Erythroferrone Modulates Iron Distribution for Fetal Erythropoiesis

Erythroferrone (ERFE) is an erythroblast-derived regulator of iron metabolism, and its production increases during stress erythropoiesis. ERFE decreases expression of the iron-regulatory hormone hepcidin to enhance iron availability for erythropoiesis 1. Pregnancy requires a substantial increase in iron availability to sustain a dramatic increase in maternal RBC volume and support fetal development. Whether maternal or fetal ERFE plays a role in regulating iron homeostasis during pregnancy is unknown. In humans, maternal ERFE concentrations were elevated in anemic pregnancies at mid gestation and delivery 2. To define the role of ERFE during iron-replete or iron-deficient pregnancy, we utilized Erfe transgenic (ETg) 3 and Erfe knockout (EKO) 1 mice. Maternal iron status of ETg, WT and EKO mice was altered by placing animals on adequate iron (100ppm) or low iron (4ppm) diet 2 weeks prior to and throughout pregnancy. ETg and WT dams were mated with WT sires to generate ETg and WT embryos while EKO dams were mated with EKO sires to generate EKO embryos. Analysis was performed at embryonic day 18.5. To examine the effect of pregnancy on ERFE expression, we compared non-pregnant females to WT dams at E18.5. Serum ERFE was mildly elevated from 0.01 to 0.2 ng/mL in iron-replete dams, but substantially elevated from 0.01 to 3.1 ng/mL in iron-deficient dams, similarly to human pregnancy 2. We next assessed iron and hematological parameters in pregnant dams with different Erfe genotypes. Under iron-replete conditions, all three groups had similar serum hepcidin, serum iron and hemoglobin concentrations, but ETg dams had 3-fold higher liver iron than WT and EKO dams, presumably because they are mildly iron-overloaded before pregnancy. On iron-deficient diet, maternal hepcidin was decreased in all three genotypes but more so in ETg dams; however, all three Erfe genotypes had similarly depleted liver iron stores, hypoferremia and anemia. MCV was the only parameter that was decreased in EKO compared to WT dams under both iron conditions. Overall, maternal ERFE played a minor role in regulation of maternal erythropoiesis and iron homeostasis, with the lack of ERFE resulting in smaller RBCs but not anemia. Among embryos, we observed a significant effect of Erfe genotype on embryo hepcidin. ETg embryos had significantly lower liver hepcidin compared to WT embryos under both iron-replete and iron-deficient conditions. Conversely, Erfe KO embryos had higher hepcidin compared to WTs under iron-deficient conditions, indicating that embryo ERFE regulates embryo hepcidin during pregnancy. Under iron-replete conditions however, all three embryo genotypes had similar hematologic parameters, and embryo liver iron was dependent on maternal iron levels, with both ETg and WT embryos from ETg dams having increased liver iron concentrations, indicating that embryo ERFE does not regulate placental iron transfer. Under iron-deficient conditions, there was no difference between ETg and WT embryos in hematological or iron parameters, and both genotypes developed iron deficiency and anemia. However, Erfe KO embryos, which had elevated hepcidin, had maldistribution of iron and worse anemia. EKO embryo liver iron concentrations were 6-fold higher compared to WT iron-deficient embryos, whereas hemoglobin was significantly decreased compared to WT iron-deficient embryos. These findings indicate that under iron-limiting conditions, embryo ERFE is important for the suppression of embryo hepcidin to ensure iron redistribution for embryo erythropoiesis. In summary, during iron replete pregnancy, ERFE plays a minor role in maternal and fetal iron homeostasis and erythropoiesis. However, in response to iron-deficiency anemia during pregnancy, ERFE is important for the redistribution of iron within the embryo to support embryo erythropoiesis. 1Kautz L et al, Nat Genet, 2014 2Delaney K et al, Curr Dev Nutr, 2020 3Coffey R et al, Blood, 2020 Disclosures Ganz: Ambys: Consultancy; Sierra Oncology: Consultancy, Research Funding; Rockwell: Consultancy; Pharmacosmos: Consultancy; Ionis: Consultancy; Protagonist: Consultancy; Intrinsic LifeSciences: Consultancy; RallyBio: Consultancy; Silence Therapeutics: Consultancy; Silarus Pharma: Consultancy; Alnylam: Consultancy; American Regent: Consultancy; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZenecaFibrogen: Consultancy; Global Blood Therapeutics: Consultancy; Gossamer Bio: Consultancy; Akebia: Consultancy, Honoraria. Nemeth: Silarus Pharma: Consultancy; Intrinsic LifeSciences: Consultancy; Protagonist: Consultancy; Vifor: Consultancy; Ionis: Consultancy.

ROS-Triggered Autophagy Is Involved in PFOS-Induced Apoptosis of Human Embryo Liver L-02 Cells

The liver is the primary target organ for perfluorooctane sulphonate (PFOS), a recently discovered persistent organic pollutant. However, the mechanisms mediating hepatotoxicity remain unclear. Herein, we explored the relationship between reactive oxygen species (ROS) and autophagy and apoptosis induced by PFOS in L-02 cells, which are incubated with different concentrations of PFOS (0, 50, 100, 150, 200, or 250 μmol/L) for 24 or 48 hrs at 37°C. The results indicated that PFOS exposure decreased cell activities, enhanced ROS levels in a concentration-dependent manner, decreased mitochondrial membrane potential (MMP), and induced autophagy and apoptosis. Compared with the control, 200 μmol/L PFOS increased ROS levels; enhanced the expression of Bax, cleaved-caspase-3, and LC3-II; induced autophagy; decreased MMP; and lowered Bcl-2, p62, and Bcl-2/Bax ratio. The antioxidant N-acetyl cysteine (NAC) protected MMP against PFOS-induced changes and diminished apoptosis and autophagy. Compared with 200 μmol/L PFOS treatment, NAC pretreatment reversed the increase in ROS, Bax, and cleaved-caspase-3 protein caused by PFOS, lowered the apoptosis rate increased by PFOS, and increased the levels of MMP and Bcl-2/Bax ratio decreased by PFOS. The autophagy inhibitor 3-methyladenine and chloroquine decreased apoptosis and cleaved-caspase-3 protein level and increased the Bcl-2/Bax ratio. In summary, our results suggest that ROS-triggered autophagy is involved in PFOS-induced apoptosis in L-02 cells.

Graphene oxide nanofilm and chicken embryo extract decrease the invasiveness of HepG2 liver cancer cells

Background The extracellular matrix (ECM) is a mosaic of various structural and functional proteins that cooperate with the cell, regulate adhesion, and consequently manage its further fate. Liver destruction is accompanied by a disruption of the physicochemical properties of the ECM which deregulates the cell–ECM interaction and can lead to uncontrolled proliferation and neoplastic transformation of cells. Therefore, it can be assumed that ECM modification and restoration of its characteristics for healthy tissue may counteract uncontrolled cell proliferation. The purpose of the presented research model was to optimise the physical characteristics of ECM by introducing a graphene oxide plane/nanofilm (nfGO) and enriching the cell environment with potentially missing proteins by adding a functional protein cocktail (chicken embryo liver extract) and determine the impact of these factors on cell–ECM cooperation and its consequences on adhesion, proliferation, and cell phase, which are factors of the invasiveness of cancer cells. Results Experiments were performed with non-cancer HS-5 cells and liver cancer cells HepG2 and C3A. The cells were divided into four groups: (1) control, (2) cultured on nfGO, (3) cultured with the addition of chicken embryo liver extract (CELE) and (4) cultured on the nfGO with the addition of CELE. CELE contained 1735 proteins; the top 57 of these proteins have been presented. The use of nfGO as well as CELE and nfGO + CELE reduced the proliferation of HepG2 cancer cells to the greatest extent; this is in contrast to non-cancer cells and also to C3A cancer cells. Furthermore, the combined use of the CELE protein cocktail and GO substrate effectively resulted in a decrease in the population of HepG2 cells in the G0/G1 phase and an increase of the population in G2/M. Molecular analysis of HepG2 cancer cells also showed an increase in the expression of genes responsible for adhesion such as focal adhesion kinase (fak), e-cadherin, and n-cadherin and a decrease in β-catenin, which is considered a proto-oncogene. Conclusions Studies have shown that both the GO surface structure on which the cells are grown as well as the presence of a multi-component natural cocktail of regulatory proteins, can modify the expression of integrins, increase adhesion and, as a consequence, proliferation and the cell cycle—entering the resting phase. For the first time, it has been documented that hepatic cancer cells of the HepG2 line under the influence of stimuli derived from mimic ECM (graphene oxide) in interaction with a unique protein complex derived from chicken liver embryo decreased of the invasiveness of cancer cells.

Graphene Oxide Nanofilm and Chicken Embryo Extract Decrease the Invasiveness of HepG2 Liver Cancer Cells

Background: The extracellular matrix (ECM) is a mosaic of various structural and functional proteins that cooperate with the cell, regulate adhesion, and consequently manage its further fate. Liver destruction is accompanied by a disruption of the physicochemical properties of the ECM which deregulates the cell-ECM interaction and can lead to uncontrolled proliferation and neoplastic transformation of cells. Therefore, it can be assumed that ECM modification and restoration of its characteristics for healthy tissue may counteract uncontrolled cell proliferation. The purpose of the presented research model was to optimise the physical characteristics of ECM by introducing a graphene oxide plane/nanofilm (nfGO) and enriching the cell environment with potentially missing proteins by adding a functional protein cocktail (chicken embryo liver extract) and determine the impact of these factors on cell-ECM cooperation and its consequences on adhesion, proliferation, and cell phase, which are factors of the invasiveness of cancer cells.Results: Experiments were performed with non-cancer HS-5 cells and liver cancer cells HepG2 and C3A. The cells were divided into four groups; (1) control, (2) cultured on GO nanofilm, (3) cultured with the addition of chicken embryo liver extract (CELE) to the medium and (4) cultured on the GO nanofilm with the addition of CELE. CELE contained 1735 proteins; the top 57 of these proteins have been presented. The use of GO nanofilm as well as CELE and nfGO + CELE reduced the proliferation of HepG2 cancer cells to the greatest extent; this is in contrast to non-cancer cells and also to C3A cancer cells. Furthermore, the combined use of the CELE protein cocktail and GO substrate effectively resulted in a decrease in the population of HepG2 cells in the G0/G1 phase and an increase of the population in G2/M. Molecular analysis of HepG2 cancer cells also showed an increase in the expression of genes responsible for adhesion such as fak (focal adhesion kinase), e-cadherin, and n-cadherin and a decrease in β-catenin, which is considered a proto-oncogen. Conclusions: Studies have shown that both the GO surface structure on which the cells are grown as well as the presence of a multi-component natural cocktail of regulatory proteins, can modify the expression of integrins, increase adhesion and, as a consequence, proliferation and the cell cycle - entering the resting phase. For the first time, it has been documented that hepatic cancer cells of the HepG2 line under the influence of stimuli derived from mimic ECM (graphene oxide) in interaction with a unique protein complex derived from chicken liver embryo decreased of the invasiveness of cancer cells.

Propagation and Molecular Characterization of Fowl Adenovirus Serotype 8b Isolates in Chicken Embryo Liver Cells Adapted on Cytodex™ 1 Microcarrier Using Stirred Tank Bioreactor

Large volume production of vaccine virus is essential for prevention and control of viral diseases. The objectives of this study were to propagate Fowl adenovirus (FAdV) isolate (UPM08136) in chicken embryo liver (CEL) cells adapted to Cytodex™ 1 microcarriers using stirred tank bioreactor (STB) and molecularly characterize the virus. CEL cells were prepared and seeded onto prepared Cytodex™ 1 microcarriers and incubated first in stationary phase for 3 h and in STB at 37 °C, 5% CO2, and 20 rpm for 24 h. The CEL cells were infected with FAdV isolate (UPM08136) passage 5 (UPM08136CELP5) or passage 20 (UPM08136CELP20) and monitored until cell detachment. Immunofluorescence, TCID50, sequencing, alignment of hexon and fiber genes, and phylogenetic analysis were carried out. CEL cells were adapted well to Cytodex™ 1 microcarriers and successfully propagated the FAdV isolates in STB with virus titer of 107.5 (UPM08136CELP5B1) and 106.5 (UPM08136CELP20B1) TCID50/mL. These isolates clustered with the reference FAdV serotype 8b in the same evolutionary clade. The molecular characteristics remained unchanged, except for a point substitution at position 4 of the hexon gene of UPM08136CELP20B1, suggesting that propagation of the FAdV isolate in STB is stable and suitable for large volume production and could be a breakthrough in the scale-up process.

Graphene Oxide Nanofilm and Chicken Embryo Extract Decrease the Invasiveness of HepG2 Liver Cancer Cells.

Background: The extracellular matrix (ECM) is a mosaic of various structural and functional proteins that cooperate with the cell, regulate adhesion, and consequently manage its further fate. Liver destruction is accompanied by a disruption of the physicochemical properties of the ECM which deregulates the cell-ECM interaction and can lead to uncontrolled proliferation and neoplastic transformation of cells. Therefore, it can be assumed that ECM modification and restoration of its characteristics for healthy tissue may counteract uncontrolled cell proliferation. The purpose of the presented research model was to optimise the physical characteristics of ECM by introducing a graphene oxide plane/nanofilm (nfGO) and enriching the cell environment with potentially missing proteins by adding a functional protein cocktail (chicken embryo liver extract) and determine the impact of these factors on cell-ECM cooperation and its consequences on adhesion, proliferation, and cell phase, which are factors of the invasiveness of cancer cells.Results: Experiments were performed with non-cancer HS-5 cells and liver cancer cells HepG2 and C3A. The cells were divided into four groups; (1) control, (2) cultured on GO nanofilm, (3) cultured with the addition of chicken embryo liver extract (CELE) to the medium and (4) cultured on the GO nanofilm with the addition of CELE. CELE contained 1735 proteins; the top 57 of these proteins have been presented. The use of GO nanofilm as well as CELE and nfGO + CELE reduced the proliferation of HepG2 cancer cells to the greatest extent; this is in contrast to non-cancer cells and also to C3A cancer cells. Furthermore, the combined use of the CELE protein cocktail and GO substrate effectively resulted in a decrease in the population of HepG2 cells in the G0/G1 phase and an increase of the population in G2/M. Molecular analysis of HepG2 cancer cells also showed an increase in the expression of genes responsible for adhesion such as fak (focal adhesion kinase), e-cadherin, and n-cadherin and a decrease in β-catenin, which is considered a proto-oncogen. Conclusions: Studies have shown that both the GO surface structure on which the cells are grown as well as the presence of a multi-component natural cocktail of regulatory proteins, can modify the expression of integrins, increase adhesion and, as a consequence, proliferation and the cell cycle - entering the resting phase. For the first time, it has been documented that hepatic cancer cells of the HepG2 line under the influence of stimuli derived from mimic ECM (graphene oxide) in interaction with a unique protein complex derived from chicken liver embryo decreased of the invasiveness of cancer cells.

Cytotoxicity study of deoxynivalenol on human embryo liver and hepatoma cell

To investigate the cytotoxicity of deoxynivalenol (DON) on human embryo liver CCC-HEL-1 and hepatoma cell line HepG2 cell models, both cell experience and metabolomic approach were studied. For the cell evaluation, cells viabilities of CCC-HEL-1 and HepG2 were decreased in both a time- and dose-dependent manner at concentration range from 0.08~10 μmol/l, after which the concentration of 1 μmol/l DON was selected for the next experiments. A higher production of reactive oxygen species (ROS) in DON treated CCC-HEL-1 cells was found after 2 h treatment compared with the HepG2 group, while ROS generation was significantly dropped after 48 h in both models. DON-treated CCC-HEL-1 and HepG2 cells displayed significantly decreased percentages of ΔΨm loss. For the metabolomic study based on liquid chromatography quadrupole time-of-flight mass spectrometry, it was notable that certain amino acids identified in the two DON-treated groups were upregulated. The pathway analysis also revealed that amino acid metabolism played a crucial role underlying DON exposure in the two studied models. Our results provided metabolic evidence that further confirmed the toxicological potential of DON to disturb amino acid and lipid metabolism in human embryo liver cells.