bluuebunny/crossref_metadata_2025_split huggingface dataset:
Aberrant Microtubule Organization and Wiskott-Aldrich Syndrome-like Defects in Platelets and Megakaryocytes of Profilin1-Deficient Mice
Authors: Stritt Simon , Bender Markus , Nurden Paquita , van Eeuwijk Judith , Zieger Barbara , Kentouche Karim , Schulze Harald , Morbach Henner , Stegner David , Heinze Katrin G , Dütting Sebastian , Gupta Shuchi , Witke Walter , Falet Hervé , Fischer Alain , Hartwig John H. , Nieswandt Bernhard 
Published: October 2019

Abstract
Wiskott-Aldrich syndrome (WAS) is a rare, X-chromosomal recessive disorder which is caused by mutations in the WAS gene and characterized by eczema, immunodeficiency and microthrombocytopenia (Thrasher and Burns, Nat Rev Immunol 2010). Interestingly, WAS protein (WASp)-deficient mice have normal-sized platelets and thus the molecular link between WAS mutations and its central hallmark microthrombocytopenia remains elusive.
Profilin1 (Pfn1) is a key actin-regulating protein that, besides actin, interacts with phosphoinositides and multiple proline-rich proteins including the WAS protein (WASp)/WASp-interacting protein (WIP) complex (Witke, Trends Cell Biol 2004; Ramesh et al., Proc Natl Acad Sci USA, 1997). Interestingly, similar to WAS patients, mice with Pfn1-null megakaryocytes/platelets suffered from microthrombocytopenia. We identified accelerated platelet clearance by macrophages and pre-mature platelet release into the bone marrow compartment as the major cause of the reduced platelet count in Pfn1-deficient mice.
Both, platelets from Pfn1-null mice and WAS patients contained abnormally organized and hyperstable microtubules. We next tested, if increased microtubule stability could account for the reduced size of Pfn1-deficient platelets. Treatment of control platelets with microtubule stabilizing toxins, such as the histone-deacetylase inhibitor trichostatin A (TSA) or taxol resulted in a decreased platelet size. This finding indicates that increased microtubule stability could account for the reduced platelet size in Pfn1-deficient mice but also in WAS patients. Based on these results we speculate that WASp might modulate Pfn1 function and dysregulation of this interaction leads to increased stability and altered organization of microtubules. In support of this, the subcellular localization of Pfn1 was altered in platelets of three WAS patients.
Together, these results reveal an unexpected function of Pfn1 as a regulator of microtubule organization and point to a previously unrecognized mechanism underlying the platelet formation defect in WAS patients.

Disclosures
No relevant conflicts of interest to declare.
The Expression of Functional Dopamine and Serotonin Receptors on Megakaryocytes
Authors: Mo Yang , Li Su-yi , Liang En-yu , Lian Qi-zhou , Meng Fan-yi 
Published: October 2019

Abstract
Monoamine dopamine (DA) and serotonin (5-HT) can affect the functions of many cells including platelets. Our previous studies have shown that 5-HT has potent effects on promoting megakaryopoiesis and platelet formation (Yang et al, Stem Cells, 2007; 2014). In this study, our hypothesis is that both DA and 5-HT receptors may have the development expression on platelets, megakaryocytes (MK) and hematopoietic stem cells (HSCs). The expression of DA receptors: DA1, DA2 and 5-HT receptors: 5-HT2A, 2B and 2C on these hematopoietic cells, and the effect of DA and 5-HT on the cell proliferation and apoptosis were investigated using RT-PCR, flow cytometry, immunofluorescence staining, annexin V, JC-1 and caspase-3 assay. Our results showed that dopamine DA1, DA2 receptors, 5-HT2A, 2B and 2C receptors were identified on human platelets, MK and HSCs using RT-PCR method. Using flow cytometry with monoclonal antibodies, these receptors were also identified on human platelets and MK. But these receptors expressed on human HSCs at a low levels. The expression of the five receptors were further confirmed on human bone marrow MK and HSC by using immunofluorescence staining. The functions of receptors were further studied in human MK and HSCs. DA at a low dose induced cell differention, but at a high dose induced cell apoptosis. This effect could be reduced by DA2 receptor blocker L-Sulpiride (L-SULP). In the other hand, 5-HT had an anti-apoptotic effect in MK cells. 5-HT also stimulated CFU-MK formation, this effect was reduced by 5-HT2R antagonist ketanserin. 5-HT also enhanced Akt phosphorylation, while addition of ketanserin compromised the 5-HT effect. 5-HT-driven MK effect was likely mediated via 5-HT2R and p-Akt. A higher number of proplatelet bearing MK was also detected in 5-HT-treated. Polymerization F-actin level was higher in 5-HT group. Adding ketanserin/PD98059, the intensity was reduced. Treated with 5-HT for 30 minutes ERK1/2 was activated. The adding of ketanserin/PD98059 to 5-HT cultures nullified the 5-HT effect. This effect was likely mediated via 5HT2R, p-ERK1/2, which leads to F-actin reorganization and proplatelet formation. This study demonstrated that the expression of DA and 5-HT functional receptors on human platelets, MKs and HSCs. DA and 5-HT may be involved in the regulation of thrombopoiesis.

Disclosures
No relevant conflicts of interest to declare.
The 1.7 Angstrom X-Ray Crystal Structure of the Porcine Factor VIII C2 Domain and Binding Analysis to Anti-Human C2 Domain Antibodies and Phospholipid Surfaces
Authors: Brison Caileen M , Mullen Steven M , Podolsky Kira , Meeks Shannon L , Spiegel Paul Clinton 
Published: October 2019

Abstract
The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity, however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the “non-classical” antibody epitopes. Furthermore, antibody-binding results illustrate that the “classical” 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 BU/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Lastly, differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with similar affinities, and the human K2227E mutant binds to phospholipid surfaces with a modestly decreased affinity.

Disclosures
No relevant conflicts of interest to declare.
p38α Is Required for Proper Proliferation of Hematopoietic Stem Cells during Stress
Authors: Karigane Daiki , Takubo Keiyo , Okamoto Shinichiro , Suda Toshio 
Published: October 2019

Abstract
Hematopoietic stem cells (HSCs) are capable of self-renewal and multilineage-differentiation during lifespan. HSCs are maintained in a quiescent state to avoid cellular senescence. Previous reports utilizing pharmacological inhibitors or shRNAs against p38MAPK suggest a pivotal role of p38MAPK-pRb-Ink4a signaling in induction of HSC senescence by hematological stress or chronological aging. However, no genetic evidence exists for p38MAPK-mediated cellular senescence in vivo. Here we report unexpected roles of the dominant isoform of p38MAPK family, p38α, in adult hematological system. p38MAPK has four isozymes, α, β, γ and δ. Among them, p38α isozyme was highly expressed in various bone marrow hematopoietic cells, and the expression level of p38α in HSCs was higher than differentiated cells (p<0.01). Phosphorylation of p38MAPK was mainly observed in multipotent progenitors but not in HSCs in steady-state hematopoiesis, in addition, physiological aging (1 year old mouse bone marrow) did not affect phosphorylation status of p38MAPK in steady state. In contrast, p38MAPK was phosphorylated in HSCs after transplantation or 5-FU treatment. Mean fluorescence index (MFI) of phosphorylation of p38MAPK in HSCs is significantly higher at day 3 post 5-FU treatment (250 mg/kg) than steady-state. MFI of phosphorylation of p38MAPK in HSCs was higher at day 1 post transplantation than steady state, and returned to normal at day 7 post transplantation. These results showed phosphorylation of p38α was immediately induced after hematopoietic demand.
p38α-deficient embryos die due to defective erythropoiesis in a non-cell-autonomous manner. Thus, we used a conditional knockout model; CAG-CreERT2:p38αfl/fl mouse to analyze the effects of p38α on adult hematopoiesis and HSCs. Expression level of p16Ink4a, one of the cellular senescence markers, was not significantly different between p38α-deficient mice and wild-type mice. Treatment of p38α-deficient mice with 5-FU exhibited defective recovery of hematopoiesis, and the survival rate were lower in p38α-deficient mice than wild-type (42.9%, N=7, p38α-deficient mice, vs 100%, wild-type, N=6, p<0.05). Loss of p38α in HSCs showed a defective transplantation capacity. Inducible loss of p38α in bone marrow chimera resulted in a gradual loss of peripheral blood chimerism of p38α-deficient cells. In addition, short-term BrdU incorporation assay showed that the cell cycle progression of p38α-deficient HSCs was suppressed (BrdU positive rate; 3.5±2.2%, N=9, p38α-deficient cells vs 6.5±2.6%, N=5, wild-type, p<0.05). Therefore, hematopoietic function was obviously lowered in p38α-deficient HSCs during hematopoietic stresses. These observations collectively support the requirement of p38α for proper proliferation of HSCs during stress hematopoiesis.

Disclosures
No relevant conflicts of interest to declare.
CD43 but Not CD41 Marks the First Hematopoietic Stem Cells in the Human Embryo
Authors: Ivanovs Andrejs , Rybtsov Stanislav , Anderson Richard A. , Medvinsky Alexander 
Published: October 2019

Abstract
Hematopoietic stem cells (HSCs) emerge during embryogenesis and maintain multilineage hematopoiesis throughout the entire lifespan of the organism. Due to limited access to human embryonic tissues, early human HSC development remains a largely unexplored area of research. A better understanding of this process may be instrumental in creating novel protocols for in vitro generation of human HSCs from pluripotent stem cells to overcome the shortage of donor cells and increase the safety of HSC transplantations in the clinic.
We have recently described the spatio-temporal distribution of HSCs in the early human embryo and established the immunophenotype of the first human HSCs (Ivanovs et al., J. Exp. Med., 2011; Ivanovs et al., Stem Cell Reports, 2014). The first human HSCs emerge in the ventral domain of the dorsal aorta, possess high regenerative potential, and reside within the CD34+VE-cadherin+CD45+c-Kit+Thy-1+Endoglin+Runx1+CD38-/loCD45RA- cell population, which harbors all HSCs and committed hematopoietic progenitors in the human aorta-gonad-mesonephros (AGM) region. Here, we aimed to find a surface marker which would further enrich for the human AGM region HSCs. We found that CD41, which is the earliest surface marker of hematopoietic commitment in the mouse embryo (Ferkowicz et al., Development, 2003; Mikkola et al., Blood, 2003), does not mark the first human HSCs (3 independent experiments). Instead, the first HSCs in the human AGM region express CD43 (4 independent experiments). Of note, it has been shown previously that during human embryonic stem cell differentiation in vitro, CD43 but not CD41 marks multipotent hematopoietic progenitors (Vodyanik et al., Blood, 2006).
Human embryonic tissues for our study were obtained after elective medical termination of pregnancy with informed written consent according to the Declaration of Helsinki. The study protocol was approved by the Lothian Research Ethics Committee. Employing magnetic cell separation and xenotransplantations, CD41- and CD43-positive and -negative cell populations were isolated from Carnegie stage 15–17 human AGM regions and individually transplanted into preconditioned adult NOD.Cg-PrkdcscidIl2rgtm1Wjl/Sz mice. Human hematopoietic contribution to the peripheral blood, bone marrow, spleen, and thymus of transplanted mice was assessed at 4 months after transplantation.
Our findings suggest that, although the process of HSC development is largely conserved between the mouse and the human, certain aspects vary substantially between the species, reflecting possible differences in the HSC specification program. We conclude that CD43 is a useful surface marker for the purification of the first human HSCs to enable further studies into molecular mechanisms underlying their development and high regenerative potential.

Disclosures
No relevant conflicts of interest to declare.
Proliferation-Independent Functions of the E2F-2 Transcription Factor during Terminal Erythroid Maturation
Authors: Swartz Kelsey , Qin Gangjian , Minella Alexander C 
Published: October 2019

Abstract
Lesions in the retinoblastoma (Rb) pathway induce defects in hematopoiesis that are typically thought to be due to inappropriate proliferative signaling from increased E2F transcriptional activity. Newer data demonstrate that E2F transcription factors have distinct roles in differentiating cells, but the key proliferation-independent functions of the E2Fs remain incompletely defined. We previously found that deregulated activity and expression of cyclin E induces defects in terminal erythroid cell maturation, using a mouse knock-in model (cyclin ET74A T393A). Because cyclin E-Cdk2 potentiates E2F transcriptional activity via Rb phosphorylation, we hypothesized that inhibiting E2F activity would rescue hyperactive cyclin E-associated erythroid cell defects. E2F-2 is specifically induced during terminal erythroid maturation; therefore, we crossed our cyclin E knock-in strain with E2F-2 knockout mice. Unlike cyclin E knock-in bone marrows that display obvious defects in erythroid cell maturation, cyclin E knock-in; E2F-2 knockout animals demonstrated normalized erythroid maturation by flow cytometry. However, these compound mutant mice remain anemic, suggesting red blood cell (RBC) maturation was not completely restored.
We studied adult E2F-2 -/- mice further, and consistent with published data, we found that they are anemic. Interestingly, we do not detect obvious terminal erythroid maturation defects in adult E2F-2-knockout bone marrows. Using CFSE-labeled erythrocyte in vivo survival experiments, we found loss of E2F-2 results diminishes survival of adult peripheral erythroid cells within syngeneic, wild-type recipients, suggesting the anemia in the E2F-2 knockout mice is due to accelerated destruction and not solely a production defect. To study the erythroid maturation program in the absence of E2F-2 in detail, we obtained fetal liver-derived hematopoietic progenitors from E2F-2 knockout versus wild-type embryos and differentiated these towards the erythroid lineage in vitro. E2F-2 deletion results in impaired erythroid maturation as evidenced by flow cytometry-based assays of cell surface marker expression, abnormal cell morphologies, and impaired enucleation. In order to identify functionally significant E2F-2 targets during erythroid maturation, we performed microarray analyses on sorted subpopulations of fetal liver-derived erythroid cells. We found widespread gene expression changes in E2F-2 knockout cells, which included but were not limited to proliferation related pathways. Furthermore, using global histone mass spectrometry analysis, we found that loss of E2F-2 results in marked alteration of histone-H3, lysine-4 methylation during erythroid differentiation. Thus, our data demonstrate that E2F-2 has both proliferation-dependent and independent functions that include the coordination of transcriptional and epigenetic programs during terminal erythroid maturation.

Disclosures
No relevant conflicts of interest to declare.
Role of Hepatocyte Growth Factor in Endothelial-Dependent Hematopoietic Stem Cell Regeneration
Authors: Zachman Derek K , Goldman Devorah C , Hamlin Kimberly L , Guha Chandan , Fleming William H. 
Published: October 2019

Abstract
Hematopoiesis is tightly regulated by the niche and highly sensitive to a variety of genotoxic stressors. The mechanisms responsible for the repair and regeneration of hematopoietic stem cells (HSCs) are poorly understood. Endothelial cells (ECs) are an essential component of the bone marrow microenvironment that provide regenerative signals for HSCs after injury; however, the identity of these signals remain largely unknown. We utilized RNA sequencing and a bioinformatics approach to identify hepatocyte growth factor (HGF) as an endothelial-derived mediator of HSC regeneration. HGF gene expression was 7.6-fold higher in arterial-derived ECs (HAEC) that regenerate functional long-term HSCs (CD150+LineageloSca1+Kit+ cells; CD150+LSK cells) compared to venous ECs (HUVEC) that support only hematopoietic progenitors. Furthermore, the HSC-supportive HAECs secreted up to 25 pg/mL HGF into conditioned media, whereas HGF was undetectable in media conditioned by HUVEC. To directly test the role of HGF in HSC regeneration, we supplemented co-cultures of irradiated bone marrow cells and primary ECs with recombinant HGF. Addition of HGF was sufficient to augment CD150+LSK cell regeneration up to 20-fold by HUVEC. This effect was abolished by treatment with the Met-specific small molecular inhibitor, PF04217903. Importantly, neither the addition of HGF alone to irradiated hematopoietic cells nor spiking HUVEC-conditioned media with HGF improved HSC regeneration in the absence of an EC monolayer. Together, these results indicate that HGF activates a signaling loop in ECs that supports HSC regeneration.

Disclosures
No relevant conflicts of interest to declare.
Histone Deacetylase 6 (HDAC6) Regulates Gene Transcription and Enucleation during Erythroid Differentiation
Authors: Li Xuehui , Yang Yurong , Lu Jianrong , McKinsey Timothy , Ji Peng , Qiu Yi 
Published: October 2019

Abstract
The deacetylation of histone and non-histone proteins by histone deacetylase (HDACs) plays a critical role in gene transcription and many other cellular processes in eukaryotic cells. Class II deacetylase HDAC6 is mainly localized in the cytoplasm and deacetylates tubulin and other cytoplasmic proteins. Earlier studies from our laboratory showed that HDAC6 can be transported to the nucleus, and can interact and deacetylate nuclear proteins, such as histones. In this study, we investigate the function of HDAC6 during erythroid differentiation. In proerythroblast cells, HDAC6 is detected in the nucleus and gradually migrates to the cytoplasm upon the induction of differentiation, indicating different HDAC6 functions during erythropoiesis. Inhibition of HDAC6 in mouse fetal liver erythroblasts impairs differentiation and enucleation. β-globin gene transcription is reduced by HDAC6 specific inhibitor, Tubastatin A, in cultured fetal erythroblasts. HDAC6 knockout mice also showed impaired globin gene transcription. HDAC6 specifically interacts with serine 2 phosphorylated Pol II and is recruited mainly at the transcribed region of β-globin, correlating with RNA Pol II recruitment. These results suggest that HDAC6 promotes erythroblast differentiation through the regulation of transcription elongation. HDAC6 also plays a role in regulating the enucleation process during the late stage of erythropoiesis. The formation of the contractile actin ring is disrupted and the enucleation process is blocked in cultured mouse fetal erythroblasts treated with the HDAC6 inhibitor. We further investigated the molecular mechanism of HDAC6 regulated enucleation. We found that HDAC6 interacts with and deacetylates mDia2, an effector of Rho GTPases that is required for erythroblast enucleation. Deacetylation of mDia2 is required for the formation of the contractile actin ring and subsequent enucleation. Altogether, our results identify the important role of HDAC6 in regulating transcription and enucleation during the different stages of mammalian erythropoiesis.

Disclosures
No relevant conflicts of interest to declare.
Loss of mDia1 Mediates the Development of Del(5q) MDS through Upregulation of the Innate Immune Response and Induction of Neutropenia
Authors: Mei Yang , Zhao Baobing , Yang Jing , Ji Peng 
Published: October 2019

Abstract
mDia1, the diaphanous homolog 1 of Drosophila in mouse, is a formin protein involving in the linear actin polymerization. Recently, our group reported that patients with del(5q) myelodysplastic syndromes (MDS) showed a significantly decreased mDia1 expression. Mice with mDia1 deficiency developed age related hematologic features mimicking human MDS. In these mice, CD14 is aberrantly overexpressed on granulocytes, which sensitized the innate immune response upon the lipopolysaccharide(LPS) injection. Importantly, chronic LPS injection accelerated the development of MDS (Keerthivasan et al Blood 2014). Here we report that 1) the mDia1 knockout (KO) mice also have a hypersensitive innate immune response to damage-associated molecular pattern molecules (DAMPs), which can be partially blocked by CD14/Toll-like receptor 4(TLR4) signaling pathway inhibitors. This is significant since release of DAMPs from necrotic or senescent cells is a common age-related phenomenon. DAMPs are also detected in cancers including MDS. Thus our study may shed lights on how the deregulated innate immune responses get involved in the pathogenesis of MDS in a more pathophysiologically relevant etiology. 2) mDia1 KO mice have an increased Gr1/Mac1 expression on the granulocytes in peripheral blood. We demonstrate that the expression levels of Gr1/Mac1 were not changed in bone marrow granulocytes, suggesting a specific up-regulation of Gr1/Mac1 levels on circulating granulocytes in mDia1 knockout mice. Mac-1, as the predominant β2 integrin on granulocytes, plays key roles in the adhesion of leukocytes to the endothelium, and regulates the cell adhesion, migration, and chemotaxis. In this respect, the mDia1 knockout mice develop serve neutropenia, which could be due to the upregulation of Gr1/Mac1 and increased binding of the cells to intercellular adhesion molecule-1 (ICAM-1). Finally, we revealed the mechanism of Gr1/Mac1 upregulation by showing that loss of mDia1 significantly affected the endocytosis of Gr1 and Mac1 on granulocytes, however, the mRNA levels of Gr1 and Mac1 were not affected. Taken together, these studies reveal a significance of loss of mDia1 in the pathogenesis of del(5q) MDS through upregulation of innate immune response and accelerated granulocyte clearance.

Disclosures
No relevant conflicts of interest to declare.