IntroductionDuring megakaryocytic (Mk) differentiation, Mk precursors switch from a mitotic to an endomitotic process characterized by DNA duplication without cytokinesis. This still poorly understood process leads to the formation of large polyploid cells with polylobulated nuclei that, in turn, give rise to platelets by cytoplasm fragmentation. 1,2 The major regulator of Mk development, Mpl ligand/ thrombopoietin (TPO), acts at all stages of megakaryocytopoiesis: commitment and proliferation of hematopoietic progenitor cells (HPCs), polyploidization of Mk precursors, and final maturation, including the formation of membrane demarcations and platelet production (reviewed in Kaushansky, 1 ZuckerFranklin and Kaushansky, 2 Zimmet and Ravid, 3 Cramer et al 4 ). However, despite these properties, TPO fails to induce in vitro a level of Mk polyploidization comparable to that observed in vivo. 5-7 Addition of either single or combined cytokines (ie, kit ligand, interleukin-3, interleukin-6) to TPO-containing cultures, although improving Mk proliferation, negatively affects cytoplasmic maturation and polyploidization. 5,6 Similarly, although erythropoietin (Epo) is considered the main growth factor stimulating erythropoiesis, additional cytokines are required at early and late erythroid (E) stages. 8 Vascular endothelial growth factor (VEGF) is a key factor for proliferation and survival of endothelial cells. [9][10][11] The VEGF family, including VEGF/VEGF-A, -B, -C, -D, and -E, 10-12 as well as the placenta growth factor (PlGF), 13 mediates angiogenic signals to endothelial cells through the binding with tyrosine kinase receptors designated VEGFR-1/Flt1, VEGFR-2/KDR/Flk1, and VEGFR-3/Flt-4. 14 VEGF is the ligand of both Flt1 and kinase domain receptor (KDR) and consists of several isoforms generated by alternative splicing of a single mRNA precursor (VEGF121, 145, 165, 189, or 206), which differ in their molecular mass and their biologic properties, such as the ability to bind heparin or heparinlike molecules on cell surface. 10,15 VEGF expression is enhanced spatially and temporally and is associated with physiologic events leading to angiogenesis in vivo, and its production is potentiated by hypoxia. 16 Studies on gene knockout mice demonstrated the physiologic role of VEGF and its receptors, as central regulators of the development of vascular and hemopoietic tissues. Flt1 knockout causes a selective defect in the assembly and organization of vasculature. 17 Lack of either VEGF or KDR gene causes major defects in both vasculogenesis and blood island formation, 18-21 suggesting the existence in embryonic life of a bipotent stem cell (SC) for hematopoietic and endothelial lineages, the hemangioblast.In postnatal life, both Flt1 and KDR are expressed at low levels on CD34 ϩ HPCs. [22][23][24][25][26][27] More important, the small fraction of CD34 ϩ Materials and methods Hematopoietic growth factors (HGFs) and culture mediaRecombinant human interleukin 3 (rhIL-3), granulomonocytic colony-stimulating factor (rhGM-C...

However, recent studies have demonstrated that circulating bone marrow-derived endothelial progenitor cells (EPCs) tightly contribute to adult blood vessel formation (4,5). The EPCs promote in vivo re-endothelization and are able to be incorporated into new vessels in animal models of hind limb ischemia (6,7). EPCs are involved in processes like myocardial ischemia and infarction, wound healing, and endogenous endothelial repair (8 -11). Furthermore, in vivo studies in animal models and in vitro studies using EPCs from type 1 diabetic patients revealed a potential role for glucotoxicity in impairing EPC function (7,(12)(13)(14).High glucose induces pathological alterations through increased formation of advanced glycosylation end product, activation of aldose reductase and protein kinase C, and increased flux through the hexosamine pathway. All of these mechanisms seem to reflect a single hyperglycemiainduced process of overproduction of superoxide anion by the mitochondrial electron transport chain (15). Superoxide inhibits the glycolytic enzyme glyceraldehyde phosphate dehydrogenase, diverting upstream metabolites from glycolysis toward the glucose-driven signaling pathways that cause hyperglycemic damage (16). These processes may be in part reduced by transketolase activation through its cofactor thiamine. In fact, both thiamine and benfotiamine have been shown to correct microvascular and macrovascular complications of diabetes, although at a different extent, blocking three major pathways of hyperglycemic damage (16 -19).Interestingly, the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway is crucial for both endothelial cell function and EPC differentiation (20 -22). The PI 3-kinase/ Akt pathway is known to direct cellular processes like differentiation and stress resistance through a tight regulation of the forkhead family of transcription factors (FoxO1/3a/4). FoxO1 and FoxO3a were recently found to play a role in angiogenesis and vasculogenesis (23-26). We and others have recently shown that both genetic and metabolic factors impair activation of the PI 3-kinase/Akt/ FoxO pathway in mature endothelial cells (27)(28)(29). In this study, we investigated the impact of glucose toxicity on the ability of EPCs to differentiate into mature endothelial cells, and we also tested benfotiamine capacity to bypass the negative effects of high glucose concentrations. RESEARCH DESIGN AND METHODSEPC isolation and culture. EPCs were obtained by isolating peripheral mononuclear cells from human blood buffy coats using Ficoll density centrifugation. Recovered cells were washed twice with PBS. Unselected mononuclear cells were plated on fibronectin-coated culture dishes (Biocoat; Becton Dickinson Labware) at a density of 10 6 cells/ml in Medium 199 (Invitrogen), supplemented with 20% fetal bovine serum, 100 units/ml penicillin/streptomycin (Invitrogen), and 0.05 mg/ml bovine pituitary extract (Invitrogen) and in From the

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Numerous transcription factors allow haematopoietic cells to respond to lineage- and stage-specific cytokines and to act as their effectors. It is increasingly evident that the interferon regulatory factor-1 (IRF-1) transcription factor can selectively regulate different sets of genes depending on the cell type and/or the nature of cellular stimuli, evoking distinct responses in each. In the present study, we investigated mechanisms underlying the differentiation-inducing properties of granulocytic colony-stimulating factor (G-CSF) and whether IRF transcription factors are functionally relevant in myeloid differentiation. Both normal human progenitors and murine 32Dcl3 myeloblasts induced to differentiate along the granulocytic pathway showed an up-regulation of IRF-1 expression. Ectopic expression of IRF-1 did not abrogate the growth factor requirement of 32Dcl3 cells, although a small percentage of cells that survived cytokine deprivation differentiated fully to neutrophils. Moreover, in the presence of G-CSF, granulocytic differentiation of IRF-1-expressing cells was accelerated, as assessed by morphology and expression of specific differentiation markers. Down-modulation of c-Myb protein and direct stimulation of lysozyme promoter activity by IRF-1 were also observed. Conversely, constitutive expression of IRF-2, a repressor of IRF-1 transcriptional activity, completely abrogated the G-CSF-induced neutrophilic maturation. We conclude that IRF-1 exerts a pivotal role in granulocytic differentiation and that its induction by G-CSF represents a limiting step in the early events of differentiation.

IRFs [IFN (interferon) regulatory factors] constitute a family of transcription factors involved in IFN signalling and in the development and differentiation of the immune system. IRF-2 has generally been described as an antagonist of IRF-1-mediated transcription of IFN and IFN-inducible genes; however, it has been recently identified as a transcriptional activator of some genes, such as those encoding histone H4, VCAM-1 (vascular cell adhesion molecule-1) and Fas ligand. Biologically, IRF-2 plays an important role in cell growth regulation and has been shown to be a potential oncogene. Studies in knock-out mice have also implicated IRF-2 in the differentiation and functionality of haematopoietic cells. Here we show that IRF-2 expression in a myeloid progenitor cell line leads to reprogramming of these cells towards the megakaryocytic lineage and enables them to respond to thrombopoietin, as assessed by cell morphology and expression of specific differentiation markers. Up-regulation of transcription factors involved in the development of the megakaryocytic lineage, such as GATA-1, GATA-2, FOG-1 (friend of GATA-1) and NF-E2 (nuclear factor-erythroid-2), and transcriptional stimulation of the thrombopoietin receptor were also demonstrated. Our results provide evidence for a key role for IRF-2 in the induction of a programme of megakaryocytic differentiation, and reveal a remarkable functional diversity of this transcription factor in the regulation of cellular responses.

Numerous transcription factors allow haematopoietic cells to respond to lineage- and stage-specific cytokines and to act as their effectors. It is increasingly evident that the interferon regulatory factor-1 (IRF-1) transcription factor can selectively regulate different sets of genes depending on the cell type and/or the nature of cellular stimuli, evoking distinct responses in each. In the present study, we investigated mechanisms underlying the differentiation-inducing properties of granulocytic colony-stimulating factor (G-CSF) and whether IRF transcription factors are functionally relevant in myeloid differentiation. Both normal human progenitors and murine 32Dcl3 myeloblasts induced to differentiate along the granulocytic pathway showed an up-regulation of IRF-1 expression. Ectopic expression of IRF-1 did not abrogate the growth factor requirement of 32Dcl3 cells, although a small percentage of cells that survived cytokine deprivation differentiated fully to neutrophils. Moreover, in the presence of G-CSF, granulocytic differentiation of IRF-1-expressing cells was accelerated, as assessed by morphology and expression of specific differentiation markers. Down-modulation of c-Myb protein and direct stimulation of lysozyme promoter activity by IRF-1 were also observed. Conversely, constitutive expression of IRF-2, a repressor of IRF-1 transcriptional activity, completely abrogated the G-CSF-induced neutrophilic maturation. We conclude that IRF-1 exerts a pivotal role in granulocytic differentiation and that its induction by G-CSF represents a limiting step in the early events of differentiation.