bluuebunny/crossref_metadata_2025 huggingface dataset:
MafG-Null Mice Show Delayed Leukocyte Engraftment Following Whole Bone Marrow Transplant.
Authors: Zafar Abu-Bakr , Li Xiao-Miao , Jorgensen Marda L. , Hu Zhongbo , Bungert Jorg , Engel James , Slayton William
Published: April 2020

Megakaryocytes, which give rise to platelets, constitute a small population of cells. However, molecular aspects of megakaryocyte development are poorly understood. MafG is the small subunit of the transcription factor, NF-E2, which plays a major role in terminal megakaryocyte differentiation and platelet release. We sought to determine the effect of impaired platelet release due to targeted ablation of the mafG gene following a hematopoietic stem cell transplant on engraftment kinetics. We performed three types transplants:C57Bl/6 Ly5.1 murine bone marrow (mafGWT/WT bm) into lethally irradiated mafG null hosts expressing green fluorescent protein, (GFP),mafG-null/GFP bm into lethally irradiated C57BL/6 mafGWT/WThosts, andmafGWT/WT GFP bm into lethally irradiated C57BL/6 mafGWT/WT hosts.

We measured peripheral cell counts, the presence of circulating donor-derived cells by flow cytometry, changes in the cellularity of the bone marrow and splenic weight, and megakaryocyte size and concentration on day 5, 7, 14, and 28 post transplant. We found:Platelet engraftment kinetics differed when mafG null animals served as donors or recipients. Specifically, platelets reached their lowest point at day 7 when mafGWT/WT or mafG null bm was transplanted into wildtype hosts. When mafGWT/WT bm was transplanted into mafG null hosts, platelets reached a low point on day 5.MafG null hosts transplant with mafGWT/WT bm had 5-fold lower circulating platelet levels than mafGWT/WT hosts at day 5.When mafGWT/WT served as donors, circulating platelets began to increase after day 7, while animals receiving mafG null donor cells had platelet counts that continued to diminish.Splenic hematopoiesis was markedly decreased when mafGWT/WT bm was transplanted into mafG null hosts. In contrast, when mafG null bm was transplanted into mafGWT/WT hosts, splenic hematopoiesis was increased 2.5 fold on day 28 compared to animals receiving mafGWT/WTdonor cells.Mortality rate was much higher in mafG null hosts.Leukocyte engraftment was delayed when mafGWT/WT bm was transplanted into mafG null hosts.

These results suggest that host factors determine the timing of the platelet nadir following bone marrow transplant. Furthermore, the mafG protein, contributes in previously unrecognized ways to splenic hematopoiesis and successful engraftment, possibly through its function in promoting megakaryocyte development and platelet release.
MicroRNA hsa-mir-155 Blocks Myeloid and Erythroid Differentiation of Human CD34+ Cells.
Authors: Georgantas Robert W. , Hildreth Richard , Morisot Sebastien , Alder Jonathan , Civin Curt I.
Published: April 2020

In a large microRNA-array and bioinformatics study, we determined all of the microRNAs (miRs) expressed by human CD34+ hematopoietic stem-progenitor cells (HSPCs) from bone marrow and G-CSF mobilized blood. When we combined miR expression data, mRNA expression data from a previous study (Georgantas et al, Cancer Research 64:4434), and data from various published miR-target prediction algorithms, we were able to bioinformaticly predict the actions of miRs within the hematopoietic system. MircoRNA hsa-mir-155 was highly expressed in CD34+ HSPCs, and was predicted by our bioinformatics database to target several HSPC-expressed mRNAs (CREBBP, CXCR4, Jun, Meis-1, PU.1, AGTRI, AGTRII, Fos, and GATA3) that encode proteins known to be involved in myeloid and/or erythroid differentiation. We used luciferase-3′UTR reporter constructs to confirm that protein expression from these mRNAs were in fact down regulated by microRNA. As an initial test of mir-155′s effect on hematopoietic differentiation, K562 cells were transduced with hsa-mir-155 lentivirus and then exposed to TPA to induce megakaryocyte differentiation, or to hemin to induce erythroid differentiation. Compared to controls, miR-155 reduced K562 megakaryocyte differentiation by ~70%, and erythroid differentiation by >90%. Thus, mir-155 appears to be sufficient to inhibit both megakayrocyte and erythroid differentiation. K562 proliferation was not affected by mir-155, showing that the differentiation block was not due to cell cycle arrest. MicroRNA hsa-mir-155-transduced human mobilized blood CD34+ cells generated >70% fewer myeloid and erythroid colonies than controls in colony forming (CFC) assays, further indicating that mir-155 blocks both myeloid and erythroid differentiation. We are currently further testing the effects of mir-155 on differentiation of CD34+ cells in vitro, and also in vivo on their ability to engraft immunodeficient mice.
The Pim-1 Kinase Is a Direct Target of the Hoxa-9 Homeodomain Protein and a Mediator of Hoxa-9’s Effects on Hematopoiesis.
Authors: Lawrence H. Jeffrey , Hu Yu-Long , Passegue Emmanuelle A. , Fong Stephen , Largman Corey
Published: April 2020

The Hoxa-9 homeodomain protein exerts dramatic effects in hematopoiesis. Loss of Hoxa-9 function in blood cells severely inhibits proliferation of primitive hematopoietic cells and impairs in vivo hematopoietic stem cell (HSC) function. The defects in Hoxa-9−/− animals are more striking than any other single or compound Hox mutation reported to date, and more dramatic than that seen in animals lacking the entire Hoxb cluster of homeobox genes, presumably because Hoxa-9 is one of the most highly expressed members of the 39-member Hox gene family in HSC. Conversely, enforced expression of Hoxa-9 markedly enhances the proliferation of primitive blood cells, expands HSC and leads inevitably to a fatal acute myeloid leukemia in mice. Despite these potent biological effects, the pathways by which Hoxa-9 acts in blood cells are unknown, and although Hoxa-9 is a DNA-binding transcription factor, no direct physiologic target genes have been identified in hematopoietic cells. Our previous gene expression profiling studies suggested that Hoxa-9 positively regulates the Pim-1 serine-threonine kinase, an oncogene with important antiapoptotic and pro-proliferative effects in blood cells. In addition, the hematological phenotypes of Hoxa-9 and Pim-1 deficient animals are strikingly similar, and include a blunted response to myeloid cytokines, T- and B-cell defects, and microcytosis of red blood cells. Using chromatin immunoprecipitation assays, we show that Hoxa-9 protein binds directly to the Pim-1 promoter/enhancer region in hematopoietic cells. Enforced expression of Hoxa-9 significantly induces Pim-1 mRNA and protein in blood cells in a DNA-binding dependent manner. Furthermore, Pim-1 protein levels are diminished in lin- blood cells from Hoxa-9−/ − fetal liver, showing that Hoxa-9 is required to maintain normal levels of Pim-1 in primitive hematopoietic cells. By real-time PCR, we find that Hoxa-9 and Pim-1 mRNA levels track together in highly purified subpopulations of long-term and short-term HSC and in common myeloid and granulocyte-monocytes progenitors (CMP/GMP). Induction of Pim-1 protein by Hoxa-9 significantly increases the phosphorylation and inactivation of the pro-apoptotic BAD protein, a known target of the Pim-1 kinase. Hoxa-9−/ − marrow cells show increased levels of apoptosis and decreased cell cycle progression and proliferation compared to wild-type cells, and these defects are significantly improved by the re-introduction of Pim-1. These data provide strong evidence that Pim-1 is a direct transcriptional target of Hoxa-9 and a key mediator of its antiapoptotic and pro-proliferative effects on primitive hematopoietic cells. Since Hoxa-9 is upregulated in many cases of human acute myeloid leukemia, Pim-1 may serve as a useful therapeutic target in Hoxa-9 driven leukemias.
Neuronal/Mitochondrial Nitric Oxide Synthase Homologous Deletion Recombinant Negative Mice (NOS1 −/−) Long-Term Bone Marrow Cultures (LTBMCs) Demonstrate Increased Longevity and Radioresistance of Derived Cell Lines.
Authors: Epperly Michael W. , Greenberger Joel S. , Cao Shaonan , Franicola Darcy , Shen Hongmei , Greenberger Emily E. , Epperly Laura D. , Zhang Xicheng
Published: April 2020

Neuronal NOS (NOS1) is localized to mitochondria. Ionizing irradiation results in influx of calcium into mitochondria stimulating production of nitric oxide (NO), and also increases production of superoxide which reacts with NO to produce peroxynitrite. Peroxidation of mitochondrial lipids, release of cytochrome C and apoptosis is directly related to mitochondrial peroxynitrite. We hypothesized that reduction of mitochondrial NO production should provide radioresistance. In addition, since ROS production is associated with aging NOS1−/− mouse LTBMCs should demonstrate greater hematopoietic longevity. LTBMCs established from NOS1 −/− mice demonstrated increased cumulative adherent cobblestone islands (adherent stem cell containing islands), production of total nonadherent cells, and cumulative day 7 and day 14 CFU-GEMM hematopoietic multi-lineage colony forming cells (over 65 weeks) compared to NOS1 +/+ controls (22 weeks) (p < 0.0001). Seven and 14 day CFU-GEMM production in nonadherent cell harvests from NOS1 −/− LTBMCs continued for 65 weeks compared to 15 weeks for NOS1 +/+ LTBMCs (p < 0.0001). Marrow stromal cell lines derived from NOS1 −/− and NOS1 +/+ culture were irradiated to doses from 0 to 800 cGy, plated in 4 well tissue culture plates, stained with crystal violet 7 days later and colonies of greater than 50 cells were counted. NOS1 −/− stromal cell lines had an increased shoulder on the survival curve compared to the NOS1 +/+ cells (n = 32.15 ± 1.21 and 10.47 ± 3.20, respectively, p=0.0026). Cell cycle analysis of NOS1 −/− and NOS1 +/+ cell lines following 10 Gy irradiation demonstrated a G1 arrest 6 hr after irradiation, in both; however, by 24 hr, NOS1 +/+ but not NOS1−/− cells resumed normal cycling. To determine whether the radioresistance of NOS1−/− cells was attributable to expected higher levels of antioxidants, cells were analyzed for glutathione (GSH) and glutathione peroxidase (GPX). NOS1 −/− cells demonstrated increased GSH compared to NOS1 +/+ cells at 0, 30 min and 24 hr following irradiation (p < 0.0001) with no significant difference in GPX before or after irradiation. NOS1 −/− compared to NOS1+/+. IL-3 dependent hematopoietic cells from NOS1 −/− LTBMCs had significantly decreased apoptosis, 24 hrs following 10 Gy irradiation (5.3 ± 2.4 vs. 14.8 ± 3.3 %, respectively, p = 0.049). Therefore, reduction of NOS1 in bone marrow increases hematopoietic longevity in LTBMCs and radioresistance of derived cell lines.
Bis(1H-indol-2yl)methanones Are Effective Inhibitors of Mutated FLT3 Tyrosine Kinase, Overcome Resistance to PKC412A In Vitro and Show Synergy with Chemotherapy.
Authors: Heidel Florian , Mirea Fian K. , Breitenbucher Frank , Dove Stefan , Huber Christoph , Bohmer Frank D. , Fischer Thomas
Published: April 2020

Inhibition of mutated FLT3 receptor tyrosine kinase has become a promising therapeutic strategy in AML. Currently, a phase III trial testing the tyrosine kinase inhibitor (TKI) PKC412A in combination with induction chemotherapy is under way. Development of resistance to PKC412A mediated by secondary FLT3 mutations has been described recently (Heidel et al., Blood 2006). This observation may have an increasing impact on duration of response in future clinical trials. Herein we investigated two novel TKIs (methanones) to determine their efficacy on FLT3-ITD transfected 32D cells as well as on primary ITD positive AML blasts.

The bis(1H-indol-2-yl)methanone compounds Cpd.98 and Cpd.102 (Mahboobi et al. J. Med. Chem. 2006) that have recently shown efficacy in proliferation assays using FLT3-ITD transfected cells were characterized. Western Blotting experiments of murine 32D-FLT3-ITD cells confirmed dose dependent dephosphorylation of FLT3-ITD and its downstream target STAT5 upon incubation with Cpd.98 and Cpd.102, respectively. To define a non-myelotoxic range of these inhibitors, bone-marrow (BM) cells of healthy donors were tested with different methanone concentrations to determine the rate of apoptosis (sub G1 fraction in cell cycle analysis). No significant increase in apoptosis was seen up to 3μM of inhibitor concentration for either compound.

Additionally, colony assays of healthy BM-cells also showed no significant reduction of colony formation up to 3μM. To determine the efficacy of these compounds in PKC412A-resistant cells, transfected 32D cells containing the previously described resistance mediating mutation (32D-FLT3-ITD-N676K) were also tested at different concentrations for induction of apoptosis. Resistance in these cells could be overcome by 300nM (Cpd.102) and 1μM (Cpd.98), respectively.

As clinical studies in AML will evaluate FLT3 inhibitors in combination with chemotherapy, we tested both compounds in vitro in combination with cytosine arabinosid (Ara-C) and daunorubicine (DNR). Both compounds showed synergistic effects in combination with chemotherapy in 32D-FLT3-ITD cells as revealed by apoptosis assays.

Primary AML blasts harbouring the FLT3-ITD length mutation were also incubated with both compounds to determine the efficacy in patient material. These experiments revealed apoptosis rates of up to 30% upon incubation for 72 hours. In conclusion bis(1H-indol-2-yl)methanone compounds overcome resistance mediated by the previously described FLT3-ITD mutation N676K in vitro and show efficacy on FLT3-ITD-positive cells alone as well as in combination with chemotherapy. Experiments evaluating toxicity in murine models are currently planned to explore a possible clinical application.
Prolonged Exposure to FLT3 Inhibitors Leads to Resistance Via Activation of Parallel Signaling Pathways.
Authors: Piloto Obdulio , Wright Melissa , Brown Patrick , Kim Kyu-Tae , Levis Mark , Small Donald
Published: April 2020

A number of tyrosine kinase inhibitors (TKI) have been developed to treat a variety of malignancies. However, continuous treatment with TKIs may select for resistant clones as has been seen with Gleevec treatment of CML. To study resistance to TKIs targeting FLT3, a receptor tyrosine kinase that is frequently mutated in AML, we developed resistant human cell lines through prolonged co-culture with FLT3 TKIs. Both FLT3 TKI sensitive and resistant cell lines exhibit inhibition of FLT3 phosphorylation upon FLT3 TKI treatment. However, FLT3 TKI resistant cell lines and primary samples often show continued activation of downstream PI3K/Akt and/or Ras/MEK/MAPK signaling pathways as well as continued expression of genes involved in FLT3-mediated cellular transformation. Inhibition of these pathways restores partial sensitivity to FLT3 TKIs. Mutational screening of FLT3 TKI resistant cell lines and primary samples failed to reveal any mutations in FLT3 or in 100 kinases/phosphatases tested but did reveal activating N-Ras mutations that were not present in the parental FLT3 TKI sensitive cell line. Taken together, these data indicate that FLT3 TKI resistant cells most frequently become FLT3 independent due to activation of parallel signaling pathways that provide compensatory survival / proliferation signals when FLT3 is inhibited. IMC-EB10, an unconjugated monoclonal antibody against FLT3, is still cytotoxic to FLT3 TKI resistant clones in vivo. An approach combining FLT3 TKIs with anti-FLT3 antibodies may prove superior and result in reduced chances of developing resistance.
Erythropoietin Modulation of Podocalyxin within an Erythroblast Niche.
Authors: Sathyanarayana Pradeep , Menon Madhu P. , Bogacheva Olga , Bogachev Oleg , Kapelle William S. , Houde Estelle , Wojchowski Don M.
Published: April 2020

Epo’s capacity to promote red cell production is ascribed largely to anti-apoptotic effects on erythroid progenitor pools. Via gene profiling of bone marrow- derived erythroblasts, Epo is now revealed to down-modulate chemokine receptor-4 (Cxcr4) and integrin alpha-4 (Itga4), and to selectively up-modulate the expression of growth differentiation factor 3 (Gdf3), oncostatin-M (Onco-M), and the sialomucin podocalyxin like-1 (PODXL). Epo dose-dependent induction of PODXL was approximately 20 fold; was discovered in KitposCD71high erythroblasts; and peaked at subsequent KitnegCD71high and Ter119pos stages. As studied using minimal knocked-in Epo receptor (EpoR) alleles, efficient PODXL expression depended upon a proximal EpoR PY343 Stat5 binding site. In mice expressing a phosphotyrosine-null EpoR-HM allele, compromised PODXL expression further correlated with an abnormal representation of anucleated red cells in marrow (and with defective Epo-induced production of PODXLpos reticulocytes). By selectively modulating Gdf3, Onco-M, Cxcr4, Itga-4 and PODXL expression, Epo is proposed to modify a unique erythroid niche, mobilize early-stage erythroblasts, and promote reticulocyte emigration to blood.
The Role of Gja1 Gap Junctions in the Bone Marrow Niche.
Authors: Anderson Nicole M. , Zirngibi Ralph , Chen Frieda , Owen Celeste , Zitano Lia , Lau Esther , Aubin Jane , Stanford William
Published: April 2020

The interplay between the stroma and hematopoietic progenitors within the bone marrow niche is critical for the homeostatic regulation of both mesenchymal and blood lineages. Gap junctions play an important role in the communication between hematopoietic supportive cells of the fetal liver and bone marrow stroma. Targeted deletion of the gap junction protein Gja1 (connexin 43) demonstrated a requirement of Gja1 for placental and cardiac function leading to neonatal lethality. Surprisingly, Gja-1 heterozygous animals demonstrated no steady state hematopoietic defects, suggesting that either Gja-1 is not critical for hematopoietic stromal cell communication or that Gja-1 hemizygosity does not functionally alter gap junctions. We have identified an N-ethyl-N-nitrosourea (ENU)-induced strain (Gja1JRT) with a dominant negative mutation in Gja1 which phenocopies the human autosomal dominant disorder oculodentodigital dysplasia (ODDD). ODDD is characterized by a variety of developmental abnormalities including syndactyly, enamel hypoplasia, craniofacial abnormalities and cardiac dysfunction. In addition to these phenotypes, Gja-1JRT mice exhibit decreased bone mass and mechanical strength as well as alterations in hematopoietic progenitor frequencies, abnormalities not previously reported in human ODDD patients (Development132: 4375–4386, 2005). We are currently performing a longitudinal analysis of 2, 4, 8 and 12 month old Gja-1JRT animals, examining hematopoietic including osteoclastic and mesenchymal/osteoblast progenitors to evaluate the dynamic interplay between these two tissues. To date, we have observed alterations in hematopoietic parameters including increased peripheral blood cell number and increased myeloid colony number at 2 months of age. At 4 months of age, Gja-1JRT mice demonstrated a significant decrease in overall clonogenic (CFU-C) cells, while some peripheral blood cell parameters remained elevated. Despite the increases in differentiated myeloid cells and their precursors at 2 months of age, analysis of hematopoietic-derived bone resorbing osteoclasts showed a reduction in osteoclasts at 2 months of age but not at later time points. Analysis of the mesenchymal compartment of the bone marrow demonstrated increased total mesenchymal progenitors (CFU-F) and osteoprogenitors (CFU-O) at 8 and 12 months of age. Thus, alterations in hematopoietic and osteoclastic cell frequencies preceded alterations in the bone marrow mesenchymal compartment in the Gja-1JRT mice. In addition to a complete longitudinal analysis, we will report on the alterations in trabecular bone, which create the niche microenvironment, and the impact of these changes on the hematopoietic stem cell frequency.
Adhesion of Hematopoietic Progenitor Cells to Human Mesenchymal Stromal Cells as a Model for Interaction between Stem Cells and Their Niche.
Authors: Wagner Wolfgang , Wein Frederik , Roderburg Christoph , Benes Vladimir , Diehlmann Anke , Krause Ulf , Frankhauser Maria , Wuchter Patrick , Volker Eckstein , Ho Anthony D.
Published: April 2020

Objective: The significant role of direct contact between hematopoietic progenitor cells (HPC) and the cellular microenvironment for maintaining “stemness” has been demonstrated. Human mesenchymal stromal cell (MSC) feeder layers represent a surrogate model for this interaction. The molecular composition of this heterotypic cell-cell contact is yet unknown.

Methods: To define this cell-cell contact between HPC and MSC, we have studied adhesion of various fractions of HPC with different preparations of MSC by using a novel assay based on gravitational force upon inversion. Adherent and non-adherent cells were then separated. Gene expression analysis by microarray (GeneChip Human Genome U133_Plus_2.0, Affymetrix) of the two populations was performed and the relationship to long-term hematopoietic culture initiating cell (LTC-IC) frequency examined.

Results: HPC subsets with higher self-renewing capacity demonstrated significantly higher adherence to MSC from human bone marrow (CD34+vs. CD34−, CD34+/CD38−vs. CD34+/CD38+, slow dividing fraction vs. fast dividing fraction). LTC-IC frequency was significantly higher in the adherent fraction than in the non-adherent CD34+ cells, thus providing evidence for specific adhesive interaction of primitive HPC with MSC. Genes coding for adhesion proteins and extracellular matrix were highly expressed in the adherent fraction compared to non-adherent CD34+ cells. These genes included fibronectin1 (FN1), cadherin11, VCAM1, connexin43 and ITGBL1. Furthermore, affinity of CD34+ cells was analyzed on human MSC isolated from bone marrow (BM), adipose tissue (AT) and cord blood (CB). Affinity to BM-MSC was significantly higher compared to AT-MSC and CB-MSC. Gene expression in different MSC preparations (BM-MSC, AT-MSC and CB-MSC) correlated in various adhesion proteins with the differences observed in affinity of HPC (including cadherin11, VCAM1, N-cadherin, ITGB1, ITGA1, ITGA5, SDF-1 and osteopontin). Western blot analysis also confirmed higher protein expression of FN1, cadherin11, N-cadherin and ITGB1 in BM-MSC compared to AT-MSC and CB-MSC.

Conclusion: MSC represent a model for the human hematopoietic niche. Primitive subsets of HPC have significantly higher affinity to BM-MSC. The essential role of specific junction proteins (cadherin11, VCAM1, N-cadherin) for stabilization of cell-cell contact is indicated by their significant higher expression on both sides of the heterotypic interaction.
Bmi1- Is a SALL4 Target Gene in Hematopoeitic and Leukemic Cells.
Authors: Ma Yupo , Yang Jianchang , Chai Li , Liu Fang , Amin Hesham M.
Published: April 2020

Bmi-1 and SALL4 are putative oncogenes modulating stem cell pluripotency and playing a role in leukemogenesis. Previously, we report that SALL4, a zinc finger transcription factor, is constitutively expressed in human acute myeloid leukemia (AML) and induces AML in transgenic mice. Here we demonstrate that transcription from the Bmi-1 promoter is strikingly activated by SALL4. The SALL4 responsiveness of the Bmi-1 promoter exhibits dose-dependent activation in a luciferase reporter gene assay. We mapped the N-terminal domain of SALL4 responsible for activation of the Bmi-1 promoter. Chromatin immunoprecipitation from a myeloid stem cell line, 32D demonstrated that SALL4 bound directly to the Bmi-1 promoter in a region involving SALL4 stimulation. The over-expression of SALL4 in the 32D cell line up-regulated Bmi-1expression and was associated with increased proliferation and inhibition of apoptosis. Deletion of one copy of SALL4 by the gene target in the mouse marrow significantly reduced Bmi-1 expression. SALL4 up-regulated Bmi-1 expression in SALL4 transgenic mice and the up-regulated levels of Bmi-1 in SALL4 transgenic mice correlated to that of disease progression from normal, to preleukemic and leukemic stages. Similar to Bmi-1, SALL4 was expressed highly in hematopoietic cells (HSCs) and was down-regulated as differentiation proceeds. These findings, when taken together, reveal a novel link between SALL4 and Bmi-1 in regulating self-renewal of normal and leukemic stem cells.
Different Kinetics and Function of Vascular Endothelial Growth Factor Recepotor-1 and −2 during Hemangioblast Development from Primate Embryonic Stem Cells.
Authors: Umeda Katsutsugu , Heike Toshio , Shinoda Gen , Niwa Akira , Arai Masato , Matsubara Hiroshi , Ma Feng , Adachi Souichi , Suemori Hirofumi , Torii Ryuzo , Shibuya Masabumi , Nakatsuji Norio , Nakahata Tatsutoshi
Published: April 2020

The close developmental association between hematopoietic and endothelial cells suggests that both lineage cells share a common precursor, the hemangioblast. The vascular endothelial growth factor (VEGF)-A system has been proven to have roles in the embryonic development of hemangioblast in mouse by the use of embryos or embryonic stem (ES) cells deprived of the genes encoding its ligand or receptors (VEGFR-1 and VEGFR-2). On the other hand, there have been only a few reports on the hemangioblast development during primate (human and monkey) embryogenesis.

We have previously demonstrated that the hemangioblast is highly enriched in the VEGFR-2high CD34+ cell fraction, differentiated from cynomolgus monkey ES cells by 6-day coculture with OP9 stromal cells. In the current study, we examined whether the VEGF-A system was involved in the development of hemangioblast induced from primate ES cells by using the coculture system. VEGFR-1 and −2 were expressed by undifferentiated monkey ES cells at low levels. The VEGFR-2low cells gradually decreased while VEGFR-2high cells could be detected on day 6. On the other hand, VEGFR-1 was constantly expressed at low levels on day 6 and thereafter. Exogenous VEGF-A165, the action of which is mediated VEGFR-1 and VEGFR-2, increased the proportion of VEGFR-2high CD34+ cells in a dose-dependent manner. Addition of VEGF-E (VEGFR-2-specific agonist) and VEGFR-1 blocking antibody resulted in the increase of VEGFR-2high CD34+ cells in a dose-dependent manner, while VEGFR-2 blocking antibody suppressed their development. Thus, VEGF-A/VEGFR-2 interaction promoted the development of hemangioblast, while VEGF-A/VEGFR-1 interaction acted as a negative regulator.

We then examined when VEGF-related signals work on VEGFR-2high cell development by changing factors on day 4. The proportion of VEGFR-2high CD34+ cells with the last 2-day VEGF-A165 and -E treatment were almost equivalent to that with continuous exposure to the VEGF. On the contrary, initial 4-day VEGF-A165 and -E treatment did not any stimulatory effects on VEGFR-2high CD34+ cell development. Our previous results show that during differentiation, Brachyury (early mesodermal marker) is first expressed on day 4, followed by the up-regulation of genes, such as SCL, LMO2, and MYB, representing hematopoietic and/or endothelial potentials on day 6. Collectively, VEGFR-2-mediated signals might contribute to the commitment and/or expansion of the hemangioblast rather than their development.

This coculture system provides an opportunity to better understand the regulative mechanisms on early hematopoietic and endothelial cell development, which remains unresolved by experiments using human embryos.
Performance of Murine Soluble Thrombomodulin (rmsTM) in a Murine Model of Endotoxin-Induced Sepsis.
Authors: Joyce David E. , Xu Haifeng , Mathur Sumeet , Castellino Francis J.
Published: April 2020

The endothelial surface protein thrombomodulin (TM) generates activated protein C (aPC) that protects from sepsis-related DIC, vascular inflammation, and apoptosis. The purpose of this study is to evaluate a novel recombinant mouse soluble TM (Eli Lilly, Indianapolis, IN) in a murine lethal endotoxin (LPS) sepsis model to determine both kinetic and survival endpoints. A dose effective in rodent reperfusion studies was employed in this sepsis model (D. Berg, Eli Lilly). C57BL6 mice were injected concomitantly with rmsTM 5 mg/kg (s.c.) and LPS 14 ug/g (i.p.) at time zero. Plasma concentrations were determined from citrated plasma at 3, 12, 24, and 48 hr, and plasma rmsTM concentrations were determined by ELISA. In vitro determinations of mouse aPC amidolytic activity and rmsTM dependent fibrinogen clotting time assays utilized rmsTM, murine thrombin, fibrinogen, and Protein C (Enzyme Research Labs). The in-vivo time-dependent mouse plasma kinetic studies confirmed plasma concentrations comparable to that achieved in human sTM antithrombotic studies. Thus, an rmsTM dose of 5 mg/kg was chosen for the 7-day LPS survival study (20% survival model). Twenty age-and weight-matched male C57BL6 mice, age 8–14 weeks, were treated with LPS 14 ug/g or LPS and rmsTM, 5 mg/kg, s.c. Survival curves were analyzed by Wilcoxon rank sum test (power 80%, alpha 2.5, p<0.05). An initial time course after rmsTM s.c. injection yielded a peak plasma rmsTM level at 3 hr (5,000 ng/mL) which persisted up to 48 hr (2,000 ng/mL). In vitro activation of aPC was saturating (2,000 ng/mL aPC with <20 ng/mL rmsTM). Prolongation of the thrombin clot time occurred at approximately 10 ug/ml of rmsTM. The LPS survival study in C57BL6 administered LPS (14 ug/g, i.p.) or LPS and rmsTM 5mg/kg s.c. demonstrated no survival difference (P=0.63). In vitro evaluation of aPC generation by rmsTM yielded ample amounts of aPC. Similarly, reduction of thrombin activity by rmsTM occurred at higher rmsTM concentrations. Thus, the lack of survival difference seen between endotoxemic mice treated with saline or adjunctive rmsTM in this model may be from the lack of thrombin inhibition, the lack of persistent (rmsTM:Thrombin) PC activation, or by more efficient clearance of aPC. Species specificity of TM is less likely a contributing factor. Further elucidation of the murine thrombin/thrombomodulin regulatory mechanism in vivo may be necessary to help explain our unexpected sTM survival results.
Augmented Effects on Neovascularization by Mixing the Conditioned Medium from Early EPCs into Late EPCs Derived from Human Cord Blood.
Authors: Yoo Eun-Sun , Ahn Jee-Young , Bae Yun-Kyung , Kong Jee Hyun , Lee Sang Min , Mun Yeung-Chul , Lee Kyoung-Eun , Seong Chu-Myong
Published: April 2020

Recently, we have reported on two different types of EPCs from human umbilical cord blood(ASH 2005, Abstract #1706). These EPCs had different biologic properties in angiogenic capabilities during the HCB ex vivo expansion. In this present study, the aim is to examine the synergism by mixing the conditioned medium from the early EPCs into the late EPCs on neovascularization. The mononuclear cells from the HCB were cultures using an EGM-2 medium with VEGF, IGF-1 and FGF for 21 days. We found early spindle-shaped cells(early EPCs), which were grown in the first week of the culture and late cobblestone shaped cells(late EPCs) which peaked in their growth in the third week of the culture. First, we compared the two types of cells in terms of phenotypic expressions and migration ability. Next, we examined their proliferation capacity and tube formations in the Matrigel plate under the conditions that the early outgrowing cells-contained medium was added to the cobblestone shaped cells. The late-appearing cobblestone shaped cells were positive for VEGFR2, VE-cadherin, CD31, CD34 and CXCR-4 but not for CD14 and CD54. These late outgrowing cells expressed high levels of mRNA on the endothelial marker genes and effectively formed capillary tubes in the Matrigel plates. The early spindle cells excreted more angiogenic cytokines and had more migratory ability. When the early spindle-shaped cell-conditioned medium was added to the late cobblestone shaped outgrowing cells, significantly higher proliferation and tube formation measured by the area and length of tubes were found. These results suggested that the two types of cells raised with different biologic properties during the ex vivo HCB expansion and the angiogenic capacity of late EPCs were augmented by a mutual interaction via the excreted cytokines from early EPCs. These finding may have potential applications for a “cell therapy” in situations such as vascular injuries (ie, hindlimb ischemia/myocardial infarction). Murine models are being tested to see whether the injections of two different EPCs will result in synergic noevascularization in our Lab.
Marrow Contributes to Kidney Vasculogenesis in Humans.
Authors: Gurevitz Stacy A. , Jorgensen Marda L. , Scott Edward W. , Cogle Christopher R.
Published: April 2020

The hematopoietic stem cell (HSC) is capable of contributing to vasculogenesis in response to ischemic injury. We have previously demonstrated that adult hemangioblast activity also exists within the capabilities of human hematopoietic stem/progenitor cells (HSC/HPC). However, we question the clinical relevance of adult hemangioblast activity. Thus, to address this question, we sought to analyze the marrow contribution to blood vessels within damaged vascular beds. Given the nephrotoxic side effects of chemotherapy and hematopoietic cell transplantation regimens, we identified kidney tissues from women (n=4) who received hematopoietic cell transplantations from male donors. Kidney tissues were stained for CD31, CD45, and XY FISH. One hundred forty vascular fields were analyzed. Fields were divided into three zones based on physiologic functions: glomerular, arteriolar (afferent & efferent), and medullary. In ever zone we identified male/marrow-derived endothelial cells contributing to kidney vasculature. However, each zone differed as to the degree of male/marrow-derived vascular contribution. In the glomerular regions, 36% of glomeruli contained one or more marrow-derived endothelial cells, as determined by orientation at the lumen edge, CD31 surface expression, and Y chromosome staining. In contrast, only 4% of arterioles contained male/marrow-derived endothelial cells. In the medulla, 9% of blood vessels contained male/marrow-derived blood vessels. The median number of marrow-derived endothelial cells in all regions was 1 male cell per blood vessel in field. In conclusion, human marrow can contribute modestly to kidney vasculature, especially in glomeruli. These results expose a potential strategy of kidney vessel repair by illuminating marrow-derived vasculogenesis within the kidney.
The Variation of Platelet Function in Gestational Diabetes Mellitus.
Authors: Li Yiqing , Yin Songmei , Nie Danian , Wang Xiuju , Xie Shuangfeng , Ma Liping , Wu Yudan
Published: April 2020

Gestational diabetes mellitus (GDM) was as glucose intolerance of different severity that onset or first recognition during the current pregnancy. GDM was a special complication of pregency and the most common cause of fetal and maternal mortality and morbidity. The etiology of this disease is not clear. Increased platelet function was common in normal pregnancy or type 2 diabetes mellitus (DM) patients in the last decade. However, when pregnancy plus type 2 DM, that is, the GDM, was the platelet function still changed? In the current study, we tested blood platelet function including platelet counts (PC), mean platelet volume (MPV) and granule membrane protein-140 (GMP-140, also known as P-selectin, CD62P) in pregnant women with GDM. CD62P was a sensitive index to evaluate platelet activation. PC and MPV would alter when platelets had been destructed significantly, so they were sensitive indexes for platelet activation too. PC was (206.02±60.23)×109/L in GDM, (228.76±56.24)×109/L in normal pregnancy, (219.76±58.14)×109/L in type 2 DM without pregnancy. MPV was 9.53±1.64fl in GDM, 8.74±1.59fl in normal pregnancy, 9.16±1.23fl in type 2 DM without pregnancy. The PC and MPV had no significant difference among GDM, normal pregnancy and type 2 DM without pregnancy (P>0.05). The expression of CD62P was higher than that of normal pregnancy or type 2 DM (41.82±12.95% vs. 26.21±7.09% or 24.18±8.79% P<0.05). These results indicate that there were much more platelet activation in GDM compared with normal pregnancy or type 2 DM, but the platelet destruction was not increased. Inhibition of activated platelet may be a new therapeutic strategy for the treatment of GDM. However, whether there was special role affecting platelet activation directly in GDM was unknown. The concrete mechanism underlying GDM platelet dysfunction need more elucidated.
The Use of Recombinant Factor VIIa (NovoSeven®) in a Patient with Bernard Soulier Syndrome and Dental Extraction.
Authors: Gerhardt Andrea , Scharf Rudiger E. , Zotz Rainer B.
Published: April 2020

Background: Bernard Soulier syndrome is a rare autosomal recessive platelet function disorder that impairs platelet adhesion to von Willebrand factor, especially at high shear flow. The disorder results from partial to complete deficiency or dysfunction of the GP Ib-IX-V receptor. Bernard Soulier syndrome typically results in prolonged bleeding time, thrombocytopenia, and giant platelets. The main clinical symptoms, evident from early childhood, include frequent episodes of epistaxis and hemorrhage associated with mucocutaneous bleeding or trauma. Specific treatment of bleeding episodes or prophylaxis for the prevention of bleeding during surgical procedures are platelet transfusions. This treatment option is not devoid of complications.

Alloimmunization by HLA antigens and antibody formation to the GPIb-IX-V complex frequently occur and, thus limit future responses to platelet transfusions. Recent patient studies have suggested that recombinant factor VIIa (rVIIa, NovoSeven®) may be an effective therapy for treatment of bleeding in patients with congenital platelet function defects and other acquired platelet disorders, including thrombocytopenia.

Case Reports: We report on our experience with rFVIIa in a 21-year-old woman with Bernard Soulier syndrome and dental extraction (2 wisdom teeth). In 2000, after a first extraction of one wisdom tooth, that was covered by administration of DDAVP and platelet transfusion, a bleeding episode occurred, requiring resurgery. This time rFVIIa was used as first line therapy. An initial dose of rFVIIa (100 μg/kg/bw) was administered 15 min. prior to surgery, followed by two doses of 90 μg/kg/bw at 120 min intervals postoperatively. In the first three days after surgery, 90μg/kg/bw rFVIIa were administered every 12 hrs. Tranexamic acid (Cyklokapron® 3 x 1 g/d) was started postoperatively and continued for 10 days. There were no postoperative bleeding episodes and no platelet transfusion was required.

In conclusion, rFVIIa in combination with tranexamic acid appears to be an effective and safe therapeutic alternative for prophylaxis of bleeding episodes in dental extraction in patients with Bernard Soulier syndrome.
Anti-CD20 Monoclonal Antibody (Rituximab) as an Optional Treatment for Intractable Acquired Glanzmann’s Thrombasthenia.
Authors: Blickstein Dorit , Lahav Judith , Molad Yair , Inbal Aida
Published: April 2020

Glanzmann’s thrombasthenia is an autosomal recessive bleeding disorder characterized by severely reduced or absent platelet aggregation in response to physiologic agonists because of quantitative or qualitative abnormalities of platelet glycoprotein IIb/IIIa (GpIIb/IIIa). Acquired Glanzmann-like thrombasthenia may develop as a result of autoantibodies specific to platelets GpIIb/IIIa and has been described in association with autoimmune disorders. Patients with the acquired form experience variable degrees of mucocutaneous bleeding, and their platelets fail to aggregate with agonists such as ADP, thrombin, collagen, or epinephrine, whereas ristocetin-induced aggregation is normal. Treatment consists of a combination of one or more of the following: corticosteroids, chemotherapy, plasma exchange and high dose intravenous immunoglobulin (IVIG). We present a 75-year old woman with SLE who developed ITP at the age of 60 and underwent splenectomy with subsequent normalization of platelet counts. However, since that operation, the patient has been admitted several times due to variable degrees of mucocutaneous bleeding, including one episode of massive gastrointestinal hemorrhage. Platelet counts were 184 to 284 X 103/μL. Laboratory evaluation revealed a bleeding time of 15 minutes (normal 0.5 to 8 minutes) and no aggregation of platelets in response to ADP, epinephrin, collagen, and thrombinbut normal ristocetin-induced aggregation. Addition of 25% of patient’s platelet poor plasma to normal whole blood revealed reduced platelet deposition by 61% using cone and plate analyzer, thus, confirming the diagnosis of acquired Glanzmann’s thrombasthenia due to autoantibodies. Because of treatment failure with corticosteroids, immunosuppession and IVIG, anti-CD20 monoclonal antibody (Rituximab) 375 mg/kg/week for 4 weeks was administered resulting in normalization of platelets aggregation and complete clinical remission. Anti-CD20 therapy might be a therapeutic option for intractable antibody-mediated acquired thrombasthenia.
Incidence and Risk Factors for Severe Thrombocytopenia in Lymphoma Patients Receiving Chemotherapy.
Authors: Huen Auris , Zhou Xiao , Teegala Shyam , Ji Yuan , Fayad Luis , Romaguera Jorge Enrique , Hagemeister F.B. , Vadhan-Raj Saroj
Published: April 2020

Background: Severe thrombocytopenia (TCP) can be a serious complication of chemotherapy (CT) in lymphoma patients (pts), however the exact incidence with the current regimens and risk factors for TCP are not well known.

Methods: A retrospective cohort study was conducted to determine the incidence of TCP, and logistic regression analysis were performed to identify the clinical and laboratory features correlated with severe TCP in lymphoma. Medical records of 538 consecutive pts out of the 1050 lymphoma pts, who were newly referred to MDACC in 2003, were reviewed.

Results: 202 pts who received > 1 cycle of treatment were included in the analysis. The total number of CT cycles (with PLT counts) for the 202 pts was 985 (median 6, range, 1–20). Grade (gr) 4 TCP (PLT nadir < 25x103/μL) was observed in 39% (79/202) of pts and 20% (196/985) of cycles. The median cycle in which gr 4 TCP occurred was 2 (range, 1 to 11), and median PLT nadir count for gr 4 TCP was 12x103/μL (3 to 24x103/μL). The regimens most commonly associated with gr 4 TCP were Hyper-CVAD/ Ara-c/Mtx (31/35=89%), ESHAP (6/7=86%), ASHAP (3/4=75%), and ICE (5/7=71%) + rituxan, and the most common histological subtypes were MCL (15/19=79%), Burkitts lymphoma (7/9=78%), T cell lymphoma (10/17=58%), and LCL (31/64=48%). Thirty two of 79 (41%) gr 4 TCP pts had bleeding as compared to 8 of 123 (7%) pts with higher PLT counts (p=0.0001). Overall, the incidence of bleeding was 20% (40/202) by pts and 6% (60/985) by cycles; however, the most incidents [95% (57/60)] were minor. 80% (63/79) of Grade 4 TCP pts received PLT transfusions, as compared to only 2% (2/123) of the rest (p<0.0001). Using multivariate logistic regression, histological gr, (highly aggressive or aggressive vs indolent: OR=10.402, 95% CI, 3.991 to 27.107, p<0.0001), baseline PLT count (≤150 vs >150x103/μL: OR=4.610, 95% CI, 1.366 to 15.560, p=0.0138), prior therapy (yes vs. no: OR=2.575, 95% CI, 1.337 to 4.961, p=0.0047), Beta2 Microglobulin (B2M) (≥2 vs <2mg/L: OR=2.846, 95% CI, 1.401 to 5.783, p=0.0038), age (>60 vs. ≤60 yrs: OR=0.479, 95% CI, 0.241 to 0.952, p=0.0356) were the most important risk factors for grade 3 or 4 TCP.

Conclusions: The incidence of severe TCP in this population is high. Baseline pt characteristics including histological gr, PLT counts, prior therapy, age and serum B2M were found to be significant risk factors predictive for chemotherapy-induced TCP (CIT). These findings could be useful to identify high risk pts for consideration of treatment approaches for prevention and treatment of CIT.
Outcome of Recombinant Factor VIIa Use for Alveolar Hemorrhage in Hematopoietic Stem Cell Transplant Recipients.
Authors: Gupta Sachin , Gupta Aditya , Jain Amit , Warneke Carla L. , Giralt Sergio , Eapen Georgie A.
Published: April 2020

Background: Hematopoietic Stem Cell Transplantation (HSCT) is being widely used as an adjunct to conventional chemoradiotherapy for many hematologic and solid malignancies and some non-malignant hematologic conditions. Alveolar Hemorrhage (AH), seen in 1 to 19% of HSCT recipients has a mortality of 70–100%. Case reports have suggested that administration of recombinant factor VIIa to patients with AH results in rapid clinical improvement and improved survival. Objectives: To study the impact of recombinant Factor VIIa administration in HSCT recipients with AH at M.D. Anderson Cancer Center.

Methods: A retrospective review of all adult patients who underwent bronchoscopy after HSCT between January 1, 2002 and December 31, 2004 for a presumptive diagnosis of AH. Outcome measures analyzed included requirement for positive pressure ventilation over the six months following bronchoscopy, the length of index hospitalization and survival. Pearson chi-squared tests, Fisher’s exact tests, and Wilcoxon rank sum tests were used for statistical analyses.

Results: Eighty seven patients were diagnosed with AH during the study period. Recombinant factor VIIa was administered to 24 patients with alveolar hemorrhage. All 24 patients received high dose steroids and intensive supportive care. Along with factor VIIa, 16 patients received platelet transfusions to keep platelet count above 50×109/L, 4 received desmopressin (DDAVP), and 3 received aminocaproic acid. Only 63% were alive at 30 days after bronchoscopy and 38% after 6 months. Overall, 17 of the 24 patients (71%) receiving factor VIIa died during the study period. Seventy five percent required positive pressure ventilation within 6 months of diagnosis. The median duration of hospitalization was 11 days (SD ± 10 days). No thrombotic complications were observed in any of these 24 patients. There were no statistically significant differences in median survival, 1 and 6-month survival, requirement for positive pressure ventilation, respiratory failure as the cause of death, or duration of hospital stay after bronchoscopy between patients with AH who received factor VIIa and those who did not (p=>0.6 for all outcomes).

Conclusion: In our study of 24 patients with AH who received recombinant factor VIIa for AH, no significant improvement in survival or alteration in the hospital course was observed. Further prospective studies are needed to clarify the role of Factor VII for this indication.