Reduced Production of JAK2V617F-positive Microparticles at Mild Hypothermia.

BACKGROUND/AIM: The Philadelphia chromosome-negative (Ph-) myeloproliferative neoplasms (MPNs) are a group of blood cancers that arise from abnormal growth of blood cells in the bone marrow. Patients with MPNs are at increased risk for life-threatening thromboembolic complications. The detection of JAK2V617F in endothelial cells (ECs) brought a new perspective to the research of thromboembolic events. However, the mechanisms by which the mutation contributes to risk have yet to be entirely understood. Consequently, the objective of this study was to investigate how JAK2V617F impacts endothelial cells by considering thermoregulation. MATERIALS AND METHODS: We applied our previously created model for EC that was genetically modified with JAK2 wild type (WT)-GFP and JAK2V617F-GFP lentiviruses; the cells were cultured for 48 h at 37°C for normothermia and 32°C for mild hypothermia. We examined the effect of thermoregulation on infection efficiency and the expression of cell surface markers, including endothelial protein C receptor (EPCR), thrombomodulin (TM), and tissue factor (TF), which are related to the coagulation pathways. Furthermore, the microparticle production from the genetically modified EC (EMPs) was analyzed. RESULTS: We found suppression of the expression of coagulation factors, including EPCR, TM, and TF in JAK2V617F positive ECs under mild hypothermia. JAK2V617F-positive ECs showed slightly higher EMP production under mild hypothermia. CONCLUSION: Although the molecular mechanisms of the thermal effects on the tumor microenvironment with JAK2V617F and its effect on EMP production and coagulation are not known yet, the therapy-oriented effect of thermoregulation might be considered in future studies.

The role of Extracellular Genomic Materials (EGMs) in psychiatric disorders.

Extracellular Genomic Materials (EGMs) are the nucleic acids secreted or released from all types of cells by endogenous or exogenous stimuli through varying mechanisms into the extracellular region and inevitably to all biological fluids. EGMs could be found as free, protein-bound, and/ or with vesicles. EGMs can potentially have immunophenotypic and/or genotypic characteristics of a cell of origin, travel to distant organs, and interact with the new microenvironment. To achieve all, EGMs might bi-directionally transit through varying membranes, including the blood-brain barrier. Such ability provides the transfer of any information related to the pathophysiological changes in psychiatric disorders in the brain to the other distant organ systems or vice versa. In this article, many aspects of EGMs have been elegantly reviewed, including their potential in diagnosis as biomarkers, application in treatment modalities, and functional effects in the pathophysiology of psychiatric disorders. The psychiatric disorders were studied under subgroups of Schizophrenia spectrum disorders, bipolar disorder, depressive disorders, and an autism spectrum disorders. EGMs provide a robust and promising tool in clinics for prognosis and diagnosis. The successful application of EGMs into treatment modalities might further provide encouraging outcomes for researchers and clinicians in psychiatric disorders.

Effect of hypoxia on HIF-1α and NOS3 expressions in CD34+ cells of JAK2V617F-positive myeloproliferative neoplasms.

PURPOSE: Myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic stem-cell diseases with excessive proliferation of one or more blood cell lines. In this study, we evaluated the effect of different oxygen concentrations on HIF-1α and NOS3 gene expression to determine the effect of the bone marrow microenvironment on JAK2V617F positive Philadelphia chromosome negative (Ph-) MPNs. PATIENTS AND METHODS: Peripheral blood mononuclear cells (MNC) of 12 patients with Ph- MPN were collected. The presence of JAK2V617F allele status was determined with allele-specific nested PCR analysis. MPN CD34+ and CD34depleted populations were isolated from MNC by magnetic beads. Separate cell cultures of CD34+/depleted populations were managed at different oxygen concentrations including anoxia (∼0%), hypoxia (∼3%), and normoxia (∼20%) conditions for 24 â€‹h. HIF-1α and NOS3 gene expression changes were examined in each population related to JAK2V617F status with real time RT-PCR. RESULT: It was revealed that relative HIF-1α and NOS3 expressions were significantly increased in response to decreased oxygen concentration in all samples. Relative HIF-1α and NOS3 expressions were found to be higher especially in CD34+ and CD34depleted populations carrying JAK2V617F mutations compared to MPN patients carrying wild-type JAK2. CONCLUSION: JAK2V617F might have specific role in HIF-1α and NOS3 regulations with respect to low oxygen concentrations in Ph- MPN. Further evaluations might reveal the effect of JAK2V617F on Ph- MPN pathogenesis in bone marrow microenvironment.

JAK2V617F-Positive Endothelial Cells Induce Apoptosis and Release JAK2V617F-Positive Microparticles

Objective: Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) have a high propensity for thrombosis, which has been attributed to increased blood counts, endothelial cell (EC) dysfunction, and inflammation. The presence of the JAK2V617F mutation in the ECs of MPN patients has been confirmed, but the consequences of EC involvement by JAK2V617F in the pathogenesis of thrombosis are unclear. Endothelial microparticles (EMPs) released from ECs play an important role in endothelial dysfunction and also in the intercellular exchange of biological signals and information. Several studies have revealed that patients with JAK2V617F and a thrombosis history have increased numbers of MPs in their circulation. Materials and Methods: The current study utilized a lentiviral transduction model of JAK2 wild type (JAK2wt) or JAK2V617F encoding green fluorescent protein (GFP) into human umbilical vein ECs to determine the effect of JAK2V617F on ECs. EC infected with JAK2V617F, JAK2WT, and only-GFP were tested after two days of culture. Results: The proteins of ECs that potentially play a role in the development of thrombosis, including endothelial protein C receptor, thrombomodulin, and tissue factor, were detected by flow cytometry analysis with no statistical significance. Increased annexin-V uptake of JAK2V617F and JAK2wt ECs compared to GFP-alone ECs was detected. The EMP production in the supernatants of the EC culture was investigated. Genotyping of the EMPs revealed the presence of genomic DNA and RNA fragments in EMP cargos. JAK2V617F-positive DNA was detected in EMPs released from JAK2V617F-infected ECs and EMPs were shown to carry the genotype of the cell of origin. Conclusion: JAK2V617F-positive EMPs were shown for the first time in the literature. This novel research provides the first evidence that EMPs might regulate neighboring and distant cells via their cargo materials. Thus, the direct effect of JAK2V617F on ECs and their functions suggests that different mechanisms might play a role in the pathogenesis of thrombosis in MPNs.

The presence of JAK2V617F mutation in the liver endothelial cells of patients with Budd-Chiari syndrome.

Patients with myeloproliferative disorders are at a high risk of developing thrombotic events. Several investigators have hypothesized that endothelial cell (EC) abnormalities might contribute to this prothrombotic state. Budd-Chiari syndrome (BCS) and portal vein thrombosis have been reported to be associated with JAK2V617F-positive hematopoiesis. We explored whether JAK2V617F was present in ECs in the vessels of polycythemia vera (PV) patients with BCS using laser capture microdissection followed by nested polymerase chain reaction or reverse-transcribed polymerase chain reaction. The ECs of the 2 BCS patients with PV were homozygous for the JAK2V617F and were shown to express transcripts characteristic of ECs but not hematopoietic cells. ECs of the other BCS patient with PV and 2 patients with hepatoportal sclerosis without PV contained exclusively wild-type JAK2. The presence of JAK2V617F in both ECs and hematopoietic cells belonging to BCS patients with PV indicate that ECs in PV are involved by the malignant process and that in a subpopulation of the patients the disease might originate from a common cell of origin for hematopoietic and ECs.

Extracellular genetic materials and their application in clinical practice.

This study reviews the possible origins, functional roles, and diagnostic applications of 'extracellular genetic material' (EGM), a novel term introduced to cover DNA, RNA, and DNA/RNA-related molecules released from all types of cells into the extracellular region. The literature on EGMs shows them to play a dual role in diverse, fine-tuning mechanisms involved in both homeostasis and pathological events, including cancerogenesis and genometastasis. Recent developments in the next-generation technology have provided successful applications of low quantities of genomic materials into the diagnostic field, yielding high sensitivity and specificity in test results. Also, the successful application of EGMs into diagnostics has afforded promising outcomes for researchers and clinicians. This study of EGM provides a deeper understanding of the subject as an area of interest, especially cell-free DNA, aiming toward the eventual development of new therapeutic applications and diagnostic strategies.

In vitro investigation of growth factors including MGF and IGF-1 in neural stem cell activation, proliferation, and migration.

Neurogenesis is mainly activated after damage in adult tissues. This destruction activates the neural stem cells (NSCs) by exiting from a quiescent state and initiating proliferation, differentiation, and migration towards the damaged area. Although studies have investigated to clarify the process of NSC biology and neurogenesis, there are still significant artifacts in understanding the primary mechanism. It is known that only a small percentage of NSC become neurons and integrate into the brain tissue after this process. The significant proportion differentiates to become either astrocytes or oligodendrocytes. Furthermore, the quiescent stem cells in the niche are mainly activated by the stimuli affect. In recent years, many studies have been conducted with varying hormones, some of which might provide neuro-stimulation effect and/or involved in the regeneration of the brain tissue and/or neuroprotection from traumatic or ischemic pathologies, including Insulin-like growth factor 1 (IGF-1), Mechano Growth Factor (MGF), Basic Fibroblast Growth Factor (FGF-2), Erythropoietin (EPO), Epidermal Growth Factor (EGF), Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF). In this study, we examined the effects of FGF-2, MGF, IGF-1, EPO, EGF, NGF, and BDNF alone or with various combinations on rat hippocampal NSC by tracking the changes in the expression of Nestin, GFAP, TUBB3, and DCX genes during 24 h (h), 72 h and 168 h time frame. The apoptosis analysis revealed that FGF-2 and FGF-2 coupled growth factors effectively protect NSCs against apoptosis, whereas MGF coupled growth factors failed in this protection. The cell cycle analysis demonstrated that these growth factors had accumulated the NSCs exit from the quiescent phase to the Mitosis phase, mostly without being long in the Synthesis Phase. Neurosphere sizes were increased with MGF, signifying MGF being effective in neural progenitor cells. The combined use of MGF with FGF-2 was more effective in postmitotic neurons than MGF alone. We have comparatively demonstrated the effect of cytokines alone and combined administration on activation, proliferation, and migration of NSCs. Although many issues are still waiting to be investigated in adult neurogenesis, neural regeneration, and adult neural stem cell biology, the results provide vital resources to the researchers that are interested in the varying effect of growth factor on NSC.

Assessment of Fetal Rhesus D and Gender with Cell-Free DNA and Exosomes from Maternal Blood.

The detection of fetal cell-free DNA (cfDNA) from maternal plasma has enabled the development of essential techniques in prenatal diagnosis during recent years. Extracellular vesicles including exosomes were determined to carry fetal DNA fragments. Considering the known difficulties during isolation and stability of cfDNA, exosomes might provide a new opportunity for prenatal diagnosis and screening. In this study, comparison of cfDNA and exosome DNA (exoDNA) for predicting the fetal sex and Rhesus D (RHD) genotype was performed by using real-time polymerase chain reaction with simultaneous amplification of sequences of SRY and RHD genes. Fetal sex and RHD were determined in 100 and 81 RHD-negative pregnant women with cfDNA and exoDNA, respectively. The gestation ages of pregnant women were between 9 and 40 weeks. The results were compared with the neonatal phenotype for gender and a serological test for RHD. The cfDNA revealed 95.75% sensitivity and 100% specificity in RHD positivity and 100% sensitivity and 95.45% specificity in SRY positivity. Cohen's agreement coefficient in the Kappa test ranged from 0.8 to 1.0 (P < 0.00001). Although the exoDNA failed to amplify 16 cases, the remaining 65 cases revealed a true estimate for both fetal RHD and SRY genes with 100% sensitivity and specificity. Successful application of exoDNA and cfDNA with real-time PCR for fetal genotyping enables this technique to be applied in the assessment of fetal RHD and gender during pregnancy, allowing initiation of early treatment methods and avoiding unnecessary interventions and cost.

Human CD34+ cells are capable of generating normal and JAK2V617F positive endothelial like cells in vivo.

Endothelial like cells (ELCs) are thought to originate from either a hierarchy of endothelial progenitor cells (EPC), monocytes or monocyte derived multipotent progenitor cells (MOMCs). In this report, the ability of CD34(+) cells to generate ELC in vivo was examined using an immunodeficient mouse transplant assay system. The Philadelphia chromosome negative (Ph(-)) myeloproliferative neoplasms (MPN) are associated with the acquired mutation, JAK2V617F. In order to further examine the ability of cord blood and JAK2V617F positive MPN CD34(+) cells to generate ELC, CD34(+) cells were transplanted into NOD/SCID mice. Cells within the livers and lungs of recipient mice had phenotypic and molecular properties of human ELC as examined using RT-PCR, flow cytometric analysis and fluorescence microscopy. These cells possessed either human wild type JAK2 or JAK2V617F indicating that they were derived from the transplanted human cells and that a fraction of such cells were involved by the malignant process. Furthermore, human CD144(+) cells isolated from the livers of recipient mice formed clusters in vitro composed of ELC, which contained either wild type JAK2 or JAK2V617F suggesting that these cells are derived from either MOMC or EPC that have an extensive proliferative capacity as well as some degree of self renewal capacity. These studies indicate that adult CD34(+) cells can be affected by JAK2V617F and that they can generate ELC which might play a role in the development of thrombosis in patients with MPN.

Effects of chromatin-modifying agents on CD34+ cells from patients with idiopathic myelofibrosis.

Idiopathic myelofibrosis (IM) is likely the consequence of both the acquisition of genetic mutations and epigenetic changes that silence critical genes that control cell proliferation, differentiation, and apoptosis. We have explored the effects of the sequential treatment with the DNA methyltransferase inhibitor, decitabine [5-aza-2'-deoxycytidine (5azaD)], followed by the histone deacetylase inhibitor, trichostatin A (TSA), on the behavior of IM CD34(+) cells. Unlike normal CD34(+) cells where 5azaD/TSA treatment leads to the expansion of CD34(+) cells and marrow-repopulating cells, treatment of IM CD34(+) cells results in a reduction of the number of total cells, CD34(+) cells, and assayable hematopoietic progenitor cells (HPC). In IM, HPCs are either heterozygous or homozygous for the JAK2V617F mutation or possess wild-type JAK2 in varying proportions. Exposure of IM CD34(+) cells to 5azaD/TSA resulted in a reduction of the proportion of JAK2V617F-positive HPCs in 83% of the patients studied and the reduction in the proportion of homozygous HPCs in 50% of the patients. 5azaD/TSA treatment led to a dramatic reduction in the number of HPCs that contained chromosomal abnormalities in two JAK2V617F-negative IM patients. IM is characterized by constitutive mobilization of HPCs, which has been partly attributed to decreased expression of the chemokine receptor CXCR4. Treatment of IM CD34(+) cells with 5azaD/TSA resulted in the up-regulation of CXCR4 expression by CD34(+) cells and restoration of their migration in response to SDF-1. These data provide a rationale for sequential therapy with chromatin-modifying agents for patients with IM.

Circulating angiogenic monocyte progenitor cells are reduced in JAK2V617F high allele burden myeloproliferative disorders.

The clinical course of patients with Philadelphia chromosome negative myeloproliferative disorder is frequently complicated by thrombotic events. Post-natal vasculogenesis has been proposed to play a critical role in angiogenesis by acting through a hierarchy of endothelial progenitor cells. Some endothelial progenitor cells have been shown to share a number of features associated with monocytes while other more primitive progenitor cells produce endothelial cells in vitro exclusively. The cells which share features of monocytes and endothelial cells have been termed angiogenic monocytes. Reduced levels of angiogenic monocyte progenitor cells have been reported to be predictive of atherosclerotic disease progression. Angiogenic monocyte progenitor cells were assayed in vitro from the peripheral blood mononuclear cells of myeloproliferative disorder patients. Angiogenic monocyte colonies were plucked and analyzed for endothelial cells and hematopoietic cell markers, JAK2V617F and their ability to incorporate into vascular endothelium following their transplantation into non-obese diabetic, severe combine immunodeficient mice. Myeloproliferative disorder angiogenic monocyte colonies that were detected were uniformly JAK2V617F positive and produced cells that expressed phenotypic markers characteristic of both monocytes and endothelial cells. Reduced numbers of angiogenic monocyte colonies were present in the blood of myeloproliferative disorder patients with a high JAK2V617F burden (>50%), (p<0.01). Transplanted angiogenic monocytes were able to contribute to the vascular endothelium of non-obese diabetic, severe combine immunodeficient mice. These studies suggest that reduced numbers of circulating angiogenic monocyte progenitors contribute to the propensity to develop thrombotic complications in myeloproliferative disorder patients.

Behavior of CD34+ cells isolated from patients with polycythemia vera in NOD/SCID mice.

OBJECTIVE: We investigated if polycythemia vera (PV) peripheral blood (PB) CD34+ cells contain cells capable of engrafting nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice and if the JAK2V617F mutational burden of these cells alters their behavior in NOD/SCID mice. MATERIALS AND METHODS: CD34+ cells isolated from patients with PV, idiopathic myelofibrosis (IM), or granulocyte colony-stimulating factor-mobilized normal donors were transplanted into sublethally irradiated NOD/SCID mice. Cells engrafted into the NOD/SCID mice were analyzed flow cytometrically using lineage-specific antibodies. Genomic DNA was extracted from granulocytes, CD34+ cells, and sorted human CD45(+) cells purified from the bone marrow cells of these mice to examine their JAK2V617F mutational burdens. RESULTS: Multilineage human cell engraftment was observed in mice transplanted with CD34+ cells from mobilized normal volunteers, IM patients and PV patients with high JAK2V617F burden, but not in mice receiving grafts from PV patients with low JAK2V617F burden. The differentiation program of engrafting PV CD34+ cells with high JAK2V617F burden was remarkably different than that of IM CD34+ cells. The JAK2V617F allele frequency in the human CD45+ cells isolated from the mice receiving CD34+ cells was lower than that observed in the CD34+ cell grafts, indicating the persistence of a JAK2V617F negative compartment of stem cells. CONCLUSION: We conclude that PB CD34+ cells from PV patients with high JAK2V617F burden and patients with IM contain NOD/SCID repopulating cells, and that differentiation program of IM and PV CD34+ cells are dramatically different.

Laser-capture microdissection and analysis of liver endothelial cells from patients with Budd-Chiari syndrome.

Myeloproliferative neoplasms (MPN) are clonal hematological malignancies that are frequently -associated with an acquired somatic mutation in JAK2 (JAK2V617F). Patients with MPN are at a high risk of developing thrombotic events. Endothelial cell (EC) abnormalities are thought to contribute to this prothrombotic state. Budd-Chiari syndrome (BCS) and portal vein thromboses have been reported to be associated with JAK2V617F positive hematopoiesis. We explored whether JAK2V617F was present in ECs within the vessels of polycythemia vera (PV) patients with BCS using laser-capture microdissection followed by nested PCR or real-time RT-PCR. The presence of JAK2V617F in both ECs and hematopoietic cells belonging to BCS patients with PV indicates that ECs from these patients are involved by the malignant process and that in this subpopulation of patients the disease may originate from a cell common to hematopoietic and endothelial cells.

Correction of the abnormal trafficking of primary myelofibrosis CD34+ cells by treatment with chromatin-modifying agents.

The abnormal trafficking of CD34+ cells is a unique characteristic of primary myelofibrosis (PMF). We have further studied the behavior of PMF CD34+ cells by examining their homing to the marrow and the spleens of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Following the infusion of PMF and normal granulocyte colony-stimulating factor-mobilized peripheral blood (mPB) CD34+ cells into NOD/SCID mice, reduced numbers of PMF CD34+ cells and granulocyte-macrophage colony-forming unit (CFU-GM) compared with mPB were detected in the marrow of these mice, whereas similar numbers of PMF and mPB CD34+ cells and CFU-GM homed to their spleens. The abnormal homing of PMF CD34+ cells was associated with reduced expression of CXCR4, but was not related to the presence of JAK2V617F. The sequential treatment of PMF CD34+ cells with the chromatin-modifying agents 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA), but not treatment with small molecule inhibitors of JAK2, resulted in the generation of increased numbers of CD34+CXCR4+ cells, which was accompanied by enhanced homing of PMF CD34+ cells to the marrow but not the spleens of NOD/SCID mice. Following 5azaD/TSA treatment, JAK2V617F-negative PMF hematopoietic progenitor cells preferentially homed to the marrow but not the spleens of recipient mice. Our data suggest that PMF CD34+ cells are characterized by a reduced ability to home to the marrow but not the spleens of NOD/SCID mice and that this homing defect can be corrected by sequential treatment with chromatin-modifying agents.

Leukemia stem cells in a genetically defined murine model of blast-crisis CML.

Myeloid leukemia arises from leukemia stem cells (LSCs), which are resistant to standard chemotherapy agents and likely to be a major cause of drug-resistant disease and relapse. To investigate the in vivo properties of LSCs, we developed a mouse model in which the biologic features of human LSCs are closely mimicked. Primitive normal hematopoietic cells were modified to express the BCR/ABL and Nup98/HoxA9 translocation products, and a distinct LSC population, with the aberrant immunophenotype of lineage(-), Kit(+/-), Flt3(+), Sca(+), CD34(+), and CD150(-), was identified. In vivo studies were then performed to assess the response of LSCs to therapeutic insult. Treatment of animals with the ABL kinase inhibitor imatinib mesylate induced specific modulation of blasts and progenitor cells but not stem- cell populations, thereby recapitulating events inferred to occur in human chronic myelogenous leukemia (CML) patients. In addition, challenge of leukemic mice with total body irradiation was selectively toxic to normal hematopoietic stem cells (HSCs), suggesting that LSCs are resistant to apoptosis and/or senescence in vivo. Taken together, the system provides a powerful means by which the in vivo behavior of LSCs versus HSCs can be characterized and candidate treatment regimens can be optimized for maximal specificity toward primitive leukemia cells.