Coronary artery disease (CAD) is a common cardiac disease and the primary cause of cardiovascular disease-related death (Agha et al., 2019; McCaffrey et al., 2021). CAD is caused by atherosclerosis or atherosclerotic occlusions of the coronary arteries (Thomas et al., 2018). Numerous environmental and genetic variables, including age, smoking habit, hypertension, dyslipidemia, obesity, diabetes, and family history, contribute to CAD (Chiu et al., 2018; Madhavan et al., 2018; Zhang H. W. et al., 2020). According to the CAD prediction model, approximately 20 million deaths and 16 million cases of reduced worker productivity were attributable to CAD in China between 2010 and 2015. Similar numbers are expected between 2000 and 2029 (Moran et al., 2008). The prevalence of CAD continues to increase, and clinical outcomes remain unsatisfactory. Although several studies have reported remarkable progress in the identification of diagnostic biomarkers for CAD in the blood (including long noncoding RNAs, methylation, and mRNAs) (Wang et al., 2020; Zhang X. et al., 2020; Chen Z. et al., 2021; Yang and Xu, 2021; Zhang L. et al., 2021) only two studies have described clinical heterogeneity in patients with different CAD severities (Zhang X. et al., 2020; Zhang B. et al., 2021). Therefore, scientific classification and targeted treatment may facilitate effective management of CAD. Current categorization approaches are based on pathological characteristics, disease development, and clinical symptoms, and the use of genotypic subgroups is developing slowly (Davies et al., 2021). The development of gene chip makes it possible to study the occurrence and development of CAD at the gene level (Franchini, 2016; Musunuru and Kathiresan, 2019).

At present, a review has reported forms of cell death that could affect CAD risk, such as apoptosis, pyroptosis, parthanatos, and autophagy, which have been previously implicated in CAD pathogenesis (Del Re et al., 2019). Additional, both ferroptosis and necroptosis are also involved in the development of CAD (Del Re et al., 2019). At present, Zhou et al.( 2021) has reported the specific mechanism of ferroptosis combined with pyroptosis in coronary atherosclerosis, and screened important marker genes. However, there is no study on ferroptosis combined with necroptosis in CAD. Ferroptosis, a novel type of cell death discovered in the last few years, is accompanied by accumulation of large amounts of iron and lipid peroxidation during the cell death process (Li et al., 2020). Furthermore, ferroptosis is an independent process triggered by the presence of harmful lipid reactive oxygen species and the consumption of polyunsaturated fatty acids (Li et al., 2020); these features distinguish ferroptosis from apoptosis, necrosis, and autophagy. Bai et al. (2020) fed apolipoprotein E (ApoE)^−/− mice a high-fat diet (HFD) to induce atherosclerosis in the presence or absence of the widely utilized ferroptosis inhibitor Ferrostatin-1 (Fer-1) and showed that Fer-1 treatment dramatically reduced atherosclerotic lesions, suggesting that ferroptosis occurs in mice with HFD-induced atherosclerosis. Further in vitro investigations using mouse aortic endothelial cells indicated that endothelial dysfunction significantly contributes to ferroptosis in the setting of atherosclerosis. Necroptosis is another newly defined form of cell death similar to necrosis and apoptosis in terms of morphology (e.g., cell swelling and rupture) (Vandenabeele et al., 2010). The necrotic features of necroptosis are marked by cell rupture and the release of immunogenic intracellular components, which activate inflammatory responses, highlighting the pro-inflammatory features of necroptosis. Vandenabeele et al. (2010) found that lipid peroxidation enhances oxidized low-density lipoprotein (ox-LDL) buildup and necroptosis. Moreover, the powerful pro-inflammatory effects of necroptosis can cause atherosclerosis. Therefore, necroptosis and ferroptosis inhibitors are promising strategies for targeting atherosclerosis, oxidative stress, and inflammatory responses. Initially, apoptosis, autophagy, and necrosis were thought to be mutually exclusive. However, recent studies have shown a delicate balance among these modes of death, suggesting that inhibition of one mode of death may increase the sensitivity of cells to activation of the other mode of death (Chen et al., 2018).

As gene chip analysis becomes more affordable, more researchers are using this approach to uncover the molecular pathways underlying the development and progression of CAD. However, most studies have only evaluated differences between CAD cases and normal controls, ignoring variances within CAD cases. Tumor samples are frequently subtyped in cancer research based on the expression patterns of ferroptosis- (Shan et al., 2021), pyroptosis- (Shao et al., 2021), DNA methylation- (Ding et al., 2019), and necroptosis-related genes (Zhao et al., 2021); such analyses can reveal intertumor heterogeneity, predict clinical endpoints, and guide treatment.

Therefore, in this study, we aimed to elucidate the mechanisms through which ferroptosis- and necroptosis-related genes contribute to CAD progression by typing CAD cases according to these genes. We also investigated the correlations between the typing results and clinical characteristics.

Figure 1 depicts the procedures used in our cohort investigation. Supplementary Table S2 lists feature information for the training datasets (GSE12288, GSE20680, and GSE20681) and validation dataset (GSE180083).

Globally, CAD is a leading cause of death, with mortality predicted to reach 23.6 million by 2030 (Zhang L. et al., 2021). CAD is caused by various genetic and environmental factors, and the cumulative influence of these variables is critical. In this study, we investigated gene expression profiles from three GEO datasets of patients with CAD and normal controls. Additionally, we effectively divided the 352 patients with CAD into two clusters (clusters A and B) for the first time based on the expression of ferroptosis- and necroptosis-related DEGs. Further research identified functional modules or pathways unique to subgroups as a result of the categorization. Subsequently, we correctly categorized the 352 patients with CAD into gene clusters A and B for the second time based on the expression of four subgroup-specific genes. There was a significant correlation between clinical features and categorization. Moreover, when compared with the two gene clusters, patients in gene cluster A had a higher CAD index and were older, suggesting that gene cluster A was associated with more severe CAD. The typing results suggested that ferroptosis and necroptosis may play critical roles in CAD. Taken together, our results showed that categorization of patients with CAD was highly correlated with clinical features and certain functional modules or pathways.

Earlier studies have only explored the heterogeneity between subtypes (Peng et al., 2019; Zhang B. et al., 2021) and have not evaluated the molecular mechanisms responsible for this heterogeneity. The current study is the first to identify differences between subtypes based on ferroptosis- and necroptosis-related genes and to explore the associations between specific pathways and clinical characteristics in individuals with CAD within specific subgroups. We further performed functional enrichment analysis of the two clusters and found that the upregulated genes in cluster A were mainly enriched in neutrophil-related biological processes. According to current research, neutrophils are short-lived phagocytic cells expressing a wide range of physiologically active enzymes, including myeloperoxidases and proteinases. Leukocytosis and neutrophilia are independent risk factors for CAD. Additionally, C-X-C chemokine motif receptor 4 (CXCR4) and its ligand C-X-C chemokine ligand 12 play roles in neutrophil egress from the bone marrow and control neutrophil recruitment to atherosclerotic lesions (Zernecke et al., 2008). Chronic CXCR4 inhibition induces neutrophilia and increases the number of neutrophils in plaques, both of which are linked to apoptosis and a pro-inflammatory phenotype, suggesting that neutrophils may have pro-inflammatory roles in atherosclerosis (Zernecke et al., 2008). Furthermore, we found that cluster A was mainly related to neutrophils by contributing to the inflammatory response in atherosclerosis, suggesting that neutrophil-related pathways, which lead to further inflammatory infiltration, could explain the differences in CAD indices between the clusters. Neutrophil activation is involved in the immunological response, neutrophil-mediated immunity, and atherosclerosis development. In this study, we observed associations among older age, neutrophil pathway activation, and disease severity in cluster A, indicating that an aberrant neutrophil pathway in older patients with CAD may result in severe disease. In addition, upregulated genes in cluster B were mainly enriched in the B-cell receptor signaling pathway and antigen receptor-mediated signaling pathway. B cells were initially detected inside the adventitia in atherosclerotic lesions, and immunoglobulin-positive cells were identified within atherosclerotic plaques (Galkina and Ley, 2007a). The functions of B cells in mediating the immune response in atherosclerosis have recently been investigated. Splenectomy was shown to exacerbate atherosclerosis in ApoE^−/− mice, accompanied by a decrease in anti-ox-LDL antibody levels. Adoptive transfer of splenic B cells from atherosclerosis-prone ApoE^−/− animals into young ApoE^−/− recipients was also shown to prevent atherosclerosis (Caligiuri et al., 2002). These findings suggest that atheroprotective immunity may expand during the development of atherosclerosis and that B cells or their immunoglobulin products may play protective roles. From these results, we inferred that the severity of CAD was lower in individuals in cluster B than in those in cluster A, mainly because of the protective effects of B cells. In addition, the typing results showed that the main genes highly expressed in cluster A were ferroptosis-related genes (e.g., NCF2, CBS, FTL, MAP3K5, MAPK14, STAT3, PGD, and TLR4); thus, ferroptosis was closely associated with the progression of CAD. Current research indicates that ferroptosis and necroptosis differ in morphological characteristics, developmental steps, and key regulators, inducers, and inhibitors. Nonetheless, accumulating evidence suggests that significant cross-talk exists between ferroptosis and necroptosis (Fearns et al., 2006; Xu et al., 2017; Maher et al., 2018; Wang et al., 2018; Proneth and Conrad, 2019; Zhou et al., 2020; Chen C. et al., 2021). In this research, although the last significant genes identified in the analysis were all related to ferroptosis, it cannot be ruled out that necroptosis plays a similar role. The mechanism of the interaction between necroptosis and ferroptosis in CAD should be further investigated. Elucidating this mechanism could provide new perspectives to support advances in CAD treatment.

In addition, with additional exploration of the mechanisms of CAD, monocytes (Lessner et al., 2002), macrophages (Stoneman et al., 2007), mast cells (Bot et al., 2007), neutrophils (Zernecke et al., 2008), T cells (Galkina and Ley, 2007b), natural killer cells (Schiller et al., 2002), and dendritic cells (Bobryshev and Lord, 1995) have been found to be closely associated with the development and progression of CAD. According to the results of our analysis by ssGSEA, the proportion of most immune cells in cluster A was significantly increased, which further explained the severity of the CAD disease in cluster A and also further confirming that the severity of CAD may be associated with degree of immune cell infiltration. Furthermore, by studying the correlations of ferroptosis- and necroptosis-related DEGs with immune cells, we were surprised to find high expression in cluster A and that all DEGs were positively correlated with the aforementioned immune cells and functioned to promote the infiltration of various immune cells. Therefore, we further confirmed the important roles of ferroptosis and necroptosis in CAD progression as reported in Dominic (Del Re et al., 2019).

In this study, we next evaluated DEGs among different subgroups and found that all four significant genes were ferroptosis-related genes. To further validate the roles of these four genes in CAD progression, we typed the four genes and found that the results of typing, clinical features, and immune cell differences between typing were highly similar to the previous typing results, demonstrating the significant roles of these four genes in CAD progression. LONP1 is an important mitochondrial target that is regulated by lipid-induced protein kinase R-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α signaling in macrophages and in lesions (Tian et al., 2011; Crewe et al., 2017). Acute cell stresses, such as hypoxia, oxidative stress, food restriction, and the unfolded protein response at the endoplasmic reticulum, have been found to upregulate LonP1 (Hori et al., 2002; Sepuri et al., 2017). Venkatesh et al. (Venkatesh et al., 2019) showed that LonP1 is an endogenous cardioprotective mediator. Furthermore, overexpression of LonP1 protects the heart from injury by limiting oxidative damage to proteins and lipids, maintaining mitochondrial redox equilibrium, and reprogramming bioenergetics by decreasing complex I content and activity. Mechanisms that enhance LonP1 expression may protect the myocardium from cardiac stress and ischemia/reperfusion damage. Thus, the abovementioned results demonstrate that upregulation of LONP1 may inhibit the progression of CAD. HSPB1 is a well-known small heat shock protein that works as an oligomer and phosphorylated dimer (Mohammad et al., 2018). In rat cardiomyocytes, HSPB1 improves the reductive activity of endogenous glutathione (GSH) reductase/GSH/GSH peroxidase and thioredoxin/peroxiredoxin antioxidant systems, confirming its role in protein oxidation resistance (Liu et al., 2019). Kraemer et al. showed that HSPB1 is upregulated and phosphorylated in ST-elevation myocardial infarction platelets. Additionally, expression of HSPB1 in cardiomyocytes is necessary for wound healing after myocardial infarction, suggesting that this protein may be a target for enhancement of repair after myocardial infarction (Wang et al., 2019). Notably, HSPB1 generates homologous oxidized HSPB1 as a result of its own unique cysteine and subsequently acts as an antioxidant in vitro (Rajagopal et al., 2015). Thus, the abovementioned results also demonstrate that upregulation of HSPB1 may inhibit the progression of CAD. The results of our study showed that LONP1 and HSPB1 were significantly upregulated in cluster B and that the severity of CAD was lower in cluster B than in cluster A, further demonstrating the protective roles of LONP1 and HSPB1 in the progression of CAD. However, this requires our follow-up further experiments to confirm the specific protective mechanism of these two genes in CAD progression.

Interestingly, our analysis also found two genes that were significantly elevated in CAD samples, CBS and TLR4. CBS is a critical enzyme involved in the trans-sulfuration pathway via catabolism of homocysteine (Hcy). Mutations in CBS have been found in homocystinuric individuals and are related to thrombosis and increased plasma Hcy levels (Gaustadnes et al., 2000). Furthermore, Hcy increases atherogenesis by promoting vascular smooth muscle cell proliferation (Tsai et al., 1996), limiting endothelial cell growth and re-endothelialization after damage, decreasing endothelial relaxation, accelerating neointimal formation (Wang et al., 1997; Wang et al., 2002), and reducing high-density lipoprotein production (Liao et al., 2006). The immunological response elicited by Hcy is intricately associated with cardiovascular disease. Thus, upregulation of CBS promotes the onset and progression of CAD. TLR4 is a member of the Toll-like receptor (TLR) family and is involved in the progression of atherosclerosis, including monocyte activation, endothelial cell injury, vascular smooth muscle cell fibrosis, and macrophage and foam cell production (Xu et al., 2001). TLR4 expression is higher in CAD plaques than in controls (Edfeldt et al., 2002). In hypercholesterolemic ApoE^−/− mice, TLR4 knockout reduces lesion size, lipid content, and macrophage infiltration (Michelsen et al., 2004; Choi et al., 2009). Additionally, in diabetic ApoE^−/− mice, administration of TLR4 antagonist reduces atherosclerotic lesions, blocks inflammatory molecule production, and decreases monocyte and macrophage content (Lu et al., 2013). Under lipid-rich conditions, endogenous TLR4 ligands activate monocytes and macrophages, causing widespread membrane ruffling, macropinocytosis, lipoprotein uptake, and foam cell production (Howell et al., 2011; Liu et al., 2012). The TLR4 signaling pathway is a potential anti-inflammatory and anti-atherosclerosis target. Importantly, TLR4 expression has been reported to be strongly correlated with the severity of CAD, as reflected by the number of coronary stenoses, and may be a clinically useful biomarker of the risk of cardiovascular disease (Shao et al., 2014). Thus, upregulation of TLR4 is involved in promoting the onset and progression of CAD. Taken together, these findings suggested that TLR4 and CBS play key roles in the development of atherosclerosis. Therefore, blocking TLR4 and CBS signaling may be beneficial in the treatment of CAD. In addition, PCA demonstrated that the subgroup-specific gene scores in cluster A or gene cluster A was higher than that in cluster B or gene cluster B. Finally, we found that the subgroup-specific upregulated genes CBS and TLR4 were significantly upregulated in the disease group in the GSE180083 dataset and had very high diagnostic efficacy. Therefore, regarding the role of CBS and TLR4 in the progression of CAD, combined with the above-mentioned related research reports and the results of this study, it is confirmed that the two play an important role in the progression of CAD. And further study of their mechanism of action will provide favorable therapeutic targets for alleviating CAD progression.

Although our findings identified potential subgroups of patients with CAD based on molecular analyses of ferroptosis- and necroptosis-related genes and revealed the characteristics of each subtype, gene expression changes are not necessarily genetically driven or could be partially genetic and partially environmental. Additional, several limitations should be acknowledged. First, some clinical follow-up information was not available for the samples; therefore, we did not consider several important factors, such as the presence of patient comorbidities, when distinguishing among subgroups. Second, the consistency of the CAD subgroups should be confirmed using further analyses. Finally, the results were acquired only through bioinformatics analysis, and the results should be confirmed in further experiments.

High expression of CBS and TLR4 in CAD was associated with increased disease severity and may be promising diagnostic markers of CAD. By contrast, LONP1 and HSPB1 may delay CAD progression. The identification of genetic subgroups of patients with CAD has improved our understanding of the pathogenesis of CAD and has facilitated the development of potential methods for disease diagnosis, classification, and prognosis evaluation.