Epo Expression Research Articles

Study of Therapeutic Effects of Olive Leaf Extract on Phenylhydrazine-Induced Anemia Mice Model

IntroductionAnemia is the most common blood disorder which affects billions of people, especially young adults and women. However, currently available treatments of anemia are with limitations and adverse effects. Therefore, natural resources have been receiving considerable attention as complementary or alternative hematinic agents in recent years. In this regard, olive leaf extract (OLE) is rich in bioactive phenolic compounds and has been reported to have anti-inflammatory, antioxidant and neuroprotective effects (Vogel et al., 2015). Our previous study showed treatment with water extract of olive leaf (WOL) for 12 days could induce erythroid differentiation in human hematopoietic stem cells (hHSCs) and showed potential in oxygen and iron homeostasis, haem metabolism, and hemoglobin (Hb) biosynthesis (Kondo et al., 2021). In the present study, we aimed to investigate the therapeutic effects of WOL on the phenylhydrazine (PHZ)-induced mice model of anemia and explore its underlying molecular mechanism.MethodsTotal 63 male ICR mice (four weeks old) were randomly divided into three groups: control, PHZ and WOL groups. Mice in the WOL group were orally administered with 150 mg/kg body weight of WOL (diluted by saline) every day, while mice in the other two groups received saline. After two weeks of WOL pretreatment, PHZ was injected intraperitoneally (60 mg/kg body weight) to PHZ and WOL groups. The mice were dissected on day0 (before PHZ injection), day1, day3, day5, and day7 after PHZ injection. We collected blood for hematologic tests during dissection and liver, kidney, intestine, bone marrow, and spleen samples. We next checked the iron homeostasis-related gene expressions by real-time PCR: hepcidin (Hamp) in liver, ferroportin (Fpn) in spleen and intestine.Results and DiscussionAfter anemia was induced, WOL group showed a significant increase in the number of reticulocytes, the erythroid progenitors, compared to that of in PHZ group on Day 5. Simultaneously, plasma iron level on Day 5 and Hb level on Day7 was significantly decreased in the WOL group compared to the PHZ group. These results suggest that during anemia, the WOL group had rapid erythropoiesis, which in turn caused a rapid consumption of Hb and iron.Additionally, mRNA expression of Hamp in liver was significantly decreased on Day 5, whereas Fpn expressions in spleen and intestine were significantly increased on Day 5 and Day 7 in the WOL group compared to the PHZ group.Hamp regulates plasma iron concentrations as well as systemic iron metabolism by interacting with its receptor Fpn, a transmembrane iron-exporter. In intestine, Fpn controls iron absorption from food intake, while in spleen, Fpn regulates phagocytosis of senescent erythrocytes by the macrophages. Decreased Hamp expression and increased Fpn expression by WOL treatment suggest increased levels of absorbed and recycled iron to meet the demand for erythropoiesis. Altogether, our findings indicate that WOL could regulate iron homeostasis in the early stage of anemia to promote the differentiation of erythroid progenitors.ConclusionOur study suggests that WOL treatment downregulates Hamp expression in liver and increased Fpn expressions in the intestine and spleen, leading to increased absorbed iron in the intestine and recycled iron in the spleen (Figure 1). Therefore, WOL may have therapeutic potential in anemia through regulating iron homeostasis and promoting erythroid differentiation. However, further studies are required to confirm these findings and to elucidate the molecular mechanisms.Our previous study showed that 12-day treatment of hHSCs with WOL could upregulate the expression of hypoxia-inducible factor 1-a (HIF1A) (Kondo et al., 2021), a key modulator of the transcriptional response to hypoxia and has been reported to regulate erythropoietin (Epo) production, which plays an important role in erythropoiesis. So, we hypothesize that WOL treatment would activate HIF1A in anemia-induced mice model, thus regulating Epo production to accelerate erythroid maturation. HIF2A is also reported to regulate Epo in kidney and Fpn in intestine to regulate erythropoiesis (Andrew J., et al., 2019).Therefore, we will investigate Epo expression in kidney, Hif1a and Hif2a expressions in kidney and intestine, and transferrin receptor (Tfrc, erythroid marker) in bone marrow. All gene expressions will be confirmed in protein levels also. [Display omitted] DisclosuresSuidasari: Nutrition Act Co. Ltd.: Current Employment. Yokozawa: Nutrition Act Co. Ltd.: Current Employment. Yamauchi: Nutrition Act Co. Ltd.: Current Employment.

Transforming growth factor-\u03b21 decreases erythropoietin production through repressing hypoxia-inducible factor 2\u03b1 in erythropoietin-producing cells

BackgroundRenal erythropoietin (EPO)-producing (REP) cells produce EPO through hypoxia-inducible factor (HIF) 2α-activated gene transcription. Insufficient EPO production leads to anemia in patients with chronic kidney disease. Although recombinant EPO is effective to improve anemia, no reliable REP cell lines limit further progress of research and development of novel treatment.MethodsWe screened Epo mRNA expression in mouse fibroblast cell lines. Small interfering RNA specific for HIF1α or HIF2α was transfected to study Epo expression in C3H10T1/2 cells. The effect of transforming growth factor-β1 (TGF-β1) on HIF-EPO axis was studied in C3H10T1/2 cells and mice.ResultsSimilar to mouse REP pericytes, C3H10T1/2 cells differentiated to α-smooth muscle actin+ myofibroblasts after exposure to TGF-β1. Specific HIF knockdown demonstrated the role of HIF2α in hypoxia-induced Epo expression. Sustained TGF-β1 exposure increased neither DNA methyltransferase nor methylation of Epas1 and Epo genes. However, TGF-β1 repressed HIF2α-encoding Epas1 promptly through activating activin receptor-like kinase-5 (ALK5), thereby decreasing Epo induction by hypoxia and prolyl hydroxylase domain inhibitor roxadustat. In mice with pro-fibrotic injury induced by ureteral obstruction, upregulation of Tgfb1 was accompanied with downregulation of Epas1 and Epo in injured kidneys and myofibroblasts, which were reversed by ALK5 inhibitor SB431542.ConclusionC3H10T1/2 cells possessed the property of HIF2α-dependent Epo expression in REP pericytes. TGF-β1 induced not only the transition to myofibroblasts but also a repressive effect on Epas1-Epo axis in C3H10T1/2 cells. The repressive effect of TGF-β1 on Epas1-Epo axis was confirmed in REP pericytes in vivo. Inhibition of TGF-β1-ALK5 signaling might provide a novel treatment to rescue EPO expression in REP pericytes of injured kidney.

Expression and significance of EPO and HIF-1α in serum and aqueous humor of patients with acute anterior uveitis

Expression and significance of EPO and HIF-1α in serum and aqueous humor of patients with acute anterior uveitis

Effects of Angiotensin II on Erythropoietin Production in the Kidney and Liver.

The kidney is a main site of erythropoietin production in the body. We developed a new method for the detection of Epo protein by deglycosylation-coupled Western blotting. Detection of deglycosylated Epo enables the examination of small changes in Epo production. Using this method, we investigated the effects of angiotensin II (ATII) on Epo production in the kidney. ATII stimulated the plasma Epo concentration; Epo, HIF2α, and PHD2 mRNA expression in nephron segments in the renal cortex and outer medulla; and Epo protein expression in the renal cortex. In situ hybridization and immunohistochemistry revealed that ATII stimulates Epo mRNA and protein expression not only in proximal tubules but also in collecting ducts, especially in intercalated cells. These data support the regulation of Epo production in the kidney by the renin–angiotensin–aldosterone system (RAS).

S1P Stimulates Erythropoietin Production in Mouse Renal Interstitial Fibroblasts by S1P1 and S1P3 Receptor Activation and HIF-2α Stabilization.

Erythropoietin (Epo) is the critical hormone for erythropoiesis. In adults, Epo is mainly produced by a subset of interstitial fibroblasts in the kidney, with minor amounts being produced in the liver and the brain. In this study, we used the immortalized renal interstitial fibroblast cell line FAIK F3-5 to investigate the ability of the bioactive sphingolipid sphingosine 1-phosphate (S1P) to stimulate Epo production and to reveal the mechanism involved. Stimulation of cells with exogenous S1P under normoxic conditions (21% O2) led to a dose-dependent increase in Epo mRNA and protein levels and subsequent release of Epo into the medium. S1P also enhanced the stabilization of HIF-2α, a key transcription factor for Epo expression. S1P-stimulated Epo mRNA and protein expression was abolished by HIF-2α mRNA knockdown or by the HIF-2 inhibitor compound 2. Furthermore, the approved S1P receptor modulator FTY720, and its active form FTY720-phosphate, both exerted a similar effect on Epo expression as S1P. The effect of S1P on Epo was antagonized by the selective S1P1 and S1P3 antagonists NIBR-0213 and TY-52156, but not by the S1P2 antagonist JTE-013. Moreover, inhibitors of the classical MAPK/ERK, the p38-MAPK, and inhibitors of protein kinase (PK) C and D all blocked the effect of S1P on Epo expression. Finally, the S1P and FTY720 effects were recapitulated in the Epo-producing human neuroblastoma cell line Kelly, suggesting that S1P receptor-dependent Epo synthesis is of general relevance and not species-specific. In summary, these data suggest that, in renal interstitial fibroblasts, which are the primary source of plasma Epo, S1P1 and 3 receptor activation upregulates Epo under normoxic conditions. This may have a therapeutic impact on disease situations such as chronic kidney disease, where Epo production is impaired, causing anemia, but it may also have therapeutic value as Epo can mediate additional tissue-protective effects in various organs.

Potential effects of angiogenesis-related factors on the severity of APAC and surgical outcomes of trabeculectomy

BackgroundEPO (erythropoietin) and PDGF (platelet derived growth factor) families are thought to be associated with angiogenesis under hypoxic condition. The sharp rise of intraocular pressure in acute primary angle closure (APAC) results in an inefficient supply of oxygen and nutrients. We aimed to measure the expression of EPO and PDGF family members in APAC eyes and demonstrate their associations with APAC’s surgical success rate.MethodsConcentrations of EPO, PDGF-AA, -BB, -CC and -DD collected in aqueous humor samples of 55 patients recruited were measured. Before operations, correlations between target proteins and IOP (intraocular pressure) were detected between APAC (acute primary angle closure) and cataract patients. Based on the post-operative follow-up, the effects of EPO and PDGF family members on the successful rate of trabeculectomy were tested.ResultsThe levels of EPO, PDGF-CC and -DD were significantly elevated in the APAC group compared to the cataract group. During the post-operative follow-up, EPO, PDGF-CC and -DD showed significant differences between the success and failure groups. In multivariable linear regression analyses, failed filtration surgery was more likely in APAC eyes with higher EPO level. The Kaplan-Meier survival plot suggested that the success rate in eyes with low EPO level was significantly higher than that in eyes with high EPO level.ConclusionThe levels of EPO, PDGF-CC and -DD were significantly elevated in failure group. EPO level correlated with preoperative IOP and numbers of eyedrops, and higher EPO level in aqueous humor is a risk factor for trabeculectomy failure. It can be a biomarker to estimate the severity of APAC and the success rate of surgery. The investigation of mechanism of EPO in APAC a may have potential clinical applications for the surgical treatment of APAC.

HBXIP accelerates glycolysis and promotes cancer angiogenesis via AKT/mTOR pathway in bladder cancer

Abnormal metabolism and uncontrolled angiogenesis are two important characteristics of malignant tumors. Although HBXIP is known to be associated with a poor prognosis for bladder cancer (BC), its effects on glycolysis and angiogenesis in BC have not been investigated. BC prognosis and relative gene expression of HBXIP were analyzed using the GEPIA, UALCAN, and STRING databases. BC cell angiogenesis and glycolysis were assessed by vasculogenic mimicry and glycolysis assay. Human umbilical vein endothelial cell (HUVEC) viability, migration, and angiogenesis were assessed by CCK8, transwell, wound healing, and tube formation assays. The results showed that HBXIP was highly expressed in BC tissues and cells. Knockdown of HBXIP expression decreased the levels of glucose uptake, lactate production, and glycolytic enzyme expression in BC cells, and decreased cell viability and migration of HUVECs. Additionally, silencing HBXIP reduced the total length of tubes and number of intersections, and EPO and VEGF protein expression in BC cells and HUVECs. Furthermore, knockdown of HBXIP expression reversed cell viability, migration, tube formation, and vasculogenic mimicry under high glucose and lactate conditions. Mechanistically, silencing of HBXIP reduced the protein expression levels of pAKT-ser473 and pmTOR, and inhibition of HBXIP, AKT, and mTOR expression decreased glycolytic enzyme protein expression. Our findings suggest that HBXIP reduces glycolysis in BC cells via regulation of AKT/mTOR signaling, thereby blocking BC angiogenesis. Collectively, this study provides a potential strategy to target HBXIP and AKT/mTOR for regulating glycolysis progression concurrently with anti-angiogenesis effects, and thereby develop novel therapeutics for the treatment of BC.

Alteration of L-Dopa decarboxylase expression in SARS-CoV-2 infection and its association with the interferon-inducible ACE2 isoform.

L-Dopa decarboxylase (DDC) is the most significantly co-expressed gene with ACE2, which encodes for the SARS-CoV-2 receptor angiotensin-converting enzyme 2 and the interferon-inducible truncated isoform dACE2. Our group previously showed the importance of DDC in viral infections. We hereby aimed to investigate DDC expression in COVID-19 patients and cultured SARS-CoV-2-infected cells, also in association with ACE2 and dACE2. We concurrently evaluated the expression of the viral infection- and interferon-stimulated gene ISG56 and the immune-modulatory, hypoxia-regulated gene EPO. Viral load and mRNA levels of DDC, ACE2, dACE2, ISG56 and EPO were quantified by RT-qPCR in nasopharyngeal swab samples from COVID-19 patients, showing no or mild symptoms, and from non-infected individuals. Samples from influenza-infected patients were analyzed in comparison. SARS-CoV-2-mediated effects in host gene expression were validated in cultured virus-permissive epithelial cells. We found substantially higher gene expression of DDC in COVID-19 patients (7.6-fold; p = 1.2e-13) but not in influenza-infected ones, compared to non-infected subjects. dACE2 was more elevated (2.9-fold; p = 1.02e-16) than ACE2 (1.7-fold; p = 0.0005) in SARS-CoV-2-infected individuals. ISG56 (2.5-fold; p = 3.01e-6) and EPO (2.6-fold; p = 2.1e-13) were also increased. Detected differences were not attributed to enrichment of specific cell populations in nasopharyngeal tissue. While SARS-CoV-2 virus load was positively associated with ACE2 expression (r≥0.8, p<0.001), it negatively correlated with DDC, dACE2 (r≤−0.7, p<0.001) and EPO (r≤−0.5, p<0.05). Moreover, a statistically significant correlation between DDC and dACE2 expression was observed in nasopharyngeal swab and whole blood samples of both COVID-19 and non-infected individuals (r≥0.7). In VeroE6 cells, SARS-CoV-2 negatively affected DDC, ACE2, dACE2 and EPO mRNA levels, and induced cell death, while ISG56 was enhanced at early hours post-infection. Thus, the regulation of DDC, dACE2 and EPO expression in the SARS-CoV-2-infected nasopharyngeal tissue is possibly related with an orchestrated antiviral response of the infected host as the virus suppresses these genes to favor its propagation.

Monotcytes/Macrophages Are Involved in Erythrocytosis Caused By Both EPO-Dependent and -Independent Activation of EPOR

Excess activation of EPO receptor (EPOR) signaling leads to erythrocytosis, which is defined as an absolute increase of red cell mass. EPO-independent and erythrocytes-intrinsic EPOR activation is induced by gain-of-function EPOR or JAK2 mutations, resulting in “primary” erythrocytosis. Erythrocytes-extrinsic EPOR activation is called “secondary” erythrocytosis, which is caused by EPOR stimulation with high level of EPO.In order to investigate primary and secondary erythrocytosis in detail, we generated two unique mouse models. LysM -Cre; Jak2 V617F knock-in heterozygous mice express constitutively active JAK2 in LysM-expressing cells, which include a small portion of HSCs and their downstream progenitors. We also generated a Alb -Cre; Rosa26 LoxP-Stop-LoxP-rtTA (LSL-rtTA);human EPAS1 double-point-mutant (DPM) mice (Alb -Cre;DPM). EPAS1 is a master regulator of EPO gene. The EPAS1-DPM has mutations in P531 and N847 sites, which are critical sites for stability and transcriptional inactivation in normoxia. This mouse expresses the DPM mutant (P531A and N847A) specifically in hepatocytes in a doxycycline-inducible manner, resulting in high EPO production in the liver. We used LysM -Cre; Jak2 V617F as an erythrocytes-intrinsic “primary” erythrocytosis model, and Alb -Cre;DPM as an erythrocytes-extrinsic “secondary” erythrocytosis model. Interestingly, both of them developed comparable degree of erythrocytosis quickly. The plasma EPO level in LysM -Cre; Jak2 V617F was significantly decreased, while that in Alb -Cre/DPM was significantly increased in comparison to the control and also to LysM -Cre; Jak2 V617F. They both exhibited splenomegaly, but spleens of Lys M-Cre; Jak2 V617F mice were significantly larger than those of Alb -Cre;DPM mice. In Alb -Cre;DPM mice, CD45- CD71+ erythroblasts were detected in the liver, whereas there were almost no erythroblasts in the liver of the control mice, suggesting that extremely high level of EPO expression in the liver causes erythropoiesis in the liver of Alb -Cre;DPM mice.Increased monocyte count has been reported in patients with erythrocytosis. Consistent with the observation in the patients, total CD11b+ CD115+ monocytes were significantly increased in PB from both LysM -Cre; Jak2 V617F and Alb -Cre;DPM mice compared to those in PB from the wild-type mice. It is also well known that macrophages play a crucial role in erythropoiesis, supplying the iron and engulfing the protruded nuclei from erythroblasts in the terminal maturation process. The complex of erythroblasts and macrophages directly interacting each other is called as “erythroblastic island (EBI)”. Thus, we next analyzed macrophages in bone marrow (BM) and spleen (SP) in our erythrocytosis models. Macrophages in SP were dramatically increased in the both models, while those in BM were not significantly altered. The percentage of EBI (multiplet F4/80+ Ter119+) in BM and SP were increased in both models.EPOR expression on the cell surface is required for EPO-EPOR signaling. To confirm the expression of EPOR by genetic approach, we combined EPOR -Cre knock-in allele and Rosa26LSL-GFP reporter allele to generate EPOR -Cre;GFP reporter mice. In this model, pro-erythroblasts and basophilic erythroblasts in BM expressed GFP as expected. We did not find GFP expression on the Macrophage and dendritic cell precursors (MDPs), common monocyte progenitors (cMoPs), mature monocytes in BM and PB monocytes. In a single-cell-gated macrophage population, we found a small fraction expressing GFP. However, FACS sorting revealed that those GFP+ cells are not macrophages alone, but macrophages with erythroblasts adhered to them. Even after adding EDTA, some of those macrophages still retain bound erythroblasts. It is possible that they could acquire activated EPOR signaling from erythroblasts within EBI through direct interaction. We are currently investigating the mechanisms for expansion of monocytes/macrophages in erythrocytosis.Collectively, here we demonstrate that both Jak2V617F and high EPO could activate EPOR signaling and expand erythroblasts and monocytes/macrophages, which might contribute to erythrocytosis phenotype in our primary and secondary erythrocytosis models. Further exploration of the roles of monocytes/macrophages in erythrocytosis could lead to development of novel therapeutic strategy for erythrocytosis by modulating their functions in EBI. DisclosuresNo relevant conflicts of interest to declare.

Placental EPO mRNA Expression Is Measurable in Very Preterm to Term Placentae

ObjectivesOur objective was to evaluate placental EPO mRNA expression in term and preterm placentae and explore maternal/neonatal/placental determinants of EPO expression. MethodsPlacental tissue was collected from pregnant adolescents (n = 82, < 19 y of age) and from women (n = 62) carrying multiple fetuses (twins = 23, triplets = 39). Placental mRNA expression was evaluated using qPCR. Hemoglobin and iron status biomarkers (SF, TBI, sTfR, Hepcidin) were measured in maternal blood at delivery and in cord blood. ResultsIn this population, women delivered between 30–42 weeks of gestation. Of the 144 women, 30% delivered prematurely (< 37 weeks, n = 44) and 69% delivered at term (≥37 weeks, n = 100). As expected, preterm placentae weighed significantly less than term placenta (293 ± 93 g vs. 544 ± 143 g, p < 0.0001). Placental EPO mRNA expression was detected in all samples collected. Placental EPO mRNA expression did not significantly differ between anemic (Hb < 13 g/dL) (n = 23) and non-anemic (n = 84) neonates (p = 0.69), nor was it significantly associated with maternal anemia at delivery (p = 0.44). Placental EPO mRNA expression was also not significantly associated with maternal (p = 0.56) or neonatal (p = 0.61) Hb concentrations, placental weight (p = 0.46) or gestational age at delivery (p = 0.39). ConclusionsWhile placental EPO mRNA was detected in all placental tissue collected between 30–42 weeks of gestation none of the iron biomarkers evaluated were significantly related to placental EPO mRNA expression. Further studies are needed to understand determinants of placental EPO mRNA and protein expression. Funding SourcesThe U.S. National Institutes of Health (NIH) National Institute of Child Health and Development (NICHD) Grant 1R21HD098864, The Gerber Foundation and USDA grants 2005–35,200-15,218 and 2009–35,200-05,171.

New insights into erythropoietin and erythropoietin receptor in laryngeal cancer tissue.

To investigate whether laryngeal cancer cells express erythropoietin (Epo) and erythropoietin receptor (EpoR) and what is their possible relationship with clinical and pathological features of the tumor.We performed immunohistochemical analysis of Epo and EpoR expression on 78 tissue samples of invasive and in situ squamous cell laryngeal carcinoma.The statistical analysis showed a weak positive and statistically significant correlation of EpoHS and EpoR HS expression levels. Epo HS and EpoR HS levels did not correlate with patient sex or age, type of diagnosis, cancer stage, histological tumor grade, presence or absence of disease recurrence, type of oncologic cancer therapy provided, or results of selected laboratory blood work. The results show a statistically significant difference in Epo expression with respect to survival.We confirmed the presence of Epo an EpoR in malignant laryngeal tumors and demonstrated the correlation between Epo expression and survival. Further studies are needed to more precisely define the role of Epo and EpoR in treatment of patients with laryngeal cancer.

Recombinant Human Growth Hormone Activates Neuroprotective Growth Factors in Hypoxic Brain Injury in Neonatal Mice.

Perinatal hypoxia severely disrupts cerebral metabolic and maturational programs beyond apoptotic cell death. Antiapoptotic treatments such as erythropoietin are suggested to improve outcomes in hypoxic brain injury; however, the results are controversial. We analyzed the neuroprotective effects of recombinant human growth hormone (rhGH) on regenerative mechanisms in the hypoxic developing mouse brain in comparison to controls. Using an established model of neonatal acute hypoxia (8% O2, 6 hours), P7 mice were treated intraperitoneally with rhGH (4000 µg/kg) 0, 12, and 24 hours after hypoxic exposure. After a regeneration period of 48 hours, expression of hypoxia-inducible neurotrophic factors (erythropoietin [EPO], vascular endothelial growth factor A [VEGF-A], insulin-like growth factors 1 and 2 [IGF-1/-2], IGF binding proteins) and proinflammatory markers was analyzed. In vitro experiments were performed using primary mouse cortical neurons (E14, DIV6). rhGH increased neuronal gene expression of EPO, IGF-1, and VEGF (P < .05) in vitro and diminished apoptosis of hypoxic neurons in a dose-dependent manner. In the developing brain, rhGH treatment led to a notable reduction of apoptosis in the subventricular zone and hippocampus (P < .05), abolished hypoxia-induced downregulation of IGF-1/IGF-2 expression (P < .05), and led to a significant accumulation of endogenous EPO protein and anti-inflammatory effects through modulation of interleukin-1β and tumor necrosis factor α signaling as well as upregulation of cerebral phosphorylated extracellularly regulated kinase 1/2 levels (ERK1/2). Indicating stabilizing effects on the blood-brain barrier (BBB), rhGH significantly modified cerebrovascular occludin expression. Thus, we conclude that rhGH mediates neuroprotective effects by the activation of endogenous neurotrophic growth factors and BBB stabilization. In addition, the modification of ERK1/2 pathways is involved in neuroprotective actions of rhGH. The present study adds further evidence that pharmacologic activation of neurotrophic growth factors may be a promising target for neonatal neuroprotection.

Regulated Basal, Inducible, and Tissue-Specific Human Erythropoietin Gene Expression in Transgenic Mice Requires Multiple cis DNA Sequences

Erythropoietin (Epo) gene expression in kidney and liver is inducible by anemia. To localize the sequences necessary for regulated expression of the Epo gene, we constructed transgenic mice containing five human Epo gene constructs and examined Epo expression under basal conditions and with anemia. Mice containing the Epo gene with 0.3 kb of 5' flanking sequence, 0.7 kb of 3' flanking sequence, and either all introns or only intron I alone were polycythemic, had Epo expression in various tissues (including non-Epo-producing tissues), and induction only in liver. In contrast, mice containing the Epo gene with 8.5 kb of 3' flanking sequence and either 9.5 or 22 kb of 5' flanking sequence had basal expression at low levels in appropriate tissues and were less likely to be markedly polycythemic. Mice with the smaller of these two constructs had induction only in the liver, whereas those with the larger construct had induction in the kidney and liver. These studies indicate that sequences sufficient for induction in the liver are located in close proximity to the Epo gene, including the immediate 5' and 3' flanking sequence and the first intron. They also indicate that sequences required for induction in the kidney are located more than 9.5 kb 5' to the gene. Furthermore, comparison of these and prior transgenic studies suggest that sequences that limit the basal expression of the Epo gene are located downstream of the gene. We conclude that multiple cis DNA sequences are required for regulated Epo gene expression.

VHL gene methylation contributes to excessive erythrocytosis in chronic mountain sickness rat model by upregulating the HIF-2α/EPO pathway

AimsHypoxia-inducible factors (HIFs) play important roles in the pathogenesis of erythrocytosis in chronic mountain sickness (CMS). von Hippel-Lindau (VHL) is a key regulator of hypoxia that can direct the poly-ubiquitylation and degradation of HIFs. Epigenetic mechanisms are believed to contribute toward adaption to chronic hypoxia. Here, we investigated the contribution and mechanism of VHL methylation in rats with erythrocytosis in CMS. Main methodsThe methylation status of VHL was measured via bisulfite sequencing PCR, while VHL, DNMT1, DNMT3α, and DNMT3β expression were assessed using real-time reverse transcription PCR and western blotting. HIF-2α and EPO expression levels in bone marrow were determined via immunohistochemical staining, and erythroid hyperplasia in bone marrow sections were observed with hematoxylin and eosin staining. Key findingsWe found that chronic hypoxia triggered erythroid hyperplasia in the bone marrow and increased the quantity of peripheral red blood cells in CMS rats. Chronic hypoxia significantly induced methylation at the CpG site in the VHL promoter, decreased VHL expression, and increased HIF-2α and EPO expression. Chronic hypoxia increased DNMT3α and DNMT3β expression, consistent with the decrease in VHL expression. The DNA methyltransferase inhibitor 5-azacytidine reduced chronic hypoxia-induced erythroid proliferation in the bone marrow of rats with CMS by suppressing VHL methylation and DNMTs expression. SignificanceOur study suggests that VHL methylation contributes toward excessive erythrocytosis in CMS by upregulating the HIF-2α/EPO pathway in the bone marrow of rats. We demonstrated that the DNMT inhibitor 5-azacytidine can attenuate erythroid hyperplasia in the bone marrow by demethylating the VHL promoter.

Changes in histidine decarboxylase expression influence extramedullary hematopoiesis in postnatal mice.

Histidine decarboxylase (HDC), histamine synthase, is expressed in hematopoietic stem cells and in lineage-committed progenitors in the bone marrow (BM). However, the role of histamine in hematopoiesis is not well described. To evaluate the role of histamine in hematopoiesis, we analyzed the changes in HDC expression at hematopoietic sites, the BM, spleen, and liver of 2-, 3-, and 6-week-old wild-type mice. We also performed morphological analyses of the hematopoietic sites using HDC-deficient (HDC-KO) mice. In wild-type adults, HDC expression in the BM was higher than that in the spleen and liver and showed an age-dependent increase. Histological analysis showed no significant change in the adult BM and spleen of HDC-KO mice compared to wild-type mice. In the liver, HDC expression was temporarily increased at 3 weeks and decreased at 6 weeks of age. Morphological analysis of the liver revealed more numerous hematopoietic colonies and megakaryocytes in HDC-KO mice compared to wild-type mice at 2 and 3 weeks of age, whereas no changes were observed in adults. Most of these hematopoietic colonies consisted of B220-positive B-lymphocytes and TER119-positive erythroblasts and were positive for the cell proliferation marker PCNA. Notably, these hematopoietic colonies declined in HDC-KO mice upon N-acetyl histamine treatment. A significant increase in the expression of hematopoiesis-related cytokines, Il3, Il7, Epo, Gcsf, and Cxcl12 mRNA was observed in the liver of 3-week-old HDC-KO mice compared to wild-type mice. These results suggest that histamine-deficiency may maintain an microenvironment suitable for hematopoiesis by regulating hematopoiesis-related cytokine expression in the liver of postnatal mice.

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.

Exercise Heat Exposure Induced Changes In Genetic Expression Before And After Heat Acclimation In Humans

PURPOSE: To evaluate changes in genetic expression of proteins that are implicated in physiological adaptation to exercise heat exposure (Hypoxia Inducible Factor -1α [HIF-1α], Erythropoietin [EPO], and Vascular Endothelial Growth Factor [VEGF]) during systemic adaptation related to Heat Acclimation (HA) in humans. METHODS: Participants (n=18, 13 males, 5 females: 30 ± 7.34 y; 173.1 ± 7.673 cm; 76.25 ±14.17 kg) underwent two Heat Tolerance Tests (HTT 1 & 2; 120 min walking, 5 km/h, 2.0% grade, 40 °C, 40% rh) with 12-14 days between tests. Participants in experimental group (HA) underwent six heat acclimation days between tests, while the control group (CON) avoided exercise heat exposure between HTT’s. Blood was drawn pre- and post- each HTT and isolated into Peripheral Blood Mononuclear Cells, then further isolated into RNA. Then, cDNA was synthesized for Quantitative Real-Time Polymerase Chain Reaction to quantify gene expression. The data was then analyzed using the ΔΔCT to acquire fold change. RESULTS: No statistical difference in resting rectal temperature was observed before HTT2 in HA (-0.13±0.36 °C) vs. CON (-.08±0.47 °C; P=0.80). No significant changes were observed between HTT1 and HTT2 in the control group for HIF-1α, EPO, or VEGF. However, it was observed that acute EPO fold change increased 10.6x; (P=0.14) from pre- to post- in HTT1 for each group. An increased fold change was also observed in both HIF-1α and EPO between HTT1 and HTT2 (3.24x; P=0.04) & (7.19x; P=0.03) respectively in the HA group. No significant changes in VEGF were found in either group. CONCLUSION: Although the HA protocol utilized in this investigations was not stressful enough to induce observable rectal temperature changes, genetic expression of HIF-1α and EPO was significantly upregulated in response to chronic exercise heat stress. Grant Info: This publication was supported by the Mountain and Plains - Educational Research Center (MAP-ERC) Pilot Grant & the High Plains Intermountain Center for Agricultural Health & Safety (HICAHS) Pilot Grant.

Effects of hypoxia environment on osteonecrosis of the femoral head in Sprague-Dawley rats.

Osteonecrosis of the femoral head (ONFH) is a disease in which the blood supply of the femoral head is interrupted or damaged, resulting in joint dysfunction. Hypoxic environments increase the expression of EPO, VEGF, and HIF causes vascular proliferation and increases the blood supply. It also causes the organism to be in a state of hypercoagulability and increases thrombosis. Therefore, the purpose of this study was to explore the occurrence of ONFH after the use of glucocorticoids (GCs) under conditions of hypoxia tolerance for a long time. Sprague-Dawley rats were fed in a hypobaric hypoxic chamber at an altitude of 4000 m, the whole blood viscosity, and plasma viscosity were determined to analyze the blood flow and hemagglutination. Western blotting, polymerase chain reaction, and immunohistochemistry were used to detect EPO, VEGF, CD31, and osteogenesis related proteins. Femoral head angiography was used to examine the local blood supply and micro-CT scanning was used to detect the structure of the bone trabecula. Under hypoxic environments, the expression of EPO and VEGF increased, which increased the local blood supply of the femoral head, but due to more severe thrombosis, the local blood supply of the femoral head decreased. Hypoxic environments can aggravate ONFH in SD rats; this aggravation may be related to the hypercoagulable state of the blood. We suggest that long-term hypoxia should be regarded as one of the risk factors of ONFH and we need to conduct a more extensive epidemiological investigation on the occurrence of ONFH in hypoxic populations.

Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation.

The detection of erythropoietin (Epo) protein by Western blotting has required pre‐purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis‐stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG‐bound epoetin β pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O2, 4 hr) caused a 400‐fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy.

Brain-specific Wt1 deletion leads to depressive-like behaviors in mice via the recruitment of Tet2 to modulate Epo expression.

Major depressive disorder (MDD) is the most common psychiatric disease worldwide. The precise molecular and cellular mechanisms underlying this disorder remain largely unknown. Wilms' tumor 1 (Wt1), a transcription factor, plays critical roles in cancer and organ development. Importantly, deletion of the 11p13 region that contains the WT1 gene is a major cause of WARG syndrome (Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation), which is characterized by psychiatric disease, including depression. However, the roles and mechanisms of WT1 in embryonic neurogenesis and psychiatric disease remain unclear. Here, we demonstrate that the brain-specific deletion of Wt1 results in abnormal cell distribution during embryonic neurogenesis, which is accompanied by enhanced proliferation of neural progenitors and reduced neuronal differentiation. Moreover, neurons exhibit abnormal morphology during cortical development following Wt1 ablation. Furthermore, Wt1cKO mice exhibit depressive-like behaviors, including immobility, despair, and anhedonia. Mechanistically, Wt1 recruits Tet2 to the promoter of erythropoietin (Epo), which results in enhanced 5-hydroxymethylcytosine (5hmC) levels and the promotion of Epo expression. Either Epo plasmid electroporation or Epo protein injection can partially restore the deficiency caused by Wt1 deletion. Importantly, administration of Epo to both embryos and adults can ameliorate the depressive-like behavior of Wt1cKO mice. In addition, WT1 plays a similar role in human neural progenitor cells (hNPCs) proliferation and differentiation. Taken together, our findings reveal the critical role and regulatory mechanism of Wt1 in embryonic neurogenesis and behavioral modulation, which could contribute to the understanding of MDD etiology and therapy.