Abstract
Endothelial dysfunction caused by endothelial cell injuries is the initiating factor for atherosclerosis (AS), and lipid peroxidative injury is one of a dominant factor for AS pathogenesis. Using RNA-seq, we compared changes in transcriptome expression before and after endothelial cell injury, and found 311 differentially expressed genes (DEGs), of which 258 genes were upregulated and 53 genes were downregulated. The protein–protein interactions (PPIs) between the genes were analysed using the STRING database, and a PPI network of DEGs was constructed. The relationship distributions among these PPIs were analysed by performing network node statistics. We found that in the top 20 DEGs with high connected protein nodes in the PPI network, 16 were upregulated and 4 were downregulated. Gene ontology (GO) functional enrichment analysis and KEGG pathway enrichment analysis on the DEGs were also performed. By comparing the upregulated expressed genes with high connected protein nodes in the PPI network to those related to endothelial cell lipid damage and repair in the GO analysis, we identified seven genes (NOX4, PPARA, CCL2, PDGFB, IL8, VWF, CD36) and verified their expression levels by real-time polymerase chain reaction. The protein interactions between the seven genes were then analysed using the STRING database. The results predicted that CCL2 interacts with NOX4, PPARα, PDGFβ and VWF individually. Thus, we examined the protein expression levels of CCL2, NOX4, PPARα, PDGFβ and VWF, and found that the expression levels of all proteins were significantly upregulated after the lipid peroxidative injury, with CCL2 and PPARα exhibiting the highest expression levels. Therefore, we investigated the interregulatory relationship between CCL2 and PPARα and their roles in the repair of endothelial cell injury. With the help of gene overexpression and knockdown techniques, we discovered that PPARα promotes the repair of endothelial cell injury by upregulating CCL2 expression in human umbilical vein endothelial cells but that CCL2 cannot regulate PPARα expression. Therefore, we believe that PPARα participates in the repair of endothelial cell lipid peroxidative injury through regulating the expression of CCL2.
Highlights
A transcriptome is a collection of all transcripts produced by a species or a specific cell type
In order to further study the regulational mechanism of endothelial cell injury and repair, we established a vascular endothelial cell injury model in vitro featuring endothelial cell lipid peroxidative injury, and used RNA-seq and relevant data analysis to identify genes involved in the repair of endothelial cell injury
RNA sequencing technology was used for a transcriptome study of endothelial cell lipid peroxidative injury, followed by protein interactions analysis on the differentially expressed genes (DEGs), protein functional research and related mechanisms
Summary
A transcriptome is a collection of all transcripts produced by a species or a specific cell type. Compared with traditional chip hybridization, transcriptome sequencing (RNA-seq) allows for the detection of the overall transcriptional activity of any species without the requirement of predesigned probes for known sequences, providing more accurate digital signals with high throughput and a broad detection range [1,2]. The development of atherosclerosis (AS) is closely related to endothelial dysfunction caused by endothelial cell injury. Increases in plasma cholesterol levels promote the oxidation of unused or unmetabolized cholesterol to produce oxycholesterols, thereby causing endothelial cell injury. Injured endothelial cells initiate a series of repair mechanisms to prevent the cell apoptosis induced by excessive oxycholesterols. The repair of injured endothelial cells is the first line of defence against AS [3,4]
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