Exogenous Erythropoietin Administration Research Articles

Low Serum Erythropoietin Levels are Associated with Fatal COVID‐19 Cases at 4,150 meters above sea level

Previous studies suggested that erythropoietin (EPO) may protect against severe COVID‐19‐induced injuries, ultimately preventing mortality. This hypothesis is based on the fact that, in addition to promoting the increase in red blood cells, EPO is an anti‐inflammatory, anti‐apoptotic and protective factor in several non‐erythropoietic tissues. Furthermore, EPO promotes nitric oxide production in the hypoxic lung and stimulates ventilation by interacting with the respiratory centers of the brainstem. Given that EPO in the blood is increased at high‐altitude, we evaluated the serum levels of EPO in critical patients with COVID‐19 at “Hospital Agramont” in the city of El Alto (4,150 masl) in Bolivia. A total of 16 patients, 15 men, one woman, with a mean age of 55.8 ± 8.49 years, admitted to the Intensive Care Unit were studied. All patients were permanent residents of El Alto, with no travel history below 3,000 masl for at least one year. Blood samples were collected upon admission to the ICU. Serum EPO concentration was assessed using an ELISA kit, and a standard technique determined hemoglobin concentration. Only half of the observed patients survived the disease. Remarkably, fatal cases showed 2.5 times lower serum EPO than survivors (2.78 ± 0.8643 mU/ml vs 7.06 ± 2.713 mU/ml; p = 0.0096), and 1.24 times lower hemoglobin levels (13.96 ± 2.56 g/dL vs 17.41 ± 1.61 g/dL; p = 0.0159). While the number of cases evaluated in this work is low, our findings strongly warrant further investigation of EPO levels in COVID‐19 patients at high and low altitudes. Our results also support the hypothesis that exogenous EPO administration could help critically ill COVID‐19 patients overcome the disease.

Low serum erythropoietin levels are associated with fatal COVID-19 cases at 4,150 meters above sea level

Previous studies suggested that erythropoietin (EPO) may protect against severe COVID-19-induced injuries, ultimately preventing mortality. This hypothesis is based on the fact that, in addition to promoting the increase in red blood cells, EPO is an anti-inflammatory, anti-apoptotic and protective factor in several non-erythropoietic tissues. Furthermore, EPO promotes nitric oxide production in the hypoxic lung and stimulates ventilation by interacting with the respiratory centers of the brainstem. Given that EPO in the blood is increased at high-altitude, we evaluated the serum levels of EPO in critical patients with COVID-19 at “Hospital Agramont” in the city of El Alto (4150 masl) in Bolivia. A total of 16 patients, 15 men, one woman, with a mean age of 55.8 ± 8.49 years, admitted to the Intensive Care Unit were studied. All patients were permanent residents of El Alto, with no travel history below 3000 masl for at least one year. Blood samples were collected upon admission to the ICU. Serum EPO concentration was assessed using an ELISA kit, and a standard technique determined hemoglobin concentration. Only half of the observed patients survived the disease. Remarkably, fatal cases showed 2.5 times lower serum EPO than survivors (2.78 ± 0.8643 mU/mL vs 7.06 ± 2.713 mU/mL; p = 0.0096), and 1.24 times lower hemoglobin levels (13.96 ± 2.56 g/dL vs 17.41 ± 1.61 g/dL; p = 0.0159). While the number of cases evaluated in this work is low, our findings strongly warrant further investigation of EPO levels in COVID-19 patients at high and low altitudes. Our results also support the hypothesis that exogenous EPO administration could help critically ill COVID-19 patients overcome the disease.

Erythropoietin Prevents Anemia and Transfusions in Extremely Premature Lambs Supported by an EXTrauterine Environment for Neonatal Development (EXTEND)

Background: We recently developed an EXTrauterine Environment for Neonatal Development (EXTEND) that provides physiologic support for premature lambs. Here, we assess the efficacy of exogenous erythropoietin (EPO) to prevent anemia and transfusions on EXTEND. Materials and Methods: Lambs were cannulated at 0.7 gestation and supported on EXTEND for up to 4 weeks. The lambs were divided into three groups: (1) No EPO, (2) Low EPO (300 U kg^–1 per day), and (3) High EPO (800 U kg^–1 per day). Daily hematocrit and weekly complete blood count were assessed. Results: The mean percentage change in hematocrit from baseline was significantly different between the groups (No EPO –23.6 ± 7.8% vs. Low EPO –16.6 ± 6.4% vs. High EPO +2.6 ± 6.6%; p = 0.02). This occurred despite a greater median number of blood transfusions in the No EPO group (5 vs. 1 vs. 0; p = 0.02). EPO administration was associated with a higher mean corpuscular volume (MCV; p < 0.01) and reticulocyte count (p = 0.02). The High EPO group was comparable to in utero control fetuses with respect to hematocrit (p = 0.49), MCV (p = 0.24), and reticulocyte count (p = 0.68). Conclusions: EPO (800 U kg^–1 per day) prevents anemia, eliminates transfusions, and restores normal red blood cell indices in premature lambs supported by EXTEND.

Erythropoietin modulation is associated with improved homing and engraftment after umbilical cord blood transplantation

AbstractUmbilical cord blood (UCB) engraftment is in part limited by graft cell dose, generally one log less than that of bone marrow (BM)/peripheral blood (PB) cell grafts. Strategies toward increasing hematopoietic stem/progenitor cell (HSPC) homing to BM have been assessed to improve UCB engraftment. Despite recent progress, a complete understanding of how HSPC homing and engraftment are regulated is still elusive. We provide evidence that blocking erythropoietin (EPO)-EPO receptor (R) signaling promotes homing to BM and early engraftment of UCB CD34+ cells. A significant population of UCB CD34+ HSPC expresses cell surface EPOR. Exposure of UCB CD34+ HSPC to EPO inhibits their migration and enhances erythroid differentiation. This migratory inhibitory effect was reversed by depleting EPOR expression on HSPC. Moreover, systemic reduction in EPO levels by hyperbaric oxygen (HBO) used in a preclinical mouse model and in a pilot clinical trial promoted homing of transplanted UCB CD34+ HSPC to BM. Such a systemic reduction of EPO in the host enhanced myeloid differentiation and improved BM homing of UCB CD34+ cells, an effect that was overcome with exogenous EPO administration. Of clinical relevance, HBO therapy before human UCB transplantation was well-tolerated and resulted in transient reduction in EPO with encouraging engraftment rates and kinetics. Our studies indicate that systemic reduction of EPO levels in the host or blocking EPO-EPOR signaling may be an effective strategy to improve BM homing and engraftment after allogeneic UCB transplantation. This clinical trial was registered at www.ClinicalTrials.gov (#NCT02099266).

The role of erythropoietin in remote renal preconditioning on hippocampus ischemia/reperfusion injury.

Remote ischemic preconditioning (RIPC) is an intriguing approach which exposes a remote organ/tissue to a non-lethal transient ischemia/reperfusion (I/R) in order to potentiate the resistance of the desired organ/tissue against the next unwanted I/R. It has been suggested that RIPC exerts its effect through neuronal and hormonal pathways. The underlying mechanisms of RIPC are obscure and should be elucidated. In this study, we induced RIPC in mice using 3 cycles of 5min ischemia alternating with 5min reperfusion of the left renal artery. Renal failure was induced in mice by intra-peritoneal (i.p.) injection of 200mg/kg body weight of gentamicin twice per day for 4 consecutive days. Global hippocampal ischemia reperfusion (I/R) was performed by bilateral carotid artery occlusion for 20min followed by reperfusion for 72h. Moreover, the retention trial of passive avoidance test was determined 72h after global ischemia. Histopathological changes of hippocampus neurons were observed using Nissl staining to detect neuronal loss. Finally, terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling (TUNEL) was performed to assess the status of apoptotic cells in the hippocampus. The results of this study suggest that renal ischemic preconditioning is a good candidate for prevention of I/R-induced hippocampal injury. However, RRPC (remote renal preconditioning) failed to exert a neuroprotective effect in mice with renal failure (RF), indicating the probable role of a humoral factor which is released from kidneys in response to ischemia. In agreement with this hypothesis, treatment of mice with rhEPO (5000IU/kg intraperitoneal) before induction of RRPC restored the neuroprotective effects of RRPC in RF mice. Accordingly, it is plausible to expect that erythropoietin is released from kidneys to act as a mediator for RRPC-induced neuroprotective effects. Renal ischemic preconditioning prevents I/R-induced hippocampal injury. In contrast, renal failure hampers protective effects of RRPC, while exogenous administration of erythropoietin (EPO) significantly prevents the inhibiting effects of renal failure.

Preclinical and preliminary clinical evaluation of genetically transduced dermal tissue implants for the sustained secretion of erythropoietin and interferon α

Protein drugs are currently delivered by bolus injection and although treatment frequently is successful, these methods also have major drawbacks, which call for the development of alternative technologies allowing prolonged delivery of these drugs. We developed a new ex vivo gene therapy platform called Transduced Autologous Restorative Gene Therapy (TARGT) for sustained long term production and secretion of autologous therapeutic proteins. A biopsy of dermal tissue taken from the patient is transduced ex vivo with a viral vector encoding the required gene under a constitutive promoter. Following measurement of protein secretion ex vivo, the transduced dermal tissue is implanted back into the patient, where it secretes the therapeutic protein into the circulation for several months or longer. A major hurdle to this approach is potential immunogenicity of the transduced tissue following implantation. In this paper we describe the preclinical and early clinical development of this technology, which allowed overcoming these hurdles. To that end, we have used the helper dependent (HD) adenoviral vector with newly designed expression cassette containing genetic elements to optimize transgene expression. Moreover, we have developed procedures for TARGT tissue implantation, with measures to improve engraftment and reduce inflammation and rejection. Implantation of human TARGT to immune-deficient SCID mice indicated long-term production of active proteins in the blood. Preliminary results of a clinical trial from two anemic end-stage renal disease patients, implanted with TARGTs expressing the human erythropoietin (EPO) gene, demonstrated prolonged secretion with physiologic blood level of the hormone and Hemoglobin maintenance in the desired range, for a period of at least five months without exogenous EPO administration. We believe that the TARGT technology has the potential to become a platform for the sustained delivery of therapeutic proteins in various clinical indications.

Patients with anaemia can shift from kidney to liver production of erythropoietin as shown by glycoform analysis

The primary production site of erythropoietin (EPO) is shifted from the liver to the kidney shortly after birth. Under conditions of lost or reduced kidney production, it is valuable to measure the production capacity of the liver. However, there is a lack of urine or serum based methods that can distinguish endogenous EPO produced in different cell types. Here is presented a method based on chromatographic interaction with the lectin wheat germ agglutinin (WGA) that can distinguish presumably liver-produced EPO, found in anaemic patients receiving epoetin and darbepoetin, from kidney-produced EPO found in healthy individuals.All the tested samples from haemodialysis patients with end-stage renal disease showed a presence of liver EPO. In some samples, the liver-produced EPO made up 90–100% of total EPO at a concentration of 8–10ng/L in urine, which indicates that the liver has a quite high production capacity, although not adequate for the degree of anaemia.This glycoform analysis has made it possible to affirm that some anaemic patients can increase their liver-production of EPO. The use of such a method can give better insight into the regulation of non-renal endogenous EPO production, a potential source of EPO intended to replace administration of exogenous EPO.

Peripheral Blood Mononuclear Cell-Derived Erythroid Progenitors and Erythroblasts Are Decreased in Burn Patients

Patients with large burns suffer from anemia of critical illness. Administration of exogenous erythropoietin is ineffective, and transfusion remains the only effective treatment. We have previously shown that erythroid precursors are decreased 1 week after burn in an animal model. Therefore, we have used a two-phase liquid culture system to quantify peripheral blood mononuclear cell (PBMC) compartment-derived erythroid progenitors (EPs) in burn patients. Institutional review board approval and informed consent were obtained. Blood samples were collected at 1 to 30 days after burn, with a mean TBSA of 37.7 ± 15.8% (n = 10; 90% men; age, 46.0 ± 18 years). Four healthy volunteers served as controls. PBMCs were isolated by Ficoll-Hypaque density-gradient centrifugation and were placed in serum-free expansion medium containing cyclosporine A (1 ng/ml), granulocyte macrophage colony-stimulating factor (20 ng/ml), stem cell factor (30 ng/ml), and interleukin-3 (5 ng/ml; phase I). On day 7, cells were reseeded in serum-free expansion medium containing erythropoietin (1 U/ml), holotransferrin (0.3 mg/ml), and stem cell factor (10 ng/ml; phase II). Aliquots from the phase II culture system on day 6 were incubated with anti-CD71, CD235a, and CD36. EPs (CD71 CD36) and erythroblast subpopulations (colony-forming unit erythroids, Proerythroblasts, and intermediate erythroblasts) were identified based on the expressions of CD71 and CD235a by flow cytometry, calculated per million expanded cells, and expressed as a percentage of controls. Total EPs were significantly decreased by days 28 to 31 after the burn (19%; P < .05). Among the erythroblast subpopulations, colony-forming unit erythroids (11%; P < .004) and proerythroblasts (24%; P < .05), were decreased significantly by days 28 to 31 after the burn. PBMCs of burn patients can be used to study impaired erythropoiesis and anemia of critical illness.

Does Erythropoietin and Its Receptor may Play a Role in Human Intervertebral Disk Degeneration?

Introduction Degeneration of lumbar intervertebral discs is considered to be a cause of low back pain and radiculopathy. Many studies have shown that intervertebral disk inflammation and axonal growth of afferent fibers innervating the disk degeneration have a principal role in this pathological process. Both mechanical compression and chemical factors within nucleus pulposus (NP) are associated with the occurrence of radiculopathy. NP contains various proinflammatory cytokines that play important roles in the chemical pathogenesis of NP-induced peripheral nerve injury. The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) is a key trigger in the pathogenesis of NP-induced inflammation and apoptosis and its inhibition can reduce NP degeneration. A class of MAPKs and p38 MAPK is phosphorylated by stress signals such as inflammatory cytokineso, smotic shock, and ischemia. Spinal cord and peripheral nerve injury cause the activation of p38 in dorsal root ganglion (DRG) neurons and spinal cord microglia. Furthermore, TNF-α phosphorylates p38 and activated p38 MAPK upregulates the biosynthesis of TNF- α. TNF and p38 are involved in neuronal cell apoptosis in peripheral nerve injuries. Erythropoietin (EPO) is a pleiotropic cytokine originally identified for its role in erythropoiesis. Recent studies have demonstrated that EPO and its receptor (EPO-R) are expressed in the central nervous system, where EPO exerts neuroprotective functions. Materials and Methods The aim of the present study was to investigate whether the resident EPO and EPO-R network is present and functional in the NP of intervertebral disk in different grade of degeneration. In this study, we analyzed 20 human NP obtained following microdiscectomy. The expressions of p-p38, TNF- α, EPO, and EPOR were assessed by immunohistochemical and immunoblotting analysis. Data were analyzed by unpaired Student t-test and Dunnett t-test (significance level, p &lt; 0.05). Results In barely degenerated disk, significantly weak expression of EPO and EPO-R was observed ( p &lt; 0.05). Accordingly, the expression of both p-p38 and TNF in the NP was significantly lower than in well-degenerated disk ( p &lt; 0.05). In clearly degenerated disc, a marked increase of expression of EPO and EPO-R was observed ( p &lt; 0.05) and a consensual increase of both p-p38 and TNF in the NP was observed ( p &lt; 0.05). Conclusion These observations suggest that the local EPO and EPO-R system is markedly engaged in the degeneration of intervertebral disk by a feasible cross-talk with network of proinflammatory cytokine such as TNF- α. These data may increase our knowledge on mechanisms underlying intervertebral disk degeneration and strongly support the possible usefulness of a therapeutic approach based on exogenous EPO administration. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared

Red Blood Cell Transfusion and Transfusion Alternatives in Traumatic Brain Injury

OPINION STATEMENT: Anemia develops in about 50% of patients hospitalized with traumatic brain injury (TBI) and is recognized as a cause of secondary brain injury. This review examines the effects of anemia and transfusion on TBI patients through a literature search to identify original research on anemia and transfusion in TBI, the effects of transfusion on brain physiology, and the role of erythropoietin or hemoglobin-based blood substitutes (HBBSs). However, the amount of high-quality, prospective data available to help make decisions about when TBI patients should be transfused is very small. Randomized transfusion trials have involved far too few TBI patients to reach definitive conclusions. Thus, it is hardly surprising that there is widespread practice variation. In our opinion, a hemoglobin transfusion threshold of 7g/dL cannot yet be considered safe for TBI patients admitted to hospital, and in particular to the ICU, as it is for other critically ill patients. Red blood cell transfusions often have immediate, seemingly beneficial effects on cerebral physiology, but the magnitude of this effect may depend in part upon how long the cells have been stored before administration. In light of existing physiological data, we generally aim to keep hemoglobin concentrations greater than 9g/dL during the first several days after TBI. In part, the decision is based on the patient's risk of or development of secondary ischemia or brain injury. An increasing number of centers use multimodal neurologic monitoring, which may help to individualize transfusion goals based on the degree of cerebral hypoxia or metabolic distress. When available, brain tissue oxygen tension values less than 15-20mm Hg or a lactate:pyruvate ratio greater than 30-40 would influence us to use more aggressive hemoglobin correction (e.g., a transfusion threshold of 10g/dL). Clinicians can attempt to reduce transfusion requirements by limiting phlebotomy, minimizing hemodilution, and providing appropriate prophylaxis against gastrointestinal hemorrhage. Administration of exogenous erythropoietin may have a small impact in further reducing the need for transfusion, but it also may increase complications, most notably deep venous thrombosis. Erythropoietin is currently of great interest as a potential neuroprotective agent, but until it is adequately evaluated in randomized controlled trials, it should not be used routinely for this purpose. HBBSs are also of interest, but existing preparations have not been shown to be beneficial-or even safe-in the context of TBI.

Erythropoietin Induction by Anemia Is Required for CFU-E Expansion During Erythroid Recovery From Sublethal Radiation Injury

AbstractAbstract 3218The massive steady-state output of the erythron makes the erythroid lineage exquisitely sensitive to clastogenic injury. While the rapid loss of reticulocytes is well-described, the response of bone marrow progenitors and precursors to sublethal irradiation and the mechanisms underlying recovery of the erythron remain poorly defined. Following 4 Gy total body irradiation (TBI) in C57Bl/6 mice, functional colony assays were utilized to study the erythroid progenitor compartment, consisting of immature day 7 erythroid burst-forming units (BFU-E) and more mature day 3 BFU-E and erythroid colony-forming units (CFU-E). Multispectral imaging flow cytometry was used to quantify the erythroid precursor compartment, consisting of proerythroblasts and progressively more mature basophilic, polychromatophilic, and orthochromatic erythroblasts. At 2 days after 4 Gy TBI, greater than 95% of erythroid progenitors and precursors in the marrow were lost. A significant decrease in peripheral reticulocyte output and a gradual 9% drop in hematocrit accompanied this marrow loss over the first 3–4 days after radiation exposure. Following this initial injury and mild radiation-induced anemia, a robust recovery of the erythron began with a significant increase in day 3 BFU-E at 5 days post-radiation immediately followed by a rapid expansion of CFU-E at 5–6 days post-radiation to 200% of unirradiated control marrow. In contrast, day 7 BFU-E only partially recovered in a gradual linear fashion. Subsequent maturation of CFU-E resulted in progressive waves of erythroid precursors, reticulocytes, and mature red cells, creating a “ripple effect” following sublethal radiation injury. These results indicate that erythroid repopulation following radiation damage is centered on specific expansion and maturation of later erythroid progenitors (day 3 BFU-E and CFU-E). Erythropoietin (EPO) is known to be the primary regulator of the erythroid lineage, and day 3 BFU-E and CFU-E form the EPO-responsive compartment of the erythron. Therefore, we hypothesized that EPO may be the primary driving force underlying day 3 BFU-E/CFU-E expansion and subsequent erythroid recovery from radiation injury. Endogenous plasma EPO levels increased 13-fold above steady-state levels at 4 days post-radiation. This spike in EPO levels preceded the CFU-E expansion seen at 5–6 days post-radiation. To specifically determine both the etiology of the endogenous EPO spike and the necessity of supra-physiologic levels of EPO for erythroid recovery from radiation injury, we performed loss-of-function studies in which mice were transfused with packed red cells post-radiation to maintain a normal hematocrit. Prevention of the mild radiation-induced anemia by transfusion also prevented the increase in endogenous EPO and significantly abrogated day 3 BFU-E/CFU-E recovery. These findings directly link radiation-induced anemia with EPO induction and erythroid lineage reconstitution. Gain-of-function studies were performed to determine whether EPO is sufficient to drive erythroid expansion after radiation injury. Administration of exogenous EPO at 1 hour post-radiation significantly advanced the timing of CFU-E expansion and subsequent recovery of the erythron. In addition, the accelerated synchronous wave of recovery following exogenous EPO very closely mirrored the physiological wave of recovery during the endogenous EPO response, indicating that the previously observed “ripple effect” is an inherent component of EPO-induced erythroid recovery from radiation injury. Finally, administration of EPO at 4 days post-radiation, at the peak of the endogenous EPO response, further enhanced CFU-E recovery to over 330% of unirradiated control levels, providing additional evidence that EPO drives expansion of irradiated CFU-E. These studies, taken together, indicate that the anemia-induced EPO response following radiation injury is both necessary and sufficient for CFU-E expansion that leads to recovery of the erythron. A better understanding of the response of the erythroid lineage to clastogenic injury will ultimately lead to improved therapies to protect and mitigate the hematopoietic system from radiation and chemotherapy damage. Disclosures:No relevant conflicts of interest to declare.

Erythropoetin as a novel agent with pleiotropic effects against acute lung injury

Current pharmacotherapy for acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is not optimal, and the biological and physiological complexity of these severe lung injury syndromes requires consideration of combined-agent treatments or agents with pleiotropic action. In this regard, exogenous erythropoietin (EPO) represents a possible candidate since a number of preclinical studies have revealed beneficial effects of EPO administration in various experimental models of ALI. Taken together, this treatment strategy is not a single mediator approach, but it rather provides protection by modulating multiple levels of early signaling pathways involved in apoptosis, inflammation, and peroxidation, potentially restoring overall homeostasis. Furthermore, EPO appears to confer vascular protection by promoting angiogenesis. However, only preliminary studies exist and more experimental and clinical studies are necessary to clarify the efficacy and potentially cytoprotective mechanisms of EPO action. In addition to the attempts to optimize the dose and timing of EPO administration, it would be of great value to minimize any potential toxicity, which is essential for EPO to fulfill its role as a potential candidate for the treatment of ALI in routine clinical practice. The present article reviews recent advances that have elucidated biological and biochemical activities of EPO that may be potentially applicable for ALI/ARDS management.

The combined effect of erythropoietin and granulocyte macrophage colony stimulating factor on liver regeneration after major hepatectomy in rats

BackgroundThe liver presents a remarkable capacity for regeneration after hepatectomy but the exact mechanisms and mediators involved are not yet fully clarified. Erythropoietin (EPO) and Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have been shown to promote liver regeneration after major hepatectomy.Aim of this experimental study is to compare the impact of exogenous administration of EPO, GM-CSF, as well as their combination on the promotion of liver regeneration after major hepatectomy.MethodsWistar rats were submitted to 70% major hepatectomy. The animals were assigned to 4 experimental groups: a control group (n = 21) that received normal saline, an EPO group (n = 21), that received EPO 500 IU/kg, a GM-CSF group (n = 21) that received 20 mcg/kg of GM-CSF and a EPO+GMCSF group (n = 21) which received a combination of the above. Seven animals of each group were killed on the 1st, 3rd and 7th postoperative day and their remnant liver was removed to evaluate liver regeneration by immunochemistry for PCNA and Ki 67.ResultsOur data suggest that EPO and GM-CSF increases liver regeneration following major hepatectomy when administered perioperatively. EPO has a more significant effect than GM-CSF (p < 0.01). When administering both, the effect of EPO seems to fade as EPO and GM-CSF treated rats have decreased regeneration compared to EPO administration alone (p < 0.01).ConclusionEPO, GM-CSF and their combination enhance liver regeneration after hepatectomy in rats when administered perioperatively. However their combination has a weaker effect on liver regeneration compared to EPO alone. Further investigation is needed to assess the exact mechanisms that mediate this finding.

Protective Effect of Erythropoietin in Renal Ischemia-Reperfusion Injury

Ischemia and/or reperfusion injury (IR) is one of the most common causes of acute renal failure. Erythropoietin (EPO), is main hematopoietic hormone that has recently been shown to exert important cytoprotective and anti-apoptotic effects in experimental I/R and toxicity models. Recent studies suggest that administration of exogenous EPO prevents ischemia-induced kidney damage. In this review we focused on anti-apoptotic, anti-inflammatory and anti-oxidant action of EPO in renal ischemia-reperfusion models and its application in nephropathies. EPO seems to be a very promising agent for protecting cellular survival during both acute and chronic ischemic and toxic nephropathies.

Anti-VEGF effects of intravitreal erythropoietin in early diabetic retinopathy

In the present study, a single intravitreal erythropoietin (EPO) to diabetic rats produced therapeutic effects on blood-retinal barrier (BRB) function and neuronal survival at different time courses of retinopathy. In parallel, the hypoxia-inducible factor 1 alpha (HIF-1 alpha) pathway has been quantitatively studied, including VEGF-A, endogenous EPO, EPO receptor (EpoR), prolyl hydroxylases (PHD1-3) and von Hippel-Lindau tumor suppressor (VHL). The mRNA levels of HIF-1 alpha, VEGF-A, endogenous EPO, PHD1-3 and VHL are all up-regulated in the diabetic retina, and suppressed by exogenous EPO. The increased protein levels of HIF-1 alpha, VEGF-A, and endogenous EPO found in diabetic retinas also have been down-regulated by exogenous EPO. The results demonstrate that the HIF-1 pathway is activated in the retina in early diabetes, but is negatively regulated by a feedback loop following the administration of exogenous EPO. Exogenous EPO at pharmacologic levels leads to suppression of VEGF and in turn, restoration of the normal functions of BRB in a time-dependent manner. In the diabetic retina, the same level of exogenous EPO that inhibits VEGF also exerted neuronal protection.

Erythropoietin-Associated CFU-E Expansion: A Novel Mechanism Responsible for Erythroid Recovery Following Sublethal Radiation.

Abstract 1483Poster Board I-506Although the synthesis of two million red blood cells every second is essential for the maintenance of steady state levels of blood cells as well as our well-being, very little is known about the sensitivity of the erythroid lineage to injury or its kinetics and mechanisms of recovery. A novel multispectral imaging flow cytometry analysis utilizing the AMNIS ImageStream and functional colony assays were utilized to study the response of the erythroid lineage in C57Bl/6 mice following 4 Gray whole body irradiation. BrdU cell cycle analysis and erythropoietin ELISA assays were also utilized to investigate erythroid recovery after radiation injury. Two days post-radiation, all erythroid progenitors and precursors in the marrow were severely depleted. Over the next week, early-stage progenitors (BFU-E) only made a partial recovery. In marked contrast, late-stage progenitors (CFU-E) dramatically expanded in numbers at 5-6 days post-radiation to greater than 200% of normal. These CFU-E subsequently generated a wave of erythroid precursors that culminated in the emergence of circulating reticulocytes beginning at 8-9 days post-radiation. Rapid expansion of the CFU-E compartment was associated with an increase in cell cycling. CFU-E expansion was also temporally associated with an acute 20-fold increase in circulating levels of erythropoietin (EPO). Additionally, exogenous EPO administration at 4 days post-radiation resulted in further expansion of CFU-E 2 days later. Interestingly, erythroid progenitors and precursors were not found in the spleen after radiation, indicating that recovery of the erythroid lineage after direct marrow injury is not associated with extramedullary erythropoiesis. We conclude that erythroid progenitors and precursors, unlike circulating erythrocytes, are exquisitely sensitive to clastogenic insults such as radiation. Furthermore, we conclude that the acute recovery of erythropoiesis following sublethal radiation injury is centered on profound expansion and robust cycling of CFU-E. The temporal association of this CFU-E expansion both with the endogenous upregulation and with the exogenous administration of EPO suggests that CFU-E expansion is due primarily to erythropoietin stimulation. We hypothesize CFU-E expansion in the marrow may represent a novel adaptive mechanism of the erythroid lineage to acute injury. Overall, a more thorough understanding of radiation-induced erythroid injury and recovery will ultimately lead to new treatments to protect and mitigate the hematopoietic system from clastogenic agents such as radiation and chemotherapy. Disclosures:No relevant conflicts of interest to declare.

The erythropoietin and regenerative medicine: a lesson from fish

Erythropoietin (EPO), the main haematopoietic growth factor for the proliferation and differentiation of erythroid progenitor cells, is also known for its angiogenic and regenerative properties. In this study, we aimed to test the regenerative effects of EPO administration in an experimental model of Sea bass (Dicentrarchus labrax) subjected to amputation of the caudal fin. Erythropoietin-treated fishes (3000 UI of human recombinant EPO-alpha immediately after cutting and after 15 days) showed an increased growth rate of their fins compared with those untreated (anova variance: P: 0.01 vs. P: 0.04). By analysing fin length at established times (15 and 30 days after cut), EPO-treated fishes always showed an increased length compared with untreated ones (T-15: 1.1 +/- 0.2 vs. 0.7 +/- 0.2 cm, P: 0.03; T-30: 1.9 +/- 0.3 vs. 1.2 +/- 0.2 cm, P: 0.01). Moreover, exogenous EPO administration induced an enormous increase in EPO-blood levels at each observation time (T-15: 2240 +/- 210 vs. 16.7 +/- 1.8 mU mL(-1), P < 0.001; T-30: 2340 +/- 190 vs. 17.1 +/- 1.9 mU mL(-1), P < 0.001), whereas these levels remained quite unmodified in untreated fishes. Immunochemical analyses performed by confocal laser scanning microscopic observations showed an increased expression of EPO-receptors and PECAM-1 (an endothelial surface marker of vessels sprout) in the regenerating tissue, whereas no signs of inflammation or fibrosis were recognisable. All these findings confirm EPO as a new factor involved in regenerative processes, also suggesting a potential, future utility for new therapeutical applications in the field of human regenerative medicine.

Erythropoietin signaling promotes transplanted progenitor cell survival

We examine the potential for erythropoietin signaling to promote donor cell survival in a model of myoblast transplantation. Expression of a truncated erythropoietin receptor in hematopoietic stem cells has been shown to promote selective engraftment in mice. We previously demonstrated expression of endogenous erythropoietin receptor on murine myoblasts, and erythropoietin treatment can stimulate myoblast proliferation and delay differentiation. Here, we report that enhanced erythropoietin receptor expression, as well as exogenous erythropoietin treatment in myoblasts, provided a survival advantage and protection against apoptosis under serum-starvation conditions. When cultured in differentiation medium, expression of the myogenic regulatory proteins shifted toward early differentiation with increased erythropoietin receptor. Expression of early myogenic differentiation proteins Myf-5 and MyoD increased, while later stage protein myogenin decreased. Transplantation of C2C12 myoblasts overexpressing truncated erythropoietin receptor showed more transplanted cell incorporation into muscle fibers in muscular dystrophy mdx mice. These cells also restored dystrophin protein expression in mdx mice at 6 wk after cell treatment that was further increased with exogenous erythropoietin administration. In summary, enhanced erythropoietin receptor expression promotes transplanted cell survival in a mouse model for myoblast transplantation and provides dystrophin expression in mice with muscular dystrophy.

Abstract 2360: Central Sleep Apnea with Nocturnal Hypoxia Promotes Increased Circulating Erythropoietin in Heart Failure

Central sleep apnea (CSA) with nocturnal hypoxia is frequent in heart failure (HF). Hypoxia causes increased circulating erythropoietin (EPO) in healthy normals. EPO promotes increased vasoconstriction and exogenous EPO administration is associated with adverse cardiovascular events. No prior studies have related EPO concentration to apnea or hypoxia due to CSA. EPO is elevated in HF patients with nocturnal hypoxia due to CSA. Ambulatory, non-anemic HF patients (n = 29) with LVEF &lt; 45% and healthy controls (n = 18) underwent polysomnography (PSG). Subjects with obstructive sleep apnea (OSA) were excluded. CSA was defined as apnea-hypopnea index (AHI) ≥ 15. Hypoxia was quantified as the proportion of sleep with arterial oxygen saturation &lt; 90% (T90%). Blood for EPO was drawn post-PSG. Other clinical characteristics were summarized from the medical record. HF subjects and controls were similar age (54 vs 60 y, p = 0.09). CSA was present in 14 HF subjects; 13 were men compared to 8 of 15 without CSA (p = 0.04). HF subjects had 42% higher mean EPO than controls (p &lt; 0.01) despite similar hemoglobin (13.9 vs 14.0 g/dL, p = 0.8). NYHA class III–IV HF subjects had 42% higher mean EPO than class I–II HF subjects (p = 0.05, figure ). EPO concentration was correlated with severity of nocturnal hypoxia by simple linear regression (r = 0.4; p = 0.02). By multivariate analysis, elevated EPO was associated with NYHA class III–IV HF and elevated AHI (p = 0.01 and 0.03, respectively; r = 0.6) after adjusting for age, gender, LVEF, renal function and hemoglobin. Nocturnal hypoxia due to CSA promotes increased endogenous EPO concentration in HF patients.

Anemia of Critical Illness

Anemia of critical illness, a commonly encountered clinical situation, is hematologically similar to that of chronic anemia, except that the onset is generally sudden. The etiology is usually multifactorial, occurring as a consequence of direct inhibitory effects of inflammatory cytokines, erythropoietin deficiency, blunted erythropoietic response, blood loss, nutritional deficiencies, and renal insufficiency. Although anemia is not well tolerated by critically ill patients, aggressive treatment of anemia can be just as detrimental as no treatment. Different types of anemia may coexist in a patient in the intensive care unit, making diagnosis and differentiation among these anemias complex, therefore requiring good diagnostic skills. Although several therapeutic options are available to treat anemia, critically ill patients often receive a transfusion, and yet, most recent studies indicate that blood transfusions in critically ill patients are associated with worse outcomes, including higher morbidity and mortality. These studies have generated interest in the administration of exogenous erythropoietin and iron therapy. Unfortunately, the accurate determination of iron status can be a rather difficult task, an undertaking that is made even more difficult by the presence of comorbid conditions that can affect the commonly used parameters for guiding iron therapy. The use of erythropoiesis-stimulating agents is rapidly gaining acceptance, although they also present potential problems of their own.