders is the primary cause of renal anemia, and that renal anemia refers to a condition in which there is no increased production of EPO and serum EPO levels remain within the reference range for healthy individuals without anemia, irrespective of the glomerular filtration rate (GFR). In other words, renal anemia is clearly identified as an "endocrine disease." It is believed that defining renal anemia in this way will be extremely beneficial for ND patients exhibiting renal anemia despite having a high GFR. We have also emphasized that renal anemia may be treated not only with ESA therapy but also with appropriate iron supplementation and the improvement of anemia associated with chronic disease, which is associated with inflammation, and inadequate dialysis, another major cause of renal anemia. In Chapter 2, which discusses the target Hb levels in ESA therapy, the guidelines establish different target levels for hemodialysis (HD) patients than for PD and ND patients, for two reasons: (i) In Japanese HD patients, Hb levels following hemodialysis rise considerably above their previous levels because of ultrafiltration-induced hemoconcentration; and (ii) as noted in the 2004 guidelines, although 10 to 11 g/dL was optimal for long-term prognosis if the Hb level prior to the hemodialysis session in an HD patient had been established at the target level, it has been reported that, based on data accumulated on Japanese PD and ND patients, in patients without serious cardiovascular disease, higher levels have a cardiac or renal function protective effect, without any safety issues. Accordingly, the guidelines establish a target Hb level in PD and ND patients of 11 g/dL or more, and recommend 13 g/dL as the criterion for dose reduction/withdrawal. However, with the results of, for example, the CHOIR (Correction of Hemoglobin and Outcomes in Renal Insufficiency) study in mind, the guidelines establish an upper limit of 12 g/dL for patients with serious cardiovascular disease or patients for whom the attending physician determines high Hb levels would not be appropriate. Chapter 3 discusses the criteria for iron supplementation. The guidelines establish reference levels for iron supplementation in Japan that are lower than those established in the Western guidelines. This is because of concerns about long-term toxicity if the results of short-term studies conducted by Western manufacturers, in which an ESA cost-savings effect has been positioned as a primary endpoint, are too readily accepted. In other words, if the serum ferritin is <100 ng/mL and the transferrin saturation rate (TSAT) is <20%, then the criteria for iron supplementation will be met; if only one of these criteria is met, then iron supplementation should be considered unnecessary. Although there is a dearth of supporting evidence for these criteria, there are patients that have been surviving on hemodialysis in Japan for more than 40 years, and since there are approximately 20 000 patients who have been receiving hemodialysis for more than 20 years, which is a situation that is different from that in many other countries. As there are concerns about adverse reactions due to the overuse of iron preparations as well, we therefore adopted the expert opinion that evidence obtained from studies in which an ESA cost-savings effect had been positioned as the primary endpoint should not be accepted unquestioningly. In Chapter 4, which discusses ESA dosing regimens, and Chapter 5, which discusses poor response to ESAs, we gave priority to the usual doses that are listed in the package inserts of the ESAs that can be used in Japan. However, if the maximum dose of darbepoetin alfa that can currently be used in HD and PD patients were to be used, then the majority of poor responders would be rescued. Blood transfusions are discussed in Chapter 6. Blood transfusions are attributed to the difficulty of managing renal anemia not only in HD patients, but also in end-stage ND patients who respond poorly to ESAs. It is believed that the number of patients requiring transfusions could be reduced further if there were novel long-acting ESAs that could be used for ND patients. Chapter 7 discusses adverse reactions to ESA therapy. Of particular concern is the emergence and exacerbation of hypertension associated with rapid hematopoiesis due to ESA therapy. The treatment of renal anemia in pediatric CKD patients is discussed in Chapter 8; it is fundamentally the same as that in adults.

We analyzed prognostic factors of response, response duration, and possible impact on survival of epoetin alpha, epoetin beta, or darbepoetin alpha (DAR) with or without granulocyte colony-stimulating factor in 403 myelodysplastic syndrome (MDS) patients. Sixty-two percent (40% major and 22% minor) and 50% erythroid responses were seen, and median response duration was 20 and 24 months according to IWG 2000 and 2006 criteria, respectively. Significantly higher response rates were observed with less than 10% blasts, low and int-1 International Prognostic Scoring System (IPSS), red blood cell transfusion independence, serum EPO level less than 200 IU/L, and, with IWG 2006 criteria only, shorter interval between diagnosis and treatment. Significantly longer response duration was associated with major response (IWG 2000 criteria), IPSS low to INT-1, blasts less than 5%, and absence of multilineage dysplasia. Minor responses according to IWG 2000 were reclassified as "nonresponders" or "responders" according to IWG 2006 criteria. However, among those IWG 2000 minor responders, response duration did not differ between IWG 2006 responders and nonresponders. Multivariate adjusted comparisons of survival between our cohort and the untreated MDS cohort used to design IPSS showed similar rate of progression to acute myeloid leukemia in both cohorts, but significantly better overall survival in our cohort, suggesting that epoetin or DAR treatment may have a favorable survival impact in MDS.

Erythropoietin (Epo) and its receptor (EpoR) are indispensable to erythropoiesis. Although roles besides angiogenesis, such as neuroprotection and heart development, have been reported for the Epo-EpoR system, the precise contribution of Epo-EpoR to these nonhematopoietic tissues requires clarification. Exploiting a GATA-1 minigene cassette with hematopoietic regulatory domains, we established 2 lines of transgene-rescued EpoR-null mutant mice expressing EpoR exclusively in the hematopoietic lineage. Surprisingly, despite the lack of EpoR expression in nonhematopoietic tissues, these mice develop normally and are fertile. As such, we could exploit them for analyzing the roles of the Epo-EpoR system in adult hematopoiesis and in nonhematopoietic tissues. These rescued lines showed a differential level of EpoR expression in erythroid cells; one expressed approximately 40%, and the other expressed 120% of the wild-type EpoR level. A colony formation assay showed that erythroid progenitors in the 2 mutant lines exhibit distinct sensitivity to Epo. The circulating Epo level was much higher in the transgenic line with a lower EpoR expression. In response to induced anemia, the plasma Epo concentrations increased in both lines. Notably, the timing of the peak of plasma Epo concentration was delayed in both lines of rescued mice compared with wild type, suggesting that, in wild-type mice, nonhematopoietic EpoR contributes to the regulation of plasma Epo concentration. We thus conclude that nonhematopoietic expression of EpoR is dispensable to normal mouse development and that the expression level of EpoR regulates erythropoiesis by controlling the sensitivity of erythroid progenitors to Epo.

Erythropoietin (EPO), a hypoxia-inducible cytokine, is required for survival, proliferation, and differentiation of erythroid progenitor cells. EPO can also stimulate proliferation and angiogenesis of endothelial cells that express EPO receptors (EPORs). In this study we investigated the EPO response of vascular endothelial cells at reduced oxygen tension (5% and 2%), in particular the effect of EPO on nitric oxide (NO) release. Endothelial nitric oxide synthase (eNOS) produces NO, which maintains blood pressure homeostasis and blood flow. We find that EPOR is inducible by EPO in primary human endothelial cells of vein (HUVECs) and artery (HUAECs) and cells from a human bone marrow microvascular endothelial line (TrHBMEC) to a much greater extent at low oxygen tension than in room air. We found a corresponding increase in eNOS expression and NO production in response to EPO during hypoxia. Stimulation of NO production was dose dependent on EPO concentration and was maximal at 5 U/mL. NO activates soluble guanosine cyclase to produce cyclic guanosine monophosphate (cGMP), and we observed that EPO induced cGMP activity. These results suggest that low oxygen tension increases endothelial cell capacity to produce NO in response to EPO by induction of both EPOR and eNOS. This effect of EPO on eNOS may be a physiologically relevant mechanism to counterbalance the hypertensive effects of increased hemoglobin-related NO destruction resulting from hypoxia-induced increased red cell mass.

Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60-75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1alpha-independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte-endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.

Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies.

We have established a heart slice primary culture, which allows us to mechanically separate distinct cardiac cell populations and assay their relative mitogenic and trophic effects on cardiac myocyte proliferation and survival. Using this system, we have found that a signal(s) from the epicardium, but not the trabeculae and endocardium, is required in embryonic day 10 (E10) chick heart slices for continued cardiac myocyte proliferation and survival. An examination of potential epicardial growth or trophic factors has revealed that blockade of either retinoic acid (RA) or erythopoietin (epo) signaling from the epicardium inhibits cardiac myocyte proliferation and survival. The blockade of cardiac myocyte proliferation following administration of an RA antagonist can be rescued by exogenous epo. Conversely, the blockade of cardiac myocyte proliferation following administration of an anti-epo receptor antisera can be rescued by exogenous RA. Thus, our findings suggest that RA and epo signals work in parallel to support myocardial cell proliferation. In addition, we have found that these factors do not act directly on myocardial cells. Rather, they induce another soluble factor(s) in the epicardium that directly regulates proliferation of cardiac myocytes. We therefore postulate that the epicardium controls normal heart growth in ventricular segments of the embryonic chick heart by secreting a cardiac myocyte mitogen whose expression (or activity) is regulated by both RA and erythropoietin signaling.

Initial clinical trials with recombinant human erythropoietin provided evidence of a quality-of-life benefit for patients with anemic end-stage renal disease who received maintenance hemodialysis. As part of a phase III clinical trial of recombinant human erythropoietin, the quality of life of patients was systematically assessed. More than 300 patients at nine dialysis centers were evaluated. A statistically significant improvement was established between baseline and second follow-up on most objective and subjective quality-of-life parameters, including energy and activity level, functional ability, sleep and eating behavior, disease symptoms, health status, satisfaction with health, sex life, well-being, psychological affect, life satisfaction, and happiness. No change was observed in ability to work or employment status. We conclude that, in addition to substantial improvement in hematologic parameters, recombinant human erythropoietin greatly enhances the quality of life of anemic patients who receive maintenance hemodialysis.

Hemopoiesis is studied in vitro mainly in semisolid culture, where hemopoietic progenitors develop into discrete colonies. We describe a liquid culture system that supports the proliferation and maturation of human erythroid progenitors. We seeded mononuclear cells from the peripheral blood (PB) of patients with beta-thalassemia in liquid medium in the presence of conditioned medium from human bladder carcinoma cells. Seven days later, RBCs, normoblasts, granulocytes, and monocytes disappeared, and the number of lymphocytes dropped considerably. In contrast, erythroid colony-forming cells increased fourfold to tenfold. The next step entailed the removal of colony-stimulating factor (CSF) and CSF-secreting cells, the exclusion of macrophages by harvesting nonadherent cells, and the lysis of T lymphocytes by treatment with monoclonal rat antihuman lymphocyte antibodies (CAMPATH-1) and complement. Reculture of the remaining cells in liquid medium supplemented with recombinant erythropoietin (EPO) resulted in the exclusive development of erythroid cells, with myeloid cells reduced to less than 2%. Stainable hemoglobin (Hb) appeared on day 3, with over 85% of the population containing hemoglobin by day 11 and the cell number increasing from 0.2 X 10(6) to 3 X 10(6) mL. By permitting the manipulation of culture conditions and components and increasing the cell yield, the liquid system may facilitate quantitative analysis of growth kinetics as well as biochemical and immunologic characterization of the developing erythroid cell.

The platelet-derived growth factor (PDGF) signaling system contributes to tumor angiogenesis and vascular remodeling. Here we show in mouse tumor models that PDGF-BB induces erythropoietin (EPO) mRNA and protein expression by targeting stromal and perivascular cells that express PDGF receptor-β (PDGFR-β). Tumor-derived PDGF-BB promoted tumor growth, angiogenesis and extramedullary hematopoiesis at least in part through modulation of EPO expression. Moreover, adenoviral delivery of PDGF-BB to tumor-free mice increased both EPO production and erythropoiesis, as well as protecting from irradiation-induced anemia. At the molecular level, we show that the PDGF-BB-PDGFR-bβ signaling system activates the EPO promoter, acting in part through transcriptional regulation by the transcription factor Atf3, possibly through its association with two additional transcription factors, c-Jun and Sp1. Our findings suggest that PDGF-BB-induced EPO promotes tumor growth through two mechanisms: first, paracrine stimulation of tumor angiogenesis by direct induction of endothelial cell proliferation, migration, sprouting and tube formation, and second, endocrine stimulation of extramedullary hematopoiesis leading to increased oxygen perfusion and protection against tumor-associated anemia.

OBJECTIVE

Erythropoietin (EPO) modulates erythropoiesis by inhibiting apoptosis in erythrocyte progenitors. Recently, EPO has been shown to be protective in experimental models of mechanical trauma, neuroinflammation, cerebral and retinal ischemia, and even in a human stroke trial. However, little is known about EPO signal transduction in vivo and the usefulness of EPO in the prevention of the chronic, purely apoptotic neuronal cell death that contributes to vision loss in glaucoma and the progression of neurodegenerative diseases.

METHODS

EPO's effects and signaling in the retinal ganglion cell axotomy paradigm were studied by Western blot analysis and immunohistochemistry, receptor expression was characterized in the retina before and after lesion. EPO was injected into the vitreous body to investigate neuroprotection of axotomized rat RGCs. Moreover, EPO's effects were studied in cultures of immunopurified retinal ganglion cells. Signal-transduction pathways transmitting neuroprotective EPO effects in vivo were characterized by the use of specific kinase inhibitors, immunohistochemistry, and Western blot analysis.

RESULTS

EPO receptors (EPORs) were expressed on RGC somata and dendrites in vivo. EPOR expression did not significantly change after axotomy. Application of EPO prevented death of neurotrophic-factor-deprived immunopurified rat RGCs in vitro, rescued axotomized RGCs in vivo, and prevented caspase-3 activation. EPO-induced Akt phosphorylation and survival-promoting EPO effects were completely abolished by inhibition of PI-3-kinase. EPO neuroprotection followed a bell-shaped dose-response curve in vitro and in vivo, whereas toxic EPO effects were never observed, even at high concentrations.

CONCLUSIONS

These data support a potential role for EPO as a therapeutic molecule against predominantly apoptotic neuronal cell death in the context of glaucoma or neurodegenerative diseases and delineate the PI-3-K/Akt pathway as the predominant mediator of EPO neuroprotection in this in vivo paradigm of neuronal cell death.

OBJECTIVE

Retinal ganglion cell (RGC) loss occurs in response to increased intraocular pressure (IOP) and/or retinal ischemia in glaucoma and leads to impairment of vision. This study was undertaken to test the efficacy of erythropoietin (EPO) in providing neuroprotection to RGCs in vivo.

METHODS

The neuroprotective effects of EPO were studied in the DBA/2J mouse model of glaucoma. Mice were intraperitoneally injected with control substances or various doses of EPO, starting at the age of 6 months and continuing for an additional 2, 4, or 6 months. RGCs were labeled retrogradely by a gold tracer. IOP was measured with a microelectric-mechanical system, and EPO receptor (EPOR) expression was detected by immunohistochemistry. Axonal death in the optic nerve was quantified by para-phenylenediamine staining, and a complete blood count system was used to measure the number of erythrocytes.

RESULTS

In DBA/2J mice, the average number of viable RGCs significantly decreased from 4 months to 10 months, with an inverse correlation between the number of dead optic nerve axons and viable RGCs. Treatment with EPO at doses of 3000, 6000, and 12,000 U/kg body weight per week all prevented significant RGC loss, compared with untreated DBA/2J control animals. EPO effects were similar to those of memantine, a known neuroprotective agent. IOP, in contrast, was unchanged by both EPO and memantine. Finally, EPOR was expressed in the RGC layer in both DBA/2J and C57BL/6J mice.

CONCLUSIONS

EPO promoted RGC survival in DBA/2J glaucomatous mice without affecting IOP. These results suggest that EPO may be a potential therapeutic neuroprotectant in glaucoma.

A novel system to study early hematopoietic development is described. This report documents the in vitro capacity of murine embryonic stem (ES) cells to differentiate into hematopoietic precursors of most, if not all, of the colony-forming cells found in normal bone marrow. This system is used to correlate the genetic expression of cytokines, their receptors, the beta-globins, and the hematopoietic cell surface markers throughout the time course of ES cell differentiation with the hematopoietic development that occurs in these cultures. Our results indicate that there is a strong transcriptional activation, in a well-defined temporal order, of most of these genes including erythropoietin (Epo), CSF-1, IL-4, beta-globins, as well as the receptors for Epo, CSF-1, and IL-4. IL-3 and GM-CSF were not expressed during the first 24 days of ES cell differentiation. In contrast, the Steel (Sl) factor (SLF) was expressed early and underwent substantial up-regulation during this differentiation, and its receptor, c-kit, was expressed relatively constantly throughout the culture period. Our results are consistent with the conclusion that SLF, Epo, IL-4, and IL-6 are important during the early stages of ES cell differentiation and hematopoietic development. Furthermore, these results argue strongly that IL-3 and GM-CSF are not critical to early hematopoiesis. This system offers a unique in vitro model for studying hematopoietic development at the earliest possible stages.

Anemia due to chronic disease or chemotherapy often is ameliorated by erythropoietin (Epo). Present studies reveal that, unlike steady-state erythropoiesis, erythropoiesis during anemia depends sharply on an Epo receptor-phosphotyrosine-343-Stat5 signaling axis. In mice expressing a phosphotyrosine-null (PY-null) Epo receptor allele (EpoR-HM), severe and persistent anemia was induced by hemolysis or 5-fluorouracil. In short-term transplantation experiments, donor EpoR-HM bone marrow cells also failed to efficiently repopulate the erythroid compartment. In each context, stress erythropoiesis was rescued to WT levels upon the selective restoration of an EpoR PY343 Stat5-binding site (EpoR-H allele). As studied using a unique primary culture system, EpoR-HM erythroblasts exhibited marked stage-specific losses in Epo-dependent growth and survival. EpoR-H PY343 signals restored efficient erythroblast expansion, and the selective Epo induction of the Stat5 target genes proviral integration site-1 (Pim-1) and oncostatin-M. Bcl2-like 1 (Bcl-x), in contrast, was not significantly induced via WT-EpoR, EpoR-HM, or EpoR-H alleles. In Kit+ CD71+ erythroblasts, EpoR-PY343 signals furthermore enhanced SCF growth effects, and SCF modulation of Pim-1 kinase and oncostatin-M expression. In maturing Kit- CD71+ erythroblasts, oncostatin-M exerted antiapoptotic effects that likewise depended on EpoR PY343-mediated events. Stress erythropoiesis, therefore, requires stage-specific EpoR-PY343-Stat5 signals, some of which selectively bolster SCF and oncostatin-M action.

Eosinophil peroxidase (EPO) has been implicated in promoting oxidative tissue injury in conditions ranging from asthma and other allergic inflammatory disorders to cancer and parasitic/helminthic infections. Studies thus far on this unique peroxidase have primarily focused on its unusual substrate preference for bromide (Br(-)) and the pseudohalide thiocyanate (SCN(-)) forming potent hypohalous acids as cytotoxic oxidants. However, the ability of EPO to generate reactive nitrogen species has not yet been reported. We now demonstrate that EPO readily uses nitrite (NO(2)(-)), a major end-product of nitric oxide ((.)NO) metabolism, as substrate to generate a reactive intermediate that nitrates protein tyrosyl residues in high yield. EPO-catalyzed nitration of tyrosine occurred more readily than bromination at neutral pH, plasma levels of halides, and pathophysiologically relevant concentrations of NO(2)(-). Furthermore, EPO was significantly more effective than MPO at promoting tyrosine nitration in the presence of plasma levels of halides. Whereas recent studies suggest that MPO can also promote protein nitration through indirect oxidation of NO(2)(-) with HOCl, we found no evidence that EPO can indirectly mediate protein nitration by a similar reaction between HOBr and NO(2)(-). EPO-dependent nitration of tyrosine was modulated over a physiologically relevant range of SCN(-) concentrations and was accompanied by formation of tyrosyl radical addition products (e.g. o,o'-dityrosine, pulcherosine, trityrosine). The potential role of specific antioxidants and nucleophilic scavengers on yields of tyrosine nitration and bromination by EPO are examined. Thus, EPO may contribute to nitrotyrosine formation in inflammatory conditions characterized by recruitment and activation of eosinophils.

OBJECTIVE

Erythropoietin (EPO) has been well characterized and shown to improve functional outcomes after ischemic injury, but EPO may also have unexplored effects on neurovascular remodeling and neuronal replacement in the neonatal ischemic brain. The current study investigates the effects of exogenous administration of EPO on revascularization and neurogenesis, 2 major events thought to contribute to neuronal replacement, in the neonatal brain after hypoxia/ischemia (H/I).

METHODS

Seven-day-old rat pups were treated with recombinant human EPO or vehicle 20 minutes after H/I and again on postischemic days 2, 4, and 6. Rats were euthanized 7 or 28 days after H/I for evaluation of infarct volume, revascularization, neurogenesis, and neuronal replacement using bromodeoxyuridine incorporation, immunohistochemistry, and lectin labeling. Neurological function was assessed progressively for 28 days after H/I by gait testing, righting reflex and foot fault testing.

RESULTS

We demonstrate that exogenous EPO-enhanced revascularization in the ischemic hemisphere correlated with decreased infarct volume and improved neurological outcomes after H/I. In addition to vascular effects, EPO increased both neurogenesis in the subventricular zone and migration of neuronal progenitors into the ischemic cortex and striatum. A significant number of newly synthesized cells in the ischemic boundary expressed neuronal nuclei after EPO treatment, indicating that exogenous EPO led to neuronal replacement.

CONCLUSIONS

Our data suggest that treatment with EPO contributes to neurovascular remodeling after H/I by promoting tissue protection, revascularization, and neurogenesis in neonatal H/I-injured brain, leading to improved neurobehavioral outcomes.

Eosinophil recruitment and enhanced production of NO are characteristic features of asthma. However, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma is established. Using gas chromatography-mass spectrometry we demonstrate a 10-fold increase in 3-nitrotyrosine (NO(2)Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bronchoalveolar lavage of severe asthmatic patients (480 +/- 198 micromol/mol tyrosine; n = 11) compared with nonasthmatic subjects (52.5 +/- 40.7 micromol/mol tyrosine; n = 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorotyrosine (ClY), selective markers of eosinophil peroxidase (EPO)- and myeloperoxidase-catalyzed oxidation, respectively, demonstrated a dramatic preferential formation of BrY in asthmatic (1093 +/- 457 micromol BrY/mol tyrosine; 161 +/- 88 micromol ClY/mol tyrosine; n = 11 each) compared with nonasthmatic subjects (13 +/- 14.5 micromol BrY/mol tyrosine; 65 +/- 69 micromol ClY/mol tyrosine; n = 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO(2)Y immunostaining in epitopes that colocalized with eosinophils. Although eosinophils from normal subjects failed to generate detectable levels of NO, NO(2-), NO(3-), or NO(2)Y, tyrosine nitration was promoted by eosinophils activated either in the presence of physiological levels of NO(2-) or an exogenous NO source. At low, but not high (e.g., >2 microM/min), rates of NO flux, EPO inhibitors and catalase markedly attenuated aromatic nitration. These results identify eosinophils as a major source of oxidants during asthma. They also demonstrate that eosinophils use distinct mechanisms for generating NO-derived oxidants and identify EPO as an enzymatic source of nitrating intermediates in eosinophils.

Erythropoietin (EPO) prevents the ischemia-induced delayed neuronal death in the hippocampal CA1 field in gerbils. EPO receptor (EPOR) is also expressed in the cerebral cortex but its function is not known. To examine whether EPO has a neuroprotective action in the cortex, EPO was infused into the cerebroventricles of stroke-prone spontaneously hypertensive rats with permanent occlusion of the left middle cerebral artery. Morris water maze test indicated that EPO infusion alleviated the ischemia-induced place navigation disability. The left (ischemic)-to-right (contralateral nonischemic) (L/R) ratio of cerebrocortical area in the EPO-infused ischemic group was larger than that in the vehicle-infused ischemic group. The occlusion caused secondary thalamic degeneration but infusion of EPO prevented the decrease in the L/R ratio of thalamic area and supported neuron survival in the ventroposterior thalamic nucleus. In situ hybridization indicated that EPOR mRNA was upregulated in the periphery (ischemic penumbra) of a cerebrocortical infarct after occlusion of the middle cerebral artery, suggesting that an increased number of EPOR in neurons facilitates the EPO signal transmission, thereby preventing the damaged area from enlarging.

Mechanical circulatory assist devices (MCADs) are increasingly utilized independently of cardiac transplantation in the management of heart failure. Though MCAD use incorporates inherent mechanical risks, the inevitable onset of chronic anemia, with its associated morbidity and mortality, is also a significant concern. MCAD support has been correlated with elevated plasma levels of inflammatory cytokines TNF-alpha, IL-1beta, and IL-6, which have separately been found to inhibit erythropoietin (Epo)-induced erythrocyte (RBC) maturation. Previous analysis of hematological parameters for MCAD-supported patients concluded that an amplified inflammatory response impedes RBC proliferation and recovery from hemolytic anemia. Additional analysis may bolster this assertion. Hemoglobin concentration (HC), RBC distribution width (RDW), mean cell volume (MCV), and cardiac index were retrospectively analysed for 78 MCAD-supported patients implanted for greater than 30 days at the University of Arizona Health Sciences Center from 1996 to 2002. Analysis confirms that the HC, a conventional marker for anemia, declines with MCAD placement and remains below the clinically defined, minimum normal value. Inversely, the RDW rises above maximum normal measure, signifying an increased fraction of juvenile RBCs. The MCV remains unchanged and within normal limits, demonstrating adequate substrate for RBC formation. MCAD performance also stabilizes as adequate perfusion returns. These results further support our previously published conclusion that a sufficient response of erythropoiesis occurs in reaction to the onset of anemia by an increased production of immature RBCs. However, the cells never fully mature and join circulation. The patient's inflammatory cytokine response to the implanted device most likely mediates the chronic MCAD-induced anemia by inhibition of Epo effects.

Recombinant adeno-associated virus (rAAV) vectors were evaluated for gene transfer into the skeletal muscle of adult immunocompetent mice. A study using a vector encoding nuclear localized beta-galactosidase (rAAV-nls-lacZ) examined: (i) the efficiency and duration of transgene expression; (ii) the status of the AAV genome in the transduced fibers; and (iii) the possibility of improving gene transfer by inducing muscle regeneration. In the absence of regeneration, the injection of 1.7 x 10(7) particles in the quadriceps resulted in gene transfer to 10-70% of myofibers. Histological analysis indicated that the vector was able to reach myofiber nuclei distant from the injection point. Cellular infiltrates were absent at early time points but became conspicuous in the vicinity of some positive fibers at 4-8 weeks and subsided by 26 weeks. Southern analysis indicated that one to three copies of the vector genome were present per cell genome equivalent. They were associated with high-molecular-weight DNA in the form of tandem oligomers or interlocked circles. Gene transfer was not facilitated in the regenerating muscle. Rather, an early inflammatory response resulted in the elimination of most positive fibers after 8 weeks. The presence of regenerated fibers with beta-galactosidase-positive nuclei suggested that myoblasts had been transduced and were able to fuse to form new fibers. Gene transfer in the absence of immune reactions against the transgene product was studied by injecting mice with a rAAV carrying the murine erythropoietin (mEpo) cDNA. Dose-dependent elevation in the hematocrit was measured for over 200 days and corresponded to 5- to 20-fold increases in plasma Epo levels. We conclude that AAV vectors efficiently and stably transduce post-mitotic muscle fibers and myoblasts in vivo.

The cytoplasmic domain of the erythropoietin receptor (EpoR) contains a membrane-distal region that is dispensable for mitogenesis but is required for the recruitment and tyrosine phosphorylation of a variety of signaling proteins. The membrane-proximal region of 96 amino acids is necessary and sufficient for mitogenesis as well as Jak2 activation, induction of c-fos, c-myc, cis, the T-cell receptor gamma locus (TCR-gamma), and c-pim-1. The studies presented here demonstrate that this region is also necessary and sufficient for the activation of Stat5A and Stat5B. The membrane-proximal domain contains a single tyrosine, Y-343, which when mutated eliminates the ability of the receptor to couple Epo binding to the activation of Stat5. Furthermore, peptide competitions demonstrate that this site, when phosphorylated, can disrupt Stat5 DNA binding activity, consistent with a role of Y-343 as a site of recruitment to the receptor. Cells expressing the truncated, Y343F mutant (a mutant with a Y-to-F alteration at position 343) proliferate in response to Epo in a manner comparable to that of the controls. However, in these cells, Epo stimulation does not induce the appearance of transcripts for cis, TCR-gamma, or c-fos, suggesting a role for Stat5 in their regulation.

Normal production of RBCs requires that the antiapoptotic protein Bcl-xl be induced at end stages of differentiation in response to erythropoietin (Epo) signaling. The critical proapoptotic pathways inhibited by Bcl-xl in erythroblasts are unknown. We used gene targeting in the mouse to evaluate the BH3-only factor Nix, which is transcriptionally up-regulated during Epo-stimulated in vitro erythrocyte differentiation. Nix null mice are viable and fertile. Peripheral blood counts revealed a profound reticulocytosis and thrombocytosis despite normal serum Epo levels and blood oxygen tension. Nix null mice exhibited massive splenomegaly, with splenic and bone marrow erythroblastosis and reduced apoptosis in vivo during erythrocyte maturation. Hematopoietic progenitor populations were unaffected. Cultured Nix null erythroid cells were hypersensitive to Epo and resistant to apoptosis stimulated by cytokine deprivation and calcium ionophore. Transcriptional profiling of Nix null spleens revealed increased expression of cell cycle and erythroid genes, including Bcl-xl, and diminished expression of cell death and B cell-related genes. Thus, cell-autonomous Nix-mediated apoptosis in opposition to the Epo-induced erythroblast survival pathway appears indispensable for regulation of erythrocyte production and maintenance of hematological homeostasis. These results suggest that physiological codependence and coordinated regulation of pro- and antiapoptotic Bcl2 family members may represent a general regulatory paradigm in hematopoiesis.