Statins modulate endothelial transcriptional profile by inhibiting EZH2

Statins protect against the development of atherosclerosis via cholesterol-dependent and –independent mechanisms. Understanding the molecular mechanisms mediating simvastatin induced atheroprotective effects is critical for designing anti-atherosclerotic agents. Here, we showed that simvastatin decreases the expression of the Polycomb methyltransferase EZH2 in endothelial cells. To better understand the influence of the simvastatin-induced EZH2 downregulation on endothelial transcriptome, we performed RNA-sequencing study to evaluate differential gene expression after overexpression of EZH2 in the presence of simvastatin treatment. We found simvastatin treatment altered a subset of genes that can be rescued with EZH2 overexpression. Therefore, simvastatin treated endothelial cells display an atheroprotective phenotype by downregulating EZH2. Overall design: Human Umbilical Vein Endothelial Cells (HUVEC, passage 3 to passage 5) were seeded in 0.2% gelatin-coated 6-well plate the day before experiment. The next day, HUVECs were treated with control adenovirus (AdCon) or EZH2 adenovirus (AdEZH2, #1371, Vector Biolab) for 24 hours, before stimulation with simvastatin (1 µM, dissolved in DMSO) for 48 hours in the presence of adenovirus. After treatment, 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.