Transcriptom analysis of normal and Slug overexpressing human endothelial cells in a 3D fibrin-gel bead angiogenesis assay

ABSTRACT: Purpose:To identify transcriptional changes in human endothelial cells (HUVEC) both during normal sprouting angiogenesis (different time points) and with Slug overexpression (different Slug expression), in a 3D fibrin-gel bead angiogenesis Methods: Angiogenic endothelial cell with different level of Slug expression (normal and overexpressing) at different stage (early/sprouting and late/lumen formation) were harvested from a 3D fibrin-gel bead angiogenesis assay. Their transcriptom profile was generated by Next Generation RNA-sequencing as single reads, performed on an Illumina HiSeq 2500 platform, using three donor HUVEC lines.At least 30 million reads per sample were sequenced. Quality control was performed on data using FASTQC(v. 0.11.2), and reads were trimmed using Trimmomatic(v.0.32) with Illumina TruSeq adapter sequences using a PHRED quality score 15 and minimum length 20 bases. Trimmed reads were then aligned to the human hg19 reference genome using the Ensembl GRCh37 annotations and post processed using Tophat2(v.2.0.12), Bowtie2(v.2.2.3) and Samtools(v.0.1.19). Expression levels were quantified both with FPKM (Fragment per kilobase per million mapped reads) using Cufflinks(v. 2.1.1) and with raw counts using HTSeq(v.0.6.1p1.). Differential analysis was done using DESeq2 and significant genes were considered (p-value < 0.05, 2 fold change). K-means clustering was performed to extract dynamic gene clusters based on differential expression between GFP and SLUG knockdown time-points. Gene set enrichment analysis (GSEA) was performed on expressed genes to identify significant gene sets across sample comparisons. Additional gene ontology analysis was performed for each cluster and reported using Metascape. Clustering was performed in R and heatmaps were generated using JavaTree. Results: Using several different gene expression analysis platforms, we found consistent and significant changes, during both normal angiogenesis and with Slug overexpression, in genes related to proliferation, migraion, cell morphology/polarity and junction regulations, all of which are consistent with a partial-EMT program. We also found changes in Notch signaling components in Slug overexpressing cells. Changes in specific genes related to EMT and Notch signaling were validated with both qPCR and WB. Conclusion: This is the first comprehensive transcriptome analysis in human angiogenic EC during normal and excessive sprouting induced by Slug expression in an in vitro 3D angiogenesis assay system. We conclude that a partial-EndoMT is necessary for normal angiogenesis and that endothelial Slug expresion level regulates Notch signaling to determines at least partly the extent of EC's commitment towards a mesenchymal identity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE154546 | GEO | 2020/08/01

REPOSITORIES: GEO