bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Mcl-1 as a Novel Target in Neoplastic Mast Cells and Demonstration of Cooperative Growth-Inhibitory Effects of mcl-1 Antisense Oligonucleotides, PKC412, and AMN107.
Authors: Aichberger Karl J. , Mayerhofer Matthias , Gleixner Karoline V. , Krauth Maria-Theresa , Derdak Sophia , Pickl Winfried F. , Wacheck Volker , Selzer Edgar , Agis Hermine , Sillaber Christian , Valent Peter
Published: April 2020

Mcl-1 is a Bcl-2 family-member that has been described to act anti-apoptotic in various myeloid neoplasms and therefore has been proposed as a potential therapeutic target. Systemic mastocytosis (SM) is a myeloid neoplasm involving myelomastocytic progenitors. We examined the expression and functional role of Mcl-1 in neoplastic mast cells (MC), to determine whether Mcl-1 could serve as a target in MC neoplasms. As assessed by RT-PCR and immunohistochemistry, primary neoplastic MC were found to express Mcl-1 mRNA and the Mcl-1 protein in a constitutive manner in all patients analyzed. Moreover, the human MC-leukemia cell line HMC-1 was found to express Mcl-1. Transfection of these cells with Mcl-1-specific antisense oligonucleotides (ASO) or an mcl-1-specific siRNA using lipofectin resulted in a reduced survival and increased percentage of apoptotic cells compared to control. The effects of mcl-1 ASO were seen with the HMC-1.1 subclone carrying the G560V c-kit mutation (mcl-1 ASO, 250 nM: 49±4% apoptotic cells compared to control: 3±2%, p<0.05; mcl-1 siRNA: 41±5% vs control: 5±3%, p<0.05) as well as with HMC-1.2 cells carrying both the G560V c-kit mutation and the D816V c-kit mutation (mcl-1 ASO, 250 nM: 36±2% apoptotic cells compared to control: 6±1%, p<0.05; mcl-1 siRNA: 30±6% vs control: 5±2%, p<0.05). Moreover, mcl-1 ASO were found to cooperate with the tyrosine kinase inhibitors (Novartis Pharma AG) imatinib, AMN107, and PKC412 in producing growth inhibition in HMC-1.2 cells. Together, these data show that Mcl-1 is a novel survival factor and attractive target in neoplastic human MC. Whether the Mcl-1-targeting concept can be developed far enough to reach clinical application remains to be elucidated.
Inhibition of Growth of Neoplastic Mast Cells by CD44 mAb A3D8 Is Associated with G1 Cell Cycle Arrest and Apoptosis.
Authors: Boehm Alexandra , Florian Stefan , Gleixner Karoline , Sonneck Karoline , Aichberger Karl J. , Derdak Sophia , Pickl Winfried F. , Valent Peter , Sperr Wolfgang R.
Published: April 2020

Recent data suggest that CD44 may serve as a new therapeutic target in AML and possibly also in other myeloid neoplasms. Systemic mastocytosis (SM) is a myeloid neoplasm characterized by abnormal growth and accumulation of mast cells (MC) in one or multiple organs. We have previously shown that normal tissue MC express CD44. In the present study, we asked whether CD44 is expressed on neoplastic human MC and whether CD44-ligation by the monoclonal antibody (mAb) A3D8 would be associated with inhibition of growth of neoplastic MC. As assessed by flow cytometry, primary neoplastic MC were found to express CD44 in all patients with SM analyzed (n=10). The human mast cell leukemia (MCL) cell line HMC-1 was also found to express CD44. As assessed by 3H-thymidine incorporation, the CD44 mAb A3D8 decreased the proliferation of HMC-1 cells in a dose-dependent manner (A3D8, 5 μg/ml: 46±26% of control=100%, p<0.05). Similar effects of A3D8 were observed with primary neoplastic MC obtained from a patient with MCL (A3D8, 5 μg/ml: 68±20%) and one with smouldering SM (A3D8, 2.5 μg/ml: 42±7% of control, p<0.05). To analyze the mechanism of A3D8-induced growth inhibition, cell survival and cell cycle distribution were analyzed. We found that CD44-ligation induces an approximately 3-fold increase in apoptotic HMC-1 cells compared to control. As assessed by flow cytometry, we were also able to demonstrate that A3D8 induces cell cycle arrest in the G1-phase. Moreover, as assessed by Western blotting, we found that incubation of HMC-1 cells with mAb A3D8 results in an increase in the cyclin-dependent kinase-inhibitor p27Kip1. In summary, our results suggest that CD44-ligation is followed by inhibition of growth of neoplastic human MC through induction of apoptosis and G1 cell cycle arrest. The latter is probably mediated by stabilization of p27Kipl. Whether targeting of CD44 in neoplastic MC in patients with high grade MC disorders is of clinical significance remains to be determined in forthcoming studies.
A Genetic Mutation on Murine Chromosome 7 Causes Thrombocytosis Associated with Thrombopoietin Excess - A Model of Jak2V617F Negative Idiopathic Thrombocytosis.
Authors: Stevenson William S. , Metcalf Donald , Alexander Warren S. , Hilton Douglas J. , Roberts Andrew W.
Published: April 2020

A substantial proportion of patients with Essential Thrombocythemia (ET) have evidence of an acquired Jak2V617F mutation that causes the myeloproliferative phenotype. Jak2V617F negative ET patients presumably have mutations in other related pathways or alternatively may have chronic thrombocytosis secondary to factors extrinsic to the hemopoietic system. This second hypothesis is supported by clonality studies that demonstrate at least 30% of women with ET have evidence of polyclonal granulocytes. As part of an ENU mutation screen to identify novel genes associated with thrombocytosis, we have studied a mutant mouse strain, called plt2, with autosomal recessive thrombocytosis associated with increased thrombopoietin production. This mutation is linked to mouse chromosome 7, and therefore is unrelated to mutations in Jak2, thrombopoietin or the thrombopoietin receptor, c-Mpl. plt2/plt2 mice have platelet counts of 2038±347x109/L (n=107) increased by 47% compared to wild-type mice (1386±223; n=107, p<0.001). This thrombocytosis was associated with increased numbers of megakaryocyte progenitor cells (plt2/plt2 28.8±4.6; n=4, Meg-CFC/5x105 cells to maximal cytokine stimulation compared to wild-type 18.3±5.5; n=4, p<0.05) and megakaryocytes (108±15; n=7 megakaryocytes per hpf compared to 70±13; n=7, p<0.01) that demonstrated a normal maturation profile based on ploidy measurement. plt2/plt2 mutant mice exhibited mild hepatomegaly (liver weight 1.6±0.2g; n=34 compared to 1.3±0.2g; n=23, p<0.001) and serum thrombopoietin levels were elevated by 76% (4930±1309pg/mL; n=18 compared to 2802±1031; n=15, p<0.001). There was no difference in relative TPO transcription between plt2/plt2 mice and wild-type mice in the liver (p=0.23), however, the TPO content of whole liver lysates was increased by 50% in the mutant mice (150%±50; n=8 per 1g liver weight compared to 100±32; n=8, p=0.03). To examine how the plt2 mutation interacts with other components of the TPO pathway, plt2/plt2 mice were intercrossed with mice lacking the TPO receptor (Mpl−/−). Mpl−/−plt2/plt2 mice have thrombocytopenia with platelet counts that are indistinguishable from Mpl−/−/ mice confirming that the plt2 mutation causes thrombocytosis by acting through the Mpl receptor. Surprisingly, Mpl+/−plt2/plt2 mice, with only one copy of the Mpl gene, had marked thrombocytosis with platelet counts of 2656±459x109/L (n=25) increased by 92% over wild-type mice suggesting an additive effect between excess thrombopoietin and Mpl heterozygosity on platelet count. We have utilized ENU mutagenesis to identify a novel mouse pedigree with heritable thrombocytosis linked to chromosome 7 that demonstrates altered cytokine production can mimic the chronic thrombocytosis that is the cardinal diagnostic feature of ET.
Effects of AMN107, a Novel Aminopyrimidine Tyrosine Kinase Inhibitor, on Human Mast Cells Bearing Wild-Type or Mutated Codon 816 c-kit.
Authors: Verstovsek Srdan , Akin Cem , Francis Giles J. , Taghi Manshouri , Huynh Ly , Manley Paul W. , Alland Leila , Dugan Margaret , Cortes Jorge E. , Hagop Kantarjian M.
Published: April 2020

Background. Majority of adult patients with systemic mastocytosis (SM) have activating mutation in codon 816 of c-kit (CD117), a receptor on the surface of mast cells. This abnormality is responsible for the pathogenesis of the disease.

Methods. We investigated the effects of a newly designed tyrosine kinase inhibitor, AMN107, by comparing its in vitro inhibitory potency on c-kit mutated mast cell lines and patient samples with that of imatinib mesylate, another tyrosine kinase inhibitor, effective in some patients with SM. Two cell lines, subclones of HMC-1 cells, were used: HMC-1560 carrying juxtamembrane domain mutation in codon 560 of c-kit, and HMC-1560, 816 carrying both codon 560 mutation and tyrosine kinase domain mutation in codon 816 of c-kit.

Results. In HMC-1560 mast cell line carrying wild-type codon 816, AMN107 was as potent as imatinib in inhibiting cellular proliferation, with IC50 values of 108 and 74 nM respectively, while in HMC-1560, 816 cell line carrying 816 mutation, neither medication had an effect. AMN107 was also as effective as imatinib in inhibiting phosphorylation of c-kit tyrosine kinase in HMC-1560 cells. The inhibition of cellular proliferation was associated with induction of apoptosis in HMC-1560 cells. AMN107 in concentrations up to 1 uM had no effect on bone marrow mast cells carrying D816V c-kit mutation obtained from patients with mastocytosis.

Conclusions. Our results suggest similar potency of AMN107 and imatinib in mast cells that carry wild-type codon 816, but no activity against codon 816 mutation carrying cells.
The Presence of the V617F Mutation Is Associated with Higher Haemoglobin, Older Age and an Increased Risk of Thrombosis in Essential Thrombocythemia.
Authors: Cheung Betty Y.Y. , Radia Deepti H. , Pantelidis Panagiotis , Yadegarfar Ghasem , Harrison Claire N.
Published: April 2020

The acquired single nucleotide valine to phenylalanine mutation (V617F) in the JAK2 tyrosine kinase is reported at variable frequencies in patients with the Philadelphia negative myeloproliferative disorders. As yet it is unclear whether its presence has an impact upon the clinical phenotype, complications or prognosis of these conditions. We have screened 87 patients under our care with myeloproliferative diseases (28 Polcythemia Vera [PV], 50 Essential Thrombocythemia [ET] and 9 Idiopathic Myelofibrosis[IMF]) using a modification of previously reported allele specific PCR on whole blood DNA (Baxter,E.J et al 2005Lancet, 365, 1054–1061) and then correlated these results with clinical data. Overall there were 50 females and 37 males with a median age of 57.4 years (range 11–92) and median follow-up of 61.4 months (1–313). V617F was detected in 26 (93%) patients with PV, 26 (52%) ET and 2 (22%) IMF. Further analysis was performed to correlate V617F status with clinical features in the patients with ET. No survival data is presented for these patients due to the inherent bias in the analysis of fresh rather than archived samples. This will be performed in a prospective manner. However, one of the V617F positive ET patients subsequently died from AML, interestingly remaining heterozygous for this mutation. When comparing ET patients with detectable V617F against those without the mutation, the following significant differences were identified at the time of diagnosis: an older age (median 49.5 vs 39.3 years, p=0.04), higher haemoglobin (median 14 vs 13g/dl, p=0.03) and documented thrombotic events (11/26 vs 2/24, p=0.01). There were no significant differences in gender, platelet count at diagnosis or duration of follow-up. Overall thrombotic events were also more frequent in V617F positive patients (16/26 vs 7/24, p=0.02). There was no difference in thrombotic events following diagnosis (5/26 vs 5/24) or in the type of thromboses (microvasular, arterial or venous). Advanced age is a well established risk factor for thrombosis in ET, hence it is possible that V617F was associated with thrombosis as a consequence of the older age of our ET patients. Multivariate analysis was limited by small patient numbers and failed to demonstrate a significant predictive impact of either age or V617F mutation upon thrombosis. Ongoing studies will hopefully provide larger numbers for further analysis.
Death-Associated Protein Kinase-2 (DAPK2) Potentiates Erythroid Progenitor Cell Apoptosis in Response to Cisplatin, and Cytokine Withdrawal.
Authors: Fang Jing , Menon Madhu P. , Tschan Mario , Torbett Bruce , Wojchowski Don
Published: April 2020

DAPK2 is a Ca++/CAM-regulated S/T kinase which when ectopically over-expressed in 293 cells, can induce membrane blebbing and apoptosis. In contrast to DAPK1 (its tumor-suppressor orthologue), DAPK2’s expression is tissue- restricted, and presently is shown to be markedly up-regulated in developing primary erythroblasts. To assess DAPK2 function in erythroid progenitor cells, wild-type and kinase-inactive K52A forms were expressed in UT7epo cells, and an siRNA lentivirus also was developed. In this Epo-dependent human erythroid progenitor cell model, wt-DAPK2 did not significantly induce apoptosis. Sensitivity to cell death due to exposure to cisplatin, however, was markedly potentiated, and altered cell morphologies were observed. These effects depended upon DAPK2’s kinase activity, and were not mediated by DAPK2-K52A. Furthermore, siRNA-mediated knock-down of DAPK2 proved to protect UT7epo erythroid progenitor cells from apoptosis due to cytokine-withdrawal. Knock-downs were confirmed at the protein level, and no such effects were exerted by negative control siRNA constructs. Within erythroid progenitor cells, DAPK2’s pro-apoptotic effects appear to be regulated by two clinically relevant cues - cisplatin exposure, and Epo deprivation. Mechanisms that regulate DAPK2, and its proposed caspase-independent apoptotic actions, presently are under investigation in primary erythroblast systems.
Randomized, Double-Blind, Placebo-Controlled Study of Darbepoetin alfa Every 3 Weeks for the Treatment of Chemotherapy-Induced Anemia.
Authors: Taylor Kerry , Ganly Peter , Charu Veena , DiBenedetto Joseph , Kracht Karolyn , Rossi Greg , Hernandez Enrique
Published: April 2020

Background: Darbepoetin alfa (Aranesp®; DA) has been shown to be safe and effective for treating chemotherapy-induced anemia (CIA). The ability to administer darbepoetin alfa every 3 weeks (Q3W) (coincident with chemotherapy) would simplify the treatment of CIA. We report results from the first multicenter, randomized, double-blind, placebo-controlled, phase 3 clinical trial evaluating efficacy and safety of fixed Q3W administration of an erythropoietic agent.

Methods: This study enrolled subjects ≥18 years, diagnosed with anemia (hemoglobin [Hb]<11g/dL) and a nonmyeloid malignancy with ≥12 weeks of planned chemotherapy. Patients (N=391) were randomized 1:1 to receive DA 300 μg or placebo Q3W for 15 weeks. Dose adjustment rules included: increase (to 500 μg Q3W) if Hb concentration was <9 g/dL at week 4 or <10 g/dL (and had <1-g/dL increase) at week 7, or decrease (dependent on previous dose) if Hb concentration was ≥13 g/dL or had ≥1-g/dL increase in any 2-week period. Efficacy was assessed by incidence of red blood cell (RBC) transfusions and achievement of target Hb of ≥11 g/dL (not exceeding 13 g/dL), consistent with ASH/ASCO, NCCN, EORTC evidence-based practice guidelines.

Results: A total of 386 randomized patients were included in the analysis. Demographic characteristics were similar between the 2 groups. Mean (SD) Hb levels at baseline were 10.03 (0.86) and 10.05 (0.92) g/dL in the placebo and DA groups, respectively. The most common tumor types were breast (23%), colon (11%), nonsmall-cell-lung cancer (10%), and hematologic malignancies (11%; 8% Non-Hodgkin’s Lymphoma). The incidence of RBC transfusions from week 5 to the end of treatment phase (EOTP) (the primary endpoint) was significantly lower for the DA group than for the placebo group (P<0.001) (see Table). Hb levels rose steadily in the DA group through approximately week 9, increasing by a mean (SD) of 1.08 (1.28) g/dL from baseline, and then remained relatively stable (see Figure). The proportion of patients achieving Hb target range from week 5 to EOTP was significantly higher for the DA group than for the placebo group (P<0.001). Dose adjustment rules helped to maintain Hb levels within target range. The safety profile of DA was consistent with that observed in previous studies. Rapid increases in Hb concentration or increases to ≥13 g/dL were not associated with adverse events.

Conclusions: Fixed Q3W administration of DA is well tolerated and effective for the treatment of CIA.

Summary of Results Placebo Darbepoetin alfa KM = Kaplan-Meier estimate Week 5 to EOTP N=185 N=181 Transfusions, KM (95% CL) (primary endpoint) 41% (34, 49) 24% (18, 30) Achievement of target Hb, KM (95% CL) 48% (41, 56) 82% (76, 88) Week 1 to EOTP N=193 N=193 Transfusions, KM (95% CL) 47% (40, 54) 30% (23, 36) Median time to target Hb, weeks (95% CL) 12 (9, 16) 6 (3, 7) Figure Figure
Effects of Mitochondrial Ferritin Expression on Tumor Iron Metabolism and Tumor Growth in Nude Mice Xenografts.
Authors: Nie Guangjun , Chen Guohua , Sheftel Alex , Pantopoulos Kostas , Ponka Prem
Published: April 2020

Mitochondrial ferritin (MtFt) is a mitochondrial iron storage protein, whose function and regulation is largely unknown. Our previous results have shown that MtFt markedly affects intracellular iron distribution and homeostasis in mammalian cells (Blood105: 2161–2167, 2005). Using tumor xenografts, we examined the effects of expression MtFt on tumor iron metabolism and growth. H1299 parental or MtFt overexpressing cells were implanted into nude mice. As compared to control tumor xenografts, the expression of MtFt dramatically reduced the implanted tumor growth. A cytosolic iron starvation phenotype in MtFt expressing tumors was revealed by increased RNA-binding activity of iron regulatory proteins (IRPs) and, concomitantly, both an increase in transferrin receptor levels and a decrease in cytosolic ferritin. MtFt overexpression also led to a decrease in both total cellular heme content and heme oxygenase-1 levels. In addition, the expression of MtFt in tumors was associated with a decrease in aconitase activity and lower frataxin protein levels. Mitochondrial iron deposition in MtFt expressing tumors was directly observed by transmission electron microscopy. The pattern of iron accumulation in MtFt overexpressing tumor cells is remarkably similar to that observed in the mitochondria of sideroblastic anemia patients. In conclusion, our study shows that MtFt expression significantly affected tumor iron homeostasis by shunting iron into mitochondria; iron scarcity resulted in partial defects in heme and iron-sulfur cluster syntheses. It is likely that deprivation of iron in the cytosol is the cause of the significant inhibition of xenograft tumor growth.
Nitric Oxide Upregulates Ferritin Synthesis Independently of Iron Regulatory Protein-Iron Responsive Element Binding Activity.
Authors: Mikhael Marc , Kim Sangwon , Schranzhofer Matthias , Mullner Ernest W. , Ponka Prem
Published: April 2020

The remarkable post-transcriptional regulation of intracellular iron homeostasis by iron regulatory proteins 1 and 2 (IRP1 and IRP2) is touted as a classic example of translational control. During low levels of intracellular iron, IRPs bind to specific nucleotide sequences known as iron responsive elements (IRE) present in the 5′-untranslated region (UTR) of ferritin (Ft) mRNA and the 3′UTR of transferrin receptor (TfR) mRNA. Such binding during conditions of low intracellular iron, blocks the translation of Ft, the ubiquitous and major iron storing protein, while TfR mRNA is stabilized, leading to increased TfR synthesis and iron uptake. High levels of intracellular iron will abrogate IRP-IRE binding activity, de-repressing Ft translation on the one hand, while causing TfR mRNA instability on the other. This system ensures that potentially hazardous iron is safely stored when it is in abundance and acquired when its levels are low. Nitric oxide (NO), a gaseous molecule involved in a myriad of functions, is known to affect the IRP system in many ways. The NO+ redox form of nitric oxide is involved in the reversible post-translational regulation of numerous proteins (including IRP2), a process called S-nitrosylation. We previously reported that NO+ causes upregulation of Ft synthesis along with IRP2 degradation. Here we report that sodium nitroprusside (SNP), an NO+ donor, causes a dramatic and rapid increase of ferritin synthesis that for a time occurs without interfering with IRP-IRE binding activities. We also show that NO+ increases the translational efficiency of ferritin mRNA as compared to treatment with ferric ammonium citrate, an iron donor. In addition, we also report that SNP does not function as an iron donor. These results indicate that NO+-mediated upregulation of ferritin synthesis is, in part, due to an IRP-independent post-transcriptional mechanism of regulation.
A New Perspective on the Natural History of Hereditary Hemochromatosis.
Authors: Waalen Jill , Beutler Ernest
Published: April 2020

A few homozygotes for the C282Y mutation of HFE manifest severe and even fatal iron-storage disease; most have only a modest increase in iron stores, show no clinical manifestations, and have no shortening of lifespan. Screening for hemochromatosis has been advocated so that therapeutic phlebotomy could be offered to the mildly affected subjects, often termed "pre-cirrhotic", presumably protecting them against progression of the disease. The rationale of this approach depends upon the implicit assumption that the natural history of the disease is one of progressive accumulation of iron and consequently ever-increasing tissue damage. In 1998 we began screening for the C282Y homozygous genotype among patients in the Kaiser Permanente Health Appraisal Clinic in San Diego. We have now followed 144 patients from this cohort of 156 C282Y homozygotes with medical records spanning up to 10 years, including 35 patients who declined phlebotomy. Data from this analysis and other sources now suggest that the natural history of the disease might be quite different than has heretofore been assumed. Our revised perspective is based upon both cross-sectional and longitudinal observations. Cross-sectional analysis of levels of serum ferritin, a surrogate for total body iron, shows little or no change with age in C282Y homozygotes. Neither are abnormal liver function tests, which occur in a small minority of homozygotes, age-related. In Denmark, longitudinal analyses of serum ferritin levels in untreated patients show no increase over as much as 20 years. Our own longitudinal data on serum ferritin levels in homozygotes who were not phlebotomized confirm this observation. Longitudinal analyses of abnormal ALT levels in homozygotes show no upward trend over a four-year period. Accordingly, it seems likely that in hemochromatosis the maximum extent of disease has been achieved in most patients by the time they reach adulthood, and that patients who are diagnosed with mild disease are unlikely to show progression.
Human Brain Endothelial Cells (HUBEC) Can Expand Both Human Bone Marrow and Cord Blood SCID-Repopulating Cells (SRC) through Cell Contact Rather Than Soluble Factors.
Authors: Takebe Naoko , Cheng Xiangfei , Farese Ann M. , Welty Emily , Meisenberg Barry , MacVittie Thomas
Published: April 2020

Human brain endothelial cells (HUBEC), a U.S. Navy proprietary cell line, was reported previously by Chute et al as a promising co-culture ex vivo expansion system for both adult bone marrow (ABM) and cord blood (CB) hematopoietic stem cells (HSC).a,b,c We report here our results of using HUBEC in ex vivo expansion and in vivo engraftment assay using NOD-SCID mice. CD34+ enriched fresh ABM was obtained using the method as described previously.a,b However, we used frozen CB and the same cytokines for both ABM and CB expansion whereas Chute et al used fresh CB and different cytokines. Ex vivo expansion studies for both ABM and CB were performed for 7 days in the HUBEC coated plates with previously reported cell density and cytokine cocktail containing GM-CSF, IL-3, IL-6, SCF, and flt-3 (GM36SF) in IMDM 10% FBS media.a HSC injections and BM harvesting of NOD-SCID mice as well as flow cytometric analysis were performed using the methods of Chute et al.a NOD-SCID mice were transplanted with limiting doses of either fresh ABM CD34+ cells or freshly thawed CB CD34+. The progeny of the identical doses of ABM CD34+ or the progeny of the identical doses of CB CD34+ cells was then transplanted. Culture with GM36SF alone resulted in a 15.5-fold and 70-fold increase in total cells, a 3.4-fold and 32-fold increase in CD34+ cells, and a 4.8-fold and 4.1-fold increase in CD34+/CD38- cells for ABM and CB, respectively. In contrast, HUBEC co-culture with GM36SF yielded a 25-fold and 48-fold increase in total cells, a 8.9-fold and 13-fold increase in CD34+ cells, and 114-fold and 106-fold increase in CD34+/CD38- cells for ABM and CB, respectively. HUBEC co-culture without GM36SF supported a 1.0-fold and 1.0-fold increase in total cells, a 0.06-fold and 0.1-fold increase in CD34+ cells, and 0.25-fold and 0.2-fold increase in CD34+/CD38- cells for ABM and CB. HUBEC co-culture with GM36SF and transwell (non-contact culture) resulted in a 20-fold and 48-fold increase in total cells, a 6-fold and 8-fold increase in CD34+ cells, and a 32-fold and 38-fold increase in CD34+/CD38- cells for ABM and CB. Overall, the transwell expansion of CD34+/CD38- population in both ABM and CB was reduced to 30% of that achieved in the contact culture. ABM CD34+ cells (5 x 105) engrafted 60% and the progeny of 5 x 105 cultured in the HUBEC monolayer with GM36SF engrafted in 90% of transplanted mice. CB CD34+ cells (1 x 104) engrafted 27% and the progeny 1 x 104 CB CD34+ cells cultured in the HUBEC monolayer with GM36SF engrafted 64% of NOD-SCID mice. SRC frequencies calculated as a 3.12-fold and 2.7-fold increase in CD34+ enriched ABM and CB, respectively, which was less than reported previously.a,b In summary, HUBEC supports and expands SRC mainly through cell-to-cell contact between HSC and endothelial cells, with HUBEC-secreted factors playing a minor role.
Ineffective Hematopoiesis in Megaloblastic Anemia Is Associated with Higher Frequency of Apoptosis in Each Lineage Immature and Mature Cells.
Authors: Oyake Tatsuo , Ito Shigeki , Kowata Shugo , Murai Kazunori , Ishida Yoji
Published: April 2020

A deficiency of Vitamin B12 is a major cause of megaloblastic anemia (MBA). Ineffective hematopoiesis is observed in MBA, characterized by cytopenia, bone marrow cells with dysplastic change and normal to hypercellularity. We reported that excessive apoptosis of each lineage CD34(+) cells was observed in myelodysplastic syndrome (MDS) in the last ASH meeting. In this study, we investigated the hypothesis that excessive apoptosis induced the ineffective hematopoiesis in MBA. We performed the three color flow cytometric analysis of bone marrow mononuclear cells in 12 MBA patients using PE labeled Annexin V, PerCP labeled anti-CD34 antibody and FITC labeled anti-each lineage antibody (anti-glycophorin A (GPA) antibody, anti-CD33 antibody and anti-CD41 antibody). The frequency of apoptosis in subpopulations of immature (CD34(+)) and each lineage (+) cells or those of mature (CD34(−)) and each lineage (+) cells were calculated as the ratio (%) of (cell number with Annexin V(+)) divided by (cell number in the subpopulation). The subpopulations include CD34(+)GPA(+) (immature erythroid), CD34(+)CD33(+) (immature myeloid), CD34(+)CD41(+) (immature megakaryocytic), CD34(−)GPA(+) (mature erythroid), CD34(−)CD33(+) (mature myeloid) and CD34(−)CD41(+) (mature megakaryocytic) cells. Much higher frequency of apoptosis was observed in each lineage CD34(+) cells in MBA (median: 23.8% (range: 10.8–43.6%) in erythroid, 43.5% (12.7–67.3%) in myeloid, 50.1% (21.0–64.1%) in megakaryocytic lineages, P< 0.05, respectively, n=12), compared to those in normal controls (8.5% (1.5–9.9%) in erythroid, 8.5% (2.2–8.8%) in myeloid, 7.7% (4.4–9.3%) in megakaryocytic lineages, respectively, n=10). While, the relatively higher frequency of apoptosis was observed in each lineage CD34(−) cells in MBA patients (median: 15.9% (range: 5.1–20.6%) in erythroid, 16.4% (5.6–23.2%) in myeloid, 16.1% (10.2–24.8%) in megakaryocytic lineages, P< 0.05, respectively, n=12), compared to those in normal controls (4.8% (1.3–6.6%) in erythroid, 2.2% (0.6–4.4%) in myeloid, 3.3% (1.5–7.1%) in megakaryocytic lineages, respectively, n=10).

These results suggest that the excessive apoptosis occurs not only in CD34(+) but also in CD34(−) cells, which induces ineffective hematopoiesis in MBA.

Figure 1. The frequency of apoptosis in CD34+ BM cells Figure 1. The frequency of apoptosis in CD34+ BM cells Figure 2. The frequency of apoptosis in CD34− BM cells Figure 2. The frequency of apoptosis in CD34− BM cells
Functional Distinctions for Smad1 and Smad5 during Hematopoiesis Revealed by the Zebrafish Model System.
Authors: McReynolds Lisa J. , Mullins Mary , Evans Todd
Published: April 2020

Smad1 and Smad5 proteins are intracellular mediators of BMP signaling, which has been implicated in regulating hemato-vascular development. Both Smad 1 and 5 are expressed widely in the developing hematopoietic mesoderm, and mutation of either mouse gene is embryonic lethal, limiting progress in defining their function in hematopoiesis. To compare the functions of these two closely related regulatory proteins, we used specific Smad5 mutants and Smad1 morpholinos in the zebrafish model. In the Smad5 mutant, piggytail (pgy), homozygous embryos are dorsalized and live until day 3–5, but have little to no blood and no circulation. Key hematopoietic regulatory genes are expressed in the pgy/pgy mutant fish, but the pattern of expression is altered in early embryos. Gata1, Gata2, Lmo2, draculin and Scl expression patterns are expanded at the five somite stage toward the more caudal region of the mutant embryos. In contrast, the expression pattern of the general mesoderm marker ‘no tail’ is not affected by loss of Smad5. The cells in the most caudal region of the mutant embryos express Gata1 precociously, indicating that they are specified along the erythroid lineage but fail to differentiate. However, Gata1 expression is lost by 48 hpf, as demonstrated in embryos carrying a gata1:gfp transgene on the pgy/pgy background. As in the pgy embryos, injection of a Smad1 morpholino at the one-cell stage leads to a dorzalized phenotype. However, in contrast to pgy embryos, Gata1 expression is maintained in the Smad1 morphants and Gata1 positive cells are found to circulate in the morphant embryos through at least 72 hpf. Our data support an essential role for Smad5 and not Smad1 in embryonic hematopoiesis. To analyze Smad5 function in the adult hematopoietic system, we established adult pgy fish that carry the gata1:gfp transgene, for analysis of blood cell profiles using flow cytometry. Blood cell populations were monitored under normal conditions and following phenylhydrazine induced hemolytic anemia in wildtype and pgy/+ fish. The pgy/+ adult fish are able to mount an erythropoietic response to hemolytic anemia. However, pgy/+ fish at two days post-treatment accumulate 30% of their erythroid progenitor population at the polychromatophilic stage, compared to 10% in wildtype. The pgy/+ fish are thus unable to reconstitute the erythrocyte population in the kidney at a rate comparable to wildtype fish. In summary, our data indicate that Smad5 has a role in the kinetics of embryonic stage erythropoiesis, distinct from the function of Smad1, and in adult erythropoiesis specifically at the transition from the proeyrthroblast to the polychromatophilic erythroblast stage.
A Role for fbxo5 in Zebrafish Myelopoiesis.
Authors: Rhodes Jennifer , Amsterdam Adam , Ho Karen , Kanki John P. , Hopkins Nancy , Look A. Thomas
Published: April 2020

The zebrafish is a powerful model system for investigating embryonic vertebrate hematopoiesis that allows for the critical in vivo analysis of cell development and lineage determination. Conservation of the variety of blood cell types and key regulatory factors between mammals and zebrafish suggests that the regulatory mechanisms directing lineage specification are similarly conserved. To identify novel genes necessary for normal myeloid cell development, we have analyzed a panel of zebrafish insertional mutants, provided by Nancy Hopkins at Massachusetts Institute of Technology (MIT), and identified mutants with deficient or abnormal distribution of myeloperoxidase (mpo), a gene specifically expressed in zebrafish granulocytes. One of the mutants identified in this screen is disrupted in fbxo5, a gene not previously identified as having a role in hematopoiesis. Using whole mount RNA in situ analysis on mutant and morpholino-injected embryos (morphants) we have determined that decreased levels of fbxo5 result in severely diminished numbers of granulocytes, although erythropoiesis appears normal. Interestingly, fbxo5 morphants have normal levels of pu.1 and l-plastin expression. Since we have previously shown that knockdown of pu.1 results in the loss of mpo-expressing cells, our data suggest that fbxo5 may function downstream or in a parallel pathway to pu.1 during granulopoiesis. To determine if the decreased number of granulocytes was due to programmed cell death, we examined the level of apoptosis in morphant versus control embryos. Whole mount acridine orange staining of apoptotic cells indicated that fbxo5 morphants had increased apoptosis, which can be mostly rescued by co-injection with p53 morpholino. However, the fbxo5/p53 double morphants continued to display a severe decrease in granulocytes, suggesting that the loss of granulocytes is not due to p53-mediated apoptosis. Fbxo5 has been examined previously in Xenopus oocytes, where the protein was found to regulate cell cycle progression by inhibiting APC. Interestingly, we have identified multiple mutants in genes that normally regulate the G2/M checkpoint and mitosis during the cell cycle all which display decreased numbers of granulocytes, suggesting a role for the regulation of the cell cycle in granulopoiesis. While further analysis is needed to determine where and how fbxo5 functions during myeloid cell development, as well as each of the genes disrupted in our additional cell cycle mutants, we have identified that fbxo5 is necessary specifically for the normal development of granulocytes.
A Novel Culture System of Human ES Cells for High Efficient Hematopoietic and Endothelial Differentiation.
Authors: Wang Zack Z. , Chen Tong , Au Patrick , Shao Ying , Jain Rakesh , Scadden David T.
Published: April 2020

Human embryonic stem cell (hESC) lines provide not only an unique system for studying human embryonic development, but also have enormous potential as a source of therapeutic tissues, including those of vascular and hematopoietic lineages. However, it is difficult to generate large-scale of relatively pure population of specific lineage cells derived from hESCs. Here, we describe a 2-dimensional (2-D) culture system for hESC differentiation to generate hematopoietic and endothelial cells in vitro without embryoid body (EB) formation (3-D). To initial differentiation, hESCs (H1 and H9 cell lines) were placed in serum-containing media for 9–10 days without supplement of additional growth factors. Gene expression analysis of 2-D differentiation indicated that Oct-4, a pluripotent gene, was decreased, while genes for GATA-2 and CD31, were increased, suggesting that differentiated hESCs contain hematopoietic and endothelial progenitors. Comparing with 3-D EB formation, 2-D differentiation was able to generate robust differentiated cells (5 to 10-fold higher). To generate a reasonable pure population of hematopoietic and endothelial precursors, CD34+ cells prior to hematopoietic commitment were isolated from 2-D differentiation by immuno-magnetic beads and cultured in the presence of hematopoietic growth factors and endothelial growth factors. After 7–10 days culture of CD34+ cells, both suspension and adherent cells emerged. The number of suspension and adherent cells was dependent on the presence of hematopoietic growth factors or/and endothelial growth factors. The suspension cells expressed hematopoietic marker, CD45, and gave rise to multi-lineage hematopoietic colonies on methylcellulose cultures. The adherent cells expressed endothelial markers, such as CD31 and Ve-cadherin, and took up Dill-labeled ac-LDL. The adherent cells rapidly formed capillary-like structures on Matrigel cultures, while the suspension cells would not. While the adherent cells were implanted into mice, they formed functional blood vessels in vivo. Our culture system paves the road for further large-scale manufacture of hematopoietic and endothelial cells derived from human ES cells. Our studies demonstrated also that CD34+ cells derived from human ES cells contain hematopoietic and endothelial progenitors.
Derivation of Lymphoid Lineage Cells from Human Embryonic Stem Cells on Co-Culture in OP9 and OP9DL1 Stromal Cells.
Authors: Mojica-Henshaw Mariluz P. , Huang Xiaosong , Spangrude Gerald J.
Published: April 2020

We have been able to direct the differentiation of CD34pos cells derived from H9 and H7 hESCs toward lymphoid lineages using OP9 or OP9DL1 feeder layers. OP9 and OP9DL1 stromal cell lines are consistently better than S17 or OP9-jagged in inducing hESC-derived CD34pos cells at different time points in culture. By day 14 in culture, about 8.7% and 7.9% of differentiated H9 hES cells harvested from co-cultures in OP9 or OP9DL1 respectively were CD34pos. The CD34pos cells in these cultures were enriched for by magnetic sorting. The clonogenic potential of differentiated hES cell suspensions and of sorted CD34pos cells were determined using methylcellulose colony assays. The highest clonogenic potential was noted after 18 days in co-culture when we obtained 142 +/− 31 and 218 +/− 38 colonies per million cells plated from OP9 and OP9DL1. Selection for CD34pos cells enriched the clonogenic activity 27-fold. Morphologic analyses of representative colonies from the methylcellulose cultures showed cells of the erythroid, myeloid, lymphoid and megakaryocytic lineages were generated. Directed differentiation of CD34pos cells towards lymphoid lineages was initiated by replating the sorted CD34pos cells onto irradiated OP9 or OP9DL1 stromal cells supplemented with stem cell factor (SCF), IL3, IL7 and Flt3L. Under these conditions, B lineage (CD19pos) and myeloid lineage (CD14pos or CD15pos) cells were observed when CD34pos cells were grown in OP9, and T lineage cells were observed when CD34pos cells were grown in OP9DL1. CD4posCD8pos cells were evident after 12 days of secondary culture in OP9DL1 and were prominent by day 20. Only a few of the CD4pos, CD8pos and CD4posCD8pos cells, however, were noted to be CD3pos. Addition of SCF + IL3, SCF + Flt3L, or SCF + Flt3L + IL7 at initiation of hESC differentiation increased the percentage of CD34pos cells and/ or increased the hematopoietic (clonogenic) potential of sorted CD34pos cells. CD34pos cells derived from hESCs grown in OP9DL1 for 14 days and replated in OP9DL1 supplemented with Flt3L + IL7 +/− SCF generated the highest percentages of CD4posCD8pos cells. Additional phenotypic analyses and functional studies of sorted CD4pos, CD8pos and CD4posCD8pos cells generated from these cultures are needed to further characterize their lymphoid properties.
The Role of Alpha Hemoglobin Stabilizing Protein (AHSP) in the Formation of Hemoglobin A.
Authors: Zhou Suiping , Weiss Mitchell J. , Gow Andrew J.
Published: April 2020

Alpha hemoglobin stabilizing protein (AHSP) binds α hemoglobin (Hb) to stabilize its structure and limit its chemical reactivity in vitro and in vivo. Addition of β Hb to α Hb-AHSP displaces AHSP to form HbA (α2β2) tetramer. Recently, we determined that binding of AHSP to free α Hb generates a novel structure in which the helix F of α Hb is disordered and the heme iron is reduced to the ferrous form and coordinated by the distal histidine (ferrous α Hb-AHSP). Over time, the heme iron within this complex oxidizes to the ferric form (ferric α Hb-AHSP) and the F helix reorders, generating a bis-histidyl heme iron that exhibits decreased capacity to catalyze the formation of reactive oxygen species. At room temperature in ambient oxygen, the conversion of ferrous to ferric α Hb-AHSP occurs over several hours. We proposed that in vivo, these complexes could serve as transient intermediates to maintain α Hb in a stable state until β Hb is available to form tetrameric HbA. The current study addresses two questions related to this hypothesis:

Does bis-histidyl ferric α Hb-AHSP form more readily under physiologic conditions? Can ferrous and ferric α Hb-AHSP complexes form functional tetrameric HbA upon reaction with β Hb?

The conversion of ferrous to ferric α Hb-AHSP is temperature-dependent with complete conversion occurring in 40 minutes at 37°C, as opposed to 5 hours at 22°C. Furthermore, the conversion is accelerated by partial oxygen saturation, increasing the likelihood that ferric-AHSP Hb will form in vivo. Using cellulose acetate gel electrophoresis, we demonstrated that β Hb displaces AHSP to form tetrameric HbA from both α Hb-AHSP complexes. The resulting tetramer from ferrous α Hb-AHSP is similar to HbA, both spectroscopically and in terms of oxygen binding. In contrast, the Hb tetramer formed from ferric α Hb-AHSP and β Hb is similar to HbY (αFeIII2βFeII2) spectroscopically. However, the α globin heme iron within this tetrameric complex appears to be occluded, as the rate of cyanide binding is reduced. Furthermore, it is resistant to reduction by sodium dithionite or methemoglobin reductase, suggesting that the α Hb retains its bis-histidyl nature. Together, these studies show that both α Hb-AHSP complexes are likely to form under physiological conditions. Upon interaction with β Hb, ferrous α Hb-AHSP forms functional Hb A tetramer. In contrast, ferric α Hb-AHSP is more chemically inert, but may not be able to form viable HbA.
Structure of the Calcium-Rich Signature Domain of Human Thrombospondin-2.
Authors: Britt C. , Bernstein Douglas A. , Annis Douglas S. , Misenheimer Tina M. , Hannah Blue-Leaf A. , Mosher Deane F. , Keck James L.
Published: April 2020

There are five members of the thrombospondin (TSP) family in vertebrates. This family of proteins comprises two separate subsets, group A and group B. Group A includes TSP-1 and TSP-2. TSP-1 is a major platelet α-granule component and contains a polymorphism that has been linked to coronary artery disease (CAD). TSP-2, although not found in platelets, is important for megakaryopoiesis. TSPs contain a signature domain that is found with high fidelity throughout the TSP family and consists of tandem epidermal growth factor (EGF)-like modules, 13 aspartate-rich calcium-binding repeats and a C-terminal lectin-like module. The signature domains of TSP-1 and TSP-2, which have three EGF-like modules, share 77% identity, without any insertions or deletions. In this study, we describe the 2.6 angstrom resolution crystal structure of the glycosylated signature domain of human TSP-2. Components of the domain interact extensively to form three striking structural features: a stalk that consists of the two N-terminal EGF-like modules, a wire that consists of the asparate-rich repeats, and a globe that consists of the lectin-like module. The third EGF-like module serves as a clasp to tie together the globe and the ends of the wire. The TSP-2 signature domain is stabilized by 30 bound Ca2+ ions and 18 disulfide bonds. The structure reveals functions of the residues at the position of the CAD-linked polymorphism in TSP-1 and many of the positions of the over 100 skeletal dysplasia-linked mutations in TSP-5. Finally, the structure allows for a better understanding of interactions between TSPs and a number of binding partners, including integrins, CD47, vonWillebrand factor, type V collagen, fibroblast growth factor, cathepsin-G and neutrophil elastase.
Heterogeneity of Endothelial Sheet Migration: Role in Angiogenic Plasticity.
Authors: Ofori-Acquah Solomon F. , Stevens Troy
Published: April 2020

The vascular system is a complex network of conduit and microvascular vessels exposed to different microenvironments that imprints unique phenotypic traits on individual endothelial cell populations. Endothelial cells in fully differentiated blood vessels in adult tissues have a quiescent phenotype characterized by an increased resistance to proliferate, migrate or undergo apoptosis. The intrinsic capacity of endothelial cells to switch from a quiescent to angiogenic phenotype however plays an important role in wound healing, several vascular proliferative disorders and tumor angiogenesis. Endothelial cells revert to an angiogenic phenotype as isolated cells in culture nonetheless they are contact inhibited at confluence reflecting their in vivo phenotype in the endothelium. In this study, we used time-lapse video microscopy to study early events in endothelial sheet migration in confluent monolayers of primary endothelial cells derived from conduit (PAECs) and microvascular (PMVECs) blood vessels. Recordings were made in a live cell chamber and were restricted to six hours to minimize the effect of proliferation on sheet migration. PMVECs at the wound edge were significantly highly spread and squamous in appearance compared to PAECs, which had a distinctly more cuboidal morphology. Majority (>95%) of PMVECs at the wound edge produced extensive lamellipodia based on morphology and dynamics that measured 25 μm ± 4 μm. By contrast, PAECs formed significantly smaller lamelipodia which extended by 8 μm ± 4 μm. On average PMVEC sheets migrated at a speed of 12.5 μm per hour covering a total distance of 75 μm ± 15 μm (n=6). Sheet migration rate in PMVECs was 3-fold faster than in PAECs (3.8 μm per hour), which covered a total distance of 23 μm ± 10 μm in the same time period. To unravel the molecular basis for this functional diversity, gene micro array analysis was performed. We identified unique transcriptional profiles for cell-cell adhesion molecules, integrins and disintegrin-metalloproteases each with a distinct role in collective cell migration. In particular, integrin alpha 7, which is a major regulator of lamellipodia formation was found to be 20-fold more abundant in PMVECs than in PAECs. This study provides molecular and functional evidence for heterogeneity of endothelial sheet migration. This central finding highlights variability in angiogenic plasticity in fully differentiated endothelial cells, which may have important ramifications for anti-angiogenesis therapy.