Benfotiamine counteracts glucose toxicity effects on endothelial progenitor cell differentiation via Akt/FoxO signaling.

Dysfunction of mature endothelial cells is thought to play a major role in both micro- and macrovascular complications of diabetes. However, recent advances in biology of endothelial progenitor cells (EPCs) have highlighted their involvement in diabetes complications. To determine the effect of glucotoxicity on EPCs, human EPCs have been isolated from peripheral blood mononuclear cells of healthy donors and cultured in the presence or absence of high glucose (33 mmol/l) or high glucose plus benfotiamine to scavenge glucotoxicity. Morphological analysis revealed that high glucose significantly affected the number of endothelial cell colony forming units, uptake and binding of acLDL and Lectin-1, and the ability to differentiate into CD31- and vascular endothelial growth factor receptor 2-positive cells. Functional analysis outlined a reduced EPC involvement in de novo tube formation, when cocultured with mature endothelial cells (human umbilical vein endothelial cells) on matrigel. To explain the observed phenotypes, we have investigated the signal transduction pathways known to be involved in EPC growth and differentiation. Our results indicate that hyperglycemia impairs EPC differentiation and that the process can be restored by benfotiamine administration, via the modulation of Akt/FoxO1 activity.

Interferon regulatory factor-2 drives megakaryocytic 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.

Autocrine-paracrine VEGF loops potentiate the maturation of megakaryocytic precursors through Flt1 receptor.

The expression/function of vascular endothelial growth factor (VEGF) receptors (VEGFR1/Flt1 and VEGFR2/KDR/Flk1) in hematopoiesis is under scrutiny. We have investigated the expression of Flt1 and kinase domain receptor (KDR) on hematopoietic precursors, as evaluated in liquid culture of CD34(+) hematopoietic progenitor cells (HPCs) induced to unilineage differentiation/maturation through the erythroid (E), megakaryocytic (Mk), granulocytic (G), or monocytic (Mo) lineage. KDR, expressed on 0.5% to 1.5% CD34(+) cells, is rapidly downmodulated on induction of differentiation. Similarly, Flt1 is present at very low levels in HPCs and is downmodulated in E and G lineages; however, Flt1 is induced in the precursors of both Mo and Mk series; ie, its level progressively increases during Mo maturation, and it peaks at the initial-intermediate culture stages in the Mk lineage. Functional experiments indicate that Mk and E, but not G and Mo, precursors release significant amounts of VEGF in the culture medium, particularly at low O(2) levels. The functional role of VEGF release on Mk maturation is indicated by 2 series of observations. (1) Molecules preventing the VEGF-Flt1 interaction on the precursor membrane (eg, soluble Flt1 receptors) significantly inhibit Mk polyploidization. (2) Addition of exogenous VEGF or placenta growth factor (PlGF) markedly potentiates Mk maturation. Conversely, VEGF does not modify Mo differentiation/maturation. Altogether, our results suggest that in the hematopoietic microenvironment an autocrine VEGF loop contributes to optimal Mk maturation through Flt1. A paracrine loop involving VEGF release by E precursors may also operate. Similarly, recent studies indicate that an autocrine loop involving VEGF and Flt1/Flk1 receptors mediates hematopoietic stem cell survival and differentiation.