The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 participate in the retention of normal hematopoietic stem cells within the bone marrow (BM) and their release into the circulation. Homing and engraftment of human stem cells in immunodeficient mice are dependent on cell surface CXCR4 expression and the production of BM SDF-1, which acts also as a survival factor for both human and murine stem cells. However, the role of SDF-1/CXCR4 interactions in the control of human acute myelogenous leukemia (AML) cell trafficking and disease progression is poorly understood. In this study, we report that although some AML cells do not express surface CXCR4, all AML cells tested express internal CXCR4 and SDF-1. Culture of AML cells with SDF-1 promoted their survival, whereas addition of neutralizing CXCR4 antibodies, SDF-1 antibodies, or AMD3100 significantly decreased it. Pretreatment of primary human AML cells with neutralizing CXCR4 antibodies blocked their homing into the BM and spleen of transplanted NOD/SCID/B2m null mice. Furthermore, weekly administrations of antihuman CXCR4 to mice previously engrafted with primary AML cells led to a dramatic decrease in the levels of human AML cells in the BM, blood, and spleen in a dose-and time-dependent manner. Interestingly, the same treatment did not affect significantly the levels of normal human progenitors engrafted into NOD/SCID mice. Taken together, our findings demonstrated the importance of the SDF-1/CXCR4 axis in the regulation of in vivo motility and development of human AML stem cells and identified CXCR4 neutralization as a potential treatment for AML.

SummaryMegakaryocytopoiesis involves the commitment of haematopoietic stem cells, and the proliferation, maturation and terminal differentiation of the megakaryocytic progenitors. Circulating levels of thrombopoietin (TPO), the primary growth-factor for the megakaryocyte (MK) lineage, induce concentration-dependent proliferation and maturation of MK progenitors by binding to the c-Mpl receptor and signalling induction. Decreased platelet turnover rates results in increased concentration of free TPO, enabling the compensatory response of marrow MKs to increased platelet production. C-Mpl activity is orchestrated by a complex cascade of signalling molecules that induces the action of specific transcription factors to drive MK proliferation and maturation. Mature MKs form proplatelet projections that are fragmented into circulating particles. Newly developed thrombopoietic agents operating via c-Mpl receptor may prove useful in supporting platelet production in thrombocytopenic state. Herein, we review the regulation of megakaryocytopoiesis and platelet production in normal and disease state, and the new approaches to thrombopoietic therapy.

A major limitation to clinical stem cellmediated gene therapy protocols is the low levels of engraftment by transduced progenitors. We report that CXCR4 overexpression on human CD34 ؉ progenitors using a lentiviral gene transfer technique helped navigate these cells to the murine bone marrow and spleen in response to stromal-derived factor 1 (SDF-1) signaling. Cells overexpressing CXCR4 exhibited significant increases in SDF-1-mediated chemotaxis and actin polymerization compared with control cells. A major advantage of CXCR4 overexpression was demonstrated by the ability of transduced CD34 ؉ cells to respond to lower, physiologic levels of SDF-1 when compared to control cells, leading to improved SDF-1-induced migration and proliferation/survival, and finally resulting in significantly higher levels of in vivo repopulation of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice including primitive CD34 ؉ /CD38 ؊/low cells. Importantly, no cellular transformation was observed following transduction with the CXCR4 vector. Unexpectedly, we documented lack of receptor internalization in response to high levels of SDF-1, which can also contribute to increased migration and proliferation by the transduced CD34 ؉ cells. Our results suggest CXCR4 overexpression for improved definitive human stem cell motility, retention, and multilineage repopulation, which could be beneficial for in vivo navigation and expansion of hematopoietic progenitors. ( IntroductionGene transfer into human hematopoietic stem cells (HSCs) may be a promising tool in the correction of a wide variety of hematopoietic and genetic disorders. HSC transplantation can be used to durably deliver these genetically modified cells to the bone marrow (BM), which in turn will release mature cells with the corrected gene into the circulation throughout life. Clinical and experimental HSC transplantation procedures mimic the physiologic process of HSC migration from the circulation into the BM occurring during late embryonic development and steady-state hematopoiesis in adults throughout life. [1][2][3] One of the disadvantages of BM transplantation is the long-lasting reduced levels of immature progenitors, including long-term culture-initiating cells (LTCICs; 1 log reduction), in the BM of patients who have received transplants compared with healthy individuals. [4][5][6] Furthermore, emerging evidence exists for impaired homing 7 and low engraftment 8 of retrovirally transduced human CD34 ϩ cells. Enhanced efficacy of HSC engraftment could improve the outcome of clinical transplantations as well as gene therapy protocols and might be achieved by modulating the ability of stem cells to home to and repopulate the recipient BM.Interactions between the chemokine stromal-derived factor 1 (SDF-1), also referred to as CXCL12, and its receptor CXCR4 play an essential role in stem cell seeding of the BM during murine embryonic development. 9,10 Moreover, we have previously demonstrated in a functional preclinical model, using nonobese diabetic/ severe ...

Objectives-Recent clinical trials use cell therapy with bone marrow (BM) cells or endothelial progenitor cells (EPCs) for ischemic syndromes. We explored the effect of BM cell-or spleen cell-derived EPC transfer on plaque size and stability markers in the apolipoprotein E knockout (apoE KO) mouse model. Methods and Results-ApoE KO mice aged 10 weeks served as recipients. Labeled BM cells and spleen cell-derivedEPCs from age-matched apoE KO mice were injected intravenously to 2 groups of recipient mice each. Additional mice served as controls receiving saline. Both protocols were repeated 3 times at 2 weekly intervals. On killing, plaque size and character were studied, lipid profile analyzed, and serum and aortic cytokines assayed. Spleen cell-derived cells contained a significantly larger number of endothelial cell precursors. Labeled EPCs and BM cells were found abundantly in the spleens, yet also in the lesions of the recipient mice. Aortic sinus lesion size was significantly increased in mice receiving BM cells (nϭ10) in the EPC-treated group (nϭ10) compared with controls (nϭ10; a 54% and a 34% increase in aortic sinus plaque area, respectively). Mice receiving EPCs exhibited plaques with larger lipid cores and thinner fibrous caps and a higher number of infiltrating CD3 cells. RT-PCR analysis of aortas revealed reduced expression of mRNA for interleukin-10 (IL-10) in both cell transfer groups. Higher serum concentrations of IL-6 and monocyte chemoattractant protein-1 were found in sera from BM recipients, whereas lower IL-10 levels were found in mice transfused with spleen-derived EPCs. 1 It is probable that factors that govern the initiation of atherosclerosis, which involves less complex cellular crosstalk, are not identical to determinants of plaque progression, in which additional matrix components and cell types are prevalent. 2 In both these processes, ECs have been proposed to play a major role forming the attachment surface on which monocytes role and adhere. ECs participate in the early fatty streak formation and in constituting the vasa vasorum network that acts to supply the inner growing neointima in more advanced lesions. These actions are regulated by expression of a set of adhesion molecules on the EC surface and by synthesis and secretion of regulatory humoral factors. 1,2 Apparently, confounding data have been provided with regard to the effect of EC on plaque progression and phenotype. Atherosclerosis is a disorder with endothelial dysfunction, and it is thus conceivable that replenishment of ECs would result in attenuated EC activation with consequent inflammation. These findings are supported by a study by Rauscher et al 3 showing that transfer of bone marrow (BM) cells from young apolipoprotein E knockout (apoE KO) mice reduces atherosclerotic plaque size. However, it appears that the angiogenesis inhibitor TNP-470 and angiostatin acting to inhibit plaque neovascularization were found to suppress atherosclerotic lesion development, 4,5 whereas vascular endothelial growth factor promote...

IntroductionHematopoietic stem cells migrate during embryonic development from the fetal liver through the blood circulation, home to the bone marrow (BM) microenvironment, and repopulate it with immature and maturing blood cells of all lineages. Similarly, in clinical and experimental stem cell transplantation protocols, hematopoietic stem cells, which are infused into the blood circulation of patients and experimental animals, home and repopulate the BM. 1 The molecular mechanisms that regulate the homing and repopulation processes are crucial for stem cell function and development. [2][3][4][5] The CXC chemokine stromal cell-derived factor 1 (SDF-1) plays a major role in migration, proliferation, differentiation, and survival of many cell types including human and murine hematopoietic stem/progenitor cells. 6,7 SDF-1 is produced by multiple BM stromal cell types and by epithelial cells in many organs 8,9 and is highly expressed by human and murine BM endothelium. [10][11][12] CXCR4, the 7-transmembrane receptor of SDF-1, is widely expressed by a variety of hematopoietic cell types, neuronal cells, and different stromal cells. 13 SDF-1 is a chemotactic agent for human lymphoid, myeloid, and immature CD34 ϩ progenitor cells. 6,7,14,15 This chemokine induces integrin-dependent adhesion of CXCR4 ϩ human T lymphocytes 16 and immature CD34 ϩ CXCR4 ϩ cells 17 under shear flow and also mediates transendothelial migration of human progenitors. 18 In vivo cell migration and localization are also mediated by SDF-1/CXCR4 interactions. Murine T cells overexpressing human CXCR4 and CD4 accumulated in the BM of transgenic mice. 19 Prevention of CXCR4 expression by introducing SDF-1 intrakine blocked in vitro migration and in vivo dissemination of a T-cell hybridoma. 20 More important, mice reconstituted with progenitor cells expressing SDF-1-intrakine suffered impaired lymphoid and myeloid hematopoiesis, whereas transplantation of progenitors overexpressing SDF-1 led to increased myeloid and B-lymphoid hematopoiesis. 21 The key role of SDF-1 and CXCR4 in embryonic development was demonstrated by knockout studies in mice. The lack of either SDF-1 or its receptor in murine fetuses results in multiple lethal defects including impaired BM hematopoiesis. [22][23][24][25] Recently, Wright and colleagues have demonstrated that SDF-1 is the sole chemokine mediating in vitro migration of purified adult murine BM stem cells. 26 This important study suggests a major role for SDF-1/CXCR4 interactions also in adult murine stem cell migration and development.We demonstrated the essential role of SDF-1/CXCR4 interactions in both homing and high-level multilineage repopulation of nonobese diabetic/severe combined immunodeficient (NOD/SCID) 10,27,28 The antihuman CXCR4-neutralizing monoclonal antibody (mAb; clone 12G5) binds CXCR4 on the first and second extracellular domain as its ligand SDF-1, interfering with SDF-1 binding and signaling. 29,30 Coinjecting enriched human CD34 ϩ cells with neutralizing anti-CXCR4 mAb blocked homing and re...

Objective: The pathological picture in ischemic tissue injury shares features with the inflammatory response. Hypoxia-mediated induction of interleukin-6 (IL-6) could set in motion the mechanisms limiting inflammation in ischemia. Intrauterine growth restriction (IUGR) represents a human model of chronic fetal hypoxia. The purpose of this study was a first-time exploration to determine whether cord blood obtained at the delivery of small-for-gestational-age (SGA) infants has increased concentrations of inflammatory markers.Study Design: Cord blood was collected from 20 SGA (term and nearterm) infants and 20 appropriate-for-gestational-age (AGA) controls. Infants exposed to maternal smoking, diabetes, maternal chronic diseases, or alcohol or drug use were excluded. Both groups had Apgar score X7 at 1 min with a normal cord pH (>7.25). Cord-serum cytokines and thrombopoietin (TPO) levels were measured by enzyme linked immunosorbent assay. C-reactive protein (CRP) was measured using a turbidometric immunoassay.Result: SGA infants had a significantly smaller birth weight than AGA controls, with a smaller gestation age by 1 week. There were significant elevations in IL-6, tumor necrosis factor (TNF-a), CRP and TPO in the SGA compared with the AGA group, which persisted in multiple regression analysis even after gestational age was taken into account. Conclusion:As hypothesized, significant increases in the cord blood concentrations of known inflammatory markers were found in SGA infants compared with the controls.

Summary. Several clinical and laboratory findings suggest the presence of a chronic hypercoagulable state in patients with b-thalassaemia major (TM). We have previously shown that isolated TM red blood cells (RBC) strongly enhance prothrombin activation, suggesting an increased membrane exposure of procoagulant phospholipids (i.e. phosphatidylserine). In this study we quantitated the procoagulant activity of RBC in TM and thalassaemia intermedia (TI) patients. We also determined the fraction of activated platelets expressing p-selectin (CD62p) or CD63 in these subjects. Both assays were performed by dual-colour flow cytometry. A significantly (P < 0 . 01) higher fraction of FITCannexin V-labelled RBC was found in TM and TI patients, compared to the controls. A highly significant correlation (P < 0 . 001) was found in TM patients between the number of RBC-bound annexin V molecules and the fraction of CD62p (p-selectin) or CD63-positive platelets. This association between annexin V binding to TM RBC and the expression of platelet activation markers was also found in individual TM patients over time. Thus, the procoagulant surface of TM RBC may accelarate thrombin generation in vivo which, in turn, triggers platelet activation.

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, participate in the retention of acute myeloblastic leukemia (AML) cells within the bone marrow microenvironment and their release into the circulation. AML cells also constitutively express SDF-1-dependent elastase, which regulates their migration and proliferation. To study the molecular events and genes regulated by the SDF-1/CXCR4 axis and elastase in AML cells, we examined gene expression profiles of the AML cell line, U937, under treatment with a neutralizing anti-CXCR4 antibody or elastase inhibitor, as compared with non-treated cells, using DNA microarray technology. Unsupervised hierarchical clustering analysis demonstrated similar gene expression profiles of anti-CXCR4 antibody or elastase inhibitor-treated cells, as compared with control. Pathway and functional analysis showed a greater tendency toward differentiation in cells under either one of both treatment modalities. Thus given, we further analyzed the effects of CXCR4 inhibition on AML cell growth and differentiation using the antagonist AMD3100. AMD3100 arrested proliferation in AML cell lines and triggered changes that mimicked differentiation, including morphological changes and the expression of myeloid differentiation antigens. Inhibition of elastase also triggered the differentiation of AML cells. Our study defines a new role for the SDF-1/CXCR4 axis in the regulation of leukemic cell survival and differentiation.