Effect of von Willebrand factor on gene expression in HUVECs
ABSTRACT: Von Willebrand factor is a paracrine/autocrine regulator of human mesenchymal stem cell adhesion to distressed/apoptotic endothelial cells. This data set examines effect of vWF on gene expression in HUVECs. HUVECs were maintained in EGM2 media and treated with vWF in Hank's buffered salt solution (HBSS).
Project description:Von Willebrand factor is a paracrine/autocrine regulator of human mesenchymal stem cell adhesion to distressed/apoptotic endothelial cells. This data set examines effect of vWF on gene expression in HUVECs. Overall design: HUVECs were maintained in EGM2 media and treated with vWF in Hank's buffered salt solution (HBSS).
Project description:We investigated the transcriptome of HUVECs under basal conditions and overexpression of Sox18 in presence or absence of small molecule protein-protein disruptor Sm4. Various RNA fractions were prepared and sequenced in order to characterise Sox18 and Sm4 responsive genes in HUVECs
Project description:Branching from conduits is a defining feature of the gas delivery systems of invertebrates (tracheae built from epithelial cells) and vertebrates (vasculature lined by endothelial cells). Here, we show that the vertebrate transcriptional repressor Tel plays an evolutionarily conserved role in angiogenesis: it is indispensable for sprouting of primary human endothelial cells and for the normal development of the Danio rerio embryo blood circulatory system. Tel controls endothelial sprouting via binding to the generic co-repressor C-terminal binding protein (CtBP). In endothelial cells, the Tel:CtBP complex temporally restricts a VEGF-mediated pulse of dll4 expression and consequently integrates VEGFR intracellular signaling and intercellular Notch-Dll4 signaling. It further refines branching by regulating expression of other factors that constrain angiogenesis such as sprouty family members and ve-cadherin. Thus, the Tel:CtBP complex moderates the balance between positive and antagonistic angiogenesis cues and thereby conditions endothelial cells for angiogenesis. Since the activity of CtBP is attuned to intracellular NADH levels, our results raise the possibility that Tel-mediated sprouting could be sensitized to the metabolic status of the tissue. Tel control of branching appears to be evolutionarily conserved since Yan, the invertebrate orthologue of Tel, is similarly required for branching morphogenesis of the invertebrate tracheae. Collectively, our work suggests that Tel is a central regulator of angiogenesis and highlights Tel and its associated networks as potential targets for the development of therapeutic strategies to inhibit pathological angiogenesis. 2 independent screens were performed testing effects of knockdown of Tel or CtBP (screen 1) or effects of VEGF-A (screen 2) on Human Umbilical Vein Endothelial Cells (HUVECs). For screen 1 we tested 3 different conditions. We established stable HUVEC cell lines which were either infected with control lentivirus (Mock), or lentivirus expressing short hairpin RNA constructs for the specific knockdown of Tel(Teli) or CtBP2 (CtBP2i). Expression in the Teli and CtBP2i cell lines was compared to expression in the Mock cell line for screen 1. For screen 2 we tested 2 conditions. We exposed HUVECs to VEGF (50ng/mL) for 30 minutes (samplename: VEGF30) and compared the transcriptome of these cells to untreated HUVECs (VEGF0). For each condition 2 independent repeats were analyzed and expression of genes was averaged for each repeat. HUVECs were grown under standard conditions (37degrees Celsius, 5% CO2) in EGM2 medium (Lonza).
Project description:The aim of this study is to determine the molecular mechanism by which indoxyl sulfate acts on endothelial cells. Endothelial cells (HUVECs) were incubated with medium containing indoxyl sulfate at concentration found in chronic kidney disease patients (1mM). Since indoxyl sulfate is a potassium salt, control medium contains KCl at 1mM. Cells were incubated during four hours in presence of indoxyl sulfate or KCl. Seven biological replicates were obtained for these two conditions. To understand how uremic solutes participate in endothelial dysfunction and cardiovascular complications of chronic kidney disease patients
Project description:Driving HUVECs toward mesenchymal fate Comparison of untreated HUVECs, HUVECs treated with TGF-B, HUVECS treated with TGF-B and oxidative stress, and comparator human dermal fibroblasts
Project description:Endothelial cells store von Willebrand factor (VWF) in rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs). WPB exocytosis is coordinated by a complex network of Rab GTPases, Rab-effectors and SNARE proteins. We have previously identified STXBP1 as the link between the Rab27A-Slp4-a complex on WPBs and the SNARE proteins syntaxin-2 and -3. In this study we investigate the function of syntaxin-3 in VWF secretion. In human umbilical vein endothelial cells (HUVECs) and in blood outgrowth endothelial cells (BOECs) from healthy controls endogenous syntaxin-3 immunolocalized to WPBs. A detailed analysis of BOECs isolated from a patient with variant microvillus inclusion disease (MVID), carrying a homozygous mutation in STX3 (STX3-/-), showed a loss of syntaxin-3 protein and absence of WPB-associated syntaxin-3 immunoreactivity. Ultrastructural analysis revealed no detectable differences in morphology or prevalence of immature or mature WPBs in control versus STX3-/- BOECs. VWF multimer analysis showed normal patterns in plasma of the MVID patient, and media from STX3-/- BOECs, together indicating WPB formation and maturation are unaffected by absence of syntaxin-3. However, a clear defect in Ca2+ and cAMP-mediated VWF secretion was found in the STX3-/- BOECs. Co-immunoprecipitation studies showed that syntaxin-3 associates with the WPB SNAREs SNAP23 and VAMP8. Our data reveal syntaxin-3 as a novel WPB-associated SNARE controlling VWF secretion and highlight the complex regulation of WPB exocytosis by multiple SNARE complexes.
Project description:Vascularization represents an important issue in bone development, fracture healing and engineering of artificial bone tissue. In the context of bone tissue engineering, it was shown that coimplantation of human primary umbilical vein endothelial cells (HUVECs) and human osteoblasts (hOBs) results in the formation of functional blood vessels and enhanced bone regeneration. Implanted endothelial cells do not only contribute to blood vessel formation, but also support proliferation, cell survival and osteogenic differentiation of coimplanted hOBs. These effects are partially mediated by direct heterotypic cell contacts. In a previous report we could show that cocultivated hOBs strongly increase the expression of genes involved in extracellular matrix (ECM) formation in HUVECs, suggesting that ECM may be involved in the intercellular communication between hOBs and HUVECs. The present study aimed at investigating whether comparable changes occur in hOBs. We therefore performed a microarray analysis of hOBs cultivated in direct contact with HUVECs, revealing 1121 differentially expressed genes. The differentially expressed genes could be assigned to the functional clusters ECM, proliferation, apoptosis and osteogenic differentiation. In summary, our data demonstrate that HUVECs provoke complex changes in gene expression patterns in cocultivated hOBs and that ECM plays and important role in this interaction.
Project description:Examine the toxic effect and molecular mechanisms of PM2.5 in primary human umbilical vein endothelial cells (HUVECs) . We used microarrays to detail the global programme of gene expression in HUVECs exposed to PM2.5 and identified distinct classes of up-regulated genes. Cultured HUVECs which were treated with PM2.5 at the concentrations of 50 μg/mL or DMEM for 24 h were collected for RNA extraction and hybridization on Affymetrix microarrays.
Project description:KLF2 and KLF4 are important transcriptional factors in endothelial cells, however their roles in statin treatment has not been elucidated. Here we report the comprehensive change of transcripts of statin treated HUVECs transfected with siRNA KLF2 or KLF4. We used repeated microarray analysis of HUVECs treated with pitavastatin for 4hours. Before statin treatment, cells were transfected with siRNA KLF2 or KLF4. HUVECs were used within the first 6 passages. For studies, HUVECs were cultivated in medium EGM2MV containing pitavastatin at a concentration of 1 micromolar.
Project description:Metastasizing tumor cells exit the vascular system through dynamic interactions with endothelial cells that line the internal surface of vessels. While extravasation is a key event within the metastatic cascade, the signals regulating tumor cell adhesion to the endothelium and their subsequent transendothelial migration are poorly understood. Here, we combined Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and phosphoproteomic analysis to identify cell-specific signaling pathways regulated between interacting breast cancer cells and endothelial cells (see PRIDE repository PXD001558). Further co-culture experiments were performed alongside the phosphoproteomic analysis in order to control for the protein quantity. These are presented here. Using SILAC, cell-specific labels were introduced into MDA-MB-231-LM2 cells (Human Breast cancer) and HUVECs (Human endothelial cells) to ensure each cell type had a distinct and traceable phosphoproteome when tumor and endothelial cells were co-cultured. To probe regulatory signaling events triggered in cancer cells following contact with endothelial cells, we labeled LM2 cells with medium or heavy isotopomers of arginine and lysine (Arg+6 Da, Lys+4 Da and Arg+10 Da, Lys+8 Da respectively). Heavy-labeled LM2 cells were collected by enzyme-free cell dissociation buffer, thereby preserving membrane proteins and adhesion receptors, and seeded onto a monolayer of light-labeled (Arg+0 Da, Lys+0 Da) HUVECs. Following 15 min of co-culture, non-adherent LM2 cells were gently removed and cancer cells that had attached to the endothelial layer were lysed together with the HUVECs. In parallel, medium-labeled LM2 cells were collected under the same conditions and maintained as suspension cells in monoculture to represent circulating tumor cells prior to any contact with the endothelium. These were then added to the harvested LM2-HUVEC co-culture in a 1:1 ratio of heavy:medium-labeled cells to provide a point of reference. Based on the SILAC labeling of the different cell populations, light-labeled peptides were assigned to HUVECs, medium-labeled peptides to monocultured LM2 cells in suspension, and heavy-labeled peptides to LM2 cells that had made contact with HUVECs. As such, the heavy/medium ratio for each peptide was used to quantify phosphorylation-dependent signaling changes occurring specifically in the LM2 cells upon contact with HUVECs. Conversely, to elucidate signaling events in HUVECs that were initiated by contacting cancer cells, light-labeled LM2 cells were seeded on top of a confluent monolayer of heavy-labeled HUVECs, while medium-labeled HUVECs were maintained in monoculture. Following 15 min of co-culture, the unattached LM2 cells were removed. Cells were lysed, mixed in a 1:1 ratio of heavy:medium HUVECs, followed by membrane fractionation and phosphoproteomic analysis as described above. A variation in phospho-peptide quantity could be due to the experimental difficulties in mixing heavy and medium labels in a 1:1 ratio or a quick regulation of the protein quantity through modification of its expression/degradation balance. Thus, we performed relative quantification of non-phosphorylated peptides in parallel with the phosphoproteomic analysis to account for changes in total protein abundance (data presented here) or correct for experimental bias. Cytoplasmic fractions were analyzed by LC-MS/MS before TiO2 enrichment and their median log2(H/M) were used for normalization of the phosphoproteomic dataset. In order to increase the number of proteins quantified in the LM2 cells, we performed a supplementary co-culture experiment and fractionated the peptides by SDS-PAGE. In order to get a confident relative quantification of Ephrin type-A receptor 2 (EPHA2), we performed 2 independent experiments followed by EPHA2 IP and LC-MSMS.