Erythropoietin Induction Research Articles

The drug-specific properties of hypoxia-inducible factor-prolyl hydroxylase inhibitors in mice reveal a significant contribution of the kidney compared to the liver to erythropoietin induction

AimsKidney disease often leads to anemia due to a defect in the renal production of the erythroid growth factor erythropoietin (EPO), which is produced under the positive regulation of hypoxia-inducible transcription factors (HIFs). Chemical compounds that inhibit HIF-prolyl hydroxylases (HIF-PHs), which suppress HIFs, have been developed to reactivate renal EPO production in renal anemia patients. Currently, multiple HIF-PH inhibitors, in addition to conventional recombinant EPO reagents, are used for renal anemia treatment. This study aimed to elucidate the therapeutic mechanisms and drug-specific properties of HIF-PH inhibitors. Methods and key findingsGene expression analyses and mass spectrometry revealed that HIF-PH inhibitors (daprodustat, enarodustat, molidustat, and vadadustat) alter Epo gene expression levels in the kidney and liver in a drug-specific manner, with different pharmacokinetics in the plasma and urine after oral administration to mice. The drug specificity revealed the dominant contribution of EPO induction in the kidneys rather than in the liver to plasma EPO levels after HIF-PH inhibitor administration. We also found that several HIF-PH inhibitors directly induce duodenal gene expression related to iron intake, while these drugs indirectly suppress hepatic hepcidin expression to mobilize stored iron for hemoglobin synthesis through induction of the EPO-erythroferrone axis. SignificanceRenal EPO induction is the major target of HIF-PH inhibitors for their therapeutic effects on erythropoiesis. Additionally, the drug-specific properties of HIF-PH inhibitors in EPO induction and iron metabolism have been shown in mice, providing useful information for selecting the proper HIF-PH inhibitor for each renal anemia patient.

Gliflozins, Erythropoietin, and Erythrocytosis: Is It Renal Normoxia- or Hypoxia-Driven?

The introduction of gliflozins in the management of type 2 diabetes mellitus leads to a better control of hyperglycemia, obesity, hypertension, dyslipidemia, and fluid retention. Most importantly, it also improves renal survival and reduces major cardiovascular events and mortality. Gliflozins were also found to induce erythropoietin (EPO) synthesis, leading to reticulocytosis and erythropoiesis. The mechanism(s) by which gliflozins induce erythropoiesis is a matter of debate. Although the canonical pathway of triggering EPO synthesis is through renal tissue hypoxia, it has been suggested that improved renal oxygenation may facilitate EPO synthesis via non-canonical routes. The latter proposes that the recovery of peritubular interstitial fibroblasts producing erythropoietin (EPO) is responsible for enhanced erythropoiesis. According to this hypothesis, enhanced glucose/sodium re-uptake by proximal tubules in uncontrolled diabetes generates cortical hypoxia, with injury to these cells. Once transport workload declines with the use of SGLT2i, they recover and regain their capacity to produce EPO. In this short communication, we argue that this hypothesis may be wrong and propose that gliflozins likely induce EPO through the documented intensification of renal hypoxia at the corticomedullary junction, related to the translocation of tubular transport from cortical segments to medullary thick ascending limbs. We propose that gliflozins, through intensified hypoxia in this region, trigger local EPO synthesis in peritubular interstitial cells via the canonical pathway of blocking HIF-prolyl hydroxylases (that initiate HIF alpha degradation), with the consequent stabilization of HIF-2 signal and an apocrinic induction of EPO in these same cells.

Drugs activating hypoxia-inducible factors correct erythropoiesis and hepcidin levels via renal EPO induction in mice

AbstractThe erythroid growth factor erythropoietin (EPO) is mainly produced by the kidneys in adult mammals and induces expansion of erythroid cells and iron use for hemoglobin synthesis. The liver also produces EPO at a lower level than the kidneys. Renal and hepatic EPO production is fundamentally regulated by hypoxia-inducible transcription factors (HIFs) in a hypoxia/anemia-inducible manner. Recently, small compounds that activate HIFs and EPO production in the kidneys by inhibiting HIF-prolyl hydroxylases (HIF-PHIs) have been launched to treat EPO-deficiency anemia in patients with kidney disease. However, the roles of the liver in the HIF-PHI–mediated induction of erythropoiesis and iron mobilization remain controversial. Here, to elucidate the liver contributions to the therapeutic effects of HIF-PHIs, genetically modified mouse lines lacking renal EPO-production ability were analyzed. In the mutant mice, HIF-PHI administration marginally increased plasma EPO concentrations and peripheral erythrocytes by inducing hepatic EPO production. The effects of HIF-PHIs on the mobilization of stored iron and on the suppression of hepatic hepcidin, an inhibitory molecule for iron release from iron-storage cells, were not observed in the mutant mice. These findings demonstrate that adequate induction of EPO mainly in the kidney is essential for achieving the full therapeutic effects of HIF-PHIs, which include hepcidin suppression. The data also show that HIF-PHIs directly induce the expression of duodenal genes related to dietary iron intake. Furthermore, hepatic EPO induction is considered to partially contribute to the erythropoietic effects of HIF-PHIs but to be insufficient to compensate for the abundant EPO induction by the kidneys.

STRUCTURAL MODIFICATIONS OF BUTYRATE REVEAL TEMPLATES FOR INTESTINAL EPITHELIAL HIF STABILIZERS

Microbiota-derived short chain fatty acids, particularly butyrate (BA), show multiple beneficial influences on health. In the colon, BA ranges from 10-20 mM and up to 99% is utilized as a metabolic fuel by the mucosa. BA plays a key role in epithelial barrier regulation, reduces inflammation, and regulates cell growth and differentiation. There are multiple mechanisms by which BA contributes to gut health, many due to its regulatory capacity for gene expression. Our group reported (PMID: 34190032) a direct influence of BA on the stabilization of the transcription factor hypoxia-inducible factor (HIF). It is known that HIF stabilization is essential for appropriate mucosal barrier regulation and the coordination of regenerative capacity in the intestine. However, BA is constantly metabolized limiting its HIF stabilization effect. This observation led to the design and investigation of BA-mimicking compounds that stabilize HIF, but may not be involved in metabolism. We hypothesize that structural modifications of BA yield analogues that stabilize HIF with better efficacy and a longer biological half-life. A library of BA derivatives was screened in search of a non-endogenous analogue with higher potency and/or longer half-life for HIF stabilization. In vitro screenings at physiologically-relevant concentrations (5 mM) were performed using intestinal epithelial cell lines (T84 and CaCO2) in normoxia to determine HIF-1a protein abundance. Various analogues stabilized HIF, with 4-mercapto-butyrate (MBA) being the most promising candidate. The best analogues were validated through the induction of classic HIF gene targets, including BNIP3 and CAIX by q-PCR. MBA exhibited higher induction of these targets compared to BA. Time-course studies revealed that MBA exhibits significantly longer half-life as observed in the stabilization of HIF and induction of gene targets for up to 72 h compared to BA (24 h). Loss of gene induction was observed in MBA treated cells expressing lentiviral shRNA against HIF1b (HIF1b KD), supporting a HIF-dependent transcription. Furthermore, it’s been widely reported that BA enhances epithelial barrier, primarily through HIF coordinated barrier protection. Among several other BA derivatives, MBA enhanced and prolonged epithelial barrier function in intestinal epithelial cells. In vivo studies in C57BL/6 mice explored the induction of HIF targets in tissue and specifically erythropoietin (EPO) in circulation following administration of BA and MBA at similar doses. These studies revealed that induction of EPO with a single dose of MBA exceeded that of native BA. In colonic and kidney tissue, GLUT1 and BNIP3 were moderately induced in vivo. Thus, a non-endogenous BA derivative (MBA) stabilizes HIF at higher potency and longer half-life than native BA and will be used as a template for the development of HIF stabilizing agents.

Non-erythropoiesis stimulating agent, non-iron therapies for the management of anemia: A scoping review.

Non-erythropoiesis stimulating agent, non-iron therapies for the management of anemia: A scoping review.

STRUCTURAL MODIFICATIONS OF BUTYRATE REVEAL TEMPLATES FOR INTESTINAL EPITHELIAL HIF STABILIZERS

Abstract Microbiota-derived short chain fatty acids, particularly butyrate (BA), show multiple beneficial influences on health. In the colon, BA ranges from 10-20 mM and up to 99% is utilized as a metabolic fuel by the mucosa. BA plays a key role in epithelial barrier regulation, reduces inflammation, and regulates cell growth and differentiation. There are multiple mechanisms by which BA contributes to gut health, many due to its regulatory capacity for gene expression. Our group reported (PMID: 34190032) a direct influence of BA on the stabilization of the transcription factor hypoxia-inducible factor (HIF). It is known that HIF stabilization is essential for appropriate mucosal barrier regulation and the coordination of regenerative capacity in the intestine. However, BA is constantly metabolized limiting its HIF stabilization effect. This observation led to the design and investigation of BA-mimicking compounds that stabilize HIF, but may not be involved in metabolism. We hypothesize that structural modifications of BA yield analogues that stabilize HIF with better efficacy and a longer biological half-life. A library of BA derivatives was screened in search of a non-endogenous analogue with higher potency and/or longer half-life for HIF stabilization. In vitro screenings at physiologically-relevant concentrations (5 mM) were performed using intestinal epithelial cell lines (T84 and CaCO2) in normoxia to determine HIF-1a protein abundance. Various analogues stabilized HIF, with 4-mercapto-butyrate (MBA) being the most promising candidate. The best analogues were validated through the induction of classic HIF gene targets, including BNIP3 and CAIX by q-PCR. MBA exhibited higher induction of these targets compared to BA. Time-course studies revealed that MBA exhibits significantly longer half-life as observed in the stabilization of HIF and induction of gene targets for up to 72 h compared to BA (24 h). Loss of gene induction was observed in MBA treated cells expressing lentiviral shRNA against HIF1b (HIF1b KD), supporting a HIF-dependent transcription. Furthermore, it’s been widely reported that BA enhances epithelial barrier, primarily through HIF coordinated barrier protection. Among several other BA derivatives, MBA enhanced and prolonged epithelial barrier function in intestinal epithelial cells. In vivo studies in C57BL/6 mice explored the induction of HIF targets in tissue and specifically erythropoietin (EPO) in circulation following administration of BA and MBA at similar doses. These studies revealed that induction of EPO with a single dose of MBA exceeded that of native BA. In colonic and kidney tissue, GLUT1 and BNIP3 were moderately induced in vivo. Thus, a non-endogenous BA derivative (MBA) stabilizes HIF at higher potency and longer half-life than native BA and will be used as a template for the development of HIF stabilizing agents.

Inhibition of Lysyl Oxidases Synergizes with 5-Azacytidine to Restore Erythropoiesis in Myeloid Neoplasms

Introduction: Limited response rates and frequent relapses during standard of care with hypomethylating agents (HMAs) in myeloid neoplasms (MN) require urgent improvement of this treatment indication. The altered bone marrow (BM) niche in MN is increasingly being recognized as a perspective therapeutic target. Upregulated lysyl oxidase (LOX/LOXL) activity in MN BM contributes to the deposition of aberrant extracellular matrix (ECM). Here, we show in pre-clinical models of MN that the inhibition of niche-associated LOX/LOXL activity in combination with 5-Azacytidine (5-AZA) restores erythropoiesis and reduces disease burden in BM samples of anemic patients with MN. Methods: Primary co-cultures of BM mesenchymal stromal cells (MSCs) and hematopoietic stem and progenitor cells (HSPCs) were established from n=31 anemic MN patients and treated with the pan-LOX/LOXL inhibitor PXS-5505, 5-AZA or PXS-5505 + 5-AZA combination (P+A) for 4 days. The differentiation potential of HSPCs in this co-culture was assessed by colony forming unit (CFU) assays and flow cytometry. In some of the experiments, MSC monolayers were pre-treated with PXS-5505 for 7 days and decellularized to obtain ECM. Subsequently, autologous BM HSPCs were cultured on ECM in the presence of 5-AZA or P+A. Patient-derived xenografts (PDX) were established in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice and treated with 3 cycles of 5-AZA, PXS-5505 (30 mg/kg on alternate days) or the combination of both (P+A). Results: MSCs of MN patients significantly overexpressed LOX/LOXL transcripts as compared to healthy age-matched controls. Remarkably, based on the CFU assay the combination of 5-AZA with the pan-LOX/LOXL inhibitor PXS-5505 improved erythroid differentiation of patient HSPCs that poorly responded to 5-AZA monotherapy. From a total of n=31 anemic patients with advanced-stage myelodysplastic syndromes, myeloproliferative neoplasia and chronic myelomonocytic leukemia, n=20 patients (65%) displayed an erythroid response to P+A combination. Of these, n=11 patients (35%) responded exclusively to P+A, whereas n=9 patients (29%) also concomitantly responded to 5-AZA monotherapy. The comparison of P+A versus 5-AZA monotherapy for improving erythroid response showed a relative risk of 0.45 with 95% confidence interval 0.24-0.80 (p=0.0103). Of note, P+A treatment facilitated differentiation of CD235a+ erythroid progenitors from HSPCs sub-clones with low mutational variant allele frequencies (VAFs). The mechanism of action underlying this drug combination required direct cellular contact of HSPCs with BM stroma components including MSCs and ECM, and is dependent on integrin signaling. Therefore, this novel drug combination represents a genuinely BM microenvironment targeting therapy. We further confirmed these results in vivo using a BM niche-dependent PDX mouse model engrafted with human MN. P+A treatment favored erythroid differentiation of human HSPCs in vivo, showing the directly improved human erythropoiesis in the total BM of PDX mice as well as increased erythroid production after human erythropoietin induction ex vivo. In addition, P+A treatment induced the greatest reduction of disease burden as assessed by decrease of spleen indices, total human engraftment rates, BM fibrosis grades and mutational VAFs. Conclusions: The response to 5-AZA in MN can be potently augmented by concomitant LOX/LOXL inhibition. These results lay out a strong pre-clinical rationale for efficacy of combination treatment of PXS-5505 with 5-AZA especially for anemic and red blood cell transfusion-dependent MN patients, which will be assessed in forthcoming clinical trials.

Butyrate Analogues Mimicking Hypoxia by the Chemical Stabilization of Hypoxia Inducible Factor (HIF)

Short chain fatty acids and specifically butyrate (BA) have been well documented to show multiple beneficial influences on health. Typically, the concentration of colonic butyrate can range from 10‐20 mM and 95‐99% is metabolized into energy by the mucosa. BA plays a key role in epithelial barrier regulation, ameliorates inflammation and regulates cell growth and differentiation. There are multiple mechanisms on how it plays such an important role in gut health, many related to its regulatory capacity for gene expression. Our laboratory recently reported a direct influence of BA on the stabilization of the transcription factor hypoxia‐inducible factor (HIF). BA acts directly and non‐competitively inhibits HIF prolyl hydroxylase 2 (PHD2), responsible for the rapid degradation of HIF in normoxia (NX). However, the efficacy of BA on HIF stabilization is limited due to its rapid metabolism as fuel. Based on these observations, we hypothesize that analogues structurally related to butyrate stabilize HIF with a longer biological half‐life. A small library of BA derivatives was screened in search of a non‐endogenous analogue with higher potency and/or half‐life for HIF stabilization. In vitro screenings at a physiologically‐relevant concentration (5 mM) were performed in intestinal epithelial cell lines T84 and CaCo2 in NX and HIF‐1a ELISA was used to determine protein abundance. Several hits were observed, with 4‐mercapto‐butyrate (SHBA) being the most promising candidate. The best BA analogues were validated through the induction of classic HIF gene targets, including BNIP3 and CaIX by q‐PCR. SHBA exhibited higher induction of these targets compared to BA. Dose response and time‐course studies were performed to compare SHBA and BA. Notably, SHBA exhibits a much longer half‐life as observed in the stabilization of HIF and induction of gene targets at least up to 72 h compared to BA (24 h). In vivo studies in C57BL/6 mice explored the induction of classic HIF targets and specifically erythropoietin (EPO) in circulation following administration of BA and SHBA at similar doses. These studies revealed that induction of EPO with a single dose of SHBA exceeded that of native BA. Thus, a non‐endogenous BA derivative (SHBA) stabilizes HIF at a higher potency and longer half‐life than BA and will be useful as a template for the development of a HIF stabilizing agent.

Effects of empagliflozin on erythropoiesis in patients with type 2 diabetes: Data from a randomized, placebo-controlled study.

Sodium-glucose cotransporter-2 (SGLT2) inhibitors have been shown to significantly reduce hospitalization for heart failure (HHF) and cardiovascular (CV) mortality in various CV outcome trials in patients with and without type 2 diabetes mellitus (T2D). SGLT2 inhibition further increased haemoglobin and haematocrit levels by an as yet unknown mechanism, and this increase has been shown to be an independent predictor of the CV benefit of these agents, for example, in the EMPA-REG OUTCOME trial. The present analysis of the EMPA haemodynamic study examined the early and delayed effects of empagliflozin treatment on haemoglobin and haematocrit levels, in addition to measures of erythropoiesis and iron metabolism, to better understand the underlying mechanisms. In this prospective, placebo-controlled, double-blind, randomized, two-arm parallel, interventional and exploratory study, 44 patients with T2D were randomized into two groups and received empagliflozin 10 mg or placebo for a period of 3months in addition to their concomitant medication. Blood and urine was collected at baseline, on Day 1, on Day 3 and after 3months of treatment to investigate effects on haematological variables, erythropoietin concentrations and indices of iron stores. Baseline characteristics were comparable in the empagliflozin (n=20) and placebo (n=22) group. Empagliflozin led to a significant increase in urinary glucose excretion (baseline: 7.3 ± 22.7g/24h; Day 1: 48.4 ± 34.7g/24h; P < 0.001) as well as urinary volume (baseline: 1740 ± 601mL/24h; Day 1: 2112 ± 837 mL/24h; P=0.011) already after 1day and throughout the 3-month study period, while haematocrit and haemoglobin were only increased after 3months of treatment (haematocrit: baseline: 40.6% ± 4.6%; Month 3: 42.2% ± 4.8%, P < 0.001; haemoglobin: baseline: 136 ± 19g/L; Month 3: 142 ± 25g/L; P=0.008). In addition, after 3months, empagliflozin further increased red blood cell count (P < 0.001) and transferrin concentrations (P=0.063) and there was a trend toward increased erythropoietin levels (P=0.117), while ferritin (P=0.017), total iron (P=0.053) and transferrin saturation levels (P=0.030) decreased. Interestingly, the increase in urinary glucose excretion significantly correlated with the induction of erythropoietin in empagliflozin-treated patients at the 3-month timepoint (Spearman rho 0.64; P=0.008). Empagliflozin increased haemoglobin concentrations and haematocrit with a delayed time kinetic, which was most likely attributable to increased erythropoiesis with augmented iron utilization and not haemoconcentration. This might be attributable to reduced tubular glucose reabsorption in response to SGLT2 inhibition, possibly resulting in diminished cellular stress as a mechanism for increased renal erythropoietin secretion.

HIF-α Prolyl Hydroxylase Inhibitors and Their Implications for Biomedicine: A Comprehensive Review.

Oxygen is essential for the maintenance of the body. Living organisms have evolved systems to secure an oxygen environment to be proper. Hypoxia-inducible factor (HIF) plays an essential role in this process; it is a transcription factor that mediates erythropoietin (EPO) induction at the transcriptional level under hypoxic environment. After successful cDNA cloning in 1995, a line of studies were conducted for elucidating the molecular mechanism of HIF activation in response to hypoxia. In 2001, cDNA cloning of dioxygenases acting on prolines and asparagine residues, which play essential roles in this process, was reported. HIF-prolyl hydroxylases (PHs) are molecules that constitute the core molecular mechanism of detecting a decrease in the partial pressure of oxygen, or hypoxia, in the cells; they can be called oxygen sensors. In this review, I discuss the process of molecular cloning of HIF and HIF-PH, which explains hypoxia-induced EPO expression; the development of HIF-PH inhibitors that artificially or exogenously activate HIF by inhibiting HIF-PH; and the significance and implications of medical intervention using HIF-PH inhibitors.

Inactivation of HIF-prolyl 4-hydroxylases 1, 2 and 3 in NG2-expressing cells induces HIF2-mediated neurovascular expansion independent of erythropoietin.

AimNG2 cells in the brain are comprised of pericytes and NG2 glia and play an important role in the execution of cerebral hypoxia responses, including the induction of erythropoietin (EPO) in pericytes. Oxygen‐dependent angiogenic responses are regulated by hypoxia‐inducible factor (HIF), the activity of which is controlled by prolyl 4‐hydroxylase domain (PHD) dioxygenases and the von Hippel‐Lindau (VHL) tumour suppressor. However, the role of NG2 cells in HIF‐regulated cerebral vascular homeostasis is incompletely understood.MethodsTo examine the HIF/PHD/VHL axis in neurovascular homeostasis, we used a Cre‐loxP‐based genetic approach in mice and targeted Vhl, Epo, Phd1, Phd2, Phd3 and Hif2a in NG2 cells. Cerebral vasculature was assessed by immunofluorescence, RNA in situ hybridization, gene and protein expression analysis, gel zymography and in situ zymography.Results Vhl inactivation led to a significant increase in angiogenic gene and Epo expression. This was associated with EPO‐independent expansion of capillary networks in cortex, striatum and hypothalamus, as well as pericyte proliferation. A comparable phenotype resulted from the combined inactivation of Phd2 and Phd3, but not from Phd2 inactivation alone. Concomitant PHD1 function loss led to further expansion of the neurovasculature. Genetic inactivation of Hif2a in Phd1/Phd2/Phd3 triple mutant mice resulted in normal cerebral vasculature.ConclusionOur studies establish (a) that HIF2 activation in NG2 cells promotes neurovascular expansion and remodelling independently of EPO, (b) that HIF2 activity in NG2 cells is co‐controlled by PHD2 and PHD3 and (c) that PHD1 modulates HIF2 transcriptional responses when PHD2 and PHD3 are inactive.

C-Reactive Protein Predicts Further Ischemic Events in Patients With Transient Ischemic Attack or Lacunar Stroke.

Patients who have experienced a first cerebral ischemic event are at increased risk of recurrent stroke. There is strong evidence that low-level inflammation as measured by high sensitivity C-reactive protein (hs-CRP) is a predictor of further ischemic events. Other mechanisms implicated in the pathogenesis of stroke may play a role in determining the risk of secondary events, including oxidative stress and the adaptive response to it and activation of neuroprotective pathways by hypoxia, for instance through induction of erythropoietin (EPO). This study investigated the association of the levels of CRP, peroxiredoxin 1 (PRDX1, an indicator of the physiological response to oxidative stress) and EPO (a neuroprotective factor produced in response to hypoxia) with the risk of a second ischemic event. Eighty patients with a diagnosis of lacunar stroke or transient ischemic attack (TIA) were included in the study and a blood sample was collected within 14 days from the initial event. Hs-CRP, PRDX1, and EPO were measured by ELISA. Further ischemic events were recorded with a mean follow-up of 42 months (min 24, max 64). Multivariate analysis showed that only CRP was an independent predictor of further events with an observed risk (OR) of 1.14 (P = 0.034, 95% CI 1.01–1.29). No association was observed with the levels of PRDX1 or EPO. A receiver operating curve (ROC) determined a cut-off CRP level of 3.25 μg/ml, with a 46% sensitivity and 81% specificity. Low-level inflammation as detected by hs-CRP is an independent predictor of recurrent cerebrovascular ischemic events.

Preeclampsia: The Interplay Between Oxygen-Sensitive miRNAs and Erythropoietin.

Changes in the oxygen partial pressure caused by a violation of uteroplacental perfusion are considered a powerful inducer of a cascade of reactions leading to the clinical manifestation of preeclampsia (PE). At the same time, the induction of oxygen-dependent molecule expression, in particular, miRNA and erythropoietin, is modulated. Therefore, the focus of our study was aimed at estimating the miRNA expression profile of placental tissue and blood plasma in pregnant women with preeclampsia using deep sequencing and quantitative RT-PCR, as well as determining the concentration of erythropoietin. The expression of miR-27b-3p, miR-92b-3p, miR-125b-5p, miR-181a-5p, and miR-186-5p, as regulated by hypoxia/reoxygenation, was significantly increased in blood plasma during early-onset preeclampsia. The possibility of detecting early PE according to the logistic regression model (miR-92b-3p, miR-125b-5p, and miR-181a-5p (AUC = 0.91)) was evaluated. Furthermore, the erythropoietin level, which is regulated by miR-125b-5p, was significantly increased. According to PANTHER14.1, the participation of these miRNAs in the regulation of pathways, such as the hypoxia’s response via HIF activation, oxidative stress response, angiogenesis, and the VEGF signaling pathway, were determined.

Erythropoietin Downregulates Red Blood Cell Clearance in Mice

Purpose Equilibrium between red blood cells (RBC) production and clearance maintains an appropriate circulating RBC biomass. During anemia or hypoxia, a well-characterized hypoxia-dependent induction of erythropoietin (EPO) synthesis leads to an increase in RBC production. At the other extremity of the RBC lifespan, age-related modifications of RBC properties are expected to be recognized by the mononuclear phagocytic system (MPS) and trigger their clearance. We reasoned that, like RBC production, RBC clearance might be physiologically regulated by hypoxia and therefore that its downregulation could contribute to maintain an appropriate RBC biomass. A mouse model was used to explore specific hypotheses on potential regulatory mechanisms involved in RBC clearance. Material and methods Two steps in vivo biotinylation was used to evaluate the impact of EPO on 3 RBC subpopulations: a young subpopulation (&amp;lt;25 days at treatment initiation) representing the RBC produced, one of intermediate age (25-34 days at treatment initiation) which is neither produced nor eliminated, and an old one (&amp;gt; 34 days at treatment initiation) that is steadily cleared. A model of RBC banking (leucocyte depleted and stored in CPDA solution) was used to evaluate the clearance after transfusion of fluorescently-labeled storage-damaged RBC by flow cytometry. Different recipient models were used to evaluate the impact of specific parameters on RBC clearance including: phlebotomy-induced anemia, normobaric hypoxia, erythropoiesis-stimulating agent (ESA) treatment (darbepoietin), splenectomy, doxorubicin-induced inhibition of erythropoiesis and EPO neutralization (anti-EPO rabbit serum) either alone or in combination. Results Decreased clearance of the oldest subpopulation was observed 2 days after ESA treatment and before the increase in RBC production (7 days). After 20 days of treatment, an increased number of RBC from the oldest subpopulation was detected in circulation confirming that senescent RBC clearance is sensitive to EPO signaling. After transfusion, clearance of storage-damaged RBC is reduced by 30% in anemic recipients when compared to non-anemic recipients. RBC clearance is significantly reduced in hypoxic non-anemic recipients, as soon as 6 hours after the initiation of hypoxia, suggesting that hematocrit per se does not affect RBC clearance. In ESA-treated non-anemic non-hypoxic mice, RBC clearance is also reduced showing that EPO signaling is sufficient. To investigate the role of the spleen in this process, splenectomy was combined with the previous models. As expected, RBC clearance was reduced by 20% in splenectomized recipients. RBC clearance is however even more decreased when splenectomy is combined with anemia, hypoxia or ESA treatment compared to splenectomized or control mice, suggesting that EPO downregulation of RBC clearance is not restricted to the spleen. Erythropoiesis inhibition did not alter the anemia-induced downregulation of RBC clearance ruling out the possibility that an erythroid factor is involved in the process. Finally, neutralization of circulating EPO not only abolishes the reduction of RBC clearance observed in anemic recipients, but also increases RBC clearance in both anemic and non-anemic recipients. Taken together these results indicate that EPO regulates RBC clearance during anemia and in steady state (Figure). Conclusion RBC clearance is downregulated during anemia/hypoxia and EPO is sufficient and necessary to mediate this physiological function. RBC clearance downregulation preceded the increase in production rate induced by ESA treatment suggesting it is a very early physiological response to maintain oxygen supply during anemia. The lifespan of a circulating RBC is therefore adaptable and could be regulated by 2 factors: the RBC pro- and anti-phagocytic properties on one side and, on the other side, the MPS level of activity and sensitivity toward these RBC properties. In case of anemia or hypoxia, increased EPO level would act on the RBC itself, on the activity/sensitivity of the MPS or both to downregulate RBC clearance until the equilibrium between oxygen need and supply is restored. Future studies will evaluate if the pathological dysregulation of this mechanism participates in the pathogenesis of anemia or, modulate transfusion efficacy and burden in chronically transfused patients. Figure Disclosures Buffet: Zimmer Biomet: Research Funding. Hermine:Celgene: Research Funding; Novartis: Research Funding; AB science: Consultancy, Equity Ownership, Honoraria, Research Funding. Amireault:Zimmer Biomet: Research Funding.

Alteration of the DNA Methylation Signature of Renal Erythropoietin-Producing Cells Governs the Sensitivity to Drugs Targeting the Hypoxia-Response Pathway in Kidney Disease Progression.

Chronic kidney disease (CKD) affects more than 10% of the population worldwide and burdens citizens with heavy medical expenses in many countries. Because a vital erythroid growth factor, erythropoietin (EPO), is secreted from renal interstitial fibroblasts [renal EPO-producing (REP) cells], anemia arises as a major complication of CKD. We determined that hypoxia-inducible factor 2α (HIF2α), which is inactivated by HIF-prolyl hydroxylase domain-containing proteins (PHDs) in an oxygen-dependent manner, tightly regulates EPO production in REP cells at the gene transcription level to maintain oxygen homeostasis. HIF2α-mediated disassembly of the nucleosome in the EPO gene is also involved in hypoxia-inducible EPO production. In renal anemia patients, anemic and pathological hypoxia is ineffective toward EPO induction due to the inappropriate over-activation of PHDs in REP cells transformed into myofibroblasts (MF-REP cells) due to kidney damage. Accordingly, PHD inhibitory compounds are being developed for the treatment of renal anemia. However, our studies have demonstrated that the promoter regions of the genes encoding EPO and HIF2α are highly methylated in MF-REP cells, and the expression of these genes is epigenetically silenced with CKD progression. This finding notably indicates that the efficacy of PHD inhibitors depends on the CKD stage of each patient. In addition, a strategy for harvesting renal cells, including REP cells from the urine of patients, is proposed to identify plausible biomarkers for CKD and to develop personalized precision medicine against CKD by a non-invasive strategy.

Induction of functional erythropoietin and erythropoietin receptor gene expression by gamma-aminobutyric acid and piperine in kidney epithelial cells

AimsThe aim of this study was to evaluate gamma-aminobutyric acid (GABA)- and piperine-induced erythropoietin (EPO) and EPO-receptor expression. Materials and methodsThe effect of GABA and piperine on cell viability was examined using kidney epithelial cells. Expression levels of EPO and EPO-R mRNA and protein were evaluated in response to GABA and piperine treatments. GABA- and piperine-mediated activation of the mitogen-activated protein kinase (MAPK) signaling pathway was investigated. Additionally, EPO function was evaluated using conditioned media containing EPO. The GABA receptor type involved in this process was identified. Key findingsMessenger RNA and protein expression levels of EPO and EPO-R significantly increased in response to treatment with GABA, piperine, or the combination of both, compared with control. GABA plus piperine synergistically enhanced EPO and EPO-R expression through p38 and c-Jun N-terminal kinase (JNK) MAPK signaling pathways, but not through the extracellular signal-regulated kinase (ERK) MAPK pathway. SB203580 and SP600125 (p38 and JNK pathway inhibitors, respectively) attenuated GABA plus piperine-induced EPO and EPO-R expression. Treatment of macrophages with EPO-containing conditioned media induced mRNA expression of interleukin (IL)-10 and nuclear factor (NF)-κB due to the interaction between EPO and EPO-R. Interestingly, GABA-induced EPO and EPO-R expression was mediated through GABAA, not GABAB, receptor activation. SignificanceThese findings demonstrate that GABA plus piperine-mediated p38 and JNK MAPK activation increases EPO and EPO-R expression, resulting in up-regulation of IL-10 and NF-κB.

PEGylated IL-11 (BBT-059): A Novel Radiation Countermeasure for Hematopoietic Acute Radiation Syndrome.

Interleukin-11 was developed to reduce chemotherapy-induced thrombocytopenia; however, its clinical use was limited by severe adverse effects in humans. PEGylated interleukin-11 (BBT-059), developed by Bolder Biotechnology, Inc., exhibited a longer half-life in rodents and induced longer-lasting increases in hematopoietic cells than interleukin-11. A single dose of 1.2 mg kg of BBT-059, administered subcutaneously to CD2F1 mice (12-14 wk, male) was found to be safe in a 14 d toxicity study. The drug demonstrated its efficacy both as a prophylactic countermeasure and a mitigator in CD2F1 mice exposed to Co gamma total-body irradiation. A single dose of 0.3 mg kg, administered either 24 h pre-, 4 h post-, or 24 h postirradiation increased the survival of mice to 70-100% from lethal doses of radiation. Preadministration (-24 h) of the drug conferred a significantly (p < 0.05) higher survival compared to 24 h post-total-body irradiation. There was significantly accelerated recovery from radiation-induced peripheral blood neutropenia and thrombocytopenia in animals pretreated with BBT-059. The drug also increased bone marrow cellularity and megakaryocytes and accelerated multilineage hematopoietic recovery. In addition, BBT-059 inhibited the induction of radiation-induced hematopoietic biomarkers, thrombopoietin, erythropoietin, and Flt-3 ligand. These results indicate that BBT-059 is a promising radiation countermeasure, demonstrating its potential to be used both pre- and postirradiation for hematopoietic acute radiation syndrome with a broad window for medical management in a radiological or nuclear event.

Prolyl-4-hydroxylase 2 and 3 coregulate murine erythropoietin in brain pericytes

AbstractA classic response to systemic hypoxia is the increased production of red blood cells due to hypoxia-inducible factor (HIF)-mediated induction of erythropoietin (EPO). EPO is a glycoprotein hormone that is essential for normal erythropoiesis and is predominantly synthesized by peritubular renal interstitial fibroblast-like cells, which express cellular markers characteristic of neuronal cells and pericytes. To investigate whether the ability to synthesize EPO is a general functional feature of pericytes, we used conditional gene targeting to examine the von Hippel-Lindau/prolyl-4-hydroxylase domain (PHD)/HIF axis in cell-expressing neural glial antigen 2, a known molecular marker of pericytes in multiple organs. We found that pericytes in the brain synthesized EPO in mice with genetic HIF activation and were capable of responding to systemic hypoxia with the induction of Epo. Using high-resolution multiplex in situ hybridization, we determined that brain pericytes represent an important cellular source of Epo in the hypoxic brain (up to 70% of all Epo-expressing cells). We furthermore determined that Epo transcription in brain pericytes was HIF-2 dependent and cocontrolled by PHD2 and PHD3, oxygen- and 2-oxoglutarate–dependent prolyl-4-hydroxylases that regulate HIF activity. In summary, our studies provide experimental evidence that pericytes in the brain have the ability to function as oxygen sensors and respond to hypoxia with EPO synthesis. Our findings furthermore suggest that the ability to synthesize EPO may represent a functional feature of pericytes in the brain and kidney.

Hematopoietic cell kinase (HCK) is a potential therapeutic target for dysplastic and leukemic cells due to integration of erythropoietin/PI3K pathway and regulation of erythropoiesis: HCK in erythropoietin/PI3K pathway

New drug development for neoplasm treatment is nowadays based on molecular targets that participate in the disease pathogenesis and tumor phenotype. Herein, we describe a new specific pharmacological hematopoietic cell kinase (HCK) inhibitor (iHCK-37) that was able to reduce PI3K/AKT and MAPK/ERK pathways activation after erythropoietin induction in cells with high HCK expression: iHCK-37 treatment increased leukemic cells death and, very importantly, did not affect normal hematopoietic stem cells. We also present evidence that HCK, one of Src kinase family (SFK) member, regulates early-stage erythroid cell differentiation by acting as an upstream target of a frequently deregulated pathway in hematologic neoplasms, PI3K/AKT and MAPK/ERK. Notably, HCK levels were highly increased in stem cells from patients with some diseases, as Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML), that are associated with ineffective erythropoiesis These discoveries support the exploration of the new pharmacological iHCK-37 in future preclinical and clinical studies.

5P - AMPKalpha1 restrains fibroblasts transformation and tumorigenesis in vivo

Adenosine monophosphate-activated protein kinase (AMPK) is a critical metabolic sensor and redox modulator. Despite evidence linking AMPK tumor suppressor functions, the role of AMPK in chromosome instability and tumorigenesis is obscure. Murine embryo fibroblasts (MEF) were isolated from AMPK alpha1 knock out (AMPK&agr;1-KO), AMPK&agr;2-KO, and wild type (WT) C57BL/6J mouse embryos, and immortalized by employing standard 3T3 protocol to investigate the mechanisms of chromosomal instability and fibroblast-mediated angiogenesis. Chromatin proteins and aneuploidy were monitored in cultured MEFs by Western blot and metaphase spread. Athymic nude mice were employed to test tumorigenesis of fibroblasts in vivo. Deletion of AMPK&agr;1, but not AMPK&agr;2, exhibited a significant reduction in centromere-specific binding proteins C (CENP-C) and elevation of Polo-like kinase 4 (PLK4), a trigger of centriole biogenesis. Both CENP-C and PLK4 may be associated with the increased aneuploid (34%-66%) and micronucleus. Allograft model data demonstrated that knock out of AMPK&agr;1 enhanced the cellular proliferation of immortalized MEFs, vascularization, and tumor development in athymic nude mice in vivo. Mechanistically, it was shown that the protein level of noncanonical nuclear factor kappa B2 (NF-&kgr;B2)/p52 was elevated in AMPK&agr;1-KO MEFs and was responsible for induction of erythropoietin (Epo) expression. Lastly, the most conclusive evidence for AMPK&agr;1-dependent inhibition of fibroblast-mediated angiogenesis as well as tumor progression was that the allograft growth of the inoculated AMPK&agr;1-KO MEFs was attenuated by treatment with Epo neutralization antibody. Taken together, these data indicate that AMPK&agr;1 activation opposes tumor development and its loss fosters tumor progression in part by controlling chromosome stability and angiogenesis that support tumor growth.