bluuebunny/crossref_metadata_2025 huggingface dataset:
Shear Stress Directly Regulates Platelet αIIbβ3 Signaling.
Authors: Feng Shuju , Lu Xin , Resendiz Julio C. , Kroll Michael H.
Published: April 2020

Integrin mechanotransduction is a ubiquitous biological process. Mechanical forces are transduced by an integrin’s ligand-bound extracellular domain through its β subunit’s cytoplasmic domain connected to the cytoskeleton. This often culminates in the activation of tyrosine kinases directing cell responses. The delicate balance between hemostasis and thrombosis requires exquisitely fine-tuned integrin function, and balance is maintained in vivo despite that the major platelet integrin αIIbβ3 is continuously subjected to frictional or shearing forces generated by laminar blood flow. To test the hypothesis that platelet function is regulated by the direct effects of mechanical forces on αIIbβ3, we examined αIIbβ3/cytoskeletal interactions in human platelets exposed to shear stress in a cone-plate viscometer. We observed that α-actinin, myosin heavy chain and Syk co-immunoprecipitate with αIIbβ3 in resting platelets, and that 120 dynes/cm2 shear stress leads to their disassociation from αIIbβ3. Shear-induced disassociation of α-actinin and myosin heavy chain from the β3 tail is unaffected by blocking VWF binding to GpIb-IX-V but abolished by blocking VWF binding to αIIbβ3. Syk’s disassociation from β3 is inhibited when VWF binding to either GpIb-IX-V or αIIbβ3 is blocked. Shear stress-induced phosphorylation of SLP-76 and its association with tyrosine phosphorylated SLAP-130 are inhibited by blocking ligand binding to αIIbβ3 but not by blocking ligand binding to GpIb-IX-V. Chinese hamster ovary (CHO) cells expressing human αIIbβ3 demonstrate calcium and shear-dependent (1 and 10 dynes/cm2) attachment to glass cover slips coated with 20 μg/ml VWF. CHO cells expressing αIIbβ3 with β3 truncated of its cytoskeletal binding domains at C-terminal residue 716 demonstrate decreased VWF-dependent adhesion and cohesion in response to 1 dyne/cm2 shear stress. These results support the hypothesis that shear stress directly modulates αIIbβ3 function, and suggest that shear-induced αIIbβ3-mediated signaling regulates platelet aggregation by directing the release of α-actinin and myosin heavy chain from the β3 tail.
The Interaction between Transmembrane Domains of Glycoprotein Ibα and Ibβ Is Critical to Efficient Expression and Assembly of the Glycoprotein Ib-IX Complex.
Authors: Mo Xi , Lu Nan , Luo Shi-Zhong , Padilla Arnoldo , Lopez Jose A. , Li Renhao
Published: April 2020

Platelet glycoprotein (GP) Ib-IX-V complex is critically involved in the early steps of platelet adhesion and activation at sites of vessel wall injury. A functional GP Ib-IX complex comprising GP Ibα, GP Ibβ, and GP IX can be expressed in transfected cells, with efficient expression on the cell surface requiring all of its three subunits. As a first step to elucidate the 3-dimensional organizing principle of this receptor complex, we probed the interactions of its transmembrane (TM) domains. Replacement of the TM domains of either GP Ibα or GP Ibβ with poly-Leu or poly-Leu-Ala sequences markedly diminished cell surface expression of the complex, as assessed by flow cytometry and western blotting of transiently transfected CHO cells. Similar replacements of the GP IX TM domain had little effect on complex expression. TM replacements in GP Ibα or GP Ibβ also affected formation of the membrane-proximal inter-subunit disulfide bond between the two subunits. A thiol-disulfide exchange assay with the TM-containing peptides dispersed in dodecylphosphocholine micelles demonstrated a direct and specific interaction between the Ibα and Ibβ TM peptides, as the peptides preferentially formed a disulfide-linked αβ hetero-dimer rather than αα or ββ homo-dimers. Replacement mutations in the Ibα TM peptide abolished formation of the hetero-dimer. Further mutagenesis studies produced several single-site mutations within the TM domains of GP Ibα and GP Ibβ that drastically lowered the level of surface expression of the GP Ib-IX complex. These mutations also disrupted formation of the inter-subunit disulfide bond in the transfected GP Ib-IX complex as well as in the context of isolated TM peptides. These results demonstrate a specific interaction between the TM domains of GP Ibα and GP Ibβ, which can be abolished by whole-domain replacement or site-specific mutations, significantly diminishing assembly and cell-surface expression of the GP Ib-IX complex.
Ceruloplasmin Is Essential for the Mobilization of Tissue Iron Stores.
Authors: Rivera Seth , Lopez Miguel , Farshidi Dina , Gabayan Victoria , Ganz Tomas
Published: April 2020

In extracellular fluid, iron is in the ferric (oxidized form) but the intracellular form is ferrous iron (reduced). The outflow of iron from cells is dependent on oxidase activity that converts ferrous to ferric iron. Iron-absorbing enterocytes possess a unique iron oxidase, hephaestin. It is presumed that the circulating hephaestin paralog ceruloplasmin fulfils this role in hepatocytes and macrophages. The DiSnA mouse lacks ceruloplasmin. We hypothesized that iron homeostasis in this mouse would be unusually dependent on dietary iron because the mouse would not be able to mobilize iron from tissue stores in hepatocytes and macrophages. We fed 4-week-old DiSnA and wildtype (WT) mice a high iron (1%) diet for 4 weeks to load tissue stores. We then switched them to an iron-deficient diet and analyzed them weekly to measure iron and hemoglobin concentrations. Even on the high iron diet, DiSnA mice had lower serum iron concentrations than WT control (32.9±17.9 vs. 53.5±17.6 μM, p=0.05) but after two weeks on the iron deficient diet, the DiSnA mice had almost undetectable serum iron (4.2±1.8 μM) whereas the WT controls had only declined slightly (43.2±9.6 μM, p<0.001). Iron saturation followed a similar trend. Neither WT nor DiSnA mice were anemic at baseline (Hgb = 13.9±0.3 and 13.9±1.1 g/dL, respectively; p=0.994) but by the end of two weeks, the DiSnA mice had developed anemia whereas the WT mice had not (Hgb = 10.4±0.5 vs. 12.6±0.5 g/dL; p<0.001). The difference in hemoglobin concentrations persisted to the 6-week timepoint (Hgb = 8.4±0.5 vs. 12.6±1.4; p<0.001). After 6-weeks on a low iron diet, iron was still present in livers and spleens of both groups. Ceruloplasmin is essential for the mobilization of iron stores to protect against iron deficiency anemia in response to periods of dietary iron deficiency.
DMT1 Mutation in a Patient with Hypochromic Microcytic Anemia: Functional Consequences and Response to Erythropoietin.
Authors: Priwitzerova Monika , Pospisilova Dagmar , Nie Guangjun , Sheftel Alex D. , Mims Martha P. , Prchal Josef T. , Divoky Vladimir , Ponka Prem
Published: April 2020

We have previously described a case of severe hypochromic microcytic anemia caused by a homozygous mutation in the divalent metal transporter 1 ( DMT1 1285G>C ). This mutation encodes for an amino acid substitution (E399D) and causes preferential skipping of exon 12 during processing of the DMT1 mRNA. To examine the functional consequences of this mutation, full length DMT1 transcript with the patient’s point mutation or a DMT1 transcript with exon 12 deleted was expressed in Chinese hamster ovary (CHO) cells. Our results demonstrate that the E399D substitution has no effect on protein expression and function. In contrast, deletion of exon 12 led to a decreased expression of the protein and disruption of its subcellular localization and iron uptake activity. We hypothesize that the residual protein in hematopoietic cells represents the functional E399D DMT1 variant, but because of its quantitative reduction, the iron uptake activity of DMT1 in the patient’s erythroid cells is severely suppressed. Because of the positive effect of erythropoietin (EPO) on the growth of patient’s BFU-Es in our in vitro studies, we have treated the patient with recombinant human EPO. The dose of 1.2 μg/kg given once a week did not lead to any improvement in hemoglobin level. However, doubling the dose of EPO (2.4 μg/kg) led to an increase in hemoglobin level from 75 to 91 g/L and to an improvement in the patient’s clinical condition.
Targeted Overexpression of the Transcription Factor XBP-1 in B Cells Promotes Plasma Cell and Lymphoplasmacytic Neoplasms in Transgenic Mice.
Authors: Carrasco Ruben D. , Sukhdeo Kumar , Protopopova Marina , German Masha , Henderson Joel , Anderson Kenneth C. , DePinho Ronald A.
Published: April 2020

The transcription factor X-box binding protein-1 (XBP-1) plays critical roles in the unfolded protein response (UPR), the differentiation of plasma cells, and the regulation of growth factor signaling pathways. XBP-1 is subject to regulation by alternative RNA processing producing XBP-1-spliced (S) and -unspliced (U) mRNAs encoding proteins with identical DNA-binding and bZIP domains yet distinct C-terminal transactivation domains. The weaker transactivation potential of XBP-1(U) has prompted speculation that it may influence XBP-1(S) activity as a transdominant mutant. While elevated XBP-1 expression has been reported in transformed cells, the relative ratios of these XBP-1 isoforms and associated physiological relevance in cancer are uncertain. Here, we assessed the differential impact of enforced XBP-1(S) versus XBP-1(U) transgene expression in the B cell lineage. Both transgenes elicited early onset antibody-dependent autoimmune disease characterized by elevated levels of serum immunoglobulin (Ig) and IL-6 production, increased numbers of marginal zone and mature follicular B cells in the spleen, and expanded mature B cell populations in the bone marrow. Notably, aged XBP-1(S) mice developed clonal plasma cell expansions, culminating in the human-equivalent of Monoclonal Gammopathy of Undetermined Significance (MGUS) or Multiple Myeloma (MM). Conversely, XBP-1(U) mice develop multi-organ lymphoplasmacytic infiltrates and, with advancing age, succumb to neoplasms resembling human Lymphoplasmacytic Lymphoma/Waldenstrom’s Macroglobulinemia (LPL/WM). These unanticipated genetic observations in the mouse were translated to human disease with documentation of elevated levels of XBP-1(S) in MM and XBP-1(U) in LPL/WM. Together, these results indicate that imbalances in XBP-1(S) and XBP-1(U) alters B cell lineage homeostasis and can drive two distinct types of lymphoplasmacytic neoplasms in vivo. The findings of this study, together with the known capacity of XBP-1 to regulate various cancer-relevant growth factor signaling pathways, predicts that epigenetic dysregulation of alternate XBP-1 RNA processing can promote age-associated B cell malignancies in humans.
Expression of the Iron Regulatory Peptide Hepcidin Is Reduced in Patients with Chronic Liver Disease.
Authors: Anderson Greg , Frazer David M. , Bridle Kim R. , Macdonald Graeme A. , Smith Jeffrey L. , Turlin Bruno , Subramaniam Nathan , Crawford Darrell H. , Powell Lawrie W. , Ramm Grant A.
Published: April 2020

Disturbances in iron metabolism often accompany liver disease in humans and hepatic iron deposition is a frequent finding. Since the peptide hepcidin, a major regulator of body iron homeostasis, is synthesised in the liver, alterations in hepcidin expression could be responsible for these effects. To investigate this possibility, we studied hepcidin expression in liver biopsies from patients with hepatitis C virus (HCV) infection, non-alcoholic fatty liver disease (NAFLD) and hemochromatosis (HC). Total RNA was extracted from the liver tissue of 24 HCV, 17 NASH and 5 HC patients, and 17 liver transplant donors (controls). The levels of mRNA for hepcidin and several other molecules involved in iron metabolism (DMT1, Dcytb, hephaestin, ferroportin, TfR1, TfR2, HFE and HJV) were examined by ribonuclease protection assay and expressed relative to the housekeeping gene GAPDH. The expression of hepcidin was significantly decreased in HCV and NASH patients relative to control liver (109±16 and 200±44 versus 325±26 respectively; P=0.008 and 0.02). We have previously reported similar findings in patients with HC, and this was confirmed in the current analysis (176±21; P=0.003). In both HCV and NAFLD patients the expression of the iron reductase Dcytb and the transferrin binding regulatory molecule TfR2 was also decreased, while the cellular iron exporter ferroportin showed a significant increase. Levels of the mRNA for the iron oxidase hephaestin were lower in HCV patients alone, while expression of the major transferrin binding molecule TfR1 was decreased only in NAFLD patients. Of particular interest was the finding that the expression of HJV (which is mutated in patients with juvenile HC) was significantly increased in NAFLD patients. No changes were seen in the expression of the iron importer DMT1 or the regulatory molecule HFE. Decreased expression of hepcidin in patients with HCV and NAFLD provides an explanation why iron homeostasis could be perturbed in these disorders. Reduced hepcidin levels would increase intestinal iron absorption and iron release from macrophages, which could contribute to hepatic iron accumulation. This in turn could lead to alterations in the expression of various proteins involved in iron transport and its regulation. Indeed most of the changes in the expression of such molecules observed in this study are consistent with this. However, the mechanisms leading to changes in the expression of hepcidin in these diseases remain to be elucidated.
Efficacy of Alpha-Lipoic Acid in Iron-Induced Oxidative Stress.
Authors: Lal Ashutosh , Suh Jung H. , Ames Bruce N.
Published: April 2020

Antioxidant supplementation could reduce organ damage and premature death in patients with iron overload. We evaluated the improvement in iron-induced oxidative stress with α-Lipoic acid (LA), a multifunctional thiol antioxidant and inducer of phase II enzymes, and compared it with benzylhydroxylamine (BH), a reducing agent that protects against free radicals. Human fibroblasts (IMR-90) were grown in regular cell culture medium or with ferric ammonium citrate (FAC). The intracellular iron content was measured by inductively-coupled plasma spectrometry (ICP). Either LA or BH was added on day 2, and cells grown to confluence. Oxidative stress was assessed by dichlorodihydrofluorescein diacetate fluorescence (DCF-fl). GSH:GSSG was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total intracellular iron content (mean±SEM, ng per 5x10^6 cells) increased from 66.95±6.51 in control cells to 262.26±4.69 after exposure to 25 μM FAC, and 556.26±21.15 after exposure to 125 μM FAC. The increase in iron-content of the IMR-90 fibroblasts caused significant rise in oxidant appearance at the highest concentration, as evidenced by 27.8±2.6% increase in steady-state DCF-fl compared with control cells (p <.001). Addition of increasing concentrations of LA attenuated iron-mediated rise in DCF-fl in dose dependent manner. LA at concentrations above 25 μM completely abolished the iron-dependent rise in DCF-fl (91.9±2.3% for FAC+LA, p <.001). BH treated cells also exhibited a dose-dependent decrease in DCF-fl, but a much higher concentration (>200 μM) was needed for complete attenuation of iron-induced rise in DCF-fl (108.3±3.4%, p <.001). These results suggest that LA is more effective than BH in ameliorating iron-mediated increase in oxidative stress. To further understand the basis for the efficacy of LA, intracellular GSH and GSSG were measured. Results show that LA improved the total GSH levels and its redox state in a dose dependent manner. LA and BH attenuated iron-mediated oxidant production, with LA exhibiting greater efficacy. This effect may be due to potent ability of LA to improve intracellular GSH levels and redox state. Our results suggest LA may be useful in reducing complications from iron overload.
Thiamine Deprivation Induces Reticulocytopenic Anemia in Slc19A2 Knockout Marrow: Evidence for Cell-Intrinsic Defect.
Authors: Montefusco Maria Claudia , Fleming Judith C. , Chick Gretchen , Neufeld Ellis J.
Published: April 2020

Megaloblastic anemia, diabetes, and sensorineural deafness characterize the autosomal recessive human disorder, Thiamine-Responsive Megaloblastic Anemia syndrome (TRMA). TRMA is due to mutations of the human SLC19A2 gene, which encodes a high-affinity thiamine membrane transporter. The murine Slc19a2 knock-out (-/-) model (Fleming et al, Mol Gen Metab; 80 (2003), 234–241) develops thiamine-dependent reticulocytopenia without megaloblastic anemia or sideroblasts, but the whole animal knockouts become ill in thiamine-depleted settings, which limit investigation of erythropoiesis. Here we investigate, by bone marrow transplantation experiments, whether the erythropoietic defect is cell-intrinsic and correlated with the correct function of Slc19a2. We conducted this study by transplanting C57BL/6J-GPIb Slc19a2−/− (−/−) or wild type (+/+) bone marrow into C57BL/6J-GPIa wild type (+/+) mice. These congenic strains were chosen to distinguish host and donor cells by GPI isozymes. After host irradiation (525 cG for 2 doses), we injected 4 X106 donor marrow cells retro-orbitally. After engraftment, confirmed by GPIa/b assay (>60 days), we switched animals to either 2mg/kg chow thiamine diet (control) or 0 mg/kg thiamine (normal mouse chow is 22 mg/kg thiamine). The transplant experiments were carried in duplicate and included a total of 53 transplant recipient mice of both sexes. After 24-day diet both +/+ and −/− groups on 0 mg/kg thiamine diet lost weight (~28%). Blood parameters changed unequally. After 24 days on 0 mg/kg thiamine diet mice transplanted with −/− bone marrow reveled a significant increment of reticulocyte mean cell volume (MCV) (23% with P< 0.01) together with a relevant decrease of red blood cell count (46% and P< 0.01). A severe leukopenia diet correlated was also observed in both groups of mice on thiamine-depleted diet. The blood parameters of mice on 2-mg/kg diet did not reveal significant changes along the entire diet period. These results confirm the involvement of Slc19a2 membrane transporter in the impaired thiamine cell uptake and allow us to say that the erythropoietic defect we first observed in Slc19A2 KO mice is not only diet related, but due to a cell-intrinsic marrow defect.

Tables. Peripheral blood parameters of bone marrow transplanted mice at baseline and after 24 days on depleted thiamine diet.

BASELINE Donor WBC RBC MCV Retic −/− mean (StDev) 20.41 (4.23) 10.5 (3.46) 61.83 (2.38) +/+ mean (StDev) 20.63 (2.36) 9.24 (1.45) 67.4 (3.03) P 0.9140 0.4257 0.0136 24 days on 0 mg/kg thiamine diet Donor WBC RBC MCV retic St Dev = Standard Deviation P = probability −/− mean (StDev) 8.53 (1.75) 6.79 (1.65) 76.18 (1.24) +/+ mean (StDev) 6.51 (3.16) 6.09 (1.37) 65.23 (5.04) P 0.3232 0.0321 0.0011
Differential Patterns of Transcriptional Protein Expression May Explain Functional Differences between Hematopoietic Progenitors Derived from Human ESC’s and Fetal Hematopoietic Tissues.
Authors: Shaaban Aimen F. , Golos Thaddeus G. , Thomson James A. , Rajesh Deepika
Published: April 2020

Recent studies suggest that development of human embryoid body-derived hematopoietic progenitor cells (EB-HPC’s) mirrors that of primitive yolk sac-derived hematopoietic progenitors (YS-HPC’s) in their expression of transcription factors known to be critically associated with hematopoietic development. However, the findings of these studies were limited to molecular characterization of heterogeneous EB cultures rather than analysis of hematopoietic lineage-committed cell subsets. We wondered: 1) if the expression patterns were consistent at the protein level in both bulk EB cultures and subsets of hematopoietic-lineage committed cells; and 2) if these expression patterns differed from those found in definitive HPC’s harvested from early fetal hematopoietic tissues. To answer these questions, we compared the intracellular expression of critical transcriptional regulatory proteins from human and rhesus EB-derived HPC’s (hEB-HPC’s and rEB-HPC’s) with hematopoietic progenitors from rhesus fetal liver (rFL-HPC’s) and rhesus placental blood HPC’s (rPB-HPC’s). Rhesus macaque fetal tissue was used in lieu of scarce human fetal tissues. Using multi-color intracellular flow cytometry we correlated the expression of transcriptional proteins (SCL/TAL1, GATA-1, GATA-2, Oct-3/4, HOXB4) with cell surface determinants of hematopoietic commitment (CD34, CD31, CD45) in hEB-HPC’s, rEB-HPC’s, rFL-HPC’s and rPB-HPC’s. Human and rhesus EB16 cells were harvested from identical culture conditions and compared to 0.34 gestation rFL-HPC’s and rPB-HPC’s. The frequency of CD45+ cells was higher in hEB-HPC’s (31%) and rPB-HPC’s (46%) compared to rFL-HPC’s and rEB-HPC’s. A much higher frequency of GATA-1 and HOXB4 expressing cells was seen in both of the fetal cell types when compared to either rhesus or human EB’s. Additionally, the frequency of GATA-2 and SCL expressing cells was comparable in all cell types. Lastly, the expression of Oct3/4 was higher in rPB-HPC’s and rFL-HPC’s when compared to bulk EB cultures. However, analysis of subsets of EB cells expressing hematopoietic antigens (CD45, CD34) revealed co-expression of Oct-3/4 on both CD45+ and CD34+ cell fractions. From these findings, we conclude:

critical differences exist in GATA-1 and HOXB4 expression between HPC’s derived from EB cultures and HPC’s harvested during early definitive fetal hematopoietic development; and human EB-derived hematopoietic progenitor cells express persistently high levels of Oct-3/4.

The differences in the expression of these critical transcriptional factors at the protein level may explain the observed functional differences between EB-derived HPC’s and definitive HPC’s from fetal and adult sources.
Slug Antagonizes p53-Mediated Apoptosis of Hematopoietic Progenitors by Repressing Puma.
Authors: Wu Wen-shu , Heinrichs Stefan , Xu Dong , Adams Jerry M. , Look A. Thomas
Published: April 2020

The discovery that Slug has a vital anti-apoptotic role in the normal response of hematopoietic progenitors to genotoxic stress compelled us to investigate the underlying cellular and molecular mechanism(s) of this effect. Here we show that, although the development of myeloid progenitors in Slug−/− mice is not impaired under steady-state conditions, their ability to repopulate the compartment after γ-irradiation is reduced. We demonstrate that the radiation-induced death of Slug−/− mice exclusively reflects bone marrow failure and show that Slug protects mice from γ-irradiation-induced death in a cell-autonomous manner. We also establish that Slug confers radioprotection by inhibiting the mitochondria-dependent apoptotic pathway activated by γ-irradiation in hematopoietic progenitors. We show that Slug acts as a transcriptional repressor by directly antagonizing p53-mediated upregulation of the BH3-only gene Puma, which was recently shown to encode a critical mediator of p53-induced apoptosis. Finally, we provide evidence for a novel feedback loop in the hematopoietic progenitor cells after DNA damage: Slug is itself induced by p53. Thus, the survival of hematopoietic progenitor cells after genotoxic stress relies on induction of Slug by p53 and Slug-mediated repression of Puma.
A Natural Variant Sequence in the AHSP Gene May Impact Severity of Beta-Thalassemia.
Authors: dos Santos Camila O. , Zhou Suiping , Albuquerque Dulcineia M. , Saad Sara , Weiss Mitchell J. , Costa Fernando F.
Published: April 2020

Alpha-hemoglobin stabilizing protein (AHSP) is an erythroid-specific molecular chaperone that binds the alpha chains of hemoglobin, preventing their precipitation and deleterious effects. Loss of AHSP exacerbates alpha globin precipitation and anemia in a murine model for beta thalassemia. In vitro, recombinant AHSP inhibits the production of reactive oxygen species (ROS) by alpha hemoglobin. To further define the role of AHSP as a modifier of beta-thalassemia, we analyzed AHSP sequences for mutations in a large population of beta-thalassemic and control subjects. A single nucleotide change, from A to T at position 12888 (Genebank accession number AC106.730.2) in the third exon of the AHSP gene was identified, converting asparagine 75 to leucine (N75I). We detected a patient who was heterozygous for both beta-thalassemia (b39/bA) and the AHSP-N75I mutation. She presented with an unusually severe anemia that required regular blood transfusion. The father is hematologically normal, without any thalassemia mutation and heterozygous for AHSP-N751. The mother is heterozygous for the beta 39 mutation with classical mild hypochromic microcytic anemia and no mutation in the AHSP gene. Of 282 unrelated control subjects tested, one (0.35%) contained the N75I mutation. Analysis of red blood cells from this subject revealed normal hemoglobin indices but a small number of Heinz bodies, suggesting hemoglobin precipitation. This finding is similar to what we observed previously in mice that are heterozygous for an AHSP null allele. To investigate how the N75I mutation affects AHSP function, we studied the biochemical properties of the recombinant mutant protein. Surface plasmon resonance showed that the binding affinity of AHSP N75I for alpha hemoglobin is normal. Importantly, compared to wild type AHSP, the N75I mutant protein exhibited significantly reduced capacity to inhibit ROS production by alpha hemoglobin. Hence, AHSP N75I may be less effective at conferring protection from oxidative-mediated damage by free alpha hemoglobin in erythrocytes. These effects, when coupled with beta thalassemia, could result in more severe anemia, implicating AHSP N75I as a potential genetic modifier.
Searching for Disease Modifiers Genes in Thalassemia.
Authors: Fucharoen Suthat , Sripichai Orapan , Winichagoon Pranee , Abel Kenneth , Braun Andreas
Published: April 2020

β0-thalassemia/HbE disease is one of the most common thalassemias in Southeast Asia. Patients with compound heterozygote for HbE and β0 mutant alleles display remarkable variability in clinical expression, ranging from nearly asymptomatic to severe, transfusion-dependent disease. It is believed that additional genetic factors modifying disease severity may account for this variability. A universal platform technology (MassARRAY) based on mass spectrometry for analyzing nucleic acids at a high level of precision and accuracy has been developed. To identify genetic modifiers influencing severity among 1060 β0-thalassemia/HbE patients, we are utilizing this technology to conduct a genome-wide association study involving approximately 110,000 gene-based single nucleotide polymorphisms (SNPs). This assay panel corresponds to SNPs with a median spacing of 10.4 kilobases, in approximately 99% of all known and predicted human genes. DNAs from approximately 200 regionally matched patients representing the extremes of mild and severe cases were included in each DNA pool. Allele frequencies for all SNPs were estimated in both pools, and those showing suggestive significant differences (p values <0.02) were selected for verification by repeated pooled DNA analysis. Approximately one-fourth of these showed reproducible allelic differences at p<0.05, and more than 600 were selected for genotyping the individual patient DNAs in order to determine precise allele and genotype frequencies. A number of SNPs showed evidence for association with disease severity, including several in reported quantitative trait loci (QTLs) associated with fetal hemoglobin HbF levels. However the most strongly associated SNPs were within a region on chromosome 11 distinct from the beta globin gene cluster, within which most analysis to date has focused.
Oxidative Stress of RBC in a Murine Model of Beta-Thalassemia Can Be Reversed by Treatment with Antioxidants.
Authors: Fibach Eitan , Amer Johnny , Rachmilewitz Eliezer , Guy Ella , Rivella Stefano
Published: April 2020

Oxidative stress is a prominent contributor to the premature destruction of RBC as well as anemia in thalassemia and sickle cell anemia. The oxidative status within RBC is maintained by the balance between oxidative systems, such as Reactive Oxygen Species (ROS), and antioxidative systems, such as reduced glutathione (GSH). Using flow cytometric methods, we previously showed that RBC obtained from patients with thalassemia (Amer et al. Eur J Haematol70:84,2003; Cytometry60:73,2004) or sickle cell anemia (Amer et al. Blood, 104:972a,2004) exhibit oxidative stress. In the present study, we assessed the extent of RBC oxidative stress and the effects of antioxidant administration, using the thalassemic mouse model Th3/+. This model closely mimics the phenotype observed in patients affected by beta-thalassemia intermedia, such as low hemoglobin levels (7 to 9 gr/dL), splenomegaly and iron overload. In addition, the mature RBC have a shorter life-span and are characterized by anisocytosis, poikilocytosis and hypochromatism.

RBC were derived from normal and thalassemic mice before and 4 hours after i.p. injection of the antioxidants N-acetyl cysteine (NAC), vitamin C (Vit. C) or tocotrinol - a mixture of vitamin E derivatives, at a dose of 150 mg/kg. Intracellular ROS was determined in dichlorofluorescin diacetate-stained RBC following stimulation with 2 mM H2O2; GSH content was assessed in RBC stained with mercury orange. Cells were analyzed by flow cytometry: RBC were gated according to size (forward light scatter) and granularity (side light scatter), their fluorescence was measured and the Mean Fluorescence Channel (MFC) was calculated. Fig. 1 shows the average MFC of ROS and GSH of normal and thalassemic mice treated or not treated with anti-oxidants (N=6 in each group).

The results show a significantly higher (2.6-fold) production of ROS and lower (three-fold) levels of GSH in RBC from the thalassemic mice versus those in RBC from normal mice. Administration of antioxidants decreased the ROS of normal and thalassemic RBC by 1.4-fold and 2.6-fold, respectively, whereas GSH levels were significantly increased both in the normal (2.7 fold) and in the thalassemic (9.4-fold) RBC. The results show that the RBC of thalassemic mice are under oxidative stress that could be ameliorated by a short antioxidant treatment. Hence, this mouse model recapitulates the oxidative stress found in thalassemic patients and can serve as a model for studying the effects of antioxidant therapy. The flow cytometry methodology used is helpful in following up the results of the treatment and in evaluating its efficacy in reducing oxidative stress.

Figure Figure
Developmental and Differential Changes of Gene Expression in Erythroid Progenitor Cells.
Authors: Cokic Vladan P. , Bhattacharya Bhaskar , Puri Raj K. , Schechter Alan N.
Published: April 2020

During erythropoiesis and human development different globin genes (α, β, γ, δ and ε) are expressed as a result of globin gene switching. We investigated globin gene expression in comparison to the expression of other genes in erythroid progenitor cells (EPC) during ontogenesis using in-house produced microarrays containing 16,659 oligonucleotides. Human primitive CD34+ cells were isolated from fetal liver (FL), cord blood (CB), adult bone marrow (BM), peripheral blood (PB) and mobilized peripheral blood (mPB), and developed into EPC in the presence of erythropoietin and other cytokines. The differentiation to EPC was confirmed by flow cytometry as 100% cells were CD71+. In microarray studies, a total of 2996 genes were highly expressed in FL, 2673 genes in CB, 2580 in mPB, 1465 in PB and 1259 in BM derived EPC. 661 of these genes were common for all type of cells. The high level of expression, beside globin genes, was observed for the following genes: transferrin receptor, proteoglycans, ALAS2, Charcot-Leyden crystal protein, nucleophosmin, eosinophil peroxidase, myeloperoxidase and ribonucleases. Most of the analyzed genes demonstrated down-regulation during ontogenesis (elastase 2, glutathione peroxidase 1, SERPINB1, nudix, mitochondrial proteins, ribosomal proteins, enthoprotin, serine proteinase inhibitor), but some showed up-regulation (hexokinase, superoxide dismutase 2, spectrin). Besides developmental changes of globin gene expression during ontogenesis, we also analyzed changes in their expression during erythropoiesis in these different tissues by quantitative PCR. Beta-globin gene expression reached the maximum levels in cells of adult blood origin: BM (176 fmol/μg) and PB (110 fmol/μg). Gamma-globin gene expression, of FL origin, had steady levels during erythroid differentiation (20 fmol/μg), whereas cord blood derived EPC demonstrated consistent up-regulation (60 fmol/μg) in contrast to cells originated from adult blood (3–15 fmol/μg at day 14th). G protein related genes and histone deacetylases were elevated in CB derived EPC, concomitant with increased gamma-globin gene expression. We also analyzed the gamma-globin induction by hydroxyurea, a well known inducer, and established which G protein-coupled receptors involved pathways are activated in PB derived EPC: dopamine receptors D1, D2 and D5, beta 2 adrenergic receptor, human DP prostanoid receptor and prostaglandin E receptor 1, as well as genes activated by cAMP/PKA, PI-3 kinase, MAP and NO/cGMP pathways. This study establishes concomitant changes in expression of globin genes and other known and/or previously unrecognized genes, which appear to be involved in erythropoiesis.
Human Beta-Globin Expression during Erythroid Cells Development in Transgenic Mice Bearing Human Beta-Globin Gene with Mutated BP1 Binding Site.
Authors: Zoueva Olga P. , Bodine David , Rodgers Griffin P.
Published: April 2020

Binding of beta protein 1 (BP1) to its site on the promoter of adult beta-globin gene has silencing effect on beta-globin transcription in vitro. To better understand the mechanism of the negative regulation of beta-globin expression by BP1 we have developed transgenic mice. Specifically, we introduced a mutated BP1 binding site into the promoter of beta-globin gene sequence of 35 kb cosmid construct. This construct containing the micro-LCR and other essential elements of human beta-globin gene cluster was microinjected into the single cell mouse embryos. To detect the differences in developmental regulation of the human beta-globin gene expression in the transgenic mice, we studied the yolk sac derived embryonic blood at embryonic day 10.5 (E10.5) and the fetal liver of mouse embryos at E13.5. In addition, we analyzed adult erythroid cells. To minimize experimental error, samples from individual animals of three transgenic lines were analyzed independently using real-time PCR assays. Levels of expression of murine alpha-globin mRNA were used as internal controls. The BP1 gene and its mouse analog Dlx4 belongs to the Distal-less family of homeobox genes, which are expressed during early development. We found that the mRNA levels of human beta-globin in transgenic mice containing mutated BP1 binding site were higher at all stages of erythroid cells development as compared with control transgenic mice bearing cosmid construct with wild type sequence of BP1 site. Particularly, we detected up to 20-fold increase in human beta-globin expression in embryonic blood at E10.5, 3-fold increase in fetal livers of transgenic mice at E13.5, and up to 1.4-fold increase in adult reticulocytes. We also found that increase in human beta-globin expression was correlated with expression pattern of murine Dlx4 which mRNA was predominantly expressed in embryonic blood at E10.5. Thus, our data indicate that transgenic mice bearing human beta-globin gene with mutated BP1 site have significantly higher human beta-globin transcripts levels in blood cells from primitive erythropoesis than control mice. These results may help develop the novel clinic approaches for the inhibition of the expression of abnormal beta-globin genes, such as sickle (hbs) and hbc.
Allogeneic Myelo-Ablative Stem Cell Transplant (SCT) as Salvage Therapy of Reduced-Intensity Conditioned (RIC) SCT: Toxicity and Outcome.
Authors: Cordonnier Catherine , Hamladji Rose-marie , Gratwohl Alois , Ljungman Per , Martino Rodrigo , Bordigoni Pierre , François Sylvie , Deconninck Eric , Buzyn Agnes , Bay Jacques-Olivier , Fouillard Loic , Kuentz Mathieu
Published: April 2020

RIC regimens are mainly used for patients who cannot benefit from the myelo-ablative (MA) approach, due to age, previous SCT, or comorbidities, based on an expected early toxicity of MA regimen. Some centers may also choose the RIC strategy in young patients because they have no facilities for the management of prolonged neutropenia. The development of RIC has been so encouraging that programs now compare MA and RIC in patients who could tolerate both approaches. However, RIC may fail, and MA SCT be secondary considered.

Objective: The goal of this retrospective survey was to assess the toxicity and outcome of MA after RIC SCT.

Patients: 17 patients (14–63 y; 6 CML, 4 AML, 2 CLL, 2 myeloma, 1 NHL, 1 CMML, 1 myelodysplastic syndrome) received a MA SCT after RIC, for relapse (n=12), graft rejection (n=4), or in a planned program (n=1), from an HLA-id sibling (n=12), a familial mismatched donor (n=1), an unrelated donor (n=3) or a cord blood (n=1). The reason for initial RIC approach was age in 4, a research program in 6, comorbidities in 3, and previous autologous SCT in 4 patients. RIC included Fludarabin (Flu)-Busulfan (BU) in 8, Flu-2Gy irradiation in 4, Cyclophosphamide (CPM)-antithymocyte globulin (ATG) in 1, and various Flu- regimens in 4. One patient had 2 consecutive RIC SCTs before MA SCT. The median delay between RIC and MA SCT was 9 months (range: 2.5–36 months). The same donor was used for the 1st and 2nd SCT in 12/17 cases. The MA transplant was conditioned with BU-CY (±VP16) in 9, Cyclo-TBI in 4, and other regimens in 4 patients. The median follow-up after MA SCT was 9 months

Results: After the MA transplant, 1 patient developed veno-occlusive disease, 2 developed interstitial pneumonia. Five patients died from relapse between 8 and 21 mo after the MA SCT. Seven (41%) died from transplant-related causes at a median of 8 months (GVHD:3; infection:1; multi-organ failure:1; thrombotic microangiopathy:1; EBV proliferative disease:1). 5/17 (29.5%) patients are alive and well 4.5 to 26 months after MA transplant. Three of them have extensive chronic GVHD while they did not develop any GVHD after RIC. The delay between the 2 transplants did not influence the outcome.

Conclusion: This survey shows that MA after RIC SCT is feasible, and considering that most of these patients have been initially eligible for RIC because of their age or comorbidities, MA SCT had an acceptable toxicity. It may be considered for patients who relapsed or rejected after RIC transplant.
Inhibition of Pathological Retinal Neovascularization by α-Defensins.
Authors: Chavakis Triantafyllos , Bdeir Khalil , Cines Douglas B. , Fogt Franz , Bdeir Yasmina , Lubkowski Jacek , Preissner Klaus T. , Hammes Hans-Peter , Economopoulou Matina
Published: April 2020

Proliferative retinopathies, such as those complicating prematurity and diabetes, are major causes of blindness. A prominent feature of these retinopathies is excessive neovascularization, which is orchestrated by the hypoxia-induced vascular endothelial growth factor (VEGF) stimulating endothelial cells, and the integrin-mediated adhesive interactions of endothelial cells with extracellular matrix components like fibronectin (FN). Recently, we demonstrated that α-defensins interfere with α5β1-FN interactions and dependent endothelial cell functions (FASEB J., 2004, 18:1306–8). Here, α-defensins were studied in hypoxia-induced proliferative retinopathy. In vitro, α-defensins specifically inhibited α5β1-integrin dependent migration of bovine retinal endothelial cells (BREC) to FN, attenuated the VEGF-stimulated increase in endothelial permeability, and blocked BREC proliferation and capillary sprout formation in three-dimensional fibrin-matrices. An upregulation of β1-integrin and FN was observed in the retinal vessels in the mouse model of hypoxia-induced retinal angiogenesis. Systemic and ocular administration of α-defensins reduced retinal neovascularization by 45% and 60%, respectively, and this effect was comparable to the inhibitory effect of α5β1-blocking antibody. α-defensins were detected in human diabetic retinas but were absent in retinas of eyes removed because of trauma. Together, these data show that α-defensins inhibit pathological retinal neovascularization in vivo and may provide a clinically efficient strategy against proliferative retinopathies.
Role of Platelets in Homing of Endothelial Progenitor Cells to the Site of Vascular Injury.
Authors: Lev Eli I. , Estrov Zeev , Aboulfatova Khatira , Harris David , Granada Juan F. , Kleiman Neal S. , Dong Jing-fei
Published: April 2020

Endothelial Progenitor Cells (EPCs) are undifferentiated bone marrow cells that are rapidly mobilized upon vascular or tissue injury. The circulating EPCs home to and differentiate at the site of vessel injury to enhance repair of damaged endothelium. Much has been learnt of how EPCs adhere to and transmigrate on endothelial cells during angiogenesis, but little is know about how EPCs home to the site where endothelial cells are denuded and subendothelium is exposed following vascular injury. We hypothesized that platelets mediate EPC homing because they are the first cells that tether and adhere to subendothelium exposed by vascular injury. To test this hypothesis, we examined the platelet-EPC interaction under static and flow conditions. Human EPCs were purified from buffy coats by cell sorting using CD133 and VEGFR-2 as the cell markers. The EPCs were then incubated with washed platelets that were either at resting state or activated by 5 mg/ml of collagen. EPC-platelet interaction was measured in the form of heterotypic aggregation between two types of cells by flow cytometry (dual labeled with FITC-CD42a and PE-CD133). We found the EPCs attached to resting platelets, however, the percentage of platelets binding to EPCs significantly increased when platelets were activated by collagen (1.02% vs. 2.76%, p < 0.05). EPC-platelet aggregation was similarly detected in whole blood and platelet-rich plasma. When perfused over a monolayer of activated platelets under a shear stress of 2.5 dyn/cm2, the CD133 positive EPCs tethered to the platelet monolayer. The tethered EPCs either firmly adhered immediately or rolled a short distance before becoming adherent. This EPC-platelet interaction was calcium-dependent and inhibited by up to 60±11.5% by a polyclonal P-selectin antibody. In comparison, the monoclonal GP Iba antibody AK2, which blocks GP Ib-VWF interaction, had no effect. These results demonstrate for the first time a significant interaction between EPCs and activated platelets both at static and flow conditions. Our findings suggest that platelets may function as carrier cells that direct EPCs to the site of vascular injury. In addition to homing, platelets, which release several growth factors known to affect EPCs, may provide a supporting matrix on which adherent EPCs proliferate and differentiate.
GAS-6 Rescues Endothelial Cells from Apoptosis through FOXO1 Inactivation.
Authors: Ganopolsky Jorge , Varnum Brian , Blostein Mark
Published: April 2020

Growth Arrest Specific gene product 6 (GAS-6), a γ-carboxylated protein expressed in quiescent fibroblasts and endothelial cells, exerts an anti-apoptotic function by binding to the receptor tyrosine kinase Axl. Recently, our laboratory has demonstrated that gas6-Axl interactions activate PI3-kinase with subsequent Akt phosphorylation during gas6-mediated protection from apoptosis. The current study explores further the mechanism by which this survival mechanism is achieved. FOXO1 is a member of the Forkhead family of transcription factors that plays a role in the expression of pro-apoptototic genes. Phosphorylation of FOXO1 at Thr24, Ser256 and Ser319 results in phospho-FOXO1 translocation from the nucleus to the cytoplasm, with consequent suppression of FOXO1 transcriptional activity and inhibition of apoptosis. In the present study we show, for the first time, that the treatment of serum-starved endothelial cells with 100 ng/ml of GAS-6 induces FOXO1 phosphorylation in a time dependent manner. Phosphorylated FOXO1 is translocated from the nucleus to the cytoplasm as evidenced by Western blot analysis of both nuclear and cytoplasmic extracts. Using fluorescence microscopy, FOXO1 is found predominantly in the nucleus during apoptosis induced by serum starvation. Upon gas6 stimulation, phosphorylated FOXO1 is translocated to the cytoplasm (see Figure 1). It is suggested that anti-apoptotic genes are then released from suppression and are thereby able to mediate cell survival. Both FOXO1 phosphorylation and translocation are suppressed by Wortmannin, a PI3-kinase inhibitor demonstrating that FOXO1 phosphorylation is PI3-kinase dependent. These results provide mechanistic insight of how gas6 rescues endothelial cells from serum-starvation-induced apoptosis.

Foxo1 distribution Foxo1 distribution
In Vitro Characterization of Umbilical Cord Blood CD133+ and In Vivo Functionality in Response to Ischemia in a Murine Hind-Limb Injury Model.
Authors: Finney Marcie R. , Greco Nicholas G. , Joseph Matthew E. , Winter Daniel G. , Bhakta Shyam , Fanning Laura R. , Kozik Margaret , Banks Jason , Huang Ying , Pompili Vincent J. , Laughlin Mary J.
Published: April 2020

Previous reports have demonstrated efficacy of cellular therapy in mediating therapeutic angiogenesis in response to ischemia. We sought to determine the potential efficacy of adult umbilical cord blood (UCB) derived selected CD133+ cells in the murine hind limb ischemia model and to characterize these cells by surface phenotype and functionality prior to injection.

Methods and Results: Mononuclear cells (MNC) from UCB were labeled with CD133+ conjugated magnetic beads, followed by automated sorting through magnetic columns (Miltenyi). Routine yield of CD133+ cells was 0.51 ± 0.2% of MNC, with a purity of 78.7 ± 2.4% (n=30). Surface expression in the UCB CD133+ population was 3.59 ± 1.49% KDR(VEGFR2), 8.66 ± 3.79% CXCR4 and 22.74 ± 2.84% CD105 compared to 7.14 ± 2.15% KDR, 28.54 ± 5.81% CXCR4 and 6.74 ± 2.07% CD105 in the UCB MNC population. Transwell plates with 5μm collagen coated filters (Costar) were used to observe chemotactic migration of MNC or CD133+ cells towards SDF-1 (100ng/mL) compared to control wells containing media alone. Following a 3 hour incubation, the cells migrating to the bottom wells were counted by flow cytometry with TruCOUNT™ tubes (BD Biosciences). MNC and CD133+ cells migration to SDF showed a 4.9 ± 2.9 and 1.8 ± 0.7 fold increase over the negative control respectively. To test vasculogenic functionality of these selected cell populations, NOD/SCID mice underwent ligation of the right femoral artery and were randomized into 3 study groups: control (endothelial media with cytokines), non-selected MNC (1 x 106 cells/mouse) or CD133+ (0.5 x 106 cells/mouse) given via intracardiac injection immediately after injury. Doppler flow measurements were taken on both limbs each week for 4 weeks and the ratio of perfusion in the ischemic/healthy limb was calculated. At 28 days, perfusion ratios were statistically higher in study groups receiving CD133+ cells from UCB, 0.55 ± 0.07 (n=8) compared to cytokine controls 0.39 ± 0.02 (n=10, p=0.019). Mice receiving MNC did not show statistically significant improvement over control animals 0.42 ± 0.06 (n=7, p=0.27).

Conclusion: Surface phenotyping was notable for increased expression of the receptor for SDF-1, CXCR4 on MNC when compared to CD133+ cells. In vitro functional assays showed that CD133+ and MNC exhibited increased chemotactic migration to SDF-1. In vivo studies showed that injection of UCB CD133+ cells improved blood flow compared with cytokines alone in the murine hind limb injury model, highlighting the vasculogenic potential of CD133+ cells from UCB.