Atherosclerosis (AS) is a chronic inflammatory disease leading to cardiovascular disease (CVD), the leading cause of morbidity and mortality globally. AS involves lipid and fibrous material deposition in arterial vessels, forming atherosclerotic plaques (APs) consisting of a fibrous cap and a lipid-rich necrotic core. AP progression leads to plaque instability and vulnerability, increasing the risk of rupture and subsequent thromboembolism, a primary cause of myocardial infarction (MI). Intraplaque hemorrhage (IPH) is a key feature of unstable plaques. While some plaques remain stable, others progress to become unstable and prone to rupture, causing severe clinical consequences. Recent high-throughput gene profiling studies have advanced our understanding of plaque pathogenesis, but identifying reliable tools to predict rupture risk remains crucial for clinical prevention. This study aimed to identify gene signatures and molecular mechanisms involved in AP progression and instability to identify novel biomarkers and therapeutic targets for AP rupture. The researchers hypothesized that specific gene expression patterns and pathways are associated with plaque progression and rupture, paving the way for novel diagnostic and therapeutic strategies.

The introduction extensively cites relevant literature establishing the global burden of atherosclerosis and its link to acute cardiovascular events. It highlights the role of plaque rupture and intraplaque hemorrhage in triggering these events. Several studies are cited detailing the pathological features of vulnerable plaques and the slow, asymptomatic progression of atherosclerosis, leading to sudden and severe clinical manifestations when rupture occurs. The review mentions prior research utilizing genomic expression studies to identify genes and pathways involved in atherosclerosis progression. This sets the stage for the current study's approach of integrating multiple datasets to identify key genes and pathways related to plaque rupture.

This study employed a multi-stage bioinformatics approach using publicly available microarray datasets from the Gene Expression Omnibus (GEO) database. Three datasets (GSE163154, GSE28829, and GSE41571) related to advanced, high-risk, and ruptured plaques were utilized. Datasets were divided into discovery and validation cohorts. In the discovery cohort (GSE163154 and GSE28829), Weighted Gene Co-Expression Network Analysis (WGCNA) was used to identify co-expression modules correlated with advanced and high-risk plaques. The Metascape database performed functional enrichment analysis of the identified modules. The validation cohort (GSE163154 and GSE41571) underwent Differential Expression Genes (DEGs) analysis using the limma package to identify differentially expressed genes between high-risk and stable plaques. Common genes from both cohorts formed the basis for further analysis. Protein-protein interaction (PPI) network analysis was performed using Metascape and ClueGO to identify hub genes and associated pathways. A miRNA-mRNA network was constructed using miRTarBase and ENCORI databases. To validate the findings in a clinical setting, peripheral blood mononuclear cells (PBMCs) from 19 patients with plaque rupture (confirmed by optical coherence tomography, OCT) and 10 controls were analyzed via RNA sequencing. Receiver operating characteristic (ROC) curve analysis assessed the diagnostic accuracy of hub genes. The expression of key genes was also validated in ox-LDL induced foam cells.

WGCNA analysis of the discovery cohort revealed the brown module in GSE28829 and the turquoise module in GSE163154 as significantly associated with advanced and IPH plaques, respectively. Functional enrichment analysis pointed to "Neutrophil degranulation" as the most significantly enriched pathway. This was validated in the DEGs analysis of the validation cohort. The intersection of genes identified in both cohorts yielded 16 hub genes clustered into three densely connected networks via PPI analysis. The "Neutrophil degranulation" pathway was consistently highlighted across analyses. A miRNA-mRNA network implicated hsa-miR-665 and hsa-miR-512-3p in regulating this pathway via PLAU and SIRPA. RNA sequencing of PBMCs from patients with plaque rupture showed significantly increased expression of PLAUR, FCER1G, PLAU, ITGB2, and SLC2A5 compared to controls. ROC analysis revealed that PLAUR, FCER1G, and PLAU had strong diagnostic power (AUC ≥ 0.8) for distinguishing patients with plaque rupture from controls, further validated using the GSE66360 dataset. In vitro experiments showed increased expression of PLAUR, FCER1G, PLAU, ITGB2, and SLC2A5 in ox-LDL-induced foam cells, supporting their role in atherogenesis.

The study's findings strongly implicate neutrophil degranulation in AP progression and rupture. Neutrophils, key players in the innate immune response, release proteins upon activation that contribute to plaque instability and rupture through mechanisms such as NET formation. The consistent identification of this pathway across multiple analyses strengthens the findings. The identification of hub genes, particularly PLAUR, FCER1G, and PLAU, provides potential new biomarkers for early diagnosis of plaque instability and rupture. PLAUR and PLAU's involvement in cell surface plasminogen activation and extracellular matrix degradation supports their role in plaque destabilization. While the role of FCER1G in AS requires further research, its association with neutrophil degranulation suggests a significant role in plaque instability. The strong diagnostic performance of these genes in both the study's RNA sequencing data and the external validation dataset (GSE66360) highlights their potential as valuable clinical biomarkers. The identification of miRNAs regulating key hub genes offers potential targets for therapeutic intervention.

This study comprehensively analyzed gene expression profiles in unstable and ruptured atherosclerotic plaques, identifying the neutrophil degranulation pathway as a key driver of plaque progression and instability. Three hub genes—PLAUR, FCER1G, and PLAU—emerged as promising biomarkers for early diagnosis of plaque rupture. Further research is needed to validate these findings in larger cohorts and explore the therapeutic potential of targeting these genes and pathways for preventing or treating atherosclerotic plaque rupture.

The study relied on bioinformatics analysis of publicly available datasets. While the findings were validated in a smaller clinical cohort, further research with larger, prospective studies is necessary to confirm the diagnostic accuracy of the identified biomarkers. The study primarily focused on gene expression and did not directly investigate the functional roles of the identified genes and pathways in plaque rupture. The study is limited by the nature of the datasets used, which may not completely capture the complexity of plaque development and rupture in vivo. Further validation in diverse populations is needed to assess the generalizability of the findings.