The kidney controls erythropoietin production in adults, and the anemia that can accompany renal failure is a major medical problem. The liver controls erythropoietin production during fetal life but is silenced shortly after birth. Erythropoietin transcription is controlled by hypoxia-inducible factor (HIF), which is inhibited by three prolyl hydroxylases (PHD1, PHD2, and PHD3). Systemic PHD2 inactivation has been found to increase renal, but not hepatic, erythropoietin production. In contrast, we show here that simultaneous genetic inactivation of all three PHD paralogs in mice reactivates hepatic erythropoietin production and stimulates red blood synthesis, suggesting that pan-PHD inhibitory drugs might be useful for the treatment of anemia caused by chronic kidney disease.

Erythropoietin (Epo) and its receptor (EpoR) are essential for proliferation, differentiation and survival of erythroid progenitors. Here, we review several mechanisms by which the EpoR can be activated. We also describe the many intracellular signal transduction pathways activated by the EpoR. None are unique to the EpoR and mutant receptors able to activate only a subset of these pathways can support erythropoiesis in EpoR-/- fetal liver cells. Furthermore, normal erythroid differentiation occurs when the EpoR is replaced by the prolactin receptor or the myeloid oncoprotein Bcr-abl. Epo and probably other growth factors are required merely to ensure the survival and proliferation of already committed progenitors.

BACKGROUND

Recent studies have suggested that endogenous erythropoietin (Epo) plays an important role in the mobilization of bone marrow-derived endothelial progenitor cells (EPCs). However, it remains to be elucidated whether the Epo system exerts protective effects on pulmonary hypertension (PH), a fatal disorder encountered in cardiovascular medicine.

RESULTS

A mouse model of hypoxia-induced PH was used for study. We evaluated right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling in mice lacking the Epo receptor (EpoR) in nonerythroid lineages (EpoR(-/-) rescued mice) after 3 weeks of exposure to hypoxia. Those mice lack EpoR in the cardiovascular system but not in the hematopoietic system. The development of PH and pulmonary vascular remodeling were accelerated in EpoR(-/-) rescued mice compared with wild-type mice. The mobilization of EPCs and their recruitment to the pulmonary endothelium were significantly impaired in EpoR(-/-) rescued mice. By contrast, reconstitution of the bone marrow with wild-type bone marrow cells ameliorated PH in the EpoR(-/-) rescued mice. Hypoxia enhanced the expression of EpoR on pulmonary endothelial cells in wild-type but not EpoR(-/-) rescued mice. Finally, hypoxia activated endothelial nitric oxide synthase in the lungs in wild-type mice but not in EpoR(-/-) rescued mice.

CONCLUSIONS

These results indicate that the endogenous Epo/EpoR system plays an important role in the recruitment of EPCs and prevents the development of PH during chronic hypoxia in mice in vivo, suggesting the therapeutic importance of the system for the treatment of PH.

The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. We have screened multiple renal and hepatic cell lines (including MDCK, LLC-PK1, BHK, WRL 68, CLCL, A704, CRFK, A498, ACHN, TCMK-1, LLC-MK2, CaKi-2, HepG2, and Hep3B) for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10(6) cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities less than 3.3 X 10(5) cells per cm2, there was little constitutive release of Epo in the medium (less than 30 milliunits per 10(6) cells in 24 hr). With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 microM cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

BACKGROUND

Residual kidney function (RKF) is associated with improved survival in peritoneal dialysis patients, but its role in hemodialysis patients is less well known. Urine output may provide an estimate of RKF. The aim of our study is to determine the association of urine output with mortality, quality of life (QOL), and inflammation in incident hemodialysis patients.

METHODS

Nationally representative prospective cohort study.

METHODS

734 incident hemodialysis participants treated in 81 clinics; enrollment, 1995-1998; follow-up until December 2004.

METHODS

Urine output, defined as producing at least 250 mL (1 cup) of urine daily, ascertained using questionnaires at baseline and year 1.

METHODS

Primary outcomes were all-cause and cardiovascular mortality, analyzed using Cox regression adjusted for demographic, clinical, and treatment characteristics. Secondary outcomes were QOL, inflammation (C-reactive protein and interleukin 6 levels), and erythropoietin (EPO) requirements.

RESULTS

617 of 734 (84%) participants reported urine output at baseline, and 163 of 579 (28%), at year 1. Baseline urine output was not associated with survival. Urine output at year 1, indicating preserved RKF, was independently associated with lower all-cause mortality (HR, 0.70; 95% CI, 0.52-0.93; P = 0.02) and a trend toward lower cardiovascular mortality (HR, 0.69; 95% CI, 0.45-1.05; P = 0.09). Participants with urine output at baseline reported better QOL and had lower C-reactive protein (P = 0.02) and interleukin 6 (P = 0.03) levels. Importantly, EPO dose was 12,000 U/wk lower in those with urine output at year 1 compared with those without (P = 0.001).

CONCLUSIONS

Urine volume was measured in only a subset of patients (42%), but agreed with self-report (P < 0.001).

CONCLUSIONS

RKF in hemodialysis patients is associated with better survival and QOL, lower inflammation, and significantly less EPO use. RKF should be monitored routinely in hemodialysis patients. The development of methods to assess and preserve RKF is important and may improve dialysis care.

Impacting a significant portion of the world's population with increasing incidence in minorities, the young, and the physically active, diabetes mellitus (DM) and its complications affect approximately 20 million individuals in the United States and over 100 million individuals worldwide. In particular, vascular disease from DM may lead to some of the most serious complications that can extend into both the cardiac and nervous systems. Unique strategies that can prevent endothelial cell (EC) demise and elucidate novel cellular mechanisms for vascular cytoprotection become vital for the prevention and treatment of vascular DM complications. Here, we demonstrate that erythropoietin (EPO), an agent that has recently been shown to extend cell viability in a number of systems extending beyond hematopoietic cells, prevents EC injury and apoptotic nuclear DNA degradation during elevated glucose exposure. More importantly, EPO employs Wnt1, a cysteine-rich glycosylated protein involved in gene expression, cell differentiation, and cell apoptosis, to confer EC cytoprotection and maintains the integrity of Wnt1 expression during elevated glucose exposure. In addition, application of anti-Wnt1 neutralizing antibody abrogates the protective capacity of both EPO and Wnt1, illustrating that Wnt1 is an important component in the cytoprotection of ECs during elevated glucose exposure. Intimately linked to this cytoprotection is the downstream Wnt1 pathway of glycogen synthase kinase (GSK-3beta) that requires phosphorylation of GSK-3beta and inhibition of its activity by EPO. Interestingly, inhibition of GSK-3beta activity during elevated glucose leads to enhanced EC survival, but does not synergistically improve protection by EPO or Wnt1, suggesting that EPO and Wnt1 are closely tied to the blockade of GSK-3beta activity. Our work exemplifies an exciting potential application for EPO in regards to the treatment of DM vascular disease complications and highlights a previously unrecognized role for Wnt1 and the modulation of the downstream pathway of GSK-3beta to promote vascular cell viability during DM.

Erythropoietin (EPO), well known for its role in stimulation of erythropoiesis, has recently been shown to have a dramatic neuroprotective effect in animal models of cerebral ischemia, mechanical trauma of the nervous system, and excitotoxins, mainly by reducing apoptosis. We studied the effect of single systemic administration of recombinant human EPO (rhEPO) on left ventricular (LV) size and function in rats during 8 weeks after the induction of a myocardial infarction (MI) by permanent ligation of the left descending coronary artery. We found that an i.p. injection of 3,000 units/kg of rhEPO immediately after the coronary artery ligation resulted, 24 h later, in a 50% reduction of apoptosis in the myocardial area at risk. Eight weeks after the induction of MI, rats treated with rhEPO had an infarct size 15-25% of the size of that in untreated animals. The reduction in myocardial damage was accompanied by reductions in LV size and functional decline as measured by repeated echocardiography. Thus, a single dose of rhEPO administered around the time of acute, sustained coronary insufficiency merits consideration with respect to its therapeutic potential to limit the extent of resultant MI and contractile dysfunction.

We have examined the effects of direct intratracheal instillation of purified eosinophil granule proteins on pulmonary function and airway responsiveness in primates. The results of this study show for the first time that installation of major basic protein (MBP) directly into the trachea of primates results in a significant and dose-related increase in airway responsiveness to inhaled methacholine. Furthermore, MBP and eosinophil peroxidase (EPO) induce a transient bronchoconstriction immediately after instillation that resolves by 1 h postinstillation. In contrast, instillation of other eosinophil granule proteins had no effect on airway responsiveness or pulmonary function. These data indicate a direct role of the eosinophil in the pathogenesis of airway hyperresponsiveness. We suggest that the MBP of human eosinophils has an effector role in the pathogenesis of airway hyperresponsiveness which may involve active interaction with resident airway tissue cells. MBP may also mediate altered lung function in various inflammatory lung diseases associated with pulmonary eosinophilia.

Hypoxia-inducible expression of the gene encoding for the glycoprotein hormone erythropoietin (EPO) is the paradigm of oxygen-regulated gene expression. EPO is the main regulator of red blood cell production and more than 100 years of research on the regulation of EPO production have led to the identification of a widespread cellular oxygen sensing mechanism. Central to this signaling cascade is the transcription factor complex hypoxia-inducible factor-1 (HIF-1). Meanwhile, it is known that HIF-1 controls more than 50 oxygen-dependent genes and is now recognized as the main regulator of oxygen homoeostasis in the body. In addition to hypoxic induction, expression of the EPO gene is tightly regulated in a tissue-specific manner. During ontogeny, production of EPO required for erythropoiesis is switched from the fetal liver to the kidneys. Here EPO is mainly synthesized in adulthood. Production of EPO has also been found in organs where it has nonerythropoietic functions: EPO is important for development of the brain and is neuroprotective, whereas it stimulates angiogenesis in the reproductive tract and possibly in other organs. Understanding oxygen and tissue-specific regulation of EPO production is of high relevance for physiology. Moreover, this knowledge might be useful for new therapies to treat human diseases.

The cardiovascular and respiratory systems play key roles in O(2) homeostasis. Physiological responses to hypoxia involve changes in gene expression that are mediated by the transcriptional activator hypoxia-inducible factor (HIF)-1. Analysis of mice heterozygous for a knockout allele at the locus encoding the O(2)-regulated HIF-1alpha or HIF-2alpha subunit has revealed that these proteins are required for multiple physiological responses to chronic hypoxia, including erythrocytosis and pulmonary vascular remodeling. In mice with partial HIF-2alpha deficiency, hypoxia-induced expression of endothelin-1 and norepinephrine is dramatically impaired, and the mice fail to develop pulmonary hypertension after 4 wk of exposure to 10% O(2). In mice with partial HIF-1alpha deficiency, the ability of the carotid body to sense and/or respond to acute or chronic hypoxia is lost. In wild-type mice, brief episodes of intermittent hypoxia are sufficient to induce production of erythropoietin (EPO), which protects the heart against apoptosis after ischemia-reperfusion, whereas in mice with partial HIF-1alpha deficiency, intermittent hypoxia does not induce EPO production or cardiac protection. Parenteral administration of EPO to rodents is sufficient to induce dramatic protection against ischemia-reperfusion injury in the heart. Thus HIF-1 mediates critical physiological responses to hypoxia, and the elucidation of these homeostatic mechanisms may lead to novel therapies for the most common causes of mortality in the US population.

In addition to the chief function of erythropoietin (Epo) in promoting erythropoiesis, some other roles have been found in the brain and uterus. We have reported that signalling pathways of Epo and Epo receptor (EpoR) are involved in the tumourigenesis of ovarian and uterine cancers. To determine whether Epo plays a similar role in other malignancies, we studied the expression of Epo in several malignant human cell lines. We found that 24 malignant human cell lines examined express Epo and EpoR regardless of their origins, types, genetic characteristics and biological properties and secrete a very small amount of Epo individually and that most of them respond to hypoxic stimuli by enhanced secretion of Epo. To determine whether the Epo-EpoR pathway operates in tumours of these cell lines, we transplanted several cell lines into nude mice and confirmed the presence of Epo-responsive sites in xenografts in which the phosphorylation of the STAT5 (signal transducer and activator of transcription) is detectable. Furthermore, in nude mice we blocked the Epo signalling in xenografts of two representative cell lines, stomach choriocarcinoma and melanoma, by i.p. injections of EpoR antagonist and found inhibition of angiogenesis and survival of tumour cells leading to destruction of tumour masses and disturbances of phosphorylation of STAT5. In contrast, Epo mimetic peptide promotes angiogenesis and tumour cell survival. These findings suggest that Epo is indispensable for the growth and viability of malignant tumour and also that the deprivation of Epo signalling may be a promising therapy for human malignancy.

Clinical development of erythropoiesis-stimulating agents (ESAs) revolutionized the management of anemia. The major clinical benefits of ESAs are effective treatment of anemia and avoidance of blood transfusion risks. Erythropoietin (EPO) interacts directly with the EPO receptor on the red blood cell (RBC) surface, triggering activation of several signal transduction pathways, resulting in the proliferation and terminal differentiation of erythroid precursor cells and providing protection from RBC precursor apoptosis. The magnitude of increase in RBC concentration in response to administration of recombinant human EPO products (rhEPO) is primarily controlled by the length of time EPO concentrations are maintained, not by the EPO concentration level. Subcutaneous (SC) EPO administration results in slower absorption than intravenous (IV) administration, leading to lower peak plasma levels and an apparent extended terminal half-life. However, SC administration requires additional needle-sticks and is associated with an increased risk of immunogenicity compared with IV administration. Multiple pathways may play a role in EPO clearance from the body. Epoetin alfa was the first rhEPO produced and approved for pharmaceutical use, followed by several related products and by newer ESAs with the same mechanism but more prolonged action. Darbepoetin alfa is a hyperglycosylated EPO analog with an extended terminal half-life and a greater relative potency compared with rhEPO at extended dosing intervals. PEGylation of EPO (addition of polyethylene glycol) has been used to further extend the terminal half-life. Also, new strategies are under investigation for stimulating erythropoiesis through activation of the EPO receptor.

Recognized as a robust cytoprotectant for multiple tissues of the hematopoietic, vascular, cardiac, and nervous systems, erythropoietin (EPO) also is considered to be an attractive therapeutic candidate to modulate inflammatory cell function and survival during neurodegenerative disorders. To this end, microglia of the central nervous system serve a complex function not only to dispense of foreign organisms and injured cells of the brain, but also to foster tissue repair and reorganization during neuronal and vascular cell insults. We therefore examined the ability of EPO to modulate microglial cell survival and the underlying signal transduction pathways that govern microglial integrity during oxygen-glucose deprivation (OGD)--induced oxidative stress. We demonstrate in the microglial cell line EOC 2 that EPO provides direct microglial protection against early and late apoptotic programs of membrane phosphatidylserine exposure and genomic DNA degradation. Furthermore, expression and activation of Akt1 is vital to the cytoprotective capacity of EPO, since pharmacological inhibition of the PI 3-K pathway or gene silencing of Akt1 expression eliminates the ability of EPO to protect microglial cells. Through Akt1 dependent mechanisms that can be abrogated through the gene silencing of Akt1, maintenance of microglial cell integrity during OGD by EPO is closely integrated with the phosphorylation and inhibition of glycogen synthase kinase-3beta activity as well as the intracellular trafficking of beta-catenin and nuclear factor-kappaB. Further work that continues to elucidate the ability of EPO to target the intricate pathways that determine inflammatory cell function and integrity may lay the ground work for new therapeutic avenues for neurodegenerative disease.

Airway inflammation is present in asthma and is thought to play a significant part in the development of airflow obstruction. In chronic obstructive pulmonary disease (COPD), neutrophilic inflammation is present in the airway lumen, whereas the submucosa displays a lymphocytic infiltrate. Less is known about the nature and mechanisms of inflammation in COPD than in asthma. Induced sputum allows noninvasive sampling of respiratory tract secretions from patients and control subjects, allowing characterization of cells and measurement of soluble markers. We exploited this technique in order to compare the presence and quantify specific markers of eosinophil and neutrophil activation in subjects with asthma or COPD, and control subjects. Differential cell counts showed significantly higher neutrophil percentages in the patients with COPD compared with other groups, while patients with asthma had higher numbers of eosinophils. The neutrophil markers myeloperoxidase (MPO), from primary granules in neutrophils, and human neutrophil lipocalin (HNL), released from secondary granules, were elevated in patients with asthma and COPD compared with control subjects but markedly more so in COPD. The difference between COPD and asthma was more marked for HNL than for MPO suggesting that HNL may be a better marker for discriminating between these conditions. Concentrations of the eosinophil granule protein, eosinophil cationic protein (ECP), and the eosinophil granule-derived enzyme, eosinophil peroxidase (EPO) were raised in the patients with asthma and those with COPD.

Given that erythropoietin (EPO) is no longer believed to have exclusive biological activity in the hematopoietic system, EPO is now considered to have applicability in a variety of nervous system disorders that can overlap with vascular disease, metabolic impairments, and immune system function. As a result, EPO may offer efficacy for a broad number of disorders that involve Alzheimer's disease, cardiac insufficiency, stroke, trauma, and diabetic complications. During a number of clinical conditions, EPO is robust and can prevent metabolic compromise, neuronal and vascular degeneration, and inflammatory cell activation. Yet, use of EPO is not without its considerations especially in light of frequent concerns that may compromise clinical care. Recent work has elucidated a number of novel cellular pathways governed by EPO that can open new avenues to avert deleterious effects of this agent and offer previously unrecognized perspectives for therapeutic strategies. Obtaining greater insight into the role of EPO in the nervous system and elucidating its unique cellular pathways may provide greater cellular viability not only in the nervous system but also throughout the body.

Multi-tissue erythropoietin receptor (EPO-R) expression provides for erythropoietin (EPO) activity beyond its known regulation of red blood cell production. This review highlights the role of EPO and EPO-R in brain development and neuroprotection. EPO-R brain expression includes neural progenitor cells (NPC), neurons, glial cells and endothelial cells. EPO is produced in brain in a hypoxia sensitive manner, stimulates NPC proliferation and differentiation, and neuron survival, and contributes to ischemic preconditioning. Mice lacking EPO or EPO-R exhibit increased neural cell apoptosis during development before embryonic death due to severe anemia. EPO administration provides neural protection in animal models of brain ischemia and trauma, reducing the extent of injury and damage. Intrinsic EPO production in brain and EPO stimulation of endothelial cells contribute to neuroprotection and these are of particular importance since only low levels of EPO appear to cross the blood-brain barrier when administered at high dose intravenously. The therapeutic potential of EPO for brain ischemia/trauma and neurodegenerative diseases has shown promise in early clinical trial and awaits further validation.

Patients with germ line mutations in the VHL tumor suppressor gene are predisposed to the development of highly vascularized tumors within multiple tissues. Loss of pVHL results in constitutive activation of the transcription factors HIF-1 and HIF-2, whose relative contributions to the pathogenesis of the VHL phenotype have yet to be defined. In order to examine the role of HIF in von Hippel-Lindau (VHL)-associated vascular tumorigenesis, we utilized Cre-loxP-mediated recombination to inactivate hypoxia-inducible factor-1alpha (Hif-1alpha) and arylhydrocarbon receptor nuclear translocator (Arnt) genes in a VHL mouse model of cavernous liver hemangiomas and polycythemia. Deletion of Hif-1alpha did not affect the development of vascular tumors and polycythemia, nor did it suppress the increased expression of vascular endothelial growth factor (Vegf) and erythropoietin (Epo). In contrast, phosphoglycerokinase (Pgk) expression was substantially decreased, providing evidence for target gene-dependent functional redundancy between different Hif transcription factors. Inactivation of Arnt completely suppressed the development of hemangiomas, polycythemia, and Hif-induced gene expression. Here, we demonstrate genetically that the development of VHL-associated vascular tumors in the liver depends on functional ARNT. Furthermore, we provide evidence that individual HIF transcription factors may play distinct roles in the development of specific VHL disease manifestations.

The growth factor erythropoietin (EPO) and erythropoietin receptors (EPOR) are expressed in the nervous system. Neuronal expression of EPO and EPOR peaks during brain development and is upregulated in the adult brain after injury. Peripherally administered EPO, and at least some of its variants, cross the blood-brain barrier, stimulate neurogenesis, neuronal differentiation, and activate brain neurotrophic, anti-apoptotic, anti-oxidant and anti-inflammatory signaling. These mechanisms underlie their tissue protective effects in nervous system disorders. As the tissue protective functions of EPO can be separated from its stimulatory action on hematopoiesis, novel EPO derivatives and mimetics, such as asialo-EPO and carbamoylated EPO have been developed. While the therapeutic potential of the novel EPO derivatives continues to be characterized in preclinical studies, the experimental findings in support for the use of recombinant human (rh)EPO in human brain disease have already been translated to clinical studies in acute ischemic stroke, chronic schizophrenia, and chronic progressive multiple sclerosis. In this review article, we assess the studies on EPO and, in particular, on its structural or functional variants in experimental models of nervous system disorders, and we provide a short overview of the completed and ongoing clinical studies testing EPO as neuroprotective/neuroregenerative treatment option in neuropsychiatric disease.

Erythropoietin (EPO) is an essential growth factor that regulates erythrocyte production in mammals. In this study, we demonstrate a novel role of EPO in regulating angiogenesis in vivo. Epo and Epo receptor (EpoR) are expressed in the vasculature during embryogenesis. Deletion of Epo or EpoR leads to angiogenic defects starting at E10.5, 2 days before ventricular hypoplasia and 3 days before the onset of the embryonic lethal phenotype. Overall, angiogenesis was severely affected in the mutant embryos: vascular anomalies included decreased complexity of the vessel networks. However, de novo vasculogenesis remained intact, consistent with the differential expression of Epo and EpoR during the early stages of embryonic development. The aforementioned angiogenesis defect can be partially rescued by expressing human EPO during embryogenesis. Moreover, Ang-1 expression is regulated by EPO/EPOR under normoxic conditions. Taken together, our results suggest important roles of EPO and EPOR in angiogenesis.

Erythropoietin (Epo) and its receptor (EpoR), critical for erythropoiesis, are expressed in the nervous system. Prior to death in utero because of severe anemia EpoR-null mice have fewer neural progenitor cells, and differentiated neurons are markedly sensitive to hypoxia, suggesting that during development Epo stimulates neural cell proliferation and prevents neuron apoptosis by promoting oxygen delivery to brain or by direct interaction with neural cells. Here we present evidence that neural progenitor cells express EpoR at higher levels compared with mature neurons; that Epo stimulates proliferation of embryonic neural progenitor cells; and that endogenous Epo contributes to neural progenitor cell proliferation and maintenance. EpoR-null mice were rescued with selective EpoR expression driven by the endogenous EpoR promoter in hematopoietic tissue but not in brain. Although these mice exhibited normal hematopoiesis and erythrocyte production and survived to adulthood, neural cell proliferation and viability were affected. Embryonic brain exhibited increased neural cell apoptosis, and neural cell proliferation was reduced in the adult hippocampus and subventricular zone. Neural cells from these animals were more sensitive to hypoxia/glutamate neurotoxicity than normal neurons in culture and in vivo. These observations demonstrate that endogenous Epo/EpoR signaling promotes cell survival in embryonic brain and contributes to neural cell proliferation in adult brain in regions associated with neurogenesis. Therefore, Epo exerts extra-hematopoietic function and contributes directly to brain development, maintenance, and repair by promoting cell survival and proliferation independent of insult, injury, or ischemia.

During neonatal hypoxic-ischemic brain injury, activation of transcription of a series of genes is induced to stimulate erythropoiesis, anti-apoptosis, apoptosis, necrosis and angiogenesis. A key factor mediating these gene transcriptions is hypoxia-inducible factor-1alpha (HIF-1alpha). During hypoxia, HIF-1alpha protein is stabilized and heterodimerizes with HIF-1beta to form HIF-1, subsequently regulating the expression of target genes. HIF-1alpha participates in early brain development and proliferation of neuronal precursor cells. Under pathological conditions, HIF-1alpha is known to play an important role in neonatal hypoxic-ischemic brain injury: on the one hand, HIF-1alpha has neuroprotective effects whereas it can also have neurotoxic effects. HIF-1alpha regulates the transcription of erythropoietin (EPO), which induces several pathways associated with neuroprotection. HIF-1alpha also promotes the expression of vascular endothelial cell growth factor (VEGF), which is related to neovascularization in hypoxic-ischemic brain areas. In addition, HIF-1alpha has an anti-apoptotic effect by increasing the expression of anti-apoptotic factors such as EPO during mild hypoxia. The neurotoxic effects of HIF-1alpha are represented by its participation in the apoptotic process by increasing the stability of the tumor suppressor protein p53 during severe hypoxia. Moreover, HIF-1alpha plays a role in cell necrosis, by interacting with calcium and calpain. HIF-1alpha can also exacerbate brain edema via increasing the permeability of the blood-brain barrier (BBB). Given these properties, HIF-1alpha has both neuroprotective and neurotoxic effects after hypoxia-ischemia. These events are cell type specific and related to the severity of hypoxia. Unravelling of the complex functions of HIF-1alpha may be important when designing neuroprotective therapies for hypoxic-ischemic brain injury.

We have previously shown the presence of erythropoietin (Epo) within the spinal fluid of normal preterm and term infants, and the presence of Epo receptor (Epo-R) in the spinal cords of human fetuses. It is not known, however: 1) whether cells within the fetal central nervous system (CNS) express Epo; 2) if so, whether this expression changes with development; 3) which cells within the CNS express Epo-R; 4) whether Epo-R expression within the CNS changes with development; and 5) whether Epo-R within the fetal CNS are functional. Expression of mRNA for Epo and Epo-R was sought by reverse transcription-PCR in mixed primary cultures of fetal spinal cords as well as NT2 and hNT cells, human cell lines of neuronal precursors and mature neurons, respectively. Epo was measured by ELISA in spent media from primary cell culture, and immunohistochemistry was used to identify Epo-R on neurons and glia in cell culture, and in brain sections. Developmental changes in Epo and Epo-R expression were sought in spinal cords and brains from fetuses of 7-24 wk postconception by semiquantitative PCR. To assess Epo-R function, NT2 cells were exposed to conditions which stimulate programmed cell death, and rescue from apoptosis by the addition of recombinant Epo was evaluated by nuclear matrix protein ELISA, cell counts, and by Klenow labeling of DNA fragments. Epo and Epo-R mRNA were expressed in mixed primary cultures of neural tissues and NT2 and hNT cells. Epo was detected by ELISA in media removed from mixed cell cultures, and immunohistochemical staining confirmed the presence of Epo-R on neurons and their supporting cells. Semiquantitative PCR revealed no significant change in expression of either Epo or Epo-R in spinal cords between 7 and 16 wk of gestation, with increased expression of Epo and Epo-R in brains from 8 to 24 wk of gestation. Epo mRNA expression from neurons doubled under conditions of hypoxia. Recombinant Epo decreased apoptotic cell death of neurons under conditions of hypoxia. Protein and mRNA for Epo and its receptor are expressed by human neurons and glial cells in spinal cord and brain during fetal development. These receptors appear to have a neuroprotective effect in conditions of hypoxia.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality in young people in industrialized countries. Although various anti-inflammatory and antiapoptotic modalities have shown neuroprotective effects in experimental models of TBI, to date, no specific pharmacological agent aimed at blocking the progression of secondary brain damage has been approved for clinical use. Erythropoietin (Epo) belongs to the cytokine superfamily and has traditionally been viewed as a hematopoiesis-regulating hormone. The newly discovered neuroprotective properties of Epo lead us to investigate its effect in TBI in a mouse model of closed head injury. Recombinant human erythropoietin (rhEpo) was injected at 1 and 24 h after TBI, and the effect on recovery of motor and cognitive functions, tissue inflammation, axonal degeneration, and apoptosis was evaluated up to 14 days. Motor deficits were lower, cognitive function was restored faster, and less apoptotic neurons and caspase-3 expression were found in rhEpo-treated as compared with vehicle-treated animals (P<0.05). Axons at the trauma area in rhEpo-treated mice were relatively well preserved compared with controls (shown by their density; P<0.01). Immunohistochemical analysis revealed a reduced activation of glial cells by staining for GFAP and complement receptor type 3 (CD11b/CD18) in the injured hemisphere of Epo- vs. vehicle-treated animals. We propose that further studies on Epo in TBI should be conducted in order to consider it as a novel therapy for TBI.

Here we describe repressible (PipOFF) as well as inducible (PipON) systems for regulated gene expression in mammalian cells, based on the repressor Pip (pristinamycin-induced protein), which is encoded by the streptogramin resistance operon of Streptomyces coelicolor. Expression of genes placed under control of these systems was responsive to clinically approved antibiotics belonging to the streptogramin group (pristinamycin, virginiamycin, and Synercid). The versatility of these systems was demonstrated by streptogramin-regulated expression of mouse erythropoietin (EPO), human placental secreted alkaline phosphatase (SEAP), or green fluorescent protein (GFP) in diverse cell lines (BHK, CHO, HeLa, and mouse myoblasts). Analysis of isogenic constructs in CHO cells demonstrated the PipOFF system gave lower background and higher induction ratios than the widely used tetracycline-repressible (TetOFF) expression systems. The streptogramin-based expression technology was functionally compatible with the TetOFF system, thus enabling the selective use of different antibiotics to independently control two different gene activities in the same cell.