Effect of Erythropoietin on the Expression of Murine Transferrin Receptor 2.

Erythropoietin (EPO) downregulates hepcidin expression to increase the availability of iron; the downregulation of hepcidin is mediated by erythroferrone (ERFE) secreted by erythroblasts. Erythroblasts also express transferrin receptor 2 (TFR2); however, the possible role of TFR2 in hepcidin downregulation is unclear. The purpose of the study was to correlate liver expression of hepcidin with the expression of ERFE and TFR2 in murine bone marrow and spleen at 4, 16, 24, 48, 72 and 96 h following administration of a single dose of EPO. Splenic Fam132b expression increased 4 h after EPO injection; liver hepcidin mRNA was decreased at 16 h. In the spleen, expression of TFR2 and transferrin receptor (TFR1) proteins increased by an order of magnitude at 48 and 72 h after EPO treatment. The EPO-induced increase in splenic TFR2 and TFR1 was associated with an increase in the number of Tfr2- and Tfr1-expressing erythroblasts. Plasma exosomes prepared from EPO-treated mice displayed increased amount of TFR1 protein; however, no exosomal TFR2 was detected. Overall, the results confirm the importance of ERFE in stress erythropoiesis, support the role of TFR2 in erythroid cell development, and highlight possible differences in the removal of TFR2 and TFR1 from erythroid cell membranes.

Stem Cell Defect in Ubiquitin-Green Fluorescent Protein Mice Facilitates Engraftment of Lymphoid-Primed Hematopoietic Stem Cells.

Transgenic mice expressing green fluorescent protein (GFP) are useful in transplantation experiments. When we used ubiquitin-GFP (UBC-GFP) transgenic mice to study the availability of niches for transplanted hematopoietic stem and progenitor cells, the results were strikingly different from the corresponding experiments that used congenic mice polymorphic in the CD45 antigen. Analysis of these unexpected results revealed that the hematopoiesis of UBC-GFP mice was outcompeted by the hematopoiesis of wild-type (WT) mice. Importantly, UBC-GFP mice engrafted the transplanted bone marrow of WT mice without conditioning. There was a significant bias toward lymphopoiesis in the WT branch of chimeric UBC-GFP/WT hematopoiesis. A fraction of immature Sca-1+ cells in the spleen of UBC-GFP mice expressed GFP at a very high level. The chimeric hematopoiesis was stable in the long term and also after transplantation to secondary recipient mice. The article thus identifies a specific defect in the hematopoiesis of UBC-GFP transgenic mice that compromises the lymphoid-primed hematopoietic stem cells in the bone marrow and spleen. Stem Cells 2018;36:1237-1248.

Low c-Kit Expression Level Induced by Stem Cell Factor Does Not Compromise Transplantation of Hematopoietic Stem Cells.

The c-Kit expression level is decreased in regenerating bone marrow, and such bone marrow performs poorly when co-transplanted with normal bone marrow. We asked whether diminished numbers of c-Kit receptors on hematopoietic stem and progenitor cells (HSPCs) after their internalization induced by the binding of the cytokine stem cell factor (SCF) would jeopardize transplantability of HSPCs. We used a battery of functional assays to evaluate the capacity of HSPCs with markedly different c-Kit expression levels to be transplanted. Surprisingly, our experiments testing the homing of transplanted HSPCs to bone marrow of recipient mice and their short-term and long-term engraftment did not reveal any defects in HSPCs with severely reduced numbers of c-Kit receptor molecules. This unexpected result can be ascribed to the fact that HSPCs exposed to SCF replace the consumed c-Kit receptors rapidly. This article demonstrates that exposure of HSPCs to SCF and diminished number of c-Kit receptors in their cell membranes do not compromise the capacity of HSPCs to reconstitute damaged hematopoietic tissue.

Epigenetic silencing of the oncogenic miR-17-92 cluster during PU.1-directed macrophage differentiation.

The oncogenic cluster miR-17-92 encodes seven related microRNAs that regulate cell proliferation, apoptosis and development. Expression of miR-17-92 cluster is decreased upon cell differentiation. Here, we report a novel mechanism of the regulation of miR-17-92 cluster. Using transgenic PU.1(-/-) myeloid progenitors we show that upon macrophage differentiation, the transcription factor PU.1 induces the secondary determinant Egr2 which, in turn, directly represses miR-17-92 expression by recruiting histone demethylase Jarid1b leading to histone H3 lysine K4 demethylation within the CpG island at the miR-17-92 promoter. Conversely, Egr2 itself is targeted by miR-17-92, indicating existence of mutual regulatory relationship between miR-17-92 and Egr2. Furthermore, restoring EGR2 levels in primary acute myeloid leukaemia blasts expressing elevated levels of miR-17-92 and low levels of PU.1 and EGR2 leads to downregulation of miR-17-92 and restored expression of its targets p21CIP1 and BIM. We propose that upon macrophage differentiation PU.1 represses the miR-17-92 cluster promoter by an Egr-2/Jarid1b-mediated H3K4 demethylation mechanism whose deregulation may contribute to leukaemic states.

Mouse models of mantle cell lymphoma, complex changes in gene expression and phenotype of engrafted MCL cells: implications for preclinical research.

Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma (NHL) associated with poor prognosis. Animal models of MCL are scarce. We established and characterized various in vivo models of metastatic human MCL by tail vein injection of either primary cells isolated from patients with MCL or established MCL cell lines (Jeko-1, Mino, Rec-1, Hbl-2, and Granta-519) into immunodeficient NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice. MCL infiltration was assessed with immunohistochemistry (tissues) and flow cytometry (peripheral blood). Engraftment of primary MCL cells was observed in 7 out of 12 patient samples. The pattern of engraftment of primary MCL cells varied from isolated involvement of the spleen to multiorgan infiltration. On the other hand, tumor engraftment was achieved in all five MCL cell lines used and lymphoma involvement of murine bone marrow, spleen, liver, and brain was observed. Overall survival of xenografted mice ranged from 22 ± 1 to 54 ± 3 days depending on the cell line used. Subsequently, we compared the gene expression profile (GEP) and phenotype of the engrafted MCL cells compared with the original in vitro growing cell lines (controls). We demonstrated that engrafted MCL cells displayed complex changes of GEP, protein expression, and sensitivity to cytotoxic agents when compared with controls. We further demonstrated that our MCL mouse models could be used to test the therapeutic activity of systemic chemotherapy, monoclonal antibodies, or angiogenesis inhibitors. The characterization of MCL murine models is likely to aid in improving our knowledge in the disease biology and to assist scientists in the preclinical and clinical development of novel agents in relapsed/refractory MCL patients.

Second bone marrow transplantation into regenerating hematopoiesis enhances reconstitution of immune system.

In bone marrow transplantation (BMT), hematopoiesis-reconstituting cells are introduced following myeloablative treatment, which eradicates existing hematopoietic cells and disrupts stroma within the hematopoietic tissue. Both hematopoietic cells and stroma then undergo regeneration. Our study compares the outcomes of a second BMT administered to mice shortly after myeloablative treatment and the first BMT, with those of a second BMT administered to mice experiencing robust hematopoietic regeneration after the initial transplant. We evaluated the efficacy of the second BMT in terms of engraftment efficiency, types of generated blood cells, and longevity of function. Our findings show that regenerating hematopoiesis readily accommodates newly transplanted stem cells, including those endowed with a robust capacity for generating B and T cells. Importantly, our investigation uncovered a window for preferential engraftment of transplanted stem cells coinciding with the resumption of blood cell production. Repeated BMT could intensify hematopoiesis reconstitution and enable therapeutic administration of genetically modified autologous stem cells.

All hematopoietic stem cells engraft in submyeloablatively irradiated mice.

Significant controversy exists regarding the impact of hematopoietic stroma damage by irradiation on the efficiency of engraftment of intravenously transplanted stem cells. It was previously demonstrated that in normal syngenic mice, all intravenously transplanted donor stem cells, present in the bone marrow, compete equally with those of the host. In this study, we comprehensively compared the blood cell production derived from transplanted donor stem cells with that from the host stem cells surviving various doses of submyeloablative irradiation. We compared the partial chimerism resulting from transplantation with theoretical estimates that assumed transplantation efficiencies ranging from 100% to 20%. The highest level of consensus between the experimental and the theoretical results was 100% for homing and engraftment (ie, the utilization of all transplanted stem cells). These results point to a very potent mechanism through which intravenously administered hematopoietic stem cells are captured from circulation, engraft in the hematopoietic tissue, and contribute to blood cell production in irradiated recipients. The damage done to hematopoietic stroma and to the trabecular bone by submyeloablative doses of ionizing radiation does not negatively affect the homing and engraftment mechanisms of intravenously transplanted hematopoietic progenitor and stem cells.

MYB transcriptionally regulates the miR-155 host gene in chronic lymphocytic leukemia.

Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In this study, we present evidence that MYB (v-myb myeloblastosis viral oncogene homolog) is overexpressed in a subset of B-CLL patients. MYB physically associates with the promoter of miR-155 host gene (MIR155HG, also known as BIC, B-cell integration cluster) and stimulates its transcription. This coincides with the hypermethylated histone H3K4 residue and spread hyperacetylation of H3K9 at MIR155HG promoter. Our data provide evidence of oncogenic activities of MYB in B-CLL that include its stimulatory role in MIR155HG transcription.

Hematopoietic stem cells survive circulation arrest and reconstitute hematopoiesis in myeloablated mice.

Hematopoietic stem and progenitor cells (HSPC) for bone marrow transplantation are currently obtained directly from living voluntary donors or from cord blood units. However, a suitable donor is not always found. Because HSPC are known for their relative resistance to hypoxia, using an experimental murine model, we explored cadaveric bone marrow (BM) as their alternative source. After donor mice were sacrificed, BM was left in intact femurs at 37°C, 20°C, or 4°C under ischemic conditions, resulting in combined oxygen and metabolic substrate shortage and the accumulation of metabolic waste products. BM cells were harvested after a set time period ranging from 0 to 48 hours. To determine the impact of delayed harvesting on the transplantability of HSPC, a competitive repopulation assay using a murine Ly5.1/Ly5.2 congenic model in 2 different settings was used: after submyeloablative (6 Gy) or myeloablative (9 Gy) total-body irradiation, Ly5.2 hosts received cadaveric Ly5.1 cells or a mixture of cadaveric Ly5.1 cells and fresh Ly5.2 cells in a 1:1 ratio. Chimerism resulting from cadaveric donor cells, followed up to 6 months after transplantation, proved that the long-term repopulation ability of HSPC was fully preserved for 2 hours, 6 hours, and 12 hours at 37°C, 20°C, and 4°C of ischemia, respectively. A colony-forming unit-spleen (CFU-S) clonogenic assay revealed a higher sensitivity of proliferating hematopoietic progenitors to ischemia compared to repopulating cells (STRC and LTRC). Flow cytometry analysis of apoptosis in cadaveric BM demonstrated that the LSK (Lin(low)Sca-1(+)c-Kit(+)) subpopulation, enriched in HSPC, contained less apoptotic and dead cells than the BM as a whole. Furthermore, the number of LSK SLAM (CD150(+)CD48(-)) and LSK SP (side population) cells (fractions highly enriched in hematopoietic stem cells) decreased in parallel with BM transplantability. As well as cadaveric BM cells, we also tested the transplantability and survival of BM cells after storage in a suspension in vitro without specific hematopoietic growth factors. HSPC did not display any decrease in transplantability after 2 days of storage at 37°C or 4 days at 4°C. A higher sensitivity of progenitors to unfavorable conditions was observed again using CFU-S and granulocyte macrophage-colony forming cell (GM-CFC) assays, especially at 37°C. This paper shows that HSPC survive the cessation of circulation for a considerable time and maintain their engraftment potential. This time is significantly extended with in vitro storage compared to the cadaveric BM.

Liver hemojuvelin protein levels in mice deficient in matriptase-2 (Tmprss6).

Mutations of the TMPRSS6 gene, encoding the serine protease matriptase-2, lead to iron-refractory iron deficiency anemia. Matriptase-2 is a potent negative regulator of hepcidin. Based on in vitro data, it has recently been proposed that matriptase-2 decreases hepcidin synthesis by cleaving membrane hemojuvelin, a key protein of the hepcidin-regulatory pathway. However, in vivo evidence for this mechanism of action of matriptase-2 is lacking. To investigate the hemojuvelin-matriptase-2 interaction in vivo, an immunoblot assay for liver membrane hemojuvelin was optimized using hemojuvelin-mutant mice as a negative control. In wild-type mice, two hemojuvelin-specific bands of 35kDa and 20kDa were detected in mouse liver membrane fraction under reducing conditions; under non-reducing conditions, a single band of approximately 50kDa was seen. Phosphatidylinositol-specific phospholipase C treatment confirmed binding of the detected protein to the cell membrane by a glycosylphosphatidylinositol anchor, indicating that the major form of mouse liver membrane hemojuvelin is a glycosylphosphatidylinositol-bound heterodimer. Unexpectedly, comparison of liver homogenates from Tmprss6+/+ and Tmprss6-/- mice revealed significantly decreased, rather than increased, hemojuvelin heterodimer content in Tmprss6-/- mice. These data do not provide direct support for the concept that matriptase-2 cleaves membrane hemojuvelin and may indicate that, in vivo, the role of matriptase-2 in the regulation of hepcidin gene expression is more complex.

Liver and muscle hemojuvelin are differently glycosylated.

BACKGROUND: Hemojuvelin (HJV) is one of essential components for expression of hepcidin, a hormone which regulates iron transport. HJV is mainly expressed in muscle and liver, and processing of HJV in both tissues is similar. However, hepcidin is expressed in liver but not in muscle and the role of the muscle HJV is yet to be established. Our preliminary analyses of mouse tissue HJV showed that the apparent molecular masses of HJV peptides are different in liver (50 kDa monomer and 35 and 20 kDa heterodimer fragments) and in muscle (55 kDa monomer and a 34 kDa possible large fragment of heterodimer). One possible explanation is glycosylation which could lead to difference in molecular mass. RESULTS: We investigated glycosylation of HJV in both liver and muscle tissue from mice. PNGase F treatment revealed that the HJV large fragments of liver and muscle were digested to peptides with similar masses, 30 and 31 kDa, respectively, and the liver 20 kDa small fragment of heterodimer was digested to 16 kDa, while the 50 kDa liver and 55 kDa muscle monomers were reduced to 42 and 48 kDa, respectively. Endo H treatment produced distinct digestion profiles of the large fragment: a small fraction of the 35 kDa peptide was reduced to 33 kDa in liver, while the majority of the 34 kDa peptide was digested to 33 kDa and a very small fraction to 31 kDa in muscle. In addition, liver HJV was found to be neuraminidase-sensitive but its muscle counterpart was neuraminidase-resistant. CONCLUSIONS: Our results indicate that different oligosaccharides are attached to liver and muscle HJV peptides, which may contribute to different functions of HJV in the two tissues.

Hematopoiesis Remains Permissive to Bone Marrow Transplantation After Expansion of Progenitors and Resumption of Blood Cell Production.

The immense regenerative power of hematopoietic tissue stems from the activation of the immature stem cells and the progenitor cells. After partial damage, hematopoiesis is reconstituted through a period of intense regeneration when blood cell production originates from erythro-myeloid progenitors in the virtual absence of stem cells. Since the damaged hematopoiesis can also be reconstituted from transplanted hematopoietic cells, we asked whether this also leads to the transient state when activated progenitors initially execute blood cell production. We first showed that the early reconstitution of hematopoiesis from transplanted cells gives rise to extended populations of developmentally advanced but altered progenitor cells, similar to those previously identified in the bone marrow regenerating from endogenous cells. We then identified the cells that give rise to these progenitors after transplantation as LSK CD48- cells. In the submyeloablative irradiated host mice, the transplanted LSK CD48- cells preferably colonized the spleen. Unlike the endogenous hematopoiesis reconstituting cells, the transplanted whole bone marrow cells and sorted LSK CD48- cells had greater potential to differentiate to B-lymphopoiesis. Separate transplantation of the CD150- and CD150+ subsets of LSK CD48- cells suggested that CD150- cells had a greater preference to B-lymphopoiesis than CD150+ cells. In the intensively regenerating hematopoiesis, the CD71/Sca-1 plot of immature murine hematopoietic cells revealed that the expanded populations of altered myeloid progenitors were highly variable in the different places of hematopoietic tissues. This high variability is likely caused by the heterogeneity of the hematopoiesis supporting stroma. Lastly, we demonstrate that during the period when active hematopoiesis resumes from transplanted cells, the hematopoietic tissues still remain highly permissive for further engraftment of transplanted cells, particularly the stem cells. Thus, these results provide a rationale for the transplantation of the hematopoietic stem cells in successive doses that could be used to boost the transplantation outcome.

Effect of erythropoietin on hepcidin expression in hemojuvelin-mutant mice.

Transcription of the hepcidin (Hamp) gene is controlled by iron stores and the rate of erythropoiesis. Functional hierarchy between these two stimuli has not yet been completely established. It is also not known whether the erythropoiesis-related downregulation of Hamp expression utilises the bone morphogenetic protein/hemojuvelin (Bmp/Hjv) pathway. Hemojuvelin-mutant (Hjv-/-) mice treated with erythropoietin (EPO) at 50IU/mouse/day for three days displayed marked decrease in Hamp mRNA, demonstrating that hemojuvelin is not an indispensable component in EPO-induced Hamp gene downregulation. Irradiation of Hjv-/- mice prevented the EPO-induced decrease of Hamp mRNA, highlighting the role of erythropoiesis in Hamp gene regulation by EPO. After a single injection of EPO, Hamp mRNA levels were not significantly changed at 6h, but decreased at 10 and 24h. Chronic bleeding decreased hepatic Bmp6 mRNA levels; however, repeated EPO treatment did not change Bmp6 mRNA, suggesting that the erythropoietic regulator(s) act independently of the Bmp/Hjv pathway. Pretreatment of C57BL/6 mice with iron (5mg/mouse) almost completely inhibited the EPO-induced decrease of Hamp mRNA. This result suggests that administration of EPO to patients with transfusional iron overload is probably not associated with the risk of additional absorption of substantial amounts of iron from the diet.

A sudden increase in partial pressure end-tidal carbon dioxide (P(ET)CO(2)) at the moment of return of spontaneous circulation.

BACKGROUND: Previous studies established that a level of partial pressure end-tidal carbon dioxide (P(ET)CO(2)) of 10 mm Hg divided patients undergoing advanced life support (ALS) into those likely to be resuscitated (values > 10 mm Hg) and those likely to die during ALS (values < 10 mm Hg). OBJECTIVE: The study tested the significance of a sudden increase in the P(ET)CO(2) in signaling the return of spontaneous circulation (ROSC) during ALS. MATERIAL AND METHODS: P(ET)CO(2) values were continuously recorded during ALS in out-of-hospital patients with cardiac arrest. Constant ventilation was maintained by an automatic device. There were 108 patients, representing two extreme outcomes of ALS, who were subdivided into two groups. The first group included 59 patients with a single ROSC followed by a stable spontaneous circulation. The second group included 49 patients with no signs of ROSC. RESULTS: ROSC was associated with a sudden increase in P(ET)CO(2) that remained significantly higher than before ROSC. P(ET)CO(2) did not rise during the entire ALS in the second group of patients without ROSC and was lower than in the first group of patients. CONCLUSIONS: In constantly ventilated patients, P(ET)CO(2) is significantly higher (about 10 mm Hg) after ROSC than before ROSC. A sudden increase in P(ET)CO(2) exceeding 10 mm Hg may indicate ROSC. Consequently, the rule of 10 mm Hg may be extended to include a sudden increase in continuously recorded P(ET)CO(2) by more than 10 mm Hg as an indicator of the possibility of ROSC.

Cell Cycle Analysis Using In Vivo Staining of DNA-Synthesizing Cells.

The thymidine analogues BrdU (5-bromo-2´-deoxyuridine) and EdU (5-ethynyl-2´-deoxyuridine) are routinely used for determination of the cells synthesizing DNA in the S-phase of the cell cycle. Availability of the anti-BrdU antibody clone MoBu-1 detecting only BrdU allowed to develop a method for the sequential DNA labelling by these two thymidine analogues for determining the cell cycle kinetic parameters.In the current step-by-step protocol, we present` two approaches optimized for in vivo study of the cell cycle and the limitations that such approaches imply: (1) determination of the cell flow rate into the G2-phase by dual EdU/BrdU DNA-labelling method and (2) determination of the outflow of DNA-labelled cells arising from the mitosis.

Altered Erythro-Myeloid Progenitor Cells Are Highly Expanded in Intensively Regenerating Hematopoiesis.

Regeneration of severely damaged adult tissues is currently only partially understood. Hematopoietic tissue provides a unique opportunity to study tissue regeneration due to its well established steady-state structure and function, easy accessibility, well established research methods, and the well-defined embryonic, fetal, and adult stages of development. Embryonic/fetal liver hematopoiesis and adult hematopoiesis recovering from damage share the need to expand populations of progenitors and stem cells in parallel with increasing production of mature blood cells. In the present study, we analyzed adult hematopoiesis in mice subjected to a submyeloablative dose (6 Gy) of gamma radiation and targeted the period of regeneration characterized by massive production of mature blood cells along with ongoing expansion of immature hematopoietic cells. We uncovered significantly expanded populations of developmentally advanced erythroid and myeloid progenitors with significantly altered immunophenotype. Their population expansion does not require erythropoietin stimulation but requires the SCF/c-Kit receptor signaling. Regenerating hematopoiesis significantly differs from the expanding hematopoiesis in the fetal liver but we find some similarities between the regenerating hematopoiesis and the early embryonic definitive hematopoiesis. These are in (1) the concomitant population expansion of myeloid progenitors and increasing production of myeloid blood cells (2) performing these tasks despite the severely reduced transplantation capacity of the hematopoietic tissues, and (3) the expression of CD16/32 in most progenitors. Our data thus provide a novel insight into tissue regeneration by suggesting that cells other than stem cells and multipotent progenitors can be of fundamental importance for the rapid recovery of tissue function.

TRAIL-induced apoptosis of HL60 leukemia cells: two distinct phenotypes of acquired TRAIL resistance that are accompanied with resistance to TNFalpha but not to idarubicin and cytarabine.

TNF-related apoptosis-inducing ligand (TRAIL) is a proapoptotic cytokine implicated in cancer cell surveillance. A potential of TRAIL as a cancer-specific therapeutic agent has been proposed, either as a single agent or in combination with chemotherapy. Prolonged exposure of TRAIL-sensitive leukemia cell line, wild-type (WT) HL60 cells to recombinant soluble TRAIL or to cytostatic agents, cytarabine and idarubicin, resulted in the establishment of resistant subclones with distinct phenotypic features. The TRAIL resistant HL60 subclones were characterized by decreased expression of TRAIL and TNFalpha death receptors. These resistant subclones had impaired activation of caspases 8 and 10 in response to TRAIL and TNFalpha, decreased TRAIL-induced nuclear translocation of NFkappaB RelA/p65, and dysregulation of the expression of several apoptosis regulators. Among the TRAIL resistant HL60 subclones we identified two separate phenotypes that differed in the expression of CD14, osteoprotegerin, and several apoptosis regulators. Both these TRAIL resistant HL60 subclones were resistant to TNFalpha, suggesting disruption of the extrinsic apoptotic pathway, but not to cytostatic agents, cytarabine and idarubicin. The concurrently derived HL60 subclones were cytarabine and idarubicin-resistant but remained sensitive to TRAIL-induced apoptosis. We identified distinct pathways for the development of HL60 leukemia cell resistance to apoptosis induction. These findings are relevant for the design of more effective strategies for leukemia therapy.

Effect of stimulated erythropoiesis on liver SMAD signaling pathway in iron-overloaded and iron-deficient mice.

Expression of hepcidin, the hormone regulating iron homeostasis, is increased by iron overload and decreased by accelerated erythropoiesis or iron deficiency. The purpose of the study was to examine the effect of these stimuli, either alone or in combination, on the main signaling pathway controlling hepcidin biosynthesis in the liver, and on the expression of splenic modulators of hepcidin biosynthesis. Liver phosphorylated SMAD 1 and 5 proteins were determined by immunoblotting in male mice treated with iron dextran, kept on an iron deficient diet, or administered recombinant erythropoietin for four consecutive days. Administration of iron increased liver phosphorylated SMAD protein content and hepcidin mRNA content; subsequent administration of erythropoietin significantly decreased both the iron-induced phosphorylated SMAD proteins and hepcidin mRNA. These results are in agreement with the recent observation that erythroferrone binds and inactivates the BMP6 protein. Administration of erythropoietin substantially increased the amount of erythroferrone and transferrin receptor 2 proteins in the spleen; pretreatment with iron did not influence the erythropoietin-induced content of these proteins. Erythropoietin-treated iron-deficient mice displayed smaller spleen size in comparison with erythropoietin-treated mice kept on a control diet. While the erythropoietin-induced increase in splenic erythroferrone protein content was not significantly affected by iron deficiency, the content of transferrin receptor 2 protein was lower in the spleens of erythropoietin-treated mice kept on iron-deficient diet, suggesting posttranscriptional regulation of transferrin receptor 2. Interestingly, iron deficiency and erythropoietin administration had additive effect on hepcidin gene downregulation in the liver. In mice subjected both to iron deficiency and erythropoietin administration, the decrease of hepcidin expression was much more pronounced than the decrease in phosphorylated SMAD protein content or the decrease in the expression of the SMAD target genes Id1 and Smad7. These results suggest the existence of another, SMAD-independent pathway of hepcidin gene downregulation.

Cell cycle and differentiation of Sca-1+ and Sca-1- hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. We analyzed the cell cycle and cell production rate in HSPCs in murine hematopoiesis. The labeling of DNA-synthesizing cells by two thymidine analogues, optimized for in-vivo use, enabled determination of the cell cycle flow rate into G2-phase, the duration of S-phase and the average cell cycle time in Sca-1+ and Sca-1- HSPCs. Determination of cells with 2n DNA content labeled in preceding S-phase was then used to establish the cell flow rates in G1-phase. Our measurements revealed a significant difference in how Sca-1+ and Sca-1- myeloid progenitors self-renew and differentiate. Division of the Sca-1+ progenitors led to loss of the Sca-1 marker in about half of newly produced cells, corresponding to asymmetric cell division. Sca-1- cells arising from cell division entered a new round of the cell cycle, corresponding to symmetric self-renewing cell division. The novel data also enabled the estimation of the cell production rates in Sca-1+ and in three subtypes of Sca-1- HSPCs and revealed Sca-1 negative cells as the major amplification stage in the blood cell development.

Expression of angiopoietic factors in normal and type-I diabetes human placenta: a pilot study.

OBJECTIVES: Morphological changes of blood vessel wall have been described in placenta from pregnancies complicated by diabetes mellitus type-I. STUDY DESIGN: We measured mRNA expression of vascular endothelial growth factor (VEGF), angiopoietin 1 and 2 (Ang-1 and Ang-2), their receptors VEGFR-1, VEGFR-2, Tie-2, fibroblast growth factor 2 (FGF-2), and its receptor FGF-2R in placental tissue of diabetes type-I patients, in normal term placenta, and endometrium of non-pregnant women by real time reverse transcriptase PCR. RESULTS: The expression of Ang-2 and VEGFR-1 mRNAs was significantly higher in placenta (P