Gene Expression Profiling of Markers of Inflammation, Angiogenesis, Coagulation and Fibrinolysis in Patients with Coronary Artery Disease with Very High Lipoprotein(a) Levels Treated with PCSK9 Inhibitors

Abstract

Besides lipids, inflammation, angiogenesis, coagulation and fibrinolysis play very important roles in coronary artery disease (CAD). We measured gene expression of the inflammatory markers interleukin (IL)-1β (IL1B) and interferon (IFN)-γ (IFNG), vascular endothelial growth factor-A (VEGF-A) (VEGFA), and coagulation and fibrinolysis markers tissue factor (TF) (F3) and plasminogen activator inhibitor-1 (PAI-1) (SERPINE) in healthy controls and CAD patients with high lipoprotein(a) (Lp(a)). The aim of our study was to identify, first, if there is a difference in these markers between controls and patients; secondly, if these markers are associated with lipids; and third, what the influence of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors is on these markers. We included 124 subjects, 27 controls and 97 patients with CAD (30 in placebo and 67 in the PCSK9 group). Blood samples were collected for lipid and gene measurement. The results showed higher expression of IL1B (p < 0.0001), VEGFA (p < 0.0001), and F3 (p = 0.018) in controls in comparison with patients. Significant correlations were observed between IL1B and lipids. Treatment with PCSK9 inhibitors increased VEGFA (p < 0.0001) and F3 (p = 0.001), and decreased SERPINE (p = 0.043). The results of our study underpin the importance of IL-1β, VEGF-A and TF in CAD as well as the effect of PCSK9 treatment on these markers.

Keywords: PCSK9 inhibitors; coagulation; coronary artery disease; fibrinolysis; inflammation; interleukin-1; lipoprotein(a); plasminogen activator inhibitor-1; tissue factor; vascular endothelial factor-A.

Figure 1

The study design. Subjects included in the study were healthy adults with no history of CAD and patients with CAD. Patients with CAD were randomized to the placebo group and the group treated with PCSK9 inhibitors, i.e., alirocumab (150 mg, N = 34 patients) or evolocumab (140 mg, N = 33 patients) for six months. Blood samples were taken at a single point for healthy controls and at the baseline and after six months of treatment for CAD patients. The lipid profile was measured and gene expression for the five studied genes (IL1B, IFNG, VEGFA, F3, SERPINE), and two reference genes (GAPDH and RPL13A) was determined.

Figure 2

Comparison of the gene expression profile between healthy controls and patients with CAD. Significant differences were obtained for IL1B, VEGFA and F3 as indicated (*** p < 0.0001, * p < 0.05, Mann Whitney U test). Shown are box and whisker plots with individual values. Gene expression data are normalized to geometric mean of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L13a (RPL13A).

Figure 3

Significant correlations between IL1B expression and lipid parameters. Significant correlations were found between IL1B and HDL cholesterol (a), triglyceride levels (b), and apolipoprotein B (ApoB) (c) in healthy controls, and between IL1B and apolipoprotein A1 (ApoA1) (d) in patients with CAD prior treatment with PCSK9 inhibitors. Scatter plots are shown with the results of Spearman correlation analysis (* p < 0.05; ρ, Spearman’s correlation coefficient). Gene expression data are normalized to geometric mean of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L13a (RPL13A).

Figure 4

Significant correlations between SERPINE expression and lipid parameters in CAD patients following PCSK9 inhibitors treatment. Significant correlations were found between SERPINE and LDL cholesterol (a) and apolipoprotein B (ApoB) (b) in patients with CAD after six months of PCSK9 inhibitors treatment. Scatter plots are shown with the results of Spearman correlation analysis (* p < 0.05; ρ, Spearman’s correlation coefficient). Gene expression data are normalized to geometric mean of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L13a (RPL13A).

Figure 5

Comparison of the gene expression profile before and after treatment with PCSK9 inhibitors in patients with CAD. Significant differences were obtained for VEGFA, F3 and SERPINE as indicated (*** p < 0.0001, ** p < 0.001, * p < 0.05, Wilcoxon signed rank test). Shown are box and whisker plots with individual values. Gene expression data are normalized to geometric mean of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L13a (RPL13A).