Project description:We and others have previously reported the paradoxical downregulation of endothelial nitric oxide synthase (eNOS, NOS3) steady-state mRNA by pharmacological histone deacetylase (HDAC) inhibitors. This microarray experiment was designed to identify novel targets of an HDAC inhibitor (trichostatin A (TSA), 500 nM, 24h, Sigma) in primary human endothelial cells (human umbilical vein endothelial cells, HUVEC). Keywords: gene expression profiling, paired samples, dichotomous conditions
Project description:We and others have previously reported the paradoxical downregulation of endothelial nitric oxide synthase (eNOS, NOS3) steady-state mRNA by pharmacological histone deacetylase (HDAC) inhibitors. This microarray experiment was designed to identify novel targets of an HDAC inhibitor (trichostatin A (TSA), 500 nM, 24h, Sigma) in primary human endothelial cells (human umbilical vein endothelial cells, HUVEC). Experiment Overall Design: HUVEC were isolated and cultured from three individuals (n=3, biological replicates). Paired samples were then treated with TSA (500nM, 24h, Sigma) or a vehicle-control. After sample preparation, hybridization to Affymetrix Human Genome Focus Arrays, image acquisition and the evaluation of several quality control metrics, summary expression measures were computed using the MAS5.0, RMA and GCRMA pre-processing strategies, respectively. The values were generated using the affy and gcrma packages of the Bioconductor Project.
Project description:Valproic acid (VPA) is a short-chain fatty acid used in the treatment of epilepsy and also considered to be an epigenetic modifier by functioning as a histone deacetylase (HDAC)-inhibitor. The aim of this study was to search for gene altered by VPA in human endothelial cells. Human umbilical vein endothelial cells (HUVEC) from five individuals were cultured in the absence or presence of 4mM VPA for 24h. Cells were cultured in EGM-2 medium and all experiments were performed in passages 1 or 2.
Project description:The endothelial transcription factor Erg (Ets Related Gene) plays an important role in homeostasis and angiogenesis by regulating many endothelial functions including survival and junction stability. Here we show that Erg regulates endothelial cell migration. Transcriptome profiling of Erg-deficient endothelial cells (EC) identified 80 genes involved in cell migration as candidate Erg targets, including regulators of the Rho GTPases. Inhibition of Erg expression in human umbilical vein endothelial cells (HUVEC) resulted in decreased migration in vitro, whilst Erg over-expression using adenovirus caused increased migration. Live-cell imaging of Erg-deficient HUVEC showed a reduction in lamellipodia, in line with decreased motility. Both actin and tubulin cytoskeletons were disrupted in Erg-deficient EC, with a dramatic increase in tubulin acetylation. Amongst the most significant microarray hit was the cytosolic histone deacetylase (HDAC)-6, a regulator of cell migration. Rescue experiments confirmed that HDAC6 mediates the Erg-dependent regulation of tubulin acetylation and actin localization. The functional role of Erg in EC was studied by gene expressing profiling using three separate HUVEC isolates treated with either control antisense or Erg-specific antisense for 24 or 48 hours.
Project description:The cAMP-dependent protein kinase A (PKA) regulates a plethora of cellular functions in health and disease. During angiogenesis, PKA activity in endothelial cells controls the transition from sprouting to vessel maturation and limits tip cell formation independently of Notch signaling. The molecular PKA targets mediating these effects remain unknown. We report a chemical genetics screen identifying endothelial-specific substrates of PKA in human umbilical vein endothelial cells (HUVEC). We identified ATG16L1, a regulator of autophagy, as novel target of PKA. Biochemical validation, mass spectrometry and peptide spot arrays revealed that PKA phosphorylates ATG16L1α at Ser268 and ATG16L1β at Ser269. The phosphorylations drive degradation of ATG16L1 protein. Knocking down PKA or inhibiting its activity increased ATG16L1 protein levels and endothelial autophagy. In vivo genetics and pharmacological experiments demonstrated that autophagy inhibition partially rescues vascular hypersprouting caused by PKA deficiency. We propose that endothelial PKA activity restricts active sprouting by reducing endothelial autophagy through phosphorylation of ATG16L1.
Project description:The endothelial transcription factor Erg (Ets Related Gene) plays an important role in homeostasis and angiogenesis by regulating many endothelial functions including survival and junction stability. Here we show that Erg regulates endothelial cell migration. Transcriptome profiling of Erg-deficient endothelial cells (EC) identified 80 genes involved in cell migration as candidate Erg targets, including regulators of the Rho GTPases. Inhibition of Erg expression in human umbilical vein endothelial cells (HUVEC) resulted in decreased migration in vitro, whilst Erg over-expression using adenovirus caused increased migration. Live-cell imaging of Erg-deficient HUVEC showed a reduction in lamellipodia, in line with decreased motility. Both actin and tubulin cytoskeletons were disrupted in Erg-deficient EC, with a dramatic increase in tubulin acetylation. Amongst the most significant microarray hit was the cytosolic histone deacetylase (HDAC)-6, a regulator of cell migration. Rescue experiments confirmed that HDAC6 mediates the Erg-dependent regulation of tubulin acetylation and actin localization.
Project description:We quantified differential microRNA (miRNA) expression in Human umbilical vein endothelial cells （HUVECs）response to Angiogenin (ANG) treatment.These data were used to determine which miRNAs are altered on ANG in Human umbilical vein endothelial cells.
Project description:ChIP-Seq profiling of human umbilical vein endothelial cells (HUVECs) overexpressing constitutively active FOXO1 to identify FOXO1 DNA binding sites and to assess changes in the histone modifications H3K4me3 and H3K27ac.
Project description:Human vein umbilical endothelial cells (HUVEC) were transfected with pre-miR control and pre-miR 146 (Ambion) in order to identify targets (direct and indirect) downregulated by miR-146a in endothelial cells. 164 transcripts were downregulated with a fold change ≥ 1.2.
Project description:We aimed to elucidate the impact of miR-520b on endothelial cell (EC) activation. To determine the potential targets of miR-520b, we performed RNA-seq analysis by transfecting miR-520b mimics in primary human umbilical vein endothelial cells (HUVECs).
