Erythropoietin Treatment In Mice Research Articles

Erythropoietin Treatment in Mice Enriches Bone Marrow Osteoclast Precursors, As Reflected By Increased Macrophage Colony-Stimulating Factor (MCSF) and Elevated Expression of Osteoclast Markers in Monocytes

The impact of erythropoietin (EPO) on erythropoiesis has long been established. However, recent studies have indicated that EPO may also exert regulatory effects on other hematopoietic lineages, including the monocyte lineage. In that respect, we have previously shown that EPO treatment in mice induces bone loss. Here, we studied the effect of EPO on bone marrow (BM) monocytes and its potential implications for osteoclastogenesis. To address the dynamic changes in monocyte populations following EPO administration, we injected EPO (180U) once, or 3 times on alternating days for one week. Mice were sacrificed 16 h after EPO injection. Our results revealed a significant 20 percent increase in the monocyte population (TER119 -, LY6G -, CD11b +) 16 hours after EPO injection, and a twofold increase in the levels of these cells after one week of 3 injections of EPO. Soluble macrophage colony-stimulating factor (MCSF) protein levels in the bone marrow were increased 1.5-fold 16 h after EPO injection, while MCSF mRNA expression levels remained unchanged. After 1-week of EPO administration (3 injections), although the global BM expression of MCSF was reduced, we observed a two-fold increase in MCSF expression levels in T cells (CD3 +). To further elucidate the functional consequences of EPO-induced monocyte expansion, we isolated monocytes from mice treated with EPO for one week. We characterized expression levels of key osteoclastic markers in these cells. Indeed, we found that in BM monocytes, EPO treatment led to the upregulation of Lymphocyte function-associated antigen 1 (LFA-1) and Receptor Activator of Nuclear Factor κ B (RANK). The former is implicated in osteoclastogenesis via its interaction with intercellular adhesion molecule-1 (ICAM−1), while the latter is the surface receptor for RANK ligand which regulates osteoclast differentiation and activation. These data suggest that EPO not only influences BM monocyte expansion, but also contributes to the regulation of osteoclast differentiation, thus linking erythropoiesis to bone homeostasis. Furthermore, we investigated the early effects of EPO treatment on the expression of transcription factors implicated in monocyte differentiation. Remarkably, we found that EPO administration for 16 hours resulted in the downregulation of KLF2 and KLF4, transcription factors involved in monocytic lineage commitment. The rapid EPO-induced expansion of the monocytic population, coupled with the EPO-regulated expression of MSCF, osteoclastic markers, and transcription factors, suggests a role for EPO in modulating monocyte-to-osteoclast differentiation. The current study opens avenues for further investigating the crosstalk between erythropoiesis, myelopoiesis, and bone homeostasis.

Neuronal nitric oxide synthase is required for erythropoietin stimulated erythropoiesis in mice.

Introduction: Erythropoietin (EPO), produced in the kidney in a hypoxia responsive manner, is required for red blood cell production. In non-erythroid tissue, EPO increases endothelial cell production of nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) that regulates vascular tone to improve oxygen delivery. This contributes to EPO cardioprotective activity in mouse models. Nitric oxide treatment in mice shifts hematopoiesis toward the erythroid lineage, increases red blood cell production and total hemoglobin. In erythroid cells, nitric oxide can also be generated by hydroxyurea metabolism that may contribute to hydroxyurea induction of fetal hemoglobin. We find that during erythroid differentiation, EPO induces neuronal nitric oxide synthase (nNOS) and that neuronal nitric oxide synthase is required for normal erythropoietic response. Methods: Wild type (WT) mice and mice with targeted deletion of nNOS (nNOS-/-) and eNOS (eNOS-/-) were assessed for EPO stimulated erythropoietic response. Bone marrow erythropoietic activity was assessed in culture by EPO dependent erythroid colony assay and in vivo by bone marrow transplantation into recipient WT mice. Contribution of nNOS to EPO stimulated cell proliferation was assessed in EPO dependent erythroid cells and in primary human erythroid progenitor cell cultures. Results: EPO treatment increased hematocrit similarly in WT and eNOS-/- mice and showed a lower increase in hematocrit nNOS-/- mice. Erythroid colony assays from bone marrow cells were comparable in number from wild type, eNOS-/- and nNOS-/- mice at low EPO concentration. Colony number increased at high EPO concentration is seen only in cultures from bone marrow cells of wild type and eNOS-/- mice but not from nNOS-/- mice. Colony size with high EPO treatment also exhibited a marked increase in erythroid cultures from wild type and eNOS-/- mice but not from nNOS-/- mice. Bone marrow transplant from nNOS-/- mice into immunodeficient mice showed engraftment at comparable levels to WT bone marrow transplant. With EPO treatment, the increase in hematocrit was blunted in recipient mice that received with nNOS-/- donor marrow compared with recipient mice that received WT donor marrow. In erythroid cell cultures, addition of nNOS inhibitor resulted in decreased EPO dependent proliferation mediated in part by decreased EPO receptor expression, and decreased proliferation of hemin induced differentiating erythroid cells. Discussion: EPO treatment in mice and in corresponding cultures of bone marrow erythropoiesis suggest an intrinsic defect in erythropoietic response of nNOS-/- mice to high EPO stimulation. Transplantation of bone marrow from donor WT or nNOS-/- mice into recipient WT mice showed that EPO treatment post-transplant recapitulated the response of donor mice. Culture studies suggest nNOS regulation of EPO dependent erythroid cell proliferation, expression of EPO receptor and cell cycle associated genes, and AKT activation. These data provide evidence that nitric oxide modulates EPO dose dependent erythropoietic response.

Effects of Erythropoietin in White Adipose Tissue and Bone Microenvironment.

Erythropoietin (EPO) is expressed primarily in fetal liver and adult kidney to stimulate red blood cell production. Erythropoietin receptor expression is not restricted to erythroid progenitor cells, and non-erythroid EPO activity includes immune response and bone remodeling. In bone fracture models, EPO administration promotes bone formation and accelerates bone healing. In contrast, in healthy adult mice, exogenous EPO-stimulated erythropoiesis has been concomitant with bone loss, particularly at high EPO, that may be accompanied by increased osteoclast activation. Other EPO-associated responses include reduced inflammation and loss of fat mass with high-fat diet feeding, especially in male mice. While EPO exhibited a sex-dimorphic response in regulation of fat mass and inflammation in obese mice, EPO-stimulated erythropoiesis as well as EPO-associated bone loss was comparable in males and females. EPO administration in young mice and in obese mice resulted in bone loss without increasing osteoclasts, suggesting an osteoclast-independent mechanism, while loss of endogenous EPO decreased bone development and maintenance. Ossicle formation of bone marrow stromal cell transplants showed that EPO directly regulates the balance between osteogenesis and adipogenesis. Therefore, during development, endogenous EPO contributes to normal bone development and in maintaining the balance between osteogenesis and adipogenesis in bone marrow stromal cells, while EPO treatment in mice increased erythropoiesis, promoted bone loss, decreased bone marrow adipogenesis, and increased osteoclast activity. These observations in mouse models suggest that the most prevalent use of EPO to treat anemia associated with chronic kidney disease may compromise bone health and increase fracture risk, especially at a high dose.

1997-P: Cross Talk between EPO and Estrogen Signaling in Regulation of Fat Mass

Erythropoietin (EPO), the cytokine that regulates erythropoiesis, contributes to metabolic regulation in rodents. The increase in red blood cell production with EPO treatment in mice was accompanied by improved glycemic control, and by attenuated body weight gain via reducing fat mass in male mice. No change in body weight was observed in mice with EPO receptor restricted to erythroid tissue, suggesting that EPO regulation of body weight is a non-erythroid response. EPO stimulated decrease in fat mass was observed in ovariectomized female mice on high fat diet, but not after estradiol supplementation or in ovariectomized female mice on normal chow, providing evidence for estrogen related gender specific EPO action in metabolic regulation beyond erythropoiesis. Here we determine the contribution of estrogen response in EPO metabolic regulation by assessing EPO treatment in estrogen receptor α knockout (ERαKO) mice. Male and female ERαKO mice exhibit decreased glucose tolerance, increased fat mass and, in female ERαKO mice, increased lean mass compared with WT control mice. Overall, EPO stimulated increase in hematocrit in male and female ERαKO mice on high fat diet was accompanied by improvement in glucose tolerance comparable to that observed in WT control mice treated with EPO. EPO treatment in male ERαKO and WT mice show significantly reduced the increase in fat mass while on high fat diet compared with saline treatment. Similarly, EPO treatment in female ERαKO mice on high fat diet show reduced fat mass gain compared to saline treatment. In contrast, EPO treatment did not affect fat mass in female WT mice on high fat diet. We also observed that on normal chow diet, EPO treatment was able to reduce fat mass in female ERαKO mice, but not in female WT mice, confirming the role of estrogen in mediating the sex-differential effect of EPO in regulating fat mass. These results are evidence of cross talk between EPO and estrogen for metabolic homeostasis and regulation of body mass in female mice. Disclosure J. Lee: None. H. Rogers: None. C.T. Noguchi: None.

2007-P: Age-Dependent Metabolic Response to Erythropoietin in Mice

Erythropoietin (EPO), the cytokine required for erythrocyte production, regulates glucose homeostasis and fat mass accumulation in animal models. For example, young (5-6 weeks) mice on high fat diet (HFD) treated with EPO for 5 weeks show a dose dependent increase in hematocrit and reduction in fat mass accumulation/weight gain. To assess potential age and sex dependent EPO metabolic response, C57/Bl6 mice beginning at 3 months, 4 months or 6 months of age were fed normal chow (NC) or high fat diet (HFD) and treated with EPO or saline for up to 6 weeks. After 3 weeks of EPO treatment, all mice exhibited the expected increase in hematocrit (hct up to 70%) that was accompanied by an improvement in glucose tolerance. Body weight gain was unchanged in the 3-month groups, while the 4- and 6-month males on HFD showed a reduced gain in body weight with 3-weeks of EPO treatment. Since earlier studies suggested an increased reduction in fat mass with extended EPO treatment, EPO treatment was continued for three more weeks. At 6 weeks EPO treatment, body weight gain was not affected in the 3-month groups on NC or HFD but was reduced in the 4- and 6-month groups on HFD in male and female mice. These differences in weight gain appear to be due to differences in fat accumulation. However, after 6 weeks EPO treatment, about 66% of the 3-month group, 70% of the 4-month group showed reduced hematocrit below baseline to low as 22% in the 3-month group and 11% in the 4-month group. Mice with the lowest hct showed the greatest impairment in glucose tolerance. In the 6-month group on HFD, EPO treatment for 6 weeks decreased body weight in both males and females. However, hct decreased in all 6-month mice treated with EPO for 6 weeks, falling as low as 15% and the improved glucose tolerance with EPO treatment seen at 3 weeks was no longer apparent. These data suggest an age dependent immune response at 6 weeks of human EPO treatment in mice and production of anti-human EPO antibodies with the potential to also reduce erythropoiesis and glucose tolerance associated with endogenous EPO. Disclosure H. Rogers: None. C.T. Noguchi: None. Funding National Institutes of Health

Erythroferrone is not required for the glucoregulatory and hematologic effects of chronic erythropoietin treatment in mice.

Erythropoietin (EPO) acts on erythroid progenitor cells to promote their survival and differentiation to mature erythrocytes. Along with this canonical role, EPO is also reported to modulate energy metabolism, resulting in improved glucose tolerance and insulin sensitivity. EPO also stimulates the production of the hormone erythroferrone (ERFE) which acts to suppress hepcidin production, thus increasing dietary iron absorption and mobilizing stored iron for use in erythropoiesis. ERFE (initially termed myonectin) was also reported have an effect on systemic lipid metabolism by promoting the clearance of nonesterifed fatty acids (NEFA) from circulation. As increased levels of circulating NEFA blunt insulin sensitivity and impair glucose tolerance, ERFE‐induced clearance of NEFA after EPO administration would have a beneficial effect on glucose metabolism. The aim of this study was to determine if the known metabolic effect of EPO treatment on glucose homeostasis is mediated by ERFE, produced in response to EPO. Mice lacking Erfe did not differ from wild‐type mice in blood lipid parameters, blood glucose, and glucose or insulin tolerance at baseline or after chronic EPO treatment. Additionally, hepcidin suppression and the response of erythrocyte parameters to chronic EPO treatment were unaffected by the absence of Erfe. These findings suggest that the known beneficial effects of EPO on glucose metabolism are not attributable to an accompanying increase in ERFE production, and that Erfe is dispensable for normal glucose homeostasis. Furthermore, our data indicate that ERFE‐independent mechanisms can suppress hepcidin in response to chronically elevated EPO levels.

Systemic Lipopolysaccharide Administration-Induced Cognitive Impairments are Reversed by Erythropoietin Treatment in Mice.

Sepsis-associated encephalopathy (SAE) is a frequent complication in critically ill patients and is associated with long-term cognitive impairments. However, the pathophysiology underlying SAE is poorly understood and the pharmacologic treatment is lacking. The purpose of the present study was to investigate the effects of erythropoietin (EPO) on cognitive impairments in an animal model of SAE induced by peripheral administration of lipopolysaccharide (LPS). Mice were randomly divided into the sham + vehicle, sham + EPO, LPS + vehicle, and LPS + EPO groups. EPO was administrated 30min after the LPS administration and daily afterward for 2days. Behavioral tests were performed on days 6 and 7 with open field and fear conditioning tests, respectively. The survival rate was estimated by the Kaplan-Meier method. The levels of proinflammatory responses, oxidative stress, and apoptosis-related markers were measured in the hippocampus at the indicated time points. The synaptic morphometry changes in the CA1 region were observed with transmission electron microscopy. Our results showed that LPS administration resulted in high mortality rate and cognitive impairments, which were accompanied by increased expressions of interleukin-1β, malondialdehyde, cleaved caspase-3, and abnormal synaptic morphometry changes in the hippocampus. Notably, EPO treatment reversed the cognitive impairments and rescued the brain pathology induced by LPS administration. In conclusion, our data suggested that treatment with EPO reduced the mortality rate and ameliorated cognitive impairments in an animal model of SAE.

Erythropoietin Increases Erythropoiesis, Metabolism and Weight Loss In Mice

▪Erythropoietin is required for erythroid progenitor cell survival, proliferation and differentiation. Increasing evidence suggests that erythropoietin treatment in mice can stimulate erythropoiesis and also affect metabolic processes in a dose dependent manner. For example, medium to high dose erythropoietin treatment (600 U/kg or 3000 U/kg) in leptin deficient obese (ob/ob) mice three times a week for three weeks or more results in the expected increase in hematocrit as well as decrease in accumulated body fat and improved glucose tolerance. Phlebotomy to maintain normal hematocrit demonstrated that erythropoietin regulation of body weight was not dependent on increased red cell mass. In non-obese wild type C57BL/6 mice, erythropoietin treatment also demonstrated the expected increase in hematocrit as well as a 15% reduction in body weight and decreased fasting blood glucose. Erythropoietin receptor is expressed at the highest level in erythroid progenitor cells. The link between increased metabolism and erythropoietin stimulated erythroid differentiation was suggested by the increased oxygen consumption rate observed in vitro in primary cultures of erythropoietin stimulated erythroid progenitor cells. Erythropoietin also stimulated glucose uptake in differentiating erythroid progenitor cells in a dose dependent manner. Glucose uptake decreased with the down regulation of erythropoietin receptor during terminal differentiation. Relatively high erythropoietin receptor expression and erythropoietin activity that may also contribute to erythropoietin metabolic activity has been observed in non-hematopoietic mouse tissue including the hypothalamus and white adipose tissue (Teng R, Gavrilova O et al., Nat Commun 2011). The hypothalamus contributes importantly to appetite regulation and mice treated with erythropoietin exhibited a decrease in food intake compared with saline control. We found that pair-feeding decreased body weight and fat mass, and improved glucose tolerance, but no more than half that observed with erythropoietin treatment, providing evidence that erythropoietin regulation of food intake accounts for only part of the metabolic response observed with erythropoietin treatment. Adipocytes isolated from white adipose tissue in erythropoietin treated mice showed an increase in oxygen consumption compared with vehicle treated or pair-fed mice. To assess the role of direct erythropoietin response of white adipose tissue in regulation of fat mass accumulation, we engineered mice with targeted deletion of erythropoietin receptor in adipose tissue. Erythropoietin treatment gave rise to the expected increase in hematocrit but resulted in a reduced decrease in body weight compared with saline treatment. These data show that erythropoietin treatment can stimulate cell oxygen consumption and can contribute to regulation of metabolism and body weight in mice. Erythropoietin receptor expression on erythroid progenitor cells provides for erythropoietin response to promote erythropoiesis and increase cell metabolic activity including glucose uptake and oxygen consumption. In non-hematopoietic tissue, erythropoietin receptor expression further contributes to erythropoietin regulated metabolic activity such as control of food intake attributed in part to hypothalamus response and modulation of fat mass affected by direct erythropoietin response in white adipose tissue. Therefore, in addition to its critical role in promoting erythropoiesis, erythropoietin can contribute to metabolic homeostasis via its activity in erythroid tissue and beyond. Disclosures:No relevant conflicts of interest to declare.

Glucose Metabolism and Erythropoietin Stimulation of Erythroid Progenitor Cells

AbstractAbstract 4232Erythropoietin (EPO) is known for its activity in stimulating erythroid progenitor cell survival, proliferation and differentiation. EPO is produced in the interstitial cells of the kidney in a hypoxic responsive manner and is used in treatment of anemia in patients with chronic kidney disease. Suggestions that EPO may affect metabolic parameters led to a recent report that high dose erythropoietin treatment in mice resulted in reduced blood glucose level in addition to increased hematocrit (Hojman et al., Plos One 2009). To examine the link between EPO and metabolic processes further, we treated wild type C57Bl/6 mice fed normal diet (11 kcal% fat) and high fat diet (45 kcal% fat) with EPO (3,000 U/kg three times a week for 3 weeks). As expected from the stimulation of erythropoiesis by exogenous EPO, both groups showed the expected increase in hematocrit up to 80%. We also found that EPO decreased fasting blood glucose level in both normal diet fed mice and high fat diet fed mice, compared with mice treated with saline. An improvement in glucose tolerance was also observed. Since hematopoietic tissue is the major target of EPO, we speculate that EPO may stimulate glucose uptake in hematopoietic tissue, especially during erythropoiesis. To examine EPO stimulation and glucose uptake in vitro, we used cultures of primary human adult erythroid progenitor cells isolated from peripheral blood. We found that with EPO stimulation, glucose uptake per cell increased with erythroid differentiation and peaked in late erythroid progenitor cells at day 8 to 10, then decreased during terminal differentiation. This pattern was observed at both 21% O2 and 5% O2. EPO increased glucose uptake in a dose dependent manner up to 10 U/ml. Cellular glucose uptake appeared to follow EpoR expression and was maximal when EpoR levels, and therefore EPO response, were highest. Glucose uptake decreased as EpoR levels decreased with the progression of erythroid maturation. These data suggest that EPO stimulation regulates glucose uptake in erythroid progenitor/precursor cells depending on the stage of erythroid differentiation and that glucose uptake is reduced in late erythroid precursor cells with the down regulation of EpoR and loss of EPO dependence as the cells differentiate into mature erythrocytes. We also examined the effect of high glucose concentration on EPO response in primary human adult erythroid progenitor cell cultures. We found that high glucose concentration in erythroid progenitor cell cultures inhibited EPO stimulated differentiating erythroid progenitor cell proliferation. The combination of EPO with high dose glucose increased hemoglobinization as determined by the increase in benzidine positive cells, suggesting premature hemoglobin accumulation and differentiation. This activity of elevated glucose level suggests that elevated blood glucose concentration contributes to the blunted EPO response, giving rise to EPO resistance. The findings from this study provide us with new insight on EPO stimulation of erythropoiesis and the potential interplay between erythropoiesis and glucose metabolism. Disclosures:No relevant conflicts of interest to declare.