Mutual mediation effects of homocysteine and PCSK9 on coronary lesion severity in patients with acute coronary syndrome: interplay with inflammatory and lipid markers

Abstract

Background

Homocysteine (Hcy) and the proprotein convertase subtilisin/kexin type 9 (PCSK9) significantly contribute to atherosclerosis (AS) as well as coronary lesion severity. Our previous work demonstrated that Hcy upregulates PCSK9, accelerating lipid accumulation and AS. A PCSK9 antagonist reduces plasma Hcy levels in ApoE^−/− mice. These findings suggest complex roles for both Hcy and PCSK9 in AS. This study investigated the mutual mediating influence of Hcy together with PCSK9 on coronary lesion severity among individuals diagnosed with acute coronary syndrome (ACS), focusing on their interplay with inflammatory and lipid-related markers.

Methods

This cross-sectional study encompassed 617 individuals diagnosed with ACS. Baseline characteristics, including inflammatory and lipid-related markers, were compared between individuals with non-severe (SYNTAX score ≤ 22) and severe (SYNTAX score > 22) coronary lesions. To evaluate both the impacts of Hcy and PCSK9 on coronary lesions severity, multivariate logistic regression along with mediation analyses were utilized. The robustness of the findings was validated by conducting subgroup analyses and sensitivity tests.

Results

Patients with severe conditions showed higher levels of Hcy, PCSK9, and inflammatory markers compared to non-severe cases. Both Hcy and PCSK9 levels were independently linked to a heightened risk of severe coronary lesions(ORs: 1.03–1.04 and 1.01–1.02, respectively, all P < 0.001). PCSK9 mediated 34.04% of Hcy’s effect on coronary lesion severity, whereas Hcy mediated 31.39% of PCSK9’s effect, indicating significant mutual mediation between these biomarkers. Subgroup analyses revealed consistent associations, with notable interactions based on creatinine levels for Hcy and gender, smoking status, and diagnosis for PCSK9. Sensitivity analyses confirmed the robustness of the mediation effects.

Conclusions

These findings emphasize the mutual mediating effects of Hcy and PCSK9 on coronary lesion severity in patients suffering from ACS. These results highlight the complex interactions between lipid metabolism and inflammation in the pathophysiology of ACS, suggesting that targeting both Hcy and PCSK9 may offer novel therapeutic strategies to mitigate severe coronary lesions among high-risk patients.

Introduction

Cardiovascular morbidity and mortality, which affect millions of people around the world, are significantly attributed to coronary artery disease (CAD) globally [1]. Among the various manifestations of CAD, acute coronary syndrome (ACS) represents the most urgent and time-sensitive medical condition, and requires immediate medical intervention to prevent irreversible myocardial damage and death [2, 3]. ACS is defined by the constriction or obstruction of coronary arteries, which is a consequence of atherosclerosis (AS), a process that leads to the formation of plaques composed of lipids, inflammatory cells, and fibrous tissue [4]. The pathogenesis of ACS involves a complex interplay of genetic factors, lifestyle, and key biological markers [5]. Among these, two key factors have gained prominence in the progression of AS and the severity of coronary lesions: homocysteine (Hcy) and proprotein convertase subtilisin/kexin type 9 (PCSK9).

An increased level of plasma Hcy (> 15µmol/L), is referred to as hyperhomocysteinemia (HHcy) [6], contribute to atherogenesis by promoting oxidative stress, impairing endothelial function, and triggering vascular inflammation [7,8,9]. A variety of investigations have established a distinct connection between elevated Hcy concentrations and increased susceptibility to CAD [10,11,12]. Nonetheless, therapies aimed at lowering Hcy levels, such as folic acid alongside vitamins B₆ and B₁₂, do not lead to a decreased risk of significant cardiovascular events [13]. The precise mechanism through which Hcy affects the severity of coronary lesions has not been fully elucidated, suggesting that its impact could be mediated through alternative biochemical pathways.

PCSK9, a serine protease, has gained attention because of its involvement in cholesterol regulation. This protein binds with low-density lipoprotein receptor (LDLR) present on hepatocyte surfaces, promoting their degradation. Consequently, this process results in LDL-cholesterol (LDL-C) elevated [14]. Elevated PCSK9 concentrations have been linked to a greater vulnerability to CAD, primarily due to its effect on lipid metabolism [15, 16]. Recent studies suggest that PCSK9 might also exert proatherogenic effects separate from its role in lipid metabolism, such as promoting inflammation and vascular calcification [17,18,19,20]. PCSK9 inhibitors have shown substantial promise in lowering LDL-C, stabilizing plaques, and improving cardiovascular outcomes [21, 22], providing a new avenue for addressing AS associated with HHcy. Notably, our previous research revealed that Hcy upregulates PCSK9 expression in macrophages, contributing to increased lipid accumulation [23]. The interplay between PCSK9 and the liver X receptor alpha (LXRα) alongside ATP binding cassette transporter A1/G1 (ABCA1/ABCG1) pathways represents a new mechanism by which HHcy hastens the progression of AS. Interestingly, the use of SBC-115,076, a targeted antagonist of PCSK9, was capable of reducing plasma Hcy levels in ApoE^−/− mice while significantly decreasing plasma PCSK9 levels, macrophage lipid accumulation, and AS severity. These findings suggest complex roles for both Hcy and PCSK9 in AS. However, the degree to which PCSK9 mediates the impact of Hcy on the severity of coronary lesions remains to be fully elucidated, necessitating further investigation into their mutual interaction and mediation.

In addition to Hcy and PCSK9, various inflammatory indicators, such as white blood cells (WBCs), neutrophils, and C-reactive protein (CRP), are integral to the progression of ACS. These markers indicate systemic inflammation, a well-established driver of AS and plaque instability [24, 25]. Lipid-related markers, including LDL-C, LDLR, and high-density lipoprotein cholesterol (HDL-C), are significantly involved in the onset and advancement of ACS [26]. However, the mediating effects of Hcy and PCSK9 on coronary severity and the interplay between these inflammatory and lipid-related factors and Hcy and PCSK9 in determining coronary lesion severity have yet to be fully explored [27, 28].

Given the established roles of Hcy and PCSK9 in ACS, this study aimed to explore their mutual mediation effects on the extent of coronary lesions in individuals diagnosed with ACS. Specifically, we sought to ascertain whether PCSK9 mediates the influence of Hcy on severe coronary lesions and vice versa while also assessing the mediating roles of other inflammatory and lipid-related factors. To achieve this goal, we conducted a cross-sectional investigation that included patients with ACS to dissect these complex interactions. This study builds on our previous work by exploring the direct and indirect effects of Hcy and PCSK9 on coronary lesion severity, aiming to clarify their roles in ACS pathophysiology. By examining the bidirectional mediation between Hcy and PCSK9, this research fills a significant gap in the literature, providing insights that may guide targeted therapeutic strategies for reducing coronary lesion severity and improving ACS outcomes.

Materials and methods

Study design and population

This single center observational cohort study adhered to the principles outlined in the Declaration of Helsinki. Additionally, the present study obtained endorsement from Ethics Committee at the General Hospital of Ningxia Medical University (Ethics Review Number: KIIL-2022-0847). Informed consent in writing was obtained from all individuals participating in the study.

This investigation consecutively enrolled individuals who received coronary angiography (CAG) for primary symptoms of acute chest pain, with the enrolment period spanning from December 2022 to December 2023. All patients diagnosed with an ACS diagnosis, regardless of whether it was a ST-elevation myocardial infarction (STEMI), non-ST-elevation myocardial infarction (NSTEMI) or unstable angina (UA), were eligible for participation [29]. The exclusion criteria implemented were: (1) being under the 18 years old; (2) having a prior diagnosis of CAD or history of CAG; (3) taking lipid-lowering medications, specifically statins or PCSK9 inhibitors, within 6 months; (4) using medications known to influence Hcy levels within 6 months, including folic acid and vitamin B₁₂ supplements; (5) having severe valvular heart disease, cardiomyopathy, arrhythmia or pulmonary heart disease; (6) having severe liver disease, severe renal disease, recent uncontrolled infection, severe thyroid dysfunction, immune system disease, or malignancy; and (7) have incomplete clinical data or who refusing to provide informed consent. Specifically, patients with severe liver disease (e.g., AST and ALT levels > 5 times the normal range, or bilirubin > 5 mg/dL), severe renal disease (e.g., eGFR < 30 mL/min/1.73 m²), or severe thyroid dysfunction (e.g., TSH > 10 mU/L for hypothyroidism or TSH < 0.1 mU/L with elevated T3 and T4 for hyperthyroidism) were excluded due to their significant effects on lipid metabolism, inflammation, and atherosclerotic processes. Considering that statin treatment could activate the sterol regulatory element-binding protein-2 (SREBP-2), resulting in co-expression of LDLR and PCSK9 [30]. Therefore, we excluded patients who had a previous medication history of statin.

A total of 617 cases were analyzed. The degree and severity of coronary lesions in individuals diagnosed with ACS were calculated using the SYNTAX score. Higher scores indicated more severe and complex coronary lesions [31]. Patients with ACS were categorized into non-severe group (SYNTAX score ≤ 22) and severe group (SYNTAX score > 22).

Data collection

Demographic basic information, such as age, gender, and details about smoking and drinking habits, were extracted from the electronic medical records. A comprehensive medical history was collected, encompassing conditions, including hypertension, atrial fibrillation, diabetes mellitus, and stroke, along with a prior CAD diagnosis or percutaneous coronary intervention (PCI). Other baseline clinical information, including initial systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR) and left ventricular ejection fraction (LVEF) were documented.

Baseline peripheral venous blood samples for measurements of PCSK9, LDLR, IL-1β (interleukin-1-beta), IL-6, MCP-1 (monocyte chemotactic protein-1), TNF-α (tumour necrosis factor-alpha), ABCA1 and ABCG1, and were collected for analyses. These samples were immediately upon admission, before any clinical intervention and prior to CAG by centrifugation at 3000 × g for a duration of 10 min at temperature of 4 ℃. The resulting isolated plasma was subsequently preserved at -80 ℃ until needed. The plasma concentrations of total PCSK9, IL-6, ABCA1, LDLR, IL-1β, MCP-1, ABCG1, and TNF-α, were quantified by enzyme-linked immunosorbent assay (ELISA), in accordance with guidelines provided by the manufacturer (refer to Supplementary material 1).

For the measurement of other laboratory parameters, additional peripheral venous blood samples, comprising cardiac troponin I (cTnI), WBCs, neutrophils, lymphocytes, monocytes, RBCs (red blood cells), platelets, N-terminal pro-brain natriuretic peptide (NT-proBNP), creatinine, D-dimer, CRP, estimated glomerular filtration rate (eGFR), and Hcy, were obtained immediately upon admission. Other parameters, including fasting plasma glucose (FPG), alanine aminotransferase (ALT), HbA1c, aspartate aminotransferase (AST), LDL-C, HDL-C, triglycerides (TG), and total cholesterol (TC), were collected after an overnight fast of 10–12 h. Standard laboratory techniques were used to test all the samples at the medical laboratory of the hospital.

Experts who were blinded to the study protocol performed the CAG. A web-based computational tool (http://syntaxscore.com/) was utilized to determine the SYNTAX score based on the preoperative angiographic images. Multivessel lesions were defined as patients having more than two coronary arteries with ≥ 50% stenosis. The initiation and selection of medications, including statins, were determined after CAG based on each patient’s coronary lesion characteristics, clinical presentation (STEMI, NSTEMI, or UA), and other risk factors in accordance with current clinical practice guidelines.

Statistical analysis

All data were conducted utilizing Empower Stats (X&Y Solutions, Inc., Boston, MA, USA) alongside R software (version 4.2.0). In this study, all presented P values are two-tailed, with a threshold of less than 5% is considered statistically significant. The baseline characteristics were expressed as means ± standard deviation (SD) for continuous variables, while proportions were utilized for categorical variables. The chi-square test and analysis of variance were used to address significant differences within the dataset. Correlations between variables, such as age, BMI, Hcy, PCSK9 and SYNTAX score, were evaluated using Pearson’s correlation coefficients and visually represented in a heatmap. Box plots show the differences between groups with and without severe lesions in Hcy and PCSK9. In addition, the difference in the PCSK9 levels in the non-HHcy and HHcy groups and the differences in the Hcy levels in the different PCSK9 groups are expressed in box plots.

The impacts of individual variables on the probability of severe lesions were assessed through univariate logistic regression models. Multivariate logistic analysis utilizing a stepwise regression approach was conducted to adjust for potential confounding variables. The odds ratios (ORs) along with their respective 95% confidence intervals (CIs) were computed. Adjusted confounders were selected based on the results of univariate analysis, literature reports and clinical experience. Variables exhibiting a variance inflation factor (VIF) greater than 10 were excluded from the analysis aimed at examining the correlation between the Hcy levels and PCSK9 levels in relation to severe lesions in patients with ACS. Hcy was considered as a continuous variable, a bivariate variable (HHcy: Hcy > 15 µmol/L) and a categorical variable (Hcy was divided into three groups by tertiles) to assess the relationship between Hcy and the presence of severe lesions. Similarly, PCSK9 was considered as a continuous variable and as a categorical variable (PCSK9 were stratified into three groups according to tertiles divisions). Generalized additive model (GAM) was utilized to define the correlation between Hcy and PCSK9 and presence of severe lesions in individuals with ACS.

Subgroup analyses based on Model 4 were also utilized to assess other factors influencing the associations between Hcy and PCSK9 levels and severe lesions. The subgroups included gender, age, BMI, smoking status, hypertension status, diabetes mellitus status, stroke status, NYHA status, diagnosis, and creatinine and AST levels. The GAM was used to assess the relationships between Hcy levels and severe lesions with different creatinine levels and between PCSK9 levels and severe lesions in different subgroups, including gender, smoking status, and diagnosis.

In addition, mediation analysis was performed to evaluate mutual mediation influences of Hcy levels and PCSK9 levels on the risk of severe lesions. Briefly, the mediating effect of Hcy on severe lesions through PCSK9 levels was examined in various adjusted models. Moreover, the mediating effect of PCSK9 levels on severe lesions through the Hcy levels was also investigated. For sensitivity analysis, we also conducted a mediation analysis including WBCs, neutrophils, monocytes, CRP, IL-1β, LDLR, TNF-α, HDL-C, MCP-1, IL-6, ABCA1, ABCG1, and LDL-C levels to examine roles of lipid and inflammatory factors.

Results

Baseline characteristics

In this study of 617 patients with ACS, a significant difference was observed between those with severe lesions, defined by a SYNTAX score above 22, and those with non-severe lesions, with a SYNTAX score of 22 or less. The average age of the study population was 61.66 ± 12.14, with 447 (72.45%) of males. As presented in Table 1, the severe lesion group included higher proportions of male patients and smokers. These patients had lower SBP and DBP (all P < 0.05) and significantly elevated levels of cardiac biomarkers such as cTnI and NT-proBNP (all P < 0.05). Inflammatory markers, including WBCs, monocytes, neutrophils, CRP, and IL-6 were significantly greater in group with severe lesion (all P < 0.05). Among the biochemical markers, the severe lesion group exhibited elevated levels of creatinine, PCSK9, and Hcy alongside decreased levels of HDL-C (all P < 0.05).

Additionally, the baseline characteristics were stratified into two groups based on HHcy status (Table S1) and into three groups based on PCSK9 tertiles (Table S2). A greater proportion of patients with HHcy were males, had a higher BMI, had higher smoking rates, and had elevated levels of cTnI, NT-proBNP, WBCs, neutrophils, monocytes, creatinine, CRP, IL-6, and PCSK9; and had lower HDL-C levels. Similarly, those in the upper tertile of PCSK9 (tertile 3), were more likely male and to engage in smoking behaviors and presented with elevated levels of cTnI, NT-proBNP, inflammatory indicators (WBCs, neutrophils, monocytes, CRP, IL-1β, IL-6, TNFα), and lipid-related markers (LDL-C, TC), and a higher SYNTAX score.

The correlation heatmap in Fig. 1 demonstrates positive correlations between the SYNTAX score and cTnI, NT-proBNP, WBCs, neutrophils, monocytes, CRP, IL-6, PCSK9, and Hcy levels and negatively correlations with HDL-C levels. A strong positive association was identified between Hcy and PCSK9, with a correlation coefficient recorded at 0.32. Figure 2 shows that both Hcy and PCSK9 levels were markedly elevated in patients exhibiting severe coronary lesions (Panels A and B). Patients with HHcy also had elevated PCSK9 levels (Panel C), and Hcy levels were highest in the group with elevated PCSK9 (Panel D).

Table 1 Baseline characteristics of patients with ACS categorized based on the SYNTAX score

Fig. 1

Heatmap illustrating the correlations between different variables

Fig. 2

Box plots for Hcy and PCSK9 in the different groups

Associations between Hcy and PCSK9 levels and the risk of severe lesions

Results of the univariate logistic regression analysis to evaluate the significance of traditional risk factors align with potential biomarkers of lipid metabolism and inflammation are displayed in Table 2. These covariates were selected for the subsequent multivariate regression analyses. WBCs were excluded due to collinearity. Male, smoking, NYHA class ≥ II, and elevated levels of cTnI, WBCs, neutrophils, monocytes, AST, CRP, IL-6, PCSK9, and Hcy were associated with a heightened likelihood of severe coronary lesions. Conversely, higher DBP, SBP, and HDL-C exhibited an inverse relationship with this risk.

Multivariate logistic regression analyses in Table 3 depicts that Hcy levels were consistently linked to a higher probability of severe coronary lesions in all the examined models. No variables were adjusted in Model 1, with OR for Hcy was 1.04 (P < 0.001). The statistical significance of this association persisted even after accounting for multiple confounding variables in Models 2, 3, and 4, with ORs ranging from 1.03 to 1.04 (all P < 0.001). Compared to those without HHcy, individuals with HHcy faced a markedly elevated risk of severe lesions, with ORs decreasing slightly but remaining significant in all models (OR: 2.67 in Model 4, P = 0.008). Trend analysis across Hcy tertiles also revealed a strong positive association with severe lesions, with the highest tertile having an OR of 3.51 (P = 0.005) in Model 4. The analysis conducted through multivariate logistic regression analysis (Table 4) indicated a notable association between higher PCSK9 and a heightened risk of severe lesions, which was consistent across all the models. In Model 4, which was adjusted for all variables, the OR for PCSK9 was 1.02 (P < 0.001). Furthermore, individuals classified in the upper tertile of PCSK9 were more than twice as likely to have severe lesions as those in the lowest tertile were, with an OR of 2.33 (P = 0.025). Trend analysis further supported this relationship, revealing a significant increase in risk with increasing PCSK9 levels. Figure 3 illustrates a positive linear relationship between higher Hcy and PCSK9 levels and the likelihood of severe coronary lesions, as represented in a GAM. The models were adjusted for multiple covariates to ensure the robustness of these associations.

Table 2 Univariate logistic regression analysis for severe lesions in patients with ACS

Table 3 Multivariate logistic regression analysis of the associations between hcy levels and severe lesions

Table 4 Multivariate logistic regression analysis of association between PCSK9 with severe lesions

Fig. 3

Associations between Hcy and PCSK9 levels and the risk of severe lesions In a generalized additive model (GAM), linear associations were observed between the levels of Hcy (A) and PCSK9 (B) in relation to the risk of severe lesions. The solid red line illustrates the probability of experiencing severe lesions, whereas the blue dashed line represents the 95% CI. This model was adjusted for various covariates, including gender, age, smoking, BMI, diabetes mellitus, stroke, hypertension, diagnosis, NYHA, LVEF, SBP, DBP, cTnI, NT-proBNP, AST, HDL-C, and LDL-C

Subgroup analysis

Figure 4 demonstrates the robust associations of both Hcy and PCSK9 levels with the risk of severe coronary lesions, as these associations remained significant across most subgroups. However, notable interactions were observed, indicating variability in these effects based on specific patient characteristics. Hcy showed a significant interaction in the creatinine subgroups. Similarly, PCSK9 exhibited significant interactions in the gender, smoking, and diagnosis subgroups. Figure 5 shows that the correlation between levels of Hcy and severe lesions was more pronounced in the presence of lower creatinine levels (Fig. 5A). Higher PCSK9 levels were particularly linked to a heightened risk of severe lesions in males, smokers, and patients diagnosed with STEMI (Fig. 5B-D).

Fig. 4

Associations between Hcy and PCSK9 levels and the risk of severe lesions according to different subgroups. All covariates were adjusted, with the exception of the stratified variable. These factors included gender, age, smoking, BMI, diabetes mellitus, hypertension, stroke, diagnosis, NYHA, LVEF, SBP, DBP, cTnI, NT-proBNP, AST, HDL-C, and LDL-C

Fig. 5

Associations of Hcy and PCSK9 levels with the risk of severe lesions according to different subgroups. All covariates were adjusted. A: Associations of Hcy and severe lesions with different creatinine levels. B-D: Associations of PCSK9 and severe lesions according to gender, smoking and diagnosis

Mediation analysis

Figure 6 demonstrates the mutual mediating effects linking Hcy and PCSK9 to severe coronary lesions. The mediation effect of PCSK9 remained significant across all the models, with the proportion of mediation ranging from 31.38 to 43.61%. High PCSK9 significantly mediated 34.04% (P < 0.001) of correlation between greater Hcy levels and the likelihood of severe lesions in a comprehensively adjusted model. The mediation role of Hcy was also significant across all the models, with the proportion of mediation ranging from 28.69 to 34.80%. In fully adjusted Model 4, Hcy accounted for 31.39% (P < 0.001) of the association between PCSK9 and the probability of severe lesions.

The sensitivity analysis outlined the mediating influences of different variables on the link between Hcy levels and presence of severe lesions (Table 5). The analysis revealed that most inflammatory and lipid-related factors, including WBCs, neutrophils, monocytes, CRP, LDLR, TNF-α, HDL-C, IL-6, ABCA1, MCP-1, ABCG1, IL-1β, and LDL-C, were not serve as significant mediators of the correlation between Hcy and occurrence of severe lesions. Similarly, Table 6 displays the sensitivity analysis concerning the mediating influence of various indicators on the relationship between PCSK9 and severe coronary artery lesions. Among the factors analyzed, WBCs, neutrophils, monocytes, and lipid-related markers such as HDL-C and LDL-C, showed minimal and nonsignificant mediating effects. Interestingly, negative mediation effects were identified for IL-1β (P = 0.044) and TNF-α (P = 0.048), indicating potential opposing influences, but these effects were small.

Fig. 6

Mutual mediation effects of Hcy and PCSK9 on severe lesions in different models A: Mediation effects of PCSK9 on the associations between Hcy and severe lesions in the different models, B: Mediation effects of Hcy on the associations between PCSK9 and severe lesions across different models. Model 1: no adjustments made Model 2: adjusted for gender and age Model 3: adjusted for gender, age, smoking, BMI, diabetes mellitus, hypertension, stroke, diagnosis, NYHA, and LVEF. Model 4: adjusted for gender, age, BMI, smoking, hypertension, diabetes mellitus, stroke diagnosis, NYHA, LVEF, SBP, DBP, cTnI, NT-proBNP, AST, HDL-C, and LDL-C

Table 5 Mediating effects of potential mediators on the association between hcy levels and severe coronary artery lesions

Table 6 Mediating effects of potential mediators on the association between PCSK9 and severe coronary artery lesions

Discussion

The current investigation revealed that increased levels of Hcy and PCSK9 are independently correlated with the severity of coronary lesions. Furthermore, notable mutual mediation effects exist between these two factors. In addition, the elevations in inflammatory and lipid markers further support the roles of Hcy and PCSK9 in determining coronary lesion severity. Subgroup analysis revealed consistent associations across most subgroups, with particularly pronounced associations in patients with lower creatinine levels for Hcy, males, smokers and those diagnosed with STEMI for PCSK9. Mediation analysis further indicated that PCSK9 mediated 34.04% of the effect of Hcy on coronary lesions, whereas Hcy contributed to 31.39% of the influence of PCSK9. Overall, this research enhances the understanding of the intricate mechanisms driving ACS and lays a theoretical foundation for future therapeutic strategies targeting both Hcy and PCSK9.

This study identified a noteworthy correlation between Hcy and coronary lesions severity in individuals diagnosed with ACS. This finding is consistent with various studies that have established Hcy as a potential risk element for AS and cardiovascular disease [10,11,12, 32]. Moreover, coronary lesions with HHcy are often associated with greater incidence rates of vulnerable plaques, thrombosis, and wider lesion areas, combined with multivessel lesions and calcifications [33,34,35]. Elevated Hcy levels have been implicated in endothelial dysfunction, oxidative stress, inflammation and vascular calcification, which are important in the pathogenesis of CAD [7,8,9, 36]. In addition to these findings, the current investigation further supports the involvement of Hcy in exacerbating coronary lesions. A notable correlation was identified between Hcy levels and various inflammatory indicators, such as WBCs, neutrophils, CRP, monocytes, and IL-6. Additionally, Hcy levels demonstrated a positive correlation with PCSK9 and inverse relationships with ABCG1 and HDL-C. These findings indicate that Hcy related coronary lesions involving inflammation and lipid metabolic pathways.

This research diverges from earlier investigations by employing a GAM to examine the relationship between Hcy levels and severe lesions, offering a deeper understanding of this association. In addition, the present study incorporated a wider array of variables within the multivariate regression and examined Hcy in three different forms: as a continuous, a binary (HHcy), and a categorical variable defined by tertiles. This comprehensive approach sought to thoroughly examine the association between Hcy and coronary lesion severity. Interestingly, the current study uncovered a link between Hcy and coronary lesion severity was particularly pronounced in patients with lower creatinine levels. This finding contrasts alongside findings from several earlier studies [37, 38], which has suggested that the cardiovascular effects of elevated Hcy might be more significant in patients with impaired renal function, because high creatinine levels often indicate reduced renal clearance and an accumulation of Hcy in the blood. However, the findings from this study indicate a different pattern, in which patients with lower creatinine levels had a stronger association between Hcy levels and coronary lesion severity. Importantly, the cohort examined in this investigation comprised a considerable percentage of individuals with normal or slightly elevated creatinine levels, which may have influenced the observed associations. Furthermore, the effects of the chronic accumulation of Hcy on coronary lesion severity may be confounded by other factors such as uraemic toxins, which also contribute to cardiovascular risk.

A notable association has been identified between elevated PCSK9 and extent of coronary lesions in individuals experiencing ACS. These findings align with several researches that have established that PCSK9 is crucial for the regulation of lipids, contributing to AS and CAD through its regulation of LDL-C levels [15, 39, 40]. Similar to previous studies, the results in the current study demonstrated positive associations between PCSK9 levels and inflammation indicators, reinforcing the notion that the role of PCSK9 extends beyond lipid metabolism to include proinflammatory effects [41]. This expanded function of PCSK9 as a mediator of both lipid metabolism and inflammation aligns with observations from additional research, which has shown its involvement in exacerbating coronary plaque formation and instability [42]. While PCSK9 inhibitors show promising effects on LDL-C reduction and plaque stabilization, real-world evidence from the EPHESUS registry revealed significant challenges in lipid-lowering therapy adherence, with treatment discontinuation primarily attributed to negative media coverage rather than adverse effects [43]. Therefore, when evaluating the therapeutic potential of PCSK9 inhibitors, treatment adherence in real-world settings should be considered as a critical factor influencing clinical outcomes.

Furthermore, this study investigated the association of PCSK9 across different subgroups and revealed that this relationship between elevated PCSK9 levels and severe coronary lesions was more pronounced in specific populations, particularly in male patients, smokers, and patients with STEMI. This subgroup analysis has rarely been addressed in previous studies, which typically focus on the overall impact of PCSK9 without exploring potential interactions with these specific patient characteristics. Interestingly, the current study revealed that PCSK9 showed positive correlation with LDL-C, and negatively associated with ABCA1, ABCG1 and HDL-C, providing evidence that the regulatory influence of PCSK9 on cholesterol efflux biomarkers ABCA1 and ABCG1, occurs independently of the LDLR pathway [44]. These findings build on and extend previous research by highlighting the complex interplay between Hcy and PCSK9, particularly in increased lipid metabolism and AS progression. Our previous research [23] demonstrated that Hcy upregulates PCSK9 expression in macrophages, resulting in heightened lipid accumulation and the development of foam cells. This interaction disrupts lipid efflux pathways via the LXRα-ABCA1/ABCG1 axis, accelerating AS in the context of HHcy [45]. Additionally, when the PCSK9 antagonist SBC-115,076 was administrated in ApoE^−/− mice, reductions in both Hcy and PCSK9 levels were noted, indicating a bidirectional relationship between Hcy and PCSK9 [23].

This current investigation further elucidated this relationship by demonstrating significant mutual mediation effects between Hcy and PCSK9 on severe coronary lesions in several adjusted models. Specifically, the results showed that PCSK9 mediated approximately 34% of the effect of elevated Hcy on severe coronary lesions, whereas Hcy mediated approximately 31% of the effect of PCSK9. These findings suggest that the interaction between these two molecules is not only bidirectional but also serves as a pivotal function in the progression of AS. Compared with earlier studies that have centered on the independent roles of Hcy or PCSK9 in AS, this research highlights the synergistic interaction between Hcy and PCSK9, providing a more comprehensive understanding of their combined effects on coronary artery disease. Moreover, although earlier research has revealed the essential role of PCSK9 in regulating lipid metabolism, the results from this present investigation highlight the importance of inflammatory processes and markers associated with lipids, such as CRP, IL-6, ABCG1, and HDL-C, in modulating the effects of Hcy and PCSK9. However, mediation analysis revealed that these inflammatory markers had minimal mediating effects on the relationships among Hcy, PCSK9, and coronary lesion severity. These findings indicates that although inflammation is a significant part in the comprehensive pathophysiology of ACS [28, 46, 47], the associations of Hcy and PCSK9 with coronary lesions are more substantial and may operate through pathways that are not entirely dependent on systemic inflammation.

When considering the complex interplay between inflammatory and lipid markers in CAD, sex-specific variations warrant attention. Important sex-specific differences exist in the pathophysiology of CAD [48]. Women demonstrate distinct features, including smaller coronary vessel diameter and higher prevalence of microvascular dysfunction. The interaction between inflammatory markers and lipid profiles differs by sex, particularly influenced by estrogen’s effects on lipid metabolism and inflammatory pathways. Post-menopausal women experience adverse changes in lipid profiles, with increased TC and TG. Understanding these sex-specific characteristics could provide additional insights into risk stratification and therapeutic approaches for CAD patients.

In the pathophysiology of ACS, thrombotic events represent a critical process interacting with inflammation and lipid metabolism in the development of severe coronary lesions. Among various thrombosis-related biomarkers, von Willebrand Factor (VWF) has emerged as a significant indicator that bridges these pathophysiological processes [49]. Recent studies have demonstrated that VWF levels are closely associated with both inflammatory responses and lipid metabolism disorders in ACS patients. While our study focused on Hcy and PCSK9 as key risk factors for severe coronary lesions, the potential interactions between these indicators and thrombosis-related factors like VWF warrant further investigation. Future studies incorporating thrombosis-related biomarkers alongside our identified indicators may provide a more comprehensive understanding of the complex mechanisms underlying severe coronary lesions in ACS patients.

Study strengths and limitations

This research highlights the mutual mediation effects of Hcy and PCSK9 with severe coronary lesions. As anti-inflammatory strategies alone may be insufficient, combining PCSK9 inhibitors with therapies aimed at lowering Hcy levels—such as folate supplementation or vitamin B therapy may provide a more comprehensive approach to reduce the severity of coronary lesions. The dual-targeted strategy of lowering both Hcy and PCSK9 not only addresses the lipid metabolism abnormalities but also disrupts the pro-atherogenic cycle driven by their mutual mediation, offering a promising avenue for reducing AS and preventing acute coronary events in patients alongside elevated Hcy and PCSK9 levels.

The current study is subject to several limitations. Primarily, the cross-sectional framework limits the ability to establish causality between elevated Hcy and PCSK9 levels and the severe coronary lesions. Second, although this study demonstrated significant mutual mediation effects between Hcy and PCSK9, it did not fully address potential confounders, such as genetic variations or lifestyle factors, including diet and physical activity, that could influence both Hcy and PCSK9 levels. This investigation excluded individuals who had previously used lipid- or Hcy-lowering medications, and adjusted for a comprehensive set of covariates; residual confounding factors cannot be completely excluded. Third, subgroup analysis in the present study revealed pronounced effects in specific populations, such as male patients, smokers, and patients with STEMI, but the relatively small sample size within these subgroups may limit the generalizability of these findings. Larger, more diverse cohorts are necessary to substantiate these findings and to explore potential gender- and lifestyle-related differences in the interplay between Hcy and PCSK9. We acknowledge the importance of apo B-containing lipoproteins in PCSK9 binding. However, due to the focus of our study on total PCSK9 levels, we did not perform separate measurements for apo B-containing lipoproteins. In addition, although this study focused on the role of inflammatory and lipid-related indicators related to the effects of Hcy and PCSK9, it did not fully elucidate the underlying molecular mechanisms involved. Further investigation is required to elucidate precise pathways through which Hcy and PCSK9 contribute to coronary lesion development. Although inflammatory parameters vary among ACS types (STEMI, NSTEMI, and UA), we did not specifically analyze their distinct profiles and impacts on coronary lesion severity. Future studies focusing on type-specific inflammatory patterns may better elucidate the pathophysiology of different ACS presentations.

Conclusions

The present study offers novel perspectives on the mutual mediating effects of Hcy and PCSK9 with the coronary lesion severity in patients diagnosed with ACS. These underscore the necessity for integrated therapeutic approaches that target both lipid metabolism and Hcy levels, as well as the inflammatory pathways involved in ACS. The results also suggest that patient-specific factors such as renal function, gender, and smoking status should be considered when developing treatment plans.