Relationships between Indicators of Lower Extremity Artery Disease and miRNA Expression in Peripheral Blood Mononuclear Cells

Abstract

Lower extremity artery disease (LEAD) is an underdiagnosed and globally underestimated vascular disease caused by the progressive and chronic formation of atherosclerotic plaques in the arteries of the lower limbs. Much evidence indicates that the abnormal course of pathophysiological processes underlying LEAD development is associated with altered miRNA modulatory function. In the presented study, relationships between miRNA expression and clinical indicators of this disease (ABI, claudication distance, length of arterial occlusion, Rutherford category, and plaque localization) were identified. MiRNA expression profiles were obtained using next-generation sequencing in peripheral blood mononuclear cells (PBMCs) of 40 LEAD patients. Correlation analysis performed using the Spearman rank correlation test revealed miRNAs related to ABI, claudication distance, and length of arterial occlusion. In the DESeq2 analysis, five miRNAs were found to be dysregulated in patients with Rutherford category 3 compared to patients with Rutherford category 2. No miRNAs were found to be differentially expressed between patients with different plaque localizations. Functional analysis performed using the miRNet 2.0 website tool determined associations of selected miRNAs with processes underlying vascular pathology, such as vascular smooth muscle cell differentiation, endothelial cell apoptosis, response to hypoxia, inflammation, lipid metabolism, and circadian rhythm. The most enriched functional terms for genes targeted by associated miRNAs were linked to regulation of the cell cycle, regulation of the transcription process, and nuclear cellular compartment. In conclusion, dysregulations of miRNA expression in PBMCs of patients with LEAD are indicative of the disease and could potentially be used in the prediction of LEAD progression.

Keywords: ankle brachial index; atherosclerosis; claudication; lower extremity artery disease; miRNA; miRNA expression; next generation sequencing; peripheral arterial disease.

Figure 1

Scatter plots depicting correlations between 19 selected miRNAs and clinical parameters of LEAD progression including (A) ankle brachial index (ABI), (B) claudication distance, and (C) length of arterial occlusion. Blue lines indicate the trend line (fitting line of the simple linear regression model).

Figure 2

Boxplots presenting normalized expression of 5 selected miRNAs differentially expressed in patients diagnosed with Rutherford category 3 (Category 3) versus patients diagnosed with Rutherford category 2 (Category 2). In the boxplots, whiskers reach extreme samples inside the 1.5 inter-quartile range, boxes range between 25% and 75% quartile, and horizontal lines inside boxes mark median values.

Figure 3

The functional network for selected miRNA sets associated with ABI (ankle brachial index), claudication distance, length of arterial occlusion and Rutherford category in studied group patients with LEAD. The network contains up to 10 the most enriched functional terms disclosed for each miRNA set along with associated miRNAs. Functional relations were obtained from the miRNet 2.0 tool using “miRNA Function” category. p value—statistical significance of enrichment.

Figure 4

Results of the functional analysis performed using the miRNet 2.0 online platform for genes regulated by 24 miRNAs associated with the studied indicators of LEAD. Up to the top 10 most enriched terms of Gene Ontology Biological Processing (GOBP), Gene Ontology Cellular Compartment (GOCC), Gene Ontology Molecular Function (GOMF), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome categories were presented. p value—p value for enrichment after adjustment for false discovery rate (FDR), the thick black vertical line represents the p = 0.05 threshold. The numbers in brackets following the name of the terms indicate the number of associated genes.