Endothelial cell is the major cell type that senses and transduces mechanosignal generated by shear stress. We have recently shown that Hippo/YAP pathway is a mechanosensitive pathway that is critical for maintaining endothelial cell homeostasis. However, the transcritpional targets and biological functions of YAP in endothelial cells remain largely unknown. To evaluate YAP-dependent gene expression in endothelial cells, we performed RNA-sequencing in YAP depleted (by transfection with by YAP siRNA) and overexpressed (by infection with YAP-S127A catalytically active adenovirus) human endothelial cells. We observed that YAP critically regulates endothelial function by modulating multiple atherosclerosis-related genes. Our study provides mechanistic insights into the question how YAP regulates endothelial function and atherosclerosis by modulating endothelial transcriptional profile.
Overall design: Human Umbilical Vein Endothelial Cells (HUVEC, passage 3 to passage 5) were seeded in 0.2% gelatin-coated dishes the day before experiment. The next day, 80% confluent HUVECs were transfected with non-targetting siRNA pool (siNC, GE Dharmacon, #D-001810-10) or YAP1 siRNA pool (siYAP1, GE Dharmacon, #M-012200) for 48 hours. In the overexpression experiment, 80% confluent HUVECs were infected with control adenovirus (Ad-NC) or YAP-S127A catalytically active adenovirus (Ad-YAP-S127A, gifted by Prof. Junichi Sadoshima, New Jersey Medical School, NJ) for 48 hours. After treatment, total RNA was isolated using the RNA-Easy Mini Plus kit (QIAGEN). High quality RNA samples (pre-assessed by Nanodrop measurements) were further processed in the Genome Research Center of the University of Rochester Medical Center. The TruSeq RNA Sample Preparation Kit V2 (Illumina, San Diego, CA) was used for next generation sequencing library construction per manufacturer’s protocols. Briefly, mRNA was purified from 100ng total RNA with oligo-dT magnetic beads and fragmented. First-strand cDNA synthesis was performed with random hexamer priming followed by second-strand cDNA synthesis. End repair and 3’ adenylation was then performed on the double stranded cDNA. Illumina adaptors were ligated to both ends of the cDNA, purified by gel electrophoresis and amplified with PCR primers specific to the adaptor sequences to generate amplicons of approximately 200-500bp in size. The amplified libraries were hybridized to the Illumina single end flow cell and amplified using the cBot (Illumina, San Diego, CA) at a concentration of 8pM per lane. Single end reads of 100nt are generated for each sample and aligned to the organism specific reference genome. Sequenced reads were cleaned using Trimmomatic-0.32 before mapping some of to the human reference genome (GRCh38.p2) with STAR-2.4.2a. Raw read counts were obtained using HTSeq and gencode 23 human gene annotations. DESeq2-1.10.1 was used to perform data normalization and differential expression analysis with an adjusted p-value threshold of 0.05. Then, Cufflinks-2.0.2 Software was used with the gencode 23 human gene annotations to perform differential expression analysis with an FDR cutoff of 0.05.
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