Introducing Circulating Vasculature-Related Transcripts as Biomarkers in Coronary Artery Disease

Abstract

Background: Atherosclerotic plaque is considered the hallmark of atherosclerotic lesions in coronary atherosclerosis (CAS), the primary pathogenesis in coronary artery disease (CAD), which develops and progresses through a complex interplay between immune cells, vascular cells, and endothelial shear stresses. Early diagnosis of CAS is critical for avoiding plaque rupture and sudden death. Therefore, identifying new CAD biomarkers linked to vessel wall functions, such as RNA molecules with their distinct signature, is a promising development for these patients. With this rationale, the present study investigated the expression level of the vascular-related RNA transcripts (lncRNA ANRIL, miRNA-126-5p, CDK4, CDK6, TGF-β, E-cadherin, and TNF-α) implicated in the cellular vascular function, proliferation, and inflammatory processes.

Methods: A case-control study design with a total of 180 subjects classified participants into two groups; CAD and control groups. The relative expression levels of the seven transcripts under study-selected using online bioinformatics tools and current literature-were assessed in the plasma of all study participants using RT-qPCR. Their predictive significance testing, scoring of disease prioritization, enrichment analysis, and the miRNA-mRNA regulatory network was investigated.

Results: The relative expression levels of all seven of the circulating vascular-related transcripts under study were statistically significant between CAD patients and controls. Receiver operating characteristic (ROC) analysis results indicated the statistical significance of all the transcripts under study with CDK4 showing the highest area under the curve (AUC) equivalent to 0.91, followed by E-cadherin (0.90), miRNA-126-5p (0.83), ANRIL (0.82), TNF-α (0.63), TGF-β (0.62), and CDK6 (0.59), in descending order. A strong association was detected between most of the transcripts studied in CAD patients with a significant Spearman's correlation coefficient with a two-tailed significance of p < 0.001. Network analysis revealed a strong relationship between the five circulating vasculature transcripts studied and their target miRNAs and miR-126-5p, but not for ANRIL.

Conclusion: The seven circulating vascular-related RNA transcripts under study could serve as potential CAD biomarkers, reflecting the cellular vascular function, proliferation, and inflammatory processes in CAD patients. Therefore, blood transcriptome analysis opens new frontiers for the non-invasive diagnosis of CAD.

Fig. 1

A heatmap illustrating the relative gene expression of the circulating vasculature-related transcripts under study. Heatmap scale from red to blue where red indicates higher expression (log2FC), and blue reflects low expression

Fig. 2

Relative gene expression of the seven circulating vasculature-related transcripts under study in CAD patients. Data are represented as median (Q1 and Q3) of log2FC in the form of whiskers and boxes. Significant p-values are red. CAD coronary artery disease

Fig. 3

Combined predictive significance by ROC curve analysis for different circulating vasculature transcripts under study. A Combined ROC curve analysis for E-cadherin, CDK4, and CDK6. B Combined ROC curve analysis for ANRIL and miR-126-5p. C Combined ROC curve analysis for TNF-α and TGF-β. ROC receiver operating characteristic

Fig. 4

Disease and prioritization scores for the transcripts under study in relation to CAD. A Disease score for the transcripts among cardiovascular diseases. Scores were calculated using GeneAnalytics tool (https://geneanalytics.genecards.org). B Prioritization score of the gene variants of the seven circulating vasculature transcripts in relation to CAD. C Average disease-causing likelihood score for the seven circulating vasculature transcripts in relation to CAD. The color scale presented from red to blue where blue reflects lower scores, and red indicates higher ones. Scores were calculated using VarElect tool (https://ve.genecards.org/). CAD coronary artery disease, CVD cardiovascular disease, Familial AA familial aortic aneurysm, Lipoprotein QTL lipoprotein quantitative trait locus

Fig. 5

Function and pathway enrichment analysis for the seven circulating vasculature-related transcripts under study according to matching score. A Top 10 biological processes. B Top 10 molecular functions. C Top 10 cellular components. D Top 10 pathways. The color scale presented from red to blue where blue reflects higher scores, and red indicates lower ones. All scores were calculated using GeneAnalytics tool (https://geneanalytics.genecards.org)

Fig. 6

Vasculature-related miRNAs-target gene network analysis for the circulating vasculature-related transcripts under study in CAD. The targets used were only the strongly validated miRNAs in the literature using ‘miRTargetLink 2.0 (https://ccb-compute.cs.uni-saarland.de/mirtargetlink2)’. A The seven circulating vasculature transcripts’ miRNA-target gene network. B The top four circulating vasculature transcripts according to ROC curve analysis miRNA-target gene network. CAD coronary artery disease, ROC receiver operating characteristic