PCSK9, a novel immune and ferroptosis related gene in abdominal aortic aneurysm neck

Abstract

The gene expression profile of abdominal aortic aneurysm (AAA) neck is not fully understood. The etiology of AAA is considered to be related to atherosclerosis and the inflammatory response, involving congenital, genetic, metabolic, and other factors. The level of proprotein convertase subtilisin/kexin type 9 (PCSK9) is related to those of cholesterol, oxidized low-density lipoprotein, and triglycerides. PCSK9 inhibitors have significant effects on lowering LDL-cholesterol, reversing atherosclerotic plaques, and reducing the risk of cardiovascular events and have been approved by several lipid-lowering guidelines. This work was aimed to investigate the potential role of PCSK9 in the neck of AAA. We extracted the expression dataset (GSE47472) containing 14 AAA patients and 8 donors and single-cell RNAseq (scRNA-seq) data (GSE164678) of CaCl₂-induced (AAA) samples from the Gene Expression Omnibus dataset. Through bioinformatics methods, we found that PCSK9 was up-regulated in the proximal neck of human AAA. In AAA, PCSK9 was mainly expressed in fibroblasts. Additionally, immune check-point PDCD1LG2 was also expressed higher in AAA neck than donor, while CTLA4, PDCD1, and SIGLEC15 were down-regulated in AAA neck. The expression of PCSK was correlated with PDCD1LG2, LAG3, and CTLA4 in AAA neck. Additionally, some ferroptosis-related genes were also down-regulated in AAA neck. PCSK9 was also correlated with ferroptosis-related genes in AAA neck. In conclusion, PCSK9 was highly expressed in AAA neck, and may exert its role through interacting with immune check-points and ferroptosis-related genes.

Figure 1

Different expression genes in AAA neck compared to normal group. (A) Volcano plot showing the up- and down-regulated genes in AAA neck (n = 14) compared with normal tissues (n = 8) from GSE47472 dataset in GEO (Adjusted p < 0.05 and |Log FC|> 1). Red dots represent up-regulation. Blue dots represent down-regulation. (B) Through using R software (version: 4.0.3) and R package ‘pheatmap’ (version 1.0.8), heat map was constructed to show the top 50 up- and down-regulated genes in AAA neck (n = 14) than donors (n = 8). Red represents up-regulation. Blue represents down-regulation.

Figure 2

The expression of PCSK9 in AAA neck and normal tissues. (A) The expression of PCSK9 in human AAA neck (n = 14) and normal tissues (n = 8) in GSE47472 dataset from GEO database. Student t-test was used to compare the different expressions. *p < 0.05. (B) The RT-qPCR results showed the PCSK9 mRNA expression in AAA neck of AAA mice model (n = 5) and normal tissues (n = 5). Student t-test was used to compare the mRNA expressions of PCSK9. ***p < 0.001.

Figure 3

Identification of the cell composition changes in AAA and cellular localization of PCSK9 by scRNA-seq data (GSE164678) analysis. (A) Separate eight cell clusters according to uniform manifold approximation and projection (UMAP) plot. (B) Cell distributions in AAA and sham samples are shown in UAMP. (C) Pie plot showing the percentages of each cell population in AAA and sham groups. (D) Top marker genes of these eight cell clusters. (E) UMAP plots showing that PCSK9 was mainly expressed in fibroblasts in AAA.

Figure 4

The expression of immune checkpoints in AAA neck and normal tissues. (A) Through using R software (version: 4.0.3) and R package ‘pheatmap’ (version 1.0.8), heat map was established to show the expression of immune checkpoints in AAA neck (n = 14) and normal tissues (n = 8) in GSE47472 dataset from GEO database. Red represents up-regulation. Blue represents down-regulation. (B) Box plots showing the expression of immune checkpoints in AAA neck (n = 14) and normal tissues (n = 8) in GSE47472 dataset from GEO database. Wilcoxon test was employed to compare the differential expression of immune checkpoints. *p < 0.05, **p < 0.01, ***p < 0.001 AAA neck vs. control group.

Figure 5

The correlation between PCSK9 and immune checkpoints in AAA neck and donor tissues. (A) Spearman’s correlation analysis of the expression data from GSE47472 showed that PCSK9 was negatively related to CTLA4. Spearman’s Rank Correlation Coefficient (Spearman r =  − 0.4816, p = 0.0232). (B) Spearman’s correlation analysis of the expression data from GSE47472 showed PCSK9 was negatively related to LAG3 (Spearman r =  − 0.4308, p = 0.0453). (C) Spearman’s correlation analysis of the expression data from GSE47472 showed PCSK9 was positively related to PDCD1LG2 (Spearman r =  − 0.4308, p = 0.0453).

Figure 6

The expression of 24 ferroptosis related genes in the neck of AAA and normal tissues. (A–C) The box plots showing the expression of ferroptosis related genes in human AAA neck (n = 14) and donor tissues (n = 8) in GSE47472 dataset. Wilcoxon test was used to compare the differential expression of ferroptosis related genes. *p < 0.05, **p < 0.01, ***p < 0.001 AAA neck vs. control group.

Figure 7

The correlation between PCSK9 and ferroptosis related genes in human AAA neck and normal tissues. Spearman’s correlation analysis of the expression data from GSE47472 from GEO database showed that PCSK9 was negatively related to ATP5MC3 (Spearman r =  − 0.5121, p = 0.0148) (A), CARS (Spearman r =  − 0.4534, p = 0.0341) (B), CISD1 (Spearman r =  − 0.6827, p = 0.0005) (C), CS (Spearman r =  − 0.4704, p = 0.0272) (D), GPX4 (Spearman r =  − 0.5144, p = 0.0143) (E), HSPA5 (Spearman r =  − 0.5630, p = 0.0064) (F), and SLC1A5 (Spearman r =  − 0.5822, p = 0.0045) (G).

Figure 8

Potential protein, miRNA, and TFs regulating PCSK9. (A) Top 15 genes positive and negative correlated with PCSK9 in AAA neck. (B) The PPI network of PCSK9 in STRING database. The meaning of the color of the connecting lines can refer to the website (https://www.string-db.org/). (C) The PPI network of PCSK9 in GeneMANIA database. (D) TF-target and miRNA-target network of PCSK9. Green: TFs; Red: miRNA; Yellow: protein that interacted with PCSK9 from PPI network; TFs: transcription factors.

Figure 9

Function enrichment analysis of PCSK9 in AAA neck. (A) GO (biological process) analysis of PCSK9 in AAA neck. (B) GO (molecular function) analysis of PCSK9 in AAA neck. (C) GO (cell component) analysis of PCSK9 in AAA neck. (D) KEGG analysis of PCSK9 in AAA neck. NES normalized enrichment score.