Stimulation Of Erythroid Progenitor Cells Research Articles

MO334ERYTHROPOIETIN-STIMULATED HUMAN ENDOTHELIAL PROGENITOR CELLS AMELIORATE INFLAMMATION AND FIBROSIS VIA INFLAMMASOME IN ISCHEMIA/REPERFUSION-INDUCED ACUTE KIDNEY INJURY

Abstract Background and Aims Ischemia/reperfusion-induced AKI (IR-AKI) is a major cause of AKI and progress to chronic kidney disease. But an effective therapeutic intervention for IR-AKI is not established yet. Erythropoietin (EPO) is a potent stimulator of erythroid progenitor cells and is significantly upregulated during hypoxia. Endothelial progenitor cells (EPCs) are derived from the bone marrow or tissue-resident cells and play major roles in the maintenance of vascular integrity and the repair of endothelial damage. So, we investigated if EPO-stimulated human EPCs could have the renoprotective effects in an IR-AKI mouse model. Method EPCs originated from human peripheral blood were cultured with EPO (10 IU/mL). Mice were assigned to sham, IR only groups, IR with EPC, and IR with EPO-treated EPC. EPCs (5x105 cells, tail vein) were administered twice at 30 min prior to bilateral renal artery occlusion, and 5 min before reperfusion, with all mice sacrificed 24 h after IR-AKI. Results Both EPCs and EPO-treated EPCs significantly attenuated the renal dysfunction associated with IR-AKI, as well as tissue injury. Apoptotic cell death and oxidative stress were significantly reduced in EPC and EPO-treated EPC mice. Expression of PCNA, ICAM-1, MCP-1 and α-SMA were also significantly reduced in EPC and EPO-treated EPC mice. Furthermore, the expression of NLRP3 and caspase-1 via the activation of NF-κB signaling pathways were significantly reduced in EPC and EPO-treated EPC mice. These results show more effective in EPO-treated EPC than EPC alone and suggest EPO might be involved in the development of EPC. Conclusion This study provides that inflammasome-mediated inflammation and fibrosis might be a potential target of EPC as a treatment for IR-AKI.

Erythropoietin Ameliorates Ischemia/Reperfusion-Induced Acute Kidney Injury via Inflammasome Suppression in Mice.

Acute kidney injury (AKI) is the most common condition in hospitalized patients. As ischemia/reperfusion-induced AKI (IR-AKI) is as a major contributor to end-stage disease, an effective therapeutic intervention for IR-AKI is imperative. Erythropoietin (EPO) is a potent stimulator of erythroid progenitor cells and is significantly upregulated during hypoxia. Here, we investigated the renoprotective effects of EPO in an IR-AKI mouse model. Mice were assigned to sham, EPO only, and IR only groups, and the IR group was treated with EPO prior to injury. EPO was administered twice at 30 min prior to bilateral renal artery occlusion, and 5 min before reperfusion, with all mice sacrificed 24 h after IR-AKI. The serum was harvested for renal functional measurements. The kidneys were subjected to histological evaluation, and the biochemical changes associated with renal injury were assessed. EPO significantly attenuated the renal dysfunction associated with IR-AKI, as well as tissue injury. Apoptotic cell death and oxidative stress were significantly reduced in EPO-treated mice. Macrophage infiltration and expression of ICAM-1 and MCP-1 were also significantly reduced in EPO-treated mice. Furthermore, the expression of inflammasome-related factors (NLRP1, NLRP3, and caspase-1 cleavage), via the activation of the COX-2 and NF-κB signaling pathways were significantly reduced following EPO treatment. To our knowledge, this is the first study to demonstrate that inflammasome-mediated inflammation might be a potential target of EPO as a treatment for ischemic AKI.

Anti-Inflammatory Effect of Erythropoietin in the TNBS-induced Colitis.

Erythropoietin is a potent stimulator of erythroid progenitor cells, which is able to inhibit NF-kB activation, due to its pleiotropic properties, thus promoting an anti-inflammatory effect. As inflammatory bowel disease is a chronic disease with reduced quality of life, and the current pharmacotherapy only induces or maintains the patient in remission, there is a crucial need of new pharmacological approaches. The main objective of this study was to evaluate the effect of erythropoietin in the TNBS-induced colitis model in mice with a normal intestinal flora. Mice with TNBS-induced colitis were treated with a daily dose of erythropoietin at 500 IU/kg bw/day and 1000 IU/Kg bw/day IP during 4 days. As to clinical symptoms/signs, erythropoietin attenuated the decreased body-weight and reduced diarrhoea and oedema of the anus registered in the non-treated mice group in a dose-dependent manner. The anti-inflammatory properties of erythropoietin in the TNBS-induced colitis were confirmed by suppression of pro-inflammatory mediators, such as TNF-α, IL-1β and MPO, as well as a significant increase in the anti-inflammatory cytokine, IL-10, was promoted. These treated mice also presented a reduction in haemoglobin faecal and ALP, suggesting a beneficial effect of erythropoietin in the haemorrhagic focus and destruction of the enterocyte associated with the colon injury induced by TNBS, respectively. The histopathological score was reduced after treatment with erythropoietin, decreasing the severity and extension of the colitis. Furthermore, renal and hepatic biomarkers, as well as haematocrit concentration, remained stabilized after treatment. In conclusion, erythropoietin reduces the inflammatory response associated with TNBS-induced colitis in mice.

Excessive Isolated Erythrocytosis Linked to Induction of SCL/Tal1 and Erythropoietin Receptor Expression.

The JAK2 V617F mutation has been detected in more than 90% polycythemia vera patients and is associated with increased erythropoietin (EPO) sensitivity or EPO independence of erythroid progenitor cells during early erythropoiesis. To determine if other molecular lesions give rise to increased erythrocytosis and realizing that the JAK2 V617F mutation may not be the initiating event in polycythemia vera, we identified an individual with isolated, excessive erythrocytosis who showed erythroid precursor hypersensitivity to EPO; no mutations on sequencing genes for erythropoietin receptor (EPO-R), VHL, or HIF-1 IRE; normal serum EPO level; normal hemoglobin function and p50; normal cardiac, hepatic and pulmonary function; no JAK2 V617F mutation and no EPO independent BFU-E. While it was not certain that this individual's erythrocytosis was lifelong, other relatives had increased hemoglobin levels. The cause of this individual's increased erythropoiesis was investigated using EPO-stimulated cultures of hematopoietic progenitor cells isolated from peripheral blood. As with normal controls, cell numbers were not increased after 12 days of incubation with low levels of EPO (<0.1 U/ml). With 1U/ml of EPO, cell proliferation was similar to control early in erythropoiesis but increased markedly after 8 days so that by day 12 cell numbers were 3-fold greater than in control cultures with 95% benzidine positive cells compared with 76% for control and 82–87% for JAK2 V617F positive erythroid progenitor cells. Analyses of transcription factor expression revealed that induction of GATA-1 and down regulation of GATA-2 were similar to control. However, SCL/Tal1 and EKLF markedly increased beginning at day 8 to 10 and continued to rise during late erythropoiesis in contrast to control and JAK2 V617F positive cultures in which SCL/Tal1 and EKLF peaked at day 10 and decreased with late erythroid differentiation. Since increased beta-globin expression is concomitant with induction of EKLF, the rise in EKLF may account for the marked increase in benzidine positive cells from our subject. Moreover, expression of EPO-R followed the continuing rise in SCL/Tal1, increasing by 3.5 fold at day 12 compared with control cultures. This high EPO-R expression is consistent with the dramatic increase in cell proliferation during late erythropoiesis. Using forced expression of SCL/Tal1, reporter gene assay and gel mobility shift analysis in erythroid cells, we determined that SCL/Tal1 is able to bind to E-boxes located 3′ of the EPO-R proximal promoter and to activate transcription. Although EPO stimulation of erythroid progenitor cells activates EPO-R, we found that forced expression of EPO-R in primary erythroid progenitor cells in the presence of EPO increases expression of GATA-1 as well as SCL/Tal1 and EKLF, providing further evidence that specific increase in SCL/Tal1 but not GATA-1 must precede induction of EPO-R in this case of excessive erythrocytosis. These data demonstrate a link between high level induction of SCL/Tal1 expression and increased erythrocytosis and suggest a mechanism that contributes to increased erythropoietin sensitivity during late erythropoiesis.

Erythropoietin reduces the development of nonseptic shock induced by zymosan in mice*

Erythropoietin is a potent stimulator of erythroid progenitor cells, and its expression is enhanced by hypoxia. In the present study, we investigated the effects of erythropoietin (1000 IU/kg subcutaneously) on the development of nonseptic shock caused by zymosan. Prospective, randomized study. University-based research laboratory. Male CD mice. Mice received either intraperitoneally zymosan (500 mg/kg, administered intraperitoneally as a suspension in saline) or vehicle (0.25 mL/mouse saline). Erythropoietin was administered at the dose of 1000 IU/kg subcutaneously, 1 and 6 hrs after zymosan administration. Organ failure and systemic inflammation in mice was assessed 18 hrs after administration of zymosan and/or erythropoietin. Treatment of mice with erythropoietin (1000 IU/kg subcutaneously, 1 and 6 hrs after zymosan administration) attenuated the peritoneal exudation and the migration of polymorphonuclear cells caused by zymosan. Erythropoietin also attenuated the lung, liver, and pancreatic injury and renal dysfunction caused by zymosan as well as the increase in myeloperoxidase activity caused by zymosan in the lung and intestine. Immunohistochemical analysis for nitrotyrosine and poly(ADP-ribose) revealed positive staining in lung and intestine tissues obtained from zymosan-treated mice. The degree of staining for nitrotyrosine and poly(ADP-ribose) was markedly reduced in tissue sections obtained from zymosan-treated mice, which received erythropoietin. In addition, administration of zymosan caused severe illness in the mice characterized by a systemic toxicity, significant loss of body weight, and a 70% mortality rate at the end of observation period (7 days). Treatment with erythropoietin significantly reduced the development of systemic toxicity, the loss in body weight, and the mortality caused by zymosan. This study provides evidence, for the first time, that erythropoietin attenuates the degree of zymosan-induced nonseptic shock in mice.

Enhanced erythropoiesis mediated by activation of the renin‐angiotensin system via angiotensin II type 1a receptor

Although clinical and experimental studies have long suggested a role for the renin-angiotensin system (RAS) in the regulation of erythropoiesis, the molecular basis of this role has not been well understood. We report here that transgenic mice carrying both the human renin and human angiotensinogen genes displayed persistent erythrocytosis as well as hypertension. To identify the receptor molecule responsible for this phenotype, we introduced both transgenes into the AT1a receptor null background and found that the hematocrit level in the compound mice was restored to the normal level. Angiotensin II has been shown to influence erythropoiesis by two means, up-regulation of erythropoietin levels and direct stimulation of erythroid progenitor cells. Thus, we conducted bone marrow transplantation experiments and clarified that AT1a receptors on bone marrow-derived cells were dispensable for RAS-dependent erythrocytosis. Plasma erythropoietin levels and kidney erythropoietin mRNA expression in the double transgenic mice were significantly increased compared with those of the wild-type control, while the elevated plasma erythropoietin levels were significantly attenuated in the compound mice. These results provide clear genetic evidence that activated RAS enhances erythropoiesis through the AT1a receptor of kidney cells and that this effect is mediated by the elevation of plasma erythropoietin levels in vivo.

Erythropoietin reduces the degree of arthritis caused by type II collagen in the mouse

Erythropoietin (EPO) is a potent stimulator of erythroid progenitor cells, and its expression is enhanced by hypoxia. The aim of this study was to investigate the effect of EPO on collagen-induced arthritis (CIA) in the mouse. CIA was induced by intradermal injection of bovine type II collagen (CII) and Freund's complete adjuvant. Starting on day 25, some of the mice with CIA received daily subcutaneous injections of EPO (1,000 units/kg). Two other groups of mice received sham treatment alone or sham treatment followed by EPO treatment, respectively. Arthritis was assessed clinically, radiologically, and histologically. Cytokine and chemokine levels were measured, and neutrophil infiltration into inflamed joints was quantitated. Immunohistochemistry studies were performed to measure protein nitrosylation. Chondrocyte apoptosis was assessed by TUNEL assay. Macroscopic clinical evidence of CIA first appeared as periarticular erythema and edema in the hind paws. The incidence of CIA was 100% by day 27 in the CII-challenged mice, and the severity of CIA progressed over a 35-day period, with radiographic evaluation revealing focal resorption of bone. Histopathologic features of CIA included erosion of the cartilage at the joint margins. Treatment with EPO starting at the onset of arthritis (day 25) ameliorated the clinical signs on days 26-35 and improved histologic status in the joints and paws. The degree of oxidative and nitrosative damage was significantly reduced in EPO-treated mice as indicated by decreased nitrotyrosine formation and poly(ADP-ribose) polymerase activation. Plasma levels of the proinflammatory cytokine tumor necrosis factor alpha were also significantly reduced by EPO treatment. In addition, EPO reduced the levels of apoptosis in chondrocytes in articular cartilage, as indicated by decreased TUNEL staining. These findings demonstrate that EPO exerts an antiinflammatory effect during chronic inflammation and is able to ameliorate the tissue damage associated with CIA.

Pretreatment with EPO reduces the injury and dysfunction caused by ischemia/reperfusion in the mouse kidney in vivo

Erythropoietin (EPO) is a potent stimulator of erythroid progenitor cells and is known to be up-regulated during states of hypoxia. Here we investigate the effects of renal ischemia/reperfusion (I/R) on the degree of renal dysfunction and injury with recombinant human EPO in mice when given as either a 3-day pretreatment, or upon reperfusion of the kidney. Mice were treated with EPO (1000 IU/kg/day subcutaneously) for 3 days, or treated with EPO (1000 IU/kg subcutaneously) upon reperfusion, and subsequently subjected to bilateral renal artery occlusion (30 minutes) and reperfusion (24 hours). At the end of experiments, the following indicators and markers of renal injury and dysfunction were measured: plasma urea, creatinine, and aspartate aminotransferase (AST), tissue myeloperoxidase (MPO) activity [for polymorphonuclear leukocyte (PMN) infiltration], and tissue malonaldehyde (MDA) levels (for tissue lipid peroxidation). Kidneys were used for histologic evaluation of renal injury. EPO was able to significantly attenuate the renal dysfunction and injury associated with I/R, as well as the tissue injury. The increase in renal MPO activity and, hence, the degree of PMN infiltration were also significantly reduced in EPO-treated mice. In addition, lipid peroxidation as a result of renal I/R injury was also attenuated in EPO-treated mice. The protection afforded by the pretreatment regime of EPO was greater than that of administering EPO as a single bolus upon reperfusion. We propose that different mechanisms underlie the protective effects seen with EPO when given as either a daily pretreatment or as a single bolus, which need to be further investigated.

Erythropoietin reduces the development of experimental inflammatory bowel disease.

Inflammatory bowel disease is characterized by oxidative and nitrosative stress, leukocyte infiltration, and up-regulation of the expression of intercellular adhesion molecule-1 (ICAM-1) in the colon. Erythropoietin (EPO) is a potent stimulator of erythroid progenitor cells, and its expression is enhanced by hypoxia. Here we investigate the effects EPO has on the development of experimental colitis. To address this question, we used an experimental model of colitis induced by dinitrobenzene sulfonic acid (DNBS). When compared with DNBS-treated mice, EPO (1000 IU/kg day s.c.)-treated mice subjected to DNBS-induced colitis experienced significantly lower rates in the extent and severity of the histological signs of colon injury. DNBS-treated mice experienced diarrhea and weight loss. At 4 days after administration of DNBS, the mucosa of the colon exhibited large areas of necrosis. Neutrophil infiltration (determined by histology as well as an increase in myeloperoxidase activity in the mucosa) was associated with up-regulation of ICAM-1. Immunohistochemistry for nitrotyrosine and poly(ADP-ribose) showed an intense staining in the inflamed colon. On the contrary, the treatment of DNBS-treated mice with EPO significantly reduced the degree of diarrhea and weight loss caused by administration of DNBS. EPO also caused a substantial reduction of the degree of colon injury, the rise in myeloperoxidase activity (mucosa), and the increase in staining (immunohistochemistry) for nitrotyrosine as well as the up-regulation of ICAM-1 caused by DNBS in the colon. Thus, treatment of rat with EPO reduces the degree of colitis caused by DNBS. We propose that EPO may be useful in the treatment of inflammatory bowel disease.

Haemoglobin during pregnancy: relationship to erythropoietin and haematinic status.

To investigate the effect of haematinic status on erythropoietin throughout pregnancy in order to assess whether haematinic supplementation is appropriate for all pregnant women or should be reserved for individuals in whom deficiency has been identified. A prospective, repeated-measures, longitudinal study of 263 women enrolled at an antenatal clinic in a district general hospital who received standard obstetric management including haematinic supplements. Haematological indices, iron status, folate and B12 status, and erythropoietin levels were measured by standard methods at scheduled intervals throughout pregnancy. The relationships between the measurements were analysed using statistical models which took into account the serial correlation between repeated measurements on the same individuals. Erythropoietin levels rose throughout pregnancy from a mean of 22.8 mU/mL at booking to 43.7 mU/mL at 38 wk. There was a significant relationship between erythropoietin levels and haemoglobin, ferritin, TIBC and folate. The relationship was strongest between erythropoietin and haemoglobin. Erythropoietin levels were influenced by parity but not maternal age. Haematinic levels, particularly iron are related to the rise in serum EPO which occurs during pregnancy. Therefore, haematinic deficiency in pregnancy will result in increased stimulation of erythroid progenitor cells by erythropoietin. While haematinic supplementation is not always prescribed throughout pregnancy, haematinic status must be assessed on an individual basis and supplementation provided as required.

Role of Antennary Structure of N-Linked Sugar Chains in Renal Handling of Recombinant Human Erythropoietin

To elucidate the role of the branched structure of sugar chains of human erythropoietin (EPO) in the expression of in vivo activity, the pharmacokinetic profile of a less active recombinant human EPO sample (EPO-bi) enriched with biantennary sugar chains was compared with that of a highly active control EPO sample enriched with tetraantennary sugar chains. After an intravenous injection in rats, 125I-EPO-bi disappeared from the plasma with 3.2 times greater total body clearance (Cltot) than control 125I-EPO. Whole-body autoradiography after 20 minutes of administration indicated that the overall distribution of radioactivity is similar, but 125I-EPO-bi showed a higher level of radioactivity in the kidneys than control 125I-EPO. Quantitative determination of radioactivity in the tissues also indicated that radioactivity of 125I-EPO-bi in the kidneys was two times higher than that of control 125I-EPO. The difference in plasma disappearance between 125I-EPO-bi and control 125I-EPO was not observed in bilaterally nephrectomized rats. The distribution of 125I-EPO-bi to bone marrow and spleen was similarly inhibited by simultaneous injection of excess amounts of either the nonlabeled EPO-bi or control EPO. These results indicate that the low in vivo biologic activity of EPO-bi results from rapid clearance from the systemic circulation by renal handling. Thus, the well-branched structure of the N-linked sugar chain of EPO is suggested to play an important role in maintaining its higher plasma level, which guarantees an effective transfer to target organs and stimulation of erythroid progenitor cells.

The role of marrow accessory cell populations in the augmentation of human erythroid progenitor cell (BFU-E) proliferation by prostaglandin E

The requirement for CD8+ T lymphocytes in the stimulation of erythroid progenitor cells by prostaglandin E (PGE) was examined. When low density bone marrow (LD-BM) or non-adherent bone marrow (NA-BM) cells were depleted of CD8+ cells the enhancing effect of PGE on BFU-E proliferation was abrogated. However, further enrichment of marrow progenitor cells by depletion of accessory cells using a cocktail of specific monoclonal antibodies, immunoadherence and fluorescence activated cell sorting with the MY10 monoclonal antibody resulted in a population of erythroid progenitor cells which were responsive to the enhancing effect of PGE despite the absence of CD8+ cells. Stepwise individual cell lineage depletion of marrow cell populations indicated that prostaglandin E enhanced erythroid burst formation in the absence of CD8+ cells provided that glycophorin-A+ cells were removed from LD-BM or NA-BM cells. These results suggest that nucleated erythroid cell populations may modulate the enhancement of BFU-E by PGE. The ability of GP-A+ cells to block the enhancement of erythroid burst formation by PGE following removal of CD8+ T cells was confirmed by readdition of conditioned medium prepared from positively selected GP-A+ marrow cells. These results expand the role of CD8+ T cells in the PGE enhancement of BFU-E proliferation and suggest another mechanism by which accessory cells regulate the proliferation of BFU-E in bone marrow.