bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transfer of Monocrotaline-Induced Pulmonary Hypertension to Healthy Mice Via Microparticles
Authors: Aliotta Jason M. , Pereira Mandy , Amaral Ashley , Sorokina Arina , Rounds Sharon I , Quesenberry Peter J. , Klinger James R. 
Published: October 2019

Abstract
Abstract 5190

Rationale:
Circulating endothelial cell-derived microparticles (MP) are altered in pulmonary arterial hypertension (PAH) but whether they are biomarkers of cellular injury or participants in disease pathogenesis is unknown. We have shown that lung-derived MP induce marrow cells to express lung-specific mRNA, protein.

Objectives:
Determine if lung, plasma-derived MP (LMP, PMP) alter pulmonary vascular endothelial or marrow progenitor cell phenotype to induce pulmonary vascular remodeling in PAH.

Methods and Main Results:
LMP, PMP isolated from monocrotaline (MCT)- or vehicle-treated mice were injected into healthy mice. Right ventricular (RV) hypertrophy and pulmonary vascular remodeling were assessed by RV-to-body weight (RV/BW), blood vessel wall thickness-to-diameter (WT/D) ratios. RV/BW, WT/D ratios were elevated in MCT vs. vehicle-injected mice (1. 99+0. 09vs. 1. 04+0. 09mg/g; 0. 159+0. 002vs. 0. 062+0. 009%). MP from MCT and vehicle-treated mice were quantitatively similar; however, MCT-LMP had higher endothelial cell mRNA expression and higher expression of mRNAs of proteins known to be abnormally expressed in PAH vs. vehicle-LMP. RV/BW, WT/D ratios were higher in mice injected with MCT-MP vs. mice injected with vehicle-MP (1. 63+0. 09vs. 1. 08+0. 09mg/g; 0. 113+0. 02vs. 0. 056+0. 01%). Lineage-depleted bone marrow cells co-cultured with MCT-MP and bone marrow cells isolated from MCT-MP infused mice had higher endothelial progenitor cell gene expression vs. cells co-cultured with vehicle-MP or cells isolated from vehicle MP infused mice.

Conclusions:
MP from MCT-injured mice have increased expression of growth factors implicated in PAH pathogenesis and induce RV hypertrophy, pulmonary vascular remodeling in healthy mice. Circulating MP may contribute to the development of MCT-induced pulmonary hypertension by inducing a pathologic phenotype in pulmonary vascular endothelial and endothelial progenitor cells.

Disclosures:
No relevant conflicts of interest to declare.
Investigation of ETV2/Etsrp Role in Lymphangiogenesis: A Potential Therapeutic Target in Lymphatic Disease
Authors: Williams Jennifer D. , Desai Sharina Palencia , Sumanas Saulius 
Published: October 2019

Abstract
Abstract 614
Lymphangiogenesis, defined as novel growth of lymphatic vessels to constitute a functional vascular network, captures great clinical interest because of its essential role such pathologic settings as inflammation, vascular malformations, and tumor metastasis. Previous observational hurdles are now overcome by use of the optically clear and genetically modifiable zebrafish embryo, which provides a vertebrate model for understanding lymphangiogenesis and for revealing putative therapeutic targets for metastatic disease and vascular/lymphatic malformations that currently have no cure.
The zebrafish ets1-related protein, Etsrp, encodes a novel ETS domain transcription factor and is an early marker for vascular endothelial progenitors. Its function is necessary and sufficient to initiate vasculogenesis and is conserved during vertebrate development. Human and murine Er71/Etv2 proteins are functional orthologs of Etsrp. However, its role in lymphatic development is unknown. Our data show that both vascular and lymphatic vessels express etsrp driven GFP transgene. To analyze Etsrp role in lymphatic development, we performed functional Etsrp knockdown by photoactivatable morpholino oligonucleotide (MO) injection into fli1:GFP; flk1:mCherry transgenic reporter zebrafish embryos whose fluorescent background allowed distinction of lymphatic from vascular vessels.
Inhibition of etsrp function at early stages resulted in the absence of both lymphatic and vascular endothelial development. Inhibition of etsrp function at 1 day-post-fertilization, after the major axial vessels have already formed, specifically affected lymphangiogenesis without affecting vascular development. The major lymphatic vessel, the thoracic duct, was absent or discontinuous in Etsrp-depleted embryos, while the major axial and secondary intersegmental blood vessels were not affected. Further analysis demonstrated that the lymphatic progenitors, parachordal lymphangioblasts, failed to migrate from the posterior cardinal vein in the absence of Etsrp function. Control morpholino-injected but not photoactivated embryos did not show significant defects in the lymphatic development, demonstrating the specificity of the observed defects. Because expression of the major VegfC receptor, VegfR3 was strongly down regulated in Etsrp MO-injected embryos, our results suggest that Etsrp initiates lymphangiogenesis by regulating VegfR3 expression. In the absence of Etsrp function, lymphangioblasts would fail to respond to VegfC signaling.
We report for the first time the requirement of Etsrp for normal lymphangioblast migration and thoracic duct development in zebrafish. Additional studies are in process to determine the mechanism of Etsrp role in lymphangiogenesis. Our results suggest that Etsrp may represent a potential novel target for chemical modulation in multiple human lymphatic disorders.

Disclosures:
No relevant conflicts of interest to declare.
Normal Hematopoiesis and Neurofibromin-Deficient Myeloproliferative Disease Require Erk
Authors: Staser Karl , Zeng Yi , Park Su-jung , Rhodes Steven D. , He Yongzheng , Shew Matthew A. , Gehlhausen Jeff , Cerabona Donna , Chen Shi , Sun Zejin , Nalepa Grzegorz , Yang Feng-chun , Clapp D. Wade 
Published: October 2019

Abstract
Abstract 704
Neurofibromatosis type 1 predisposes individuals to the development of juvenile myelomonocytic leukemia, a fatal myeloproliferative disease (MPD). In genetically engineered murine models, nullizygosity of Nf1, a tumor suppressor gene which encodes a Ras GTPase activating protein, results in hyperactivity of Raf-Mek-Erk in hematopoietic stem and progenitor cells (HSPCs). Activated Erk1/2 phosphorylate kinases and transcription factors with plethoric mitogenic roles in diverse cell types. However, genetic studies examining Erk1/2's differential and/or combined control of normal and Nf1-deficient myelopoiesis are lacking. Moreover, prior studies relying on chemical Mek-Erk inhibitors have reached conflicting conclusions in normal and Nf1-deficient mice. Here, we show that while single Erk1 or Erk2 disruption does not grossly compromise myelopoiesis, dual Erk1/2 ablation rapidly ablates granulocyte and monocyte production, diminishes progenitor cell number, and prevents HSPC proliferation. Intriguingly, genetic disruption of Erk1/2 in the context of Nf1 nullizygosity (Mx1Cre+Nf1flox/floxErk1−/−Erk2flox/flox) fully protects against the genesis of MPD. Collectively, we identify a fundamental requirement for Erk1/2 signaling in normal and Nf1-deficient hematopoiesis, elucidating a critical hematopoietic function for Erk1/2 while genetically validating highly-selective Mek-Erk inhibitors for the attenuation of a leukemia that is otherwise resistant to traditional therapy.

Disclosures:
No relevant conflicts of interest to declare.
Recombinant Circularly Permuted TRAIL (CPT) for the Treatment of Relapsed or Refractory Multiple Myeloma: An Open-Label, Multicenter Phase II Clinical Trial
Authors: Chen Wenming , Qiu Lugui , Hou Jian , Zhao Yaozhong , Pan Ling , Yang Shifang , Leng Yun , Xi Hao , Zhang Xuejun , Cui Junsheng , Wei Na 
Published: October 2019

Abstract
Abstract 78

Background:
Circularly permuted TRAIL (CPT) is a recombinant mutant of human Apo2L/TRAIL developed by Beijing Sunbio Biotech Co., Ltd. as a targeted therapy for multiple myeloma and other hematologic malignancies. CPT is a dual pro-apoptotic receptor agonist that directly activates both pro-apoptotic receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). CPT selectively induces apoptosis in a variety of cancer cells, while sparing most normal cells in preclinical models.

Objective:
CPT has shown preliminary activities for patients with relapsed or refractory multiple myeloma (Rel/Ref MM) in phase I study. The aim of this phase II study is to investigate the effect and safety of CPT for Rel/Ref MM patients.

Methods:
In this multi-center, open-label, single arm phase II study, twenty-seven Rel/Ref MM patients were enrolled to receive CPT treatment alone at the dose of 2.5 mg/kg/day intravenously, once daily for 14 consecutive days of each 21-day cycle. Clinical responses to CPT after 2 cycle's treatment were assessed by an independent review committee according to the criteria of the European Group for Blood and Marrow Transplantation (EBMT).

Results:
Among the 27 patients, one patient (3.7%) achieved near complete response (nCR) and eight patients (29.63%) exhibited partial response (PR). The overall response rate (ORR) was 33.3%. The median PFS was not reached within 2 cycle's treatment. The drug related adverse events (≥10%) included fever, AST/ALT elevation, leucopenia, neutropenia, rash, thrombocytopenia, and LDH elevation. The severe adverse events were occurred in 3 patients (11%), one of them was CPT-related liver injury. Anti-drug antibodies were detected in 6 patients without reduced efficacy or increased toxicities.

Conclusions:
The CPT is a very effective and well-tolerated new agent for Rel/Ref MM, and it is worthy to be further studied.

Disclosures:
Yang: Beijing Sunbio Biotech Co. Ltd.: Employment. Cui:Beijing Sunbio Biotech Co. Ltd.: Employment.
CHD7 Negatively Regulates Hematopoietic Stem Cell Potency
Authors: Hsu Jingmei , Huang Hsuan-Ting , Lee Chung-Tsai , Yu Shuqian , Gerber Scott , Zon Leonard I. , Speck Nancy A. 
Published: October 2019

Abstract
Abstract 850
The molecular mechanisms governing HSC homeostasis and potency are incompletely understood. Runx1-CBFb is required for HSC formation in the embryo and for maintaining normal HSC and progenitor cell frequencies in the adult. To identify chromatin regulatory proteins that function with Runx1-CBFb during hematopoiesis, we performed co-immunoprecipitation and mass spectrometry using a FLAG-tagged CBFb protein, and as a control a FLAG-CBFb (G61A/N104A) mutant that cannot bind Runx1. Multiple unique peptides of the ATP-dependent chromatin remodeling enzyme Chromodomain Helicase DNA binding protein 7 (CHD7) were immunoprecipitated specifically with FLAG-CBFb. Autosomal dominant CHD7 mutations are associated with CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, and Ear abnormalities and deafness). Several subunits of the nucleosome remodeling and histone deacetylase (NuRD) complex were also identified, including p66a, p66b, MTA2, and histone binding protein RBBP4. Hence Runx1-CBFb may associate with distinct CHD7 and NuRD containing complexes to regulate HSC function.
The CHD7 interaction with Runx1 requires the Runx1 transactivation domain, which is critical for all stages of hematopoiesis. The interaction of CHD7 with Runx1 does not require heterodimer formation with CBFb. Runx1+/−; Chd7+/− double heterozygous mice are born in a sub-Mendelian ratio, indicating that Runx1 and Chd7 interact genetically. Knockdown of chd7 in zebrafish embryos increased the number of runx1 expressing cells specified in the AGM, and this effect is cell autonomous as determined by blastula transplantation. Downstream lineages such as myeloid and erythroid cells were also expanded. Consistent with these results, conditional pan-hematopoietic deletion of Chd7 in mice with Vav1-Cre did not cause mature lineage differentiation defects. Methylcellulose colony forming assays of CHD7 deficient cells resulted in increased granulocyte/monocyte progenitors. Although CHD7 deficient bone marrow had a normal frequency of CD48− CD150+ Lin−Sca-1+c-kit+(LSK) phenotypic long term repopulating HSCs (LT-HSCs), it has a three fold higher frequency of functional LT-HSCs as determined by whole bone marrow limiting dilution transplants. By serial transplantation, a higher percentage of tertiary recipient mice were engrafted with CHD7 deficient bone marrow cells compared to wild type. In summary, our study identifies CHD7 as a novel epigenetic regulator of HSC and progenitor function that functions with Runx1 and members of the NuRD complex to negatively regulate HSC numbers and potency.

Disclosures:
Zon: Fate Therapeutics: Founder Other.
Loss of Krüppel-Like Factor 4 (KLF4) Impairs the Self-Renewal of Leukemic Stem Cells
Authors: Park Chun Shik , Shen Ye , Yamada Takeshi , Suppipat Koramit , Lacorazza H. Daniel 
Published: October 2019

Abstract
Abstract 911
The identification of pathways that differentially regulate the self-renewal in normal hematopoietic stem cells (HSC) and leukemic stem cells (LSC) is critical for effective treatment of relapsed leukemia without affecting normal hematopoiesis. Krüppel-like factor 4 (KLF4) is a bi-functional transcription factor that can either activate or repress transcription and thus function as an oncogene or a tumor suppressor depending on the cellular context. We have previously shown that loss of KLF4 leads to increased self-renewal of HSC in serial bone marrow transplantation. In this work, we investigated whether KLF4 also regulates self-renewal in LSCs using a mouse model of somatic deletion of KLF4 in hematopoietic cells (Klf4fl/fl Vav-iCre+) and the BCR-ABL-induced chronic myeloid leukemia (CML) model. Mice transplanted with wild type bone marrow (BM) cells transduced with retrovirus carrying BCR-ABL (p210) (n=18) showed an expansion of myeloid cells (GFP+ Gr1+) with a median survival of 19 days. In contrast, mice transplanted with KLF4-deficient BM cells transduced with BCR-ABL retrovirus (n=12) exhibited prolonged survival with a median survival of 27 days (p<0.0011). Progression of leukemia was monitored in peripheral blood, BM and the spleen by flow cytometry. Mice transplanted with KLF4-deficient cells showed expansion of myeloid leukemic cells (GFP+ Gr1+) in the first two weeks after BM transplant, which was followed by a progressive loss of myeloid cells and an expansion of B cells (GFP+ B220+). In control group, 90% of leukemic mice succumbed from CML whereas more than 50% of KLF4-deficient leukemic mice developed mixed CML and B-ALL leukemia. These results suggest that loss of KLF4 impairs the maintenance of BCR-ABL-induced CML, while allowing expansion of BCR-ABL positive B cells (GFP+ B220+). Since KLF4 deletion prevented continuous expansion of myeloid leukemic cells (GFP+ Gr1+), we postulated that KLF4 may be critical to LSC maintenance. Thus, we analyzed the frequency of LSCs (GFP+ Lin–c-Kit+ Sca-1+and GFP+ Lin– c-Kit+ Sca-1+Flt3–) in BM and the spleen. We found that LSCs were significantly reduced in recipients of BCR-ABL-transduced KLF4-deficient BM cells 18 days after transplantation (n=6, p <0.001). These studies demonstrate that KLF4 has different roles in the maintainenance of LSCs and normal HSCs and that inactivation of KLF4 provides a therapeutic strategy for eradicating LSCs without damaging normal HSC.

Disclosures:
No relevant conflicts of interest to declare.
ULK1-Hsp90-Cdc37 and AMPK: Novel Insight Into the Regulation of Mitophagy
Authors: Joo Joung Hyuck , Kundu Mondira 
Published: October 2019

Abstract
Abstract 987
Autophagy plays an important role in maintaining mitochondrial integrity, directing lysosome-mediated destruction of cellular cargo, including damaged or dysfunctional mitochondria. Flux through the autophagy pathway is rapidly induced to promote survival in response to metabolic or proteotoxic stress resulting from exposure to noxious environmental cues, such as starvation, hypoxia or heat stress and likely contributes to the increase in mitochondrial turnover observed under each of these conditions. Dysregulation of this process has been linked to the pathogenesis of diseases, including anemia, diabetes, neurodegeneration and cancer.
Atg1 is a serine-threonine kinase that directs the autophagy machinery to appropriate cargo in responses to changes in the availability of carbon and nitrogen in yeast. Ulk1, one of the mammalian homologues of Atg1, is required for starvation-induced autophagy and clearance of mitochondria in terminally differentiating erythroid cells. The function of Ulk1 is also regulated by AMP dependent Kinase (AMPK)-mediated phosphorylation, however, the precise molecular consequence of this post-translational modification has not been explored. Our preliminary findings indicate that AMPK phosphorylates Ulk1 during red blood cell maturation and in response to mitochondrial uncoupling, and that this phosphorylation is critical for mitochondrial clearance. Therefore, we sought to use these systems to explore the mechanism by which AMPK phosphorylation regulates Ulk1 function.
We previously demonstrated that the stability and kinase activity of Ulk1 depends on its physical interaction with Hsp90 and the kinase-specific co-chaperone, Cdc37. Hsp90 is an abundant chaperone that directs the maturation and activation of a restricted group of metastable proteins, typically kinases and signaling molecules, and orchestrates a broad response to cellular stress. Here, we demonstrate that AMPK phosphorylation of Ulk1 does not affect Ulk1 kinase activity, but instead promotes its release from Hsp90 and its localization to damaged mitochondria. Preliminary studies indicate that the serine-proline rich domain of Ulk1, which contains at least 4 residues that are phosphorylated by AMPK, is an intrinsically disordered domain. We hypothesize that phosphorylation of Ulk1 by AMPK stabilizes a predicted alpha-helical structure within this domain and contributes to release of Hsp90. These findings are important because they provide significant insight into the regulation and function of Ulk1, a protein involved in mitochondrial turnover during red blood cell maturation and in proliferating cells.

Disclosures:
No relevant conflicts of interest to declare.
Stress-Mediated Activation of Dormant Hematopoietic Stem Cells In Vivo
Authors: Trumpp Andreas , Essers Marieke 
Published: October 2019

Abstract
Abstract SCI-41
Maintenance of the blood system is dependent on dormant hematopoietic stem cells (HSCs), which are characterized by pluripotency and lifelong self-renewal capacity. In order to both maintain a supply of mature blood cells and not exhaust HSCs throughout the lifespan of the organism, most adult HSCs remain deeply quiescent during homeostasis, and only a limited number are cycling at any given time. The balance between self-renewal and differentiation of HSCs is controlled by external factors such as chemokines and cytokines, as well as by interactions of HSCs with their niche environment. The transcriptome of dormant CD34-CD150+CD48-LSK- HSCs significantly differs from that of active HSCs with the same phenotype, while the latter are highly similar to MPP1 progenitors which express CD34. One of the genes differentially expressed is the cylindromatosis (CYLD) gene, which encodes a negative regulator of the NF-κB signaling pathway. HSCs failing to express functional CYLD show various defects associated with a disturbed balance between dormant and active HSCs, suggesting a role for NF-κB signaling in establishing dormancy in HSCs. In addition, our studies have recently shown that the cytokine interferon-α (IFNα) very efficiently activates dormant HSCs in vivo. Within hours after treatment of mice with IFNα, HSCs exit G0 and enter an active cell cycle. In general, IFNα is produced in response to viral infections by cells of the immune system, and plays an important role in the antiviral host defense. We now questioned whether endogenous IFNα is also produced in response to other types of bone marrow stress and whether this affects the proliferation rate of HSCs. To monitor IFNα production in the bone marrow in vivo, we have generated MxCre ROSA-R26-EYFP mice and found that treatment with both the chemotherapeutic agent 5-FU as well as the endotoxin LPS leads to the production of IFNα in the vicinity of HSCs and progenitors. In addition, LPS treatment in vivo induced a strong increase in HSC cycling. Surprisingly, since mice lacking the IFNα receptor (Ifnar−/−) still respond to LPS, this effect is independent of IFNAR signaling. Strikingly, LPS-induced HSC activation correlated with increased expression of Sca-1, similar to what occurs upon IFNα treatment. Moreover, as for IFNα, the upregulation of SCA-1 is required for LPS-induced proliferation, since Sca-1−/− mice fail to respond to LPS stimulation. In summary, these data suggest that not only virus-inducible IFNα, but also infections by gram-negative-bacteria-produced LPS induce cycling of progenitors and otherwise dormant HSCs.

Disclosures:
No relevant conflicts of interest to declare.
Prostaglandin E Synthase Is Upregulated By Gas6 during Cancer-Induced Venous Thromboembolism
Authors: Aghourian Meghedi N , Lemarie Catherine A , Bertin Francois-Rene , Blostein Mark D 
Published: October 2019

Abstract
Venous thromboembolism (VTE) is the most common morbid complication related to cancer and its treatments. Although malignancies are characterized by a hypercoagulable state leading to VTE, the pathophysiology of this state has not been well studied. Growth arrest specific 6 (Gas6) is a protein that has pro-coagulant properties. Gas6 deficient mice develop smaller venous thrombi as compared to wild type mice, and express less tissue factor in the endothelium when challenged with thrombotic stimuli. We hypothesize that Gas6 may be involved in cancer-induced venous thrombosis. In order to test this hypothesis, venous thrombi were induced in wild type (WT) and Gas6 null (-/-) mice injected with M27 murine lung cancer cell lines. Thrombus size was measured using ultrasonography, thrombus weight and histology. We observed that WT mice with cancer developed larger thrombi than their healthy counterparts (p<0.05). However, these larger thrombi induced by cancer were not seen in Gas6-/- mice, suggesting that Gas6 has a pathophysiologic role in promoting malignancy associated VTE. Whole genome microarray analysis was then used to identify differential gene expression in WT and Gas6-/- endothelial cells co-cultured with M27 murine lung carcinoma cells. Microarray analysis revealed that prostaglandin E synthase (PTGES) was increased in WT endothelial cells but not in Gas6-/- cells co-cultured with M27. These results were confirmed using real-time PCR and immunofluorescence staining (p<0.05). In WT endothelial cells, PTGES expression was regulated through ERK1/2 phosphorylation. We also show that co-culture of WT endothelial cells with M27 augments the secretion of PGE2, the enzymatic product of PTGES. PGE2 activates platelets in vitro after binding to its receptor, EP3. In vivo, EP3 receptor antagonism reversed the effect of cancer-induced thrombosis in WT mice. These results show that Gas6, through upregulation of PGE2, contributes to cancer-induced VTE.

Disclosures
No relevant conflicts of interest to declare.