1. Introduction

Atherosclerosis (AS) is a common cardiovascular disease with a high mortality and morbidity rate due to chronic inflammatory changes caused by the accumulation of cholesterol on the walls of blood vessels over time. Atherosclerosis mainly occurs in medium and large arteries, characterized by the formation of multifocal atherosclerotic plaques on the inner wall of arteries [1–4]. Atherosclerotic plaques are mainly composed of lipids, necrotic cells, and some inflammatory factors, which are divided into stable and unstable plaques according to the stability of the plaques. The rupture of unstable atherosclerotic plaques often leads to serious consequences, including ischemic stroke and acute myocardial infarction. Some studies have found that more than 70% of fatal coronary thrombosis is caused by the rupture of unstable plaques [5, 6]. In-depth elucidation of the molecular mechanism of unstable plaque formation to achieve early recognition and intervention is of great significance to prevent severe clinical complications. However, the specific molecular mechanisms involved in the instability of atherosclerotic plaques are very complicated and have not been fully reported. It is imperative to conduct in-depth research on it.

Currently, only a few studies have reported the possible factors involved in the formation and rupture of unstable plaques. For example, Bao et al.’s study suggested that CD5L, S100A12, and CKB were involved in regulating the stability of atherosclerotic plaques [7]. Other studies reported that visfatin could act as an inflammation mediator and ultimately aggravated the instability of atherosclerotic plaques [8, 9]. Liu et al. reported the downregulation of IGFBP6 expression in unstable carotid atherosclerotic plaques and indicated that it might be an available prediction marker for vulnerable plaques [10]. Besides, a previous study has confirmed that EFEMP1, BGN, and RILP may have potential diagnostic value for atherosclerotic plaque rupture [11]. Another study suggested that the stability of atherosclerotic plaques may be related to oxidative stress of cells, and the oxidative stress caused by excessive reactive oxygen species in cells plays an important role in atherosclerosis. In carotid plaques, SIRT6 can promote the occurrence of oxidative stress through various ways, and it is obviously overexpressed in unstable carotid plaques, suggesting that the process of oxidative stress is a dangerous factor in the stability of plaques. Summarily, many studies have identified various factors that may contribute to plaque instability. However, it is worth noting that the above research and analysis on plaque instability are mostly limited to the transcriptome level. Existing studies have confirmed the difference in cell composition between ruptured and stable plaques [12–14]. Also, the underlying mechanism of macrophages and foam cells involved in atherosclerotic plaque instability and rupture has been partially characterized [15, 16]. Numerous studies have reported that monocytes play an essential role in atherosclerotic, but monocyte-associated genes’ effect on plaque instability is still not very clear [17–21]. Therefore, it is essential to explore the clinical significance and potential mechanisms of monocyte-associated genes in atherosclerotic plaque instability through the combined single-cell sequencing and transcriptomics data analysis.

In this study, we combined single-cell sequencing data analysis, differential expression analysis, and WGCNA analysis to obtain significant monocyte-associated candidate genes related to atherosclerotic plaque instability. Furthermore, we detected the expression status of the obtained candidate genes in monocytes and unstable plaques and explored its diagnostic significance. Finally, GSEA, GSVA, coexpression analysis, etc., were used to explore the underlying mechanism preliminarily.

2. Materials and Methods

3. Results

3.1. Extraction of Monocyte-Related Genes

To obtain monocyte-related genes in atherosclerotic plaques, we first integrated and quality-controlled the single-cell sequencing data from the six samples included in GSE159677. The top 1500 variable genes are shown (Figure 1(a)). Further, the normalization of single-cell sequencing data was carried out, and 15 principal components (p value < 0.05) were used for subsequent analysis (Figures 1(b) and 1(c)). This study identified 10 cell clusters using the Seurat package, and cluster 2 was classified as monocytes (Figure 1(d)). We subsequently extracted cell cluster-associated specific genes and presented the top 10 genes for each cell cluster in a heatmap (Figure 1(e)). The extracted 239 monocyte-related genes will be used in subsequent studies.

3.2. Extraction of Genes Associated with Plaque Instability Phenotype

We utilized WGCNA analysis to identify modules significantly associated with the phenotype of unstable atherosclerotic plaques. Module genes were considered to be significantly associated with plaque instability. The parameters were set as follows: (1) MergeHeightCut = 0.25, (2) scale-free R² = 0.9, (3) minimum size of module genes = 30, and (4) soft-thresholding power = 11. Ultimately, we obtained eight nongrey modules, among which the turquoise module containing 1323 genes had the strongest positive correlation with the unstable plaque phenotype (R² = 0.40, p-value = 5.3e − 52) (Figures 2(a)–2(d)). Genes included in this module will be selected for conducting further analysis.

3.3. Identification of 4 Significant Monocyte-Associated Candidate Genes Related to Plaque Instability

PCA analysis revealed significant differences in gene expression between stable and unstable plaques (Figure 3(a)). In our current study, a total of 1221 DEGs were identified from the merged dataset, which may play an important role in the development of unstable plaques (Figure 3(b)). The heatmap showed the top 20 upregulated and downregulated genes (Figure 3(c)). Only overlapping genes identified in single-cell sequencing data analysis, WGCNA analysis, and differential expression analysis will be considered significant monocyte-associated candidate genes related to plaque instability. We intersected the genes identified by the three approaches to obtain 4 overlapping genes, including CXCL3, FPR1, GK, and LST1, which will be used as candidate genes for follow-up research (Figure 3(d)).

3.4. Overexpression of Four Candidate Genes and Its Promising Diagnostic Significance

In our study, we determined the expression status of four candidate genes (CXCL3, FPR1, GK, and LST1) and corresponding diagnostic significance in unstable plaques. Our study showed that these candidate genes had moderate and above discriminative capability and could effectively discriminate between stable and unstable plaques. Besides, we detected the overexpression of the candidate genes in unstable atherosclerotic plaques in the merged dataset (Figures 4(a)–4(d)). Not only that, but we also detected their upregulated expression in monocytes (Figures 5(a)–5(d)). In conclusion, our study suggested that the expression of four candidate genes was significantly upregulated, which has a certain diagnostic value for unstable plaques.

3.5. Functional Enrichment Analysis of DEGs

We performed GO enrichment analysis and KEGG pathway analysis through the online DAVID database. GO enrichment analysis had three parts, including cellular components (CC), biological process (BP), and molecular function (MF). For BP, DEGs were mainly enriched in signal transduction, inflammatory response, immune response, etc. (Figure 6(a)). Regarding CC, DEGs were mainly enriched in the plasma membrane, integral component of membrane, etc. (Figure 6(b)). In terms of MF, DEGs were significantly enriched in protein binding, protein homodimerization activity, etc. (Figure 6(c)). In addition, KEGG pathway analysis showed that these genes were particularly related to the PI3K-Akt signaling pathway, pathways in cancer, and focal adhesion (Figure 6(e)).

3.6. Gene Set Enrichment Analysis (GSEA)

Herein, GSEA was carried out to explore underlying pathways involved in the development of unstable plaques, which may participate in the regulation of plaque instability. Our results suggest that multiple signaling pathways may be involved, including “HALLMARK_INFLAMMATORY_RESPONSE” and “HALLMARK_PI3K_AKT_MTOR_SIGNALING” (Figure 7).

3.7. Identification and Functional Analysis of Overlapping Coexpressed Genes (CEGs) of Candidate Genes

We identified 11 overlapping CEGs of the candidate genes (CXCL3, FPR1, GK, and LST1) in the merged dataset. Figure 8 shows the correlation between the candidate genes and 11 overlapping CEGs. Further, functional analysis from the GeneMANIA database suggested that these genes were closely related to regulating inflammatory responses (Figure 9(a)). From the protein interaction analysis of STRING database, we can see that there is a close relationship between related genes (Figure 9(b)).

3.9. Evaluation of Protein Interaction and Correlation Analysis of PIK3R5 Gene

We analyzed each candidate gene at the level of biological process (BP) and cellular component (CC) and got the protein similarity score of the candidate genes. In the box diagram, the line in the box indicates the median value of functional similarity, and it can be seen that the score of FPR1 is the highest (Figure 10(a)). In the correlation analysis diagram, FPR1 is positively correlated with PIK3R5 and other overlapping genes significantly (Figures 10(b)–10(e)).

4. Discussion

Atherosclerosis (AS) is an occlusive artery disease that usually occurs in middle-aged and older adults, which seriously threatens patients’ health and life quality [24, 25]. In particular, unstable atherosclerotic plaques rupture, leading to blood embolism, which can lead to a series of serious clinical consequences that can be fatal for patients [26, 27]. It is therefore critical to explore biomarkers associated with plaque instability and to take targeted interventions and preventive measures to reduce fatal clinical complications. In this study, we sought to combine single-cell sequencing data with transcriptome data to elucidate the clinical significance and potential mechanisms of monocyte-associated genes in atherosclerotic plaque instability.

As we all know, monocyte recruitment in the vascular wall plays an important role in the development of atherosclerosis [28, 29]; however, there are relatively few studies about the role of monocyte-associated genes on atherosclerosis. Our current study identified four (CXCL3, FPR1, GK, and LST1) significant monocyte-associated candidate genes related to atherosclerotic plaque instability using single-cell sequencing data analysis, WGCNA analysis, and differential expression analysis methods. We found that these candidate genes were significantly overexpressed in unstable plaques compared with stable plaques. Single-cell sequencing data analysis indicated that the expression of these genes was upregulated in monocytes. There are some other studies that support our findings. Gargalovic et al. found that CXCL3 might contribute to the inflammatory effects of oxidized1-palmitoyl-2-arachidonyl-sn-3-glycero-phosphorylcholine in the atherosclerotic lesion, which is crucial for the recruitment of monocytes [30]. Autoradiography and immunohistochemical analysis in one study has confirmed abundant FPR1 expression in atherosclerotic lesions [31]. Many studies have shown that GK is an essential enzyme in the formation of triacylgycerol [32, 33], which contributes to the development and progression of atherosclerosis [34, 35]. Furthermore, it was found that the candidate genes have the upper-middle ability to distinguish the stable and unstable plaques (AUC > 0.850 for all), which suggests that the four upregulated monocyte-associated candidate genes may be underlying biomarkers for early identification and intervention of unstable plaques.

Meanwhile, our study was also devoted to exploring the underlying mechanisms of the candidate genes in atherosclerotic plaque instability. Previous studies have reported that CXCL3, FPR1, and LST1 were closely associated with inflammation and oxidative stress, while GK played a central role in adipogenesis and gluconeogenesis [25–28]. CXCL3 is a well-known proinflammatory gene [36, 37]. FPR1 serves as a receptor that mediates oxidative stress signaling in the mammalian cell [38, 39, 40]. A study found that LST1 regulates inflammatory response in a model of inflammatory disease [41]. As for GK, it also shows a potential relationship with oxidative stress [42]. Previous studies have reported that these candidate genes were closely associated with inflammation and oxidative stress. It is consistent with our previous study that a high functional similarity was found among the candidate genes, which indicates that these candidate genes may act on several similar pathways. In our study, GO and KEGG analyses suggested that DEGs might be related to inflammation response and the PI3K-Akt signaling pathway. GSEA indicated that multiple pathways, including “HALLMARK_PI3K_AKT_MTOR_SIGNALING” and “HALLMARK_INFLAMMATORY_RESPONSE,” were excessively activated in unstable plaque. GSVA showed that these two pathways were also significantly activated in high expression samples of the candidate genes. Furthermore, coexpression analysis suggested that these candidate genes may be related to regulating inflammatory response. In conclusion, existing evidence showed that the candidate genes were likely to affect the atherosclerotic plaque instability through the inflammatory response and PI3K/Akt/mTOR signaling pathways, a well-known pathway relating to oxidative stress. Previous studies have shown that inflammation played an important role in the progression of atherosclerosis, which may be involved in driving plaque instability [43–46]. Another study showed that it could affect the initial recruitment of white blood cells to the final rupture of unstable atherosclerotic plaques [47]. In addition, numerous studies have reported its crucial role in the rupture of atherosclerotic plaque [48–51]. Similarly, our current study also indicated that the candidate genes were closely related to the excessive activation of inflammatory response in unstable plaques, which may be one of the potential mechanisms that aggravated the instability of atherosclerotic plaques. From the existing evidence, we speculate that the crosstalk between the candidate genes and PI3K/Akt/mTOR signaling pathway may activate inflammatory response leading to atherosclerotic plaque instability.

However, it was undeniable that our study conclusions may also have certain limitations due to the insufficient sample size, which required further in vivo and in vitro experimental verification.

5. Conclusions

Taken together, this study shows that the upregulated CXCL3, GK, FPR1, and LST1 may be effective biomarkers for early identification and intervention of unstable atherosclerotic plaques. Besides, we speculate that they may play a synergistic role with CEGs in regulating inflammation and PI3K/AKT/mTOR signaling pathways, thereby aggravating the instability of atherosclerotic plaques and promoting plaque rupture.

Conflicts of Interest

The authors declare that they have no competing interests.

Authors’ Contributions

YF Dai and J Liu conceived and designed the study. WT Qin, RG Liang, and YF Dai were responsible for the data analysis. WT Qin and F Gan were responsible for writing the draft. J Li and XM Lai made contributions to data collecting and collating. YF Dai and Jie Liu revised and proofread the manuscript. All of the authors have reviewed and approved the final manuscript. Wentao Qin, Fu Gan, and Riguan Liang contributed equally to this work.