Project description:The goal of this study was to reveal SNAI1 function in angiogenesis. SNAI1 is up-regulated by VEGF stimulus. Using RNASeq method (single-ended 150-bp sequencing on Illumina Hi-seq 3000 instrument), we measured the transcriptional difference between siSNAI1 or NC-transfected HUVECs.
Project description:IL-6 family cytokines as OSM and CNTF modify angiogenesis to different degree. This study investigates to which extend treatment of endothelial cells with those cytokines can modify VEGF induced angiogenic reactions. Human umbilical vein endothelial cells (HUVECs) were stimulated for 24h by cytokines followed by RNA harvesting and processed for RNA sequencing.
Project description:The role of STAT3 signaling induced by stimulation of IL-6 cytokine family members such as OSM and CNTF on the angiogenic response of endothelial cells is still not fully understood. To assess the impact of this pathway on angiogenesis, this study compares human umbilical vein endothelial cells (HUVECs) with or without STAT3 knock-down introduced by siRNA and stimulation with VEGF, OSM+VEGF and CNTF+VEGF in the presence of the CNTF receptor (CNTFR).
Project description:In order to identify new genes potentially involved in angiogenesis process, we performed a deep-sequencing transcriptome analysis of HUVECs stimulated with recombinant VEGF-A, introducing a difference with respect to the several previous studies. Primary ECs are normally cultured in vitro using an endothelial growth medium (EGM) constituted by an endothelial basal medium (EBM) supplemented with 2% fetal bovine serum (FBS) and a mix of growth factors and reagents, typically represented by basic Fibroblast Growth Factor (bFGF), Insulin-like Growth Factor 1 (IGF1), Epidermal Growth Factor (EGF), heparin, hydrocortisone, ascorbic acid and the same VEGF-A. In previous studies, HUVECs or other primary ECs were starved in EBM with low FBS concentration before the stimulation with VEGF-A. Instead of starvation, we grown HUVECs in complete EGM-2 medium except for VEGF-A to maintain the best condition for in vitro survival and propagation of primary ECs. After 24 hours of VEGF-A deprivation, VEGF-A was re-added to the medium and the stimulation was performed for six hours.
Project description:Purpose: The goals of this study are to compare VEGF-treated HUVECs with or without Verteporfin (VP) pretreatment transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: After serum-starving for 12 hours, HUVECs were divided into two group: VEGF and VEGF+VP. Cells from VEGF+VP group were pretreated with VP (4 μM) for 2 hours and then all cells were treated with VEGF (200 ng/mL) for another 24 hours. Subsequently, total RNA from HUVECs were prepared using Trizol reagent and mRNA library was constructed. RNA-sequencing: RNA-sequencing was carried out by BGI (Beijing Genomic Institute, ShenZhen, China). SOAPnuke software (v1.5.2) was used to filter the data for RNA-sequencing and then these data were mapped to the reference genome using HISAT2 software (v2.0.4). The clean reads were aligned to the gene set by Bowtie2 (v2.2.5). The expression level of genes was then measured by RSEM software (v1.2.12). The heatmap of top 40 different expression genes was drawn according to the gene expression with FPKM (fragment per kilobase of transcript per million). Reactome (https://www.reactome.org/) enrichment analysis of different expression genes was undertaken and the significant levels of terms and pathways were corrected by Q value. Results: The statistical results of significant DEGS confirmed that approximately 7204 genes of the transcripts showed differential expression between the VEGF group and VEGF+VP group, with a fold change ≥1.5 and p value <0.05. Altered expression of 20 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to angiogenesis. Data analysis with GO analysis and GSEA analysis revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the first detailed transcriptomic analysis of VEGF treated HUVECs with or without VP treatment, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles.
Project description:Endothelial cells (ECs) are phenotypically heterogeneous mainly due to their dynamic epigenetic status. VEGF, the best-known angiogenic factor, activates calcium-NFAT signalling following acute angiogenic gene transcription. Here, we evaluated the global mapping of VEGF-mediated dynamic transcriptional events with a particular focus on major histone-code profiles using ChIP-seq. Remarkably, the regulatory regions of angiogenic transcription factors exclusively acquired embryonic stem-like bivalent histone marks after the VEGF stimulus. Moreover, the newly discovered NFAT-associated epigenome modifier, PTIP in the COMPASS complex, directed gene transcription via MLL3/4-enrichment which overwhelmed the polycomb-brakes. The non-canonical polycomb1 variant, PRC1.3, specifically bound to and allowed the transactivation of PRC2-enriched bivalent angiogenic genes until conventional PRC1 association and gene silencing. Knockdown of these epigenome modifiers abrogated postnatal aberrant neovessel formation via selective inhibition of acute angiogenic bivalent gene transcription. Collectively, the reported dynamic epigenome landscape in ECs may support the development of advanced therapeutic strategies against various vasculopathies.
Project description:Human umbilical vein vascular endothelial cells (HUVECs) are crucial for angiogenesis that benefits functional recovery after cerebral infarction. This study aims to investigate the mechanisms underlying the effects of vascular endothelial growth factor (VEGF) on HUVECs.
Project description:Human umbilical vein vascular endothelial cells (HUVECs) are crucial for angiogenesis that benefits functional recovery after cerebral infarction. This study aims to investigate the mechanisms underlying the effects of vascular endothelial growth factor (VEGF) on HUVECs. HUVECs were treated with 16 ng/mL VEGF165 for 4 days
Project description:HUVECs (human umbilical cord vein endothelial cells) are treated with the angiogenic factors VEGF-A (vascular endothelial growth factor-A) and PlGF (placental growth factor) in low or high serum media.
Project description:Purpose: The goal of this study is to compare endothelial transcriptome affected by OASL knockdown under basal or stimulated conditions by utilizing RNA-seq. Methods: Endothelial mRNA profilies of siCTL or siOASL transfected HUVECs were generated by illumina sequencing method, in duplicate. Following an optimized data analysis workflow, RNA sequence reads were counted using StringTie-2.1.3b and used to identify genes differentially expressed between siCTL- and siOASL-transfected HUVECs treated with or without TNFα and IFNγ. Results: DESeq2 was used to identify genes differentially expressed between siCTL- and siOASL-transfected HUVECs under basal or stimulated conditions. Genes showing <10 read counts total were pre-filtered. Conclusions: Our study represents the first analysis of endothelial mRNA affected by OASL knockdown with biologic replicates, generated by RNA-seq.