Interaction of the v-rel protein with an NF-kappa B DNA binding site

The avian reticuloendotheliosis virus T contains within its genome the oncogene rel. The expression of this gene is responsible for the induction of lymphoid tumors in birds. Recently, the rel gene was shown to be related to the p50 DNA binding subunit of the transcription factor complex NF-kappa B. Binding sites for the NF-kappa B complex are found in the enhancer regions of a number of genes, including the immunoglobulin kappa gene and the human immunodeficiency virus long terminal repeat. In this communication we identify an activity from avian reticuloendotheliosis virus T-transformed avian lymphoid cells that binds in an electrophoretic-mobility-shift assay to an NF-kappa B binding site from the kappa enhancer. This activity contains proteins immunologically related to rel, as detected by polyclonal and monoclonal antibodies directed against v-rel. In a DNA affinity precipitation assay using the NF-kappa B site from the human immunodeficiency virus long terminal repeat, v-rel and several other proteins were identified. These data suggest that oncogenic transformation by v-rel is the result of an altered pattern of gene expression

Molecular pharmacology of VEGF-A isoforms: binding and signalling at VEGFR2

Vascular endothelial growth factor-A (VEGF-A) is a key mediator of angiogenesis, signalling via the class IV tyrosine kinase receptor family of VEGF Receptors (VEGFRs). Although VEGF-A ligands bind to both VEGFR1 and VEGFR2, they primarily signal via VEGFR2 leading to endothelial cell proliferation, survival, migration and vascular permeability. Distinct VEGF-A isoforms result from alternative splicing of the Vegfa gene at exon 8, resulting in VEGFxxxa or VEGFxxxb isoforms. Alternative splicing events at exons 5–7, in addition to recently identified posttranslational read-through events, produce VEGF-A isoforms that differ in their bioavailability and interaction with the co-receptor Neuropilin-1. This review explores the molecular pharmacology of VEGF-A isoforms at VEGFR2 in respect to ligand binding and downstream signalling. To understand how VEGF-A isoforms have distinct signalling despite similar affinities for VEGFR2, this review re-evaluates the typical classification of these isoforms relative to the prototypical, “pro-angiogenic” VEGF165a. We also examine the molecular mechanisms underpinning the regulation of VEGF-A isoform signalling and the importance of interactions with other membrane and extracellular matrix proteins. As approved therapeutics targeting the VEGF-A/VEGFR signalling axis largely lack long-term efficacy, understanding these isoform-specific mechanisms could aid future drug discovery efforts targeting VEGF receptor pharmacology. View Full-Tex

The Role of Monocytes in Angiogenesis and Atherosclerosis

New vessel formation inside the arterial wall and atherosclerotic plaques plays a critical role in pathogenesis of heart attacks and strokes. The 2 known mechanisms resulting in the formation of new vessels within the plaque are local ischemia and inflammation. Blood monocytes play an important role in both processes. First, they express receptors for vascular endothelial growth factor and some of them may serve as circulating ancestors of endothelial cells. Second, monocytes are associated with inflammation by synthesis of inflammatory molecules following their activation (e.g., after stimulation of Toll-like receptors). Neovascularization is a reparative response to ischemia, and includes 3 processes: angiogenesis, arteriogenesis, and vasculogenesis. Angiogenesis, the formation of new capillary vessels is known to occur in response to a hypoxic environment. The interaction between leukocytes and vascular wall via overexpression of various molecules facilitates the migration of inflammatory cells into the plaque microenvironment. Monocytes are intimately involved in tissue damage and repair and an imbalance of these processes may have detrimental consequences for plaque development and stability. Importantly, monocytes are comprised of distinct subsets with different cell surface markers and functional characteristics and this heterogeneity may be relevant to angiogenic processes in atherosclerosis. The aim of this review article is to present an overview of the available evidence supporting a role for monocytes in angiogenesis and atherosclerosis

Decoding the regulatory network of early blood development from single-cell gene expression measurements.

Reconstruction of the molecular pathways controlling organ development has been hampered by a lack of methods to resolve embryonic progenitor cells. Here we describe a strategy to address this problem that combines gene expression profiling of large numbers of single cells with data analysis based on diffusion maps for dimensionality reduction and network synthesis from state transition graphs. Applying the approach to hematopoietic development in the mouse embryo, we map the progression of mesoderm toward blood using single-cell gene expression analysis of 3,934 cells with blood-forming potential captured at four time points between E7.0 and E8.5. Transitions between individual cellular states are then used as input to develop a single-cell network synthesis toolkit to generate a computationally executable transcriptional regulatory network model of blood development. Several model predictions concerning the roles of Sox and Hox factors are validated experimentally. Our results demonstrate that single-cell analysis of a developing organ coupled with computational approaches can reveal the transcriptional programs that underpin organogenesis.We thank J. Downing (St. Jude Children's Research Hospital, Memphis, TN, USA) for the Runx1-ires-GFP mouse. Research in the authors' laboratory is supported by the Medical Research Council, Biotechnology and Biological Sciences Research Council, Leukaemia and Lymphoma Research, the Leukemia and Lymphoma Society, Microsoft Research and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust - MRC Cambridge Stem Cell Institute. V.M. is supported by a Medical Research Council Studentship and Centenary Award and S.W. by a Microsoft Research PhD Scholarship.This is the accepted manuscript for a paper published in Nature Biotechnology 33, 269–276 (2015) doi:10.1038/nbt.315

A molecular roadmap of the AGM region reveals BMP ER as a novel regulator of HSC maturation

In the developing embryo, hematopoietic stem cells (HSCs) emerge from the aorta-gonad-mesonephros (AGM) region, but the molecular regulation of this process is poorly understood. Recently, the progression from E9.5 to E10.5 and polarity along the dorso-ventral axis have been identified as clear demarcations of the supportive HSC niche. To identify novel secreted regulators of HSC maturation, we performed RNA sequencing over these spatiotemporal transitions in the AGM region and supportive OP9 cell line. Screening several proteins through an ex vivo reaggregate culture system, we identify BMP ER as a novel positive regulator of HSC development. We demonstrate that BMP ER is associated with BMP signaling inhibition, but is transcriptionally induced by BMP4, suggesting that BMP ER contributes to the precise control of BMP activity within the AGM region, enabling the maturation of HSCs within a BMP-negative environment. These findings and the availability of our transcriptional data through an accessible interface should provide insight into the maintenance and potential derivation of HSCs in culture.Peer reviewe

Emergence of human angiohematopoietic cells in normal development and from cultured embryonic stem cells

Human hematopoiesis proceeds transiently in the extraembryonic yolk sac and embryonic, then fetal liver before being stabilized in the bone marrow during the third month of gestation. In addition to this classic developmental sequence, we have previously shown that the aorta-gonad-mesonephros (AGM) embryonic territory produces stem cells for definitive hematopoiesis from 27 to 40 days of human development, through an intermediate blood-forming endothelium stage. These studies have relied on the use of traditional markers of human hematopoietic and endothelial cells. In addition, we have recently identified and characterized a novel surface molecule, BB9, which typifies the earliest founders of the human angiohematopoietic system. BB9, which was initially identified with a monoclonal antibody raised to Stro-1(+) bone marrow stromal cells, recognizes in the adult the most primitive Thy-1(+) CD133(+) Lin(−), non-obese diabetic—severe combined immunodeficiency disease (NOD–SCID) mouse engrating hematopoietic stem cells (HSCs). In the 3- to 4-week embryo,BB9expression typifies a subset of splanchnopleural mesodermal cells that migrate dorsally and colonize the ventral aspect of the aorta where they establish a population of hemogenic endothelial cells. We have indeed confirmed that hematopoietic potential in the human embryo, as assessed by long-term culture-initiating cell (LTC-IC) and SCID mouse reconstituting cell (SRC) activities, is confined to BB9-expressing cells. We have further validated these results in the model of human embryonic stem cells (hESCs) in which we have modeled, through the development of hematopoietic embryoid bodies (EBs), primitive and definitive hematopoieses. In this setting, we have documented the emergence of BB9(+) hemangioblast-like clonogenic angiohematopoietic progenitors that currently represent the earliest known founders of the human vascular and blood systems

Hormone-regulated v-rel estrogen receptor fusion protein: reversible induction of cell transformation and cellular gene expression

We describe the construction of a v-rel estrogen receptor fusion protein (v-relER) which allows the regulation of v-rel oncoprotein activity by hormone. In the presence of estrogen, v-relER readily transformed primary chicken fibroblasts and bone marrow cells in vitro. In both cell types, v-rel-specific transformation was critically dependent on the presence of estrogen or the estrogen agonist 4-hydroxytamoxifen (OHT). Withdrawal of estrogen or application of an estrogen antagonist, ICI164,384 (ICI) caused a reversal of the transformed phenotype. We also demonstrate that the v-relER protein binds to NF-kappa B sites in an estrogen-dependent manner, thereby showing that sequence-specific DNA binding of v-relER is critical for the activation of its transforming capacity. In transient transfection experiments, we failed to demonstrate a clear repressor or activator function of the v-rel moiety in v-relER. However, in v-relER-transformed bone marrow cells, estrogen and OHT induced elevated mRNA levels of two cellular genes whose expression is constitutive and high in v-rel-transformed cells. These results suggest that v-rel might exert part of its activity as an activator of rel-responsive genes