Metabolomics unveils the exacerbating role of arachidonic acid metabolism in atherosclerosis

Abstract

Atherosclerosis is a complex vascular disorder characterized by the deposition of lipids, inflammatory cascades, and plaque formation in arterial walls. A thorough understanding of its causes and progression is necessary to develop effective diagnostic and therapeutic strategies. Recent breakthroughs in metabolomics have provided valuable insights into the molecular mechanisms and genetic factors involved in atherosclerosis, leading to innovative approaches for preventing and treating the disease. In our study, we analyzed clinical serum samples from both atherosclerosis patients and animal models using laser desorption ionization mass spectrometry. By employing methods such as orthogonal partial least-squares discrimination analysis (OPLS-DA), heatmaps, and volcano plots, we can accurately classify atherosclerosis (AUC = 0.892) and identify key molecules associated with the disease. Specifically, we observed elevated levels of arachidonic acid and its metabolite, leukotriene B4, in atherosclerosis. By inhibiting arachidonic acid and monitoring its downstream metabolites, we discovered the crucial role of this metabolic pathway in regulating atherosclerosis. Metabolomic research provides detailed insights into the metabolic networks involved in atherosclerosis development and reveals the close connection between abnormal metabolism and the disease. These studies offer new possibilities for precise diagnosis, treatment, and monitoring of disease progression, as well as evaluating the effectiveness of therapeutic interventions.

Keywords: arachidonic acid; atherosclerosis; exacerbating role; metabolism; metabolomics.

FIGURE 1

Schematic illustration of metabolomics unveils the exacerbating role of arachidonic acid metabolism in atherosclerosis. (A) Collection of serum samples derived from patients with atherosclerosis and serum obtained from an atherosclerosis model constructed using high-fat-fed APOE mice. (B) Elucidation of the mechanism of atherosclerosis through metabolomics analysis, depicted in a flowchart. (C) Illustration of the synthesis and detection of the metabolomics matrix on atherosclerosis investigation. (D) Post-metabolomics analysis, encompassing discrimination, heatmap generation, volcano plot visualization, and enrichment analysis.

FIGURE 2

Classification efficacy of clinical samples. Utilizing (A) principal component analysis (PCA) and (B) orthogonal projections to latent structures discriminant analysis (OPLS-DA), metabolic profiling was performed on serum samples from 38 individuals with atherosclerosis and 35 healthy subjects, yielding classification plots. (C) The identified feature mz molecules were utilized to distinguish serum samples from the aforementioned groups, differentiating between the 38 individuals with atherosclerosis and the 35 healthy subjects. (D) The volcano plot exhibits upregulated and downregulated factors for serum samples from the 38 individuals with atherosclerosis and the 35 healthy subjects.

FIGURE 3

Construction and validation of the atherosclerosis model. (A) Methodology and Procedures for Establishing Atherosclerosis. (B) Masson’s Staining of the Control Group and (C) Masson’s Staining of the Atherosclerosis Group. Analysis of Oil Red O staining (D) in the control group and (E) in the atherosclerosis group.

FIGURE 4

Analysis of potential biomarkers before and after the development of atherosclerosis. (A) Western blotting analysis of P16 and P53 in samples from the atherosclerosis model and control group. (B) Comparison of P16, and (C) P53 between the atherosclerosis group and the control group. (D) Comparison of P53 mRNA expression levels between the atherosclerosis group and the control group.

FIGURE 5

Classification efficacy of serum samples from animal modelling of atherosclerosis compared to the control group. (A) Metabolic profiling of serum samples from 32 individuals with modelled atherosclerosis and 32 control subjects using principal component analysis (PCA), and (B) orthogonal projections to latent structures discriminant analysis (OPLS-DA), resulting in classification plots. (C) The identified feature mz molecules were utilized to discriminate between serum samples from the 38 individuals with atherosclerosis and 35 healthy subjects. (D) The volcano plot illustrates upregulated and downregulated factors in serum samples from the 32 individuals with modelled atherosclerosis and 32 control subjects.

FIGURE 6

Screening of biomarkers for atherosclerosis and corresponding diagnosis capability. (A) Summarizing the selected panel of biomarkers derived from the integration of metabolomics results from clinical samples and animal models, enabling discrimination between atherosclerosis and the control group with an area under the curve (AUC) of 0.892. (B) Significant elevation of arachidonic acid molecules in atherosclerosis. (C) Arachidonic acid molecules were used for discrimination between atherosclerosis and the control group, yielding an AUC value of 0.714. (D) Marked increase of leukotriene B4 molecules in atherosclerosis. (E) Leukotriene B4 molecules were used for discrimination between atherosclerosis and the control group, resulting in an AUC value of 0.684.

FIGURE 7

Analysis of disrupted metabolic pathways in atherosclerosis. (A) Enrichment analysis and (B) interconnections between the dysregulated metabolic pathways closely associated with atherosclerosis, derived from the metabolomics analysis of the aforementioned clinical biosamples and animal models.

FIGURE 8

Validation of the pivotal role of arachidonic acid metabolism disruption in atherosclerosis. (A, B) Analysis of Sirius-red staining before and after the administration of arachidonic acid inhibitor in atherosclerotic mice. (C) Analysis of plaque area before and after inhibitor administration, demonstrating effective mitigation of atherosclerosis upon inhibitor administration. (D) Significant upregulation of 5-lipoxygenase (5-LO) and LTC4 synthase (LTC4S) in the atherosclerosis group compared to the control group.