Recently, erythropoietin (EPO) receptors and synthesis of EPO have been identified in the brain. To clarify the effects of EPO on neuronal cells, we investigated the effects of EPO on Ca2+ uptake, intracellular Ca2+ concentration, membrane potential, cell survival, release and biosynthesis of dopamine, and nitric oxide (NO) production in differentiated PC12 cells, which possess EPO receptors. EPO (10(-12)-10(-10) M) increased 45Ca2+ uptake and intracellular Ca2+ concentration in PC12 cells in a dose-related manner; these increases were inhibited by nicardipine (1 microM) or anti-EPO antibody (1:100 dilution). EPO induced membrane depolarization in PC12 cells. After a 5-day culture without serum and nerve growth factor (NGF), viable cell number decreased to 50% of that of the control cells cultured with serum and NGF. EPO (10(-13)-10(-10) M) increased the number of viable cells cultured without serum and NGF; this increase was blunted by nicardipine or anti-EPO antibody. Incubation with EPO (10(-13)-10(-10) M) stimulated mitogen-activated protein kinase activity in PC12 cells. EPO (10(-13)-10(-10) M) increased dopamine release from PC12 cells and tyrosine hydroxylase activity; these increases were sensitive to nicardipine or anti-EPO antibody. Following a 4-h incubation with EPO (10(-14)-10(-10) M), NO production was increased, which was blunted by nicardipine and anti-EPO antibody. In contrast, maximal NO synthase activity was not changed by EPO. These results suggest that EPO stimulates neuronal function and viability via activation of Ca2+ channels.

Sera of 25 healthy controls and 75 patients suffering from myelodysplastic syndromes (MDS) were investigated for serum concentration of interleukin-1 alpha (IL-1 alpha), IL-3, IL-6, granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), erythropoietin (Epo), and tumor necrosis factor-alpha (TNF-alpha). According to French-American-British (FAB) classification, 21 refractory anemia (RA), seven refractory anemia with ring sideroblasts (RARS), 15 chronic myelomonocytic leukemia (CMML), 12 refractory anemia with excess of blasts (RAEB), and 20 RAEB in transformation (RAEBt) were examined. TNF-alpha levels were inversely correlated with lower levels of hemoglobin concentration (r = -0.31, p = 0.005), irrespective of the requirements for transfusion in anemic MDS patients. Significant differences in TNF-alpha levels between CMML (26.2 +/- 5.9 pg/ml) and the FAB subgroups (16.1 +/- 1.6 pg/ml) were detected. There was an overall inverse relationship between the level of erythropoietin and the degree of anemia, but a wide range of Epo response between patients with similar hemoglobin concentrations. Serum levels of IL-1 alpha and GM-CSF were undetected in most of the patients. In 57% of the samples there were detectable levels of G-CSF, without a correlation of the serum levels with blood cell counts, nor with any of the FAB subcategories. Overall, 29% and 25% of the patient sera exhibited elevated IL-3 and IL-6 levels, respectively. There was no correlation of the serum levels with any of the blood counts, other cytokines, nor FAB subcategories. In conclusion, simple negative feedback mechanism between a specific cytokine and the production of blood cells seems not to be the case in MDS, except for red cell production and erythropoietin concentration. Our data may suggest the involvement of TNF-alpha in the pathogenesis of anemia in MDS.

The transcription factor AP1 has been implicated in the induction of apoptosis in cells in response to stress factors and growth factor withdrawal. We report here that AP1 is necessary for the induction of apoptosis following hormone withdrawal in the erythropoietin (EPO)-dependent erythroid cell line HCD57. AP1 DNA binding activity increased upon withdrawal of HCD57 cells from EPO. A dominant negative AP1 mutant rendered these cells resistant to apoptosis induced by EPO withdrawal and blocked the downregulation of Bcl-XL. JunB is a major binding protein in the AP1 complex observed upon EPO withdrawal; JunB but not c-Jun was present in the AP1 complex 3 h after EPO withdrawal in HCD57 cells, with a concurrent increase in junB message and protein. Furthermore, analysis of AP1 DNA binding activity in an apoptosis-resistant subclone of HCD57 revealed a lack of induction in AP1 DNA binding activity and no change in junB mRNA levels upon EPO withdrawal. In addition, we determined that c-Jun and AP1 activities correlated with EPO-induced proliferation and/or protection from apoptosis. AP1 DNA binding activity increased over the first 3 h following EPO stimulation of HCD57 cells, and suppression of AP1 activity partially inhibited EPO-induced proliferation. c-Jun but not JunB was present in the AP1 complex 3 h after EPO addition. These results implicate AP1 in the regulation of proliferation and survival of erythroid cells and suggest that different AP1 factors may play distinct roles in both triggering apoptosis (JunB) and protecting erythroid cells from apoptosis (c-Jun).

Erythropoietin (Epo) is secreted from the liver and kidney, where Epo production is strictly regulated at the transcriptional level in a hypoxia- and/or anemia-inducible manner. Here, we examined the in vivo function of the enhancer located 3' to the Epo gene (EpoE-3'). Reporter transgenic-mouse analyses revealed that the EpoE-3' enhancer is necessary and sufficient for the liver-specific and hypoxia-responsive expression of the gene after embryonic day 14.5 (E14.5). However, the enhancer is dispensable for Epo gene expression in the kidney and early-stage embryonic liver. Genetic removal of EpoE-3' from the endogenous Epo gene resulted in mice with severe anemia at late embryonic and neonatal stages due to defects in hepatic erythropoiesis, but early hepatic and splenic erythropoiesis was not affected. The mutant mice recover from the anemia in the juvenile period when major Epo production switches from the liver to the kidney. These results demonstrate that EpoE-3' is necessary for late hepatic erythropoiesis by specifically supporting paracrine production of Epo in the liver. In contrast, Epo production in the kidney utilizes distinct regulatory machinery and supports erythropoiesis in the bone marrow and spleen in adult animals.

We examined the value of transferrin concentrations in estimating nutritional status as determined by the subjective global assessment (SGA) score. Fifty-nine hemodialysis patients (37 men and 22 women, aged 59+/-16 years, dialyzed for 3.6+/-3.9 years) were selected by predetermined criteria. All received erythropoietin (EPO) and oral iron therapy. SGA evaluation was conducted twice by both a dietitian and a physician. Serum iron, total iron-binding capacity (TIBC; which is linearly correlated with transferrin), transferrin saturation ratio, ferritin, albumin, total protein, and cholesterol were measured. Twenty-seven (46%) patients were well nourished (group A), 20 (34%) were moderately nourished (group B), and 12 (20%) were poorly nourished (group C) according to the SGA. TIBC values were 276+/-47 mg/dL, 217+/-54 mg/dL, and 176+/-41 mg/dL, respectively (P < 0.00001), and thus directly correlated with the state of nutrition. The relationship between TIBC and nutritional status was independent of age and number of years on hemodialysis. Serum ferritin values were 104+/-93 ng/mL, 161+/-154 ng/mL, and 363+/-305 ng/mL, respectively (P < 0.0003), and thus inversely correlated with the state of nutrition. Transferrin saturation ratios were slightly higher in the severely malnourished patients. The number of years on dialysis were a determinant of nutritional status. These values were 2.4+/-2.4 years for group A, 3.9+/-4.0 years for group B, and 5.7+/-3.9 years for group C (P < 0.05). The average age of the poorly nourished patients was 10 years older than the well-nourished patients. Serum iron values were lower but transferrin saturation ratios were higher in the severely malnourished patients. The required EPO doses were higher in the poorly nourished patients. We suggest that transferrin values are superior to other laboratory tests in assessing nutrition and will supplement SGA criteria. Serum ferritin may be useful as a predictor of illness. Older patients who have been on dialysis longer warrant special concern. Malnutrition may be an indicator of EPO resistance in dialysis patients. Finally, since a decreased TIBC level in poorly nourished patients may erroneously increase the transferrin saturation ratio, our findings may have implications in making the diagnosis and treatment of anemia and iron deficiency in malnourished dialysis patients.

Despite numerous publications and clinical trials, the results of treatment of recalcitrant chronic plantar fasciitis with extracorporeal shockwave therapy (ESWT) still remain equivocal as to whether or not this treatment provides relief from the pain associated with this condition. The objective of this study was to determine whether extracorporeal shock wave therapy can safely and effectively relieve the pain associated with chronic plantar fasciitis compared to placebo treatment, as demonstrated by pain with walking in the morning. This was set in a multicenter, randomized, placebo-controlled, double-blind, confirmatory clinical study undertaken in four outpatient orthopedic clinics. The patients, 114 adult subjects with chronic plantar fasciitis, recalcitrant to conservative therapies for at least 6 months, were randomized to two groups. Treatment consisted of approximately 3,800 total shock waves (+/-10) reaching an approximated total energy delivery of 1,300 mJ/mm(2) (ED+) in a single session versus placebo treatment. This study demonstrated a statistically significant difference between treatment groups in the change from baseline to 3 months in the primary efficacy outcome of pain during the first few minutes of walking measured by a visual analog scale. There was also a statistically significant difference between treatments in the number of participants whose changes in Visual Analog Scale scores met the study definition of success at both 6 weeks and 3 months posttreatment; and between treatment groups in the change from baseline to 3 months posttreatment in the Roles and Maudsley Score. The results of this study confirm that ESWT administered with the Dornier Epos Ultra is a safe and effective treatment for recalcitrant plantar fasciitis.

The effect of ammonium chloride was determined on a culture of CHO cells transfected with the human erythropoietin (EPO) gene. Cell growth was inhibited above a culture concentration of 5 mM NH(4)Cl with an IC-50 determined to be 33 mM. The specific production of EPO increased with the addition of NH(4)Cl above 5 mM. At 10 mM NH(4)Cl, the final cell density after 4 days in culture was significantly lower but the final yield of EPO was significantly higher. This appeared to be due to continued protein production after cell growth had ceased. The metabolic effects of added NH(4)Cl included higher specific consumption rates of glucose and glutamine and an increased rate of production of alanine, glycine, and glutamate. The EPO analyzed from control cultures had a molecular weight range of 33-39 kDa and an isoelectric point range of 4.06-4.67. Seven distinct isoforms of the molecule were identified by two-dimensional electrophoresis. This molecular heterogeneity was ascribed to variable glycosylation. Complete enzymatic de-glycosylation resulted in a single molecular form with a molecular mass of 18 kDa. Addition of NH(4)Cl to the cultures caused a significant increase in the heterogeneity of the glycoforms as shown by an increased molecular weight and pI range. Enzymatic de-sialylation of the EPO from the ammonia-treated and control cultures resulted in identical electrophoretic patterns. This indicated that the effect of ammonia was in the reduction of terminal sialylation of the glycan structures which accounted for the increased pI. Selective removal of the N-glycan structures by PNGase F resulted in two bands identified as the O-glycan linked structure (19 kDa) and the completely de-glycosylated structure (18 kDa). The proportion of the O-linked glycan structure was reduced, and its pI increased in cultures to which ammonia was added. Thus, the glycosylation pattern altered by the presence of ammonia included a reduction in terminal sialylation of all the glycans and a reduction in the content of the O-linked glycan. The addition of a sialidase inhibitor to the cultures had no effect on the ammonia-induced increase in EPO heterogeneity. Also, the effect of ammonia on glycosylation could not be mimicked using the weak base chloroquine in our system.

The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line. However, a successful application of changing process conditions implies a profound understanding of the provoked physiological changes within the cells. In a systematic approach we varied the dissolved oxygen tension (DOT), pH, and temperature of CHO cultures in controlled bioreactors and investigated the impact on growth, productivity, metabolism, product quality and cell cycle distribution using a recombinant CHO cell line expressing the highly glycosylated fusion protein Epo-Fc. We found the reduction of cultivation temperature and the reduction of (external) pH to exert the most significant effects on process performance by mainly reducing cell growth and metabolism. With respect to the cell line used we identified a set of parameters capable of affecting cell proliferation in favor of an increased specific productivity and total product yield. The well directed alteration of the process environment has emerged as a tool adequate for further process optimization applying a biphasic cultivation strategy.

Chuvash polycythemia (CP) is an autosomal recessive condition that is endemic in the Russian mid-Volga River region of Chuvashia. We previously found that CP patients may have increased serum erythropoietin (EPO) levels, ruled out linkage to both the EPO and EPO receptor (EPOR) gene loci, and hypothesized that the defect may lie in the oxygen homeostasis pathway. We now report a study of five multiplex Chuvash families which confirms that CP is associated with significant elevations of serum EPO levels and rules out a location for the CP gene on chromosome 11 as had been reported by other investigators or a mutation of the HIF-1 alpha gene. Using a genome-wide screen, we localized a region on chromosome 3 with a LOD score >2. After sequencing three candidate genes, we identified a C to T transition at nucleotide 598 (an R200W mutation) in the von Hippel-Lindau (VHL) gene. The VHL protein (pVHL) downregulates the alpha subunit of hypoxia-inducible factor 1 (HIF-1 alpha), the main regulator of hypoxia adaptation, by targeting the protein for degradation. In the simplest scenario, disruption of pVHL function causes a failure to degrade HIF-1 alpha resulting in accumulation of HIF-1 alpha, upregulation of downstream target genes such as EPO, and the clinical manifestation of polycythemia. These findings strongly suggest that CP is a congenital disorder of oxygen homeostasis.

We used RNAase protection assays to measure low levels of erythropoietin messenger RNA (EPO mRNA) in the organs of unstimulated rats, and to compare basal and stimulated levels of EPO mRNA in the kidneys and extrarenal organs of rats rendered uremic by subtotal nephrectomy, with pair-fed controls. Using this sensitive assay, EPO mRNA was measured in the kidneys of unstimulated control animals and was detectable, at lower levels, in the liver and lung. After exposure to hypoxia, there was a 150-fold increase in renal EPO mRNA. Hepatic EPO mRNA was also greatly increased and accounted for 39 +/- 10% of the total. The small quantity of EPO mRNA in lung did not increase, but EPO mRNA became detectable in spleen. Animals subjected to subtotal nephrectomy became uremic and anemic (hematocrit 0.32 +/- 0.04 vs. 0.43 +/- 0.04 in controls, P = 0.002), but serum EPO concentrations were not significantly increased (32 +/- 9 vs. 24 +/- 6 mU/ml, P = 0.14). However, after hypoxic exposure, uremic animals increased serum EPO concentrations greatly, although the response was less than in controls (349 +/- 82 vs. 1009 +/- 238 mU/ml, P = 0.002). After hypoxia, extrarenal EPO mRNA levels in uremic animals were similar to controls. In particular, the large hepatic potential for EPO mRNA synthesis was unchanged but accounted for a greater proportion (84 +/- 5%) of the total EPO mRNA. The renal EPO mRNA content was reduced, as expected, after subtotal nephrectomy, but increased 50-fold after hypoxia. In this model of chronic renal failure, despite anemia, a large potential for EPO synthesis exists in liver and remnant kidney.

To better understand how humans adapt to hypoxia, the levels of hemoglobin (Hb), serum erythropoietin (Epo), and vascular endothelial growth factor (VEGF) were measured in 106 patients with severe obstructive sleep apnea-hypopnea syndrome. The results indicated that temporal hypoxic stimulation increases Hb. Furthermore, a minor increase in Epo and a substantial increase in VEGF were found. The induction in patients with severe sleep apnea was greater than that reported in other types of hypoxia. (Blood. 2001;98:1255-1257)

To investigate structure-function relationships of erythropoietin (Epo), we have obtained cDNA sequences that encode the mature Epo protein of a variety of mammals. A first set of primers, corresponding to conserved nucleotide sequences between mouse and human DNAs, allowed us to amplify by polymerase chain reaction (PCR) intron 1/exon 2 fragments from genomic DNA of the hamster, cat, lion, dog, horse, sheep, dolphin, and pig. Sequencing of these fragments permitted the design of a second generation of species-specific primers. RNA was prepared from anemic kidneys and reverse-transcribed. Using our battery of species-specific 5' primers, we were able to successfully PCR-amplify Epo cDNA from Rhesus monkey, rat, sheep, dog, cat, and pig. Deduced amino acid sequences of mature Epo proteins from these animals, in combination with known sequences for human, Cynomolgus monkey, and mouse, showed a high degree of homology, which explains the biologic and immunological cross-reactivity that has been observed in a number of species. Human Epo is 91% identical to monkey Epo, 85% to cat and dog Epo, and 80% to 82% to pig, sheep, mouse, and rat Epos. There was full conservation of (1) the disulfide bridge linking the NH2 and COOH termini; (2) N-glycosylation sites; and (3) predicted amphipathic alpha-helices. In contrast, the short disulfide bridge (C29/C33 in humans) is not invariant. Cys33 was replaced by a Pro in rodents. Most of the amino acid replacements were conservative. The C-terminal part of the loop between the C and D helices showed the most variation, with several amino acid substitutions, deletions, and/or insertions. Calculations of maximum parsimony for intron 1/exon 2 sequences as well as coding sequences enabled the construction of cladograms that are in good agreement with known phylogenetic relationships.

Baculovirus vectors are efficient tools for gene transfer into mammalian cells in vitro. However, in vivo gene delivery by systemic administration is hindered by the vector inactivation mediated by the complement system. To characterize further the gene transfer efficacy of baculovirus we examined the vector transduction efficiency in skeletal muscle. Vectors expressing vesicular stomatitis virus glycoprotein (VSV-G) in the viral envelope were generated by inserting the VSV-G coding sequence downstream of the polyhedrin promoter. Two viruses were constructed to carry either the Escherichia coli beta-galactosidase (beta-Gal) gene or the mouse erythropoietin (EPO) cDNA cloned downstream of the cytomegalovirus immediate-early promoter and enhancer. The greater gene transduction efficiency of the Bac-G-betaGal vector was confirmed by comparing the beta-Gal expression level in a variety of human and mouse cell lines with that obtained on infection with Bac-betaGal, a vector that lacks VSV-G. Similarly, a 5- to 10-fold increase in beta-Gal expression between Bac-G-betaGal and Bac-betaGal was observed when mouse myoblasts and myotubes were infected. The same increase in beta-Gal expression was detected on injection of the Bac-G-betaGal vector in the quadriceps of BALB/c and C57BL/6 mice. In contrast, a 2-fold difference in transduction was observed between these two vectors in DBA/2J mouse strain. Last, expression of EPO cDNA was detected for at least 178 days in DBA/2J mice on Bac-G-EPO injection into the quadriceps whereas EPO expression declined to normal values by 35 days postinfection in BALB/c and C57BL/6 mice. Thus, these results indicate that baculovirus may be considered a useful vector for gene transfer in mouse skeletal muscle and that persistence of expression may depend on the mouse strain used.

Oxygen (O₂) tension has emerged as a major regulator of stem cell (SC) biology. Low O₂ concentrations that are toxic to mature cells can confer advantage to stem and early progenitors, while superoxide stress remains a constant threat in aerobic biology and may be partially avoided through sequestration of SCs in the relatively hypoxic stem or regenerative niche. Using primary retina-derived retinal progenitor cells (RPCs) and the R28 progenitor cell line in vitro, we show that RPCs are sensitive to hydrogen peroxide (H₂O₂) induced damage and resistant to moderate levels of low oxygen stress (1% O₂). Under hypoxic conditions, multipotent RPCs upregulate Epo receptors, and Epo, along with insulin, protects against both superoxide- and severe hypoxia- (0.25% O₂) induced apoptosis through activation of the canonical PI3K/Akt/mTOR pathway. This survival advantage is sensitive to inhibitors of PI3K and mTOR. We further demonstrate phosphorylation of the p70S6 ribosomal kinase, a downstream mediator of PI3K/Akt/mTOR and translational activator. Overall, these data confirm that RPCs are sensitive to superoxide stress and resistant to hypoxia and that this resistance is mediated in part by Epo. They further suggest that manipulation of RPCs ex vivo prior to ocular delivery, or the in vivo delivery of exogenous survival factors at the time of cell implantation, could enhance the success of regenerative therapies aimed to restore sight.

Neonatal necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of preterm infants. Increased intestinal epithelium permeability is an early event in NEC pathogenesis. Autophagy and apoptosis are induced by multiple stress pathways which may impact the intestinal barrier, and they have been associated with pathogenesis of diverse gastrointestinal diseases including inflammatory bowel disease. Using both in vitro and in vivo models, this study investigates autophagy and apoptosis under experimental NEC stresses. Furthermore this study evaluates the effect of erythropoietin (Epo), a component of breast milk previously shown to decrease the incidence of NEC and to preserve intestinal barrier function, on intestinal autophagy and apoptosis. It was found that autophagy and apoptosis are both rapidly up regulated in NEC in vivo as indicated by increased expression of the autophagy markers Beclin 1 and LC3II, and by evidence of apoptosis by TUNEL and cleaved caspase-3 staining. In the rat NEC experimental model, autophagy preceded the onset of apoptosis in intestine. In vitro studies suggested that Epo supplementation significantly decreased both autophagy and apoptosis via the Akt/mTOR signaling pathway and the MAPK/ERK pathway respectively. These results suggest that Epo protects intestinal epithelium from excessive autophagy and apoptosis in experimental NEC.

OBJECTIVE

Gene-modified mesenchymal stromal cells (MSC) provide a promising tool for cell and gene therapy-based applications by potentially acting as a cellular vehicle for protein-replacement therapy. However, to avoid the risk of insertional mutagenesis, targeted integration of a transgene into a 'safe harbor' locus is of great interest.

METHODS

We sought to determine whether zinc finger nuclease (ZFN)-mediated targeted addition of the erythropoietin (Epo) gene into the chemokine [C-C motif] receptor 5 (CCR5) gene locus, a putative safe harbor locus, in MSC would result in stable transgene expression in vivo.

RESULTS

Whether derived from bone marrow (BM), umbilical cord blood (UCB) or adipose tissue (AT), 30-40% of human MSC underwent ZFN-driven targeted gene addition, as determined by a combination of fluorescence-activated cell sorting (FACS)- and polymerase chain reaction (PCR)-based analyzes. An enzyme-linked immunosorbent assay (ELISA)-based analysis of gene-targeted MSC expressing Epo from the CCR5 locus showed that these modified MSC were found to secrete a significant level of Epo (c. 2 IU/10(6)cells/24 h). NOD/SCID/gammaC mice injected with ZFN-modified MSC expressing Epo exhibited significantly higher hematocrit and Epo plasma levels for several weeks post-injection, compared with mice receiving control MSC.

CONCLUSIONS

These data demonstrate that MSC modified by ZFN-driven targeted gene addition may represent a cellular vehicle for delivery of plasma-soluble therapeutic factors.

Transforming growth factor beta 1 (TGF-beta 1) has been shown to be associated with active centers of hematopoiesis and lymphopoiesis in the developing fetus. Therefore, the effects of TGF-beta 1 on mouse hematopoiesis were studied. TGF-beta 1 is a potent inhibitor of IL-3-induced bone marrow proliferation, but it does not inhibit the proliferation induced by granulocyte/macrophage, colony-stimulating factor (CSF), granulocyte CSF, and erythropoietin (Epo). TGF-beta 1 also inhibits IL-3-induced multipotential colony formation of bone marrow cells in soft agar, which includes early erythroid differentiation, while Epo-induced terminal differentiation is unaffected. In addition, IL-3-induced granulocyte/macrophage colonies were inhibited; however, small clusters of differentiated myeloid cells were consistently seen in cultures containing IL-3 and TGF-beta 1. Thus, TGF-beta 1 selectively inhibits early hematopoietic progenitor growth and differentiation but not more mature progenitors. TGF-beta 1 is also a potent inhibitor of IL-3-dependent and -independent myelomonocytic leukemic cell growth, while the more mature erythroid and macrophage leukemias are insensitive. Therefore, TGF-beta 1 functions as a selective regulator of differentiating normal hematopoietic cells, and suppresses myeloid leukemic cell growth.

Cerebral vasospasm and the resulting cerebral ischemia occurring after subarachnoid hemorrhage (SAH) are still responsible for the considerable morbidity and mortality in patients affected by cerebral aneurysms. Mechanisms contributing to the development of vasospasm, abnormal reactivity of cerebral arteries and cerebral ischemia after SAH have been intensively investigated in recent years. It has been suggested that the pathogenesis of vasospasm is related to a number of pathological processes, including endothelial damage, smooth muscle cell contraction resulting from spasmogenic substances generated during lyses of subarachnoid blood clots, changes in vascular responsiveness and inflammatory or immunological reactions of the vascular wall. A great deal of experimental and clinical research has been conducted in an effort to find ways to prevent these complications. However, to date, the main therapeutic interventions remain elusive and are limited to the manipulation of systemic blood pressure, alteration of blood volume or viscosity, and control of arterial dioxide tension. Even though no single pharmacological agent or treatment protocol has been identified which could prevent or reverse these deadly complications, a number of promising drugs have been investigated. Among these is the hormone erythropoietin (EPO), the main regulator of erythropoiesis. It has recently been found that EPO produces a neuroprotective action during experimental SAH when its recombinant form (rHuEPO) is systemically administered. This topic review collects the relevant literature on the main investigative therapies for cerebrovascular dysfunction after aneurysmal SAH. In addition, it points out rHuEPO, which may hold promise in future clinical trials to prevent the occurrence of vasospasm and cerebral ischemia after SAH.

OBJECTIVE

To study the role of some soluble factors in the process of angiogenesis that accompanies multiple myeloma (MM).

METHODS

The concentrations of three well-known angiogenic peptides, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) were evaluated by an ELISA method. All of these factors were measured in the plasma obtained from peripheral blood (PB) and bone marrow (BM) aspirates of 34 patients affected by plasma cell disorders. This series included one patient with a solitary extramedullary plasmacytoma, 17 patients with MM at diagnosis, and 16 with previously treated MM.

RESULTS

In all the patients, the concentration of each angiogenic factor was higher in bone marrow than in peripheral blood. Mean values of the three angiogenic factors in BM or in PB were lower in stage I than stage II-III. One patient with extramedullary solitary myeloma had high levels of VEGF and bFGF but this increase was not found in the other 6 patients with extramedullary disease when compared with patients without extramedullary disease. VEGF and bFGF did not correlate with each other while HGF showed a weak correlation with VEGF and a stronger one with bFGF. Moreover, VEGF correlated with features of disease activity, such as C-reactive protein, and 2-microglobulin, while both bFGF and HGF showed an inverse correlation with albumin level. No correlation was found between VEGF, bFGF and HGF levels and age, M protein level, osteolytic lesions, or percentage of BM plasma cells. Since angiogenic factors may be released by normal cells in response to hypoxia, we also evaluated erythropoietin (EPO) levels (which correlate with the hypoxic stimulus) both in PB and BM plasma of these patients but none of the measured angiogenic factors correlated with EPO levels. Interpretation and Conclusions. Several soluble factors may play a role in the angiogenic activity described in MM but their contribution to the progression of disease may be different. The finding of higher levels of these factors in BM than in PB might indicate that the bone marrow environment is their major source. Concentrations of angiogenic factors parallel the activity of disease and are independent of the hypoxic stimulus.

This review summarizes published as well as preliminary data on the biology of erythropoietin (Epo) in the developing and mature human central nervous system (CNS). Both Epo receptor (Epo-R) and Epo gene expression underlie developmental changes and a brain-specific regulation. These features suggest a different role of Epo in normal brain development than in neuroprotection and neuronal tissue repair after brain injury. Epo concentrations in the cerebrospinal fluid may have primary paracrine effects. While the transport of Epo across the intact blood brain barrier (BBB) is generally limited in humans, systemically produced or administrated Epo may cross during BBB dysfunction. Summarized data of the in vivo and in vitro effects of Epo in the CNS show significant neuroprotective and neurotrophic effects of this molecule. These effects are mediated by several mechanisms, including the activation of a variety of genes and their consecutive protein production. Therapeutic strategies involving activation of the CNS Epo-R are discussed, including the potential use of Epo mimetic peptides.

Recombinant human erythropoietin (rhEpo) has proven to be remarkably safe and effective for treatment of anemias, primarily those secondary to renal disease and malignancy. Despite the worldwide use of rhEpo, concerns about its cost, the need for frequent parenteral administration, and the development of anti-Epo antibodies have prompted development of improved agents to stimulate erythropoiesis. Three strategies appear to be particularly promising. The half-life of Epo in the circulation can be prolonged by the addition of N-linked carbohydrate groups, by formation of adducts with polyethylene glycol, and by preparation of Epo multimers. Second, mimetic peptides can effectively trigger signal transduction at the Epo receptor, thereby boosting red-cell production. Finally, the hypoxia inducible transcription factor (HIF) can be pharmacologically induced by oral agents, resulting in enhanced expression not only of endogenous Epo but also of other genes important in the regulation of erythropoiesis.

The cytoplasmic domain of the cloned erythropoietin (EPO) receptor (EPOR) contains no protein kinase motif, yet addition of EPO to EPO-responsive cells causes an increase in protein-tyrosine phosphorylation. Here we show that addition of EPO or interleukin-3 (IL-3) to an IL-3-dependent cell line expressing the wild-type EPOR causes a small fraction (less than 5%) of total cellular EPOR to shift in gel mobility from 66 to 72 kDa, due at least in part to phosphorylation. Using biotinylated EPO as an affinity reagent, we show that the 72-kDa species is greatly enriched on the cell surface. To demonstrate that a protein kinase activity associates with cell surface EPOR, cells were incubated with biotinylated EPO and then cross-linked with a thiol-cleavable chemical cross-linker. The avidin-agarose-selected complexes were incubated with [gamma-32P]ATP. After in vitro phosphorylation and denaturation without reducing agent, both antiphosphotyrosine and anti-EPOR antibodies immunoprecipitated labeled 72-kDa EPOR and an unidentified 130-kDa phosphoprotein (pp130), indicating that a protein kinase is associated with cell surface EPOR and that a fraction of the EPOR was phosphorylated on tyrosine residues either in the cells or during the cell-free phosphorylation reaction. Under reducing conditions, the 72-kDa phosphorylated EPOR but not pp130 was immunoprecipitated with an anti-EPOR antibody, suggesting that the pp130 is bound to the EPOR by the thiol-cleavable chemical cross-linker. Previously, we showed that deletion of the 42 carboxy-terminal amino acids of the EPOR allows cells to grow in 1/10 the normal EPO concentration, without affecting receptor number or affinity. Two carboxy-terminal truncated EPO receptors that are hyperresponsive to EPO were poorly phosphorylated during the in vitro reaction, suggesting that the carboxy-terminal region of the EPOR contains a site for phosphorylation or a site for interaction with a protein kinase. Our data suggests that phosphorylation or interaction with a protein kinase in the carboxy-terminal region may down-modulate the proliferative action of the EPOR.

Purified natural (n) and recombinant (r) murine (mu) mast cell growth factor (MGF, a c-kit ligand) were evaluated alone and in combination with r human (hu) erythropoietin (Epo), rhu granulocyte-macrophage colony-stimulating factor (rhuGM-CSF), rhuG-CSF, and/or rhuM-CSF for effects in vitro on colony formation by multipotential (colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]), erythroid (burst-forming unit erythroid [BFU-E]) and granulocyte-macrophage (CFU-GM) progenitor cells from normal human bone marrow. MGF was a potent enhancing cytokine for Epo-dependent CFU-GEMM and BFU-E colony formation, stimulating more colonies and of a larger size than either rhu interleukin-3 (rhuIL-3) or rhuGM-CSF. MGF, especially at lower concentrations, also acted with rhuIL-3 or rhuGM-CSF to enhance Epo-dependent CFU-GEMM and BFU-E colony formation. MGF had little stimulating activity for CFU-GM colonies by itself, but in combination with suboptimal to optimal amounts of rhuGM-CSF enhanced the numbers and the size of CFU-GM colonies in an additive to greater than additive manner. While we did not detect an effect of MGF on CFU-G colony numbers stimulated by maximal concentrations of rhuG-CSF, MGF did enhance the size of CFU-G-derived colonies. MGF did not enhance the activity of rhuM-CSF. In a comparative assay, maximal concentrations of rmu and rhuMGF were equally effective in the enhancement of human bone marrow colony formation, but rhuMGF, in contrast to rmuMGF, did not at the concentrations tested enhance colony formation by mouse bone marrow cells. MGF effects on BFU-E, CFU-GM, and CFU-GEMM may be direct acting ones as MGF-enhanced colony formation by these cells in highly enriched progenitor cell populations of CD34 HLA-DR+ and CD34 HLA-DR+CD33- sorted cells in which greater than or equal to 1 of 2 cells was a BFU-E plus CFU-GM plus CFU-GEMM. MGF appears to be an early acting cytokine that preferentially stimulates the growth of immature hematopoietic progenitor cells.

Mesenchymal stem cells (MSCs) can ameliorate symptoms in several neurodegenerative diseases. However, the toxic environment of a degenerating central nervous system (CNS) characterized by hypoxia, glutamate (Glu) excess and amyloid beta (Abeta) pathology may hamper the survival and regenerative/replacing capacities of engrafted stem cells. Indeed, human MSC (hMSC) exposed to hypoxia were disabled in (i) the capacity of their muscarinic receptors (mAChRs) to respond to acetylcholine (ACh) with a transient increase in intracellular [Ca(2+)], (ii) their capacity to metabolize Glu, reflected by a strong decrease in glutamine synthetase activity, and (iii) their survival on exposure to Glu. Cocultivation of MSC with PC12 cells expressing the amyloid precursor protein gene (APPsw-PC12) increased the release of IL-6 from MSC. HMSC exposed to erythropoietin (EPO) showed a cholinergic neuron-like phenotype reflected by increased cellular levels of choline acetyltransferase, ACh and mAChR. All their functional deficits observed under hypoxia, Glu exposure and APPsw-PC12 cocultivation were reversed by the application of EPO, which increased the expression of Wnt3a. EPO also enhanced the metabolism of Abeta in MSC by increasing their neprilysin content. Our data show that cholinergic neuron-like differentiation of MSC, their functionality and resistance to a neurotoxic environment is regulated and can be improved by EPO, highlighting its potential for optimizing cellular therapies of the CNS.