Abstract 1963: Endothelial Cell-Derived Microparticles Interact with Regenerating Cells and Transfer Biological Information

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Nikos Werner ◽  
Nadine Heiermann ◽  
Hao Liu ◽  
Fritz Horlbeck ◽  
Georg Nickenig
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Peripheral Blood ◽  
Intracellular Transport ◽  
Mononuclear Cells ◽  
Biological Information ◽  
Annexin I ◽  
And Migration

Apoptosis of endothelial cells (ECs) leads to the development of endothelial dysfunction, which itself is strongly associated with poor cardiovascular prognosis. EC apoptosis can be quantified using flow-cytometry-based enumeration of circulating EC-derived microparticles (EMP) within peripheral blood. Bone marrow-derived endothelial progenitor cells (EPC) contribute to EC regeneration and are an important predictor for cardiovascular mortality. We postulate that EC apoptosis with concomitant release of EMP is an important signal for regenerating cells to initiate EC repair. EMP were generated from human coronary arterial endothelial cells (HCAEC). Proteomic analysis showed that the identified proteins represent cytoskeleton/cytoskeleton-binding proteins, proteins involved in intracellular transport/signalling, and protein folding. In addition, proteins related to apoptosis, proliferation, and migration were identified. HCAEC/EPC were able to incorporate EMP in an annexin I/ phosphatidylserine receptor-dependent way. Incubation of mononuclear cells with EMP lead to an enhanced conversion into early outgrowth EPC. EMP co-incubation also changed the phenotype of cultivated Langerhans-like dendritic cells into an immature phenotype. Co-cultivation of ECs and EPC with EMP prevented TNF-alpha induced cell apoptosis. Migration of EPC was enhanced in response to EMP. Finally, we measured EPC liberation from bone marrow into peripheral blood. Intravenous treatment of C57bl6 mice with EMP enhanced the number of sca-1/flk-1 positive EPC within peripheral blood compared to control. The number of CD31+/Annexin+ EMP and CD34 + /KDR + EPC was determined in 40 patients with coronary artery disease. The number of circulating EMP correlated with EPC function (p<0.001, r=0.601). EMP and EPC seem to substantially interact in an annexin I/PSR dependent way. EMP influence EPC biology in vitro and induces EPC mobilization in vivo. We speculate that the described interaction of EMP with EPC enhance the homing process of EPC within the area of EC damage.

Chronic Lymphocytic Leukemia-Derived Exosomes Stimulate Cells From The Microenvironment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3683-3683
Author(s):  
Jerome Paggetti ◽  
Guy J. Berchem ◽  
Etienne Moussay
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Bone Marrow Microenvironment ◽  
Lymphocytic Leukemia ◽  
Target Cells ◽  
And Migration

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in the blood and the primary lymphoid organs of long-lasting, mature, but non-functional B lymphocytes. Although CLL B cells can survive for long time periods in vivo, cells are undergoing apoptosis relatively quickly in vitro. This spontaneous apoptosis and their sensitivity to drugs is strongly reduced in presence of bone marrow mesenchymal stem cells (MSC) and endothelial cells (EC), which provide anti-apoptotic stimuli to CLL cells via direct contact or secretion of soluble factors. We recently reported the first profiling of circulating miRNA obtained from plasma of CLL patients (Moussay et al., PNAS, 2011). Specific miRNAs were found at higher level in the plasma of CLL patients compared to healthy donors. Exosomes, which are small extracellular vesicles of 50-150 nm originating from endosomes, are now known to efficiently transport nucleic acids and transfer mRNA, microRNA and proteins to target cells. Therefore, exosomes constitute a new component of intercellular communication and their role in CLL remains totally unknown. The specific miRNA signature from plasma of CLL patients combined with our observations that primary CLL B cells can transfer vesicles to MSC through 0.4 µm culture inserts in vitro prompted us to investigate whether CLL B cells secrete exosomes that could modify cells of the bone marrow microenvironment to produce tumor growth promoting factors locally in order to favor their own survival. We isolated, purified and characterized exosomes derived from CLL cell lines, primary cells culture supernatants and plasma from CLL patients. Proteins, mRNA and microRNAs contents were evaluated by high-throughput methods (LC-MS, microarrays) revealing in particular the presence of oncogenic molecules. In vitro, purified CLL-exosomes were found to rapidly enter target cells (already after 1h in MSC and endothelial cells) and to transfer proteins and miRNA. Flow cytometry showed that transferred proteins were expressed at cell surface. Luciferase reporter assay confirmed that miRNAs were efficient in targeting cellular mRNA. Exosomes could also be taken up ex vivo and in vivo by mouse bone marrow cells. Functionally, CLL-exosomes activated key signaling pathways (PI3K, AKT, and MAPK) Immunoblotting indicated the rapid phosphorylation of kinases after 5 min of incubation with CLL-exosomes and the subsequent activation of the canonical NF-kB pathway. We also observed that CLL-exosomes modulated gene expression in target cells among which cytokines (BAFF, IL-6, and IL-8), chemokines (CCL2/MCP-1, CCL5/RANTES, and CXCL1), and other factors involved in cell adhesion and migration (ICAM-1 and MMP-1). These factors were also secreted in the supernatants of MSC and EC as detected by antibody arrays. Exosomes were also shown to increase MSC and EC proliferation, to stimulate actin remodeling, cell migration and to enhance EC angiogenic capabilities (tube formation and aortic ring assays). In conclusion, CLL-exosomes contain pro-oncogenic molecules and strongly affect key functions of MSC and EC which are critical component of the bone marrow microenvironment. Activation of these cells by CLL-exosomes led to release of cytokines/chemokines and oncogenic factors that could promote angiogenesis and also favor leukemic cells survival and migration. Our findings may lead to applications in both diagnosis and therapy development. Molecules identified at the surface or inside CLL-exosomes may be further used as cancer biomarkers. Finally, the description of cell-to-cell communication mechanisms will generate opportunities of innovative therapeutic strategies and confirms the crucial role of exosomes in the development of CLL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals THE ORIGIN AND KINETICS OF MONONUCLEAR PHAGOCYTES

1968 ◽  
Vol 128 (3) ◽  
pp. 415-435 ◽  
Author(s):  
Ralph van Furth ◽  
Zanvil A. Cohn
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Calf Serum ◽  
Peritoneal Macrophages ◽  
Mononuclear Phagocytes ◽  
Sterile Inflammation

The origin and turnover of efferent populations of mouse mononuclear phagocytes has been described. Mononuclear phagocytes were defined as mononuclear cells which are able to adhere to glass and phagocytize. In vitro labeling studies with thymidine-3H showed that monocytes in the peripheral blood and peritoneal macrophages do not multiply and can be considered end cells in a normal, steady state situation. However, the mononuclear phagocytes of the bone marrow appear to be rapidly dividing cells. This conclusion was supported by in vivo labeling experiments. A peak of labeled mononuclear phagocytes of the bone marrow was found 24 hr after a pulse of thymidine-3H. This was followed, 24 hr later, by a peak of labeled monocytes in the peripheral blood. From these experiments it was concluded that the rapidly dividing mononuclear phagocytes of the bone marrow, called promonocytes, are the progenitor cells of the monocytes. Labeling studies after splenectomy and after X-irradiation excluded other organs as a major source of the monocytes. Peak labeling of both the blood monocyte and peritoneal macrophages occurred at the same time. A rapid entry of monocytes from the blood into the peritoneal cavity was observed, after a sterile inflammation was evoked by an injection of newborn calf serum. These data have led to the conclusion that monocytes give rise to peritoneal macrophages. No indications have been obtained that mononuclear phagocytes originate from lymphocytes. In the normal steady state the monocytes leave the circulation by a random process, with a half-time of 22 hr. The average blood transit time of the monocytes has been calculated to be 32 hr. The turnover rate of peritoneal macrophages was low and estimated at about 0.1% per hour. On the basis of these studies the life history of mouse mononuclear phagocytes was formulated to be: promonocytes in the bone marrow, → monocytes in the peripheral blood, → macrophages in the tissue.

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

scholarly journals Transcription factor Gfi-1 induced by G-CSF is a negative regulator of CXCR4 in myeloid cells

Blood ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2276-2285 ◽  
Author(s):  
Maria De La Luz Sierra ◽  
Paola Gasperini ◽  
Peter J. McCormick ◽  
Jinfang Zhu ◽  
Giovanna Tosato
Keyword(s):  
Bone Marrow ◽  
Dna Sequences ◽  
Peripheral Blood ◽  
Cell Function ◽  
Myeloid Cell ◽  
Cxcr4 Expression ◽  
Negative Regulator ◽  
Hematopoietic Stem

The mechanisms underlying granulocyte-colony stimulating factor (G-CSF)–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood remain elusive. We provide evidence that the transcriptional repressor growth factor independence-1 (Gfi-1) is involved in G-CSF–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood. We show that in vitro and in vivo G-CSF promotes expression of Gfi-1 and down-regulates expression of CXCR4, a chemokine receptor essential for the retention of hematopoietic stem cells and granulocytic cells in the bone marrow. Gfi-1 binds to DNA sequences upstream of the CXCR4 gene and represses CXCR4 expression in myeloid lineage cells. As a consequence, myeloid cell responses to the CXCR4 unique ligand SDF-1 are reduced. Thus, Gfi-1 not only regulates hematopoietic stem cell function and myeloid cell development but also probably promotes the release of granulocytic lineage cells from the bone marrow to the peripheral blood by reducing CXCR4 expression and function.

Abstract 697: Mobilization of Oct-4 + Bone Marrow-Derived Very Small Embryonic-Like Stem Cells in Patients with Acute Myocardial Infarction

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Wojciech Wojakowski ◽  
Magda Kucia ◽  
Boguslaw Machalinski ◽  
Edyta Paczkowska ◽  
Joanna Ciosek ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Stem Cells ◽  
Bone Marrow ◽  
Pluripotent Stem Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Small Cells ◽  
Acute Mi

Bone marrow-derived CD34 + CXCR4 + progenitor cells are mobilized into peripheral blood early in acute myocardial infarction (MI). Adult murine bone marrow contains population of small CD34 + lin − CD45 − CXCR4 + cells expressing markers of pluripotent stem cells (PSC) SSEA, Oct-4 and Nanog. This population of very small embryonic-like cells (VSEL) has unique morphology (small size 2– 4 μm, large nucleus, euchromatin) and capability to form embrioid bodies (EB). Murine EB-derived cells can in vitro differentiate into cells from all three germ layers including cardiomyocytes. We hypothesized that in patients with acute MI small cells expressing the VSEL immunophenotype and PSC markers are present in bone marrow and mobilized into peripheral blood. Blood samples (20 mL) from 18 patients with acute MI were obtained after 12 hours, 2 and 5 days after symptoms onset. Bone marrow samples (20 mL) were obtained from 2 patients with acute MI and 3 healthy volunteers. Mononuclear cells were isolated using hypotonic lysis and samples were analyzed by FACS. Mobilization of following cell populations was confirmed: hematopoietic lin − CD45 + CXCR4 + , lin − CD45 + CD133 + , lin − CD45 + CD34 + and non-hematopoietic (VSEL) lin − CD45 − CXCR4 + , lin − CD45 − CD133 + , lin − CD45 − CD34 + . Analysis of the cell number using lymphocyte gate showed more significant increase of CD45 + (hematopoietic) populations of lin − CD34 + , lin − CD133 + and lin − CXCR4 + cells. After gating for small events (VSEL size range) we found more significant mobilization of small, non-hematopoietic populations of lin − CD34 + , lin − CD133 + and lin − CXCR4 + cells (Table ). The expression of PSC markers (Oct-4, Nanog, SSEA-1) in VSEL was confirmed using real-time RT-PCR. Conclusion: We report for the first time that acute MI is associated with mobilization of non-hematopoietic VSELs expressing pluripotent stem cells markers.

scholarly journals Anti-CD38-blocked ricin: an immunotoxin for the treatment of multiple myeloma [see comments]

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 3017-3025 ◽  
Author(s):  
VS Goldmacher ◽  
LA Bourret ◽  
BA Levine ◽  
RA Rasmussen ◽  
M Pourshadi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Tumor Cell Line ◽  
Covalent Attachment ◽  
Myeloma Cells ◽  
Colony Forming Units

Abstract We report the development of a potent anti-CD38 immunotoxin capable of killing human myeloma and lymphoma cell lines. The immunotoxin is composed of an anti-CD38 antibody HB7 conjugated to a chemically modified ricin molecule wherein the binding sites of the B chain have been blocked by covalent attachment of affinity ligands (blocked ricin). Conjugation of blocked ricin to the HB7 antibody has minimal effect on the apparent affinity of the antibody and no effect on the ribosome-inactivating activity of the ricin A-chain moiety. Four to six logs of CD38+ tumor cell line kill was achieved at concentrations of HB7-blocked ricin in the range of 0.1 to 3 nmol/L. Low level of toxicity for normal bone marrow (BM) granulocyte-macrophage colony- forming units (CFU-GM), burst-forming units-erythroid (BFU-E), colony- forming units-granulocyte/erythroid/monocyte/macrophage (CFU-GEMM) cells was observed. Greater than two logs of CD38+ multiple myeloma cells were depleted from a 10-fold excess of normal BM mononuclear cells (BMMCs) after an exposure to HB7-blocked ricin under conditions (0.3 nmol/L) that were not very toxic for the normal BM precursors. HB7- blocked ricin was tested for its ability to inhibit protein synthesis in fresh patients' multiple myeloma cells and in normal BMMCs isolated from two healthy volunteers; tumor cells from four of five patients were 100-fold to 500-fold more sensitive to the inhibitory effect of HB7-blocked ricin than the normal BM cells. HB7 antibody does not activate normal resting peripheral blood lymphocytes, and HB7-blocked ricin is not cytotoxic toward these cells at concentrations of up to 1 nmol/L. The potent killing of antigen-bearing tumor cells coupled with a lack of effects on peripheral blood T cells or on hematopoietic progenitor cells suggests that HB7-blocked ricin may have clinical utility for the in vivo or in vitro purging of human multiple myeloma cells.

scholarly journals Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4102-4109 ◽  
Author(s):  
CI Civin ◽  
G Almeida-Porada ◽  
MJ Lee ◽  
J Olweus ◽  
LW Terstappen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Population ◽  
Mononuclear Cells ◽  
Cell Subset ◽  
Fetal Sheep ◽  
Adult Human

Abstract Data from many laboratory and clinical investigations indicate that CD34+ cells comprise approximately 1% of human bone marrow (BM) mononuclear cells, including the progenitor cells of all the lymphohematopoietic lineages and lymphohematopoietic stem cells (stem cells). Because stem cells are an important but rare cell type in the CD34+ cell population, investigators have subdivided the CD34+ cell population to further enrich stem cells. The CD34+/CD38-cell subset comprises less than 10% of human CD34+ adult BM cells (equivalent to < 0.1% of marrow mononuclear cells), lacks lineage (lin) antigens, contains cells with in vitro replating capacity, and is predicted to be highly enriched for stem cells. The present investigation tested whether the CD34+/CD38-subset of adult human marrow generates human hematopoiesis after transfer to preimmune fetal sheep. CD34+/ CD38- cells purified from marrow using immunomagnetic microspheres or fluorescence-activated cell sorting generated easily detectable, long- term, multilineage human hematopoiesis in the human-fetal sheep in vivo model. In contrast, transfer of CD34+/CD38+ cells to preimmune fetal sheep generated only short-term human hematopoiesis, possibly suggesting that the CD34+/CD38+ cell population contains relatively early multipotent hematopoletic progenitor cells, but not stem cells. This work extends the prior in vitro evidence that the earliest cells in fetal and adult human marrow lack CD38 expression. In summary, the CD34+/ CD38-cell population has a high capacity for long-term multilineage hematopoietic engraftment, suggesting the presence of stem cells in this minor adult human marrow cell subset.

scholarly journals Recombinant human megakaryocyte growth and development factor stimulates thrombocytopoiesis in normal nonhuman primates

Blood ◽  
1995 ◽  
Vol 86 (1) ◽  
pp. 54-59 ◽  
Author(s):  
AM Farese ◽  
P Hunt ◽  
T Boone ◽  
TJ MacVittie
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Growth And Development ◽  
Nonhuman Primates ◽  
Normal Male ◽  
Platelet Counts ◽  
Development Factor ◽  
Development In Vitro

Megakaryocyte growth and development factor (MGDF) is a novel cytokine that binds to the c-mpl receptor and stimulates megakaryocyte development in vitro and in vivo. This report describes the ability of recombinant human (r-Hu) MGDF to affect megakaryocytopoiesis in normal nonhuman primates. r-HuMGDF was administered subcutaneously to normal, male rhesus monkeys once per day for 10 consecutive days at dosages of 2.5, 25, or 250 micrograms/kg of body weight. Bone marrow and peripheral blood were assayed for clonogenic activity and peripheral blood counts were monitored. Circulating platelet counts increased significantly (P < .05) for all doses within 6 days of r-HuMGDF administration and reached maximal levels between day 12 and day 14 postcytokine administration. The 2.5, 25.0, and 250.0 micrograms/kg/d doses elicited peak mean platelet counts that were 592%, 670%, and 449% of baseline, respectively. Bone marrow-derived clonogenic data showed significant increases in the concentration of megakaryocyte (MEG)- colony-forming unit (CFU) and granulocyte-erythroid-macrophage- megakaryocyte (GEMM)-CFU, whereas that of granulocyte-macrophage (GM)- CFU and burst-forming unit-erythroid (BFU-e) remained unchanged during the administration of r-HuMGDF. These data show that r-HuMGDF is a potent stimulator of thrombocytopoiesis in the normal nonhuman primate.

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