Project description:A key step in angiogenesis is the upregulation of growth factor receptors on endothelial cells. Here we demonstrate that a small regulatory microRNA, miR-296 has a major role in this process. Glioma cells and angiogenic growth factors elevate the level of miR-296 in primary human brain microvascular endothelial cells in culture. The miR-296 level is also elevated in primary tumor endothelial cells isolated from human brain tumors compared to normal brain endothelial cells. Growth factor-induced miR-296 contributes significantly to angiogenesis by directly targeting the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) mRNA, leading to decreased levels of HGS and thereby reducing HGS-mediated degradation of the growth factor receptors VEGFR2 and PDGFR-β. Furthermore, inhibition of miR-296 with antagomirs reduces angiogenesis in tumor xenografts in vivo. Keywords: comparitive miRNA analysis 3 biological replicates (HBMVECs) are compared to 3 biological replicates (HBMVECs exposed to U87 glioma cells)
Project description:From two donors of human umbilical vein endothelial cells, in vitro cell lines were established. Both cell lines were grown in vitro until irreversible growth arrest was observed (replicative senescence). Total RNA from young (replicating) cells as well as senescent cells was harvested and used for hybridization of microRNA chips (MRC) from TU Graz based on Sanger miRBase 9.2
Project description:Class IIa histone deacetylases (HDACs) are signal-responsive regulators of gene expression involved in vascular homeostasis. To investigate the differential role of class IIa HDACs for the regulation of angiogenesis, we used siRNA to specifically suppress the individual HDAC isoenzymes. Among the HDAC isoforms tested, silencing of HDAC5 exhibited a unique pro-angiogenic effect evidenced by increased endothelial cell migration, sprouting and tube formation. Consistently, overexpression of HDAC5 decreased sprout formation, indicating that HDAC5 is a negative regulator of angiogenesis. The anti-angiogenic activity of HDAC5 was independent of MEF2 binding and its deacetylase activity, but required a nuclear localization indicating that HDAC5 might affect the transcriptional regulation of gene expression. To identify putative HDAC5 targets, we performed microarray expression analysis. Silencing of HDAC5 increased the expression of fibroblast growth factor 2 (FGF2) and angiogenic guidance factors including Slit2. Antagonization of FGF2 or Slit2 reduced sprout induction in response to HDAC5 siRNA. ChIP assays demonstrate that HDAC5 binds to the promoter of FGF2 and Slit2. In summary, HDAC5 represses angiogenic genes, like FGF2 and Slit2, which causally contribute to capillary-like sprouting of endothelial cells. The de-repression of angiogenic genes by HDAC5 inactivation may provide a useful therapeutic target for induction of angiogenesis. Experiment Overall Design: 6 samples: 3x siSCRAMBLED transfected HUVEC (control) + 3x siHDAC5 transfected HUVEC 24h after transfection
Project description:As susceptibility to many adult disorders originates in utero, we here hypothesized that fetal sex influences gene expression in placental cells and produces functional differences in human placentas. We found that fetal sex differentially affects gene expression in a cell-phenotype dependent manner among all four placental cell-phenotypes studied: cytotrophoblasts, syncytiotrophoblasts, arterial endothelial cells and venous endothelial cells. The markedly enriched pathways in males were identified to be signaling pathways for graft-versus-host disease as well as the immune and inflammatory systems, both supporting the hypothesis that there is reduced maternal-fetal compatibility for male fetuses. Our study is the first microarray study investigating sexual dimorphism in purified and characterized somatic cells from a single human tissue, the placenta, that underlines the importance of considering fetal sex as an independent variable in any work using human placenta. Arterial and venous endothelial cells were isolated from eight different placentas, four of each sex. A total of ten placentas were used for isolation of cytotrophoblasts and six for syncytiotrophoblasts, with equal numbers from each sex.
Project description:Laminar shear stress due to constant blood flow is known to play a critical role in maintaining vascular health. In contrast, endothelial cell senescence appears to be closely associated with the incidence of vascular disorder. In an attempt to identify functional biomarkers for age-related vascular health/disease, the present study investigated differential gene expression of young and senescent human umbilical vein endothelial cells (HUVECs) under static and laminar shear stress. We used a cDNA microarray method to compare gene expression profiles of young and senescent HUVECs under static and laminar shear stress conditions. Experiment Overall Design: Senescent cells were prepared by continuous subculture in vitro, and a cone-and-plate device was used to impose laminar shear stress onto cells. Young and senescent cells were exposed to laminar shear stress or maintained under static conditions. Total mRNA was extracted and gene expression profiles were analyzed by cDNA microarray.
Project description:Biologic characterization of SB-559457 (SB), a non-peptidyl hydrazone class of thrombopoietin receptor (Mpl) agonist, revealed toxicity towards human leukemia cells. Anti-proliferative effects followed by significant, non-apoptotic, cell death within 72 hours occurred in 24/26 AML, 0/6 ALL, and 3/6 CML patient samples exposed to SB, but not recombinant human thrombopoietin (rhTpo), in liquid suspension culture. Further investigation revealed increased phosphorylation of p70S6/S6 kinases in SB, but not in rhTpo, treated cells. Expression profiling of cells exposed to SB vs rhTpo revealed statistically significant, ~2-fold changes in GAPDH and REDD1 gene expression, confirmed by QRT-PCR. These genes, induced in energy or hypoxia stressed cells, have been implicated in cell death pathways, and may provide important clues to the mechanism of SB induced, leukemic cell death. These results suggest that nonpeptidyl, hydrazone class Mpl agonists may be clinically useful anti-leukemic agents by virtue of their combined thrombopoietic and anti-leukemic effects. Experiment Overall Design: Primary cells collected from 5 patients with acute myeloid leukemia (AML) were stimulated stimulated with MPL receptor agonist (SB559457) or recombinant human Tpo (rhTpo) for 6 hours. Next, RNA was isolated from these cells. RNA was used for gene profile analyis to compare genes regulated in AML cells by SB559457 verus rhTpo. For each sample half of the cells were stimulated with Mpl agonist and half with rhTpo.
Project description:Angiogenesis, the formation of new capillaries by sprouting from preexisting vessels, is mainly induced by VEGF-A. To identify genes which are induced by VEGF-A in endothelial cells, HUVEC were starved and induced by VEGF-A165 for 30, 60 and 150min. RNA of induced and uninduced cells was isolated and subjected to microarray analysis using Affymetrix microarray. Experiment Overall Design: Human umbilical vein endothelial cells were grown in complete EGM-2 medium in dense culture for four days without change of medium and before the stimulation in EGM-2 medium without growth factors over night. The cells were stimulated with 100ng/ml VEGF-A165 for 30, 60 and 150 min. RNA was isolated with Trizol according to the manufacturer's instructions.
Project description:Long non-coding RNAs (lncRNAs) have emerged as critical regulators of gene expression and chromatin modifications, with important functions in development and disease. Here we sought to identify and functionally characterize lncRNAs critical for vascular vertebrate development with significant conservation across species. Genome-wide transcriptomic analyses during human vascular lineage specification enabled the identification of three conserved novel lncRNAs: TERMINATOR, ALIEN and PUNISHER that are specifically expressed in pluripotent stem cells, mesoderm and endothelial cells, respectively. Gene expression profiling, alongside RNA immunoprecipitation coupled to mass spectrometry, revealed a wide range of new molecular networks and protein interactors related to post-transcriptional modifications for all three lncRNAs. Functional experiments in zebrafish and murine embryos, as well as differentiating human cells, confirmed a developmental-stage specific role for each lncRNA during vertebrate development. The identification and functional characterization of these three novel non-coding provide a comprehensive transcriptomic roadmap and shed new light on the molecular mechanisms underlying human vascular development. shRNA knock down of lncRNAs followed by DNA methylation profiling